TRENDS IN

RADIOPHARMACEUTICALS
(ISTR-2005)

VOLUME 2
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 PROCEEDINGS SERIES

TRENDS IN
RADIOPHARMACEUTICALS
(ISTR-2005)

PROCEEDINGS OF AN INTERNATIONAL SYMPOSIUM

ORGANIZED BY THE INTERNATIONAL ATOMIC ENERGY AGENCY
AND HELD IN VIENNA, 14–18 NOVEMBER 2005

In two volumes

VOLUME 2

INTERNATIONAL ATOMIC ENERGY AGENCY

VIENNA, 2007
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International Symposium on Trends in Radiopharmaceuticals (2005 :

Vienna, Austria)
Trends in radiopharmaceuticals : ISTR-2005 : proceedings of an
international symposium / organized by the International Atomic
Energy Agency and held in Vienna, 14–18 November, 2005. —
Vienna : IAEA, 2007.
p. ; 24 cm. (Proceedings series, ISSN 0074–1884)
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1. Radiopharmaceuticals — Congresses. 2. Nuclear medicine —

Congresses. I. International Atomic Energy Agency. II. Series:
Proceedings series (International Atomic Energy Agency).

IAEAL 07–00497
 FOREWORD

The growth of nuclear medicine depends on advances in

radiopharmaceutical development and discovery, as well as improvements in
instrumentation. The field of radiopharmaceuticals has witnessed continuous
evolution thanks to the contributions of scientists from diverse disciplines such
as chemistry, physiology and pharmacology. The IAEA has been supporting
activities in the field of radiopharmaceuticals, which has resulted in significant
capacity building in the above fields in Member States. Many Member States
have developed manufacturing facilities through technical cooperation projects
for the large scale production of radiopharmaceuticals which helped the growth
of nuclear medicine in those countries. IAEA efforts through coordinated
research projects have also helped in advancing research activities in the
development and utilization of new products in many Member States.
The International Symposium on Trends in Radiopharmaceuticals
(ISTR-2005) was organized in order to provide scientists and professionals
from 70 countries working in the field of radiopharmaceuticals and related
sciences with the opportunity to present their research to an international
audience. Sessions covered the most relevant topics of radiopharmaceuticals
chemistry, including radionuclide production, radiochemical processing,
manufacturing and quality control, quality assurance, latest advances in
radiopharmaceuticals research, good manufacturing practices and regulatory
aspects. On the basis of the invited presentations, papers and panel discussions
during ISTR-2005, several areas of possible future international cooperation
were identified.
This publication comprises two volumes and constitutes a record of the
symposium and includes a summary as well as invited papers presented. A
CD-ROM containing the unedited contributed papers which were presented in
the two poster sessions of the symposium is included in volume 2.
The IAEA gratefully acknowledges the contribution made by the various
participants to the success of this symposium, in particular to M.M. Vora for his
technical editing of the papers.
 EDITORIAL NOTE

The Proceedings have been edited by the editorial staff of the IAEA to the extent
considered necessary for the reader’s assistance. The views expressed remain, however, the
responsibility of the named authors or participants. In addition, the views are not
necessarily those of the governments of the nominating Member States or of the
nominating organizations.
Although great care has been taken to maintain the accuracy of information
contained in this publication, neither the IAEA nor its Member States assume any
responsibility for consequences which may arise from its use.
The use of particular designations of countries or territories does not imply any
judgement by the publisher, the IAEA, as to the legal status of such countries or territories,
of their authorities and institutions or of the delimitation of their boundaries.
The mention of names of specific companies or products (whether or not indicated
as registered) does not imply any intention to infringe proprietary rights, nor should it be
construed as an endorsement or recommendation on the part of the IAEA.
The authors are responsible for having obtained the necessary permission for the
IAEA to reproduce, translate or use material from sources already protected by
copyrights.
 CONTENTS OF VOLUME 2

PHARMACOLOGY AND THERAPEUTIC

RADIOPHARMACEUTICALS (SESSION 8)

Biological and chemical evaluation of various radiocolloids used for

clinical radiosynovectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
G.A. Jánoki, L. Balogh, A. Polyák, D. Máthé, K. Kőrösi, R. Király

Targeted radiotherapy with alpha particle emitting radionuclides . . . . . . . 25

M.R. Zalutsky, O.R. Pozzi, G. Vaidyanathan

DOTA-Tyr3-octreotate labelled with 177Lu and 131I . . . . . . . . . . . . . . . . . . . 39

V. Lungu, D. Niculae, D. Chiper, M. Radu

177
Lu labelled nitroimadzoles and nitrotriazoles for possible use
in targetedtherapy of hypoxic tumours . . . . . . . . . . . . . . . . . . . . . . . . . 51
T. Das, S. Chakraborty, A. Mukherjee, S. Banerjee, G. Samuel,
H.D. Sarma, M. Venkatesh

Labelling and biological evaluation of anti-CD20 for treatment of

non-Hodgkin’s lymphoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
P. Oliver, A. Robles, V. Trindade, P. Cabral, V. Tortarolo,
A. Nappa, G. Rodriguez, H. Balter

THERAPEUTIC RADIOPHARMACEUTICALS (SESSION 9)

177
Lu-DOTA-J591 monoclonal antibody: Chemistry, toxicity, dosimetry
and clinical efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
S.J. Goldsmith, S. Vallabhajosula, M.I. Milowsky, D.M. Nanus,
N.H. Bander

Radiolabelled somatostatin analogues for radionuclide therapy

of tumours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
M. de Jong, D. Kwekkeboom, R. Valkema, E. Krenning

Iodine whole body scan, thyroglobulin levels, 99mTc MIBI scan and
computed tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
N.Ö. Küçük, S.S. Gültekin, G. Aras, E. İbiş
99m
Tc-MIBI and 131I scintigraphy in the follow-up of differentiated
thyroid carcinoma (DTC) patients after surgery . . . . . . . . . . . . . . . . . 123
S. Sergieva, T. Hadjieva, V. Botev, A. Dudov

Results of knee radiosynoviortesis in haemophilic and rheumatoid

arthritic patients with 32P colloid of local production . . . . . . . . . . . . . 135
V.E. Soroa, M.H. Velázquez Espeche, C. Giannone, G. Naswetter,
H. Caviglia, G. Galatros

PET RADIOPHARMACEUTICALS (SESSION 10)

Alternative methods of making [11C]amides: Application to the

preparation of 5-HT1A receptor radioligands . . . . . . . . . . . . . . . . . . . . 147
V.W. Pike, S.Y. Lu, J. Hong, J.L. Musachio, J.A. McCarron

[18F]fluoroethylated and [11C]methylated PET tracers for research and

routine diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
M. Mitterhauser, W. Wadsak

A novel finding: Anti-androgen flutamide kills androgen independent

PC-3 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
F. Al-Saeedi

A semi-automated [13N]NH3 production module: Design, quality control

and optimization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
A.R. Jalilian, P. Rowshanfarzad, M. Sabet, M. Mirzaii, A. Ziaee,
D. Sardari

RADIOIODINE RADIOPHARMACEUTICALS (SESSION 11)

Production of radioiodines with medical PET cyclotrons . . . . . . . . . . . . . . 209

J.J. Čomor, G.-J. Beyer, G. Pimentel-Gonzales

New developments in radioiodinated radiopharmaceuticals for SPECT

and radionuclide therapy: [123I]/[131I] labelled L- and
D-phenylalanine analogues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
M. Bauwens, J.J.R. Mertens, T. Lahoutte, K. Kersemans, C. Gallez,
A. Bossuyt
Comparison of 131I-TYR3-octreotate and 131I-DOTA-TYR3-octreotate:
The effect of DOTA on pharmacokinetics and stability . . . . . . . . . . . 243
E.B. de Araújo, E. Muramoto, L.T. Nagamati, J.S. Caldeira Filho,
R.M. Couto, C.P.G. Silva

Iodine labelled diethylstilbestrol (DES) of high specific activity:

A potential radiopharmaceutical for therapy of estrogen receptor
positive tumours and their metastases?. . . . . . . . . . . . . . . . . . . . . . . . . 253
T. Fischer, H. Schicha, K. Schomäcker

18
F RADIOPHARMACEUTICALS AND AUTOMATION OF
SYNTHESIS (SESSION 12)
18
F based radiopharmaceuticals and automation of synthesis . . . . . . . . . . . 265
P.K. Garg, S. Garg

Rapid method for radiofluorination of pyridine derivatives:

Prosthetic groups for labelling bioactive molecules. . . . . . . . . . . . . . . 283
I. Al Jammaz, B. Al Otaibi, H. Ravert, J. Amartey

1-[18F]fluoroethyleneglycol-2-nitroimidazoles:
A novel class of potential hypoxia PET markers . . . . . . . . . . . . . . . . . 295
R.J. Abdel-Jalil, M. Übele, W. Ehrlichmann, W. Voelter,
H.-J. Machulla

Radiosynthesis and in vivo evaluation in melanoma-bearing mice of

O-(2-[18F]fluoroethyl)-L-tyrosine as a tumour tracer . . . . . . . . . . . . . 297
Mingwei Wang, Duanzhi Yin, Yongxian Wang

CYCLOTRON BASED RADIONUCLIDES AND GENERATORS

(SESSION 13)

Production of radionuclides with a cyclotron. . . . . . . . . . . . . . . . . . . . . . . . . 311

D.J. Schlyer

Perspectives for the large scale production of radiolanthanides with

medical potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
G.-J. Beyer, H.L. Ravn, U. Köster

Production of 123I-MIBG IPEN-CNEN/SP . . . . . . . . . . . . . . . . . . . . . . . . . . 349

M.F. de Barboza, V. Sciani, R. Herrerias, M.M.N. Matsuda,
 N.T.O. Fukumori, L.C.A. Sumiya, H. Matsuda, A.A. Souza,
M.M. Goes, J.T. Pires, J. Mengatti, C.P. Gomez da Silva

A new 82Sr–82Rb generator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

A. Bilewicz, B. Bartoś, R. Misiak, B. Petelenz

Cyclotron production of 103Pd via proton induced reactions on

a 103Rh target. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
M. Sadeghi, H. Afarideh, G. Raisali, M. Haji-Saeid

The status and potential of new radionuclide generators providing

positron emitters to synthesize new targeting vectors for PET . . . . . 367
F. Roesch, K.P. Zhernosekov, D.V. Filosofov, M. Jahn,
M. Jennewein

RADIOPHARMACY (SESSION 14)

Nuclear pharmacy practices in the United States of America . . . . . . . . . . . 385

K. Ozker

Regulatory aspects of hospital radiopharmacy and clinical trials . . . . . . . . 391

A.A. Soylu

Standardization and quality control of an in-house formulation of

99m
Tc(V)-DMSA in tumour imaging and assessment of tumour
biology: Work in progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
P.S. Choudhury, N.C. Goomer, A. Gupta, D.C. Doval, T. Kataria,
A.K. Vaid, P.K. Sharma

Development of centralized radiopharmacies in Spain:

A successful experience in Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
I. Oyarzábal, R. Jiménez-Shaw

Chairpersons of Sessions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421

Secretariat of the Symposium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
Programme Committee. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
List of Participants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
PHARMACOLOGY AND THERAPEUTIC
RADIOPHARMACEUTICALS

(Session 8)

Chairpersons

C. DECRISTOFORO
Austria

K.K. SOLANKI
IAEA
.
 BIOLOGICAL AND CHEMICAL EVALUATION OF
VARIOUS RADIOCOLLOIDS USED FOR CLINICAL
RADIOSYNOVECTOMY

G.A. JÁNOKI, L. BALOGH, A. POLYÁK, D. MÁTHÉ, L. KŐRÖSI,

R. KIRÁLY
“Fodor József” National Centre of Public Health, “FJC” National
Research Institute for Radiobiology and Radiohygiene,
Budapest, Hungary
Email: janoki@hp.osski.hu

Abstract

Radiopharmaceuticals used for radiosynovectomy are the following: 90Y-citrate

colloid, 90Y-silicate colloid, 169Er-citrate colloid, 166Ho-phytate and 188Re-tin colloid.
Radiochemical purity and stability in all cases were higher than 99.0%. The particle sizes
of the radiocolloids were different; the mean values were as follows: 90Y-citrate colloid:
3.1 µm, 90Y-silicate colloid: 0.9 µm, 167Er-citrate colloid: 1.1 µm, 166Ho-phytate: 0.66 µm,
188
Re-tin colloid: 0.6 µm. During biodistribution studies in rabbits the activity values in
various organs (liver, blood, spleen, kidneys, lymph nodes, skeleton) outside of the
injected knee ranged up to 10% of injected dose (ID) even after 14 days since injection.
Activity values retained in the knee were in the range 95–87% of ID. Activity recoveries
during animal studies were also high during the initial time period (90–95%) and a week
after injection only dropped slightly below 90%. Owing to the favourable intra-articular
retention and very low leakage values in all cases, the calculated absorbed dose showed
high values in the target synovial surface (~40 Gy), low effective dose (5.3 mGy) and
also low whole body absorbed dose (1.9 mGy) values. According to preclinical data
obtained during this study, the authors concluded that all the colloid radiopharmaceuti-
cals tested showed very low leakage and that organs only accumulate insignificant
amounts of radioactivity. This leads to very favourable dosimetric calculations.

1. INTRODUCTION

Radiation synovectomy is a technique whereby a beta emitting radio-

pharmaceutical is delivered into the affected synovial compartment in order to
treat rheumatoid arthritis. Beta emitting radiocolloids are widely used for this
purpose. The ideal radionuclide would possess beta emission with sufficient
energy for a maxiumum tissue penetration of 5–10 mm, gamma emission
suitable for gamma camera imaging, a short half-life and ready availability. An

3
 JÁNOKI et al.

198
Au colloid was first used for radiocolloid synovectomy and this isotope has
been continously investigated. The main drawbacks of an 198Au colloid are the
411 keV gamma emission which creates an unnecessary radiation hazard, the
small particle size, which results in excessive loss from the joint space by
lymphatic drainage, and the high radiation doses to the proximal lymph nodes.
Other radiopharmaceuticals [1–17] that have been developed, studied
and registered for human use are listed in Table 1. The different radiation
properties of each therapeutic isotope determine their respective use regarding
the joint size. The authors studied the preparation and biological use of colloids
labelled with isotopes 90Y, 166Ho, 169Er and 188Re which are applicable for
treating the various joints for radiosynoviorthesis.

TABLE 1. RADIONUCLIDE USED FOR RADIATION

SYNOVECTOMY

Max.b– Tissue penetration depth

Radionuclide and T½ G energy (mm)
energy
pharmaceutical form (d) (KeV)
(MeV) Max: Min:
32
P colloid 14.4 1.71 7.9 2.6
198
Au colloid 2.7 0.96 411 3.9 1.2
165
Dy-FHMA 0.1 1.29 95 5.7 1.3
90
Y-Ca-oxalate 2.7 2.26 11.0 3.6
ferric hydroxide
citrate colloid
silicate
166
Ho-hydroxyapatite 1.2 1.85 81 8.5 2.1
-FHM-phytate
153
Sm-hydroxy MA 1.9 0.811 103 3.1 0.7
169
Er-citrate colloid 9.4 0.35 1.0 0.3
186
Re colloid 3.7 1.07 137 3.6 1.2
-rhenium sulphide
188
Re-tin colloid 0.7 2.12 155 11.0 2.1
-sulphur colloid
-microsphere

4
 SESSION 8

2. MATERIALS AND METHODS

Compounds tested during this study were as follows:

90
Y-citrate colloid (CIS bio YMM-1), 90Y-silicate colloid (GE Amersham
Health YAS-2P), 169Er-citrate colloid (CIS bio ERMM-1) and 188Re-tin colloid
were prepared in the “Fodor József” laboratory using methods published
previously [3], 166Ho-phytate suspension was the test compound obtained from
the Isotope Institute Ltd (Hungary).

2.1. Methods for determination of activity ratio bound to colloid form

In the case of the 90Y-citrate-silicate colloid, the TLC method was used to
determine colloid bound activity. Merck Silicagel 5553 layer, solvent containing
64 mL n-Propanol, 16 mL water, 20 mL methanol and 1 g of tartaric acid were
used and 1–5 µL volume of sample was deposited.
The Rf value of radiocolloids was zero. For in vivo stability study 100 µL
of radiocolloid was inoculated in 3 mL of rabbit synovial fluid at 37ºC for 24 h
and 5 d. After inoculation, colloid bound activity ratios were determined by
using the same TLC method used earlier.

2.2. Methods used for particle size determination of various radiocolloids

The analytical instrument (DynaPro) used in the authors’ experiments is

a product of Proteinsolutions Inc. (United States of America). The sample is
illuminated by a semiconductor laser of ~830 nm wavelength. The light
scattered by the sample in the cell is collected and guided via a fibre optic cable
to an actively quenched, solid state single photon counting module. The
photons are then converted to electrical pulses and correlated. The DynaPro
analyses the timescale of the scattered light intensity fluctuations by a mathe-
matical process called autocorrelation. The translational diffusion coefficient of
the molecules in the sample cell is determined from the decay of the intensity
autocorrelation data. The hydrodynamic radius of the sample is then derived
from the translational diffusion coefficient, using the Stokes-Einstein equation.
In the authors’ experiments, 160 nm and 240 nm polystyrene calibration
standards (Bangs Laboratories Inc., Serial No. 5692) were measured in parallel
with the samples.

2.3. Biodistribution study

New Zealand white male laboratory rabbits weighing 2–3 kg were

purchased. Rabbits were individually housed in 60 cm × 80 cm × 80 cm metal

5
 JÁNOKI et al.

cages. Environmental conditions in the animal room were maintained at an

average temperature of 20.5ºC (ranging from 18.6–23.8ºC), an average relative
humidity of 55% (ranging from 32% to 66%), and a 12 h light/12 h dark
photoperiod. The rabbits had ad libitum access to fresh standard rodent feed
manufactured and purchased by Biofarm Kft., Hungary. The rabbits had ad
libitum access to fresh tap water, provided daily in glass bottles with stainless
steel sipper tubes. Feed certification was performed by the manufacturer.
Water analysis was performed by the “Fodor József” National Centre for
Public Health, National Institute of Public Health Water Laboratories
(Budapest, Hungary) and included dissolved solids, conductivity, microbial
content (heterotrophic plate count) and heavy metals. The animals were accli-
matized for a minimum of 5 d. General health status was monitored by the
veterinarian in charge.
Cage side observation of general health, behaviour and appearance was
made at least once daily. The rabbits were under continuous observation for
any clinical signs for at least 1 h after dosing. Any clinical signs were recorded.
The animals were weighed at receipt, before dosing and before being sacrificed.
The animals used for the study were received in good health and were
clinically free from any apparent abnormalities or disease. A veterinarian
examined the animals within 24 h of receipt and deemed them healthy and free
of gross abnormalities.
Prior to dose administration, the animals were weighed and anaesthetized
by intramuscular administration of 100 mg ketamine hydrochloride/kg b.w.
(SBH-Ketamin, SBH Kft., Hungary) and 2 mg xylazine/kg b.w. (Primazin,
Alfasan B.V., Netherlands). This resulted in an anaesthetic and immobile state
of at least 30 min.
To assess the needle positioning during the injections, some rabbits were
checked by X ray (BV 22, Müller Co., Germany) and ultrasound (SpinelVET
2000, Echoson Ltd, Poland) imaging. The needle was found to be correctly
positioned in the knee joint cavity with the proposed injection method as
described in Section 2.4.

2.4. Dose administration

An aliquot of 100 µL of the supplied test article suspensions was injected

into the knee joint cavity. After induction of general anaesthesia, the hair
above the knee joint was shaved and the skin cleaned and disinfected with
iodine spray. The knee joints were punctured in slight flexion through the first
third of the median patellar ligament with an analytical syringe with fixed
needle (Hamilton Corp., USA) under aseptic conditions. The rabbits received

6
 SESSION 8

an activity in the range 105–500 µCi (5.6–18.5 MBq)/100 µL of the test article
into their knee joint randomly selected.
As previous experiments in the NRIRR have shown, in the case of
gamma camera (Nucline X-Ring/R, Mediso Ltd, Hungary) imaging of 90Y
bremsstrahlung in a fully open energy window centred at 140 keV, a vial with
0.55 MBq and a vial containing 25 MBq of activity placed at the same detector
can be distinguished visually by the operator. Thus, correctness of the injections
was controlled with the use of a gamma camera, the exclusion criterion being
the appearance of a second spot of activity near the injection site. Immediately
after dosing, the animal was placed in the ventrodorsal position above the
detector and the scintigraphic image was checked. In the case of two or more
spots appearing on screen, the fact was recorded and the animal was discarded
from the experiment. If the activity spot was unique and in the region of the
knee, the injection was deemed to be correct. Figure 1 shows an example of
correct injection.

2.5. Sample collection, processing and analysis

At the end of the study, the rabbits were anaesthetized again by intra-
muscular administration of 100 mg ketamine/kg b.w. (SBH-Ketamin, SBH Kft.,
Hungary) and 2 mg xylazine/kg b.w. (Primazin, Alfasav B.V., Netherlands). The
animals were sacrificed 5–10 min after anaesthesia induction by intracardial
injection of 0.5 mL/kg b.w. of a specific veterinary drug for euthanasia, T 61® ad

FIG. 1. Ventrodorsal whole body scintillation image of a correctly injected rabbit. The
only spot appearing corresponds to the injection site at the knee.

7
 JÁNOKI et al.

us. vet. (Intervet B.V., Netherlands). All the animal handling methods used in
the study were in compliance with all applicable sections of the Hungarian
Laws No. XXVIII/1998 and LXVII/2002 on the protection and welfare of
animals.
Animals were sacrificed at 6, 24 and 48 h and 5 d post-injection. The
samples of the injected knees (with 1 cm of femoral and tibial bone outside the
capsule) were collected. The samples were washed in physiological saline, dried
with absorbent paper and weighed after collection. Samples were collected in
containers labelled according to animal identification, organ, collection time
points and date.
Samples were placed in individually identified porcelain crucibles. The
wet net weight of the samples was calculated from weight measurements made
on the empty crucible and containing the sample. Samples were desiccated in a
thermodryer at 200°C for 24 h and ashed in a laboratory furnace (OH-63,
OMSZOV, Hungary) at 600°C for 12 h. The net weight of each ash sample was
determined by measuring the crucibles with and without ashes with an
analytical balance to four digits.
Concentrated HNO3 was added to the ash samples and the solution of
each sample was brought to a fixed 10 mL volume. This solution was incubated
for at least 12 h at room temperature and homogenized by a Vortex mixer
several times during dissolution. Immediately after the last homogenization,
1 mL aliquots of the sample solutions were transferred to plastic measurement
test tubes in triplicate.

2.6. Immobilization of the injected knee

After the dose administration and a 30 min totally immobile period due
to general anaesthesia, the injected knee of the rabbits in the immobilized
group was held by a PVC plastic splint formed individually for the animals and
fixed by tape and bandage to hold the knee at semi-flexion, to facilitate the
keeping of a natural seated position of the rabbit in the cage. This immobili-
zation splint was kept on the knees for 24 h to ensure proper and indulgent
immobilization of the joint, as demonstrated by preliminary experiments at the
NRIRR.

2.7. Radioactivity measurements

Before sample measurements, the device, a NaI(Tl) crystal gamma scintil-

lation counter with automatic sample changer and multichannel signal
amplifier–analyser (NZ-310, Gamma, Hungary), was checked for linearity of
bremsstrahlung measurements and sensitivity. Radioactivity was determined in

8
 SESSION 8

triplicate sample solutions. It was found that the device measurements are
linear in the range of 750–2694 kBq for 1 mL sample volumes and 60 s
measurements.

2.8. Statistical methods for data analysis

Standard deviations (SD) of data were calculated using n-1 as the degree
of freedom for multiplying n in the divider of the appropriate formula.
Comparison of the groups was performed using the two-tailed Student’s t-test
and the Mann-Whitney U-test. ANOVA calculations were made to compare
the different organ uptake values at the different time points. On the basis of
biodistribution data, retention times, pharmacokinetic curves and the absorbed
doses were calculated using Prism 4.0 and MIRDOSE 3.1 softwares.

3. RESULTS

3.1. Radiochemical purity study

The results of radiochemical purity and stability data of the five

compounds studied are given in Table 2.

3.2. Particle size determination

A summary of particle size determinations are given in Figs 2 and 3. The

results for standard colloids are also shown.

TABLE 2. COLLOID BOUND ACTIVITY OF

RADIOPHARMACEUTICALS USED FOR RADIOSYNOVECTOMY

Colloid bound activity (%)

Compounds
in saline in synovial fluid
90
Y-citrate colloid 99.2 99.8
90
Y-silicate 99.7 99.2
167
Er-citrate colloid 99.3 99.5
166
Ho-phytate 99.2
188
Re-tin colloid 97.3

9
 JÁNOKI et al.

FIG. 2. Colloid size distribution of registered radiopharmaceuticals available for radio-

synovectomy.

FIG. 3. Colloid size distribution of research radiopharmaceuticals used for radiosynovectomy.

10
 SESSION 8

4. BIODISTRIBUTION STUDY

90
4.1. Y-citrate/silicate colloid

Biodistribution of these two 90Y labelled colloids are seen in Tables 3 and 4.

167
4.2. Er-citrate colloid

In the present study, the remaining activity in rabbit knee after intra-
articularly injected 167Er-citrate colloid suspension was determined. At three
different time points (6 h, 2d, 8 d) knee, bone and blood were sampled and
measured by liquid scintillation techniques. Radiochemical purity of the
filtered batch used in the present work was high throughout the study (>99%).
Activity values retained in the knee as injected dose per cent were as
follows:

6 h: 90.8 ± 0.36%; 2 d: 87.2 ± 0.05%; 8 d: 80.07 ± 0.04%.

TABLE 3. SUMMARY OF DISPOSITION OF 90Y-CITRATE COLLOID AT

DIFFERENT TIME POINTS IN ORGANS AFTER INTRA-ARTICULAR
INJECTION
(%ID/whole organ)

Time
6h 24 h 5d 8d 13 d
point

Dose %
in whole Ave. ± SD Ave. ± SD Ave. ± SD Ave. ± SD Ave. ± SD
organ

Blood 0.158 0.386 0.083 0.120 1.454 2.606 0.885 2.168 0.018 0.030
Liver 0.318 0.307 0.301 0.437 0.749 1.009 0.272 0.196 0.082 0.183
Spleen 0.014 0.017 0.029 0.030 0.050 0.043 0.099 0.051 0.046 0.048
Kidney 0.120 0.106 0.061 0.047 0.205 0.095 0.248 0.372 0.082 0.077
Testes 0.006 0.010 0.029 0.032 0.008 0.008 0.055 0.069 0.000 0.000
Ing. 0.006 0.008 0.015 0.016 0.000 0.001 0.075 0.064 0.059 0.081
lymph
node
Whole 1.396 2.495 0.050 0.050 3.252 1.023 6.300 2.969 0.150 0.196
skeleton
Knee 95.984 2.479 94.265 1.733 91.282 2.721 88.116 4.851 87.600 2.074

11
 JÁNOKI et al.

TABLE 4. SUMMARY OF DISPOSITION OF 90Y-SILICATE COLLOID AT

DIFFERENT TIME POINTS IN ORGANS AFTER INTRA-ARTICULAR
INJECTION
(%ID/whole organ)

Time point 6h 24 h 48 h 5d

Dose %
in whole Ave. ± SD Ave. ± SD Ave. ± SD Ave. ± SD
organ

Blood 0.025 0.030 0.277 0.379 0.008 0.011 0.076 0.155

Liver 0.033 0.026 0.012 0.005 0.005 0.010 0.064 0.066
Popl. lymph. 0.085 0.194 0.002 0.005 0.001 0.001 0.003 0.005
node
Ing. 0.010 0.006 0.007 0.006 0.000 0.001 0.006 0.010
lymph node
Whole 2.673 0.946 1.814 0.782 0.368 0.122 5.013 2.615
skeleton
Knee 100.106 10.978 91.480 8.592 84.451 13.522 78.894 13.320

Low level leakage was documented also with the very low blood activity
which was always less than 0.01%ID and with the low whole skeleton uptake
which ranged 1.7–4.3%ID.
None of the rabbits used in this experiment showed any chemical or
radiation toxicity signs, including any local intolerance.

188
4.3. Re-tin colloid

Retention of 188Re-tin colloid in the synovial space of the rabbits was

observed up to 72 h.
No leakage to the inguinal lymph node (the predilection site of the
accumulated outflow of radioactivity) or other lymph nodes could be observed.
Figure 4 presents static scintigraphic images of the knee regions of a rabbit at 3,
24, 48 and 72 h after injection into the knee. The knee:knee radiopharmaceu-
tical uptake ratios were stable over time. In whole body scans, no other activity
accumulation was seen anywhere in the body (e.g. in the predilection sites such
as the thyroid gland and the gastric mucosa). The scans of healthy rabbits
receiving only intra-articular 188Re perrhenate solution revealed uptake in the
predilection sites — stomach mucosa and thyroid gland were visualized in the
images, most prominently 3 h after injection (Fig. 5). Later, healthy rabbit scans

12
 SESSION 8

FIG. 4. Static images of the knee of a rabbit taken at 3, 24, 48 and 72 h after injection.
Differences in background are due to the automatic cut-off level setting of the camera
software. Rabbit contours were generated from the acquisition data by using the Inter-
View© software.

demonstrated a very fast excretion of perrhenate via the kidneys and bladder
48 and 72 h post-injection; only background activity could be observed in
healthy rabbits receiving perrhenate eluate only.

166
4.4. Ho-phytate biodistribution

The intra-articularly injected 37 MBq/0.1 mL 166Ho-phytate solution is

retained at the injection site. Results 6, 24, 72 and 168 h after injection showed
that 88.5–98.5% of injected dose remains in the knee sample. Leakage was in
the range 1.5–11.5%. Only liver, kidney, lung and blood samples contained low
amounts of activity. Because no lymph nodes contained measurable activity the
authors conclude that only low levels of leakage occurred by the hematogene
process. Figure 6 shows 166Ho-phytate biodistribution 48 h after injection.
Even after 168 h of injection the levels were: knee: 92%; liver: 2.8% and
kidney: 1.5%; with cummulative activities: urine 1.19% and faeces 2.23%.

13
 JÁNOKI et al.

FIG. 5. Comparison of biodistributions of the same amount and activity of intra-articular

perrhenate eluate (A) and 188Re-tin colloid solution (B) in rabbit, 3 h post-injection.

168 h after injection

Knee: 92%
Liver: 2.8%
Kidney: 1.5%

Cummulative activity
In urine: 1.19%
In faeces: 2.23%

166
FIG. 6. Ho-phytate distribution in rabbit 48 h after intra-articulation injection.

14
 SESSION 8

4.5. Dosimetric evaluation

The residence time of various radiocolloids tested are shown in Table 5.

The doses delivered to the knee are summarized in Table 6. The knee doses
were calculated with the MIRD unit density spheres method (the authors
considered the adult knee a 300 g sphere of 8.19 cm diameter). Absorbed
radiation dose of various organs by the MIRD method is presented in Table 7.
The dose calculations were performed using the standard MIRD method
(MIRD pamphlet no.1 Society of Nuclear Medicine, 1976) and Olinda 1.0
software.

TABLE 5. RESIDENCE TIMES IN THE KNEE FOR DIFFERENT

COLLOID RADIOPHARMCEUTICALS
90 90 90
Y-citrate Y-citrate Y-silicate 169Er-citrate 188
Re-tin 166
Ho-phytate

Observation 13 d 5d 5d 19 d 3d 7d
time
T1/2 2.67 d 2.67 d 2.67 d 9.4 d 0.71 d 1.12 d
Residence time (h)
Knee 282.072 112.081 103.152 395.042 10.647 157.380

TABLE 6. SUMMARY OF KNEE DOSES AFTER INTRA-ARTICULAR

APPLICATION OF DIFFERENT RADIOCOLLOIDS
90 90
Y-citrate Y-citrate 90Y-silicate 169Er-citrate 188Re-tin 166
Ho-phytate
Target organ colloid (13 d) colloid (5 d) colloid (5 d) colloid (19 d) colloid (3 d) colloid (7 d)
(Gy/MBq) (Gy/MBq) (Gy/MBq) (Gy/MBq) (Gy/MBq) (Gy/MBq)

Knee 0.487 0.194 0.178 0.0778 0.0156 0.206

15
 JÁNOKI et al.

TABLE 7. ESTIMATED ABSORBED RADIATION DOSE

INTRA-ARTICULAR APPLICATION
(phantom: 70 kg adult)
90 90
Y-citrate Y-citrate 90Y-silicate 169Er-citrate 188
Re-tin 166Ho-phytate
Target organ colloid (13 d) colloid (5 d) colloid (5 d) colloid (19 d) colloid (3 d) colloid (7 d)
(µSv/MBq) (µSv/MBq) (µSv/MBq) (µSv/MBq) (µSv/MBq) (µSv/MBq)

Adrenals 11.800 5.580 0.796 0.119 7.720 8.680

Brain 11.800 5.580 0.796 0.119 7.680 7.640
Breasts 11.800 5.580 0.796 0.119 7.390 7.520
Gall bladder 11.800 5.580 0.796 0.119 7.550 9.290
wall
LLI wall 11.800 5.580 0.796 0.119 7.630 7.530
Small intestine 11.800 5.580 0.796 0.119 7.580 7.690
Stomach wall 11.800 5.580 0.796 0.119 7.500 7.720
ULI wall 11.800 5.580 0.796 0.119 7.550 7.820
Heart wall 11.800 5.580 0.796 0.119 7.560 87.400
Kidney 890.000 5.580 0.796 0.119 7.570 243.000
Liver 323.000 160.000 8.800 0.119 7.520 552.000
Lungs 11.800 5.580 0.796 0.119 7.550 126.000
Muscle 11.800 5.580 0.796 0.119 7.560 7.660
Ovaries 11.800 5.580 0.796 0.119 7.610 7.540
Pancreas 11.800 5.580 0.796 0.119 7.610 8.320
Red marrow 636.000 125.000 187.000 111.000 119.000 226.000
Osteogenic cells 1280.000 252.000 374.000 991.000 259.000 595.000
Skin 11.800 5.580 0.796 0.119 7.430 7.440
Spleen 521.000 5.580 0.796 0.119 7.530 7.680
Testes 11.800 5.580 0.796 0.119 7.460 7.330
Thymus 11.800 5.580 0.796 0.119 7.500 7.630
Thyroid 11.800 5.580 0.796 0.119 7.590 7.520
Urinary bladder 11.800 5.580 0.796 0.119 7.520 7.390
wall
Uterus 11.800 5.580 0.796 0.119 7.570 7.470
Total body 91.500 22.600 20.700 16.700 19.700 49.100
Effective dose 227.000 36.600 34.800 43.100 28.500 117.000
equivalents
(µSv/MBq))
Effective dose 121.000 29.900 27.200 23.300 23.000 82.000
(µSv/MBq))

16
 SESSION 8

5. DISCUSSION

5.1. Discussion of literature data

Synovectomy by intra-articular application of β emitting radioisotopes

(radiation synovectomy) was introduced in 1952 by Fellinger et al. [18] for
treatment of an inflamed synovial membrane. Since that time a number of
radiopharmaceuticals have been studied [19–23]. In Europe recently, the
following compounds are registered and used to treat various sized joints:
90
Y-silicate, 90Y-citrate colloids, 186Re-sulphide and 169Er-citrate. Today, radio-
synoviorthesis is an alternative or even supplementary therapeutic approach to
pharmacotherapy for the treatment of patients suffering from painful, inflam-
matory or severe degenerative joint diseases [24–28].
After intra-articular injection, radiocolloids are phagocytosed by the
superficial synovial cells. Owing to irradiation, necrosis of the superficial
synovial layer is observed from the first day on. Early animal studies in rabbits
demonstrated the uptake of the radiocolloid by the synovial membranes and
homogenous distribution throughout the entire tissue by autoradiography.
After injection of 0.59 MBq (16 µCi) of 88Y (the isotope chosen for its
gamma radiation which increases counting precision), a study reported that
87–100% of the injected yttrium is recovered in the articulation after 7 d.
Another study showed that 24 h after intra-articular injection of 3.7–37 MBq of
90
Y-citrate colloid, 0.2% of the activity is recovered in the blood and 0.4% and
0.13% in urine and faeces respectively. The problem of radioactivity leakage
from the joint was experienced during the human clinical practice. Several
studies have suggested that particle size is critical in limiting the leakage of
radiocolloids from the synovial joint [7, 12, 27]. Attempts to quantify the
amount of leakage have given values of 5–10% at 24 h after administration and
between 15–25% of injected dose at 5 d after application of small-sized
(10–100 nm) 90Y-silicate particles. Even from moderate leakage, the radiation
absorbed dose to normal organ (e.g. liver, regional lymph nodes) has raised the
level of concern because of the unnecessary radiation burden.
There have been only a few animal studies to determine the amount of
leakage occurring during radiation synovectomy using 90Y colloids or other
radiopharmaceutical products [7, 12, 29–31].
Dedicated animal studies to determine all factors affecting the amount of
leakage, stability of colloid in vitro and in synovium, including particle size and
size distribution of colloid, biodistribution after intra-articulation injection in a
large number of animals at different time points and presented as ID% in
whole organ (lymph node, skeleton, blood, liver and knee) have not been
published until now. The authors’ designed studies allowed monitoring and

17
 JÁNOKI et al.

comparison of extra-articular leakage and biodistribution of different

registered and experimental products. Their experimental works were based on
detailed protocol which covered all aspects of the work dealing with experi-
mental animal testing, biodistribution of radiopharmaceuticals and, finally,
handling of low level radioactive waste.
During housing, anaesthesia and sacrifice of experimental animals,
current laws on protection and welfare of animals were followed. Activity
measuring devices and other tools used during experiments were calibrated
and proven to be sensitive and precise. For each animal, the quality (success) of
intra-articular administration was followed and documented by gamma camera
scintigraphy, X ray or ultrasound image.
Immobilization of the group of animals was also managed. PVC plastic
splints were formed individually for each animal and fixed by tape and bindings
to hold the knee of the rabbits in a semi-flexed position.
The testing facility at the “Fodor József” National Centre of Public
Health, “Frederic Joliot-Curie” National Research Institute for Radiobiology
and Radiohygiene, the Diagnostic and Therapeutic Isotope Laboratory, has no
GLP status but during the experiments and reports the GLP rules were
followed.
The compiled study set-up allowed determination of the volume of
leakage and the amounts of various organ uptake, if any occurred.

5.2. Discussion of the authors experiments

The radiochemical purity and stability studies (in vitro and in synovial
fluid) of different radiocolloids showed very uniform results. In all cases, the
rate of colloid bound activity always exceeded 99% throughout the whole
experiment. The mean particle size of this colloid was 0.6–3.1 µm and after
intra-articular injection into 149 rabbits the animals showed no sign of chemical
or radiation toxicity. No local intolerance was reported. The injection
technique developed and practiced was controlled by XR, US and scintigraphy
and allowed personnel to control any leakage relating to the incorrectly
positioned needle in the knee joint cavity.
The distribution of injected activity in various organs outside the knee
(this value is equivalent to leakage activity) such as the liver, spleen, kidneys,
testes, blood, lymph nodes and skeleton was very low; in most cases it was
around 1% ID during the whole study.
The long stability of radiocolloids is exemplified by 90Y-citrate colloid
where applied activity values retained in the knee between 6 h and 13 d ranged
from 93.61 ± 1.67% to 86.80 ± 1.59% of injected activity in the non-
immobilized group and 95.10 ± 0.91% to 87.68 ± 2.26% in the immobilized

18
 SESSION 8

group of animals. The non-significant difference occurring between the

immobilized and non-immobilized groups can be explained by the influence of
anaesthesia and animal movement behaviour. The anaesthetic state in both
groups was at least 30 min. This is generally followed by a reconvalescent
period, usually connected to restricted moving. This can be a time long enough
for the colloid to bind into the joints. The higher blood perfusion of the joints in
rabbits can account for a shortened leakage time window than in humans.
Owing to behavioural characteristics, rabbits tend to redistribute their weight
onto the untreated legs after any treatment. This means that in any case, an
animal will move and charge less its treated leg and will rather use the
remaining three limbs. The self-restriction of movement in the treated leg will
be present in all animal models of joint treatment. The moving space available
for a rabbit in a standard 60 cm × 80 cm × 80 cm cage is enough to provide for
the habitual moving needs of the animal. However, these natural movements
are restricted in a small space with a lower amplitude of movement. The lack of
higher angle flexion and extension of the joint can result in a normal blood
perfusion. There is no increased blood flow (consequently the lymph flow also
remains normal) that could lead to higher leakage. The investigators found no
activity in the splints, thus there is no evidence of misinjection or increased
pressure in the joint space.
The activity recovery during studies was also high at early time points
(90–95%); only 13 d after injection did it drop slightly below 90%.
On the basis of the favourable intra-articular retention and very low
leakage values, the calculated absorbed dose showed high values in the target
synovial surface (~40 Gy) and low effective dose (5.3 mGy) and also low whole
body absorbed dose (1.9 mGy) values.
In the scientific literature, Noble et al. [8] and Davis et al. [11] reported
studies about the animal evaluation of various radiocolloid agents. They
determined the leakage of radioactivity from the rabbit synovial pouch and
found that it is dependent on the size and resistance to degradation of the
various colloids (e.g. 99mTc-FeHMAA, MAA, phytate, 90YCa-oxalate, 90Y-ferric
hydroxide). In their work, Noble et al. [8] reported leakage values of 0.3–65%
ID after 24 h of intra-articular injection of 99mTc labelled diagnostic radio-
pharmaceuticals. Davis et. al [11] published results that showed that their
in-house prepared 90Y-oxalate and 90Y-ferric hydroxide macro-aggregates were
sized in the range 1–10 µm, and therefore the leakage was less, 5 ± 2%.

19
 JÁNOKI et al.

6. CONCLUSION

In their review article, Kampen et al. [24] reported a literature

compilation about the leakages of different radiopharmaceuticals. The
reviewed literature showed the leakage value for small-sized colloids (<30 nm),
as 198Au colloid, is up to 48% of ID, leading to a 50–150 Gy radiation dose to
the involved regional lymph nodes.
In the case of 90Y, they also report a radiation dose of more than 100 Gy
to regional lymph nodes. They explained that, owing to a lack of strict size
control of the colloidal radionuclide, some very small particles (fines) seem to
leave the joint and lead to this excessive leakage.
Moreover, a colloidal solution of 90Y of pH<6 may contain free 90Y ions
which can be easily drained from the treated joint. In these cases, besides the
problem of whole body (non-target) radiation burden, leakage of radionuclide
out of the treated joint will lead to a significant reduction of the radiation dose
imported to the synovial surface, which may fall to 60% and thus constitutes a
drop in the probability of clinical success.
If one summarizes literature information about the ‘ideal’ radiopharma-
ceutical for radiosynovectomy, the following have to be mentioned:

(a) The energy of the ß– particles must be high enough to penetrate the whole
depth of the inflamed synovial membrane.
(b) The colloid particle size should be between 1 and 5 µm.
(c) The hardness of particles (resistance to in vivo dissolution, enzymatic
action or mechanical stress) are also important.

In the light of literature to date, the evaluated 90Y-citrate/silicate colloid,

169
Er-citrate colloid, 166Ho-phytate and 188Re-tin colloid possess features that
are very close to literature advice. The leakage activity value of up to 2 weeks is
around 10% ID, and organs accumulate only insignificant amounts of radio-
activity and this leads to very favourable dosimetric calculations. The authors
believe that the reported data of this lege artis developed and evaluated
preclinical studies proved and support the efficient and safe use of radiocolloid
injection in humans.

ACKNOWLEDGEMENTS

The authors would like to thank M. Pállai, Z. Suhajda, K. Haller and

N. Fésüs for technical assistence and M. Kovács for preparation of the
manuscript.

20
 SESSION 8

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Persistent synovitis treated with radiation synovectomy using Yttrium-90: a retro-
spective evaluation of 83 procedures for 45 patients, British Journal of Rheuma-
tology 36: pp. 861 – 869, (1997).
[22] WUNDERLICH, G., et al., Preparation and biodistribution of Rhenium-188
labeled albumin microspheres b 20: a promising new agent for radiotherapy,
Applied Radiation and Isotopes 52: pp. 63 – 68, (2000).
[23] HEUFT-DORENBOSCH, L.L.J., DE VET, H.C.W., VAN DER LINDEN, S.,
Yttrium radiosynoviortheses in the treatment of knee arthritis in rheumatoid
arthritis: A systematic review, Ann. Rheum. Diss. 59 (2000) 583–586.
[24] KAMPEN, W.U., BRENNER, W., CZECH, N., HENZE, E., Intraarticular appli-
cation of unsealed beta-emitting radionuclides in the treatment course of inflam-
matory joint diseases, Curr. Med. Chem. Anti-inflamatory & Anti-allergy Agents
1 (2002) 77–87.
[25] VUORELA, J., SOKKA, T., PUKKALA, E., HANNONEN, P., Does Yttrium
radiosynovectomy increase the risk of cancer in patients with rheumatoid
arthritis?, Ann. Rheum. Dis. 62: pp. 251 – 253, (2003).
[26] EUROPEAN ASSOCIATION OF NUCLEAR MEDICINE, EANM procedure
guidelines for radiosynovectomy, Eur. J. Nucl. Med. BP12 – PB16 Vol . 30, No. 3,
March (2003).
[27] ERSELCAN, T., et al., Lypoma arborescens; successfully treated by Yttrium-90
radiosynovectomy, Annals of Nuclear Medicine, Vol. 17, No. 7, pp. 593 – 596,
(2003).
[28] SILVA, M., LUCK, J.V., LLINÁS, A., Chronic hemophilic synovitis: the role of
radiosynovectomy, Treatment of Hemophilia, 33, April (2004).
[29] MÄKELÄLA, O., et al., Experimental radiation synovectomy in rabbit knee with
holmium-166 ferric hydroxide macroaggregate, Nucl. Med. Biol. 29 (2002) 593–
598.
[30] WANG, S.-J., et al., Histologic study of effects of radiation synovectomy with
rhenium-188 microsphere, Nucl. Med. Biol. 28 (2001) 727–732.

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[31] UNNI, P.R., CHAUDHARI, P.R., VENKATESH, M., RAMAMOORTHY, N.,

PILLAI, M.R.A., Preparation and bioevaluation of 166Ho labelled hydroxyapatite
(HA) particles for radiosynovectomy, Nucl. Med. Biol. 29 (2002) 199–209.
[32] MÁTHÉ, D., et al., Preliminary studies with 188Rhenium-tin colloid for radiation
synovectomy: preparation, size determination, in-vivo distribution, effects and
dosimetry studies, Nuclear Medicine Review Vol 5., No 2., (2002)

23
.
 TARGETED RADIOTHERAPY WITH ALPHA
PARTICLE EMITTING RADIONUCLIDES

M.R. ZALUTSKY, O.R. POZZI, G. VAIDYANATHAN

Department of Radiology, Duke University Medical Center,
Durham, North Carolina,
United States of America
Email: zalut001@mc.duke.edu

Abstract

One of the most important considerations in the development of radiopharma-

ceuticals for the targeted radiotherapy of cancers is the selection of the type of radiation
emitted during the decay of the radionuclide. Alpha particle emitters have emerged as a
promising approach for certain clinical applications such as compartmentally spread
disease and neoplasms present in the blood. An attractive feature of radionuclides
decaying by the emission of α particles is that they offer the prospect of matching the
cell specific reactivity of targeting vehicles, such as receptor avid peptides and mono-
clonal antibodies, with radiation with a range of only a few cell diameters. In addition,
α particles have important radiobiological advantages when compared with conven-
tional external beam radiotherapy and β particles including a more potent cytotoxic
effectiveness, with sterilizing potential nearly independent of oxygen concentration,
dose rate and cell cycle position. This review summarizes the current status of targeted
radiotherapy with α particle emitting radionuclides. Because they have reached the
stage of clinical investigation, monoclonal antibodies labelled with the promising α
particle emitting radionuclides 213Bi, 225Ac and 211At will be highlighted.

1. INTRODUCTION

Targeted radiotherapy involves the use of a molecular carrier such as a

receptor avid compound or an antibody to deliver a radionuclide to malignant
cell populations. This emerging therapeutic strategy has a number of potential
advantages that compensate for its relatively complex nature. Compared with
conventional external beam radiation treatment, targeted radionuclide therapy
offers the prospect of delivering lethal radiation doses to tumour cells more
selectively while leaving neighbouring normal cells intact. Unlike conceptually
similar targeted therapeutics employing toxins or chemotherapeutics as the
cytotoxic agent, radionuclides do not require intracellular localization to be
effective. Indeed, one of the attractions of radionuclide therapy is the existence

25
 ZALUTSKY et al.

of radiation with quite different dimensions of effectiveness, ranging from

subcellular (Auger electrons) to hundreds of cell diameters (β particles). One
of the attractive features of a particles for targeted radiotherapy is their inter-
mediate tissue range equivalent to only a few cell diameters. As a consquence
of this property, as well as the radiobiological advantages discussed in the next
section, interest in the utilization of a particle emitters for targeted radio-
therapy has blossomed [1, 2].
Although the potential utility of a particle emitters for targeted radio-
therapy has been appreciated for many years, translation of this concept into
the clinical domain has been slow. Many of the reasons for this are in the realm
of radiopharmaceutical chemistry. One problem has been the poor availability
of a particle emitters with radionuclide decay and chemical properties that
have practical potential for cancer treatment. Another is the need for labelling
methods that provide sufficient stability in the in vivo environment to be
suitable for patient studies. This is a major challenge for radionuclides with
multiple a particle emitting progeny because each has its own chemical
behaviour. Finally, an important consideration for therapeutic radiopharma-
ceutical chemistry that is particularly relevant for a particle emitters is the
potentially deleterious effects of radiolysis on both labelling chemistry and
product stability.

2. RATIONALE FOR ALPHA EMITTERS

The best utilization of targeted radionuclide therapy is problably not for

the treatment of large tumours, which might be best treated by conventional
approaches, but rather, in minimal residual disease settings in which cancerous
deposits are small, perhaps even subclinical [3]. For this reason, radiations
which are well matched to the dimensions of micrometastatic disease might be
of particular value. In that regard, Humm [4] compared the fraction of decay
energy that would be deposited in spherical tumours for a high energy
b emitter (90Y), a low energy b emitter (131I) and an b emitter (211At). In
1000 mm diameter tumours, the calculated absorbed fractions were 0.097,
0.54 and ~0.9, for the particulate emissions of 90Y, 131I and 211At, respectively;
for 200 mm diameter lesions, absorbed fractions of 0.015, 0.17 and ~0.5 were
calculated. Extrapolating to single cell conditions, which are found in diseases
such as neoplastic meningitis, the calculated energy deposition advantage for
211
At compared with 90Y is about one thousand [5].
An important consequence of the relatively short range of a particles is
that this characteristic, in combination with their high energy (4–9 MeV for the
radionuclides of interest for targeted radiotherapy), imparts a high linear

26
 SESSION 8

energy transfer (LET) quality to this radiation. Yttrium-90 emits high energy
b particles that have a mean LET of about 0.2 keV/mm; in comparison, the LET
of clinically relevant a particle emitters is several orders of magnitude higher,
about 100 keV/mm. The radiobiological implications of high LET radiation are
perhaps the most compelling rationale for pursuing a particle emitters for
therapy [6]. Of primary importance is the fact that the relative biological effec-
tiveness of high LET a particles is considerably higher than b particles or
standard external beam radiation. Radiation at 100 keV/mm is particularly
cytotoxic because the distance between ionizing events at this LET is nearly
identical to that between DNA strands, increasing the probability of creating
highly cytotoxic double DNA strand breaks. The predicted exquisite cytotox-
icity of targeted a particle emitting radiopharmaceuticals has been validated
experimentally with a variety of radionuclides, carrier molecules and human
cancer cell lines [7]. In addition, the conditions under which high LET radiation
is maximally effective are relatively wide ranging, not being compromised by a
lack of oxygen, cell cycle stage or dose rate.

3. ALPHA PARTICLE EMITTING RADIONUCLIDES

Even though more than 100 a particle emitting radionuclides exist, to

date, less than 10 of them have received serious attention for targeted radio-
therapy applications. This can be attributed in part to the fact that most a
particle emitters are part of natural decay chains with multiple progeny radio-
nuclides, necessitating the development of strategies that can compensate for
the often divergent chemical behaviour of the radioactive parent and progeny.
To date, the a particle emitters that have been utilized for clinical investigations
include 45.6 min 213Bi, 7.2 h 211At and 11.4 d 223Ra, while 61 min 212Bi, 4.2 h
149
Tb and 10 d 225Ac have been explored in cell culture and animal models of
human cancer, and clinical studies with 225Ac are commencing. The physical
properties of these radionuclides and their radioactive progeny are
summarized in Table 1.
The a particle emitters under investigation fall into two general groups –
those with half-lives of about 1–7 h and emitting about 1 a particle per decay
(213Bi, 211At, 212Bi and 149Tb) and those which are part of natural decay chains
with half-lives of about 10 d and emitting multiple a particles per decay (223Ra
and 225Ac). The first group presents practical problems with regard to radio-
nuclide supply to locations distant from the site of production, time available
for radiopharmaceutical synthesis and adaptability to the exigencies of admin-
istering therapeutics to cancer patients. The main problem with the second
group is in devising strategies that will maximize the probability that all of the

27
 ZALUTSKY et al.

TABLE 1. ALPHA PARTICLE EMITTING RADIONUCLIDES FOR

TARGETED RADIOTHERAPY

α particle energy Yield per 100

Radionuclide Progeny Half-life
(MeV) decays
149
Tb 4.15 h 3.97 17
211
At 7.21 h 5.87 42
211
Po 516 msec 7.44 58
212
Bi 61 min 6.05 36
212
Po 298 nsec 8.78 64
213
Bi 45.6 min 5.84 36
213
Po 4.2 μsec 8.38 64
225
Ac 10 d 5.75 100
221
Fr 4.9 min 6.36 100
217
At 32.3 msec 7.07 100
213
Bi 45.6 min 5.84 2
213
Po 4.2 μsec 8.38 98
223
Ra 11.4 d 5.64 100
219
Rn 4.0 sec 6.75 100
215
Po 1.78 msec 7.39 100
211
Pb 36.1 min 1.37(â)
211
Bi 2.17 min 6.55 100

radionuclide progeny decay in close proximity to the originally targeted

tumour cells and do not migrate to normal organs, causing significant toxicities.
A wide variety of molecular carriers have been investigated for use in
tandem with a particle emitting radionuclides, including monoclonal
antibodies, peptides, bone seeking complexes as well as receptor and
transporter avid molecules. As with all types of radiopharmaceutical devel-
opment, the selection of the radionuclide must take into account the pharma-
cokinetics of the carrier molecule. Monoclonal antibodies have been the most
widely investigated tumour targeting vehicle; however, as noted above, none of
the a particle emitters currently under investigation has a half-life in the range
of 1–2 d, which would be best suited to the pharmacokinetics of antibody
molecules. As noted below, this has lead investigators to focus on clinical
settings, such as compartmental administration or tumours in the blood, where
more rapid delivery of the labelled antibody to malignant cells can be accom-
plished.

28
 SESSION 8

4. TERBIUM-149

One of the least explored a particle emitting radionuclides is 149Tb, which

was first proposed for therapeutic applications by Allen and Blagojevic
10 years ago [8]. A significant limitation of this 4.15 h half-life radiolanthanide
is that a particle emission is associated with only 17% of its decays. A potential
problem is that with such a low a particle yield, effective killing of cancer cells
will require the targeting of a molecule expressed at a high density because of
limitations in the specific activity of the labelled molecule that can be produced
by current chemistries.
An additional shortcoming of 149Tb for targeted radiotherapy is limited
radionuclide availabilty. Terbium-149 has been produced at the ISOLDE
isotope separator facility at CERN using a tantalum foil target irradiated with
1.0 or 1.4 GeV protons [9]. After surface ionization and collection of the A =
149 isobars, the 149Tb was isolated in nearly 100% radionuclidic purity. A
variety of antibodies has been labelled successfully in high yield with 149Tb
using the SCN-CHX-A’-DTPA bifunctional chelate via procedures adapted
from those originally developed for other radiometals and these immuno-
conjugates have been shown to be cytotoxic to human cancer cells in vitro [2].
Two recent papers have explored the therapeutic potential of 149Tb
labelled mAbs in both cell culture and murine models of human cancers. The
first involved labelling of a truly tumour specific antibody, d9, which binds
specifically to the mutated delta form of the E-cadherin molecule [10]. This
mutated molecular target is found only on cancer cells such as diffuse type
gastric cancers but not on normal tissues. The d9 mAb was labelled with 149Tb
via SCN-CHX-A’-DTPA in nearly quantitative yield with an immunoreactive
fraction of about 30% and a specific activity of about 100 MBq per mg. The
149
Tb labelled d9 immunoconjugate was able to inhibit the proliferation by 50%
of tumour cells expressing the d9 mutant target at an activity concentration of
444 kBq/mL. However, there were two negative aspects to this study. Killing of
cells expressing the wild type E-cadherin molecule (i.e. those not expressing the
d9 molecular target) were killed just as efficiently, suggesting that the cytotoxic
effect was non-specific. Furthermore, even with high activity concentrations of
149
Tb labelled d9 mAb, cell survival could not be reduced below about 20%
suggesting that antigen saturation occurred prior to the achievement of total
cell kill.
The therapeutic potential of 149Tb labelled Rituximab® has also been
evaluated. This is the same anti-CD20 chimeric mAb that when labelled with
90
Y is utilized for the treatment of patients with lymphoma. Groups of SCID
mice (n = 4–9) received 5 × 106 Daudi leukemia cells and only 2 d later were
treated with: 5 mg Rituximab, 300 mg Rituximab, 5 mg Rituximab labelled with

29
 ZALUTSKY et al.

5.55 MBq 149Tb, or saline control [11]. At 120 d, tumour free survival was
observed in all but one animal receiving the labelled mAb but none of the
animals receiving cold mAb or vehicle alone. Although these results at first
glance appear to be encouraging, this interpretation must be made with
caution. Treatment only two days after injection of tumour cells presents a
rather favourable model in which problems related to homogeneous delivery of
radionuclide are minimized. Additionally, particularly for a model of this type,
the most critical control was not performed, namely, a 149Tb labelled non-
specific control mAb.
In summary, the principal attractions of 149Tb as an a particle emitter for
targeted radiotherapy are its 4.15 h half-life, which could be a good match for
smaller molecular weight tumour avid compounds, as well as its adaptability to
radiochemistries already developed for other radiometals such as 90Y and
177
Lu. On the other hand, the limited availablity of 149Tb and its low a particle
abundance are signficant disadvantages. Finally, the lack of compelling
evidence that specific and selective tumour cell kill can be achieved with a 149Tb
labelled molecule is also of concern.

5. RADIUM-223

Radium-223 is a naturally occurring radionuclide that is derived from the

decay of 235U. The decay properties of 11.4 d half-life 223Ra as well as those of its
principal progeny are summarized in Table 1. From a commercial perspective,
the long half-life of 223Ra is a significant advantage because it facilitates distri-
bution and, provided radiolysis associated problems can be avoided, allows for
a longer product shelf life. However, it presents a major challenge to the
radiopharmaceutical chemist because strategies must be devised to trap the
223
Ra labelled therapeutic and its radioactive progeny at the tumour site for
prolonged time periods in order to maximize tumour dose and minimize
toxicity to normal tissues.
For radiopharmaceutical purposes, the optimal method of supplying 223Ra
is from a generator with 21.8 y 227Ac serving as the radionuclide parent. The
227
Ac is produced in a reactor by irradiation of 226Ra, which produces 227Ra,
which in turn decays to the desired 227Ac. The 227Ac undergoes b decay to 18.7 d
227
Th, which decays by a particle emission to 223Ra. An efficient generator
system has been developed by Algeta (Oslo, Norway) [12]. The 227Ac and 227Th
are immobilized on an actinide selective resin and the 223Ra is isolated as
223
RaCl2.
With the exception of a study using sterically stabilized liposomes as a
carrier system for entrapping 223Ra [13], the main focus of endoradiothera-

30
 SESSION 8

peutic efforts with 223Ra has involved exploiting the natural affinity of radium
for bone for the treatment of skeletal metastases. An a emitter could be ideal
for this type of clinical application because the shorter particle range,
compared with b emitters, should greatly increase the capability of delivering
high radiation doses to bone metastases while minimizing excessive dose to
radiation sensitive bone marrow, which otherwise could be dose limiting.
Because 89Sr was used routinely in the past as a bone seeking radiophar-
maceutical, the biodistribution and radiation dosimetry of 223Ra has been
compared to that of 89Sr, with both being administered as their chloride salts
[14]. The uptake of 223Ra in the bone was significantly higher than that of 89Sr,
reaching a level of 40% injected dose per gram in the femur at 24 h post-
injection. Importantly, no significant decline in bone retention was seen over
the course of the 14 d experiment and redistribution of progeny from the bone
was quite small. Accumulation of 223Ra was also observed in soft tissues, partic-
ularly the spleen and the kidneys. However, femur:soft tissue radiation dose
ratios were extremely favourable, generally greater than 1000:1. Dosimetry
calculations modelled dose to bone and bone marrow and indicated that 223Ra
should offer significant advantages compared with 89Sr with regard to
minimizing bone marrow radiation dose.
Preclinical evaluation of 223Ra has also been performed in rats including
those with skeletal metastases [15]. Initital studies confirmed the bone seeking
properties of the radiopharmaceutical, with femur:kidney, femur:liver and
femur:spleen ratios of 590:1, 620:1 and 810:1 observed 24 h after injection. The
femur:bone marrow ratio increased from 6.5:1 at 24 h to >15:1 at 14 d. For the
therapy studies, rats were injected with 1 × 106 MT-1 human breast cancer cells
into the left ventricle and then treated one week later. Control animals had to
be killed 20–30 d later because of tumour induced paralysis while 36 or 40% of
rats receiving 10 or 11 kBq 223Ra had significantly longer symptom free survival
over the 67 d follow-up period. No sign of bone marrow toxicity was observed,
indicating that this radiopharmaceutical warranted evaluation as an endoradio-
therapeutic in cancer patients.
Recently, the results of the first clinical trial of 223Ra in patients with
skeletal metastases were reported [16]. In this phase I trial, 25 patients
(10 breast cancer, 15 prostate cancer) were treated in cohorts of five patients at
doses of 46, 93, 163, 213 or 250 kBq per kg body weight. The labelled
compound was well tolerated with myelosuppression being mild and reversible.
No dose limiting toxicites were observed at any dose level. Median survival for
the 25 patients was greater than 20 months which is encouraging; however, the
lack of a control group makes it difficult to draw meaningful conclusions about
the survival benefit of this treatment. Such a comparison will be done in due

31
 ZALUTSKY et al.

course, in a phase II randomized trial in prostate cancer patients with bone

metastases [16].

6. BISMUTH-213

Bismuth-213 has a 45.6 min half-life and a particle emission is associated

with each of its decays, either directly to 2.2 min 209Tl (2%) or after b decay to
4.2 msec 213Po (98%), followed by a emission to 3.25 h 209Pb. A radionuclide
generator system for supplying 213Bi has been developed using 10 d 225Ac as the
parent. The system has been scaled up and optimized so that it is capable of
providing clinical dose levels of 213Bi [17, 18]. The generator is based on an
AGMP-50 column that is eluted with 0.1M HCl/NaI and is a reasonable source
of 213Bi provided that the 225Ac parent is available at a reasonable cost.
A variety of tumour specific mAbs and fragments have been labelled with
213
Bi, with the labelling most frequently being accomplished via the bifunc-
tional trans-cyclohexyldiethlenetriaminepentaacetic acid SCN-CHXA’-DTPA
chelator. For example, the anti-prostate specific membrane antigen (PSMA)
mAb J591 was labelled with 213Bi and demonstrated to be highly effective in
killing human prostate carcinoma cells grown in culture, as tumour spheroids
and as subcutaneous tumour xenografts grown in athymic mice [19, 20]. Intra-
peritoneal injection of a 213Bi labelled mAb reactive with a tumour specific
mutant E-cadherin molecule was reported to be effective in treating a perito-
neally spread gastric cancer in mice [21]. Other types of application involving
213
Bi labelled antibodies or fragments have been directed at fungal infections
[22] and bone marrow conditioning prior to transplantation [23, 24]. Strategies
to compensate for the short half-life of this radionuclide are vital to the use of
213
Bi and in that regard, pretargeting [25] and use of small scFv fragments [26]
have been investigated.
The first a emitting radiotherapeutic to be evaluated in a clinical trial was
213
Bi labelled HuM195, a humanized mAb that binds to the anti-CD33 antigen
that is over expressed on human leukemia cells [27]. Gamma camera imaging
documented the rapid uptake of 213Bi activity in liver and spleen, which also
express the target antigen. A phase I trial was performed on 18 patients with
relapsed and refractory acute myelogenous leukemia or chronic myelomono-
cytic leukemia. The patients received 3–7 doses of 213Bi labelled HuM195 at
total administered activities ranging from 602 to 3515 MBq [28]. The ratios of
radiation absorbed dose to potential sites of tumour (bone marrow, liver and
spleen) and whole body were about three orders of magnitude higher than
those observed in previous trials when b emitting radionuclides were used.
Large leukemia volume reductions were achieved in many patients; however,

32
 SESSION 8

no complete remissions were observed. Nonetheless, this study demonstrated

that a particle endoradiotherapy in patients is a feasible treatment approach.

7. ACTINIUM-225

A number of significant problems result from the short half-life of 213Bi

and these have decreased enthusiasm for using this radionuclide for targeted
radiotherapy. First, delivery of the 213Bi labelled radiopharmaceutical to the
tumour must be accomplished very rapidly in order to achieve acceptable
tumour-to-normal organ radiation absorbed dose ratios. Second, decay during
radionuclide purification, radiopharmaceutical synthesis and transport of the
labelled compound to the clinic drastically decreases the activity available for
use. This has lead investigators to seek alternative radiometal a particle
emitting radionuclides and 10 d 225Ac has emerged as a promising a emitter for
radioimmunotherapeutic applications. A radionuclide generator has been
developed for supplying 225Ac from a 229Th parent following a two column
based purification procedure [29].
As shown in Table 1, four a particles are produced as a consequence of
each 225Ac decay, which could lead to very effective cell killing, provided that
all of the decays occurred within the tumour. However, accomplishing this will
be a formidable challenge because of the different chemical characteristics of
all the 225Ac progeny. A ligand system that can be utilized to form actinium
mAb complexes that are stable under in vivo conditions has yet to be identified
[30–32]. Furthermore, the ligand must form chemically stable complexes with
all of the 225Ac progeny [33], which include radionuclides of francium, astatine
and bismuth — elements with diverse chemistries. An additional daunting
problem is that the energy of the a particle recoil nuclei is sufficient to break
chemical bonds in the progeny radionuclide complexes, releasing these radio-
nuclides into the circulation.
An innovative tactic for minimizing the problems noted above relies on
the use of receptor or antigens that are rapidly internalized into tumour cells as
molecular targets for 225Ac labelled radiopharmaceuticals [34, 35]. With this
strategy, the egress of the progeny radionuclides from the initial site of 225Ac
localization might be reduced because even if they dissociated from the chelate,
they could be trapped within the tumour cell because of their charge. Using a
DOTA bifunctional macrocycle to accomplish initial 225Ac labelling, promising
therapeutic responses have been obtained with several internalizing mAbs in
various murine xenograft models.
Concern exists about utilization of this strategy in patients because in
most cases only a small fraction (<5%) of the injected dose of radiopharmaceu-

33
 ZALUTSKY et al.

tical localizes in tumour. Thus, release and redistribution of progeny radio-

nuclides could occur after the vast majority of 225Ac decays which do not take
place inside cancer cells. Recently, the toxicity and pharmacokinetics of 225Ac
labelled Hu195, an mAb reactive with the CD33 molecule found on human
leukemias, were evaluated in cynomolgus monkeys [36]. The main toxic effect
that was observed was damage to the renal tubules and the labelled mAb was
deemed to be safe at a dose of 28 kBq/kg. Investigational new drug approval
was obtained by the Sloan Kettering group for a phase I clinical trial of 225Ac
labelled Hu195 in patients with leukemia.

8. ASTATINE-211

Astatine-211 has many attractive characteristics for targeted a particle

radiotherapy and has been the radionuclide of choice for this purpose in the
authors’ laboratory. Their rationale for this perspective is that 211At has a half-
life (7.2 h) that is long enough for multistep synthetic procedures and is also
compatible with the pharmacokinetics of a wide range of molecules, including
peptides, mAbs and small organic molecules. Each decay of 211At yields an a
particle, either by direct a emission to 207Bi (42%), or by electron capture decay
to 520 msec 211Po (58%) followed by a emission (Table 1). An important
practical consequence of the electron capture branch, which results in the
emission of 77–92 keV polonium X rays, is that 211At distribution can be
determined in animals or patients using either conventional nuclear medicine
planar or single photon emission tomographic imaging devices [37, 38].
Astatine-211 is most frequently produced by cyclotron bombardment of
natural bismuth metal targets with a particles via the 209Bi(a,2n)211At reaction.
Particularly for clinical radiotherapy applications, it is critical to utilize an
incident a particle energy that minimizes the production of 8.1 h 210At, a radio-
nuclide that decays to 210Po, a 138 d a emitter that could cause bone marrow
toxicity. For this reason, incident beam energies for 211At production are
generally kept below 29 MeV. Separation of 211At from the bismuth metal
target is generally accomplished using a variety of modifications of the dry
distillation procedure described by Friedman et al. [39]. Internal cyclotron
target systems have been developed that permit the production of clinically
useful levels of 211At [40]. Using this type of target, the authors have been able
to produce 6.6 GBq of 211At after a 4 h irradiation with a 55 mA beam of
28 MeV a particles [41].
Because astatine is a halogen, radioiodination methods can often be
adapted for use with 211At; however, an important exception is protein and
peptide labelling by electrophilic radiohalogenation of constituent tyrosine

34
 SESSION 8

residues. The most common tactic for labelling biomolecules with 211At is via an
astatodemetallation reaction and a wide variety of 211At labelled molecules
have been synthesized and evaluated as potential targeted radiotherapeutics
[7]. The authors’ own efforts in the small molecule arena have included 211At
labelled meta-iodobenzylguanidine analogues, biotin conjugates, peptides,
bisphosphonates and thymidine analogues [7]. By far the most active area of
research both at the authors’ institution and elsewhere has been in evaluation
of 211At labelled mAbs and mAb fragments as a particle emitting targeted
radiotherapeutics. Promising results have been obtained with 211At labelled
immunoconjugates directed at many types of cancer including brain, ovarian,
osteosarcoma, melanoma and non-Hodgkins lymphoma [42–48]. A clinical trial
of 211At labelled chimeric 81C6 mAb administered directly into surgically
created tumour resection cavities is currently under way at the authors’
institution and the results thus far have been very encouraging [49].

9. CONCLUSIONS

Targeted radiotherapy with a particle emitting radionuclides is an

attractive approach for cancer therapy. Recent studies at several institutions
have documented the feasibility of this approach in patients. Ongoing research
in radionuclide production and radiochemistry, as well as in tumour and
molecular biology, should further advance the field and perhaps ultimately lead
to radiopharmaceuticals that are both highly effective and practical.

ACKNOWLEDGEMENTS

Work performed in the authors’ laboratory was supported by grants from

the National Institutes of Health and the US Department of Energy.

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38
 DOTA-TYR3-OCTREOTATE LABELLED WITH 177Lu
AND 131I
A comparative evaluation

V. LUNGU*, D. NICULAE*, D. CHIPER*, M. RADU**

*Radiopharmaceuticals and Labelled Compounds Department

Email: vlungu2000@yahoo.com

** Nuclear Medicine Department

“Horia Hulubei” National Institute for Physics and Nuclear Engineering

Bucharest, Romania

Abstract

The chemical structure of somatostatin receptor ligand 1,4,7,10-tetraazacyclodo-

decane-N,N’,N’,N’’’-tetraacetic acid-Tyr3-octreotate (DOTA-Tyr3-TATE), provides the
means for radiolabelling with halogen, by electrophilic substitution, to the Tyr3 residue
and with metal, by a coordination mechanism, to the DOTA chelator. In this study, the
DOTA-Tyr3-TATE was radiolabelled with 177Lu and 131I of high radiochemical purity
and specific activity. The in vitro study regarding the competitive and the saturation
binding assays were performed using rat brain cortex membrane. The IC50 value was
determined as 4.74nM for natLu-DOTA-Tyr3-TATE and the Kd value was 142.8pM for
177
Lu-DOTA-Tyr3-TATE. The biodistribution data of 177Lu-DOTA-Tyr3-TATE and
DOTA-131I-Tyr3-TATE in HRS1 (hepato-colangiom carcinomas) tumour bearing rats,
show that the 177Lu-DOTA-Tyr3-TATE is more stable and has better uptake than
DOTA-131I-Tyr3-TATE. Furthermore, the competitive localization index of 177Lu-
DOTA-Tyr3-TATE is three times higher than that obtained for DOTA-131I -Tyr3-TATE.
The results of work based on comparative experiments suggest that 177Lu-DOTA-Tyr3-
TATE could be an effective targeted radiotherapy agent of SSTR tumours.

1. INTRODUCTION

There is a great interest in the radiolabelling of somatostatin analogues or

their bioconjugates with the aim of developing new radiopharmaceuticals for
targeted diagnosis [1–3] and therapy [4–8] of cancer as well as in vitro
radioassays of somatostatin receptors. In the authors’ experiments, the
biological and pharmacological properties of the receptor binding sequence of

39
 LUNGU et al.

DOTA-Tyr3-TATE, after radiolabelling with 131I and 177Lu, were investigated.

The DOTA-131I-Tyr3-TATE and 177Lu-DOTA-Tyr3-TATE products were
studied individually and in competition.

2. MATERIALS AND METHODS

2.1. Materials

All chemicals were purchased from Fluka Chemical and Sigma Aldrich.
The 177LuCl3 and Na131I, with specific activities of 45 Ci/mg and
1600 Ci/mg respectively, were purchased from Nordion Canada.
DOTA-Tyr3-TATE and Sandostatin were obtained from the IAEA and
from Pichem Austria.
Wistar rats (150–200 g) were used for the preparation of cortex membranes
and Lewis rats were used for biodistribution studies. The HRS1 tumour bearing
rats were prepared in the Institute of Oncology Bucharest, Romania.
Radioactive samples were counted on Robotron and Spectroscaler
gamma counters.

2.2. Methods

2.2.1. Preparation of DOTA-131I-Tyr3-TATE

Experiments were performed taking into account the regularly used

therapeutic dose of 100 mCi 131I and 100 mg DOTA-Tyr3-TATE; the molar
ratios of DOTA-Tyr3-TATE to 131I and DOTA-Tyr3-TATE to chloramine-T
being 1.7 and 0.09, respectively.
The samples of DOTA-131I-Tyr3-TATE with the activity of 10 mCi were
prepared. The synthesis method was as follows. A solution (5–10 mL) of Na131I
(10 mCi) was added to a solution containing 10 mg DOTA-Tyr3-TATE in 50 mL
0.01M PBS, pH7.4. To this reaction mixture 19.7 mg of chloramine-T in 20 mL
0.1M PBS were added, followed by 1–3 min stirring at room temperature. The
reaction was stopped by the addition of 39.8 mg sodium methabisulphite
dissolved in 10 mL 0.1M PBS. 5 mg of 3-hydroxy-4-aminobenzoic acid (HABA),
for each 10 mCi DOTA-131I-Tyr3-TATE, were added for mixture stabilization.
The RCP was estimated by PC and ITLC using BuOH:acetic acid:water
(5:2:1) as solvent; the Rf of DOTA-131I-Tyr3-TATE is 0.65–0.70, while the Rf of
I+ is 0 and Rf of I- is 0.9–1.0. The addition of 5 mg HABA to DOTA-131I-Tyr3-
TATE has a scavenger effect, maintaining a high radiochemical purity of the
radiocompound at 4–8oC, up to 6 d.

40
 SESSION 8

2.2.2. Preparation of 177Lu-DOTA-Tyr3-TATE

54 mCi 177LuCl3 in 0.05N HCl, 45 Ci/mg specific activity, was diluted to

100 mL with 0.05N HCl .
The samples consisting of 10 mg DOTA-Tyr3-TATE in 50 mL acetate
buffer 0.4M, pH4.5, were labelled with 10 mCi 177LuCl3 in 20 mL 0.05N HCl.
The vials containing the reaction mixture were incubated for 30 min at 80oC.
After incubation and cooling, 5 mg of HABA, as radiolytic stabilizer, was
added. The stabilizer was added after the radiolabelling process because it is
subjected to a certain degree of thermal decomposition. The concentration of
the stabilizer is calculated for 10 mCi 177Lu-DOTA-Tyr3-TATE.
The RCP of 177Lu-DOTA-Tyr3-TATE was checked by PC and ITLC
using different solvents: in the 0.1M Na-citrate, pH5, the labelled peptide
migrated from the origin with Rf = 0.67, while the free radionuclide migrated
with the solvent front (Rf = 1); in other solvent, 10% NH4COOCH3:MeOH
(30:70), the labelled peptide migrated to Rf 0.76–0.80 while free radionuclide
remains at the origin.

2.2.3. Preparation of SST membrane receptor from rat brain cortex

Rat brain cortex membranes were prepared according to a published

method [10].

2.2.4. Competition binding assays of DOTA-131I-Tyr3-TATE

The competition binding assay was performed using rat brain cortex
membrane (50 mg protein). 35 000–40 000 counts/min of 125I-Tyr3-octreotide
(970 Ci/mM) were added in each test tube in the presence of increasing concen-
tration of DOTA-natI-Tyr3-TATE (synthesized in the same conditions as
DOTA-131I-Tyr3-TATE): 0.06; 0.13; 0.34; 0.68; 1.00; 2.72; 10.00; 57.80; 100nM in
total volume of 300 mL 50mM HEPES (pH7.6, 0.3% BSA, 5mM MgCl2, 10mM
bacitracin). The samples were incubated for 2 h at room temperature; the
incubation was stopped by addition of ice cold buffer (1 mL, 10mM HEPES,
150mM NaCl, pH7.6).
The suspension was rapidly filtered over glass fibre filters (Whatman GF/B)
presoaked in binding buffer using a Millipore multifiltration apparatus. The
filters were rinsed with buffer (4 × 2 mL) and filter activity was measured on a
NaI(Tl) gamma counter.

41
 LUNGU et al.

2.2.5. Competition binding assays of 177Lu-DOTA-Tyr3-TATE

The binding affinity of natLu-DOTA-Tyr3-TATE was measured in rat

brain cortex membrane. 20 000 counts/min of 125I-Tyr3-octreotide was displaced
with the following increasing concentrations of natLu-DOTA-Tyr3-TATE
(synthesized under the same conditions as 177Lu-DOTA-Tyr3-TATE): 0.03;
0.14; 0.45; 0.70; 3.80; 12.80; 30.80; 170; 500nM. The obtained samples were
processed using the same procedure (Section 2.2.4).

2.2.6. Saturation binding assays of DOTA-131I-Tyr3-TATE

The saturation binding experiments of DOTA-131I-Tyr3-TATE were

performed using rat brain cortex membrane. For total binding assay, the
following concentrations of DOTA-131I-Tyr3-TATE were prepared: 0.06; 0.13;
0.34; 0.68; 1.00; 2.72; 10.00; 57.80; 100nM in 50 mL binding buffer, 50 mL
radioligand solution of corresponding concentration and 200 mL rat brain
cortex membrane homogenate containing 40 mg protein. For the non-specific
series, the authors used 20 mL binding buffer plus 30 mL of unlabelled peptide
as competitor (Sandostatin) (1mM in the reaction vial), instead of 50 mL buffer.
The tubes were incubated for 2 h at room temperature and then binding was
interrupted by rapid filtration through a glass fibre filter presoaked with the
binding buffer. Filters were washed with binding buffer, dried and counted
using a NaI(Tl) gamma counter.

2.2.7. Saturation binding assays of 177Lu-DOTA-Tyr3-TATE

A similar method (Section 2.2.6) was used. For the experiments regarding
saturation binding assay of 177Lu-DOTA-Tyr3-TATE, the following concentra-
tions of 177Lu-DOTA-TATE were prepared: 0.05; 0.15; 4.5; 13.5; 40.5; 120.5;
361.5nM for total and non-specific binding assay. A method similar to that
described in Section 2.2.6 was used for the processing of the obtained samples
and acquisition of data.

2.2.8. Experimental animal models

The hepatom RS1 (HRS1), hepato colangiom carcinoma, was obtained as

a cell culture from the Institute of Oncology, Bucharest. The solid tumour was
obtained by subcutaneous injection of 107 cells into 5 week old female Lewis
rats (150–200 g). Once the tumours had grown to ~1 cm3, they were serially
propagated by subcutaneous injection of 0.2 mL of a 20% (w/v) tumour

42
 SESSION 8

suspension, prepared by mincing tumours in 0.9% NaCl. Ten days prior to

radiobiological studies, the following lots of HRS1 bearing rats were prepared:

(a) Lot for the biodistribution studies of 177Lu- DOTA-Tyr3-TATE;

(b) Lot for the biodistribution studies of DOTA-131I-Tyr3-TATE;
(c) Lot for the biodistribution competitive studies of 177Lu- DOTA-Tyr3-
TATE and DOTA-131I-Tyr3-TATE;
(d) Lot for the biodistribution experiments of 177Lu-DOTA-Tyr3-TATE and
DOTA-131I-Tyr3-TATE in the presence of SSTR blocking agent.

2.2.9. Biodistribution experiments of 177Lu- DOTA-Tyr3-TATE and DOTA-131I-

Tyr3-TATE either alone or in competition

For individual biodistribution studies of 177Lu- DOTA-Tyr3-TATE and

DOTA-131I-Tyr3-TATE, tumour bearing rats from lots (a) and (b) were IV
injected with 0.2 mL radioactive solutions containing 50 mCi 177Lu-DOTA-
Tyr3-TATE and DOTA-131I-Tyr3-TATE, respectively, corresponding to
approximately 0.68 mg DOTA-Tyr3-TATE. The tumour bearing rats from lot
(c), for competitive biodistribution studies, were IV injected with 35 mCi 177Lu-
DOTA-Tyr3-TATE and 15 mCi DOTA-131I-Tyr3-TATE as a cocktail solution
containing 0.68 mg DOTA-Tyr3-TATE, 0.2 mL.
At specific time points, the tumours as well as various tissues (blood,
liver, spleen, kidney, stomach, small intestine, large intestine, adrenals,
pancreas, thyroid, pituitary gland, bone and lung) were removed, weighed and
their radioactivity determined.
The tumour bearing rats from lot (d), for biodistribution of 177Lu-DOTA-
Tyr3-TATE and DOTA-131I-Tyr3-TATE in the presence of SSTR blocking
agent, 0.2 mL containing 50 mCi 177Lu-DOTA-Tyr3-TATE and DOTA-131I-
Tyr3-TATE, were IV injected, corresponding to 0.68 mg DOTA-Tyr3-TATE
and 150 mg Sandostatin as blocking agent. After 1 h post-injection, the tumours
and SSTR expressive tissues were removed, weighed and their radioactivity
determined.

3. RESULTS AND DISCUSSION

3.1. In vitro binding assay

The experimental results regarding the SSTR receptor binding assays of

the prepared radiopeptides indicate the biological properties of the receptor
binding sequence of DOTA-Tyr3-TATE after radiolabelling with 131I and 177Lu.

43
 LUNGU et al.

3.1.1. Competition binding assays of DOTA-131I-Tyr3-TATE and 177Lu-DOTA-

Tyr3-TATE

The competition binding data obtained from the experiments (Sections

2.2.4 and 2.2.5) using rat brain cortex membranes were analysed using the
Prism-2 program (GraphPad software). The representative competition curves
are shown in Figs 1 and 2. The IC50 values were determined to be 1.28nM for
DOTA-natI-Tyr3-TATE and 4.74nM for natLu-DOTA-Tyr3-TATE.

Best-fit values
35000 BOTTOM 239.5
TOP 32796
30000
LOGEC50 -8.893
25000 EC50 1.280e-009
Mean cpm

Std. Error
20000
BOTTOM 1815
15000 TOP 3338
LOGEC50 0.1711
10000

5000

0
-10 -9 -8 -7

log[somatostatin](M)

FIG. 1. Competitive binding curve of DOTA-natI-Tyr3-TATE.

14000

12000

10000
Mean (CPM)

8000

6000

4000

2000

0
-10 -9 -8 -7 -6

Log([somatostatin])

FIG. 2. Competitive binding curve of natLu-DOTA-Tyr3-TATE.

44
 SESSION 8

3.1.2. Saturation binding assays of DOTA-131I-Tyr3-TATE and 177Lu-DOTA-

Tyr3-TATE

Radioactive measurements for total and non-specific binding were made

experimentally (Sections 2.2.6 and 2.2.7) and were analysed using the Prism-2
program for determination of the equilibrium dissociation constant, Kd, for
both DOTA-131I-Tyr3-TATE and 177Lu-DOTA-Tyr3-TATE as shown in Figs 3
and 4.

Best-fit values
0.35
Slope -0.006346 ± 0.0006098
0.30 Y-intercept when X=0.0 0.3568 ± 0.02275
X-intercept when Y=0.0 56.22
0.25
1/slope -157.6
Bound/Free

0.20

0.15

0.10

0.05

0.00

10 20 30 40 50 60 70
-0.05
Bound (pM)
FIG. 3. Saturation binding curve of DOTA-131I-Tyr3-TATE.

0.6

0.5

0.4
Bound/Free

0.3

0.2

0.1

0.0
0 20 40 60

Bound (pM)
177
FIG. 4. Saturation binding curve of Lu-DOTA-Tyr3-TATE. Fit line: y = A + Bx:
A = 0.48 ± 0.03; B = -0.007 ± 0.0008 pM.

45
 LUNGU et al.

The Kd values of radiopeptides were found to be 157.6 for DOTA-131I-

Tyr3-TATE and 142.8 for 177Lu-DOTA-Tyr3-TATE.

3.2. Biodistribution studies

3.2.1. Biodistribution of 177Lu-DOTA-Tyr3-TATE in HRS1 tumour bearing rats

In Table 1 are presented the biodistribution data of 177Lu-DOTA-Tyr3-

TATE for all studied time points. The blood clearance is fast. The uptake of
177
Lu-DOTA-TATE in SSTR expressed tissues is high up to 24 h and decreases
with each time point of the investigation. The uptake of 177Lu-DOTA-Tyr3-
TATE in tumour increases between 24–72 h post-injection.
Figure 5 shows the decreasing biodistribution of 177Lu-DOTA-Tyr3-
TATE in SSTR expressed normal tissues and increasing, with a maximum in
the 24–72 h time range of biodistribution of 177Lu-DOTA-Tyr3-TATE in HRS1
tumour. The bone uptake of 177Lu-DOTA-Tyr3-TATE was high but shows a
linear decrease, indicating the possibility of decomposition after IV injection,
with delivery of free 177Lu. With this view, the biodistribution of 177LuCl3 in

TABLE 1. BIODISTRIBUTION OF 177Lu-DOTA-Tyr3-TATE IN

SELECTED ORGANS EXPRESSED IN %ID/g

Organ 3h 24 h 48 h 72 h 168 h

Blood 0.72 ± 0.47 0.04 ± 0.03 0.04 ± 0.01 0.03 ± 0.01 0.03 ± 0.00
Liver 0.68 ± 0.15 0.44 ± 0.11 0.40 ± 0.05 0.28 ± 0.13 0.18 ± 0.01
Spleen 0.20 ± 0.11 0.14 ± 0.05 0.07 ± 0.01 0.03 ± 0.05 0.03 ± 0.00
Kidney 2.12 ± 0.23 1.90 ± 0.78 1.55 ± 0.82 1.56 ± 0.02 1.23 ± 0.05
Stomach 1.95 ± 0.42 1.25 ± 0.03 1.08 ± 0.32 0.94 ± 0.08 0.79 ± 0.01
Small intestine 0.92 ± 0.37 0.89 ± 0.13 0.57 ± 0.22 0.30 ± 0.10 0.17 ± 0.01
Large intestine 2.66 ± 0.14 2.02 ± 0.10 1.73 ± 0.10 1.99 ± 0.21 0.73 ± 0.02
Adrenal 0.71 ± 0.32 0.76 ± 0.51 0.75 ± 0.16 0.50 ± 0.11 0.35 ± 0.01
Pancreas 7.79 ± 0.08 4.10 ± 0.92 3.67 ± 0.67 2.01 ± 0.31 1.50 ± 0.02
Thyroid 1.92 ± 0.21 2.09 ± 0.14 0.82 ± 0.13 0.97 ± 0.08 0.73 ± 0.10
Pituitary 0.37 ± 0.13 0.57 ± 0.17 0.39 ± 0.11 0.34 ± 0.02 0.12 ± 0.01
Bone 9.54 ± 0.27 5.40 ± 0.72 2.79 ± 0.53 1.72 ± 0.31 1.05 ± 0.15
Lung 0.24 ± 0.11 0.17 ± 0.08 0.12 ± 0.05 0.05 ± 0.01 0.02 ± 0.01
Tumour 3.22 ± 2.15 6.31 ± 3.02 4.18 ± 3.42 4.07 ± 2.58 0.61 ± 0.29

46
 SESSION 8

9
3h 24 h 48 h 72 h 168 h
8

7
ID (%) / organ (g)

0
Blood Liver Kidney Adrenals Pancreas Tumor

organ

FIG. 5. Biodistribution of 177Lu-DOTA-Tyr3-TATE in SSTR2 expressed tissues (adrenals,

pancreas and tumour) in comparison with the biodistribution of 177Lu-DOTA-Tyr3-TATE
in transport, metabolic and elimination tissues.

bone was tested. The results (see Fig. 6.) show a progressive accumulation of
177
Lu, with the maximum value in the 4–24 h range. The high level of radio-
activity was maintained for the duration of the experiment; the kinetics of
177
LuCl3 appear different from those obtained for 177Lu-DOTA-Tyr3-TATE.

Lu chloride Lu peptide

18

16

14
ID (%) / organ (g)

12

10

0
3h 24 h 48 h 72 h 168 h

Time

FIG. 6. Bone uptake of 177Lu-DOTA-Tyr3-TATE and 177LuCl.

47
 LUNGU et al.

3.2.2. Competitive biodistribution studies of 177Lu-DOTA-Tyr3-TATE and

DOTA-131I-Tyr3-TATE

Because the uptake in the non-tumour tissues and the clearance of 177Lu-
131
and I-DOTA-TATE were similar, the present study examines whether one
radioproduct is more stable and has more uptake than the other in tumour
bearing rats co-injected with 177Lu-DOTA-TATE and 131I-DOTA-TATE.
The competitive localization index (CLI) was defined:

%ID/g 177 Lu-DOTA-TATE in tumour

177
Lu-CLI =
%ID/g 177 Lu-DOTA-TATE in blood

131 %ID/g 131 I-DOTA-TATE in tumour

I-CLI=
%ID/g 131 I-DOTA-TATE in bllood

177
The results (see Table 2 and Fig. 7) show that Lu-CLI is higher than
131
I-CLI (~3 times).

177
TABLE 2. COMPETITIVE LOCALIZATION INDEX OF Lu-DOTA-
Tyr3-TATE AND DOTA-131I-Tyr3-TATE IN TUMOUR

CLI 3h 24 h 48 h 72 h 168 h
177
Lu-CLI 4.87 157.75 104.50 69 20.38
131
I-CLI 1.42 103.00 29.81 6.38 3.08

3.2.3. Biodistribution of 177Lu-DOTA-TATE and DOTA-131I-Tyr3-TATE in

HRS1 tumour bearing rats with and without co-administration of SSTR2
blocking agent

The results show a smaller uptake of the radiopeptides in SSTR

expressed tissues and tumour in rats injected with a blocking agent than in
those animals where a blocking agent was absent (see Table 3).

48
 SESSION 8

CLI 177Lu-T/177Lu-Bl 131I-T/131I-Bl

180

160

140

120

100

80

60

40

20

0
3h 24 h 48 h 72 h 168 h

Time (h)

FIG. 7. Competitive uptake of 177Lu-DOTA-Tyr3-TATE and DOTA-131I-Tyr3-TATE.

TABLE 3. BIODISTRIBUTION OF 177Lu-DOTA-Tyr3-TATE AND DOTA-

131
I-Tyr3-TATE IN HRS1 TUMOUR BEARING RATS WITH AND
WITHOUT A BLOCKING AGENT
177 177 131 131
Organ Lu Lu, block I I, block
Blood 0.96 ± 0.05 1.12 ± 0.04 1.02 ± 0.03 1.35 ± 0.01
Liver 0.82 ± 0.01 0.98 ± 0.05 0.38 ± 0.07 0.42 ± 0.03
Kidney 2.91 ± 0.03 3.41 ± 0.06 8.63 ± 0.01 9.37 ± 0.03
Pituitary 0.93 ± 0.15 0.38 ± 0.02 2.12 ± 0.12 0.51 ± 0.34
Adrenal 0.89 ± 0.32 0.12 ± 0.10 3.42 ± 0.24 0.48 ± 0.61
Pancreas 3.72 ± 0.33 0.95 ± 0.12 2.66 ± 0.04 0.62 ± 0.10
Tumour 3.01 ± 0.15 0.72 ± 0.04 2.24 ± 0.05 1.98 ± 0.32

4. CONCLUSIONS

The authors’ studies demonstrate that the DOTA-Tyr3-TATE can be

labelled with both 131I and 177Lu. The IC50 and Kd values for DOTA-131I-Tyr3-
TATE and 177Lu-DOTA-Tyr3-TATE places these products in the range of
radiopeptides with high binding affinity for somatostatin expressive receptors.

