STATISTICAL APPROACHES For Dissoprofile Copmarision

STATISTICAL APPROACHES TO PERFORM
DISSOLUTION PROFILE COMPARISONS
Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III
Chemie, Pharmazie, Bio- und Werkstoffwissenschaften
der Universität des Saarlandes
von
José David Gómez Mantilla
Saarbrücken
2013
Tag des Kolloquiums: 16.12.2013
Dekan: Prof. Dr. Volkhard Helms
Berichterstatter: Prof. Dr. Claus-Michael Lehr
apl. Prof. Dr. Ulrich Schäfer
Vorsitz: Prof. Dr. Hans Maurer
Akad. Mitarbeiter: Dr. Martin Frotscher

Life is not a hundred meter sprint; life is an
ultra-marathon with hurdles, and it is all
about not losing rhythm with the obstacles.
–Vicente
Casabó -
(1958-2013)
May you rest in peace,
Dear professor.
“The aim of science is not to open the door
to infinite wisdom, but to set a limit to infinite
error.”
–Bertolt Brecht –
Life of Galileo
Table of Contents
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Table of Contents
1. Short Summary ....................................................................................................... 1

2. Kurzzusammenfassung ......................................................................................... 2
3. Introduction ............................................................................................................. 3
3.1. Bioequivalence and Biowaiving ............................................................................... 4
3.2. Drug Dissolution Tests .............................................................................................. 6
3.3. Modeling Drug Dissolution........................................................................................ 7
3.4. Dissolution Profile Comparisons.............................................................................. 9
3.5. In vitro-in Vivo Correlation Models ........................................................................ 11
3.6. Permeability and d/p-systems ................................................................................ 13
3.7. Aim of this Work ....................................................................................................... 14
4. Two New, Nonparametric Test for Statistical Comparison of Dissolution
Profiles................................................................................................................... 16
4.1. Abstract ............................................................................................................ 17
4.2. Introduction ...................................................................................................... 18
4.3. Methods ........................................................................................................... 22
4.3.1. Dissolution Models ............................................................................................... 22
4.3.2. Data Simulation .................................................................................................... 22
4.3.2.1. Reference and Test Formulations ................................................................. 22
4.3.2.2. Intrinsic and Residual Variability .................................................................... 22
4.3.2.3. Sampling Time Points ...................................................................................... 25
4.3.3. Dissolution Profile Comparison Tests ............................................................... 25
4.3.3.1. f2 Similarity Factor ............................................................................................ 25
4.3.3.2. f2 Bootstrap Confidence Interval (f2 CI) ....................................................... 26
4.3.4. Two New Nonparametric Tests for Statistical Comparison of Dissolution
Profiles ................................................................................................................... 27
4.3.4.1. Permutation Test .............................................................................................. 27
4.3.4.2. Tolerated Difference Test ................................................................................ 29
4.3.5. Robustness Explorations .................................................................................... 31
4.3.6. Power Explorations .............................................................................................. 31
4.3.7. Effect of Statistical Independence and Sample Size ...................................... 32
4.3.8. Software ................................................................................................................. 33
4.4. Results and Discussion .................................................................................... 33
Table of Contents
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4.4.1. Statistical Robustness ......................................................................................... 33
4.4.2. Effect of Statistical Independence and Sample Size on Robustness .......... 33
4.4.3. Statistical Power ................................................................................................... 36
4.4.4. Flexibility of TDT ................................................................................................... 41
4.4.5. Effect of Independence and Sample Size on Power ...................................... 43
4.4.6. Time Sample Strategies ...................................................................................... 46
4.4.7. Experimental Design Driven Strategy for Performing Optimal Dissolution
Profile Comparisons............................................................................................. 49
4.5. Conclusions...................................................................................................... 52
5. Tailor-Made DPC-tests for comparing ER Formulations Using IVIVC Models 53
5.1. Abstract ............................................................................................................ 54
5.2. Introduction ...................................................................................................... 55
5.3. Methods ........................................................................................................... 58
5.3.1. General Strategy .................................................................................................. 58
5.3.2. In Vitro-In Vivo Correlation Models (IVIVC models) ....................................... 58
5.3.3. Test and Reference Formulations ..................................................................... 63
5.3.4. Simulations and Bioequivalent Studies ............................................................ 63
5.3.5. Dissolution Profile Comparisons Tests (DPC-tests) ....................................... 64
5.3.6. Statistical Power Explorations ............................................................................ 64
5.4. Results ............................................................................................................. 64
5.4.1. Illustrative Example .............................................................................................. 64
5.4.2. Bioequivalent and Similarity Spaces ................................................................. 65
5.4.3. Customization of TDT .......................................................................................... 67
5.4.4. Effect of Drug & Formulation .............................................................................. 67
5.4.5. Effect of BE trials Conditions and DPC-tests Conditions on BE-space and
Sim-space.............................................................................................................. 69
5.4.6. BE-space Compared to TDT to MDT and MRT .............................................. 72
5.5. Discussion ........................................................................................................ 72
5.6. Conclusions...................................................................................................... 79
6. Prediction of Equivalence in a Combined Dissolution and Permeation System
using customized DPC-tests ............................................................................... 80
6.1. Abstract ............................................................................................................ 81
6.2. Introduction ...................................................................................................... 82
6.3. Materials and Methods ..................................................................................... 86
Table of Contents
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6.3.1. Mathematical Modeling ....................................................................................... 86
6.3.2. Construction of Equivalent Space (Eq-space) ................................................. 88
6.3.3. Dissolution Profile Comparisons ........................................................................ 89
6.3.5. Formulation Optimization .................................................................................... 89
6.4. Results ............................................................................................................. 90
6.4.1. Model Fitting of the Combined Dissolution and Permeation Data ................ 90
6.4.2. Equivalent Space ................................................................................................. 91
6.4.4. Formulation Optimization ................................................................................. 92
6.5. Discussion ........................................................................................................ 96
6.6. Conclusions.................................................................................................... 100
7. Summary and Outlook ....................................................................................... 101
8. Abbreviations ...................................................................................................... 106
9. Annexes ............................................................................................................... 107
10. Scientific Output ................................................................................................. 120
11. Curriculum vitae ................................................................................................. 121
12. Bibliography ........................................................................................................ 123
13. Acknowledgments .............................................................................................. 133
Short Summary
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1. Short Summary
Comparative dissolution testing is extensively used as a tool to evaluate equivalency
between oral solid dosage forms. However the current official procedure to compare
dissolution profiles, the f2 similarity factor, lacks solid statistical foundation and the
statistical uncertainty is unknown. Additionally the limits to declare in vitro similarity with
the current methodology (f2 ≥ 50) are arbitrary and not bound to any biopharmaceutical
property; therefore it cannot be considered as a good predictor of the in vivo
performance of formulations, especially in the case of extended release formulations.
The aim of this work was to design, develop and explore two new statistical tests for
comparing dissolution profiles. These tests have more statistical foundations than the
current methodologies and exhibit the flexibility to be customized for a specific
formulation. One test, the tolerated difference test (TDT) can be tailored to detect
differences in the release profiles of extended release formulations that represent a lack
of bioequivalence (or a significant difference in the performance in a combined
dissolution permeability system).
Customization of the dissolution profile comparison tests for ER formulations was
possible by using the TDT without sacrificing its statistical properties (known and
acceptable statistical uncertainty).
In summary, a new approach to design and perform dissolution profile comparisons
under typical principles of statistical experimental design is described. Four
demonstrative examples of comparisons of ER formulations are presented.
1
Kurzzusammenfassung
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2. Kurzzusammenfassung
Vergleichende Untersuchungen des Auflösungsverhaltens werden häufig zur
Bestimmung der Äquivalenz von oralen, festen Arzneiformen eingesetzt. Die derzeitige
offizielle Prüfung, der f2-Test, verwendet hierfür den sogenannten similarity factor f2,
dem jedoch eine solide statistische Grundlage fehlt und dessen statistische
Unsicherheit nicht bekannt ist. Darüber hinaus sind bei diesem Test die Grenzen für die
in-vitro-Ähnlichkeit (f2 < 50) willkürlich gewählt und nicht an biopharmazeutische
Eigenschaften gebunden. Daher kann diese Methode nicht als eine gute Vorhersage für
das in-vivo-Verhalten der untersuchten Arzneiformen angesehen werden. Dies gilt
insbesondere für Arzneiformen mit verlängerter Wirkstofffreigabe.
Das Ziel dieser Arbeit war die Entwicklung zweier neuer statistischer Tests zum
Vergleich von Auflösungsprofilen. Im Vergleich zu momentan angewendeten Methoden
besitzen diese beiden neuen Tests eine fundierte statistische Basis und verfügen über
die Flexibilität, für spezifische Formulierungen angepasst werden zu können. Einer
dieser Tests, der Tolerated Difference Test (TDT), kann so angepasst werden, dass
solche Unterschiede zwischen den Dissolutionsprofilen von verlängert freisetzenden
Formulierungen identifiziert werden, die auf mangelnde Bioäquivalenz hinweisen.
Im Rahmen der vorliegenden Arbeit wird ein neuer Ansatz zur Entwicklung und
Durchführung von Vergleichstests für Dissolutionsprofile beschrieben, die auf Prinzipien
des statistischen experimentellen Designs basieren. Die Anwendungsmöglichkeiten
werden anhand von vier Beispielen für verlängert freisetzende Formulierungen
dargestellt.
2
Introduction
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3. Introduction
After a drug formulation proves to be safe and effective in human trials, it is crucial to
demonstrate that later formulations, following changes during drug development, post-
approval stages, or production by generic manufacturers, possess the same efficacy
and safety profile of the original formulation [1-3]. Because human experiments to prove
efficacy and safety are highly costly, time consuming and in some cases ethically
questionable, the pharmaceutical industry is constantly searching for effective
surrogates for judging therapeutic equivalence of pharmaceutically equivalent drug
products. One widely accepted and official procedure to ensure efficacy and safety of
new formulations is the assessment of Bioequivalence (BE), in which an absence of
significant differences in the rate and extent to which the active ingredient become
available at the site of drug action must be demonstrated [4-9]. However, under certain
conditions in vitro testing has been accepted as a sufficiently reliable surrogate for an in
vivo BE study [10, 11]. When in vitro testing is accepted as a surrogate, the therapeutic
equivalence of drug formulations is assured by comparison of their dissolution profiles.
In this chapter, the theory of in vitro equivalence testing and in vivo BE is reviewed, as
well as the conditions and procedures by which in vitro testing is considered as a
reliable surrogate for an in vivo BE.
3
Introduction
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3.1. Bioequivalence and Biowaiving
Testing the bioequivalence between a product, called the Test formulation, and a
suitable comparator, the Reference formulation, in a pharmacokinetic (PK) study with a
limited number of subjects is one way of demonstrating equivalence without having to
perform a clinical trial involving many patients [8].
Two products are considered bioequivalent when the rate and extent of absorption of
the Test formulation do not show a significant difference from the rate and extent of
absorption of the Reference formulation when administered at the same molar dose of
the active pharmaceutical ingredient (API) under similar experimental conditions [12].
As a prerequisite both formulations must have the same pharmaceutical form and the
same qualitative and quantitative composition in active substances.
In bioequivalence studies, plasma concentrations obtained after administration of the
Test formulation are contrasted to those of a Reference formulation. Pharmacokinetics
(PK) parameters as maximum plasma concentration (Cmax) and Area Under the Curve
(AUC) from both formulation are compared through 90% confidence intervals (90% CI )
around the ratio of the estimated geometric means between the contrasted formulation.
Acceptance criteria used by most regulators are that the 90% CI of the Test/Reference
geometric mean ratios for AUC and Cmax should fall within 80-125% limits [4, 6-8, 12].
Most regulatory entities recommend that a BE study enroll at least 12 healthy volunteers
to ensure reliable estimates but some jurisdictions suggest 18 or 24. Differences among
guidelines to establish BE vary from one jurisdiction to another not only in the
recommended number of subjects but also in: 1) use of volunteers or patients, 2)
4
Introduction
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administration of food, 3) PK parameters to be compared, 4) measurement of parent
drug or metabolite, and 5) strength to be investigated [12, 13].
When the excipients of a formulation do not affect the absorption of the API, the API is
not a prodrug, does not have a narrow therapeutic index and is not intended to be
absorbed in the oral cavity, in vitro testing has been accepted as a sufficiently reliable
surrogate for an in vivo BE study [10, 11]. The regulatory acceptance of in vitro testing
as a reliable surrogate for an in vivo BE is referred as “biowaiver” [10, 11].
Requirements for granting a biowaiver of an in vivo BE study depend on the class of
drug, type of formulation, the type of post-approval change and the information
available. Drugs are normally classified according to their solubility and intestinal
permeability. In 1995, Amidon et. al. [14] introduced the biopharmaceutical classification
system (BCS) which classifies drugs as follows.
 Class I : high solubility – high permeability
 Class II : low solubility – high permeability
 Class III : high solubility – low permeability
 Class IV: low solubility – low permeability
For immediate release (IR) solid oral dosage forms of BCS class I drugs demonstration
of ≥ 85% dissolution in one or several media in 15 min is normally enough for conceding
a biowaiving of BE studies in the case of post-approval changes of minimal impact [11].
Debate is still open as to whether biowaiving could be accepted for IR formulations of
other BCS class drugs and guidelines differ in this point [10, 13]. Likewise, biowaiving of
ER is not granted in all jurisdictions and in those where it is accepted a IVIVC model
must be employed to justify the decision.
5
Introduction
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3.2. Drug Dissolution Tests
In vitro dissolution studies are commonly used in drug manufacturing to monitor process
control, minor formulation changes and manufacturing site changes [15]. Originally, the
dissolution test was used primarily as a formulation development tool and as a quality
control test for determining that the API would dissolve in vivo [16]. Currently,
dissolution is the only test that indicates if a dosage form will dissolve in the patient and
is accepted as an indicator of the ability of the dosage form to release the API and
enable it to become available at its site of action [17]. For that reason dissolution tests,
at first exclusively used as quality control test, have been emerging as a surrogate
equivalence tests for certain categories of orally administered pharmaceutical products
[8].
The most commonly employed dissolution test methods are the basket method (USP
Apparatus 1) and the paddle method (USP Apparatus 2) [18]. These methods are
simple, robust, well standardized regarding volumes and agitation, and used worldwide
[19]. Recently a flow-through cell system (USP apparatus 4) that aims to mimic sink
conditions has also received much attention because of its flexibility for research and
development. This dissolution technique has been proven to be reproducible and
robust, which is an important characteristic for dissolution testing [20].
Dissolution testing should be carried out under physiological conditions which normally
includes temperature of 37 ± 0.5°C and aqueous medium with pH range 1.2 to 6.8. The
inclusion of biorelevant media is also possible and advances in developing more
biorelevant dissolution methodologies have been continuously made [21-27], and have
been identified as a major priority [28, 29].
6
Introduction
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3.3. Modeling Drug Dissolution
The quantitative analysis of the data obtained from dissolution tests is deeper when
mathematical formulas that express the dissolution results as a function of some
characteristics of the dosage forms are used. In some cases, these mathematic models
are derived from the theoretical analysis of the release process. In most of the cases a
mechanistic expression is not available and some empirical equations have proven to
be suitable [30]. Drug dissolution from solid dosage forms has been described by kinetic
models in which the accumulated mass dissolved is a function of time.
Dissolution data is most commonly (but not always) described by the following
mathematical dissolution models:
- Zero order kinetics [31]:
equation 1
Where is the accumulated mass is dissolved at time t and is the mass dissolved
at infinite time, and k0 is the zero order release constant. According to this model the
drug is released is at a constant rate.
- Firs order kinetics [32]:
equation 2
In which the rate of drug release is proportional to the remaining (not released) quantity
of drug in the formulation. And k is the proportional constant.
7
Introduction
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- Higuchi Model [33] :
equation 3
Where b is the Higuchi dissolution constant. This expression attempts to describe a
nonlinear release kinetics with higher release rates at the beginning of the dissolution
test.
- Korsmeyer model [34]:
equation 4
This model is a semi-empirical expression in which k is a constant incorporating
structural and geometric characteristics of the drug dosage form, n is the release
exponent, indicative of the drug release mechanism, and the release rate is proportional
to the remaining drug in the formulation. Values of indicate Fickian release,
values of indicate an anomalous (non-Fickian or coupled
diffusion/relaxation) drug release, and values of indicate a case II (relaxation-
erosion controlled) drug release.
- Peppas model (equation 5) [35]
equation 5
In which is the diffusional constant, is the relaxation constant and is the
diffusional exponent, the latter depends on the geometrical shape of the releasing
device through its aspect.
8
Introduction
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- Weibull model [36, 37] [38]:
( )
( )
equation 6
Where , and β is a shape parameter that can also indicate the release mechanism
[38].
- Hill model [39]:
equation 7
In which t50 is the time at which 50% of the drug is released from the formulation and
is a shape parameter. Another mechanistic expressions such as Noyes-Whitney,
Nernst-Brunner, and Hixson-Crowell has been widely used to describe drug dissolution
of solids but have very limited applicability in describing release from complete dosage
forms [40].
3.4. Dissolution Profile Comparisons
When the biowaiving of drug formulations is possible, the therapeutic equivalence of
drug formulations is assured by in vitro comparison of dissolution profiles. The term
similarity has been employed to describe the lack of difference between dissolution
profiles from two different sources (formulations) and it is normally established by using
a similarity factor [2, 19, 41, 42] presented in 1996 by Moore and Flanner [41] (equation
8) to compare dissolution curves. They introduced this method as especially
recommended for use in stability studies and optimization during product development
and scale-up. Since f2 has been proposed, several publications have explored the
advantages and disadvantages of f2 [30, 43, 44] and some modifications, such as the
9
Introduction
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constructions of confidence intervals have been proposed [45, 46]. Presently, f2 is
employed and recommended by regulatory authorities for scale-up and post-approval
changes; however, the level of confidence of the method is uncertain and several
publications have shown it to have low statistical power [30, 43].
{[ ∑ ( ) ] } equation 8
In equation 8, Rt is the mean of the dissolved drug from the Reference batch at time t, T t
is the mean of the drug dissolved from the Test batch at time t, n in the number of time
points and wt is a weight factor that can be used to enhance the influence of particular
time points. If the calculation yields f2 ≥ 50, similarity of R and T is declared.
FDA guidelines [19] recommend testing 12 tablets of each batch. Theoretically, if the
difference in drug dissolution between R and T is exactly 10% at every time point, the
value of f2 is 50; if the differences are >10% (at every time point), f2 becomes smaller
than 50, and if the differences are smaller (<10%) f2 becomes larger than 50. However,
values of f2 above 50 can be obtained with differences greater than 10% at some
particular time points if the differences at the other points are small enough to
compensate for the larger differences; thus, the basis for choosing a value of 50 as the
rejection criterion is questionable. To alleviate this problem, Moore and Flanner included
the weight factor wt in the expression; however, there is no clarity on how to employ the
weight factor and it can highly favor (intentionally or unintentionally) either similarity or
non-similarity. The FDA guidelines [19] also mention the weight factor but allow the
researcher to decide whether to use it or not. Another disadvantage of using f2 is that
the arithmetic mean is very susceptible to extreme values and this may result in large
differences between individual tablets being ignored.
10
Introduction
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Apart from f2, several methodologies for comparing dissolution profiles have been
described [30, 47]:
Adaptations of single value comparisons of level B parameters (area under curve, mean
dissolution time, time to reach 85% of dissolution etc.) have not been well accepted
because it is often not possible to properly include the information for every time point in
such comparisons.
Multivariate analysis [48, 49], requires assumptions that are difficult to fulfill. Moreover, it
is questionable whether comparison of dissolution profiles should be consider as
multivariate problem, because the same variable is measured repeatedly over time.
Model-dependent methods have also been used, but these rely highly on fitting to a
specific dissolution model, and in some cases such a model is not available. Moreover,
model-dependent methods are still bound to multivariate distances with the
aforementioned problems [50]. Factors as f2 which are easy to be implemented have
been widely employed, but normally lack scientific justification [30] or statistical support.
3.5. In vitro-in Vivo Correlation Models
ER formulation are dosage forms usually designed to achieve safer and more constant
in vivo concentrations of the administered drug or to decrease the administration
frequency to improve compliance in the patient [29].
Currently, biowaiver of ER drug formulations can only be granted when an IVIVC model
is available. These IVIVC models are defined as a predictive mathematical model
describing the relationship between an in vitro property of a dosage form and a relevant
in vivo response [2]. The term correlation in IVIVC is due to the fact that in some cases
11
Introduction
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the strength of the relationship between parameters derived from the in vitro and in vivo
studies are quantified by the Pearson correlation [15].
An acceptable IVIVC requires that the in vitro dissolution and in vivo release or
dissolution behavior of a dosage form should be either similar or have a scalable
relationship to each other. IVIVC could only be established when the factor controlling
the appearance of the drug in the blood flow is linked with the formulation (slow release)
and not with any physiological limiting factor (for example permeability) [51].
IVIVC have been divided into three groups:
Level A: The entire in vivo time course (normally plasma concentrations) is predicted
from the in vitro data.
Level B: Correlation between a statistical moment of the in vitro dissolution (mean
dissolution time MDT) and a statistical moment of the in vivo plasma levels (mean
residence time).
Level C: Single point relationship between a dissolution parameter and a PK parameter.
Traditionally IVIVC models have been performed through the use of convolution and
deconvolution processes to find mathematical functions able to connect the in vitro and
in vivo mathematical functions. However, these methodologies are not based on
biopharmaceutical principles or mechanistic expressions that allow a more detailed
description of the in vivo situation. Recently some mechanistic IVIVC models have been
introduced to overcome this weakness [52, 53].
12
Introduction
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3.6. Permeability and d/p-systems
Apart from the release kinetics the bioavailability of an orally administered drug is also
determined by the intestinal permeability. However, these two properties are normally
evaluated separately in vitro which restricts the possibility of studying the effects of drug
formulations on absorption. Therefore, a tool to simultaneously study in vitro dissolution
and permeability can be of great utility during the drug formulation development process
to study the effect of drug release and excipients on oral absorption in an easy and
inexpensive way, which ultimately can lead to an optimization of the drug formulation.
Several approaches have been described to study dissolution and absorption
simultaneously in the same experimental apparatus [54-59], however, the only set-up
that allows the evaluation of complete solid oral dosage forms in an open system using
dynamic flow conditions is the one developed by Motz et al [60]. Moreover, this
combined dissolution and permeation system (d/p system) has been improved to allow
continuous measurements of the drug concentrations in the different compartments of
the device [61] and continuous monitoring of the Caco-2 cells monolayer integrity by
measuring the transepithelial electrical resistance (TEER) [62]. The d/p-system has also
been recently adjusted to perform experiments with artificial membranes [63].
13
Introduction
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3.7. Aim of this Work
The current official procedure to compare dissolution profiles, the f2 similarity factor,
lacks solid statistical foundation. The level of statistical uncertainty when this
methodology is employed is therefore unknown. Additionally, the limits to declare in vitro
similarity with the current methodology (f2 ) are arbitrary, and as they are not
bound to any biopharmaceutical property, they cannot be considered as a good
predictor of the in vivo performance of formulations, especially when ER formulations
are considered. On the other hand, two major characteristics are needed in statistical
tests for performing dissolution profile comparison: high statistical power and flexibility
to perform in a variety of scenarios. Because these two properties are not likely to be
fulfilled by the same test, two separate tests, each with one of the aforementioned
properties, were designed in this thesis as a new methodology to compare dissolution
profiles. The intention in this respect was to design, develop and explore new
dissolution profile comparison tests (DPC-tests) with more statistical basis that the
current methodologies (f2 similarity factor), and to link the limits of rejection of these
DPC-tests with significant differences in relevant biopharmaceutical properties, so as to
achieve a greater predictive power of the in vivo performance of formulations. In more
detail, the major aims of this thesis were:
 To design, present and explore the statistical robustness and statistical power of
two new tests based on nonparametric permutation test theory. The first test,
called permutation test (PT), was designed to detect small differences in
dissolution profiles and very stringent to confer similarity. The second test, called
14
Introduction
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tolerated difference test (TDT), designed with the required flexibility to be
customized according to the requirements of any particular case.
 To develop drug-specific DPC-test for three ER formulations (metformin,
diltiazem and pramipexole) using IVIVC models, computer simulated BE trials
and permutation tests. It was intended that these customized tests should be
able to detect, at a known level of certainty, differences in release profiles
between ER formulations that represent a lack of BE.
 To investigate the effect of DPC-test conditions, BE-trial conditions, and
drug/formulation properties in the determination of biorelevant limits of the DPC-
test.
 To apply the concept of DPC-test customization in a combined dissolution
permeability system (d/p-system) in order to identify as in vitro similar, only
formulations that would not differ significantly in the permeated amount achieved
in the permeability module of that system.
15
Two New, Nonparametric Test for Statistical Comparison of Dissolution Profiles
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4. Two New, Nonparametric Test for Statistical Comparison of

