Biomedical Chemistry - Current Trends and Developments

Nuno Vale
Biomedical Chemistry: Current Trends and Developments

Nuno Vale
Biomedical Chemistry:
Current Trends and
Developments
Managing Editor: Anna Rulka
Language Editor: Reuben Hudson & Michael Jones

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Part of Walter de Gruyter GmbH, Berlin/Munich/Boston
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which means that the text may be used for non-commercial purposes, provided credit is given to
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Copyright © 2015 Nuno Vale and chapters’ contributors
ISBN 978-3-11-046875-5
e- ISBN 978-3-11-046887-8
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Cover illustration: © Thinkstock / Garsya

Contents
Preface XI
Section 1: Chemical Principles in Drug Design and Discovery
1.1 Functional Groups of Biomolecules and their Reactions 2

1.1.1 Functional Groups in Biological Systems 2
1.1.2 Acids and Bases Versus Electrophiles and Nucleophiles 5
1.1.3 Stereoisomerism and Chirality 8
1.1.3.1 Cis/trans Isomerism 8
1.1.3.2 Chirality and Enantiomerism 9
1.1.4 Common Mechanisms in Biological Chemistry 10
1.1.4.1 Nucleophilic Substitution Reactions 10
1.1.4.1.1 SN2 – Bimolecular Nucleophilic Substitution 10
1.1.4.1.2 SN1 ‒ Unimolecular Nucleophilic Substitution Reactions 11
1.1.4.1.3 Phosphate Group Transfer – the Grey Area of Nucleophilic Substitutions
in Biological Systems 12
1.1.4.2 Electrophilic Addition Reactions 13
1.1.4.2.1 Synthesis of α-Terpineol ‒ Intramolecular Addition 14
1.1.4.3 Aromatic Substitutions 15
1.1.4.3.1 Electrophilic Aromatic Substitution 16
1.1.4.3.2 Nucleophilic Aromatic Substitutions 17
1.1.4.3.3 Hallucinogen Synthesis ‒ Aromatic Substitution on Fungi 17
1.1.4.4 Eliminations Reactions 18
1.1.4.4.1 E1 ‒ Unimolecular Elimination 18
1.1.4.4.2 E2 ‒ Bimolecular Elimination 19
1.1.4.4.3 E1cB ‒ Unimolecular Elimination through Conjugate Base 19
1.1.4.5 Nucleophilic Carbonyl Addition Reactions 20
1.1.4.5.1 Nitrofurantoin ‒ a Semicarbazone 22
1.1.4.6.1 Aspirin ‒ Esterifications and Transesterifications 24
1.1.1.4.7 Carbonyl Condensation Reactions 25
1.1.4.7.1 Aldol Reaction 25
1.1.4.7.2 Claisen Condensation 26
1.1.4.7.3 Aldolases ‒ Stabilization Strategies 27
1.1.4.8 Oxidations and Reductions 28
1.1.4.8.1 Disulfide Bridges ‒ Oxidized Thiols 31
1.1.5 The Organic Mechanisms of Biological Transformations 32
1.1.5.1 Cis/trans-Isomers Interconversion in the Vision Pathway 32
1.1.5.2 Metabolism of Fatty Acids ‒ β-Oxidation Pathway 33
1.1.5.3 Penicillin ‒ a Strong Acylating Agent 35
1.1.5.3.1 Transpeptidase Mechanism 35
1.1.5.3.2 Transpeptidase Inhibition 36
1.1.5.3.3. Penicillin Biosynthesis 37
1.1.5.4 NAD+ − a Classical Coenzyme 39
1.1.5.5. FAD − a More Versatile Coenzyme 40
1.1.5.6 Biotin and Carboxylation Reactions 41
References 42
1.2 Designing Covalent Inhibitors: A Medicinal Chemistry Challenge 44

1.2.1 Introduction 44
1.2.2 Designing Safer Covalent Inhibitors 45
1.2.3 Case Study 1: Michael Acceptors to Treat Infectious Diseases 48
1.2.3.1 K777 Inhibitor 50
1.2.3.2 Rupintrivir (AG7088) 51
1.2.4 Case Study 2: From Covalent Inhibitors to Hybrid Drugs 51
1.2.4.1 Hybrid Compounds Containing an Electrophilic Warhead 52
1.2.4.2 Hybrid Compounds Containing a Masked Electrophilic Warhead 54
1.2.5 Conclusions 56
References 57
Section 2: Chemical Basis of Drug Action and Diseases
2.1 Pharmacokinetics and Bioanalysis to Improve Drug Development 62

Abbreviations 62
2.1.2 Pharmacokinetic on Drug Discovery and Development Process 65
2.1.2.1 ADME/Pharmacokinetic Evaluation on Early Drug Discovery Phases 66
2.1.2.2 Pharmacokinetic Evaluation on Drug Development Phases 76
2.1.3 Bioanalysis and Validation Requirements on DDD 81
2.1.4 Bioanalysis & Pharmacokinetics, a Synergistic Partnership on DDD 88
2.1.4.1 Bioanalytic Support of In Vitro ADME Studies 107
2.1.4.2 Bioanalytic Support of In Vivo ADME/Pharmacokinetic Studies 110
References 113
2.2 Translational Research in Endocrinology and Neuroimmunology Applied to

Depression 119
2.2.1 Major Depressive Disorder 119
2.2.2 The Stress Response 120
2.2.2.1 The CRH System and the Stress Response 120
2.2.2.2 The Locus Ceruleus Norepinephrine (LC-NE) System and Other Central
Nervous System (CNS) Structures that Modulate the Stress System 121
2.2.2.3 The Immune System 122
2.2.3 The Effect of Chronic Stress and MDD in Dysregulating the Core Stress
System 123
2.2.4 Summary and Conclusions 125
References 125
2.3 Understanding the Metabolic Syndrome Using a Biomedical Chemistry

Profile 132
2.3.2 Natural Mineral-rich Waters and MetSyn 133
2.3.3 Magnesium and MetSyn/MetSyn Features – Associated Mechanisms
134
2.3.4 Calcium and MetSyn/MetSyn Features – Associated Mechanisms
137
2.3.5 Potassium and MetSyn/MetSyn Features – Associated Mechanisms
138
2.3.6 Bicarbonate and MetSyn/MetSyn Features – Associated Mechanisms
139
2.3.7 Magnesium, Calcium, Potassium and Bicarbonate versus Sodium
140
2.3.8 Conclusion 140
References 141
2.4 Brain Neurochemistry and Cognitive Performance: Neurotransmitter

Systems 148
2.4.1 Monoaminergic Neurotransmission and Cognition 148
2.4.2 Glutamate Neurotransmission and Cognition 154
2.4.3 GABAergic Neurotransmission and Cognition 160
2.4.4 Gliotransmitters 164
2.4.5 Cognitive Enhancement 165
References 166
Section 3: Strategies to Develop New and Better Drugs
3.1 Amino Acids and Peptides in Medicine: Old or New Drugs? 178
3.1.1.1 Amino acids: biological and chemical concepts 178
3.1.2 Amino Acids and Drug Development 182
3.1.2.1 Rationale for Drug Design 182
3.1.2.2 Amino Acid Prodrug in Drug Delivery 185
3.1.2.3 L-type Amino Acid Transporter 192
3.1.2.4. Variability of Amino Acid Application to Exclusive Drugs 194
3.1.3 Peptides for Biomedicine 197
3.1.3.1 Antimicrobial Peptides (AMPs) 198
3.1.3.1.1 AMPs: Mechanism of Action and Peptide Families 199
3.1.3.1.2 α-Helical Peptides without Cys Residues 201
3.1.3.1.3 Peptides Containing Disulfide Bridges 202
3.1.3.1.4 Peptides Rich in Pro, Gly, His, Arg and Trp Residues 202
3.1.3.3 Peptides: Scaffolding Materials in Tissue Engineering 206
3.1.3.3.1 Peptide-based Biopolymers 206
3.1.3.3.2 Strategies to Create Scaffolds as Instructive Extracellular
Microenvironments for Tissue Engineering - Peptide-conjugated
Polymers to Mimic Natural ECM 207
3.1.3.3.3 Peptide-based Biomaterials Responsive to Environmental Cues 208
3.1.3.3.4 Self-assembling Peptides as Biomaterials 209
3.1.3.4 Therapeutic Peptides and Market 211
3.1.3.4.1 Chemical Strategies to Improve Peptide Biological Activity 211
3.1.3.4.2 Market 213
References 216
3.2 Targeting Calcium-mediated Excitotoxicity in the CNS 229

3.2.2 Glutamate and Glutamate Receptors 230
3.2.2.1 AMPA Receptors 231
3.2.2.2 Kainate Receptors 232
3.2.2.3 NMDA Receptors 232
3.2.3 The Role of Calcium in Normal Neuronal Biochemistry 233
3.2.4 Excitotoxicity 234
3.2.5 The Role of Calcium in Excitotoxic Neuronal Biochemistry 234
3.2.6 Diseases that are Potentially Exacerbated by Calcium-mediated
Excitotoxicity 235
3.2.6.1 Amyotrophic Lateral Sclerosis (ALS) 235
3.2.6.2 Multiple Sclerosis (MS) 236
3.2.6.3 Alzheimer’s Disease (AD) 237
3.2.6.4 Huntington’s Disease (HD) 238
3.2.6.5 Stroke 238
3.2.6.6 Parkinson’s Disease 239
3.2.6.7 Traumatic Brain or Spinal Cord Injury 240
3.2.7 Why not Fully Block Calcium Entry via Pharmacological Agents? 240
3.2.8 Emerging Targets for Reducing Calcium-mediated Excitotoxicity 241
3.2.9 Conclusions and Outlook 242
References 242
3.3 Strategies for Conversion of Peptides to Peptidomimetic Drugs 245
3.3.1 Peptides as Starting Points in Drug Discovery 245
3.3.1.1 Strategy for the Development of Peptidomimetics 246
3.3.1.1.1 Property Elucidation 247
3.3.1.1.2 Structure–Activity Relationship 249
3.3.1.1.3 Bioactive Conformation 249
3.3.1.1.3.1 Secondary Structure Mimetics 251
3.3.2 A Case study of Rational Peptide Lead Optimization: Development of
Small and Constrained Peptides Targeting the Substance P 1-7 Binding
site 256
3.3.2.1 SP1-7 and its Binding Site 257
3.3.2.2 SAR and Truncation Studies of SP1–7 and EM-2 258
3.3.2.2.1 Strategy 258
3.3.2.2.2 Structure–activity Relationship 258
3.3.2.2.3 Effects of SP1–7 and its Analogs 263
3.3.2.3 Design and Synthesis of Small Constrained H-Phe-Phe-NH2 Analogs
264
3.3.2.3.1 Strategy 264
3.3.2.3.2 Structure–activity relationship and ADME properties 265
3.3.3 Conclusion 269
References 271
3.4 Synthetic Vs. Natural Bioactive Compounds Against Tropical Disease

275
3.4.2 Early History of Malaria Treatment; Quinine and Artemisinin 275
3.4.3 Post World War II and the Development of Synthetic Anti-malarials
276
3.4.4 Modern Efforts in Antimalarial Drug Development 276
3.4.4.1 Quinine, 4-Aminoquinolines, and Quinoline Methanols 277
3.4.4.2 8-Aminoquinolines 279
3.4.4.3 Artemisinins and other Endoperoxides 280
3.4.4.4 Repurposed Drugs 281
3.4.5 Natural Products in the Treatment of Malaria 281
3.4.6 Considerations for Anti-parasitic Drug Development 282
References 286
3.5 Current Trends and Developments for Nanotechnology in Cancer 290

3.5.2 Drug Delivery Nanosystems in Cancer Therapy 294
3.5.2.1 Controlled Drug Delivery 294
3.5.2.2 Stimuli-responsive Controlled Drug Delivery Systems 296
3.5.2.3 Combination Therapy 301
3.5.3 Cancer Targeting 303
3.5.3.1 Passive Targeting 305
3.5.3.1.1 The Fundamentals of Passive Targeting and the EPR Effect 305
3.5.3.1.2 Physicochemical Properties of Nanoparticles Affecting the Passive
Targeting 305
3.5.3.1.3 Challenges and Future Prospects of Passive Targeting 308
3.5.3.2 Active Targeting 310
3.5.3.2.1 The Fundamentals of Active Targeting 310
3.5.3.2.2 Factors Affecting Tumor Active Targeting 310
3.5.3.2.3 Ligands for Tumor Active Targeting 312
3.5.3.2.3.1 Monoclonal Antibodies 312
3.5.3.2.3.2 Proteins and Peptides 313
3.5.3.2.3.3 Aptamers 314
3.5.3.2.3.4 Small Molecules 315
3.5.4 Nanotechnology and Immunotherapy 316
3.5.4.1 Nano-based Cancer Immunotherapy 317
3.5.4.2 Nanoparticulate Adjuvants for Cancer Immunotherapy 319
3.5.4.3 Nanoparticle Based DC Targeting for Cancer Immunotherapy 320
3.5.5 Cancer Imaging, Diagnostics and Multifuctional Nanosystems 324
3.5.6 Conclusions and Future Prospects 327
References 328
Index 343
Preface
Biomedical Chemistry focuses on creating molecules that help advance the
understanding and treatment of diseases. Basic chemical ideas and determination
of disease etiology are approached by developing techniques to ensure optimum
interaction between drugs and human cells.
This book provides readers with an understanding of how fundamental chemical
concepts are used to combat some diseases. The authors explain the interdisciplinary
nexus of chemistry with biology, physics, pharmacy and medicine. The results of
chemical research can be applied to understand chemical processes in cells and in
the body, and new methods for drug transportation.
Biomedical Chemistry: Current Trends and Developments is an excellent resource
for students and researchers in health-related fields with frontier topics in medicinal
and pharmaceutical chemistry, organic chemistry and biochemistry.
Nuno Vale
University of Porto, Portugal
Section 1: Chemical Principles in Drug Design and
Discovery
Diana C. G. A. Pinto, João M. P. Pereira, Artur M. S. Silva*1
1.1 Functional Groups of Biomolecules and their
Reactions
Abstract: This chapter starts with a general introduction on some concepts needed to
understand the reactivity of organic functional groups. Elementary reaction mecha-
nisms are then presented according to their functionality with relevant biological
examples. These reactions explain the vast majority of transformations involving bio-
molecules. Finally, two examples on the application of the presented concepts are
given, namely the metabolism of fatty acids and reactivity of penicillin. Both of these
examples call for various types of reactions showing the diversity and simplicity of
biological transformations when analysed step by step.
1.1.1 Functional Groups in Biological Systems
The main definition of a functional group in organic chemistry books is as a chemically

reactive group of atoms within a molecule that contribute to its characteristic
reactivity. Functionality is usually regarded as “implying the presence of heteroatoms
and/or unsaturation, but it would not be helpful to attempt to define precisely the
limits of application of the term” (IUPAC, Commission on Nomenclature of Organic
Chemistry, 1993).
Functional group reactivity may be changed by the presence of other neighbouring
functional groups but usually behaves uniformly in every molecule where it can be
found. There are several common functional groups that are related to families of
organic compounds according to their structural features. However, from those
functional groups only a few are found in biological systems (Table 1.1.1). The types
of bonding found in these functional groups may be explained by the existence of
various hybrid atomic orbitals of the carbon atom created from combination of the
one 2s and the three 2p orbitals (Table 1.1.2).
Diana C. G. A. Pinto, João M. P. Pereira, Artur M. S. Silva: Departamento de Química da Universidade

de Aveiro, 3810-193 Aveiro, Portugal, *E-mail: artur.silva@ua.pt
© 2015 Diana C. G. A. Pinto, João M. P. Pereira, Artur M. S. Silva

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
Functional Groups in Biological Systems 3
Table 1.1.1: Common functional groups present in biomolecules. In parentheses are the names of the
families of compounds, where the group has the highest priority in the compound.
C C
C=C double bond (alkenes)
aromatic ring (arenes)
C C C
C
O N
OH
R-oxyalkyl (ethers)
hydroxyl (alcohols) amino (amines)
C S
C C C S C
SH S
sulfhydryl (thiols/mercaptanes) sulfide (thioethers/sulfides) disulfide (disulfides)
O O
N
C
C C
C C
H C C
formyl (aldehydes)
dialkylcarbonyl (ketones)
imino (imines)
O O O
C C C C
C OH C O C N
carboxyl (carboxylic acids) R-oxycarbonyl (ester) aminocarbonyl/carboxamide