49
 LUNGU et al.

The biodistribution data show that the 177Lu-DOTA-Tyr3-TATE is more

stable and has higher uptake than DOTA-131I-Tyr3-TATE and the CLI of 177Lu
DOTA-Tyr3-TATE is threefold higher than that obtained for DOTA-131I-Tyr3-
TATE. The results of work based on the comparative experiments suggest that
the 177Lu-DOTA-Tyr3-TATE could be an effective targeted radiotherapy agent
for SSTR tumours.

ACKNOWLEDGEMENTS

This work was financially supported by the National Research

Programme of Health VIASAN, Project No. 367/2004 and by the IAEA
through the Coordination Research Programme on Comparative Evaluation of
Therapeutic Radiopharmaceuticals (contract no. 12122/R0).

REFERENCES

[1] WEN, PING LI, et al., DOTA-D-Tyr – Octreotate: A Somatostatine analogue for
labeling with metal and halogen radionuclides for cancer imaging and therapy,
Bioconjugate Chem. 13 (2002) 721-728.
[2] BAKKER, W.H., et al., Iodine-131 labelled octreotide: not an option for somato-
statine receptor therapy, Eur. J. Nucl. Med. 23 7 (1996) 775-781.
[3] MAINA, T., et al., [99mTc] Demotate, a new 99mTc-based [Tyr3] octreotate
analogue for the detection of somatostatine receptor – positive tumours: synthesis
and preclinical results, Eur. J. Nucl. Med. 29 6 (2002) 742-753.
[4] KWEKKEBOOM, D.J., et al., [177Lu-DOTA0,Tyr3] octreotate: comparison with
[111In-DTPA0]octreotide in patients, Eur. J. Nucl. Med. 28 9(2001) 1319-1325.
[5] LEWIS, J.S., et al., Toxicity and dosimetry of 177Lu-DOTA-Y3-octreotate in a rat
model, Int. J. Cancer 94 (2001) 873-877.
[6] LEWIS, J.S., et al., Radiotherapy and dosimetry of 64Cu-TETA-Tyr3 -Octreotate
in a somatostative receptor-positive, tumor-bearing rat model, Clinical Cancer
Research 5 (1999) 3608-3616.
[7] VALKEMA, R., et al., Long-term folow-up of renal function after peptide
receptor radiation therapy with 90Y-DOTA0, Tyr3 – Octreotide and 177Lu-DOTA0,
Tyr3 -Octreotate, J. Nucl. Med. 46 1 (2005) 995-1065.
[8] KWEKKEBOOM, D.J., et al., Overview of results of peptide receptor radionu-
clide therapy with 3 radiolabeled somatostatin analogs, J. Nucl. Med. 45 11 (2004)
1-5.
[9] RAYNOR, K., REISINE, T., Analogs of somatostatin selectively label distinct
serbtypes of somatostatin receptor in ratbrain, J. Pharmacol Exp. Ther. 251 (1989)
510-517.

50
 177
Lu LABELLED NITROIMADZOLES AND
NITROTRIAZOLES FOR POSSIBLE USE IN
TARGETED THERAPY OF HYPOXIC TUMOURS

T. DAS*, S. CHAKRABORTY*, A. MUKHERJEE*, S. BANERJEE*,

G. SAMUEL*, H.D. SARMA**, M. VENKATESH*

*Radiopharmaceuticals Division
Email: meerav@apsara.barc.ernet.in

** Radiation Biology and Health Sciences Division

Bhabha Atomic Research Centre,

Mumbai, India

Abstract

In the authors’ attempt at the development of potential therapeutic agents for

targeting hypoxic tumours, two potent tumour avid substrates, metronidazole (a
5-nitroimadazole) and sanazole (a nitrotriazole derivative), were radiolabelled with
177
Lu after their conjugation with suitable bifunctional chelating agents. Lutetium-177 is
presently being considered as an excellent radionuclide for the development of targeted
agents for tumour therapy owing to its suitable nuclear decay characteristics and the
possibility of its production with reasonably high specific activity and high radionuclidic
purity using moderate flux reactors. In both cases, radiolabelling yields of >95% were
achieved under optimized reaction conditions and the radiolabelled conjugates showed
excellent stability at room temperature. Biodistribution studies carried out in Swiss mice
bearing fibrosarcoma tumours showed selective accumulation of activity in tumour with
high tumour/blood and tumour/muscle ratios within 1 h post-injection. No accumulation
of injected activity was observed in any of the vital organs/tissues of the animals.

1. INTRODUCTION

The presence of hypoxic cells in tumours is considered to be a major

factor limiting the efficacy of tumour diagnosis and radiotherapy [1]. The
interesting observation that nitroimidazole derivatives have a tendency to be
accumulated in the hypoxic regions led to the possibility of these compounds
being considered for use as hypoxia markers [2, 3]. In the authors’ attempt to
develop a potential agent for targeted tumour therapy, metronidazole, a
5-nitroimadazole, and sanazole, a nitrotriazole derivative, documented earlier

51
 DAS et al.

as potent tumour avid substrates, are chosen as the carrier molecules [4, 5].
Lutetium-177, which is fast emerging as a promising radionuclide for targeted
therapy owing to its suitable decay characteristics (T1/2 = 6.73 d, Eβ(max) =
497 keV and Eγ = 113 keV (6.4%), 208 keV (11%)) and favourable production
logistics (σ = 2100 b for 176Lu(n,γ)177Lu), was identified as the radioisotope of
choice [6]. The relatively longer half-life of 177Lu provides logistical advantages
for facilitating supply to places far away from the reactors and assumes signifi-
cance for those countries having limited reactor facilities for isotope
production. Moreover, 177Lu can be produced in adequate specific activity for
in vivo targeted therapy applications by a simple (n,γ) process using an
enriched 176Lu target due to its very high neutron capture cross-section (σ =
2100 b) [6, 7]. Since direct incorporation of 177Lu in either of the aforemen-
tioned nitroimidazole or nitrotriazole moiety is not feasible, indirect incorpo-
ration of 177Lu through a suitable bifunctional chelating agent (BFCA) was
envisaged. As it is well documented that the lanthanide complexes of poly-
azamacrocycles exhibit very high thermodynamic stability and excellent kinetic
inertness, it is quite logical to choose those ligands as the BFCAs of choice [8].
For the present study, metronidazole was coupled with para-aminobenzyl-
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-amino-benzyl-
DOTA) while the sanazole derivative [(N-2’(carboxyethyl)-2-(3’-nitro-1’-tri-
azolyl)acetamide] was conjugated with 1,4,7,10-tetraaza-1-(4’-aminobenzyl-
acetamido)-cyclododecane-4,7,10-triacetic acid (p-amino-DOTA-anilide). The
present paper describes the radiolabelling of both the conjugates with 177Lu in
high yields and the pharmacokinetic behaviour of the developed radiochemical
agents in suitable tumour bearing animal models.

2. EXPERIMENTAL

2.1. Materials

Metronidazole was obtained as a gift from a local pharmacy college. Sanazole

derivative [(N-2’(carboxyethyl)-2-(3’-nitro-1’-triazolyl)acetamide] was
obtained from the Radiation Biology and Health Sciences Division of the
Bhabha Atomic Research Centre. The p-amino-benzyl-DOTA and p-amino-
DOTA-anilide were procured from Macrocyclics (United States of America).
Dicyclohexylcarbodiimide was purchased from Aldrich chemical company
(USA). Dioxane was distilled and dried as per reported procedure [9]. All the
other chemicals were purchased from reputable local manufacturers and were
of analytical grade. The Lu2O3 (60.6% enriched in 176Lu) powder used as the
target for irradiation was procured from Isoflex (Russian Federation).

52
 SESSION 8

The radionuclidic purity of 177Lu produced was determined by recording

gamma ray spectra using a HPGe detector (EGG Ortec/Canbera detector)
coupled to a 4K multichannel analyser system. A 152Eu reference source,
obtained from Amersham Inc., USA, was used for both energy and efficiency
calibration of the detector. All other radioactivity measurements were carried
out using a well-type NaI(Tl) scintillation counter unless otherwise mentioned,
keeping the base line at 150 keV and a window of 100 keV, thereby utilizing the
208 keV gamma photon of 177Lu.
Flexible silica gel plates used for carrying out thin layer chromatography
(TLC) studies were obtained from Bakerflex Chemical Co., Germany.
Whatman 3 MM chromatography paper (United Kingdom) was used for paper
chromatography (PC) studies. The high performance liquid chromatography
(HPLC) system used was obtained from JASCO, Japan (PU 1580). All the
solvents used for HPLC studies were of HPLC grade and purchased from
reputable local manufacturers. The solvents were degassed and filtered prior to
use. Fourier transform infrared (FT-IR) spectra were recorded by using a Jasco
FT/IR-420 spectrophotometer. Proton NMR spectra were recorded on
300 MHz Varian VXR 300S spectrometer.

2.2. Production of 177Lu

Lutetium-177 was produced by thermal neutron bombardment of an

isotopically enriched (60.6% in 176Lu) Lu2O3 target at a thermal neutron flux of
3 × 1013 n·cm–2·s–1 for 14 d. Following irradiation, the target was dissolved in 1M
HCl by gentle warming. The resultant solution was evaporated to near dryness
and reconstituted in double distilled water. The assay of the activity as well as
the determination of specific activity and radionuclidic purity of the 177Lu
produced was carried out by recording gamma ray spectra using a HPGe
detector coupled to a 4K multichannel analyser system. Energy and efficiency
calibrations of the detector were carried out using a standard 152Eu source.

2.3. Synthesis of the conjugates

2.3.1. Metronidazole-p-amino-benzyl-DOTA

The metronidazole-p-amino-benzyl-DOTA conjugate was synthesized by

a two-step reaction. Metronidazole was first converted to its carboxylic acid
derivative by oxidation using alkaline KMnO4. To a solution of metronidazole
(500 mg, 2.92mM) in 5 mL 10% aqueous Na2CO3, KMnO4 (580 mg, 3.67mM)
dissolved in 10 mL of double distilled water was added slowly under constant
stirring at 4–5°C. The reaction was continued for another 4 h at 4–5°C and then

53
 DAS et al.

left overnight at room temperature (25°C). The reaction mixture was filtered
and the filtrate was acidified and extracted in diethyl ether. The oxidized
product, 2-[N-(2’-methyl-5’-nitro)imidazolyl]ethanoic acid, was obtained by
evaporating the solvent under vacuum. In the second step, 2-[N-(2’-methyl-5’-
nitro)imidazolyl]ethanoic acid (3 mg, 0.0162mM), p-NH2-benzyl-DOTA.4HCl
(10 mg, 0.0152mM) and DCC (3 mg, 0.0146mM) were stirred in dioxan for 20 h
at room temperature to obtain the desired conjugate. After the completion of
reaction, the reaction mixture was filtered and the filtrate was evaporated. The
crude product thus obtained was purified by preparative TLC using 3%
ammonium hydroxide in methanol as the eluting solvent.

2.3.2. Sanazole-p-amino-DOTA-anilide

The coupling between sanazole derivative [(N-2’(carboxyethyl)-2-(3’-

nitro-1’-triazolyl)acetamide] and p-amino-DOTA-anilide was achieved by a
single step procedure. In a typical reaction, the sanazole derivative (5 mg,
0.0205mM), p-amino-DOTA-anilide (13 mg, 0.0203mM) and DCC (5 mg,
0.0243mM) were dissolved in 2–3 mL of dry dioxane and the resulting mixture
was stirred at room temperature for 24 h. After completion of the reaction, the
precipitate was separated from the supernatant liquid by filtration and dried.
The crude product thus obtained was purified by preparative TLC using 2%
ammonium hydroxide in methanol as the eluting solvent. Both the conjugates
were characterized by FT-IR and proton NMR spectroscopy.

2.4. Preparation of 177Lu labelled conjugates

For preparation of the 177Lu complexes of the synthesized conjugates, a

stock solution of the conjugate was prepared in 0.1M NH4Ac buffer (pH~5).
Then, 20 μL of 177LuCl3 (~25 MBq of 177Lu, 0.2 μg Lu) was added to 100 μL of
the stock solution of the conjugate (containing 100 μg sanazole-p-amino-
DOTA-anilide or 25 μg metronidazole-p-amino-benzyl-DOTA conjugate) and
the volume of the reaction mixture was made up to 200 μL by addition of 0.1M
NH4Ac buffer (pH~5). The reaction mixture was incubated at 50°C for 1 h
maintaining its pH at ~5. Various reaction parameters, such as ligand concen-
tration, pH, incubation time and temperature were optimized in order to
obtain maximum radiolabelling yield.

2.5. Characterization of 177Lu labelled conjugates

The radiolabelled conjugates were characterized by PC using 50%

aqueous acetonitrile and TLC using 10% ammonium acetate:methanol (1:1, v/v)

54
 SESSION 8

as the eluting solvents. The radiolabelled conjugates were further purified by

HPLC (C-18 reversed phase column (25 cm × 0.46 cm)) using water (A)
acetonitrile (B) with 0.1% trifluoroacetic acid as the mobile phase and
employing a gradient elution technique (0–4 min: 95% A, 4–6 min: 95–80% A,
6–9 min: 80–40% A, 9–12 min: 40% A, 12–18 min: 5% A). The flow rate was
maintained at 1 mL/min and the elution was monitored by detecting the
radioactivity signal using a NaI(Tl) detector.

2.6. Biodistribution studies

The in vivo biological evaluations of 177Lu labelled conjugates were

carried out on Swiss mice bearing fibrosarcoma tumours. Fibrosarcoma cells
were prepared in normal saline (106 cells/mL) and 200 μL of the cell suspension
was injected subcutaneously into each Swiss mouse, the weight of which varied
around 20–25 g. The animals were observed for visible tumours. At the end of
two weeks, tumours of ~1 cm diameter were observable. The radiolabelled
conjugates (~100 μL, 7–8 MBq) were injected into the tumour bearing animals
through the tail vein. The animals were sacrificed by cardiac puncture post-
anesthesia at 1 h, 3 h and 24 h post-injection. Various organs and tumours were
excised following sacrifice and the radioactivity associated with each organ/
tissue was measured using a flat type NaI(Tl) counter. The percentage of
injected dose (%ID) in various organs/tissues and tumours was calculated from
the above data and expressed as percentage injected dose per gram (%ID/g) of
organ/tissue. The activity excreted was indirectly determined from the
difference between ID and the %ID accounted for in all the organs. All the
animal experiments were carried out in strict compliance with the relevant
national laws relating to the conduct of animal experimentation.

3. RESULTS AND DISCUSSION

3.1. Production of 177Lu

Approximately 185 TBq/g (5 × 103 Ci/g) of 177Lu activity was obtained at

6 h post-end of bombardment when a 60.6% enriched Lu2O3 target was
irradiated at a thermal neutron flux of 3 × 1013 n·cm–2·s–1 for 14 d. The radio-
nuclidic purity of 177Lu produced was 99.985% as estimated by analysing the
gamma ray spectrum; 177mLu (T1/2 = 160.5 d) being the sole radionuclidic
impurity detected. The major gamma peaks were observed at 72, 113, 208, 250
and 321 keV, all of which correspond to the photopeaks of 177Lu [6]. For
determining the radionuclidic impurity burden in the 177Lu produced due to

55
 DAS et al.

177m
Lu, the activity of 177Lu was allowed to decay completely (45–65 d, ~8–10
half-lives of 177Lu) and the gamma ray spectrum was recorded. The average
level of radionuclidic impurity burden was found to be 150 nCi of 177mLu /1 mCi
of 177Lu (5.5 kBq /37 MBq) at end of bombardment.

3.2. Characterization of the conjugates

The conjugates were characterized by FT-IR and proton NMR

spectroscopy.

3.2.1. Metronidazole-p-amino-benzyl-DOTA

FT-IR (KBr, ν cm-1): 3425, 2925, 2849, 1733, 1692, 1514, 1469, 1385.
1
H-NMR, CD3OD (δ ppm): 1.29 [3H, s, metronidazole CH3], 3.56-3.63
[15H, m, DOTA cyclic CH2], 3.67-3.74 [8H, m, N(DOTA)-CH2-COOH], 4.08-
4.20 [2H, m, CH(DOTA)-CH2-Ph], 4.94 [2H, s, N(metronidazole)-CH2-CO],
7.44-7.49 [2H, m, p-amino-benzyl-DOTA ArH], 7.58-7.62 [2H, m, p-amino-
benzyl-DOTA ArH], 8.03 [1H, s, metronidazole H].
The peak positions and integrations observed in the 1H-NMR spectrum
of the conjugate were consistent with the structure of the expected compound.
In addition to the protons assignable to the DOTA moiety, the presence of the
peaks at δ = 1.29 and 4.94 corresponding to the protons of the metronidazole
moiety and the deshielded proton of the imidazole ring of metronidazole
observable at δ = 8.03 provided further evidence towards the desired
derivatization.

3.2.2. Sanazole-p-amino-DOTA-anilide

FT-IR (KBr, ν cm-1): 3443, 3096, 2966, 2853, 1682, 1631, 1555.
1
H-NMR (CD3OD, δ ppm): 3.40-3.78 [16H, m, cyclic CH2], 4.11-4.24 [6H,
m, N(DOTA)-CH2-COOH] 4.30-4.38 [2H, m, N(DOTA)-CH2-CO-NH], 4.76
[2H, dd, J = 2.2 Hz & 14.3 Hz, sanazole-N-CH2-CO], 5.11 [4H, dd, J = 9.9 Hz &
23 Hz, -CO-CH2-CH2-CO], 8.07 [2H, bs, CONH-aromatic H], 8.09 [1H, s,
sanazole H], 8.11-8.13 [2H, m, CONH-aromatic H].
The peak positions, multiplicities and integrations observed in the high
resolution 1H-NMR spectrum of the conjugate were consistent with the
structure of the expected compound. Appearance of the peaks at δ = 5.11 and
4.76 corresponding to the protons of the sanazole moiety indicates the desired
conjugation. The deshielded proton of the triazole ring of sanazole observable
at δ = 8.09 provides further evidence towards the desired derivatization.

56
 SESSION 8

3.3. Characterization of 177Lu complexes

The radiolabelled conjugates were characterized by a combination of PC

and TLC systems. In PC using 50% aqueous acetonitrile as the eluting solvent,
it was observed that both 177Lu labelled conjugate and 177Lu labelled BFCA
exhibited Rf = 0.7–0.8, while uncomplexed 177Lu remained at the point of
spotting (Rf = 0). Therefore, this technique could be used to determine the
percentage radioactivity due to uncomplexed 177Lu in the reaction mixture. On
the other hand, in TLC and 10% ammonium acetate:methanol (1:1, v/v), it was
observed that only 177Lu labelled BFCA moved towards the solvent front with
Rf = 0.7, while both the uncomplexed 177Lu as well as the 177Lu labelled
conjugate did not show any appreciable movement from the point of spotting
(Rf = 0 and 0–0.1, respectively). The percentage radioactivity due to the 177Lu
labelled BFCA in the reaction mixture could therefore be determined by
employing this technique. Hence, a combination of PC and TLC techniques
enabled the determination of the radiolabelling yield.
Determination of the accurate radiochemical purities of the 177Lu
labelled conjugates was achieved by employing HPLC. The 177Lu labelled
metronidazole-BFCA conjugate exhibited a retention time of 675 s while the
uncomplexed 177Lu and 177Lu-BFCA complex were eluted out with retention
times of 184 s and 250 s, respectively. On the other hand, 177Lu labelled
sanazole-BFCA conjugate exhibited a retention time of 420 s while 177Lu
labelled BFCA eluted out in 330 s under identical conditions. The HPLC
chromatograms of the radiolabelled conjugates are shown in Fig. 1 (a) and (b).

FIG. 1. (a) HPLC pattern of 177Lu labelled metronidazole-BFCA conjugate, (b) HPLC
pattern of 177Lu labelled sanazole-BFCA conjugate.

57
 DAS et al.

3.4. Optimization of the complexation yield

Several reaction parameters, such as concentration of the conjugates, pH

of the reaction mixture, incubation time and temperature, were varied
extensively in order to maximize the complexation yield. A maximum
complexation of ~97% was obtained when 25 μg of the metronidazole-BFCA
conjugate was incubated at 50°C for a period of 1 h at pH~5, while ~98%
complexation was obtained using 100 μg of the sanazole-BFCA conjugate
under identical conditions.

3.5. Stability of the 177Lu labelled conjugates

The stability of the 177Lu labelled conjugates was studied by incubating

the radiolabelled conjugates prepared under optimized conditions, employing
the standard quality control techniques mentioned earlier. It was observed that
both radiolabelled conjugates exhibited excellent stability as the radiochemical
purities of the complexes remained unaltered for 7 d at room temperature.

3.6. Biodistribution studies

To determine the tumour specificity of the 177Lu labelled conjugates, the

radiolabelled preparations were injected in Swiss mice bearing fibrosarcoma
tumours. The results of the biodistribution studies for 177Lu labelled metro-
nidazole-BFCA conjugate and sanazole-BFCA conjugate are tabulated in
Tables 1 and 2, respectively. The results show moderate tumour uptake of
0.73–0.88% of the injected activity per gram (ID/g) within 1 h post-injection.
Around 90% of the injected activity was observed to clear via the renal route
with insignificant accumulation in other major organs/tissues except the liver at
this time point. Clearance of the activity from all the organs at 24 h post-
injection is evident from the >98% excretion observed at this time point. The
tumour uptake was observed to decrease gradually over time and at 24 h post-
injection, 0.18–0.45% of the injected activity was retained in the tumour.
However, as the activity cleared out rapidly from all the non-target organs, the
tumour to blood (4.0–5.2 at 1 h post-injection and 18.0–22.5 at 24 h post-
injection) and tumour to muscle (4.6–12.2 at 1 h post-injection and 15.0–18.0 at
24 h post-injection) ratios at all the time points studied (1–24 h) were observed
to be quite high.
Though imaging hypoxia using diagnostic radiopharmaceuticals is a quite
well explored field, reports describing therapeutic agents making use of
nitroimidazoles as hypoxia markers are not well documented. Therefore, it is

58
 SESSION 8

TABLE 1. BIODISTRIBUTION PATTERN OF 177Lu LABELLED

METRONIDAZOLE-P-AMINO-BENZYL-DOTA CONJUGATE IN
SWISS MICE BEARING FIBROSARCOMA TUMOUR

%ID/g
Organ
1h 3h 24 h

Blood 0.14 (0.03) 0.02 (0.00) 0.02 (0.01)

Liver 0.52 (0.01) 0.18 (0.09) 0.07 (0.03)
Intestine 0.57 (0.04) 0.59 (0.13) 0.07 (0.01)
Kidney 1.72 (0.45) 0.56 (0.13) 0.61 (0.06)
Stomach 0.15 (0.05) 0.04 (0.03) 0.03 (0.01)
Heart 0.09 (0.05) 0.02 (0.02) 0.01 (0.01)
Lung 0.26 (0.14) 0.05 (0.03) 0.04 (0.03)
Muscle 0.06 (0.05) 0.04 (0.02) 0.06 (0.03)
Bone 0.16 (0.07) 0.13 (0.06) 0.08 (0.02)
Spleen 0.13 (0.08) 0.09 (0.04) 0.02 (0.01)
Tumour 0.73 (0.13) 0.56 (0.27) 0.45 (0.02)
Excretion 96.38 (0.90) 97.63 (0.68) 98.95 (0.28)

Note: Figures in parentheses show standard deviations. Three animals were used for
each time point studied.

pertinent to draw a comparison with respect to the tumour uptake and target to
non-target ratios of the radiolabelled conjugates described in the present paper
with a few standard agents intended for use in hypoxia imaging, such as 99mTc-
BMS181321 [10] and 99mTc-BRU59-21 [11], which indicates that the developed
agents exhibit superior tumour to blood and tumour to muscle ratios at the
time points studied.

4. CONCLUSION

The preparation and preliminary biodistribution studies of two tumour

avid 177Lu labelled conjugates are described in the present paper. Lutetium-177
was produced in ~185 Tbq/g (~5 × 103 Ci/g) specific activity and excellent radio-
nuclidic purity in moderate thermal neutron flux using an enriched (60.6% in
176
Lu) Lu2O3 target. Metronidazole and sanazole were successfully coupled
with polyazamacrocyclic BFCAs and subsequently radiolabelled with 177Lu of

59
 DAS et al.

TABLE 2. BIODISTRIBUTION PATTERN OF 177Lu LABELLED

SANAZOLE-P-AMINO-DOTA-ANILIDE CONJUGATE IN
SWISS MICE BEARING FIBROSARCOMA TUMOUR

Organ %ID/g

1h 3h 24 h

Blood 0.22 (0.10) 0.10 (0.04) 0.01 (0.00)

Liver 1.34 (1.05) 1.34 (0.13) 0.38 (0.03)
Intestine 0.36 (0.03) 0.26 (0.09) 0.17 (0.15)
Kidney 2.76 (0.62) 1.33 (0.31) 1.01 (0.12)
Stomach 0.30 (0.08) 0.20 (0.10) 0.06 (0.05)
Heart 0.22 (0.09) 0.04 (0.04) 0.03 (0.02)
Lung 0.50 (0.19) 0.13 (0.06) 0.03 (0.01)
Muscle 0.19 (0.14) 0.04 (0.00) 0.01 (0.00)
Spleen 0.24 (0.07) 0.09 (0.02) 0.07 (0.03)
Tumour 0.88 (0.20) 0.43 (0.12) 0.18 (0.02)
Excretion 89.55 (2.32) 94.77 (1.64) 97.69 (0.98)

Note: Figures in parentheses show standard deviations. Three animals were used for
each time point studied.

high radiochemical purity (>95%). The radiolabelled conjugates exhibited

excellent stability in storage at room temperature for up to 7 d. Biodistribution
studies carried out in Swiss mice bearing fibrosarcoma tumours revealed good
tumour uptake (0.73–0.88% ID/g at 1 h post-injection) with favourable tumour
to blood (4.0–5.2 at 1 h post-injection and 18.0–22.5 at 24 h post-injection) and
tumour to muscle (4.6–12.2 at 1 h post-injection and 15.0–18.0 at 24 h post-
injection) ratios.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge V. Venugopal, Director, Radio-

chemistry and Isotope Group for his keen interest and constant encour-
agement. The authors are grateful to C.K.K. Nair, Radiation Biology and
Health Sciences Division, BARC and to V.T. Kagiya, Health Research
Foundation, Kyoto, Japan for the kind gift of sanazole. The authors
acknowledge the help received from the staff members of the Animal

60
 SESSION 8

Experimentation Facility during animal experiments. The authors are grateful

to S.V. Thakare and K.C. Jagadeesan for their valuable help in carrying out the
irradiations of Lu targets.

REFERENCES

[1] NUNN, A., LINDER, K., STRAUSS, H.W., Nitroimidazoles and imaging
hypoxia, Eur. J. Nucl. Med. 22 (1995) 265.
[2] BROWN, J.M., Hypoxic cell radiosensitizers: What next?, Int. J. Radiat. Oncol.
Biol. Phys. 16 (1989), 987.
[3] CHAPMAN, J.D., Hypoxic sensitizers: Implications for radiation therapy, N.
Engl. J. Med. 301 (1979) 1429.
[4] DAS, T., et al., 99mTc-labeled modified metronidazole: A potential agent for
imaging hypoxia, Nucl. Med. Biol. 30 (2003) 127.
[5] SHIBAMOTO, Y., et al., Evaluation of various types of new hypoxic cell sensi-
tizers using the EMT6 single cell-spheroid-solid tumor system. Int. J. Radiat. Biol.
52 (1987) 347.
[6] FIRESTONE, R., Table of isotopes (Shirley, V.S., Ed.), John Wiley and Sons, New
York (1996).
[7] PILLAI, M.R.A., CHAKRABORTY, S., DAS, T., VENKATESH, M., RAMA-
MOORTHY, N., Production logistics of 177Lu for radionuclide therapy. Appl.
Radiat. Isot. 59 (2003)109.
[8] LIU, S., EDWARDS, D.S., Bifunctional chelators for therapeutic lanthanide radi-
opharmaceuticals, Bioconj. Chem. 12 (2001) 7.
[9] VOGEL, A.I., Textbook of practical organic chemistry, Longman Scientific
Group, London (1994).
[10] BALLINGER, J.R., MIN-KEE, J.W., RAUTH, A.M., In vitro and in vivo evalua-
tion of a technetium-99m labeled 2-nitroimidazole (BMS 181321) as marker of
tumor hypoxia, J. Nucl. Med. 37 (1996) 1023.
[11] MELO, T., DUNCAN, J., BALLINGER, J.R., RAUTH, A.M., BRU59-21, a
second-generation 99mTc-labeled 2-nitroimidazole for imaging hypoxia in tumors,
J. Nucl. Med. 41 (2000)169.

61
.
 LABELLING AND BIOLOGICAL EVALUATION OF
ANTI-CD20 FOR TREATMENT OF NON-HODGKIN’S
LYMPHOMA

P. OLIVER, A. ROBLES, V. TRINDADE, P. CABRAL,

V. TORTAROLO, A. NAPPA, G. RODRIGUEZ, H. BALTER
Radiopharmacy Department, Nuclear Research Center,
Faculty of Sciences,
Montevideo, Uruguay
Email: poliver@cin.edu.uy

Abstract

A radiopharmaceutical based on the use of Mab anti-CD20 labelled with 131I and
188
Re is proposed for the treatment of non-Hodgkin’s lymphoma. The antibody has been
successfully used alone as well as associated with cytotoxic drugs, therefore encouraging
the present research. The radionuclides were chosen on the basis of their decay proper-
ties and availability. Labelling techniques employed were previously used with other
monoclonals. Oxidation with chloramine-T was used for 131I labelling and SnF2·2H2O as
the reducing agent for 188Re. Non-specific precipitation and Sephadex purification were
used for primary control and extraction. Quality control procedures including thin layer
as well as high performance liquid chromatography were undertaken. Biodistribution in
mice as well as affinity characteristics in a source rich in CD20 antigens were also
studied. Stability over time was estimated. It was concluded that the labelling of anti-
CD20 with ß emitters of therapeutic interest, in this case 131I and 188Re, gave reliable
results by simple and efficient methodologies, yielding products compatible with clinical
radioimmunotherapy. Quality control methods for the evaluation of radiochemical
purity showed good reproducibility with short bench time. Immunoaffinity studies
showed binding dependency on membrane antigen concentration and good specificity
of binding was demonstrated by inhibition with unlabelled anti-CD20. Use of
membranes, stable at -80ºC for more than 6 months instead of concentrated short lived
leucocytes, has shown excellent reproducibility and therefore they are convenient at
production centres distant from blood banks. Biodistributions were useful to determine
the normal pattern and kinetics of uptake and excretion.

1. INTRODUCTION

The CD20 antigen is a non-glycosilated transmembrane phosphoprotein

35-37kD, expressed on the cell surface of normal B and pre-B but not in differ-
entiated normal plasma cells. It is overexpressed in neoplastic B cells; it then
being a good target for the therapeutical use of mab anti-CD20.

63
 OLIVER et al.

The anti-CD20 monoclonal chimeric humanized murine antibody

(Rituximab) has been successfully applied for the treatment of non-Hodgkin’s
lymphoma. However, upon labelling of the mab anti-CD20 with ß emitters
such as 90Y or 131I, the therapeutic efficacy is significantly increased due to the
radiological effects of ionizing radiation [1].
The authors’ objective was to develop reliable and efficient methods for
labelling anti-CD20 with ß emitters of therapeutic interest, and simple and
rugged quality control methods to evaluate radiochemical purity, biological
performance and immunoaffinity assessment.
Iodine-131 and 188Re have been used for the labelling of anti-CD20 as two
attractive alternatives due to their decay properties and availability (131I: Eßmax
= 0.63 MeV, Eγ = 0.364 MeV, T1/2 = 8 d; 188Re: Eßmax = 2.2 MeV, Eγ = 0.155 MeV,
T1/2 = 17 h, generator produced). Labelling of anti-CD20 was optimized
following the oxidation procedure of chloramine-T in the case of 131I [2] and
the synthesis of 188Re(IV) complex with the previously reduced monoclonal
antibody [3]. Quality control of the species obtained was done by physico-
chemical methods, including ITLC-SG and HPLC, non-specifc protein precipi-
tation, biological distribution in normal mice and immunoaffinity studies with
membrane antigens extracted from isolated leucocytes.

2. MATERIALS

Monoclonal antibody anti-CD20, 10 mg/mL, (Rituximab, Mabthera) from

Roche. Na131I, pH7–11, highly concentrated >100 mCi/mL (>3.7 GBq/mL) from
Tecnonuclear, Argentina. Na188ReO4 from 188W–188Re alumina generator
system with 24 h since previous elution, 500 mCi (18.5 GBq) from Oak Ridge
National Laboratory, ARCAL LII Programme.
Protein pak column SW300 (Waters). Gel permeation Sephadex G25 fine
short column PD10 (Pharmacia). Chloramine-T, phosphate buffer, BSA, NaCl,
methyl ethyl ketone, sodium tartrate, stannous fluoride, gentisic acid,
ITLC-SG, ammonia, ethanol, water.
Oven, magnetic stirrer, homogenizer, ionizing chamber calibrator
(Capintec CRC-7, United States of America), solid crystal NaI(Tl) low
efficiency gamma counter (ORTEC, USA), coaxial NaI(Tl) automatic high
efficiency gamma counter (Compac-120, Picker, USA), HPLC system with
radiometric and UV detectors (Varian Associates, model 5000), refrigerated
high speed and low speed centrifuges (Heraeus, Kendro, Germany and
IEC-CENTRA 8, USA).

64
 SESSION 8

3. METHODOLOGY

3.1. Labelling with iodine

The 131I- was introduced in one tyrosyl residue of the protein chain by
adding 28 MBq to 20 µg of anti-CD20 at pH7.4 and 10 µL of chloramine-T
(0.13 µg/µL). After 1 min reaction time at room temperature, the yield was
determined by protein precipitation with trichloroacetic acid solution (10%)
and purification was done by gel permeation with Sephadex G-25 eluting with
phosphate saline buffer 50mM, 0.2% BSA. Specific activity and iodine incorpo-
ration were determined.

3.2. Labelling with rhenium

For the labelling with 188Re, anti-CD20 was first reduced by incubation
with 2-mercaptoethanol to expose sulphydril groups and then purified by gel
permeation over a PD10 column. Fractions of reduced antibody were pooled
and formulated as kit for instant labelling. Each kit contained 1 mg anti-CD20;
82.8 mg of sodium tartrate; 1.67 mg of stannous fluoride and 0.25 mg gentisic
acid. For the labelling, sodium perrhenate (1.5–1.9 GBq), previously acidified,
was added to the kit and then incubated for 1 h at room temperature. The
radiochemical purity of 188Re-anti-CD20 was evaluated by ITLC-SG using
MEK and saline as solvents and by saturated ITLC-SG strips (BSA 5%) using
EtOH-NH4OH-H2O (2:1:5). It was also evaluated by HPLC using an SW300
protein Pak column and eluting with phosphate buffer 0.01M, pH7.4 at
1.0 mL/min. Specific activity was determined.

3.3. Affinity studies

Affinity studies were performed on leucocytes and on extracted

membrane antigens. Isolated CD20 antigen membrane preparations were
developed using a pool of concentrated leucocytes from the blood bank. After
initial centrifugation at 500g for 10 min at 4°C, the supernatant yellow layer
containing the leucocytes was separated, homogenized and centrifuged again at
500g to eliminate debris in the precipitate from the supernatant CD20 activity.
Isolation of membrane antigens was achieved by centrifugation at 26 800g for
60 min. Final protein concentration was determined by Lowry. The recovered
pool was aliquoted and stored at –80°C. Immunoaffinity was evaluated by
specific binding of tracer to membrane preparations ranging from 0.25 mg/mL
to 30 mg/mL, using an excess of (1.5 × 104 and 1 × 105) unlabelled monoclonal
anti-CD20 as two levels of non-specific binding, followed by calculating the

65
 OLIVER et al.

maximum binding capacity. Inhibition studies were conducted by incubating a

fixed membrane concentration of 1 and 4 mg/mL for 188Re-anti-CD20 and
131
I-anti-CD20 respectively, with increasing concentrations of unlabelled
anti-CD20 (0.3–3 mg/mL) and 250 Bq of tracer. All incubations of membrane
assays were done overnight at 4°C. For assays of intact leucocytes, 1 × 106 and
2.5 × 106 cells per tube were incubated with a similar amount of tracer and
unlabelled antibody for 1 h at 37°C. Data were analysed and fitted by Prisma
GraphPad software.

3.4. Biological studies

Biodistribution studies at 4, 18 and 24 h post-injection were carried out in

CD1 normal mice by intravenous administration of 9.3–55.5 MBq of 188Re-anti-
CD20. Organs and tissues of interest were removed and weighed and the radio-
activity counted. Results were expressed as per cent injected dose per organ
(%ID).

4. RESULTS

Radioiodination yields of anti-CD20 ranged from 43% to 82% and

specific activity was over 30 µCi/µg (1.11 MBq/µg), depending upon the amount
of monoclonal and the activity concentration of the radionuclide. The best
results were obtained when excess monoclonal and high concentrations of
activity were present. Figure 1 shows the typical purification profile.
The labelling with 188Re gave a radiochemical purity higher than 95% for
up to 3 h elapsed time and a specific activity of 40–50 µCi/µg (1.48–1.85 MBq/µg).
HPLC analysis revealed an Rt of 8.2 ± 0.3 min for the native mab anti-
CD20, while for 188ReO4- it was 10.6 ± 1 min.
Specific binding of 131I-anti-CD20 to membrane antigens increased as a
function of membrane concentration and reached 20.2 ± 0.5% for a total
protein content of 16.5 mg/mL (Fig. 2). Maximum binding capacity was 15 ± 2%
(n = 3). Inhibition of binding to membranes (4 mg/mL) was 67.2 ± 1% when 290
µg (5.22µM) of unlabelled anti-CD20 was added (Fig. 3). The IC50 value was
12.7nM.
Specific binding of 188Re-anti-CD20 to membranes reached 46 ± 1% for a
protein content of 33 mg/mL (Fig. 4). Maximum binding capacity was 17 ± 2%
(n = 3). Inhibition of binding to membranes (1 mg/mL) was 66 ± 5% when
12.6 µM (700 µg) of unlabelled anti-CD20 was added (Fig. 5). The IC50 was
determined as 13.3nM.

66
 SESSION 8

45

40

35

30
act (%)

25

20

15

10

0
5 2 5 5 5 8 5 11 .5 14
0. 3. 6. 9. 12

eluate (mL)

FIG. 1. Purification profile of mab anti-CD20 labelled with 131I on PD10 columns.

2000

1500
B (cpm)

1000

500

0
0 4 8 12 16

CD20 membranes (mg/mL)

FIG. 2. Binding of 131I anti-CD20 to increasing amounts of membrane antigens isolated
from leucocytes.

The biological distribution showed high urinary elimination at 24 h (59%)

while intestinal excretion was 10%. Negligible uptake by thyroid and stomach
(less than 0.7% and 1.9%, respectively) was observed.

67
 OLIVER et al.

4000

3000
B (cpm)

2000

1000

0
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

Anti-CD20 (mg/mL)

FIG. 3. Inhibition of the binding of 131I-anti-CD20 to membrane antigens by unlabelled

anti-CD20.

100

80

60
% Binding

40

20

0
0 5 10 15 20 25 30 35
CD20 Membranes (mg/mL)

B max 100ug aCD20 700ug aCD20

FIG. 4. 188Re-anti-CD20 binding to increasing amounts of membrane antigens isolated

from leucocytes.

68
 SESSION 8

8000

6000
B (cpm)

4000

2000

0
0 1 2 3 4

Anti-CD20 (mg/mL)

FIG. 5. Inhibition of the binding of 188Re-anti-CD20 to membrane antigens by unlabelled

anti-CD20.

FIG. 6. Per cent injected dose of 188Re-anti-CD20 in CD1 mice at 4, 18 and 24 h.

69
 OLIVER et al.

5. CONCLUSIONS

The labelling of anti-CD20 with ß emitters of therapeutic interest, in this

case 131I and 188Re, gave reliable results by simple and efficient methodologies
yielding products compatible with clinical radioimmunotherapy. In particular,
the radioiodination results were satisfactory even though purification was
always needed. Quality control methods for evaluation of radiochemical purity
showed good reproducibility with short bench time.
Immunoaffinity studies showed binding dependency to membrane
antigen concentration and good specificity of binding demonstrated by
inhibition with unlabelled anti-CD20.
Both tracers displayed high affinity in their binding to antigen
membranes with better results in the case of 188Re owing to its higher specific
activity. Nevertheless, it is considered that with a better quality and higher
specific activity of 131I than the one used in the present work, both tracers
would be equivalent.
Use of membranes, stable at -80ºC for more than 6 months instead of
concentrated short lived leucocytes, has shown excellent reproducibility and
therefore they are convenient at production centres distant from blood banks.

ACKNOWLEDGEMENTS

Partially supported by the IAEA, Arcal LII, and PEDECIBA Quimica.

REFERENCES

[1] JUWEID, M.E., Radioimmunotherapy of B cell Non-Hodgkin’s lymphoma: from

clinical trials to clinical practice, J Nucl Med (2002) 43:1507-1529.
[2] ROBLES, A.M., BALTER, H., OLIVER, P., WELLING, M., PAUWELS, E.K.J.,
Improved Radioiodination of biomolecules using exhaustive chloramine-T oxida-
tion, Nucl. Med. Biol. (2001) 28:999-1008.
[3] OLIVER, P., et al., Anti-CD20-188Re: Labelling and biological performance,
Nucl. Med. Review (2005) 8: 19-20.

70
THERAPEUTIC RADIOPHARMACEUTICALS

(Session 9)

Chairpersons

J. HARVEY TURNER
Australia

M. DONDI
IAEA
.
 177
Lu-DOTA-J591 MONOCLONAL ANTIBODY:
CHEMISTRY, TOXICITY, DOSIMETRY AND
CLINICAL EFFICACY
RIT of prostate cancer using 177Lu-J591 anti-PSMA antibody

S.J. GOLDSMITH, S. VALLABHAJOSULA, M.I. MILOWSKY,

D.M. NANUS, N.H. BANDER
Department of Radiology, Medicine and Urology,
New York Presbyterian Hospital and Weill Cornell Medical College of
Cornell University,
New York, United States of America
Email: svallabh@med.cornell.edu

Abstract

Prostate specific membrane antigen (PSMA) is a transmembrane antigen

virtually restricted to prostate tissue and the expression of which is increased in prostate
carcinoma. Indium-111 capromab pendetide (ProstaScint®) is an antibody specific to
the intracellular epitope of PSMA. For the past several years, the authors have been
engaged in the development and evaluation of a radiolabelled humanized monoclonal
antibody, J591, specific to an extracellular epitope of PSMA. After demonstrating that
J591 is internalized, the authors developed 90Y and 177Lu labelled DOTA-J591 for radio-
immunotherapy of prostate cancer. These radiometals remain localized in tumour foci
(as well as other tissues including non-specific hepatic uptake) compared to 131I labelled
proteins. In contrast to 90Y, 177Lu is a radiometal with a low energy β− emission as well as
a γ photon emission that makes it convenient to demonstrate localization and quantify
biodistribution and turnover externally. Metastatic prostate carcinoma, in particular, is
an ideal target for radioimmunotherapy since chemotherapeutic or other medical
therapies are not ideal or appropriate. All human studies were approved by the institu-
tional IRB and were performed as a phase I dose escalation study under an IND.
Initially, different doses of J591 were used to assess the influence of total protein content
on biodistribution. From these studies, it was determined that 10 mg/m2 total protein per
patient was sufficient to reduce non-specific organ uptake, potentially optimizing
tumour access to labelled antibody. With 177Lu-J591, dose limiting haematological
toxicity was observed at 2590 MBq/m2. In 35 patients, the appearance and severity of
myelotoxicity correlated with the calculated bone marrow radiation absorbed dose.
Response in terms of significant decreases in PSA, bone flare phenomena followed by
improvement in bone scan findings have been observed but only in a single dose trial;
relapse has occurred suggesting that further evaluation with multidose administration at
<MTD or therapy in combination with radiosensitizing chemotherapy may be necessary.

73
 GOLDSMITH et al.

1. INTRODUCTION

In the last 10 years, among a number of radionuclides emitting β−

particles, 131I and 90Y have emerged as the primary choices for developing
radiopharmaceuticals for therapy. Both of these nuclides, however, have
advantages and disadvantages [1]. Iodine-131 has lower energy β− particles and
longer physical half-life compared to 90Y. The radioiodinated molecules are
dehalogenated in vivo and the free radioiodide and the iodinated peptide
fragments are washed out of tissues and excreted in the urine. In contrast, when
90
Y is bound to peptides and proteins via a bifunctional chelate, the radio-
labelled complex is stable in vivo and 90Y is trapped within the cell, leading to
higher accretion and retention by the tumour. Iodine-131 has γ photons
(0.364 MeV) useful for biodistribution and dosimetry studies. Since 90Y does
not emit γ photons, 111In labelled antibodies are generally used as chemical and
biological surrogates to study biodistribution and estimate radiation dosimetry
of 90Y labelled antibodies. Some recent studies, however, have shown that there
are significant differences in biodistribution between 111In and 90Y labelled
agents.

177
1.1. Lu for therapy

In recent years, 177Lu, a β− emitting lanthanide radiometal with excellent

physical properties (Table 1), has emerged as an ideal and appropriate radio-
nuclide for therapy [2]. Unlike 90Y, 177Lu has gamma photons that are useful for
biodistribution and dosimetry studies. Chemically, yttrium and lutetium metals
favour the +3 oxidation state, similar to indium, but there are minor differences
in the solution and coordination chemistries among these metals.
The theoretical maximum specific activity of 177Lu is 4070 GBq/mg
(110 Ci/mg). At present, 177Lu is produced in reactors by neutron capture on
natural Lu or enriched 176Lu (direct method) and 176Yb (indirect method).

TABLE 1. RADIONUCLIDES FOR THERAPY

Half-life β− energy (MeV) γ energy Range in tissue (mm)

Nuclide (d) Max. Ave. (MeV) Max. Ave.
90
Y 2.67 2.28 0.935 No γ 12.0 2.76
177
Lu 6.7 0.497 0.133 208 (11%)
131
I 8.04 0.61 0.20 364 (81%) 2.4 0.40

74
 SESSION 9

The maximum achievable specific activity of 177Lu produced via neutron

irradiation of 176Yb strongly depends on the percentage of 175Lu, 176Lu and
174
Yb stable isotopes in the material to be irradiated. The ‘indirect’ route could
provide no carrier added 177Lu free of 177mLu (T½ = 160 d) radionuclidic
contaminant. Processing and dispensing of 177Lu from the ‘direct’ route is
straightforward and efficient, while processing and purification from the
indirect route is time consuming and expensive [3].
The 177Lu (specific activity = 740–1110 GBq/mg) used in the authors’ J591
antibody studies was produced using the direct method at the Missouri
University Research Reactor facility. Radionuclide purity was evaluated by
gamma spectroscopy to determine the amount of 177Lu present in the samples.
A typical sample of 177Lu contains 59–64 ppm of 177mLu, which corresponds to
approximately 0.006% [4].

1.2. Prostate specific membrane antigen (PSMA)

In prostate cancer, the most well-established, cell surface antigen yet

identified is PSMA [5, 6]. Since it is not secreted into the blood stream, PSMA
is an ideal antigen for targeted therapy. PSMA is a highly prostate restricted
type II integral membrane cell surface glycoprotein expressed by all prostate
cancers and expression levels progressively increase in more poorly differen-
tiated, metastatic and hormone refractory cancers [7–10]. PSMA was originally
thought to be prostate specific, but recent studies have shown that it is also
expressed by small intestine epithelial (brush border) cells, proximal renal
tubule cells and salivary glands [11] and the level of expression in these normal
tissues, however, is 100–1000 fold less than in prostate tissue. Immunohisto-
chemical studies have shown that PSMA is also expressed by vascular
endothelial cells of numerous solid tumour malignancies. However, PSMA is
not expressed by normal vascular endothelium in benign tissues or in
neoplastic epithelial cells of non-prostate malignancies [12, 13]. These data
suggest that metastatic prostate carcinoma, in particular, is an ideal target for
radioimmunotherapy because of the restricted expression of PSMA, the
pattern of metastatic involvement often involving micrometastases in lymph
nodes and bone marrow and the general lack of suitable chemotherapeutic or
other medical therapy [14].
PSMA is a 100 000 kDa molecular weight protein that spans the cell
membrane [5, 15]. The first 19 amino acids (from the N-terminus) lie within the
cytoplasm, followed by a membrane spanning domain of 24 amino acids, in turn
followed by a large extracellular domain of 707 amino acids [15].

75
 GOLDSMITH et al.

1.3. Anti-PSMA monoclonal antibodies (mAb)

A murine mAb, 7E11 that binds to an intracellular epitope of the PSMA

protein [16] labelled with 111In (Capromab pendetide or ProstaScint®) is an
FDA approved imaging agent for identification of soft tissue metastases in
patients with prostate cancer [17, 18]. Owing to its intracellular binding site,
however, 7E11 binds only dead or dying PC cells [19].
The J591 is an anti-PSMA mAb that binds with high affinity to the extra-
cellular domain of PSMAext and is rapidly internalized [12, 20]. The murine
antibody J591 was de-immunized to allow for repeated dosing. J591 de-
immunization involved genetic engineering into a human IgG1 with identical
specificity and affinity as its murine counterpart with the added capability to
induce ADCC with human immune effector cells [21, 22].

177
1.4. Lu-DOTA-J591

The clinical grade de-immunized J591 mAb was produced under GMP
conditions at Lonza Biologics (Slough, United Kingdom) and supplied in 5 mL
of phosphate buffer, pH7.0 containing 5 mg/mL of antibody. The J591 antibody
was covalently linked with the chelating agent, 1,4,7,10-tetraazacyclododecane-
N,N’,N’’,N’’’-tetraacetic acid (DOTA) (Goodwin Biotech, Plantation, United
States of America) as previously reported [22]. The sterile pyrogen free clinical
material, DOTA-J591 mAb in 0.3M ammonium acetate buffer, pH7.0 (8 mg/mL)
was provided by BZL Biologics (Framingham, USA).
The DOTA-J591 mAb was labelled by incubating the antibody in an
ammonium acetate buffer (1M, pH7.0) with 177Lu chloride as previously
described [23]. 177Lu-DOTA-J591 mAb (177Lu-J591) was purified by gel
filtration and sterilized by membrane (0.2 μm) filtration prior to administration
into patients. Labelling efficiency and radiochemical purity were determined
using ITLC SG and 5mM DTPA solution as solvent. Immunoreactivity of
177
Lu-J591 was determined by the Lindmo method using PSMA positive
LNCaP tumour cells [24, 25]. Immunoreactivity was well preserved, even at
0.5–0.7 GBq/mg of specific activity.

2. CLINICAL STUDIES

2.1. Patient population

Eligible patients had a prior histological diagnosis of prostate cancer with

evidence of recurrent or metastatic disease as defined by a rising prostate

76
 SESSION 9

specific antigen (PSA) and/or abnormal radiological studies including bone

scan, computed axial tomography and/or magnetic resonance imaging.
Patients were required to have a PSA ≥1.0 at the time of entry with three
consecutive rising PSA values over a period of ≥2 weeks. Additional require-
ments included: platelet count ≥150 000/mm3 and neutrophil count ≥2000/mm3;
a bone marrow biopsy demonstrating ≤10% replacement by tumour on a
unilateral sample or a mean of ≤25% replacement by tumour on bilateral
samples. A total of 35 patients received 177Lu treatment; 19 patients received a
single dose while 16 patients received 2–3 doses.

2.2. Dose escalation

In a dose escalation phase I clinical, patients received 177Lu activity in the

range 370–2775 MBq/m2 (10–75 mCi/m2). Additional unconjugated, cold J591
antibody was added to give a constant protein dose of 10 mg/m2 with the 177Lu
dose. The final radiolabelled J591 mAb was diluted to 20 mL with physiological
saline solution and was infused intravenously over a period of 5 min.

2.2.1. Retreatment

Patients were considered eligible for up to 2 or 3 retreatments with 177Lu-

J591 at 6 week intervals if their platelet and neutrophil count recovery was
satisfactory (platelet count >70% of the baseline platelet count of the prior
treatment cycle with a minimum recovery to at least 75 × 109/L and ANC >80%
of the baseline ANC of the prior treatment cycle with a minimum recovery to
1.3 × 109/L). Patients who experienced any grade >3 non-haematological
toxicity in a prior treatment cycle were ineligible for retreatment. Retreatment
consisted of patients receiving the same 177Lu dose as their initial cycle.

2.3. Imaging studies: Tumour targeting of 177Lu-J591

In order to determine the plasma clearance kinetics following infusion of

177
Lu-J591, 8–10 venous blood samples were obtained over a period of 12–14 d.
To assess the biodistribution, total body images were obtained within 1 h post-
infusion (day 0) and again at 4 additional time points over the next 2 weeks
(e.g. 1, 3, 6–9 and 13–14 d). The gamma camera images were obtained using a
dual head ADAC (ADAC, Milpitas, USA) or GE gamma camera (GE,
Milwaukee, USA) fitted with an appropriate collimator. All 5 scans for each
patient were obtained with the same gamma camera. SPECT studies of the
abdomen, pelvis and/or areas of suspected metastatic lesions were performed
on days 2–3 and/or 6–7 in selected patients.

77
 GOLDSMITH et al.

2.4. Toxicity evaluation

Dose limiting toxicity (DLT) was defined as the following: haemato-

logical toxicity consisting of grade 4 thrombocytopenia (platelet <10 × 109/L)
and/or grade 4 neutropenia (ANC <0.5 × 109) lasting >5 d; and other toxicity
consisting of any grade ≥3 non-haematological toxicity attributable to 177Lu or
90
Y labelled J591. The NCI CTEP Common Toxicity Criteria, version 2.0, was
utilized. The maximum tolerated dose (MTD) was defined as the dose level at
which 0/6 or 1/6 patients experience a DLT with the next higher dose level
having ≥2 patients experiencing DLT. Once the MTD was reached, at least 6
patients were to be evaluated at that dose level. The stopping point for single
doses and the subsequent doses were the same. DLT and MTD were
determined with multiple doses using the same criteria that were used for
single doses.

3. PHARMACOKINETICS AND RADIATION DOSIMETRY

Following intravenous administration of 177Lu-J591, there is a bi-

exponential plasma clearance; <20% of activity had a fast component with a T½
of <3 h. The remaining 80% cleared from plasma slowly with an average T½ of
44 ± 15 h. The percentage of injected radioactivity in the total urine collected
over a period of 3 d is 7.3 ± 2.8%.
Whole body gamma camera images showed that within 24 h post-
injection, 177Lu radioactivity was predominantly in the blood pool as seen by
the increased activity in the heart and major blood vessels. Subsequently, there
was a decrease in blood pool activity with a gradual accumulation of activity in
liver, spleen, kidney and bone or bone marrow. Starting from day 2, there was
some gastrointestinal activity. Images on days 6 and 7 clearly show that 177Lu is
very effective in identifying the metastatic lesions with a very high target/
background contrast.
Radiation absorbed dose estimates (mGy/MBq) for a number of target
organs from 177Lu-J591 were estimated using the MIRD technique. The liver
receives the highest dose followed by spleen and kidney. The liver is the critical
organ, with a radiation absorbed dose of 2.10 ± 0.60 mGy/MBq. The dose to
bone marrow is 0.32 ± 0.1 mGy/MBq.

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 SESSION 9

177
4. Lu-J591 TUMOUR TARGETING

Among the 35 patients receiving 177Lu-J591 mAb, 30 (86%) had

metastatic disease detected on imaging studies following administration of
treatment dose. Specifically, 21 (60%) patients had bone only metastases, 6
(17%) had soft tissue only metastases and 3 (9%) had both bone and soft tissue
disease. In all patients, all known sites of metastatic disease were successfully
imaged by 177Lu-J591 scintigraphy as shown in Fig. 1. Patients receiving
multiple doses were imaged one week after dose 2 and, where applicable,
dose 3. In all of these cases, tumour targeting was seen on serial images.

5. HAEMATOLOGICAL TOXICITY

Among patients receiving a single dose of 177Lu-J591, thrombocytopenia

and neutropenia were dose related and this is summarized in Table 2. One of the
six patients at the 2590 MBq/m2 dose level and one of the three patients at the
2775 MBq/m2 dose level experienced dose limiting (grade 4) platelet toxicity.

Anterior Posterior

FIG. 1. Tumour localization of 177Lu-J591 (on day 6) in a patient with prostate cancer.

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 GOLDSMITH et al.

TABLE 2. HAEMATOLOGICAL TOXICITY FOLLOWING 177Lu-J591

ADMINISTRATION

Dose Pts Thrombocytopenia grade Neutropenia grade

(MBq/m2) (n) 0 1 2 3 4 0 1 2 2 4

370 3 3 3
555 3 2 1 3
1110 5 2 2 1 2 2 1
1665 5 1 3 1 2 1 2
2220 3 1 1 1 1 2
2590 6 1 4 1 1 2 1 2
2775 3 2 1 2 1

Most of the remaining patients at these two dose levels experienced grade 3
platelet toxicity. With the 177Lu-J591 antibody, the 2590 MBq/m2 dose level was
determined to be the MTD. Post-treatment platelet counts decline generally at
2.5–3 weeks with platelet nadirs occurring at 4–5 weeks thereafter followed by a
recovery phase. In all the three subjects, at 2775 MBq/m2, the fractional decrease
in platelets as a function of time (days) is shown in Fig. 2. The mean platelet
counts returned to 80–90% of their pre-treatment values over 2–3 weeks.

FIG. 2. Haematological toxicity (thrombocytopenia) in patients with prostate cancer

(P1–P3) following treatment with a single dose (2775 MBq/m2) of 177Lu-J591.

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 SESSION 9

5.1. Haematological toxicity following retreatment

Multiple doses of 1110 MBq/m2 were well tolerated in 10 patients; 6

received 2 doses and 4 received 3 doses. The median time between doses 1 and 2
was 64 d (range 42–238 d) and between doses 2 and 3 was 53 d (range: 50–55 d).
The 4 patients who received 3 doses totalling 3330 MBq/m2 did so over a period
of 98–126 d. The fractional decrease in platelets following the three doses at
1110 MBq/m2 is shown in Fig. 3(a). The platelet recovery was very good in
three patients while the fourth patient had grade 4 platelet toxicity and began a
different therapy owing to disease progression, prior to being able to assess
toxicity. At 1665 MBq/m2, two of the three patients developed prolonged
grade 3 platelet toxicity, each requiring three platelet transfusions after the
second dose. Thrombocytopenia in one of the patients who received two doses
is shown in Fig. 3(b).

5.2. Correlation of haematological toxicity with bone marrow radiation dose

With 177Lu-J591, haematological toxicity increased directly with the dose

177
of Lu. Similarly, the fractional decrease in platelets also gradually increased
as the 177Lu dose is increased. However, with ANC, no dose–response
relationship was seen in the range 370–1665 MBq/m2. The fractional decrease
in platelets following administration of 177Lu-J591 correlated very well with

FIG. 3(a). Haematological toxicity (thrombocytopenia) in patients with prostate cancer

(P1–P4) following treatment with three doses (1110 MBq/m2) of 177Lu-J591.

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 GOLDSMITH et al.

FIG. 3(b). Haematological toxicity (thrombocytopenia) in a patient with prostate cancer

following treatment with two doses (1665 MBq/m2 each) of 177Lu-J591.

177
Lu dose (r = 0.88) and the bone marrow radiation dose based on blood
radioactivity (Fig. 4 (a) and (b)).

6. FORMATION OF HUMAN ANTI-HUMAN ANTIBODIES

Human anti-human antibody assays were negative throughout the trial,

including those patients receiving multiple doses. In the patients who received
multiple doses, there was no change in the rate of drug clearance or tumour
targeting on scans with sequential doses although, in some cases, it appeared
that increased uptake and/or additional lesions were seen on the later antibody
scan(s) consistent with disease progression (data not shown).

7. ANTI-TUMOUR RESPONSE

All 35 patients in this trial had abnormal, rising PSAs and 7 patients had
measurable disease. None of the 7 patients with measurable disease had an
objective tumour response nor a ≥50% PSA decline. On the basis of PSA, 14
patients demonstrated progressive disease (PSA increase of >25%) after

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 SESSION 9

FIG. 4(a). Correlation of fractional decrease in platelets with the total treatment dose
(MBq) of the 177Lu-J591 antibody.

FIG. 4(b). Correlation of fractional decrease in platelets with bone marrow radiation
absorbed dose (cGy) following treatment with the 177Lu-J591 antibody.

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 GOLDSMITH et al.

treatment while 21 of the 35 patients had evidence of biological activity. Four

patients had >50% PSA declines lasting 3+ to 8 months, and 16 patients had
PSA stabilization (<25% increase from baseline) of >28 d. The median
duration of PSA stabilization was 60 d with a range of 28–601+ d.

8. DISCUSSION

The de-immunized J591 mAb is the first radiolabelled antibody specific

to the extracellular domain of PSMA to be tested as a radiotherapeutic in
patients with prostate cancer. The authors have previously documented that
radiolabelled J591 binds with high affinity (1nM) to PSMA and that the PSMA
antibody complex is internalized, thereby delivering the radionuclide only to
the interior of the targeted cancer cells [20, 22]. In nude mice bearing PSMA
positive xenografts, the authors demonstrated the tumour specific localization
of the J591 antibody labelled with 131I and 111In. Autoradiographic studies
suggested that J591 preferentially accumulates in the areas of viable tumour
[26]. In the same animal model, the authors have also compared the biodistri-
bution of 111In, 90Y and 177Lu labelled DOTA-J591 mAbs and reported that the
biodistribution of 177Lu and 90Y-J591 were similar. However, the uptake and
retention of 111In activity in the liver and spleen were significantly higher [27].
In PSMA positive prostate cancer xenografts, RIT with single and multiple
treatments demonstrated significant tumour reduction and prolongation of
average life span with both 90Y and 177Lu labelled J591 antibodies [28].
In patients with prostate cancer, the authors have performed several
phase I dose escalation studies to evaluate the safety and toxicity of RIT using
111
In, 90Y and 177Lu labelled de-immunized DOTA J591 mAb [6, 14, 29, 30].
Following intravenous administration of radiolabelled J591, the plasma
clearance of 111In-J591 (44.2 ± 14) and 177Lu-J591 (43.6 ± 16) are quite similar
[31]. Between these two tracers, statistically no significant differences were
observed in biological half-life, area under the curve, maximum blood concen-
tration at time zero, volume of distribution and clearance. The imaging studies,
however, showed that there were minor differences in the biodistribution of
111
In and 177Lu labelled J591 [31]. During the first week, both tracers showed a
gradual accumulation in the liver and by day 6, the amount of 111In activity was
25% higher compared to that with 177Lu (p <0.05). Radiation dosimetry
estimates for 90Y-J591 calculated from 111In or 177Lu data were mostly similar
and show that the liver is the critical organ, followed by the spleen and kidney.
The biodistribution studies in human subjects suggest that 177Lu may be a
potential alternative for estimating the pharmacokinetics and biodistribution
of 90Y labelled radiopharmaceuticals [31].

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177
8.1. Lu-J591 tumour targeting

The authors have previously published their experiences with regard to

imaging studies using 111In-J591, which clearly demonstrated excellent tumour
targeting in PC patients without prior selection for PSMA expression [32]. In
this trial, using the same preparation of DOTA-J591 and a similar patient
population, the authors also studied the value of 177Lu-J591 imaging studies to
assess tumour targeting. The gamma emission from 177Lu allows direct imaging
and dosimetry determinations, thereby eliminating the need for a surrogate
isotope such as 111In for scintigraphy purposes. As in the authors’ previous
trials, patients in this trial were not pre-screened or selected for PSMA
expression. Nevertheless, as previously observed with the J591 antibody,
targeting in this trial was excellent, further confirming immunopathological
studies indicating that all prostate cancers are PSMA positive and, therefore,
potential candidates for J591 targeted monoclonal antibody vehicles. In this
trial, all known lesions clinically defined on bone scan and computed
tomography or magnetic resonance imaging were detectable on planar J591
images [30]. In at least three cases, bone lesions were apparent on the J591 scan
prior to becoming evident on the conventional bone scan. Sequential imaging
in patients who received multiple doses continued to localize in tumour sites
with no change in pharmacokinetics or biodistribution, consistent with
laboratory assays which demonstrated that the J591 antibody is not
immunogenic and that human anti-human antibody development has not
occurred.

177
8.2. Lu-J591 toxicity

DLT in this trial, as in RIT trials in general, was limited to myelotoxicity.

The MTD of a single dose of 177Lu-J591 was 2590 MBq/m2. This MTD is
significantly higher than the MTD of 647.5 MBq/m2 that the authors found with
90
Y using the same DOTA-J591 preparation in a similar patient population
[29]. This finding likely relates to the lower energy and range of 177Lu resulting
in less bystander radiation to the marrow. The longer half-life, lower energy
and shorter range of the beta emission of 177Lu, relative to 90Y, provide
theoretical advantages in prostate cancer where the metastases tend to be small
volume sites in marrow rather than bulky sites. Ultimately, of course, a higher
MTD is irrelevant unless it results in an improved therapeutic ratio. There is
limited prior experience with RIT in prostate cancer and no previous RIT trials
have studied 177Lu labelled antibodies in prostate cancer. The MTD of
2590 MBq/m2, however, compares well with that of 131I labelled mAbs in other
RIT trials in prostate and other cancers. While 177Lu and 131I have similar decay

85
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characteristics, use of the latter is compromised in this antigenic system as, after
internalization, 131I labelled antibody is rapidly dehalogenated and diffuses out
of tumour cells while radiometals such as 177Lu or 90Y remain sequestered
within the targeted tumour cells.
In this study, as in the authors’ 90Y-J591 trial, no clear relationship was
found between toxicity and a history of prior chemotherapy treatment.
Similarly, no correlation was found between toxicity and prior radiotherapy nor
between toxicity and the extent of bone marrow involvement by cancer [30].
Similar observations in prostate cancer were made by Knox et al. [33] and
O’Donnell et al. [34]. The non-haematological toxicity in RIT trials in general
and in this 177Lu-J591 trial was minimal and not dose limiting. Radiation
dosimetry calculations indicate a radiation dose to liver is approximately
1088 cGy at the MTD of 2590 MBq/m2. In addition, in this 177Lu-J591 trial, no
significant hepatotoxicity was seen in patients who received multiple doses,
including 8 patients who received cumulative doses between 3330–4440 MBq/m2.
Radiation doses to kidney and spleen were also well within acceptable limits
and no related organ toxicity was noted [31].
In patients who were treated with 177Lu-J591, a strong correlation was
observed between myelotoxicity and the amount of administered 177Lu dose or
the bone marrow absorbed radiation dose [35]. In contrast, the correlation
between myelotoxicity and marrow radiation absorbed dose or the adminis-
tered total MBq dose has been poor with 90Y-J591. The similarly weak
association between myelotoxicity and bone marrow radiation absorbed doses
based on blood has been previously reported for both 131I and 90Y labelled
mAbs. The authors believe that the demonstration of the predictability of
myelotoxicity based on bone marrow radiation dose with 177Lu, but not with
the 90Y radionuclide, may help us understand the importance of the energy of
radiation and the relative in vivo stability of the radionuclide–antibody
complex in the overall assessment of radiation dose and myelotoxicity.

177
8.3. Lu-J591 retreatment

Sixteen of the 35 patients in 177Lu trial received multiple doses. Two doses
of 1665 or 2220 mCi/m2, totalling 3330–4440 MBq/m2, proved to be quite toxic,
with 3 of 5 patients experiencing prolonged and incomplete platelet recovery.
Two or more doses of 1110 MBq/m2, however, were well tolerated and
4 patients received cumulative doses of 3330 MBq/m2, almost 30% higher than
the single dose MTD [30]. In this study, each dose was administered after
allowing for haematological recovery from the prior dose. It therefore took
3–4 months to administer the three doses, resulting in a higher cumulative dose
but lower dose rate. While there may be advantages to the higher cumulative

86
 SESSION 9

dose, the time required to deliver this dose using this regimen may be more
than offset by unrelenting tumour progression. Given the kinetics of platelet
decline and recovery, a dose interval of 14–17 d may allow a two dose regimen
that might result in a higher cumulative dose than a single dose regimen to be
given over a shorter period of time than attempted in this trial. Such a schedule
would result in the onset of platelet recovery from the first dose coinciding with
platelet decline from the second dose thereby resulting in a longer but
shallower nadir than with a single MTD dose. Such a dose schedule remains to
be explored.

9. CONCLUSION

The 177Lu-J591 is well tolerated, non-immunogenic, can be administered

in multiple doses and targets PC metastases with sensitivity and specificity.
Having determined the single dose MTD of 2590 MBq/m2 and the tolerability
of multiple doses of 177Lu-J591, phase 2 trials are being performed to assess
anti-tumour activity in both the single and multiple dose formats. Although no
patients in this trial had an objective measurable disease response (PR or CR),
in the authors’ 90Y trial there was a correlation of PSA response with
measurable disease response indicating that PSA was a reasonable measure of
anti-tumour activity in the RIT setting. In the 177Lu trial, 4 patients had PSA
declines of ≥50% and 16 patients had PSA stabilization suggesting that 177Lu-
J591 may have biological activity. This merits exploration in a phase 2 trial.
Additional studies can evaluate the combination of radiolabelled J591 plus
chemotherapy, such as docetaxel, an agent active in prostate cancer and known
to have radiosensitizing properties.

ACKNOWLEDGEMENTS

The following individuals are acknowledged for their substantial contri-

bution to this trial: P.J. Kothari, K.A. Hamacher, M. Cobham, RN, F. Berger,
RN, and M. Joyce, NP, and the technical staff in Nuclear Medicine and the
General Clinical Research Center.

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[16] HOROSZEWICZ, J.S., et al., Monoclonal antibodies to a new antigenic marker

in epithelial prostatic cells and serum of prostatic cancer patients, Anticancer Res.
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[17] PETRONIS, J.D., et al., Indium-111 capromab pendetide (ProstaScint) imaging to
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antibodies specific for the extracellular domain of prostate specific membrane
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[23] VALLABHAJOSULA, S., et al., Radiolabeled J591 antibody specific to prostate
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[26] SMITH-JONES, P.M., et al., Radiolabeled monoclonal antibodies specific to the
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nude mice bearing LNCaP human prostate tumor. J. Nucl. Med. 44 (2003) 610.
[27] SMITH-JONES, P.M., et al., Comparative biodistributions of 111In-DOTA-J591
and 177Lu-J591 in nude mice bearing LNCaP tumors, J. Nucl. Med. 42 (2001) 241.
[28] VALLABHAJOSULA, S., et al., Radioimmunotherapy of prostate cancer in
human xenografts using monoclonal antibodies specific prostate specific
membrane antigen (PSMA): Studies in nude mice, The prostate 58 (2004) 145.
[29] MILOWSKY, M.I., et al., Phase I trial of 90Y-labeled anti-PSMA monoclonal
antibody J591 for androgen-independent prostate cancer, J. Clin. Oncol. 22 (2004)
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[30] BANDER, N.H., et al., Phase I trial of 177Lutetium-labeled J591, a monoclonal
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[31] VALLABHAJOSULA, S., et al., Pharmacokinetics and biodistribution of 111In

and 177Lu labeled J591 antibody specific to prostate specific membrane antigen:
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46 (2005) 634.
[32] BANDER, N.H., et al., Targeting metastatic prostate cancer with radiolabeled
monoclonal antibody J591 to the extracellular domain of prostate specific
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[33] KNOX, S.J., et al., Yttrium-90-labeled anti-CD20 monoclonal antibody therapy of
recurrent B-cell lymphoma, Clin. Cancer Res. 2 (1996) 457.
[34] O’DONNELL, R.T., et al., Combined modality radioimmunotherapy for human
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Prostate 50 (2002) 37.
[35] VALLABHAJOSULA, S., et al., Prediction of myelotoxicity based on bone
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90
 RADIOLABELLED SOMATOSTATIN ANALOGUES
FOR RADIONUCLIDE THERAPY OF TUMOURS

M. DE JONG, D. KWEKKEBOOM, R. VALKEMA, E. KRENNING

Department of Nuclear Medicine,
Erasmus MC,
Rotterdam, Netherlands
Email: m.hendriks-dejong@erasmusmc.nl

Abstract

Molecular imaging and therapy are rapidly developing and will become important
topics in medicine in the 21st century. Radiolabelled peptides that bind to receptors
form an important class of radiopharmaceuticals for tumour diagnosis and therapy. The
specific receptor binding property of the peptide can be exploited by labelling with
radionuclide and using the radiolabelled peptide as a vehicle to guide the radioactivity
to tumours expressing a particular receptor. The high affinity of the peptide for the
peptide–receptor complex facilitates high uptake of the radiolabel in receptor
expressing tumours, while its relatively small size facilitates rapid clearance from blood,
resulting in low background radioactivity. The use of radiolabelled peptides is growing
rapidly due to these favourable characteristics, their low antigenicity and ease of
production. Receptor binding peptides labelled with gamma radiation emitters or
positron emitters enable non-evasive, whole body visualization. This process is referred
to as peptide receptor scintigraphy and is being used to detect, stage and plan the
therapy of receptor expressing tumours and also to follow tumours after therapy. In
addition, labelled with a therapeutic beta emitter these peptide molecules have the
potential to destroy receptor expressing tumours, an approach referred to as peptide
receptor radionuclide therapy (PRRT). To date, five somatostatin receptor subtypes
(sst1–sst5) have been identified and cloned. The diagnostic accuracy of 111In labelled
octreotide to visualize tumour lesions after intravenous injection has been determined
in a large series of patients with sst2 positive, mostly neuroendocrine tumours. Most
interesting is the successful application of somatostatin analogues in PET, after labelling
with positron emitters. The next logical step was to try to label these with therapeutic
radionuclides and to treat receptor positive tumors with peptide receptor radionuclides.
So far, 90Y and 177Lu are the most frequently used radionuclides in PRRT. The second
generation somatostatin analogue DOTA-Tyr3-octreotide can form a stable complex
with 90Y. In rats with subcutaneous, CA20948 pancreatic tumours, 90Y-DOTA-Tyr3-
octreotide, effectively controlled tumour growth. Studies to determine the therapeutic
efficacy of 90Y-DOTA-Tyr3-octreotide in cancer patients are ongoing at various institu-
tions and show most promising rates of complete plus partial remission. With the devel-
opment of new somatostatin analogues that bind with high affinity receptors on

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tumours, the available tools for radionuclide imaging and therapy of these tumours have
increased significantly.

1. INTRODUCTION

The 14 amino acid peptide somatostatin plays an important role in the

physiological regulation of hormones and organs in the body. Somatostatin
effects are mediated by high affinity G-protein coupled membrane receptors,
the integral membrane glycoproteins. Five different human somatostatin
receptor subtypes have been cloned [1–3]. Somatostatin binds to all subtypes
with high affinity.
The finding that somatostatin inhibits hormone secretion of various
glands led to the use of somatostatin in the treatment of symptoms due to
diseases with overproduction of hormones. The native peptide somatostatin is
itself unsuitable for treatment, as after intravenous administration it has a very
short half-life due to rapid enzymatic degradation. Therefore, somatostatin
analogues that are more resistant to enzymatic degradation were synthesized.
The molecule was modified in various ways with preservation of the biological
activity of the original molecule, resulting in, for example, the 8 amino acids
containing the somatostatin analogue octreotide, having a long and therapeuti-
cally useful plasma half-life. The affinity of the different somatostatin
analogues for these subtypes differs considerably. Octreotide for example binds
with high affinity to the somatostatin receptor subtype 2 (sst2), and with lower
affinities to sst5 and sst3. It shows no binding to sst1 or sst4 [2, 4].
Neuroendocrine gastro-entero-pancreatic (GEP) tumours, which
comprise pancreatic islet cell tumours, non-functioning neuroendocrine
pancreatic tumours and carcinoids are usually slow growing. When metasta-
sized, the widely used treatment with stable somatostatin analogues results in
reduced hormonal overproduction and symptomatic relief in most cases [5–8].
Treatment with somatostatin analogues, whether or not in combination with
Interferon-alpha, is however seldom successful in terms of CT or MRI assessed
tumour size reduction [9].
An interesting application of these peptides in nuclear medicine is the use
of radiolabelled analogues for tumour scintigraphy after intravenous injection.
The diagnostic peptide [111In-DTPA]octreotide (OctreoScan, 111In-pentetre-
otide) was approved by the FDA on 2 June 1994 for scintigraphy of patients
with these somatostatin receptor positive tumours. As soon as the success of
peptide receptor scintigraphy for tumour visualization became clear, the next
logical step was to try to label these peptides with therapeutic radionuclides

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 SESSION 9

and to perform peptide receptor radionuclide therapy (PRRT) of receptor

positive GEP tumours.

2. [111In-DTPA]OCTREOTIDE

The molecular basis for the use of radiolabelled octreotide in scintigraphy

and radionuclide therapy is receptor mediated internalization and cellular
retention of the radionuclide. Internalization of radiolabelled [DTPA]octre-
otide in somatostatin receptor positive tumours and tumour cell lines has been
investigated [10–12]. It appeared that this process is receptor specific and
temperature dependent. Receptor mediated internalization of [111In-
DTPA]octreotide results in degradation to the final radiolabelled metabolite,
111
In-DTPA-D-Phe, in the lysosomes [13]. This metabolite is not capable of
passing the lysosomal and/or other cell membrane(s) and will therefore stay in
the lysosomes, causing the long retention time of 111In in sst2 positive (tumour)
cells. Internalization of [111In-DTPA]octreotide is especially important for the
radionuclide therapy of tumours when radionuclides emitting therapeutic
particles with very short path lengths are used, such as those emitting Auger
electrons. These electrons are only effective at a short distance of only a few
nanometres up to micrometres from their target, the nuclear DNA. Recently,
Hornick et al. [14] and Wang et al. [15] described in vitro cellular internaliz-
ation, nuclear translocation and DNA binding of radiolabelled somatostatin
analogues, which significantly increased after prolonged exposure. Indium-111
labelled peptides are therefore suitable for both scintigraphy and radionuclide
therapy, all the more so as the decay of Auger electron emitters has recently
been shown to lead to a ‘bystander’ effect, an in vivo, dose independent
inhibition or retardation of tumour growth in non-radiotargeted cells by a
signal produced in Auger electron labelled cells [16].

2.1. Clinical studies with [111In-DTPA]octreotide

Initial studies with high dosages of [111In-DTPA]octreotide in patients

with metastasized neuroendocrine tumours were encouraging, although partial
remissions were exceptional. Fifty patients with somatostatin receptor positive
tumours, of which 26 had GEP tumours, were treated with multiple doses of
[111In-DTPA]octreotide in Rotterdam [17, 18]. Forty patients were evaluable
after cumulative doses of at least 20 GBq up to 160 GBq. The therapeutic
effects found in the patients with GEP tumours were no partial remissions,
minor remissions (i.e. a decrease in tumour size of 25–50%, as measured on CT
scans) in 5 patients, and stabilization of previously progressive tumours in

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11 patients, underscoring the therapeutic potential of Auger emitting radio-

labelled peptides. The toxicity was generally mild bone marrow toxicity.
However, 3 out of the 6 patients who received more than 100 GBq developed a
myelodysplastic syndrome or leukemia. Therefore, 100 GBq was considered
the maximum tolerable dose. With a renal radiation dose of 0.45 mGy/MBq
(based on previous studies), a cumulative dose of 100 GBq will lead to 45 Gy
on the kidneys, twice the accepted limit for external beam radiation. However,
no development of hypertension, proteinuria, or significant changes in serum
creatinine or creatinine clearance were observed in the patients, including the
2 patients who received 106 and 113 GBq of [111In-DTPA]octreotide without
renal protection by infusion with amino acids (see further) over a follow-up
period of 3 and 2 years, respectively. These findings show that the radiation of
the short range Auger electrons originating from the cells of the proximal
tubules is not harmful for the renal function. The decrease in serum inhibin B
and concomitant increase of serum FSH levels in males indicate that sperma-
togenesis was impaired.
At the Louisiana State University Medical Center in New Orleans, a
clinical trial was performed to determine the effectiveness and tolerability of
therapeutic doses of [111In-DTPA]octreotide in patients with GEP tumours
[19–21]. GEP tumour patients who had failed all forms of conventional therapy,
with worsening of tumour related signs and symptoms and/or radiographically
documented progressive disease, an expected survival of less than 6 months
and somatostatin receptor expression on the tumour as determined by the
uptake on a 222 MBq [111In-DTPA]octreotide scan, were treated with at least
2 monthly 6.6 GBq intravenous injections. Twenty-seven GEP (24 carcinoid
neoplasms with carcinoid syndrome and 3 pancreatic islet cells) patients were
accrued. Clinical benefit occurred in 16 (62%) patients. Objective partial
responses on CT occurred in 2 (8%) patients. The following transient grades 3/4
side effects were observed, respectively: leucocytes: 1/1; platelets: 0/2;
haemoglobin: 3/0; bilirubin: 1/3; creatinine: 1/0; neurological: 1/0. The renal
insufficiency in one patient was probably not treatment related but due to pre-
existent retroperitoneal fibrosis. Transient liver toxicity was observed in
3 patients with widespread liver metastases. Myeloproliferative disease and/or
myelodysplastic syndrome had not been observed in the 6 patients followed up
for 48+ months. It was concluded that two doses (6.6 GBq each) of [111In-
DTPA]octreotide were safe, well tolerated and improved symptoms in 62% of
patients, with 8% partial radiographic responses and increased expected
survival in GEP cancer patients with somatostatin receptor expressing
tumours.
Both series had relatively high numbers of GEP cancer patients who were
in a poor clinical condition upon study entry. Also, many had progressive

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disease when entering the study. Although in both series favourable effects on
symptomatology were reported, CT assessed tumour regression was only
observed in rare cases.
Stokkel et al. [22] recently reported on a study that determined the effect
of [111In-DTPA]octreotide therapy in patients with progressive radioiodine
non-responsive thyroid cancer. Therapeutic effects were determined in relation
to [111In-DTPA]octreotide uptake by tumour localizations assessed on pre-
treatment diagnostic [111In-DTPA]octreotide scans. Eleven patients, selected
on positive pretreatment diagnostic scans, were treated with up to four fixed
doses of 7400 MBq [111In-DTPA]octreotide with an interval of 2–3 weeks
between the doses. In 44% of the patients, stable disease was achieved up to 6
months after the first treatment. These four patients had relatively low
pretreatment thyroglobulin values, representing limited metastasized disease.
It was therefore concluded that treatment with high doses of [111In-
DTPA]octreotide in differentiated thyroid cancer can result in stable disease in
a subgroup of patients, whereas low pretreatment thyroglobulin value, repre-
senting a small tumour load, might be a selection criterion for treatment.
It was concluded that 111In coupled peptides are not ideal for PRRT
because of the small particle range and therefore short tissue penetration.
Consequently, various research groups have aimed to develop somatostatin
analogues that can be linked via a chelator to a therapeutic radionuclide.
DOTA is a universal chelator capable of forming stable complexes with such
metals as 111In, 67Ga, 68Ga, 86Y and 64Cu for imaging as well as with 90Y and with
radiolanthanides such as 177Lu for receptor mediated radionuclide therapy
[23–25]. In addition, new somatostatin analogues were synthesized to improve
receptor affinity [4, 26].

3. OTHER SOMATOSTATIN ANALOGUES

After 111In, the next radionuclide investigated for PRRT was 90Y, emitting
ß particles with a high maximum energy (2.27 MeV) and a long maximum
particle range. The first somatostatin analogue radiolabelled with 90Y and
applied for PRRT in animals and patients was [90Y-DOTA,Tyr3]octreotide, in
which, in comparison with octreotide, the phenylalanine residue at position 3
has been replaced with tyrosine; this makes the compound more hydrophilic
and increases the affinity for sst2, leading to higher uptake in sst2 positive
tumours both in preclinical studies and in patients [27, 28].
The next analogue investigated in preclinical radionuclide therapy studies
was [177Lu-DOTA,Tyr3]octreotate. This somatostatin analogue has a very high
affinity for sst2 [4] and after radiolabelling with 177Lu high tumour uptake was

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 DE JONG et al.

FIG. 1. SPECT/CT image (NanoSPECT, BioScan, United States of America) of a rat

bearing small CA20948 tumours on flank and injected with [177Lu-DOTA,Tyr3]octreotide.

seen, even very small tumours could be visualized (Fig. 1) and very good anti-
tumour effects were found in therapeutic studies in animal models [29–31].
Lutetium-177 emits gamma radiation with a suitable energy for imaging and
therapeutic β particles with low to medium energy (maximum 0.50 MeV), so
the same compound can be used for imaging and dosimetry and radionuclide
therapy, thus obviating the need for a pretherapeutic dosimetric study. The
approximate range of the β particles is 20 cell diameters, whereas the range of
those emitted by 90Y is 150 cell diameters. Less ‘cross-fire’ induced radiation
damage in the radiosensitive renal glomeruli (see below) can therefore be
expected with 177Lu. Also, in comparison with 90Y, a higher percentage of the
177
Lu radiation energy will be absorbed in very small tumours and
(micro)metastases [24, 29, 32].

3.1. Reduction of renal uptake

One problem arising during radionuclide therapy may be caused by the

uptake and retention of radioactivity in the radiosensitive kidneys; small

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 SESSION 9

radiolabelled peptides in the blood plasma are filtered through the glomerular
capillaries in the kidneys and subsequently partly reabsorbed by, and retained
in, the proximal tubular cells, thereby reducing the scintigraphic sensitivity for
detection of small tumours in the perirenal region and the possibilities for
radionuclide therapy. It was shown that the renal uptake of radiolabelled
octreotide in rats could be reduced by positively charged amino acids, such as
lysine and arginine. About a 50% reduction could be obtained by single
intravenous administration of 400 mg/kg of L- or D-lysine [33, 34]. Therefore,
during PRRT, an infusion containing the positively charged amino acids
L-lysine and L-arginine can be given during and after the infusion of the
radiopharmaceutical, in order to reduce the kidney uptake. Various protocols
have been described, resulting in up to a 55% reduction in renal uptake of
radioactivity, thereby allowing a higher administered dose [35–38]. The
authors’ preferred protocol comprises a combination of lysine and arginine.
Patients receive a 1 L infusion (500 mL of L-lysine HCl 5% plus 250 mL of
L-arginine HCl 10% plus 250 mL saline, brought to pH7.4). This infusion lasts
4 h; it starts 30 min prior to the radiopeptide injection and a constant infusion
rate is used throughout the infusion period [35].

3.2. Clinical studies with [90Y-DOTA,Tyr3]octreotide

Various multicentre phase 1 and phase 2 PRRT trials have been

performed using these 90Y and 177Lu labelled somatostatin analogues. Otte et al.
[39–41] and Waldherr et al. (University Hospital, Basel, Switzerland) reported
different phase 1 and phase 2 studies in patients with neuroendocrine GEP
tumours. Otte et al. [39–41] described a study in which patients received four or
more single doses of [90Y-DOTA,Tyr3]octreotide with ascending activity at
intervals of approximately 6 weeks (cumulative dose 6.12 ± 1.3 GBq/m2) with
the aim of performing an intra-patient dose escalation study. In total, 127 single
treatments were given. In 8 of these 127 single treatments, total doses of
≥3.7 GBq were administered. In an effort to prevent renal toxicity, two patients
received Hartmann-Hepa 8% amino acids (including lysine and arginine)
solution during all therapy cycles, while 13 patients did so during some but not
all therapy cycles; in 14 patients no solution was administered during the
therapy cycles. Of the 29 patients, 24 showed no severe renal or haematological
toxicity (toxicity ≤grade 2 according to the National Cancer Institute grading
criteria). These 24 patients received a cumulative dose of ≤7.4 GBq/m2. Five
patients developed renal and/or haematological toxicity. All five patients
received a cumulative dose of >7.4 GBq/m2 and had received no Hartmann-
Hepa 8% solution during the therapy cycles. Four of the five patients
developed renal toxicity; two of these patients showed stable renal insufficiency

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and two required haemodialysis. Two of the five patients exhibited anaemia
(both grade 3) and thrombocytopenia (grades 2 and 4, respectively). Twenty of
the 29 patients had disease stabilization, two a partial remission, four a
reduction of tumour mass of <50% and three a progression of tumour growth.
Waldherr et al. reported several phase 2 studies in patients with neuro-
endocrine tumours [42–44]. The patients received four or more single doses of
[90Y-DOTA,Tyr3]octreotide with ascending activity at intervals of approxi-
mately 6 weeks. Observed renal or haematological toxicity was ≤grade 2
according to the National Cancer Institute grading criteria. The cumulative
dose was ≤7.4 GBq/m2. Complete and partial responses obtained in different
studies amounted to 24%. In addition, distinct protocols were compared: in the
above mentioned studies, patients received four injections of 1.85 GBq/m2
(at intervals of around 6 weeks), while in another study two injections of
3.7 GBq/m2 were administered at an interval of 8 weeks. Interestingly, the
results from the last study were the most impressive. A higher percentage of
complete responses plus partial remissions (24% after four injections versus
33% after two injections) was found, while side effects were not significantly
different [44]. It should be emphasized, however, that this was not a
randomized trial comparing two dosing schemes.
Forrer et al. recently described the results of a study with [90Y-
DOTA,Tyr3]octreotide in which 116 patients with metastatic neuroendocrine
tumours were included [45]. All patients were pre-therapeutically staged with
morphological imaging procedures and with somatostatin receptor scintig-
raphy. The scintigraphy was positive in all cases. The patients were treated with
6–7.4 GBq/m2 body surface. In this study, complete remissions were found in
4%, partial remissions in 23%, stabilization in 62% and progressive disease in
11%. A significant reduction of symptoms was found in 83%. No serious
adverse event occurred and the toxicity was acceptable [45].
Chinol et al. from the European Institute of Oncology (Milan, Italy)
described dosimetric and dose finding studies with [90Y-DOTA,Tyr3]octreotide
with and without the administration of kidney protecting agents [46]. No major
acute reactions were observed up to an administered dose of 5.6 GBq per cycle.
Reversible grade 3 haematological toxicity was found in patients injected with
5.2 GBq, which was defined as the maximum tolerated dose per cycle. None of
the patients developed acute or delayed kidney nephropathy, although follow-
up was short. Partial and complete remissions were reported by the same group
in 28% of 87 patients with neuroendocrine tumours [47]. In more detailed
publications from the same group, Bodei et al. [36, 38] report the results of a
phase 1 study of 40 patients with somatostatin receptor positive tumours, of
whom 21 had GEP tumours. Cumulative total treatment doses were in the
range 5.9–11.1 GBq, given in two treatment cycles. Six of 21 (29%) patients had

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tumour regression. Median duration of the response was 9 months. Bodei et al.
also recently evaluated the efficacy of [90Y-DOTA,Tyr3]octreotide therapy in
metastatic MTC patients with a positive [111In-DTPA]octreotide scintigram,
progressing after conventional treatments. Twenty-one patients were retro-
spectively evaluated after therapy, receiving 7.5–19.2 GBq in 2–8 cycles. Two
patients (10%) obtained a CR, while stable disease was observed in 12 patients
(57%); 7 patients (33%) did not respond to therapy. The duration of the
response was in the range 3–40 months [38].
Another study with [90Y-DOTA,Tyr3]octreotide (OctreoTher, 90Y-SMT-
487) was the phase 1 Novartis study performed in Rotterdam, Brussels and
Tampa, which aimed to define the maximum tolerated single and four cycle
doses of [90Y-DOTA,Tyr3]octreotide and in which patients received escalating
doses up to 14.8 GBq/m2 in 4 cycles or up to a 9.3 GBq/m2 single dose, without
reaching the maximum tolerated single dose [18, 48, 49]. The cumulative
radiation dose to kidneys was limited to 27 Gy. All patients received amino
acids concomitant with [90Y-DOTA,Tyr3]octreotide for kidney protection.
Three patients had dose limiting toxicity: 1 liver toxicity, 1 thrombocytopenia
grade 4 (<25 × 109/L) and 1 myelodysplastic syndrome. Four out of 54 (7%)
patients who had received their maximum allowed dose had partial remission
and 7 (13%) minor remission. The median time to progression in the
44 patients who had either stable disease, minor remission or partial remission
was 30 months. An important observation in this study was a clear dose–
response relation; the percentage reduction in tumour volume increased with
increasing tumour radiation dose (up to about 600 Gy) [50]. Prior chemo-
therapy was predisposed to haematological toxicity. Renal toxicity was mild in
these patients, with individualized dosimetry and amino acid infusion for
kidney protection. Despite the differences in the protocols used, the rate of
complete plus partial responses seen in the various aforementioned [90Y-
DOTA,Tyr3]octreotide studies consistently exceeds that obtained with [111In-
DTPA]octreotide (see above).