Dissolution Profiles
Parts of this Chapter were published in:
Gomez-Mantilla JD, Casabo VG, Schaefer UF, Lehr CM. Permutation Test (PT) and Tolerated
Difference Test (TDT): Two new, robust and powerful nonparametric tests for statistical
comparison of dissolution profiles. Int J Pharm. 2013;441(1-2):458-67.
The author of the thesis made the following contribution to the publication:
 Design and construction of the DCP-tests
 Design, Performance and interpretation of simulations.
 Writing the Manuscript.
16
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4.1. Abstract
The most popular way of comparing oral solid forms of drug formulations from different
batches or manufacturers is through dissolution profile comparison. Usually, a similarity
factor known as (f2) is employed; However, the level of confidence associated with this
method is uncertain and its statistical power is low. In addition, f2 lacks the flexibility
needed to perform in special scenarios. In this study two new statistical DPC-tests
based on nonparametrical permutation test theory are described, the permutation test
(PT), which is very restrictive to confer similarity, and the tolerated difference test (TDT),
which has flexible restrictedness to confer similarity, are described and compared to f2.
The statistical power and robustness of the tests were analyzed by simulation using the
Higuchi, Korsmeyer, Peppas and Weibull dissolution models. Several batches of oral
solid forms were simulated while varying the velocity of dissolution ( from 30 mins to
300 mins to dissolve 85% of the total content) and the variability within each batch (CV
2% to 30%). For levels of variability below 10% the new tests exhibited better statistical
power than f2 and equal or better robustness than f2. TDT can also be modified to
distinguish different levels of similarity and can be employed to obtain customized
comparisons for specific drugs. In conclusion, two new methods, more versatile and
with a stronger statistical basis than f2, are described and proposed as viable
alternatives to that method. Additionally, an optimized time sampling strategy and an
experimental design-driven strategy for performing dissolution profile comparisons are
described.
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4.2. Introduction
Comparing time profiles for dissolution data or for any other type of data is a complex
statistical challenge. The highly correlated nature of this type of data, which exists in
spite of its mostly unknown mechanisms, the many types of curves observed in
dissolution profiles, the high variability combined with the finite nature of the variable
(≤100%), and the fact that two curves may cross producing both positive and negative
differences, make it difficult to determine whether two curves should be regarded as
similar or different, and therefore represent a major barrier to an adequate solution to
this problem [30, 41, 64]. When a variable is measured over time and compared under
two or more conditions, a simple and commonly used technique is to compare the value
of the variable at one or two particular time points and to test hypotheses about
differences in the variable between the different conditions at these precise time points
[65]. Although this approach is adequate in a broad variety of situations, it fails, when
the major interest lies in the kinetic of the process, as when drug dissolution profiles are
compared.
Drug dissolution assays of oral solid dosage forms are designed to predict the
performance of these formulations in the gastro-intestinal tract (GIT) and ultimately
provide information about the bioavailability of oral formulations. The information
obtained at each time point can be crucial because the absorption of drugs varies
across the GIT due to the different membrane properties of the mucosal cells, the local
microclimate and, the presence or absence of transporters, enzymes and other
substances ([66], [67], [68] and [69]). Because of this, comparisons using data obtained
at only one or two time points are insufficient. Comparisons of areas under the curve
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are also inadequate because two curves can have very similar areas under the curve
but present important differences at single time points, especially if the two profiles
cross [41]. To date, there is no satisfactory statistical tool, either for dissolution profiles
or for other types of data that completely solves this particular problem.
In 1996, Moore and Flanner [41] described the use of an expression that they called f2
(equation 8) to compare dissolution curves. Since f2 has been proposed, several
publications have explored the advantages and disadvantages of f2 [30, 43, 44] and
some modifications, such as the constructions of confidence intervals have been
proposed [45, 46]. Presently , f2 is employed and recommended by regulatory
authorities for scale-up and post-approval changes; in addition it can be used to waive
clinical bioequivalence studies (at least under certain conditions) for immediate release
and modified release solid formulations [19],[2, 4]. However, the level of confidence of
the method is uncertain and several publications have shown it to have low statistical
power [30], [43].
Apart from f2, several methodologies for comparing dissolution profiles have been
described [30] [47] like adaptations of single value comparisons of level B parameters,
(area under curve, mean dissolution time, time to reach 85% of dissolution etc.), or
multivariate analysis [48, 49], and model-dependent methods. However these
methodologies have not been accepted by the industry because of its statistical and
conceptual limitations (section 3.4). Factors as f2 which are easy to implement has
been widely employed, but normally lack scientific justification [30] or statistical support.
Most available statistical tests are designed to detect differences, rather than to prove
similarities, and a lack of difference does not necessarily imply similarity. However,
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demonstration of a lack of difference with quantified and adequate type-I and type-II
errors would provide a more solid statistically method for detecting similarities than a
method based on subjective limits.
The power of a statistical test is the probability that the test will reject the null hypothesis
(in this case similarity) when the null hypothesis is false (i.e. the probability of not
committing a type-II error, or making a false negative decision). With the help of
dissolution models (equations 1-7), scenarios when the null hypothesis is false can be
generated (differences in the value of one or more parameters), and the power of the
tests can be evaluated. The more powerful a test is, the smaller difference it can detect
in the value of model parameters.
A more powerful DPC-test (able to detect small differences between two profiles) would
be very valuable for comparing the dissolution profiles of formulations containing drugs
with very narrow therapeutic windows and/or drugs classed as II, III and IV in the
Biopharmaceutical Classification for which in vivo bioequivalence can require a more
strict, almost identical in vitro similarity [14]. It can also be postulated that for a
transporter substrate (active transport or efflux) of a transporter present in enterocytes,
the effective concentration in the intestinal lumen may play a decisive role in
determining the bioavailability of the compound. Very powerful statistical tests are
needed, indeed, to detect small differences in dissolution profiles to assure similarity of
two products from different manufacturers or from the same manufacturer after a major
or minor change in production technology. In a large number of cases, the bioavailability
of two different drug products with the same active molecule will be very similar if their
dissolution profiles, evaluated under the relevant conditions [21], are very similar.
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On the other hand, for some compounds, large differences in dissolution profiles are
necessary to produce significant differences in bioavailability, and a test less strict than
f2 is also needed[70] [44]. In general, a flexible DPC-test that offers variable power
according to specific needs, but still retains adequate levels of robustness and statistical
uncertainty, is highly desirable.
Aware of the expectations that dissolution and drug release will play an even wider role
in regulating quality generic drug products in the future [71], two major characteristic are
needed in statistical tests for dissolution profile comparison: High statistical power and
flexibility, as these two properties are not likely to be fulfilled by the same test, two
separate tests, each with one of the mentioned properties may be an adequate solution.
In this study two new statistical DPC-tests based on nonparametric permutation test
theory are presented, and their ability to satisfy the above mentioned requirements
(more restrictiveness and more flexibility) is assessed. The first, called permutation test
(PT), is capable of detecting small differences in dissolution profiles and is very exigent
to confer similarity. The second one, called tolerated difference test (TDT), the level of
exigency to confer similarity can be modified to detect large or small differences
according to the requirements of any particular case. Both tests were explored in terms
of statistical robustness and power and were compared to f2 and bootstrap 95%
Confidence Intervals of f2 (C.I.) [45, 46].
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4.3. Methods
4.3.1. Dissolution Models
Dissolution data were simulated following five different mathematical dissolution
models. i.e., the Higuchi model (equation 3) [33], the Korsmeyer model (equation 4)
[34], the Peppas model (equation 5) [35] , the Weibull model (equation 6) [36, 37] [38]
and the Hill model equation 7 [39].
4.3.2. Data Simulation
4.3.2.1. Reference and Test Formulations
Reference formulations were modeled as follows:
Higuchi Model:
Korsmeyer Model: ;
Peppas Model: ; ;
Weibull Model: ; ;
Hilll Model: ; ;
4.3.2.2. Intrinsic and Residual Variability
Test formulations were modeled by varying the models parameters around those of the
Reference formulations to obtain a wide range of dissolution profiles (85% of labeled
drug dissolved in 30 to 300 min). For each individual tablet, intrinsic (parameters of the
model) and residual variability (experimental error), variability was included. Intrinsic
variability was included for all parameters.
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equation 9
Where is the parameter for the i-th tablet, is the parameter of the batch and ƞ is the
intra-batch variability with mean value zero and variance ( ( ))
The residual variability (experimental) was described by:
equation 10
Where is the simulated dissolved amount (%) of drug from the i-th tablet at the j-th
sample time, is the predicted dissolved amount (%) of drug from tablet i at the j-th
sample time and is the residual (experimental) variability with mean value zero and
variance ( ( )). In summary, for each single tablet one or more individual
parameter are calculated according to equation 9, the number of parameters depends
on the model and, ranges from one parameter in the Higuchi model to three parameters
in the Peppas Model. Finally, a predicted value is calculated for each time point
according to the models employed (equations 3-7, section 3.3) and the residual
variability is also incorporated according to equation 10.
According to equation 9equation 10), the variability within the batches is due to the
values of and employed in each simulation. For every model (equation 3equation
7) several combinations of and at different T85 were studied to address the effects
of these values on variability. For each combination of T85, and 10.000 batches
were simulated and the CV at each time point for every batch was analyzed. The 95%
percentile of all the measured CV’s was recorded as CV95 as a measure of global
variability.
As can be observed in Figure 4.1, no significant differences in CV95 were found for
batches with different T85 for data following the Higuchi model at different T85, the same
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effect was observed for all the models; however, visualization is not as simple because
more than one is present in each case.
Figure 4.1. Intrinsic and residual variability of the simulated batches (Higuchi Model).
Setting up of intrinsic and residual variability in the simulated tablets batches according to equation 9-
10) Contour Plot of cv for different combinations of and . The result were almost identical for
batches of tablets with different t85 under Higuchi model.
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4.3.2.3. Sampling Time Points
For every condition, a set of values for 12 tablets was generated. Time points were
established according to the following scheme:
t85 ≤ 40 minutes sampling every 5 minutes
40 < t85 < 60 minutes sampling every 10 minutes
60 ≤ t85 < 90 minutes sampling every 15 minutes
90 ≤ t85 < 150 minutes sampling every 20 minutes
t85 ≥ 150 minutes sampling every 30 minutes
where t85 = time at which 85% of labeled drug is dissolved. According to the current
guidelines [19] only one time point with average dissolved drug higher to 85% is
considered.
4.3.3. Dissolution Profile Comparison Tests
In a typical dissolution profile comparison (Reference vs. Test) two Matrices (Reference
and Test) of data points; R (m × n) and T(m × n) are evaluated, being m the number of
tablets (normally 12) and n the number of time points sampled. The data for every tablet
are expressed as a vector of length n which length is defined by the number of time
points sampled in the dissolution profile.
4.3.3.1. f2 Similarity Factor
f2 similarity factor was calculated according to equation 8. If the calculation yielded f2 ≥
50 similarity of R and T was declared.
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4.3.3.2. f2 Bootstrap Confidence Interval (f2 CI)
Bootstrap 95% Confidence Intervals (CI) of f2 were calculated similar to ones described
in the literature [45] [46]. Initial simulations were performed to establish the number of
repetitions to be used. As shown in Figure 4.2, 5000 repetitions produced acceptable
estimations and allowable computation time.
Figure 4.2. Simulations for determining the number of bootstrap repetitions to employ. The
variance of the estimator is reduced increasing the number of repetitions. For a very large number of
repetitions the bootstrapping estimates will converge to one value of CI-lower limit. It was observed
that after 5000 repetitions, any estimation is not farther than 0.5 units from the converged value at
large repetitions (200.000). To evaluate the impact of this difference the number of rejections for CI-
lower limit < 50 and for CI-lower limit < 49 were recorded in all the experiments and compared. There
was no significant change (less than 1%) in robustness or power by this modification.
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4.3.4. Two New Nonparametric Tests for Statistical Comparison of
Dissolution Profiles
4.3.4.1. Permutation Test
Figure 4.3 illustrates the procedure for the permutation test (PT). In this procedure, the
mathematical distance D0, a square difference between means at every time point is
used. This value is stored as the Original Distance or D0. Data from every tablet can be
represented as a vector of length n, a set of 2 × m vectors now representing the data of
the Reference and Test batches. The first subset of m vectors represents the Reference
batch and the last subset of m vectors represents the Test batch. After D 0 is calculated,
the vectors are randomly sampled without replacement. In this way, each vector is
relocated randomly in new Reference or Test subsets, creating two new (m x n)
matrices Ri and Ti. The same distance D between Ri and Ti is calculated and the value
is stored as Di. This cycle is repeated 5000 times and an empirical distribution of the Di
values (5000 values of Di) is built. According to a predetermined type I error (typically,
alpha = 0.05), a rejection value that is greater than the 1-alpha percent of all Di values
in this empirical distribution is calculated. If the profiles are similar, D0 is expected to be
bellow this rejection value; D0 above the rejection value indicates lack of similarity
between the two profiles.
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Figure 4.3. Methodology of PT. Illustrative representation of PT methodology, In this case the
number of tablets m = 12, first D0, is calculated between the two profiles. Then each vector is
randomly located in new reference or test subsets creating two new (m × n) matrices Ri and Ti . The
same distance D is calculated between Ri and Ti and the value is stored as Di . This cycle is repeated
5000 times or more (500,000 in this example) and an empirical Distribution of the Di values (all the
500,000 values of Di) is built. According to an established type I error (alpha = 0.05), a rejection
value is indicated in this empirical distribution. A and B show distribution of Di for similar and not
similar profiles respectively, because it is an empirical distribution the shape is similar but not
identical, it can be observed than in not similar profiles D0 is bigger than the rejection value and
therefore similarity hypothesis is rejected.
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4.3.4.2. Tolerated Difference Test
This test is based on a tolerated difference ( ) in dissolution between two tablets at
each time point. Following the concept that there is some difference in percentage of
dissolved drug that can be tolerated, this test attempts to statistically prove whether the
differences between the Reference and Test samples exceed the predetermined
tolerated difference or not.
Having at any time point the dissolved drug for m tablets from the Reference and m
from the Test
Rt1, Rt2, Rt3, . . . , Rtm ; Rti= dissolved drug of i-th tablet from the Reference at time t
Tt1, Tt2, Tt3, . . . , Ttm, ; Tti= dissolved drug of i-th tablet from the Test at time t
differences between all Rti and Tti are evaluated, and the number of events for which this
difference is greater than the established tolerated difference ( ) is counted.
For a single time point, under the null hypothesis the random variable
Dd, the number of events in which difference is greater than , has a discrete distribution
easy to calculate. For several time points the same procedure is followed but the
statistic Dd is expressed as:
∑
equation 11
Where Di = the sum of differences greater than at the i-th time point. In this work,
values of = 5 (TDT-1) and = 10 (TDT-2) were analyzed. The distributions of for m
= 12 and n = (1,2,3, . . . ,12) are shown in Figure 4.4.
For m = 12 or less the exact discrete distribution of can be calculated without
difficulty for values of n ≤ 5. For any increment in the value of n the computational time
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required to calculate the distribution of is m 2 times longer, and not easily shortened
by using parallel computing. In this work we calculated for m =12 the exact distribution
of for values of n = (1, 2, 3, 4 and 5). Distributions for higher values of n were built by
simulation with 100.000.000 repetitions. This value was sufficient to produce a
distribution that differed less than 10e-5% from the exact distribution for the case of n =
5. (Table of rejection values for TDT is in A.1)
Figure 4.4. Discrete distribution of Dd for different values of n for 12 tablets. n represents the
number of time points sampled in the comparison, the figure presents the probability of all the 144/n
values, increasing n produces more leptokurtic shapes and narrower rejection values. For example for
alpha = 0.05, rejection values are 101 for n = 1, 86.25 for n = 4 and 81.0 for n = 10. Proximity of
points must not be confused with continuous distribution.
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4.3.5. Robustness Explorations
Under conditions of similarity, in which the Reference and the Test formulations have
equal parameters values in the dissolution models employed. Pairs of Reference-Test
batches were generated at different levels of variation. Every pair of batches was
compared using the four procedures described (f2, CI, TDT and PT). At every level of
variation 5000 pairs of batches were generated and the percentage of rejections (no
similarity) was evaluated for each method. Ideally, under conditions that satisfy the null
hypothesis (in this case, similarity), a robust statistical test does not increase the level of
rejections at increasing levels of variation. In the best case, the level of rejections
should be constant and very similar to the set type I error of the test (normally 5%) in
order to quantify uncertainty. Variation in the models were generated including intrinsic
and residual variability, the 95% percentile of all the measured CV’s at all-time points
was recorded as CV95 as a measure of global variability. In preliminary experiments,
stable (no difference with increment in repetitions) values of percentage of rejections
were found at 2000 repetitions, internal validation with sets of 2000 from the 5000
repetitions were also made and there was no difference in the results.
4.3.6. Power Explorations
Under conditions of non-similarity (different parameters values in the model employed)
pairs of Reference-Test batches were generated at different levels of variation. As in the
Robustness analysis, each pair of batches was compared using the four procedures
described (f2, CI, TDT and PT). Differences in parameters were designed to produce
values of t85 ranging from 60 to 300 minutes. For Korsmeyer, Peppas and Weibull
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models in which more than one parameter describes the kinetic of the process,
differences in single parameters (keeping the others constants) and bidirectional
differences (varying two parameters simultaneously) were explored. At every condition,
5000 pairs of batches were generated and the percentage of rejections (%detections of
no similarity) was evaluated for each method. More powerful tests are expected to
detect smaller differences in the parameters used. As for the robustness experiments,
stable values of percentage of rejections for both robustness and power were found at
2000 repetitions in preliminary experiments.
4.3.7. Effect of Statistical Independence and Sample Size
It must be considered that equation 11 is completely operative only if all are
independent and identically distributed (iid) which may be not the case in a typical
dissolution profile, because only one determination is allowed for each tablet and for
each time point, this implies that for a typical case of 12 observations at 5 different time
points, 60 individual tablets must be evaluated under independent conditions (iid-
conditions). Although this may seem fatiguing, it could be well worth the effort in order to
reduce or completely avoid in vivo studies. Therefore, all explorations were made under
normal conditions (typical dissolution profile with questionable independence) as well as
under independent conditions (iid-conditions). The options of using only 6 and 3
observations per time point (options requiring 30 or 15 tablets, respectively, for 5 time
points), were also evaluated. To simulate iid-conditions, a new tablet (with the same
parameters and intrinsic and residual variability) was generated to estimate the
dissolved drug at each time point; n × m tablets are needed and each tablet was
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evaluated just one time. Results were obtained under both conditions for all the
comparison tests to evaluate the influence of independence in the comparisons.
Additionally, sample size of n = 6 and n = 3 tablets were generated under iid-conditions
to evaluate the influence of sample size in the comparisons.
4.3.8. Software
All the analyses, simulations and statistical tests were performed using the R software
environment for statistical computing and graphics (version 2.14.2. R Development core
Team 2013).
4.4. Results and Discussion
4.4.1. Statistical Robustness
All presented tests showed good robustness for standard (CV95 ≤ 0.1) conditions. Figure
4.5 shows that the percentage of rejections remains under 0.05 for values of CV 95 ≤ 0.12
and sample size n = 12 tablets for all the tests and models. The robustness of the tests
was always in the same order (from less robust to more robust) i.e., TDT<CI<f2<PT.
4.4.2. Effect of Statistical Independence and Sample Size on
Robustness
Sample size and iid-conditions did not affect significantly the robustness of the tests, in
all cases the rejection levels remained within the desired limits (≤0.05) for values of
CV95 ≤ 0.1 as shown for the Korsmeyer model in Figure 4.6 The summary of the effect
of statistical independence and sample size for all models is display in Table 4.1
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Robustness of f2 in not affected by iid-conditions but evidently reduced with smaller
sample sizes. Robustness of CI is evidently increased under iid-conditions, and
evidently reduced with smaller sample sizes.
Figure 4.5. Robustness comparison of the presented tests under different dissolution
models. In each model, pairs of similar batches were generated (batches with the same parameter
values in equations 2-5) and the percentage of rejections is measured at different levels of variation
(CV95). Dotted line at 5% indicates the ideal percentage of rejections. All of the tests have acceptable
levels of Rejections for values of cv ≤ 0.12. f2, and PT show ideal levels of rejection for values of cv ≤
0.3 in all the models.
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Robustness of TDT is slightly increased under iid-conditions and evidently increased
with smaller sample sizes. Robustness of PT Is not affected by iid-conditions or smaller
sample size except for n ≤ 3 where the total number of possible permutations does not
allow useful comparisons.
For conditions of high variability (CV95 ≥ 0.2) just f2 and PT showed acceptable levels
of rejections. PT was the only test in which the level of rejections remained under 5% for
CV95 ≥ 0.3 in all the models and conditions studied.
Figure 4.6. Effect of iid-conditions and sample size on test robustness (Korsmeyer model).
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Table 4.1. Effect of iid conditions and batch size in Robustness for all the models and tests
Model & Test Effect of iid conditions Effect of smaller sample