(amides)
O O O O
C C C P
C P O
C S C O
O O
O
sulfanyl O
carbonyl (thioesters) alkyl phosphate (phosphates) acyl phosphate (mixed
anhydrides)
4 Functional Groups of Biomolecules and their Reactions
Table 1.1.2: Possible orbital combinations in the carbon atom, their spatial configuration and geometry
with inter-bond angles.
Combination Hybrid result Geometry
1 x 2s + 3 x 2p 4 x sp3
tetrahedral
1 x 2s + 2 x 2p 3 x sp2
trigonal planar
1 x 2s + 2 x 2p 2 x sp
linear
All of the hybrid orbitals may be used to form σ (sigma) molecular orbitals by fusion
with s or with other hybrid orbitals from other atoms (according to Molecular Orbital
Theory). If there are remaining 2p orbitals in the carbon atoms (in the case of sp2
and sp hybrid orbitals) they are used to form π (pi) molecular orbitals by lateral
combination with other adjacent 2p orbitals (Fig. 1.1.1). A simple bond is formed with
a single σ-bond; the double bond is formed with a σ-bond and a π-bond; the triple
bond is formed with a σ-bond and two π-bonds.
Figure 1.1.1: A simplified representation of the spatial distribution of two adjacent p orbitals (a) and
their combination into a π bond (b) between two sp2 carbon atoms. The π-bonds in a triple bond
would be along orthogonal planes to each other.
Acids and Bases Versus Electrophiles and Nucleophiles 5
These hybridisations have several consequences, such as the electron density of a

π-bond lying above and below the plane of the bonding atoms (Fig. 1.1.1), resulting
in greater exposure for a reaction. Simultaneously, with the increasing s character of
the hybrid orbital:
–– the formed bond length decreases;
–– the polarity of a C-H bond increases;
–– breaking a bond between carbon and a more electronegative atom is more dif-
ficult (e.g. the C-O bond in isopropanol is easier to cleave than the C-O bond in
isopropenol).
The electronegativity of an element can also be an important factor to explain some

functional groups reactivity. For instance, alcohols, ethers, amines, thiols, sulfides,
disulfides and phosphates (Table 1.1.1) all have a carbon forming a single bond with a
more electronegative atom, causing the carbon to bear a partial positive charge (δ+).
These modifications affect both the σ- and π-bonds, although in the case of some
π-bonds the resonance effect should also be considered. The carbonyl group can be
classically treated as a resonance hybrid represented by two resonance structures
(Scheme 1.1.1), which contributes to the reactivity of the compounds that have this
functional group (aldehydes, ketones, carboxylic acids, esters, thioesters, amides,
acyl phosphates). Resonance is possible whenever movement of electrons are allowed
within the same molecule without movement of atoms.
O O O δ−
or
C C C δ+
Scheme 1.1.1: Carbonyl group resonance structures and equivalent notation, explicitly showing the
bond polarity with partial charges.
1.1.2 Acids and Bases Versus Electrophiles and Nucleophiles
Acids and bases are very important in biological transformations, as most require
some form of acid or basic catalysis to occur. The simplest acid-base theory is the
Brønsted-Lowry theory, which states that acids are molecules that donate protons
(hydrogen ions, H+) and bases/alkalis are molecules that accept protons. For example,
a carboxylic acid can donate a proton to a base, such as an amine, in a reversible
proton-transfer reaction (Scheme 1.1.2).
O H O H
+ N
+ N H
1
R OH R H R O R1 H
acid base conjugate base conjugate acid
Scheme 1.1.2: Example of a proton transfer reaction. This specific reaction explains why it is difficult
for condensations to happen directly between an amine and a carboxylic acid, as the non-ionic
forms of these molecules are more reactive (Chapter 1.1.4.5).
Acids can differ in their ability to donate protons, being classified as strong or weak
according to the extent of deprotonation. A strong acid will have a stable conjugate
base (or weak conjugate base), resulting in ready donation of a proton. Table 1.1.3 lists
the acidity of some typical functional groups (water and ammonium acidity are also
given for comparison). The acidity is measured by the acidity constant, Ka or by its pKa
(Scheme 1.1.3), where a stronger acid has a smaller pKa and a weaker acid has a larger
pKa. The same approach can be applied to bases and their strength.
HA + H2 O A- + H3 O+
Scheme 1.1.3: Acidity constant and pKa.
The problem with the Brönsted/Lowry definition is that it only covers the compounds
that donate or accept protons. The more general and widely used model is the Lewis
definition. A Lewis acid is a molecule that accepts a pair of electrons and a Lewis base
is a molecule that donates a pair of electrons. To accept electrons, a Lewis acid must
have a vacant low-energy orbital. As a consequence, many species, including H+ itself,
metal cations such as Mg2+ and Zn2+, and neutral species such as boron trifluoride
(BF3) and carbon dioxide (CO2) are Lewis acids.
Lewis acids and bases are involved in many biological reactions, such as the
transformation of carbon dioxide into hydrogen carbonate (Scheme 1.1.4). Lewis
bases use unshared electrons to form new bonds with other atoms and are usually
referred as nucleophiles (“nucleus-loving”, versus Lewis acids as electrophiles,
“electron-loving”). The terms “electrophile” and “nucleophile” are commonly used
Acids and Bases Versus Electrophiles and Nucleophiles 7
in organic and bioorganic transformations. Electrophiles are either positively charged

or neutral and have at least one positively polarised, electron-poor atom. Conversely,
nucleophiles are either negatively charged or neutral and have a lone pair of electrons
that can be donated.
Table 1.1.3: Relative acidity strengths of some functional groups and common reactants.
Functional group Example pKa

carboxylic acid O 4.76
CH3COH
ammonium 9.26
NH4+
alkylthiol CH3SH 10.3
alkylammonium ion 10.66

CH3NH3+
β-keto ester O O 10.6
CH3CCH2COCH3
water H 2O 15.74
alcohol CH3CH2OH 16.0
ketone O 19.3
CH3CCH3
thioester O 21
CH3CSCH 3
ester O 25
CH3COCH3
δ− O
O carbonic
δ+ anhydrase
HO + C C
HO O
O δ−
Lewis base Lewis acid
Scheme 1.1.4: Example of a biochemical reaction involving a Lewis acid and a Lewis base. Reversible
conversion of carbon dioxide to hydrogen carbonate can occur rapidly in the active site of a carbonic
anhydrase, the equilibrium being regulated by factors such as pH.
1.1.3 Stereoisomerism and Chirality
Stereoisomers having the spatial orientation of bonds as their unique difference,

maintaining the same composition of atoms and bonds.
1.1.3.1 Cis/trans Isomerism
Cis/trans stereoisomerism exists in alkenes (double bonds) and saturated rings

(cycloalkanes), the latter because the π-bonds and σ–bonds of saturated rings cannot
rotate freely. As a consequence, a group in one side of the double bond or saturated
ring cannot change sides without breaking bonds. This causes different properties in
compounds with opposite configurations. A double bond presents a cis-configuration
if equal substituents are on the same side and a trans-configuration if they are on
opposite sides (Fig. 1.1.2). This type of isomerism is particularly relevant for the
physical properties of compounds, with differences being greater if polar groups are
present. Reactivity is also affected, as cis compounds tend to have larger strain on the
bonds making them easier to break.
Although the cis/trans designation is more common, it is worth of mentioning that
Z/E designation is also used in some cases. The general designation of Z/E isomerism
for stereoisomers follows simple rules of priority established by Cahn, Ingold and
Prelog which allow unequivocal classification of each isomer. In this nomenclature,
substituents with equal priority on the same side of the double bond or saturated
ring give the Z-isomer, whereas in opposite sides give the E-isomer. Nevertheless, it is
important to highlight that in biological systems the vinylic systems present, almost
always, hydrogen atoms attached to the vinylic carbons.
O O
H O
HO OH
cis trans
HO C
C C
C OH
H H
O H
maleic acid fumaric acid
m.p. 135oC m.p. 287oC
Figure 1.1.2: Maleic and fumaric acid structures with their respective melting points, highlighting the
importance of cis-trans isomerism for physical properties.
Stereoisomerism and Chirality 9
1.1.3.2 Chirality and Enantiomerism
A compound is defined as chiral if it does not possess any planes of symmetry. This
implies that two chiral molecules, constructed in such a way that one is the reflection
of the other upon a plane, are not superimposable. In this case, these two molecules
are classified as enantiomers. One can refer to carbons as asymmetrical or chiral
whenever all of its substituents are unique, and are also referred to as stereogenic
centers (stereocenters), as they can cause stereoisomerism in the molecule.
A molecule can have more than one stereocenter, such as 1,2-dimethylcyclopropane
with two asymmetric carbons. Molecules that differ in the configuration of one or
more (but not all) stereocenters, whilst being stereoisomers but not enantiomers,
are called diastereomers. Having that in mind, it follows that any of the trans-1,2-
dimethylcyclopropane enantiomers is a diastereomer of cis-1,2-dimethylcyclopropane,
since it is equivalent to switching only one of the methyl groups. The cis-stereoisomer
does not have enantiomers because it has a plane of symmetry. Structures such as cis-
1,2-dimethylcyclopropane are called a meso structures.
plane of
reflection
H H
H 3C
CH 3
CH2 H2C
H3C CH 3
H H
Figure 1.1.3: trans-1,2-Dimethylcyclopropane is an example of a molecule with enantiomers. The 3D

representation (with hydrogen atoms omitted) shows that the enantiomers do not overlap (the asym-
metric carbons are in black).
It is worthy to note that enantiomers only differ by reflection, so their physical

properties are exactly the same because the steric hindrances and dipolar moments
of each molecule are the same. However, one physical property that distinguishes
each enantiomer is its optical rotation: when illuminated, as a solid or in solution,
chiral molecules rotate the plane of polarization of light transmitting through it.
One of the enantiomers rotates this plane clockwise by some amount dependant on
concentration and path length of the light through the sample. The other enantiomer
will rotate light by the same amount but anticlockwise. Racemic mixtures (racemates)
are mixtures with same amount of both enantiomers of a compound and have a null
optical rotation.
Biological systems are highly stereospecific ‒ in general, only one stereoisomer
is reactive towards an enzyme or a certain receptor. As a result, racemate resolution
is of extreme importance for drug synthesis, particularly when one enantiomers has a
negative effect. This problem is often solved by two different approaches:
1. Enzymatic resolution: the racemate is transformed in such a way that the
resulting product may be then cleaved by an enzyme (e.g. lyase). Because of the
stereospecificity of the enzyme, only one of the compounds is cleaved, enabling
separation of cleaved and uncleaved product.
2. Diastereomeric resolution: the racemate is made to react with a specific,
enantiomerically pure reagent (e.g. L-tartaric acid, to form an ester). The obtained
products are diastereomers, which unlike enantiomers possess different
properties and are therefore separable. After separation, the reverse reaction is
performed to return the original enantiomers.
1.1.4 Common Mechanisms in Biological Chemistry
Reactions that occur in living organisms follow the same rules of those occurring
in the laboratory. The solvent, temperature and almost certainly the catalyst can be
different, but the fundamental reaction mechanisms are the same. So conveniently,
common organic reaction mechanisms can be used to understand the equivalent
biological transformations.
1.1.4.1 Nucleophilic Substitution Reactions
Nucleophilic substitution reactions occur when a group attached to an sp3 carbon

is substituted for a more nucleophilic one. These reactions may follow two similar
mechanisms ‒ bimolecular and unimolecular ‒ but with very different implications
for biological systems in terms of stereochemistry.
1.1.4.1.1 SN2 – Bimolecular Nucleophilic Substitution
This type of mechanism (Scheme 1.1.5) is called a bimolecular nucleophilic substitution

(SN2) since the determining step involves the reaction of two species, the nucleophile
and the substrate (electrophile species).
Common Mechanisms in Biological Chemistry 11
Scheme 1.1.5: General reaction for an SN2 reaction mechanism.
The following happens in one concerted step: a nucleophile (N) attacks the carbon
atom as a more electronegative group (X) leaves, with inversion of stereochemistry
through an unstable transition state. Note that N can be a neutral protic nucleophile
that deprotonates after the substitution. This type of mechanism is typical of primary
alkyl halides substitutions. One reaction often performed in a laboratory is an
O-methylation using methyl iodide. The oxygen atom (of an alcohol, for example) acts
as nucleophile substituting the iodide which is a very good leaving group.
1.1.4.1.2 SN1 ‒ Unimolecular Nucleophilic Substitution Reactions

Unimolecular nucleophilic substitutions (SN1) occur when a carbocation intermediate
is stable enough to be transiently formed. In this case, the rate determining step
involves reaction of only one species: the substrate where the substitution will take
place (Scheme 1.1.6).
Scheme 1.1.6: General reaction for a SN1 reaction mechanism.
A carbocation may be easily formed on tertiary carbons because the carbocation is

stabilized through inductive effect by vicinal carbons (R = alkyl or aryl groups).
With this in mind, the more electronegative moiety is able to heterolytically cleave
its bond to the carbon atom. Since the carbocation is planar, there is no preference
for the nucleophile on which side to attack, resulting in a mixture of enantiomers
if the product in question in chiral. In enzymes, this does not happen because the
active sites are chiral themselves, restricting addition to only one side. This type of
mechanism is typical of tertiary alkyl halides substitutions and allylic phosphates. For
example, geranyl diphosphate cleaves at the C-O bond and the corresponding allylic
carbocation, well stabilized by resonance, is attacked by water which deprotonates
to produce geraniol (Scheme 1.1.7).
Scheme 1.1.7: SN1 reaction mechanism of geranyl diphosphate forming geraniol.
1.1.4.1.3 Phosphate Group Transfer – the Grey Area of Nucleophilic Substitutions in

Biological Systems
Nucleophilic substitutions are not restrained to carbon atoms. Phosphate and acyl
group transfer reactions, key pieces in metabolic pathways, are also nucleophilic
substitution reactions although with some differences. In the phosphorylation of
glucose a phosphate group is transferred from ATP to glucose with a phosphorus
atom undergoing a nucleophilic substitution (Scheme 1.1.8).
Scheme 1.1.8: Glucose phosphorylation.

In this reaction, the phosphorus electrophilicity is reinforced trough chelation with

two Mg2+ ions present in the phosphoryl transferase enzyme. In the complex, the
most representative resonance structure places a positive formal charge in the P atom
and negative charges on the O atoms. This makes the tetrahedral phosphate easy to
be attacked by the oxygen atom from the 6-hydroxyl group in glucose, while a base
captures the released proton. The mechanism shown in Scheme 1.1.8 is a simplified
version of what has been observed. The preferred reaction path is not well defined in
biological systems, since it is highly dependent on the nature of the nucleophile and
the enzyme scaffold. Intermediates may or may not be involved. Either a pentavalent
trigonal bipyramidal (associative intermediate) or a metaphosphate (dissociative
intermediate) intermediate may be formed (Scheme 1.1.9). A concerted reaction
mechanism has also been observed, where the substitution is done smoothly in one
step (i.e. SN2 like).
-O O-
O
R P R 1
P
- O O-
O
a b
Scheme 1.1.9: a) Phosphorane intermediate ‒ analogue of the activated complex ‒ in red are the
axial positions (collinear) and in blue are the equatorial positions (coplanar); b) metaphosphate
(planar, stabilised by resonance) ‒ analogue of a carbocation.
1.1.4.2 Electrophilic Addition Reactions
Electrophilic reagents can react with compounds that are electron rich in certain
exposed regions, from which alkenes are a typical example. In these systems, the π
bond results from overlapping of p orbitals and provides regions of increased electron
density above and below the plane of the molecule. π electrons are more loosely bound
than those of a σ-bond so they can interact more easily with a positively charged
electrophilic species, forming a new σ-bond and a carbocation (Scheme 1.1.10). This
in turn rapidly reacts with a nucleophile to form another σ-bond. In this case, the
nucleophile is either an anion or a neutral moiety with free pairs of electron that will
become neutral again eliminating a group or an atom.
E Nu
C C E C C E C C Nu
Scheme 1.1.10: Electrophilic addition reaction mechanism.
1.1.4.2.1 Synthesis of α-Terpineol ‒ Intramolecular Addition

In the following example we can see the simplified (without enzyme interactions)
biosynthetic mechanism of α-terpineol from linalyl diphosphate, which occurs
through an electrophilic addition (Scheme 1.1.11).
Scheme 1.1.11: Synthesis of α-terpineol from linalyl diphosphate.
It should be noted that in this intramolecular addition, the electrophile is generated

via diphosphate ion (PPO-) elimination. The diphosphate ion leaves easily because
it is itself a stable anion and the generated carbocation is also stable, as it is an
allylic carbocation stabilized by resonance. The delocalized positive charge turns the
terminal carbon into a strong electrophile capable of adding to the double bond; the
deficiency of electrons in the electrophile does not always coincide with the atom that
will be attacked. Biological examples of electrophilic reactions occur frequently in
metabolic processes, such as in the β-oxidation pathway of the fatty acid metabolism
(Chapter 1.1.5).
1.1.4.3 Aromatic Substitutions
In simple terms, aromaticity may be described as a chemical property that arises from
delocalization of electrons in a ring. These electrons may be provided by conjugated
unsaturation, lone pair electrons or even orbitals; the system becomes aromatic when
agreeing with the Huckel rule, which are state that the number of the total conjugated
electrons must be 4n+2 electrons. It is a particularly strong form of resonance
stabilization making aromatic compounds more stable than expected otherwise. The
orbital alignment required for aromatic stabilization turns the aromatic (aryl) moieties
planar (Figs. 1.1.4 and 1.1.5). Because of the intermediate character of its bonds (Fig.
1.1.4) the reactivity of aromatic compounds is not identical to the reactivity of other
unsaturated compounds.
Figure 1.1.4: Clarification of the aromatic stabilization of benzene. Hypothetically, cyclohexatriene

would have two different types of bonds: simple and double bonds, but spectroscopic data show
that all bonds are equivalent. The bonds in benzene have an intermediate character between a
simple and a double bond with the electrons delocalized (density evenly distributed).
Figure 1.1.5: Cytosine is an example of a biomolecule exhibiting aromaticity in its imidic acid form.
Furan is an example of a heteroaromatic compound where a lone pair of electrons of the heteroatom
is delocalized into the ring to allow aromaticity. The Fig. explicitly shows all the resonance forms in
the rings of the compounds.
1.1.4.3.1 Electrophilic Aromatic Substitution