3.3. Clinical studies with [177Lu-DOTA,Tyr3]octreotate

Most promising is the use of [177Lu-DOTA,Tyr3]octreotate. In patients, it

was found that the uptake of radioactivity, expressed as a percentage of the
injected dose of [177Lu-DOTA,Tyr3]octreotate was comparable to that of [111In-
DTPA0]octreotide for kidneys, spleen and liver, but was three- to fourfold
higher for 4 of 5 tumours [37]. Therefore, [177Lu-DOTA,Tyr3]octreotate
potentially represents an important improvement because of the higher
absorbed doses that can be achieved in most tumours with about equal doses to
potentially dose limiting organs and because of the lower tissue penetration

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range of 177Lu when compared with 90Y, which may be especially important for
small tumours [24].
The first treatment effects of [177Lu-DOTA,Tyr3]octreotate therapy were
described in 35 patients with neuroendocrine GEP tumours, who had a follow-
up of 3–6 months after receiving their final dose [51]. Patients were treated with
dosages of 3.7, 5.6, or 7.4 GBq [177Lu-DOTA,Tyr3]octreotate, up to a final
cumulative dose of 22.2–29.6 GBq, with treatment intervals of 6–9 weeks. The
effects of the therapy on tumour size were evaluable in 34 patients. Three
months after the final administration a complete remission was found in one
patient (3%), partial remission in 12 (35%), stable disease in 14 (41%) and
progressive disease in 7 (21%), including 3 patients who died during the
treatment period. The side effects of treatment with [177Lu-DOTA,Tyr3]octre-
otate were few and mostly transient, with mild bone marrow depression the
most common finding. In a more recent update of this treatment in 76 patients
with GEP tumours [52], complete remission was found in 1 patient (1%),
partial remission in 22 (29%), minor remission in 9 (12%), stable disease in
30 (39%), and PD in 14 patients (18%). Six out of 32 patients who had initially
stable disease or tumour regression after the therapy and who were also
evaluated after 12 months (mean 18 months from therapy start) became
progressive; in the other 26 the tumour response was unchanged. Median time
to progression was not reached at 25 months from start of therapy. Serious side
effects in the whole group of patients who had been treated or were being
treated up to that moment consisted of myelodysplastic syndrome in a patient
who had had chemotherapy with alkylating agents two years before entering
the study and renal insufficiency in another patient who had had unexplained
rises in serum creatinine concentrations in the year preceding the start of
therapy and who had a urinary creatinine clearance of 41 mL/min when
entering the study. Tumour regression was positively correlated with a high
uptake on the [111In-DTPA]octreotide scintigram, limited hepatic tumour mass
and high Karnofsky performance score.
In patients with progressive metastatic (or recurrent) differentiated
thyroid carcinoma (DTC) who do not respond to radioiodine therapy or do not
show uptake on radioiodine scintigraphy, treatment options are few. As these
tumours may express somatostatin receptors, PRRT using somatostatin
analogues might be effective and therefore the authors evaluated the
therapeutic efficacy of [177Lu-DOTA,Tyr3]octreotate in patients with DTC. In
addition, the uptake of radioactivity in the tumours was studied in relation to
treatment outcome. Five patients with DTC were treated with 22.4–30.1 GBq
of [177Lu-DOTA,Tyr3]octreotate. Three patients had Hürthle cell thyroid
carcinoma, one patient had papillary thyroid carcinoma and one had follicular
thyroid carcinoma. The uptake on [177Lu-DOTA,Tyr3]octreotate scintigraphy

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was compared with the uptake on the pre-therapy [111In-DTPA]octreotide

scintigram. After the last treatment with [177Lu-DOTA,Tyr3]octreotate, one
patient had stable disease as the maximum response, whereas the other two
patients had minor remission and partial remission, respectively. The response
in papillary thyroid carcinoma and follicular thyroid carcinoma was stable
disease and progressive disease, respectively. The patients with minor remission
and partial remission had the highest [177Lu-DOTA,Tyr3]octreotate versus
[111In-DTPA]octreotide uptake ratios. The authors concluded that in patients
with progressive DTC with no therapeutic option left and with sufficient
uptake of [111In-DTPA]octreotide in the tumour lesions, [177Lu-
DOTA,Tyr3]octreotate therapy can be effective. This finding is especially
important in patients with Hürthle cell thyroid carcinoma, as these patients
cannot benefit from radioiodine therapy because of non-iodine avid lesions at
diagnosis [53].

3.4. Comparison of the different treatments

Treatment with radiolabelled somatostatin analogues is a promising new

tool in the management of patients with inoperable or metastasized neuro-
endocrine tumours. The results that were obtained with [90Y-DOTA,Tyr3]octre-
otide and [177Lu-DOTA,Tyr3]octreotate are very encouraging, although a
direct, randomized comparison between the various treatments is lacking.
Also, the reported percentages of tumour remission after [90Y-
DOTA,Tyr3]octreotide treatment vary. This may be due to several causes:
(i) the administered doses and dosing schemes differ; some studies use dose
escalating schemes, whereas others use fixed doses; and (ii) there are several
patient and tumour characteristics that determine treatment outcome, such as
the amount of uptake on the [111In-DTPA]octreotide scintigram, the estimated
total tumour burden and the extent of liver involvement. Therefore, differences
in patient selection may play an important role in determining treatment
outcome. Other factors that can have contributed to the different results that
were found in the different centres performing trials with the same compounds
may be differences in tumour response criteria and centralized versus decen-
tralized follow-up CT scoring. Therefore, in order to establish which treatment
scheme and which radiolabelled somatostatin analogue or combination of
analogues is optimal, randomized trials are needed.

3.5. [90Y-DOTA]lanreotide

Virgolini et al. developed an 111In–90Y labelled somatostatin analogue,

[DOTA]lanreotide, for tumour diagnosis and therapy [54–57]. They described

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that 111In labelled [DOTA]lanreotide bound with high affinity to hsst2, hsst3,
hsst4 and hsst5 and with lower affinity to hsst1 expressed on COS7 cells, making
it a universal receptor binder [58]. However, Reubi et al. found in vitro in cell
lines transfected with the different somatostatin receptor subtypes and that
whereas [90Y-DOTA]lanreotide had a good affinity for the sst5, it had a low
affinity for sst3 (IC50 290nM) and sst4 (IC50 > 10 000nM) [4]. Froidevaux et al.
[59] concluded from their comparison study of, among other things,
[DOTA,Tyr3]octreotide and [DOTA]lanreotide in rats that radiolabelled
[DOTA,Tyr3]octreotide has more potential for clinical application than
[DOTA]lanreotide.

3.6. Clinical data with [90Y-DOTA]lanreotide

Lanreotide labelled with 90Y was the second analogue used for clinical
PRRT studies at different centres in the MAURITIUS trial [56]. In this study,
cumulative treatment doses of up to 8.58 GBq [90Y-DOTA]lanreotide were
given as a short term intravenous infusion. Treatment results in 154 patients
indicated minor responses in 14%. No severe, acute or chronic haematological
toxicity or changes in renal or liver function parameters due to [90Y-
DOTA]lanreotide were reported. In two-thirds of patients with neuroendo-
crine tumour lesions, [90Y-DOTA,Tyr3]octreotide showed a higher tumour
uptake than [90Y-DOTA]lanreotide, which can be explained by the lower
affinity of [90Y-DOTA]lanreotide for sst2.

3.7. Patient characteristics

Despite differences in amounts and types of radioactivity that are admin-

istered, there are also shared patient and disease characteristics as well as
similar exclusion and inclusion criteria between the various studies with
radiolabelled somatostatin analogues.
Needless to say, all protocols share the feature that the patients’ tumours
have to show uptake on the diagnostic [111In-DTPA]octreotide scintigram. In
some studies, the amount of uptake has at least to equal that of normal liver
tissue, in others it is required to be more than that. Most studies also require a
patient life expectancy of at least 3–6 months, which makes sense if the admin-
istration of the total cumulative dose takes several months and one of the
studies’ objectives is to evaluate tumour response. Also, as in all studies with
new treatment modalities, other, accepted, treatments have to be exhausted
(i.e. in case of neuroendocrine GEP tumours) and that tumours have to be
inoperable or metastasized. Because of the absorbed radiation doses, especially
to the kidneys and the bone marrow, most studies require certain minimum

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kidney function and haematological parameters for study entry and for each
new administration. Lastly, the patients have to meet a minimum performance
score as they are isolated in a nuclear medicine ward for a variable time,
depending on national laws on radiation protection.

4. OPTIONS TO IMPROVE PRRT

From animal experiments it can be inferred that 90Y labelled somatostatin

analogues may be more effective for larger tumours, whereas 177Lu labelled
somatostatin analogues may be more effective for smaller tumours, but their
combination may be the most effective [24]. Therefore, apart from comparisons
between radiolabelled octreotate and octreotide, and between somatostatin
analogues labelled with 90Y or 177Lu, PRRT with combinations of 90Y and 177Lu
labelled analogues should also be evaluated. Apart from the combination of
analogues labelled with different radionuclides, future efforts to improve this
therapy will also mean increasing the somatostatin receptor number on the
tumours, studying the effects of radiosensitizers as well as developing new
peptide analogues. An interesting example is [DOTA, 1-Nal3]octreotide, which
has a high affinity for sst2, sst3 and sst5 [60]. This compound may allow the
PRRT of tumours which do not bind octreotide and octreotate with high
affinity, i.e. sst3 and sst5 positive tumours.

5. CONCLUSION

Treatment with radiolabelled somatostatin analogues is a promising new

tool in the management of patients with inoperable or metastasized
(neuro)endocrine tumours. Symptomatic improvement may occur with all
111
In, 90Y, or 177Lu labelled somatostatin analogues that have been used for
PRRT. In particular, the results that were obtained with [90Y-DOTA,Tyr3]octre-
otide and [177Lu-DOTA,Tyr3]octreotate are very encouraging in terms of
tumour regression. Also, if kidney protective agents are used, the side effects of
this therapy are few and mild and the duration of the therapy response for both
radiopharmaceuticals is more than two years. These data compare favourably
with the limited number of alternative treatment approaches.

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.
 IODINE WHOLE BODY SCAN, THYROGLOBULIN
LEVELS, 99mTc MIBI SCAN AND COMPUTED
TOMOGRAPHY
Results in patients with lung metastasis from differentiated
thyroid cancer

N.Ö. KÜÇÜK, S.S. GÜLTEKIN, G. ARAS, E. İBIŞ

Department of Nuclear Medicine,
Ankara University Faculty of Medicine,
Ankara, Turkey
Email: n.ozlem.kucuk@medicine.ankara.edu.tr

Abstract

Correlation of the 131I whole body scan (131I WBS), 99mTc-sestamibi (Tc-MIBI)
scans, computed tomography (CT) and the value of routine follow-up for 131I WBS and
thyroglobulin levels were assessed in patients with differentiated thyroid cancer (DTC)
lung metastasis. Pulmonary metastasis was detected with 131I WBS, increased thyroglob-
ulin levels and/or other positive radiological findings in 32 patients out of 583 with DTC.
Iodine-131 WBS, thyroglobulin level assessment and/or CT were performed in the
diagnosis and follow-up of the patients with lung metastasis. A Tc-MIBI scan was
performed on 19 randomly chosen patients. Nineteen out of 32 patients had lung meta-
stasis before they received the first 131I treatment. Pulmonary metastasis was observed
in the first 131I WBS of all the patients except one; whereas no pulmonary metastasis was
detected in CT in 3/32. The final 131I WBS became normal in 13/32. Thyroglobulin levels
diminished in 21/32 and were elevated in 3/32. Iodine-131 WBS continued to be
abnormal in 2 out of 3 patients with increased thyroglobulin levels but became normal in
1 patient whose CT still demonstrated macronodulary lesions. Thyroglobulin levels did
not change significantly in 8/32. Iodine-131 WBS became normal in 5/8 and 4/5 showed
micronodules in their CT scans. Metastasis was detected in 12/19 patients who had
Tc-MIBI scans, 18/19 showed metastasis in 131I WBS and 17/19 in CT. Of the 7 patients
without the sign of metastasis in Tc-MIBI scintigraphy, 1 was negative in terms of meta-
stasis in 131I WBS and 1 in CT. Fibrosis was observed in 2/32 patients in CT. One patient
developed dedifferentiation decided by negative 131I WBS and positive CT. It was
concluded that the 131I WBS and thyroglobulin levels are the most important parameters
in the evaluation of lung metastasis in DTC. CT is an additional effect to 131I WBS and
thyroglobulin level, on the other hand, MIBI imaging alone may not be enough to detect
these metastases.

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1. INTRODUCTION

Thyroid cancer represents 90% of all endocrine malignancy but it

represents less than 1% of all malignancies. Metastatic disease develops in
7–23% of patients with differentiated thyroid carcinoma (DTC) [1, 2]. However,
distant metastasis at the time of initial diagnosis is observed in only 1–4% [3].
Lungs are the most frequent distant localization of metastasis from DTC [4].
Complete surgical resection of the thyroid (total or near total thyroid-
ectomy) and post-operative ablation therapy with 131I involve routine
treatment approaches for patients with DTC. The patients are followed up
after thyroidectomy and radioiodine therapy by serial 131I whole body scan (131I
WBS) and serum thyroglobulin measurements [5]. Technetium-99m-sestamibi
whole body scan (Tc-MIBI WBS) is useful, especially for the evaluation of
patients with negative 131I WBS and elevated thyroglobulin levels (non-
functional disease) in DTC. Furthermore, the Tc-MIBI scan has advantages
over the 131I scan, which are: (a) no necessity to discontinue hormonal therapy
when performing scintigraphy (b) Tc-MIBI provides better quality images and
(c) undertaking scintigraphic examination is easy and saves time. On the basis
of these favourable considerations, the use of Tc-MIBI WBS has been
suggested as an alternative to 131I WBS for the evaluation of the metastatic
disease in DTC patients [6–8].
This retrospective study researched the correlation and value of the 131I
WBS, Tc-MIBI scan, computed tomography (CT) and thyroglobulin levels in
the assessment of the lung metastasis in patients with DTC. The use of routine
thyroglobulin level measurements and 131I WBS combined as a gold standard
in the verification and follow-up of lung metastasis in patients without any
distant organ metastasis after successful thyroid remnant ablation were
evaluated.

2. MATERIALS AND METHODS

2.1. Patients

A total of 583 patients with DTC were admitted to the Department of

Nuclear Medicine of Ankara University Medical School between 1985 and
2004 and these patients were examined retrospectively. They were evaluated by
clinical examination and 99mTc pertechnetate thyroid scintigraphy. Routine
nuclear medicine methods (131I WBS, 201Tl or 99mTc-MIBI scans, thyroid
function tests) and when necessary some biochemical tests and radiological
studies (chest X ray, neck USG, neck and thorax CT, etc.) were performed for

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 SESSION 9

the evaluation and follow-up of patients. Of the patients, 32 had lung

metastasis. Histopathologically, papillary carcinoma in 15/32 patients, follicular
carcinoma in 13/32 patients and mixed type in 4/32 patients were observed.
Ages at the first diagnosis of the 32 patients ranged from 22 to 79 years (mean:
58 + 19 years, 15 female and 17 male). The duration of follow-up was in the
range 36–240 months. Clinical, pathological and treatment related character-
istics are presented in Table 1.

2.2. Surgery

All 32 patients except one underwent a total or near total thyroidectomy.

Lymph node dissections at the initial surgery were performed for 12 patients.
Nine patients underwent operations twice for an adequate operation.

TABLE 1. CLINICAL, PATHOLOGICAL AND TREATMENT

RELATED CHARACTERISTICS OF THE PATIENTS WITH LUNG
METASTASIS FROM DTC

Characteristic n = 32 %

Gender

Male 17 53.12
Female 15 46.87

Median age at the diagnosis (years)

58 + 19
Surgery

None 1 3.12
Total or near total thyroidectomy 31 96.87
Lymph node dissection 12 37.50

Histology

Papillary 15 46.87
Follicular 13 40.62
Mix type 4 12.50

Lung metastasis at the diagnosis

Yes 19 59.37
No 13 40.62

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2.3. The algorithm of treatment and follow-up

The patients with DTC who had undergone total thyroidectomy received
a fixed dose of 131I for the ablation of thyroid remnants. Post-treatment WBS
(PTWBS) was performed six days after the ablation therapy. Periodical
thyroglobulin measurement and DWBS were used for the follow-up of patients
after successful ablation. Tellurium-201 or Tc-MIBI is used for diagnosis and
follow-up in the case of negative DWBS and elevated thyroglobulin levels. The
authors could not undertake F-18 FDG scanning of the patients owing to the
absence of a PET camera in the department. The algorithm of treatment and
follow-up used is given in Fig. 1.

131
2.4. I therapy and PTWBS

The patients were given a high fixed ablative dose of 131I. L-thyroxin
replacement therapy withdrawal and low iodine diet protocols were applied for
4 weeks before the 131I therapy. TSH levels were increased at least 30 ng/mL.
A total of 100–1450 mCi (3.7–53.65 GBq) 131I was given to each patient. In the
presence of persistent functioning lung metastasis, radioiodine therapy was
repeated periodically (at least 6 months after the therapy).
PTWBS planar and spot images were obtained in anterior and posterior
projections. It was performed on the sixth post-treatment day using a large field
of view gamma camera equipped with a high energy (peak energy centred on
360 keV with a 20% energy window) parallel hole collimator (GE 4000iXC-T/
STARCAM, GE Medical Systems, Milwaukee, United States of America).
PTWBS was used only for the purpose of verification of the metastatic disease.
All the patients were researched about positive uptake on the PTWBS. These
patients with increased pulmonary activity were referred to the Department of
Thoracic Diseases and evaluated by bronchoscopy in order to confirm the
metastasis.

2.5. Routine follow-up protocol

Suppressive hormonal therapy (L-thyroxin) was started 48–72 h after the

radioiodine therapy. Diagnostic 131I WBS (DWBS) and thyroglobulin level
were obtained after the first treatment for the first follow-up in 6 months and
both 3 and 6 months, respectively. Thyroid hormone withdrawal and low iodine
diet protocols were applied for 4 weeks. The DWBS was performed with a
5 mCi (185 MBq) dose of 131I. DWBS, planar and spot images were acquired
with anterior and posterior projections at 24 and 72 h (early and late images)
using a GE 4000iXC-T/STARCAM gamma camera equipped with a high

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FIG. 1. The algorithm of treatment and follow-up.

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 KÜÇÜK et al.

energy collimator. Patients with normal DWBS were evaluated with periodic
follow-up, at first once a year and then every two or three years within a ten
year period. However, thyroglobulin levels of all the patients were obtained
every sixth month. The thyroglobulin levels were measured by the immuno-
radiometric method over the period 1985–2000 and by the chemiluminescence
method after 2000. Suppressive hormonal therapy was resumed after each 131I
WBS. All the patients were evaluated regarding positive pulmonary uptake on
the DWBS.

2.6. Tc-MIBI scan

The Tc-MIBI WBS was carried out on 19 patients who were randomly
chosen. The authors have used Tc-MIBI in since 1990 and it took a long time to
start using it routinely for the patient group. Therefore, not all patients in the
group have had Tc-MIBI scans. The authors performed the 201Tl WBS on some
patients but did not include them in this study because their number was small.
A Tc-MIBI WBS was performed on the patients during suppressive hormonal
therapy. Tc-MIBI (15–20 mCi (555–740 MBq), CARDIO-SPECT, Medi-
Radiopharma Ltd, Hungary) was injected intravenously. Images were obtained
20–30 min later. MIBI WBS planar and spot images were obtained in anterior
and posterior projections with a large field of view gamma camera equipped
with a low energy (peak energy centred on 140 keV with a 15% energy
window), high resolution collimator (Siemens ECAM Dual Head Variable
Systems, Siemens Medical Solutions, Illinois, USA). The patients were
examined for the similar positive pulmonary uptake regions in Tc-MIBI and
131
I WBS.

2.7. CT

CT was performed when pulmonary metastasis was detected by positive

131
I WBS and/or increasing thyroglobulin levels. In most of the patients, CT was
not performed in order to follow up. All patients underwent conventional CT
for the examination of metastatic lesions without the use of contrast media in
order not to affect the radioiodine therapy. The CT equipment used did not
have high resolution properties. The CT images were obtained with a 7 mm
slice thickness starting from the apex of the lungs. All CT images were taken in
the supine position. The patients were evaluated in terms of metastatic
pulmonary lesions and the lesions were classified according to size (macro-
nodule >10 mm).

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3. RESULTS

Nineteen out of 32 patients had lung metastasis before the first 131I
treatment. Thirteen out of 32 patients exhibited lung metastasis during the
follow-up period. All patients were given radioiodine therapy. Thyroid
remnant ablation was successful in the first DWBS in 22/32 patients. Six out of
32 patients had other distant organ metastases than the lungs. Four of the 6
patients only had lung and bone metastases. Pulmonary metastasis was
observed in the 131I WBS of all the patients except one (31/32, 96.8%), whereas
CT showed pulmonary metastasis to be absent in 3/32 patients.
The last DWBS was negative in 13/32 patients (40.6%). DWBS was
positive in 19/32 patients. In their initial examination, 27/32 patients had
thyroglobulin levels higher than 30 ng/mL (84.4%) and 5/32 patients had
thyroglobulin lower than 30 ng/mL (15.6%). In their final examination,
20/32 patients had thyroglobulin levels higher than 30 ng/mL (62.5%) and
12/32 patients had thyroglobulin levels lower than 30 ng/mL. The final
thyroglobulin levels began to change in 7/32 patients. In 8/32 patients the
thyroglobulin level was lower than 30 ng/mL plus a negative 131I WBS.
However, one patient had thyroglobulin values higher than 30 ng/mL who
had been negative for 131I WBS. Four out of 32 patients had thyroglobulin
levels less than 30 ng/mL (15.6%) and positive 131I WBS. Thirteen out of 32
patients had negative 131I WBS after a final diagnostic 131I WBS.
Thyroglobulin levels were lower in 21/32 and elevated in 3/32 patients.
The 131I WBS continued to be abnormal in 2 out of 3 patients with increased
thyroglobulin levels but became normal in one patient whose CT still demon-
strated macronodular lesions. Thyroglobulin levels did not change signifi-
cantly in 8/32. Iodine-131 WBS became normal in 5/8 and 4/5 showed
micronodules in their CTs. Fibrosis was observed in 2/32 patients in CT. One
patient developed dedifferentiation with negative 131I WBS and positive CT.
Metastasis was discerned in 12/19 patients who underwent Tc-MIBI
WBS, 18/19 showed metastasis in 131I WBS and 17/19 in CT (Table 2). Of the

TABLE 2. PATIENTS WITH SERUM THYROGLOBULIN LEVELS

>30 ng/mL WITH RESPECT TO DIFFERENT MODALITIES
131
Finding I WBS Tc-MIBI scan CT

Positive 18 12 17
Negative 1 7 2
% 94.7 63.1 89.4

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 KÜÇÜK et al.

seven patients showing no sign of metastasis in Tc-MIBI WBS, one was

negative in terms of metastasis in 131I WBS and one in CT. Of the 3/4 patients
who had negative 131I WBS and thyroglobulin levels higher than 30 ng/mL
during last follow-up examination, scans were performed by Tc-MIBI WBS. Of
the patients, two (2/3) had positive Tc-MIBI scans. In contrast, the patient who
had negative 131I WBS and a thyroglobulin level higher than 30 ng/mL during
initial follow-up had a negative MIBI scan.

4. DISCUSSION

Lungs are the most frequent distant localization of metastasis due to DTC
(1–4%) [4]. The 131I WBS and thyroglobulin levels play an important diagnostic
role in the evaluation of lung metastasis in DTC [9, 10]. Lung metastases were
observed in the 131I WBS of all the patients except one, who had a very high
thyroglobulin level and a negative 131I WBS.
Serum thyroglobulin level measurement is the most sensitive and specific
marker of DTC patients, but increased thyroglobulin concentration alone is not
enough when there is a large thyroid remnant. The thyroglobulin levels used in
the retrospective study were measured by an immunoradiometric method over
the period 1985–2000 and by chemiluminescence after 2000. The cut-off values
were 30 ng/mL in the first period and 5 ng/mL in the second period and the
values above these were considered for the assessment of metastatic disease.
Although it is reported that the chemiluminescence method is more sensitive
compared to the immunoradiometric method [19], a cut-off value of 30 ng/mL
was selected in this retrospective study because both methods were used over
the long duration of the analysis period.
Initial thyroglobulin concentrations determined after total thyroidectomy
or near total thyroidectomy with lung metastasis in 32 patients off thyroxin and
before 131I therapy or scanning showed that 15.6% (5/32) of patients had initial
levels less than 30 ng/mL and, in a contrary manner, 84.4% (27/32) had initial
levels higher than 30 ng/mL. Filesi et al. [11] reported that in their series of
patients, 66.7% of those with metastases had their initial thyroglobulin levels
higher than 60 ng/mL. David et al. [7] reported that 46% of patients with
thyroid remnants or metastases had their initial serum thyroglobulin values
higher than 30 ng/mL. In the authors’ group, 84.4% of patients with lung
metastases had their initial thyroglobulin levels higher than 30 ng/mL. In
addition, Filesi et al. reported metastases were observed in the initial 131I WBS
in 47.8% of patients with thyroglobulin values of less than 60 ng/mL; 61.3% of
patients with the initial 131I WBS negative for metastases had thyroglobulin
levels greater than 60 ng/mL. In contrast to the finding, David et al. determined

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only 2.7% of scan negative subjects had initial serum thyroglobulin levels
higher than 30 ng/mL. The authors’ retrospective findings suggested that 3.1%
of patients had both 131I WBS negative and thyroglobulin levels higher than
30 ng/mL. It might be associated with thyroid remnants and/or other distant
metastases. Thyroid remnant ablation was successful in the first DWBS in 22/32
patients and 6/32 patients had other distant organ metastases out of lung in the
study. In addition, the study showed that 16.5% of patients who had initial 131I
scan positive for metastases had thyroglobulin levels of less than 30 ng/mL. The
authors consider that the thyroglobulin cut-off level must be reduced to below
30 ng/mL. David et al. reported that with 131I ablation and long term thyroxin
suppression, the level of thyroglobulin tended to fall. In the study, final
thyroglobulin concentrations were determined in order to measure initial
thyroglobulin concentrations under the same conditions. The authors observed
that 37.5% (12/32) of patients had final thyroglobulin levels of less than 30 ng/mL
and, conversely, 62.5% (20/32) had final thyroglobulin levels higher than 30 ng/mL.
Initial thyroglobulin levels varied from 0.5 to 13 454 (average: 882.6) ng/mL.
Final thyroglobulin levels were measured from 0.5 to 4795 (average: 519).
Consequently, declining thyroglobulin levels were observed in 65.6% (21/32) of
the patients. Furthermore, final thyroglobulin values had less than 30 ng/mL in
37.5% (12/32) of the patients (Fig. 2).
The combination of routine thyroglobulin measurement and 131I WBS
can be used as a gold standard in the diagnosis and follow-up of lung metastasis
in DTC patients without any determined distant organ metastasis and after
successful thyroid remnant ablation. Increased activity in the lung region in the

FIG. 2. Thyroglobulin levels are presented in 32 patients with lung metastases from DTC.

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 KÜÇÜK et al.

PTWBS is considered as pulmonary metastasis. These patients were referred to

the Department of Thoracic Diseases and bronchoscopic methods were used to
confirm the presence of metastatic disease. The patients were divided into two
groups according to whether their thyroglobulin levels were higher or lower
than 30 ng/mL after L-T4 replacement therapy had been stopped. It is thought
that the group with thyroglobulin higher than 30 ng/mL can represent the high
risk group for the effective treatment of the disease [1, 11]. By doing this the
authors were able to evaluate how effective routine thyroglobulin determi-
nation can be in the follow-up of patients as a preparameter prior to the
multistep, multiparameter evaluation of progression. At the beginning of the
study there were 27 patients with thyroglobulin levels higher than 30 ng/mL
and 5 patients with thyroglobulin levels of less than 30 ng/mL. In the last
evaluation there were only 20 patients with thyroglobulin levels higher than
30 ng/mL. The last DWBS were negative in 13 of these 20 patients. DWBS was
negative in 8 and positive in 4 of 12/32 patients with thyroglobulin less than
30 ng/mL in the final evaluation. In two of three patients with thyroglobulin
progression, DWBS continued to be positive in the final evaluation. These
results show that thyroglobulin measurement and 131I WBS cannot yet be
evaluated as a gold standard in the follow-up of DTC patients with pulmonary
metastasis. However, depression of the serum thyroglobulin level can be
considered as an obvious sign that the patient benefits from radioactive iodine
therapy although selecting a lower cut-off value will increase the sensitivity and
the chance of a better correlation. Although it was not easy to evaluate the
efficacy of treatment, the authors were able to gather valuable information on
deciding if the high dose cumulative radioactive iodine therapy would result in
long term stability in the patient group by using thyroglobulin measurement
and DWBS together.
The Tc-MIBI scan has been useful especially for the evaluation of
patients with a negative 131I scan and elevated thyroglobulin levels (non-
functional disease) in DTC. Furthermore, the Tc-MIBI scan has advantages
over the 131I scan, these being (a) no necessity to discontinue hormonal therapy
when performing scintigraphy (b) MIBI provides better quality images and (c)
undertaking scintigraphic examination is easy and saves time.
On the basis of these favourable considerations, the use of Tc-MIBI WBS
has been suggested as an alternative to 131I WBS for the evaluation of the
metastatic disease in DTC patients [6–8]. However, there are different opinions
about the sensitivity of Tc-MIBI WBS in DTC. Sundram et al. showed Tc-
MIBI WBS had a sensitivity comparable to 131I WBS [12]. On the other hand,

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Dadparvar et al. [13] found a poor sensitivity (36%) but a high specificity
(89%) for Tc-MIBI WBS compared with 131I WBS. Miyamoto et al. reported
that Tc-MIBI WBS did not discern pulmonary metastases in more patients
than the 131I scan (75% and 85%, respectively). The authors’ findings suggested
that, compared with 131I WBS, Tc-MIBI WBS was less sensitive (94.2% and
63.2%, respectively) in detecting lung metastasis with DTC patients.
It has been reported that the sensitivity of thoracic CT is about 80% [14].
To the contrary, some studies have shown that a CT scan can detect 3 mm
peripheral and 6 mm central nodules, although it still fails to discern the diffuse
interstitial type of lung metastases in patients with DTC [15]. The authors
found that CT detected lung metastases in 29/32 patients (90.6%) in their
series. Nevertheless, their findings suggest that, compared with 131I WBS, CT
was less sensitive in detecting lung metastasis with DTC patients. It may be
difficult to interpret data in an area of the neck already submitted to surgery.
However, the authors also found that fibrosis was observed in 2/32 patients in
CT. Dedifferentiation was observed in one patient decided by negative 131I
WBS and positive CT, owing to the fact that CT was an additive effect to the
131
I WBS and the thyroglobulin level.
FDG PET scanning may be useful in localizing distant metastases,
especially when there is no radioiodine uptake. These metastases may be
located in the mediastinum or other distant areas [16, 17]. FDG uptake was
also detected more frequently in patients with poor DTC, in whom no
detectable 131I uptake could be demonstrated. FDG PET cannot supersede the
131
I scan. However, several investigators reported that FDG PET and 131I WBS
played complementary roles in the detection of recurrent metastatic DTC [17].
The authors were not able to use PET scanning because this modality was not
available in the department.
In their study, the authors chose to evaluate retrospective data with the
help of previous data, instead of statistically assessing various parameters
affecting the survival period. Their aim in doing so was to see if there was a
treatment mode which would result in progress in the patients. Margo et al.
analysed patient, tumour and treatment related factors and their relation to
disease specific survival using statistical tests. They found that at the age of
45 years or more, a site other than lung only or bone only and symptoms at the
time of diagnosis are associated with poorer outcomes [18]. In addition, Ronga
et al. reported that a young age at diagnosis and radioiodine uptake by
metastases are the most important factors positively affecting survival time.
They found that radioiodine therapy, also with high cumulative radioiodine 131I
activity, can lead to longer survival time or complete recovery [4]. The authors
found a prolongation of the disease free period and progress in the parameters
followed in those of their patient group who showed radioactive iodine

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accumulation and were younger than 45 years old at the first diagnosis. Six of
8/32 patients who showed progress in the parameters followed (DWBS,
thyroglobulin level less than 30 ng/mL) were younger than 45 years old and
needed fewer cumulative doses than the other two patients. The authors
believe that radioactive iodine therapy should continue to be given to the
patients because metastatic disease at least slows down the course of the
disease, although it may not appear to cure the disease in persistent or
recurrent cases.

5. CONCLUSION

For lung metastasis detection and follow-up after total thyroidectomy, the
131
I WBS and thyroglobulin levels were the most important parameters. CT has
an additive effect to 131I WBS. The authors’ findings suggested that, compared
with 131I WBS, Tc-MIBI WBS was less sensitive (94.2% and 63.2%, respec-
tively) in detecting lung metastasis with DTC patients. MIBI imaging alone
might not be enough to detect these metastases.

REFERENCES

[1] SCHLUMBERGER, M., et al., Long-term results of treatment of 283 patients

with lung and bone metastases from differentiated thyroid carcinoma. J Clin
Endocrinol Metab 1986; 63:960–967.
[2] RUEGEMER, J.J., et al., Distant metastases in differentiated thyroid carcinoma:
a multivariate analysis of prognostic variables. J Clin Endocrinol Metab
1988;67:501–507.
[3] HOIE, J., STENWIG, A.E., KULLMANN, G., LINDEGARD, M., Distant
metastases in papillary thyroid cancer. A review of 91 patients. Cancer 1988;61:1–6.
[4] RONGA, G., et al., Lung metastases from differentiated thyroid carcinoma. Q j
nucl med mol ımagıng 2004;48:12-9.
[5] OZATA, M., et al., Serum thyroglobulin in the follow-up of patients with treated
differentiated thyroid cancer. J Clin Endocrinol Metab. 1994;79:98–105.
[6] NEMEC, J., et al., Positive thyroid cancer scintigraphy using technetium-99m
methoxyisobutylisonitrile. Eur J Nucl Med. 1996;23:69–71.
[7] NG ENG, C.D., SUNDRAM, F.X., SIN, E.A., 99mTc-Sestamibi and 131I Whole-
Body Scintigraphy and Initial Serum Thyroglobulin in the Management of
Differentiated Thyroid Carcinoma. J Nucl Med 2000; 41:631–635.
[8] MIYAMOTO, S., KASAGI, K., MISAKI, T., ALAM, M.S., KONISHI, J.,
Evaluation of technetium- 99m-MIBI scintigraphy in metastatic differentiated
thyroid carcinoma. J Nucl Med. 1997;38:352–356.

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[9] MAZZAFERRI, E.L., MASSOLL, N., Management of papillary and follicular

(differentiated) thyroid cancer: new paradigms using recombinant human
thyrotropin. Endocrine-Related Cancer (2002) 9 227–247.
[10] PACINI, F., et al., Diagnostic 131-iodine whole-body scan may be avoided in
thyroid cancer patients who have undetectable stimulated serum thyroglobulin
levels after initial treatment. Journal of Clinical Endocrinology and Metabolism
(2002) 87 1499–1501.
[11] FILESI, M., SIGNORE, A., VENTRONI, G., MELACRINIS, F.F., RONGA, G.,
Role of initial iodine-131 whole-body scan and serum thyroglobulin in
differentiated thyroid carcinoma metastases. J Nucl Med. 1998;39:1542–1546.
[12] SUNDRAM, F.X., GOH, A.S.W., ANG, E.S., Role of technetium-99m sestamibi
in localisation of thyroid cancer metastases. Ann Acad Med Singapore. 1993;
22:557–559.
[13] DADPARVAR, S., et al., Clinical utility of technetium-99m methoxisobutyliso-
nitrile imaging in differentiated thyroid carcinoma: comparison with thallium-201
and iodine-131 scintigraphy and serum thyroglobulin quantitation. Eur J Nucl
Med. 1995;22:1330– 1338.
[14] LORENZEN, J., et al., Chest X ray: routine indication in the follow-up of
differentiated thyroid cancer? Nuklearmedizin 1998; 37:208–212.
[15] PIEKARSKI, J.D., et al., Chest computed tomography in patients with
micronodular lung metastases of differentiated thyroid carcinoma. Int J Radiat
Oncol Biol Phys. 1985;11(5):1023-7.
[16] WANG, W., et al., 18F-2-Floro-2-D-glucose positron emission tomography
localizes residual thyroid cancer in patients with negative diagnostic 131I iodine
whole body scans and elevated serum thyroglobulin levels. J Clin Endocrinol
Metab. 1999;84(7):2291-302.
[17] SHIGA, T., et al., Comparison of (18)F-FDG, (131)I-Na, and (201)Tl in diagnosis
of recurrent or metastatic thyroid carcinoma. J Nucl Med. 2001;42(3):414-9.
[18] SHOUP, M., et al., Prognostic indicators of outcomes in patients with distant
metastases from differentiated thyroid carcinoma. J Am Coll Surg.
2003;197(2):191-7.

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 99m
Tc-MIBI AND 131I SCINTIGRAPHY IN THE
FOLLOW-UP OF DIFFERENTIATED THYROID
CARCINOMA (DTC) PATIENTS AFTER SURGERY

S. SERGIEVA*, T. HADJIEVA**, V. BOTEV*, A. DUDOV*

*Sofia Cancer Centre

Email: sergieva_s@yahoo.com

**UH “Queen Giovanna”

Sofia, Bulgaria

Abstract

The MIBI scan has been reported to be a highly sensitive imaging technique for
the detection of differentiated thyroid carcinoma (DTC) metastases that have lost the
capability to uptake 131I. The purpose of this study was to evaluate, retrospectively, the
value of the 99mTc-MIBI scan and 131I whole body scintigraphy using thyroglobulin (Tg)
levels as a basis for comparison. A total of 84 patients with DTC (47 cases with papillary,
18 cases with follicular and 19 cases with papillary–follicular) were assessed. All of them
had undergone total or near total thyroidectomy and received radioiodine treatment for
ablation of post-surgical residual thyroid tissue. They were examined after 4 weeks
L-thyroxin withdrawal in the follow-up of DTC. Planar and whole body images were
acquired at 15 min and 180 min after IV application of 99mTc-MIBI (555–740 MBq) and
at 48 h after post-operative administration of 131I (111–185 MBq). Serum Tg assays were
performed to clarify the presence of residual recurrent malignancy. The 131I scan was
positive in 55 patients, showing thyroid remnants in 31 cases, lymph node metastases in
24 cases, pulmonary metastases in 6 cases and bone lesions in 2 cases. In 18 patients the
131
I scan was negative, Tg was undetectable, and therefore the patients were considered
tumour free. In 11 patients the 131I scan was negative while serum Tg was increased.
These false negative results were observed predominantly in cases with less differenti-
ated metastatic cells, especially after several courses of high dose 131I therapy. The 99mTc-
MIBI scan revealed the presence of lymph node and/or lung metastases (non-func-
tioning metastases) in 9 of them; false negative results were obtained in 2 cases. Serum
Tg was increased in all patients with local lymph node and distant metastases, visualized
by 131I or by 99mTc-MIBI, but also in 18 patients with thyroid remnants only. Considering
the 131I scan as the most specific standard procedure the authors conclude that the
combined 99mTc-MIBI scintigraphy and serum Tg assay appear to be an alternative to
radioiodine diagnostic imaging to demonstrate the extent of the disease in cases with
DTC and elevated Tg.

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1. INTRODUCTION

It is widely accepted that the 131I scan is the most specific diagnostic
procedure, a ‘gold standard’ in follow-up of patients with differentiated thyroid
carcinoma (DTC) after surgery. Depending on the histological type and
tumour stage, local recurrence, lymph node metastases or distant metastases
may be present or may develop during follow-up [1]. The most reliable
parameter for tumour recurrence or metastatic disease is a raised thyroglobulin
(Tg) level. With the introduction of lipophilic cationic tracers such as 99mTc–
sestamibi (MIBI) and 99mTc-tetrofosmin and, especially, fluorine-18 fluorode-
oxyglucose positron emission tomography (FDG PET) it has become obvious
that the use of only whole body 131I scintigraphy (131I-WBS) leads to serious
underestimation of the extent of disease in patients with DTC and elevated Tg
[2–4].
The 99mTc-MIBI scan has been reported to be a highly sensitive imaging
technique for detection of DTC metastases that have lost the capability to
uptake 131I [5–7].
The purpose of the present study was to evaluate, retrospectively, the
value of the 99mTc-MIBI scan and 131I-WBS using Tg levels as a basis for
comparison.

2. MATERIALS AND METHODS

2.1. Patients

A group of 84 patients (63 females and 21 males) with an age range of

17–74 years (mean: 43.5 years) with DTC were assessed. Forty-seven cases
exhibited the papillary variant of DTC, 18 cases had follicular and 19 cases had
papillary–follicular cancer.

2.2. Methods

All of the patients had undergone total or near total thyroidectomy and
received radioiodine treatment for ablation of post-surgical residual thyroid
tissue, in a total activity range of 3.3–7.0 GBq (90–190 mCi).
Routine follow-up examination was conducted 6 months post-ablation
therapy, after 4 weeks of L-thyroxin withdrawal. Serum Tg was measured. A
serum level above 4 ng/mL was considered abnormal when the patients were in
the hypothyroid state. All patients were asked to adhere to a low iodine diet
until the study had been completed.

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131
I-WBS images were acquired at 48 h after oral administration of 111–
185 MBq radioiodine on Toshiba GCA gamma camera at a speed of 7.5 cm/min
with a 1.024 × 512 matrix. Additional planar views from the neck, chest and
abdominal regions were also acquired as necessary using a HEAP collimator.
In 15 patients post-therapeutic 131I-WBS was carried out 5 d after treatment
with high dose radioiodine.
In 11 patients with a negative 131I scan and increased level of serum Tg,
additional 99mTc-MIBI scintigraphy was carried out. Planar images were
performed at 15 min and 180 min after IV application of 555–740 MBq 99mTc-
MIBI with a LEHR collimator.
All scintigraphy findings were compared to US, CT or MRT data.

3. RESULTS

The 131I-WBS was positive in 55 patients. Scintigraphy data showed

thyroid remnants in 31 cases. Thirteen patients had normal levels of Tg, but in
another 18 cases serum Tg was increased before radioiodine treatment. Six
months to one year after the ablation therapy of these patients 131I-WBS
revealed reduced or ablated remnant with a corresponding decrease in Tg level
in all of them (Figs 1 and 2).
Lymph node metastases were visualized in 24 cases with elevated serum
Tg levels (17 had hot spots in the neck region and 7 in the neck/upper
mediastinum regions) (Fig. 3).
In 19 patients with positive 131I-WBS for lymph node metastases,
visualized by sonography, 131I therapy was carried out (Fig. 4). In the other 5

(a) (b)

FIG. 1. A female patient (68 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam et 131I therapiam – pT4pNxM0”; Tg 11.55 ng/mL. (a) 131I-WBS was
positive for a remnant in the region of operative cicatrise. (b) After a second course of
radioiodine therapy 131I-WBS showed a reduced residual thyroid tissue; Tg 2.68 ng/mL.

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(a) (b)

FIG. 2. A female patient (76 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam – pT1pN0M0. Status post-131I therapiam”; Tg 2.05 ng/mL. (a)
Planar and (b) 131I-WBS showed totally ablated remnant after radioiodine treatment.

cases cervical lymph node dissection was performed with histological confir-
mation for metastatic infiltration (Fig. 5).
Six patients had positive 131I-WBS for pulmonary metastases and 2 had
bone lesions, confirmed by CT and/or MRI (Figs 6 and 7). After therapy with a
high dose of radioiodine, a partial therapeutic response and decreased level of
serum Tg were obtained during the follow-up in 5 of them with lung metastases.

(a) (b) (c)

FIG. 3. (a) A female patient (33 years) with the clinical diagnosis “Ca gl.thyreoideae.
Status post-thyreoidectomiam et 131I therapiam – pT1pN1M0”; Tg 23.74 ng/mL. 131I-WBS
was positive for a remnant in the region of operative cicatrise and metastatic infiltration of
left cervical lymph nodes. (b) A female patient (23 years) with the clinical diagnosis “Ca
gl.thyreoideae. Status post-thyreoidectomiam et 131I therapiam – pT4pN1M0”; Tg 115.32
ng/mL. 131I-WBS was positive for metastatic process in the region of the left cervical
lymph nodes and upper mediastinum. (c) A female patient (22 years) with the clinical
diagnosis “Ca gl.thyreoideae. Status post-thyreoidectomiam et 131I therapiam –
pT2pN1bM0”; Tg 1548.0 ng/mL. 131I-WBS was positive for a recurrence in the region of
operative cicatrice and metastatic infiltration of left and right cervical lymph nodes.

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(a) (b)

FIG. 4. A male patient (38 years) with the clinical diagnosis “Ca gl.thyreoideae. Status post-
thyreoidectomiam et 131I therapiam – pT2pN1M0”; Tg 28.98 ng/mL. (a) 131I-WBS was
positive for a metastatic infiltration of right cervical lymph nodes. (b) After radioiodine
therapy 6 months later 131I-WBS was negative for lymph node metastases; Tg 4.05 ng/mL.

In 1 case with lung lesions and 2 with bone metastases, there was no
answer to the therapy and they showed disease progression (Fig. 8).
In 11 patients, the 131I scan was negative while the serum Tg was
increased. These false negative results were observed predominantly in cases
with lymph node and pulmonary metastases, especially after several courses of
high dose 131I therapy. The 99mTc-MIBI scan revealed the presence of lymph
node and/or lung lesions, which have lost the capability to uptake 131I in 9 of

(a) (b) (c)

FIG. 5. A male patient (34 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam et 131I therapiam – pT4pN1M0”. (a) First 131I-WBS was positive
for a remnant and metastatic infiltration of left cervical lymph nodes; Tg 19.36 ng/mL. (b)
Second 131I-WBS performed 6 months later was positive for a reduced remnant and
persistence of metastatic infiltration of left cervical lymph nodes; Tg 9.23 ng/mL. (c) 131I-
WBS, performed after extirpation of metastatic cervical lymph node in the right and the
third course of radioiodine therapy, showed background radioactivity in the neck.

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FIG. 6. A male patient (50 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam et 131I therapiam – pT4pN1M1”; Tg 1350 ng/mL. 131I-WBS was
positive for a recurrence and diffuse lung metastases.

these patients (Figs 9–11). In 2 cases with elevated Tg, the 99mTc-MIBI scan and
the 131I-WBS were negative.
Serum Tg was increased in all patients with local lymph node lesions and
distant metastases, visualized either by 131I or by 99mTc-MIBI, but also in 18
patients with thyroid remnants only (Figs 12 and 13).

(a) (b)

FIG. 7. A female patient (50 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam et 131I therapiam – pT3pN1M1”; Tg 1341 ng/mL. 131I-WBS was
positive for multiple lung and bone metastases, visualized in anterior (a) and posterior (b)
positions.

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(a) (b)
FIG. 8. A female patient (70 years) with the clinical diagnosis “Ca gl.thyreoideae. Status post-
thyreoidectomiam et 131I therapiam – pT2pN0M0”; Tg 294 ng/mL. 131I-WBS was positive for
multiple lung metastases, visualized before application of high 131I activity (a); Tg 194 ng/mL.
There was no answer to the radioiodine treatment on the control 131I-WBS (b); Tg 203.4 ng/mL.

In three cases, additional lymph node metastatic lesions were visualized

on the 131I-WBS performed 5 d after administration of a high therapeutic 131I
activity compared to the diagnostic imaging with a low 131I dose (Fig. 14).

4. DISCUSSION

Accurate staging of local, regional and distant metastases is extremely

important for treatment and prognosis of patients with thyroid cancer. Most of
these lesions can be managed successfully by surgery and/or radioiodine

(a) (b)

FIG. 9. A female patient (54 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam – pT3pN1aM0. Status post-131I therapiam”; Tg 139.7 ng/mL. (a)
131
I-WBS showed background radioactivity in the neck. (b) 99mTc-MIBI showed an
intensive uptake of the tracer in the region of left cervical lymph nodes, significant for
metastatic infiltration.

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(a) (b) (c)

(d) (e)

FIG. 10. A female patient (72 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam et lymphadenectomiam – pT4pN2M0. Status post-131I therapiam
et TGT”; Tg 10.19 ng/mL. (a) 131I scan showed background radioactivity in the neck
region. (b) 99mTc-MIBI showed an intensive uptake of the tracer in the region of left
cervical lymph nodes, significant for metastatic infiltration. (c) Pulmonary metastases,
visualized in the CT. (d) 131I-WBS showed a high level of background radioactivity in the
gastric and intestinal regions. (e) 99mTc-MIBI showed intensive uptake of the tracer in the
left and right lung, visualized in the anterior and posterior positions, significant for an
active proliferation.

therapy, therefore, early detection of recurrent or metastatic events is

critical [8].
As Tg is produced only by normal or malignant thyroid tissue, it should
not be detectable in ablated patients [9]. An increase in the Tg level indicates
the presence of neoplastic tissue. In order to decide upon the most appropriate
therapeutic regimen in the case of elevated Tg, it is necessary to identify as

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(a) (b)

FIG. 11. A female patient (70 years) with clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam et 131I therapiam – pT1pN0M0. Lymph metastases colli dextra.
Status post-extirpationem”. (a) 131I-WBS showed background radioactivity in the neck.
(b) 99mTc-MIBI showed an intensive uptake of the tracer in the region of the right sub-
clavicular and cervical lymph nodes, significant for metastatic infiltration; Tg 123.2 ng/mL.

many of the lesions as possible and to ascertain whether they have the
capability ability to store iodine [3, 8, 9]. Negative 131I-WBS in patients with
elevated Tg may be explained by methodological difficulties or by the degree
of differentiation of the neoplastic cells. These false negative results were
observed predominantly in cases with less differentiated metastatic cells,
especially after several courses of high dose 131I therapy. The authors’ results
showed that 99mTc-MIBI scintigraphy has a high sensitivity in revealing
metastatic lesions that have lost the capability to uptake radioiodine. Others
reported that the 99mTc-MIBI scan has a higher sensitivity for detection of local
recurrences, lymph nodes and bone metastases, but lower sensitivity in

FIG. 12. A female patient (51 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam et 131I therapiam – pT4apN0M0”; Tg 14.05 ng/mL. 131I-WBS was
positive for a remnant in the region of operative cicatrice.

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 SERGIEVA et al.

FIG. 13. A female patient (70 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post thyreoidectomiam et 131I therapiam – pT2pN0M0”; Tg 194 ng/mL. 131I-WBS was
positive for multiple lung and liver metastases.

revealing thyroid remnants and diffuse lung metastases as compared to the 131I-
WBS [10, 11].
By using the 99mTc-MIBI scan instead of the 131I-WBS it is not necessary
to stop thyroid replacement treatment. It is very important for patients who
cannot tolerate the withdrawal of replacement therapy.
In three cases, additional metastatic lesions in the cervical region were
visualized on the post-therapeutic 131I-WBS compared to the diagnostic
imaging obtained before radioiodine treatment. These data suggest that a
negative diagnostic 131I-WBS does not necessarily indicate a lesion lacking the
capability to accumulate iodine [3, 12]. There is also evidence that ‘blind’ high
dose administration of radioiodine has a beneficial therapeutic effect in these
cases [8, 13]. Therapeutic effect of ‘blind’ radioiodine treatment is indicated by
a significant reduction in the serum Tg level. On the other hand, this practice
has been questioned by other groups, mainly due to a lack of long term
randomized trials [14].
In conclusion, considering the 131I scan as the most specific standard
procedure, the combined 99mTc-MIBI scintigraphy and serum Tg assay appears

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(a) (b) (c)

FIG. 14. A male patient (72 years) with the clinical diagnosis “Ca gl.thyreoideae. Status
post-thyreoidectomiam et 131I therapiam – pT4apN0M0”; Tg 75.15 ng/mL. (a) Diagnostic
131
I-WBS was positive for a recurrence in the region of operative cicatrice, visualized on
the CT scan (b). In (c), 131I-WBS performed 5 d after administration of a high dose of
therapeutic radioiodine showed intensive uptake in the region of cervical lymph nodes
significant for metastatic process.

to be an alternative substitute for radioiodine diagnostic imaging to

demonstrate the extent of the disease in cases with DTC and elevated Tg.

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thyroglobulin and negative diagnostic scan, J Clin Endocrinol Metab 80 (1995)
1488-1492.
[14] BIERMANN, M., SCHOBER, O., Should high hTg levels in the absence of
iodine uptake be treated?- Aginst. Eur J Nucl Med 30 (2003) 160-163.

134
 RESULTS OF KNEE RADIOSYNOVIORTHESIS IN
HAEMOPHILIC AND RHEUMATOID ARTHRITIC
PATIENTS WITH 32P COLLOID OF LOCAL
PRODUCTION

V.E. SOROA*,**, M.H. VELÁZQUEZ ESPECHE**, C. GIANNONE*,**,

G. NASWETTER**, H. CAVIGLIA***, G. GALATROS***

*Centro de Medicina Nuclear,

Comisión Nacional de Energía Atómica
Email: soroa@cnea.gov.ar

** Hospital de Clínicas,
University of Buenos Aires

***Traumatology and Orthopedics,

Hospital Municipal J.A. Fernández, Hemophilic Foundation

Buenos Aires, Argentina

Abstract

The objective of this study was to assess the effects of radioactive treatment in
knee joints with refractory synovitis in haemophilic patients, using a colloidal suspen-
sion of 32P, a pure beta emitter, developed domestically. Results were then compared
with those from chemical synovectomy. A population of rheumatoid arthritis (RA)
patients was treated and compared against intra-articular steroids and systemic drugs.
Fifty-eight male haemophilic patients, aged 4–52 years, were treated. Nine of them had
re-injections (67 procedures). Adults received 37–74 MBq; children of 2–6 years
received one third the adult’s activity; 6–10 years received one half the activity, whereas
10–16 years were injected with three quarters the activity given to adults. Anti-haemo-
philic factors (AHF) therapy, clinical examination as well as a pre-3-phase MDP scan
were registered and followed-up with MDP scans through 9 months. The intra-articular
therapies for either 32P in 44 patients or the antibiotic Rifampicin-99mTc macroaggre-
gates in 14 patients were monitored in the gamma camera with 32P bremsstrahlung
emission, searching for leakage. Twelve RA patients were studied: six received 32P and
the others intra-articular corticoids. Comparison of RoIs in treated knees during soft
tissue scintigraphies in pre- and post-third MDP control shows knee improvement. Joint
motion increased. Bleeding episodes, as well as requirements of AHF in 80% of the
radiosinovectomies, diminished. Intra-articular Rifampicin treatment requires several

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 SOROA et al.

injections. Outcomes in RA lasted 3 months and were less promising. Radiosinovectomy

in haemophilic patients with one injection provides 3–6 months relief. The paediatric
benefit from radiosinovectomy outweighs potential radiation hazards. Radiosinovec-
tomy is a safe, cost effective alternative therapy in emerging nations, where availability
of AHF is difficult and expensive.

1. INTRODUCTION

Haemophilia is a worldwide disease, affecting males including young

children; management of the associated arthropathy is compromised in
emerging countries by high cost (injection of anti-haemophilic factors (AHF))
and limited availability of specialized treatments. Intra-articular injections of
chemicals (such as osmic acid) or of antibiotics (Rifampicin) have been applied
to ameliorate haemophilic chronic recurrent synovitis and control joint
bleeding [1]. If unsuccessful, an alternative could be surgical intervention with
synovium removal. The latter is not an option in the haemophiliac patient,
because of possible haemorrhage [2]. Moreover, requirements of AHF for
haemostatic preparation rose to 100% during surgery. This elevates health
costs and requires prolonged hospitalization and kinesic therapy for the
intervened joint.
In rheumatoid arthritis (RA), an auto-immune disease, the compromised
painful joints are predominantly small ones. Systemic treatment must be
prescribed (aspirin, non-steroidal and steroidal anti-inflamatory anti-metabo-
lites, penicillamine or tumour necrosis factor α blocker). Local intra-articular
corticoid injections have also been used [2].
Radiosinovectomy is a procedure used to destroy the synovium with an
intra-articular injection of appropriate β colloids. The physical characteristics
of the β radiopharmaceutical will provide the indication of how to treat
different types of joint. New developments of colloid particles such as
hydroxide, citrates, silicates or macroaggregates of appropriate size, to be
phagocytosized without signs of inflammation and labelled with different β
emitters have reduced the unwanted joint leakage and the radiation exposure
to different organs [3].
The aim of this study was to assess the effects of radioactive treatment in
large joints, using a colloidal suspension of 32P, a pure β emitter, developed
domestically [4, 5]. The amelioration of synovial inflammation with radio-
sinovectomies was evaluated against the alternative treatments in the studied
pathologies, also taking into account the cost–benefit ratio to justify the present
option.

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 SESSION 9

2. MATERIALS AND SUBJECTS

Characteristics of 32P: T1/2 14.3 d; max. β energy 1.71 MeV; max. tissue
penetration 7.9 mm (mean penetration 2.2 mm). The authors’ local radio-
pharmacy, BACON Laboratories, supplied sterile 32P enriched gelatine
chromic phosphate colloid sized 100–200 nm, with 97% radiochemical purity
and a specific activity of 20 µCi/mg 32P [4, 6].
Radiosinovectomies were performed using 32P colloid in 58 male
haemophilic patients sent by the Hemophilic Foundation, aged 4–52 years.
Nine of them had retreatments (67 procedures), either in the same or in the
contralateral knee. The doses used were found to be safe and are based on the
results of several trials [5, 7]. Adults were injected with 37–74 MBq; children of
2–6 years of age received one third the activity of the adult; 6–10 year olds
received one half the activity of an adult, whereas 10–16 year olds were injected
with three quarters the activity given to adults. When children were overweight
by more than 20% from normal (for age and height), one quarter more activity
from the adult dose was added. Informed consent from adults or from the
children’s parents was obtained.
Patients were included in this study only if several knee episodes had
occurred. Exclusion criteria: large Bakers cysts, grades IV–V arthropaties, skin
infections of the joint area and bleeding at the time of the radiosinovectomies
[8]. Documentation of patients’ haemophilic history (severity, haemophilia A
or B), AHF therapy, number of bleedings, pain (visual analogue scale in 10
steps) range of articular movement and clinical examination were registered as
well as a pre-3-phase MDP scan in case report forms (CRF). Patients were
followed-up with the 3-phase bone scans through 1, 3, 6, 9 and 12 months. If
required, joint aspiration was carried out. The puncture sites for intra-articular
therapy for either the radiosinovectomies 32P in 44 patients or the antibiotic
Rifampicin–99mTc macroaggregates (4 MBq) in 14 patients were monitored
with the gamma camera.
Twelve RA patients were studied: six received 32P therapy and the other
six intra-articular corticoids. Clinical, blind evaluation (state of joint
involvement, pain, motility, requirements of AHF, corticoids or analgesics) was
registered in follow-up charts. For intra-articular chemical or corticoid injection
therapy, 4 MBq of 99mTc macroaggregates was added in order to obtain gamma
camera images and to blind the evaluating team of which patient received
radiotherapeutic treatment
To quantify the remission of the lesion over time, a remission rate index
(RR) was defined as the rate of severity of the lesion at the baseline and at each
control after treatment (months 3, 6, 9).

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 SOROA et al.

The severity at each control was defined as the product of the extension
of the lesion (area of the lesion expressed in pixels) and the increase in count
density at the lesion relative to a background normal area. This definition for
the relative count density was a means to establish afterwards an RR independ-
ently of the acquisition parameters and the total activity applied at each study:

SI(t) = {[dL(t) – dR(t)] / dR(t)} × N(t)

where for each planar image, SI(t) is the severity index at time t, with t = 0
(baseline), or month 3, or month 6 and so on, after treatment and:

N(t): area of the lesion [RoI in pixels] at time t.

dL(t): average counts/pixels in the lesion RoI at time t.

dR(t): average count density in the reference background selected in the

same image at time t.

The RR at time t is defined as:

RR(t) = SI(t=0) / SI(t)

A successful treatment implies RR(t) >1 while RR(t) <1 implies disease
progression.
A clinical criterion for recovery was considered based on the fall of lesion
detectability with time for responders owing to lack of contrast, or in other
words, an increasing standard error (inversely proportional to the square root
of the count statistics at the lesion RoI). As a result, the authors accepted
clinical recovery as RR(t) values >1.10 whereas RR(t) values less than 0.90
define disease progression. Between both limits, patients were considered as
being without clinical improvement or non-responders.

2.1. Therapeutic protocol for haemophilia and RA

Procedure undertaken in a clean room. Intra-articular injection

performed with sterile instruments. 32P phosphate chromic colloid unit dose
was drawn in a laminar flow hood. Intra-articular injection was in accordance
to patient age, joint (volume) and body weight [4, 7]. Synovial or haematic
content was previously evacuated from the compromised joint. The injection
was performed under gamma camera control. Knee and large joints were the
only ones that could be treated due to the characteristics of 32P colloid [9].

138
 SESSION 9

Saline flushing was carried out before the needle was withdrawn. The treated
joint was manipulated through a full range of motion to distribute the radio-
colloid throughout the joint space [1, 6, 8]. 32P bremsstrahlung emission in 201Tl
photopeak settings with a 25% window was used in the gamma camera for
early and late 24–48 h imaging to monitor extra-articular leakage [6]. After the
procedures, immobilization with a plaster and relative rest for 72 h followed
[8, 10]. Twenty-four h urine collections were obtained from 3 haemophilic
patients and counted in a β scintillation counter.
Figure 1 shows an example of a successfully treated haemophilic child
32
with P colloid injection in the left knee followed through 48 h images, to
which 99mTc markers or flood sources were added in order to provide
anatomical landmarks. Acquisition was obtained with bremsstrahlung
emission.

2.2. Follow-up

CRF, with a similar set of questions as before treatment, entailed

registering the number of subsequent episodes, diminished bleeding episodes,
pain score, range of movement, joint circumference and AHF requirements.
The MDP scan was obtained with an evaluation on the 2-phase at 1, 3, 6 and 12
months. In the pre-32P and third bone scans, irregular/similar RoIs were drawn
on the treated knee and in a background area over the contralateral thigh. The
analyses took into account the background, density, counts and number of
pixels, and then a formula was applied to obtain the severity index and the RR
in the radiosinovectomy treated knee (see the example in Fig. 2), the location
of RoIs in lesion and background, both in the baseline MDP scan and in the 3
month MDP images. If the RR was greater than unity, it was considered a
treatment success [6].

3. RESULTS

For the haemophilic patients, there were neither local burns, systemic
effects, nor leakage registered during 32P treatment (see the example in Fig. 1).
Intra-articular Rifampicin procedure required frequent injections (5–9) to
obtain a similar outcome as with 32P. Comparison of RoIs in treated knees
during soft tissue scintigraphies in pre- and post-third MDP control showed
knee improvement when the RR was >1. The follow-up evaluation demon-
strated an increase in joint motion, between 10–30%, diminished articular
volume and less requirement and frequency for the use of AHF in 80% of the

139
 SOROA et al.

(a)

(b)

FIG. 1. Intra-articular 32P colloid injection in the left knee of a haemophilic patient. (a)
Upper quadrant of the gamma camera image at 4 h, to which a contour of a 99mTc marker
was added around the left leg. The lower quadrant is the 24 h registration, plus a 99mTc
flood source underneath the patient. (b) 48 h images with no leakage in the iliac fossa or
thorax.

radiosynovectomies (54/67 procedures), thus lowering health costs dramati-

cally. No 32P counts were observed in the 24 h urine collections of the 3 patients.
Outcomes in RA lasted 2–3 months and were not so promising. In Fig. 2,
the authors present an RA patient where the low RR obtained proves the
unwanted outcome.

140
 SESSION 9

FIG. 2. An RA patient in which the RR = 0.69 proved an unsuccessful treatment.

Figure 3 shows plots of the RR of the haemophiliac knees treated with 32P
colloidal injection against the results of the Rifampicin injection. It clearly
demonstrates that the RR exceeds 1.5 in more than 80% of the RS.

FIG. 3. Comparable RR of 32P treatment with those from chemical synovitis in haemo-
philic patients.

141
 SOROA et al.

4. CONCLUSIONS

One intra-articular knee RS in haemophilic patients provides around

3–6 months relief of symptoms after treatment with locally produced 32P
colloid. Bleeding episodes diminished in the RS treated knees and the range of
movement increased [5–7]. Younger patients had a greater likelihood of
successful outcome [11].
In RA, a maximum of a 3 month pain palliative effect was documented.
RA is predominantly a pathology of small joints, but the high beta penetration
of 32P colloid allowed only the knees to be treated with this radiotherapeutic
alternative, making its use less beneficial.
Extra-articular leakage was not detected in any of the RS procedures (67
in haemophilia plus 6 in RA).
RS turned out to be a safe, cost effective alternative outpatient therapy
for use in emerging nations and could be considered as an initial procedure for
haemophilic haemartrosis where AHF is not readily available or is expensive.

ACKNOWLEDGEMENTS

The authors wish to thank L.B. Questa and T. Bonavita providing gamma
camera images, as well as N. Moretti in charge of the kinesic therapy and the
coordination of all the haemophiliac patients received by the Hemophilic
Foundation.
We thank R. Ughetti from the Radiopharmacist Section in BACON
Laboratories S.A.I.C. for the provision of 32P β colloid. This study was
supported by the IAEA (CRP).

REFERENCES

[1] MOLHO, P., et al., A retropspective study on chemical and radioactive synovec-
tomy in severe haemophilia patients with recurrent hemarthrosis, Hemophilia 5
(1999) 115-123.
[2] SCHNEIDER, P., FARAHATI, J., REINERS, C., Radiosynovectomy in Reuma-
tology, Orthopedics, and Hemophilia, J. Nucl. Med. 46 (2005) 11 (Suppl.1) 48 S-54
S.
[3] CLUNIE, G., LUI, D., CULLUM, I., EDWARDS, J.C.W., ELL, P., Samarium-
153-particulate hydroxyapatite radiation synovectomy: Biodistribution data for
chronic knee synovitis, J. Nucl. Med. 36 (1995) 51-57.

142
 SESSION 9

[4] ANGHELERI, L.J., Rapid method for obtaining colloidal suspension of phos-
phorous-32 as chromic phosphate, Proc of 2nd Intl. Conf on Peaceful uses of
Atomic Energy, UN, Geneva (1957).
[5] SOROA, V.E., Radiosynoviorthesis with P-32 colloid is still a helpful therapeutic
practice in haemophilia, J. Nucl. Med. 45 (2004) Abstract Book Supplement
abstract) 147.
[6] SOROA, V.E., et al., Effects of radiosynovectomy with P-32 colloid therapy in
haemophilia and rheumatoid ar-thritis, Cancer Biotherapy & Radiopharmaceuti-
cals 20 (2005) 3, 344-348.
[7] RIVARD, G.-E., et al., Synoviorthesis with colloidal Phosphorous-32 Chromic
Phosphate for the Treatment of Hemophilic Arthropathy, JB & JS 76-A (1994) 4,
482-488.
[8] CLUNIE, G., FISCHER, M., EANM Procedure Guidelines for Radiosynovec-
tomy. Eur. J. Nucl. Med. 30 (2003) BP12-BP16.
[9] JOHNSON, L.S., YANCH, J.C., SHORTKOFF, C.L., BARNES, A.L., SLEDGE,
C.B., Beta-particle dosimetry in radiation synovectomy, Eur. J. Nucl. Med. 22
(1995) 977-988.
[10] WINFIELD, J., CRAWLEY, J.C., HUDSON, E.A., FISHER, M., GUMPEL, J.M.,
Evaluation of two regimens to immobilise the knee after injections of 90yttrium,
British Med. J. 1(1979) 986-987.
[11] SIEGEL, H.J., LUCK, J.V., SIEGEL, M., QUINONES, C., The clinical utilization
of 32Pchromic phosphate radiosynovectomy in Hemophilia outcome analysis of
125 procedures, Proc. Orthopedic Surgeon Annual Meeting (2000) Poster PE152
(abs).

143
.
PET RADIOPHARMACEUTICALS

(Session 10)

Chairpersons

H.-J. MACHULLA
Germany

M.C. LEE
Republic of Korea
.
 ALTERNATIVE METHODS OF MAKING
[11C]AMIDES: APPLICATION TO THE
PREPARATION OF 5-HT1A RECEPTOR
RADIOLIGANDS

V.W. PIKE, S.Y. LU, J. HONG, J.L. MUSACHIO, J.A. McCARRON

Molecular Imaging Branch, National Institute of Mental Health,
National Institutes of Health,
Bethesda, Maryland,
United States of America
pikev@mail.nih.gov

Abstract

Many ligands for brain 5-HT1A receptors contain an amide group that is subject to
hydrolysis in vivo. In the development of radioligands for use with positron emission
tomography (PET), labelling in the carbonyl function of an amide group may be advan-
tageous for avoiding radioactive metabolites that would readily enter the brain to
confound PET receptor measurements. Several methods of labelling secondary and
tertiary amides in their carbonyl functions with 11C (T1/2 = 20.4 min) have been
developed over the past two decades or so. These methods include reaction of a
[carbonyl-11C]acid chloride, [carboxyl-11C]magnesium halide carboxylate or
[carboxyl-11C]acid with an amine or reaction of [11C]carbon monoxide with an amine
plus an aryl halide, alkyl halide or aryl triflate. Some of these processes are successfully
promoted with microwaves, palladium complexes, light or thermally intitated radicals.
These methods are surveyed here and especially exemplified from research on the
development of 5-HT1A receptor radioligands for brain imaging applications with PET.

1. INTRODUCTION

The amide group is widely found in drug-like ligands for neurotransmitter

receptors from which radioligands [1, 2] are sometimes developed for imaging
these receptors in the brain using positron emission tomography (PET), either
in clinical research [3] or in drug development [4, 5]. An important consider-
ation for radioligand development is the molecular position at which a positron
emitting isotope, frequently 11C (T1/2 = 20.4 min), should be introduced. Careful
choice of position may avoid radioactive metabolites that could enter the brain
to confound PET measurements of radioligand binding to the target receptor.
Amides are often metabolized in phase 1 by simple hydrolysis in the liver and/

147
 PIKE et al.

or other organs to produce the parent amine and carboxylic acid in the blood.
Generally, simple carboxylic acids, because they are ionized at physiological
pH7.4, do not enter the brain to a great extent, whereas the brain penetration
of amines, though subject to many factors, can be very appreciable. Hence, for
a potential PET radioligand containing an amide linkage, introduction of the 11C
label on the carbonyl side of the amide group, and even in the carbonyl entity,
may be advantageous. This is strikingly so [6, 7] for PET imaging of human
brain 5-HT1A receptors with 11C labelled WAY-100635 (1), where labelling in
the carbonyl function has been shown to provide a far more sensitive
radioligand [8] than labelling in the methoxy group [9] (Fig. 1). Labelling in the
methoxy group gives rise to a radioactive amine, 11C labelled WAY-100634 (2)
which readily enters the brain to bind both specifically and non-specifically,
whereas labelling in the carbonyl function avoids this radioactive metabolite
and instead gives rise to [11C]cyclohexanecarboxylic acid which enters the brain
only to a low and transient extent (Fig. 2). Such metabolic considerations create
a need for effective methods of labelling amides in their carbonyl functions
with cyclotron produced 11C, which is nearly always produced from the
14
N(p,α)11C reaction as either [11C]carbon dioxide or [11C]methane [10].
Various methods of labelling secondary and tertiary amides in the
carbonyl function with 11C have been developed over the last two decades or
so, and these methods are surveyed here. Although these methods have very
wide applicability, they are mainly exemplified in this survey through their
previous and ongoing applications in producing antagonist and agonist type

H
R N
O N O
N
N N R'

1, WAY-100635, R = Me, R' = c.hexyl-CO 5, (-)-NPPC

2, WAY-100634, R = Me, R' = H OMe
3, DWAY, R = H, R' = c.hexyl-CO
4, p-MMPF, R = Me, R' = 4-F-C6H4-CO
O
N N F
NH
6, S14506
FIG. 1. Labelling with 11C.

148
 SESSION 10

[methoxy-11C]1 [carbonyl-11C]1

Hydrolysis in vivo Hydrolysis in vivo

11
CO2H CO2H
[methoxy-11C]2 + 2 +
A B

FIG. 2. The metabolic fate of [methoxy-11C]1 (A) and [carbonyl-11C]1 in humans (B).

PET radioligands for 5-HT1A receptors. Key considerations with respect to the
various methods to be discussed here are their isotope efficiency (i.e. radio-
chemical yields), speed and ability to deliver high specific radioactivity. If a
radioligand is to be obtained in adequately high activity and specific radio-
activity for PET investigations, then generally no more than two half-lives of
11
C (i.e. a total of 40 min) may be taken for radiosynthesis, purification and
formulation. Useful labelling methods typically comply with this time
constraint.

2. LABELLING VIA [CARBONYL-11C]ACID CHLORIDES

A wide range of [carbonyl-11C]acid chlorides has been prepared by the

general method of carboxylation of a Grignard reagent (RMgX, X = halogen)
with cyclotron produced [11C]carbon dioxide followed by chlorination of the
adduct (Fig. 3). The 11C-carboxylation reaction has to be controlled in order to
avoid further reaction of the adduct or acid with the Grignard reagent, which is
necessarily present in excess. Judicious choice of the halogen in the Grignard
reagent, reagent concentration, temperature and reaction time often result in
efficient carboxylation to give the initial adduct. Chlorination with high boiling

i ii iii
11
CO2 R11COOMgX R11COCl R11CONR'R''

FIG. 3. Synthesis of [carbonyl-11C]acid chlorides for conversion into [carbonyl-

11
C]amides. Typical conditions: (i) RMgX (X = Cl or Br) in diethyl ether solution for
R = lower alkyl or aryl, or immobilized in tube for R = aryl or c.Hex, RT, 2 min; (ii)
phthaloyl dichloride and 2,6-di-t-butylpyridine for R = lower akyl or aryl, or thionyl
chloride for R = aryl, c.Hex, heat, 5–8 min; (iii) primary or secondary amine (R’R”NH),
solvent (e.g. THF, CH2Cl2), 0-37° C, 5–8 min. Reported specific radioactivity (exemplified
by [carbonyl-11C]3 [23, 24]): 74 GBq/μmol.

149
 PIKE et al.

point phthalolyl dichloride in the presence of an involatile base allows volatile

[11C]acid chlorides (e.g. R11COCl, R = Me, Et, Pr, c.Pr or c.Bu) to be
transferred cleanly out of the heated reaction mixture in a nitrogen stream. The
[11C]acid chloride may then be trapped within an amine solution to generate
the desired [carbonyl-11C]amide, usually in a moderate decay corrected radio-
chemical yield (RCY) [11–12].
Where the required [11C]acid chloride is not very volatile (e.g. R11COCl,
R = c.Hex, Ar), reaction with the amine partner may be performed in situ
[13, 14]. However, this method may pose a severe separation challenge. Hence,
an alternative technique has been devised to limit the amount of material that
needs to be separated [15]. A narrow plastic (e.g. polypropylene) tube is coated
with an almost dry film of the Grignard reagent. 11C-carboxylation is achieved
by controlled passage of the cyclotron produced [11C]carbon dioxide into the
tube and the generated adduct washed out with a solution of thionyl chloride
into a solution of the amine partner. This method has been automated and
applied routinely to the production of [carbonyl-11C](1) ([11C]WAY) from
[carbonyl-11C]cyclohexanecarbonyl chloride [16]. Some laboratories prefer to
use the one pot process and this method has also been automated for the
production of [11C]WAY [17, 18]. Practical aspects with regard to the regular
production of [11C]WAY, by either the one pot or ‘immobilized’ Grignard
reagent procedure, have been discussed in an EC sponsored workshop and the
key conclusions published [19].
Desmethyl-WAY (DWAY, 3), may also be labelled in a similar manner to
WAY without need for protection of the phenolic hydroxyl group [20]. In fact,
[11C]DWAY is superior to [11C]WAY as a PET radioligand for 5-HT1A
receptors in monkey and human subjects [21]. Several analogues of WAY have
also been labelled similarly, either from [carbonyl-11C]cyclohexanecarbonyl
chloride [22–23] or one of the various [carbonyl-11C]benzoyl chlorides [24].
These analogues include p-MPPF (4), which it may be noted has been exploited
extensively as a 5-HT1A receptor radioligand in humans with a longer lived 18F
(T1/2 = 109.7 min) radiolabel [25].
This method can deliver acceptably high specific radioactivities if care is
taken to exclude atmospheric carbon dioxide during the preparation and use of
the Grignard reagent.

3. LABELLING VIA [CARBONYL-11C]MAGNESIUM HALIDE

CARBOXYLATES

Aubert et al. [26] have reported the rapid one pot synthesis of aliphatic
[carbonyl-11C]amides in moderate RCYs (15–60%) by direct treatment of

150
 SESSION 10

i ii
11
CO2 R11COOMgX R11CONR'R''

FIG. 4. Preparation of aliphatic [carbonyl-11C]amides by reaction of [carboxyl-

11
C]magnesium halide carboxylates with amines. Typical conditions: (i) RMgX (R = lower
alkyl; X = Cl or Br ), THF or diethyl ether, 0° C, 3–8 min; (ii) R’R’’NH (primary or
secondary amine with R’ and R”aliphatic or alicyclic, THF, 70° C, 1–10 min and then aq.
HCl or aq. NH4Cl at 0° C). Specific activity: no carrier added.

[11C]magnesium halide carboxylates with amines in THF in the presence of 2.5

equivalents of alkylmagnesium halide (Fig. 4). The 5-HT1A agonist (-)-NPCC
(5) has been labelled succesfully with this type of procedure [Lu et al.,
unpublished results]. Aniline, however, failed to give a labelled amide by this
route under thermal conditions [31].
With the application of microwaves, this approach has been extended to
encompass the preparation of a [carbonyl-11C]amide having a benzoyl moiety
(4-F-C6H411CO) in up to 45% RCY in 10 min (Fig. 5) [27]. This method has
been applied to label the 5-HT1A receptor agonist, S14506 (6) in the carbonyl
function with 11C in 10–18% overall RCY [28].

4. LABELLING VIA [CARBOXYL-11C]ACIDS

The controlled carboxylation of organometallic reagents, such as

Grignard reagents or organolithiums, with [11C]carbon dioxide followed by
hydrolysis has been exploited for generally efficient radiosynthesis of a very
wide range of aliphatic and aryl [carboxyl-11C]acids [29]. In non-radioactive
chemistry, many methods, aside from conversion into acid chlorides, are known
for activating acids for amide formation. However, such methods have been
adapted only sparsely for preparing [carbonyl-11C]amides. Rogers et al. [30]

i ii
11
CO2 Ar11COOMgX Ar11CONR'R''

FIG. 5. Preparation of aryl [carbonyl-11C]amides by reaction of [carboxyl-11C]magne-

sium halide carboxylate with amine. Typical conditions: (i) 4-F-C6H4MgX, THF, RT,
6 min; (ii) R’R’’NH (primary arylamine or primary or secondary aliphatic amine), THF,
microwaves, 70–130° C, 2–10 min, and then aq. H2SO4. Specific radioactivity: no carrier
added.

151
 PIKE et al.

11 i,ii iii, iv v
CO2 Ar11COOH Ar11COIm Ar11CONR'R"

FIG. 6. Preparation of aryl [carbonyl-11C]amides from a [carboxyl-11C]benzoic acid by

activation with carbonyldiimidazole. Typical conditions: (i) ArMgBr, 5 min; (ii)
imidazole-HCl (Im HCl), 1 min; (iii) bromine, 2 min; (iv) carbonyldiimidazole, 5 min; (v)
R’R’’NH (e.g. piperidine), 5 min. Specific radioactivity: no carrier added.

demonstrated activation of [carboxyl-11C]benzoic acids with carbonyldiimi-

dazole for one pot amide synthesis (Fig. 6). As for many one pot radiosyntheses
with 11C, separation of the radioactive product from unused reagents and by-
products was challenging. In this work, an aryl by-product was produced that
was incompletely resolved from labelled amide during HPLC. This separation
problem was circumvented by brominating the aryl byproduct with bromine,
before activation of the labelled acid. The desired [carbonyl-11C]amide was
then obtained pure in >80% RCY in a preparation time of 22 min.

5. LABELLING VIA [11C]CARBON MONOXIDE

5.1. Palladium mediated

Carbon monoxide has a versatile transition metal mediated chemistry for

the introduction of the carbonyl function into organic compounds, including
amides. [11C]carbon monoxide of high specific activity may be produced
efficiently from [11C]carbon dioxide by on-line reduction over heated zinc [31]
or molybdenum [32]. The low solubility of [11C]carbon monoxide in organic
solvents had impeded its application in radiosynthesis until techniques were
developed quite recently to overcome this obstacle, including recirculation
[33], the use of high pressure miniature autoclaves [34, 35] and reversible
entrapment as a borane complex [36, 37]. Synthia Lab Systems AB (Uppsala,
Sweden) has shown that its miniature high pressure apparatus may be
automated for radiation safe radiosynthesis with [11C]carbon monoxide.

5.1.1. From aryl halides

Kihlberg and Långström first demonstrated the potential of palladium

mediated reactions of [11C]carbon monoxide with aryl and benzyl halides and
primary and secondary amines for the preparation of biologically active
[carbonyl-11C]amides in almost quantitative RCYs (Fig. 7) [38].

152
 SESSION 10

11 i
CO Ar11CONR'R"

FIG. 7. Preparation of [carbonyl -11C]amides from [11C]carbon monoxide, aryl or

benzyl halides halides and primary or secondary amines. Typical conditions: (i) aryl or
benzyl halide, Pd(PPh3)4, 1,4-dioxane, primary or secondary alkyl amine, 130–150° C,
5 min. Reported specific activities: = 1000 GBq/μmol [43]; = 1250 GBq/μmol [45].

In an extension of this work, less reactive amines were activated with

lithium bis(trimethylsilyl)amide, resulting in greatly improved RCYs over
reactions conducted without activation [39]. Several analogues of (1) have been
labelled in this manner [B. Långström, personal communication]. Alterna-
tively, addition of 1,2,2,,6,6-pentamethylpiperidine to the reactions also
increased RCYs for less reactive amines, such as methylamine [40].

5.1.2. From aryl triflates

Rahman et al. [41] have shown that aryl triflates may serve well in place
of aryl halides in the palladium mediated radiosynthesis of [carbonyl-
11
C]amides from [11C]carbon monoxide. Lithium bromide facilitates the
reactions which may be performed in the miniaturized autoclave described by
Synthia AB. A variety of [carbonyl-11C]amides was prepared from aryl triflates
and primary or secondary aliphatic amines or aniline in RCYs of 2–63% from
5 min reaction times (Fig. 8).
This method has been applied successfully to the preparation of several
candidate radioligands for the peripheral benzodiazepine receptor (PBR) in
RCYs ranging from 10 to 55% and with high specific radioactivities (200–900
GBq/μmol) [42].

11 i
CO Ar11CONR'R"

FIG. 8. Preparation of [carbonyl-11C]amides from [11C]carbon monoxide, aryl triflates

and amines. Typical conditions: (i) Pd(PPh3)4, LiBr, THF, aryl triflate, primary or
secondary alkyl amine or aniline, 150° C, 5 min. Reported specific radioactivity: 200–900
GBq/μmol [47].

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 PIKE et al.

i
11
CO R11CONR'R''

FIG. 9. Preparation of [carbonyl-11C]amides via photoinitiated carbonylation with

[11C]carbon monoxide using amines and alkyl iodides. Typical conditions: (i) N-methyl
pyrolidinone, primary or secondary aliphatic amine or primary aromatic amine, alkyl
bromide or iodide or aryl iodide, hv, 400 s. Reported specific activity: 192 GBq/μmol [48].

5.2. Radical carbonylation

5.2.1. Photoinitiation

The use of palladium to mediate the insertion of [11C]carbon monoxide

into amides and other carbonyl compounds is restricted when competing
β-hydride elimination is possible in the electrophile. For this reason, this
approach is inapplicable to the radiosynthesis of [11C]WAY since an
appropriate electrophile (cyclohexyl halide) would have a β-hydrogen.
Recently, photoinitiated radical carbonylation was shown to be successful for
the preparation of [carbonyl-11C]amides and alkyl halides bearing β-hydrogens,
such as ethyl iodide and cyclohexyl bromide, from [11C]carbon monoxide,
amines and alkyl halides [43] (Fig. 9). In particular examples, conversions of
[11C]carbon monoxide in fast reactions (e.g. 400 s) may reach up to 95%, with
RCYs of labelled amides reaching 74%.
This method has been adapted to the one step synthesis of [11C]WAY in
40–50% RCY in 30 min synthesis time (Fig. 10) [44].

5.2.2. Thermal initiation

Recently, it was briefly reported that the preparation of a [carbonyl-

11
C]amide from [11C]carbon monoxide, alkyl halide and amine may be achieved
through thermal generation of free radicals [45]. In a preliminary finding, a
30% conversion of [11C]carbon monoxide and a 19% RCY of [carbonyl-
11
C]amide was achieved. Further development of this approach may provide
practical advantages over the corresponding photoinitiated process, since these
reactions may be conducted simply in a ‘windowless’ miniature autoclave.

i
11
CO [11C]WAY

FIG. 10. One step preparation of [11C]WAY from [11C]carbon monoxide. Conditions: (i)
WAY-100634 (2), base, c.hexyl iodide, hv, 5 min. Specific radioactivity: no carrier added.

154
 SESSION 10

i ii
11
COCl2 N Cl N
11 11
C C
O O
FIG. 11. Synthesis of a [carbonyl-11 C]amide via [11C]phosgene and a [carbonyl-
11
C]carbamoyl chloride. Sample conditions: (i) 2,4-dimethoxybenzyl-tetrahydroisoquino-
line, dichloromethane, 20° C; (ii) Ph2CuMgBr.BrMgCN, THF, -30° C, 5 min then sat. aq.
NH4Cl. Specific radioactivity: no carrier added.

6. LABELLING VIA [CARBONYL-11C]CARBAMOYL CHLORIDES

[11C]phosgene may be obtained by different methods from cyclotron

produced 11C. The most effective, especially with regard to achieving high
specific radioactivity, is the conversion of cyclotron produced [11C]methane into
[11C]carbon tetrachloride with subsequent oxidation [46]. Lemoucheux et al.
[47] have shown that [11C]carbamoyl chlorides can be formed efficiently (RCY
= 76%) by reactions of tertiary amines with [11C]phosgene and in one example
(Fig. 11) that such a [11C]carbamoyl chloride may be converted with high RCY
(54%) into a [carbonyl-11C]amide by treatment with an organometallic reagent
(cyanocuprate, or a Grignard reagent in the presence of a nickel catalyst).
However, it is recognized that the two step catalyzed production of
[11C]phosgene requires a high level of skilled maintenance for reliability and
hence this method is unlikely to supplant the simpler alternatives.

7. CONCLUSIONS

Several useful and alternative methods are now known for the versatile
and efficient labelling of secondary and tertiary amides in their carbonyl
functions with cyclotron produced 11C and these are finding extensive
application in the preparation of PET radioligands for 5-HT1A receptors and
other targets (e.g. opiate receptors [11, 17, 42], α1-adrenoceptors [15], σ1
receptors [42], CK receptors [42], PBR [47] and MAO [42]).

ACKNOWLEDGEMENTS

This work was supported by the Intramural Research Program of the

National Institutes of Health (National Institute of Mental Health).

155
 PIKE et al.

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.
 [18F]FLUOROETHYLATED AND [11C]METHYLATED PET
TRACERS FOR RESEARCH AND ROUTINE DIAGNOSIS

M. MITTERHAUSER, W. WADSAK
Department for Nuclear Medicine, Medical University of Vienna,
Vienna, Austria
Email: markus.mitterhauser@meduniwien.ac.at

Abstract

Development of new tracers for PET is a prerequisite for the success of this
technique as a tool for routine diagnosis. Routine PET is mainly used for oncological,
cardiological and neurological/psychiatric purposes. Two important characteristics of
the radiotracers are specificity and selectivity, requiring strong focus on research in this
field. Also necessary is a wide understanding of the mechanisms involved in the uptake
process for the improvement of the status of existing radiotracers, with consideration
given to saturation processes or whether there are enzymes or receptors involved as
targets. Radiopharmacological aspects such as metabolic stability and the behaviour of
these potential radioactive metabolites should also play a pivotal role in the design of
the new radiotracers. Additionally, the feasibility of the preparation method should be
considered for routine demands. There are few methods for the introduction of the 18F
into molecules, including electrophilic substitution, nucleophilic substitution and simple
isotopic exchange. The preparations and formulations play a pivotal role in the selection
of new radiotracers. Owing to the short half-lives of 18F (~109 min) and 11C (~20 min),
only fast and reproducible methods can be used for these radiosyntheses. Additionally,
the preparation method can significantly influence the stability of the radiotracer in
vivo. The paper addresses aspects of the preparation and application of the newly
developed radiopharmaceuticals and discusses differences and improvements, and also
drawbacks to the routine tracers. Emphasis will be put on tracers for the dopamine
transporter, for the GABA receptor, the serotonin receptor (5HT1A) and the µ-opioid
receptor. Also, new attempts for the imaging of adrenocortical pathologies ([11C]-MTO
and [18F]-FETO) will be discussed as examples for innovative oncological tracers.

1. HISTORICAL BACKGROUND

The history of radiopharmaceuticals is rather short, since radioactivity

was not detected until the end of the 19th century. The first nuclear medical
examinations of the thyroid were conducted in the 1940s and in 1958 the first
clinical application of a positron emitting nuclide (i.e. 15O) was described [1].
But only after considerable improvements in the instrumentation were

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 MITTERHAUSER and WADSAK

implemented did positron emission tomography (PET) become of interest as a

diagnostic technique for use in the commuinity. Ido et al. presented the first
synthesis of 2-[18F]fluoro-2-deoxy-D-glucose (FDG) in 1978 [2] but it was the
significant improvements made by Hamacher and Coenen at the research
centre Juelich (Germany) [3] which led to the continued success of this
compound. Even today, FDG is by far the most important PET radiopharma-
ceutical, accounting for approximately 90% of all clinical PET studies
worldwide [4]. In recent years, more specific and selective tracers have been
developed and labelled with different PET nuclides, which provides further
insight into oncological, cardiological and neurological relationships.

2. PET NUCLIDES

Positron emitters can be found throughout the entire chart of nuclides but
only few display suitable physical properties while being capable of being
produced under simple conditions. The most important PET nuclides for
clinical applications are summarized in Table 1. Fluorine-18 is the most widely
used nuclide since it combines a relatively long half-life (109.7 min) with the
possibility of production in a small, medical cyclotron.

3. LABELLING REACTIONS WITH 18F

Figure 1 illustrates the labelling techniques possible with 18F. Depending

on the starting material, e.g. [18F]fluoride or [18F]F2 gas, respectively,

TABLE 1. IMPORTANT PET NUCLIDES

Nuclide Production Half-life

F-18 (F-) 18
O (p,n) 18
F 109.7 min
20 18
F-18 (F2) Ne (d,α) F 109.7 min
14 11
C-11 N (p,α) C 20.4 min
16 13
N-13 O (p,α) N 10.0 min
14 15
O-15 N (d,n) O 2.0 min
64 64
Cu-64 Ni (p,n) Cu 12.7 h
86 86
Y-86 Sr (p,n) Y 14.7 h
76 76
Br-76 Se (p,n) Br 16.0 h
68 68
Ga-68 Ge/ Ga generator 67.6 min

162
 SESSION 10

Labelling with F-18

Direct Labelling Indirect Labelling

no-carrier-added carrier-added Fluoroalkylations Other prostethic

labelling using labelling using (e.g. Fluoroethylations) groups
[18 F]fluoride [18 F]F2 -gas
Labelling of peptides,
proteins and antibodies
2-[18 F]Fluoro-2- 6-[18 F]Fluoro-L- O-(2-[18 F]Fluoroethyl)-
using linker systems
deoxy-D-glucose Dopa L-Tyrosin
(FDG) (FDOPA) (FET)

FIG. 1. Labelling with 18F.

nucleophilic and electrophilic reactions are possible. Complementary to the

direct radiofluorination reactions, fluorinated synthons can be produced which
are subsequently bound to the target molecules. Fluoroalkylations – and
especially fluoroethylations – represent the most important class for this
indirect labelling technique. Important examples are given in the black boxes.

4. FLUORALKYLATIONS

Since many biologically active compounds contain alkylic side chains, e.g.
methyl and ethyl groups, these structural units may be targets for the affixation
of a radiolabel. In fact, many compounds have been labelled with a [11C]methyl
group for PET (vide infra). Thus, the development of [18F]fluoroalkylated
tracers was the logical consequence. A variety of different fluoroalkylating
agents have been developed so far: [18F]bromofluoromethane [5, 6], [18F]fluoro-
iodomethane [7], 2-[18F]bromofluoroethane [8, 9], 2-[18F]tosyloxyfluoroethane
[10, 11], 3-[18F]bromofluoropropane [12–14], 3-[18F]fluoroiodopropane [14, 15]

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 MITTERHAUSER and WADSAK

and 3-[18F]tosyloxyfluoropropane [16]. The thus labelled synthons are

restricted to small alkyl chains to avoid too large a structural difference. The
most important fluoroalkylated tracers, already introduced into clinical appli-
cation, are [18F]FET (O-(2-[18F]fluorethyl)-l-tyrosin) [17] and [18F]FP-CIT (2β-
carbomethoxy-3β-(4-iodophenyl)-8-(3-[18F]fluorpropyl)nortropan) [13, 18, 19].