Size
Higuchi f2 + --
Higuchi CI ++ --
Higuchi TDT - -
Higuchi PT () ( )*
Korsmeyer f2 () --
Korsmeyer CI ++ --
Korsmeyer TDT + ++
Korsmeyer PT () ( )*
Peppas f2 + --
Peppas CI ++ --
Peppas TDT ++ ++
Peppas PT () ( )*
Weibull f2 () --
Weibull CI ++ --
Weibull TDT () ++
Weibull PT () ( )*
+ : Slight increase in Robustness
++: Evident increase in Robustness
- : Slight decrease in Robustness
--: Evident decrease in Robustness
(): No apparent effect
( ) * For batch size = 3, level of rejections of PT remain at 0.03 at all levels of CV95 in all the models.
For bigger batch sizes there was no effect in Robustness for batch size. See discussion for detailed
explanation.
4.4.3. Statistical Power
Figure 4.7 illustrates how, under the Higuchi model, the level of rejections increases in
all the tests when the difference between bTest and bReference becomes greater, in other
words, when the simulated Reference and Test batches are more different. As shown
here for the Higuchi model with sample size n = 12 and no-iid conditions, PT was the
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most powerful test, followed by TDT, CI and f2. These results were very similar
regardless of the T85 of the Reference used in the simulations (Figure 4.8. A-B).
Analogous results were obtained under the other models for differences in single
parameters (Figure 4.7 B-D). The magnitude of the differences detected for single
parameters under all the models are summarized in Table 4.2.
Again, for conditions of high variability (CV95 ≥ 0.2) PT was the only test in which the
statistical power and robustness are not so severely compromised due to an increase in
variability (Figure 4.8.C).
The capacity of the tests to detect simultaneous differences in more than one parameter
(Power) is shown in figure 4.9, In this Power contour plots, two parameters are varied
simultaneously (X and Y axis) and the combination of differences in these parameters
required by each test to reach a power ≥ 0.8 is represented by a point on the contour
plot. More powerful tests are able to detect smaller combination of differences with a
power ≥ 0.8 (points closer to the origin on the diagram). Again, in these cases, PT was
the most powerful test, detecting the smallest combination of differences (Points closer
to the origin of the contour plots) of the parameters studied, followed by TDT, CI and
finally f2 under all the models employed, highlighting that in the Peppas model, TDT and
CI have very similar statistical power.
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Figure 4.7. Power Comparison of the presented tests. In A (Higuchi Model), Percentage of
rejections (Power) Vs Difference (%) in bTest according to equation 3 bReference was set at 9 (T85 ≈ 90
mins) and bTest varying from 7(T85 ≈ 150 mins) to 11 (T85 ≈ 60 mins). In B (Hill Model), Percentage of
rejections (Power) Vs Difference (%) in t50_test according to equation 4 t50-Reference was set at 1.605 h
and nReference at 1.85 (T85 ≈ 240 mins) and t50-Test varying from 1.605 to 2.3554 h (T85 ≈ 240 mins to
T85 ≈ 360 mins). For Peppas Model (C), Percentage of rejections (Power) Vs Difference (%) in k d-Test ,
according to equation 5, kr-Reference was set at 0.6 and kd-Reference at 4.4 (T85 ≈ 130 mins) and kd-Test
varying from 4.4 to 6.5 (T85 ≈ 130 mins to T85 ≈ 90 mins). For Hill Model (D), Percentage of rejections
(Power) Vs Difference (%) in t50-Test , according to equation 7, t50-Reference was set at 1.605 h and
nReference at 1.85 (T85 ≈ 240 mins) and t50-Test varying from 1.605 to 3.405 h, (T85 ≈ 160 to T85 ≈ 520
mins).
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Figure 4.8. Power comparisson of the presented (Higuchi model). A and B present data from
typical variability conditions (CV95 =0.1) form Reference formulations with different T85. According to
equation 3, in A bReference was seted at 9 (T85 ≈ 90 mins) and bTest varying from 7(T85 ≈ 150 mins) to
11 (T85 ≈ 60 mins). In B bReference was seted at 7 (T85 ≈ 150 mins) and bTest varying from 5 (T85 ≈ 300
mins) to 9 (T85 ≈ 90 mins). In C condtions are equal to B but in high variability conditions (CV95
=0.2).
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Table 4.2. Detectable differences in single parameters with each test. For each paramater of
each model, the minimum detectable difference (in percentage of the parameter) for each test (with
power ≥ 0.8) is presented, the correspondent difference in t85 produced by the difference in the
parameter is also displayed.
Model Parameter f2 CI TDT PT
Higuchi b 16.6% 14.4% 12.22% 6.6%