A strong electrophile may capture electrons from the ring, forming a very unstable
intermediate. Since the loss of aromaticity is energetically unfavourable, a nucleophile
does not add to the cation. Instead, the ring eliminates a proton (Scheme 1.1.12).
Scheme 1.1.12: Mechanism of an electrophilic aromatic substitution reaction.
The reactivity of the aromatic ring is enhanced if electron donating (hydroxyl, amino
and alkoxyl) groups are attached to the ring, increasing the electron density and
making the attack to the electrophile easier.
1.1.4.3.2 Nucleophilic Aromatic Substitutions

Nucleophilic aromatic substitutions are also possible when electron withdrawing
groups are attached to the ring (e.g. nitro group and carbonyl moieties) allowing it to
accommodate a carbanion, even though aromatic rings are already dense in electrons.
The leaving group also needs to be quite electronegative to leave easily and drive the
reaction forward (Scheme 1.1.13). The exact mechanism varies dependently on the
leaving group having some parallels to normal nucleophilic substitution.
Scheme 1.1.13: General reaction of a nucleophilic aromatic substitution.
1.4.3.3 Hallucinogen Synthesis ‒ Aromatic Substitution on Fungi

Ergot fungi produce a variety of alkaloids often with strong hallucinogenic effects
upon consumption. The first pathway-specific step in their synthesis is the alkylation
of tryptophan by dimethylallyl diphosphate obtaining the dimethylallyl tryptophan
(DMAT) (Scheme 1.1.14). This step consists of an electrophilic aromatic substitution,
catalysed by DMAT synthase.
Scheme 1.1.14: Reaction mechanism for the synthesis of DMAT from tryptophan and dimethylallyl
phosphate.
1.4.4 Eliminations Reactions
An elimination reaction is a type of reaction upon which there is a net elimination

of a molecule from another. To clarify this point, look at the example shown in
Scheme 1.1.15.
Scheme 1.1.15: Dehydrogenation of ethane to form ethene through two different mechanisms:
proton-hydride transfer and a radical mechanism, both leading to the elimination of what is equi-
valent to a hydrogen molecule (the arrows without origin represent electron transfers to/from other
molecules).
A dehydrogenation reaction involves the net elimination of a hydrogen molecule, but

does not necessarily release a hydrogen molecule. It may proceed through abstraction
of a proton connected to one of the carbons followed by transfer of a hydride from the
other. It may also follow a radical mechanism by hydrogen atom abstraction from both
carbons (Scheme 1.1.15). In a dehydrogenation, the elimination is oxidative because
the oxidation state of each carbon atoms goes from -3 to -2, a net molecular change of
+2 (Chapter 1.1.4.8).
In mechanistic terms, non-oxidative eliminations may occur in various steps
having its variations similarly to the nucleophilic substitutions. In all of the following
cases, the reactions shown are [1,2]-eliminations, meaning that the reaction implicates
electron movement between two vicinal carbons. Other types of eliminations may
occur involving more distant carbon atoms and heteroatoms ‒ [1,3] in decarboxylations
or [1,4] in aldol condensations (Chapter 1.1.4.6).
1.1.4.4.1 E1 ‒ Unimolecular Elimination

An E1 reaction mechanism occurs when a relatively stable carbocation may be formed
by elimination of a stable anion (X-), such as a chloride ion (Cl-) for a good leaving group.
The only rate determining (slow) step is the dissociation of the leaving group to form a
carbocation (hence a unimolecular reaction). A base (B) captures the proton released
from one carbon away of the carbocation formed in the E1 reaction mechanism. It is
worthy to note that the E1 mechanism competes with the SN1 mechanism since the
nucleophile (B) may react directly at the halogenated carbon (substitution) or at the
neighbouring hydrogen atom (elimination). Steric hindrance both at the base and at
the carbocation, as well as stronger bases promote elimination.
H B H
C C C C C C + BH+
X + X-
Scheme 1.1.16: General E1 reaction mechanism.
1.1.4.4.2 E2 ‒ Bimolecular Elimination
B + BH+
C C C C
+ X-
X
Scheme 1.1.17: General E2 reaction mechanism.
E2 mechanisms occur through a concerted transfer of a set of electron pairs from the
base to the more electronegative group (X), the latter leaving as its anion and a vicinal
proton is transferred to the base (Scheme 1.1.17). This mechanism is preferred if no
stable ionic intermediate can be formed. The double bond is formed in a step involving
the two reagents, thus the elimination mechanism is bimolecular. It is noteworthy
that the E2 mechanism competes with the SN2 mechanism, as both reactions involve a
base and an electronegative leaving group. More steric hindrance and stronger bases
favour an E2 mechanism over SN2.
1.1.4.4.3 E1cB ‒ Unimolecular Elimination through Conjugate Base
H BH+ X-
B
C C C C C C
X X
Scheme 1.1.18: General E1cB reaction mechanism.

E1cB mechanism is the symmetric version of the E1 mechanism. First, the proton
is removed from the main molecule to form its conjugate base, a carbanion, which
promotes the elimination of the electrophilic group. This mechanism is preferred
whenever a carbanion intermediate is stabilized, in most of the cases by resonance
(Scheme 1.1.18). This mechanism is particularly relevant in biological transformation,
as it is by far the most frequent elimination mechanism because of the high occurrence
of carbonyl compounds which form relatively stable carbanions.
1.1.4.5 Nucleophilic Carbonyl Addition Reactions
This type of reaction happens between a nucleophile and a carbonyl group where a
pair of electrons from the nucleophile is transferred to the carbonyl carbon (Scheme
1.1.19).
δ−
O O
Nu
+
Cδ C
R R
Nu R R
1 2 1 2
Scheme 1.1.19: General simplified scheme for a nucleophilic addition reaction
If the nucleophile is neutral and gives a pair of electrons then it will acquire a positive
charge, which in turn is compensated by a deprotonation of the introduced group
(as in the formation of imines; Scheme 1.1.20c). If the nucleophile is anionic, no
positive charge is generated. In both cases, a negative charge is generated at the most
electronegative atom, the carbonyl oxygen, which is usually neutralized through
protonation (Scheme 1.1.20). Scheme 1.1.20 illustrates some of typical nucleophilic
addition reactions of different nucleophiles to aldehydes and ketones.
All the reactions depicted in Scheme 1.1.20 are called “direct” or [1,2]-additions.
A special case of nucleophilic addition reactions is the Michael or [1,4]-conjugate
addition. This reaction is quite frequent in biochemical pathways and consists of
a nucleophile addition to the β–position of an α,β-unsaturated carbonyl system
(Scheme 1.1.21).
a b c d
- Nu = RNH2
Nu = H Nu = ROH Nu = H2N-NH2
O O O O
C C R C R C NH 2
H O N N
H H2 H2
OH OH OH OH
C C R C R C NH 2
H O N N
H H
alcohol hemiacetal carbinolamine carbinolhydrazine

+ROH
-H2O -H2 O
-H2 O
OR
C R C R C NH2
O N N
acetal imine hydrazone
Scheme 1.1.20: Nucleophilic addition reactions: a) with a hydride ion as nucleophile; b) with an
alcohol as nucleophile; c) with an amine as nucleophile; and d) with a hydrazine as nucleophile.
Scheme 1.1.21: General scheme for a Michael or [1,4]-conjugate addition reaction.

As with electrophilic addition reactions, the nucleophile may be added to any of

the p orbitals, leading to the formation of enantiomer mixtures (in the case of α,β-
unsaturated ketones, monosubstituted at β-position or disubstituted with two
different groups). However, enzymes can be enantioselective and produce only one
enantiomer.
1.1.4.5.1 Nitrofurantoin ‒ a Semicarbazone

Semicarbazones (Scheme 1.1.22) are a family of compounds classified as imine
derivatives, originating from the action of semicarbazines on aldehydes or ketones
(instead of amines). The –NH2 group of a semicarbazide is akin to a primary amino
group. Thus, the reaction mechanism is the same as with other nucleophilic carbonyl
addition reactions.
Scheme 1.1.22: General scheme for a semicarbazone synthesis.
There are some semicarbazones with pharmacological interest, such as nitrofurantoin

(non-systematic name). Nitrofurantoin is a nitrofuran-based antibiotic considered
an essential medicine by the World Health Organization. Scheme 1.1.23 presents the
mechanism of the nitrofurantoin synthesis from 5-nitrofuran-2-carbaldehyde and
1-aminoimidazolidine-2,4-dione.
Scheme 1.1.23: Synthesis of Nitrofurantoin from 5-nitrofuran-2-carbaldehyde and

1-aminoimidazolidine-2,4-dione.
The reaction steps are the same as in any other nucleophilic carbonyl addition reaction
followed by dehydration:
1. nucleophilic attack of the nucleophilic amino moiety to the electrophilic carbo-
nyl carbon;
2. deprotonation of the positive nitrogen atom and protonation of the negative
oxygen atom;
3. elimination of water by protonation of the hydroxyl group and deprotonation at
the nitrogen atom.
This synthetic route as a whole is a condensation reaction: two molecules produce a

larger molecule with the loss of a small molecule, in this case water. In fact, imine and
hydrazone synthesis are condensations too, but were introduced as additions (see
above) since the condensation product is often readily generated from the unstable
addition product unless very strict reaction conditions are used.
1.1.1.4.6 Acyl Substitution Reactions

Acyl substitutions are another class of reactions involving carbonyl groups (Scheme
1.1.24). An acyl substitution reaction is favoured over a simple addition whenever
an electronegative group is attached to the carbonyl group. This property is a main
feature presented by carboxylic acids and their derivatives.
Scheme 1.1.24: General mechanism for an acyl substitution reaction.
The first step of this mechanism is shared with nucleophilic addition reactions
and consists in the nucleophilic addition to the carbonyl group. The second step is
a regeneration of the carbonyl group with elimination of the anion. It is important
to note that the whole reaction is reversible unless some product stabilization is
provided, such as if the leaving group is a stable anion, which is unlikely to be able
to attack the carbonyl again. Acyl phosphates are considered activated analogues of
carboxylic acids, as the leaving phosphate is a very stable anion and it is unlikely
that a better leaving group is attached to a carbonyl group. This shifts the equilibrium
towards product formation. On the other hand, a simple carboxylic acid would have
the hydroxyl as leaving group which is a strong nucleophile capable of adding onto
the carbonyl again. This is the reason why esters, thioesters and acyl phosphates
play a major role in promoting substitution reactions within biological systems. In
the laboratory environment it is more common the use of carboxylic acid anhydrides
and acyl chlorides or bromides. The resulting anions (carboxylates and halogens,
respectively) are very weak bases promoting the completeness of the reaction. Because
of that, these compounds are very sensitive to hydrolysis thus rendered useless in
biological systems.
1.1.4.6.1 Aspirin ‒ Esterifications and Transesterifications

Aspirin, acetylsalicylic acid or, by its systematic name, 2-acetoxybenzoic acid, is a
nonsteroidal anti-inflammatory drug that inhibits the formation of prostaglandins
and thromboxanes by inactivating cyclooxygenases (COXs). It is synthesized by
esterification of salicylic acid with acetic anhydride (Scheme 1.1.25). One of its
action pathways involves the acetylation of a serine residue in the enzyme through a
transesterification reaction (Scheme 1.1.26).
Esterification is an acyl substitution reaction where an acyl group is transferred
to the oxygen atom of the alcohol. In the synthesis of aspirin, acetic anhydride is
used for efficiency reasons as it is a much better acetylating agent than acetic acid
(Scheme 1.1.25).
Scheme 1.1.25: Simplified mechanism for the synthesis of aspirin from salicylic acid and acetic anhy-
dride. This reaction is normally performed under acid catalysis with sulfuric or phosphoric acids.
Transesterifications are acyl substitution reactions where the nucleophile is an

alcohol and the electrophile is an ester. Essentially, the alkoxyl moiety in the ester
is substituted by another. In the case of aspirin, the alcohol/nucleophile is the side
chain of the serine residue in the COX enzyme which substitutes the alcohol moiety of
the salicylic acid (Scheme 1.1.26).
Scheme 1.1.26: Simplified mechanism of the transesterification reaction of aspirin with the serine
residue.
1.1.1.4.7 Carbonyl Condensation Reactions

As we have seen before, a condensation reaction joins two molecules together to form
a bigger one and liberates a small one (e.g. water, methanol, acetic acid). Carbonyl
condensations occur with two carbonyl compounds. This type of reaction is a very
important one, adding to the chemical versatility that carbonyl groups grant to a
system. Carbonyl condensations are essentially nucleophilic substitutions that allow
easy carbon-carbon bond formation in relatively mild conditions (certainly within
the reach of an enzyme), adding chain formation and polymerization to the list of
enzyme-catalysed reactions.
1.1.4.7.1 Aldol Reaction

An aldol reaction involves carbonyl compounds such as aldehydes or ketones in
which at least one species has an α-proton (Scheme 1.1.27). Although it is not formally
a condensation reaction unless dehydration happens, it is often named as aldol
condensation in biochemical fields. The “proper” aldol condensation product though
is the result of a dehydration of the formed aldol.
Scheme 1.1.27: General mechanism for an aldol condensation reaction. The aldol adduct suffers a
dehydration for which the mechanism is not specified since the precise step sequence may vary
according to catalyst used.
First of all, the enolate is generated from a ketone or an aldehyde by an α-deprotonation

with a base. The enolate is a strong nucleophile and a nucleophilic addition occurs
unto an aldehyde (or ketone) acting as electrophile. The resultant anion, the aldolate,
is protonated to produce the aldol, a β-hydroxy-aldehyde or ketone. The aldol may
then eliminate a water molecule yielding an α,β-unsaturated aldehyde or ketone
(Scheme 1.1.27).
1.1.4.7.2 Claisen Condensation

Claisen condensation is the base-catalysed condensation reaction of an ester
with another carbonyl compound taking place through the mechanism depicted in
Scheme 1.1.28.
Scheme 1.1.28: General mechanism for a Claisen condensation reaction.

Firstly, the enolate is generated from an ester by α-deprotonation with a base. The
enolate performs an acyl substitution followed by elimination of the alcoxyl group,
giving a β-keto ester (Scheme 1.1.28).
1.1.4.7.3 Aldolases ‒ Stabilization Strategies

In order to better perform aldol additions, enzymes create stabilized intermediate
forms that provide a lower energy reaction path (Scheme 1.1.29). In the case of class II
fructose-biphosphate aldolases, a zinc(II) ion is used to further polarize the carbonyl
C=O bond of dihydroxyacetone phosphate (DHAP). The latter deprotonates easily, as
the enolate intermediate is stabilized by the zinc ion. The enolate then attacks the
carbonyl carbon of the glyceraldehyde 3-phosphate (GA3P or GAP), producing the
addition product fructose biphosphate. This reaction is completely stereospecific as
with all enzyme catalysed reactions this statement in parenthesis should be in the
description of Scheme 1.1.29. The reverse reaction (hydrolysis) is also catalysed by
another enzyme.
Scheme 1.1.29: Mechanism of an aldol addition in a class I fructose-biphosphate aldolase.
Class I fructose-biphosphate aldolases have another stabilization mechanism that

does not resemble a regular aldol reaction, though the final product is the same
(Scheme 1.1.30). The mechanism occurs in the following abbreviated steps:
1. A carbinolamine is formed by nucleophilic addition of a lysine residue to DHAP
2. The carbinolamine eliminates water through acid/base catalysis, forming an
enamine
3. The enamine acts as an enol (through analogous resonance structures) and adds
to GAP
4. Water is added to the resulting iminium ion (hydrolysis is the reverse of the imine
synthesis)
5. The carbonyl and lysine residue are regenerated from the new carbinolamine.
Scheme 1.1.30: Simplified mechanism of an aldol addition in a class II fructose-biphosphate

aldolase.
1.1.4.8 Oxidations and Reductions
Oxidation and reduction reactions, or simply redox reactions, are a complex class of
reactions with diverse mechanisms.
The procedure for determining the oxidation state of a carbon atom is similar to
other compounds (Fig. 1.1.6):
1. For each bond to less electronegative atoms, such as a hydrogen atoms, count
as -1
2. For each bond to more electronegative atoms, such as oxygen atoms, count as +
3. Bonds between carbon atoms do not affect the oxidation state, unlike other
elements.
H H H CH3 H OH H OPO3 2- O
C C C C C
H H H H H H H3C H H H
-4 -3 -2 -1 0
H SH Cl CH 3 O O
C C C C
H 3C NH2 H3C OH H3C OH H 2N OH
+1 +2 +3 +4
Figure 1.1.6: Compounds with the oxidation state of the highlighted carbon indicated below each
structure. In order: methane, ethane, methanol, ethyl phosphate, formaldehyde, (R)-1-aminoethane-
1-thiol, 1,1-dichloroethan-1-ol, acetic acid and carbamic acid.
The simplest redox reaction in a biological system is the oxidation of an alcohol to a

carbonyl compound. In a laboratory, a metal in a high oxidation state is usually used
as oxidant, where it attaches to the oxygen of the alcohol then acts as a leaving group
with an E2-like mechanism (Scheme 1.1.31).
Scheme 1.1.31: General mechanism for the oxidation of an alcohol to a carbonyl compound with a
metal or its complex (M). Note that the M leaves in a lower oxidation state.
In the case of an aldehyde, the carbonyl group may be oxidized to carboxyl by

nucleophilic attack of water generating a hydrated aldehyde, in which one of the
hydroxyl groups is then oxidized to carbonyl yielding the carboxyl group.
In biological systems however, the majority of the hydroxyl/carbonyl redox reactions

involve the coenzyme NAD+ (oxidized nicotinamide adenine dinucleotide) or NADP+
(oxidized nicotinamide adenine dinucleotide phosphate) in lieu of a metal catalyst
(Fig. 1.1.7). The oxidation reaction occurs in just one step: as a base captures the proton
attached to the oxygen atom, a hydride is transferred to NAD+ and the carbonyl group
is generated (Scheme 1.1.32). The hydride transfer is a simple conjugate nucleophilic
addition, as the ion is a strong nucleophile. The reduction reaction is simply the
reverse of the oxidation process (Scheme 1.1.33).
Figure 1.1.7: Structure of NAD+ and NADH. The structures of NADP+ and NADPH are identical with
exception of the highlighted hydroxyl group (in magenta), which is replaced by a phosphate group.
Scheme 1.1.32: Mechanism of an alcohol oxidation by NAD+ or NADP+ ‒ the base may be provided by
residues of an enzyme.
Scheme 1.1.33: Mechanism of an alcohol reduction of a carbonyl group by NAD or NADP, in this case
of an acetylated acyl carrier protein (ACP), an important part in the fatty acid synthesis.
1.1.4.8.1 Disulfide Bridges ‒ Oxidized Thiols