5. FLUOROETHYLATIONS

Fluoroethylations represent the most important class amongst the fluoro-

alkylations since: (1) fluoroethylating agents can be easily produced from
commercially available substances and (2) the fluoroethyl group is sterically
close to methyl and ethyl groups. Targets for fluoroethylations are amine [20–25],
hydroxylic [17, 26, 27], mercapto [28, 29] and carboxylic [30–35] moieties.
2-[18F]tosyloxyfluoroethane is widely used since it is easy to prepare, very
stable and suitable for a variety of compounds [36]. On the other hand, it is:
(1) not as reactive as 2-[18F]fluoroethyltriflate [37, 38]; (2) sensitive to some
solvents and bases [37]; (3) not a selective agent [39]; and (4) complex to purify
– a semi-preparative HPLC is unavoidable. Hence, microwave enhanced
conditions were proposed that increased the selectivity and the radiochemical
yields [40].
2-[18F]bromofluoroethane can also be produced rapidly. Thus, a lot of
effort was put into investigations to optimize the yields and quality of this inter-
mediate compound by addition of sodium iodide and application of solid phase
extraction for purification [38, 41, 42].

6. LABELLING REACTIONS WITH 11C

Carbon-11 is commonly prepared as carbon dioxide in the cyclotron.

Subsequently, this active precursor is converted into: (1) methyl iodide for
methylations; (2) [11C]HCN, [11C]COCl2 or [11C]CHO for special applications;
or (3) it is used directly for the synthesis of carbonyls or carboxyls via corre-
sponding Grignard reactions. The major radiosynthetic pathways are illustrated
in Fig. 2.

6.1. Methylations

[11C]methyl iodide may be prepared from [11C]carbon dioxide by

reduction to [11C]methanol and then treatment with a source of HI. The
reduction is carried out either by catalytic hydrogenation or more often by

164
 Labelling with C-11

[11 C]CO2 [11 C]CO

Methylations Carbonyls, Carboxyls special applications

methyl iodide methyl triflate Grignard reagents [11C]HCN [11C]COCl2

11 11
[ C]CH3I [ C]CH3OTf RMgX
[11C]CHO

[S-methyl-11C]-L- [11C]Raclopride [C1-11C]Acetate [Carbonyl-11C]WAY-

Methionine (MET) (RAC) (ACE) 100635 (WAY)

FIG. 2. Labelling with 11C.

lithium aluminium hydride (LiAlH4). As the carbon dioxide, methanol and

methyl halide are all volatile, separation from non-volatile impurities is easily
achieved by distillation or gas chromatography. [11C]methyl iodide has also
been prepared (60 ± 10% radiochemical yield) from [11C]methane through
substitution by iodine [43]. Larsen et al. [44] have reported the automated on-
line preparation of [11C]methyl iodide in 1.0 Ci quantities. [11C]methane and
iodine in helium were recirculated through a tube at 700–750°C, removing the
iodomethane being formed. Methyl triflate has been introduced as an even
faster methylating agent [45–47]. As mentioned earlier, radiolabelled methyl
triflate is generally obtained by the reaction of methyl halide with silver triflate;
for example, a gas stream containing [11C]methyl iodide impinges upon silver
triflate absorbed upon graphite at loadings as high as 50% at 170–200°C.
Recently, an excellent review on this topic was presented by Bolton [48].
In Table 2, an overview of the most commonly used [11C]methylated PET
radiopharmaceuticals is given. In the following section, a more detailed insight
into four applications of the authors’ fluoroethylation concept is given.

6.2. [18F]FE@CIT

In the last decade, radiolabelled cocaine analogues based on ß-CIT have

proven indispensable for the imaging of the dopamine transporter (DAT).

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 MITTERHAUSER and WADSAK

TABLE 2. IMPORTANT [11C]METHYLATED PET

RADIOPHARMACEUTICALS

Tracer Abbreviation Application References

[S-methyl-11C]-L-methionine MET Brain tumours 69, 70

11
[ C]metomidate MTO 11ß-hydroxylase 58–60
(adrenal cortex)
[11C]flumazenil FMZ, Central 52–54
Ro 15-1788 benzodiazepine
receptors (GABAA)
(R)-[N-methyl-11C-]PK11195 PK11195 Peripheral 71, 72
benzodiazepine
receptor
[11C]Patrick-Emond-substance 2I PE2I Dopamine transporter 73
11
[ C]raclopride RAC D2 receptor 74
11
[ C]FLB 457 FLB 457 D2 receptor 75, 76
[11C]N,N-Dimethyl-2-(2-amino- DASB Serotonin transporter 77, 78
4-cyanophenylthio)-benzylamine
[11C]carfentanyl CFN µ-opioid receptor 63–65
11
[N-Methyl- C]-6-OH-BTA-1 PIB ß-amyloid plaques 79
(AD)

Alterations of the DAT can be associated with neurodegenerative and neuro-

psychiatric disorders, including Parkinson’s disease, depression, attention deficit
hyperactivity disorder, Huntington’s chorea and schizophrenia. A multitude of
cocaine analogues have been synthesized to date [9, 13, 30, 48–51]. Among these
the so-called WIN compounds exhibit a 2–200-fold higher affinity for the DAT
than cocaine. Some of these compounds have been labelled with 11C or 18F and
were used for PET. However, further improvements in their pharmacodynamic
and pharmacokinetic features are desirable. An important improvement, yielding
in higher affinity to the DAT versus serotonin transporter (SERT) can be achieved
by a simple replacement of the carboxylic methyl ester group in ß-CIT by a
fluoroethyl ester [48, 49]. The preparation and ex vivo evaluation of this new
ß-CIT-analogue – [18F]FE@CIT – is discussed. Precursor and standard were
prepared from ß-CIT and analysed by spectroscopic methods. Yields of precursor
and standard preparation were 61% and 42%, respectively. [18F]FE@CIT was
prepared by distillation of [18F]bromofluoroethane ([18F]BFE) and reaction with
(1R-2-exo-3-exo)8-methyl-3-(4-iodo-phenyl)-8-azabicyclo[3.2.1]octane-2-carboxylic

166
 SESSION 10

acid. After 10 min at 150°C, the product was purified using a C-18 SepPak. The
radiosynthesis evinced radiochemical yields of >90% (based on [18F]BFE), the
specific radioactivity was >416 GBq/µmol. An average 30 µA·h cyclotron
irradiation yielded more than 2.5 GBq [18F]FE@CIT (Figs 3–5).

Me Me Me
N N N O F/[18F]
COOMe a) COOH b) O
I I c) I

1 2 3

FIG. 3. A scheme for the synthesis of FE@CIT and [18F]FE@CIT. Reagents and condi-
tions: (a) 6N HCl, reflux; (b) standard synthesis: 2-fluoroethanol, DMAP, EDCI, dichloro-
methane; (c) radiosynthesis: TBAH, DMF, [18F]BFE, 150°C.

100

80
rc. yield [%]

60

40 % [18F]FE@CIT
20 % [18F]BFE
0
0 20 40 60 80 100 120 140 160 180 200
Temp. [°C]

FIG. 4. Showing the dependence of the radiochemical yield of [18F]FE@CIT and

[18F]BFE on reaction temperature (5mM, 20 min, means ± SD; n = 4; P = 0.95).

100
rc. yield [18F]FE@CIT [%]

5mM
80

60

2mM
40

20

0 1mM
0 10 20 30
time [min]

FIG. 5. Showing the dependence of the radiochemical yield of [18F]FE@CIT on the

amount of precursor (150°C; means ± SD; n = 4; P = 0.95).

167
 MITTERHAUSER and WADSAK

For the ex vivo bioevaluation, 20 male Sprague-Dawley rats were

sacrificed at 5, 15, 30, 60 and 120 min after infection. Organs were removed,
weighed and counted. For autoradiographic experiments, transversal brain
slices of about 100 µm were prepared. The ex vivo evaluation showed the
highest brain uptake in striatal regions followed by the thalamus and
cerebellum. The highest striatum to cerebellum ratio was 3.73 and the highest
thalamus to cerebellum ratio was 1.65. Autoradiographic images showed good
and differentiated uptake in striatal regions with a good target to background
ratio (Fig. 6).
In conclusion, [18F]FE@CIT was prepared via the well-established distil-
lation method and followed the expected biodistribution routes. Precursor and
standard syntheses were fast and simple with good yields. Optimum reaction
conditions are 10 min at a temperature of 150°C with a precursor concentration
of ≥5mM. Uptake in DAT rich regions was high with striatum to thalamus/
hypothalamus ratios in the upper range of comparable DAT tracers. Ex vivo
bioevaluation together with ex vivo autoradiographic findings support the
further evaluation of [18F]FE@CIT for DAT-PET.

5,0
Striatum
P=0.02
* *
Thalamus/Hypothalamus
4,0
P=0.03
P=0.05 P=0.002
3,0
DVR

n.s.
2,0

1,0

0,0
5min 15min 30min 60min 120min 120min

FIG. 6. Showing distribution volume ratios (DVR) representing tissue to cerebellum

ratios at various time points (%ID/g; means ± SD; n = 4; P = 0.95). The * denotes values
for [123I]FP-CIT for reasons of comparison. Statistical P-values are given for DVR
(striatum) versus DVR (thalamus/hypothalamus) and were determined via t-test for
comparison of two means from independent (unpaired) samples with α = 0.05.

168
 SESSION 10

6.3. [18F]FE@FMZ

Benzodiazepines are used as sedative, anxiolytic, hypnotic, anti-

convulsant and muscle relaxant drugs. These drugs evolve their action via a
special binding pocket on the central GABA receptor (CBR). Various diseases,
such as epilepsy, Huntington’s disease, Alzheimer’s disease or schizophrenia
can be caused by alterations of the CBR. Thus, imaging and quantification of
CBRs with PET can be helpful for the diagnosis of neurological and psychiatric
diseases. [11C]flumazenil, a highly selective benzodiazepine antagonist is the
most extensively used GABAA ligand for PET so far [52–54] (Fig. 7).
To overcome half-life disadvantages of 11C, a [18F] labelled flumazenil
derivative, 2´-[18F]fluoroflumazenil, was developed and biologically evaluated
with respect to the GABAA receptor. In contrast to the presented analogue
FEFMZ [21, 55, 56], FE@FMZ is radiolabelled at the original ethylester group
and the radiosynthesis followed the distillation method. FE@FMZ was also
presented by a group of Korean scientists, in a one pot reaction with direct [18F]
fluorination of the tosylated ethylester precursor [57].
The organ with the highest uptake was the pituitary gland. Brain uptake
was high and followed the order cortex>thalamus>cerebellum>rest of brain.
Fluoroflumazenil displaced [3H]flumazenil binding from membrane GABAA

R1 R2
FMZ Ethyl- Methyl-
FEFMZ Ethyl- 2-Fluoroethyl
FE@FMZ 2-Fluoroethyl- Methyl-
N-desmethyl FFMZ 2-Fluoroethyl- H-
Precursor H- Methyl-

FIG. 7. Structures of various analogues of FMZ.

169
 MITTERHAUSER and WADSAK

receptors with an IC50 value (3.5nM) comparable to that of flumazenil (2.8nM).

The presented data confirm the potential of [18F]FFMZ for PET imaging of the
GABA-ergic system (Figs 8–10).

6.4. [18F]FE@ETO

The proposed synthesis of [18F]FE@ETO allows the production of

sufficient amounts of this new PET tracer to serve 1–2 patients with an overall
synthesis time below 80 min.
The 11β-hydroxylase (CYP11B1, P45011β) plays an important role in the
biosynthesis of cortisol and aldosterone and has been shown to be a good target
for the in vivo imaging of adrenocortical incidentalomas in nuclear medicine.
[11C]metomidate (MTO), a potent inhibitor of this enzyme, is used for routine
PET imaging of adrenocortical pathology [58–60]. [18F]FE@ETO, (the
[18F]fluoroethyl ester of etomidate, (R)-1-(1-phenylethyl)-1H-imidazole-5-
carboxylic acid, 2‘-[18F]fluoroethyl ester), an analogue of [11C]MTO and
[11C]ETO, was chosen due to the suspected similarity of the pharmacokinetic
and pharmacodynamic properties and was prepared in a two step procedure. In

FIG. 8. Competitive binding curves: displacement of [3H]FMZ binding by FFMZ and

FMZ. Membranes of rat forebrains were incubated with 2nM [3H]FMZ in the absence or
presence of 100µM diazepam (to calculate the 100% control value) or various concentra-
tions of drugs as indicated. Data were analysed by non-linear regression with a curve
fitting computer software package (GraphPad Prism 3.0, San Diego; mean values ± SD
from three different experiments each performed in triplicate).

170
 SESSION 10

1,80

1,60 5min 30min

1,40
Tissue/blood ratio

1,20

1,00

0,80

0,60

0,40

0,20

0,00
x

n
us

)
(*
te

M
lu

ra
m

Z
or

F
el

tb

FM
a

]F
C

b
al

8F
es
re

FE
Th

Ce

[1

]
8F
n
ai

[1
br

n
ai
le

br
ho

le
W

ho
W

FIG. 9. Showing tissue to blood ratios of brain regions: cerebellum, cortex, thalamus,
whole brain and rest of brain (* data taken from Ref. [55]).

0,7
Cerebellum

Cortex

Thalamus
0,6
Rest brain

Whole brain
%I.D./g

0,5

0,4

0,3
0 10 20 30 40 50 60
Time [min]

FIG. 10. Showing time–activity curves of FFMZ for various brain regions.

171
 MITTERHAUSER and WADSAK

the first step, [18F]fluoride was reacted with 2-bromoethyl triflate using the
kryptofix/acetonitrile method to yield 2-bromo-[18F]fluoroethane ([18F]BFE)
(Fig. 11). In the second step, [18F]BFE was reacted with the tetrabutylam-
monium salt of (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid to yield
[18F]FE@ETO, a novel inhibitor of the 11b-hydroxylase.
Male Sprague-Dawley rats were injected with 1.73–3.06 MBq of
FE@ETO into a tail vein after venodilatation in a 40°C water bath. Eighteen
rats were sacrificed by exsanguination from the abdominal aorta in deep ether
anaesthesia after 10 (n = 6), 30 (n = 6) and 60 min (n = 6) and the organs
removed, weighed and counted. For binding experiments, rat cerebellar
membranes were incubated for 90 min at 4°C in TC-50 buffer, 150mM NaCl,
and 2nM of [3H]flunitrazepam in the absence or presence of 10µM diazepam or
various concentrations of ETO, MTO and FE@ETO.
In vivo evaluation evinced very high uptake in the adrenal glands (7.52 ±
1.19% ID/g at 30 min), followed by lung (1.18 ± 0.19% ID/g, 10 min), liver (0.59
± 0.13% ID/g, 10 min) and duodenum (0.7 ± 0.29% ID/g, 60 min) (Figs 12–14).
No defluorination or fluoroethyl-ester cleavage was observed. As etomidate-
analogues have been used as anesthetics for a long time and the way of action is
explained GABA-ergic [61, 62], the authors in addition evaluated binding
parameters on GABA receptors.
Brain regions have been compared and showed highest relative uptake in
the cortex (2.34) followed by rest brain (2.13), cerebellum (1.96) and thalamus
(1.0, reference value). FE@ETO and ETO were able to increase the binding of
[3H]flunitrazepam with similar potencies and to a comparable extent (Fig. 15).
FE@ETO shows characteristics suitable for the imaging of adrenocortical
pathology with PET. Binding experiments on GABA receptors demonstrate a
comparable effect of FE@ETO and ETO (Fig. 16). Hence, FE@ETO possibly

Br [18F]fluoride / K2CO3 / K2.2.2 / acetonitrile Br

18
TfO 90°C, 10min F
[18F]BFE

anhydrous DMF
O O
Br 135°C-150°C, 20-30min
+
+ TBA O - N 18 O N
18 F
F
[18F]BFE N N

precursor (TBA-salt) [18F]FETO

FIG. 11. The reaction scheme for the preparation of [18F]FE@ETO.

172
 SESSION 10

9 ,0

8 ,0

7 ,0

1 0 m in 3 0 m in 6 0 m in
6 ,0

5 ,0
% ID/g

4 ,0

3 ,0

2 ,0

1 ,0

0 ,0

nd
s
d

um

lon

als
t

art
s

ng

r
x

h
um

um

s
er
ain

le

Fa

mu
ey
mu

rt e

e
oo

lee

ac
sc

Liv

yro est
gla
Lu
He
ren
br

Co
en
ell

ec

dn
om
Bl
Co

Fe
Mu

Sp
ala

T
re b

Ca
od
st

Ki

id
Ad
St
Th

Re

Du
Ce

Th
FIG. 12. The values counted in various organs at different time points after application of
1.73–3.06 MBq FE@ETO. Bars express values as per cent injected dose per gram organ
(%ID/g).

FIG. 13. Effects of ETO, MTO and FE@ETO on [3H]flunitrazepam binding to

membranes from rat cerebellum. Membranes were incubated with 2nM [3H]fluni-
trazepam in the absence or presence of 10µM diazepam or various concentrations of
drugs as indicated. Data represent mean values ± SD from three different experiments
each performed in triplicate.

173
 MITTERHAUSER and WADSAK

100

80

FETO ETO MTO

relative binding (%)

60

40

20

0
Adrenal Liver Heart Kidney Brain

FIG. 14. Relative binding of FE@ETO, ETO and MTO to various rat tissues; normalized
to adrenal binding (100%).

300

250

200
relative binding (%)

150

100

50

0
Thalamus Cerebellum Cortex Rest Brain

FIG. 15. Calculated ratios of brain regions: cerebellum, cortex and rest brain calculated as
per cent of thalamic uptake.

174
 SESSION 10

100

80
relative binding (%)

FETO ETO MTO

60

40

20

Adrenal Liver Heart Kidney Brain

FIG. 16. Organs with the highest uptake. Ratios are expressed as %ID/g organ/blood at
the time of highest uptake (lung, liver 10 min; adrenals 30 min; duodenum 60 min).

could also be used to elucidate the function, dynamics and kinetics of narcotic
drugs with PET.

6.5. [18F]FE@CFN

Activation of the µ-opioid receptor (μ-OR) by an agonist such as

morphine causes analgesia, sedation, reduced blood pressure, itching, nausea,
euphoria, decreased respiration, miosis and decreased bowel motility often
even leading to constipation.
Although tolerance to respiratory depression develops relatively quickly,
it is the single most adverse side effect of opioid use. In medicine, opioids are
widely used as effective analgesics for severe pain (www.opioids.com). PET
imaging of the µ-OR is still restricted to [11C]carfentanil ([11C]CFN, [63–65])
and used in areas of alcohol abuse and detoxification, therapeutic effectiveness
in heroin addicts, pain activation or temporal lobe epilepsy.
Recent findings demonstrated that µ-OR imaging with PET could, beside
its application in the human brain, also become a very useful tool in cardiology,
providing a better insight into cardiovascular pathophysiology, especially in the
ischemic heart and in arrhythmias. Furthermore, cardiovascular effects of drug
abuse and chronic µ-OR stimulation could be evaluated and quantified [66–68].
Therefore, the authors’ aim was the reliable radiosynthesis of a [18F]fluorinated
CFN derivative and its preliminary bioevaluation in rats. The [18F]fluoroethyl

175
 MITTERHAUSER and WADSAK

ester of carfentanil, [18F]FE@CFN (2-[18F]fluoroethyl 4-[N-(1-oxopropyl)-N-

phenylamino]-1-(2-phenylethyl)-4-piperidinecarboxylate), and its corre-
sponding inactive standard compound were prepared by fluoroethylation of
desmethyl-CFN acid (4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-
piperidine-4-carboxylic acid) with 2-bromo-[18/19F]fluoroethane in dimethyl-
formamide (Figs 17 and 18).
Purification of [18F]FE@CFN was achieved via solid phase extraction.
Whole body biodistribution was investigated in rats and specific binding was
measured autoradiographically in brain slices (Table 3).
[18F]FE@CFN was prepared with excellent purity (>98%) and yields
sufficient for routine PET imaging. In rats, considerable uptake was observed
in ileum, lung, kidney and heart. Uptake in the rat brain peaked at 5 min
(0.21% ID/g). On autoradiographic slices, the highest uptake was seen in the
olfactory bulb and cerebral cortex whereas almost no uptake was observed in
the cerebellum (Fig. 18).

7. CONCLUSION

The presented considerations are meant to be a basis for discussions on

the value of newly developed methods. Originally, the presented method was
established for the preparation of [18F]FET. Since fluoroethylations become
increasingly used in PET routine diagnosis and research, the method has to be

O Br [18F]fluoride / K2CO3 / K2.2.2 / acetonitrile Br

F3C O S 18
1. 90°C, 10 min F
O

2-Bromoethyltriflate [18F]BFE

Br anhydrous DMF
N N
18 + N N
150°C, 20 min
2. F
O O
O O
O- O
TBA+
18
F

[18F]BFE CFN-acid, TBA-salt (1b) [18F]FE@CFN (2a)

FIG. 17. The reaction scheme for the production of [18F]FE@CFN.

176
 SESSION 10

TABLE 3. VALUES IN VARIOUS ORGANS AT DIFFERENT TIME

POINTS AFTER ADMINISTRATION OF 0.5–1.5 MBq [18F]FE@CFN AS
PER CENT INJECTED DOSE PER GRAM ORGAN
(%ID/g; mean values ± SD; n = 5)

Tissue 5 min 15 min 30 min 60 min

Blood 0.30 ± 0.05 0.18 ± 0.03 0.17 ± 0.02 0.14 ± 0.02

Brain 0.21 ± 0.03 0.17 ± 0.06 0.11 ± 0.04 0.14 ± 0.10
Carcass 0.18 ± 0.05 0.21 ± 0.04 0.25 ± 0.03 0.25 ± 0.02
Colon 0.33 ± 0.10 0.26 ± 0.07 0.43 ± 0.12 0.37 ± 0.05
Fat 0.07 ± 0.04 0.28 ± 0.12 0.62 ± 0.19 0.60 ± 0.11
Femur 0.31 ± 0.03 0.28 ± 0.05 0.26 ± 0.02 0.26 ± 0.04
Heart 0.66 ± 0.06 0.37 ± 0.08 0.29 ± 0.04 0.19 ± 0.01
Ileum 0.95 ± 0.29 3.43 ± 0.50 3.16 ± 0.50 1.14 ± 0.20
Kidney 2.22 ± 0.51 1.10 ± 0.20 1.04 ± 0.07 0.61 ± 0.04
Liver 0.98 ± 0.34 1.71 ± 0.47 1.85 ± 0.18 1.72 ± 0.38
Lung 2.34 ± 0.43 0.96 ± 0.23 0.67 ± 0.07 0.39 ± 0.03
Muscle 0.27 ± 0.04 0.23 ± 0.04 0.16 ± 0.02 0.13 ± 0.01
Pituitary gland 2.40 ± 0.39 3.16 ± 0.70 3.15 ± 0.60 2.66 ± 1.10
Tail 0.46 ± 0.20 0.65 ± 0.15 0.44 ± 0.12 0.40 ± 0.13

integrated in the standard repertoire of radiopharmaceutical preparations.

Table 2 was drawn up with special reference to tracers that may serve as
templates for future fluoroethylations. As a matter of fact, the concept of a
‘me too’ generation of analogous radiotracers opens a new perspective for PET
centres without on-site radiochemistry units. The derived fluoroethylated
compounds normally provide comparable characteristics with respect to their
kinetic and dynamic behaviour, but it always has to be taken into account that
there is a significant chemical difference to the parent compounds. Therefore,
even minor structural changes may cause significant changes in their in vivo
behaviour. Some fluoroethyl esters are described as similar tools for PET
imaging as compared to their methylated relatives, e.g. FE@ETO [31, 33], some
others completely change the biological profile such as FE@FMZ [32, 34].
Nevertheless, the concept of fluoroethylations is a valuable tool for the rising
demand of innovative radiotracers in satellite PET centres.
Since there are a variety of methods described for the preparation of
fluoroethylated radiopharmaceuticals (vide supra), the method of choice for a

177
 MITTERHAUSER and WADSAK

100 a

rc. yield [%]

80
60
40
20
0
0 20 40 60 80 100 120 140 160
temperature [°C]

100 b
rc. yield [%]

80
60
40
20
0
0 5 10 15
precursor concentration [mM]

100 c
rc. yield [%]

80
60
40
20
0
0 5 10 15 20
time [min]

FIG. 18. The evaluated reaction conditions for the preparation of [18F]FE@CFN.

specific synthetic demand has to be evaluated separately for each single case.
For example, the addition of NaI has been reported to increase the radio-
labelling yields [38, 80], but in the authors’ experience with fluoroethylated
esters, this effect is reversed (a significant reduction of the labelling yield was
observed) [32].
In conclusion, fluoroethylations represent an important and valuable
strategy to widen the list of available PET radiotracers without the necessity of
synthesizing compounds ‘from scratch’. A major drawback of the method is
hidden in the fact that fluoroethylated compounds do not exist in nature and
are rarely evaluated drugs. This fact demands de novo biomedical evaluations
prior to their use for human PET applications in routine diagnosis. Fluoroethyl-
ation is a method with restrictions but once established it can serve as a valuable
tool for the evaluation and development of new and innovative PET tracers.

178
 SESSION 10

cerebellum

cerebral cortex

brain stem
olfactory bulb midbrain

FIG. 19. Showing a typical rat mid-brain slice.

ACKNOWLEDGEMENTS

The authors are indebted to Profs Kletter and Dudczak, Drs Ettlinger,
Lanzenberger and Mien, and all the staff at the PET centre Vienna. Parts of the
presented work were supported by two research grants from the Austrian
National Bank (Jubiläumsfonds der Österreichischen Nationalbank), project
numbers 8263 (W. Wadsak) and 11439 (M. Mitterhauser).

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 A NOVEL FINDING: ANTI-ANDROGEN FLUTAMIDE
KILLS ANDROGEN INDEPENDENT PC-3 CELLS
A radiolabelled methyl-choline incorporation into tumour
cells

F. AL-SAEEDI
Nuclear Medicine Department, Faculty of Medicine,
Kuwait University (Health Sciences Center),
Safat, Kuwait
Email: Fatimas@hsc.edu.kw

Abstract

[Methyl-11C]-choline was introduced to image many types of cancer, especially

prostate cancer. Al-Saeedi et al. reported that the incorporation of [Methyl-3H]-choline
into breast tumour (MCF-7) cells correlated strongly with proliferation as determined
by [Methyl-14C]-thymidine uptake. Also, Al-Saeedi et al. showed that the chemotherapy
using MCF-7 cells treated with 5-Fluorouracil (5-FU) induced modulation in [Methyl-
3
H]-choline incorporation and certain mechanisms for this modulation were reported.
In this study, the androgen dependent prostate tumour (LNCaP) cells were treated with
the well known pure anti-androgen drug, flutamide, for 3 d. The cells were then
incubated with [Methyl-3H]-choline for 10 min to detect the effect of flutamide on both
cell proliferation and choline incorporation. At the same time, a preliminary work was
established using androgen independent PC-3 cells treated with flutamide as controls in
this study. PC-3 cells were treated with a range of doses of flutamide, inhibiting growth
by 20–70%. Treated and control cells were incubated with [Methyl-3H]-choline for 10
min, then in non-radioactive medium to simulate the rapid blood clearance of [Methyl-
11
C]-choline tracer in control and treated PC-3 cells, and then extracted with organic and
aqueous solvents to determine its effect on the intracellular distribution of this tracer.
The results were interesting in that they showed that flutamide killed the androgen inde-
pendent prostate cancer cells, PC-3, and the mechanisms responsible for flutamide
induced modulation on [Methyl-3H]-choline incorporation are reported. The PC-3 cell
proliferation was inhibited by flutamide. In addition, treatment of PC-3 cells with
flutamide for 3 d resulted in a buildup of cells in the S phase and [Methyl-3H]-choline
incorporation per a cell was found to be decreased in treated as opposed to untreated
cells. In conclusion, flutamide inhibits PC-3 cell proliferation by a certain mechanism
(unknown) other than the well-known androgen receptor mechanism, which accord-
ingly induced modulation in [Methyl-3H]-choline incorporation into the PC-3 cells.

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1. INTRODUCTION

Many studies suggest that changes in the uptake of PET tracers [1–3] such
as 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and 18F-fluorothymidine (18F-
FLT) after chemotherapy (4, 5), and radiotherapy (6, 7), compared with
pretreatment uptake can reflect the efficacy of the drug and the tumour
responses to chemotherapy treatment [8]. 18F-FDG-PET has been widely used
to investigate many types of cancer such as breast cancer and colorectal cancer
after chemotherapy [9]. However, 18F-FDG imaging was found to be limited in
the detection of localized prostate cancer because of excessive excretion of the
radionuclide into the urine, masking any lower pelvic region lesions [10]. The
evaluation of the efficacy of the treatment of men with prostate cancer is
mainly based on post-treatment levels of the prostate specific antigen. In
addition, digital rectal examination and conventional imaging techniques are
used but they are not sensitive enough to detect a local recurrence. [Methyl-
11
C]-choline PET scan provides a sensitive metabolic and non-invasive imaging,
which is not dependent on anatomical distortions. Recently, [Methyl-11C]-
choline was introduced to image prostate cancer [1], and in addition to evaluate
the post-treatment of the localized prostate cancer [11]. Flutamide is often used
as part of the initial treatment of prostate cancer. In this study, the questions of
whether flutamide treatment inhibits cell proliferation and whether
modulation of [Methyl-3H]-choline incorporation is induced or not in PC-3
cells were investigated.

2. MATERIALS AND METHODS

2.1. Materials

2.1.1. Cell culture and media

The human prostate cancer cells (PC-3 cells) were obtained from the
American Type Culture Collection (ATCC).

2.1.2. Radiolabelled compounds

[Methyl-3H]-choline chloride (specific activity: 2.92 TBq/mmol) was

obtained from Amersham Pharmacia Biotech UK Ltd (Buckinghamshire,
United Kingdom).

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2.1.3. Chemicals and reagents

All chemical reagents used were supplied by Sigma-Aldrich (UK) unless

otherwise specified. Ultima Gold scintillant fluid was obtained from Meridian.
Culture flasks and 96 well plates were obtained from Nunc (Denmark).

2.2. Methods

2.2.1. Cell culture and cell media

PC-3 drug sensitive/wild type cells were grown in Dulbecco’s Modified

Eagles medium (DMEM) supplemented with 20 U/mL penicillin, 20 µg/mL
streptomycin and 10% foetal calf serum. All cells were cultured in monolayers
in 25 cm2 flasks in triplicate, incubated at 37°C (in 5% CO2:95% air), and
allowed to grow exponentially to 70% confluency.

2.2.2. Drug dose and choline incorporation

PC-3 cells were treated with flutamide drug added in concentrations of 0,

5 and 10nM in triplicate in 25 cm2 flasks and incubated for 3 d.

2.2.3. Cell viability assays

Cell viability was assessed using both the MTT assay and the trypan blue
exclusion methods. For MTT assay, cells were seeded in flat bottomed 96 well
tissue culture plates at a concentration of 1 × 105 cells/mL medium in a volume
of 200 µL per well and were allowed to grow to 70% confluency before adding
drugs. The plates were set up for controls and different drug concentrations,
then incubated in a humidified atmosphere with 5% CO2:95% air at 37°C.
After reaching 70% confluency, different concentrations of flutamide were
added separately into triplicate plates of PC-3 cells and were incubated for 3 d,
after which the medium was pipetted out and new medium was added in a final
volume of 170 µL per well. Then, 30 µL of MTT (5 mg/mL in phosphate
buffered saline (PBS)) was added to each well and incubation of the cells
continued. After 4 h, the medium was carefully pipetted out, leaving the
adherent cells and precipitate in the wells. A volume of 200 µL of DMSO was
then added to each well with gentle mixing for 20 min to dissolve the
precipitate and the appearance of crystals was checked under the microscope.
Then plates were read in a 96 well plate scanner (ELISA plate reader) at dual
filter wavelengths of 540 and 690 nm.

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2.2.4. Trypan blue exclusion method

In another set of experiments, cells were grown to 70% confluency in

25 cm2 flasks in triplicate, followed by addition of 0, 5 and 10nM flutamide to
PC-3 cells for 3 d. Dilution was made just prior to counting to prevent viable
cells from absorbing the stain and so appearing non-viable. Cells were
harvested by trypsinization, neutralized with medium, and washed with PBS.
Trypan blue was added to suspended cells at a concentration of 0.4% weight/
volume (w/v) for 3–5 min. Live cell numbers were determined by counting with
a haemocytometer and expressed as a percentage of their controls. These
viability tests were used to determine the optimum treatment concentration
and the 50% inhibitory concentration (IC50) for flutamide drug to be used in
the subsequent choline incorporation experiments. PC-3 cells were incubated
in triplicate in 25 cm2 flasks with 37 kBq/mL medium/flask [Methyl-3H]-choline
for 10 min at 37°C, then the pulse chase and efflux method and flow cytometry
were carried out as described below.

2.2.5. [Methyl-3H]-choline pulse chase and efflux

Cells were incubated in triplicate for 10 min at 37°C with 37 kBq [Methyl-
3
H]-choline/mL of DMEM medium in 25 cm2 flasks, washed 6 times with ice
cold PBS, then incubated with non-radioactive DMEM medium for 10 min.
The cells were washed once with ice cold PBS, trypsinized with 0.5 mL trypsin,
neutralized with 0.5 mL medium and centrifuged at 10 000g for 5 min at 4°C.
Activity in the media was counted using a liquid scintillation counting analyser
(LSC-1900CA; Tri-CARB; Packard). Cell pellets were washed once with PBS
to remove extracellular protein and the intracellular location of [Methyl-3H]-
choline determined by extraction with buffer and organic solvents.

2.2.6. Extraction of radiolabelled metabolites and lipids

Metabolites were extracted from cells using a chloroform/methanol Tris

buffer solvent system [12]. Cells were pelleted in eppendorf tubes to which was
added 1 mL of methanol and 0.5 mL of chloroform, which was then left at 4°C
for 60 min, after which 0.5 mL chloroform was added to the suspension
followed by 0.5 mL Tris buffer (10mM, pH7.0) with thorough mixing. After
centrifugation at 1000g for 15 min at 4°C, the upper (aqueous) and lower (lipid)
phases separated and a sample of each counted for radioactivity. Protein assay
content was determined on the cell debris located at the interface of the two
phases using standards prepared from a solution of 1.4 mg/mL BSA after
solubilization with 1M NaOH and neutralization with HCl.

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2.2.7. Flow cytometry

Cells harvested by trypsinization were washed twice in ice cold PBS then
resuspended in 100 µL PBS. A volume of 1 mL of ice cold 70% ethanol was
added to the cell suspension whilst vortexing and the cells left overnight at
-20°C for flow cytometry. For flow cytometry analysis, cell number was adjusted
to 1 × 106 cells/sample. A volume of 1 mL of PI/RNAse and triton x-100
staining buffer was added to the cells and the suspension incubated for 20 min,
protected from light at room temperature. Flow cytometry was performed
using a 488 nm laser on a FACSCalibur flow cytometer (Becton Dickinson) and
CELLQuest software (Becton Dickinson) equipped for fluorescence detection,
forward, 90° angle light scatter and doublet discrimination.

3. RESULTS

3.1. Cell viability assays

Figure 1 shows the treatment effect, viability and IC50 for flutamide on
PC-3 cells using MTT (%MTT, n = 12) and trypan blue (%TB, n = 3) tests
compared to controls. The IC50 for flutamide on PC-3 cells was 10nM.
Therefore, PC-3 cells were treated with 10nM flutamide for 3 d in the
subsequent experiments.

PC-3 Cells Treated with Flutamide for 3 days

100
Viability (%)

75

50

25

0
0 10 20 30 40 50 60 70 80 90 100
Concentration (nM)

FIG. 1. The viability tests using MTT and trypan blue after 3 d exposure to flutamide of
PC-3 cells. Cell viability is expressed as the mean ± SD of the values obtained from three
independent experiments of TB (open circles) and from 12 replicates of MTT 96 well
plates (closed circles).

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 AL-SAEEDI

3.2. Drug dose and choline incorporation

Figure 2 shows DNA histograms of PC-3 cells incubated with flutamide

for 3 d. The IC50 for flutamide, 10nM, induced accumulation of DNA in the S
phase of PC-3 cells.
[Methyl-3H]-choline incorporation and DNA synthesis (S phase) in PC-3
cells treated with 0, 5 and 10nM flutamide for 3 d (Table 1) demonstrated that
whilst treatment of PC-3 cells with flutamide showed a significant cell accumu-
lation in the S phase compared to their controls, [Methyl-3H]-choline uptake
declined significantly (P < 0.0001) at 10nM concentrations compared with
control levels.

4. DISCUSSION

[Methyl-11C]-choline has been recently introduced as a novel tumour

seeking PET tracer [1, 13], especially to image and stage prostate cancer [11,
14, 15]. This study demonstrates a decrease in cell viability, as well as an
accumulation of cells in the S phase of the cell cycle after the treatment of PC-3
cells with flutamide. Here, some possible mechanisms of flutamide in relation
to cell proliferation will be discussed.

G1 S G2 G1 S G2

FIG. 2. DNA histograms of PC-3 cells alone (left) and when incubated with 10nM
flutamide for 3 d (right).

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TABLE 1. [METHYL-3H]-CHOLINE INCORPORATION (n = 6) AND

CORRESPONDING S PHASES (n = 3) OF PC-3 CELLS AFTER
EXPOSURE TO 0, 5 AND 10nM FLUTAMIDE
(the data are expressed as the mean dpm/µg protein ± SD (*P < 0.0001 compared
with controls))

Flutamide (nM) Choline uptake S phase

0 114.21 ± 0.57 (n = 6) 30.03 ± 0.58 (n = 3)

5 93.98 ± 0.97 (n = 6) 30.54 ± 0.30 (n = 3)
10 65.95 ± 0.72 (n = 6)* 61.81 ± 1.33 (n = 3)*

Prostatic cancer consists of cells that are sensitive to or dependent on

hormones, androgen sensitive, such as the human prostate cancer LNCaP cell
lines, and others that are hormone independent, androgen insensitive, PC-3
cells. The latter cells express low levels of androgen receptors. Accordingly,
PC-3 cells do not respond to either androgens or anti-androgens and have been
used extensively as a model for androgen independent and hormone non-
responsive prostate cancer [16–19]. In this study, the effect of flutamide was
investigated using PC-3 cells as controls. Control and treated cells were
incubated with [Methyl-3H]-choline incorporation for 10 min. Studying choline
incorporation in PC-3 cells is useful particularly if condsideration is given to the
fact that the onset of cancer progression results in the primary tumours being
composed predominantly of androgen insensitive cells that are no longer
affected by anti-androgens or androgen deprivation [20]. The preliminary study
showed that PC-3 cells were killed by flutamide. Flutamide is a well-known
anti-androgen drug. Its mechanism of action includes a competitive inhibitor of
testosterone and dihydrotestosterone for the AR, interfering with the
androgen action [21], and in the author’s study its mechanism may involve
other unknown mechanisms.
The present study demonstrates a novel finding that flutamide inhibits
PC-3 cell proliferation, which was associated with a build-up of cells in the S
phase and a dose dependent decrease of [Methyl-3H]-choline incorporation.
From the two viability tests (cytotoxic analysis), the IC50 for flutamide after 3 d
in PC-3 cells was found to be 10nM. A reduction in choline uptake following
therapy has been reported in several studies [5, 22]. DeGrado et al. [23]
reported that fluorocholine PET uptake, which closely mimics choline uptake
by normal tissues and prostate cancer was markedly reduced in patients
rescanned during androgen deprivation therapy. Here, PC-3 cells plus flutamide
suggests that flutamide acts by another mechanism other than the well-known
one that prevents the interaction of testosterone and dihydrotestosterone with

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the AR in the prostate. Inaloz et al. [24] have shown that the administration of
flutamide in rats inhibits skin growth and proliferation in parallel with a
decrease in the epidermal growth factor, a key hormone for skin development,
and is known to stimulate cell proliferation and differentiation in a wide range
of tissues. Flutamide may result in the inhibition of epidermal growth factor
action in PC-3 cells, leading to the inhibition of the cells proliferation and their
DNA synthesis, and result in the subsequent alteration to their cellular
phospholipid metabolism, especially choline incorporation after the treatment
with flutamide, and this is may involve the inhibition of phospholipase D
activity.
Another possibility could be mediated through oestrogen receptors. For
example, Kawashima et al. [25] reported that in PC-3 cells both the cell growth
and the AR activity were remarkably inhibited by tamoxifen (TAM) at 50µM
and flutamide at 5–50µM. They reported that anti-oestrogens modulate the
transactivation activity of the AR in prostate cancer cells. Although the most
prominent characteristic of prostate cancer is its androgen dependence,
oestrogen seems to be involved. In the human prostate, ERα is localized in the
stromal tissue of the prostate and associated with the progression, metastasis
and the hormone refractory phenomenon of the prostate cancer [26, 27]. It was
reported that both ERα and ERβ were expressed in PC-3 cells [28]. The
activation of ERβ by its putative ligands 5α, androstane-3β and 17β-diol was
reported to suppress the growth of the ventral prostate [29]. Activation of such
a receptor may be involved in inhibition of cell proliferation and subsequently
reduced choline incorporation in PC-3 cells treated with flutamide.
Flutamide may act through similar mechanisms to those involved in the
inhibitory effect of TAM, the widely used anti-oestrogen therapy for breast
cancer, which are unclear at present. Flutamide may act through caspase
activation. Some studies reported that anti-oestrogen, raloxifen induces
apoptosis in androgen independent prostate cancer cells through caspase
activation [30] and TAM inhibits prostate cancer by inhibiting protein kinase C
followed by the induction of p21(waf1/cip1) [31]. Kiss and Crilly [32] reported
that in an ER deficient multidrug resistant subline of MCF-7 human breast
carcinoma cells, clinically relevant concentrations of TAM inhibited the uptake
and phosphorylation of choline and, as a result, the synthesis of corresponding
phospholipids. Here, PC-3 cells treated with flutamide showed dose dependent
reduced [Methyl-3H]-choline uptake, reflecting a decrease in phospholipid
synthesis. Moreover, flutamide can induce apoptosis in PC-3 cells through the
formation of free radicals [33], which will inhibit the ability of cells to
synthesize new DNA and ultimately reduce its choline uptake. In this study
flutamide inhibited cell growth and proliferation as well as [Methyl-3H]-choline
incorporation in the AR negative PC-3 cells in vitro. Currently, the study is

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extended to investigate many different concentrations of flutamide in both

LNCaP cell lines and PC-3 cell lines. Moreover, since the in vitro system differs
from the in vivo systems owing to the presence of non-tumour tissue, hypoxic
regions, other androgenic, oestrogenic, or any other hormonal or anti-
hormonal activities, and other factors, the extrapolation of these findings to the
in vivo systems has to be carried out with caution.

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[16] KAIGHN, M.E., NARAYAN, K.S., OHNUKI, Y., LECHNER, J.F., JONES, L.W.,
Establishment and characterization of a human prostatic carcinoma cell line (PC-
3), Invest Urol 17(1) (1979) 16-23.
[17] WARE, J.L., PAULSON, D.F., MICKEY, G.H., WEBB, K.S., Spontaneous metas-
tasis of cells of the human prostate carcinoma cell line PC-3 in athymic nude mice,
J Urol 128(5) (1982) 1064-7.
[18] KOZLOWSKI, J.M., et al., Metastatic behavior of human tumor cell lines grown
in the nude mouse, Cancer Res 44(8) (1984) 3522-9.
[19] PIZZI, H., et al., Androgen regulation of parathyroid hormone-related peptide
production in human prostate cancer cells, Endocrinology 144(3) (2003) 858-67.
[20] CATALONA, W.J., Management of cancer of the prostate, N Engl J Med 331(15)
(1994) 996-1004.
[21] BRUFSKY, A., et al., Finasteride and flutamide as potency-sparing androgen-
ablative therapy for advanced adenocarcinoma of the prostate, Urology 49(6)
(1997) 913-20.
[22] FULHAM, M.J., et al., Mapping of brain tumor metabolites with proton MR
spectroscopic imaging: clinical relevance, Radiology 185(3) (1992) 675-86.
[23] DeGRADO, T.R., et al., Synthesis and evaluation of 18F-labeled choline as an
oncologic tracer for positron emission tomography: initial findings in prostate
cancer, Cancer Res 61(1) (2000) 110-7.
[24] INALOZ, H.S., KETANI, M.A., INALOZ, S.S., YILMAZ, F., KETANI, S., The
effects of sialoadenectomy & flutamide on skin development, Clin Exp Obstet
Gynecol 27(3-4) (2000) 231-4.
[25] KAWASHIMA, H., et al., Effect of anti-estrogens on the androgen receptor
activity and cell proliferation in prostate cancer cells, Urol Res. (2004).
[26] GRIFFITHS, K., Estrogens and prostatic disease. International Prostate Health
Council Study Group, Prostate 45(2) (2000) 87-100.
[27] BONKHOFF, H., FIXEMER, T., HUNSICKER, I., REMBERGER, K.,
Estrogen receptor expression in prostate cancer and premalignant prostatic
lesions, Am J Pathol 155(2) (1999)641-7.

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[28] LAU, K.M., LaSPINA, M., LONG, J., HO, S.M., Expression of estrogen receptor
(ER)-alpha and ER-beta in normal and malignant prostatic epithelial cells: regu-
lation by methylation and involvement in growth regulation, Cancer Res 60(12):
(2000) 3175-82.
[29] WEIHUA, Z., et al., A role for estrogen receptor beta in the regulation of growth
of the ventral prostate, Proc Natl Acad Sci U S A 98(11) (2001) 6330-5.
[30] KIM, I.Y., et al., Raloxifene, a mixed estrogen agonist/antagonist, induces
apoptosis in androgen-independent human prostate cancer cell lines, Cancer Res
62(18) (2002) 5365-9.
[31] ROHLFF, C., et al., Prostate cancer cell growth inhibition by tamoxifen is associ-
ated with inhibition of protein kinase C and induction of p21(waf1/cip1), Prostate
37(1) (1998) 51-9.
[32] KISS, Z., CRILLY, K.S., Tamoxifen inhibits uptake and metabolism of
ethanolamine and choline in multidrug-resistant, but not in drug-sensitive, MCF-
7 human breast carcinoma cells, FEBS Lett 360(2) (1995) 165-8.
[33] NUNEZ-VERGARA, L.J., FARIAS, D., BOLLO, S., SQUELLA, J.A., An elec-
trochemical evidence of free radicals formation from flutamide and its reactivity
with endo/xenobiotics of pharmacological relevance, Bioelectrochemistry 53(1)
(2001) 103-10.

193
.
 A SEMI-AUTOMATED [13N]NH3 PRODUCTION
MODULE: DESIGN, QUALITY CONTROL AND
OPTIMIZATION

A.R. JALILIAN*, P. ROWSHANFARZAD*, M. SABET**,

M. MIRZAII*, A. ZIAEE***, D. SARDARI***

*Cyclotron and Nuclear Medicine Department

Email: ajalilian@nrcan.org

** SSDL and Health Physics Department

Nuclear Research Center for Agriculture and Medicine,

Atomic Energy Organization of Iran,
Karaj

*** Science and Research Branch, Azad Islamic University,

Tehran

Islamic Republic of Iran

Abstract

The compound [13N] NH3 was prepared using a home-made, cost effective
prototype module meeting pharmaceutical criteria. The 16O(p,α)13N nuclear reaction was
chosen as the best method of 13N production. High purity natural water was bombarded
by 18 MeV protons with a current intensity of 8 μA. A production yield of 13.5 mCi·μA-1·h-1
was obtained. Chemical modification of [13N]nitrogen oxides into [13N]NH3 was accom-
plished using in-house made De Varda’s catalyst. Chemical, radiochemical and radionu-
clide purity controls performed on the final product showed high radiopharmaceutical
purity in the form of [13N]NH3 produced by a prototype production module. [13N]NH3
was formulated in normal saline media under appropriate pH and sterile conditions.
Microbial and LAL tests were performed on the final product in order to assure its
safety for administration to experimental subjects.

1. INTRODUCTION

Improvements in positron emission tomography (PET) have encouraged

many research groups to prepare different radiopharmaceuticals following

195
 JALILIAN et al.

administration to humans. [13N]NH3 is probably the only clinically used 13N

radiotracer (positron energy = 1.19 MeV, tissue range = 5.4 mm, half-life =
9.965 min). Its short half-life can be an advantage, allowing the possibility of
performing repeated experiments on the same patient.
No matter what the target is, the chemical process should afford the
reduced form of nitrogen atom, i.e. NH3. The most abundant form of this
radiotracer in the biological environment is NH4+ which can be used in the wall
motion studies of the heart [1], local cardial perfusion [2], study of revasculari-
zation after heart transplants [3] and viability tests after myocardial infarction [4].
The authors have been interested in the domestic preparation and quality
control of PET radiopharmaceuticals for ultimate use in clinics [5–7]. In this
study, one of the simplest routes of production, i.e. irradiation of natural high
purity water by protons, has been targeted as well as manufacturing of a
prototype [13N]NH3 production module.
Regarding the irradiation of natural water for the production of
[13N]NH3, there are some possible chemical reactions. In the first step, oxgen is
turned into a nitrogen radical that can scavenge protons from water, producing
peroxide. In the next step, the peroxides attack [13N]NH3 and finally nitroxides
will be produced:

13
N + H-OH Æ 13NH3

13
NH3 + HOH Æ 13NH4++ OH–

13
NH4+ ….Æ 13NO3– + 13
NO2–

Using anion retardation resins, the anions can be removed from the target
solution. Therefore, by the addition of various radical scavengers such as
ethanol or acetic acid, the [13N]NH3 compound will be the major product. The
use of reducing agents such as De Varda’s catalyst or Ti salts [8] may be the
alternative.

2. EXPERIMENTAL

The chemicals were purchased from Aldrich Chemical Company,

Germany. All chemicals were recrystallized repeatedly before use. Radio thin
layer chromatography (RTLC) was run on polymer backed silica gel (F 1500/
LS 254, 20 cm × 20 cm, TLC Ready Foils Schleicher & Schuell). A mixture of
acetone–propionic acid–saturated brine (1:2:4) was used as eluent. The radio-
chromatogram scanner was coupled to a HPGe detector (Canberra germanium

196
 SESSION 10

detector; model GC1020-7500SL). The step motor was installed to count a

0.4 cm section each 30 s through the slot of a shielded chamber.

2.1. Preparation of De Varda’s catalyst for routine production

Aluminium or copper turnings (50 g) were washed in a 2% solution of

SDS (3 × 100 mL), followed by rinsing with double distilled H2O (3 × 500 mL).
The washed turnings were heated separately in a vacuum chamber at 200ºC for
2 h. The turnings were then dispersed in acetone (200 mL) and irradiated in an
ultrasound water bath for 1 h and were finally washed by a mixture of
acetone:water (1:1) in order to remove organic impurities (3 × 250 mL) and
were dried at 200ºC for 4 h. A mixture of both of the above turnings and
commercially available zinc powder (Al:Zn:Cu; 9:1:10) were mixed in a rotary
motor under reduced pressure at 50–70ºC and the final mixture was dispensed
in 5 g portions and to each was added 2 ± 0.1 g of NaOH. The portions were
capped carefully under N2 gas and kept for the production process. De Varda’s
catalyst containing vials were capped and autoclaved for routine use.

2.2. Production of carrier NH3 in the production module

A solution of 1% KNO3 (2 mL) was injected carefully into a De Varda’s

batch using a flow of He gas. The evolution of gases (NH3, H2O vapour) started
rapidly after treatment due to the reduction of the NO3- anion to NH3 exother-
mically. The vapour was finally trapped in a sterile normal saline vial (5 mL)
and placed in an ice water bath. An outlet was placed over the last vial in order
to control the pressure and this is connected to an exhaust equipped with a
charcoal filter. The final solution was checked using colorimetric assay to
detect the presence of the NH4+ cation using freshly prepared Nessler’s reagent
(K2[HgI4]). A concentration of about 1000 ± 10 ppm was produced in a single
run starting from the carrier NO3 anion.

2.3. Selection of the production parameters

In this research, 16O(p,α)13N was selected as the best nuclear reaction for
the production of 13N, using natural water as the target material, owing to the
small amount of produced 18O resulting in 18F, that could be easily separated by
physical methods and also for cost effectiveness. Other metal impurities such as
51
Cr, 52Mn, 55Co, 56Co, 57Co,58Co and 57Ni radioisotopes were also produced as a
result of proton irradiation of the windows and target body, but they could be
easily removed in the radiochemical or physical separation process. The
optimum proton beam energy was calculated and types of possible impurities

197
 JALILIAN et al.

were predicted using previous experimental data given in the literature which
showed that the maximum production yield can be achieved with the lowest
level of radioactive impurities at 18 MeV proton energy.

2.4. Preparation of [13N]-nitrate/nitrite anions

Nitroxy anions were prepared by 18 MeV proton bombardment of a

double distilled H2O sample (1.7 mL) prepared by passing a distilled sample
through the ion retardation resins, followed by a reverse osmosis step that
finally reached a maximum conductivity of 0.05 ± 0.001 μsiemens. The sample
was irradiated for 15–20 min by 18 MeV protons in an all-silver target in a 30
MeV cyclotron at NRCAM. Cooling was performed using a pressurized flow of
He gas.

2.5. Control of radionuclide purity

Gamma spectroscopy of the irradiated target before radiochemical

processing was carried out by a HPGe detector coupled with a Canberra™
multichannel analyser. The peaks were observed and the area under the curve
was counted for 1000 s. After the reduction and purification steps in the
module, the final injectable solution was checked for radionuclide purity as
shown in Fig 1.

FIG. 1. Gamma spectroscopy of the final injectable solution.

198
 SESSION 10

2.6. Chemical purity control

The presence of zinc cation was checked by visible colorimetric assay.

Even at 5 ppm of standard zinc concentration, the pinkish complex is visible to
the naked eye, while the author’s test sample remained similar to the blank.
The amount of copper cation was checked in the final solution using colour
formation of acidic dithizone reagent reacting with gold dilutions based on a
previously reported colorimetric method.

2.7. Radiochemical purity of [13N]NH3

RTLC was performed using a mixture of acetone–propionic acid–

saturated brine (1:2:4) as the mobile phase for both the target liquid and final
solutions (Fig. 2). The radiochromatogram showed a major and distinct
radiopeak at Rf = 0.80, using an in-house made radiochromatogram scanner
coupled to a HPGe detector. The step motor was installed to count a 0.4 cm
section each 30 s through the slot of a shielded chamber. The NO3 and NO2
anions were eluted at Rf = 0.45. The radiochemical yields (higher than 98% in
each case, n = 9) were determined by the comparison of NO3 and NO2 anions
with the major radiopeak at Rf = 0.80 for 13N–NH3.

TABLE 1. AMOUNTS OF CATION IMPURITIES CHECKED IN THE

FINAL SAMPLE

Present work USP EP

Cation Origin Reagent
(ppm) (ppm) (ppm)

Ag+ Target body Dithizone 3 3 5

3+
Al De Varda Dithizone 6 5 8
2+
Zn De Varda Dithizone 1.5 5 10
2+
Cu De Varda Dithizone 0.5 5 6
NH4+ Impurities Nessler’s 5 2 4
Ni2+ Havar Dimethylglyoxime 2 4 6
2+
Fe Havar Bi-pyridyl 5 5 5
3+
Cr Havar 8-Hydroxy-quinoline 3 4 8
Co3+ Havar 8-Hydroxy-quinoline 4 5 10

199
 JALILIAN et al.

14000 12000

13000
11000
12000
10000
11000
9000
10000

9000 8000

8000 7000
A.U.C

A.U.C.
7000 6000
6000
5000
5000
4000
4000

3000 3000

2000 2000

1000 1000
0
0
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chromatogram Slices
Chromatogrm Slices

FIG. 2. RTLC of target solution in acetone–propionic acid–saturated brine (1:2:4) as the

mobile phase for both the target liquid (left) and final solution (right).

2.8. Half-life measurement

In order to ensure the presence of 13N in the final solution and the
absence of other PET radionuclides, activity measurements were performed on
radiopharmaceutical samples (3.2 ± 0.1 mCi) and the activities were plotted
versus time (Fig. 3).

3.5

2.5
Activity (mCi)

1.5

0.5

0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)

FIG. 3. Decay curve of the final NH3 sample.

200
 SESSION 10

80
16 15
O(p,pn) O
70

Cross Section (mb) 60

50

40

30

20
M.G.Albouy et al 1962
M.Sajjad et al - 1985
10
S.W.Kitwanga et al - 1989

0
15 25 35 45 55 65 75 85 95 105 115 125 135 145
Proton Energy (MeV)

FIG. 4. Comparison of the experimental data for the 16O(p,α)13N reaction.

3. RESULTS AND DISCUSSION

The experimental data given in the literature for the 16O(p,α)13N nuclear
reaction were compared in order to select the optimum proton beam energy
(Fig. 4) [9]. It is clearly obvious that all the results with the exception of one
report are in good agreement.
The best proton energy for this reaction is 18 MeV. At this energy, the
16
O(p,pn)15O nuclear reaction which also takes place has been reported by
many reseach groups [10–12] (Fig. 5). The probability of 15O production is

200
16 13
O(p,α) N M.G.Albouy et al - 1962

180 M.Sajjad et al - 1986

H.A.Hill et al - 1961
160
Furukawa et al - 1960

Whitehead et al - 1985
140
Cross-Section (mb)

S.W.Kitwanga et al - 1989
120 W.Gruhle et al - 1977

S.Takacs et al - 2003
100
P.Guazzoni et al - 1971

80

60

40

20

0
5 10 15 20 25 30 35 40 45
Proton Energy (MeV)

FIG. 5. Comparison of the experimental data for the 16O(p,pn)15O reaction.

201
 JALILIAN et al.

higher at energies above 18 MeV. The SRIM nuclear code was used for target
thickness determination [13].
[13N]NH3 was prepared by 18 MeV proton bombardment of the natH2O
target. The target was bombarded with a current intensity of 8 μA for 20 min.
The chemical separation process was based on a distillation method. The
resulting activity of [13N]NH3 was 36 mCi at the end of bombardment and the
production yield was 13.5 mCi·μA-1·h-1 (Fig. 6).
The production yield obtained in this study was comparable to other
published data. The yield was calculated in the hot cell, at 10 m distance from
the target room using a long PE hose that imposed a percentage of activity loss
through the line. The ion exchange chromatography method proposed in some
previous publications was rather time consuming. Radiochemical separation
was performed by a one step reduction of nitroxy anions in a sealed disposable
chamber containing in-house made De Varda’s catalyst.
The reaction took place as soon as the target aqueous content was added to
the catalyst chamber equipped with a small stirrer rod and in less than 40–60 s the
maximum formation of NH3 took place with a yield of more than 95%. Quality
control of the product was performed in two steps. Radionuclide control

FIG. 6. [13N]NH3 production module details: (1) aluminium window, (2) target outlet, (3)
proton beam, (4) cooling gas, (5) target inlet, (6) irradiated liquid, (7) silver body target,
(8) Havar window, (9) 6-wedge valve, (10) module inlet valve, (11) catalytic reduction unit
outlet, (12) catalytic reduction unit inlet, (13) De Varda’s catalyst, (14) gas buffer chamber,
(15) 0.22 μm microbial filter, (16) dose calibrator, (17) final sterile solution, (18) over
pressure outlet, (19) excess ammonia chemical trap.

202
 SESSION 10

1000

Production Yield (mCi/uA)

100

10 S.Takacs et al - 2003

M.Sajjad et al - 1986

1 N.N.Krasnov et al - 1969

M.Sajjad et al - 1985

This Work
0.1

0.01

0.001
6 9 12 15 18 21 24 27 30 33
Proton Energy (MeV)

FIG. 7. Comparison of the literature data with the production yield in the present work.

13
showed the presence of 511 keV gamma energy, originating from N, and
purity was higher than 99%.

3.1. Chemical control

Owing to the exposure of target material’s constituent metals, the

possibilty of any chemical impurities must be considered. Since the target body
contained the Havar window (mostly containing Fe, Co, Cr, Ni) and silver, the
colorimetric assay for the detection of these elements was checked according to
USP and EP limitations.
As a result of the use of De Varda’s catalyst, the possibility of vaporization
of the catalyst elements and contamination of the final solution must be taken
into account. Finally, the amount of carrier ammonia in the final preparations
due to the presence of nitrate salt impurities was checked using Nessler’s test.

3.2. Radiochemical purity

Owing to the formation of nitrite/nitrate anions in the target liquid and

consequent formation of ammonium ion after the reduction step, the amounts
of each of these components was checked by RTLC before and after the
reduction step. A mixture of acetone–propionic acid–saturated brine (1:2:4)
was used as the eluent. The ammonium cation migrated to Rf = 0.8 while the
anions stayed at a lower Rf of 0.45 [14].

203
 JALILIAN et al.

3.3. Radionuclide purity

As a result of the interaction of protons with the target material (target

body and windows), the possibilty of other nuclear reactions was also
considered.

3.4. Rival nuclear reactions of natural water and proton beam

As a result of the use of a high purity natural water sample, the presence
of O nuclide in natural oxygen (0.038%) could lead to the formation of 18F via
18

the 18O(p,n)18F nuclear reaction. By selection of the best proton energy, this
possiblity was minimized, but the possibilty of forming 18F ionic forms in the
target still remained. It was shown that if the De Varda alloy pretreatment were
not performed properly, the presence of impurities in the starting materials,
such as organic traces, etc., could produce amounts of volatile 18F-fluorinated
wastes that could be distilled from the reaction vessel and lead to the contami-
nation of the final product. By carefully taking into account the probabilities in
a normal run, the half-life determination of the final sample could be an
important criterion for the presence of long half-life PET radionuclides such as
18
F that could not be identified by gamma spectroscopy. A half-life determi-
nation diagram is illustrated in Fig. 3.

3.5. Biological controls

The Pyrogen test was performed using a commercial LAL kit (sensitivity
0.125 EU/mL, Charles River Endosafe Co., United States of America).
Microbial/fungal tests showed suitable pharmaceutical sterility.

4. CONCLUSION

The method used in this research for the production and chemical
separation of [13N]NH3 was quite simple and cost effective, while previous
studies given in the literature have reported a production yield similar to the
author’s work (Fig. 7). Total labelling and formulation of [13N]NH3 took about
2 min, with a yield of 98%. A significant specific activity (≈1200 Ci/mmol) was
formed via production of [13N]nitroxy anions. No unlabelled and/or labelled
by-products were observed upon RTLC analysis of the final preparations by
chemical determination tests. The radiopharmaceutical was usable in aqueous
solution for at least 25 min and no significant amounts of other radioactive
species were detected by RTLC. Trace amounts of other PET radionuclides

204
 SESSION 10

(≈1%) were detected by RTLC showing a radiochemical purity of higher than

99% for [13N]NH3. In contrast to other [13N]NH3 production methods, the
authors’ module represents an easy, rapid, cost effective and fully automated
method meeting the suitable quality requirements for a radiopharmaceutical
production system. Disposable units, hose lines and filters represent a GMP
directed production module for ultimate use of this tracer in the country.

REFERENCES

[1] MASUDA, D., et al., Improvement of regional myocardial and coronary blood
flow reserve in a patient treated with enhanced external counterpulsation: evalu-
ation by nitrogen-13 ammonia PET. Jpn Circ J 1999 May; 63(5): 407-411.
[2] PHELPS, M.E., HOFFMAN, E.J., RAYBOUND, C., Factors with affect cerebral
uptake and retention of N-13 NH3. Stroke 1977; 8: 694-702.
[3] KITSIOU, A.N., et al., 13N-ammonia myocardial blood flow and uptake: relation
to functional outcome of asynergic regions after revascularization, J Am Coll
Cardiol 1999 Mar; 33(3): 678-686.
[4] SAWADA, S., et al., Interobserver and interstudy variability of myocardial blood
flow and flow-reserve measurements with nitrogen 13 ammonia-labeled positron
emission tomography. J Nucl Cardiol 1995 Sep-Oct; 2(5): 413-422.
[5] JALILIAN, A.R., et al., Production Iranian Journal of Nulear Medicine, 12, 13-24,
1998.
[6] JALILIAN, A.R., J. Pharmacy & Pharmaceutical Sciences, 3(1) January-April,
2000. 114-124.
[7] JALILIAN, A.R., et al., Iranian Journal of Nulear Medicine, 21, 49-62, 2004.
[8] AKUTSU, Y., et al., Jpn Circ J 1997 Aug; 61(8): 665-672.
[9] SAJJAD, M., et al., Radiochimica Acta 1986; 39: 165-168.
[10] ALBOUY, M.G., et al., Spallation of oxygen by protons with 20 to 150 MeV, J
Phys Lett 1962; 2:306.
[11] KITAWANGA, S.W., LeLEUX, P., LIPNIK, P., VANHORENBEECK, J.,
Production of N-13 radioactive nuclei from 13C(p,n) or 16O(p,α) reactions, J Phys
Rev/C 1989 ; 40: 35.
[12] SAJJAD, M., LAMBRECHT, R.M., WOLF, A.P., Cyclotron isotopes and radiop-
harmaceuticals, Investigation of some excitation functions for the preparation of
15O, 13N, 11C, J Radiochimica Acta 1985; 38: 57.
[13] ZIEGLER, J., The code of SRIM- the Stopping and Range of Ions in Matter,
Version 2000.XX, (2000).
[14] GATLEY, S.J., et al., Int J Rad Appl Instrum [A], 1991; 42(9): 793-796.

205
.
RADIOIODINE RADIOPHARMACEUTICALS

(Session 11)

Chairpersons

H.S. BALTER
Uruguay

G.-J. BEYER
Switzerland
.
 PRODUCTION OF RADIOIODINES WITH MEDICAL
PET CYCLOTRONS

J.J. ČOMOR*, G.-J. BEYER**, G. PIMENTEL-GONZALES***

*Laboratory of Physics, Vinča Institute of Nuclear Sciences,

Belgrade, Serbia

** Division of Nuclear Medicine, University Hospital of Geneva,

Geneva, Switzerland

*** National Institute of Oncology & Radiobiology, Bedado,

Havana, Cuba

Abstract

The authors discuss the possibility of producing radioiodine with mass numbers
120, 123 and 124 via proton reactions on enriched TeO2 targets with medical PET cyclo-
trons under three aspects: yield and impurity considerations, technical feasibility and
radiopharmaceutical chemistry. The thick target yield for the XXXTeO2(p,n)XXXI process
is of the order of 2.5 × 109 atoms and relatively independent of the mass number. Conse-
quently, the (p,n) process could be a suitable alternative to the well-known 124Xe(p,x)
method — large scale production technology for 123I. Concerns regarding radionuclide
impurities do not play a role any longer, because of availability of the target material
with an enrichment close to 100% without increased costs. The standard medical PET
cyclotrons and a standardized separation technique can be used for the production of all
three isotopes: 120I, 123I and 124I in high purity via the (p,n) reaction. A suitable target
consists of a platinum disc carrying about 200–300 mg of TeO2. A dedicated COmpact
Solid Target Irradiation System (COSTIS) has been developed, which is now available
commercially. Irradiation conditions are: 13–15 MeV protons, 20 µA/cm2 beam intensity.
Thus, 10 GBq or 1.5 GBq batches of 123I or 124I, respectively, are practically available
using a PET cyclotron. The radioiodine is separated from irradiated TeO2 targets using
a thermochromatographic process. The TeO2 targets cycled in this way can be reused
immediately for the next production run without further treatment. A corresponding
good manufacturing practice conform separation module has now been developed
(TERIMO = TEllurium based RadioIodine production MOdule) and is available
commercially. The losses of target material for one cycle is negligible (<0.2 mg/cycle).
Local in-house production of radioiodine has the advantage of obtaining an iodinated
radiopharmaceutical compound. Preliminary examples along this line will be presented.
The technology described is identical for all three mentioned iodine isotopes with the
mass numbers 120, 123 and 124.

209
 ČOMOR et al.

1. INTRODUCTION

Positron emission tomography (PET) has become the technique of choice

in modern nuclear medical diagnostics. The vast majority of PET diagnostic
procedures are currently carried out by radiopharmaceuticals based on the
radionuclide 18F and to a much lesser extent by 11C, 13N and 15O. The short half-
lives of these radionuclides is limiting the applicability of PET to procedures
that are based on tracer molecules with rapid biodistribution and metabolism.
However, there are a number of alternative positron emitting radionuclides
with significantly longer half-lives which might be used for PET provided that
they are produced on-site as they are not yet widely available from commercial
suppliers. Among them, the most promising radionuclides, which can be
produced by a compact medical cyclotron delivering typically 13–20 MeV
proton beams (i.e. by (p,n) nuclear reactions) are: 64Cu, 76Br, 86Y, 94mTc, 120gI,
and 124I [1].
According to current trends in radiopharmacy and nuclear medicine the
positron emitting radionuclides 120gI and 124I are of utmost interest [2]. The long
half-life of 124I (4.18 d) allows for timely quantitative biodistribution and
metabolic studies. It has been shown that PET with 124I labelled tracers is
particularly useful in oncology [3]. In addition to common functional visuali-
zation, quantitative PET imaging of 124I tracer analogues of 131I labelled
therapeutic radiopharmaceuticals can be used for patient specific dosimetric
calculations for 131I radiotherapy planning. On the other hand, the 120gI gained
attention due to the similarity of its half-life to that of 18F and its usefulness in
quantifying the biodistribution of 123I radiopharmaceuticals.
Taking into account the growing interest in the application of 120gI and
124
I, every effort made to improve their production technologies is justified.
Owing to the identical radiochemical behaviour of isotopes, the technologies
developed for these two radioiodines are also suitable for the production of
123
I, an important radionuclide for single photon emission computed
tomography (SPECT), which is still not widely used in the developing countries
owing to logistical problems and its high price. The local production of 123I in
PET centres operating small medical cyclotrons could boost the application of
this invaluable radionuclide in nuclear medicine.
Even though a number of papers have already been published discussing
various aspects of radioiodine production paths by small medical cyclotrons
[2, 5–21], there is no consensus yet as to what technology would be the most
suitable, particularly if one considers the current good manufacturing practices
(GMP) applicable to the production of radiopharmaceuticals. In the case of
production of radioiodines, radiation safety is another significant issue, due to
the high volatility of certain iodinated species. The paper presents a detailed

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description of a GMP compliant technology for the production of 120gI, 123I and
124
I, based on a COmpact Solid Target Irradiation System (COSTIS) and on a
TEllurium oxide based RadioIodine separation MOdule (TERIMO).

2. NUCLEAR REACTIONS SUITABLE FOR RADIOIODINE

PRODUCTION

Taking into account the performances of common compact cyclotrons for

medical radionuclide production (13–20 MeV protons and 6–10 MeV
deuterons), the only suitable nuclear reactions for radioiodine production are
the (p,n) reactions on enriched tellurium isotopes. Figure 1 shows the excitation
functions of nuclear reactions leading to the three most important radioiodines.
One can easily see that the cross-sections of all three reactions, and conse-
quently their production rates (in terms of atoms per second and µA of beam
current), are very similar, meaning that their production technology might be
essentially the same concerning the irradiation conditions. The optimal
conditions that would give the highest radionuclidic purity and still sufficiently
high yields for all three radioiodines are indicated in Figs 1 and 2. It is
important to note that most of the compact medical cyclotrons can provide
proton beams of sufficient energy (14.9 MeV) for the optimal production of the
radioiodines.

FIG. 1. Excitation functions of proton induced nuclear reactions on tellurium

isotopes [14, 22]. The shaded regions represent the most suitable energy range for radio-
iodine production.

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 ČOMOR et al.

FIG. 2. Stopping of protons in the target disc used for radioiodine production. The
thickness of the TeO2 layer is 0.6 mm; after passing this layer the protons are stopped in the
platinum backing.

The radionuclidic purity of radioiodines used for nuclear medical applica-

tions is an important aspect for the selection of the appropriate production
technology. In general, pure γ emitting impurities do not significantly disturb the
PET images (they slightly decrease the signal to noise ratio), provided that the
energies of the most prominent lines are below 511 keV. However, in the case
of 123I, the other radioiodines increase the radiation dose received by the patient
and the high energy γ lines decrease the resolution of the SPECT images.
The most critical radionuclidic impurities for 123I are 124I, 126I and, surpris-
ingly, 130I. These impurities can be produced via (p,n) reactions from the 124Te,
126
Te and 130Te impurities in the enriched 123Te target material. The levels of
these target impurities were still relatively high in the 1980s. The composition
of a typical, close to 70% enriched 123TeO2 material and the obtained
composition of an 123I product after bombardment with 15 MeV protons is
shown in Table 1 [10].
The 130I contamination causes deterioration in the SPECT image due to
the five intense and highly energetic gamma transitions of this radionuclide.
Since its half-life is similar to that of 123I, the 130I content does not change with
time, whereas those of 124I and 126I increase with time due to the decay of 123I.
Consequently, the most critical impurity in the target material is the 124Te
isotope as it is hard to separate from 123Te. Thus, the absolute isotopic purity of

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TABLE 1. COMPOSITION OF NATURAL TELLURIUM AND

TYPICALLY ENRICHED 123TeO2 IN THE 1980s AND THE
COMPOSITION OF AN 123I PREPARATION OBTAINED IN
BOMBARDMENT WITH 15 MeV PROTONS
(data taken from Ref. [10])

Mass number 120 122 123 124 125 126 128 130

Natural abundance 0.096 2.60 0.908 4.816 7.14 18.95 31.69 33.80
Target material composition 0.01 2.3 67.3 11.7 3.9 5.7 5.0 4.1
Radionuclide composition 100 2.3 0.04 0.26 2.1

the enriched 123Te is not the most important quality parameter of a target
material; it is much more important to have a very small 124Te/123Te ratio [23].
Nowadays, the manufacturer of enriched isotopes can provide a 123Te target
material with a 124Te/123Te ratio of 6 × 10-4 [24]. With such a high purity target
material and compact PET cyclotrons, it is possible to produce, via the (p,n)
process, 123I having all the quality parameters (radionuclidic purity as well as
radionuclide concentration) comparable to the high purity 123I product
obtained in the well-known large scale 124Xe(p,x)123I production process. The
(p,n) process, however, cannot compete with respect to the productivity.
The chemical form of the target material is another important factor for
the design of the radioiodine production technology. In general, metallic
targets are preferred due to their better heat conduction and mechanical
stability. However, in the case of tellurium, targets in the form of TeO2 are the
most suited chemical form for two reasons: first, the vapour pressure of
tellurium oxide is lower than that of metallic tellurium at the same temper-
ature, thus less expensive target material is lost during irradiation and thermal
processing; second, tellurium oxide targets might be reused immediately after
thermochromatographic separation of the produced radioiodines, without
troublesome wet chemical processing of the irradiated targets.

3. COST EFFECTIVE TECHNOLOGY FOR RADIOIODINE

PRODUCTION WITH MEDICAL CYCLOTRONS

Even though compact medical cyclotrons can deliver rather high currents
of proton beams (50–100 µA are common), the large scale production of radio-
iodines with them is not feasible as the beam power acceptance of the tellurium
oxide targets is limited. Practical experiences have shown that a p-beam density

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 ČOMOR et al.

of 20 µA·cm-2 seems to be about the limit. If one distributes the p-beam over a
larger area, aiming to increase the productivity, one creates the problem of
inconvenience in the following separation process. Nonetheless, batches of the
order of 3.7 GBq 124I have already been obtained on a routine basis using the
technology described in this paper. The investment needed for such a small
scale production is reasonably low, i.e. the usage of sophisticated target stations
and an automated rabbit target transport system is not really needed and not
justified. However, the automation of the post-irradiation target processing is
justified, as automation can provide reproducibility, repeatability and reliability
required by current GMP for the production of radiopharmaceuticals.

3.1. Targetry

As a result of the specific requirements for local production of radio-

iodines by compact medical cyclotrons, a COmpact Solid Target Irradiation
System (COSTIS) has recently been developed [18]. The target station has
been designed in such a way that it can be connected to virtually any existing
cyclotron using internal beams for irradiation. Using custom made flanges it
can also be connected to any external beam line as well. The principal scheme
of COSTIS is presented in Fig. 3, while Fig. 4 shows two typical installations on

a b

1 2 3 4 5 6 7 8

FIG. 3. (a) Simplified cross-sectional view of COSTIS: (1) quick connection flange; (2)
water cooled collimator with graphite insert; (3) insulating alumina rings; (4) helium
cooling cavity; (5) helium inlet; (6) platinum target disc with TeO2 in its central groove; (7)
water jet; (8) cooling water inlet; (9) cooling water outlet; (10) helium outlet; (11) window
foil. (b) Loading of the target coin into COSTIS.

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 SESSION 11

COSTIS
a b

FIG. 4. COSTIS installed on a GE PETrace (a) and IBA Cyclone 18/9 (b) cyclotrons
(courtesy of IBA Molecular Imaging).

common compact medical cyclotrons. The beam passes through a water cooled
graphite collimator, which forms a circular beam 12 mm in diameter. The next
element is a window foil separating the helium cooling loop from the vacuum.
The helium stream in this loop cools the window foil as well as the front face of
the target disc. Finally, a water jet cools the back face of the target disc.
The target is a 2 mm thick platinum disc, 24 mm in diameter with a
circular groove, 12 mm in diameter, in its centre. This groove is filled with the
target material, for instance with isotopically enriched TeO2, which is melted
into the groove. Platinum is selected as a metal with a sufficiently high thermal
conductivity, which is also chemically inert and not heavily activated by the
beam.
The target disc is loaded manually before the irradiation. It is locked in
position by two pneumatic actuators during the irradiation. By reversing the
action of these actuators, the irradiated target disc is released into a shielded
transport container conveniently placed below COSTIS before the irradiation.
COSTIS has been designed as a compromise between high power
metallic Te targets, which require troublesome post-irradiation radiochemical
processing, and compact low power TeO2 targets, which allow for easy
separation of the produced radioiodine by thermal chromatography. After dry
distillation of iodine from the melted oxide and cooling down of the target disc,
it can be immediately reused for the next production cycle without any further
preparation. This makes the production process simple and cost effective, even
though the yields are limited by the beam power that such an oxide target may
withstand, which is typically of the order of 350 W. Since January 2005, COSTIS
is commercially available from IBA S.A., Louvain-la-Neuve, Belgium.

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 ČOMOR et al.

3.2. Thermochromatographic apparatus

The simplest way of releasing radioiodine from the irradiated TeO2

targets is by thermochromatography, since after this process the targets can be
immediately reused for a new production cycle [2]. Assuming that the diffusion
process is the time determining step for the overall release of radioiodine from
TeO2 melts, the release kinetics of radioiodine may be described by the
following relation [16]:

p2
ln
t 1/ 2 = 216 h 2
p D

where t1/2 is the release half-life, D is the diffusion coefficient and h is the
thickness of the TeO2 layer. However, if convection inside the liquid phase is the
time determining step, one can use the following simple equation for describing
the relation between fractional release (F) and the release time (t) [16]:

F ( t ) = e - m 1t

where the empirical factor µ1 describes the desorption process from the surface.
The radioiodine release kinetics from TeO2 targets have been extensively
studied [16] and it became evident that the release rate is proportional to the
target thickness to diameter ratio, provided that circular targets are employed.
From the dependence of the release half-time on this ratio one can easily
determine that the release half-time for targets designed to be irradiated on
COSTIS is 26.3 s (see Fig. 5); thus 97% of the produced radioiodine will be
released within just 2 min. This is of utmost importance, as the vapour pressure
of molten TeO2 is not negligible, thus the faster the radioiodine release is, the
shorter the time the target has to be kept in a molten state and the lower the
losses of expensive enriched target material. The loss of target material during
the thermochromatographic process has to be taken into account as a potential
source of contamination of the final product as well.
The apparatus for thermochromatographic separation of radioiodine
from the irradiated TeO2 is shown schematically in Fig. 6. It has a quartz target
holder for positioning of the target discs, a fast electric heater, a thermocouple
for temperature measurements, an alumina trap for adsorbing tellurium oxide
vapours and a quartz bulb containing the trapping solution. The iodine vapours

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 SESSION 11

100000

Radioiodine release half-time, sec

10000

1000

100

COSTIS + TERIMO operating point

10
0.01 0.1 1 10
TeO2 target thickness/diameter ratio

FIG. 5. Release half-time of radioiodine from molten TeO2 at 800°C as a function of

target thickness to diameter ratio. O denotes experimental data from [16], + denotes the
operating point of targets designed for irradiation in COSTIS (0.6 mm thick TeO2 layer,
12 mm in diameter).

are carried through the apparatus via sterile filtered air. The quartz apparatus is
shown in Fig. 7.
The quartz oven is integrated into a GMP compliant module for carrying
out the whole procedure of radioiodine separation. The scheme of the module
is presented in Fig. 8. The operation of the module starts with loading the target
coin onto the quartz target holder and closing the quartz apparatus. The rest of
the process is carried out fully automatically, controlled by an industrial PLC
based control system and monitored on a notebook computer.

°C

heater thermocouple

air in

air out TeO2 target melted on a platinum disk

Al2O3 trap for TeO2 vapours

solution for trapping radioiodine

FIG. 6. Schematic view of quartz apparatus for thermochromatographic separation of

radioiodine from the irradiated TeO2 targets.

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 ČOMOR et al.

heater

air in

target holder

quartz bulb with the trapping solution

FIG. 7. Quartz apparatus for thermochromatographic separation of radioiodine.

First, the quartz bulb is loaded with a pre-adsorption solution from

reservoir B1, then the target is heated up to 550°C in order to clean the target
of any volatile contamination it might have picked up during its irradiation and
transport (the quartz apparatus is purged by a stream of air during the whole
thermochromatographic separation process). Then, the pre-adsorption
solution which trapped the impurities is transferred to the waste container, the
quartz bulb is washed with the content of reservoir B2 and then the absorption
solution from reservoir B3 is transferred to the quartz bulb. The target is then
heated up until it melts and the radioiodine is released and becomes trapped in
the absorption solution whose volume can be less than 1 mL. After the short
period during which the target’s temperature is above the melting point
(2 min), the system is purged further with the carrier gas (air) for a few more
minutes at a lower temperature that is safe for the target material, in order to

V6
F1 F2 Quartz furnace F5
V11 V10 V7
Air in Target

V9
B6 Waste V12
C1
T F3 F4
B1 B2 B3 B4 B5 V8
R1
N1 N2 P1
D1
V1 V2 V3 V4 V5
B7 Product Air out

FIG. 8. Technological scheme of TERIMO: (F1–F4) sterile filters; (F5) iodine trap; (B1–
B6) process reservoirs; (B7) sterile vial for the product; (N1, N2) sterile needles; (V1–V11)
solenoid operated valves; (V12) air flow regulator; (P1) vacuum pump; (R1) trapping
vessel; (C1) programmable temperature controller; and (D1) radioactivity detector.

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 SESSION 11

trap the iodine that is still adsorbed at different surfaces inside the furnace and
on the alumina filter that retains TeO2 traces. The trapping solution containing
the radioiodine in the form of iodide is then transferred to the sterile product vial
passing through a 0.2 μm sterile filter. Finally, the quartz bulb is washed twice
with the content of the reservoirs B4 and B5 in order to prepare it for the next run.
The operation of all of the elements of the module are logged into a file
that can be visualized on the screen of the computer (see Fig. 9) and it can be
printed out as well and attached to the production protocol.
Instead of an alkaline iodine trapping solution, one may load the quartz
bulb with a solution containing certain precursors. This way, one may directly
produce certain radiopharmaceuticals during the release of the radioiodine
from the target. This is possible owing to the fact that the radioiodine carried by
the air from the hot target to the quartz bulb is in the form of a highly reactive
iodine radical (I*) with clear electrophylic properties. The possibility of
producing o-iodo-hippuric acid and m-iodo-benzylguanidine directly during
the thermochromatographic process has already been reported [16].

FIG. 9. Synoptic view of the graphical user interface running on a notebook computer
while the PLC controls the operation of the module.

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 ČOMOR et al.

The technology described (irradiation of enriched TeO2 targets using

COSTIS installed on common medical cyclotrons and performing the thermo-
chromatographic separation of produced radioiodine using the automated
module TERIMO) provides batches of over 10 GBq or 1.5 GBq for 123I and
124
I, respectively. Iodine-124 batches of 3.7 GBq are already routinely produced
using this technology. By carefully performing the thermochromatographic
separation process, the loss of target material during one production cycle
(batch) is negligible (<0.2 mg/cycle).
The module is of compact design and consists of three main parts: (1) the
module itself that fits into a mini-cell for handling open radioactive materials;
(2) the control system that can be installed below the common hot cells; and (3)
the notebook computer providing the user interface that communicates with
the control system using the TCP/IP protocol.

4. CONCLUSION

Despite the fact that the production of radioiodines by cyclotrons has a

long history, there is still no technology available that would allow for cost
effective small and medium scale production of radioiodines by compact
medical PET cyclotrons following the GMP requirements. The technology
described in this paper, which is based on a compact target station (COSTIS)
that can be installed on virtually any cyclotron and on a GMP compliant
module for radioiodine separation from the irradiated TeO2 targets
(TERIMO) is so far the closest approach to the ideal situation. With a
relatively small investment one may produce 123I and 124I in quantities
exceeding the in-house needs of a nuclear medical centre for routine applica-
tions, not to speak of clinical research. The quality of 123I produced by this
technology is comparable to the quality of 123I produced by the well-known
124
Xe based technology.

ACKNOWLEDGEMENTS

This work was partially supported by the Ministry of Science and

Environmental Protection of Serbia through the realization of the TESLA
Project (project code 101247).

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REFERENCES

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[7] BEYER, G.J., DAM, C., ODRICH, H., PIMENTEL, G., Production of 123I at the
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124
Te(p,2n)123I reaction at a compact cyclotron, Appl. Radiat. Isot. 32 (1981) 581.
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Induced Nuclear Reactions On Natural Tellurium and Enriched 122Te: Production
of 123I via the 122Te(d,n)123I-Process, Int. J. Appl. Radiat. Isot. 34 (1983) 1425.
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Isotopenpraxis 24 (1988) 297.
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[13] CLEM, R.G., LAMBRECHT, R.M., Enriched 124Te targets for production of 123I
and 124I, Nucl. Inst. Meth. Phys. Res. A 303 (1991) 115.
[14] HOHN, A., COENEN, H.H., QAIM, S.M., “Nuclear Data Relevant to the
Production of 120gI via the 120Te(p,n)-Process at a Small-Sized Cyclotron”, Appl.
Radiat. Isot. 49 (1998) 1493.
[15] SHEH, Y., et al., Low energy cyclotron production and chemical separation of
“no carrier added” iodine-124 from a reusable, enriched tellurium-124 dioxide/
aluminum oxide solid solution target, Radiochim. Acta 88 (2000) 169.
[16] BEYER, G.-J., PIMENTEL-GONZALES, G., Physicochemical and radiochem-
ical aspects of separation of radioiodine from TeO2-targets, Radiochim. Acta 88
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[17] KUDELIN, B.K., GROMOVA, E.A., GAVRILINA, L.V., SOLIN, L.M., Purifi-
cation of recovered tellurium dioxide for re-use in iodine radioisotope produc-
tion, Appl. Radiat. Isot. 54 (2001) 383.
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Proceedings of the Ninth International Workshop on Targetry and Target Chem-
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[19] ČOMOR, J.J., STEVANOVIĆ, Ž., RAJČEVIĆ, M., KOŠUTIĆ, Đ., “Modeling of
thermal properties of a TeO2 target for radioiodine production”, Nucl. Instrum.
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[22] INTERNATIONAL ATOMIC ENERGY AGENCY, Charged Particle Cross-
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topes and Monitor Reactions, IAEA-TECDOC-1211, IAEA, Vienna (2001).
[23] TIKHOMIROV, A.V., “Enriched stable isotopes for 123I production: Pathways
and prospects”, J. Radioanal. Nucl. Chem. 257 (2003) 157.
[24] KUDELIN, B.K., SOLIN, L.M., JAKOVLEV, V.A., “Experience in iodine-123
production via p,n reaction. Synthesis and Applications of Isotopically Labeled
Compounds”, Vol. 7, Proc. 7th Intern. Symp., Dresden, Germany 18-22 June 2000,
PLEISS, U., VOGES, R. (Eds), J.Wiley & Sons Ltd., p. 37.

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 NEW DEVELOPMENT IN RADIOIODINATED
RADIOPHARMACEUTICALS FOR SPECT AND
RADIONUCLIDE THERAPY: [123I]/[131I] LABELLED
L- AND D-PHENYLALANINE ANALOGUES
M. BAUWENS, J.J.R. MERTENS, T. LAHOUTTE, K. KERSEMANS,
C. GALLEZ, A. BOSSUYT
ICMID/BEFY, Vrije Universiteit Brussel,
Brussels, Belgium
Email: jjmerten@vub.ac.be

Abstract

[123/125I]-2-Iodo-L-phenylalanine and [123/125I]-2-Iodo-D-phenylalanine, with the

radioiodine atom in the 2 position of the aromatic ring, were evaluated as potential
specific tumour tracers for SPECT. The tracers were obtained with an overall radio-
chemical yield of at least 98% and a purity of >99% in one pot kit conditions. The
tracers were evaluated in vitro using R1M rhabdomyosarcoma cells as the cancer cell
model. The initial uptake of [125I]-2-Iodo-D-phenylalanine is slower than that of the L-
analogue but from 20 min the uptake as a function of time is the same. The uptake of the
D- analogue is proven to occur by the same LAT transport system used by L amino acid
and to be satiable, resulting in a Km value of 32µM comparable with the L- analogue.
Although both compounds showed an apparent accumulation over 24 h, no incorpora-
tion was noticed. In vivo, the biodistribution of [123/125I]-2-Iodo-L- and D-phenylalanine
in R1M rhabdomyosarcoma bearing rats was measured by dynamic planar imaging.
[99mTc]-PP planar imaging was performed to correct for blood pool activity. [123I]-2-
Iodo-D-phenylalanine showed a DUR value of 2.9 at 30 min p.i., while for [123I]-2-Iodo-
L-phenylalanine, the DUR value reached 3.2. Blood clearance of the tracers occurred
through the kidneys to the bladder, resulting in low tracer activity in the abdomen. The
activity in the brain was also low. Specific tumour uptake was confirmed using [99mTc]-
PP and by displacement with L-phenylalanine. In rats, both tracers showed a high in vivo
stability up to 2 h p.i., almost no free radioiodide and no other labelled metabolites were
observed. The characteristics of the biodistribution make [123I]-2-Iodo-L- and D-pheny-
lalanine promising tumour specific tracers for SPECT. The long term retention of the
activity in the tumours coupled to a profound clearance from blood and tissue make
these tracers candidates for radionuclide therapy with 131I.

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 BAUWENS et al.

1. INTRODUCTION

Nuclear medicine became molecular imaging. The use of ‘smart’ radio-

pharmaceuticals for the imaging of the biochemical changes that come with any
disease has allowed a worldwide recognition of the usefulness of scintigraphic
imaging in medicine and biology. The latter requires molecules of biological
interest, especially for functional imaging of metabolism, tumours and neuro-
transmission functions using single photon emission computed tomography
(SPECT). An effective labelling of compounds of biological interest is required
for the preparation of these radiopharmaceuticals and therefore the radio-
labelling methodology is considered to be one of the pillars on which nuclear
medicine rests. Often, labelling with 123I is preferred as the introduction of
99m
Tc multidentate complexing groups (Tc I, III or V) into small molecules can
dramatically change the biological activity.
As 123I is currently still rather expensive, ‘almost quantitative yields’ are
required. Daily routine preparations in clinic of 123I radiopharmaceuticals
require one pot kit synthesis, as simple as the routine 99mTc kits (mix and heat),
or an automated system with a fast mini-column purification system. For the
first, the Cu+ assisted nucleophilic exchange in reducing aqueous conditions is
well suited, while for the second, the eletrophilic substitution with the highly
lipophilic tri-Butyl-Sn as the leaving group offers a good alternative.
Radiopharmaceutical research is matching a new development by
designing targeted radiotracers that allow non-invasive imaging of specific gene
expression, gene products or protein function in vivo. This approach makes it
possible to monitor changes non-invasively at the molecular level, during the
natural history of disease and/or after therapy.
There is an increasing interest in tumour specific SPECT tracers for
follow-up of tumours after surgery and irradiation and/or chemotherapy, but
not in FDG-mimics as [18F]-FDG is not specific enough as it shows high uptake
in inflamed tissue. Moreover, when the compound is highly retained in the
tumour, the 131I analogues are an alternative for coupled radionuclide therapy.
MIBG, developed in the 1980s and still in use, is such an example as when
labelled with 123I it is used as a receptor based (adrenergic receptors)
myocardial and tumour tracer, and when labelled with 131I it is used for radio-
nuclide therapy of neuroblastoma, pheochromocytoma and other tumours.
Within the broad spectra of diagnostics and coupled radionuclide therapy,
radiolabelled amino acid analogues can fulfil an important role. Amino acid
transporters are proteins that mediate amino acid influx and efflux across cell
membranes [1]. Their functions are to supply the cells with amino acids, to
transport amino acids across barriers (intestine, kidney, blood–brain barrier,
placenta, etc.) and to terminate synaptic transmission through reuptake of

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excitatory amino acids. Initially, the amino acid transporters were classified on
the basis of their functional and pharmacological characteristics. They differ in
substrate selectivity and Na+ dependency of the transport, and also in tissue
expression and subcellular localization according to their specific role.
Furthermore, the physiological role type can also vary according to the cell or
tissue where it is expressed.
The L transporter is a major nutrient transport system responsible for
Na+ independent transport of large neutral amino acids including synthetic
amino acids by an obligatory exchange mechanism coupled to an anti-port
system [6]. The heterodimeric LAT transport system contains 2 subunits, a
heavy chain 4F2/CD98 and a light chain named LAT1 or LAT2.
LAT1 expression was scarcely detected in non-tumour areas [7–9] but
highly expressed (up-regulated) in proliferating tissues, in particular malignant
tumours, as it plays a critical role in cell growth and proliferation [9]. A
remarkable characteristic of system L and the hLAT-1 amino acid uptake is its
broad substrate selectivity, which enables the transport system to accept amino
acid related compounds, such as D-amino acids and cancer drugs like
Melphalan [10]. LAT2 on the other hand has a high level of expression in the
small intestine, kidney, placenta and brain and in the epithelia and blood–tissue
barriers [11]. It transports all of the isomers of neutral alpha amino acids by
facilitated diffusion; however it does not transport D-amino acids. Previously, it
was supposed that the amino acid tracers had to be incorporated into the
tumour cell proteins [21]. More recently, Langen et al. [22] and Lahoutte et al.
[24] showed that it is the increased L mediated transport of amino acid
analogues into tumour cells and not necessarily the incorporation that is
needed for efficient tumour imaging and follow-up. Langen et al. [25],
moreover, have demonstrated that the expression of L transporters and related
reversible uptake depend on the proliferation rate of human glioma cells. The
authors’ group recently developed 2-I-L-tyrosine [31], which shows a very high
tumour selectivity, better than [18F]-FDG which is taken up considerably in the
brain and inflammatory tissue, and is not retained in the kidney as shown by
Tamemasa, Goto et al. and Takeda et al. [32–34]. They showed uptake of [14C]
labelled D-Leucine, D-Alanine and D-Tryptophane in mice tumour and human
tumour derived tumour cells induced in nude mice, without being able to
explain this phenomenon at that time. They suggested that D-Leucine was
transported by the same transporter as L-Leucine, but that the isomers were
supposed to bind to different parts of the transporter. This transporter was later
defined as the L transporter. In 1985, Meyer et al. [1] showed that both the L
and D forms of the [11C]-methionine could accumulate in human brain
tumours. Since then all the development of, and studies with, radioactively

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labelled amino acids for tumour diagnosis with PET and SPECT were focused
on the L-enantiomeric form.
The knowledge of the molecular biology of the LAT transporter system
drove the authors to explore the D-analogues of tyrosine and phenylalanine,
having also noticed that [14C]-D-phenylalanine and D-phenylalanine were
taken up in R1M cells by the LAT1 transport system [35]. This paper describes
the evaluation of two new tracers L- and D-[123I]-2-I-phenylalanine in R1M
tumour bearing rats by scintigraphic imaging.