t85 44% 35% 27.77% 10%
Korsmeyer n 9% 6.75% 6.5% 2.75%
t85 33% 28.32% 27.53% 12.25%
k 16.6% 14.4% 12.22% 6.6%
t85 44% 35% 27.77% 10%
Peppas Kd 40.91% 29.55% 27.27% 15.90%
t85 23.08% 17.36% 16.15% 9.81%
Kr 43.75% 34.37% 36.46% 16.6%
t85 25.47% 21.17% 22.17% 11.54%
Weibull a 39.17% 30% 20.83% 14.17%
t85 35.64% 29.52% 22.3% 16.19%
B 10% 7.67% 6% 3.67%
t85 39.51% 32.55% 26.87% 17.76%
Hilll T50 34.57% 29.90% 20.55% 7.13%
t85 38.33% 32.91% 25% 10%
n 86.5% 81.1% 43.24% 10.8%
t85 33.5% 31.66% 22.08% 5%
Consistent with the results for robustness, the power of the CI and TDT tests under high
variability conditions was rather poor, f2 performance was slightly better but still not
acceptable, and PT showed the best performance in this scenario although its power
was significantly decreased compared to low variability conditions (Figure 4.9).
The demand for a very powerful and robust statistical tool, able to detect small
differences in dissolution profiles can be satisfied with the introduced PT. PT was able
to detect with statistical power ≥0.8 the smallest differences in each model parameters,
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normally more than two times smaller than the differences detected with the same
power with f2 (Table 4.1). For example, in Korsmeyer model, PT was able to detect
differences of 4% in the kinetic constant while f2 is able to detect just differences
greater than 20%, this can represent a 10% detectable difference in T85 with PT
against a 40% detectable difference in T85 with f2.
As we have shown, PT can be used to compare profiles even with high levels of
variation, moreover, PT allows the user to choose the level of statistical uncertainty.
Furthermore, this test is not especially sensitive to the sample size employed in the
comparisons, provided that the sample size is greater than n = 3 (due to the
permutation nature of PT, the sample size of n = 3 highly compromised the power of the
test and should not be employed). A sample size of n = 6 could be used without
significantly altering its good performance compared to a sample size of n = 12. PT
appears ideal for situations in which high similarity should be proven, e.g., in cases of
drugs with a narrow therapeutic window, or with low permeability and/or solubility or
susceptible of intestinal transport or metabolism, and currently there is no test as
powerful and robust that can do so with similar statistical consistency.
4.4.4. Flexibility of TDT
As previously mentioned, in some situations, however, detection of significant but small
differences in dissolution profiles may not be the objective and a more tolerant and
flexible test is needed. This flexibility to vary the tolerated in order to detect larger or
smaller differences in dissolution profiles is precisely one of the designed properties of
the TDT test.
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Figure 4.9. Bidirectional power exploration of the tests. Contour plots of power ≥ 0.8 for the
tests. The combination of differences in two parameters required by each test to reach a power ≥ 0.8
is represented by a point in the contour plot. In A (Korsmayer Model), according to equation 4.
kReference was set at 7.5 and nReference at 0.5 (T85 ≈ 130 mins) and kTest and nTest varying from 7.5 to 8.2
and 0.5 to 0.54 respectively (T85 ≈ 150 mins to T85 ≈ 65 mins). For Peppas Model (B), according to
equation 5, kr-Reference was set at 0.6 and kd-Reference at 4.4 (T85 ≈ 130 mins) and kr-Test kd-Test varying
from 0.6 to 0.8 and 4.4 to 6 respectively (T85 ≈ 130 mins to T85 ≈ 80 mins). For Weibull Model (C),
according equation 6, BReference was set at 0.75 and kd-Reference at 0.03 (T85 ≈ 250 mins) and kd-Test and
BTest varying from 0.03 to 0.045 and 0.75 to 0.82 respectively (T85 ≈ 250 mins to T85 ≈ 120 mins). In
D (Hill Model), according to equation 7. t50-Reference was set at 1.605 h and nReference at 1.85 (T85 ≈ 240
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mins) and t50-Test varying from 1.605 to 3.405 h, and nTest varying from 1.85 to 3 (T85 ≈ 160 to T85 ≈
520 mins).
Flexibility of TDT is shown for TDT in Figure 4.10 in which TDT with and are
compared to f2. It can be appreciated that increasing the value of decreases the
power of the test, in this particular case TDT with was more powerful than f2,
while TDT with was less powerful than f2.
In addition TDT takes into account information on every tablet at every single point and
does not rely on measures of central tendency as do f2, CI and PT, therefore, the
analysis it provides may be more comprehensive than those of the other tests.
4.4.5. Effect of Independence and Sample Size on Power
AS previously stated, the underlying principle of the TDT demands that the data from
every time point of every tablet be independent and identically distributed (iid-
conditions). Effects of iid-conditions were analyzed and compared with no-iid conditions
to determine how necessary iid-conditions are to a proper performance of the test. The
effect of iid was shown to be of no practical importance because, in all the cases and
models studied, the differences in power between iid and no-iid conditions were typically
less or equal to 5% (Figure 4.1 and Table 4.3). In principle, the TDT test will perform
similarly under no-iid conditions or iid-conditions and the former may be preferred for
convenience (a smaller number of tablets is needed).
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Figure 4.10. Flexibility of TDT. Power Comparison of f2 and TDT with two different values of . For
Peppas Model, according to equation 5, kr-Reference was set at 0.6 and kd-Reference at 4.4 (T85 ≈ 130 mins)
and kr-Test kd-Test varying from 0.6 to 0.8 and 4.4 to 6 respectively (T85 ≈ 130 mins to T85 ≈ 80 mins.
Although iid-conditions had a minor effect on the power of the three tests, this did not
alter the relative power of the tests (TDT-1 > f2 > TDT-2) in any of the studied models.
The robustness of TDT for was good and even better for higher values of .
The effect of sample size on the power of the tests is also summarized in table 4.3 for
each test and each dissolution model; in general, smaller sample sizes reduced the
power of PT and TDT and increase the power of CI and f2. According to standard
statistical theory, the power of a test increase with sample size and should not be
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increased by reduction in sample size as happened with f2 and CI in these simulations,
it shows the limitations of the f2 similarity factor.
Figure 4.11. Effect of iid-conditions on DPC-tests Power. Power Comparison of f2 and TDT with
two different values of under iid and no-iid conditions under Weibull model. according to equation 6,
BReference was set at 0.75 and aReference at 0.03 (T85 ≈ 250 mins) and aTest and BTest varying from 0.03 to
0.045 and 0.75 to 0.82 respectively (T85 ≈ 250 mins to T85 ≈ 120 mins)
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Table 4.3. Effect of iid-conditions and sample size in statistical power for all the tests.
Test &
Condition f2 CI TDT PT F2 CI TDT PT
Model iid iid iid iid S.S.S. S.S.S S.S.S. S.S.S.
Higuchi () - - ( )* + ++ -- -**
Korsmeyer - - f + + ++ -- -**
Peppas () - f + f () - -**
Weibull () () () () () + - -**
Hill () () () () () + - -**
iid : Independent identically distributed.
S.S.S. : Smaller Sample Size
+ : Slight increase in Power
+ + : Evident increase in Power
- : Slight decrease in Power
-- : Evident decrease in Power
() : No apparent effect
f : Fluctuating. A different effect (slightly increase or decrease) at different zones of the diagrams
-*For batch size = 3, level of rejections of PT remain at 0.03 at all values of the test parameters, it is
not and effect of batch size in general but of this very small batch size in particular.
4.4.6. Time Sample Strategies
Figure 4.7 B and C show some apparent discontinuities in the power curves of f2, CI
and TDT tests. For example, in Figure 4.7 B for nReference = 0.5, the power of f2, CI and
TDT first increases continuously at increasing values of n Test (Korsmeyer model), but at
nTest = 0.5275 (difference of 5.25%) the power of the three tests is reduced. This
unexpected phenomenon was identified as an artifact due to sampling times. The time
sampling scheme was designed to be as realistic as possible (intervals of 5, 10, 15, 20,
or 30 min. see section 4.3.2.3). According to this rules, solving equation 5 for nTest =
0.52625, t85=100.8161, samples must be collected at 6 time points (20,40,60,80,100
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and 120 min), in contrast, when nTest = 0.5275, t85 is 99.72 and just 5 time points need to
be sampled (20,40,60,80 and 100 min). This reduction in the number of sampling time
points can produce a 50% decrease in power in the f2, CI and TDT tests. To counteract
this effect, an optimized sampling scheme was developed. In optimized sampling, the
number of time points is fixed, and t85 (the smaller between Reference and Test) is
divided into equidistant time points with t85 as the last time point, for example, fixing 6
time points for a t85 = 95 min, the time points are: 15.833, 31.667, 47.5, 63.333, 79.167
and 95 min. Optimized sampling with 6 and 5 points, respectively, was employed in the
analysis (Figure 4.12). In either case of such optimized sampling the discontinuity in
power was no longer present.
No significant difference was found between results obtained using 5 or 6 time points,
confirming that the apparent discontinuity is due to the sampling strategy and not to the
number of time points sampled. These finding suggest that optimized sampling should
be employed as a first option for dissolution profile comparisons.
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Figure 4.12. Effect of time sampling strategy on statistical power (Korsmeyer model). Power
Comparison of f2, CI and TDT with three different sampling schemes at typical variability conditions
(CV95 =0.1). According to equation 3, kReference was set at 7 and nReference at 0.5 (T85 ≈ 150 mins) and
kTest varying from 7.5 to 8.2 and nTest fixed at 0.5.
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4.4.7. Experimental Design Driven Strategy for Performing Optimal
Dissolution Profile Comparisons
Combining the information presented here with basic principles of experimental design,
we propose an experimental design driven strategy for performing optimal dissolution
profile comparisons; this strategy is illustrated in Figure 4.13.
 If the goal is to detect small differences (Table 4.2) in dissolution profiles, PT
must be employed with a sample size greater or equal to n = 6 and a standard
time sampling can be employed.
 If a less strict comparison is needed or if there is no certainty about the degree of
similarity that can be accepted, the following procedure should be followed:
o Preliminary experiments must be conducted to determine the t 85 of the
Reference and Test formulations and to fit the dissolution data to one or
several models, (including models not presented in this work).
o The minimum detectable difference (the difference to be considered as not
similarity), must be determined either, by finding the adequate difference
at t85 or t50 (time at which 50% of the labeled drug is dissolved) or a
combination of both, or ideally, through either an IVIVC model [72]
indicating the differences in dissolution that may lead to differences in
bioavailability, [73] or by fitting the dissolution data for preliminary
experiments to available dissolution models [30], [74-76] and estimating
the difference in parameters acceptable as similar. This step is the most
complicated and the most susceptible to produce under- or over-
estimation of the detectable difference due to personal interpretation.
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o After the acceptable difference is determined and the dissolution model
selected, simulations with TDT must be done to determine the value of
and sample size at which the determined minimal difference in parameters
or t85 or possible combinations is detected with an acceptable statistical
power (power of 0.8 or higher is recommended) always using an
optimized time sampling strategy.
o Preferably, effects of iid-conditions on variability, robustness and power of
TDT should be addressed in simulation or laboratory experiments.
o Finally, the dissolution assays must be performed under the conditions
(iid/no-iid-conditions, and sample size) found and the statistical
comparison must be done using TDT.
In this way the flexibility of TDT is used to customize a comparison test (setting a
specific value) able to detect the differences in dissolution that can produce a
difference in the bioavailability/bioequivalence of the formulations. The procedure
described may seem arduous compared to the current f2 standard, however, it follows
the typical procedure employed in any experimental design aimed at detecting
significant differences with a quantified statistical uncertainty and known type-I and
type-II errors. The procedure involves the following steps: i)preliminary data analysis,
ii)determination of minimum detectable acceptable difference, iii)determination of
sample size according to a desired level of power and robustness and
iv)experimentation and statistical computation.
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Figure 4.13. Experimental Design Driven Strategy to Perform Optimal Dissolution Profile
Comparisons. Diagram flow of the proposed strategy to Perform Optimal Dissolution Profile
Comparisons, Each stage of the presented strategy corresponds to a stage of a typical experimental
design lustrated in the right.
Due to the simulated nature of the data presented here, experimental verification of the
lack of effect for iid-conditions and examples of how to customize TDT with specific
formulations are recommendable. Also, evaluation of additional available expressions
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for modeling drug dissolution [74], including models for controlled released mechanisms
[75, 76] might be an obvious subject for future studies.
4.5. Conclusions
Two new statistical tests, the permutation test (PT) and the tolerated difference test
(TDT), are presented for dissolution profile comparison in which type-I and type-II errors
can be quantified, and have a stronger statistical basis than the current alternatives
(e.g., the f2 similarity factor). The two new tests showed acceptable robustness at
standard conditions of variation (CV95 ≤ 0.1). PT was the most robust and powerful test
in all the conditions studied (even in conditions of high variability CV 95 ≤ 0.2 and reduced
sample sizes). This test is strongly recommended for identifying small differences in
dissolution. For , TDT showed good robustness and very good power in all the
conditions studied.
The impact of iid-conditions in TDT was not particularly large, therefore the more usual
no-iid conditions could be employed (experimental confirmation of this is still pending).
The possibility to modify the value of confers great versatility on TDT and allows it to
be customized for any specific formulation. To make the best use of the two new tests,
a strategy to design and perform a dissolution profile comparison is presented under
typical premises of statistical experimental design. Finally, it was shown that optimized
time sampling should be employed when possible to avoid artificial discontinuities in the
statistical power of the tests, except for PT which is not susceptible to this effect.
52
Tailor-Made DPC-tests for comparing ER Formulations Using IVIVC Models
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5. Tailor-Made DPC-tests for comparing ER Formulations