Disulfide bridges are essential in a protein to allow stable structural scaffolds. They
are S‒S bonds between cysteine residues (Scheme 1.1.34). Note that the sulfur loses
a bond to a hydrogen and gains one to another sulfur atom, so the formation of this
linkage is a redox reaction with thiols being oxidized. The oxidant (to be reduced) is a
glutathione dimer (GSSG), which consists in two glutathione molecules connected by
a disulfide bridge (Scheme 1.1.35).
Scheme 1.1.34: Disulfide bridge formation.
Scheme 1.1.35: Mechanism of disulphide linkage formation by a glutathione dimer, releasing two
reduced glutathione molecules.
1.1.5 The Organic Mechanisms of Biological Transformations
Previously, we highlighted that common organic reaction mechanisms can be used

to understand the biosynthetic pathways. Herein some illustrative examples are
presented.
1.1.5.1 Cis/trans-Isomers Interconversion in the Vision Pathway
It is common knowledge that vitamin A, retinol (Scheme 1.1.36), plays an important

role in our vision. Although the sequence of reactions and detailed transformations are
not in the scope of this chapter, it is remarkable that a simple oxidation and change in
configuration is ultimately responsible for a complex process such as vision. Cis/trans
isomerase enzymes, through a cysteine residue, are responsible for this isomerization.
The next step is Schiff base (imine) formation through the reaction of retinal with
a lysine residue of the protein opsin to produce rhodopsin, which isomerizes upon
absorption of visible light. This change in geometry causes an electrical signal that
is sent to the brain. Finally, the hydrolysis of the imine linkage regenerates the opsin
protein and (11E)-retinal (Scheme 1.1.36).
Scheme 1.1.36: The chemistry of vision.

The Organic Mechanisms of Biological Transformations 33
1.1.5.2 Metabolism of Fatty Acids ‒ β-Oxidation Pathway
The catabolism of fatty acids (saturated or unsaturated) starts with chemical activation
by esterification with coenzyme A (Scheme 1.1.37), with the following steps occurring
in an acyl-CoA synthase:
1. The carboxylate acts as a nucleophile to attack the double P‒O bond in ATP;
2. The diphosphate group leaves, resulting in an activated acid in the form of a
mixed anhydride (acyl AMP). (PPi is a good leaving group as it is stable and not
nucleophilic)
3. Another acyl substitution is performed on the mixed anhydride by the thiol
moiety in CoA (a moderate nucleophile). Again, this is facilitated because AMP is
a good leaving group.
Scheme 1.1.37: Activation of a fatty acid via esterification.
With the thioester (activated fatty acid) formed, β-oxidation may now occur through a
sequence of dehydrogenation, hydration and dehydrogenation reactions:
1. The Cα-Cβ single bond is oxidised to a double bond, with flavin adenine nucleotide
(FAD) as the oxidant. This step occurs in a family of acyl-CoA dehydrogenases
where the products are FADH2 (reduced FAD) and α,β-unsaturated acyl-CoA. The
mechanistic details of this step are not yet fully resolved, though it is known that
one of the α protons may be first attacked by 376-Glu and a β-hydride is abstracted
from the fatty acid by FAD.
acyl CoA a ,b-unsaturated acyl CoA
(thioester) FAD FADH2
O O
H2 H
C C
C SCoA C SCoA
H2 H
2. Enoyl-CoA hydratase performs a nucleophilic addition of water to the unsaturated

double bond. The hydroxyl (nucleophile) is added exclusively in the β-position to
the carbonyl since the enzyme is stereospecific.
3. The formed hydroxymethylene group is oxidised to a carbonyl group with

a β-hydroxyacyl-CoA dehydrogenase. This reaction yields an NADH ion, formed
from the coenzyme NAD+ acting as the oxidant, and the β-ketoester.
4. A retro-Claisen reaction cleaves the β-ketothioester yielding two thioesters,

producing the initial acyl-CoA shortened by two carbons and acetyl-CoA (this
proceeds to the citric acid cycle, ultimately being oxidized to CO2).
The pathway is repeated until all of the fatty acid is oxidised. If, during the cleavage
process, a cis-oriented unsaturation is encountered, the stereochemistry of the double
bond is switched to trans by an isomerase and the reaction sequence continues as
for saturated fatty acids, since the enoyl-CoA hydratase is stereospecific for a trans-
configuration.
1.1.5.3 Penicillin ‒ a Strong Acylating Agent
Historically, penicillin antibiotics are one of the most important classes of antibiotics
and are worth studying since many different reactions in its synthesis and mode of
action are involved. The essential feature in penicillin class antibiotic’s structure is
shown in Fig. 1.1.8. Penicillins are part of the bigger class of β-lactam antibiotics,
named because the β-lactam ring is the active site of the compound. A lactam is a
cyclic amide, and the β means there are 2 carbons in between the O and N atoms in
their structure.
Figure 1.1.8: On top, the penicillin core structure is depicted. The β-lactam ring is highlighted in
magenta, and in yellow the thiazolidine ring. R is a variable group and the structures on the bottom
are those of the named antibiotics in this group. There are many other known possible groups that
improve certain properties (absorption, hydrolysis, resistance, etc.).
1.1.5.3.1 Transpeptidase Mechanism

Penicillin acts by irreversibly inhibiting the enzyme transpeptidase, which catalyses
the formation of a peptide bond between an alanine and a terminal glycine of
different polypeptides present in the cell wall. This halts the fabrication of new
peptide crosslinks in the peptidoglycan layer, which degrades and eventually leads to
cytolysis. The following steps constitute the catalysis mechanism of transpeptidase:
1. One peptide chain is linked to the enzyme via an acyl substitution reaction. The
nucleophile is the hydroxyl from a serine residue of the transpeptidase and the
leaving group is the C-terminal of alanine.
2. Another acyl substitution reaction involves a second peptide chain that displaces
the link to the hydroxyl of a serine residue resulting in the two peptide chains
being linked.
1.1.5.3.2 Transpeptidase Inhibition

As seen above, the core transformation in the transpeptidase reaction is an acyl
substitution reaction. Penicillin (Scheme 1.1.38) is similar to the normal transpeptidase
substrates, so it mimics the substrate and binds irreversibly to the enzyme active site.
What makes penicillin so effective is that the β-lactam ring is under considerable
strain, making the reaction irreversible. The thiazolidine ring further increases the
strain by distorting the bonds and removing resonance stabilization. The β-lactam in
anionic form is also protected from hydrolysis so the absorption is more efficient.
Scheme 1.1.38: Mechanism of the acyl substitution reaction occurring with penicillin and the serine
residue from transpeptidase.
1.1.5.3.3. Penicillin Biosynthesis

Penicillin biosynthesis begins with the formation of the tripeptide L-δ-(α-
aminoadipoyl)-L-cysteinyl-D-valine (ACV) (Scheme 1.1.39) by condensation of three
amino acids catalysed by ACV synthase. ACV is then processed by isopenicillin-N
synthase, an enzyme of the oxyreductase family, with the following mechanistic steps
(Scheme 1.1.40):
1. Attachment of the cysteine thiol moiety by displacing a water ligand
2. Oxidation of Fe(II) to Fe(III) by molecular oxygen, creating a radical species
3. Intramolecular hydrogen transfer, oxidation of the thiol to thioaldehyde
(extremely reactive due higher dipole moment than that of an aldehyde) and
reduction of Fe(III) to Fe(II)
4. Amide deprotonation by the hydroperoxide ligand and nucleophilic addition of
the nitrogen to the thiol with oxidation of Fe(II) to Fe(IV). This transformation
results in the lactam ring formation
5. Radical formation by hydrogen abstraction by the oxide (turns into an hydroxide
ligand) with reduction of Fe(IV) to Fe(III)
6. Radical attack of the sulfur atom, closing the thiazolidine ring and reducing
Fe(III) to Fe(II)
7. Displacement of the sulfur by water to restore the enzyme active site and release
isopenicillin-N.
Scheme 1.1.39: Equation for the synthesis of ACV.

Scheme 1.1.40: Mechanism for the synthesis of isopenicillin-N by the action of isopenicillin-N
synthase.
The isopenicillin-N may then proceed to be epimerised by isopenicillin-N epimerase

(Scheme 1.1.41). This enzyme acts by α-deprotonation of the amino adipoyl moiety
followed by protonation on the opposite side of the plane of the formed carboanion.
Scheme 1.1.41: Reaction scheme of the epimerisation of isopenicillin-N.
Substitutions of the side chain can also be performed by penicillin acylase (PA) to
give other types of penicillin. PA catalyses the hydrolysis and synthesis of the amide
bond to some side chain. The use of this enzyme in vitro enables the formation of
semi-synthetic penicillin.
1.1.5.4 NAD+ − a Classical Coenzyme
An enzyme cannot catalyse oxidation reactions unless a coenzyme is present. In some

sense, the enzyme’s role is to hold the substrate and coenzyme together to facilitate
the oxidation reaction. One of the most commonly used coenzymes is nicotinamide
adenine dinucleotide (NAD+) (Fig. 1.1.9). There is evidence that sirtuins are proteins
related to several diseases and are NAD+ dependent.
Figure 1.1.9: Structure of nicotinamide adenine dinucleotide (NAD+).
The most known intervention of coenzyme NAD+ is its role in the ethanol metabolism,
where it acts as a common hydride acceptor (Scheme 1.1.42).
Scheme 1.1.42: Mechanism of ethanol oxidation in cells.

1.1.5.5. FAD − a More Versatile Coenzyme
Flavin adenine dinucleotide (FAD) (Fig. 1.1.10) is another coenzyme used in oxidation
reactions and is normally associated tightly to a protein, usually designated as a
flavoprotein. For example, in chorismate synthase, FAD is linked through the flavin
ring C(2)=O to one protein histidine residue (His106) (Scheme 1.1.44). FAD is considered
a more versatile coenzyme than NAD+, because it can participate in oxidation
reactions by several different mechanisms. For example, oxidation of dihydrolipoate
involves a nucleophilic attack on C-4a whereas oxidation of an amino acid involves a
nucleophilic attack on the N-5 position (Scheme 1.1.43).
Figure 1.1.10: Structure of flavin adenine dinucleotide (FAD).
Scheme 1.1.43: Examples of FAD versatility in oxidation reactions.

Furthermore, FAD oxidation mechanisms are controversial because in some proposals,

such as the chorismate synthase reaction (Scheme 1.1.44), the authors suggest the
involvement of ionic and radical structures.
Scheme 1.1.44: Chorismate synthase reaction.
1.1.5.6 Biotin and Carboxylation Reactions
Biotin-dependent enzymes are common in living organisms and are involved in

carboxylation reactions. Biotinylation occurs by the addition of a biotin molecule to a
specific lysine residue (Fig. 1.1.11).
Figure 1.1.11: Biotin and enzyme-bound biotin.

The carboxylation catalysed by biotin-dependent enzymes use carbonate (HCO3⁻)

as source of the carboxyl group, ATP to activate it and Mg2+ to decrease the overall
negative charge. The mechanism involves a nucleophilic attack of the biotin moiety
on the activated carbonate, resulting in the formation of carboxybiotin (Scheme
1.1.45). Nucleophilic attack by the substrate on carboxybiotin results in the transfer
of the carboxyl group from biotin to the substrate, as shown in Scheme 1.1.45 with
acetyl-CoA.
Scheme 1.1.45: Acetyl-CoA carboxylation mechanism.
References
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Francisca Lopes, Maria M. M. Santos and Rui Moreira*2
1.2 Designing Covalent Inhibitors: A Medicinal
Chemistry Challenge
1.2.1 Introduction
The design of enzyme inhibitors is one of the most attractive research topics in
Medicinal Chemistry. In particular, covalent inhibitors provide the opportunity of
combining concepts of chemical reactivity and mechanisms of organic reactions with
the structural features required for optimal molecular recognition in order to obtain
the appropriate reactivity and selectivity profile towards the desired enzyme target.
Typically, these inhibitors present an electrophilic functionality capable of reacting
irreversibly with catalytic amino acid residues containing a nucleophilic group (e.g.
serine, threonine and cysteine) (Powers, 2002; Santos, 2007). In spite of its tremendous
potential (Robertson, 2007; Potashman, 2009), designing selective covalent inhibitors
remains a challenging task as the electrophilic groups present in many inhibitor
structures can also react with other macromolecules leading to deleterious (off-
target) events, or can be scavenged by ubiquitous low-molecular-weight nucleophiles
such as glutathione leading to sub-optimal drug concentration at the site of action
(Johansson, 2012). However, there are several examples of covalent inhibitors that are
widely used drugs, including acetyl salicylic acid (the active ingredient of Aspirin),
orlistat (anti-obesity drug) and ampicillin (antibiotic) (Fig. 1.2.1). Many of these drugs
were not originally designed as irreversible inhibitors and their exact mechanism of
action was often discovered afterwards. A recent example is the case of clopidogrel
(antiplatelet agent), which was found to require activation by cytochrome P450 in the
liver to generate an active metabolite containing a free thiol capable of reacting with
a cysteine residue of adenosine 5´-diphosphate (ADP) receptor to form a covalent
disulfide adduct (Fig. 1.2.1). Overall, nearly 30% of the enzymes that are inhibited by
marketed drugs are irreversibly inhibited via covalent modification, which highlights
the therapeutic potential of covalent inhibitors (Robertson, 2005; Robertson, 2007;
Johansson, 2012).
Francisca Lopes, Maria M. M. Santos and Rui Moreira: Research Institute for Medicines (iMed.ULis-
boa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal,
*Email: rmoreira@ff.ul.pt
© 2015 Francisca Lopes, Maria M. M. Santos and Rui Moreira