2. EXPERIMENTAL

2.1. Materials and methods

All the conventional products mentioned were at least analytical or

clinical grade and obtained from Sigma-Aldrich. The solvents were of HPLC
quality (VWR, Belgium).

2.1.1. Synthesis and radiosynthesis

The synthesis of 2-I-D-phenylalanine/2-I-L-phenylalanine was achieved

as already described [27].
Radioiodination with 123I-, 125I- or 131I- (10–30 µL) on 1.0 mg 2-iodo-L-
phenylalanine or 2-iodo-D-phenylalanine was performed by Cu+ assisted
isotopic exchange under acidic and reducing conditions (0.2 mg CuSO4, 2.5 mg
citric acid, 0.5 mg SnSO4, 1.3 mg gentisic acid in 565 µL; 60 min at 100°C) [17].
The reaction mixture was drawn up in a syringe containing the appropriate
amount of ‘make-up solution’ (tri-sodium citrate dihydrate, 71mM) to render
the solution isotonic and to adjust the pH to at least 4. The reaction mixture
was filtered through a 0.22 µm Ag filter (Millipore, Belgium) to remove the
free 123I-/125I- and through a sterile 0.22 µm filter (Millipore, Belgium) into a
sterile vacuum vial. Quality control was achieved by HPLC and Sep-pak C18
(Waters, Belgium). Radiolabelling of both tracers with 123I- or 125I- resulted in a
radiochemical purity of >99% and a specific activity of 65 GBq/mmol [123I]
labelling and 11 GBq/mmol [125I] labelling.

2.1.2. [99mTc]-Pyrophosphate

Technescan PYP (Mallinckrodt Medical) ([99mTc]-PP) was prepared

according to producer guidelines. [99mTc] was obtained from Mallinckrodt.

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 SESSION 11

2.1.3. Quality control

Quality control was achieved by HPLC, using a C8-column (LichrosPher

100RP8 (5 µm), Lichrocart 125-4) and 10/90 MeOH/H2O containing 1mM
NH4Ac as the mobile phase at 1.0 mL/min while monitoring UV absorption
(Hitachi UV detector, 254 nm) and radioactivity (NaI(Tl) detector (Harshaw
chemie)).
Chiral chromatography was performed on a chiral column (5 µm
Chirobiotic T (Astec) column (150 mm × 4.6 mm)) using 80/20 v/v% methanol/
H2O containing 20mM NH4Ac at a flow of 1 mL/min. In these conditions a
complete separation of the chiral isomers was obtained. The capacity values
(K’) of L-2-I-Phe and D-2-I-Phe were 2.7 min and 3.8 min, respectively. No
transformation of the chirality was observed.

2.2. Reverse transcriptase PCR

Reverse transcriptase PCR was performed using BioTaq RED (Bioline)

according to manufacturer’s guidelines. The amplification of the cDNA strands
was performed using the following conditions: (1) pre-cycle: 94°C for 3 min,
60°C for 1 min, 72°C for 2 min; (2) 26 cycles of 94°C for 1 min, 60°C for 1 min,
72°C for 2 min; (3) hold: 72°C for 10 min. Gel electrophoresis was performed
on a 1.5% agarose/EtBr gel at 250 V and 250 mA for 15 min. The entire PCR
product was loaded onto the gel; Hyperladder IV (Bioline) was used as a
marker. Actin was used as a positive control of the method.

2.3. In vitro experiments

2.3.1. Cell cultures

R1M rhabdomyosarcoma cells (VUB) were cultivated as described [27].

2.3.2. Experimental procedure

All in vitro experiments were carried out in 6-well plates (VWR), using at
least three wells for each data point. Cells were counted by means of a Bürker
counting chamber. Influx and efflux were studied both in a Na+ containing
buffer (HEPES+: pH7.4; 100mM NaCl (VWR), 2mM KCl (Sigma), 1mM
MgCl2 (VWR), 1mM CaCl2 (VWR), 10mM Hepes (Sigma), 5mM Tris (VWR),
1 g/L glucose (VWR) and 1 g/L bovine serum albumin (Sigma)), a Na+ free
buffer (HEPES-: pH7.4; 100mM choline-Cl (Sigma), 2mM KCl, 1mM MgCl2,
1mM CaCl2, 10mM Hepes, 5mM Tris, 1 g/L glucose and 1 g/L bovine serum

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albumin) and MEM buffer (pH7.2, containing essential and non-essential

amino acids of which 1.2mM of amino acids known to be transported by the L
transport system). The process was terminated by physical withdrawal of the
buffer and washing three times with ice cold phosphate buffered saline (PBS).
Subsequently, the cells were detached from the well with 2 mL of 0.1M NaOH
(VWR). The radioactivity of the samples was counted using a gamma counting
system (Cobra-inspector 5003, Canberra Packard, Meriden, CT, United States
of America).
Time and concentration dependency: For the kinetic studies, the cells were
incubated for periods ranging from 1 min to 24 h in 1 mL of MEM under
constant partial CO2 pressure (5%) containing 37 kBq [125I]-2-I-D-Phe or [125I]-
2-I-L-Phe. Throughout the 24 h period the viability of the cells was checked
using the trypan blue method and also the 15 min uptake of ³H-L-Phe was
measured to indicate whether the number of transporters remained constant
during the 24 h cell growth.
For concentration dependency, uptake was measured at 1 min uptake
with concentrations varying from 0.01 to 0.2mM. The data were fitted to the
Michaelis-Menten relation and the apparent Km, Vmax and Ki values were
calculated from Eady-Hofstee and Hanes-Woolf and Lineweaver-Burk (LWB)
plots. ‘Apparent Km’ is calculated from Vo conditions, i.e. uptake after 1 min
where for the larger part only influx has to be considered.
Using [³H]-L-Phe/L-Phe (Amersham Biosciences/Sigma) as a reference
molecule, the type of competition and Ki value of 2-I-D-Phe and 2-I-L-Phe
were determined using a double reciprocal LWB plot. The concentration of the
inhibitors was 0.1 mM.
Inhibition of [125I]-2-I-D-Phe influx: The cells were incubated with 37 kBq
125
[ I]-2-I-D-Phe for 1 min in HEPES+ and HEPES- buffer supplemented with
8mM L-Phe, 8mM BCH (2-amino-2-norbornane-carboxylic acid, a LAT
transport system specific inhibitor) or 8mM MeAIB (methyl amino isobutyric
acid, an A/ASC transport system specific inhibitor).
Trans-stimulation of [³H]-L-Phe and [125I]-2-I-D-Phe efflux: The cells
were incubated with 37 kBq [³H]-L-Phe or 37 kBq [125I]-2-I-D-Phe for 15 min in
HEPES- buffer. The incubation medium was removed and the cells were
washed three times with ice cold PBS. Subsequently, HEPES- buffer containing
0.1mM L-Phe or 2-I-D-Phe was added. The efflux medium was removed after
1 min, after which the cells were washed three times with ice cold PBS,
detached with 0.1M NaOH, suspended and counted.
Incorporation into cell proteins: The cells were incubated with the
radioactive amino acid (37 kBq [125I]-2-I-D-Phe) for a period ranging from 15
min up to 24 h in MEM under constant CO2 partial pressure (5%) using the
GENBOX system. After removal of the radioactive solution, precipitation of

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 SESSION 11

proteins was performed by adding 2 mL of 20% trichloroacetic acid, intense

mixing (vortex) followed by cooling for 30 min at 0°C. After two sessions of
repeated centrifugation and washing, 2 mL of 0.1M NaOH was added and the
radioactivity counted.

2.4. In vivo experiments

2.4.1. Laboratory animals

Water and food was ad libidum during the experimental period. For the
tumour model, male Wag/Rij rats (n = 6) (Bioservices, Netherlands) were
injected subcutaneously in the right flank (armpit region) with 15 × 106 R1M
rhabdomyosarcoma cells.
Imaging experiments with [123I]-2-Iodo-D-phenylalanine, [123I]-2-Iodo-L-
phenylalanine and [99mTc]-PP were performed 6 h p.i. of the R1M cells.
During all imaging experiments, the animals were anaesthetized intra-
peritoneal with 350 µL (21 mg) of a solution containing 60 mg pentobarbital
per mL (Nembutal, 60 mg/mL, Ceva Santé Animale, Belgium). For the biodis-
tribution experiments by dissection, the animals were killed without sedation
by cervical dislocation and the organs of interest were dissected.
All tracers were injected intravenously into the penis vein. The study
protocol was approved by the ethical committee for animal studies at the
authors’ institution. Guidelines of the National Institute of Health principles of
laboratory animal care (NIH publication 86-23, revised 1985) were followed.

2.4.2. Dynamic planar imaging (DPI).

Imaging was performed using a gamma camera (Philips) in planar mode

equipped with a high resolution parallel hole collimator. All images were
acquired into 128 × 128 matrices (3.2 zoom factor, pixel size 1.5 mm) and with a
photopeak window set at 15% around 159 keV. The injected activity was also
calculated as the amount of radioactivity in the syringe before and after
injection (Capintec CRC-15R, Ramsey, NJ, USA). To allow semi-quantifi-
cation of the results of the ROI analysis, the activity in the total body (counts)
20 min p.i. was regarded as the injected amount of radioactivity. ROIs were
drawn around the tumour, the contra-lateral background area, both kidneys,
heart, brain, bladder, thyroid and total body. The ROI of the heart was used as
a measure of blood pool activity. Owing to partial volume effects, the data can
be overestimated [18]. The tracer uptake was expressed as DUR (differential
uptake ratio; average counts per pixel of the region of interest divided by the
average counts per pixel inside the total body).

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Imaging was performed for crossed (two by two) experiments with a 2 d

interval using four R1M bearing Wag/Rij rats. DPI was started immediately
after injection of 18.5 MBq [123I]-2-Iodo-D-phenylalanine or [123I]-2-Iodo-L-
Phe and continued up to 90 min for all rats. To test the tumour retention of the
compounds, a static image of 10 min was acquired at 0.5, 24 and 48 h p.i.
Tumour retention was calculated relative to the time point 0.5 h p.i. as (counts/
pixel24h or 48h / counts/pixel0.5h ) (decay corrected).
In a separate experiment 2 rats were injected with 200 µL of a 20mM L-
Phe solution (2.2 mg/kg) 60 min after the administration of the D-isomer.
[99mTc]-Pyrophosphate was used to measure the relative blood pool
distribution in order to correct the uptake of radioactivity in the tumour as well
as the rest of the animal for blood pool activity. In this experiment, all rats were
injected with 37 MBq [99mTc]-PP. Planar images of 10 min were acquired 15 min
p.i. The ratios tumour to heart and organ to heart were calculated to correct for
blood pool activity in the tumour or organ.

2.4.3. Detection of metabolites

Blood was collected in an EDTA coated vial at different time points;

1 mL of blood was centrifuged at 3000g for 1 min for quantitative plasma
separation. To 75 µL of plasma successively 125 µL of 9‰ NaCl solution and
200 µL of 20% trichloroacetic acid were added. After 5 min of centrifugation at
3000g, the supernatant was collected and filtered through a 0.22 µm filter. In a
final step, 125 µL of 1M NaAc and 325 µL 20/80 MeOH/H2O containing 1mM
NH4Ac was added. The samples were analysed on RP-HPLC (C8-column
(LichrosPher 100RP8 (5 µm), Lichrocart 125-4, λ = 254 nm) (Alltech, Belgium)
with 20/80 MeOH/H2O containing 1mM NH4Ac as mobile phase at 1.0 mL/min
while monitoring UV absorption (Hitachi UV detector, 254 nm) and radio-
activity (NaI(Tl) detector (Harshaw Chemie)).

3. RESULTS

3.1. Synthesis and radiolabelling of 2-I-D-phenylalanine/2-I-L-phenylalanine

2-I-D-Phe and 2-I-L-Phe purity was at least 99%. Radiolabelling of both

tracers with 123I- or 125I- resulted in a radiochemical purity of >99% and a
specific activity of 65 GBq/mmol ([123I] labelling) and 11 GBq/mmol ([125I]
labelling). Chiral chromatography showed that no chiral modification occurred
during the labelling. The sterile and isotonic [123I]-2-I-D-Phe solution was
injected in the rats within 2–4 h after the preparation. The [125I] labelled

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 SESSION 11

compound was stored at 4°C. HPLC analysis during the long duration of the
experiments revealed the quality of the preparation to be stable for at least a
month. After that period, a small amount of free radioiodide was observed
(2–3%). This was quantitatively removed by filtering the solution through a
sterile 0.22 µm Ag membrane filter.

3.2. In vitro results

The in vitro evaluation of 2-I-L-Phe has been described in detail in Ref. [27].

3.2.1. Time and concentration depend on kinetics and affinity

The initial uptake of [125]-2-I-D-phenylalanine in R1M cells in MEM

buffer is slower than that of [125I]-2-I-L-Phe (Fig. 1). The uptake of both
compounds at longer times, up to 24 h, shows a slight accumulation which is not
due either to incorporation (the iodinated phenylalanine analogues are not
incorporated into the cell proteins) or to the increase in cell numbers (the
results are expressed per million cells or amount of transporters (3H-L-Phe
uptake check)).
The Ki calculated from the LWB plot representing the inhibition of the
uptake of [³H]-L-PHE/L-PHE in R1M cells in HEPES- medium (Fig. 2)
resulted in a value of 50 ± 10µM. In the same conditions, 2-I-L-Phe showed a Ki
value of 35 ± 10µM.

FIG. 1. [125]-2-I-D-Phe and [125]-2-I-L-Phe uptake time kinetics in R1M cells in MEM
buffer: mean ± SD (n = 9).

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 BAUWENS et al.

3,50E+09

L-Phe
3,00E+09
0.1 mM 2-I-L-Phe as inhibitor
1/mass uptake L-Phe (1/mol)

2,50E+09
0.1 mM 2-I-D-Phe as inhibitor

2,00E+09

1,50E+09

1,00E+09

5,00E+08

0,00E+00

-1,00E+04 1,00E+04 3,00E+04 5,00E+04 7,00E+04 9,00E+04 1,10E+05

1/[L-Phe] (1/M)

FIG. 2. Inhibition of [³H]-L-Phe/L-Phe uptake by D-2-I-Phe, D-Phe and 2-I-L-Phe:

LWB plot data: mean ± SD (n = 3).

3.2.2. Transporter type characterization

The PCR coupled gel electrophoresis of the R1M cell line is depicted in
Fig. 3 and clearly shows the presence of potentially functional LAT1 and LAT2
transport systems proven by the presence of the bands for hLAT1, hLAT2 and
light chain 4F2hc. The actin band is used for positive control.

FIG. 3. Gel electrophoresis of LAT1 light chain (LAT1), LAT2 light chain (LAT2),
CD98 heavy chain (CD98hc) and actine (Actine).

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 SESSION 11

The uptake of [125I]-2-I-D-Phe in HEPES+ (Na+ presence) and HEPES-

(Na+ absence) was not significantly different and the uptake was almost quanti-
tatively reduced (2% of the original value) by the presence of 8mM BCH, a
specific inhibitor of the L transport system, and also by 8mM of L-phenyla-
lanine. Moreover, 8mM MeAIB (a specific inhibitor of the Na+ dependent
A/ASC transport system) had no influence on the uptake. Figure 3, repre-
senting a double reciprocal LWB plot, reveals that the inhibition of [³H]-L-Phe
by D-2-I-Phe is competitive and also by 2-I-L-Phe and D-phenylalanine (which
were earlier proven to be LAT transported amino acids). The 0.1mM 2-I-D-
Phe caused a net efflux of 26% of the initial uptake of [³H]-L-Phe within 1 min
while the efflux of [125I]-2-I-D-Phe can be stimulated by BCH and L-Phe.

3.3. In vivo results

3.3.1. In vivo stability

HPLC analysis of the blood plasma showed that within the first 2 h p.i. of
123
[ I]-2-I-D-Phe, neither metabolites nor free radioiodide could be significantly
detected. For [123I]-2-I-L-Phe in the same conditions, about 4% free
radioiodide and no metabolites were found.

3.3.2. DPI

3.3.2.1. Tumour uptake and biodistribution

The biodistribution of [123I]-2-Iodo-D-Phe and [123I]-2-Iodo-L-Phe was

compared with that of the blood flow tracer [99mTc]-PP. The tumour/heart ratio
values for both radioiodinated phenylalanine analogues showed a value of
about 2.0 at 15 min whereas it only reached a value of 0.2 for [99mTc]-PP. Figure
4 shows a composite image of the biodistribution of [123I]-2-I-D-Phe and [123I]-
2-I-L-Phe in a R1M tumour rat at 20–30 min p.i. Table 1 depicts the corre-
sponding average DUR values of the tumours and different organs. The uptake
in the pancreas zone is not calculated as with DPI the pancreas could not be
accurately distinguished from the liver or lower stomach region.
The uptake in the tumours in periods up to 40 min for both [123I]-2-Iodo-
D-Phe and [123I]-2-Iodo-L-Phe is shown in Fig. 5. At the maximum uptake,
DUR values of 3.01 ± 0.40 and 3.40 ± 0.40 are obtained for [123I]-2-Iodo-L-Phe
and [123I]-2-Iodo-D-Phe, respectively. The apparent difference between the
time activity curves is not statistically significant (p = 0.20, Wilcoxon Signed
Rank Test on average values of 30 min).

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 BAUWENS et al.

FIG. 4. Composite image (20–30 min) of a R1M rhabdomyosarcoma tumour bearing rat
p.i. of 18.5 MBq [123I]-2-I-D-Phe (left) and [123I]-2-I-L-Phe (right). Image: gray scale
gamma 1.0, the scale is set to the maximum in the tumour.

Figure 6 shows that the injection of 200 µL of a 20mM L-Phe solution (2.2
mg L-Phe per kg at 60 min) displaces about 25% of [123I]-2-I-D-phenylalanine
activity from the tumour. A re-accumulation occurs after displacement as
shown by the positive slope up from 65 min.

TABLE 1. AVERAGE DUR VALUES AT 20–30 min p.i. OF [123I]-2-Iodo-D-

Phe AND [123I]-2-Iodo-L-Phe
(data = mean ± SD (n = 4))

[123I]-2-Iodo-D- [123I]-2-Iodo-L-
DUR value
phenylalanine phenylalanine

Tumour 2.9 ± 0.4 3.2 ± 0.4

Left kidney 1.9 ± 0.1 1.5 ± 0.1
Heart 1.5 ± 0.1 1.6 ± 0.1
Muscle 0.6 ± 0.1 0.7 ± 0.1
Bladder 3.7 ± 0.3 2.1 ± 0.3
Thryoid Not detectable Not detectable
Brain 1.0 ± 0.1 1.2 ± 0.1

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 SESSION 11

4,0

3,5
[123I]-2-I-L-Phe
3,0

[123I]-2-I-D-
2,5
Phe
DUR

2,0

1,5

1,0

0,5

0,0
0 5 10 15 20 25 30 35 40 45 50

time (min)

FIG. 5. DUR tumour values of [123I]-2-I-D-Phe and [123I]-2-I-L-Phe as a function of

time. Data = mean ± SD (n = 4).

3.3.2.2. Clearance

Figure 7 shows that the clearance of [123I]-2-Iodo-D-phenylalanine from

the blood through the kidneys to the bladder is faster than that of [123I]-2-Iodo-
L-phenylalanine. In the early time-frames, the increase of activity in the

3,5

2,5

2
DUR

1,5

0,5

0
0 10 20 30 40 50 60 70 80 90

tim e (m in)

FIG. 6. DUR values of [123I]-2-I-D-Phe as a function of time. At 60 min p.i., a high

concentration of L-Phe was injected. Data = mean (n = 2).

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 BAUWENS et al.

8
D - Bladder D - Kidney D - Heart

7 L - Bladder L - Kidney L - Heart

5
DUR

0
0 5 10 15 20 25 30 35 40 45

time (min)

FIG. 7. Uptake (DUR) of [123I]-2-I-D-Phe and [123I]-2-I-L-Phe as a function of time in

kidney, heart and bladder. Data = mean ± SD (n = 4).

bladder as a function of time is much higher for the D-isomer than for the L-
isomer (the slope of the curve at 5–10 min is 0.27 DUR values/min for the D-
isomer and 0.096 DUR values/min for the L-isomer), resulting in a twofold
amount of activity in the bladder for the D analogue compared with 2-I-L-Phe
at the end of imaging.

3.3.3. Long term biodistribution and retention

Interestingly, for both tracers there is a retention in the tumour over a

long time period (48 h). Table 2 shows that a high amount of radioactivity
remains in the tumour for a longer time while the DUR values show an
apparent increase. Figure 8 shows the biodistribution at 48 h with a clear
contrast of the tumour vis a vis the background activity.

4. DISCUSSION

Reverse transcriptase PCR gel electrophoresis (Fig. 3) clearly shows the

presence of the mRNA of hCD98hc, hLAT1 and hLAT2 in the R1M cell line,
allowing a functional LAT1 and LAT2 transport system. The band width and

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TABLE 2. DUR VALUES AND PER CENT RETENTION OF

[123I]-2-I-D-Phe AND [123I]-2-I-L-Phe AT 30 min AND 24 AND 48 h p.i.
(data = mean ± SD (n = 4))

Tumour (30 min) Tumour (24 h) Tumour (48 h)

DUR
DUR % Retention DUR % Retention DUR % Retention

[123I]-2-I-D-Phe 2.9 ± 0.4 3.5 ± 0.3 92 ± 10 3.7 ± 0.3 77 ± 3

123
[ I]-2-I-L-Phe 3.2 ± 0.4 3.5 ± 0.5 91 ± 10 3.7 ± 0.3 73 ± 7

brightness indicate a greater presence of the LAT1 transport system compared

with the LAT2 transport system.
The same intercept of the different lines in the LWB plot (Fig. 2) obtained
for L-Phe, 2-I-D-Phe and 2-I-L-Phe reveals that all these compounds enter the
R1M cells in vitro by the same transport system(s).
No Na+ dependence was found, while the influx of [125I]-D-2-I-Phe was
completely inhibited by 8mM BCH. The influx of 0.1mM D-2-I-Phe caused an
efflux of 26% of the initial [³H]-L-Phe uptake while [125I]-D-2-I-Phe efflux,
although a smaller amount, was stimulated by 0.1mM L-Phe and BCH. These
characteristics are compliant with the exclusive utilization of the LAT system
as an obligatory 1/1 antiport system.

FIG. 8. Composite images of a R1M rhabdomyosarcoma tumour bearing rat p.i. of 18.5
MBq [123I]-2-I-D-Phe (left) and [123I]-2-I-L-Phe (right) at 48 h p.i. Image: gray scale
gamma 1.0, the scale is set to the maximum in the tumour.

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 BAUWENS et al.

The difference in initial uptake kinetics (Fig. 2) between 2-I-D-Phe and

2-I-L-Phe might be due to the fact that the D form only shows a high affinity
for the LAT1 system and not for LAT2. This is supported by the finding that
in cell types expressing only LAT1, no difference in uptake is found between
2-I-D-Phe and 2-I-L-Phe (V. Kersemans, unpublished).
The increase in non-incorporated uptake and apparent retention during
stimulated efflux of the radioiodinated phenylalanine analogues can be due to
a significant difference in affinity of the intracellular part of the transport
protein for the iodinated compounds compared with the natural amino acids
present. It is known that the LAT systems show a relatively symmetrical broad
selectivity but strongly asymmetrical substrate affinity, such that the intra-
cellular amino acid pool controls their exchange activity [22, 23]. The increase
in non-incorporated uptake in vitro as well as a less stimulated efflux than
L-Phe can be due to a significant difference in affinity between the radio-
iodinated D analogue and the natural L amino acid present in the tumour cells.
The in vivo short time kinetics (0–40 min) of tumour uptake show no
significant difference for [123I]-2-I-L-Phe and [123I]-2-I-D-Phe (about 10%
higher for the L-enantiomer). The tumour uptake was considerably higher than
blood flow, measured with [99mTc]-PP. The high tumour specific uptake could
be displaced for 25% by injection of a high dose of natural L-Phe, pointing at
the involvement of the LAT transport system. There is a re-accumulation of
[123I]-2-I-D-Phe in the tumour after displacement (Fig. 6) indicated by the
positive slope up from 65 min.
Over longer time periods (24 and 48 h), a high retention of radioactivity
in the tumour is noticed. This retention can be compared with the ‘apparent
accumulation’ discussed in the in vitro part as the uptake in vivo occurs by the
same transporter type and mechanism.

5. CONCLUSION

The uptake of [123/125I]-2-I-D-Phenylalanine and [123I]-2-I-L-Phenyla-

lanine occurs in vitro as well as in vivo exclusively and with a high affinity via
the LAT1 system which is overexpressed in many tumour cell lines. Imaging of
R1M tumour bearing rats shows specific and fast tumour uptake and an
appropriate biodistribution, fast clearance from blood and abdomen and low
brain uptake, making [123I]-2-I-D-Phenylalanine and [123I]-2-I-L-Phenylalanine
promising tracers for specific tumour imaging within and outside the brain
using SPECT. The long term retention of the activity in the tumours coupled to
a profound clearance from blood and tissue make these tracers candidates for
radionuclide therapy with 131I.

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ACKNOWLEDGEMENTS

The authors thank the FWO Vlaanderen and GOA-VUB for their
financial support.

REFERENCES

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241
.
 COMPARISON OF 131I-TYR3-OCTREOTATE AND 131I-
DOTA-TYR3-OCTREOTATE: THE EFFECT OF DOTA ON
PHARMACOKINETICS AND STABILITY

E.B. DE ARAÚJO, E. MURAMOTO, L.T. NAGAMATI,

J.S. CALDEIRA FILHO, R.M. COUTO, C.P.G. SILVA
Institute of Energetic and Nuclear Research, IPEN-CNEN,
São Paulo, Brazil
Email: ebaraujo@ipen.br

Abstract

The authors compared the biodistribution, and in vivo and in vitro stabilities of
131
I-Tyr3-octreotate and 131I-DOTA-Tyr3-octreotate. The peptides were radioiodinated
by the chloramine T method and high radiochemical yields were obtained (greater than
97%). Both labelled compounds showed high stability when incubated in human plasma
at 37°C. The 131I-Tyr3-octreotate showed significant hepatic uptake and biliary excretion.
The biodistribution of 131I-DOTA-Tyr3-octreotate, however, can be compared with the
distribution of radiometal labelled octreotide analogues.

1. INTRODUCTION

The introduction of radiolabelled somatostatin analogues for peptide

recetor imaging and peptide receptor radiotherapy of neuroendocrine cancer
has provided a primary focus of interest in nuclear medicine. The 111In-DTPA-
D-Phe1-octreotide (OctreoScan) became the first radiopeptide to be routinely
used for scintigraphy of somatostatin receptor positive tumours [1].
The introduction of the metal chelator DOTA (1,4,7,10-tetraazacyclodo-
decane-1,4,7,10-tetraacetic acid) initiated a marked improvement in the
stability of the radioconjugates, allowing the incorporation of a variety of
radionuclides, such as 90Y and 177Lu, for receptor mediated therapy and 68Ga
and 64Cu for positron emission tomography [2].
90
Y-DOTA-octreotide was tested in several clinical studies for use in
treatment [3, 4]. One of the most recent developments is the introduction of
177
Lu-DOTA-Tyr3-octreotate, in which the carboxy terminal threoninol has
been replaced with the natural amino acid threonine, yielding a very high
SSTR2 affinity [5].
For sst-target diagnosis and radiotherapy, radiohalogens offer a broad
spectrum of suitable isotopes for single photon emission tomography (123I),

243
 DE ARAÚJO et al.

positron emission tomography (18F) and peptide receptor therapy (131I, 125I and
211
At). Consequently, 123I labelled Tyr3-octreotide was the first compound to be
used for the imaging of somatostatine receptor positive tumours [6].
However, the experience gained with radioiodinated sst ligands showed
that the diagnostic and therapeutic usefulness of these ligands was limited by
their unfavourable biokinetics, in vivo deiodination and resulting dosimetry.
Owing to fast hepatic uptake and biliary clearance, most of the tracers showed
high abdominal background acitivity and fast blood clearance, leading to low
tumour uptake. Additionally, they exhibited low tumour retention, which was
often attributed to fast intracellular degradation of the tracers and subsequent
extracellularization [7].
In this paper, the authors prepared radioiodinated octreotates, 131I-Tyr3-
octreotate and 131I-DOTA-Tyr3-octreotate, with high radiochemical yield.
Although the DOTA chelating group was not necessary to the radioiodination
procedure, the authors evaluated the influence of the chelating group on
biodistribution, particularly on hepatic uptake, biliary excretion and renal
clearance. Tumour uptake was evaluated in nude mice bearing AR42J tumour.

2. MATERIALS AND METHODS

2.1. Reagents

DOTA-Tyr3-octreotate was provided from piChem by the IAEA and the

3
Tyr -octreotate was purchased from Anaspec (EUA). All other reagents were
purchased from Sigma-Aldrich. [131I]NaI was obtained from Nordion
(Canada).

2.2. Radiolabelling

Radiolabelling of Tyr3-octreotate and DOTA-Tyr3-octreotate with 131I-

was performed using the chloramine T method. A solution of 10 μg of peptide
in 40 μL of PBS (0.05M phosphate buffered saline, pH7.5) was transferred to
the reaction vial. After addition of 10 μL (74–111 MBq) of radioiodine
solution and 5 μL of chloramine T solution (1 mg/mL PBS), the cap was
carefully stirred and the labelling reaction was allowed to proceed for 3 min
at room temperature. To the reaction mixture, 5 μL of sodium metabisulphite
solution (2 mg/mL PBS) was introduced as a reducing agent. Different molar
peptide to radionuclide ratios were applied to the labelling of DOTA-Tyr3-
octreotate with 131I.

244
 SESSION 11

2.3. Quality control

Radiochemical purity was determined by HPLC (Waters) using an RP

C18 column (4.2 mm × 50 mm, 5 μm, Waters) with UV (230 nm) and radio-
activity (Packard Canberra) detection, flow rate of 0.5 mL/min with a linear
gradient of 40–80% (v/v) methanol in 50mM sodium acetate buffer (pH5.5) for
20 min, maintained for another 25 min. Free radioiodine was also determined
by horizontal zone electrophoresis (Amersham Pharmacia) on Whatman 1
paper, 0.05M barbital buffer, pH8.6, 300 V, 40 min.

2.4. In vitro stability

The in vitro stabilities of 131I-Tyr3-octreotate and 131I-DOTA-Tyr3-

octreotate were evaluated after incubation in human plasma at 37°C. Radio-
chemical purity was determined 1, 4 and 24 h after incubation using the electro-
phoresis procedure.

2.5. Animal studies

Biodistribution studies of 131I-Tyr3-octreotate and 131I-DOTA-Tyr3-

octreotate were performed on normal Swiss mice and nude mice bearing
AR42J rat pancreatic tumours. About 540 kBq/0.1 mL of the respective radio-
pharmaceutical were injected into the tail vein. The animals were sacrificed at
different time points post-injection and the organs of interest were dissected.
Tissue samples were weighed and the radioactivity was measured using a
gamma counter (Packard). All experiments were carried out following the
principles of laboratory animal care.

3. RESULTS AND DISCUSSION

3.1. Radioiodination

Labelled peptides were obtained with radiochemical purities exceeding

95%, as determined by the electrophoresis method.
The authors investigated different molar peptide to radionuclide ratios in
order to obtain mono-iodinated peptides to be applied in the biodistribution
studies, considering that the di-iodinated peptide no longer binds to the
somatostatin receptor, as previously reported [8].
The HPLC profile of the 131I-DOTA-Tyr3-octreotate obtained when using
a molar peptide to radionuclide ratio of 2.73 (7.4 MBq 131I/μg peptide)

245
 DE ARAÚJO et al.

produced only one radiochemical species with a retention time of 22.73 min
(Fig.1). When using a molar peptide to radionuclide ratio of 0.54 (37 MBq
131
I/μg peptide), a second radiochemical species can be observed in the
HPLC profile (Fig. 2), with a retention time of 24.9 min, probably related to
the di-iodinated form of the peptide.

FIG. 1. HPLC profile of 131I –DOTA-Tyr3-octreotate molar peptide to radionuclide ratio

of 2.73 (7.4 MBq/μg).

FIG. 2. HPLC profile of 131I-DOTA-Tyr3-octreotate molar peptide to radionuclide ratio

of 0.54 (37 MBq/μg).

246
 SESSION 11

3.2. In vitro studies

In vitro studies show clearly the high level of stability of the iodinated
ligands even after 24 h of incubation in human plasma (Table 1).

3.3. Biodistribution studies

Biological distribution studies were performed with radiolabelled

peptides obtained in a peptide to radionuclide ratio of 2.73 with radiochemical
purity exceeding 97%, which obviated the need to undertake the SepPak
purification procedure. However, as with other radioiodinated peptides and
proteins labelled on constituent tyrosine residues, it was important to study the
possibility of dehalogenation in vivo.
The biodistribution data for 131I-Tyr3-octreotate (Table 2) was similar to
that reported for 123I-Tyr3-octreotide in rats [9] and for 125I-Tyr3-octreotide and
125
I-Tyr3-octreotate in nude mice [7]. The 131I-Tyr3-octreotate (Table 2) was
extracted rapidly from the blood via hepatobiliary excretion, resulting in high
liver uptake (2.28 ± 0.78%ID/g 1 h p.i.) and increasing intestinal uptake to 1
and 4 h p.i. (12.30 ± 4.36%ID/g for the small intestine and 14.85 ± 0.83%ID/g
for the large intestine). The 131I-DOTA-Tyr3-octreotate was predominantly
excreted via the kidneys. Nevertheless, renal activity accumulation for this
compound was similar to that of 131I-Tyr3-octreotate.

TABLE 1. IN VITRO STABILITY OF 131I-Tyr 3-OCTREOTATE AND

131
I-DOTA-Tyr3-OCTREOTATE IN HUMAN PLASMA AT 37°C

Radiochemical purity (%)

Labelled peptide
Immediately 1h 4h 24 h
131
I-Tyr3-octreotate 98.42 ± 0.32 98.43 ± 0.53 96.79 ± 1.01 95.76 ± 0.10
131 3
I-DOTA-Tyr -octreotate 95.41 ± 0.51 93.80 ± 0.80 92.40 ± 0.55 91.05 ± 0.55

247
 TABLE 2. BIODISTRIBUTION OF 131I-Tyr3-OCTREOTATE AND 131I-DOTA-Tyr3-OCTREOTATE IN NORMAL

248
SWISS MICE
131 131
I-Tyr3-octreotate I-DOTA-Tyr3-octreotate
Tissue Dose/g (%) Dose/g (%)

1h 4h 24 h 1h 4h 24 h

Total blood 3.17 ± 0.32 0.81 ± 0.12 0.103 ± 0.006 2.56 ± 0.27 1.19 ± 0.13 0.020 ± 0.004
Liver 2.28 ± 0.78 0.42 ± 0.06 0.161 ± 0.034 0.70 ± 0.05 0.36 ± 0.05 0.088 ± 0.018
Spleen 0.76 ± 0.25 0.37 ± 0.11 0.114 ± 0.044 0.57 ± 0.06 0.27 ± 0.04 0.057 ± 0.018
Stomach 3.51 ± 1.67 1.94 ± 1.02 0.093 ± 0.032 3.32 ± 0.45 2.02 ± 0.59 0.19 ± 0.05
Int. (small) 12.30 ± 4.36 1.33 ± 0.96 0.045 ± 0.015 1.48 ± 0.13 0.57 ± 0.12 0.41 ± 0.19
Int. (large) 0.96 ± 0.58 14.85 ± 0.83 0.103 ± 0.032 0.44 ± 0.08 2.18 ± 0.25 1.90 ± 0.82
Kidney 12.54 ± 0.51 9.77 ± 3.46 3.752 ± 1.183 12.18 ± 0.86 9.86 ± 1.00 1.60 ± 0.22
Muscle 0.47 ± 0.24 0.13 ± 0.06 0.022 ± 0.002 0.26 ± 0.02 0.13 ± 0.03 0.03 ± 0.01
DE ARAÚJO et al.

Brain 0.08 ± 0.01 0.024 ± 0.003 0.007 ± 0.001 0.08 ± 0.03 0.04 ± 0.010 0.006 ± 0.003
Heart 0.66 ± 0.11 0.20 ± 0.08 0.049 ± 0.020 0.50 ± 0.10 0.20 ± 0.05 0.019 ± 0.003
Lung 1.59 ± 0.40 0.45 ± 0.14 0.013 ± 0.021 0.80 ± 0.42 0.49 ± 0.22 0.052 ± 0.005
Thyroid * 0.49 ± 0.07 1.04 ± 0.20 0.878 ± 0.304 0.55 ± 0.10 1.23 ± 0.17 0.29 ± 0.06
Adrenals* 0.020 ± 0.007 0.007 ± 0.004 0.002 ± 0.0002 0.012 ± 0.003 0.008 ± 0.001 0.0014 ± 0.0005
Pancreas 0.87 ± 0.17 0.23 ± 0.05 0.023 ± 0.008 1.11 ± 0.52 0.79 ± 0.12 0.030 ± 0.010
* % dose organ; values are mean ± SD (n = 6).
 SESSION 11

Tracer uptake in the pancreas, tumour and adrenal were similar for both
compounds (p = 0.01) after 1 h p.i. (Table 3). Tumour to blood ratios at 1 h p.i.
were similar for both compounds but tumour to liver and tumour to intestine
ratios were superior to 131I-DOTA-Tyr3-octreotate (Table 4).
Both labelled peptides presented low uptake in thyroid, which suggests
low in vivo dehalogenation of the compounds.
The distribution pattern of 131I-Tyr3-octreotate was similar to that
reported for 123I-Tyr3-octreotide in rats [9], with relatively high activity levels in
liver and intestine. The biodistribution of 131I-DOTA-Tyr3-octreotate, however,
can be compared with the distribution of radiometal labelled octreotide
analogues in mice [10, 11].

TABLE 3. BIODISTRIBUTION OF 131I-Tyr3-OCTREOTATE AND

131
I-DOTA-Tyr3-OCTREOTATE IN NUDE MICE BEARING AR42J RAT
PANCREATIC TUMOURS
131
I-Tyr3-octreotate 131
I-DOTA-Tyr3-octreotate
Dose/g (%) Dose/g (%)
Tissue
1h 24 h 1h 24 h

Total blood 2.12 ± 0.49 0.085 ± 0.035 2.93 ± 0.32 0.124 ± 0.006
Liver 2.07 ± 0.73 0.17 ± 0.03 1.34 ± 0.09 0.149 ± 0.009
Int. (small) 8.75 ± 1.87 0.061 ± 0.001 2.78 ± 0.60 0.067 ± 0.022
Muscle 0.37 ± 0.12 0.024 ± 0.009 0.42 ± 0.21 0.039 ± 0.013
Thyroid * 0.28 ± 0.13 0.516 ± 0.031 0.54 ± 0.17 1.19 ± 0.31
Adrenals* 0.021 ± 0.010 0.002 ± 0.001 0.018 ± 0.004 0.003 ± 0.001
Pancreas 0.78 ± 0.05 0.038 ± 0.001 1.15 ± 0.29 0.047 ± 0.013
Tumour 1.10 ± 0.45 0.18 ± 0.08 1.73 ± 0.01 0.13 ± 0.01
* % dose organ; values are mean ± SD (n = 3).

TABLE 4. TUMOUR TO TISSUE RATIOS AT 1 h p.i. OF

131
I-Tyr3-OCTREOTATE AND 131I-DOTA-Tyr3-OCTREOTATE IN NUDE
MICE BEARING AR42J RAT PANCREATIC TUMOURS
131
Ratio I-Tyr3-octreotate 131
I-DOTA-Tyr3-octreotate

Tumour to blood 0.51 0.59

Tumour to liver 0.53 1.29
Tumour to intestine (small) 0.13 0.62
Tumour to muscle 2.97 4.12

249
 DE ARAÚJO et al.

Although DOTA is not necessary for the radioiodination procedure, the

chelating group seems to decrease the lipophilicity as evidenced by the low
uptake in liver and intestines of 131I-DOTA-Tyr3-octreotate. In contrast to 131I-
Tyr3-octreotate, which was eliminated via the hepatobiliary route, the DOTA
analogue was predominantly cleared by the kidneys. The 131I-DOTA-Tyr3-
octreotate also presented better tumour to non-tumour ratios, especially for
liver and intestine, which are well known to be critical organs for scintigraphy
(Table 4).
Radiometal labelled somatostatin derivatives often show longer tumour
retention, compared to radioiodinated somatostatin analogues, due to intra-
cellular trapping of the radionuclide or radiolabelled metabolites [7]. Despite
this, the results obtained in this study with the 131I-DOTA-Tyr3-octreotate were
promising and suggest the applicability of the compound for SPECT imaging or
therapy using, respectively, 123I or 131I in labelling procedures.

ACKNOWLEDGEMENTS

This work was supported by IPEN-CNEN and IAEA.

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 SESSION 11

[8] BAKKER, W.H., et al., Iodine-131 labelled octreotide:not an option for somato-
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251
.
 IODINE LABELLED DIETHYLSTILBESTROL (DES)
OF HIGH SPECIFIC ACTIVITY: A POTENTIAL
RADIOPHARMACEUTICAL FOR THERAPY OF
ESTROGEN RECEPTOR POSITIVE TUMOURS AND
THEIR METASTASES?
T. FISCHER, H. SCHICHA, K. SCHOMÄCKER
Department of Nuclear Medicine,
University of Cologne,
Germany
Email: thomas.fischer@uk-koeln.de

Abstract

Diethylstilbestrol (DES) is a well-known, non-steroidal estrogen with higher

affinity to the estrogen receptor (ER) than the natural hormone estradiol itself. Radio-
actively labelled DES would be a useful tool for therapy of ER positive mamma carci-
nomas and their metastases. Particularly with Auger electron emitters, high cytotoxic
potential combined with only slight side effects can be expected. In the present work,
DES was labelled by a new method, which allows the synthesis of *I-DES with a higher
yield and higher specific activity than achievable with former methods. Binding affinity
and cytotoxic effects on MCF-7 mamma carcinoma cells, depending on radioactivity
concentration applied and location of decay (nucleus or cell surface), were tested.
Different iodine isotopes (123I, 125I, 131I) bound to DES or in the form of iodide were
compared with regard to apoptosis, necrosis and viability. Also, the radiation protective
effects of the radical scavenger vitamin C were tested. In animal experiments with
tumour bearing mice the biodistribution of 123I-DES was investigated. Results showed
significantly lower viability of cells exposed to the Auger electron emitters 123I and 125I
than those tested in the presence of the ß emitter 131I. All radionuclides induced apoptosis.
The amount of apoptosis was different for all nuclides: 131I-DES < 125I-DES < 123I-DES.
In the form of iodide, no increase of apoptosis could be detected. Necrosis did not occur
in the radioactivity concentration range observed, only secondary necrosis — a late
phase of apoptosis — was found. In the presence of vitamin C, a significant reduction of
apoptosis was observed, which points to an induction mechanism mainly via free
radicals. The 123I-DES showed a high tumour uptake of 42% ID/g in mice, which could
be blocked by co-application of ‘cold’ estrogens. Tumour/background ratios were
excellent (tumour/blood = 16.5, tumour/liver = 7.8). *I-DES, in connection with Auger
electron emitting radionuclides, seems to be a favourable candidate with a high
cytotoxic potential for therapy of ER positive tumours and their metastases.

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 FISCHER et al.

1. INTRODUCTION

The most common methods for therapy of mamma carcinomas involve

the surgical removal of the tumour and/or hormone therapy. If these
treatments are not possible (e.g. small metastases), proliferation inhibiting
therapy forms such as chemotherapy and radiation, which cause several
unpleasant side effects, are used. Therefore, it would be very advantageous to
find other, more specific therapy options.
Radioactive labelled estrogens, especially in connection with Auger
electron emitters, which have only a very short range (1–10 nm) of radiation,
are excellent candidates with which to achieve high specific cytotoxicity, in
combination with a low degree of side effects [1–5]. Until the 1970s, the use of
Auger electron emitters in cancer therapy was discussed [6] and their
impressive cytotoxic potential could be demonstrated [7–22].
Diethylstilbestrol (3,4-Di-(4‘-hydroxyphenyl)-hexene-3) (DES) is a well-
known, non-steroidal estrogen [23] with higher affinity to estrogen receptor
(ER) than the natural hormone estradiol itself [24, 25]. Therefore, radioactively
labelled DES would be a promising compound for therapy of ER positive
mamma carcinomas and their metastases. Certainly, DES was radioiodinated
years ago and its biodistribution was investigated [26–32]. Because of the low
specific activity, biodistribution data were disappointing. Recently, however, a
modified radioiodination method for DES has been published [33], which
allows synthesis of *I-DES with much higher specific activity and radiochemical
purity than achievable with former methods. Its affinity for ER remained high
after labelling [33].
For testing its cytotoxic effects on MCF-7 mamma carcinoma cells,
different iodine isotopes bound to DES or in the form of iodide were compared
with regard to apoptosis, necrosis and viability. Last but not the least, the first
animal experiments with tumour bearing mice were carried out.

2. MATERIALS AND METHODS

DES was iodinated by chloramin T in methanolic solution according to

the literature [33]. Purification and quality control were carried out with
reversed phase HPLC (column: Hypersil ODS, 250 mm × 4 mm, 10 µm, eluent
A: methanol G, eluent B: water G, gradient: 20% A to 70% A within 5 min,
flow: 1 mL/min, UV detection: 254 nm).
MCF-7 tumour cells (DKFZ, Heidelberg, Germany) were cultivated in
Dulbecco’s Modified Eagle Medium (GIBCO, Grand Island, New York), as

254
 SESSION 11

well as Dulbecco’s Medium without Phenol Red. The cells’ ER status was
evaluated with the ER-ICA test from ABBOTT.
For investigation of cell viability, the cells were incubated with a
tetrazolium salt (cell proliferation reagent WST-1, Roche Diagnostics), which
was cleaved by succinat tetrazolium reductase, an enzyme of the respiratory
chain in the membrane of mitochondria. The reaction product was a coloured
formazan, which could be measured by optical methods. For the experiments,
100 µL Dulbecco’s Modified Eagle Medium containing 1 × 104 MCF-7-cells was
transferred to each well of a microtitre plate (MTP) with a flat bottom. One
row was kept empty. The cells were incubated in the MTP for 24 h at 37°C and
under the above mentioned atmospheric conditions. Then to each well, 100 µL
*
I-DES or *I-iodide of different concentrations between 0.1 MBq/mL and 5
MBq/mL was added. One row of eight wells was only filled with 100 µL of
medium for control. The row that was kept without cells was now filled with
200 µL medium in each well. All set-ups were made fourfold to eightfold. The
cells were now incubated for 18 h under the mentioned conditions and then
mixed with 20 µL WST-1-reagent. After 2 h incubation at 37°C, the extinction
of the samples was determined with the ELISA reader at 450 nm (reference
filter: 620 nm).
For determination of apoptosis or necrosis, the Cell Death Detection
ELISA from Roche Diagnostics was used. The amount of apoptotic cells in
comparison to a control group can be measured by detection of DNA
fragments by special antibodies included in the kit. According to whether
detection of those fragments occurs before or after cell lysis, it will be possible
to distinguish between necrosis and apoptosis. Measured DNA fragments in
the supernatant before cell lysis were due to apoptosis, detected DNA
fragments after lysis indicated necrosis. In the experiment, 100 µL DMEM
containing 1 × 104 MCF-7 cells was transferred to each well of a flat bottomed
MTP. The cells were incubated in the MTP for 24 h at 37°C and under the
above mentioned atmospheric conditions. Samples, that should be tested in the
presence of vitamin C, were mixed with 20 µL of ascorbic acid solution (0.01
mg/mL or 0.1 mg/mL giving 0.001 mg/mL or 0.01 mg/mL in the well). Then, to
each well (in the presence or absence of vitamin C) 100 µL *I-DES or *I-iodide
in concentrations of between 0.1 and 5 MBq/mL was added. Eight wells were
filled with 100 µL of medium instead of radioactive material. All other samples
were carried out fourfold to eightfold.
The MTPs were incubated for 18 h under the usual conditions and then
centrifuged at 200g. A 20 µL sample of each liquid was transferred for
measurement of the amount of necrosis to another streptavidine coated MTP
from the Cell Death Detection ELISAPLUS kit. Now, the residual liquid in the
first MTP was carefully removed and 200 µL of lysis buffer placed instead in

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 FISCHER et al.

every well. After 30 min incubation at room temperature, the plate was
centrifuged again at 200g and then 20 µL of the liquid was transferred to a
second streptavidine coated MTP for determination of apoptosis. For
background measurement, some wells were filled with 20 µL of incubation
buffer. In addition, some positive controls were also carried out.
Animal experiments were carried out with male tumour bearing DBA/2
mice (Charles River). Three groups of animals (n = 5 in each group) were
injected with: (a) 1.5 MBq 123I-DES, (b) 7 µmol DES-2-P + 2 MBq 123I-DES, (c)
7 µmol Estradiol + 2 MBq 123I-DES. Three hours after IV injection, the animals
were sacrificed and organs, tissues and blood were measured in a well counter.

3. RESULTS

In Figs 1–3, the relative metabolic activity of succinat tetrazolium

reductase (an enzyme of the respiratory chain in mitochondria membrane) is
presented, depending on the applied *I-DES or *I-iodide concentration. For all
three nuclides, a significant difference can be observed between *I-DES and *I-
iodide in the declining cell viability at increasing radioactivity concentrations.
When *I-DES labelled with the Auger electron emitters is used, the viability of
MCF-7 cells decreases very rapidly according to increasing radioactivity

120

I-123-DES
I-123
100
reductase activity in % of control group

80

60

40

20

0
0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5
activity concentration [MBq/ml]

FIG. 1. Relative metabolic succinat tetrazolium reductase activity in MCF-7 cells after 18 h
incubation with 123I-DES or 123I- in per cent enzyme activity in control group not exposed
to radioactivity (n = 8 per point, SD = ±15–20%).

256
 SESSION 11

120
I-125-DES
I-125
reductase activity in % of control group 100

80

60

40

20

0
0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5
activity concentration [MBq/ml]

FIG. 2. Relative metabolic succinat tetrazolium reductase activity in MCF-7 cells after
18 h incubation with 125I-DES or 125I- in percent enzyme activity in control group not
exposed to radioactivity (n = 8 per point, SD = ±15 – 20%).

120

I-131-DES
I-131
100
reductase activity in % of control group

80

60

40

20

0
0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5
activity concentration [MBq/ml]

FIG. 3. Relative metabolic succinat tetrazolium reductase activity in MCF-7 cells after 18 h
incubation with 131I-DES or 131I- in per cent enzyme activity in control group not exposed to
radioactivity (n = 8 per point, SD = ±15–20%).

257
 FISCHER et al.

concentration. To compare these different gradients of viability decrease, the

value V50 can be introduced. It is the activity concentration at which the
viability decreases to 50% of the initial viability in the control group. The V50
values for the Auger electron emitters bound to DES were V50(123I-DES) = 0.8
MBq/mL and V50(125I-DES) = 0.5 MBq/mL. A little less effective is the ß
emitter with V50(131I-DES) = 1.7 MBq/mL and the highest concentrations are
needed for comparable effects with the nuclides applied in the form of iodide
(V50(123I-iodide) ≈ 1 MBq/mL, V50(125I-iodide) ≈ 1 MBq/mL, and V50(131I-
iodide) ≈ 4 MBq/mL.
Apoptosis increased (see Figs 4–6) with rising activity concentrations up
to 35-fold for 123I-DES, 10-fold for 125I-DES and threefold for 131I-DES
compared with a control group. Necrosis increased parallel to apoptosis
(results not shown). Presumably this was not really necrosis, but a late phase of
apoptosis.
Animal experiments (Fig. 7) showed high specific uptake in prostate and
tumour, which could be blocked by estrogen or Honvan (DES-2-P).

40

35

30
enrichment factor

25

20

15

10

0
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
activity concentration [MBq/ml]
I-123-DES I-123-Iodid
I-123-DES+0,001 mg/ml Vitamin C I-123-DES+0,01 mg/ml Vitamin C
I-123-Iodid I-123-DES+0,001 mg/ml Vitamin C
I-123-DES+0,01 mg/ml Vitamin C

FIG. 4. Apoptosis accumulation factors of MCF-7 cells incubated with different radio-
activity concentrations of 123I-DES or 123I-iodide in comparison to cells of an untreated
control group (n = 4–8 per point, incubation time: 18 h, SD = ±15%).

258
 SESSION 11

12

10

enrichment factor 8

0
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
activity concentration [MBq/ml]

I-125-DES I-125-Iodid
I-125-DES+0,001 mg/ml Vitamin C I-125-DES+0,01 mg/ml Vitamin C
I-125-Iodid I-125-DES+0,001 mg/ml Vitamin C
I-125-DES+0,01 mg/ml Vitamin C

FIG. 5. Apoptosis accumulation factors of MCF-7 cells incubated with different radio-
activity concentrations of 125I-DES or 125I-iodide in comparison to cells of an untreated
control group (n = 4–8 per point, incubation time: 18 h, SD = ±15%).

4
enrichment factor

0
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
activity concentration [Bq/ml]

I-131-DES I-131-Iodid
I-131-DES+0,001 mg/ml Vitamin C I-131-DES+0,01 mg/ml Vitamin C
I-131-Iodid I-131-DES+0,001 mg/ml Vitamin C
I-131-DES+0,01 mg/ml Vitamin C

FIG. 6. Apoptosis accumulation factors of MCF-7 cells incubated with different radio-
activity concentrations of 131I-DES or 131I-iodide in comparison to cells of an untreated
control group (n = 4–8 per point, incubation time: 18 h, SD = ±15%).

259
 FISCHER et al.

120
Blood
100 Liver
Spleen
80 Kidney
Muscle
60 Femur
Thyroid
40 Prostate
Intestines
20
Tumor
Urine
0
I-123-DES I-123-DES + I-123-DES + E
HON.

FIG. 7. Biodistribution (in %ID/g) of 123I-DES in tumour bearing male mice with and
without co-application of HONVAN™ (DES-2P) or estradiol.

4. CONCLUSIONS

Radioactively labelled DES is a promising compound for therapy of ER

positive mamma carcinomas and their metastases. This compound showed, in
vitro, a high cytotoxic potential to destroy ER positive tumour cells very specif-
ically. Biodistribution experiments were also very encouraging.

REFERENCES

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125
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[3] ADELSTEIN, S.J., The Auger process: a therapeutic promise? Am. J. Roentgen
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[8] BLOOMER, W.D., et al., 5-(I-125)-iododeoxyuridine as prototype for radionu-

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estimation of radiation risk, Int. J. Nucl. Med. Biol. 8 (1981) 171.
[13] BLOOMER, W.D., et al., Therapeutic implications of iodine-125 cytotoxicity, Int.
J. Radiat. Oncol. Biol. Phys. 8 (1982) 1903.
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Effects on the survival curve of radioiodine incorporated into DNA, Radiat. Res.
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[16] FEINENDEGEN, L.E., et al., DNA strand breakage and repair in human kidney
cells after exposure to incorporated iodine-125 and cobalt-60 ?-rays, Curr. Top.
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[17] DESOMBRE, E.R., et al., Therapy of estrogen receptor-positive micrometastases
in the peritoneal cavity with Auger-electron-emitting estrogens - theoretical and
practical considerations, Acta Oncol. 39 (2000) 659.
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IUdR in mammalian cells: implication of localized energy deposition by Auger
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coli and bacteriophage T1, Int. J. Radiat. Biol. 21 (1972) 167.
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decay of iodine-125 in bacteriophages, Int. J. Radiat. Biol. 27 (1975) 553.
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des 16?-[125I]-Iodöstradiol-3,17?, Nuklearmedizin 37 (1998), 134.
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tion of prolactin synthesis by antiestrogens in vitro, J. Biol. Chem. 258 (1983) 4741.
[25] KORACH, K.S., et al., Estrogen receptor stereochemistry: ligand binding and
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[27] TUBIS, M., et al., The distribution of 131I labeled diethylstilbestrol diphosphate in
animals and men with observations on its potential use as a scanning agent of the
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nach intravenöser Injektion, Gynäk. Rdsch. 19 (1979) 30.
[30] MELLER-REHBEIN, B., et al., 123I-Diethystilbestrolphophate-a potential tracer
for receptorimaging and therapy ? Eur. J. Nucl. Med. 21 (1994) 878 (abstract).
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Basel (1997) 333-342.
[32] SCHOMÄCKER, K., et al., Biokinetics of I-123-Diethylstilbestrol (DES) in
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[33] FISCHER, T., et al., Labelling, purification, and receptor affinity of radioactive
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262
 18
F RADIOPHARMACEUTICALS
AND AUTOMATION OF SYNTHESIS

(Session 12)

Chairpersons

V.W. PIKE
United States of America

D. SOLOVIEV
Switzerland
.
 18
F BASED RADIOPHARMACEUTICALS AND
AUTOMATION OF SYNTHESIS
New 18F radiopharmaceuticals

P.K. GARG, S. GARG

Department of Radiological Sciences,
Wake Forest University Medical Center,
Winston Salem, North Carolina,
United States of America
Email: pgarg@wfubmc.edu

Abstract

Fluorine-18 is one of the most commonly used positron emitting isotopes for
clinical and research needs with a physical half-life of 110 min. PET isotopes deposit
higher radiation absorbed dose than nuclear medicine isotopes. Because of their rela-
tively short half-life, larger quantities of these isotopes are used at the start of synthesis.
Therefore, increased shielding and remote automated synthesis are essential for their
safe handling. Unlike other radiopharmaceuticals, it is not practical to produce PET
radiopharmaceuticals at a central location for subsequent distribution to clinical and
research facilities around the country. This limitation compels various academic and
research facilities to manufacture their own PET radiopharmaceuticals for in-house use.
For multiple reasons, 18F fluorodeoxyglucose ([18F]FDG) is one of the most commonly
used radiopharmaceuticals. The synthesis of [18F]FDG has been optimized and auto-
mated, thus allowing independent laboratories to produce this radiopharmaceutical safely.
Nonetheless, these laboratories should acquire resources and expertise to fulfil ever
increasing regulatory requirements for the safe production and usage of PET radio-
pharmaceuticals. In addition to [18F]FDG, a wide array of new and novel radiotracers is
being developed to explore various biological processes. This paper emphasizes the fact
that it is possible to accomplish research and fulfil clinical needs within an academic
setting with modest resources. A careful assessment of the need for due diligence in
radiation safety issues is very important for the longevity of any PET research endeavour.

Among various positron emitting isotopes, 18F has gained wider

popularity. This is largely because of its favourable physical half-life (110 min),
allowing multistep synthesis and the capability to transport labelled radiotracer
to sites remote from the synthesis laboratory and cyclotron facilities. Continued
efforts in developing novel and innovative isotope production targetry now
allow production of large quantitites of 18F fluoride, which in turn makes it
practical to produce multicurie quantities of 18F fluorodeoxyglucose
([18F]FDG). This advancement eased the work load and beam time on the

265
 GARG and GARG

cyclotron while meeting the increased clinical demands. Production of larger

batches of [18F]FDG also helped in reducing the overall cost of producing this
radiopharmaceutical.
Despite a large inventory of radiopharmaceuticals for positron emission
tomography (PET), [18F]FDG remains the leading radiopharmaceutical for
oncology, and cardiac and neurological applications. Although demand for
[18F]FDG depends on the patient load and clinical practices, an average busy
PET scanning facility may require ~5–15 doses of [18F]FDG. Because of the
short half-life of 18F (T1/2 = 110 min), it is not practical to cover the clinical
needs for an entire day from one production batch of [18F]FDG. Therefore,
production of multiple batches per day may be essential. Currently, a typical
[18F]FDG production laboratory may utilize in excess of 3000 mCi of 18F
fluoride, whereas laboratories with smaller [18F]FDG consumption may use
200–1000 mCi of 18F fluoride isotope. Safe and effective handling of such large
amounts of radioisotopes mandates careful design of the laboratory and
procurement of necessary equipment and accessories. The automated synthesis
of [18F]FDG is well described and several automated synthesis modules are
now available on the market. Nonethelesss, a careful assessment of compati-
bility of these modules with local expertise and needs is important.
Currently, [18F]FDG plays a significant role in PET imaging. The future of
PET depends on the availability of additional radiopharmaceuticals with
higher specificity and selectivity to various diseases. Numerous neuroreceptor
ligands are labelled with C-11, a significant number of ligands are also labelled
with 18F for neuroimaging, and oncological and cardiac applications. In this
paper, the major emphasis is on the 18F labelled radiopharmaceuticals and their
safe and effective handling options.
The last five years have been a boom for the advancement in PET
technology and its applications. During this period, PET scanners have
improved in terms of their spatial resolution and subsequently advanced to a
CT–PET fusion technology. This advancement not only helped improve image
quality but also provided efficient and high throughput screening to handle
increased patient volumes. While these advancements are helpful for an
improved and effective diagnosis, they underscore the need for newer and
improved PET ligand agents. Although, additional inventory of PET imaging
agents would not necessarily increase the quantities of 18F handled by radio-
chemistry personnel, it does provide a challenge to accommodate multiple
synthesis protocols with varying chemistries within the constraints of a typical
PET research and clinical facility. Although the synthesis of [18F]FDG is now
routine and well standardized, the synthesis of other radiotracers could be
more complex and involved. For the last several years, the authors’ research
group has been engaged in the development of PET tracers for a variety of

266
 SESSION 12

applications. One of the approaches pursued was to design newer PET imaging
molecules using templates for exisiting drugs. To that end, the authors
developed 18F labelled p-fluorobenzylguanidine [1, 2], 18F labelled monoclonal
antibodies and their fragments [3, 4] and peptides [5], and 18F labelled
androgens [6] and various other neuroimaging agents The authors derived
these newer molecules from templates that have been tested and used in
clinical or preclinical studies using other imaging modalities. The other benefit
of this approach was that the biological properties of the parent analogues were
known. Therefore, successful translation of these molecules for PET imaging
was straightforward. Some of the molecules that were produced and evaluated
over the last few years are briefly described.
Meta-iodobenzylguanidine (MIBG; Fig. 1) was first developed as a
norepinephrine (NE) mimic to detect and treat neuroendocrine tumours.
Successful use of MIBG in the detection of neural crest tumours and cardiac
sympathetic innervations led the authors to develop the 18F labelled analogue,
i.e. 18F p-fluorobenzylguanidine ([18F]PFBG, Fig. 1) for PET imaging. The
structures of PFBG, MIBG and NE are shown in Fig. 1 for comparison and to
highlight the similarity in their structures.
The synthesis of [18F]PFBG and their initial in vitro and in vivo results
have already been reported [2, 7, 8]. In small animals, [18F]PFBG shows higher
specificity for the target tissues compared to that with MIBG [1, 8].
Encouraged by these studies, the authors explored the potential of [18F]PFBG
in detecting myocardial infarct in a dog model. As shown in Fig. 2, [18F]PFBG
images acquired 16 d post-infarction lack 18F accumulation in the area of
infarction despite reperfusion of the myocardium from newly formed collateral

HO H2N
N

HO HN
NH2
OH
HN
norepinephrine (NE) guanethidine

18
F
H
N NH2 H
I N NH2

NH
NH

meta-iodobenzylguanidine para-fluorobenzylguanidine
(MIBG) (PFBG)

FIG. 1. Structure of MIBG and PFBG, and their resemblance to NE.

267
 GARG and GARG

FIG. 2. PET images using [18F]PFBG (innervation tracer) and 13N ammonia (perfusion
tracer) in dog heart. Left panel shows well perfused heart prior to surgery and a distinct
tracer uptake deficit 2 d post-surgery scans. The right panel shows continued [18F]PFBG
uptake deficit 16 d post-surgery.

blood vessels [9]. As shown in the left hand graphic of Fig. 2, [18F]PFBG and
13
N ammonia both showed an area of marked decrease in accumulating both
radiotracers 2 d post-surgery compared with control (pre-surgery scans). The
[18F]PFBG accumulation deficit persisted even 16 d post-surgery as shown for
two dogs in Fig. 2, right hand panel. These observations suggest that blood
vessel ligation not only leads to perfusion deficit, but may have caused damage
to the myocardium that was not detectable from blood perfusion scans, but
which was quite evident on [18F]PFBG scans.
A graphical representation of these results is shown in Fig. 3. The graph
presents radioactivity uptake in the area of infarction using 13N ammonia for
myocardial perfusion and using [18F]PFBG to assess innervation. A sharp
decrease in both the tracers’ uptake was noticed in the area of infarction when
images were acquired 2 d post-surgery: [18F]PFBG 59% of control values; 13N
ammonia 61% of control values. Although perfusion deficit resolved 16 d post-
surgery as seen from 13N images, [18F]PFBG deficit remained unchanged even
23 d post-surgery (Fig. 3). These observations suggest that the [18F]PFBG
accumulates in tissues that were damaged during infarction and the damage
existed three weeks after surgery despite recovery of blood perfusion [9, 10].
[18F]PFBG also shows promise for its applications in oncology. When
tested on dogs with spontaneous pheochromocytoma, [18F]PFBG showed a
rapid and high uptake at tumour sites. PFBG accumulation peaked within
minutes of injection [11] and remained constant for the duration of the study.

268
 SESSION 12

FIG. 3. Graphic representation of normalized uptake of 13N ammonia and [18F]PFBG in

the area of myocardial infarction for pre-infarct, 2, 16 and 23 d post-infarction studies.

A coronal view of the PET scan from one of the dogs with pheochromocytoma
acquired after injecting with [18F]PFBG is shown in Fig. 4. The [18F]PFBG
localized in the right adrenal gland mass within the first few minutes and
remained constant. A rapid clearance of radioactivity from the blood pool and
a persistent retention of tracer within the area of the adrenal mass were
observed from time activity curves generated from region of interest analysis.
PET images of dogs with spontaneous pheochromocytomas utilizing
[18F]PFBG showed an SUV of 20–45 [11].
The authors further evaluated [18F]PFBG utility in a diabetic rat heart
model. Diabetes was induced by injecting streptozotocine (55 mg/kg i.p.) in
male Sprague Dawley rats. One week later, blood glucose levels were

FIG. 4. [18F]PFBG accumulation in the area of adrenal mass of a dog with suspected
pheochromocytoma at 20–30 min post-injection of [18F]PFBG.

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30 MIN
3 60 MIN

% Injected dose/g
*
2
*

0
n=5 NORMAL DIABETIC
p <0.05

FIG. 5. [18F]PFBG uptake in diabetic and normal rat hearts at 30 and 60 min p.i.

monitored and rats with glucose levels of >200 mg/dL were used for the study.
A separate group of rats with no diabetes was used as control. As shown in
Fig. 5, a higher [18F]PFBG accumulation was observed in diabetic rat hearts at
30 min followed by rapid and significant washout of activity from the heart by
60 min. In comparison, [18F]PFBG uptake and washout was less dramatic in
normal rat hearts. These observations indicate a reduced retention of
[18F]PFBG in the vesicles, perhaps due to reduced NE transporter acitivy in
diabetic rat heart. These observations are similar to those reported earlier
using MIBG in the same model [12].
One of the probable causes of failed or ineffective radiation therapy is the
presence of hypoxia in tumours. These hypoxic areas are radioresistant and
also respond poorly to chemotherapy and other therapeutic interventions.
2-nitroimidazoles have been shown to possess excellent radiosensitization
properties. These compounds are taken up by the tumours and under hypoxic
conditions they are metabolized via a 2 electron reduction pathway. The
reduced species remains trapped within the hypoxic areas. If developed, 2-
nitroimidazole compounds could help in the assessment of the tumour hypoxia
fraction. Therefore, misonidazole, a first generation radiation sensitizer was
one of the first 2-nitroimidazoles to be labelled with 18F for its use as a hypoxia
imaging agent [13, 14]. Although the high lipophilicity of this molecule causes
low target tissue to blood ratios (<1.6), imaging experiments showed promise
[15]. Encouraged from these studies, newer 18F labelled 2-nitroimidazoles are
being developed to obtain a hypoxia imaging agent with better imaging charac-
terisitics and pharmacokinetics. Some of the recently developed compounds
include 18F FAZA [16], 18F EF [17, 18] and 18F PK 110 [19]. Unfortunately, most
of these compounds continue to possess high lipophilicity, leading to slow
clearance from the blood pool and confounding image analysis.

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Recently, the authors’ group designed a water soluble 2-nitroimidazole

analogue using the pimonidazole template. A microPET image of a mouse with
human colorectal carcinoma tumour xenograft using 18F PIMO is shown in
Fig. 6. As evident from such microPET images, radioactivity is cleared rapidly
via urinary excretion from the body and the tumour showed high retention of
the radiotracer throughout the study. A general lack of radioactivity from most
normal organs signifies the potential of this compound as a useful hypoxia
imaging agent with improved imaging characteristics.
Prostate cancer represents a significant health problem worldwide for the
male population. Early diagnosis and detection of this disease ensures effective
treatment. Nonetheless, besides the PSA test, no advance imaging modality
exists that can provide early detection and localization of this disease to aid in
selecting appropriate treatment regimens for better therapeutic outcome.
[18F]FDG has been ineffective in the detection and localization of this disease.
Recently, 18F choline [20], 18F FDHT [21] and 18F FMDHT [6] have been
developed. In mice bearing prostate tumour xenografts, the tumour uptake of
18
F choline was similar to that of [18F]FDG, whereas in patients with prostate
cancer, 18F choline detected more lesions and showed higher SUVs than did
[18F]FDG. Use of [18F]FDHT in prostate cancer patients showed an avid
uptake in tumour areas on PET images [22]. Nonetheless, a rapid metabolic
degradation of this compound was seen in humans.
The authors recently developed a metabolically stable 18F androgen
(FMDHT) that exhibits a high affinity for tumours expressing androgen
receptors [6]. In their preliminary studies, the authors observed a selective

FIG. 6. MicroPET image of a mouse bearing HT-29 human colorectal tumour xenograft.
A 30 min frame image acquired 90–120 min after injecting with 18F pimonidazole.

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uptake and distribution pattern of this tracer in mice and rats. As with most
steroidal compounds, including [18F]FDHT, the clearance of [18F]FMDHT
occurred via the GI track in mice and rats.
The authors further assessed the potential of [18F]FMDHT as a prostate
imaging agent using microPET. The imaging characteristics of [18F]FMDHT
were assessed using microPET imaging of an androgen dependent LnCAP
tumour xenograft in mice. As shown in Fig. 7, the tumour was clearly visible on
the microPET image. Additional studies were carried out using [18F]FMDHT
and comparing its imaging characteristics with other currently available PET
imaging agents. Figure 8 shows a head-on comparison of [18F]FMDHT images
with [18F]choline and [18F]FDG in the same mice bearing the LnCAP tumour
xenograft. As evident from these images, a better visualization of tumour was

FIG. 7. MicroPET image of a mouse bearing the LnCAP tumour xenograft

FIG. 8. MicroPET image of a mouse bearing the LnCAP tumour xenograft. Among the
three ligands used, more intense uptake of radioactivity was evident with [18F]FMDHT
compared with [18F]choline or [18F]FDG.

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 SESSION 12

observed using [18F]FMDHT. Imaging characristics of [18F]FMDHT as assessed

from these studies were far superior to those of other imaging agents.
The other area of significance for PET imaging is to diagnose breast
cancer and to follow the therapy outcome in those patients. Although
[18F]FDG has been successful in imaging breast tumours and their recurrence,
development of receptor based ligand, for example [18F]fluoroestradiol, would
provide information on the hormonal status of the tumours. Therefore,
developing this tracer offers advantages over [18F]FDG which include
localizing breast cancer sites, predicting the response to tumour therapy and
assessing the estrogen blocking effect of hormonal therapies such as the
tamoxifen and aromatase inhibitors. Several research groups are involved in
developing such PET imaging agents with clinical potential. Some of the initial
clinical studies performed using these tracers show encouraging results [23, 24].
In addition, several 18F labelled amino acids have been developed as PET
imaging probes that utilize the active amino acid transport mechanism for their
accumulation in target sites. Several 18F PET imaging agents are under investi-
gation by various researchers. An 18F labelled fluoromethyl tyrosine has shown
a higher SUV in brain tumours than that seen with [18F]FDG [25].
Because of the relatively short half-life of 18F, its role in utilizing the
monoclonal antibodies (MAb) and other bioactive molecular probes has been
ignored for a long time. Continuing their work on developing novel radiohalo-
genation methods, the authors explored the possibility of labelling MAbs with
18
F [4]. Using their newly developed labelling technique, they successfully
labelled MAbs with 18F in good radioconjugation yields and within time-frames
compatible with a short half-life isotope while retaining the immunoreactivity
of the labelled MAb [4, 26]. In tumour bearing mice, 18F labelled MAb showed
a significant uptake in the tumours within 2–4 h p.i. [3]. The authors further
assessed the utility of 18F labelled MAb to detect tumours in dogs with radio-
labelled MAbs and MAb fragments. A PET scan using a 18F labelled TP-3 Fab
antibody fragment was successful in detecting spontaneous osteosarcoma in
dogs [4, 27].
Besides its role in oncology, PET imaging plays a significant role in the
detection and monitoring of various neurological disorders. In their continued
efforts to develop novel nortropane analogues for brain receptor imaging
studies, the authors developed a 18F labelled N-(3-fluoropropyl)-2-β-
carbomethoxy-3-β-(4-bromophenyl)nortropane (FPCBT) as a potentially
useful ligand for dopamine transporter (DAT) imaging. This compound has a
binding affinity (Ki) of 3.3nM, 36.4nM, and 196nM for the DAT, SERT and
NET, respectively. The in vivo properties assessed in non-human primates
showed preferential uptake of this tracer in DAT rich areas of the brain (Fig. 9).

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STRIATUM CEREBELLUM
18
FIG. 9. PET image of a babboon brain after injecting F FPCBT. An intense uptake in
striatal area and minimal to negligible uptake in cerebellum are evident.

A rapid uptake of this tracer in the brain with highest accumulation in the
striatum was observed. Despite excellent accumulation in the striatum, a
modest striatum to cerebellum ratio (St:Cb = 3.3) observed for this ligand
reduced the enthusiasm to pursue it. A large collection of 18F labelled
radiotracers exist for neuroreceptor imaging. Recently developed tracers
include 18F ACF and 18F AFM for the SERT [28], 18F reboxitine analogue to
image NET [29] and 18F Fallypride for D2 dopamine receptor imaging [30].
While 18F compounds and molecular imaging probes are being developed
at a fierce pace, their handling and usage in radiochemical synthesis requires
particular care. In general, a positron emitting isotope imparts 2–10 fold higher
radiation doses to personnel than those received from the isotopes commonly
used for nuclear medicine imaging. For example, a 1 mCi unshielded gamma
emitting nuclear medicine isotope produces an exposure of 0.2–2.2 R/h at 1 cm
from the surface (depending on gamma energies) while exposure to a 1 mCi
PET isotope at 1 cm would be ~5.8 R/h, signifying a need for more careful and
cautious handling of PET isotopes. Because of a short physical half-life, larger
quantities of these isotopes are routinely used for radiochemical synthesis, thus
further increasing the risk of higher exposure. Therefore, it is important to use
extra caution and to develop safe handling practices in the laboratories. An
automated or remote handling procedure for the delivery of the radioisotopes
and for the synthesis of PET radiopharmaceuticals is essential to minimize
personnel exposure. Although several manufacturers are developing
automated synthesis boxes, their application is mainly limited to carrying out
synthesis of ligands that are fully evaluated and closer to routine use. It is
important to highlight that a number of PET tracers with somewhat
complicated chemistries are still synthesized manually with limited or no

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automation. Once evaluated and the synthesis optimized, it is likely that the
synthesis of those molecules can be adapted to the existing modules.
There are several synthesis modules that are now commercially available
for [18F]FDG production. One can now produce multiple batches of [18F]FDG
with one set-up routine in the beginning. Although multiple radiotracers
utilizing similar chemistries can be produced using an [18F]FDG box, it would
be difficult to adapt new ligand synthesis to these modules without the help of
experienced radiochemistry personnel. Therefore, there is a growing need for
more flexible automation sytems.
Because of the different chemistries involved, it is difficult to develop an
automated synthesis module for each individual ligand. Therefore, a large
number of research groups design their own semi-automated synthesis modules
that are either operated by remote electric valves or by programmable logic
controllers. The complicated aspect of this option is that these modules are
custom tailored for individual needs and are often prone to failure. Since these
modules are highly individualized, it is difficult for others to adapt and use
them with any reproducibility and reliability. One of the low cost automated
synthesis boxes commercially available is shown in Fig. 10. Although all the
synthesis functions are automated and streamlined, they are customized for the
synthesis of a particular radiotracer. Several manufacturers have commercial
modules that are now available to perform simple to complex radiochemical
syntheses. Most of these modules are computer controlled and adapt well to a

FIG. 10. An automated synthesis module for producing 18F fluorobromo methane from
18
F fluoride.

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11
FIG. 11. A fully automated synthesis module housed in a hot cell for producing C
labelled radiopharmaceuticals.

hot cell, cave, or even mini-cells. One of the examples is shown in Fig. 11. This
particular unit is used for the synthesis of 11C labelled molecules. A large
number of neuroreceptor ligands and other 11C radiopharmaceuticals use 11C
methyl iodide as a common synthone. Therefore, a number of molecules can be
synthesized using this module. There are several advantages of these
commercial modules; the user does not need to learn the mechanics behind the
module and the procedures are mostly standardized by the manufacturers. The
three major categories of modules that are available in the market are: a 11C
methyl iodide production and methylation module; 18F nucleophilic fluori-
nation modules and 18F electrophilic fluorination modules. Although not all
synthesis can be accomplished using one of the available modules, they are
optimized for the production of most commonly used radiopharmaceuticals. It
is important to remember that some of the fully automated modules are quite
expensive and could cost well above US $100 000. Nonetheless, it does provide
an option to establish reliable and regulatory compliant production of radio-
pharmaceuticals. Using automated synthesis modules is by far the most
effective, safe and practical approach to manufacture radiopharmaceuticals.
The production of PET radiopharmaceuticals is becoming increasingly
valuable and indispensable. Unlike a decade ago, at present, one has multiple
choices for the safe and effective handling of PET isotopes. It is imperative that
radiopharmaceutical laboratory design should consider addressing require-
ments of their local regulatory agencies and personnel safety. All PET research
radiopharmaceutical laboratories should require a properly shielded

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workplace. The level of shielding and automation need depends on workload,

resources and expertise. There are several options available to minimize
personnel exposure in a PET radiochemistry laboratory. The most primitive
but practical option is the lead lined fume hood or a lead cave. A typical lead
cave is shown in Fig. 12. The work area is covered with lead bricks and radio-
chemistry synthesis apparatus lies inside the lead lined area. The operator can
see the inside operations through lead glass windows. The reagents are added
from outside using syringes and slider valves that are located outside the lead
lined area and which are easily accessible without entering the radiation field.
The lead glass windows allow monitoring of the reactions without having to
lean over the lead wall. These lead caves are suitable to house a computer
controlled synthesis box or a remotely controlled procedure where minimal or
no operator interventions are necessary inside the work area. The use of these
lead lined hoods is not well suited for developmental work since access is
difficult and the potential for exposure is quite high. In addition, allowable
quantities of radioactivity usage in one of these lead caves should be restricted
if lacking ceiling shielding.
The next option is the use of hot cells (Fig. 13). The hot cell concept is
similar to that described for the lead cave. The hot cells are primarily lead caves
but provide front and side panels for easy access. In addition, these cells are

FIG. 12. Fume hood converted to lead lined cave for remote handling of radioisotopes.
The reagents are added from outside using syringes and slider valves (located on upper
right hand corner of lead wall) and push buttons (on right side sash) to the reaction vessel
located behind the lead wall.

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FIG. 13. A typical hot cell fitted with robotic arms (manipulators). The front door is
closed for radiochemistry operations. The front panel provides easy access to various
valves or other optional procedures.

shielded at the top and the bottom ends which significantly reduces the
radiation exposure in and around the radiochemistry laboratory. The hot cells
provide easy access to inside work space for preparatory work before the
synthesis and for easy cleanup after use. During the synthesis, all doors and side
panels are closed to minimize radiation exposure to laboratory personnel.
These hot cells are ideal to house synthesis boxes and automated procedures.
Depending on the option purchased, the final product can be delivered either
to a lead lined side drawer or accessed from a side panel without opening the
hot cell doors. It is advisable to wait for the radioactivity to decay before
opening the hot cell doors for cleanup or for setting up for reuse. There are
several manufacturers that sell hot cells internationally. Since this is a large
investment, it is advisable to match the options that a particular vendor
supplies and the site requirements for safe and effective operation before
finalizing a hot cell vendor. It should not be assumed that all hot cell vendors
will provide the same options, designs and accessories.
A large number of laboratories acquire robotic arms (manipulators) to
help with remote handling of radioactivity within the hot cell (Fig. 14). If semi-
automated synthesis or manual interventions are anticipated during the radio-
synthesis, it is strongly advised to have manipulators installed in the hot cells
(Figs 14 and 15). As shown in Figs 14 and 15, the operator works with the
manipulator arms from outside the hot cell. The arms aid in moving and
transfering radioactivity from one place to another and activating or
proceeding with the next process as needed, similar to that performed during
manual processing. Once the skill to operate these manipulators is mastered,

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FIG. 14. Operation of robotic arms (manipulators) from outside for safe and effective
radiochemical handling.

FIG. 15. Inside view of the hot cell while operator is working with the manipulators from
outside the hot cell.

they are helpful in accomplishing manual synthesis steps remotely and safely.
In addition, this option facilitates safe handling of multicurie quantities of
radioactivity while maintaining safety. These manipulators provide convenient
work access within the hot cell area while the doors are closed (Fig. 15). There
are several vendors manufacturing manipulators that work in hot cells and
most of them ship these items internationally. Most hot cell vendors can retrofit
these manipulators if not purchased during the hot cell acquisition or installation.

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If deferring the manipulator purchase at the time of hot cell purchase, it is

advisable to opt for manipulator hole cut-out plugs. The manufacturer will
make the housing insert for manipulators and will cover these with lead plugs
to retain their integrity while providing the flexibility to install manipulators by
removing these plugs as needed at a later date.
Hot cell manufacturers have responded to the ever increasing regulatory
requirements for the production of safe and effective radiopharmaceuticals.
Stringent pharmaceutical quality control criteria can now be met with HEPA
filter options available with some hot cells. Once the radiopharmaceutical is
prepared, it is collected in a sterile vial under a totally sterile environment and
further doses can be taken using dose drawing options available from certain
hot cell manufacturers.
It is important to develop newer and novel radiotracers to advance the
field of radiopharmaceutical chemistry. The descriptive path is to explore the
potential of various radiotracers while the goal is to develop radiotracers with
clinical relevance. To achieve that goal, one would need to combine expertise in
medicinal chemistry, organic chemistry, radiochemistry, radiation safety and
regulatory compliance with a good understanding of clinical issues. It is
important to bridge the gap between preclinical testing of the newly developed
radiotracers and carefully and cautiously testing and using these new imaging
agents in humans.
In conclusion, production of new and novel radiopharmaceuticals is
simpler and reproducible now, depite increased demands requiring
manufacture of larger and multiple batches of these radiopharmaceuticals.

REFERENCES

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[2] GARG, P.K., GARG, S., ZALUTSKY, M.R., Synthesis and preliminary evalua-
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[3] GARG, P.K., GARG, S., BIGNER, D.D., ZALUTSKY, M.R., Selective localiza-
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[6] GARG, P.K., LABAREE, D.C., HOYTE, R.M., HOCHBERG, R.B., [7α-
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[13] VALK, P.E., MATHIS, C.A., PRADOS, M.D., GILBERT, J.C., BUDINGER, T.F.,
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fluorine-18-fluoromisonidazole and positron emission tomography. J. Nucl. Med.,
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[17] MAHY, P., et al., Preclinical validation of the hypoxia tracer 2-(2-nitroimidazol-1-
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[19] GARG, P.K., DeGRAFF, W., GARG, S., ZALUTSKY, M.R., MITCHELL, J.B.,
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[20] DeGRADO, T.R., et al., Synthesis and evaluation of (18)F-labeled choline

analogs as oncologic PET tracers. J Nucl Med, 42: 1805-1814, 2001.
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receptor ligands in baboons. J Nucl Med, 37: 1009-1015, 1996.
[22] LARSON, S.M., et al., Tumor localization of 16beta-18F-fluoro-5alpha-dihy-
drotestosterone versus 18F-FDG in patients with progressive, metastatic prostate
cancer. J Nucl Med, 45: 366-373, 2004.
[23] MORTIMER, J.E., et al., Positron emission tomography with 2-[18F]Fluoro-2-
deoxy-D-glucose and 16alpha-[18F]fluoro-17beta-estradiol in breast cancer:
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[24] MANKOFF, D.A., et al., [18F]fluoroestradiol radiation dosimetry in human PET
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[25] INOUE, T., et al., 18F alpha-methyl tyrosine PET studies in patients with brain
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[26] ZALUTSKY, M.R., GARG, P.K., JOHNSON, S.H., COLEMAN, R.E., Fluorine-
18 antimyosin monoclonal antibody fragments: preliminary investigation in a
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1506-1513, 1994.
[28] HUANG, Y., et al., A new positron emission tomography imaging agent for the
serotonin transporter: synthesis, pharmacological characterization, and kinetic
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nylamine ([11C]AFM). Nucl Med Biol, 31: 543-556, 2004.
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 RAPID METHOD FOR RADIOFLUORINATION OF
PYRIDINE DERIVATIVES: PROSTHETIC GROUPS
FOR LABELLING BIOACTIVE MOLECULES

I. AL JAMMAZ*, B. AL OTAIBI*, H. RAVERT**, J. AMARTEY*

*Cyclotron and Radiopharmaceuticals Department,

King Faisal Specialist Hospital and Research Centre,
Riyadh, Saudi Arabia
Email: jammaz@kfshrc.edu.sa

** Division of Nuclear Medicine and Radiation Health Sciences,

The Johns Hopkins Medical Institutions,
Baltimore, Maryland,
United States of America

Abstract

Ethyl 2-[18F]fluoro-4-pyridine and ethyl 6-[18F]fluoro-3-pyridine carboxylates

were synthesized by catalyzed nucleophlic no-carrier-added radiofluorination.
Treatment of the ethyl-2-(N,N,N-trimethylammonium)-4-pyridine- and ethyl-6-(N,N,N-
trimethylammonium)-3-pyridine carboxylate.treflate precursors with radiofluoride and
Kryptofix 2.2.2 in anhydrous acetonitrile at 95°C provided these radiofluorinated inter-
mediates with a greater than 90% radiochemical yield within 2 min reaction time. These
intermediates served as precursors to obtain the activated N-succinimidyl 2-[18F]fluoro-
4-pyridine and 6-[18F]fluoro-3-pyridine carboxylate esters for efficient coupling to
amine functions in bioactive molecules. This technique was used to radiofluorinate a
model chemotactic peptide (N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys). Biodistribution
studies in normal CBA/J mice revealed very rapid clearance through the renal system.

1. INTRODUCTION

Synthesis of fluorinated aromatic compounds has been achieved by one

of three main routes: (1) fluorination by molecular F2, (2) fluorination by the
Schiemann reaction and (3) nucleophilic substitution on a precursor with a
good leaving group. The first two methods suffer from several drawbacks that
have led to limited success [1–3]. Another method of limited utilization has
been halogen–halogen exchange. The main drawbacks of this method are the
elevated temperature and long reaction time which preclude its use on many

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molecules [4, 5]. Furthermore, the short half-life of 18F imposes an additional
time constraint on the application of such a method. The efficiency and
simplicity of the nucleophilic fluoride substitution has been accepted and
applied for the production of several radiopharmaceuticals from precursor
substrates bearing a good leaving group [6, 7]. As part of their on-going
research effort to develop prosthetic precursors for the radiohalogenation of
bioactive molecules such as peptides, the authors have synthesized N-succin-
imidyl activated esters of the 2-[18F]fluoro-4-pyridine and 6-[18F]fluoro-3-
pyridine carboxylates. These intermediates were then used to label a potent
chemotactic peptide [N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys] and a biodistri-
bution study in normal CBA/J mice was investigated.

2. EXPERIMENTAL

The chemicals used in the study were all analytical reagent grade
purchased from Aldrich (St. Louis, United States of America) and were used
without further purification unless stated. Acetonitrile was kept over molecular
sieves. Sep-Pak cartridges were purchased from Waters-Millipore (USA). Thin
layer chromatographic (TLC) sheets were purchased from Gelman Sciences
Inc. (Ann Arbor, USA). High performance liquid chromatography (HPLC)
analysis was carried out on Econosil C-18 reversed phase columns (semi-
preparative, 250 mm × 10 mm or analytical, 250 mm × 4.6 mm). The solvent
system used for the latter was non-linear gradient (eluant A, water with 0.1
TFA; eluant B, acetonitrile/water, 3/1 v/v with 0.1% TFA; gradient, 0–90% B,
90–0% B and 90–10% B over 10 min each at a flow rate of 1.5 mL/min).
A Jasco (Tokyo, Japan) chromatographic system equipped with a variable
wavelength ultraviolet monitor and in tandem with a Canberra flow through
radioactivity detector was used. Ultraviolet absorption was monitored at
254 nm. Chromatograms were acquired and analysed using BORWIN
software. Melting points were determined on a Thomas Hoover Unimelt
capillary melting point apparatus. Mass spectroscopy was run on a Quattra
electrospray mass spectrometer (ES-MS).

2.1. Ethyl-6-(N,N,N-trimethylammonium)-3-pyridine carboxylate.treflate (6b)

Compounds (2a,b), (3a,b), (4a,b), (5a,b), (6a), (7a) and (8a) in Figs 1 and
2 were all synthesized utilizing the methods reported by Stöcklin et al. [8] and
Amartey et al. [9, 10]. Compound (5b), (250 mg, 1.25 mmol) was dissolved in
dry dichloromethane (3 mL) and was purged with nitrogen for 5 min. Methyl-
trifluoromethane sulphonate (150 uL, 1.25 mmol) was added through a rubber

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 SESSION 12

septum. The mixture was stirred at ambient temperature overnight. The

reaction mixture was then concentrated under reduced pressure to obtain a
yellowish paste. Upon treatment with cold ether, white crystals of the triflate
(6b) separated out. The crystals were filtered and washed with cold ether and
dried in a desiccator.

2.2. 6-fluoro-3-methylpyridine (7b)

In a plastic flask kept at ice–salt bath temperature, Olah’s reagent [11]

(4 mL, pyridine:HF 30:70% w/w) was used to dissolve compound (1b) (1.0 g,
9.25 mmol). To control the rise in temperature, sodium nitrite (0.7 g, 10.1 mmol)
was added in small portions while stirring. The mixture was continuously
stirred at ambient temperature overnight followed by quenching in crushed ice.
The excess acid was carefully neutralized with sodium hydroxide (5.0M). The
product was then extracted with ether (2 × 50 mL) and dried over anhydrous
sodium sulphate. Evaporation of the ether yielded an oily material, which was
used immediately.