Using IVIVC Models
Parts of this chapter were submitted for publication in a peer-reviewed journal:
Gomez-Mantilla JD, Casabo VG, Lehr T, Schaefer UF, Lehr CM. Identification of
Nonbioequivalent Extended Release Formulations by Tailor-Made Dissolution Profile
Comparisons Using In Vitro-In Vivo Correlation Models.
The author of the thesis made the following contribution to the publication:
 Writing the Manuscript.
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5.1. Abstract
Current procedures for performing dissolution profile comparisons are restricted to
mathematical distances (such as the f2 similarity factor) in which limits for declaring
similarity or non-similarity are fixed, drug-unspecific and not based on any
biopharmaceutical criteria. This problem and the lack of strong statistical basis, hinder
the application of DPC-tests for evaluating similarity of ER formulations. This study
aimed to develop drug-specific DPC-tests, able to detect differences in release profiles
between ER formulations that represent a lack of BE. Dissolution profiles of Test
formulations were simulated using the Weibull and Hill models. Differential equations
based in vivo-in vitro correlation (IVIVC) models were used to simulate plasma
concentrations. BE trial simulations were employed to find the formulations likely to be
declared bioequivalent and nonbioequivalent (BE-space). Customization of DPC-tests
was made by adjusting the delta of the tolerated difference test (TDT) described in the
previous chapter. This delta value was tailored for three ER formulations (3.6 for
metformin, 5.95 for diltiazem and 3.45 for pramipexole) to detect with a statistical power
≥ 80%, differences in release profiles identified as biorelevant limits
(nonbioequivalence). The Impact of the dissolution profile comparisons conditions, BE-
trial conditions, and drug properties in the determination of biorelevant limits were
investigated. The other described DPC-test, the permutation test (PT), showed excellent
statistical power. All the formulations declared as similar with PT were also
bioequivalent. Similar case-specific studies may support the biowaiving of ER drug
formulations based on customized DPC-tests.
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5.2. Introduction
The pharmaceutical industry is constantly searching for effective surrogates for judging
therapeutic equivalence of pharmaceutically equivalent drug products. One widely
accepted and official procedure to ensure efficacy and safety of new formulations is the
assessment of BE, in which it an absence of significant differences in the rate and
extent to which the active ingredient become available at the site of drug action must be
demonstrated [4-9].
In cases where the excipients of a formulation do not affect the absorption of the active
pharmaceutical ingredient (API), the API is not a prodrug, does not have a narrow
therapeutic index and is not intended to be absorbed in the oral cavity, in vitro testing
has been accepted as a sufficiently reliable surrogate for an in vivo BE study [10, 11].
The regulatory acceptance of in vitro testing as a reliable surrogate for an in vivo BE is
referred as “biowaiver” [10, 11].
Requirements for granting a biowaiver of an in vivo BE study depend on the type of
drug, type of formulation, the type of post-approval change and the information
available. For IR solid oral dosage forms of BCS class I drugs (highly permeable and
soluble), demonstration of ≥ 85% dissolution in one or several media in 15 min is
normally enough for conceding a biowaiving of BE studies in the case of post-approval
changes of minimal impact [11]. Debate is still open as to whether biowaiving could be
accepted for IR formulations of other BCS class drugs and guidelines differ in this point
[10].
The therapeutic equivalence of drug formulations is assured by in vitro comparison of
dissolution profiles. The term similarity has been employed to describe the lack of
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difference between dissolution profiles from two different sources (formulations) and it is
normally established by using the f2 similarity factor [2, 19, 41, 42]. In order to grant a
biowaiver for ER formulations or for higher impact changes [1] in IR formulations,
guidelines normally demand that similarity of profiles is demonstrated through the f2
similarity factor (equation 8). Additionally, in the case of ER, a validated IVIVC model
must be available. However, the limits of rejection of f2 (≤ 50) are not justified by any
mechanistic or biopharmaceutical reasons. As it is an empirical and fixed limit, it is
unlikely to exhibit the specific discriminatory power required in all scenarios in which it is
currently used. Moreover, recent publications have stated that on the one hand, f2 may
classify formulations that are nonbioequivalent as similar [30, 44], while on the other
hand f2 can also be over-discriminative in some cases [32, 70, 77]. Additionally, several
publications have recognized some major statistical and conceptual limitations of f2,
including, uncertain level of confidence, low statistical power, lack of biopharmaceutical
or statistical reasons, lack of flexibility to perform in different scenarios and poor
statistical consistency [30, 43, 44, 77, 78].
This rigidity of f2, being too restrictive in some cases and too liberal in others, ratifies
that dissolution tests are only physical tests until they are linked to in vivo performance
of the formulations tested [28, 79], and further highlights the need to identify dissolution
limits which ensure clinical quality for each particular case [32]. Advances in developing
more biorelevant dissolution methodologies are continuously being made [21-27], and
have been identified as a major priority [28, 29]. However, statistical tools to compare
dissolution profiles have not been improved in the last years, despite the fact that a
more rigorous application of statistics to understand and incorporate variability and
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uncertainty has also been identified as a priority in order to achieve a better integration
of biopharmaceutics and quality for patient benefit. [80].
It was described in the previous chapter a new strategy to perform case-by-case
dissolution profile comparisons [78], including two new statistical tests for comparing
drug dissolution profiles; the PT, a very powerful and strict test to confer similarity, and
the TDT, a flexible test in which the limits of rejection can be varied according to a
desired level of tolerance without affecting its statistical properties. These two tests
have the advantage of a better uncertainty quantification (Type I and Type II errors) and
better statistical consistency of their estimators.
Using validated IVIVC models, plasma concentrations achieved by different
formulations can be simulated from their dissolution profiles [72, 81, 82]. It is further
possible using such models, to find what difference in dissolution, or in other words
which formulations are likely to produce differences in-vivo large enough to be
considered as nonbioequivalent [83]. We can then customize DPC-tests to declare non-
similarity at levels of dissolution differences at which nonbioequivalence is expected.
This study aimed first, to develop drug-specific DPC-tests for three ER formulations
(metformin, diltiazem and pramipexole) using IVIVC models, computer simulated BE
trials and permutation tests. These customized tests should be able to detect, at a
known level of certainty, differences in release profiles between ER formulations that
represent a lack of BE. Secondly, we aimed to investigate the effect of Dissolution
Profile comparisons conditions, BE-trial conditions, and drug/formulation properties in
the determination of biorelevant limits.
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5.3. Methods
5.3.1. General Strategy
The strategy used to identify bioequivalent and nonbioequivalent formulations is
illustrated in Figure 5.1 taking pramipexole as example. In-vitro dissolution profiles
were simulated for several formulations by modifying the Weibull model parameters and
PK profiles of the formulations were generated using IVIVC models. Through BE
simulation studies, nonbioequivalent formulations were detected. Once the BE-spaces
were delimited, TDT a DPC-test, was customized to declare as non-similar, the
formulations that were likely to be nonbioequivalent. The strategy used to investigate
the effect of drug/formulation properties was similar (Figure 5.2). Starting with the
same dissolution profile different PK profiles can be generated by varying the IVIVC
model input parameters. Investigation of the effect of such variation on the BE-space for
the theoretical drug is then possible.
5.3.2. In Vitro-In Vivo Correlation Models (IVIVC models)
Two published differential-equation-based IVIVC models were used for analysis. For
diltiazem and metformin (Figure 5.3.a), a one compartment pharmacokinetic model with
a first order rate elimination was employed for describing plasma concentrations in
which the rate of in-vivo input is connected to the rate of in-vitro dissolution through a
functional dependency that allows inclusion of time scaling, time shifting and absorption
window [52].
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Figure 5.1. General strategy used to build the BE-spaces.
( ) ( ) ( ) equation 12
Where is the time-scaling factor, is the scaling factor, is the dissolution rate
and ( ) accounts for the variability of the in-vivo absorption as the drug moves
along the gastrointestinal tract, including a truncated absorption at time :

( )
( ) ( )
equation 13
The dissolution rate ( ) was described by the Hill function [39] described in equation 7
Of which the differential expression is:
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( ) equation 14
( )
Where ( ) is the fraction (%) of drug released at time , is the time at which 50%
of the drug is released from the formulation and is a shape parameter. Data were
generated to reproduce the data of Gillespie [84] for diltiazem and the data of Balan and
co-workers [85] for metformin.
Figure 5.2. strategy used to investigate the effect of drug/formulation properties) on the
determination of equivalent formulations. Starting with the same dissolution profile different PK
profiles can be generated by varying the IVIVC model input parameters.
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Figure 5.3. Schematic representation of the biopharmaceutic/pharmacokinetic models for

the IVIVC of dialtiazem and metformin (a) and pramipexole (b).
For pramipexole (Figure 5.3.b), a two compartments model with first order absorption
and elimination was used for describing plasma concentrations [53], in which the
dissolution rate was described by the Weibull function described in equation 6 [36-38] of
which differential expression is:
( ) ( ) ( )
equation 15
Where ( ) is the fraction (%) of drug released at time , is the dissolution constant
and is the shape parameter. The relationship between the and
was modeled by where is a scale factor representing
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the increment in the in-vivo dissolution. of ( ) was used in all
simulations.
For the purpose of this study only differential equations-based IVIVC methods were
included, because of the more mechanistic nature of these models [29, 86, 87]. All
parameters employed are listed in Table 5.1.
Table 5.1. Population pharmacokinetic models parameters used in the IVIVC models.
Pramipexole I Diltiazem Metformin
BCS I I III
Kel (h-1) 0.087 (13) 0.138 (10) 0.23 (10)
tlag (h) 0.22 (66.3) 0.57 (10) 0.86 (10)
tcut (h) NA 6.36 (20) 4.77 (20)
Ka (h-1) 5.26 (91.8) NA NA
V1 (L) 351 (14.1) NA NA
V2 (L) 60.9 (10) NA NA
CLD (L/h) 33.2 (10) NA NA
Parameters are listed with the IIV in parenthesis. Kel, elimination constant; tlag, lag time; tcut,
absorption window; Ka, absorption constant; V1, V2, volumes of distribution in the central and
peripheral compartments respectively. CLD, Apparent distribution clearance, NA: Parameter not used
in that model.
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5.3.3. Test and Reference Formulations
Reference formulations were modeled as follows:
Metformin: ; (Hill Model, equation 7)
Diltiazem: ; (Hill Model, equation 7)
Pramipexole: ; (Weibull Model, equation 6)
Test formulations were generated by varying simultaneously the two dissolution model
parameters (t50 and n or and ) from -95% to 200% around those of the Reference
formulation. Variability (CV 10%) was included at all dissolution points to mimic
experimental data.
5.3.4. Simulations and Bioequivalent Studies
Simulations of plasma concentration were conducted in the R software environment
(version 2.14.2. R Development core Team 2013) using the models detailed in the
previous section. Inter individual variability (IIV) was include for each parameter (Table
5.1) to fit the reported experimental variability [52, 53] including an overall CV of area
under the curve (AUC)0-∞ and Cmax of 15%. In total, 1000 BE crossover simulated
studies per scenario were conducted. In each study, 12 healthy volunteers were
generated by Monte Carlo simulations. Each volunteer received an oral dose of the Test
and Reference formulation with a wash-out period between the administrations. AUC0-∞
and maximum plasma concentration (Cmax) were calculated from the generated plasma
concentrations. BE between formulations was determined by calculating 90%
confidence intervals (90%CI) of the ratio between Test and Reference means after log-
transformation of AUC0-∞ and Cmax. The formulations were considered bioequivalent if
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the 90%CI of AUC0-∞ and Cmax ratios were contained within the acceptance interval of
80.00 – 125.00%.
5.3.5. Dissolution Profile Comparisons Tests (DPC-tests)
Dissolution profiles from the Reference and Test formulations were compared using the
f2 similarity factor (Equation 7) and two recently described tests, PT and TDT [78]. As
described in section 4.3.3.
5.3.6. Statistical Power Explorations
Sets of 12 Reference formulation tablets and 12 Test formulations tablets were
generated under conditions of non-similarity, when different model parameters were
employed for the Reference and Test formulations. Each pair of batches was compared
using f2, TDT and PT. At every condition, 5000 sets of Reference-Test were generated
and the percentage of rejections (%detections of no similarity) was evaluated. More
powerful tests are expected to detect smaller differences in the parameters used. In all
cases equidistant sample points were sampled from which the last sample point was the
smaller t85 (time to reach 85% release) of the two formulations.
5.4. Results
5.4.1. Illustrative Example
In Figure 5.1.b, results from pramipexole are outlined as an example. Test-1 is
bioequivalent to the Reference formulation by AUC and Cmax. Formulation Test-2 is
bioequivalent to the Reference formulation by AUC but not by C max and formulation
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Test-3 is not bioequivalent to the Reference formulation by either AUC or C max. The BE-
space delimits the bioequivalent formulations from the nonbioequivalent. TDT was
customized to declare as non-similar, formulations that are likely to be
nonbioequivalent. To investigate the effect of drug/formulation properties the same
dissolution profile (Test-1) was employed. Different PK profiles can be generated by
varying the IVIVC model input parameters. In Figure 5.2, using the same Test
formulation (equal kd and β), two different PK profiles are generated by using different
Ka values. The Cmax BE-space for the theoretical drug with a smaller ka was reduced
(yellow BE-space) and the Test formulation is no longer bioequivalent to the Reference
formulation by Cmax. When the larger ka is used the same Test formulation is
bioequivalent to the Reference formulation (inside the red BE-space).
5.4.2. Bioequivalent and Similarity Spaces
For each drug, Test formulations were compared to the Reference formulation in their
dissolution profiles and plasma levels. Figure 5.4 illustrates the effect of dissolution
parameters on BE and similarity. The X and Y axes show the difference in each one of
the dissolution parameters of the Test formulation compared to the Reference
formulation. When the difference in the two parameters is zero (coordinates 0,0 in the
contour plots) the Test and Reference formulations are the same. Response surfaces
for AUC and Cmax display the Test formulations (combination of dissolution parameters)
with probability ≥ 80% of being nonbioequivalent by AUC or C max respectively. Density
contour plots displaying probability of rejection from 0 to 100% for AUC and Cmax are
shown in the annexes (A.2-A.7). Response surfaces for f2 and PT delimit the
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combination of dissolution parameters at which the probability of declaring non-
similarity, using the corresponding test, is ≥ 80% (Sim-space).
Figure 5.4. BE-Spaces of the three formulations. Contour lines for AUC and Cmax display the Test
formulations (combination of dissolution parameters) with probability ≥ 80% of being declaring
nonbioequivalent by AUC or Cmax respectively. Striped zones display the nonbioequivalent formulations
declared as similar with f2. a) Metformin, b) Diltiazem and c) Pramipexole
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PT was the most powerful test to declare as non-similar, Test formulations with changes
in the dissolution model parameters compared to those of the Reference formulation.
Consequently, formulations declared as similar with PT were always also bioequivalent
and differences in Cmax and AUC from the Test and Reference formulations were less
than 1.5%. For all three drugs, f2 showed less power than PT, and several formulations
declared as similar with f2 were not bioequivalent.
5.4.3. Customization of TDT
Dissolution profiles of the generated formulations were compared using TDT at different
values of . Figure 5.5 shows the different similarity space (Sim-space) of TDT at
increasing values of for the pramipexole formulation. A of 3.45 is the maximum at
which all the formulations declared as similar with this test are also bioequivalent. A of
3.6 for metformin and 5.95 for diltiazem were found following the same procedure (A.8-
A.9). This value represents the average tolerated difference (in %) between two
formulations at any time point to produce bio-equivalent formulations under both criteria,
AUC and Cmax.
5.4.4. Effect of Drug & Formulation
The effect of changes in ka, kel (CL), t-lag, V1, V2 and CLD (model II in Figure 5.3) on the
BE-space of the pramipexole formulation was studied. Changes in V 1, V2 and CLD of
ten-fold had no effect on the BE-space (A.10). Changes in ka of 10-fold had no
apparent effect on the BE-space of AUC or Cmax.
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Reductions in ka of 50 fold or bigger showed an increasing reduction in the BE-space of
Cmax (Figure 5.6.a). Changes in t-lag had a direct impact in the BE-space of both AUC
and Cmax (Figure 5.6.b-c), when t-lag was not considered (t-lag =0) the BE-space was
increased. For a t-lag of 2 hours, the BE-space was reduced to 25% of the original area.
Changes in Kel had no effect on the BE-space of AUC. A slight effect in the BE-space of
Cmax by changes in kel was observed, however, there was no change in the total area of
the BE-space (less than 5%), but a small modification in the shape, regardless if the k el
was increased or decreased. The same effect was observed for metformin and
diltiazem (A.11-A.12). Studied effects are summarized in Table 5.2.
Figure 5.5. Customization of TDT. Sim-Spaces of TDT at different values of . for pramipexole
formulations.
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Table 5.2. Effect of volunteers sample size and drug parameters on the BE-space
Pramipexole Diltiazem Metformin
AUC Cmax AUC Cmax AUC Cmax
Increase sample size (volunteers) ↑ ↑ ↑ ↑ ↑ ↑
Change Kel ↔ ↑↓ ↔ ↑↓ ↔ ↑↓
Increase t-lag ↓ ↓ NA NA NA NA
Reduce ka ↔ ↓ NA
NA NA NA
Change V1 ↔ ↔
NA NA NA NA
Change V2
↔ ↔ NA NA NA NA
Change CLD
↔ ↔ NA NA NA NA
↑: Increase BE space; ↓: Reduce BE Space; ↑↓: Change BE space, some zones are augmented and
some are reduced but no total increase or decrease; ↔ : No apparent effect; NA: Parameter not used
in that model.
and c) effect of tlag on Cmax BE-Space.
5.4.5. Effect of BE trials Conditions and DPC-tests Conditions on BE-
space and Sim-space
BE trials simulations were performed with different numbers of patients to study the
effect of sample size in the BE-space, Figure 5.7.a shows the increment in the BE-
space, in both AUC and Cmax, at increasing numbers of patients. Nevertheless, the
increment in the area from 12 to 18 patients was less than 3%, and the reduction in the
BE-space from 12 to 6 patients was less than 5%.
The Sim-space was sensitive to the number of points used in the dissolution profile
comparison, a reduction in Sim-space (increasing statistical power) at higher number of
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time points was detected (Figure 5.7.b). These effects of number of patients and of time
points were also observed for the metformin and diltiazem formulations (A.13-A.14).
Figure 5.6. Effect of drug/formulation parameters in the determination of BE-Space. a) effect