Designing Safer Covalent Inhibitors 45
Figure 1.2.1: Drugs acting as covalent inhibitors including their protein target(s) with active-site nuc-
leophiles. Arrows indicate the reaction of the enzyme’s nucleophilic residue with the electrophilic
site at the drug or its metabolite resulting in covalent modification of the target.
1.2.2 Designing Safer Covalent Inhibitors
Several approaches have been adopted by researchers in academia and pharmaceutical

companies to overcome the liabilities associated with covalent inhibitors. A widely
adopted strategy to develop highly selective enzyme inhibitors has been the design of
mechanism-based or suicide inhibitors (Powers, 2002; Lucas, 2013). These are substrate
analogs containing a poor electrophile moiety that are activated by the catalytic
machinery of the target enzyme to generate a more electrophilic species capable of
reacting with a nucleophile in the active site, leading to irreversible inhibition of
the enzyme. This challenging approach has led to the discovery of suicide inhibitors
(Fig. 1.2.2), which are in clinical use such as vigabatrin, an anticonvulsivant agent
designed to irreversibly inhibit the GABA transaminase. Other suicide inhibitors such
as tranylcypromine (monoamine oxidase inhibitor, antidepressant) and selegiline
(monoamine oxidase inhibitor, antiparkinsonian) had their mechanism of action
discovered serendipitously.
A second widely used strategy is to modulate the reactivity of the electrophilic
site in the inhibitor and to optimize the molecular recognition towards the target
enzyme. This approach was successfully used to develop selective Michael acceptors
inhibitors towards cysteine proteases expressed by several viruses and parasites
that are crucial for the development and infectiveness of these infectious agents.
Molecular hybridization, where two different pharmacophores are joined through
a linker (Meunier, 2008), has also emerged as a useful tool in medicinal chemistry
to modulate the reactivity and improve selectivity of toxic compounds, including
46 Designing Covalent Inhibitors: A Medicinal Chemistry Challenge
covalent irreversible inhibitors. These approaches will be dealt in more detail in the
following sections.
N
H 2N CO2 H NH2
Vigabatrin Tranylcypromine Selegiline
Figure 1.2.2: Mechanism-based covalent inhibitors. For detailed description on their mechanism of
action see Silverman, 1992.
Fragment-based methods have been developed to rapidly discover selective irreversible

covalent inhibitors of cysteine proteases with tempered reactivity. Typically, an initial
assessment of the intrinsic reactivity of a panel of low-molecular weight Michael
acceptors is performed using papain as a model cysteine-dependent enzyme (Santos,
2007), allowing the selection of the most effective warhead (Kathman, 2014). In this
way, a fragment with the most specific binding affinity, rather than the most reactive
fragment, might be identified for future optimization.
More recently, the concept of reversible covalent inhibitors has emerged as
a powerful approach to avoid toxicity issues often associated to the formation of
irreversible covalent adducts with off-targets. Although frequently designed to
inactivate conserved, catalytically essential cysteines, covalent inhibitors can
also achieve maximal selectivity among related targets by exploiting the intrinsic
nucleophilicity of poorly conserved, solvent-exposed non-catalytic cysteines
(Singh, 2011). Elegant work developed by Taunton and collaborators paved the way
to establish the chemical basis for designing reversible, cysteine-targeted covalent
inhibitors. Based on a report that revealed that simple thiols reacted instantaneously
with 2-cyanoacrylates at physiological pH, although the corresponding products could
not be isolated or structurally characterized (Pritchard, 1968), Taunton hypothesized
that this scaffold underwent Michael-type conjugate addition via a rapid-equilibrium
process (Serafimova, 2012). Using a simple model reaction of different Michael
acceptors with β-mercaptoethanol (BME) monitored by NMR, the group showed
that, while compounds with a single electron-withdrawing group (e.g. 3-phenyl
acrylonitrile, Fig. 1.2.3A) led to a stable adduct, their counterparts with two electron-
withdrawing groups (e.g. ethyl 3-phenyl-2-cyanoacrylate, Fig. 1.2.3A) formed an adduct
that rapidly reverted to the starting material upon ten-fold dilution with phosphate
saline buffer (PBS). Combining this result with the predicted binding orientation of
the pyrrolopyrimidine scaffold, similar to the irreversible fluoromethylketone-based
inhibitor developed previously by the same group for the C-terminal domain of the
Designing Safer Covalent Inhibitors 47
p90 ribosomal protein S6 kinase RSK2, led to the discovery of the first reversible
covalent kinase inhibitor (Fig. 1.2.3B) (Serafimova, 2012). Additional scaffolds were
discovered de novo using a fragment-based ligand approach, where a library of low
molecular-weight cyanoacrylamides was screened against several kinase assays
(Fig. 1.2.3B) (Miller, 2013).
Figure 1.2.3: A) Thiol reactivity of electron-deficient olefins; B) Reversible covalent inhibitors that
selectively target the non-catalytic cysteine-436 present in the C-terminal domain of the p90 riboso-
mal protein S6 kinase RSK2.
The reversibility of thiol addition to electron-deficient olefins relates to the propensity

of the resulting adduct to undergo β-elimination via an E1cB mechanism (Fig. 1.2.4). A
kinetic study to determine the β-elimination rates of BME from the adduct highlighted
the structural features required to design reversible covalent inhibitors (Krishnan,
2014). Remarkably, the rates were shown to correlate inversely with the computed
proton affinity of the corresponding carbanions, suggesting that the acidity of the
proton at the α-position of the adduct provides the driving-force for the β-elimination
(Fig. 1.2.4). In this way, a feasible method is now available to fine-tune the intrinsic
reversibility of the thiol-Michael reaction in a predictable way.
Figure 1.2.4: β-Elimination half-lives for the adducts from electron-deficient acrylonitriles and
β-mercaptoethanol (BME) – adapted from Krishnan, 2014.
1.2.3 Case Study 1: Michael Acceptors to Treat Infectious Diseases
Electrophilic compounds that act as irreversible enzyme inhibitors are generally

considered unsuitable drugs by medicinal chemists because the covalent binding
might lead to off target effects, and many compounds are metabolically unstable
(Wilson, 2013). However, this mode of action is quite common in approved drugs
and biologically active molecules that inhibit enzymes (Johansson, 2012). In fact,
several electrophilic compounds containing a Michael acceptor were described as
potent cysteine protease inhibitors (Powers, 2002; Santos, 2007). Typically, Michael
acceptor cysteine protease inhibitors have a peptide component (or mimic) that
binds to subsites of the cysteine protease target. These compounds have considerable
potential utility for therapeutic intervention in a variety of diseases, such as malaria,
Chagas’ disease and the common cold.
Case Study 1: Michael Acceptors to Treat Infectious Diseases 49
Peptidyl Michael acceptor inhibitors were introduced by Hanzlik and co-workers

as specific irreversible inhibitors of the cysteine protease papain (Hanzlik, 1984;
Thompson, 1986; Liu, 1992). Following that work, several vinyl sulfones and α,β-
unsaturated carbonyl derivatives have been developed as highly potent inhibitors for
many other cysteine proteases (Fig. 1.2.5) (Olson, 1999; Roush, 2001; Kumar, 2012;
Graczyk, 1999; Ekici, 2006; Glória, 2011; Dragovich, 2003; Tan, 2013).
Figure 1.2.5: Selected examples of Michael acceptors that act as cysteine protease inhibitors.
These compounds inhibit cysteine proteases by forming covalent bonds with the
active site thiol of cysteine proteases. In general, they are stable, and need the catalytic
machinery of the cysteine proteases for activation. Of all the compounds developed,
two Michael acceptor cysteine protease inhibitors, K-777 and Ruprintrivir (Fig. 1.2.6),
have entered clinical trials.
Figure 1.2.6: Cruzain inhibitor K777 and human rhinovirus 3C protease inhibitor Rupintrivir.
1.2.3.1 K777 Inhibitor
Cruzain, a cysteine protease that belongs to clan CA, is a key protease required for
the survival of Trypanosoma cruzi, the ethiologic agent of Chagas’ disease (McGrath,
1995; Wilkinson, 2009). For that reason, a promising group of drug leads for Chagas’
disease is cysteine protease inhibitors targeting cruzain. In 1998, McKerrow’s
group reported a dipeptide vinyl sulfone, K-777 (Fig. 1.2.6), that rescued mice from
a lethal Trypanosoma cruzi infection by inhibition of cruzain (Engel, 1998). Proof
of mechanism came from the crystal structure of inhibitor K777 bound to cruzain,
where the catalytic cysteine residue (Cys25) is covalently linked to the β-carbon of the
vinylsulfone (Fig. 1.2.7). K777 was shown to be safe and efficacious in animal models
of acute and chronic Chagas disease (Doyle, 2007; Barr, 2005). In particular, this vinyl
sulfone protected Beagle dogs from cardiac damage during infection by T. cruzi (Barr,
2005). The results of preclinical trials showed that K777 was non-mutagenic, well-
tolerated, and demonstrated efficacy in models of acute and chronic Chagas’ disease
in both mice and dogs (Kerr, 2009). On the basis of these results the inhibitor entered
clinical trials, but in 2013 the clinical assays were stopped due to tolerability findings
at low dose in primates and dogs (http://www.dndi.org/diseases-projects/portfolio/
k777.html).
Case Study 2: From Covalent Inhibitors to Hybrid Drugs 51
Figure 1.2.7: The crystal structure of inhibitor K777 bound to cruzain (pdb 2OZ2). The insert shows
the interaction of K777 with the primary recognition site (S2) and secondary recognition sites (S1 and
S3) of cruzain.
1.2.3.2 Rupintrivir (AG7088)
The human rhinovirus 3C protease (3CP) belongs to clan PA(C). Because the rhinoviral
protease (3CP) is specific to rhinovirus, inhibitors of this protease should have few
side effects in humans. Several compounds containing a Michael acceptor moiety
were described as potent Human rhinovirus (HRV) 3C protease inhibitors (Santos,
2007). Specifically, Rupintrivir (also known as Ruprintrivir or AG7088) was developed
by Agouron Pharmaceuticals to treat HRV infections (Fig. 1.2.6). Rupintrivir is an α,β-
unsaturated carbonyl that irreversibly inhibits the HRV 3C protease and shows potent,
broad-spectrum anti-HRV activity in vitro (Hayden, 2003). The phase I and phase II
results showed good safety and pharmacokinetic profiles. Although it successfully
completed the initial Phase II trials (Hayden, 2003), due to lack of efficacy in natural
infection studies, further development of Rupintrivir was stopped in PhaseII/III trials
(http://www.drugdevelopment-technology.com/projects/ag7088/).
1.2.4 Case Study 2: From Covalent Inhibitors to Hybrid Drugs
The design of hybrid compounds is a widely used strategy to improve activity or to

overcome the emergence of resistance to treatments infectious diseases. It has been
successfully used to optimize the targeting of reactive cysteine protease covalent
inhibitors in the field of parasitic diseases.
Falcipain-2 (FP-2) and falcipain-3 (FP-3) are cysteine proteases, located in the digestive
vacuole of the malaria parasite and involved in the digestion of host hemoglobin
essential for the survival of Plasmodium falciparum, the causing agent of the most
lethal form of malaria. Rosenthal and co-workers demonstrated that inhibition
of these enzymes allowed the cure of murine models of the disease and thus they
constitute a validated target for its treatment. Generally, the inhibitors of falcipains
have a backbone recognition element coupled with an electrophilic warhead such
as aldehydes, fluoro methyl ketones, vinyl esters, vinyl sulfones (Fig. 1.2.6), and
chalcones that can react with the catalytic cysteine residue (Shenai, 2003). However,
the intrinsic reactivity of many of these warheads has prompted the development
of hybrid compounds in order to modulate reactivity, improve selectivity and to
avoid the emergence of resistant strains. Several hybrid compounds were prepared
combining endoperoxides with different inhibitors of hemoglobin digestion. The goal
was to bring together two drugs that can act in the digestive vacuole by two different
mechanisms of action and taking advantage of the iron (II) coming from the digestion
of the host hemoglobin (Meunier, 2008).
1.2.4.1 Hybrid Compounds Containing an Electrophilic Warhead
In one of the first studies of hybrid compounds containing an electrophilic warhead,

Capela et al. reported the synthesis of hybrids comprising artemisinin and dipeptidyl
vinyl sulfones (Fig. 1.2.8) (Capela, 2009). These were designed to act in the parasite
food vacuole − where host hemoglobin is decomposed to provide nutrients − via
endoperoxide activation by Fe(II) and falcipain inhibition. All compounds were very
potent against the P. falciparum W2 strain, with IC50 values in the low nanomolar
range. Some of the compounds showed also superior activity than chloroquine or
artemisinin, the golden standards to treat malaria, against four additional resistant
P. falciparum strains with different phenotypes. However the hybrid compounds
were only moderately active against FP-2 in the low micromolar range. The authors
ascribed the antiplasmodial activity of the compounds to the size of the artemisinin
moiety inside the active site of falcipain, which might preclude optimal binding to the
enzyme.
Using the same rationale, Oliveira et al. synthesized 1,2,4,5-tetraoxane-based
hybrid molecules (Fig. 1.2.8) containing dipeptidyl vinyl sulfone partners to deliver
the FP-2 inhibitor once activated in the parasitic food vacuole (Oliveira, 2013). These
compounds showed excellent antimalarial activity against both chloroquine-sensitive
and chloroquine-resistant strains of P. falciparum. In spite of the low in vitro inhibitory
activity against FP-2, the hybrid compounds inhibited hemoglobin digestion inside the
parasites at low nanomolar concentrations, which was confirmed by the microscopic
observation of swelling of the parasite digestive vacuoles. This result is consistent
with a Fe(II)-triggered process inside the parasite that leads to the delivery of a potent
vinyl sulfone (Fig. 1.2.9). Importantly, the authors were able to detect the intact parent
vinyl sulfone in infected erythrocytes 48h post-treatment with the hybrid compounds,
which strongly suggests that the vinyl sulfone inhibitor remained within infected
erythrocytes long after the fast-acting endoperoxide moiety had exerted its effects.
These results indicate that the intrinsic activity of the vinyl sulfone partner can be
masked, suggesting that a tetraoxane-based delivery system offers the potential to
attenuate the off-target effects of known drugs (e.g. irreversible enzyme inhibitors
such as Michael acceptors).
Artemisinin-based hybrids Tetraoxane-based hybrids
Electrophilic warhead
H O
O O H
O N SO 2Ph
N
H
O O O O O
O Ph
O
1,2,4,5-tetraoxane-vinyl sulfone hybrid
O O Br
H O O N
N S H
O N Ph N
H N N
O O O N
O
Ph
Artemisinin-vinyl sulfone hybrid 1,2,4,5-tetraoxane-pyrimidine nitrile hybrid
Figure 1.2.8: Structures of endoperoxide-based hybrids containing an electrophilic warhead for

cysteine protease inhibition.
The hybrid approach was extended to tetraoxane derivatives designed to deliver

FP-2 inhibitors based on peptidomimetic pyrimidine nitriles. Nitriles inhibit cysteine
proteases by forming a reversible thioimidate intermediate resulting from the
nucleophilic attack of the catalytic cysteine residue (Ehmke, 2011; Ehmke, 2012).
Several heterocyclic and peptidomimetic compounds containing a nitrile warhead
displayed excellent inhibitory activity against falcipain-2 and against cultured P.
falciparum (Ehmke, 2011; Coterón, 2010). The pyrimidine nitrile tetraoxane hybrids
(Fig. 1.2.8) displayed potent nanomolar activity against three strains of P. falciparum
and FP-2, combined with low cytotoxicity. These hybrid compounds showed also
significant effects on liver stage model in vitro using P. berghei infecting Huh-7 human
hepatome cells and one derivative also showed in vivo efficacy when compared with
a non-treated control group (Oliveira, 2014). As with their vinyl sulfone counterparts,
the pyrimidine nitrile tetraoxane hybrids were shown to deliver the corresponding
peptidomimetic pyrimidine nitrile inside the parasite upon activation by Fe(II).
Figure 1.2.9: Tetraoxane-vinylsulfone hybrid compounds and their activation by Fe(II) from host
hemoglobin in infected erythrocytes (Oliveira, 2013). Insert: delivery of the Michael acceptor (vinyl
sulfone) via iron binding to oxygen-2 leads to the swelling of the parasitic digestive vacuole.
1.2.4.2 Hybrid Compounds Containing a Masked Electrophilic Warhead
Based on Roche’s antimalarial arteflene, O’Neill reported a prodrug approach

consisting of an endoperoxide containing cysteine protease inhibitors in a latent form.
After entering the ferrous-rich food vacuole of the malaria parasite, the molecule will
be fragmented by iron (II) releasing various antiparasiticidal elements (Fig. 1.2.10).
These compounds exhibit significant antimalarial activity, in the low nanomolar
range, and they can act by two different mechanisms, cytotoxicity exerted by the
C-radicals species formed by the activation of the peroxide, and inhibiting falcipains
due to the effect of the carbonyl inhibitor released in the activation process (O’Neill,
2004). They demonstrated that in the presence of Fe(II) the compounds liberated a
chalcone and additional potential parasitocidal chemical species. Furthermore, they
proved, by LC-MS analysis of pro-drug exposed parasites, that these systems are
capable of liberating a chalcone in the living parasites (O’Neill, 2004).
Figure 1.2.10: Fragmentation of arteflene-related peroxide to secondary carbon centered radical

species and protease inhibitor, active against falcipain-2 from P. falciparum. Adapted from O’Neill,
2004.
The same group extended the concept to a new type of hybrids combining a 1,2,4-
trioxolane moiety with peptidic cysteine protease inhibitors. They demonstrated that
in the presence of Fe(II) the 1,2,4-trioxolane scaffold decomposes into a potentially
toxic carbon radical that alkylates the heme in vitro, and a Michael acceptor that acts
as a cysteine protease inhibitor (Fig. 1.2.11). The most potent compound of the series
presented an IC50 value in the low nanomolar region (35 nM) against the 3D7 strain of
P. falciparum. Remarkably, although the same compound showed inhibitory activity
of FP-2 in the submicromolar range (0.5 µM), the corresponding aldehyde released
upon Fe(II) activation (Fig. 1.2.11) was shown to inhibit FP-2 with an IC50 value of
16 nM, thus confirming 1,2,4-trioxolane scaffold as a site-specific delivery system for
electrophilic aldehydes and ketones (Gibbons, 2010).
Figure 1.2.11: Fragmentation of 1,2,4-trioxolane to secondary carbon centred radical species and
protease inhibitor, active against falcipain-2 from P. falciparum. Adapted from Gibbons, 2010.
1.2.5 Conclusions
Designing selective covalent inhibitors remains one of the most attractive areas of
Medicinal Chemistry, where concepts of Organic Chemistry and Biochemistry are
used to discover compounds that react preferentially with the active site of the target
enzyme. The chemical basis to design irreversible and reversible covalent inhibitors
is now well established, providing the medicinal chemist with a variety of solutions to
modulate intrinsic reactivity and reduce the likelihood of toxicity issues. The chemical
toolbox is further expanded with the concept of molecular hybridization to modulate
the reactivity of electrophilic warheads or even to mask these chemical functionalities.
This chemical toolbox is already delivering interesting drug candidates into the
pipelines of pharmaceutical companies, small biotech and academic laboratories.
Acknowledgements: The authors thank Fundação para a Ciência e Tecnologia