2.3. 6-fluoro-3-pyridine carboxylic acid (8b)

Compound (7b), (0.25 g, 2.25 mmol) was added to a solution of potassium

permanganate (1.5 g, 4.5 mmol) in water (10 mL). The mixture was made
alkaline by adding few drops of sodium hydroxide (5M) and was then heated to
reflux for 5 h. The unreacted permanganate was reduced with sodium thiosul-
phate. The solid manganese dioxide formed was filtered off and the filtrate was
then concentrated to approximately 50 mL. Acidification with concentrated
hydrochloric acid precipitated the fluorinated nicotinic acid. The product was
filtered, washed with cold water and dried to obtain a white solid material.

2.4. Ethyl 2-fluoropyridine-4-carboxylate (9a) and ethyl 6-fluoropyridine-3-

carboxylate (9b)

Compound (8a), (50 mg, 0.35 mmol) was dissolved in absolute ethanol
(2 mL) followed by the addition of concentrated sulphuric acid (100 μL). The
mixture was heated in a sealed vial overnight at 60°C. The reaction mixture was
carefully basified using ammonium hydroxide while cold. The product was then
extracted with ether (2 × 10 mL) and dried over anhydrous sodium sulphate.
Evaporation of the ether yielded an oily greenish material, which was solidified
by cooling. The same procedure was used to produce ethyl 6-fluoropyridine-3-
carboxylate (9b) starting from 6-fluoropyridine-3-carboxylic acid (8b).

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2.5. N-succinimidyl 2-fluoropyridine-4-carboxylate (SFPa, 10a) and

N-succinimidyl 6-fluoropyridine-3-carboxylate (SFPb, 10b)

To a mixture of 2-fluoropyridine-4-carboxylic acid (8a) (50 mg,

0.35 mmol) and TEA (100 μL, 0.71 mmol) dissolved in acetonitrile (300 μL), O-
(N-succinimidyl)-tetramethyluronium tetrafluoroborate (TSTU) (128 mg, 0.43
mmol) was added and heated in a heating block for 1 h at 90°C. The reaction
mixture was diluted with hexane/ethyl acetate 7/3 v/v (1 mL) and passed
though a Sep-Pak silica cartridge. Then the product was eluted by hexane/ethyl
acetate 7/3 v/v (5 mL). After the solvent was evaporated to dryness, white
crystals separated and were dried under vacuum. The same procedure was used
to produce N-succinimidyl 6-fluoropyridine-3-carboxylate (SFPa, 10b) starting
from 6-fluoropyridine-3-carboxylic acid (8b).

2.6. Chemotactic peptide-2-fluoropyridine-4-carboxylate conjugate (peptide-

SFPa, 11a) and chemotactic peptide-6-fluoropyridine-3-carboxylate
conjugate (peptide-SFPb, 11b)

Chemotactic peptide, (4.15 mg, 5 μmol) was dissolved in 9:1 acetonitrile:

DMF (100 µL). This was followed by the addition of compound (10a) (2.0 mg,
8.4 μmol). Enough triethylamine (TEA) solution in acetonitrile was added to
attain pH9. The reaction mixture was heated in a heating block for 20 min at
95°C. The reaction mixture was diluted with water (1 mL) and mixed well. The
solution was then loaded onto a Sep-Pak C-18 cartridge, washed with water
(5 mL), blow dried with air and the conjugate (11a) eluted with ethanol (1 mL).
After the solvent was evaporated to dryness, white crystals separated, which
were dried under vacuum. The same procedure was used to produce
chemotactic peptide-6-fluoropyridine-3-carboxylate conjugate (peptide-SFPb,
11b) starting from N-succinimidyl 6-fluoropyridine-3-carboxylate (10b).

2.7. Radiosynthesis

Aqueous [18F]-fluoride was produced by the 18O (p,n) 18F reaction. The
fluoride activity (5–20 mCi, 185–740 MBq) was trapped in Kryptofix 2.2.2 (5 mg)
and potassium carbonate (1 mg) in acetonitrile/water solution (950 μL/50 μL), then
dried by azeotropic distillation with aliquots of acetonitrile. The solid residue was
resolubilized in 0.2 mL of CH3CN containing the required amount of precursor
ethyl-2-(N,N,N-trimethylammonium)-4-pyridine carboxylate.triflate (6a) or
ethyl-6-(N,N,N-trimethylammonium)-3-pyridine carboxylate.triflate (6b). The
reaction mixtures were heated in a capped 2 mL reaction vial at 95°C and
fractions were taken for chromatographic analysis at 2, 5, 8 and 10 min. The

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 SESSION 12

intermediates ethyl 2-[18F]fluoro-4-pyridine and 6-[18F]fluoro-3-pyridine

carboxylates (12a,b) were extracted with ether (2 × 1 mL) and passed through
a Sep-Pak silica cartridge. Each layer was dried with a steady stream of
nitrogen. For subsequent hydrolysis, residues were resolubilized with
acetonitrile (500 μL) followed by the addition of sodium hydroxide (100 μL,
1M) and heated at 95°C for 5 min. After acidification with hydrochloric acid
(100 uL, 1M), reaction mixtures were dried by azeotropic distillation with
aliquots of acetonitrile in the presence of TEA (10 μL). For activation,
solutions of O-(N-succinimidyl)-tetramethyluronium tetrafluoroborate (TSTU,
5 mg) in acetonitrile (100 μL) were added and heated for 15 min at 95°C.
Reaction mixtures were diluted with hexane (3 mL) before passing through a
Sep-Pak cyano cartridge. Finally, N-succinimidyl 2-fluoropyridine-4-
carboxylate ([18F]SFPa) (14a) and N-succinimidyl 6-fluoropyridine-3-
carboxylate ([18F]SFPb) (14b) were eluted from the cartridge with hexane/ethyl
acetate (8/2, v/v).
The SFPa,b solutions which contain TEA were dried and the chemotactic
peptide (50 μg) in acetonitrile:DMF (9:1) was added and then heated for 15 min
at 95°C. The peptide reaction mixtures were diluted with water (1 mL) and
passed through a Sep-Pak C-18 cartridge. Finally, peptide-[18F]SFPa,b
conjugates (15a,b) were eluted with absolute ethanol (1 mL). For the determi-
nation of stability in plasma, peptide-[18F]SFPa,b conjugates (100 µL, 20 µCi)
were incubated with human plasma (500 µL) in duplicate at 37°C for 2 h. This
was followed by precipitation using a mixture of acetonitrile/ethanol (1:1 v/v)
and centrifuging at 5000 rpm for 5 min. The supernatant layer was then
analysed by HPLC to determine the stability in plasma.

2.8. In vivo biodistribution

The biodistribution was performed in normal female mice to ascertain the

in vivo distribution profile of the radiotracer peptide-[18F]SFPa (15a). Mice
(CBA/J, 25–30 g) were injected via the tail vein with 0.1 mL of the radiotracer
formulated in saline containing 2% absolute ethanol. Each dose contained 20
µCi (740 kBq) of radioactivity. Animals were sacrificed at 5, 30 and 120 min
post-injection and organs and tissues of interest were dissected, weighed and
assayed for radioactivity. The percentage injected dose per gram was then
calculated for all tissues using a stored sample of the injection solution to
estimate the total dose injected per mouse. The animal biodistribution
experiments were performed in accordance with institutional, national and
international regulations governing the safe and humane use of laboratory
animals in research.

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3. RESULTS AND DISCUSSION

3.1. Organic chemistry

The synthesis of the precursor triflates (6a and 6b) needed for the
radiofluorination reactions is outlined in Fig. 1, whereas the scheme shown in
Fig. 2 was used to synthesize the reference compounds (8a,b, 9a,b, 10a,b and

CH3 CH3 COOH

HBr/Br2 KMnO4

NaNO2
N NH2 N Br N Br

(1) (2) (3)

HN(CH3)2

COOCH2CH3 COOCH2CH3 COOH

CF3SO3CH3 Ethanol

H2SO4
+ CH3 CH3 CH3
N N N N N N
H3C
CH3 CH3 CH3
(6) (5) (4)

FIG. 1. Synthesis of ethyl-2-(trimethylammonium)-4-carboxylate (6a) and ethyl-6-

(trimethylammonium)-3-carboxylate (6b) triflates.

CH3 CH3 COOH

Olah's
reagent KMnO4

N NH2 N F N F

(1) (7) (8)

H2SO4
TSTU/TEA
Ethanol

O
O
O N O
Peptide-NH O COOCH2CH3

Peptide / TEA

N F N F N F

(11) (10) (9)

FIG. 2. Synthesis of the reference compounds 2-fluoro-4-pyridine carboxylate (a) and 6-

fluoro-3-pyridine carboxylate (b).

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 SESSION 12

11a,b). These compounds were characterized by physical, chromatographic and

spectral data and were in agreement with the anticipated structures.
Methyltrifluoromethane sulphonate was used to convert compound (5b)
to the precursor ammonium.triflate (6b) as white crystals with a 35% yield;
melting point = 139–141°C. The calculated molecular mass of C11H17N2O2 (the
cationic portion of the molecule) was 209.26. This was in agreement with the
found ES-MS [M + 1]+ = 210. The 1Hnmr spectroscopic data obtained for this
precursor (400 MHz, CD3CN, with TMS as the reference) is as follows: δ1.40
(t, 3H, -O-CH2CH3), δ 3.55 (s, 9H, N-(CH3)3), δ 3.46 (q, 2H, -O-CH2CH3), δ 8.11
(d, 1H, H5), δ 8.27 (d, 1H, H5) and δ 8.77 (s, 1H, H2). Compound (7b) was
obtained as an oily material with a very good yield (83%) by diazotization of
the starting material (1b) with Olah’s reagent. Permanganate oxidation of this
oily intermediate furnished the corresponding acid (8b) also with an excellent
yield (94%) as a white solid, melting point = 150–152°C. The molecular mass
calculated for C6H4FNO2 was 141 and the ES-MS found was [M + 1]+ = 142.
1
Hnmr spectroscopic data for the acid 8b is as follows: (DMSO) δ 7.33 (d, 1H,
H5), δ 8.44 (d, 1H, H4), δ 8.77 (s, 1H, H2). Compounds 8a and 8b were
converted to their corresponding ethyl esters using the classical esterification
method to give compounds 9a and 9b with good yields (55% and 50%, respec-
tively) as oily materials which solidified upon cooling. The calculated molecular
mass of C8H8FNO2 was 168 and the ES-MS found was [M + 1]+ = 169 for both
compounds 9a and 9b. 1Hnmr spectroscopic data for 9a and 9b are as follows:
(CDCl3) (9a) δ1.15 (t, 3H, -O-CH2CH3), δ 3.34 (q, 2H, -O-CH2CH3), δ 9.23 (s,
1H, H2), δ 9.34 (d, 1H, H5), δ 10.2 (d, 1H, H5), and (9b) δ1.20 (t, 3H, -O-
CH2CH3), δ 3.46 (q, 2H, -O-CH2CH3), δ 6.75 (d, 1H, H4), δ 8.17 (d, 1H, H5), δ
8.82 (s, 1H, H2). The activated ester intermediates 10a and 10b were obtained
as white crystals by the activation of the corresponding acids (8a and 8b) using
TSTU as described above. Chemical yields for 10a and 10b were 47% and 45%,
respectively. Both activated esters decomposed when the temperature reached
121–123°C. The calculated molecular mass of C10H7FN2O4 was 238.17. This
was in agreement with the found ES-MS [M + 1]+ = 239 for both esters. The
amide linked targeted fluorinated chemotactic peptides were obtained as white
solids by conjugation of compounds 10a and 10b with chemotactic peptide. The
overall yields were greater than 80%. Chemical purity was found to be greater
than 98% without HPLC purification as confirmed by analytical HPLC. Both
fluoropyridine-peptide conjugates showed molecular ion at 948, corresponding
to the [M + 1]+ ion as expected.

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3.2. Radiochemistry

The synthetic approaches for preparation of the chemotactic peptide-

[18F]SFPa,b conjugates (15a,b) (Fig. 3) entailed several reaction sequences. The
key precursors ethyl-2-(N,N,N-trimethylammonium)-4-pyridine carbox-
ylate.triflate (6a) or ethyl-6-(N,N,N-trimethylammonium)-3-pyridine carbox-
ylate.triflate (6b) were treated using classical catalyzed nucleophlic substitution
on an ammonium leaving group with no-carrier-added radiofluoride reported
earlier [9, 10]. The radiochemical yields of the intermediates ethyl 2-[18F]-fluoro-
pyridine and 6-[18F]-fluoro-3-pyridine carboxylates (12a,b) were found to be
greater than 90% at all reaction times ranging from 2 to 10 min as determined
by HPLC and confirmed by TLC-SG developed with the ethyl
acetate:methanol 8:2 (v/v) solvent system. It was observed that between 2 and
10 min reaction time there was no significant change in the radiochemical yield
(Table 1). Similarly, increasing the amount of the triflate precursors from 2 to
10 mg was not advantageous. In order to eliminate or reduce the concentration
of most polar impurities, both ester intermediates were extracted by ether and
followed by passing through a Sep-Pak silica cartridge prior to the conversion
to the corresponding acids. It was also necessary to convert both ethyl ester
intermediates to the corresponding acid prior to activation with TSTU. This led
to a quantitative conversion of the ester intermediates to the corresponding
[18F]SFPa,b esters (14a,b) in a short period of time and at relatively lower

COOCH2CH3 COOCH2CH3 COOH

Kryptofix 222/K2CO3 NaOH
18
H F/CH3CN
+ CH3 18 18
N N N F N F
H3C
(6) CH3 (12) (13)

TSTU/TEA

O
O
O N O
Peptide-NH O

Peptide / TEA

18 18
N F N F

(15) (14)

FIG. 3. Radiosynthesis of 2-[18F]fluoro-4-pyridne carboxylate (a) and 6-[18F]fluoro-3-

pyridine carboxylate (b) compounds.

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 SESSION 12

TABLE 1. RADIOCHEMICAL YIELDS OF THE INTERMEDIATES

ETHYL 2-[18F]FLUORO-4-PYRIDINE AND 6-[18F]FLUORO-3-
PYRIDINE CARBOXYLATES

Time (min) Ethyl 2-[18F]fluoro-4-pyridine Ethyl 6-[18F]fluoro-3-pyridine

5 mg carboxylates carboxylates

2 91.9 ± 1.2 93.5 ± 1.8

5 91.0 ± 3.1 91.4 ± 2.6
8 92.8 ± 1.9 95.4 ± 0.5
10 92.1 ± 4.3 93.0 ± 2.1
Precursor (mg)
5 min.
2 91.5 ± 2.2 91.5 ± 0.8
5 92.0 ± 1.9 93.8 ± 1.4
8 92.3 ± 2.9 94.3 ± 3.4
10 89.0 ± 3.1 90.1 ± 4.1

temperature. Because of the ionic character of TSTU, the authors have been
able to purify [18F]SFPa,b esters (14a,b) by adsorption on Sep-Pak cyano
cartridge, followed by elution with hexane/ethyl acetate (8/2, v/v) and replacing the
laborious HPLC purification. The radiochemical yields ranged between 60 and
70% (decay corrected) with a preparation time of about 60 min. Radiochemical
purities of both [18F]SFPa,b esters (14a,b) were always greater than 98% as
determined by HPLC and confirmed by TLC-SG using ethyl acetate:methanol
(8:2) as a mobile phase. The retention times and Rf values for all intermediates
and the final product are shown in Table 1. The dry residues of [18F]SFPa,b
(14a,b) were reacted with the chemotactic peptide in DMF and acetonitrile in
the presence of TEA for 15 min at 95°C. The difference between the
unlabelled peptide and the conjugates permitted the isolation of the peptide-
[18F]SFPa,b conjugates (15a,b) by passing through a Sep-Pak C-18 cartridge and
eluting with absolute ethanol (1 mL). Hence, the Sep-Pak purification
technique is amenable to automation and holds considerable promise as a rapid
and simple method for the radiofluorination of bioactive molecules with high
specific activity, a prerequisite for studying low capacity and saturable sites.
The overall radiochemical yields ranged between 40 and 50% (decay
corrected) with a preparation time of about 80 min. Radiochemical purities of
both peptide-[18F]SFPa,b conjugates (15a,b) were greater than 98% as
determined by HPLC and confirmed by TLC-SG.

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TABLE 2. RETENTION TIME OF THE INVESTIGATED SPECIES

Retention time
Compound Rf values
(min)

Ethyl 2-[18F]fluoro-4-pyridine 9.45 0.7–0.8

carboxylate
Ethyl 2-[18F]fluoro-4-pyridine 9.37 0.7–0.8
carboxylate
2-[18F]fluoro-4-pyridine carboxylic acid 7.27 0.0–0.2
18
6-[ F]fluoro-3-pyridine carboxylic acid 7.27 0.0–0.2
18
[ F]-N-succinimidyl-2-fluoro-4- 7.20 0.4–0.5
pyridine carboxylate
[18F]-N-succinimidyl-6-fluoro-3- 7.10 0.4–0.5
pyridine carboxylate
2-[18F]fluoro-4-pyridine carboxylate 10.90 0.0
chemotactic peptide conjugate
6-[18F]fluoro-3-pyridine carboxylate 10.92 0.0
chemotactic peptide conjugate

3.3. Biodistribution studies

The biodistribution data in normal CBA/J mice are shown in Table 3.

Generally, the peptide-[18F]SFPa (15a) appears to clear fast from most of the
tissues. This trend is similar to that seen with the same chemotactic peptide
when coupled with [18F]fluorinatedbenzene [12]. However, the initial uptake of
peptide-[18F]SFPa in the liver and the kidney was fivefold lower for the former
and almost twice as high for the latter than reported for the [18F]fluorinated-
benzene-peptide. These differences are mainly attributed to the higher
hydrophilicity of the peptide-[18F]SFPa in comparison with [18F]fluorinated-
benzene-peptide. In addition, the lower bone activity for peptide-[18F]SFPa can
be due to the high stability of the carbon–fluorine bond.

4. CONCLUSION

The authors have developed a ‘synthetic’ figure leading to the

procurement of key precursors needed for radiofluorination of the hydrophilic
pyridine carboxylic acids. The radiofluorination reactions proceeded in very

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 SESSION 12

TABLE 3. BIODISTRIBUTION OF THE CHEMOTACTIC PEPTIDE-2-

[18F]FLUOROPYRIDINE-4-CARBOXYLATE CONJUGATE IN
NORMAL MICE

Organ/tissue 5 min 30 min 120 min

Blood 3.1 ± 1.2 0.4 ± 0.3 0.2 ± 0.1

5.0 ± 0.1 0.9 ± 0.1 0.1 ± 0.1
Liver 2.6 ± 1.2 0.4 ± 0.3 0.1 ± 0.1
14.2 ± 1.3 1.6 ± 0.3 0.4 ± 0.2
Lung 2.1 ± 1.8 0.3 ± 0.2 0.2 ± 0.1
4.5 ± 0.4 0.9 ± 0.1 0.6 ± 0.2
Kidney 39.2 ± 2.3 18.0 ± 1.9 4.9 ± 2.5
26.0 ± 1.3 10.9 ± 0.7 0.9 ± 0.2
Intestine 2.5 ± 1.8 0.4 ± 0.3 0.13 ± 0.1
4.5 ± 0.3 6.9 ± 0.6 0.7 ± 0.2
Heart 2.4 ± 1.4 0.2 ± 0.04 0.2 ± 0.03
2.7 ± 0.3 0.4 ± 0.04 0.11 ± 0.1
Muscle 2.7 ± 2.2 0.5 ± 0.2 0.3 ± 0.2
1.4 ± 0.1 0.3 ± 0.1 0.1 ± 0.1
Bone 0.7 ± 0.4 0.6 ± 0.2 0.8 ± 0.3
3.9 ± 1.1 1.4 ± 0.6 0.4 ± 0.2
Spleen 2.8 ± 2.1 0.6 ± 0.3 0.2 ± 0.1
3.0 ± 0.2 0.6 ± 0.1 0.4 ± 0.5

The values are average of %dose/g ± standard deviation for n = 4. Italicized results
reported by Vaidyanathan and Zalutsky [12].

high yields (>90%) and in a relatively short ‘synthetic’ time (2 min). These
acids were subsequently converted to their N-succinimidyl activated esters
followed by conjugation with chemotactic peptide as a model through their
lysine moieties. Sep-Pak purifications make this technique amenable to
automation and hold considerable promise as a rapid and simple method for
the radiofluorination of bioactive molecules. Tissue distribution in normal
CBA/J mice showed that the peptide-[18F]SFPa was excreted predominantly
(as expected) through the renal system.

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ACKNOWLEDGEMENTS

The authors wish to thank M. Al-Amoudi for the MS analysis. This

project was supported by the Research Centre of the King Faisal Specialist
Hospital & Research Centre (RAC # 2040027).

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otracer for studying nicotinic acetylcholine receptors (+/-)-exo-2-(2-[18F]fluor-5-
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[6] BERRIDGE, M., TEWSON, T., Chemistry of fluorine-18 radiopharmaceuticals,
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[7] HAKA, M., KILBOURN, M., WATKINS, D., TOORONGIAN, S., Aryltrimeth-
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[9] AL JAMMAZ, I., AL OTAIBI, B., AMARTEY, J., Synthesis of 2-[18F]-fluoroi-
sonicotinic acid hydrazide: Potential radiotracers for tuberculosis, J. of Labeled
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sonicotinic acid hydrazide and initial biological evaluation, J. of Nucl. Med. Biol.
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[11] OLAH, G., et al., Synthetic methods and reactions. 63. Pyridinium poly (hydrogen
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 1-[18F]FLUOROETHYLENEGLYCOL-2-
NITROIMIDAZOLES: A NOVEL CLASS OF
POTENTIAL HYPOXIA PET MARKERS

R.J. ABDEL-JALIL*, M. ÜBELE**, W. EHRLICHMANN**,

W. VOELTER***, H.-J. MACHULLA**

*Chemistry Department,
Faculty of Science,
Hashemite University,
Zarka, Jordan
Email: jalil@hu.edu.jo

** Radiopharmazie,
PET-Zentrum,
Universitätsklinikum Tübingen

***Abteilung für Physikalische Biochemie des

Physiologisch-chemischen Instituts der Universität Tübingen

Tübingen, Germany

Several hypoxia markers contain a nitroimidazole moiety as the reactive

chemical species, e.g. 18FNIM [1], 18FETNIM [2], 18FMISO [3] or 18FAZA [4]. It
is believed that these imaging agents primarily enter tissue by diffusion and are
trapped due to radical formation and subsequent intracellular reaction in the
case of decreased oxygen concentration [5]. In particular, the blood brain
barrier permeability is thought to play a crucial role in the cerebral uptake of
the marker within hypoxia tissues [6].
Recently, new hypoxia PET markers [7] have been developed with
higher lipophilicity compared to 18FMISO to enhance the blood brain barrier
permeability while avoiding excess non-specific biodistribution. However,
none of the developed markers offered improved biological properties over
18
FMISO, a well-accepted hypoxic agent. This unsuccesful enhancement of
trapping may be due to the lack of an oxygen atom in the β-position to the
2-nitroimidazole ring in these markers [6].
In an attempt to develop new hypoxia markers exhibiting rapid localization
in hypoxic tissue, the authors present the synthesis of new hypoxia PET markers
with higher lipophilicity than 18FMISO and the required β-oxygen atom (Fig. 1).

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 ABDEL-JALIL et al.

(a) (b)
N N OTs N N X
N NH O O
n n
NO2 (1) NO2 NO2
X=F X = 18F
2a n = 1 3a n = 1 4a n = 1
b n=2 b n=2 b n=2
c n=3 c n=3 c n=3

a) TsO-(CH2CH2O)n = 2, 3, 4-Ts, Et3N, DMF, rt, 4 d; b) (i) CsF, [bmim][BF4], CH3CN, 100 °C, 1h or
(ii) n-Bu4NF, THF, rt, 5d; 18F-radiolabeling: (iii) K18F/Kryptofix 2.2.2., CH3CN, 80 °C, 10 min.

FIG. 1. Synthetic scheme for 2-nitroimidazole derivatives.

REFERENCES

[1] LIM, J.L., BERRIDGE, M.S., Appl. Radiat. Isot. 44 (1993) 1085-1091.
[2] TOLVANEN, T., et al., J. Nucl. Med. 43 (2002) 1674-1680.
[3] KAEMAERAEINEN, E.-L., KYLLOENEN, T., NIHTILAE, O., BJOERK, H.,
SOLIN, O., J. Label. Compd. Radiopharm. 47 (2004) 37-45.
[4] REISCHL, G., et al., J. Nucl. Med. 43 (2002) 364.
[5] MACHULLA, H.-J., Imaging of Hypoxia, Kluwer Acad. Publishers, Netherlands,
1999.
[6] MATHIAS, C.J., et al., Life Sci. 41 (1987) 199-206.

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 RADIOSYNTHESIS AND IN VIVO EVALUATION IN
MELANOMA-BEARING MICE OF O-(2-
[18F]FLUOROETHYL)-L-TYROSINE AS A TUMOUR
TRACER
MINGWEI WANG, DUANZHI YIN, YONGXIAN WANG
Radiopharmaceutical Centre,
Shanghai Institute of Applied Physics, CAS,
Shanghai, China
Email: wmwnuclear@163.com

Abstract

Radiolabelled amino acid O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET), a clinical

potential PET tracer for metabolic imaging of tumour, was synthesized from direct
nucleophilic displacement reaction of the precursor N-BOC-(O-2-tosyloxyethyl)-L-
tyrosine methyl ester with the activated [18F]fluoride ion, followed by acidic and basic
hydrolysis of the protective groups. The biological evaluation of [18F]FET was investi-
gated with biodistribution studies and qualitative autoradiography (QARG) in B16
melanoma-bearing mice and its activity–time curve was investigated in normal mice.
The total synthesis time was within 60 min and the radiochemical yield was about 45%
(not decay corrected) and a radiochemical purity of more than 95% after simplified
solid phase extraction. [18F]FET showed rapid and high uptake and long retention in
tumour as well as low uptake in the brain. The ratios of tumour to brain, tumour to
muscle, tumour to blood and tumour to skin of [18F]FET were 4.13 ± 0.38, 2.01 ± 0.20,
2.03 ± 0.12 and 3.40 ± 0.14, respectively at 30 min post-injection and 3.63 ± 0.44, 2.85 ±
0.32, 2.80 ± 0.76 and 4.26 ± 0.63, respectively at 60 min post-injection. The QARGs
demonstrated remarkable accumulation of [18F]FET in melanoma with high contrast. In
conclusion, [18F]FET was synthesized via a convenient route and it could be a very
practical PET tracer for brain tumour imaging and a probable alternative for peripheral
tumour imaging.

1. INTRODUCTION

2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) is the most important 18F

labelled radiopharmaceutical used worldwide as a positron emission
tomography (PET) tracer for tumour diagnosis [1, 2]. However, its inherent
limitations for brain tumour diagnosis are also well known, such as the false
positive findings due to the uptake of the normal brain tissues and inflam-
matory and infectious lesions [3–5] and low imaging contrast for brain tumour

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diagnosis [6, 7]. During the search for new PET tracers, radiolabelled amino
acids were widely studied and their powerful clinical potential as tumour PET
tracers in neuro-oncology and for peripheral tumours such as lymphoma
demonstrated [8]. More attention has been paid to the positron emitter 18F
labelled amino acids owing to its advantages over 11C for PET and 123I for
SPECT [9], of which the most typical example is O-(2-[18F]fluoroethyl)-L-
tyrosine ([18F]FET), an analogue of tyrosine developed recently [3, 10]. It was
synthesized by a nucleophilic displacement reaction with much higher radio-
chemical yield than L-[18F]FT [11, 12] and [18F]FMT [13, 14], which were both
derived from an electrophilic substitution reaction. Originally, [18F]FET was
prepared by a two-step reaction consisting of fluorination of 1,2-
bis(tosyloxy)ethane and fluoroethylation of unprotected L-tyrosine, then using
HPLC [10] or simple solid phase extraction [15] to separate the final product.
However, the existing two-step synthesis of [18F]FET appears cumbersome
because of the two separate purifications involved in the process. Later, one-
step syntheses of [18F]FET via direct nucleophilic radiofluorination of corre-
sponding precursors were developed by Hamacher and Coenen [16] and
Wanga et al. [17].
In this paper, the authors present a novel synthesis method of [18F]FET that
they have developed from easily available precursor to improve the radiochemical
yield and to simplify the purification procedure. The feasibility of [18F]FET as a
PET tracer for brain and probably peripheral tumour imaging was evaluated in
B16 melanoma-bearing mice via biodistribution and autoradiography.

2. MATERIALS AND METHODS

2.1. General

Aminopolyether Kryptofix 222 (4,7,13,16,21,24 hexaoxa-1,10-diazabi-

cyclo [8.8.8]hexacosan, K2.2.2.) and dry acetonitrile were purchased from Acros,
tetrabutylammonium fluoride trihydrate (TBAF·3H2O) from Fluka, N-BOC-
L-tyrosine methyl ester (N-BOC-L-Tyr-OMe) from GL Biochem (Shanghai)
Ltd, trifluoroacetic acid from Merck and 1,2-bis(tosyloxy)ethane from TCI. Other
chemicals, including sodium hydroxide, potassium carbonate, ammonium
chloride, ethyl acetate, acetonitrile and chloroform were of spectral analysis
grade or HPLC grade and obtained from the Shanghai Chemical Company
(China). All of the chemicals and solvents were used directly without further
purification. Sep-Pak Silica Plus cartridge for solid phase extraction and mini-
vials were purchased from Waters Corporation, United States of America and

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 SESSION 12

Alltech Associates, Inc., USA, respectively. GF254 silica gel plate based on glass
came from the Huiyou Silica Development Co. Ltd, Yantai, Shangdong, China.
1
H NMR assays were performed on an AVANCE 500 NMR spectrometer
(BRUKER). Mass spectra were recorded on a MicroMass GCT CA 055 mass
spectrometer (Agilent LCMSD-SL, Agilent Technologies, Palo Alto, USA).
Autoradiography was performed on a Packaged Cyclone Storage Phosphor
System with storage phosphor scanner and screen (Perkin Elmer, USA).
Radioactivity was measured with either a radioactivity counter (FJ-391A2,
Beijing Nuclear Instruments, Beijing, China) or radioimmune gamma counter
(SN-697, Rihuan Photoelectric Instruments Co. Ltd, Shanghai, China). Thin
layer chromatography (TLC) was performed using an imaging scanner
(AR-2000, Bioscan, USA) or visualized by UV lamp (Anting Electronic Instru-
ments, Shanghai, China). HPLC was carried out on a Dionex summit system
equipped with a P680 HPLC pump (Dionex, USA), PDA-100 photodiode
array detector, flow count detector (Bioscan, USA), analytical Vydac C18
column (10 μm, 2.5 mm × 250 mm, Shimadzu Corporation, Japan).
No-carrier-added aqueous [18F]fluoride ion was supplied by Amersham
Kexing Pharmaceuticals Co. Ltd.

2.2. Synthesis of [18F]FET

2.2.1. Preparation of the radiofluorinated precursor

To a solution of N-BOC-L-tyrosine methyl ester (366 mg) in dry MeCN

(10 mL) was added a solution of 1,2-bis(tosyloxy)ethane (580 mg) in dry MeCN
(15 mL) and K2CO3 (30 mg) and the reaction mixture was stirred under reflux.
After 3 h, the solvent was removed under reduced pressure and the residue
redissolved in saturated aqueous NH4Cl solution and extracted with AcOEt.
The organic phase was dried over MgSO4, filtered and solvent removed under
reduced pressure. The residue was purified by chromatography on silica gel
eluted with CH2Cl2 and CHCl3/Et2O (2:1, vol/vol) to afford N-BOC-(O-2-
tosyloxyethyl)-L-tyrosine methyl ester as yellow oil. MS-EI (m/z,) 493 (M+,
0.8), 437 (0.6), 420 (1.1), 392 (0.3), 377 (1.1), 376 (4.3), 360 (0.5), 332 (0.2), 305
(59), 264 (0.2), 248 (1.1), 219 (0.2), 199 (100), 188 (1.0), 155 (17), 134 (2.2), 117
(1.4), 107 (6.4), 91 (35), 90 (6.0), 88 (5.3), 77 (0.5), 65 (3.4), 59 (3.4), 57 (9.7), 41
(1.6) 1H-NMR (CDCl3/TMS)δ = 7.83–7.81 (d, J = 8Hz, 2H), 7.36–7.34 (d, J
= 8Hz, 2H), 7.01–7.00 (d, J = 8Hz, 2H), 6.73–6.71 (d, J = 8Hz, 2H), 4.55–4.52 (t,
J = 6Hz, 1H), 4.36–4.35 (m, 2H), 4.13–4.11 (m, 2H), 3.71 (s, 3H), 3.06–2.97 (m,
2H), 2.46 (s, 3H), 1.42 (s, 9H).

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 WANG et al.

2.2.2. Direct nucleophilic radiofluorination

No-carrier-added aqueous [18F]fluoride ion was treated as previously

described [18, 19] to afford the activated 18F-–K2.2.2.–K2CO3 complex. The
synthesis route of [18F]FET was determined as in Fig. 1, with slight modifica-
tions made according to the literature [16, 17].
To the above complex was added a solution of N-BOC-(O-2-
tosyloxyethyl)-L-tyrosine methyl ester (5 mg) in anhydrous acetonitrile
(0.7 mL) and the mixture was heated to 130°C for 30 min. The reaction mixture
was dried by evaporation under reduced pressure with a stream of N2 gas.

2.2.3. Solid phase extraction and removal of protective groups

After cooling to room temperature with a cold nitrogen stream, the dry
residue was redissolved in dichloromethane (1.5 mL). The solution was passed
through a Sep-Pak Silica Plus cartridge preconditioned with diethyl ether and
then eluted with diethyl ether (3 mL). The eluate was dried again with a stream
of N2 gas. Trifluoroacetic acid (0.5 mL) was added to the dry residue and
maintained at room temperature for 5 min. Afterwards, the solvent was
evaporated under a continuous N2 flow. To the residue was added 1 mol/L
aqueous NaOH solution (0.5 mL), followed by heating for 10 min at 80°C to
conduct hydrolysis. The reaction mixture was neutralized with a 1 mol/L
solution of HCl (0.5 mL). After adding a certain volume of phosphate buffer
saline (PBS, pH7.4), the solution was passed through a sterile 0.22 µm
membrane filter to afford the isotonic [18F]FET injection solution.

2.2.4. Quality analyses

The radiochemical purity of [18F]FET was determined using TLC and

HPLC. TLC was performed on gel plate based on glass, using CH3CN/H2O
(95/5, vol/vol) as the developing agent. The mobile phase of HPLC was

COOMe COOH
a), b)
TsO 18
NHBoc F NH2
O O

N-BOC-(O-TsE)-L-Tyr-OMe [18F]FET

FIG. 1. Reaction conditions: a) 18F-–K2.2.2.–K2CO3, CH3CN, 130°C, 30 min; (b) Sep-Pak

Silica Plus cartridge; TFA, RT, 3 min; NaOH/H2O, 90°C, 5 min.

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 SESSION 12

ethanol/water/acetic acid/ammonium acetate (10/87.5/2.25/0.25, by volume) at

a flow rate of 1 mL/min.

2.3. Animal experiments

The animal experiments were performed according to the guidelines of

the Shanghai Institute of Pharmaceutical Industry (SIPI). The female C57BL/6
mice bearing B16 melanoma (weighing 20–22 g) and normal mice were also
supplied by SIPI. All the animals were kept in cages with standardized
conditions of light, asepsis and free access to water and food. Mice were
inoculated with B16 melanoma cell in the front right armpit. Experiments were
performed 8–10 d after inoculation of the tumour cells. At this time, the
tumours weighed 300–800 mg and a tumour diameter was about 0.5–1.0 cm.

2.3.1. Biodistribution studies

The tumour-bearing mice were injected with 2.90–3.70 MBq (80–100 μCi)
of [ F]FET in 100 μL of PBS through the tail vein. Three mice were sacrificed
18

at each time point by extirpation of eyeball at 5, 10, 30, 60, 90 and 120 min post-
injection. After, the dissection was carried out and the tissue samples of
interest, including tumour, brain, liver, heart, lung, spleen, kidney, stomach,
small intestine, pancreas, muscle, bone, skin and blood, were collected. All
samples were weighed and the radioactivity was measured using a SN-697 type
radioimmune gamma counter, applying a decay correction. Counts were
compared with those of standards and the results were expressed as a
percentage of injected radioactivity dose per gram of tissue (%ID/g).

2.3.2. Qualitative autoradiography (QARG)

Another three groups of model mice (n = 3 in each group) were also

injected with 2.90–3.70 MBq (80–100 μCi) of [18F]FET in 100 μL of PBS into
the tail vein and sacrified at 10, 30 and 60 min post-injection for QARG.
QARG was performed using non-sectioned whole body autoradiography. In
other words, the intact, completely bleeding carcases of each group were placed
directly onto the multisensitive storage phosphor screen for exposure of 5 min
in the imaging cassette at room temperature, without cutting into micron thick
sections. Then, the storage phosphor screens were read by the storage
phosphor scanner with the resolution of 300 DPI to acquire the phosphor
image for the qualitative observation of tumour uptake of [18F]FET.

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 WANG et al.

2.3.3. Activity–time correlation of blood

Before the above animal experiments, three normal mice were injected in
the tail vein with [18F]FET for the investigation of activity–time correlation of
blood. 5 μL of blood was collected by cutting the tail at 1, 3, 5, 10, 15, 30, 45, 60,
90, 120, 150 and 180 min post-injection followed by measurement of the radio-
activity of blood at each time point. The activity–time correlation of blood was
expressed as a function of time with radioactivity per μL of blood and analysed
with DAS 1.0 drug analysis software.

3. RESULTS AND DISCUSSION

3.1. Synthesis of [18F]FET

Originally, [18F]FET was prepared by a two-step reaction consisting of

fluorination of 1,2-bis(tosyloxy)ethane and fluoroethylation of unprotected
L-tyrosine, then using HPLC [10] or simple solid phase extraction [15] to
separate the final product. However, the existing two-step synthesis of
[18F]FET appeared cumbersome because of the two separate purifications
involved in the process. Later, one-step syntheses of [18F]FET via direct
nucleophilic radiofluorination of corresponding precursors were developed by
Hamacher et al. [16] and Wanga et al. [17].
In this paper, [18F]FET has been prepared from the nucleophilic substi-
tution of the above tosylated precursor with [18F]fluoride ion in CH3CN at
130°C for 30 min followed by acidic and basic hydrolysis as shown in Fig. 1. The
total synthesis time was about 50 min and a high radiochemical yield was
recorded (45% on average, no decay corrected) and high radiochemical purity
(more than 95%). Purification via a Sep-Pak Silica Plus cartridge prior to
hydrolysis was used instead of HPLC after hydrolysis, which resulted in the
simplification of the preparation and the shortening of the whole synthesis
time.

3.2. Biodistribution studies

The biodistribution results of [18F]FET in B16 melanoma-bearing mice

are summarized in Table 1. [18F]FET showed very fast accumulation into the
whole body with a peak occurring at 10 min after IV administration. The radio-
activity in the blood of [18F]FET injected mice decreased from 12.6 ± 3.8 at 10
min post-injection to 2.0 ± 0.3 at 180 min post-injection. The uptakes of
[18F]FET in all organs decreased with time from 10 min post-injection, e.g.

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TABLE 1. BIODISTRIBUTION OF [18F]FET IN ORGANS OF B16

MELANOMA-BEARING MICE
(n = 3, mean ± SD)

Uptake (%ID/g)
5 min 10 min 30 min 60 min 90 min 120 min 150 min 180 min
Tumour 12.7 ± 6.6 19.4 ± 5.6 16.3 ± 5.7 14.6 ± 0.7 12.9 ± 2.0 8.2 ± 3.8 2.4 ± 0.1 2.0 ± 0.1
Skin 5.0 ± 3.6 7.5 ± 1.8 4.8 ± 0.2 3.4 ± 1.4 2.5 ± 1.2 1.9 ± 0.0 1.0 ± 0.1 1.3 ± 0.2
Brain 2.4 ± 1.3 4.3 ± 0.7 3.9 ± 0.6 4.0 ± 0.0 3.5 ± 0.3 3.4 ± 1.8 2.5 ± 0.3 2.0 ± 0.1
Liver 11.3 ± 4.4 14.2 ± 1.5 7.5 ± 0.5 4.8 ± 0.4 4.2 ± 0.3 2.9 ± 0.6 1.8 ± 0.7 1.5 ± 0.7
Heart 12.5 ± 4.5 14.3 ± 2.6 8.1 ± 0.6 5.3 ± 0.4 4.7 ± 0.5 3.0 ± 0.8 2.4 ± 0.4 1.8 ± 0.5
Lung 10.9 ± 3.9 14.4 ± 2.6 8.6 ± 0.6 5.6 ± 0.6 4.7 ± 0.6 3.3 ± 1.0 2.7 ± 0.2 1.8 ± 0.7
Spleen 9.3 ± 5.1 15.0 ± 1.7 8.4 ± 0.8 6.0 ± 0.5 5.8 ± 0.6 3.5 ± 0.9 3.4 ± 0.9 2.0 ± 0.7
Kidney 15.4 ± 6.5 14.9 ± 1.8 9.3 ± 0.1 6.6 ± 0.6 5.7 ± 0.9 3.7 ± 0.7 3.3 ± 0.7 2.1 ± 0.2
Intestine 9.7 ± 4.3 12.8 ± 1.6 7.3 ± 0.8 5.0 ± 1.0 5.0 ± 0.6 5.0 ± 0.3 2.5 ± 0.6 2.0 ± 0.2
Pancreas 27.5 ± 3.7 59.8 ± 9.1 41.1 ± 8.1 32.9 ± 8.6 26.4 ± 9.2 21.1 ± 7.0 15.8 ± 7.8 11.1 ± 1.5
Muscle 9.5 ± 4.9 12.2 ± 2.4 8.1 ± 0.6 5.1 ± 0.5 4.6 ± 0.5 3.0 ± 0.6 2.8 ± 0.4 1.9 ± 0.0
Bone 10.0 ± 4.1 17.7 ± 1.7 14.6 ± 3.3 11.9 ± 1.1 11.3 ± 1.0 10.9 ± 3.8 9.6 ± 0.8 8.2 ± 1.2
Stomach 10.3 ± 5.8 10.7 ± 1.3 6.0 ± 1.2 5.9 ± 0.8 5.8 ± 1.8 4.4 ± 1.3 3.3 ± 0.5 2.5 ± 0.2
Blood 12.0 ± 3.8 12.6 ± 3.8 8.0 ± 0.6 5.2 ± 0.2 5.0 ± 1.4 2.8 ±0.6 2.7 ± 0.3 2.0 ± 0.3

19.4 ± 5.6, 4.3 ± 0.7 and 59.8 ± 9.1 at 10 min post-injection to 2.0 ± 0.1, 2.0 ± 0.1
and 11.1 ± 1.5 at 180 min post-injection in the tumour, brain and pancreas,
respectively. [18F]FET showed a certain volume of bone uptake and retention
of [18F]FET in the pancreas was higher than that in the other organs as
previously reported [10, 17].
The ratios of T/B, T/M, T/Bd and T/S of [18F]FET were 4.13 ± 0.38, 2.01 ±
0.20, 2.03 ± 0.12 and 3.40 ± 0.14 at 30 min post-injection and 3.63 ± 0.44, 2.85 ±
0.32, 2.80 ± 0.76 and 4.26 ± 0.63 at 60 min post-injection. The higher T/B ratios
indicated the usefulness of [18F]FET as a PET tracer for brain tumour imaging.
Moreover, [18F]FET was also shown to be a promising PET tracer for
peripheral tumour imaging with great potential due to the relatively high ratios
of T/M, T/Bd and T/S.

3.3. QARG

The accumulation of [18F]FET in the tumour was further confirmed by in

vivo QARGs as shown in Fig. 2. The uptake of [18F]FET in B16 melanoma was

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 WANG et al.

10 min 30 min 60 min

FIG. 2. QARG via non-sectioned whole body autoradiography in the B16 melanoma-
bearing mice after intravenous injection of [18F]FET. The arrow indicates the location of
the tumour.

visualized vividly at 30 min post-injection and maintained enough contrast of

tumour-to-counterparts up to 60 min post-injection. These observations were
highly consistent with the results obstained from the biodistribution studies.
QARGs showed that the biodistribution patterns of [18F]FET correlated with
the implantation positions of the tumours.
Actually, [18F]FET has been investigated to diagnose peripheral tumour
by PET imaging [20]. Here, QARG was performed using the non-sectioned
whole body autoradiography method which has not, to the authors’ knowledge,
been used to characterize in vivo biological behaviour of radiolabelled
substances to date. Generally, conventional autoradiographs are carried out by
frozen section autoradiography using a freeze microtome to cut the tissues of
interest, such as the brain or whole body, into sections of thickness ~10–30 μm
[21]. The whole procedure is very cumbersome and time consuming, which is a
disadvantage for short lived positron emitters such as 18F and 11C. It is
necessary to acquire quantitative information regarding the distribution of the
receptor in the brain or the accumulation of each organ in the whole body for
related tracers. However, when qualitative visualization of accumulation of
tracers in tumour tissue is the main purpose, QARG should be taken as a
selective method. Qualitative evaluation of tumour uptake could be achieved
rapidly by QARGs within about 30 min of acquisition of carcases injected
with tracers. Certainly, its limitations are also obvious compared with conven-
tional autoradiographs and small animal PET (microPET), availability of
which is rather restricted at present [22].

3.4. Blood activity–time correlation

The blood radioactivity curve of [18F]FET is depicted in Fig. 3. The

analysis of the results implied that the activity–time correlation of blood in
mice was consistent with a two-component model with a short distribution

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 SESSION 12

3000

2500

2000
cpm/uL

1500

1000

500

0
0 20 40 60 80 100 120 140 160 180 200
time (min)

FIG. 3. Blood activity–time correlation of [18F]FET in mice.

phase (T1/2α = 3.7 min) and long clearance phase (T1/2β = 448.0 min). Combined
with the above results of biodistribution and QARGs, this suggested that
[18F]FET might be a rapid PET imaging agent for tumour diagnosis.

4. CONCLUSION

[18F]FET was directly synthesized from the tosylated precursor of N-

BOC-(O-2-tosyloxyethyl)-L-tyrosine methyl ester within 60 min with a good
radiochemical yield (45% on average, not decay corrected) and radiochemical
purity (more than 95%). Biological evaluation of [18F]FET in B16 melanoma-
bearing mice showed significantly high ratios of T/B, T/M, T/Bd and T/S. The
results demonstrated that [18F]FET could be used as a useful PET tracer for
brain tumour imaging and probably for peripheral tumour imaging.

ACKNOWLEDGEMENTS

The authors are very grateful to Amersham Kexing Pharmaceuticals Co.

Ltd for the supply of no-carrier-added aqueous [18F]fluoride ion. Furthermore,
they appreciate the excellent technical support in the animal experiments
provided by the technologists at their institution.

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 WANG et al.

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CYCLOTRON BASED RADIONUCLIDES
AND GENERATORS

(Session 13)

Chairpersons

M.M. VORA
Saudi Arabia

M. HAJI-SAEID
IAEA
.
 PRODUCTION OF RADIONUCLIDES WITH A
CYCLOTRON

D.J. SCHLYER
Department of Chemistry,
Brookhaven National Laboratory,
Upton, New York,
United States of America
Email: schlyer@bnl.gov

Abstract

The production of radioisotopes for use in biomedical procedures such as diag-

nostic imaging and/or therapeutic treatments may be achieved through nuclear
reactions from charged particle bombardment using an accelerator. The goal is to get
the target material into the beam, keep it there during the irradiation and then remove
the product radionuclide from the target material efficiently and quickly. The specific
design of the cyclotron target is what allows one to achieve this goal. For every radio-
nuclide, there are usually nearly as many target designs as there are people producing
the isotope. The design and use of cyclotron targets can be a very complex problem and
involves the use of physics, chemistry and engineering in order to produce a target which
is reliable and efficient. This paper addresses how basic principles of physics, engi-
neering and chemistry apply to radionuclide production with an accelerator. The
concept of power density applied to cyclotron targets, various means of heat removal
from the target, and the efficient extraction and separation of the desired product radio-
nuclide from both the target material and the other radioisotopes present is discussed.

1. INTRODUCTION

Nuclear medicine offers one of the safest ways to diagnose and/or treat a
number of serious, life threatening diseases including cancer. It does so without
adverse effects on normal organs and without the debilitating side effects of
some of the more common treatments and extended hospital stays. Each day,
thousands of patients with cancer, heart disease and other illnesses receive a
radioisotope injection either for diagnosis or for treatment. Radioisotopes and
radiopharmaceuticals, which are at the heart of nuclear medicine, are used in
the United States of America alone in almost 40 000 procedures every day, and
in more than 100 million laboratory tests each year [1–4]. Radioisotopes for
these uses are produced either by neutron bombardment of a target material in
nuclear reactors or from charged particle bombardment in particle accelerators.

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 SCHLYER

In accelerators, the typical charged particle reactions utilize protons, deuterons

and helium nuclei (3He++ and α~ particles) [5].
One clear advantage that accelerators possess is the fact that, in general,
the target and product are different chemical elements. This makes it possible
to find a suitable chemical or physical means of separation and to achieve high
specific activities. The ability to control the energy of the bombarding particle
can also lead to fewer radioisotopic impurities by selecting the optimum energy
window for irradiation.
The availability of accelerators fits into several categories. First there are
university based cyclotrons that are typically multiparticle machines with
energies around 30–50 MeV. Then there are the hospital based machines, which
are generally dedicated to the production of the standard PET radioisotopes
(11C, 13N, 15O and 18F). These cyclotrons accelerate protons in the 10–19 MeV
range, and some also produce deuterons with an energy of about half that of
the proton (5–9 MeV). The cyclotrons used by industry for large scale
production are typically 30 MeV proton only machines, although there are
some using lower energies for dedicated production of some radioisotopes [6].

2. NUCLEAR REACTIONS

2.1. Compound nucleus

There is a wide variety of nuclear reactions which are used in an

accelerator to produce the artificial radioactivity. The energies of the
bombarding particles which are used range from a few MeV to hundreds of
MeV [7]. One of the most useful models for nuclear reactions is the compound
nucleus model original introduced by Bohr in 1936. In this model, the incident
particle is absorbed into the nucleus of the target materials and the energy is
distributed throughout the compound nucleus. In essence, the nucleus comes to
some form of equilibrium before decomposing with the emission of particles.
These two steps are considered to be independent of one another. It doesn’t
matter how the compound nucleus got to the high energy state, the evaporation
of the particles will be independent of the way in which it was formed. The total
amount of excitation energy contained in the nucleus will be given by the
equation:

MA
U= T + Sa
MA + Ma a

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 SESSION 13

where: U = excitation energy

MA = mass of the target nucleus

Ma = mass of the incident particle

Ta = kinetic energy of the incident particle

Sa = binding energy of the incident particle in the compound nucleus

The nucleus can decompose along several channels as shown in Fig. 1.

2.2. Q values

There is a minimum energy below which a nuclear reaction will not occur
except by tunnelling effects. When the compound nucleus decomposes, the
kinetic energy of all the products may be either greater or less than the total
kinetic energy of all the reactants. If the energy of the products is greater, then
the reaction is said to be exoergic. If the kinetic energy of the products is less
than the reactants, then the reaction is endoergic. The magnitude of this
difference is called the Q value. If the reaction is exoergic, Q values are
positive.

Q= Â D(react) - Â D(prod)

ELASTIC
a + A SCATTERING

INELASTIC
a + A SCATTERING
a A Aa

b + NUCLEAR
B
REACTION 1

b + c + D NUCLEAR
REACTION 2

FIG. 1. Compound nucleus and reaction pathways.

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 SCHLYER

Q value Threshold

15O 5.1 MeV 0 Mev

n
12C 13.6 MeV 0 Mev
α
t
d + 14N 16O 13N -4.3 MeV 4.9 Mev
n+p
14N -2.2 MeV 2.5 Mev
γ

16O 20.7 MeV 0 Mev

FIG. 2. Diagram of the reactions channels and their associated Q values and threshold
energies for the reaction of a deuteron with a 14N nucleus.

An example of the possible reaction pathways is shown in Fig. 2 along

with their corresponding Q values and reaction thresholds. The energy changes
in a nuclear reaction are large enough that changes in the mass of the reactants
and products are observable.
The threshold energy is defined as the minimum projectile energy
necessary to satisfy mass energy and momentum conservation. The incident
particle energy must be sufficient to overcome the Coulomb barrier and to
overcome a negative Q of the reaction. Particles with energies below this
barrier have a very low probability of reacting.

2.3. Coulomb barrier

The Coulomb barrier is a measure of the nucleus–nucleus charge

repulsion. The Coulomb barrier is given by the relationship.

(Z p e)(Z T e) Z pZ T e 2 0.90Z p Z T
Vcoul = = ª MeV
d Rp + RT p + AT )
(A 1/3 1/3

where: Vcoul = Coulomb barrier

Zp = atomic number of the particle

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 SESSION 13

ZT = atomic number of the target

Rp = atomic radius of the particle

RT = atomic radius of the target

Ap = atomic mass of the particle

AT = atomic mass of the target

Since the compound nucleus must carry off some kinetic energy, the
Coulomb barrier measured in the laboratory will be corrected by the amount:

ÈA ˘
lab
Vcoul = Í CN ˙ Vcoul
Î AT ˚

where ACN is the mass of the compound nucleus and AT is the mass of the
target.
Thus, the energy required to induce a nuclear reaction increases as the Z
of the target material increases and as the Z of the incident particle increases.
For many low Z materials it is possible to use a low energy accelerator, but for
high Z materials, it is necessary to increase the particle energy [8].

2.4. Nuclear reaction cross-section

The nuclear reaction cross-section represents the total probability that a

compound nucleus will be formed and that it will decompose in a particular
channel. This parameter is often symbolized by the Greek letter σ and has the
units of barns (10-24 cm2). The number of reactions occurring in one second is
given by the relation [8]:

dn = I0NAdsσAB

where: dn = number of reactions occurring in one second

I0 = number of particles incident on the target in one second

NA = number of target nuclei per gram

ds = thickness of the material in grams per cm2

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 SCHLYER

σab = cross-section expressed in units of cm2

In practical applications, the thickness ds of the material can be

represented by a slab of thickness s, thin enough that the cross-section can be
considered constant. NAds is then the number of target atoms in a 1 cm2 area of
thickness s. If the target material is a compound rather than a pure element,
then the number of nuclei per unit area is given by the expression:

FAC¡
NA =
AA

where: NA = number of target nuclei per gram

FA = fractional isotopic abundance

C = concentration in weight

ℑ = Avogadro’s number

AA = atomic mass number of nucleus A

3. PRACTICAL PRODUCTION OF RADIOISOTOPES

3.1. Thick target yields

The cross-section is always a function of energy and therefore the yield of

the reaction y(E) is also a function of energy. When the cross-sections σ(E) of
all reactions concerned are known, the thick target yield Y(E, ΔE) for each
radionuclide can be calculated by either numerical or analytical integration of
y(E), as a function of both incident projectile energy E and energy loss ΔE of
beam in the target itself.
E
Y(E, DE) = Ú
E - DE
y(E)dx

where: Y(E,ΔE) = yield between the energy E and E-ΔE

ΔE = energy lost as the beam passes through a distance x of the target

y(E) = yield of a particular radionuclide at a particular energy E

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 SESSION 13

This definition holds in the raw approximation of a monochromatic beam

of energy E, in which the integrand y(x) represents the thin target excitation
function.

3.2. Saturation factors

The rate of production is of course affected by the fact that the resulting
nuclide is radioactive and is thus undergoing radioactive decay. For short lived
nuclides, the competition between formation and decay will come to
equilibrium after sufficiently long bombardment times. This point is called
saturation, meaning that no matter how much longer the irradiation occurs the
production rate is equal to the rate of decay and no more product will be
formed.
The rate of formation in this case is given by:

R = Nλ/(1-e-λt)

where: R = rate of formation of nuclei

N = number target nuclei present at the end

l = decay constant

The term in the denominator is often referred to as the saturation factor

and accounts for the competition of the production of nuclei due to the particle
reaction and the radioactive decay of the nuclei which have been produced. It
is clear why the assumption had to be made that the beam current was nearly
constant since a variation in the beam current would affect the relative number
of nuclei being created versus the number being destroyed by decay.
If this relation is substituted back into the cross-section equation then the
result is:

È Al N i ˘
s i = 2.678 x 10 -10 Í - lt ˙
Î Ir x(1 - e ) ˚

where: σi = cross-section for process (mb)

A = atomic mass of the target (amu)

λ = decay constant for species i (sec-1)

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 SCHLYER

ρ = density of the target material (g/cm3)

t = time of irradiation (sec)

x = thickness of the target (cm)

I = beam current (μA)

It should be noted that the time of irradiation and the decay constant are
both given in the seconds. This is due to the fact that the beam current is
defined in microamperes (μA) which is 6.2 × 1012 charges/second.

3.3. Practical production times

At shorter irradiation times, the fraction of product generated is related

to the saturation factor given by (1-e-λt), where λ. is the decay constant for the
decaying nuclide and t the bombardment time. It is evident that an irradiation
equivalent to one half-life would result in 50% saturation. The practical
production limit of a given radionuclide is then in most part determined by the
half-life of the isotope. It is relatively easy to come near saturation for the
production of 15O with a 2 min half-life, but it is not reasonable to irradiate a
target for the production of 18F to near the point of saturation because of the
times involved.

3.4. Radionuclidic purity

One of the basic facts of life in radioisotope production is that it is not

always possible to eliminate the radionuclidic impurities even with the highest
isotopic enrichment and the widest energy selection. An example of this is
given in Fig. 3 for the production of 123I with a minimum of 124I impurity [9–12].
As can be seen from Fig. 3, it is not possible to eliminate the 124I impurity
from the 123I using a proton on 124Te reaction because the 124I is being made at
the same energy. All that can be done is to minimize the 124I impurity by
choosing an energy interval where the production of 124I is near a minimum. In
this case, proton energies higher than about 20 MeV will give a minimum of 124I
impurity.

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 SESSION 13

FIG. 3. Cross-sections for the production of 123I and 124I from 124Te using the 124Te(p,n)124I
and 124Te(p,2n)123I nuclear reactions.

3.5. Specific activity

Specific activity is the fraction of radiolabelled molecules relative to the

total number of molecules and is usually expressed as a unit of radioactivity per
mole of compound. Specific activity is a critically important property in the
preparation of radiotracers. It is particularly important in PET, where the
radionuclide is incorporated into a radiotracer that is used to probe some
physiological process in which very small amounts of the native biomolecule
are used. When carrying out these studies, such as probing the number of
receptors or the concentration of an enzyme, considerations of the total mass of
compound injected become even more important [13, 14]. There is, of course,
an ultimate limit to specific activity when nothing but the radioactive atoms or
radiolabelled molecules are present. The characteristics of the same radio-
isotopes are shown in Table 1.
There is a clear need to reach reliable production of high specific activity
radiopharmaceuticals, as present day tracers and certainly future tracers
include receptor/transporter ligands that are potent. In order for PET to be a
true tracer technique, receptor occupancy with the radiolabelled tracer should
be kept below 5% in order to avoid pharmacological or pharmacodynamic
effects.

319
 SCHLYER

TABLE 1. CHARACTERISTICS OF SOME RADIOISOTOPES

Nuclide Half-life (min) Decay mode Maximum specific activity (theoretical)

C-11 20.4 100%+ 9220 Ci/μmole (341 TBq/µmole)

+
N-13 9.98 100% 18 900 Ci/μmole (700 TBq/µmole)
O-15 2.03 100%+ 91 730 Ci/μmole (3394 TBq/µmole)
+
F-18 109.8 97% 1710 Ci/μmole (63.4 TBq/µmole)
+
Cu-62 9.74 99.7% 19 310 Ci/μmole (714 TBq/µmole)
+
Ga-68 68.0 89% 2766 Ci/μmole (102 TBq/µmole)
Br-75 96.0 75.5%+ 1960 Ci/μmole (73 TBq/µmole)
+
Rb-82 1.25 95.5% 150 400 Ci/μmole (5565 TBq/µmole)
+
I-122 3.62 75.8% 51 950 Ci/μmole (1922 TBq/µmole)
+
I-124 6019.2 23.3% 31 Ci/μmole (1.15 TBq/µmole)

4. CYCLOTRON TARGETS

4.1. Types of target

The goal of a cyclotron target is to get the target material into the beam,
keep it there during the irradiation and then remove the product radionuclide
from the target material efficiently and quickly. The specific design of the
cyclotron target is what allows one to achieve this goal. Unless care is taken in
the design and fabrication of the target, the production of the radioisotope can
be far from optimal and may even be impossible. Although the underlying
nuclear phenomena are very well known, target behaviour in the form of yield,
maximum beam current and obtainable specific activity varies within and
across different users. Much of this variation can be attributed to less than
optimal matching of the targets and their operating conditions to the actual
cyclotrons used. There is a close interplay between beam parameters (size,
intensity, emittance, orientation and energy) and the performance of a given
target. This variation is seen even within the range of commercial target/
cyclotron combinations. A great deal of useful information about targets and
target chemistry can be found in the proceedings of the International
Workshop on Targets and Target Chemistry. These proceedings are available
on-line by courtesy of TRIUMF at http://www.triumf.ca/wttc/proceedings.html
This is a very valuable resource for all who are concerned with the production
of radioisotopes.

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 SESSION 13

Targets can be made for solids, liquids and gases. There are some
advantages and disadvantages to each of these states of matter. The gases are
usually easy to get in and out of the target and the separation of the radioactive
product from the target material is often simple when compared to solids or
liquids. However, gas targets suffer from such effects as beam density reduction
and in-target chemistry. Liquid targets often boil and solid targets may vaporize
and the radioisotopes may be difficult to separate from the target material after
irradiation. Many of these problems are a direct result of the heating of the
target by the beam.

4.2. Power deposition

One of the main concerns in targets is the deposition of power in the

material during irradiation. If the power deposited exceeds the ability of the
target to remove the heat, the target will eventually be destroyed or the target
material will be melted, volatilized or reduced in density to the point where the
yield will be drastically reduced. In liquid targets, the material may boil and
thereby reduce the average density. In gaseous targets, the density of the gas is
reduced in the beam strike area. All these effects are a result of the increased
temperature in the beam strike area and this in turn is a result of the power
deposited by the beam as it passes through matter.

Power (W) = 1 mAΔE (MeV)

The power deposited in the material is the beam current in microamps

multiplied by the energy loss in MeV and the result is the number of watts
deposited.
The exact position of the heat deposition will depend on the dE/dx
(stopping power) of the beam in the target material with most of the heat being
deposited near the end of the particle range in the Bragg peak. A simple
approximation for the stopping power is given by the relation:

- dE 4p z2 e4 ¡Z 2m0 V2
= ln
dx m0 V2 A I

where: dE/dx = energy loss per unit length

z = atomic number of the projectile

e = elementary charge 4.803 × 10-10 (erg-cm)1/2

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 SCHLYER

m0 = electron rest mass (g)

V = relativistic projectile velocity (cm-sec-1)

ℑ = Avogadro’s number

Z = atomic number of the target material

I = adjusted ionization potential of the target material (eV)

Some additional helpful approximations are that the relativistic velocity

is given by the relation:

E 9
V = 1.384 10 cm/s
m

where: E = particle energy in MeV

m = particle atomic mass number

The other useful approximations for the adjusted ionization potential are:

I = 13 Z (eV) if Z ≤ 13

I = 9.76 Z + 58.8 Z-0.19 (eV) if Z > 13

The stopping power of particles other than protons is given by the

relationships:

deuterons Sd(E) = Sp(E/2)

tritons St(E) = Sp(E/3)

3
He S (E) = 4Sp(E/3)

4
He S(E) = 4Sp(E/4)

4.3. Heat transfer

In order to have a useful accelerator target for the production of a radio-

nuclide, it is necessary to remove the heat generated by the passage of the

322
 SESSION 13

beam. There are three modes of heat transfer which are active in targets —
conduction, convection and radiation. Radiation is only a significant mode of
heat loss at high temperatures (>500ºC). Gases and liquids can transfer heat via
convection and conduction. Heat transfer in solids is somewhat simpler than in
other media since the heat usually flows through the target matrix mainly by
conduction.
Once the heat has been transferred to the cooled surface of the target, it
will usually be removed by a fluid such as water flowing against the back of the
target. Most of the problems arise at the interfaces, where there are discontinu-
ities in the heat transfer, such as where the target material meets the backing
material or where the backing material meets the cooling water. The more
efficient the transfer design at these interfaces, the better the heat transfer will
be and the less likely one is to have problems with loss of target material or
damage to the target during the irradiation.
In cyclotron targetry, it is usual to have both free convection and forced
convection. In a gas or liquid target during irradiation, the heating of the fluid
inside the target body will cause free convection currents to be set up which will
aid in removing heat from the fluid and decrease the effects of density reduction.
It is also usual to have some forced convective flow of gas over the front entrance
window to the target. For many reasons, this gas is often helium. Since this gas
has very low viscosity, it is very efficient in cooling the front window.
There are several factors which contribute to the maximum beam
currents which may be run with a solid target. Just as is the case with gas and
liquid targets, the beam density is a determining factor. If there are ‘hot’ spots
in the beam, the highest current at which the target may be run will be much
lower than if the beam has a uniform profile.
As a simple example of heat transfer in a solid target, use can be made of
a thallium target which can be considered as a three layer system facing the
accelerator vacuum on one side and the coolant fluid on the other as shown in
Fig. 4 [15].

Layer 1: The 203Tl deposit has a physical thickness of 80 × 10-4 cm and is

denoted by ‘a’ in the diagram. In this layer, 201Pb is produced by the
203
Tl(p,3n)201Pb nuclear reaction using 30 MeV protons hitting the target at a θ°
beam–target angle. Owing to excitation, ionization and bremstrahlung, protons
lose kinetic energy in this layer which is converted into heat. Assuming the
current density (μA/mm2) is constant over the whole surface area, the total
heat production rate qTl (J/s) and the heat production rate per unit of volume
q’’’Tl (J·cm-3·s-1) in this layer are related as:

q Tl = q ’’’
Tl aS

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 SCHLYER

Cu-coolant
Layers Interface

1 2 3

Tl Cu-b Cu-c

VACUUM

Coolant Fluid
cc
30 MeV protons
(Turbulent flow)
aa bb
I( A)

a b c

FIG. 4. The three layer system showing the target material on the far left with the coolant
on the far right.

Layer 2: The Cu–b layer has a physical thickness of b cm. In this layer protons
emerging from the Tl layer are stopped completely. The heat production rate
qCu and the heat production rate per unit volume q’’’Cu in this layer are linked
by:

q Cu = q ’’’
Cu bS

Layer 3: The Cu–c layer has a physical dimension of c cm and merely serves as
a mechanical support for the Tl layer. The total heat (qT) produced in the layers
is transferred to the coolant fluid through this layer by conduction.

qT = qTl + qCu

At the Cu–c/coolant interface, the heat is transferred to the coolant only

by convection, i.e. no subcooled nucleate boiling occurs and the bulk
temperature of the coolant is a constant (Tw).
In steady state conditions, the heat and heat transfers result in a
temperature profile represented in Fig. 5, where:

T = temperature

Tm = maximum temperature at the vacuum/Tl interface

Ti = temperature at the Tl/Cu–b interface

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 SESSION 13

T
Cu-coolant
T Tl Cu-b Cu-c
m Interface
T
i
T
b

T
c

a b c

T
w x

FIG. 5. Temperature profile through the target during irradiation.

Tb = temperature at the Cu–b/Cu–c interface

Tc = temperature at the Cu–c/coolant interface

Tw = temperature of the coolant

The exact temperatures can be calculated if the heat transfer parameters

are known. The integrity of the thallium layer is extremely important since this
will dramatically affect the transfer of heat through the layer as well as the
stopping power of the layer.

4.4. In target chemistry

The chemistry occurring inside a cyclotron target is the basis for the
chemical products which are produced during irradiation. The reaction of the
highly excited nucleogenic atom with the surroundings during the de-excitation is
the determining factor by which radiolabelled molecules will be formed. Many of
the chemical reactions occurring were first studied using hot atom chemistry.
However, the conditions inside the production target are quite different from a
typical hot atom experiment. In the case of a hot atom experiment, the beam
current is usually less than 1 μA and the gas is at a pressure of much less than
100 kPa. In a normal production gas target, the beam current may be 20 or 30 μA
(or higher with the newer targets) and the pressure up to 7 × 106 Pa. However, in
many cases the results from hot atom experiments have been very successful in
explaining the product distributions from production gas targets. The state of the
matter inside a cyclotron target of course depends on the state of the matter

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 SCHLYER

FIG. 6. View inside a gas target (left) during irradiation where the highly excited and
ionized gas molecules emit light and heat. Water target (right) shows light emission from
the highly excited molecules (with perhaps a small contribution from Cherenkov radia-
tion) as the beam slows in the water. The effects of boiling can also be observed in the
picture in the second band of light near the top of the target as the beam passes through the
water vapour and is stopped in the water behind the bubble. Gas target picture courtesy of
S.-J. Heselius.

being bombarded. In a gas target, the gas is highly ionized and ion–molecule, as
well as highly endothermic, reactions are occurring. A view inside the target
during irradiation is shown in Fig. 6 [16–18].
Nearly any chemical species can be formed in this ionic soup. When other
gases are present, either as contaminants or as additives, the situation becomes
very complex. In most cases the final product distribution will be determined
by the thermodynamics of the situation since there is more than enough energy
to overcome the kinetic activation barriers which would place constraints on
the product distribution at lower temperatures.

5. SEPARATION OF RADIOISOTOPES FROM TARGETS

5.1. Gas and liquid targets

The separation of the radioisotope from the target material in gas and
liquid targets is usually a simple matter of trapping the desired compound. In
the case of a liquid target, this can be a resin or a solid support such as the
trapping of fluoride ion on a resin column after irradiation of 18O water [19]. In
the gas target, the desired radioisotope can be reacted with some other
compound while still in the gas stream, as is the case with carbon dioxide
produced in a nitrogen gas target where the [11C]CO2 can be transformed into
[11C]methanol in lithium aluminium hydride [20, 21].

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 SESSION 13

5.2. Solid targets

Many of the radionuclides used in the modern practice of nuclear

medicine can be produced in available cyclotrons using solid targets. Further
material on some radioisotopes can be found in Ref. [22].
For most solid targets, electroplating is the best method for producing
targets that will withstand high beam currents and yet can be easily processed,
although pressed powder targets and salt targets are in common usage. Good
thermal contact between the target material and the cooling plate allows beam
currents to be higher and by using electrochemical processing and recovery of
the material, the processing is greatly simplified.
There are several prerequisites for the successful preparation of a solid
target. The following is a list of the physiochemical requirements for a solid
target [23]:

• The layer must be homogeneous over the entire surface area;

• The layer must adhere strongly to the carrier at the temperatures typical
of those achieved during irradiation;
• The layer must be smooth (not spongy), dense (no occlusions nor
vacuoles) and stress free;
• The layer must be free of any organic plating additives (complexing
agents or surfactants).

6. CONCLUSION

Development of new and clinically useful radioisotopes and radiophar-

maceuticals (tagged compounds) is the single most important contributor to
the progress and growth of the field of nuclear medicine. Diagnostic imaging,
using techniques such as single photon emission tomography and positron
emission tomography (PET), and the measurement of in vivo organ function,
physiology, or biochemistry, have become indispensable tools in both clinical
research and in patient work-up and management and in drug research and
development [24]. Nuclear medicine provides patients with the chance of early
diagnosis and a more tailored course of treatment. Effective treatment of
disease, in particular cancer, using appropriate radioisotopes, is becoming a
reality. The speciality is expanding, with specific PET and single photon
emission tomography radiopharmaceuticals allowing for an extension from
functional process imaging in tissue to pathological processes and nuclide
directed treatments. PET is an example of a technique that has been shown to

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yield the physiological information necessary for clinical oncology diagnoses

based upon altered tissue metabolism [25].
Most PET drugs are currently produced using a cyclotron at locations
that are in close proximity to the hospital or academic centre at which the
radiopharmaceutical will be administered. The evolution of PET radiopharma-
ceuticals has introduced a new class of ‘drugs’ requiring production facilities
and product formulations that must be closely aligned with the scheduled
clinical utilization. The production of the radionuclide in the appropriate
synthetic form is but one critical component in the manufacture of the finished
radiopharmaceutical.

ACKNOWLEDGEMENTS

This research was carried out at Brookhaven National Laboratory under

contract DE-AC02-98CH10886 with the US Department of Energy.

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[6] SCHLYER, D.J., (1987) Production of Short-lived Radiopharmaceuticals for
PET. Nuclear Instruments and Methods in Physics Research B24/25 925-927.
[7] GANDARIAS-CRUZ, D., OKAMOTO, K., (1988) Status on the Compilation of
Nuclear Data for Medical Radioisotopes Produced by Accelerators, IAEA
Report INDC(NDS)-209/GZ.
[8] DECONNINCK, G., (1978) Introduction to Radioanalytical Physics, Nuclear
Methods Monographs No.1 Elsevier Scientific Publishing Co. Amsterdam.

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[9] GUILLAUME, M., LAMBRECHT, R.M., WOLF, A.P., (1975) Cyclotron

production of 123Xe and high purity 123I: A comparison of tellurium targets. Inter-
national Journal of Applied Radiation and Isotopes 26, 703-707.
[10] LAMBRECHT, R.M., WOLF, A.P., “Cyclotron and short-lived halogen isotopes
for radiopharmaceutical applications”, Radiopharmaceuticals and Labelled
Compounds (Proc. Int. Conf. Copenhagen, 1973), Vol. 1, IAEA, Vienna (1973)
275–290.
[11] QAIM, S.M., STÖCKLIN, G., (1983) Production of some medically important
short-lived neutron deficient radioisotopes of halogens. Radiochimica Acta, 34,
25-40.
[12] CLEM, R.G., LAMBRECHT, R.M., (1991) Enriched 124Te targets for production
of 123I and 124I. Nuclear Instruments and Methods A303, 115-118.
[13] FOWLER, J.S., WOLF, A.P., Working against time. Rapid radiotracer synthesis
and imaging the human brain. Accounts of Chem Res 1997; 30:181-8.
[14] DANNALS, R., RAVERT, H.T., WILSON, A.A., WAGNER, H.N., Special
problems associated with the synthesis of high specific activity carbon-11 labeled
radiotracers. In: Emran AM, editor. New Trends in Radiopharmaceutical
Synthesis, Quality Assurance and Regulatory Control. New York: Plenum Press,
1991:21-30.
[15] VAN DEN WINKEL, P., Private communication 2004.
[16] HESELIUS, S.-J., LINDBLOM, P., SOLIN, O., (1982) Optical studies of the
influence of an intense ion beam on high pressure gas targets. International
Journal of Applied Radiation Isotopes 33, 653-659.
[17] HESELIUS, S.-J., SCHLYER, D.J., WOLF, A.P., (1989) A Diagnostic Study of
Proton-beam Irradiated Water Targets, Appl. Radiat. Isot., Int. J. Radiat. Appl.
Instrum. Part A, 40 663-669.
[18] WIELAND, B.W., SCHLYER, D.J., WOLF, A.P., (1984). Charged Particle Pene-
tration in Gas Targets Designed for Accelerator Production of Radionuclides
Used in Nuclear Medicine. Int. J. Appl. Radiat. Isot. 35, 387-396.
[19] SCHLYER, D.J., BASTOS, M.A., ALEXOFF, D., WOLF, A.P., Separation of
[18F]fluoride from [O-18] water using anion exchange resin. Int J Appl Radiat Isot
[A] 1990; 41:531-3.
[20] LANGSTROM, B., LUNDQVIST, H., The preparation of [11C]methyl iodide and
its use in the synthesis of [11C]methyl-L-methionine. Int J Appl Radiat Isot 1976;
27:357-63.
[21] LANGSTROM, B., et al., Synthesis of compounds of interest for positron
emission tomography with particular reference to synthetic strategies for 11C
labeling. Acta Radiol Suppl 1990; 374:147-51.
[22] INTERNATIONAL ATOMIC ENERGY AGENCY, Standardized High
Current Solid Targets for Cyclotron Production of Diagnostic and Therapeutic
Radionuclides, Technical Reports Series No. 432, IAEA, Vienna (2004).

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[23] VAN DEN BOSSCHE, B., FLORIDOR, G., DECONINCK, J., VAN DEN
WINKEL, P., HUBIN, A., Steady-state and pulsed current multi-ion simulations
for a thallium electrodeposition process, Journal of Electroanalytical Chemistry
531 (1) p.61-70 (2002).
[24] WAHL, R., editor, Principles and Practice of Positron Emission Tomography,
Lippincott Williams & Wilkens, Philadelphia, 2002.
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Sussex: John Wiley & Sons, 2003.

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 PERSPECTIVES FOR THE LARGE SCALE
PRODUCTION OF RADIOLANTHANIDES
WITH MEDICAL POTENTIAL

G.-J. BEYER*, H.L. RAVN**, U. KÖSTER**

*Cyclotron Unit, Division of Nuclear Medicine,

University Hospital of Geneva

**CERN ISOLDE

Geneva, Switzerland
Email: gerd.beyer@hcuge.ch

Abstract

The new developments in systemic radionuclide therapy based on chelated

peptides calls for metallic radioisotopes with new characteristics. Especially important
for new applications is the high specific activity of radiotracers and their availability in
therapeutic amounts. The rare earth radionuclides play a dominant role in this context,
because of their diversity of radiation properties and half-lives. Presently used methods
in production of isotopes of interest have reached their technical limitations, and the
progress in systemic radionuclide therapy is limited by availability of radionuclides with
the desired characteristics. These radionuclides are either only or best produced in high
energy spallation and fragmentation reactions. This paper discusses the opportunity for
industrial scale production of such new radioisotopes for future medical use in a fully
parasitic or prime user mode. The target and ion source techniques developed for
producing high purity mass separated radioactive ion beams at CERN ISOLDE is a
well-documented new type of rapid, efficient, continuous and automatic radiochemical
separation. Its key element, the electromagnetic isotope separator on-line (ISOL),
allows the efficient production of very pure samples of almost all radioactive isotopes
with the highest possible specific activity, i.e. the carrier free form. Until now these tech-
niques have exclusively been used to make radioactive ion beams of short lived species
for scientific purposes. It is now generally recognized that simplified variants of these
techniques will also allow harvesting samples of longer lived, high purity and carrier free
radionuclides as byproducts from the spent target material, spent beam absorbers or,
eventually, from dedicated on-line target stations. Although current emphasis tends to
be limited to producing ‘exotic’ radioisotopes, the authors believe that the time has
come to prepare for more rational and large scale industrial radioisotope production
methods using the ISOL target techniques. The future production sites should be put in
synergy with either the planned upgrade of CERN ISOLDE and/or one of the major
new physics research facilities planned for using GeV proton beams of MW power such

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as EURISOL, neutrino factories, spallation neutron sources or accelerator driven

systems where the production rates will be orders of magnitude higher than those at
current facilities.

1. INTRODUCTION

New achievements in the development of biospecific tracer molecules,

such as bioconjugated monoclonal antibodies and peptides, call for metallic
radionuclides showing suitable radiation properties for different aspects of
therapy. Depending on the tissue to be targeted, there is a need for metallic
radionuclides of suitable half-life and with a large variety of different decay
properties. The group of the rare earth elements (lanthanides and the other
elements of the Group IIIB: Sc and Y) is particularly interesting since it contains
more than 700 radionuclides amongst which can be found all the kinds of decay
properties desirable. For the following reasons, the entire group of rare earth
elements plays and will continue to play an important role in the R&D of radio-
pharmaceuticals for therapy as well as in their future clinical application [1]:

(i) Owing to the chemical similarity of the rare earth elements we are
practically allowed to handle, the radionuclides of this group serve as
‘homologues’ in a standard protocol for the therapy.
(ii) The chemical similarity of the lanthanides provides a unique possibility to
study relationships between physicochemical molecule parameters and
the biological response without changing the basic tracer molecule. A large
number of suitable radionuclides may be used simultaneously in a most
efficient way for this kind of R&D work.
(iii) The radionuclides of the rare earth elements provide an almost universal
variety of half-life and form of radiation such as single photon emitters
for single photon emission tomography (SPECT), beta emitters for
therapy with a large range of beta energies, positron emitters for positron
emission tomography (PET), an alpha emitter (149Tb), as well as several
interesting Auger electron emitters.

With regard to the first aspect, it has already been shown that 90Y and
177
Lu are used with the same peptide conjugate without changes in the
protocol. As regards the second aspect, mixtures (cocktails) of several
radiolanthanides allow a form of ‘fine tuning’ in tracer development, especially
for therapy, as reported, for example, in Ref. [2]. Finally, the third aspect opens
the door to individual in vivo dosimetry using PET based on the homologues
positron emitter available in the group.

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A selection of radionuclides of the rare earth elements is shown in

Table 1, including their main potential for an application in diagnosis, for

TABLE 1. SELECTION OF SUITABLE RADIONUCLIDES OF THE

RARE EARTH ELEMENTS WITH POTENTIAL USE IN NUCLEAR
MEDICINE AND RELATED R&D

Diagnosis Therapy
R&D
SPECT PET b– a e
87g 44 88 47 149 165
Y Sc Y Sc* Tb Er
147 85 173m 90 225
Eu Y Ce Y Ac/
progeny
147 86 139 142
Gd Y Ce Pm
149 134 141 143
Gd Ce/La Ce Pm
155 138 143 149
Tb Nd/Pr Pm Pm*
157 140 144 153
Dy Nd/Pr Ce/Pr Sm*
167 142 144 156
Tm Sm/Pm Pm Eu
169 152 145 159
Yb Tb Sm Gd
145 161
Eu Tb
146 166
Gd/Eu Ho*
147 169
Nd Er
148 177
Gd Lu*
149
Eu
152
Eu
153
Gd
159
Dy
168
Tm
170
Tm
171
Lu
172
Hf/Lu
173
Lu
174
Lu
Note: The generator–parent nuclides listed under the PET isotopes are also pure Auger
electron emitters if one neglects the positrons of the short lived progeny nuclides.
The b– nuclides for therapy labelled with an asterisk (*) show gamma transitions
suitable for SPECT. The nuclides highlighted in bold are those that are already
routinely used in the nuclear medical practice.

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therapy or for certain R&D work. The following criteria were used in selecting
the isotopes:

(a) Nuclear medical imaging using gamma cameras or SPECT requires from
the radionuclide proper single photon radiation in the energy range of
about 100–300 keV (the ideal would be 140 keV, the photon energy of
99m
Tc, the most widely used radionuclide in classical nuclear medical
imaging with SPECT). The half-life should be between several hours and
about a month.
(b) PET requires the positron decay mode, with positron branching, as high
as possible and with a gamma contribution as low as possible.
(c) For therapeutic application it is desirable to have three types of radio-
nuclide. For treatment of manifested solid tumour nodes, beta emitting
radionuclides are useful, without gamma radiation, that could contribute
to uncontrolled whole body dose of the patient. However, an
accompanied gamma radiation suitable for SPECT imaging (100–300
keV) is an advantage. The half-life of the beta emitting isotopes should
preferably be between two days and about two weeks. Alpha emitting
nuclides are useful for treatment of single cancer cells in circulation
(targeted alpha therapy). Fortunately, there is one suitable isotope (149Tb)
in this class of elements. Owing to their chemical similarity, 225Ac has also
been included into the discussion. Finally, Auger electron emitters are
demanded for targeting the DNA inside a cancer cell directly. In
principle, all radionuclides that decay via EC mode are Auger electron
emitters. The criteria for the nuclides of this group are the half-life (<1
month), available purity of the isotope (specific activity) and the absence
of accompanying gamma radiation.
(d) Radionuclides for research distinguish themselves from others by having a
suitable half-life of between a week and a year; they must also have very
suitable characteristic gamma lines that allow them to be detected easily in
a mixture of several isotopes and beta radiation should be absent for
easier handling. Essentially, all the SPECT isotopes are suitable for R&D
as well.

The nuclear medical community is well aware of the technical difficulties

in meeting the fast growth in this demand. Consequently, alternative
production routes, ways of cooperation in new large basic physics facilities and
new techniques such as mass separation are presently under discussion in order
to benefit from the potential of upcoming advanced nuclear centres with their
powerful accelerator installations [3].

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2. STATUS OF COMMERICAL PRODUCTION OF

RADIOLANTHANIDES

As can be seen from Table 1, there are only a few radionuclides of the
rare earth elements that are commercially available today. A review on the
general production of radiolanthanides has recently been given by Roesch [1].
There are, in principle, three main production routes: (i) radioactive decay
(90Y), (ii) neutron induced reactions performed in reactors, leading to the
neutron rich nuclides, and (iii) charged particle induced reactions, leading
mainly to the neutron deficient nuclides.
The 90Y may be seen as an exception, because this isotope is generated in
the decay from the long lived fission product 90Sr, that is, one of the more
dangerous fission products. A high degree of technical effort and a correspond-
ingly high level of investment was needed in order to set up a GMP conform
production technology that is absolutely safe from the point of view of radio-
protection and that meets the high requirements of radionuclidic purity.
The other nuclides highlighted in Table 1, 153Sm, 166Ho and 177Lu, are
generally produced in reactors via the (n,γ) process, leading to preparations
that are primarily not carrier free. According to Culter et al. [4], the contents of
required nuclides in the irradiated target are 1.4%, 0.43% and 18% for 153Sm,
166
Ho and 177Lu, respectively (conditions: MURR Reactor, n flux density = 3 ×
1014 cm-2·sec-1, 155 h irradiation time, using highly enriched target materials). In
a few cases one can nevertheless obtain non-carrier added (n.c.a.) preparations
in the (n,γ) process, if the required product is generated from the radioactive
decay of the primary (n,γ) product. Examples are 149Pm, 151Pm, 161Tb, 166Ho
(after double neutron capture) and 177Lu [5]. The high specific activity 177Lu is
produced from irradiated Yb targets according to Lebedev et al. [6]. The
double neutron capture process in combination with the beta decay provides
some possibilities for making a few radiolanthanides in n.c.a. quality. Examples
are 166Ho or 156Eu. The latter isotope can be made in reasonable quantities
from 154Sm according to:

154
Sm (n,γ) 155Sm → 155Eu (4.96 a) (n,γ) 156Eu. ( σ1 = 5.5 b) ( σ2 = 4040 b)

Owing to the high formation cross-section, the long lived intermediate

product is burned down efficiently to the required 156Eu. These examples
should simply illustrate that radiochemistry provides certain possibilities for
making rare earth isotope products in higher qualities. Nevertheless, we are
confronted with the problem that it only allows deriving one product from each
target.

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A similar situation occurs in accelerator based production routes [1].

Cyclotrons provide increased possibilities for making radiolanthanides.
Generally, carrier free products can be obtained since the production process is
generally combined with a change in the nuclear charge of the target element.
Usually, the amount of target material needed is larger, mainly because of
smaller formation cross-sections in the charged particle induced reactions. The
larger quantities of target material require greater effort in their radiochemical
separation, since usually a carrier free product has to be separated from a
neighbouring ‘macro’ amount of target material. Even if this separation can be
carried out in an efficient way, the problem of ‘one target, one product’
remains.
A more rational approach would be to separate the radiolanthanides
from fission products. At least the ß- emitting rare earth nuclides for the
lanthanides up to Eu/Gd and for Y may be considered as products. Yttrium-91
has been routinely produced from fission in the Department of Radioisotopes
in the Rossendorf Research Centre [7]. Praseodymium-143 has been separated
at high quality and used as a DTPA complex to measure local blood flow in eye
surgery using mini-Si-detector probes. Even 153Sm has clearly been obtained in
the Sm fraction of fission produced radiolanthanides. However, consideration
needs to be given to the significant dilution of the product nuclei resulting from
the presence of other isotopes of the same element produced simultaneously,
including the stable isotopes. In the case of Sm these isotopes would be 154Sm
(stable), 152Sm (stable), 151Sm (91a) and 149Sm (stable). The isotope 150Sm is
shielded by 150Nd. This gives the advantage that from one target it would be
possible to harvest several elements. However, a mass separation step would be
needed if the production of high quality preparations was required.

3. HIGH ENERGY PROTON INDUCED REACTIONS

Nowadays, the discovery, production and study of new and existing

radioactive isotopes are done by means of radioactive ion beam (RIB) facilities
as shown in Fig. 1. In particular, those based on the isotope separator on-line
(ISOL) attached to an intense accelerator beam are of interest here. They are
very efficient and their use of thick targets (mole/cm2) results in extremely high
production rates of nuclei with half-lives ranging from minutes to weeks. These
radionuclides are less interesting for nuclear physics studies, but of potential
interest for medical applications. Such facilities are optimized for the
production of the most short lived nuclei far from the line of stability for
nuclear physics studies and need nuclear reactions with high energy particles

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FIG. 1. Layout of the world leading ISOL facility CERN ISOLDE.

for their production. Apart from the cost, this has important technical conse-
quences. The reactions have many exit channels so that the resulting product
mixture is very complex both chemically and isotopically. In order to make
continuously pure samples of interesting short lived exotic species, a number of
particular fast procedures for their purification has been developed especially
for the rare earth elements.
These new isotope production techniques are centred on an electromag-
netic mass separation that, in conjunction with a chemically selective release
from the target and/or the ion source, allows production of mono-isotopic ion
beams with high efficiency. By collection of the continuous ion beam of long
lived nuclei, the strength of the high purity radioisotopes obtained for medical
research has been thoroughly demonstrated and reviewed, in particular with
respect to the biomedical research done with carrier free lanthanides, mainly
performed within the experimental programme at ISOLDE [8–12]. In this
paper, emphasis is made of the virtues of the ISOL production and separation
methods that have been developed in conjunction with electromagnetic mass
separation and the possibility for their general use in the large scale production
of almost any existing radionuclide of interest.
These procedures are now ripe for application in the commercial radio-
isotope production process where they could rapidly be introduced as an
additional purification step in the existing production lines for making carrier
free variants of currently used medical isotopes. For production of more

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uncommon isotopes of similar high quality, the present ISOL facilities only
allow supplying the samples needed for biomedical research. In future, they
may become widely available as by-products of a number of planned new large
basic physics research facilities on-line to GeV proton accelerators in the MW
class, such as EURISOL [13, 14]. Cooperation with one of these constitutes an
opportunity for their large scale industrial production. This may be done either
in an off-line mode, independent of the physics programme where the radio-
nuclides are harvested from the spent targets, or ultimately from dedicated on-
line target stations optimized for the longer lived medical radioisotopes.
The onset of the spallation reaction at proton or light ions at energies
>100 MeV opens up a vast range of neutron deficient nuclei while the almost
full range of neutron rich nuclei may be produced by the same particles or by
fast neutron induced fission of 232Th or 238U. For years this has been exploited in
the RIB facilities for production and study of the most short lived species [15].
As shown in Fig. 2 these scientifically interesting nuclei located at the extremes
of the production curve are accompanied by a huge amount of longer lived
nuclei found at the top of the yield curve. It can be seen that production rates of
up to 1013 atoms/s may be reached in the future facilities. They result from the
tens of millibarn formation cross-sections in conjunction with the use of very
thick targets (moles/cm2).
These production methods have only rarely been used for supplying
nuclear medicine research and not at all for commercial production. The
reason is not only the difficult access to the energetic particle beams and their

1.E+14

1.E+13
Production rate [atoms/s]

1.E+12
Gd
1.E+11

Dy
1.E+10
Tb
1.E+09

1.E+08
135 140 145 150 155 160 165 170
Mass

FIG. 2. Production rates at EURISOL for some rare earth isotopes which could be
retrieved from the 30 cm long Hg converter target irradiated with a 5 mA, 1 GeV proton
beam.

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 SESSION 13

present modest intensities but also a lack of suitable radiochemical methods.

The many exit channels of these reactions result in very complex reaction
mixtures both chemically and isotopically as seen in the example shown in
Fig. 2. To separate an isotope of interest out of this large amount of target
material requires a very high chemical selectivity in conjunction with a mass
separation.
The authors believe that the time has come to establish more rational and
larger scale industrial radioisotope production using these reactions since the
long lived residues may in the future become available for free as a by-product
from the MW targets needed for production of short lived species in a number
of planned facilities.

4. NEW PRODUCTION METHODS DERIVED FROM ISOL

TECHNIQUES

It has been a major challenge to transfer rapidly and continuously the

reaction products brought to rest in a thick target into an accelerated ion beam
of short lived nuclei as illustrated in Fig. 3. The sensitivity to impurities, the 10–8
mol/s low throughput of many ISOL ion sources and the short delay
requirement does not allow using conventional aqueous phase chemistry to
separate the radioactive element from the target material. Instead, new physico-
chemical methods based on high temperature targets were developed (see for
example Ref. [16]). They allow the mass transfer to the ion source of the
wanted species to be effected via diffusion in the solid state and vacuum subli-
mation. Production of a very broad range of radioactive ion beams is currently

Integrated
Acceleration target and ion
to 60 keV source unit
Electromagnetic
mass separation

1 GeV Proton beam

Beams of singly charged

mono-isotopic sprecies at
60 keV energy

FIG. 3. The principle of the on-line mass separator.

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 BEYER et al.

FIG. 4. Elements for which radioisotopes can be produced in carrier free form using the
ISOL technique [15].

available for the elements shown in Fig. 4 by combining the release of the
products from refractory compounds kept at high temperature with an ion
source of a mass separator [15].
A typical combined target and ion source unit [17] developed for each
element or group thereof is shown in Fig. 5. The mass separation assures a
purification from any other produced isotope of the same element of >103, i.e

FIG. 5. A typical ISOLDE target and ion source unit as used for production of the rare
earth elements.