of ka on BE-Spaces, b) effect of tlag on AUC BE-Space, c) effect of tlag.
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Figure 5.7. Effect of BE trials conditions and DPC-tests conditions on BE-Space and Sim-
Space. a) effect of number of volunteers included in BE-trials on BE-Spaces, b) effect dissolution
sampled time points on Sim-Space.
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5.4.6. BE-space Compared to TDT to MDT and MRT
Mean dissolution time (MDT) and mean residence time (MRT) were calculated for all
the reference formulations and compared to the BE-space. Surface responses of MDT
and MRT are depicted in Figure 5.8 for pramipexole formulations. There is no overlap
between the MDT or MRT response surfaces with the BE-space. Test formulations with
the same MRT or MDT of the Reference formulation could be nonbioequivalent.
Likewise, formulations with very different values of MDT and MRT are included in the
BE-space. Similarly, BE-space did not match with the surface spaces of MDT or MRT
for metformin or diltiazem (A.15-A.16).
5.5. Discussion
In this study, using IVIVC models, we aimed to design case-specific DPC-tests which
were able to identify formulations likely to be bioequivalent in-vivo as in-vitro similar. As
expected, the differences in dissolution necessary to produce nonbioequivalent
formulations vary from one drug-formulation to another, depending on the drug and
formulation. f2 failed to associate the in vitro similarity of two formulations with their
comparative in-vivo performance in the three cases investigated. Moreover, f2 declared
as similar not only formulations that are likely to be bioequivalent in vivo, but also
formulations that are likely to be nonbioequivalent in vivo (Figure 5.4). This observation
is in agreement with one of the criticisms of f2 [30, 32] of lack of flexibility to perform in a
wider range of cases. We propose the need to analyze individually each formulation,
and customize case-specifically, the limits of rejection of DPC-tests based on the type of
formulation and the drug.
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Figure 5.8. Comparison of BE-Spaces with a) MDT and b) MRT.
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TDT is a DPC-test specifically designed to display this desired flexibility, by varying the
δ value, without compromising its statistical properties (known type I and II errors). We
were able to associate TDT limits of rejection with an in-vivo property (BE) of the tested
formulations. Test formulations declared as similar to the Reference formulation using
this customized DPC-test are expected to be also bioequivalent and consequently to
retain the same efficacy and safety profile.
Having a customized DPC-test offers some technological and regulatory advantages;
From the point of view of the manufacturer, once a DPC-test is customized, dissolution
profile similarity can be used as a critical quality attribute (CQA) [88], for routine quality
control, as a tool for post-approval changes, or to assure quality consistence of the
same formulation manufactured in a new facility [88, 89].
From the regulatory point of view, an established DPC-test of an innovator formulation
represents a fast, cheap and reliable protocol with clear acceptance criteria to test new
generic formulations, reducing the costs and time of these submissions and potentially
avoiding unnecessary in-vivo BE studies.
PT was the most restrictive test to confer similarity of dissolution profiles, typically,
formulations declared as similar with this test did not differ in AUC and Cmax more than
1.5% assuming the same patient with no intra-occasion variability. PT should be a more
suitable test for monitoring similarity as a CQA because it would be more sensitive to
detect CQA changes.
Ideally, all the bioequivalent formulations should be declared as similar, however, a total
overlap of the BE-space and the Sim-space was not achieved with the investigated
tests. Nevertheless, according to risk management principles [80, 90], the reduction of
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risk caused to patients by declaring as similar formulations that are not bioequivalent,
must be considered as a higher priority than reducing manufacturer risk of declaring as
non-similar formulations that are bioequivalent. TDT allows maximization (Figure 5.5)
of the overlap between BE-space and Sim-space without compromising the patient risk.
It has been stated that ER formulations can produce flip flop-like kinetics when the
apparent ka < kel (kel/ka ratio > 1), and could lead to miscalculations of the PK
parameters [91], For pramipexole we observed reductions in the Cmax BE-space for
values of ka ≤ 0.4 h-1 (kel/ka ratio = 0.2). Calculating the BE-space for theoretical drugs
with different kel and ka, the reduction in the Cmax BE-space due to reduction in ka was
produced only at kel/ka ratios of 0.2 or higher (A.17). This seems to be the limit at which,
the ka is small enough, in comparison with the kel, to reduce the Cmax in the PK profile.
This aspect should be accounted for in the design of ER formulations, since a slow
enough release can have in practical terms the same effect as reducing the ka of the
drug. We suggest that this flip flop-like phenomenon could be present at kel/ka ratios
smaller than 1 (i.e. 0.2).
The BE-spaces of Cmax from the diltiazem and pramipexole (Figure 5.4 and A.2-A.7 )
formulations were more affected for changes in the Y axis (shape parameter) than for
changes in the X axis (speed parameter). It is appropriate to set DPC-tests sensitive to
changes in the shape parameter because it is related to the release mechanism [28,
87], and small changes in this in-vitro mechanism may have a larger in-vivo impact.
For the metformin formulation, the AUC BE-Space and the Cmax BE-space were affected
by changes in both, the speed and shape parameters, mainly because of the narrow
absorption window (tcut = 4.77h). The δ value of TDT for this formulation was smaller
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compared to the one for Diltiazem (Both using the same IVIVC model) supporting the
concept that DPC-tests to compare formulations of drugs with small absorption windows
must be very restrictive to declare similarity.
The δ value of TDT for the metformin formulation (BCS Class III drug with narrow
absorption window, tcut = 4.77h) was smaller compared to the one for Diltiazem (Both
using the same IVIVC model), supporting the concept that DPC-tests to compare
formulations of drugs with small absorption windows must be very restrictive to declare
similarity. Metformin is a BCS class III drug, experiencing poor bioavailability (40-60%)
[92, 93] and low permeability in-vivo (2.96-4.5 5 cm-7/s ) [92] and in-vitro (1.4 – 5 cm-7/s)
[94], this low permeability was included in the model (as smaller S1) and manifested as
a slower progression (compared to diltiazem a BCS class I) in the plasmatic
concentration versus percentage of dissolved drug relationship (A.18), reflected not only
in a larger tmax (4.7 h vs 3.6 h for diltiazem) but also in the fact that the 50% of C max in
plasma is reached only after the 80% of the drug its released (Compared to 45% for
diltiazem) .
Absorption is recognized as the rate-limiting step for BCS class III drug formulations [95,
96], however, for the metformin formulation, test formulations with faster release
(smaller t50 and larger ) than the Reference formulation, generated plasma profiles with
higher Cmax and AUC, resulting in declaration of nonbioequivalence. Formulations with
slower release (larger t50 and smaller ) were also declared as nonbioequivalent,
generating plasma profiles with smaller Cmax and AUC. These results indicate that in the
case of ER products, dissolution may also play a substantial role in the in-vivo
performance of BCS class III formulations.
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The input function (equation 12) used in the metformin and diltiazem model, limits the
potential processes that can be included in the model. For example, differential
absorption across the GI tract, and concentration-dependent permeability mediated by a
saturable process have been reported for metformin [92], and cannot be completely
included in this particular model. However, these effects are not expected to be of high
relevance in formulations of the same high dose (250 mg), unless the release is
radically slow, in such case, the PK profile would be restricted more for the short
absorption window, which is included in the model, than for the effect of saturable
transport. The customized DPC-test should still be able to declare as similar only
bioequivalent formulations. Notwithstanding, more mechanistic models, as the one used
for pramipexole are preferable and necessary to develop in order to increase the
knowledge of these types of characterizations and formulation comparisons.
MDT and MRT are statistical moments of the dissolution and PK-profile respectively,
which have been proposed as predictors of the in-vivo performance of a formulation [72,
97]. When we compared the surface responses of these moments with the BE-spaces
(Figure 5.8 and A.15-A.16) we found that MDT and MRT are not useful to discriminate
between nonbioequivalent and bioequivalent formulations. Nonbioequivalent
formulations can yield values of MDT and MRT identical or very similar to the MDT and
MRT of the Reference formulation, and bioequivalent and nonbioequivalent formulations
can yield the same values of MDT and MRT. We also observed that formulations with
different MRT yield the same MDT and vice versa (A.19), showing that a correlation of
these two moments is not possible in the investigated cases.
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The observed increment in the BE-spaces when the volunteer sample size is
incremented is in agreement with the fact that due to inter-individual variability, it is
more likely to declare BE of two formulations when more volunteers are included in a
BE trial. Likewise, the Sim-space was reduced when the number of time points sampled
in the dissolutions profiles was increased. These results manifest that conclusions about
BE or similarity can be affected by the specific setup of each particular comparison.
Precise limits must be fixed in order to standardize comparisons and rejections criteria.
Based on these results we propose that a number between 6 and 10 equidistant time
points must be sampled in the dissolution comparisons, as a good predictor of BE
studies with 12 individuals.
The biggest limitation of the introduced case-by-case DCP-tests customizations is that it
requires a validated IVIVC model for each formulation, and in the case of generic drugs,
serious harmonization efforts should be made to share these IVIVC models between
agencies and manufacturers. The computational effort and knowledge required also
currently impose real constraints on the wide diffusion and implementation of the
strategy presented here. Besides, it is still questionable which is the most suitable
procedure to declare BE [13, 98-101] and which other mathematical expressions could
model drug release more mechanistically [40, 74] .
The strategy presented in this study of setting limits of rejection of dissolution similarity
according to probabilities of presenting nonbioequivalence, can be refined, improved
and applied to other drug-formulations when more IVIVC mechanistic models become
available. For BCS class III drugs, further studies are required before analyzing the
possibility of biowaving ER formulations of these drugs [102]. However, we propose that
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under a deep case-specific analysis, biowaiving of ER of BCS class I drug formulations
may be possible through customized DPC-tests.
5.6. Conclusions
In the present study, we have customized a DPC-test for three different ER
formulations, linking limits of rejection with an in vivo attribute: the high probability of
being nonbioequivalent. According to these simulations, formulations that can prove to
be similar with the Reference formulation under the established conditions are likely to
be bioequivalent. Established conditions were TDT with δ of 3.6, 5.95, and 3.45 for
metformin, diltiazem and pramipexole respectively and sampling at least six time points
in the release profiles. Once a specific test is developed for a particular formulation, this
DPC-test can be used to explore post-approval changes by the manufacturer or to
evaluate BE between products from different manufacturers, decreasing the need for
future human BE studies and reducing costs of production. PT was the most powerful
DPC-test and differences in Cmax and AUC produced by formulations declared as similar
with PT were less than 1.5% in all cases. Tlag and ka were the drug/formulation
parameters that influenced BE-space to the greatest degree. For the investigated
cases, MDT or MRT were not suitable to detect bioequivalent formulations. Similar
case-specific studies may support the biowaiving of ER drug formulations based on
customized DPC-test.
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Prediction of Equivalence in a Combined Dissolution and Permeation System using customized
DPC-tests
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6. Prediction of Equivalence in a Combined Dissolution and
Permeation System using customized DPC-tests
The following author contributed to this chapter: José David Gómez-Mantilla, Sandra P.
Gantzsch, Dominik Selzer, Thorsten Lehr, Ulrich F. Schaefer, Claus- Michael Lehr.
The author of the thesis made the following contribution to this chapter:
 Developing the mathematical model and fitting. (With contribution of D. Selzer).
 Construction of the equivalent and similar spaces and customization of the DCP-
test.
 Design, performance and interpretation of the formulation optimization part.
 Writing the chapter.
Sandra P. Gantzsch manufactured the tablets and performed the experiments in the
d/p-sytem.
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6.1. Abstract
An apparatus for combined determination of dissolution and permeability (d/p-system)
was previously described by our group and tested with propranolol IR and ER
formulations. In this study we developed a mathematical model able to predict the
permeated amount of propranolol from an ER formulation in the d/p-system. After
considering this propranolol ER formulation as a Reference formulation, we were able to
predict which dissolution profiles would lead to significant differences in permeability
compared to the Reference formulation. Formulations that did not lead to significant
differences in the calculated permeated amount were considered equivalent to the
Reference formulation. Subsequently a DPC-test was customized to identify only
equivalent formulations as in vitro similar. It was possible to group all the equivalent
formulations in a set called the equivalent space. After customization, it was shown that
a TDT with will classify as in vitro similar to the Reference formulation only
formulations that were also equivalent. Additionally two examples of computer-assisted
formulation optimization were introduced. However, the need of further improvement in
the model and the limitations of the conclusions are highlighted.
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6.2. Introduction
The bioavailability of an orally administered drug is largely determined by the speed at
which the drug crosses the gastrointestinal mucosa and reaches the blood stream. The
speed at which a drug crosses a mucosa or a membrane is known as permeability and
can be experimentally measured in vivo with humans and animals [103-106], or in vitro
using cell lines and artificial membranes [107-112]. Due to its high morphological and
physiological resemblance to intestinal cells and its spontaneous polarization into
monolayers, the Caco-2 cell line model [113] is a very common procedure to study in
vitro permeability. This model is useful to study transcellular and paracellular transport
of drugs as well as transport events mediated by transporters [114]. Altogether,
experiments performed with Caco-2 cells are considered the golden standard for in
vitro prediction of intestinal drug permeability and absorption.[107, 115].
However, permeability experiments based on Caco-2 cells are normally performed with
totally dissolved substances (drug and additives if used), which restricts the possibility
of studying the effects of complete drug formulations on absorption. Furthermore, donor
concentrations used in these experiments are normally constant, arbitrarily chosen and
do not represent appropriately the in vivo situation, in which the drug concentration at
the apical side of the enterocytes is variable and depends on the release out of the drug
formulation.
As it has been stated in sections 3, 4.2 and 5.2, drug release from a formulation can
also play an important role in the absorption and bioavailability of drugs administered as
oral solid forms. Therefore, a tool to simultaneously study in vitro dissolution and
permeability can be of great utility during the drug formulation development process to
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study the effect of drug release and excipients on oral absorption in an easy and
inexpensive way, which ultimately can lead to an optimization of the drug formulation.
A d/p-system that allows the evaluation of complete solid oral dosage forms in an open
system using dynamic flow conditions was presented by Motz et al [60]. This d/p system
has been improved to allow continuous measurements of the drug concentrations in the
different compartments of the device [61] and continuous monitoring of the Caco-2 cells
monolayer integrity by measuring the transepithelial electrical resistance (TEER) [62].
The d/p-system has also been recently adjusted to perform experiments with artificial
membranes [63].
The d/p-system described by Motz et al [60] is schematically shown in Figure 6.1 and
consists of two main parts. The dissolution module part is a flow through dissolution cell
(USP apparatus 4). It is connected in line with the second part, the permeation module
which enables the setup of a Transwell® with a Caco-2 cell monolayer. The apparatus
includes an automated sampling and detection devices using a sequential injection
analysis (SIA). As dissolution and permeation require different flow rates the dissolution
and permeation modules are connected with each other by a stream splitter.
As it was stated in sections 3 and 5.2, declaring in vitro similarity of ER formulations is
still an unresolved problem and methodologies able to link in vitro dissolution to an in
vivo performance variable are highly desirable. Therefore, having two solid oral dosage
forms of the same drug and strength, this d/p-system can help to predict whether a
similar in vivo performance from the two formulations is expected or not. Moreover, after
applying an adequate mathematical model of the d/p-system, a computational tool can
be generated to predict the permeated amounts in the basolateral compartment in this
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device from any dissolution profile obtained with the USP 4 apparatus. Therefore, when
comparing the dissolution profiles between two formulations, it could be possible to
predict in which cases the differences in release kinetics are large enough to produce
significant differences in the permeability compartment of the d/p-system.
Figure 6.1 Schematic illustration of the d/p-system according to [60-63]. A, B and D are the
sampling ports for apical, dissolution and basolateral compartments. KRB is the working buffer (Krebs
Ringer Buffer), UV-VIS and PMT-FL are detector for UV and fluorescence respectively. EVOM is an
epithelial voltohmmeter for TEER monitoring.
An ER formulation is usually designed pursuing two main objectives: 1) achieving safer
and more constant in vivo concentrations of the administered drug, or 2) decreasing the
administration frequency to improve compliance of the patient [29]. In both cases a
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release as slow as possible is desired. The d/p-system complemented with the strategy
presented here to predict equivalent formulations can be used to establish how slow the
released of an ER can be without compromising its in vivo bioavailability. This would be
expected to be proportional to the permeated amount in the system. Additionally,
formulation optimization can be performed by predicting what formulations would lead to
the largest amount of permeated drug in the permeation compartment or would have a
slow release without compromising bioavailability, represented in this device by the
permeated amount in the permeation module.
The aim of this work was first to efficiently model the data from the ER propranolol
tablets in the d/p-system and secondly to identify through simulations, what formulations
would be equivalent (in terms of permeated amount in the basolateral compartment (B))
to the ER tablets analyzed experimentally (considered as the Reference formulation).
This group of equivalent formulations was considered as the equivalent space (eq-
space). Once the critical differences in dissolution were established, TDT, a DPC-test,
was customized to identify as in vitro similar only dissolution profiles from formulations
with high probabilities of being equivalent to the Reference formulation. Additionally,
nonlinear optimization was employed to predict what formulation would lead to the
largest amount of permeated drug in the B compartment. The same method was
employed to predict what formulation from the eq-space could have the slowest release.
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6.3. Materials and Methods
All the experimental data were taken from the work of Gantzsch et al [63], in summary,
ER tablets with 10 mg propranolol were prepared according to Motz et al [60],
Caco-2 cells following an established protocol [60, 61] Passages 61 – 70 within 21 – 25
days after seeding were used for experiments. Only Transwells ® showing TEER values
above 300 Ω*cm² were used.
Experiments were performed in an automated apparatus (d/p-system) [60-62]. Two
propranolol tablets were inserted in the flow-through dissolution cell for each
experiment. At certain defined time points, sampling took place at the apical (A) and
basolateral (B) compartment of the FTPC. For a detailed description see [63] .
6.3.1. Mathematical Modeling
Concentration of the drug in the Dissolution module was described by the Weibull
function (equation 6, section 3.3). Fitting of the Weibull function was performed using a
nonlinear least square fit. The dose from the formulation was considered as an
unknown due to the variability of propranolol content in the tablets (CV = 13%).
The transit through the d/p-system was modeled through a two-compartment pharmacokinetic model
according to
Figure 6.2.
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Figure 6.2 Illustration of the pharmacokinetic two compartments model for d/p system.
Change in D (mass in the dissolution module) was described by equation 16:
( ) ( )
equation 16
In which and are defined as in equation 6, is the flow rate leaving the
dissolution port, and is the volume of the dissolution module ( ).
Change in A (mass in the apical Compartment) was described by equation 17:
equation 17
is the absorption constant of the drug through the basolateral compartment, is the flow rate leaving
the apical compartment, and is the volume of the apical compartment. ( ). The factor describes the
split of into and shown in
Figure 6.2. A tlag of 4 minutes was considered between compartments D and A
according to times measured experimentally.
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Change in B (mass in the basolateral compartment) was described by equation 18:
equation 18
6.3.2. Construction of Equivalent Space (Eq-space)
The strategy used to identify equivalent formulations was similar to the one described in
section 5.3.1 and illustrated in Figure 6.3. In vitro dissolution profiles were simulated
for several formulations by modifying the Weibull model parameters and the permeated
amounts were generated using the model described in the previous section.
Inter-individual variability (IIV) was include for each parameter in the model to fit the
reported experimental variability. In total, 1000 equivalence simulated studies per
scenario were conducted. In each study, 12 tablets of the Reference formulation and 12
tablets of the Test formulations were generated by Monte Carlo simulations.
Equivalence between formulations was determined by calculating 90% confidence
intervals (90%CI) of the ratio between Test and Reference means (n=12) after log-
transformation of the permeated amount. The formulations were considered equivalent
if the 90%CI of the ratios were contained within the acceptance interval of 80.00 –
125.00%. Eq-space was delimited by bounding the Reference formulations with high
chances of being non-equivalent ( ). Once the Eq-space was delimited, TDT, a
DPC-test, was customized to declare the formulations that are likely to be non-
equivalent as non-similar, using the same procedure as in section 5.4.3.
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Figure 6.3 Strategy for building the equivalent spaces and customize a DPC-test.
6.3.3. Dissolution Profile Comparisons
Dissolution profiles from the Reference and Test formulations were compared using the
DPC-tests described in section 4.3.4.
As described in section 5.4.3 Sim-spaces of TDT at increasing values of were
constructed in order to find the maximum at which all the formulations declared as
similar uisng this test are also equivalent.
6.3.5. Formulation Optimization
Using the same simulated dissolution profiles from Test formulations as in the previous
section, the cumulative permeated amount was calculated for each Test formulation.
The formulations with larger calculated permeated drug amounts were considered as
the optimized formulations. Similarly the t85 of all the equivalent formulations was
calculated to identify the equivalent formulations with the longest t85.
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6.4. Results
6.4.1. Model Fitting of the Combined Dissolution and Permeation Data
Dissolution data was modeled by the Weibull function and after fitting the
average profile was described by equation 6 with ,
and . Data presented as a
mean . The corresponding fitting plot is shown in Figure 6.4. For the
permeation into the basolateral compartment, a permeation constant
was calculated. The fitting plot is shown in Figure 6.5.
Figure 6.4 Fitting of the data from dissolution module. Experimental data (•) and predicted
values (solid line), data presented as mean and range .
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Figure 6.5 Fitting of the data from the basolateral module. Experimental data (•) and predicted
values (solid line), data presented as mean and range .
6.4.2. Equivalent Space
Figure 6.6.a shows the in vitro dissolution profiles of the experimental formulation, for
this purpose considered the Reference formulation and the dissolution simulated
profiles for several Test formulations. On Figure 6.6.b, the contour plot displays the
probability of a Test formulation for being non-equivalent (in terms of permeated
amount) to the Reference formulation. It can be observed that Test formulations 1-3
have high probabilities of being non-equivalent, while Test formulation 4 has a low
probability of being non-equivalent. Similar to section 5.4.2, the Eq-space is delimited by
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separating the formulations based on a probability of them being non-equivalent.
All the formulations declared as similar with PT presented differences in the permeated
amount compared to the Reference formulation of less than 0.8%.
Figure 6.7 shows two different similarity spaces (Sim-space) for TDT at different values
of . A of 8 is the maximum at which all the formulations declared as similar with this
test are also equivalent in terms of permeated amount of drug in the d-p system. This
value represents the average tolerated difference (in %) between two formulations at
any time point to produce equivalent formulations for the criteria of permeated amount.
6.4.4. Formulation Optimization
Figure 6.8 shows the calculated permeated amounts in the d/p-system for the
formulations used as test formulations in previous experiments (Figure 6.6). The
formulation with release kinetics described by a Weibull function with
and showed the largest calculated permeated amount ( ) and is
represented in the contour plot as Max PA. Its dissolution profile can be observed in
Figure 6.10 in comparison to the Reference formulation. Similarly, Figure 6.9 displays
the difference in release kinetics of the equivalent and non-equivalent formulations
expressed as t85. The formulation with a release kinetics described by
and was the equivalent formulation with the longest t85 ( ) and is
denoted in the contour plot as Max t85.
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Figure 6.6 Construction of the Eq-space and identification of non-equivalent formulations.