(Portugal) for financial support through iMed.ULisboa (UID/DTP/04138/2013) and grant
PTDC/SAU-FAR/118459/2010, and the dedication and enthusiasm of our co-workers
who were involved in some of the reactions described herein. MMMS acknowledges
FCT, “Programa Operacional Potencial Humano” and the European Social Fund for
the IF Programme (IF/00732/2013)
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Section 2: Chemical Basis of Drug Action and
Diseases
Ana Fortuna, Gilberto Alves, Amílcar Falcão3
2.1 Pharmacokinetics and Bioanalysis to Improve
Drug Development
Abbreviations
ADME – absorption, distribution, metabolism and excretion;

APCI – atmospheric pressure chemical ionization;
AUC – area under the drug concentration-time curve;
BA/BE – bioavailability/bioequivalence;
BCRP − breast cancer resistance protein;
Caco-2 − human colon adenocarcinoma cell line;
CL − clearance;
Cmax − maximum concentration;
CV − coefficient of variation;
CYP − cytochrome P450;
DDD − drug discovery and development;
DDI − drug-drug interactions;
EMA − European Medicines Agency;
ESI − electrospray ionization;
FDA − Food and Drug Administration;
GC − gas chromatography;
HPLC − high performance liquid chromatography;
HTS − high-throughput screening;
ICH − International Conference on Harmonisation;
IS − internal standard
LC − liquid chromatography;
LLE − liquid-liquid extraction;
LLOQ − lower limit of quantification;
*Corresponding author: Amílcar Falcão: Laboratory of Pharmacology, Faculty of Pharmacy, University

of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal;
CNC – Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal,
E-mail: acfalcao@ff.uc.pt
Ana Fortuna: Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciên-
cias da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal; CNC – Centre for Neurosci-
ence and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
Gilberto Alves: CNC – Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517
Coimbra, Portugal; CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av.
Infante D. Henrique, 6200-506 Covilhã, Portugal
© 2015 Ana Fortuna, Gilberto Alves, Amílcar Falcão

Introduction 63
LOD − limit of detection

MDCK − Madin-Darby canine kidney cell line;
MIST − metabolites in safety testing;
MRM − multiple-reaction monitoring
MS − mass spectrometry;
MS/MS − tandem mass spectrometry;
NCEs − new chemical entities;
PAMPA − parallel artificial membrane permeability assay;
Papp − apparent permeability;
PepT1 − oligopeptide transporter;
QC − quality control;
Q-TOF − quadrupole coupled with time-of-flight mass analyzer;
RE − relative error;
RSD − relative standard deviation;
SIM − selective ion monitoring;
SPE − solid-phase extraction;
SRM − single-reaction monitoring;
t1/2 − elimination half-life;
TDM − therapeutic drug monitoring;
tmax − time to reach the Cmax;
UGT − UDP glucuronosyltransferase;
UHPLC − ultra-high pressure liquid chromatography;
UV − ultraviolet;
Vss − apparent volume of distribution at steady state.
2.1.1 Introduction
The first decade of the 21st century was a challenging period for the pharmaceutical
industries focused on drug research. Several changes in technology and market
dynamics have occurred over the last years and they inexorably revolutionized
the drug discovery and development (DDD) process. The tremendous progress
in biomedical knowledge and the completion of the human genome sequencing
project have accelerated the discovery of the molecular basis of human diseases
and have allowed the identification of important new biological therapeutic targets.
In addition, the synergistic interaction between rational drug design, recombinant
biotechnology and combinatorial chemistry prompted a fast increment of the number
of new chemical entities (NCEs) with molecular characteristics propitious to interact
with specific biological targets (Ohlmeyer, 2010).
Despite this exponential increase in the number of NCEs, pharmaceutical
industry is suffering from a lack of productivity considering the number of new drugs
that have successfully reached the market (Keserü, 2009). According to recent news, it
64 Pharmacokinetics and Bioanalysis to Improve Drug Development
is estimated that only one in ten drug candidates that enter the clinical development
phase will reach the market and the success rate for drugs to be approved considering
all the therapeutic areas is only 11% (Tamimi, 2009).
The current climate of downward pressure on healthcare costs combined with
the need of expensive technologies and greater information about drugs under
development prompted pharmaceutical companies to develop paradigm-shifting
strategies and tactics in order to cut drug development costs, fill the depleting product
pipelines and reverse the trend of dropping productivity. A well-succeeded front-
loading approach consisted on selecting, in the early drug discovery stage, the drug
candidates that are not only pharmacologically active but also those having desirable
pharmacokinetic properties. It is particularly disheartening when the in vitro potency
is achieved, but the lead compound fails in vivo because, for instance, it is so short-
lived that its clinical benefits would be minimal. In fact, since the pharmaceutical
industries embraced the early pharmacokinetic screening of drug candidates, the
attrition rates of DDD were substantially improved and, therefore, it is currently
straightforward to make a decision on the pharmacokinetic desirability of each drug
candidate in the beginning of DDD processes and not only later in the non-clinical
and clinical development stages (Saxena, 2008; Gallo, 2010). This demand expanded
the investigation on pharmacokinetics into the initial phases of drug discovery and
several in silico, in vitro and in vivo models have been developed and improved to
optimize the pharmacokinetic behavior of drug candidates at different stages of DDD.
Moreover, pharmacokinetic principles are also applied during the post approval
period of a drug, particularly for therapeutic drug monitoring (which has recently
been defined as therapeutic drug management, TDM), evaluation of line-extension
medicinal products and generic formulations, particularly through bioavailability/
bioequivalence (BA/BE) studies.
Taking into account these new attempts, a close involvement between
pharmacokinetics and bioanalysis (defined as the determination of drug
concentrations on biological specimens) became required in order to obtain data
with a high quality level and allow an adequate interpretation and ultimately rational
decision-making. On the other hand, the increasing number of NCEs that must be
investigated and the application of pharmacokinetic screening at initial phases of
DDD increases the number of samples that must be analyzed and, consequently,
new bioanalytical techniques with higher sensitivity, selectivity and speed have
emerged. Indeed, decades ago, high performance liquid chromatography (HPLC)
coupled with ultraviolet (UV) or fluorescence detection and gas chromatography
(GC) with mass spectrometry (MS) ensured the acquisition of plasma concentration
data to define drug exposure in animal tests and clinical studies. In current practices,
liquid chromatography (LC) coupled to MS or tandem mass spectrometry (MS/MS) are
becoming essential due to their higher throughput capacity to identify and quantify
the parent drugs and metabolites in biological matrices, enabling fast and effective
decision-making. Furthermore, new strategies for sample preparation have also been
Pharmacokinetic on Drug Discovery and Development Process 65
thoroughly developed to support the high-throughput analysis of samples, including

automated online sample preparation with instrumental analysis.
It is important to acknowledge that although the high-throughput strategy is
well established in all DDD phases, it has different emphases and, consequently,
each stage of DDD places different requirements on the bioanalytical assays used
to provide information. Briefly, in the initial phases of DDD, straightforward, fast,
efficient and high-throughput analytical methods are needed without requiring a
fully regulated validation. As the drug candidate moves to development stages, the
quality of bioanalytical methods must be improved and their accuracy, precision and
reliability are regulated by governmental requisites.
Thus, in this chapter the role of pharmacokinetics in each stage of the DDD
process and the general characteristics of the bioanalytical methods developed
and employed to assess the pharmacokinetic properties of drug candidates will be
discussed. Additionally, owing to the recent evolution of the international guidelines
on bioanalytical method validation and its increasingly stricter sanctions on drug
development and post-marketed studies, a section of this chapter is devoted to
overview the most important regulatory guidelines. Moreover, due to the significant
technological advances continuously reached in this field, recent bioanalytical
strategies that promote greater speed and productivity in bioanalysis during the
pharmacokinetic studies are also addressed.
2.1.2 Pharmacokinetics on Drug Discovery and Development Process
The pharmacokinetic principles applied during the process of DDD have undergone a
revolution in the last decades, as it was understood that the efficacy of any successful
drug depends not only on its pharmacological potency but also on its absorption,
distribution, metabolism and excretion (ADME) characteristics.
Over the time, it became clear that drug candidates with low and/or high variable
bioavailability and/or short half-life in animal studies were frequently prevented
to progress to clinical trials. Furthermore, poor pharmacokinetic properties of new
drug candidates in phase I clinical trials accounted for their clinical failure, while the
incidence of severe (toxic) adverse side effects resulting from drug metabolism and
drug-drug interactions (DDI) observed during phase II and III clinical trials may lead to
the termination of potential new drugs or the withdrawal of marketed drugs. Although
ADME studies are undeniably important throughout the stages of (non)-clinical drug
development, some of the aforementioned failures could have been avoided if the
pharmacokinetic deficits of the drug candidate were identified earlier in the discovery
phase. The recognition that understanding the ADME processes of test compounds
would potentially prevent their unsuccessful progression into clinical development
phases expanded the ADME/pharmacokinetics interest from its traditional role as
non-clinical safety support towards the early stage of drug discovery. Today, ADME/
pharmacokinetic studies are considered essential from drug discovery to phase IV

clinical development and their main goals on each phase are summarized in Table
2.1.1. To attain these objectives, a range of in silico, in vitro and in vivo approaches have
been developed and optimized as it will be further explained.
2.1.2.1 ADME/Pharmacokinetic Evaluation on Early Drug Discovery Phases
As projects advance from the beginning of drug discovery (hit identification) to

the last stage of drug discovery, which is named lead characterization and mostly
performed in vivo, the required throughput (number of candidates to be considered)
declines but assay predictability (content of information) increases proportionally.
Most importantly, during the early and middle stages of drug discovery (including
hit identification, lead identification and optimization), high-throughput screening
(HTS) assays are utilized, allowing to test hundreds of compounds to be tested per day
(Pereira, 2007; Carlson, 2008; Di, 2008; Wan, 2009).
In current practice, even before NCEs are synthesized, in silico mathematical
models have been employed to predict the pharmacokinetic attributes of compounds,
preventing the synthesis and screening of compounds with obvious liabilities.
Based on mathematical and statistical relationships between the physicochemical
and molecular characteristics of the NCEs and their pharmacokinetic properties,
recent in silico models are commercialized as software that predict pharmacokinetic
parameters: Meteor®, MetabolExpert® and MetaSite® are examples of commercial
metabolic fate/stability predictors; GastroPlus® and IDEA® perform pharmacokinetic
simulations of the rate and extent of absorption in gastrointestinal tract (Parrott, 2002;
Kuentz, 2006). Although in silico models have higher throughput ADME screening
ability, saving more time and effort than in vitro and in vivo models, they can hardly
be accurate enough to replace real circumstances (Lavè, 2007; Burton, 2010; Gallo,
2010).
Thus, during the phases prior to lead characterization where the throughput is
high, the potential pharmacokinetic features are mainly addressed by in vitro tools.
On the other hand, in the last phase of drug discovery, more comprehensive in vitro
and in vivo models may provide a definitive assessment of overall drug disposition of
the best compounds previously screened. Table 2.1.2 summarizes the major in vitro
model systems most frequently employed for ADME/pharmacokinetic analysis in
the early stages of drug discovery programs and their respective objectives. At the
initial stages of drug discovery, ADME screening is conducted in a high-throughput
mode and includes, but it is not limited to, determinations of apparent permeability
[using parallel artificial membrane permeability assays (PAMPA) and/or cell lines
such as human colon adenocarcinoma (Caco-2) or Madin-Darby canine kidney cells
(MDCK)], plasma protein binding, human or animal liver microsomal stability, and
identification of the reactive metabolites. Most of these in vitro models are generally
Table 2.1.1: The role of pharmacokinetics in different stages of drug discovery and development
process (Panchagnula, 2000; Chien, 2005; Bhogal, 2008; Zhang, 2012).
Stages Objectives Purpose of pharmacokinetic

studies
Drug Discovery The global aim is to choose the optimum candidates considering their
pharmacokinetic characteristics in connection with their intrinsic activity and
potency and particularly:
Predict in vitro ADME of NCEs; Screen NCEs according to their
absorption;
Understand the mechanisms underlying the
ADME of NCEs; Identify the main elimination
pathways and metabolic enzymes
involved;
Foresee the plasma protein

binding of NCEs;
Predict their potential to develop

drug-drug interactions;
Determine NCE bioavailability in rodent In vivo fast exposure and
species. pharmacokinetic screen of the
NCEs.
Predict pharmacokinetic parameters.
Pre-clinical The global aim is to evaluate, in laboratory animal models, the
Development pharmacokinetics and toxicokinetics of the most promising compounds found
on the drug discovery stage and:
Characterize the pharmacokinetic profile Determination of compound full
of the parent compound and its main exposure in animals;
metabolites;
Determine absolute and relative
Demonstrate biologic activity and safety in bioavailability;
laboratory animal models;
Evaluate dose proportionality;
Investigate the food effect on
Assess the margin of safety
pharmacokinetics.
based on efficacy concentration
Select the best formulation for drug exposure and exposure data from
and provide confidence that the predicted toxicokinetic studies;
drug effects are achievable via the chosen
Determine the best formulation
route of administration;
for drug exposure;
Establish safe and/or efficient starting doses
Predict compound
and dosing regimens for clinical trials;
pharmacokinetics in humans;
Determine whether a drug is deemed to be
Integration of pharmacokinetics
suitable for administration to humans on the
and pharmacodynamics data to
basis of an acceptable risk assessment.
understand the pharmacology in
animals from different species;
Continued
Table 2.1.1: The role of pharmacokinetics in different stages of drug discovery and
development process (Panchagnula, 2000; Chien, 2005; Bhogal, 2008; Zhang, 2012).

studies
Investigate whether the compound is safe Tolerability and safety over a
and well tolerated in humans; range of doses;
Phase I
Assess pharmacokinetics and Measurement of pharmacokinetic
pharmacodynamics in humans and parameters using single and
investigate the pharmacokinetic attributes of multiple doses;
the compound;
Establishing whether plasma
Evaluate if the compound should move on to concentration increases
further development phases; in proportion to the dose
administered in humans;
Establish dosing range and dosage regimens.
Evaluate the influence
of age, gender, genetic
characteristics and food on drug
pharmacokinetics in humans.
Demonstrate efficacy in the intended Quantitative determination of

population; the distribution of a compound in
Phase II bodily fluids and tissues;
Identify attributes of the compound in target
population compared to the existing therapy; Investigate absolute and relative
bioavailability;
Identify the metabolites and evaluation of
their contribution to the biological profile of Establishing a clear dose-
the compound; response relationship;
Evaluate sex, food and polymorphism Assessment of the influence

influence in drug therapeutic and safety of gender and food on
profiles. pharmacokinetic profiles;
Assessment of the influence of

genetics in drug metabolizing
enzymes on pharmacokinetic
profiles.
Continued
Table 2.1.1: The role of pharmacokinetics in different stages of drug discovery and
development process (Panchagnula, 2000; Chien, 2005; Bhogal, 2008; Zhang, 2012).

studies
Demonstrate safety and efficacy for clinical Determination of the
use; pharmacokinetic profile in the
Phase III target population;
Studies at patient subgroups at potential risk
to adjust dose regimens; Determination of the
pharmacokinetic profile in
Confirm dose/exposure response relationship subgroups of target population;
in target population and subpopulations.
Determination of the
pharmacokinetic profile of the
final formulation of the drug
candidate;
Determination of a clear dose-

response relationship in target
population and subpopulations.
Dosage form improvements; Determination of the
pharmacokinetic profile of new
Phase IV Change drug formulation (modified release formulations;
preparations, line extensions).
Determination of the
pharmacokinetic profile of
modified drugs designed to
extend patent life;
Determination of eventual
influence of other drugs on the
pharmacokinetic profile;
ADME, absorption, distribution, metabolism and excretion; NCE, new chemical entity
amenable to miniaturization and automation, improving the evaluation capacity

of HTS assays (Mayr, 2009). For ADME/pharmacokinetic analysis in the late stage
of drug discovery, major methodologies include protein binding (as well as blood–
plasma partitioning and plasma stability), hepatocyte stability, metabolic enzyme
phenotyping and human cytochrome P450 enzyme (CYP) inhibition/induction.
Independently of the precise nature of the screening technology, all methodologies
must generally follow five criteria when designed and implemented during the
discovery program (Fretland, 2010; Volpe, 2010):
Table 2.1.2: Major in vitro model systems used to investigate the absorption, distribution and
metabolism of new chemical entities in initial phases of drug discovery.
In vitro model General characteristics Applications