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 SESSION 13

FIG. 6. Schematic layout of the RILIS at ISOLDE [6].

the typical achievable mass separation enhancement factor. The chemical

separation factor from any produced isobar can reach the same order of
magnitude. This is particularly true for the recently developed resonance
ionization laser ion sources (RILIS) [18] of which the principle is shown in
Fig. 6 [13]. Combined with the high temperature target, it allows the conversion
of the order of 10% of the nuclei produced in the target into particularly pure
beams of the elements shown in Fig. 7.
Since there is no need for a high speed of release as in the nuclear
research application, the extreme temperatures that shorten the lifetime of the
target oven and ion source can be avoided. Cost effective variants of these new
radiochemical techniques developed for the ISOL RIB facilities are already
available and ready to be transferred to the industry for production of the
above mentioned high quality radioisotopes.
Although operating specialized target units on-line may become the
ultimate solution for continuous production of selected radionuclides, use of
the ISOL methods in an off-line mode seems to be the most appropriate.
In this way, the irradiated reaction material charged into the target and
ion source unit may be obtained from a variety of sources ranging from conven-
tional (n,γ) reaction products to samples abundantly available in a high energy
accelerator laboratory. These range from target units or samples of refractory

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elements ionized with ISOLDE RILIS

1 2
H tested ionization scheme He
3 4 5 6 7 8 9 10
Li Be possible ionization scheme (untested) B C N O F Ne
11 12 13 14 15 16 17 18
Na Mg Al Si P S Cl Ar
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe
55 56 57 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86
Cs Ba La Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn
87 88 89 104 105 106 107 108 109 110 111 112
Fr Ra Ac Rf Db Sg Bh Hs Mt

58 59 60 61 62 63 64 65 66 67 68 69 70 71
Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu
90 91 92 93 94 95 96 97 98 99 100 101 102 103
Th Pa U Np Pu Am Cm Bk Cf Es Fm Md No Lr

FIG. 7. Elements for which the RILIS ionization scheme has been tested at ISOLDE or
other RILIS facilities using copper vapour pump lasers.

target materials irradiated in the spent beam absorbers to samples separated

from any other radioactive waste and brought into an ion source friendly form.
An overview of radiochemical techniques related to the separation of
lanthanides is given in Ref. [19]. The idea of processing materials that are
normally considered to be radioactive waste in order to separate ‘high tech’
isotope products for research and medical application has already been demon-
strated [20]. A typical example of a new, highly desirable product that such a
facility could mass produce is the often discussed 82Sr/82Rb generator which has
extremely high isotopic purity. It is presently not available since it needs off-
line Sr isotope separation, the feasibility of which has already been demon-
strated at ISOLDE [21, 22].
The use of the described ISOL methods brings a number of other
advantages. The non-destructive high temperature separation allows the
targets or the target materials to be used for many irradiations. This reduces the
waste flow and makes it almost free of liquids. The carrier free radioactive
isotopes are delivered as an ion beam which conveniently allows their
collection by implantation in any substrate optimized for the efficient labelling
of the pharmaceutical product [11] or production of very compact (mm3)
isotope generators [23].
The broad product spectrum, in combination with the mass separation,
allows the simultaneous collection of a large number of interesting radionuclei
with these high quality parameters. This is a powerful tool to support
systematic research activities in radiopharmaceutical development [8].

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5. MEDICAL ISOTOPES FROM FUTURE HIGH POWER

ACCELERATOR DRIVEN PROJECTS

Modern accelerators with GeV proton beams in the multi-MW power

class are today on our horizon to serve as drivers for a number of planned
research and energy producing purposes. They range from various neutrino
sources over RIB facilities to accelerator driven systems for nuclear waste
management. Common to them all is the need to convert the energy of the
beam into a flux of various types of short lived particle by stopping it in a
converter target consisting of an element with high Z. As a by-product, the
spallation reaction yields a vast spectrum of useful radionuclides. Accumulated
in the converter target they all represent an interesting production opportunity
if they are harvested by means of the above mentioned methods.
Since the project group of the recently finished EURISOL Report [24] has
decided to make provisions for associating industrial isotope production with its
operation, examples taken from this project are discussed below. With its ‘proton
to neutron converter’ target and several target stations connected to mass
separators, it represents perhaps the most elegant source for providing the raw
material needed to produce these ‘high tech’ medical isotope products. An
associated laboratory described in Ref. [13] would constitute a unique potential
for industrial production of essentially all existing radioisotopes of interest.
The main source of radioisotopes could be the spallation neutron source
needed to decouple the high power deposited by a proton beam from the target
and the ion source for production of fission fragment beams. As shown in Fig. 8
[25], such a target may consist of a mercury flow that serves both as neutron
source and heat transfer medium. Samples extracted from the Hg target and
cooling loop allow parasitic harvesting to the physics experiments’ mixed
spallation products well suited for subsequent refinement in an off-line mass
separator.
In Table 2, the calculated production rates in the EURISOL converter
target of a number of interesting radionuclides are given. These are only a few
cases representative of different applications that serve to illustrate the
potential of the proposed isotope production mode using the Hg target. They
compare very favourably with other technologies, where any exist for
producing sufficient quantities of the radioisotopes concerned. (Included for
comparison is the production rate of the most important fission product, 99Mo,
extensively used as a generator for 99mTc, of which the production is not
optimal with the Hg target undergoing mainly spallation reactions). There are
a number of radionuclides (especially from the lanthanides) with great
potential for therapeutic application [8].

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 BEYER et al.

FIG. 8. Schematic layout of a high power target module. A jet of mercury is injected via
an annular nozzle that allows coaxial injection of the multi-MW, 1 GeV proton beam.
Neutrons from the (p,xnyp) reaction in the Hg then interact with the surrounding hot UCx
fission target matrix, from which the radioactive atoms produced emerge for ionization
and subsequent acceleration.

A number of long lived isotopes were included that are suitable for use in
generators, thus making short lived isotopes available on-site in a hospital
without running facilities for direct production. Examples are 188W, 82Sr, 68Ge
and 44Ti, but this list is not exhaustive. Finally, the authors have included some
radioisotopes of the light elements needed for various applications in industry
and research. These radionuclides are usually difficult to obtain (32Si, 26Al and
28
Mg), but could be produced with relative ease in connection with the
EURISOL facility.
In addition, the possibility of access to samples collected from the
radioactive beam spectrum, spent targets, as well as material activated in the
spent beam absorbers, would give free access to an unlimited variety of both
conventional and new radioisotopes.

6. CONCLUSION AND OUTLOOK

The mass separation technology derived from the RIB-ISOL facilities

constitutes a technically very attractive new operational step ready to be
integrated into large scale medical isotope production. It makes available

344
 SESSION 13

TABLE 2. ESTIMATED PRODUCTION RATES AT EURISOL FOR A

NUMBER OF INTERESTING NUCLIDES WHICH COULD BE
RETRIEVED FROM THE Hg TARGET [13]

another dimension in terms of the quality of isotopes prepared, namely, the

highest possible specific activity and isotopic purity.
Used either on-line or off-line to modern accelerator beams, it opens up
in addition a vast range of other useful isotopes presently in demand, but so far
not available for medical application. By applying these techniques to the
abundant long lived residues that will become available as by-products from
the targets of future high power accelerator driven physics projects, they may
be made available on an industrial production scale.

345
 BEYER et al.

The delivery of these high quality radioisotopes in the form of an

energetic ion beam has opened up a number of new techniques for production
of radiopharmaceutical products by means of ion implantation.

REFERENCES

[1] ROESCH, F., FORSSELL-ANDERSON, E., Radiolanthanides in Nuclear

medicine, Metal Ions in Biological Systems, Volume 42: Metal Complexes in
Tumor Diagnosis and as Anticancer Agents, A.Siegel and H.Siegel (eds.), Marcel
Decker, Inc., New York - Basel 2004, pp.77-108.
[2] BEYER, G.J., et al., The influence of EDTMP-concentration on the biodistribu-
tion of radio-lanthanides and 225Ac in tumor bearing mice, Nuclear Medicine and
Biology 24 (1997) 367.
[3] RIVARD, M.J., et al., The US national isotope program: Current status and
strategy for future success, Appl- Rad. And Isotopes 63 (2) (2005) 157-178.
[4] CULTER, C.S., et al., Cancer Biotherapy & Radiopharmaceuticals 15 (6) (2000)
531.
[5] MIKOLAJCZAK, R., PARUS, J., Prospects for carrier-free lanthanide produc-
tion in the nuclear reactor, COST D18/0004/Working group Meeting, Athens
(Greece), May 20-22 (2004).
[6] LEBEDEV, N.A., NOVGORODOV, A.F., MISIAK, R., BROCKMANN, J.,
ROESCH, F., Radiochemical separation of no-carrier-added 177Lu as product via
the 176Yb(n,γ)177Yb ◊ 177Lu process, Appl.Radiat.Isotopes. 53 (2000) 421.
[7] JANTSCH, K., et al., 30 Jahre Radioaktive Präparate Rossendorf, ROTOB &
ISOCOMMERZ Booklet, D.Dörr, H.Schmidtke and I.Hein (eds.) Rossendorf
1988.
[8] BEYER, G.J., Radioactive Ion Beams for Biomedical Research and Application,
Hyperfine Interaction 129 (2000) 529-553.
[9] BEYER, G.J., Radioactive ion beams for biomedical research and nuclear
medical application, in: Advanced Technology and Particle Physics (ICATPP-7),
Villa Olmo, Como (Italy), Oct.15-19, 2001, World-Scientific, New York, pp.504-
511.
[10] BEYER, G.J., et al., Spallation produced 167Tm for medical application,
in:Medical Radionuclide Imaging 1980, IAEA Vienna, IAEA-SM-247/60 (1981)
Vol.1, pp.587-598.
[11] BEYER, G.J., RUTH, T.J., The Role of Electromagnetic Isotope Separators in
the Production of Radiotracers for Bio-Medical Research and Nucler Medical
Applications, Nucl. Instr. and Meth. B204 (2003) 694-700.
[12] BEYER, G.J., et al., Targeted Alpha Therqapy (TAT) in vivo – direct evidence for
single cancer cell kill using 149Tb-Rituximab, Eur.J. of Nuclear Medicine and
Molecular Imaging 33 (4) (2004) 547-554.
[13] RAVN, H.L., et al., Appendix C in [24] http://www.ganil.fr/eurisol/Final_Report/
APPENDIX-C.pdf

346
 SESSION 13

[14] BEYER, G.J., KOESTER, U., RAVN, H.L., Medical Isotope Program at a Multi-
MW proton Driver, Proc. Workshop on Physics with a Multi-MW Proton Source,
May 25-27, 2004, CERN (Switzerland).
[15] RAVN, H.L., ALLARDYCE, B.W., On-line mass separators, in Treatise on Hevy
Ion Science, Vol.8, D.A.Bromley (ed.), Plenum Press, New York 1988.
[16] BEYER, G.J., NOVGORODOV, A.F., ROESCH, F., RAVN, H., Spallation
Produced Radioisotopes for Nuclear Medical Applicatioon, in Data Requirement
for Medical Radio-Isotope production, Tokyo (Japan) April 20-24, IAEA Vienna,
(1987), Isotopenpraxis 25 (1) (1989) 2-10.
[17] KÖSTER, U., ISOLDE target and ion source chemistry, Radiochimica Acta 89
(2001) 749-756.
[18] KÖSTER, U., Resonance ionization laser ion sources, Nucl. Phys. A701 (2002)
441c-451c.
[19] BEYER, G.J., HERRMANN, E., TYRROFF, H., Gewinnung tragerfreier Radio-
nuklide der Lanthaniden (Production of carrier-free radionuclides of the lantha-
nides), Isotopenpraxis 13 (6) (1977) 193-203.
[20] NOVGORODOV, N.A., et al., Isolation of lanthanide and hafnium radioisotopes
from a massive Ta-target irradiated with 1 GeV protons, Mid-Term Evaluation
Workshop on LanthanideChemistry for Diagnosis and Therapy, F.Roesch (ed.),
COST Chemistry Action D 18, Heidelberg (Germany), 22-25 July 2002, p.29.
[21] BEYER, G.J., ROESCH, F., RAVN, H.L., A high purity 82Sr/82Rb generator,
CERN-Report, CERN-EP/90-91 (1990)
[22] YUSHKEVICH, Y.V., et al., A high efficient ion source with surface ionization
for the determination of micro-traces of radioactive Sr isotopes, Report JINR
Dubna P13-94-213 (1994), Annual Report, Inst.Nuclear Chemistry, University
Mainz 1993/1994.
[23] BEYER, G.J., RAVN, H.L., A new type of 81Rb/81mKr generator made by ion
implantation, Appl. Radiat. Isot. 35 (1984) 1075.
[24] The EURISOL Report, A feasibility study for a European Isotope-Separation-
On-Line Radioactive Ion Beam Facility, http://www.ganil.fr/eurisol/
Final_Report.html
[25] RAVN, H.L., Advanced target concepts for production of radioactive ions and
neutrino beams, Nucl. Instr. and Meth. B204 (2003) 197-204.

347
.
 PRODUCTION OF 123I-MIBG AT IPEN-CNEN/SP

M.F. DE BARBOZA, V. SCIANI, R. HERRERIAS,

M.M.N. MATSUDA, N.T.O. FUKUMORI, L.C.A. SUMIYA,
H. MATSUDA, A.A. SOUZA, M.M. GOES, J.T. PIRES,
J. MENGATTI, C.P. GOMEZ DA SILVA
Instituto de Pesquisas Energéticas e Nucleares IPEN-CNEN,
São Paulo, Brazil
Email: mbarboza@ipen.br

Sodium [123I]iodide is obtained in a Cyclone 30 (IBA) at IPEN-CNEN/SP.

The production process uses the following nuclear reaction: 124Xe
(p,2n)→123Cs→123Xe→123I. The 124Xe gas is highly enriched (>99.8%) which
results in ultra pure 123I end product. The water cooled target (internal volume
= 75 mL) is machined from aluminium alloy. In front of the target there are two
helium cooled molybdenum windows and an alignment system consisting of a
pair of four sector collimators. The irradiation is performed with protons of
30 MeV energy and an effective beam current on target of 60–70 µA. The 124Xe
transference from the storage bottle to the target and the recovery of the gas
after irradiation and return to the bottle is made cryogenically with liquid
nitrogen through stainless steel pipes. The [123I] activity on the wall of the target
is rinsed with sterile water and the [123I] active solution (60–70 mL) is
transferred to the hot cell. With a 124Xe gas pressure (without proton beam) of
2 bars, about 220 MBq/µA of 123I at end of bombardment was obtained.
The labelling process is based on the copper(I) assisted exchange radio-
iodination methods using MIBG sulphate, (NH4)2SO4 and CuSO4 at 165–170ºC
for 30 min. Some groups have reported that radiochemical purity after the
exchange radioiodination was sufficient and that no further purification was
needed. From these data, the preliminary conclusion is that 1% benzyl alcohol
and a low temperature (–10ºC) are effective stabilizers of 123I-MIBG solutions
over 24 h. Addition of 1% benzyl alcohol and storage at 4ºC resulted in a
fourfold reduction of the formation rate of free [*I]. Amartey [1] concluded
that temperature and benzyl alcohol had a slight cumulative effect in retarding
decomposition and the product remained above 90% for over 7 d at the lower
specific activities [2, 3].
After the reaction time has elapsed, the vial is cooled to room
temperature and a sterile saline–benzyl alcohol 1% solution is added. The
volume is adusted until a desirable radioactive concentration is achieved. The
active solution is sterilized through a 0.22 μm Millipore filter. After quality
control aproval, the final product is delivered to nuclear medicine centres.

349
 DE BARBOZA et al.

The radiochemical and radionuclide tests of 123I are determined with

Whatmann 3MM paper (1.5 cm × 12 cm) in 85% MeOH (Rf *I- = 0.75 and Rf
*IO3- = 0.40) and by γ ray spectroscopy using a HPGe detector, before the
labelling procedure. The radiochemical impurity of 123I–MIBG is evaluated in a
fast paper chromatographic system: Whatman 3MM (1 cm × 8 cm), in
n-butanol, acetic acid and water (5:2:1) as a solvent.
The values are: Rf 123I–MIBG = 1.0 and free [123I] iodide = 0.0 [4, 5]. The
retention of 123I solution in a strong anionic resin is greater than 99% and the
recovery of 123I–Na is more than 97% of total activity in 3 mL of 0.02N NaOH.
The radionuclide purity of 123I–Na and the radiochemical purity of 123I–MIBG
are >98% and >97%, respectively, in 90% of all routine production during a
24 h period at low temperature, without any purification step (Tables 1 and 2).
The SD is less than 1.0%.
The microbiological analysis is determined in a different culture medium
which is incubated both at room temperature and at 33 ± 2ºC. The apirogenicity
is evaluated using the ‘in vitro’ Limulus test. The method was developed,
validated and simplified to extend it to large scale productions at the IPEN-
CNEN/SP radiopharmacy centre.
During 2004, 129.5 GBq of 123I–Na and 48 GBq of 123I–MIBG in 37
batches, respectively, were distributed to approximately 28 hospitals and
nuclear medicine centres in Brazil.

TABLE 1. RADIOCHEMICAL PURITY OF 123I –Na AND 123I –MIBG IN

PAPER CHROMATOGRAPHIC SYSTEM
123 123
Batch (n = 37) I–Na I–MIBG

M 98.89 97.68
SD 0.26 0.35

TABLE 2. STABILITY OF 123I –MIBG KEPT AT LOW TEMPERATURE

Batch (n = 37) 1h 24 h

M 98.81 97.72
SD 0.30 0.58

350
 SESSION 13

REFERENCES

[1] AMARTEY, J.K., AL-JAMMAZ, I., LAMBRECHT, R.M., An efficient batch

preparation of high specific activity [123I] and [124I] mBGI., Appl. Radiat. Isot. 54
(2001) 711–714.
[2] MERTENS, J., GYSEMANS, M., “Cu1+ assisted nucleophilic exchange radiohal-
ogenation: application and mechanistic approach”, New Trends in Radiopharma-
ceutical Synthesis, Quality Assurance, and Regulatory Control (EMRA, A.M.,
Ed.), Plenum Press, New York (1991) 53.
[3] NEVES, M., PAULO, A., PATRICIO, L., A kit formulation of [123I]-meta-
iobenzylguanidine (MIBG) using Cu (I) generated “in-situ” by sodium disul-
phide, Appl. Radiat. Isot. 43 (1992) 737.
[4] ALMEIDA, M.A., DE BARBOZA, M.F., COLTURATO, M.T., The synthesis of
131
I-MIBG. In Cox, P.H. & Touya, E. News Perspectives in Nuclear Medicine. Pt 2,
p.185, Gordon & Breach Science Publishers (1985).
[5] ROSSOUW, D.T., Routine Production and Quality Control of 123I-labeled mBGI
at NAC, Appl. Radiat. Isot. 43 (1992) 1301-1302.

351
.
 A NEW 82Sr–82Rb GENERATOR

A. BILEWICZ, B. BARTOŚ
Institute of Nuclear Chemistry and Technology,
Warsaw
Email: abilewicz@ichtj.wew.pl

R. MISIAK, B. PETELENZ
Institute of Nuclear Physics,
Cracow

Poland

Owing to the similarity of rubidium and potassium cations, the radionuclide

82
Rb, a positron emitter, has been used in nuclear medicine to characterize
myocardial perfusion with high sensitivity and specificity [1–3]. The advantage of
82
Rb PET versus classical SPECT with 201Tl is the short half-life of 82Rb (T1/2 = 75 s)
which allows one to scan patients every 10 min and to reduce the exposure of
patients to radiation. Additionally, 82Rb is a generator produced from the longer
lived parent radionuclide 82Sr (T1/2 = 25.55 d), which also permits clinical PET
studies in hospitals which do not have expensive on-site cyclotrons. Numerous
methods for the manufacture of 82Sr–82Rb generator have already been described
[4, 5]. All these procedures, however, suffer from various limitations, e.g.
complicated multistep separation of 82Sr from the rubidium target and insignificant
radiation resistance of the organic extractans and ion exchange resins. To get
around these disadvantages the authors used an inorganic ion exchanger —
cryptomelane MnO2 which has a tunnel framed structure with exchangeable alkali
or alkali earth cations. The average tunnel diameter is 280 pm, therefore the
sorbent is selective for those cations with crystal ionic radii of 130–150 pm, e.g. K+,
Rb+, Ba2+ and Ra2+. To find the optimum conditions for Rb+–Sr2+ separations, the
distribution coefficients (Kd) of Rb+ and Sr2+ on cryptomelane MnO2 were
determined as a function of HNO3 concentration. The influence of the HNO3
concentration on the Kd for Sr2+ and Rb+ on cryptomelane MnO2 was studied. It
was observed that Kd for Rb+ even at 1M HNO3 is very high. This confirms the high
affinity of cryptomelane MnO2 for cations with ionic radii close to 150 pm. For Sr2+,
whose ionic radius is lower (118 pm), Kd decreases with increasing concentration of
H+ ions. For the efficient separation of the Rb+–Sr2+ pair, 0.5 mol/dm3 HNO3 was
chosen as the optimal solution, wherein the Kd for Rb+ on cryptomelane MnO2 is
greater than 104, while for Sr2+ it is close to 1. This allows one to perform a simple
and quantitative separation of 82Sr from the irradiated rubidium target.

353
 BILEWICZ et al.

The 82Sr isotope was produced in the AIC-144 cyclotron located in the
Institute of Nuclear Physics, Cracow. In the pilot experiment, a target of 0.133 g
RbCl of natural isotopic abundance (72.17% 85Rb, 27.83% 87Rb) was irradiated
for 4 h with the internal proton beam of 48 MeV and 0.5 μA. At this energy,
proton activation of the natural rubidium target leads to direct or indirect
formation of 82,83,85Sr and 82,83,84,86Rb isotopes. The radionuclides detected by
gamma spectrometry in the irradiated target are presented in Table 1. After the
8 d waiting period, which is enough to allow decay of 83Sr, the RbCl target was
dissolved in 0.5M HNO3 solution. Next, the solution was passed through the
cryptomelane MnO2 column bed. The inactive rubidium (target material) and
83,84,86
Rb were quantitatively adsorbed on the cryptomelane MnO2. The
effluent from the column was made alkaline with 1M NaOH to pH6–8.
Afterwards, the strontium radionuclides from the neutralized solution were
loaded on top of the SnO2(aq) bed. The inorganic ion exchanger (tin oxide)
was prepared by acidification of sodium stannate solution according to the
procedure described in Ref. [6]. The 82Rb formed from decay of 82Sr was eluted
from the column by 0.9% NaCl (physiological saline). The elution was
performed every 10 min. The radionuclide purity of the effluent was measured
by gamma spectroscopy after the decay of 82Rb. Additionally, the decay curves
of the effluent fractions were also measured. The 82Sr and 85Sr breakthroughs
measured by gamma spectroscopy were lower than the established limits. After
passing 1 L of 0.9% NaCl through the column, no significant breakthrough was
observed either by gamma spectrometry or by analysis of the decay curves. The
half-life of the eluted 82Rb determined from the decay curve measured for
more than 6 expected half-lives, is identical with the value reported in the
literature.

TABLE 1. RADIONUCLIDES DETECTED IN THE natRbCl TARGET

AFTER IRRADIATION WITH A 48 MeV PROTON BEAM

Radionuclide T1/2 (d) Activity (MBq) Nuclear reaction

82 85
Sr 25.5 6.49 Rb(p,4n) 82Sr
83 85
Sr 1.35 4.45 Rb(p,3n) 83Sr
85 85
Sr 64.8 8.55 Rb(p,n) 85Sr
87
Rb(p,3n) 85Sr
83
Rb 86.2 18.60 83
Sr(EC, β+)→83Rb
84 85
Rb 32.9 14.29 Rb(p,pn) 84Rb
86 85
Rb 18.7 18.66 Rb(n,γ) 86Rb

354
 SESSION 13

REFERENCES

[1] ANDERSON, C.J., WELCH, M.J., Chem. Rev. 99 (1999) 2219.

[2] PARKASH, R., DEKEMP, R.A., RUDDY, T.D., J. Nucl. Card. 11 (2004) 440.
[3] EPSTEIN, N.J., et al., Appl. Radiat. Isotop. 60 (2004) 921.
[4] BRIHAYE, C., GUILLAUME, M., COGNEAU, M., J. Biophys. Med. Nucl. 6
(1982) 151.
[5] VALLABHAJOSULA, S., et al., J. Nucl. Med. 22 (1981) 76.
[6] CLEARFIELD, A., Inorganic Ion Exchange Materials, CRC Press Inc., Boca
Raton, Florida (1982) p.144.

355
.
 CYCLOTRON PRODUCTION OF 103Pd VIA PROTON
INDUCED REACTIONS ON A 103Rh TARGET

M. SADEGHI, H. AFARIDEH, G. RAISALI

Cyclotron Department, Nuclear Research Centre
for Agriculture & Medicine,
P.O. Box 31585-4395,
Karaj, Islamic Republic of Iran
Email: msadeghi@nrcam.org

M. HAJI-SAEID
International Atomic Energy Agency,
Vienna

Abstract

Electroplated rhodium was employed as the target for cyclotron production of

103
Pd. The electrodeposition of rhodium metal on a copper backing experiments were
performed in acidic sulphate media using RhCl3·3H2O, Rh2(SO4)3 (recovered from
hydrochloric acid solution) and also the commercially available Rhodex plating baths.
The paper discusses development of a high current density (2.4 A/cm2) electrodissolution
system that allows solubilization of rhodium fragments, powder, and pieces of foil and
wire in the presence of hydrochloric acid and chlorine gas. An electrodissolution
apparatus was found better than other dissolution methods in terms of personnel
shielding and 103Pd yield. The ion exchange column chromatography method was simple
and effective for the purification of 103Pd.

1. INTRODUCTION

Prostate cancer is a common malignancy in men in the Western world. In

recent years, improvement in biochemical diagnostic methods and the availa-
bility of a wide range of treatment options have increased the number of
patients surviving free of disease. It has been noted that the proportion of
patients treated by permanent brachytherapy is rapidly increasing (more than
40 000 in 1998 in the United States of America) and reached 50% in 2006 [1].
The favourable results of permanent implants may, however, not be repro-
ducible if strict treatment procedures and patient selection guidelines are not
followed. For this reason, regular updates on recommendations are published

357
 SADEGHI et al.

by the American Brachytherapy Society [1], which also reports on the large
number of clinical studies and dosimetry problems [2–12]. Essentially, two
radionuclides, namely 125I and 103Pd, are used for this technique. As early as
1958, 103Pd was proposed by Harper et al. [13] for interstitial implantation. It
was not until 1987 that encapsulated 103Pd sources became commercially
available in the USA, where a company now operates more than 10 dedicated
accelerators to produce this nuclide [14]. Recently, a manufacturer in Europe
also brought its patented type of 103Pd seed implants to the world market.
The accelerator production method for 103Pd used nowadays is based on
the irradiation of rhodium metal with rather low energy protons via the
reaction 103Rh (p,n)103Pd, followed by a default chemical separation of the
radionuclide from the expensive target material. Harper et al. [15] and
Lagunas-Solar et al. [16] described some early procedures.
An alternative production and purification route for 103Pd employing
silver targets has been proposed by Fassbender et al. [17].
Irradiated rhodium metal targets (plated layers, foils and wires) have
been frequently dissolved by sodium bisulphate fusion (time consuming,
complex medium), by gold tetrachloroaurate oxidation (very expensive, time
consuming) and by alternating current electrodissolution in hydrochloric acid.
Up to now the latter method has been recommended for the solubility of foils
(not applicable for rhodium powder, wires or fragments) [18, 19]. A new, high
current density electrodissolution technique resulting in quantitative solubility
of the target material in acid has been developed.
Since rhodium is a precious metal, it is, therefore, essential that it be
recovered from the processed solution of the radiochemical separation and
reused for preparation of the electrodeposition bath. The electrodeposition of
Rh using Rhodex baths gives quite acceptable quality for irradiation purposes.
However, after electrodissolution and radiochemical separation, rhodium is
present as chloride complexes in about 6M HCl solutions and not suitable for
direct use in the Rhodex bath. The investigations were, therefore, conducted to
evaluate the cycle of recovery/electrodissolution/electrodeposition for routine
production of 103Pd.

2. EXPERIMENTAL

The production of 103Pd is mainly achieved via the nuclear reaction 103Rh
(p, n)103Pd, which is well suited to low energy cyclotrons [20]. The production of
this radionuclide in the Islamic Republic of Iran is highly important, therefore
the Cyclone-30 cyclotron (IBA, Belgium) at NRCAM was employed. This
work was also partially supported by the IAEA. The solid targetry system in

358
 SESSION 13

this cyclotron is made up of a pure copper backing on which the target

materials are electrodeposited. To take full benefit of the excitation function
and to avoid the formation of the radionuclide impurity 101Pd, the proton
entrance energy should be 18 MeV [20]. The physical thickness of the rhodium
layer is chosen in such way that for a given beam/target angle geometry the
particle exit energy should be 6 MeV. According to the SRIM code, the
thickness has to be 475 µm for 90° geometry. To minimize the thickness of the
rhodium layer (and hence lowering the cost price per target), a 6° geometry is
preferred, in which case a 48 µm layer is recommended. Identification and
assay of gamma ray emitting radionuclides were carried out using gamma
spectroscopy with a high purity germanium (HPGe) detector (Canberra™
model GC1020-7500SL).

2.1. Target fabrication

Rh target preparation from sulphate plating baths

To prepare the sulphate baths, the hydrated rhodium oxide (Rh2O3(aq))

was used which in turn had been recovered from hydrochloride acid solution
containing rhodium chloride complexes. Therefore, the procedure included two
parts:

(1) Recovery of Rh as rhodium oxide from chloride solution

The hydrochloric acid solution containing Rh was primarily obtained

from the electrodissolution of the irradiated rhodium target on which a radio-
chemical separation of 103Pd had been performed. This solution was passed
through a 0.45 µm filter (HVLP, Millipore), the filtrate evaporated to near
dryness, 300 mL water added to the residue and the pH of the solution adjusted
to 10–10.5 with 10M NaOH until a yellow, colloidal solution of Rh2O3(aq) was
formed.
To improve the filterability of the yellow Rh2O3(aq) precipitate, the
solution was allowed to digest for 24 h at 50°C under gentle stirring and then
passed through a Bleu Band filter paper (Schleich & Scheull 589). The
precipitate on the filter was washed several times with water to remove most of
the adsorbed Cl- ions. Since some of the yellow precipitate had gone through
the filter paper, the filtrate was passed through a second 0.45 µm filter. The
Rh2O3(aq) on the filters was left in air for 24 h to dry, followed by grinding into
a fine powder, then dried for 48 h under vacuum at room temperature and
finally weighed.

359
 SADEGHI et al.

(2) Electrodeposition of Rh on the Cu backings

To prepare the plating solution, 5.7 g of the recovered hydrated rhodium

oxide (for 4 targets with a thickness of about 48 µm) was transferred into a
100 mL beaker with a magnetic stirring bar and 10 mL of 95% sulphuric acid
was then carefully introduced into the beaker. The beaker was covered with a
watch glass. The solution was heated to 350°C under gentle stirring until SO2
fumes evolved and thereafter heating continued for 15 min. A brownish yellow
solution was then obtained. The solution was added to 300 mL of water in a
600 mL beaker, passed through a 0.45 µm filter paper and the pH adjusted to
1.0–2.0. Five grams of sulphamic acid was then added to the solution and
diluted to 450 mL with water. The electrolyte solution was transferred into the
plating vessel and a direct current applied to the electrodes. The electrodepo-
sition was carried out at 60°C under bi-directional stirring (1000 rpm, 8s/8s) for
24 h using a current density of 8.55 mA/cm2.

2.2. Electrodissolution

The electrodissolution system consists of a cylindrical upper body

(diameter 70 mm, height 120 mm) and a conical lower part (height 30 mm)
ending in a 12 mm diameter circular window allowing the attachment of the
filter combination. The latter consists of a classical G-4 glass frit fitted with an
end glass tube allowing removal and recirculation of the solution by means of a
high flow rate (1 L/min) peristaltic pump. This loop contains water coolant
(glycol) to allow removal of heat produced during the electrochemical
dissolution of the Rh. The G-4 supports a home-made graphite fibre (diameter
100 mm, 360 holes of 0.5 mm) clamped in a ring of copper (to ensure electrical
contact) and a Perspex ring. Above the filter combination, a supply
compartment allows introduction of reagents that is also done by means of
peristaltic pumps. The upper graphite ring electrode (thickness 2 mm, external
diameter 100 mm, window diameter 20 mm) is also mounted in a copper/
Perspex ring combination 30 mm above the graphite filter. Sealing of the
system is obtained by means of O-rings and by clamping different parts. The
upper window of the unit is closed by means of a Perspex cover fitted with
nipples that allow the escape of nitrogen oxide and excess chlorine. The latter
are absorbed in sodium hydroxide solution to avoid contamination of the
environment (Fig. 1).
The main problem in the 103Pd radiochemical stage is dissolution of target
material due to extremely low chemical reactivity of rhodium metal towards
acids, alkalis and other corrosive reagents. The Cu carrier was dissolved by flow
rate controlled introduction of nitric acid into the vessel holding the vertically

360
 SESSION 13

FIG. 1. Electrodissolution set-up.

mounted target and the Rh layer was not dissolved. The resulting Cu(NO3)2/
HNO3 solution was removed by filtration through a glass/graphite filter
combination whereby the Rh fragments are collected on the filter and the
vessel walls. Rhodium fragments were washed with water and removed. A
mixture of 12M hydrochloric acid and chlorine gas was introduced into the
vessel. Electrochemical dissolution of the Rh was done by applying a high AC
density (2.4 A/cm2) between the electrographite filter and a perforated circular
upper graphite electrode mounted at an appropriate distance from the carbon
filter.

2.3. Separation of carrier free 103Pd

The Cu/Rh/Pd separation was achieved using a Dowex1X8 (Cl-)/100–200

mesh column (1.5 cm × 10 cm). Copper was eluted with 0.03M HCl, rhodium
with 6M HCl and palladium with a 1:1 mixture of 0.5M NH3/NH4Cl [21].

3. RESULTS AND DISCUSSION

Electroplated rhodium targets can be prepared from home-made or

commercially available sulphate or chloride baths containing appropriate
plating additives. When home-made plating solutions are used, the addition of
1% sulphamic acid is recommended. As a plating technique, constant current (DC

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 SADEGHI et al.

or AC) electrolysis at elevated temperature (40–60°C) can be applied. As the

plating current efficiency is less than 100%, plating to depletion (>98% rhodium
deposition) is recommended. To estimate the quality of the electroplated rhodium
targets, some criteria had to be taken into account such as homogeneity,
morphology, visual appearance of the rhodium layer and thermal shock.
The homogeneity of the rhodium layer is important as it may seriously
affect the production rate of 103Pd. This was determined by measuring the
thickness of several parts of the layer by micrometer and calculating the
standard deviation of the data.
All electrodeposited Rh target layers were examined in morphology by a
scanning electron microscopy (using a Joel model JSM 6400 at an accelerating
voltage of 20 kV). The photomicrographs were then compared with each other
and also with the ones obtained from the Rhodex solution. The evaluation of
the quality of the layers was achieved by comparison of the photomicrographs
in terms of size and form of the Rh nuclei and the extent to which they
overlapped each other; the smaller, more spherical and the more overlapped
nuclei were considered to be good plating qualities.
The thermal shock tests involved heating the target up to 500°C (the
temperature that the Rh layer can experience during a high current irradiation)
for 1 h followed by submersion in cold (15°C) water. The absence of crack
formation and peeling of the rhodium layers indicated good adhesion.
To increase the Rh dissolution rate and to avoid boiling of solution, closed
loop circulation through a water (glycol) cooler is introduced. To increase the
dissolution rate, chlorine gas was introduced between two graphite fibres.
Experimental data of dissolutions are summarized in Table 1. The
experiments were done up to 30 A current and with hydrochloric acid concen-
trations of 6M and 12M.

TABLE 1. DISSOLUTION TRIALS WITH FRAGMENTED RHODIUM AND

PIECES OF FOIL AND WIRE
(effective area of electrographite = 16.5 cm2)

W(Rh) J Acidity t T Residual Rh Dissolved Rh Chlorine

(g) (A/cm2) (N) (min) (°C) (g) (%) gas

0.4901 1.5 6 180 95 0.1102 77.5 no

0.4903 1.8 6 180 75 0.0978 80.0 no
0.4922 1.8 12 210 85 0.0580 88.2 no
0.4935 1.8 6 210 75 0.0076 98.46 yes
0.4907 1.8 12 210 75 0.0052 98.93 yes
0.7310 2.4 12 240 85 0.0072 99.01 yes

362
 SESSION 13

There are impurities such as 102m,102,101mRh in this solution. On the basis of

differences in the affinity of doubly charged PdCl42- and triply charged RhCl63-,
the anion exchange method was used for the separation of palladium from
rhodium. After eluting with 100 mL 0.03M HCl, which is intended to remove
Cu2+ and other metal ions, such as Zn2+and Fe3+, which adhere to the resin; the
resin was eluted 120 mL 6M HCl at a 4 mL/min flow rate. The elution was
continued for a total volume of 120 mL and 30 mL of distilled water was then
used to remove the HCl remaining in the column. The mixed NH3+NH4Cl(1:1)
eluant (100 mL) was used to release 103Pd from the resin. After anion exchange
with Dowex1X8 (Cl-)/100–200 mesh and taking a gamma ray spectrum by
HPGe detector, the 103Pd purity obtained was more than 99% (Figs 2 and 3).

4. CONCLUSIONS

The rhodium target for production of 103Pd can be prepared by the

electrodeposition technique. For the purpose, either a chloride electroplating
bath through RhCl3(aq) or sulphate bath through Rh2O3(aq) (recovered from
hydrochloric acid solution), H2SO4 and sulphamic acid can be used. The former

FIG. 2. Gamma ray spectrum of an irradiated 103Rh target after electrodissolution, other
peaks include the Pb detector shielding fluorescence X rays.

363
 SADEGHI et al.

103
FIG. 3. HPGe spectrum of radiochemically separated Pd. No other peaks have been
detected in the γ spectrum.

bath is recommended for low current beam irradiation (up to 200 µA) and the
latter for current beam irradiation of higher than 200 µA.
The high current density results in a high production rate of the
electroactive chlorine species that rapidly dissolves the rhodium present at the
working electrode. The dissolution rate depends on parameters such as current,
concentration of HCl and weight of rhodium. In general, the magnitude of these
parameters is time dependent. The optimum conditions of the electrodissolution
were as follows: 12N HCl solution, AC density higher than 2.4 A/cm2,
temperature 85°C and bubbling chlorine gas, in which case the dissolution rate
was greater than 99%.
Recovery of 103Pd from rhodium solution was achieved by ion exchange
column with Dowex1X8 resin and NH3+NH4Cl (1:1) as eluant; the obtained
103
Pd purity was more than 99%.

REFERENCES

[1] NAG, S., BEYER, D., FRIEDLAND, J., GRIMM, P., NATH, R., Int. J. Radiat.
Oncol. Biol. Phys. 44 (1999) 789.
[2] MERRICK, G.S., BUTLER, M.W., DORSEY, A.T., LIEF, J.H., Int. J. Radiat.
Oncol. Biol. Phys. 44 (1999) 1111.

364
 SESSION 13

[3] PRETE, J.J., et al., Int. J. Radiat. Oncol. Biol. Phys. 40 (1998) 1001.
[4] LING, C.C., Int. J. Radiat. Oncol. Biol. Phys. 23 (1992) 81.
[5] MESSING, E., et al., Int. J. Radiat. Oncol. Biol. Phys. 44 (1999) 801.
[6] BLASKO, J.C., WALLNER, K., GRIMM, P.D., J. Urol. 154 (1995) 1096.
[7] BLASKO, J.C., et al., Urol. Clin. North Am. 23 (1996) 633.
[8] PORTER, A.T., BLASKO, J.C., GRIMM, P.D., REDDY, S.M., RAGDE, H.,
Californian Cancer J. Clin. 45 (1995) 165.
[9] DATTOLI, M., WALLNER, K., SORACE, R., J. Brachyther. Int. 13 (1997) 347.
[10] RAGDE, H., et al., Semin. Surg. Oncol. 13 (1997) 438.
[11] STOCK, R.G., STONE, N.N., TARBERT, A., Int. J. Radiat. Oncol. Biol. Phys. 41
(1998) 101.
[12] WHITTINGTON, R., et al., Int. J. Radiat. Oncol. Biol. Phys. 44 (1999) 1107.
[13] HARPER, P., LATHROP, K., BALDWIN, L., Ann. Surg. 148 (1958) 606.
[14] PORAZZO, M.S., et al., Int. J. Radiat. Oncol. Biol. Phys. 23 (1992) 1033.
[15] HARPER, P.V., LATHROP, K., NEED, J.L., ORNL-LR-DWG 51564 (1961) 124.
[16] LAGUNAS-SOLAR, M.C., AVILA, M.J., JOHNSON, P.C., Appl. Radiat. Isot.
38 (1987) 151.
[17] FASSBENDER, M., NORTIER, F.M., SCHROEDER, I.W., VAN DER WALT,
T.N., Radiochimica Acta 87 (1999) 87.
[18] BOX, W.D., In ORNL-3802 UC-23-Isotopes-Industrial Technology TID-450.39th,
(1964), p.29.
[19] LAGUNAS-SOLAR, M.C., AVILA, M.J., JOHNSON, P.C., Int. J. Appl. Radiat.
Isot. 38(2) (1987) 151.
[20] HERMANNE, A., SONCK, M., FENYVESI, A., DARABAN, L., Nucl. Inst. &
Meth. In Phys. Res. B, 170, (2000) 281.
[21] ZHANG, C., WANG, Y., ZHANG, Y., ZHANG, X., Applied Radiation &
Isotopes, 55, 441 (2001).

365
.
 THE STATUS AND POTENTIAL OF NEW
RADIONUCLIDE GENERATORS PROVIDING
POSITRON EMITTERS TO SYNTHESIZE NEW
TARGETING VECTORS FOR PET

F. ROESCH*, K.P. ZHERNOSEKOV*, D.V. FILOSOFOV**,

M. JAHN*, M. JENNEWEIN*

*Institute of Nuclear Chemistry, University of Mainz,

Mainz, Germany
Email: frank.roesch@uni-mainz.de

**Joint Institute of Nuclear Research, LNP,

Dubna, Russian Federation

Abstract

The 68Ge/Ga generator (68Ge, T½ = 270.8 d) provides a cyclotron independent

source of positron emitting 68Ga (T½ = 68 min, ββ+ branching = 89%), which can be used
for coordinative labelling. Recently, tumour imaging using 68Ga labelled DOTA conju-
gated peptides became one of the most exciting approaches to diagnose neuroendocrine
and other tumours and metastases because (i) octreotide derivatives with high affinity
and selectivity to somatostatin receptor expressing tumour cells are available, (ii)
syntheses of DOTA conjugated targeting vectors are straightforward due to the kit type
labelling, and (iii) PET/CT scanners perfectly correlate morphological and functional
parameters. However, for labelling of biomolecules via bifunctional chelators, 68Ga(III)
as eluted initially needs to be preconcentrated and purified from 68Ge(IV), Zn(II),
Ti(IV) and Fe(III). The paper describes a system for the simple and efficient handling of
the 68Ge/68Ga generator eluates with a microchromatography column filled with about
50 mg of a cation exchange resin. Chemical purification and volume concentration of
68
Ga are carried out in an 80% acetone/0.15M HCl solution. Finally, more than 97% of
68
Ga is obtained in 400 µL of a 98% acetone/0.05M HCl solution. The initial 68Ge
contamination of the eluate was reduced by a factor of 1000. Contents of Zn(II), Fe(III)
and Ti(IV) were reduced significantly. Consequently, the processed fraction can be used
directly for the synthesis of radiopharmaceuticals. The developed system represents a
simple and efficient way of labelling DOTA conjugated biomolecules with generator
produced 68Ga(III). [68Ga]DOTATOC and [68Ga]DOTANOC were successfully used in
a series of human somatostatin receptor expressing tumour diagnoses with PET/CT.

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 ROESCH et al.

1. INTRODUCTION

Radionuclide generator systems continue to play a key role in providing

both diagnostic and therapeutic radionuclides for various applications in
nuclear medicine, oncology and interventional cardiology. Key advantages of
the use of radionuclide generators include reasonable cost, the convenience of
obtaining the desired radionuclide progeny on demand and the availability of
the radionuclide progeny in high specific activity, no carrier added form.
Although many parent/progeny pairs have been evaluated as radionuclide
generator systems, in particular for the application of labelled PET radiophar-
maceuticals (Table 1), there are only a relatively small number of generators
which are currently in routine clinical and research use [1].
Those generators can be categorized according to the half-life of the
progeny nuclide. The short lived progeny cover half-lives of a few minutes. As
the short half-lives do not allow radiochemical synthesis, these systems are
relevant for perfusion imaging exclusively. The longer lived progeny, on the
other hand, provide the potential for development of labelled radiopharmaceu-
ticals. Recently, the 68Ge/Ga and 72Se/As systems have found impressive appli-
cation, but also the 44Ti/44Sc generator represents a promising system.
The 68Ge/Ga generator (68Ge, T½ = 270.8 d) provides a cyclotron
independent source of positron emitting 68Ga (T½ = 68 min, ββ+ branching =
89%), which can be used for coordinative labelling. Recently, tumour imaging
using 68Ga labelled DOTA conjugated peptides became one of the most
exciting approaches with which to diagnose neuroendocrine and other tumours
and metastases because (i) octreotide derivatives with high affinity and
selectivity to somatostatin receptor expressing tumour cells are available, (ii)
syntheses of DOTA conjugated targeting vectors are straightforward due to the
kit type labelling, and (iii) PET/CT scanners perfectly correlate morphological
and functional parameters. Radiolabelled peptides designed for tumour
receptor targeting are of great interest for diagnostic imaging and/or radio-
nuclide therapy [2]. Somatostatin analogues such as DOTA-DPhe1-Tyr3-
octreotide (DOTATOC) and DOTA-DPhe1-Nal3-octreotide (DOTANOC)
labelled with radiogallium (66,67,68Ga) show high potential for diagnosis of
somatostatin receptor expressing tumours, improved binding affinity and very
promising properties in vivo [3, 4]. In particular, if PET/CT is used, a highly
accurate diagnosis is provided.

368
 SESSION 13

TABLE 1. RADIONUCLIDE GENERATORS WITH POTENTIAL FOR

PET

Parent Progeny
Generator system
β+branch Eβ+
T½ T½ Application
(%) (MeV)
82 82
Sr Rb 25.6 d 1.27 min 95.0 1.41 Perfusion
140 140
Nd Pr 3.37 d 3.39 min 51.0 0.544 Perfusion
118 118
Te Sb 6.00 d 3.6 min 74.0 0.882 Perfusion
122 122
Xe I 20.1 h 3.6 min 77.0 1.09 (Labelling)
128 128
Ba Cs 2.43 d 3.62 min 69.0 0.869 Perfusion
134 134
Ce La 3.16 d 6.4 min 63.0 0.756 Perfusion
62 62
Zn Cu 9.26 h 9.74 min 97.0 1.28 Labelling, perfusion
52 52m
Fe Mn 8.28 d 21.1 min 97.0 1.13 Perfusion
68 68
Ge Ga 270.8 d 1.135 h 89.0 0.74 Labelling, perfusion
110 110m
Sn In 4.1 h 1.15 h 62.0 0.623 Labelling
44 44
Ti Sc 47.3 a 3.927 h 94.0 0.597 Labelling
72 72
Se As 8.4 d 1.083 d 88.0 1.02 Labelling

2. AIM

The commercially available 68Ge/68Ga radionuclide generator (Fig. 1)

based on a TiO2 phase (Cyclotron Co., Obninsk, Russian Federation) recently
became an object for evaluation and can be successfully used for 68Ga3+
recovery. This type of generator allows the elution of more than 50% of the
activity of 68Ge generated in the first year with 5–7 mL of 0.1M HCl. However,
the eluate contains long lived 68Ge (10-2%, increasing with time or frequency of
generator use), small amounts of Zn(II) as generated from the decay of 68Ga,
Ti(IV) and Fe(III), and can contain radiolysis products. It thus should not be
used for labelling directly.
One approach to overcome these problems (i.e. of processing 68Ga(III)
eluate) was described recently [3, 5] using an anion exchanger. The initial 0.1M
HCl eluate must be mixed with concentrated HCl in order to achieve a 20 mL
solution of an overall HCl concentration of 5.5M. Under these conditions,
Ga(III) can be adsorbed on an anion exchanger such as [GaCl4]– and eluted
with H2O in low volumes (>0.5 mL). However, this strategy does not allow
direct loading of the 68Ga(III) activity on the resin from 0.1M HCl and thus

369
 ROESCH et al.

FIG. 1. Scheme of the 68Ge/68Ga radionuclide generator system and problems related to
the direct use of the generator eluate for medical directions.

adds an additional step. Moreover, it does not provide purification of Ga(III)

from Zn(II) and Fe(III). Finally, residual acid amounts might lead to an uncon-
trollable low pH value of the final fraction. Further approaches include
microwave supported labelling [6] or fractionation of the eluates [7].
The aim of authors’ work is to develop an efficient and simplified system
for processing of generator produced 68Ga3+ adequate for clinical requirements.
It involves (i) preconcentration of the eluted 68Ga, (ii) purification of the eluted
68
Ga, (iii) isolation of the purified 68Ga in a form useful for labelling
(acceptable pH and volume), (iv) labelling and preparation of an injectable
68
Ga labelled radiopharmaceutical and (v) construction of a corresponding
apparatus with a capability in the design for an automated ‘module’.
The key step is in the direct transfer of the initial 0.1M HCl 68Ga(III)
eluate on a cation exchanger. Analysis of cation exchanger distribution coeffi-
cients with Bio-Rad AG 50W-X8 in hydrochloric acid–acetone medium [8]
shows that Ga(III) can be eluted from the resin with minimum volume and low
acid amount, with Ga(III) purified from Ge(IV), Zn(II), Ti(IV) and even
Fe(III). This approach was used to develop a processing of generator produced
68
Ga3+ for labelling of biomolecules containing appropriate bifunctional
chelators.

370
 SESSION 13

3. MATERIALS AND METHODS

68
3.1. Ge/68Ga radionuclide generator

Commercial generators based on a TiO2 phase adsorbing 68Ge(IV) were

obtained from the Cyclotron Co., Obninsk, Russian Federation. In the present
study, 20 and 30 mCi systems were used. According to its technical certification,
not less than 50% of 68Ga generated can be eluted with 5 mL 0.1M HCl in the
first year of operation, decreasing not less than 25% after 3 years or after 200
elutions. The breakthrough of 68Ge is described as less than 0.01% within three
years of operation or during the first 200 elutions.

3.2. Radiometals used for ion exchange distribution measurements

Ga(III): 110 MBq of 68Ga in 7 mL of 0.1M HCl were obtained from a 1

year old 20 mCi generator after more than 200 elutions. The absolute activity of
68
Ga was determined using a curie meter. As its 18F position was used, the
activity shown was corrected for the different positron branching of 18F and
68
Ga (96.9% versus 89%).
Ge(IV): The activity of 68Ge in the 68Ga eluate used was about 170 kBq.
The absolute activity of 68Ge was analysed by γ spectrometry using an HPGe
detector about 2 d after the corresponding radionuclide generator elution.
These samples indicate a constant level of 68Ga as generated by the percentage
of co-eluted 68Ge.
Fe(III): 59Fe was produced by a neutron capture reaction on natural iron,
whereby 198 mg of iron oxide (Fe2O3) was irradiated for 50 d at the HMI
neutron source BER II at 1.6 × 1014 n·cm-2·s-1, yielding 440 MBq 59Fe. The iron
oxide was dissolved in HNO3 solution and after evaporation transferred into
appropriate solutions. The activity of 59Fe was analysed by γ spectrometry using
a HPGe detector.
Mn(II): 54Mn was co-obtained with 59Fe in an activity of 0.25 MBq per
1 MBq of 59Fe. The activity of 54Mn was analysed by γ spectrometry using a
HPGe detector.
Zn(II): 69Zn was produced with specific activity of ~700 kBq/mg by
irradiation of 380 μg of 68Zn (Zn(NO3)2, >98% isotopically enriched 68Zn) for
6 h at the TRIGA II reactor Mainz at a neutron flux of 4 × 1012 cm-2·s-1. The
target was subsequently dissolved in 0.1M HCl. The activity of 69Zn was
analysed by γ spectrometry using a HPGe detector.

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 ROESCH et al.

3.3. Chemicals and equipment

Analytical reagent grade chemicals and Milli-Q water (18.2 MΩ⋅cm) were
used. A Bio-Rad AG 50W-X8 cation exchanger minus 400 mesh was preferred
to prepare a microchromatography column. DOTATOC was kindly provided
by Novartis Pharma AG.
For labelling reactions, 11 mL glass vials, Mallinckrodt and reaction
vessels PP, Brand were used. A heating block with a 1.5 cm lead thickness for
radiation adsorption was built using 24 V PTC heating elements. For processing
of the labelling product, C-18 cartridges, Phenomenex Strata-X Tubes, 30 mg
were used. Sterile filtration was done on a 0.22 µm MILLEX®GV membrane
filter. For quality control, TLC (aluminium sheets, silica gel 60, eluent 0.1M
Na3citrate) and HPLC (Machery Nagel column, Nucleosil 5 C18-AB, 250 mm ×
4 mm; eluent, 20% AcCN, 80% TFA 0.01% in H2O, 1 mL/min) were used.

4. EXPERIMENTS

4.1. Distribution of metallic cations

A microchromatography column was prepared using 53 mg of the resin.

The distribution of various cations on this cation exchanger column was
investigated. First, 68Ga in 7 mL of 0.1M HCl was loaded dynamically (within
1–2 min) on the chromatography column. In the second step, the column was
eluted with acetone–HCl solutions of 80% acetone and HCl concentrations of
0.1M, 0.15M and 0.2M. The volume of these mixtures ranged from 0.6 to
5.0 mL. The remaining solution was removed by air. Thirdly, the 68Ga was
eluted in 0.4 mL of a 98% acetone–0.05M HCl solution with a 2 min pause after
the column filling. The column was finally reconditioned with 1 mL 4M HCl
and 1 mL H2O.
Thus, 5 fractions were obtained for analysis of their content of the various
chemical and radiochemical impurities:
(1) 7 mL 0.1M HCl, (2) 80% acetone–HCl solution, (3) 98% acetone–
0.05M HCl solution, (4) 4M HCl (this fraction represents the amounts of metals
remaining after the elution with the 98% acetone–0.05M HCl solution), (5) H2O.
Using the same protocol, the distribution of 59Fe, 54Mn and 69Zn, containing
83 μg and 130 μg of Fe(III) and Zn(II), respectively, was determined. In addition,
the distribution of Ti(IV) was investigated in order to estimate the distribution,
eventually co-eluted within the 68Ga fraction. Thus, 20 µg Ti(IV) in 5 mL 0.lM
HCl was processed the same way. The distribution of Ti(IV) in the different
fractions was studied using an Elan 5000 ICP-MS (Perkin-Elmer).

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 SESSION 13

4.2. Labelling

In the case of the new 30 mCi 68Ge/68Ga generator, about 600–750 MBq
68
of Ga was obtained with 7 mL of 0.1M HCl. In addition, in order to obtain
about 1400 MBq, two 30 mCi generators were combined and eluted with 12 mL
of 0.1M HCl in a cascade scheme. These fractions were processed and applied
for labelling. After preconcentration and purification of the initial generator
eluates on the microchromatography column, 68Ga(III) was eluted with the
400 µL 98% acetone–0.05M HCl solution (2 × 10-5 mol HCl). This fraction was
used directly for labelling. For labelling with 68Ga(III), DOTA-octreotides
(DOTATOC, DOTANOC) and desferrioxamine-B-succinyl-octreotide
(DFOOC) were used. All labelling reactions were carried out at temperatures
of ~98°C. Kinetics of the syntheses were recorded up to 15 min by taking
aliquots of 1 µL at 1, 2, 5, 10, 15 min.
Two approaches were studied, namely 68Ga labelling with or without
additional buffer solutions. The processed activity was added to 4.5 mL pure
H2O in a standard glass reagent vial (11 mL, Mallinckrodt) containing 7–14 nmol
DOTATOC. To achieve higher specific activity 0.5–0.7 mL 1 molal HEPES,
pH4.0–4.1 (according to Ref. [6]) was added to 2 mL reaction vessels (PP,
Brand equipped with a vent) containing 2–5 nmol DOTATOC.

4.3. Purification of primary labelling products

The reaction mixture was passed through a small C-18 cartridge

(Phenomenex Strata-X Tubes, 30 mg). After washing the cartridge with H2O
(aqua ad iniectabilia), the 68Ga labelled peptides were recovered with 200–400 µL
of pure ethanol.

4.4. Quality control

Quality control was performed using TLC and HPLC. For HPLC, an
aliquot was dissolved in TFA 0.1% in H2O and used for analysis. Acetone
content was studied by a gas chromatography HP 6890 series GC system.

4.5. Synthesis for clinical application

Priorities for routine synthesis of 68Ga-DOTATOC were safety and

highest overall yield. The following scheme was developed for routine synthesis
of 68Ga-DOTATOC (Fig. 2). A microchromatography column with about
50 mg of the cation exchanger was prepared using two three-way valves. The
68
Ge/Ga radionuclide generator was connected to the column. PEEK capillary

373
 ROESCH et al.

tubing connected the reagent vials in a heating block. The column could be
eluted using a standard single use syringe (3) and was connected to the waste
vial (2). Syntheses were performed in pure water as described above using
14 nmol DOTATOC. After purification of the product on the C-18 cartridge, the
ethanol eluate containing the pure 68Ga-DOTATOC was dissolved in 5–10 mL
0.9% saline solution and sterilized by filtration through a 0.22 µm membrane
filter. Routine quality control was performed rapidly by TLC.

5. RESULTS

68
5.1. Ge/68Ga radionuclide generator performance
( Ga yield, 68Ge breakthrough)
68

All generator systems utilized provided stable recovery of 68Ga of >50%

(up to 80% in the first instance). More than 90% of the activity available can be
eluted within the first 5 mL of 0.1M HCl. A 68Ge breakthrough of less than
0.01% of the actual 68Ge activity was detected for all 68Ge/Ga generation within
the first year of operation. This reflects the technical characteristics (see
Section 3) of the producer. Up to 0.05% of 68Ge could be detected in the eluate
of an ‘old’ generator with >200 elutions.

5.2. Eluate purification: Chemical and radiochemical purities

Relative distribution of 68Ga(III), 68Ge(IV), Zn(II), Ti(IV), Fe(III) and

Mn(II) on a microchromatography column (53 mg AG 50 W-X8 200–400 mesh)
in hydrochloric acid–acetone media have been evaluated systematically. The
cation exchanger provides almost quantitative adsorption of more than 99% of
68
Ga(III) from the 0.1M HCl solution. Less than 2% of 68Ga(III) is lost in
applying the 80% acetone–0.05M HCl solution. After all the purification steps,
more than 97% of 68Ga(III) could be recovered in the 98% acetone–0.05M HCl
solution. The scheme is illustrated in Fig. 2.
68
Ge(IV) passes through the column in 0.1M HCl and is additionally
washed with 80% acetone–HCl solutions. The processed 68Ga fraction finally
contains <0.01% of 68Ge relative to the initial eluate. Thus, a 68Ge decontami-
nation factor of >1000 was achieved.
The purification step with 80% acetone–0.15M HCl allows the significant
reduction of the amount of Zn(II) to less than 10-3% and Fe(III) up to 11%
(see Fig. 3). Both Ti(IV) and Mn(II) could be eluted from the resin mainly in
4M HCl and only 0.1% of Ti(IV) and 10% of Mn(II) were detected in the
processed 68Ga fraction.

374
 SESSION 13

FIG. 2. Scheme of the generator associated post-processing resulting in a 68Ga labelling

unit.

5.3. Volume

Finally, the volume of the isolated, chemically and radiochemically

purified 68Ga fraction is 400 µL with HCl amounts of 2 × 10-5 mol.

5.4. Time

With application of the scheme described above (Fig. 2), processing of the
eluate requires only 4 min.

5.5. Labelling

The preconcentrated and purified 68Ga(III) eluted from the resin with
400 µL of the 97.6% acetone–0.05M HCl mixture was used for labelling
reactions. For labelling in pure water, up to 700 MBq 68Ga(III) was added to
4–4.5 mL (preheated) H2O in standard reagent vials containing 7–14 nmol
DOTATOC. The amount of HCl contained in the final eluate solution (400 µL
98% acetone–0.05M HCl = 2 × 10-5 mol H+) provided an overall pH of 2.30
(±0.05). No incorporation was achieved at higher or lower pH. At ~98°C, the
radiolabelling yield was over 95% within 10 min. Variable radiochemical yield

375
 ROESCH et al.

FIG. 3. Purification factors achieved within the generator associated post-processing

(cf. FIG. 2).

and increased adsorption on the glass surface were detected if less than 14 nmol
of peptide was used. Incorporation also dropped with decreasing reaction
temperature.

5.6. Specific activity

Specific activities of up to 40 MBq/nmol could be achieved using pure

water as the solvent of DOTATOC. In this case, the processed 68Ga eluate
(2 × 10-5 mol HCl, up to 1400 MBq) was added to 0.5–0.7 mL 1 molal HEPES
solutions of pH4.0–4.1 in 2 mL reaction vessels containing 2–4 nmol
DOTATOC, the radiochemical yield was up to 88% and the specific activity up
to 450 MBq/nmol at ~99°C within 10 min.

5.7. Purification

Radiochemical purity over 99% was achieved by processing the labelling

fraction on small C-18 cartridges independent of the initial labelling yield
(cf. Fig. 4). However, essential losses of the peptide on the RP phase occurred if
less than 4 nmol of DOTATOC was used. After incubation of the reaction
mixture at ~98°C for 10 min, the overall acetone content was about 5 µg. The

376
 SESSION 13

FIG. 4. Purification and pre- and post-purification quality control of 68Ga radiopharma-
ceuticals associated to the post-processing and synthesis scheme.

final product after processing on C-18 contained not more than 0.15 µg of
acetone.

6. DISCUSSION

For labelling of biomolecules via bifunctional chelators, 68Ga(III) as

eluted initially needs to be preconcentrated and purified from 68Ge(IV),
Zn(II), Ti(IV) and Fe(III). The authors describe a system for simple and
efficient handling of the 68Ge/68Ga generator eluates with a microchromato-
graphy column filled with about 50 mg of a cation exchange resin (Bio-Rad AG
50W-X8) as the main component. Chemical purification and volume concen-
tration of 68Ga are carried out in an 80% acetone–0.15M HCl solution. Finally,
more than 97% of 68Ga are obtained in 400 µL of a 97.6% acetone–0.05M HCl
solution. The initial 68Ge contamination of the eluate was reduced by a factor of
1000. Contents of Zn(II), Fe(III) and Ti(IV) were reduced significantly.
Consequently, the processed fraction can be used directly for the synthesis of
radiopharmaceuticals.
Simple labelling protocols were used for routine preparation of 68Ga-
DOTATOC as a prototype of a 68Ga labelled PET radiopharmaceutical. The

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reaction was carried out in pure water using 14 nmol of DOTATOC and
resulted in high incorporation and high overall yield. Up to 400 MBq of
DOTATOC with specific activity up to 40 MBq/nmol could be obtained after
processing on C-18 with a new 30 mCi generator.
Within 20 min, an injectable radiopharmaceutical such as 68Ga-
DOTATOC can be prepared with specific activities of up to 450 MBq/nmol.
While standard specific activities of 40 MBq/nmol are quite acceptable for
clinical diagnosis using 68Ga-DOTATOC, increased specific activities can be
required for clinical application of DOTA-peptides with potential pharmaco-
logical side effects [7].
The application of C-18 cartridges for final purification seems to be an
essential step which provides high radiochemical purity independent of
labelling yield. Furthermore, additional quality control might be avoided.
Thus, a rapid, simple and chemically efficient processing of generator
produced 68Ga(III) was developed. The process guarantees safe preparation of
injectable 68Ga-DOTATOC (or other 68Ga labelled radiopharmaceuticals) for
routine application and can be successfully used in the clinical environment.
Simple equipment for routine preparation of injectable 68Ga-DOTATOC was
installed in various nuclear medicine departments equipped with a PET/CT.
More than 1000 patients with known neuroendocrine tumours were involved in
initial systematic studies to establish and evaluate a clinical protocol. Both
[68Ga]DOTATOC and [68Ga]DOTANOC were successfully used in a series of
human somatostatin receptor expressing tumour diagnoses with PET/CT. The
developed system represents a simple and efficient way for the labelling of
DOTA conjugated biomolecules with generator produced 68Ga(III).
Moreover, a variety of other 68Ga labelled compounds might be synthesized for
many other applications. The current level of radiochemical development and
radiopharmaceutical investigation clearly indicates a significant potential for
the 68Ge/Ga generators, for applications in basic research and for routine
application in state of the art nuclear medicine. In particular, for countries or
medical centres not yet running medical cyclotrons and/or not yet owning a
sophisticated organic radiopharmaceutical production infrastructure, the avail-
ability of the 68Ge/Ga generator might help to initiate PET chemistry develop-
ments and patient diagnoses using PET.
As the developed scheme guarantees safe preparation of injectable 68Ga
labelled DOTA conjugates for routine application (easy to automate) it has
been successfully used in clinical environments. Using the authors’ system, over
the last year several German clinical centres have been involved in establishing
clinical protocols for application of 68Ga-DOTATOC and similar compounds in
more than 500 patients. The clinical impact is extraordinarily high. Using PET
and in particular PET/CT, the diagnosis of neuroendocrine tumours and

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 SESSION 13

metastases is significantly superior to the current state of the art approaches

using 111In-DTPA-octreotide and SPECT.
Owing to the low costs of the 68Ge/Ga generators and to the simple kit
type of labelling, this approach is straightforward and to be adopted in clinical
centres not owning a cyclotron for in-house productions of 11C and 18F, and/or
not owning a rather complicated and expensive organic labelling chemistry
facility in conjunction with the required hot cell and automated synthesis
modules. Some other features of the generator system are of additional
importance:

Diagnosis:

• Per generator elution, up to three patients can be diagnosed.

• The generator can be eluted every 3 or 4 h at almost 100% yield, allowing
clinical applications of up to 4 or 5 elutions per day with up to 20 patient
studies per day.
• A generator once installed can work one year or more and more than
500 PET studies might be performed.
• The price per patient study thus will be very low.
• Owing to the 68 min half-life of 68Ga, patients do not need to stay in the
hospital at all.

Therapy:

• As many of the therapeutic targeting vectors today are designed for

labelling with trivalent metallic cations (90Y, 153Sm, 177Lu, etc.), 68Ga PET
scans could become a standard approach for pre-and post-therapeutic
screening.

Furthermore, supposing that the biomedical targeting approach will

require longer lived positron emitters, there are two other PET radionuclide
generators.
One is the 44Ti (T½ = 47 a)/44Sc (T½ = 3.927 h) radionuclide generator
which provides a very similar chemical system. The longer lived progeny 44Sc,
however, presents a physical half-life adequate to biological investigations,
where the 68 min half-life of 68Ga appears to be too short. Similar to 68Ga(III),
the 44Sc(III) action needs to be adequate to label many of the already existing
targeting vectors containing bifunctional chelators for trivalent cations. Last
year, the authors started a programme on the development of this system.
The second is the 72Se/As system which provides a 26 h positron emitter
with 88% positron branching. The authors recently developed the basic

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radiochemistry for this generator and have shown the potential of longer lived
radioarsenic to image the targeting parameters of monoclonal antibodies.
In addition, in particular if targeting vectors such as monoclonal
antibodies are used instead of small peptides, the authors recently proposed the
radioarsenic isotopes 72As (T½ = 26 h, 88% ββ+ branching) and 74As (T½ = 17.78 d,
29% ββ+ branching). As a proof of principle, they successfully tested the
hypothesis that a new chimeric IgG3 monoclonal antibody ch3G4 (Tarvacin®)
directed against anionic phospholipids and labelled with radioactive arsenic
isotopes can be used for the vascular targeting and molecular imaging of solid
tumours in rats in vivo. For generators using no carrier added 72Se, the authors
described the distillation of AsCl3, while Se remains in non-volatile compounds
in the residue, and developed a solid phase extraction system with 72Se fixed as
metallic Se. Systematic chemical investigations on the labelling chemistry of no
carrier added radioarsenic are currently being developed prior to the
application of 72As labelled compounds [9–12].
Thus, the potential of PET radionuclide generator systems seems to fit
excellently with the strategy of the IAEA in facilitating the development of
modern medical imaging technologies in the developing countries.

REFERENCES

[1] RÖSCH, F., KNAPP, F.F., “Radionuclide generators”, Handbook of Nuclear

Chemistry, Vol. 4 (VÉRTES, A., NAGY, S., KLENCSÁR, Z., RÖSCH, F., Eds),
Kluwer Academic Publishers, The Netherlands (2003) 81-118.
[2] MÄCKE, H.R., GOOD, S., “Radiometals (non-Tc, non-Re) and bifunctional
labeling chemistry”, Handbook of Nuclear Chemistry, Vol. 4 (VÉRTES, A.,
NAGY, S., KLENCSÁR, Z., RÖSCH, F., Eds), Kluwer Academic Publishers, The
Netherlands (2003) 279-314.
[3] HOFMANN, M., et al., Biokinetics and imaging with the somatostatin receptor
PET radioligand 68Ga-DOTATOC: preliminary data, Eur. J. Nucl. Med. (2001)
28:1751–1757.
[4] WILD, D., et al., DOTA-NOC, a high-affinity ligand of somatostatin receptor
subtypes 2, 3 and 5 for labelling with various radiometals, Eur. J. Nucl. Med.
(2003) 30: 1751–1757.
[5] MEYER, G.-J., MÄCKE, H.R., SCHUHMACHER, J., KNAPP, W.H.,
HOFMANN, M., 68Ga-labelled DOTA-derivatised peptide ligands, Eur. J. Nucl.
Med. (2004) 31: 1097–1104.
[6] VELIKYAN, I., BEYER, G.J., LÅNGSTRÖM, B., Microwave-Supported Prepa-
ration of 68Ga Bioconjugates with High Specific Radioactivity, Bioconjugate
Chem. (2004) 15: 554-560.

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[7] BREEMAN, W.A.P., et al., Radiolabelling DOTA-peptides with 68Ga, Eur. J.

Nucl. Med. (2004) 32: 478–1104.
[8] STERLOW, F.W.E., VICTOR, A.H., VAN ZYL, C.R., ELOFF, C., Distribution
coefficient and cation exchange behavior of elements in hydrochloric acid-
acetone, Anal. Chem. 1971; 43: 870-876.
[9] JENNEWEIN, M., SCHMIDT, A., NOVGORODOV, A.F., QAIM, S.M.,
RÖSCH, F., A no-carrier-added 72Se/72As radionuclide generator based on distil-
lation, Radiochim Acta 92 (2004) 245-249.
[10] JENNEWEIN, M., et al., A new method for radiochemical separation of arsenic
from irradiated germanium oxide, Appl Radiat Isot 63 (2005) 343–351.
[11] JENNEWEIN, M., et al., A no-carrier-added 72Se/72As radionuclide generator
based on solid phase extraction, Radiochim Acta 93 (2005) 579-583.
[12] JENNEWEIN, M., HERMANNE, A., MASON, R.P., THORPE, P.E., RÖSCH, F.,
A new method for the labelling of proteins with radioactive arsenic isotopes, Nucl
Instr Methods B, 2006, in press.

381
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 RADIOPHARMACY

(Session 14)

Chairpersons

N.G. HARTMAN
United Kingdom

E. JANEVIK-IVANOVSKA
The Former Yugoslav Republic of Macedonia
.
 NUCLEAR PHARMACY PRACTICES IN THE UNITED
STATES OF AMERICA

K. OZKER
Medical College of Wisconsin,
Milwaukee, Wisconsin,
United States of America
Email: ozker@mcw.edu

Abstract

There are more than 450 nuclear pharmacies in the United States of America.
Approximately 80% of these are centralized nuclear pharmacies operated by three
major companies: Cardinal Health, Tyco Healthcare/Mallinckrodt and GE Healthcare.
There are 88 independent facilities and two additional companies specialized in PET
radiopharmaceuticals: CTI/PETNET and Eastern Isotopes. Institutional nuclear phar-
macies, representing 20% of the radiopharmacies in the USA, prepare multidose radio-
pharmaceuticals in university or hospital settings. All commercial nuclear pharmacies
are licensed by a state board of pharmacy and operate under the supervision of an
authorized nuclear pharmacist. The Nuclear Regulatory Commission requires that all
nuclear pharmacists be certified pharmacists, completing an approved programme
consisting of 200 h of didactic and 500 h of practical training. Recently, requirements for
aseptic compounding and dispensing of radiopharmaceuticals have been developed by
the United States Pharmacopoeia (USP <797>). The Society of Nuclear Medicine
communicated with the USP in late 2004 regarding unique situations specific to the
preparation of radioactive compounds (such as radiation exposure, shielding require-
ments and contamination risks) that would make it difficult to comply fully with the new
USP regulations. The USP <797> Sterile Compounding Committee subsequently
approved revisions and exemptions for radiopharmaceutical compounding.

Currently, radiopharmaceutical preparation in the United States of

America is mostly performed by centralized radiopharmacies (CRPh). CRPh
provide single patient unit doses in shielded containers to their customers. The
advantages of this practice to nuclear medicine providers are: minimization of
radiation exposure; simplification of regulatory requirements including
paperwork; and reduction of risk to staff, patients and the public. In general,
procurement, preparation and dispensing of radiopharmaceuticals is provided
by CRPh, which makes a wide variety of products available for immediate
delivery. Dose preparation, labelling, quality control, record keeping and
radioactive waste retrieval are all performed by the CRPh. These services

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reduce the responsibilities of the nuclear medicine facility, including those

involving risk management, quality assurance and regulatory standards in an
efficient and cost effective manner. The capability to acquire agents on the
same day from CRPh eliminates the necessity of having certain expensive
supplies available at the imaging location, particularly a 99Mo/99mTc generator
[1].
In a high volume nuclear medicine facility, a hospital radiopharmacy
(HRPh) may be more cost effective compared to purchasing unit doses from a
CRPh. Preparation of radiopharmaceuticals in-house can save money since a
large number of patient doses may be obtained from a single multidose vial.
The number of studies performed each day (and thus the number of injected
doses) must be high enough to justify the cost of staffing, supplies and quality
assurance procedures. Currently, there are more than 450 radiopharmacies in
the USA of which CRPh represent about 80% of these, operated by three
major companies: Cardinal Health, Tyco Healthcare/Mallinckrodt and GE
Healthcare. Cardinal Health (formerly Syncor) has the largest network of
facilities following its merger with Geodax Imaging (19 pharmacies) in July
2005 [2]. There are two other CRPh companies dedicated solely to PET radio-
pharmaceuticals: CTI/PETNET (39 pharmacies) and Eastern Isotopes
(9 pharmacies). There are 88 independent commercial CRPh laboratories and
about 70 institutional radiopharmacies located in hospitals or universities [1, 2].
All commercial CRPh are licensed by a state board of pharmacy and
operated under the supervision of an authorized nuclear pharmacist. The
possession, handling, dispensing and waste management of radiopharmaceu-
ticals are regulated by the Nuclear Regulatory Commission (NRC) or an
agreement state regulatory agency such as the State Board of Radiation
Protection. The NRC requires that all nuclear pharmacists be certified pharma-
cists, completing an approved programme consisting of 200 h of didactic and
500 h of practical training involving radiopharmaceuticals [3]. Various
programmes offer either on-site classroom training or remote education. The
first graduate level radiopharmacy programme was taught at the University of
Southern California, from which graduated 210 students between 1968 and
1986. The Purdue University programme has been in operation since 1972. The
University of New Mexico and the University of Arkansas jointly developed a
remote learning programme which is currently available on the internet. A
syllabus for nuclear pharmacy training published by the American Pharmaceu-
tical Association in 1995 as a guide for educational institutions and educators
(faculty/preceptors) is based on the NRC requirements for authorized nuclear
pharmacist training. The NRC requirement for authorized nuclear pharmacy
programmes contains 200 class room hours covering the following topics:

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radiation physics and instrumentation, radiation protection, mathematics

related to radioactivity, radiation biology and radiopharmaceutical chemistry.
Some 500 h of practical training in a radiopharmacy under the supervision
of an authorized nuclear pharmacist is also required. Nuclear pharmacy
certificate programmes are currently offered at The University of Arkansas,
The Massachusetts College of Pharmacy, Mercer University, The University of
New Mexico, The University of North Carolina, The Medical University of
South Carolina, The University of Oklahoma, Purdue University and Temple
University [1]. Several nuclear pharmacy companies, such as Cardinal Health,
have established their own training programmes [2].
NRC requirements for radiopharmacies are based on radiation safety
principles to minimize patient, occupational and public radiation exposure.
Additionally, guidelines for aseptic compounding and dispensing of radiophar-
maceuticals developed by the United States Pharmacopoeia (USP) have
recently become enforceable by the Food and Drug Administration (USP
Chapter <797> regulations effective 1 January 2004). All healthcare facilities
that prepare sterile products for medical use, including biological agents,
diagnostic agents, drugs, nutrients and radiopharmaceuticals, must comply with
these regulations. USP <797> describes pharmacies as having one of three risk
levels: low, medium or high [4].

(1) Low risk conditions are where all compounding with aseptic manipula-
tions occur entirely with ISO Class 5 (old class 100) or better air quality
using only sterile ingredients, products, components and devices.
(2) Medium risk conditions include multiple individual or small doses of
sterile products that are compounded or pooled to prepare a
compounded sterile product that will be administered either to multiple
patients or to one patient on multiple occasions. USP <797> implies that
medium risk compounding must also take place in an ISO Class 5 or
better air environment.
(3) High risk conditions include the use of non-sterile ingredients,
components or devices and sterile ingredients, components or devices
which are exposed to air quality inferior to ISO Class 5. In this case, the
compound must subsequently be sterilized in an ISO Class 5
environment.

Radiopharmaceuticals are considered compounded sterile products

according to USP <797>. All sterile compounding is to be performed in an ISO
Class 5 environment using a laminar airflow workbench. The area surrounding
the location where the compounding and/or sterilization occur is called the
buffer zone (clean room). Buffer zones should meet at least an ISO Class 7

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 OZKER

level (old class 10 000) of cleanliness. Buffer zone surfaces must be smooth,
impermeable, readily cleanable and capable of sanitization. Low and medium
risk environments must have controlled ISO level Class 5 cleanliness as well as
a buffer zone and an additional ante area (where gowning and hand washing
are carried out) which does not necessarily need to be separated by a physical
wall. High risk environments must have the same features as the low and
medium risk sites but the additional ante area must be separated from the
buffer zone by a physical barrier [4, 5].
USP <797> regulations are currently enforceable by the Food and Drug
Administration and the State Board of Pharmacy, Medicine and Nursing.
Healthcare institutions must begin to comply with the requirements of USP
<797> over a specific timeline. The Society of Nuclear Medicine communicated
with the USP in late 2004 about unique situations specific to the preparation of
radioactive compounds (such as radiation exposure, shielding requirements
and contamination risks) that would make it difficult to comply fully with the
USP regulations. The USP Sterile Compounding Committee met in October
2004 and approved the following revisions to USP <797> [6]:

(1) Currently official <797> requires positive pressure for all sterile
compounding, but that is wrong for radioactive and other hazardous
drugs.
(2) Direct visual inspection of highly radioactive CSPs is not required.
(3) The 99mTc/99Mo generator systems shall be stored and eluted (operated)
under conditions recommended by their manufacturers and applicable
state and federal regulations.
(4) Three or fewer sterile products may be prepared in lower than ISO Class
5 air when there is no direct contact contamination and administration
begins within 1 h and is completed within 12 h of preparation.

The Sterile Compounding Committee reviews received comments, then

determines whether additional revision is necessary before the next version is
published in the Pharmacopeial Forum as an Interim Revision Announcement
which bears a date for official USP adoption. The next <797> will appear either
in an annual USP revision, e.g. USP 29 in 2006 or in one of the two semi-annual
supplements to each annual USP revision [6].
On the basis of the latest approved revisions of USP <797>, there are still
several uncertainties regarding exemptions for radiopharmaceutical
compounding that can affect the design of a nuclear pharmacy.
The Joint Commission on Accreditation of Healthcare Organizations
(JCAHO) has begun to survey hospitals for compliance with USP <797>.
JCAHO expects healthcare organizations to have performed a risk assessment/

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 SESSION 14

gap analysis and established an action plan for achieving compliance by

January 2005, and to have implemented interim steps to mitigate the impact of
non-compliance, assure sterility in compounding by July 2005 and to have
completed action plans and achieved full compliance by January 2008 [7].

REFERENCES

[1] AMERICAN PHARMACIST ASSOCIATION, Radiopharmaceuticals, Nuclear

Medicine and Nuclear Pharmacy: An Overview. In: Kowalsky RJ, and Falen SW.
Radiopharmaceuticals in Nuclear Pharmacy and Nuclear Medicine 2nd ed.
American Pharmacist Association, (2004) 1-15.
[2] CARDINAL HEALTH NUCLEAR PHARMACY SERVICES,
www.nps.cardinal.com/NPS/content/nucpharm/index.asp. Accessed on
September 19, 2005.
[3] BOARD OF PHARMACEUTICAL SPECIALTIES, Nuclear Pharmacy
Practice Guidelines. Website Available at: www.bpsweb.org. Accessed on
September 19, 2005.
[4] US PHARMACOPEIA, The United States Pharmacopeial Convention, Inc. U.S.
Pharmacopeia 27. Chapter <797>, Pharmaceutical Compounding – Sterile Prepa-
rations, Rockville, MD: U.S. Pharmacopeial Convention, Inc., (2003).
[5] US PHARMACOPEIA, The United States Pharmacopeial Convention, Inc. U.S.
Pharmacopeia 28, Chapter <797> Pharmaceutical Compounding – Sterile Prepa-
rations, Rockville, MD: U.S. Pharmacopeial Convention, Inc., (2004).
[6] US PHARMACOPEIA, The United States Pharmacopeial Convention, Inc.
Website Available at: www.usp.org/standards/proposed 797 revisions.html.
Accessed on June 24, 2005.
[7] AMERICAN HOSPITAL ASSOCIATION, JCAHO clarifies expectations and
timelines in compliance with USP <797> Website Available at: www.aha.org/ashe/
codes/jcaho/clarify_usp797.html. Accessed on July 25, 2005.

389
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 REGULATORY ASPECTS OF HOSPITAL
RADIOPHARMACY AND CLINICAL TRIALS

A.A. SOYLU
Nuclear Medicine Department,
Faculty of Medicine, Ankara University

Monrol Radiopharmaceutical Company

Ankara, Turkey
Email: ayfersoylu@yahoo.com

Abstract

The number of clinical research studies is increasing as scientists concentrate their

efforts on diagnosing, treating and preventing diseases by increasing knowledge about
human health. The progress of nuclear medicine is heavily dependent on development
of new radiopharmaceuticals. Many research studies exist relating to new
radiopharmaceuticals, but it is a known fact that the number of studies involving human
application of these investigated products is comparatively low. While the main problem
is financial, owing to the insufficiency of the market, overregulation of clinical studies on
humans and uncertainties on how these regulations should apply to
radiopharmaceuticals compared to regular pharmaceutical products has always been
another issue. The purpose of this paper is to discuss the main regulations on clinical
trials in general and to evaluate certain points specific to clinical studies involving
radiopharmaceuticals, and at the same time assess the existing radiopharmacy
legislation.

1. INTRODUCTION

The Declaration of Helsinki, published in 1964 [1] by the World Medical

Association and entitled Ethical Principles for Medical Research Involving
Human Subjects, and its revised forms (1975, 1983, 1989, 1996, 2000 and 2004)
and clarification notes (2002, 2004), should constitute the basis for all medical
research on human beings with a set of recommendations that guide
responsible physicians all over the world. Special caution should also be given
to the welfare of animals used for research and to the environment in which the
trial is conducted .

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 SOYLU

2. CLINICAL TRIAL

A clinical trial may be defined as: “Any investigation in human subjects

intended to discover or verify the clinical, pharmacological or
pharmacodynamic effects of a drug, identify any adverse reactions related to a
drug, study the absorption, distribution, metabolism and excretion of the drug
with the object of ascertaining its safety and/or efficacy” [2]. Clinical trials may
either be commercially funded or non-commercial. Studies set up for
commercial goals are usually sponsored by industrial companies for the
purpose of drug development and registration. It takes years for a new drug to
be put on the market and results obtained only by authorized clinical trials can
be relied on for marketing authorization of an investigational medicinal
product (IMP). Non-commercial clinical trials are generally conducted by
academia and use existing medicinal products to optimize treatment regimes or
establish new techniques with the same products. But there are occasions
where a new pharmaceutical product is tried on patients by non-commercial
investigators. Many studies involving the use of radiopharmaceuticals on
human beings fall into this category. Investigators who are authorized health
professionals conducting non-commercial trials must be responsible for all
administrative, financial and legal obligations and must assume the human
subject protection requirements completely, just as with commercial sponsors.
The regulations apply equally to commercial and non-commercial trials.

3. IMP

A general definition of IMP would be: “A pharmaceutical form of an

active substance or placebo being tested, or to be tested, or to be used, as a
reference in a clinical trial, including a medicinal product which has a
marketing authorization but is, for the purposes of the trial, used or assembled
(formulated or packaged) in a way different from the authorized form, or used
for an indication not included in the summary of product characteristics under
the authorization for that product, or used to gain further information about
the authorized form” [2].

4. RADIOPHARMACEUTICALS

Radiopharmaceuticals may be argued to be different from classical drug

products in being radioactive and carrying a small amount of material in a small
volume of injection. A radiopharmaceutical is applied to a patient usually only

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once or a few times in their lifetime. In general, these products spend a short
time in the body and no biological effect is expected to occur by the intro-
duction of a radiopharmaceutical to a human being. On-site preparation and
immediate use may be other aspects of radiopharmaceuticals differing from
regular drug products. In spite of these arguments, radiopharmaceuticals are
considered as medicinal products [3] and any radiopharmaceutical used in a
clinical trial is therefore subject to all the legislation regarding IMP.
The radioactive nature of radiopharmaceuticals also makes it necessary
to follow the regulations related to ionizing radiation. Euratom Directive 97/43,
entitled Protection of Individuals Against the Dangers of Ionizing Radiation in
Relation to Medical Exposure applies to exposure of healthy individuals or
patients voluntarily participating in medical or biological, diagnostic or
therapeutic, research programmes [4].

5. GOOD CLINICAL PRACTICE (GCP)

The European Directive on Good Clinical Practice in Clinical Trials —

2001/20/EC [2] together with its supplements [5] constitute the main
regulations in Europe which apply to all IMP, including radiopharmaceuticals.
This directive, which became fully operational on 1 May 2004, has many
implications covering various issues on trials such as the suitability of investi-
gators and facilities, the quality of the IMP, informed consent from the subjects,
ethics committees and the European Clinical Trials Database. The main
purpose of the Directive on clinical trials is to guide the EU Member States on
the implementation of good clinical practice in the conduct of clinical trials on
medicinal products for human use by adopting laws, regulations and adminis-
trative provisions.
The European Commission Directive 2005/28/EC [5] is one of the recent
supplements of Directive 2001/20/EC strengthening the legal basis and estab-
lishing more specific terms for GCP. This new directive clarified some questions
regarding the use of unauthorized products in clinical trials and was to be
implemented by the Member States by 29 January 2006.
GCP is a set of minimum standards for clinical trials which ensure the
protection of the rights, welfare and safety of human subjects and the validity of
collected data and reported results. It also covers issues related to the design,
conduct, performance, auditing, recording and reporting of clinical trials. An
individual who participates in a clinical trial either as a recipient of the IMP or
as a control is the ‘subject’ of this trial. The subjects can be healthy volunteers
or patients. The rights, safety and well-being of the trial subjects are the most

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important considerations and should prevail over the interests of science and
society.

6. CLINICAL TRIALS

All clinical research studies involving humans must be designed

according to a ‘protocol’, which is a document describing the objectives, design,
methodology, statistical considerations and organization of that specific trial
and must have been approved by the related regulatory bodies before the trial
commences. Detailed preclinical studies should have been carried out,
covering both clinical and non-clinical aspects of the IMP. In studies with
classical pharmaceutical products, the preclinical work is followed by phase I
clinical studies if the available information is found to be satisfactory. The
studies are usually carried out on a small number (50–100) of healthy
volunteers, starting with a dose as low as possible and then increasing the dose
until the desired effect is reached. During phase II, the investigational product
is given to 200–400 patients with the indicated disorder. In general, a larger
number of patients are involved in phase III of the project, some receiving a
placebo, for a valid statistical evaluation of the results. The situation may be
different for clinical trials involving the use of radiopharmaceuticals; studies on
healthy volunteers may be omitted in order to avoid unnecessary radiation
exposure to the public and the trial may continue with a small number of
patients after adequate preclinical evidence has been obtained.
The Directive 2001/83/EC [3], which is also related to medicinal products
for human use and was implemented on 1 May 2004, does not directly regulate
clinical trials but clearly states that any application for market authorization of
a medicinal product in the EU should be accompanied by a complete dossier
containing the documents related to the results of the clinical trials carried out
on that product. 2004/27/EC [6] is an amendment of this directive and gives
more details on clinical trial file preparation, documentation and similar issues.

7. GOOD MANUFACTURING PRACTICE (GMP)

Directives 2001/20/EC, 2001/83/EC and 2003/94/EC [7], which is an

extension of GMP to IMP, clearly require that IMP activities for human use be
carried out in licensed premises in accordance with GMP and the competent
authorities are required to inspect these activities. A ‘qualified person’ must be
assigned to ensure that the GMP requirements are met for the IMPs released.
The qualified person must be the holder of a diploma, certificate or other

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evidence of formal qualification in pharmacy, chemistry, medicine, biology or

related life science and must have gained experience in similar work and
acquired the necessary skills [3].
Many non-EU countries have their national regulations set similarly and
they are being harmonized by the efforts of the International Conference on
Harmonization (ICH). Guideline CPMP/ICH/135/95 [8], entitled Good
Clinical Practice has been adopted by the Committee for Medicinal Products
for Human Use. It is decided that results of clinical trials which have been
carried out in accordance with this guideline should be accepted by Australia,
Canada, the EU, Japan, the Nordic countries and the United States of America.

8. CONCLUSION

The above discussion should make it clear that use of unauthorized

radiopharmaceuticals on human beings is certainly subject to permission being
granted by the competent authorities. This covers home-made radio-
pharmaceuticals and any changes in the method of preparation of a licensed
radiopharmaceutical including dividing up and packaging or presentation.
Even the use of an authorized radiopharmaceutical for a different indication or
given at a different dosage or use of a different route of administration other
than that registered on the authorization are considered as cases which need
permission. The only exception are the clinical studies which involve standard
application of approved radiopharmaceuticals to assess the efficacy of a
treatment (e.g. bone scan used for monitoring in a chemotherapy trial), which
are not considered as clinical trials and thus there is no need to go through the
procedures for gaining clinical trial permission for such projects.
The recent amendments on the other hand, state that authorization will
not be required for a process involving reconstitution prior to administration of
the product or packaging if these are done in hospitals, health centres or clinics
by pharmacists or qualified persons and if the IMP is intended to be used
exclusively in that institution. The procedures for importing IMPs from other
countries are also simplified. Trials with products having marketing authoriza-
tions and manufactured or imported in accordance with EU rules are permitted
to be carried out if they are conducted on patients with the same indication
specified in the marketing authorization. However, guidance is needed which
specifies how to protect the subjects in such applications.
Owing to the unique nature of radiopharmaceuticals, various organiza-
tions have been working on defining a set of current GMP conditions, specifi-
cally developed for radiopharmaceuticals. The Radiopharmacy Group of the
European Association of Nuclear Medicine has prepared Draft Guidelines on

395
 SOYLU

Current Good Radiopharmacy Practices for Radiopharmaceuticals in Nuclear

Medicine-GRPP and Draft Guidelines on Current Good Manufacturing
Practices for Positron Emission Tomography and Other Locally Produced
Radiopharmaceuticals-PET GMP [9].
Similar efforts follow in the USA where the Food and Drug Adminis-
tration (FDA) has recently announced a proposed rule for the production of
PET drugs [10] and also draft guidance providing additional information about
the proposed regulation when it becomes a final rule [11]. The proposed rule is
intended to set the minimum standards for the production and testing of PET
radiopharmaceuticals and guide the producers on achieving these require-
ments. Although these current GMP conditions differ greatly from those of
non-PET drugs, the aim is, of course, to ensure that the best quality PET
radiopharmaceutical was available for the patient regardless of the purpose
and place of production and the person who carried out the production.
The finalization of the rule and the guidance by the FDA may be
expected to help speed up the international harmonization procedures.