On the left the color scale of the probability of being nonequivalent to the Reference formulation.
Formulations with high probabilities of being nonequivalent are located in the purple area.
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Figure 6.7. Sim-space and Customization of TDT Sim-Spaces of TDT at different values of . for
propranolol formulations.
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Figure 6.8. Counter plots of the calculated permeated amounts in the d/p-sytem. Max Pa,
points to the formulation of largest calculated permeated amount ( ).
Figure 6.9. Counter plots of the t85 of the formulations in combination with the equivalent
space of the d/p-sytem. Max t85 points to the formulation with the maximum possible t 85 (223 min)
that is inside the equivalent space.
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Figure 6.10. Dissolution profiles of the optimized formulations.
6.5. Discussion
It is highly desirable to link dissolution tests to in vivo performance of the formulation
tested [28, 79]. In section 5 it was presented how to do make that link when and IVIVC
model is available. However in early stages of drug development an IVIVC may not be
always available and a fast and inexpensive system to study the effect of formulation on
permeability can be of great use. In this study we also evaluated the potential of this
d/p-system as a tool to predict equivalency of different formulations with a given
Reference formulation as well as for computer-assisted formulation optimization.
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It was possible to predict what formulations would be equivalent to the Reference
formulation in terms of permeated amount in compartment B. In general, only
formulations with very slow release kinetics lead to a calculated permeated amount
significantly lower than the reference formulation. In agreement with the results in
sections 4.4.3 and 5.4.2 and , PT showed very good statistical power and all the
formulations declared as similar with this test differ less than 0.8% in the calculated
permeated amount for the basolateral compartment. That reinforces the potential of this
test in the sphere of quality by design where tools which detect small changes in a
critical quality attribute (in this case dissolution) are highly needed.
In the sphere of generic drugs, the regulatory agencies could use the strategy
presented here to detect if an ER Test formulation is equivalent to an ER Reference
formulation in the market. Moreover, by customizing a TDT DPC-test, a simple in vitro
dissolution test, without the permeability module may be sufficient to prove equivalency
between two ER formulations. Previously, it must be proven that, for one, the excipients
of the formulation do not affect the absorption of the drug, for two, the drug should not
be a prodrug and it should not have a narrow therapeutic index or be intended to be
absorbed in the oral cavity. Nevertheless, in order to achieve this level of reliance on the
information generated by this strategy, it must be first verified that the d/p-system is a
good representation of the in vivo case, which only can be concluded with further
experiments. Likewise, the mathematical model can be improved to include the whole
geometry of the GI tract and the inferences about the in vivo case would be more
reliable. More generally, at least for ER formulations of BCS class I drugs like
propranolol, in which absorption is expected to be driven by the release kinetics, the
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strategy presented here is a reasonable approximation that can be improved step by
step to identify equivalent formulations.
If the objective of the formulation development is to design an oral solid dosage form
with the highest possible bioavailability, it is of advantage to predict what formulation
would exhibit the largest permeated amount in a d/p-system. Likewise, if the objective of
the formulation development is to design a formulation with the slowest possible release
to achieve safer plasma concentration or decrease administration frequency without
sacrificing bioavailability, it is of advantage to predict which of the formulations of the
eq-space has the slowest release. We presented in this work a computer-assisted tool
to help in the formulation optimization process. This tool can be employed to maximize
the permeated amount of drug in the d/p-system which ultimately could represent a
maximized absorbed drug in vivo. Similarly it can be used to find the equivalent
formulation with the slowest possible release kinetics or in general an equivalent
formulation with a specific desired kinetics. Nonetheless, this method is constrained by
the same limitations as in the case of identifying equivalent formulations (the uncertainty
of how good the d/p-system mimics the in vivo case), but also retains the same improve
opportunities.
All the simulations were performed mimicking the typical experiment in the d/p-system
which is performed for no more than 4 hours to assure the integrity of the Caco-2 cell
monolayer. This time restriction is mathematically equivalent to an absorption window of
4 hours and may not mimic properly the in vivo case. Using the mathematical model it
would be possible to explore longer absorption windows that are not possible
experimentally. Nevertheless, It must be considered that the results obtained from eq-
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space, customization of TDT as well as formulation optimization are valid only for an
absorption window of four hours and that different absorption windows may lead to
different results.
The mathematical model presented here can also be used to detect which setup
variables (flow rates and volumes) have a stronger effect on the concentrations in the
apical and basolateral compartments, therefore it can be determined whether the
discrimination power of the d/p-system, i.e. the capacity to transform differences in
dissolution into differences in the permeated amounts can be increased by a different
setup. Also, deeper explorations can be performed to check, by simulations, if the
system can be improved by mounting more permeability units after the port D to
simulate a segment of the intestine exposed to decreasing concentrations of the drug.
In general this mathematical model can be used to explore different alternatives of the
apparatus setup to optimize it or to mimic better the in vivo case.
The presented mathematical model described reasonably well the experimental data.
However, it is based on limited data (only one formulation) with high variability in all the
ports sampled. Therefore conclusion must be drawn carefully and considering that the
model must be improved considerably by including more experimental data.
Additionally, the step in the splitter pump can be modeled differently by including
expressions that describe the diffusion in the transit compartments, since the transit
volume in the connecting tubes is almost 10 mL and the transit time is between 4 and 7
minutes.
99
DPC-tests
______________________________________________________________________________
6.6. Conclusions
A mathematical model was successfully developed to describe the experimental data of
a d/p-system. Taking the tablets evaluated experimentally as a Reference formulation,
the mathematical model was used to predict which formulations would be equivalent in
terms of the permeated amount in the basolateral compartment to the Reference
formulation. The group of equivalent formulations was called the eq-space, and it was
possible to customize the DPC-test TDT in such a way that when the customized test (a
TDT with ) declares a formulation as in vitro similar to the Reference formulation,
these formulations will most likely be equivalent regarding their permeated amount in
the basolateral compartment. Through linear optimization it was possible to identify the
equivalent formulations which would exhibit the longest possible t85 or the largest
amount of permeated amount in the basolateral compartment. The mathematical model
can also be used to evaluate modifications in the apparatus setup and configuration.
However, the model must be further improved, mainly by the inclusion of more
experimental data and better description of the transit between the compartments.
100
Summary and Outlook
______________________________________________________________________________
7. Summary and Outlook
Dissolution testing has been widely used as a global indicator of the ability of a dosage
form to release API, and in doing so enable it to reach its site of action. Furthermore,
comparative dissolution testing has been explored as a tool to evaluate equivalency
between formulations. However, the current official procedure to compare dissolution
profiles, the f2 similarity factor, lacks solid statistical foundation and the level of
statistical uncertainty is unknown. Moreover, the limits of the f2 similarity factor to
declare in vitro similarity (f2 ≥ 50) not derived from any specific biopharmaceutical
property. Such a test therefore cannot be considered as a good predictor of in vivo
performance, especially in the case of ER formulations. The aim of this work was to
design, develop and explore new DPC-tests with stronger statistical basis than the
currently employed methodologies f2 similarity factor, and to link the limits of rejections
of these new DPC-tests with significant differences in important biopharmaceutical
properties with more predictive power of in vivo formulation performance. Examples of
DPC-test customization have been presented for three ER formulations (metformin,
diltiazem and pramipexole) using IVIVC models; one example of DPC-test
customization for a propranolol ER formulation using a d/p-system has been also
illustrated. In summary:
Exploration of the two new DPC-Tests
 Two new statistical tests, the PT and the TDT, have been presented for
dissolution profile comparison. In these tests both type-I and type-II errors can be
quantified; both tests also have stronger statistical basis than the current
101
Summary and Outlook
______________________________________________________________________________
alternative (e.g., the f2 similarity factor). The two new tests showed acceptable
robustness at standard conditions of variation (CV95 ≤ 0.1).
 A strategy to design and perform a dissolution profile comparison under typical
principles of statistical experimental design has been presented.
 An optimized time sampling strategy was introduced in the current work. This
strategy should be employed when possible in DPC-tests to avoid artificial
discontinuities in the statistical power. The exception to the recommended use of
such a strategy is in the case of PT which is not susceptible to this effect.
Customization of DPC-Tests
 A DPC-test, TDT, was customized for three different ER formulations and its
limits of rejection linked with an in vivo attribute: the high probability of being
nonbioequivalent. Formulations that proved to be similar to the Reference
formulation using a customized DPC-test (TDT with δ of 3.6, 5.95, and 3.45 for
metformin, diltiazem and pramipexole respectively) are likely to be bioequivalent.
Such a result demonstrates a clear application of formulation-specific DPC-test in
the exploration of post-approval changes by a manufacturer, or the evaluation of
BE between products from different manufacturers. The use of such DPC-tests
could decrease the need for future human BE studies and reduce costs of
production.
 Tlag and ka were found to be the drug/formulation parameters that influenced BE-
space to the greatest degree for the pramipexole ER formulation.
102
Summary and Outlook
______________________________________________________________________________
 MDT or MRT were not suitable for bioequivalence prediction in the case of the
ER formulations investigated (metformin, diltiazem and pramipexole).
 The presented work leads to the suggestion that case-specific studies, using
IVIVC or d/p-systems may support the biowaiving of ER drug formulations, based
on customized DPC-tests.
Application of DPC-test customization in a d/p-system
 A mathematical model was successfully developed to describe the experimental
data of a d/p-system. This mathematical model could be used to predict which
formulations would be equivalent to a Reference formulation in terms of the
permeated amount in the basolateral.
 By establishing in vitro similarities between Test and Reference formulation, a
customized TDT ( ) could be used to identify formulation likely to be
equivalent with respect to the permeated amount in the basolateral compartment
of the d/p-system.
 A computer assisted tool for formulation optimization has been presented which
allow for the design of formulations with larger permeated amounts in the d/p-
system and longer t85.
Properties of PT
 PT was the most robust and powerful test in all the conditions studied (even in
conditions of high variability CV95 ≤ 0.2 and reduced sample sizes). Differences in
Cmax and AUC produced by formulations declared as similar with PT were less
103
Summary and Outlook
______________________________________________________________________________
than 1.5% for the metformin, diltiazem and pramipexole ER formulations. This
test is therefore strongly recommended for identifying small differences in
dissolution.
 In the case of the propranolol ER formulation, PT also showed excellent
statistical power. All the formulations declared as similar with this test in the d/p-
system differed by less than 0.8% in the calculated permeated amount in the
basolateral compartment of the system.
Outlook
While the work presented in this thesis shows a promising application of both PT and
TDT, it must be considered that more experimental confirmation is needed to prove the
suitability of the new DPC-tests as alternatives for performing dissolution profile
comparison. In particular, experimental verification of the lack of effect for iid-conditions
(section 4.3.7) is necessary to ensure an adequate performance of the TDT under the
standard (no iid-conditions).
Another step that should be explored is the linking of customized DPC-tests with clinical
outputs or pharmacodynamics biomarkers instead of BE probabilities. This could be
achieved using pharmacokinetic pharmacodynamic (PK/PD) modeling which, of course
demands a deeper understanding of the pharmacology of the drug as well as a higher
level of detail in the mathematical modeling.
The mathematical model employed to describe the data from the d/p-system has many
opportunities for improvement. Specifically the inclusion of more experimental data and
a better description of the transit between the compartments should be considered.
104
Summary and Outlook
______________________________________________________________________________
Similarly, attractive opportunities such as the exploration of formulation and apparatus
optimization, and inclusion of data from drugs which are substrates of active
transporters remain open for further development.
It should be emphasized that the excellent statistical power exhibited by the PT is a very
promising outcome of this thesis. It is worth exploring the potential of PT as a quality by
design tool due to its ability to detect even small differences in dissolution profiles.
When dissolution profiles are used as a critical quality attribute (CQA), the performance
of the formulation will not be compromised as long as similarity is declared by PT. Also
knowledge of a manufacturing process can be improved by checking the influence of
different process variables (compression pressure, granulation time, percentage of
polymer etc.) on this CQA, and identifying the changes in these variables required to
produce a non-similar dissolution profile.
105
Abbreviations
______________________________________________________________________________
8. Abbreviations
90% CI 90% confidence intervals