Absorption in vitro models
PAMPA HTS assay rapidly performed in 96-well Screen NCEs in accordance
plates; to their absorption ability by
Papp measurement through a transcellular passive diffusion;
hydrophobic filter impregnated with Prediction of human intestinal
lipids; fraction absorbed.
Compound quantification by UV/VIS
spectrophotometry;
Good predictability and easily to be
automated.
Caco-2 cell line Cells have human origin and encompass Screen NCEs in accordance
many characteristics of intestinal to their permeability through
epithelium; Caco-2 monolayer;
Cells are grown on semi-porous filters in Evaluation of absorption and
6-, 12-, 24- or 96-well plates; prediction of human intestinal
Papp is determined from apical to permeability;
basolateral sides and vice-versa. Understand mechanisms
Compound quantification by HPLC. underlying intestinal
absorption;
Identification of NCEs that are
substrates or inhibitors of the
intestinal efflux transporter,
P-glycoprotein.
Identify efflux transporters
involved in compounds
intestinal absorption.
Toxicity studies.
Ussing chamber Uses intestinal membrane from mice or Screen NCEs in accordance
rats; to their permeability through
Segments of small Intestine are animal intestinal membrane;
mounted in the chamber with Evaluation of absorption and
oxygenated buffer surrounding it for understand the mechanisms
approximately 3 h. underlying intestinal
Papp is determined from apical to absorption
basolateral sides and vice-versa. Identify efflux transporters
Compound quantification by HPLC involved in compounds
with simple sample preparation for intestinal absorption.
bioanalysis. Evaluation of specific
transport mechanisms,
enhancing strategies on a
mechanistic basis;
Evaluation of toxicity of
compounds.
Transporter in vitro models
Caco-2 Caco-2 cells undergo enterocytic Evaluate the efflux transport

differentiation and become polarized via P-glycoprotein and BCRP.
in culture (usually for 21 days), Identification of
resembling human intestinal epithelium P-glycoprotein substrates and
in transporter expression and tight inhibitors in drug discovery.
junction formation. Evaluation of drug-drug
interaction potential.
Bi-directional studies and compound
quantification by HPLC with simple
sample preparation for bioanalysis.
Determination of efflux coefficient.
Transfected MDCK cell line MDCK are previously transfected with Evaluate the efflux transport
P-glycoprotein gene to over-express the via P-glycoprotein.
transporter. Identification of
P-glycoprotein substrates and
5 days of incubation is usually required inhibitors in drug discovery.
to initiate the studies. Evaluation of drug-drug
Bi-directional studies and compound interaction potential.
quantification by HPLC with simple
sample preparation for bioanalysis.
Determination of efflux coefficient.
Membrane vesicles Membrane vesicles prepared from Assess the transporter

organs (such as liver, kidney and mechanisms in liver, kidney
intestine) that naturally express high and intestine.
concentrations of transporters or from
transfected cell lines (such as MDCK)
that over-express a single transporter.
This technique allows separate
preparations of the blood side of cell
membranes (liver- sinusoidal, kidney
and intestine basolateral) and luminal
side of cell membranes (liver-bile
canalicular, the intestine and kidney-
brush-border).
Protein binding in vitro models
Ultrafiltration A semi-permeable membrane separates The simplest and fastest

an upper chamber where drug protein method for determining the
solution is placed from a lower chamber. concentration of the drug that
Centrifugation (approximately 2000 is not binding to the plasma
x g) is applied to separate the bound proteins during:
and unbound fractions due to the high - Drug discovery;
molecular size of compound-protein - Clinical therapeutic drug
complex which cannot pass through the monitoring;
membrane. - Clinical pharmacodynamic
The free drug concentration in the and pharmacokinetic studies.
lower chamber and the total drug
concentration prior to ultrafiltration are
determined and then used to determine
the extent of protein binding.
Commercially available 96-well
ultrafiltration apparatus.
HPLC is frequently employed.
Equilibrium Dialysis Equilibrium dialysis is based on the Frequently used in drug

principle of the diffusion of solutes discovery in order to
along a concentration gradient across a determine the percentage
semi-permeable membrane. of drug binding to plasma
The protein solution containing drug proteins maintaining a true
is placed in one chamber while buffer equilibrium during the whole
is placed in the opposing chamber. experiment.
The unbound drug passes through
the membrane and at equilibrium,
the unbound drug will be at equal
concentrations on both sides of the
membrane while the bound drug
will remain in the protein chamber.
Equilibration times are long (typically
12–48 h).
HPLC is used to quantify the free and
total drug concentrations.

Metabolic in vitro models
Expressed enzymes Expressed enzymes are concentrated Conduct reaction-phenotyping
enzymes with high activity toward for drug candidates and
a specific substrate, they are often identify non-CYP microsomal
used as ‘‘bioreactors’’ to generate or cytosolic enzymes often
metabolites. involved in drug metabolism.
Using the generated metabolites that Identify the relative
are collected as chromatographic contribution of each CYP
fractions, it is possible to test for their isoform to the total hepatic
pharmacological activity. clearance.
Identify the involvement of
CYPs in metabolic pathways.
Predict drug-drug interaction
potential.
Microsomes They are the most widely used sub- Determine intrinsic clearance,
cellular fractions for drug metabolism a useful parameter for
studies, with the advantages of being facilitating the screening
inexpensive and easy to handle while process for stable compounds
containing the major drug metabolism and for establishing an in vitro
enzymes, e.g., CYPs and UGTs. correlation between animals
It is necessary to supplement the sub- and humans.
cellular fractions with enzymes co- Identification of the main
factors to initiate the various enzymatic metabolites.
reactions. By including or excluding Assess extra-hepatic
certain co-factors, one can pinpoint metabolism and further
the involvement of certain metabolic strengthen the in vitro
pathways for a given drug candidate. prediction of total body
clearance by using
Compounds are quantified after pre- microsomes from various
defined times of incubation by HPLC. tissues.
Both reversible inhibition and time- Microsomal assays are the

dependent inhibition studies provide default assays for metabolism
information on the possibility of the and DDI studies at the drug
drug candidate being a perpetrator discovery stage.
of drug-drug interaction for a co-
administered drug.

Cell culture and cell lines Primary cultures of hepatocytes: Identification of the
They carry enzymes and co-factors metabolites generated.
at physiological concentrations and Predict drug clearance;
provide a drug metabolism environment Predict DDI potential of drug
that closely mimics the in vivo candidates.
conditions; however they have a limited ‘Gold standard’ for conducting
life span and differentiation ability. CYP induction studies before
Immortalized human hepatic tumor cell (non)-clinical investigations.
lines (HepG2 or HepaRG)
They present lower cost and higher
ease of storage than primary culture
of hepatocytes, making them more
suitable as a HTS methodology. However
as they exhibit some morphological
changes regarding in vivo models that
must be considered during the analysis
of the results.
Compounds are quantified after pre-

defined times of incubation by HPLC.
Three-dimensional hepatic Different cells-types besides hepatic Predict hepatic clearance;

models cells are seeded in the same scaffold Mainly used to predict drug
and may include fibroblasts, Kupffer hepatic toxicity.
cells, vascular and biliary duct
endothelial cells in order to maintain
the liver-cells homeostasis.
Although allow better cell-cell matrix
interactions, resembling better in vivo
functions and morphology than cell
culture/lines, CYP expression may vary
according to experimental conditions.
BCRP, breast cancer resistance protein; Caco-2, human colon adenocarcinoma cell line; CYP, cytochrome
P450; DDI, drug-drug interaction; HPLC, high performance liquid chromatography; HTS, high throughput
screening assay; MDCK, Madin-Darby canine kidney cell line; PAMPA, parallel artificial membrane
permeability assay; Papp, apparent permeability; UGT, UDP glucuronosyltransferase.
–– Relevance: the result of the screen should have good concordance with the cor-
responding in vivo properties of the drug, requiring validation of the screen with
standard compounds of known animal or human performance;
–– Effectiveness: the cut-off criterion should eliminate a substantial fraction of com-
pounds without eliminating every compound, otherwise the screen would merely
add a useless extra step that delays the discovery process;
–– Speed: the experimental procedure must be fast enough to keep pace with the
input rate of new compounds from chemistry;
–– Robustness: the experimental procedure must be applicable to a wide variety of
chemical structures;
–– Accuracy and reproducibility: any screening procedure has an inevitable charac-
teristic error rate, because it is usually necessary to sacrifice some accuracy or
precision to achieve the desired speed. Thus, when a large number of compounds
is carried through a particular screen model, some of the compounds will be clas-
sified incorrectly. False positives are tolerable, but false negatives may be lost
forever if the failure eliminates them from further testing.
It is also essential to regularly validate the in vitro models against in vivo data to ensure
that decisions based on in vitro results are sufficiently predictive (Burton, 2010).
Once exhibiting these characteristics, in vitro assays contribute to understanding
the underlying mechanisms of drug absorption and disposition, which are invaluable
to establishing structure-activity relationships to guide subsequent chemical synthesis
of new drugs, avoiding potential obstacles in drug development. It is also important
to highlight that in vitro data are in current practice recognized as good predictors of
in vivo end-points. Even the Food and Drug Administration (FDA) accepts the
importance of in vitro data, as it can inclusively avoid certain clinical studies (FDA,
2000, 2003, 2006). For instance, since a negative result in CYP induction for in vitro
human-based studies using primary hepatocyte cultures always seems to lead to a
negative induction in human clinical studies, FDA typically waives clinical DDI studies
if the drug candidate is tested as negative in a human in vitro CYP induction study.
However, the relative simplicity of in vitro systems for studying ADME/
pharmacokinetics in comparison to real in vivo drug behavior means that absolute
correlations between in vitro and in vivo findings are often difficult to establish
routinely, particularly for humans. For this reason, in vitro screens are frequently
viewed as a means to rank compounds for further study rather than for outright
rejection, so the in vivo pharmacokinetic studies performed in animals during lead
characterization remain crucial after in vitro evaluation.
Without requiring full pharmacokinetic characterization, the in vivo studies
performed at this stage ensure that lead compounds have appropriate pharmacokinetic
properties to be evaluated in non-clinical pharmacology and safety studies (Table 2.1.1).
In fact, when combined with in vitro results, they quickly assess the bioavailability of a
compound, giving a good indication of the suitability for advancement to pre-clinical
and clinical trials. The current trend is to use rodents (mice and rats) as first animal
species to test drug exposure because they are less expensive and require smaller
amount of test compound. These in vivo pharmacokinetic studies in rodents include
single oral and intravenous pharmacokinetic studies and provide information such as
drug oral bioavailability, distribution, clearance and duration of exposure for a drug
and its metabolites. Thus, they can help to identify some limitations of a new chemical
series regarding their ADME characteristics, such as low absorption or high clearance,
leading to undesirable pharmacokinetic profiles. Subsequently, in vitro models such as
Caco-2 can be used to optimize absorption of compounds from the same chemical series.
The microsomal stability assay can also be applied to select more stable compounds.
Although a continuous demand for in vivo pharmacokinetic studies remains
at discovery stages, most of these studies are still conducted in a traditional, low-
throughput manner in many pharmaceutical companies. Furthermore, compound
quantities as well as practical and ethical issues preclude in vivo studies performed
at a sufficient rate to investigate the high number of new drug candidates (Balani,
2005). Conventional pharmacokinetic studies include the intravenous and oral
administration of each compound to groups of 6−8 animals with blood samples taken
at 6−12 different points of time (Alves, 2008), requiring a high number of animals,
time and intensive bioanalysis. Consequently, in order to make timely and meaningful
contributions in early discovery programs, in vivo pharmacokinetic analysis has been
efficiently modified and accomplished through recent strategies such as pooling of
plasma samples and fewer sampling time points per study, reducing the number of
samples analyzed per each compound (Table 2.1.3). Interestingly, similar pooling
strategies have also been applied in order to determine brain concentrations and
estimate the concentration ratios in relation to plasma levels (Amore, 2010).
2.1.2.2 Pharmacokinetic Evaluation on Drug Development Phases
The most promising compounds found in discovery stages are then evaluated in at
least two relevant animal species to obtain pharmacodynamic, pharmacokinetic, and
toxicological information. The animal species are chosen according to the previous in
vitro analysis.
The extensive pharmacokinetic studies performed at this stage include single
and multiple dose administration with several ascending doses, metabolic profiling
and evaluation of drug potential to induce/inhibit drug-metabolizing enzymes.
Major objectives of these studies are depicted in Table 2.1.1. Compartmental and non-
compartmental methods are used to determine multiple pharmacokinetic parameters,
including the maximum concentration (Cmax), time to reach the Cmax (tmax), plasma
drug concentration-time curve (AUC), apparent volume of distribution at steady state
(Vss), clearance (CL), elimination half-life (t1/2) and absolute or relative bioavailability,
providing characterization of the pharmacokinetic profiles for a test compound.

Table 2.1.3: Comparison between the strategies employed to increase the throughput of in vivo pharmacokinetic studies during drug discovery stages (Ward,
2001; Cheung, 2005; Li, 2013).
Strategy General characteristics Advantages Disadvantages Application

Cassette dossing Co-administration of several Allows obtaining complete Compounds must be compatible within the same Triage compounds based
(or N-in-one compounds (≈ 5−10) to a single pharmacokinetic profiles dosing solution that is administered; on their oral exposure.
dossing) animal; for each compound in Difficulties in preparing a convenient formulation
Collection of plasma samples over each animal. of multiple compounds;
the study time course; Provides a faster Co-administration of NCEs may increase
Measurement of concentrations assessment of in vivo significantly the potential to develop DDI;
of each compound in a single run exposure levels; Complexity in bioanalysis of multiple analytes in
by high precision bioanalytical Requires fewer animals the same run.
methods. and bioanalysis resources.
Snapshot Compounds are dosed Reduced time analysis; No inter-animal variability data available; Triage compounds based
Pharmacokinetic individually as in conventional Savings in number of Only truncated AUC0-5h is obtained; on their oral exposure;
Study pharmacokinetic studies; animals and analytical Sample dilution and higher LLOQ; Investigate structure-
Oral administration; resources in relation to Complex bioanalysis and longer bioanalytical activity relationships.
Decrease the number of samples rapid pharmacokinetic method development time if samples from animals
analyzed per compound, usually studies; dosed with different compounds are pooled;
using only two animals per No DDI interference; Difficult to adapt to mice due to the small sample
compound and collecting samples Complete pharmacokinetic volume.
up to 5 h (4 time points). profiles for each
Sample pooling (also named compound.
cassette analysis) consists on
combining equal volumes of
plasma from the same time point
from each animal;
Pharmacokinetic on Drug Discovery and Development Process
Sample pooling can be done

with samples of animals dosed
individually with different
compounds.
77
Continued
Table 2.1.3: Comparison between the strategies employed to increase the throughput of in vivo pharmacokinetic studies during drug discovery stages
78
(Ward, 2001; Cheung, 2005; Li, 2013).
Strategy General characteristics Advantages Disadvantages Application