REFERENCES

[1] WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI,

Ethical Principles for Medical Research Involving Human Subjects, Helsinki,
Finland, June 1964.
[2] EUROPEAN COMMISSION, Directive 2001/20/EC of the European Parliament
and of the Council of 4 April 2001 on the approximation of the laws, regulations
and administrative provisions of the Member States relating to the implemen-
tation of good clinical practice in the conduct of clinical trials on medicinal
products for human use (Official Journal L 121, 1/5/2001 p. 34 – 44).
[3] EUROPEAN COMMISSION, Directive 2001/83/EC of the European Parliament
and of the Council of 6 November 2001 on the Community code relating to
medicinal products for human use (Official Journal L 311, 28/11/2001 p. 67 - 128).
[4] EUROPEAN COMMISSION, Council Directive 97/43 Euratom, Health
Protection of Individuals Against the Dangers of Ionizing Radiation in Relation
to Medical Exposure ,The Council of the European Union (Official Journal L 180,
09/07/1997 p. 22 – 27).
[4] EUROPEAN COMMISSION, Commission Directive 2005/28/EC of 8 April 2005
laying down principles and detailed guidelines for good clinical practice as regards
investigational medicinal products for human use, as well as the requirements for
authorisation of the manufacturing or importation of such products (Official
Journal L 91, 09/04/2005 p. 13 - 19).
[6] EUROPEAN COMMISSION, Amended by Directive 2004/27/EC of the
European Parliament and of the Council of 31 March 2004 amending Directive
2001/83/EC on the Community code relating to medicinal products for human use
(Official Journal L 136, 30/4/2004 p. 34 - 57).

396
 SESSION 14

[7] EUROPEAN COMMISSION, Commission Directive 2003/94/EC of 8 October

2003 laying down the principles and guidelines of good manufacturing practice in
respect of medicinal products for human use and investigational medicinal
products for human use (Official Journal L 262, 14/10/2003 p. 22 - 26).
[8] INTERNATIONAL CONFERENCE ON HARMONIZATION, Note for
Guidance on Good Clinical Practice, ICH/135/95, Committee for Medicinal
Products for Human Use , July 1996 .
[5] EUROPEAN JOURNAL OF NUCLEAR MEDICINE, The Draft Guidelines
on Good Radiopharmacy practices for Radiopharmaceuticals (GRPhP) in
Nuclear Medicine and the Draft Guidelines on Good Manufacturing Practices for
Positron Emission Tomography (PET) Radiopharmaceuticals, EJNM (2003)
30:BP63-BP72.
[6] UNITED STATES FOOD AND DRUG ADMINISTRATION, Current good
Manufacturing Practice for Positron Emission Tomography Drugs : draft for
proposed rule by USFDA ; 21 CFR Parts 210,211,212 (Fed Reg V 70, no 181, Sept
20 2005 p 55038-55062).
[7] UNITED STATES FOOD AND DRUG ADMINISTRATION, PET Drug
Products-Current Good Manufacturing Practice-Draft Guidance, Sept 2005,
USFDA (CDER).

397
.
 STANDARDIZATION AND QUALITY CONTROL OF
AN IN-HOUSE FORMULATION OF 99mTc(V)-DMSA IN
TUMOUR IMAGING AND ASSESSMENT OF
TUMOUR BIOLOGY: WORK IN PROGRESS

P.S. CHOUDHURY*, N.C. GOOMER**, A. GUPTA*,

D.C. DOVAL***, T. KATARIA+, A.K. VAID***, P.K. SHARMA*

* Department of Nuclear Medicine

Email: pschoudhury@rgci.org

** Department of Medical Oncology

*** Department of Radiation Oncology

Rajiv Gandhi Cancer Institute and Research Centre

+
Regional Centre for Radiopharmaceuticals (BRIT)

New Delhi, India

Abstract

Various in-house formulated radiopharmaceuticals have been used for tumour

imaging with varied success. In order to achieve clinically reproducible results, the purity
and stability of the formulation needs to be standardized. The objective of the present
study was to reproduce a simple but optimum method for in-house formulation of
99m
Tc(V)-DMSA from the commercially available DMSA(III) kits for its use as an
agent for assessing tumour biology. The authors used two commercially available kits of
DMSA(III): one from the Board of Radiation & Isotope Technology (BRIT) and the
other from Amersham. Both kits were formulated as per the manufacturer’s protocol.
The authors determined the radiochemical purity using TLC (T-6145 TLC precoated
plated silica gel with a 24 nm fluorescent indicator on polyester supplied by Sigma/
Merck) with a solvent containing n-butanol acetic acid water (3:2:3 v/v). Free pertech-
netate levels were measured using TLC-SG in saline. Per cent labelling of DMSA and
free pertechnetate was calculated and the ratios of DMSA(III) and DMSA(V) were
estimated. These were carried out for a period of 3 h. Observations showed that the
radiochemical purity of DMSA(V) for the Amersham kit was optimum at about 1 h
post-labelling. The same effect was observed in 15 min in the kit that was supplied by
BRIT. The time of optimum radiochemical purity was reduced to 15 min by bubbling
oxygen through the preparation for 20 min and attributed to oxidation of tin. Human

399
 CHOUDHURY et al.

biodistribution studies showed predominantly renal pelvicalyceal excretion in a radio-

chemically pure preparation with physiological concentration in the bladder. The
cardiac blood pool showed significant tracer washout in 3–4 h. A pilot study carried out
has so far shown a maximum tumour concentration with adequate target to non-target
ratio occurring by 3 h post-injection in patients with histologically proven lung carci-
noma.

1. INTRODUCTION

Malignant transformation leads to major biochemical changes within a

cell. These include modifications of the energy metabolism of the cell such as
glucose and other substrate utilization, protein synthesis and expression of
receptor and antigens. These biochemical changes are difficult to map by
conventional morphological imaging studies and require the use of tracers
which can assess tumour viability. Standardization of a particular method of
formulation is important to achieve the highest radiochemical purity and
stability over an optimum time duration and thereby achieve the desired
clinical results. The paper reports on the standardization, quality control and
initial clinical results of one such radiopharmaceutical — 99mTc(V)-DMSA.

2. MATERIALS AND METHODS

2.1. Kits used

The authors formulated 99mTc(V)-DMSA from two commercially

available kits of DMSA(III). One of the kits was procured from the Board of
Radiation & Isotope Technology (BRIT) India (kit 1) and the other was from
Amersham Inc., United Kingdom (kit 2).

2.2. Kit preparation

Formulation of the BRIT kit was done by adding 7 mg of NaHCO3 in

0.2 mL of water for injection and 99mTcO4- simultaneously to the reaction vial.
The kit contains 1 mg of DMSA and 0.3 mg of SnCl2·2H2O. An equal volume of
air was withdrawn from the reaction vial. Mixing followed by incubation was
done for 15 min.
Formulation of the Amersham kit (1 mg DMSA, 0.42 mg of SnCl2·2H2O,
0.7 mg ascorbic acid, 2.9 mg sodium chloride and 50 mg inositol) was done by

400
 SESSION 14

adding 0.2 mL 7.5% w/v NaHCO3 + 99mTcO4-. Mixing, followed by incubation,

was done for 15 min.

2.3. Analysis of the preparation

Radiochemical purity was determined by a combination of two chroma-

tographic systems using TLC (T-6145 TLC precoated plated silica gel with 254
nm fluorescent indicator on polyester supplied by Sigma, silica gel 60F 254 TLC
aluminium strip supplied by Merck) with a solvent containing n-butanol:acetic
acid:water (3:2:3 v/v). Free pertechnetate levels were measured using TLC–SG
in saline. Test samples were applied by 1 mL syringe 2 cm from the bottom of
the chromatography strips. The length of the strip was 15 cm and the width was
0.5 cm. The strips were marked every 1 cm and placed in prepared ascending
chromatography development jars. The strips were removed after 10 cm
elution and dried. Strips were cut every 1 cm for the butanol–acetic acid–water
system. The strip from the saline system was cut into two equal portions. Both
the systems were then counted in a well counter. Per cent labelling of DMSA
and free pertechnetate was calculated and the ratios of DMSA(III) and
DMSA(V) were calculated. These were carried out for over 3 h. The same
procedure was repeated on multiple occasions and the reproducibility was
verified. On the basis of the authors’ observations, the oxygen bubbling method
was tried with the Amersham kit where pure hospital grade oxygen was
bubbled through the preparation for 20 min. Chromatographic methods to
assess radiochemical purity were performed as above. The above chromato-
graphic procedures were reproduced on multiple occasions with kits from
different batches.

2.4. Biodistribution studies and clinical trials

Limited clinical trials in the form of a pilot study were carried out in the
patients of biopsy proven lung carcinoma during staging workup to evaluate
the tumour concentrating capability of the radiopharmaceutical and the in vivo
kinetics of the tumour. Whole body imaging was performed with a gamma
camera. 740 MBq of the prepared 99mTc(V)-DMSA was injected intravenously
and images were aquired immediately, at 1, 2 and 3 h. SPECT of the region of
primary malignancy was performed. The radiochemical purity was assessed by
chromatography concomitantly.

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 CHOUDHURY et al.

3. RESULTS

3.1. Analysis of the kits

Different batches of both the kits were used for chromatographic studies.
It was observed that the unbound (free) 99mTcO4 (technetium pertechnetate) in
both kits was less than 5%. This was also substantiated in the imaging studies.
Biodistribution and initial clinical studies showed predominantly renal pelvi-
calyceal excretion and bladder concentration. No tracer concentration was
seen in the salivary glands, thyroid gland or stomach, suggesting the absence of
significant unbound 99mTcO4 in the preparation. Radiochemical purity in both
the kits was more than 90% with the method of formulation used. However,
the incubation time taken to achieve this purity was variable in both the kits.
The kit supplied by BRIT (India) took 15 min of incubation time (Fig. 1),
whereas the Amersham kit required about 1 h of incubation time (Fig. 2) to
achieve the same purity. Significant amounts of DMSA(III) were found in the
latter preparation after 15 min of incubation (Fig. 3). On bubbling with hospital
grade oxygen for 20 min, the desired radiochemical purity was achieved in 15
min (Fig. 4). Both kits were stable for at least 3 h (Figs 5–7 and Tables 1–3).

15 m in.post preparation
on-Kit 1

200
20
150
15
ounts
k count

100
10 Series2
50
0
1 3 5 7 9 11
cm s

FIG. 1. Radiochemical purity 92.34%.

402
 SESSION 14

1 hour post preparation -Kit 2

200000
150000
counts

100000 Series2
50000
0
1 3 5 7 9 11
cm s

FIG. 2. Radiochemical purity 91%.

15 m in post preparation-Kit 2

20
k counts

15
10 Series2
5
0
1 3 5 7 9 11
cm s

FIG. 3. Radiochemical purity 50.36%.

403
 CHOUDHURY et al.

15 m in w ith oxygen bubbling

40
k counts

30
20 Series2
10
0
1 3 5 7 9 11
cm s

FIG. 4. Radiochemical purity 90%.

3 hours post preparation-Kit 1

80
60
counts

40 Series2
20
0
1 3 5 7 9 11
cm s

FIG. 5. Radiochemical purity 92%.

404
 SESSION 14

3 hrs post preparation-kit 2

300000
Counts

200000
Series2
100000

0
1 3 5 7 9 11
cm s

FIG. 6. Radiochemical purity 93%.

4 hrs . pos t pre paration w ith oxygen

bubbling
Kit- 2

12
10
8
Counts

6 Series2
4
2
0
1 3 5 7 9 11
cm s

FIG. 7. Radiochemical purity 93%.

405
 CHOUDHURY et al.

TABLE 1. CHROMATOGRAPHIC ANALYSIS OF THE KITS*

(15 min incubation, no oxygen bubbling)

Kit 99m - Reduced/hydrolysed 99mTcO2 DMSA(III) DMSA(V)

TcO4
1 <5% <5% <5% 90–98%
2 <5% <5% Approx 50% Approx 50%

* The above results reflect the overall performance of a number of samples of both the
kits from different batches. The pH of the preparations was 8.5–9.0. The Rf values for
99m
Tc(III)-DMSA and 99mTc(V)-DMSA were 0.3 and 0.6–0.7, respectively.

TABLE 2. CHROMATOGRAPHIC ANALYSIS OF THE KITS*

(1 h incubation, no oxygen bubbling)

Kit 99m - Reduced/hydrolysed 99mTcO2 DMSA(III) DMSA(V)

TcO4
1 <5% <5% <5% 90–98%
2 <5% <5% <5% >90%

* The above results reflect the overall performance of a number of samples of both the
kits from different batches. The pH of the preparations was 8.5–9.0. The Rf values
for 99mTc(III)-DMSA and 99mTc(V)-DMSA were 0.3 and 0.6–0.7, respectively.

TABLE 3. CHROMATOGRAPHIC ANALYSIS OF THE KITS*

(15 min incubation, no oxygen bubbling)

Kit 99m - Reduced/hydrolysed 99mTcO2 DMSA(III) DMSA(V)

TcO4
1 Not performed
2 <5% <5% <5% >90%
* The above results reflect the overall performance of a number of samples of both the
kits from different batches. The pH of the preparations was 8.5–9.0. The Rf values
for 99mTc(III)-DMSA and 99mTc(V)-DMSA were 0.3 and 0.6–0.7, respectively.

406
 SESSION 14

3.2. Biodistribution and clinical studies

An initial clinical trial (pilot study) in 10 diagnosed cases of lung

carcinoma showed adequate concentration in the tumour and coexistent bone
metastasis in 3 cases. Imaging at 3 h post-injection appears to be the optimum
point at which to achieve a good target to non-target ratio for interpretation of
the images.
Evaluation of in vivo tumour biology in the above cases was done post-
treatment (Figs 8–10). The clinical and morphological response correlated well
with the corresponding scintigraphy findings.

4. DISCUSSION

In routine clinical practice the authors have been preparing 99mTc(V)-

DMSA simply by adding 0.2 mL of 7.5% w/v NaHCO3 + 99mTcO4- simultane-
ously to the commercially available DMSA(III) kits. This method has been
described in the literature, with slight modifications between different studies,
and a radiochemical purity of >95% has been achieved [1–5]. However, similar

FIG. 8. Pretreatment 99mTc(V)-DMSA scan in a 63 year-old male showing tumour con-

centration in a case of lung carcinoma involving right lower lobe.

407
 CHOUDHURY et al.

99m
FIG. 9. Tc(V)-DMSA scan in the same patient post-treatment (concurrent chemo-
therapy and external radiation) showing residual disease.

radiochemical purity could not be reproduced by Washburn et al., who

reported a radiochemical purity of <77% with this method. Although the
authors have not undertaken prior chromatographic studies, their clinical
results and biodistribution of the formulated radiopharmaceutical, by this
method, have been inconsistent (unpublished data). It has been stated that the
stannous concentration of the renal DMSA kit can play a role in the radio-
chemical purity of the 99mTc(V)-DMSA preparation. The kit supplied by BRIT
contains 0.3 mg of SnCl2·2H2O and it would be possible to achieve >95% purity
by adding only NaHCO3 in 15 min. On the other hand, the kit supplied by
Amersham contained 0.42 mg of SnCl2·2H2O and 0.7 mg ascorbic acid in
addition. With the same method of formulation, the authors could achieve the
same level of radiochemical purity but with an extended incubation time. The
reason for this could be attributed to the presence of ascorbic acid which stops
tin from being oxidized. It has been stated that oxidation of tin is responsible
for the conversion of DMSA(III) to DMSA(V). Kumar [7] aimed to improve
the radiochemical purity to 100% and also determine whether stannous
oxidation plays an important role in the process of conversion of DMSA(III) to
DMSA(V). He studied the change in radiochemical purity in response to

408
 SESSION 14

FIG. 10. Post-treatment CT scan of the chest in the same patient showing residual disease
corresponding to the scintigraphic abnormality.

varying amounts of tin and oxygenation time. On bubbling compressed oxygen

for 5 min he could achieve 100% purity in 20 min. Increasing the bubbling time
resulted in a corresponding decrease in the time needed to achieve the same
purity. In the study, the authors could also achieve the same purity by adding
the oxygen bubbling method to their protocol in kit 2. This enabled the
oxidation of excess tin and facilitated the conversion by countering the effect of
the added ascorbic acid in this process.
It has been reported that the pH of the preparation could also be a
determining factor in achieving a high radiochemical purity for DMSA(V).
Oh et al. obtained a radiochemical yield of 95 ± 1.2% within 10 min and a
preparation which was stable for 7 h. The radiochemical purity was 92 ± 1.5%
at pH9. However, at a lower pH the yield was below 90% and a higher reaction
time was required to achieve a purity of >90%. In the study, the measured pH
after formulation was in the range 8.5–9. Biodistribution studies (Fig. 11)
showed predominantly renal pelvicalyceal excretion in a radiochemically pure
preparation with physiological concentration in the bladder. Cardiac blood
pool concentration showed significant washout over time. Clinical studies
reported in the literature have predominantly focused on tumour detection,

409
 CHOUDHURY et al.

99m
FIG. 11. Normal biodistribution of Tc(V)-DMSA in a radiochemically pure prepa-
ration.

410
 SESSION 14

mostly in medullary carcinoma thyroid and bone metastasis [9–12]. The authors
are attempting to study tumour biology with an emphasis on detection,
response to treatment and follow-up. Initial clinical results have been encour-
aging.

5. CONCLUSION

The authors have so far been able to standardize and reproduce an

optimum radiochemically pure formulation of 99mTc(V)-DMSA. The BRIT kit
formulation achieves a faster conversion to DMSA(V) and achieves good
tumour concentration in a radiochemically pure preparation. The Amersham
kit formulation without oxygen bubbling requires a longer incubation time for
the same process. The radiochemical yield and stability of both kits were found
to be the same. This formulation has been shown to concentrate in the active
tumour and the current study suggest 3 h post-injection as being the optimum
imaging time to achieve a good target to non-target ratio in the lesions.

ACKNOWLEDGEMENT

This study was supported by a grant ( IAEA E1.30.28 ) from the IAEA in
the framework of the coordinated research project entitled Standardization
and Quality Control of In-house Prepared Radiopharmaceuticals in Nuclear
Oncology.

REFERENCES

[1] OHTA, H., et al., A new imaging agent for medullary carcinoma of the thyroid, J
Nucl Med 25 (1984) 323-325.
[2] WESTERA, G., GADZE, A., HORST, W., A convenient method for the
preparation of 99mTc(V)-dimercaptosuccinic acid [99mTc(V)-DMSA], J Appl
Radiat Isot 36 (1985) 311-312.
[3] CHAUHAN, U.P.S., et al., Evaluation of a DMSA kit for instant preparation of
99m
Tc(V)-DMSA for tumour and metastasis scintigraphy, Nucl Med Biol 19 (1992)
825-830.
[4] BABBAR, A., KASHYAP, R., CHAUHAN, U.P.S., A convenient method for the
preparation of 99mTc labelled pentavalent DMSA and its evaluation as a tumour
imaging agent, J Nucl Med Biol 35 (1991) 100-104.
[5] RAMAMOORTHY, N., et al., Preparation and evaluation of 99mTc(V)-DMSA
complex: Studies in medullary carcinoma of thyroid, Eur J Nucl Med 12 (1987)
623-628.

411
 CHOUDHURY et al.

[6] WASHBURN, L.C., BINIAKIEWICZ, D.S., MAXON, H.R., Possible

preparation of 99mTc(V)-DMSA by a simple modified method using a commercial
kit for 99mTc(III)-DMSA, Nucl Med Biol 22 (1995) 689-691.
[7] KUMAR, V., Evaluation of stannous oxidation in the preparation of ultra-high
purity 99mTc(V)-DMSA, Nucl Med Commun 22 (2001) 1261-1266.
[8] OH, S.J., et al., Simple and reliable preparation of pentavalent 99mTc m-dimerap-
tosuccinic acid at alkaline pH without oxygen bubbling, Nucl Med Commun 22 (6)
(2001) 613-616.
[9] SAHIN, M., et al., Evaluation of metastatic bone disease with pentavalent 99mTc
dimercaptosuccinic acid: a comparison with whole body scanning and 4/24 hour
quantitation of vertebral lesions, Nucl Med Commun 21(3) (2000) 251-258.
[10] LAM, A.S., et al., Pentavalent 99mTc DMSA imaging in patients with bone
metastasis, Nucl Med Commun 18(10) (1997) 907-914.
[11] AMBRUS, E., et al., Value of 99mTc MIBI & 99mTc(V)DMSA scintigraphy in
evaluation of breast mass lesions, Anticancer Res 17 (1997) 1599-1605.
[12] HIRANO, T., et al., Preparation & clinical evaluation of technetium-99m dimer-
captosuccinic acid for tumour scintigraphy, Eur J Nucl Med 21(1) (1994) 82-85.

412
 DEVELOPMENT OF CENTRALIZED
RADIOPHARMACIES IN SPAIN: A SUCCESSFUL
EXPERIENCE IN EUROPE

I. OYARZÁBAL, R. JIMÉNEZ-SHAW
Molypharma, S.A.,
Madrid, Spain
Email: rjs@molypharma.es

Abstract

Good manufacturing practice requirements applied to radiopharmaceuticals

compounding and unit dose preparation were seen as an opportunity when they were
made compulsory by law in Spain during the mid-1990s. An innovative company was set
up to develop centralized radiopharmacies or ‘nuclear pharmacies’, as they are known
in the United States of America, with units designed for a safe and efficient operation
with respect to product and personnel and which fulfil radioactive and pharmaceutical
criteria. A quality management system has been implanted and a computerized manage-
ment system supports the operation of each unit, helping the operator in their work,
preventing mistakes and human error, and keeping control of record and traceability.
An e-business system based upon the internet allows customers to place an order for
their needs and provides additional information.

1. INTRODUCTION

In Spain, 650 000 nuclear medicine procedures are performed every year
in more than 120 nuclear medicine departments in public and private hospitals.
The doses to be administered to patients were traditionally prepared by
hospital personnel in small hot laboratories in the departments. However,
during the 1990s new legislation was developed that triggered the need for a
change in the model. The change from radiotracers to the new concept of
radiopharmaceuticals led to pharmaceutical requirements for the devel-
opment, fabrication, commercialization and preparation of radioactive
compounds and their administration to patients. Additionally, regulatory
authorities imposed quality assurance rules in nuclear medicine practices.
As regards dose preparation and administration, those legal require-
ments established new specific criteria derived from compliance with good
manufacturing practices (GMPs) in several areas such as the following:

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 OYARZÁBAL and JIMÉNEZ-SHAW

— Premises and equipment;

— Qualification of personnel;
— Quality systems with procedures, records and documentation control;
— Traceability.

Implementation of all these changes not only meant significant

investments in terms of time and resources but also important modifications in
habits and practices developed over many years. Several initiatives were
launched to cope with the need for change, the concept of centralized radio-
pharmacies being among them, particularly in regions with a high density of
nuclear medicine departments where scale economies make these pharmacies
efficient.
Currently, a completely new system governs the preparation of unit doses
of radiopharmaceuticals in the country:

— 45% of the unit doses are prepared and supplied from 6 centralized
radiopharmacies;
— 20% of the unit doses are prepared in hospital radiopharmacies but
managed and operated by an external contractor;
— 35% of the unit doses are prepared in hospital radiopharmacies managed
by hospital personnel.

The significant penetration of centralized radiopharmacies should be

stressed. This makes Spain the European country in which the pharmacies have
progressed the most, in spite of its different population density rates. They are
owned and operated by private companies that have taken the initiative to
bring this preparation and distribution system to the Spanish radiopharmaceu-
tical sector.

2. INNOVATIVE CONCEPT

Spanish centralized radiopharmacies can be considered as an innovative

sector in Europe, as they offer hospitals the possibility of outsourcing their
compounding needs to an external contractor in order to save costs and focus
on their patients.
Molypharma’s innovation in this field can be seen in three features:

(1) Common system of radiopharmacies: In order to provide a better service

and obtain synergies, the company has developed several radiophar-
macies with a central administrative hub that provides software and other

414
 SESSION 14

support. Design, systems and operation of each unit are practically

identical.
(2) E-business: The company has developed an e-business system in order to
provide customers with an easy method to set up error free orders and to
guarantee traceability. This e-business system is composed of:
— Software that processes orders, issues invoices and offers information
through the internet located at the company’s headquarters; and
— Other software that manages every step of the pharmacy production
located at each unit.
Both softwares are interconnected via the internet.
(3) Quality: In order to focus all the activities on their customer’s needs and
complying with the strictest regulations, the company established an ISO
9001 certified quality management system and designed their units and
operations according to GMP standards.

Operation of the first centralized radiopharmacy started in 2000. There

were three operating units as of 2005.

3. UNIT DESIGN AND LAYOUT

Each unit has been designed to meet both GMPs and radioactive regula-
tions. These are conflicting regulations, as GMPs call for the protection of the
product, requiring positive pressure to avoid possible contamination of the
product, while radioactive regulations establish protection for the operator and
the environment, requiring negative pressure. This apparent contradiction is
solved in the company’s units through having a pressure cascade, the laminar
air flow open cabins being in negative pressure in relation to the clean room
where they are located. The clean room is in positive pressure over the
reception and expedition rooms in order to avoid any possible contamination.
The laminar airflow open cabins are shielded to protect the operator and
include a dose calibrator activimeter connected to the computer system.
Labelling and unit dispensing is done within the cabins. Operators are
comfortably seated. Generators are eluted in a special cabinet.
The unit also has conventional laboratory equipment and rooms, quality
control, air conditioning and filters, as required by pharmaceutical and
radioactive regulations.

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 OYARZÁBAL and JIMÉNEZ-SHAW

4. QUALITY MANAGEMENT SYSTEM

Quality is of paramount importance to Molypharma in all its activities in

such a way that:

— The quality of all its products and services is controlled. Molypharma

controls the quality of its product with the maximum efficacy and safety
and under conditions for continued improvement.
— The client’s needs and expectations are always met satisfactorily.
— The operations at its facilities are always carried out under optimum
radiological safety and protection conditions.

To carry out its activities, the company has adopted a quality management
system based on processes in accordance with the standard UNE-EN-ISO
9001:2000 requirements. The quality management system has been certified by
AENOR, the Spanish Association of Standardization and Certification. The
company also holds a licence for use of the ‘Madrid Excelente’ seal of
guarantee, issued by the regional government of Madrid and based on the
EFQM model of management.
The quality management system has the same quality manual and
standard operating procedures for each unit. These standard operating
procedures include reception, inventory and registry of materials,
compounding and quality control of each radiopharmaceutical, expedition of
materials, and maintenance, verification and calibration of equipment. There
are also general standard operating procedures for audits, customers and
process flow management, etc.
Quality assurance performance is measured regularly by a set of indexes.
One of the most important indexes is punctuality. It measures the percentage of
on-time deliveries. This index is measured every month and it is compared with
the goal of achieving a 99% success rate. In some units, the average punctuality
index is better than 98%.

5. E-BUSINESS SYSTEM

Customers can place their orders easily through the internet. The only
software needed is an internet navigator, either Netscape or Explorer. Each
hospital can have several users with their own user name and password. The
access is made through a secure connection.
The order web page is user friendly. The only field that must be typed in is
the name of the doctor making the prescription. Scroll bars manage all the

416
 SESSION 14

other fields. Once the product is selected from a different list for each
customer, it is only necessary to set activity at the calibration time and delivery
time and date. The system automatically proposes the agreed delivery time.
Each order can have different lines, each line being a different product.
Filling in the name in the patient field is an option for the customer. The
database is designed to comply with the confidentiality required for these types
of data. Some of Molypharma’s customers leave it blank and others put in
either the real name of the patient or another kind of patient identification,
such as a clinical history number. Each order is automatically downloaded to
the computer system located in each radiopharmacy, avoiding possible human
errors.
In addition to order management, this e-business system (known as
Molysic) can handle a lot of valuable information for the customer. Customers
can check the status of pending orders and orders already delivered and have
access to a set of predesigned queries such as a list of products and a list of
delivered activity by date and isotope, etc. This query is very useful for
complying with the requirements of radioactive regulatory authorities.
Customers can rapidly have access to the identity of each unit dose. Just
by typing the identification number of a single dose, the system delivers the
following information: order number, container identification, delivery note
number, worker identification (who prepared the dose), identification of eluted
and labelled vials, expiry date, volume, patient identification, etc. The system
provides all this information in an error free way, as a bar code reader controls
every step of preparation.
Molyfact is the invoicing side of the e-business system. It gets all the
information from Molysic and allows the company to invoice each hospital with
their own requests. It can invoice for each delivery or over some defined
period, such as a week, a month, and so on. It also provides each customer with
statistics about its consumption in euros, not in units.

6. RADIOPHARMACY MANAGEMENT SYSTEM

The management system of Molypharma’s radiopharmacies, Udemon, is

automated from receipt of on-line orders to the issuing of the final product, and
covers all the intermediate steps of the compounding of radiopharmaceuticals.
Udemon also generates the documentation required for transport and delivery
and:

— Ensures complete traceability of the product, from the supplier’s details

to those of the dosage delivered to the client.

417
 OYARZÁBAL and JIMÉNEZ-SHAW

— Identifies each single dose via bar coded labels and all the data required
by current legislation.
— Minimizes the possibility of human error, owing to the extensive use of
bar codes on vials, containers, processes and operators.

Udemon has three main modules: (i) administrative, (ii) compounding

and (iii) communication.
The administrative module deals with personnel identification, customer
specifications, supplier identification and product portfolio. Other important
submodules are orders to suppliers, orders from customers, inventory control
and production control.
The compounding module makes it possible to operate the central
radiopharmacy very efficiently from a personnel point of view as every minor
step is automated. The main submodules are elution, which controls each
generator, vial labelling, quality control, syringe dispensing, and label and
documentation printing. Each excursion, which can involve several deliveries
to different hospitals, has its computer generated consignment note. Each
delivery has a delivery note, a list of unit doses per container and an optional
second set of labels to be added to the clinical history.
Every step of the production process is controlled by bar codes. Bar codes
are applied to each vial and generator that arrives at the pharmacy, to each
worker involved in every step and to each intermediate vial of the elution or
labelled product. Bar codes are also applied to unit doses and containers,
avoiding the possibility of sending a dose to another customer in error.
The communication submodule exchanges information with the central
Molysic e-business system. It downloads customers orders and uploads
suppliers orders and other information required for traceability and invoicing.

7. HUMAN RESOURCES

Each unit has two specialists in radiopharmacy. To obtain this qualifi-

cation in Spain, the person must be either a chemist or a pharmacist and to
have completed a two year residency in a hospital. The company provides
additional education in quality, GMPs, customer relations, software and
management.
Each unit has several technicians that have taken a two year course after
high school. They also receive further education in the company. The number
of technicians per unit depends on the number of doses to be prepared per day
and on the opening hours.

418
 SESSION 14

Internal communication is facilitated by an intranet application accessible

through a virtual private network that is available to all personnel. It contains
information on quality and production indices, quality systems and procedures,
practical company information and additional features. It also permits
exchange of opinions through a discussion forum. A satisfaction personnel
survey is performed every year, its results being published on the intranet.

8. DISTRIBUTION

Unit dose syringes are conditioned inside a sterilized envelope with their
label. Several unit doses can be delivered in a type ‘A’ certified package. The
company has designed and certified that the package contains a 4 mm lead
shield covered by a stainless steel container (127 mm × 127 mm × 270 mm),
with handle, reinforced hinges and closure system, two-piece expanded
polystyrene padding, plastic drum, metallic lid, closure ring and safety
strapping.
The pharmacy also manages the waste of some hospitals. Hospital
personnel dispose of used syringes in a biohazard plastic container. This
container is situated within a protective shield. After the container is closed
and measured as an exempt radioactive package, it is sent to the pharmacy’s
waste room. Once it is considered non-radioactive, it is disposed of as conven-
tional biohazard waste by a specialized company.
In order to focus on the radiopharmacy business and to provide the best
possible service to the customer, physical distribution is subcontracted to a
specialized multimodal transport company that operates in several dangerous
goods sectors. The transport company has its own vans and drivers. This
scheme requires very close collaboration between the radiopharmacy company
and the transport company, and several common procedures have been
developed and implemented.
A hospital representative, usually a nuclear medicine technician, signs
delivery notes, indicating the actual time of receipt. This time is introduced
subsequently into the system to obtain the punctuality index.

9. CONCLUSION

Development of a GMP standard network of centralized radiophar-

macies in Spain has been an achievement based on the innovative implemen-
tation of an e-business system with extensive use of internet and computer
software and hardware, as well as a quality management system. These systems

419
 OYARZÁBAL and JIMÉNEZ-SHAW

are interconnected with an instructed staff and a customer focused strategy,

making for a well-balanced organization.
Hundreds of radiopharmaceutical unit doses are efficiently compounded
outside of the hospital every day. These unit doses are delivered on time, error
free, and with high quality and assured traceability, providing a smooth and
valuable hospital radiopharmacy relationship.

420
 CHAIRPERSONS OF SESSIONS

Session 1 S. GOMEZ DE CASTIGLIA Argentina

F.F. KNAPP, Jr. United States of America
Session 2 A. DUATTI Italy
J. KÖRNYEI Hungary
Session 3 I. CARRIO Spain
E.K.J. PAUWELS Netherlands
Session 4 R. ALBERTO Switzerland
M. VENKATESH India
Session 5 R.S. KAMEL IAEA
N. RAMAMOORTHY IAEA
Session 6 M. JEHANGIR Pakistan
R. MIKOLAJCZAK Poland
Session 7 A. VERBRUGGEN Belgium
M.R.A. PILLAI IAEA
Session 8 C. DECRISTOFORO Austria
K.K. SOLANKI IAEA
Session 9 J. HARVEY TURNER Australia
M. DONDI IAEA
Session 10 H.-J. MACHULLA Germany|
M.C. LEE Republic of Korea
Session 11 H.S. BALTER Uruguay
G.-J. BEYER Switzerland
Session 12 V.W. PIKE United States of America
D. SOLOVIEV Switzerland
Session 13 M.M. VORA Saudi Arabia
M. HAJI-SAEID IAEA
Session 14 N.G. HARTMAN United Kingdom
E. JANEVIK-IVANOVSKA The Former Yugoslav
Republic of Macedonia

421
 SECRETARIAT OF THE SYMPOSIUM

M.R.A. PILLAI Scientific Secretary

K.K. SOLANKI Scientific Co-Secretary
R. PERRICOS Conference Services
L. BARRIOS Conference Assistance
R.J. BENBOW Proceedings Editor

PROGRAMME COMMITTEE

M. DONDI
M. HAJI-SAEID
R.S. KAMEL
M.R.A. PILLAI
N. RAMAMOORTHY (Chairperson)
K.K. SOLANKI

422
 LIST OF PARTICIPANTS

Abdel-Jalil, R.J. Chemistry Department,

Faculty of Sciences and Arts,
Hashemite University,
P.O. Box 330127,
13133 Zarka, Jordan
Fax: +96253403333
Email: jalil@hu.edu.jo

Abdul Rahman, A. Technical Service Division,

Malaysian Institute for Nuclear,
Technology Research (MINT),
Bangi, 43000 Kajang,
Selangor Darul Ehsan, Malaysia
Fax: +60389282992
Email: anwar@mint.gov.my

Abedin, M.Z. Institute of Nuclear Science

& Technology,
Atomic Energy Research
Establishment,
Ganakbari, P.O. DEPZ,
Savar, Dhaka, Bangladesh
Fax: +88028613051
Email: ripd_inst@yahoo.com

Afroz, S. Centre for Nuclear Medicine

and Ultrasound,
Dhaka Medical College Hospital,
Bangladesh Atomic Energy
Commission (BAEC),
Dhaka-1000, Bangladesh
Email: nmcdhaka@agni.com

Al Rayyes, A.H. Cyclotron and Medical

Radioisotopes Division,
Atomic Energy Commission,
P.O. Box 6091,
Damascus, Syrian Arab Republic
Fax: +963116112289
Email: aalraies@aec.org.sy

423
 LIST OF PARTICIPANTS

Al-Azzawi, H.M. Directorate of Chemistry and

Petrochemical Industry,
Ministry of Science and Technology,
P.O. Box 0765, Al-Jadrya,
Baghdad, Iraq
Email: hisham_alazzawi@yahoo.com

Alberto, R. Institute of Inorganic Chemistry,

University of Zurich,
Winterthurerstrasse 190,
8057 Zurich, Switzerland
Fax: +41446356802
Email: ariel@aci.unizh.ch

Ali, O.I. National Agency for Food and

Drug Administration and Control,
NAFDAC Headquarters,
Plot 2032 Olusegun Obasanjo Way,
Wuse Zone 7,
Abuja, Nigeria
Fax: +23495241461
Email: aliotib@yahoo.com

Al-Jammaz, I. Cyclotron & Radiopharmaceutical,

MBC-03,
King Faisal Specialist Hospital
and Research Centre,
P.O. Box 3354,
Riyadh 1211, Saudi Arabia
Fax: +96614424743
Email: jammaz@kfshrc.edu.sa

Al-Nuzal, S.M.D. Directorate of Chemistry and

Petrochemical Industry,
Ministry of Science and Technology,
P.O. Box 0765, Al-Jadrya,
Baghdad, Iraq
Email: saadidhaher@yahoo.com

424
 LIST OF PARTICIPANTS

Al-Saeedi, F.J. Nuclear Medicine Department,

Faculty of Medicine,
Kuwait University,
P.O. Box 24923,
13110 Safat, Jabria, Kuwait
Fax: +9655338936
Email: fatimas@hsc.edu.kw

Alwan, I.F. Directorate of Chemistry and

Petrochemical Industry,
Ministry of Science and Technology,
P.O. Box 0765, Al-Jadrya,
Baghdad, Iraq
Email: rchpimost@most-Iraq.com

Andráskó, M. Institute of Isotopes Co. Ltd,

Konkoly Thege M. Str. 29–33,
1121 Budapest, Hungary
Fax: +3613959070
Email: andrasko@izotop.hu

Andreae, F. piCHEM Research & Development,

Kahngasse 20,
8045 Graz, Austria
Fax: +43316681711/4
Email: andreae@pichem.at

Antalffy, M. Institute of Isotopes Co. Ltd,

Konkoly Thege M. Str. 29–33,
1121 Budapest, Hungary
Fax: +3613959070
Email: antalffy@izotop.hu

Aras, G. Department of Nuclear Medicine,

Faculty of Medicine,
Ankara University,
06100 Ankara, Turkey
Fax: +903123620897
Email: okucuk@medicine.ankara.edu.tr

425
 LIST OF PARTICIPANTS

Arrechedera Mejias, L.I. Facultad de Farmacia,

Universidad Central de Venezuela,
Los Chaguaramos,
Apartado Postal 47008,
Caracas 1041-A, Venezuela
Fax: +582126052707; +582126052701
Email: arrechel@camelot.rect.ucv.ve

Artl, A. piCHEM Research & Development,

Kahngasse 20,
8045 Graz, Austria
Fax: +43316681711/4
Email: artl@pichem.at

Asikoglu, M. Department of Radiopharmacy,

Faculty of Pharmacy,
Ege University,
35100 Izmir, Turkey
Fax: +902323885258
Email: asikoglum@pharm.ege.edu.tr

Avila Sobarzo, M.J. Chilean Nuclear Energy Commission,

Amunátegui 95,
P.O.Box 188-D,
6500687 Santiago, Chile
Fax: +5623646277
Email: mavila@cchen.cl

Balter, H.S. Centro de Investigaciones

Nucleares,
Facultad de Ciencias,
Igua 4225,
11400 Montevideo, Uruguay
Fax: +59825250895
Email: jbalter@cin.edu.uy

Banerjee, S. Radiopharmaceuticals Division,

RLG Building,
Bhabha Atomic Research Centre,
Trombay, Mumbai 400 085, India
Fax: +912225505151
Email: sharmila@apsara.barc.ernet.in

426
 LIST OF PARTICIPANTS

Baranyai, L. Institute of Isotopes Co. Ltd,

Konkoly Thege M. Str. 29–33,
1121 Budapest, Hungary
Fax: +3613959070
Email: barlajos@izotop.hu

Bayomy, T. Nuclear Medicine Department,

Arab Contractors Medical Centre (ACMC),
Gabal Al Akhdar,
P.O. Box 9033,
Nasr City, 11765 Cairo, Eqypt
Fax: +2026821624
Email: tamerbayomy@hotmail.com

Benz, L.M. Hans Wälischmiller GmbH,

Schießstattweg 16,
88677 Markdorf, Germany
Fax: +497544951499
Email: lb@hwm.com

Beran, M. Nuclear Physics Institute,

Academy of Sciences,
250 68 Rez, Prague,
Czech Republic
Fax: +420220940151
Email: beran@ujf.cas.cz

Bercik, I. BIONT a.s.,

Karloveska 63,
84229 Bratislava, Slovakia
Fax: +421220670748
Email: bercik@biont.sk

Beyer, G.-J. Cyclotron Unit,

University Hospital of Geneva,
24 rue Micheli-du-Crest,
1211 Geneva-14, Switzerland
Fax: +41223727585
Email: gerd.beyer@hcuge.ch

427
 LIST OF PARTICIPANTS

Bilewicz, A. Institute of Nuclear Chemistry

and Technology,
Dorodna 16,
03-195 Warsaw, Poland
Fax: +48228111532
Email: abilewicz@ichtj.wew.pl

Bortoleti de Araújo, E. Instituto de Pesquisas,

Energéticas e Nucleares,
Av. Prof. Lineu Prestes 2242,
Cidade Universitária,
BR-05508-000 Sao Paulo, SP,
Brazil
Fax: +551138120253
Email: ebaraujo@ipen.br

Boucekkine, N. Department of Nuclear Medicine,

CHU Bab El Oued,
Boulvard Said Touati,
16000 Bab El Oued,
Algiers, Algeria
Fax: +21321965101
Email: nadiaboucekkine@yahoo.fr

Bozóky, Z. “Fodor József” National

Centre of Public Health,
National Research Institute for
Radiobiology and Radiohygiene,
Anna Utca 5,
1221 Budapest, Hungary
Fax: +3614822012
Email: bozoky@hp.osski.hu

Bruskin, A. SSC Institute of Biophysics,

Ministry of Health,
Zhivopisnaya Street 46,
123182 Moscow,
Russian Federation
Fax: +70951903590
Email: a_bruskin@mail.ru

428
 LIST OF PARTICIPANTS

Bucher, C. THP Medical Products,

Vertriebs GmbH,
Shuttleworthstrasse 19,
1210 Vienna, Austria
Fax: +4312928280-88
Email: ch.bucher@thp.at

Burkart, W. Department of Nuclear

Sciences and Applications,
International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: w.burkart@iaea.org

Cabral Gonzalez, P. Catedra de Radioquimica,

Facultad de Quimica,
Universidad de la República,
General Flores 2124,
P.O. Box 1157,
11800 Montevideo, Uruguay
Fax: +59829241906
Email: pcabral@fq.edu.uy

Cañellas, C.O. Tecnonuclear S.A.,

Arias 4149,
1430, Buenos Aires, Argentina
Fax: +541145456005
Email: canellas@tecnonuclear.com.ar

Carrio, I. Nuclear Medicine Department,

Hospital Sant Pau,
Avenida Padre Claret 167,
08025 Barcelona, Spain
Fax: +34932919409
Email: icarrio@santpau.es

Cepelová, Z. BIONT a.s.,

Karloveska 63,
84229 Bratislava, Slovakia
Fax: +421220670686
Email: capelova@biont.sk

429
 LIST OF PARTICIPANTS

Cetto, A.M. Department of Technical Cooperation,

International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
A-1400 Vienna, Austria
Fax: +43126007
Email: a.cetto@iaea.org

Chakraborty, M. Laboratory Nuclear Medicine Section,

c/o Tata Memorial Hospital,
Bhabha Atomic Research Centre,
Annex Building, Jerbai Wadia Road,
Parel, Mumbai 400 012, India
Fax: +912224171872
Email: mayukh29@rediffmail.com

Cheng, Zuoyong Nuclear Power Institute of China,

P.O. Box 436,
Chengdu 610041, China
Fax: +862885868504
Email: mrchengzy@yahoo.com.cn

Choudhury, P.S. Department of Nuclear Medicine,

Rajiv Gandhi Cancer Institute & Research
Centre — Sector 5,
New Delhi 110085, India
Fax: +911127051037
Email: pschoudhury@rgci.org

Cojocaru-Toma, M. Ministry of Health,

Institute of Pharmacy,
Post Code MD 2028,
2/1 Korolenko str.
Chisinau, Moldova
Fax: +37322737448
Email: inf_isf@rambler.ru

Comor, J.J. Laboratory of Physics (010),

Vinca Institute of Nuclear Sciences,
P.O. Box 522,
11001 Belgrade, Serbia
Fax: +381114447963
Email: jcomor@vin.bg.ac.yu

430
 LIST OF PARTICIPANTS

Cortes-Blanco, A. Spanish Agency for Medicines

and Healthcare Products,
C/Alcala 56,
28071 Madrid, Spain
Fax: +34918225161
Email: acortesb@agemed.es

Crudo, J.L. Division Radiofármacos,

Centro Atomico Ezeiza,
Presb. Gonzalez y Aragon 15,
(B1802AYA) Ezeiza,
Buenos Aires, Argentina
Fax: +541167798288
Email: jlcrudo@cae.cnea.gov.ar

Çuçi, T. Institute of Nuclear Physics,

P.O. Box 85,
Tirana, Albania
Email: triumfcuci@yahoo.com

Das, T. Radiopharmaceuticals Division,

Bhabha Atomic Research Centre,
Trombay, Mumbai 400 085, India
Fax: +912225505345
Email: tdas@apsara.barc.ernet.in

de Jong, M.H. Department of Nuclear Medicine,

Erasmus MC,
Rotterdam, Netherlands
Email: m.hendriks-dejong@erasmusmc.nl

Decristoforo, C. Clinical Department

of Nuclear Medicine,
Medical University Innsbruck,
Anichstrasse 35,
6020 Innsbruck, Austria
Fax: +4351250422659
Email: clemens.decristoforo@uibk.ac.at

Djokic, D. Laboratory for Radioisotopes,

Vinca Institute of Nuclear Sciences,
P.O. Box 522,
11001 Belgrade, Serbia
Fax: +381112438134
Email: djokici@vin.bg.ac.yu

431
 LIST OF PARTICIPANTS

Dondi, M. Department of Nuclear

Sciences and Applications,
International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: m.dondi@iaea.org

Duatti, A. Laboratory of Nuclear Medicine,

Department of Radiology,
University of Ferrara,
Via L. Borsari 46,
44100 Ferrara, Italy
Fax: +39532236589
Email: dta@unife.it

Ebrahimi-Fakhari, F. Chemistry Department,

Philipps University Marburg,
Hans-Meerwein Strasse,
35032 Marburg, Germany
Fax: +4964212863427
Email: ebrahimi@staff.uni-marburg.de

El Haouzi, M. Laboratoire National du

Contrôle des Médicaments,
Ministère de la Santé,
B.P. 6206,
Rabat, Morocco
Fax: +21237681931
Email: melhaouzi@sante.gov.mo

Esiashvili, S. National Cancer Centre,

Sairme Street B/L 6, Apt.15,
0194 Tbilisi, Georgia
Email: sesiash@yahoo.com

Faintuch, B.L. Radiopharmacy Centre,

Institute of Energetic and
Nuclear Research (IPEN/CNEN),
Av. Professor Lineu Prestes 2242,
Cidade Universitária,
BR-05508-900 Sao Paulo, SP,
Brazil
Fax: +551138169257
Email: faintuch@ipen.br

432
 LIST OF PARTICIPANTS

Farstad, B. Isotope Laboratories,

Institute for Energy Technology,
P.O. Box 40,
2027 Kjeller, Norway
Fax: +4763803021
Email: brit.farstad@ife.no

Ferro-Flores, G. Instituto Nacional de

Investigaciones Nucleares (ININ),
Kilometro 36.5,
Carretera Mexico-Toluca,
52045 Municipio de Ocoyoacac,
Mexico
Fax: +525553297306
Email: gff@nuclear.inin.mx

Figols de Barboza, M. Instituto de Pesquisas

Energéticas e Nucleares,
Avenida Professor Prestes 2242,
Cidade Universitária,
BR-05508-000 Sao Paulo, SP,
Brazil
Fax: +551138169257
Email: mbarboza@ipen.br

Fischer, T. Department of Nuclear Medicine,

University of Cologne,
Kerpener Strasse 62,
50924 Cologne, Germany
Fax: +492214786777
Email: thomas.fischer@uk-koeln.de

Flores de la Torre, J.A. Universidad Autonoma de

Zacatecas,
Unidad Academica de Estudios Nucleares,
Cipres # 10 FRACC La Penuela,
98068 Zacatecas, Mexico
Fax: +525553297322
Email: arman_do19@hotmail.com

433
 LIST OF PARTICIPANTS

Fresvig, M. GE Healthcare,
Amersham Health,
P.O. Box 4220 Nydalen,
0401 Oslo, Norway
Fax: +4723186025
Email: marianne.fresvig@ge.com

Freud, A. Nuclear Research Centre NEGEV,

Israel AEC,
P.O. Box 9001,
Beer-Sheva 84190, Israel
Fax: +97286567015
Email: freud@netvision.net.il

Gandomkar, M. Radioisotope Deparment,

Nuclear Research Centre,
Atomic Energy Organization of Iran,
P.O. Box 11365-3486,
Tehran, Islamic Republic of Iran
Fax: +98218020887
Email: msgandomkar@yahoo.com

Garg, P.K. PET Center,

Center for Biomolecular Imaging,
Wake Forest University Medical Center,
Medical Center Blvd,
Winston-Salem, NC 27157,
United States of America
Email: pgarg@wfubmc.edu

Geets, J.-M. Ion Beam Applications s.a.,

Chemin du Cyclotron 3,
1348 Louvain-la-Neuve, Belgium
Fax: +3210475958
Email: geets@iba.be or piccoli@iba.be

Gibson, P.N. Institute for Health and

Consumer Protection,
Joint Research Centre,
Via E. Fermi 1,
21020 Ispra (VA), Italy
Fax: +390332785388
Email: neil.gibson@jrc.it

434
 LIST OF PARTICIPANTS

Giubbini, R.M.T. Spedali Civili di Brescia,

Medicina Nucleare,
Piazza Spedali Civili 1,
25100 Brescia, Italy
Email: giubbini@spedalicivili.brescia.it

Gjerde, H. Amersham Health,

P.O. Box 4220 Nydalen,
0401 Oslo, Norway
Fax: +4723186024
Email: hallvard.gjerde@ge.com

Gniazdowska, E. Institute of Nuclear Chemistry

and Technology,
Dorodna 16,
03-195 Warsaw, Poland
Fax: +48228111532
Email: egniazdo@ichtj.waw.pl

Gomez de Castiglia, S.I. Centro Atómico Ezeiza,

Comisión Nacional de Energía Atómica,
Avenida Del Libertador 8250,
1429 Buenos Aires, Argentina
Fax: +541167798288
Email: silgomez@cae.cnea.gov.ar

Gourni, E. National Centre for Scientific

Research (NCSR) “Demokritos”,
P.O. Box 60228,
15310 Aghia Paraskevi,
Athens, Greece
Fax: +302106522661
Email: egourni@yahoo.gr

Gunawardana, C. Lady Ridgeway Hospital for

Children,
Dr. Danister de Silva Mawatha,
Colombo 08, Sri Lanka
Fax: +9412691521
Email: chamly2000@yahoo.com

435
 LIST OF PARTICIPANTS

Haji-Saeid, S.M. Department of Nuclear

Sciences and Applications,
International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: m.haji-saeid@iaea.org

Hansson, L.K. Medical Products Agency,

P.O. Box 26,
751-03 Uppsala, Sweden
Fax: +4618548566
Email: lena.hansson@mpa.se

Harbo, B.T. Isotope Laboratories,

Institute for Energy Technology,
P.O. Box 40,
2027 Kjeller, Norway
Fax: +4763803021
Email: bente.tange.harbo@ife.no

Hartman, N.G. Addenbrooke’s Hospital,

Hills Road,
Cambridge CB2 2QQ,
United Kingdom
Email: nh289@cam.ac.uk

Hawerkamp, A. Eurotope GmbH,

Robert-Rössle-Strasse 10,
13125 Berlin, Germany
Fax: +4930941084160
Email: andrea.hawerkamp@eurotope.de

Hazra, D.K. Nuclear Medicine Unit,

S.N. Medical College Agra,
Soami Bagh,
Agra 282003, India
Fax: +91562226524
Email: hazra@sancharnet.in

Hong, Seong-Seok KIRAMS,

Korea Cancer Centre Hospital,
215 Gongneung Dong, Nowon-ku,
Seoul, Republic of Korea
Email: mkh@kcchsun.kcch.re.kr

436
 LIST OF PARTICIPANTS

Husbyn, M. GE Healthcare,
Amersham Health,
P.O. Box 4220 Nydalen,
0401 Oslo, Norway
Fax: +4723186025
Email: mette.husbyn@ge.com

Iller, E. Radioisotope Centre,

POLATOM,
05-400 Otwock-Swierk, Poland
Fax: +48227180351
Email: e.iller@polatom.pl

Ishfaq, M.M. Isotope Production Division,

Pakistan Institute of Nuclear Science
and Technology (PINSTECH),
P.O.Box 1482,
Islamabad, Pakistan
Fax: +92519290275
Email: mishfaq@pinstech.org.pk

Jahren, G. GE Healthcare,
Amersham Health,
P.O. Box 4220 Nydalen,
0401 Oslo, Norway
Fax: +4732186026
Email: grete.jahren@ge.com

Jalilian, A.R. Cyclotron Department,

Nuclear Research Centre for
Agriculture & Medicine,
P.O. Box 31585-4395,
Karaj, Islamic Republic of Iran
Fax: +982624411106
Email: ajalilian@nrcam.org

Janevik-Ivanovska, E. Medical Faculty,

Institute of Pathophysiology
and Nuclear Medicine,
Vodnjanska 17,
1000 Skopje,
The Former Yugoslav Republic of Macedonia
Fax: +38923147203
Email: janevik@yahoo.com

437
 LIST OF PARTICIPANTS

Janoki, Gy.A. Frederic Joliot-Curie National Institute

for Radiobiology and Radiohygiene,
Anna Utca 5,
1221 Budapest, Hungary
Email: janoki@hp.osski.hu

Janota, B. Radioisotope Centre,

POLATOM,
05-400 Otwock-Swierk, Poland
Fax: +48227180351
Email: b.janota@polatom.pl

Jehangir, M. Isotope Production Division,

Pakistan Institute of Nuclear Science
and Technology (PINSTECH),
P.O.Box 1482,
Islamabad, Pakistan
Fax: +92519290275
Email: mustansar@pinstech.org.pk

Jiménez-Shaw, R. Molypharma,
Dr. Severo Ochoa 37-4-1,
28100 Alcobendas,
Madrid, Spain
Fax: +34914905740
Email: rjs@molypharma.es

Jovanovic, S. Faculty of Sciences,

University of Montenegro,
P.O. Box 211,
81000 Podgorica, Serbia
Fax: +38181244608
Email: bobo_jovanovic@yahoo.co.uk

Jurina, V. Public Health Authority

of the Slovak Republic,
Trnavska cesta 52,
82645 Bratislava, Slovakia
Fax: +421244372619
Email: jurina@uvzsr.sk

438
 LIST OF PARTICIPANTS

Moghaddam, K.K. Cyclotron Department,

Nuclear Research Centre for
Agriculture & Medicine,
Rajaee Shahr,
P.O. Box 31585-4395,
Karaj, Islamic Republic of Iran
Fax: +982624411106
Email: kkamali@nrcam.org

Kamel, R. Department of Technical Cooperation,

International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: r.kamel@iaea.org

Kassai, Z. BIONT a.s.,

Karloveska 63,
84229 Bratislava, Slovakia
Fax: +421220670748
Email: kassai@biont.sk

Khanna-Hazra, P. S.N. Medical College,

Soami Bagh,
Agra 282003, India
Fax: +915622226524
Email: hazra@sancharnet.in

Kibwage, I.O. Faculty of Pharmacy,

College of Health Sciences,
University of Nairobi,
P.O. Box 19676,
Nairobi 00202, Kenya
Fax: +254202711132
Email: okibwage@uonbi.ac.ke

Kim, Yu-Seok KIRAMS,

Korea Cancer Centre Hospital,
215 Gongneung Dong, Nowon-ku,
Seoul, Republic of Korea
Email: mkh@kcchsun.kcch.re.kr

439
 LIST OF PARTICIPANTS

Kiondo, P.M. Nuclear Medicine Unit,

Mulago Hospital,
P.O.Box 7051,
Kampala, Uganda
Fax: +25641532591
Email: pmkiondo@med.mak.ac.ug

Knapp, F.F. Nuclear Medicine Program,

Oak Ridge National Laboratory,
Building 4501, MS 6229,
P.O. Box 2008,
Bethel Valley Road,
Oak Ridge, TN 37831-6229,
United States of America
Fax: +18655746226
Email: knappffjr@ornl.gov

Knopp, R. Eurotope GmbH,

Robert-Rössle-Strasse 10,
13125 Berlin, Germany
Fax: +4930941084160
Email: roger.knopp@eurotope.de

Környei, J. Institute of Isotopes Co. Ltd,

Konkoly Thege M. Str. 29–33,
1121 Budapest, Hungary
Fax: +3613959070
Email: kornyei@izotop.hu

Környeiné Kovács, G. “Uzsoki” Hospital,

Department of Nuclear Medicine,
Uzsoki str. 29,
1145 Budapest, Hungary
Fax: +3614673772
Email: kornyei@uzsoki.hu

Korsak, A. Radioisotope Centre,

POLATOM,
05-400 Otwock-Swierk, Poland
Fax: +48227180351
Email: a.korsak@polatom.pl

440
 LIST OF PARTICIPANTS

Kovac, P. BIONT a.s.,

Karloveska 63,
84229 Bratislava, Slovakia
Fax: +421220670748
Email: kovac@biont.sk

Kudelin, B.K. V.G. Khlopin Radium Institute,

28, 2nd Murinski Avenue,
194021 St. Petersburg,
Russian Federation
Fax: +78122476181
Email: kudelin@atom.nw.ru

Kuecuek, N.O. Department of Nuclear Medicine,

Faculty of Medicine,
Ankara University,
06100 Ankara, Turkey
Fax: +903123620897
Email: n.ozlem.kucuk@medicine.ankara.edu.tr

Kugel, D.J. St. Luke’s Hospital,

801 Ostrum St.,
Bethlehem, PA 18015,
United States of America
Fax: +16109237224
Email: debikugel@aol.com

Laznicek, M. Faculty of Pharmacy,

Charles University,
Heyrovskeko 1203,
500 05 Hradec Kralove,
Czech Republic
Fax: +420495514373
Email: laznicek@faf.cuni.cz

Laznickova, A. Faculty of Pharmacy,

Charles University,
Heyrovskeho 1203,
500 05 Hradec Kralove,
Czech Republic
Fax: 420495518002
Email: laznicko@faf.cuni.cz

441
 LIST OF PARTICIPANTS

Lee, Myuna-Chul Department of Nuclear Medicine,

Seoul National University Hospital,
28 Yongon-dong, Chongno-gu,
Seoul 110-744, Republic of Korea
Fax: +8227457690
Email: mychlee@yahoo.com

Leyva Montaña, R. Centro de Isotopos,

AENTA, CITMA,
Ave. Monumental y Carr. La Rada km 3½,
San José de Las Lajas,
Apartado Postal 3415,
Havana, Cuba
Fax: +5378661821
Email: rene@centis.edu.cu

Lipka, R.J. OBRI POLATOM,

Woj Mazowieckie,
05-400 Otwock-Swierk, Poland
Email: treborl_pl@yahoo.com

Lungu, V. Institute of Physics and Nuclear

Engineering “Horia Hulubei”,
Str. Atomistilor 407,
P.O. Box MG 6,
76900 Bucharest, Magurele, Romania
Fax: +4014231701
Email: vlungu2000@yahoo.com

Luo, Zhifu China Institute of Atomic Energy,

P.O. Box 275 ext. 12,
Beijing 102413, China
Fax: +861069358952
Email: lzhf@iris.ciae.ac.cn

Macásek, F. BIONT, a.s.,

Karloveska 63,
84229 Bratislava, Slovakia
Fax: +421220670748
Email: macasek@biont.sk

442
 LIST OF PARTICIPANTS

Machulla, H.-J. Radiopharmacy PET Centre,

University of Tübingen,
Röntgenweg 15,
72076 Tübingen, Germany
Fax: +497071295264
Email: machulla@uni-tuebingen.de

Malhotra, A. Department of Nuclear Medicine,

All India Institute of Medical Sciences,
New Delhi 110029, India
Fax: +911126588663
Email: arun190@hotmail.com

Malja, S. Institute of Nuclear Physics,

P.O. Box 85,
Tirana, Albania
Fax: +3554362596
Email: smalja@albmail.com

Marujo Marques, F. Instituto Tecnológico e Nuclear,

Estrada nacional 10,
Apartado 21,
2686-953 Sacavém, Portugal
Fax: +351219941455
Email: fmarujo@itn.pt

Mat Ail, N. Pharmacy Division,

Kuala Lumpur Hospital,
Jalan Pahang,
50386 Kuala Lumpur, Malaysia
Fax: +60320930293
Email: mhh@cimb.com.my

Máthé, D. “Fodor József” National Centre

of Public Health,
National Research Institute for
Radiobiology and Radiohygiene,
Anna utca 5,
1221 Budapest, Hungary
Fax: +3614822012
Email: mdomokos@hp.osski.hu

443
 LIST OF PARTICIPANTS

Mather, S.J. Imperial Cancer Research Fund,

Department of Nuclear Medicine,
St. Bartolomew’s Hospital,
51–53 Bartholomew Close,
London EC1 7BE, United Kingdom
Fax: 441717963907
Email: stephen.mather@cancer.org.uk

Melichar, F. Nuclear Physics Institute,

Academy of Sciences,
250 68 Rez, Prague,
Czech Republic
Fax: +42026875481
Email: melichar@ujf.cas.cz

Mertens, J.J. Radiopharmaceutical Chemistry/BEFY,

Vrije Universiteit Brussel,
Cyclotron Building,
Laarbeeklaan 103,
1090 Brussels, Belgium
Fax: +3223053131
Email: jjmertens@vub.ac.be

Mesa Dueñas, N. Instituto de Nefrología (INEF)

“Dr. Aberlardo Buch Lopez”,
Ave. 26 y Rancho Boyeros Cerro,
10600 Havana, Cuba
Fax: +5378812413 or +5372041188
Email: mninef@infomed.sld.cu

Miceva Ristevska, S. Institute of Pathophysiology

and Nuclear Medicine,
Medical Faculty,
Vodnjanska 17,
1000 Skopje,
The Former Yugoslav Republic of Macedonia
Email: svetlana@ukim.edu.mk

Mikolajczak, R. Radioisotope Centre,

POLATOM,
05-400 Otwock-Swierk, Poland
Fax: +48227180350
Email: r.mikolajczak@polatom.pl

444
 LIST OF PARTICIPANTS

Mishra, A.K. Division of Cyclotron and

Radiopharmaceutical Sciences,
Institute of Nuclear Medicine
and Allied Sciences,
Brig. S.K. Mazumdar Road,
Timarpur, New Delhi 110054, India
Fax: ++911123919509
Email: akmishra@inmas.org

Mititelu, M.R. Central Clinical Emergency

Military Hospital,
Cal Plevnei 134, Sector 6,
010242 Bucharest, Romania
Fax: +4022223670
Email: ralunuclear@yahoo.com

Mitterhauser, M. Department of Nuclear Medicine,

Allgemeines Krankenhaus Universitätskliniken,
Währinger Gürtel 18-20,
1090 Vienna, Austria
Fax: +431404001559
Email: markus.mitterhauser@meduniwien.ac.at

Naidoo, C. Radionuclide Production Group,

IThemba Labs,
P.O. Box 722,
Somerset West 7129, South Africa
Fax: +27218433901
Email: clive@tlabs.ac.za

Nano, G. Dompe SpA,

Via Campo di Pile SNC,
Z.1. Pile,
67100 L’Aquila, Italy
Fax: +390862338219
Email: nano@dompe.it

Napaporn, T. Department of Radiology,

Division of Nuclear Medicine,
Siriraj Hospital Medical School,
Bangkok 10700, Thailand
Fax: +6624127165
Email: sintj@mahidol.ac.th

445
 LIST OF PARTICIPANTS

Niculae, D. National Institute for Physics and Nuclear

Engineering “Horia Hulubei”,
Atomistilor Street 407,
P.O. Box MG-6,
76900 Bucharest – Magurele, Romania
Fax: +4021454440
Email: radphy@ifin.nipne.ro

Novotny, D. Hans Wälischmiller GmbH,

Rossendorfer Ring 42,
01328 Dresden, Germany
Fax: +493512663410
Email: waelischmiller_dresden@t-online.de

Ocak, M. Department of Pharmaceutical Technology,

Faculty of Pharmacy,
Istanbul University,
34116 Istanbul, Turkey
Fax: +902125306734
Email: melocak@yahoo.com

Oliver, Y.P. Centro de Investigaciones Nucleares,

Facultad de Ciencias,
Igua 4225,
11400 Montevideo, Uruguay
Fax: +59825250895
Email: poliver@cin.edu.uy

Narbutt, J. Institute of Nuclear Chemistry

and Technology,
Dorodna 16,
03-195 Warsaw, Poland
Fax: +48228111532
Email: jnarbut@ichtj.waw.pl

Özer, A.Y. Department of Radiopharmacy,

Faculty of Pharmacy,
Hacettepe University,
06100 Ankara, Turkey
Fax: +903123114777
Email: ayozer@hacettepe-edu.tr

446
 LIST OF PARTICIPANTS

Ozker, K. Medical College of Wisconsin,

9200 W. Wisconsin Avenue,
Milwaukee, WI 53226,
United States of America
Fax: +14147713460
Email: ozker@mcw.edu

Pasquali, M. Laboratory of Nuclear Medicine,

Department of Radiology,
University of Ferrara,
Via L. Borsari 46,
44100 Ferrara, Italy
Fax: +39532236589
Email: dta@unife.it

Pauwels, E.K.J. Department of Nuclear Medicine,

Leiden University Medical Centre,
C4-075,
Albinusdreef 2,
2333 ZA Leiden, Netherlands
Fax: +31715266751
Email: ernestpauwels@gmail.com

Pawlak, D. Radioisotope Centre,

POLATOM,
05-400 Otwock-Swierk, Poland
Fax: +48227180351
Email: d.pawlak@polatom.pl

Pedersen, B. Danish Medicines Agency,

Axel Heidesgade 1,
2300 Københans, Denmark
Email: bp@dkma.dk

Perewusnyk, G. Swiss Federal Office of Public Health,

Division of Radiation Protection,
3003 Bern, Switzerland
Fax: +41313228383
Email: gloria.perewusnyk@bag.admin.ch

Pereyra Molina, V.E. Nuclear Medicine National Institute,

C. Mayor Rafael Zubieta #1555,
Miraflores, La Paz, Bolivia
Fax: +59122112784
Email: inamen@entelnet.bo

447
 LIST OF PARTICIPANTS

Persson, B.E. Medical Products Agency,

P.O. Box 26,
751-03 Uppsala, Sweden
Fax: +4618548566
Email: bengt.persson@mpa.se

Pike, V.W. Molecular Imaging Branch,

National Institute of Mental Health,
Building 101 RM B3 C346A,
10 Center Drive,
Bethesda, MD 20892-1003,
United States of America
Fax: +13014805112
Email: pikev@mail.nih.gov

Pillai, M.R. Department of Nuclear

Sciences and Applications,
International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: M.R.A.Pillai@iaea.org

Pimentel, G.J. National Institute of Oncology

and Radiobiology,
Calle 29 E y 29,
Vedado Plaza,
10400 Havana, Cuba
Email: gilmara@infomed.sld.cu

Pirmettis, I. Institute of Radioisotopes,

NCSR “Demokritos”,
Patriarchou Grigoriou and Napoeleos,
15310 Aghia Paraskevi,
Athens, Greece
Fax: +302106524480
Email: ipirme@rrp.demokritos.gr

Poramatikul, N. Radioisotope Production Programme,

Office of Atoms for Peace (OAP),
16 Vibhavadee Rangsit Road,
Chatujak,
Bangkok 10900, Thailand
Fax: +6625620127
Email: nipavan@oaep.go.th

448
 LIST OF PARTICIPANTS

Prats Capote, A. Centre for Clinical Research,

34 Street no. 4501e/ 45 y 47,
Kholy, Playa,
11300 Havana, Cuba
Fax: +5372043298
Email: anais.prats@infomed.sld.cu

Pruszynski, M. Institute of Nuclear Chemistry

and Technology,
Dorodna 16,
03-195 Warsaw, Poland
Fax: +48228111532
Email: mprusz@ichtj.waw.pl

Rajec, P. BIONT a.s.,

Karloveska 63,
84229 Bratislava, Slovakia
Fax: +421220670748
Email: rajec@biont.sk

Rakiás, F. National Institute of Pharmacy,

P.O. Box 450,
1372 Budapest, Hungary
Fax: +3612663245
Email: rakias.ferenc@ogyi.hu

Ramamoorthy, N. Department of Nuclear

Sciences and Applications,
International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: n.ramamoorthy@iaea.org

Razbash, A.A. Cyclotron Co. Ltd,

1, Bondarenko Square,
Obninsk,
Kaluga Region 249033,
Russian Federation
Fax: +70843997048
Email: razbash_isotop@obninsk.com

449
 LIST OF PARTICIPANTS

Rey Rios, A.M. Catedra de Radioquimica,

Facultad de Quimica,
Universidad de la República,
General Flores 2124,
P.O. Box 1157,
11800 Montevideo, Uruguay
Fax: +59829241906
Email: arey@fq.edu.uy

Robles, A.M. Centro de Investigaciones Nucleares,

Facultad de Ciencias,
Igua 4225,
11400 Montevideo, Uruguay
Fax: +59825250895
Email: anamar@cin.edu.uy

Rodriguez, G. Centro de Investigaciones Nucleares,

Facultad de Ciencias,
Igua 4225,
11400 Montevideo, Uruguay
Fax: +59825250895
Email: grodri@cin.edu.uy

Roesch, F. Institut für Kernchemie,

Johannes Gutenberg Universität,
Fritz-Strassmann-Weg 2,
55128 Mainz, Germany
Fax: +4961313924692
Email: frank.roesch@uni-mainz.de

Rossbach, M. Department of Nuclear

Sciences and Applications,
International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: m.rossbach@iaea.org

Rossi, G. ENEA UTS FIS-ION,

C.R. Casaccia,
S.P. Anguillarese 301,
00060 Rome, Italy
Fax: +3930484874
Email: rossig@casaccia.enea.it

450
 LIST OF PARTICIPANTS

Rossouw, D.D. IThemba Labs,

P.O. Box 722,
7129 Somerset West,
South Africa
Fax: +27218433901
Email: niel@tlabs.ac.za

Sadeghi, M. Cyclotron Department,

Nuclear Research Centre for
Agriculture & Medicine,
P.O. Box 31585-4395,
Karaj, Islamic Republic of Iran
Fax: +982614411106
Email: msadeghi@nrcam.org

Salouti, M. Radioisotope Department,

Nuclear Research Centre,
P.O. Box 11365-3486,
Tehran, Islamic Republic of Iran
Fax: +98218020887
Email: saloutim@yahoo.com

Santos, R.G. Centre for Development of

Nuclear Technology (CDTN),
Av. Prof. Mario Werneck s.n.,
CP 941,
BR-30123-970 Belo Horizonte,
Minas Gerais, Brazil
Fax: +553134993380
Email: santosr@cdtn.br

Satpati, D. Radiopharmaceuticals Division,

Bhabha Atomic Research Centre,
Mumbai 400085, India
Fax: +912225595371
Email: drishtys@apsara.barc.ernet.in

Saw, M.M. Department of Nuclear Medicine

and PET Centre,
Block 2, Basement 1,
Singapore General Hospital,
Outram Road,
169608 Singapore
Fax: +6563230735
Email: maungmaungsaw@hotmail.com

451
 LIST OF PARTICIPANTS

Sawe, S.F. Tanzania Atomic Energy Commission (TAEC),

P.O. Box 743,
Arusha,
United Republic of Tanzania
Fax: +255272509709
Email: shovisawe@hotmail.com

Schlyer, D. Department of Chemistry,

Brookhaven National Laboratory,
Upton, NY 11973-5000,
United States of America
Email: schlyer@bnl.gov

Sedda, A.F. ENEA UTS FIS-ION,

C.R. Casaccia,
S.P. Anguillarese 301,
00060 Rome, Italy
Fax: +3930484874
Email: anqioco.sedda@casaccia.enea.it

Sergieva, S. Sofia Cancer Centre,

Kv. “Mladost” 1,
Blvd. Andrey Saharov 1,
P.O. Box 54,
Sofia 1756, Bulgaria
Fax: +3592761530
Email: sergieva_s@yahoo.com

Sevastyanov, Yu.G. Cyclotron Co. Ltd,

1, Bondarenko Square,
Obninsk,
Kaluga Region 249033,
Russian Federation
Fax: +70843997048
Email: cyclotron@obninsk.com

Siddig, S.M.A. Institute of Nuclear Medicine, Molecular

Biology and Oncology (INMO),
University of Gezira,
P.O. Box 20,
Wad Medani, Sudan
Fax: +249511843862
Email: siddig@Email.com

452
 LIST OF PARTICIPANTS

Singh, N. Institute of Nuclear Medicine

and Allied Sciences,
Brig. S.K. Mazumdar Road,
Timarpur, Delhi 110054, India
Email: namsingh1@yahoo.com

Sirandoni Riquelme, G. Regional Hospital of Temuco,

Guillermo MacDonald 02551,
Barrio Ingles,
Temuco, IX Region, Chile
Fax: +5624296660
Email: gsirandoni@araucaniasur.cl

Sobreira, A.C. Rem Indústria e Comércio Ltda,

Rua Columbus 282,
05304-010 Sâo Paulo, SP,
Brazil
Fax: +551138316922
Email: anacelia@rem.ind.br

Solanki, K.K. Department of Nuclear

Sciences and Applications,
International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: k.solanki@iaea.org

Solodyannikova, O.I. Department of Nuclear Medicine,

Institute of Oncology,
Lomonosov Str. 33/43,
Kiev-22, Ukraine
Fax: +380442590182
Email: oko@mail.ru

Soloviev, D. Cyclotron Unit,

University Hospital of Geneva,
24 rue Micheli-du-Crest,
Geneva-14, Switzerland
Fax: +41223727585
Email: dmitri.soloviev@hcuge.ch

453
 LIST OF PARTICIPANTS

Soroa, V.E. Radiobiology — Centro de Medicina Nuclear,

Comisión Nacional de Energía Atómica,
Avenida Del Libertador 8250,
1429 Buenos Aires, Argentina
Fax: +541148052123
Email: tate@cnea.gov.ar

Soylu, A.A. Department of Nuclear Medicine,

Faculty of Medicine,
Ankara University,
06100 Cebeci Ankara, Turkey
Fax: +903123620897
Email: ayfersoylu@yahoo.com

Suzuki, K. Department of Medical Imaging,

Research Centre for Charged
Particle Therapy,
National Institute of Radiological
Sciences (NIRS),
9-1, Anagawa 4-chome,
Inage-ku, Chiba-shi 263,
Japan
Fax: +81432063261
Email: kazutosi@nirs.go.jp

Tandon, P. Radiological Physics & Advisory Division,

Bhabha Atomic Research Centre,
CT&CRS Building,
Anushaktinagar,
Mumbai 400 094, India
Fax: +912225519209
Email: ptandon_barc@rediffmail.com

Tendilla del Pozo, J.I. Instituto Nacional

de Investigaciones Nucleares (ININ),
Kilometro 36.5,
Carretera Mexico-Toluca,
52045 Municipio de Ocoyoacac,
Mexico
Fax: +5553297316
Email: jtp@nuclear.inin.mx

454
 LIST OF PARTICIPANTS

Thakur, M.L. Radiopharmaceutical Research,

Thomas Jefferson University,
1020 Locust Street, Suite 359,
Philadelphia, PA 19107,
United States of America
Fax: 215-923-9245
Email: mathew.thakur@jefferson.edu

Tiskevicius, S. Nuclear Medicine Department,

Vilnius University Institute of Oncology,
Santariskiu 1,
2021 Vilnius, Lithuania
Fax: +37052720164
Email: sigitas.tiskevicius@delfi.lt

Trabelsi, M. Centre national des sciences et

technologies nucléaires,
Pôle technologique,
Sidi Thabet,
2020 Ariana, Tunisia
Fax: +21671537555
Email: moez.trabelsi@cnstn.rnrt.tn

Tran Van Quy Vietnam Atomic Energy Commission,

59 Ly Thuong Kiet Street,
Hanoi, Vietnam
Fax: +84494234133
Email: tranquyru@yahoo.com

Trtic-Petrovic, T. Laboratory of Physics (010),

Vinca Institute of Nuclear Sciences,
P.O. Box 522,
11001 Belgrade, Serbia
Fax: +381114447963
Email: ttrtic@vin.bg.ac.yu

Turner, J.H. Fremantle Hospital,

University of Western Australia,
Alma Street,
Fremantle WA 6160, Australia
Fax: +61894312889
Email: harvey.turner@health.wa.gov.au

455
 LIST OF PARTICIPANTS

Tzanopoulou, S. Institute of Biology,

National Centre for Scientific
Research (NCSR) “Demokritos”,
P.O. Box 60228,
15310 Aghia Paraskevi,
Athens, Greece
Fax: +302106511767
Email: tina@bio.demokritos.gr

Vallabhajosula, S. Nuclear Medicine,

New York Presbyterian Hospital and Weill
Medical College of Cornell University,
525 East 68th Street,
STARR 2-21 New York, NY 10021,
United States of America
Fax: +2127469010
Email: svallabh@med.cornell.edu

Vandecapelle, M. Federal Agency for Nuclear Control (FANC),

Ravenstein Street 36,
1000 Brussels, Belgium
Fax: +3222892112
Email: marleen.vandecapelle@fanc.fgov.be

Venkatesh, M. Radiopharmaceuticals Division,

Bhabha Atomic Research Centre,
Mumbai 400085, India
Fax: +912225505151 or +91225505345
Email: meerav@apsara.barc.ernet.in

Verbruggen, A.M. Laboratory of Radiopharmaceutical Chemistry,

University Hospital,
Gasthuisberg-Radiopharmacy,
Herestraat 49,
B-3000 Leuven, Belgium
Email: alfons.verbruggen@farm.kuleuven.ac.be

Verdera Presto, E.S. TECHI S.A.,

Bulevar Espana 2729,
Apartamento 701,
Montevideo, Uruguay
Fax: +59822008060
Email: sverdera@hotmail.com

456
 LIST OF PARTICIPANTS

Verk, T. Department of Radiophysics,

Institute of Oncology,
Zaloska 2,
1000 Ljubljana, Slovenia
Fax: +38614319108
Email: tverk@onko-i.si

Volkova, O.N. “Techsnabexport”,

Staromonetny per. 26,
119180 Moscow, Russian Federation
Fax: +70952302638
Email: tenex@online.ru

von Wenzel-Obholzer, K.S. Department of Nuclear Medicine,

Dr. A. Bernard May Cancer Care Centre,
P.O. Box 8650,
Windhoek, Namibia
Fax: +264612033263
Email: kvwo@iafrica.com.na

Vora, M.M. Cyclotron and Radiopharmaceuticals

Department,
King Faisal Specialist Hospital
and Research Centre,
MBC-03, P.O. Box 3354,
Riyadh 11211, Saudi Arabia
Fax: +9664424743
Email: vora@kfshrc.edu.sa

Wang, Mingwei Radiopharmaceutical Centre,

Shanghai Institute of Applied Physics, CAS,
2019 Jialuo Road,
Jiading,
Shanghai 201800, China
Fax: +862159554696
Email: wmwnuclear@163.com

Wang, Yongxian Radiopharmaceutical Centre,

Shanghai Institute of Applied Physics, CAS,
2019 Jialuo Road,
Jiading,
Shanghai 201800, China
Fax: +862159556884
Email: yongxianw@163.com

457
 LIST OF PARTICIPANTS

Wastiel, C. Institut Universitaire Radiophysique,

Grand Pré 1,
1007 Lausanne, Switzerland
Fax: +41216233435
Email: claude.wastiel@chuv.ch

Westera, G. Nuclear Medicine,

Centre for Radiopharmaceutical Science,
University Hospital Zürich,
8091 Zürich, Switzerland
Fax: +41442554428
Email: gerrit.westera@dmr.usz.ch

Xie, Wucheng China Atomic Energy Authority (CAEA),

8A Fucheng Road,
Haidian District,
Beijing 100037, China
Fax: +861088581511
Email: xiewch@caea.gov.cn

Yassine, T. Radioisotope Division,

Department of Chemistry,
Atomic Energy Commission,
P.O. Box 6091,
Damascus, Syrian Arab Republic
Fax: +963116112289
Email: tyassin@aec.org.sy

Zaknun, J. Department of Nuclear

Sciences and Applications,
International Atomic Energy Agency,
Wagramer Strasse 5, P.O. Box 100,
1400 Vienna, Austria
Fax: +43126007
Email: j.zaknun@iaea.org

Zalutsky, M.R. Department of Radiology,

Duke University Medical Center,
P.O. Box 3808,
Durham, NC 27710,
United States of America
Fax: +19196847121
Email: zalut001@mc.duke.edu

458
 LIST OF PARTICIPANTS

Zasepa, M. Institute of Nuclear Chemistry

and Technology,
Dorodna 16,
03-195 Warsaw, Poland
Fax: +48228111532
Email: mkz@ichtj.waw.pl

Zhang, Jianfeng National Institute for

Radiological Protection,
CDC,
2 Xinkang Street,
Deshengmenwai,
Beijing 100088, China
Fax: +861062012501
Email: zhangjianfengbj@hotmail.com

Zitko-Krhin, M. Department for Nuclear Medicine,

University Medical Centre,
Zaloska 7,
1525 Ljubljana, Slovenia
Fax: +38615222237
Email: mojca.zitko@kclj.si

Zolle, I. Ludwig-Boltzmann Institute

of Nuclear Medicine,
Zimmermanngasse 22/8,
1090 Vienna, Austria
Fax: +4314057198
Email: ilse.zolle@univie.ac.at

Zuvic, M. Clinical Hospital Centre,

Department of Nuclear Medicine
and Radiation Protection,
Kispaticeva 12,
10000 Zagreb, Croatia
Fax: +38512421874
Email: mzuvic@kbc-zagreb.hr

Zyuzin, A. Advanced Cyclotron Systems Inc. (ACSI),

7851 Alderbridge Way,
Richmond, BC V6X 2A4,
Canada
Fax: +16042761495
Email: azyuzin@advancedcyclotron.com

459
.
 AUTHOR INDEX

Abdel-Jalil, R.J.: (2) 295 Bouyoucef, S.F.: (1) 167

Abedon, M.Z.: (1) 241 Bouziotis, P.: (1) 185
Adelfang, P.: (1) 349 Cabral, P.: (2) 63
Afarideh, H.: (2) 357 Caldeira Filho, J.S.: (2) 243
Al Jammaz, I.: (2) 283 Cardi, C.A.: (1) 113
Al Otaibi, B.: (2) 283 Caviglia, H.: (2) 135
Alberto, R.: (1) 355 Chakraborty, S.: (1) 285; (2) 51
Al-Saeedi, F.: (2) 183 Chiewvit, S.: (1) 83
Amartey, J.: (2) 283 Chiper, D.: (2) 39
Andonovski, A.: (1) 65 Choudhury, P.S.: (2) 399
Andy Hor, T.S.: (1) 395 Chura-Chambi, R.M.: (1) 375
Aras, G.: (2) 109 Chuttani, K.: (1) 31
Archimandritis, S.C.: (1) 185 Ciavaro, M.: (1) 217
Arteaga de Murphy, C.: (1) 55 Čomor, J.J.: (2) 209
Aruva, M.R.: (1) 113 Cosgrove, J.: (1) 333
Badillo-Almaraz, V.E.: (1) 333 Couto, R.M.: (2) 243
Balogh, L.: (2) 3 Czerwinski, M.: (1) 367
Balter, H.: (1) 43 da Silva, C.P.G.: (2) 243, 349
Balter, H.: (2) 63 Das, T.: (2) 51
Bander, N.H.: (2) 73 de Araújo, E.B.: (2) 243
Banerjee, S.: (1) 197; (2) 51 de Barboza, M.F.: (2) 349
Bapat, K.: (1) 197 de Castiglia, S.G.: (1) 217
Barto , B.: (2) 353 de Jong, M.: (2) 91
Bashar, K.M.: (1) 249 Decristoforo, C.: (1) 21
Bauwens, M.: (2) 223 Djorgoski, I.: (1) 65
Béhé, M.: (1) 21 Doval, D.C.: (2) 399
Bellazoug, K.: (1) 167 Duanzhi Yin: (2) 297
Benchelaghem, B.: (1) 167 Duatti, A.: (1) 171
Benlabgaa, R.: (1) 167 Dudov, A.: (2) 123
Bequet, M.: (1) 205 Dumrongpisutikul, S.: (1) 83
Berger, M.L.: (1) 383 Ehrlichmann, W.: (2) 295
Beyer, G.-J.: (2) 209, 331 Faintuch, B.L.: (1) 375
Bhatnagar, A.: (1) 95 Fani, M.: (1) 185
Bilewicz, A.: (2) 353 Fernandez, E.: (1) 205
Blauenstein, P.: (1) 375 Ferro-Flores, G.: (1) 55
Bossuyt, A.: (2) 223 Fettich, J.: (1) 183
Botev, V.: (2) 123 Filosofov, D.V.: (2) 367
Boucekkine, N.: (1) 167 Fischer, T.: (2) 253

461
 AUTHOR INDEX

Fiszman, G.: (1) 217 Khelifa, A.: (1) 167

Flores de la Torre, J.A.: (1) 333 Khiewvan, B.: (1) 83
Fukumori, N.T.O.: (2) 349 Király, R.: (2) 3
Galatros, G.: (2) 135 Knapp, Jr., F.F.: (1) 301, 333
Gallez, C.: (2) 223 Konior, M.: (1) 323
Garayoa, E.G.: (1) 375 Kőrösi, L.: (2) 3
Garg, P.K.: (2) 265 Köster, U.: (2) 331
Garg, S.: (2) 265 Kothari, K.: (1) 197
Garland, M.A.: (1) 301 Kováč, P.: (1) 265
Giannone, C.: (2) 135 Krenning, E.: (2) 91
Giubbini, R.M.T.: (1) 103 Küçük, N.Ö.: (2) 109
Goes, M.M.: (2) 349 Kumar, N.: (1) 31
Goldman, I.: (1) 349 Kunze, S.: (1) 355
Goldsmith, S.J.: (2) 73 Kwekkeboom, D.: (2) 91
Goomer, N.C.: (2) 399 Lago, G.: (1) 43
Gourni, E.: (1) 185 Lahoutte, T.: (2) 223
Gültekin, S.S.: (2) 109 Leyva, R.: (1) 205
Gupta, A.: (2) 399 López, A.: (1) 43
Habeche, M.: (1) 167 Loudos, G.: (1) 185
Hadjieva, T.: (2) 123 Lu, S.Y.: (2) 147
Haji-Saeid, M.: (2) 357 Lungu, V.: (2) 39
Hammerschmidt, F.: (1) 383 Macá ek, F.: (1) 265
Hanzal, S.: (1) 167 Machulla, H.-J.: (2) 295
Haque, M.A.: (1) 241 Malik, S.A.: (1) 149
Hernández, A.: (1) 205 Markovic, S.: (1) 183
Herrerias, R.: (2) 349 Máthé, D.: (2) 3
Hong, J.: (2) 147 Matsuda, H.: (2) 349
Ýbiș, E.: (2) 109 Matsuda, M.M.N.: (2) 349
Iller, E.: (1) 323 McCarron, J.A.: (2) 147
Ishfaq, M.M.: (1) 249 Mechken, F.: (1) 167
Jahn, M.: (2) 367 Mekni, A.: (1) 365
Jalilian, A.R.: (2) 195 Meléndez-Alafort, L.: (1) 55
Janevik-Ivanovska, E.: (1) 65 Mengatti, J.: (2) 349
Jánoki, G.A.: (1) 65; (2) Mertens, J.J.R.: (2) 223
Jehangir, M.: (1) 149, 249 Miceva-Ristevska, S.: (1) 65
Jennewein, M.: (2) 367 Mikolajczak, R.: (1) 323
Jiménez-Shaw, R.: (2) 413 Milenkov, V.: (1) 65
Kai, Y.Y.: (1) 395 Milowsky, M.I.: (2) 73
Karczmarczk, U.: (1) 323 Mingwei Wang: (2) 297
Kataria, T.: (2) 399 Mirzadeh, S.: (1) 301
Kersemans, K.: (2) 223 Mirzaii, M.: (2) 195

462
 AUTHOR INDEX

Mishra, A.K.: (1) 31 Ramamoorthy, N.: (1) 349

Mishra, P.: (1) 31 Ramírez, F.: (1) 55
Misiak, R.: (2) 353 Ratanawichitrasin, A.: (1) 83
Mittal, G.: (1) 95 Ravert, H.: (2) 283
Mitterhauser, M.: (2) 161 Ravindranath, T.: (1) 95
Moghaddam, K.K.: (1) 271 Ravn, H.L.: (2) 331
Monroy-Guzmán, F.: (1) 333 Repse, S.: (1) 183
Morganti, L.: (1) 375 Reyes, O.: (1) 205
Mouas, F.: (1) 167 Robles, A.: (1) 43; (2) 63
Mukherjee, A.: (2) 51 Rodríguez, G.: (1) 43; (2) 63
Mundwiler, S.: (1) 355 Rodríguez-Cortés, J.: (1) 55
Muramoto, E.: (1) 375; (2) 243 Roesch, F.: (2) 367
Musachio, J.L.: (2) 147 Roohi, S.: (1) 149
Mushtaq, A.: (1) 149 Rowshanfarzad, P.: (2) 195
Nagamati, L.T.: (2) 243 Sabet, M.: (2) 195
Nanus, D.M.: (2) 73 Sadeghi, M.: (2) 357
Nappa, A.: (2) 63 Saidi, M.: (1) 365
Narbutt, J.: (1) 367 Samuel, G.: (2) 51
Nascimento, N.: (1) 375 Santiago, N.: (1) 205
Naswetter, G.: (2) 135 Sardari, D.: (2) 195
Niculae, D.: (2) 39 Sarma, H.D.: (1) 197; (2) 51
Nizakat, H.: (1) 249 Satpati, D.: (1) 197
Obenaus, E.: (1) 217 Saw, M.M.: (1) 395
Oliver, P.: (1) 43; (2) 63 Sawlewicz, K.: (1) 323
Oyarzábal, I.: (2) 413 Schibli, R.: (1) 367, 383
Ozker, K.: (2) 387 Schicha, H.: (2) 253
Panwar, P.: (1) 31 Schlyer, D.J.: (2) 311
Paravatou, M.: (1) 185 Schomäcker, K.: (2) 253
Pauwels, E.K.J.: (1) 43 Schubiger, P.A.: (1) 375
Pedraza-López, M.: (1) 55 Sciani, V.: (2) 349
Perera, A.: (1) 205 Sergieva, S.: (2) 123
Petelenz, B.: (2) 353 Shanthly, N.: (1) 113
Pike, V.W.: (2) 147 Sharma, P.K.: (2) 399
Pimentel, G.: (1) 205; (2) 209 Sharma, R.: (1) 31
Pires, J.T.: (2) 349 Sharma, R.K.: (1) 31
Polyák, A.: (2) 3 Singh, A.K.: (1) 95
Pozzi, O.R.: (2) 25 Singh, N.: (1) 95
Prats, A.: (1) 205 Snoj, M.: (1) 183
Radu, M.: (2) 39 Soroa, V.E.: (2) 135
Raisali, G.: (2) 357 Souza, A.A.: (2) 349
Rajec, P.: (1) 265 Soylu, A.A.: (2) 391

463
 AUTHOR INDEX

Spingler, B.: (1) 355 Vilar, J.: (1) 43

Staniszewska, J.: (1) 323 Voelter, W.: (2) 295
Stichelberger, M.: (1) 383 von Guggenberg, E.: (1) 21
Sumiya, L.C.A.: (2) 349 Vora, M.M.: (1) 227
Sundram, F.X.: (1) 395 Wadsak, W.: (2) 161
Taweepraditpol, S.: (1) 83 Welling, M.M.: (1) 43
Thakur, M.L.: (1) 113 Wickstrom, E.: (1) 113
Tojinda, N.: (1) 83 Xanthopoulos, S.: (1) 185
Tortarolo, V.: (2) 63 Xiobing, T.: (1) 113
Trabelsi, M.: (1) 365 Yongxian Wang: (2) 297
Trindade, V.: (1) 43; (2) 63 Zalutsky, M.R.: (2) 25
Übele, M.: (2) 295 Zasepa-Lyczko, M.: (1) 367
Vaid, A.K.: (2) 399 Zelek, Z.: (1) 323
Vaidyanathan, G.: (2) 25 Zemoul, S.A.: (1) 167
Valkema, R.: (2) 91 Zhang, K.: (1) 113
Vallabhajosula, S.: (2) 73 Zhernosekov, K.P.: (2) 367
Varvarigou, A.D.: (1) 185 Ziaee, A.: (2) 195
Velásquez Espeche, M.H.: (2) 135 Zitko-Krhin, M.: (1) 183
Venkatesh, M.: (1) 197, 285; (2) 51 Zolle, I.: (1) 383
Verbruggen, A.M.: (1) 3

464