A Apical compartment
API Active pharmaceutical ingredient (s)
AUC Area Under the Curve
B Basolateral compartment
BCS Biopharmaceutical classification system
BE Bioequivalence
BE-space Bioequivalent space
Cmax Maximum plasma concentration
CQA critical quality attribute
D Dissolution port in the d/p-system
d/p-system Dissolution permeability system
DPC-test Dissolution profile comparison test(s)
Eq-space Equivalent space
EVOM Epithelial volt ohm meter
FTPC Flow-through permeation cell
GI Gastro-intestinal
GIT gastro-intestinal tract
iid-conditions Independent identically distributed
IR immediate release
KRB Krebs ringer buffer
MDT Mean Dissolution Time
MRT Mean Residence Time
PK Pharmacokinetic (s)
PK/PD pharmacokinetic pharmacodynamic
PT Permutation test
Sim-space Similarity space
t85 Time required to release 85% of the drug from an oral solid dosage form
TDT Tolerated difference test
TEER Transepithelial electrical resistance
106
Annexes
______________________________________________________________________________
9. Annexes
Time Points
4 5 6 7 8 9 10
(1-α)
0.9 83.00 82.00 81.00 80.43 79.88 79.44 79.00
0.95 86.25 84.80 83.67 82.71 82.13 81.44 81.00
0.975 89.00 87.20 85.83 84.86 84.00 83.33 82.70
0.99 92.00 90.00 88.33 87.14 86.25 85.44 84.70
0.995 94.25 91.80 90.17 88.86 87.75 86.78 86.10
0.999 98.50 95.80 93.67 92.14 90.88 89.78 88.90
0.9995 100.25 97.20 95.00 93.43 92.00 90.89 90.00
A.1. Rejection Values for TDT for several time points and uncertainty.
A.2. AUC BE-Space Metformin.
107
Annexes
______________________________________________________________________________
A.3. Cmax BE-Space Metformin.
A.4. AUC BE-Space Diltiazem
108
Annexes
______________________________________________________________________________
A.5. Cmax BE-Space Diltiazem
A.6. AUC BE-Space Pramipexole
109
Annexes
______________________________________________________________________________
A.7. Cmax BE-Space Pramipexole
A.8. Customization of TDT (Metformin)
110
Annexes
______________________________________________________________________________
A.9. Customization of TDT (Diltiazem)
111
Annexes
______________________________________________________________________________
A.10. BE-Spaces under different values of V1, V2, CLD.
112
Annexes
______________________________________________________________________________
A.11. BE- Spaces at different values of kel. (Pramipexole).
A.12. BE-Spaces at different values of kel. (Metformin).
113
Annexes
______________________________________________________________________________
A.13. Effect of Number of volunteers on BE-Space (Metformin)
A.14. Effect of Number of volunteers on BE-Space (Diltiazem)
114
Annexes
______________________________________________________________________________
A.15. Comparison of BE-Spaces to a)MDT and b)MRT (Metformin).
115
Annexes
______________________________________________________________________________
A.16. Comparison of BE-Spaces to a)MDT and b)MRT (Diltiazem).
116
Annexes
______________________________________________________________________________
A.17. Reduction in the Cmax BE-Space at different values of Clearance. The reduction is
significant when the kel/ka ratio is 0.2 or higher. The AUC BE-Space is not affected (lower right).
117
Annexes
______________________________________________________________________________
A.18. Comparison of PK models of Metformin and Diltiazem. Dissolution, PK profile, and

relationship between dissolution and concentration in the central compartment.
118
Annexes
______________________________________________________________________________
A.19. Relationships between Sim-Space and MDT and MRT. Several formulations can have the
same MDT but different MRT for both metformin and diltiazem, no relationship was observed bwtween
the BE-Space or the Sim-space and MDT or MRT.
119
Scientific Output
______________________________________________________________________________
10. Scientific Output
Original Papers
J.D. Gomez-Mantilla, V.G. Casabo, U.F. Schaefer, C.M. Lehr, Permutation Test (PT)
and Tolerated Difference Test (TDT): Two new, robust and powerful nonparametric
tests for statistical comparison of dissolution profiles, Int J Pharm, (2012).
J.D. Gomez-Mantilla, V.G. Casabo, U.F. Schaefer, T. Lehr, C.M. Lehr, Identification of
Non-Bioequivalent Extended Release Formulations by Tailor-Made Dissolution Profile
Comparisons Using In Vitro-In Vivo Correlation Models, (Submited).
Conference Abstracts
J.D. Gomez-Mantilla, V.G. Casabo, U.F. Schaefer, C.M. Lehr, Permutation Test (PT)
and Tolerated Difference Test (TDT): Two new, robust and powerful nonparametric
tests for statistical comparison of dissolution profiles
8th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical
Technology, Istanbul, Turkey, March 2012
J.D. Gomez-Mantilla, V.G. Casabo, U.F. Schaefer, T. Lehr, C.M. Lehr, Tailor-made
dissolution profile comparisons using In Vivo-In Vitro Correlation Models
22nd Population Approach Group Europe (PAGE), June 2013, Glasgow, Scotland
120
Curriculum vitae
______________________________________________________________________________
11. Curriculum vitae
José David Gómez Mantilla

18.06.1979
Bogotá, Colombia
Doctoral Thesis
Education 10/2010 – 12/2013
Department of Biopharmaceutics and Pharmaceutical Technology
Saarland University, Saarbrücken, Germany
Thesis Project: “statistical approaches to perform dissolution profile
comparisons”.
Research Internship
10/2009 - 05/2010
Universidad de Valencia, Valencia, Spain
Specialization in Statistics
07/2004 - 07/2006
Universidad Nacional de Colombia, Bogotá, Colombia
Thesis Project: “Robustness exploration of F-permutation, kruskal-Wallis and F-
Anova tests on biomedical ranges of interest”.
Degree in Pharmacy
02/1997 - 07/2003
Thesis Project: ”Immunological evaluation in Chronic Mucocutaneous
Candidiasis patients”.
Universidad de Antioquia, Medellín, Colombia

Young Researcher
01/2002-8/2002

Professional Teaching Assistant
Experience 01/2007-09/2009
Sun Pharmaceutical Service LTD.

Pharmaceutical Care Director
03/2004-12/2004
121
Curriculum vitae
______________________________________________________________________________
Schering-Plough S.A.
In-Process Control Inspector
05/2003-12/2003
Cooperativa de Medicamentos de Cundinamarca

Pharmaceutical Services Coordinator
01/2003-05/2003
National Army of Colombia

12/1995-12/1996 (Mandatory Military Service)
Awards and Colciencias Scholarship 01/2013-Present

Holder of a Colombian Government Scholarship
Recognitions to pursue a Doctoral Degree Abroad.
DAAD Scholarship 05/2010-Present

Holder of a DAAD Scholarship to pursue a Doctoral Degree in Germany.
Free Tuition for Outstanding Records 2005

Universidad Nacional de Colombia, Department of Statistics.
First Class Honours for Outstanding Records 1997

Universidad Nacional de Colombia, Department of Pharmacy.
Best National Exam Score 1995

Antonio Nariño High School, Bogotá.
122
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132
Acknowledgments
______________________________________________________________________________
13. Acknowledgments
Since the first moment I understood what achieving a PhD means it became my
dream to pursuit one. I feel extremely lucky for the chance I had during these years
to focus only on my research question and the freedom, guide, encouragement and
support and fun that I found on the way.
I can only thank Prof. Claus-Michael Lehr, not only for inviting me to his group, but
also for encouraging me constantly to follow my academic appetites and keep trying
harder. Also for all the fruitful scientific discussions in our project meetings and all
his personal dedication to find collaborators for our projects and solutions to my
problems. I can only condense my sincere gratitude by saying that every time after a
meeting with him I was happy to have more work and that extra motivation was
constantly helpful.
I am more than thankful to apl. Prof. Ulrich Schäfer for his personal concern in my
career and for all his contribution to my work. I fairly admire his passion for detail
and for digging deeper to find plausible answers. It was a luxury to count on
someone with such enthusiasm to acquire and transmit knowledge and such
extraordinary expertise in the laboratory and in the discussion of theoretical
principles.
I thank also Jr. Prof. Thorsten Lehr for his great disposition to collaborate in my work
and all the numerical and non-numerical fun that we had together in the last time. It
was one a very happy coincidence to find someone in the middle of my PhD
experience with such involvement in the field I was craving to explore and that I
definitely want to keep discovering.
133
Acknowledgments
______________________________________________________________________________
I also thank Prof. Vicente Casabó for his collaboration in my project and his
unforgettable lessons. I was very motivated to meet for the first time another
pharmacist with such a level of passion for mathematics and statistics. Undoubtedly
his loss was a sad and bitter moment of the last year.
To Domink Selzer and Sandra Gantztsch for the nice collaboration in the work on
the final chapter of this thesis.
To DAAD and Colciencias (Colombia) for the financial support during these years. In
particular to Silke Hamacher from DAAD for her great help and personal attention to
my case.
My open thankfulness to Prof. Marta Fontanilla of my University in Colombia for the
help, encouragment and understanding.
Thanks to the Secretaries of the group Karin, Sarah and Isabelle for their help in the
practical matters that surrounded this experience.
To Sarah Gordon, Christiane Mathes and Nico Mell for the language assitance in
this thesis.
These years in Germany have been without a doubt a huge cultural experience and I
consider myself very privileged for all the things I have had the chance to learn from
so many people. I am very thankful to my colleagues from the group who gave me
the opportunity to integrate my culture into theirs and were very empathetic to invite
me to a long list of interesting activities and were always happy to join me in any
social suggestion. Particularly I thank Clemens Tscheka for all the kilometers of
sports, all the skiing, and his unconditional friendship.
134
Acknowledgments
______________________________________________________________________________
It was never in my plans to do theater in Spanish in Germany, but it turned out to be
a great chance to meet nice people and get to know myself better. I thank sincerely
all the members of the theater group “Los Mutantes” for all the nice moments of
disconnecting from everything and recharging energy, specially to Lina Ruiz, who
also helped me to get installed in this city since the first week of my stay.
To Augusto Salazar and Anna Kühn for bringing more happiness to my German
adventure and exciting plans for the future.
To my Montagpola friends Miguel Granados, Fidel Ramírez and José Manuel Brito,
for letting me grow with them and acquire together a new vision of life and
meaningfulness. Thank you for pushing me to widen my reading tastes and for being
a guarantee of a nice and interesting conversations and help with my problems
during these years. Muchas Gracias!
To Champetica for giving me new reasons for daydreaming.
Finally I want to say that I have the luck to come from a country where my mother´s
last name stays into mine during my whole life and this long name that sounds
strange to many people is for me a little tribute to her efforts and sacrifices. I will
never be able to return all the things she has done for me. Because of her I had
access to a very good middle education and she was also the person who
encouraged me the most to be curious about science, nature, mathematics and
foreign languages and to keep searching and demanding more from myself. She
was also the person who gave me the discipline to work intensively on unresolved
problems and woke me up early in the morning when necessary to keep on trying.
Definitely, I would have never dreamed about a PhD without her. Gracias Mamá!
135
Acknowledgments
______________________________________________________________________________
136

STATISTICAL APPROACHES For Dissoprofile Copmarision

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STATISTICAL APPROACHES For Dissoprofile Copmarision

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STATISTICAL APPROACHES TO PERFORM

DISSOLUTION PROFILE COMPARISONS

zur Erlangung des Grades

des Doktors der Naturwissenschaften

der Naturwissenschaftlich-Technischen Fakultät III

Chemie, Pharmazie, Bio- und Werkstoffwissenschaften

der Universität des Saarlandes

Dekan: Prof. Dr. Volkhard Helms

Berichterstatter: Prof. Dr. Claus-Michael Lehr

apl. Prof. Dr. Ulrich Schäfer

Vorsitz: Prof. Dr. Hans Maurer

Akad. Mitarbeiter: Dr. Martin Frotscher

ultra-marathon with hurdles, and it is all

about not losing rhythm with the obstacles.

May you rest in peace,

to infinite wisdom, but to set a limit to infinite

1. Short Summary ....................................................................................................... 1

Comparative dissolution testing is extensively used as a tool to evaluate equivalency

property; therefore it cannot be considered as a good predictor of the in vivo

performance of formulations, especially in the case of extended release formulations.

current methodologies and exhibit the flexibility to be customized for a specific

of bioequivalence (or a significant difference in the performance in a combined

dissolution permeability system).

Customization of the dissolution profile comparison tests for ER formulations was

acceptable statistical uncertainty).

In summary, a new approach to design and perform dissolution profile comparisons

under typical principles of statistical experimental design is described. Four

demonstrative examples of comparisons of ER formulations are presented.

Vergleichende Untersuchungen des Auflösungsverhaltens werden häufig zur

in-vitro-Ähnlichkeit (f2 < 50) willkürlich gewählt und nicht an biopharmazeutische

das in-vivo-Verhalten der untersuchten Arzneiformen angesehen werden. Dies gilt

insbesondere für Arzneiformen mit verlängerter Wirkstofffreigabe.

Vergleich von Auflösungsprofilen. Im Vergleich zu momentan angewendeten Methoden

die Flexibilität, für spezifische Formulierungen angepasst werden zu können. Einer

solche Unterschiede zwischen den Dissolutionsprofilen von verlängert freisetzenden

Formulierungen identifiziert werden, die auf mangelnde Bioäquivalenz hinweisen.

Durchführung von Vergleichstests für Dissolutionsprofile beschrieben, die auf Prinzipien

des statistischen experimentellen Designs basieren. Die Anwendungsmöglichkeiten

werden anhand von vier Beispielen für verlängert freisetzende Formulierungen

approval stages, or production by generic manufacturers, possess the same efficacy

questionable, the pharmaceutical industry is constantly searching for effective

surrogates for judging therapeutic equivalence of pharmaceutically equivalent drug

new formulations is the assessment of Bioequivalence (BE), in which an absence of

equivalence of drug formulations is assured by comparison of their dissolution profiles.

well as the conditions and procedures by which in vitro testing is considered as a

reliable surrogate for an in vivo BE.

suitable comparator, the Reference formulation, in a pharmacokinetic (PK) study with a

limited number of subjects is one way of demonstrating equivalence without having to

perform a clinical trial involving many patients [8].

same qualitative and quantitative composition in active substances.

In bioequivalence studies, plasma concentrations obtained after administration of the

Test formulation are contrasted to those of a Reference formulation. Pharmacokinetics

recommended number of subjects but also in: 1) use of volunteers or patients, 2)

drug or metabolite, and 5) strength to be investigated [12, 13].

as a reliable surrogate for an in vivo BE is referred as “biowaiver” [10, 11].

Requirements for granting a biowaiver of an in vivo BE study depend on the class of

system (BCS) which classifies drugs as follows.

 Class I : high solubility – high permeability

 Class II : low solubility – high permeability