Rapid Identical to cassette analysis No DDI interference; No inter-animal variability data available; To get full
Pharmacokinetic but six animals are usually used Compounds are analyzed Labor intensive in-life portion; pharmacokinetic
study per compound and samples separately; No savings on in-life dosing, sampling or number parameters : CL, Vss,
are collected up to 24 h (6 time Amendable for of animals. Cmax, AUC, t1/2 and F.
points). automation;
The six samples from the same Suitable to obtain full
collection time are pooled; set of pharmacokinetic
Oral and intravenous parameters;
administration. Probable to reveal no
sample dilution and low
LLOQ.
AUC, area under the curve; CL, systemic clearance; Cmax, maximum concentration; DDI, drug-drug interaction; F, systemic bioavailability; LLOQ, lower limit of
quantification; NCEs, new chemical entities; t1/2, half-life time; Vss, apparent volume of distribution at steady state
Pharmacokinetics and Bioanalysis to Improve Drug Development
In parallel, toxicity tests are always performed in this stage and include genotoxicity,
safety pharmacology in all biological systems, reproductive toxicology in male and
female animals and long-term carcinogenicity. In this context, toxicokinetics, which is
defined as the generation of pharmacokinetic data under the conditions of the toxicity
studies themselves, is required as an integral component of non-clinical toxicity
studies (Guideline ICH S3A). The primary objective of toxicokinetics is to describe the
systemic exposure achieved in animals and its relationship to the dose level and the
time course of the toxicity study. These objectives are mainly achieved by measuring
the plasma (or whole blood or serum) concentrations of the parent compound and/
or metabolite(s) through time and determining the plasma (or whole blood or serum)
AUC and Cmax. Subsequently, toxicokinetics relates the exposure achieved in toxicity
studies with the toxicological findings to assess the relevance of these results on
human clinical safety. Toxicokinetics is thus recognized as an integral part of non-
clinical testing programs that enhances the value of the toxicological data generated,
not only to understand the toxicity results but also to predict the risk and safety in
humans. Together with bioavailability evaluation, metabolite profile determination
and plasma drug levels associated with toxicity, toxicokinetics determines the
choice of the no observed adverse effect level, which is essential to define the human
equivalent dose and the maximum recommended starting dose in first-in-human
clinical trials (FDA, 2005).
At non-clinical development, disposition studies are also performed using
radiolabeled compounds administered to animals to provide useful mass balance
data, first-pass metabolism from vascular and portal vein cannulation animal models,
biliary excretion conducted on bile-duct cannulated animals and tissue distribution,
where the organs can be removed and drug quantified in order to evaluate whether drug
accumulation may be involved. In all these studies, identification and quantification
of drug (radiolabeled or otherwise) and main metabolites is required, implying the
application of bioanalytical techniques adequately developed and validated for that
purpose.
Clinical development initiates with the first administration of the drug to humans
and all the stages are strictly based on specific guidelines from the International
Conference on Harmonisation (ICH), European Medicines Agency (EMA) and FDA (ICH,
1997; EMA, 1998 2008; FDA, 2008a; Milton, 2009). The main goal of phase I clinical
trials is to investigate whether the compound is safe and well tolerated in humans
(Table 2.1.1). Pharmacokinetic properties must also be evaluated and the development
of the drug can be stopped if its elimination half-life is found to be too short, too
long or if it has poor bioavailability. Phase I clinical trials usually start with single
sub-therapeutic dose studies which are escalated gradually followed by multiple
dose studies and if the drug is not well tolerated, it is dropped from development.
These closely-monitored trials help define the safe dosing range and whether the
compound should move on to further development phases (Duff, 2007). Thus, from
the pharmacokinetic point of view, absorption and elimination phases of the plasma
concentration-time curve must be fully characterized and all metabolites must be

fully resolved, identified and quantified, demanding analytical techniques with
enough sensitivity and selectivity to quantify the small amounts present in biological
samples. Indeed, regulatory guidance documents have highlighted not only the
importance of the identification and structure elucidation of drug metabolites, but also
their early quantification in clinical development in order to address three important
requirements: determination of whether metabolites contribute significantly to the
overall pharmacology of a drug; assessment of safety coverage of metabolites and
understanding the DDI potential of the drug and its metabolites (FDA, 2008b; 2012;
Zhu, 2009; Frederick, 2010; EMA, 2012). The general key factors to be considered for
these metabolites are relative quantity (major or minor metabolites), pharmacological
activity, likelihood to be formed via reactive intermediates and whether they result
from phase I or phase II metabolic reactions.
Thus, the FDA guidance on Safety Testing of Drug Metabolites (FDA, 2008b)
defines a major metabolite as that which AUC value is higher than 10% of the AUC
of the parent drug at steady-state conditions. The DDI guidance also proposes that
human metabolites present at ≥ 25% of the parent drug’s exposure (given by AUC)
should be investigated in vitro for their DDI potential (EMA, 2012; FDA, 2012).
Considering Table 2.1.1, the pharmacokinetic analysis in phase II clinical trials
is employed to assess the dose/exposure response and dose ranging studies are
carried out to establish the effective doses for phase III clinical trials. On the other
hand, phase III clinical trials can involve up to several thousands of patients to create
an adequate database for assessing the efficacy and safety profile of a compound
and to enable accurate drug labeling. Pharmacokinetic analysis is particularized
for special sub-populations, including patients with impaired renal or hepatic
functions (EMA, 2004a, 2005) as well as pediatric and elderly populations (EMA,
2004b). Hundreds of sites around the world participate in the study to get a large
and diverse group of patients, implying a vast number of samples to be handled
and analyzed.
Even after receiving approval to be marketed, a drug must continue to be evaluated
by an appropriate pharmacovigilance system. Indeed, as a much larger number of
patients begin to use the drug, companies must continue to monitor it carefully in
order to evaluate long-term safety and the effect of the new drug in specific subgroups
of patients.
Importantly, although several new drugs are brought to the market and help
fight many diseases, ADME processes of a drug may differ among individuals,
resulting in differences in the relationship of drug plasma concentrations and dosage
administered. Since inter-individual differences may occur due to differences in
age, weight, genetic polymorphism, co-morbid diseases and DDI, drug posology
regimen is frequently individualized and TDM plays a very important role in this
personalized medicine field. Its major objective is to ensure that drug concentrations
are within a defined optimal therapeutic range to maximize drug efficacy, minimize
Bioanalysis and Validation Requirements on DDD 81
drug toxicity and reduce therapeutic costs. In practice, it involves the measurement
of drug concentrations in plasma or serum samples, and the interpretation of these
results in an attempt to adjust the dose to that particular patient. Not all drugs are
candidates for TDM, as it must meet the following criteria: (1) a narrow therapeutic
range; (2) significant inter-individual variability in systemic exposure at a given dose;
(3) a clear relationship between blood exposure and clinical effect; and (4) a validated
bioanalytical method to measure drug concentration in plasma or serum.
2.1.3 Bioanalysis and Validation Requirements on DDD
As described in the previous section, in an attempt to diminish the attrition rate of DDD
process, the pharmaceutical industry has put more emphasis on pharmacokinetic
evaluation in the beginning of NCEs discovery. Consequently, bioanalysis emerged as
an essential support tool to characterize ADME/pharmacokinetics of NCEs through all
stages of DDD and not only in the development ones. Around the world, bioanalysis
shares the main goal of quantifying drugs (and/or metabolites) in biological matrices,
ensuring that high-quality data for valid scientific decision making is obtained.
For that, several initiatives are taken in day-to-day laboratory practices, such as
various kinds of validation, inclusion of independent quality control (QC) samples
or calibration curves mimicking real samples as close as possible. Considering the
distinct objectives and techniques employed in each stage of DDD to characterize
ADME/pharmacokinetics, it is evident that bioanalysis must be adapted to each
particular case, requiring different sample preparation and analytical methodologies
which do not need the same quality validation results.
Table 2.1.4 suggests the general characteristics of bioanalytical methods and
the validation requirements for each DDD stage. In opposition to the regulated
bioanalysis, which is focused on the rigorous development and validation of highly
specific bioanalytical methods for a selected drug candidate in a particular biological
fluid, the most striking feature of discovery bioanalysis is its breadth and diversity
of in vitro and in vivo studies. Consequently, a high number of sample matrices are
generated and a high number of different NCEs are simultaneously measured. As a
result, the throughput, capacity and turnaround requirements are generally much
higher in discovery phases in order to make timely decisions and process the elevated
number of samples. At this stage, early screening tests conducted in vitro and in vivo
can be performed following either good laboratory practices or not, with no real need
to input significant resources in an attempt of developing a rugged and fully validated
method. Instead, only some parameters, such as accuracy, precision and selectivity
must be contained under certain well-accepted boundaries to guarantee that the
results can be used for critical decision making. Thus, at drug discovery stages, the
ideal bioanalytical assay should be simple, straightforward, rapid, largely applicable,
efficient and allow significant throughput in order to be a successful screening tool
Table 2.1.4: Bioanalysis and method validation requirements suggested during drug discovery and development.
82
Stage Characteristics Objectives Validation
Drug Discovery • Very fast • Absorption screening (n-octanol/water, PAMPA, • Early study of detection and chromatographic
• High-throughput Caco-2 cells ) conditions
• Straightforward • Metabolic stability screening (microsomes, • Stability assays of stock solution
• Moderate quality data hepatocytes) • Former sample extraction procedure
• In vitro cytochrome P450 inhibitory screening • Selectivity (no sample endogenous interferences,
(microsomes, human recombinant enzymes) no interference of co-administered leads in cassette
• Plasma protein binding (ultrafiltration, dosing and/or cassette analysis)
equilibrium dialysis) • Calibration curve (low number of standards, n = 3-5)
• In vivo pharmacokinetic screening (cassette • Preliminary quantification range
dosing, pooling samples) • Reasonable precision and accuracy
Preclinical Drug • Fast • Pharmacokinetic characterization in rodents, •Sample extraction procedure optimization
Development • Moderate-throughput dogs, monkeys (single and multiple dose studies, •Selectivity (no sample endogenous interferences, no
• High quality data absolute bioavailability) interference of parent drug/metabolites)
• Route-dependent pharmacokinetic disposition •Internal standard selection
(intravenous/oral) •Defined range of standard calibration curve
• Toxicokinetics characterization of parent drug •LLOQ and ULOQ establishment
and its metabolites in toxicology species (dose/ •Intra and interday precision and accuracy assessment
exposure levels, estimating the first dose to (3-5 validation runs)
human exposure) •Effect of dilution on precision and accuracy

• In vivo drug-drug interaction potential •Extraction recovery assessment (parent drug/
metabolites and internal standard)
• Stability experiments to cover sample handling and
storage conditions

Table 2.1.4: Bioanalysis and method validation requirements suggested during drug discovery and development.
Continued
Stage Characteristics Objectives Validation
Clinical Drug • As fast as possible • Pharmacokinetics in healthy humans, target • Finalization of extraction, chromatographic
Development • Low-throughput patient population and special populations and detection conditions (based on anticipated
• Very high quality data (paediatric, geriatric, hepatic and renal concentrations following the lowest dose in humans)
impairment) • Full validation is required: selectivity, linearity, LLOQ,
• Food effect and relative bioavailability (solution/ LOD, accuracy, precision, recovery, parent drug/
suspension vs solid dosage form) metabolites stability experiments
• Drug-drug interactions with agents commonly co- • Verification of robustness/ruggedness of the assay
prescribed or with narrow safety window
Caco-2, human colon adenocarcinoma cell line; LLOQ, lower limit of quantification; LOD, limit of detection; PAMPA, parallel artificial membrane permeability
assay; ULOQ, upper limit of quantification.
Bioanalysis and Validation Requirements on DDD
83
capable of quantifying several NCEs subjected to the in vitro and in vivo tests performed
in initial screening experiments.
As the drug candidate enters to the development stages, matrix complexity
increases as well as the rigor needed to accurately quantify the NCEs in biological
samples and then estimate the pharmacokinetic parameters. The throughput of
the method may decrease, but the quality of data must be higher and validation
should be formalized and mandated as per the required norms. The most widely
accepted guidelines for validation of bioanalytical methods, particularly in
the pharmaceutical and medical sciences, are the Technical Requirements for
Registration of Pharmaceuticals for Human Use guideline Q2(R1) issued by the
ICH (2005), the Guidance for Industry, Bioanalytical Method Validation by the FDA
(2001) and the Guideline on Bioanalytical Method Validation recently issued by the
European Medicines Agency (EMA) in 2011. Typical validation parameters and the
requirements of each guideline are presented in Table 2.1.5. Selectivity is referred to as
the ability of a bioanalytical method to unequivocally differentiate and quantify the
analytes of interest in the presence of other sample components. Endogenous matrix
components, metabolites, and decomposition products or even co-prescribed drugs
include the most common interferences. Thus, it is necessary to establish that the
chromatographic signal produced is only due to the analyte and not as a result of the
co-elution of other compounds. A bioanalytical method is considered to be selective if
the lack of response in the blank biological matrix at the retention time of the analytes
is demonstrated at the lower limit of quantification (LLOQ). The determination of
method sensitivity, linearity, precision and accuracy is required by the three guidelines
and usually performed at three concentration levels in several replicates (typically
three to five). Linearity should be demonstrated within the defined calibration range
with the quality of the calibration curve as an aspect of the greatest importance in
bioanalytical method validation. In fact, as Almeida et al. (2002) stated, the quality of
bioanalytical data is highly dependent on the quality of the calibration curve used to
extrapolate the analyte concentrations in unknown samples. The calibration curve is
the relationship between instrumental response and the concentrations of the analyte
and it must be generated for each analyte, using a sufficient number of calibration
standards (Table 2.1.5). Precision, which expresses the closeness degree among
individual measures of an analyte when the same procedure is applied repeatedly to
multiple aliquots of a homogenous matrix, is frequently performed on the same day
(intra-day precision or repeatability) and over different days (intermediate precision
or inter-day precision), both expressed by the relative standard deviation (RSD),
which is the absolute value of coefficient of variation (CV). Accuracy, which describes
the closeness between the mean experimental data obtained by the method and the
corresponding nominal concentration, is also determined intra- and inter-daily and
is expressed by the relative error (RE), as a percentage, which is the ratio between
experimental and nominal concentrations, or by bias, which is the deviation from the
nominal value as a percentage. Matrix effects, carry-over and dilution integrity have

Table 2.1.5: The ICH, FDA and EMA validation parameters and respective limits instituted.
VALIDATION ICH FDA Limits EMA Limits

PARAMETER
Selectivity √ 6 blank samples No interferences 6 blank samples No interferences
LLOQ √ 6 replicates Precision ≤ 20% 6 replicates Precision ≤ 20%

Accuracy ± 20% Accuracy ± 20%
LOD √ NR NR
ULOQ NR NR Intra and inter-day (n = 6) Precision ≤ 20%

Accuracy ± 20%
Calibration curve 5 calibration 6-8 calibration Accuracy ± 15%a ≥ 6 calibration concentration levels Accuracy ± 15% a
linearity concentration levels concentration levels
Range √ NR Defined between LLOQ and ULOQ
Accuracy 3 concentration levels 3 concentration levels Accuracy ± 15% 4 concentration levelsb Accuracy ± 15% c
(Bias %) 3 replicates for each one 5 replicates for each one 5 replicates for each one
Precision 3 concentration levels 3 concentration levels Precision ≤ 15% 4 concentration levelsb Precision ≤ 15%
(RSD or CV %) 3 replicates for each one 5 replicates for each one 5 replicates for each one
With-in run and between-run
Recovery (%) NR 3 replicates Precise and consistent NR
Carry over effect NR NR Blank sample analysis following the Interference should be
high concentration standard ≤ 20% of LLOQ and
5% for internal
standard
Bioanalysis and Validation Requirements on DDD
85
Table 2.1.5: The ICH, FDA and EMA validation parameters and respective limits instituted.
Continued
86
VALIDATION ICH FDA Limits EMA Limits

PARAMETER
Dilution Integrity NR NR Matrix spiked with analyte Precision ≤ 15%
concentration > ULOQ and diluted with Accuracy ± 15%
blank matrix
5 replicates per dilution factor
Matrix Effects (%) NR NR 6 lots of blank matrix Precision ≤ 15%
Robustness √ NR NR
Stability NR Stability of stock and working solution of Stability of stock and working solution of standards, Stability
standards, Stability of the analyte in matrix of the analyte in matrix includes:
includes: long-term storage, short-term stability at room temperature
long-term storage, short-term temperature or sample processing temperature, post-preparative stability
stability, post-preparative stability, freeze–thaw at room temperature or under the storage conditions to be
cycle. used during the study (dry extract or in the injection phase),
freeze–thaw cycle.
Mean concentration at each level within ± 15% of nominal
concentration.
a
Limits defined for all calibration standards with exception of LLOQ (in LLOQ ± 20%);
b
One must correspond to the LLOQ
c
At LLOQ ± 20%
√, Parameter required but its limits are not referenced; LLOQ, Lower limit of quantification; LOD, Limit of detection; NR, Not required; RSD, relative standard
deviation (corresponds to the absolute value of coefficient of variation); ULOQ, Upper limit of quantification.
Bioanalysis and Validation Requirements on DDD 87
been recently integrated in the guideline issued by EMA as well as stability evaluation.
From a practical point of view, these parameters can be estimated from the analysis of
QC samples, which represent the future real samples.
During pre-clinical development, bioanalytical data may integrate the regulatory
submission process and influence clinical trials design, and, therefore, the method
may differ from the original assay of the discovery phase. Frequently, an internal
standard (IS) is added to samples in order to compensate errors that can occur during
sample preparation and chromatographic analysis, ensuring the reliability of analyte
quantification. Validation experiments should be performed maintaining constant
IS concentration, where the chromatographic response is given by analyte/IS peak
area or height ratio. A stable isotope-labeled derivative of the parent compound is
increasingly being used as IS. However, when not possible, a compound structurally
similar to the analyte and sharing identical behaviors during the entire bioanalytical
protocol is used.
It is noteworthy that various animal species and matrices are frequently
investigated within pre-clinical studies, requiring significant effort to fully validate
all the assays if they are independently developed to each species/matrix. In these
situations, a partial validation or cross validation may be sufficient to manage time
and resources more efficiently at this stage. Using cross validation, the analyst will rely
on the ability of the full validation protocol established in the matrix of one species
to predict the concentrations of NCE in a similar matrix of other species. However, the
scope of partial validation must be justified and certain parameters must be assessed
separately in the sample type that is being subjected to cross validation (FDA, 2001;
EMA, 2011), including stability and selectivity. In essence, although a significant
shortcut of the procedures is undertaken, it cannot compromise either the data quality
or the data integrity.
As the molecule advances into clinical trials, the assays developed to analyze
human samples need to be more sensitive, rugged and robust to some small variations
in matrices. Particularly in phase I clinical trials, high sensitivity is demanded to
ensure that the lowest effective doses can be identified. In fact, if the samples are from
first-time-into-humans studies, resolution is more important than throughput since
the number of samples is low but the fate of the compound is unknown, requiring
complete resolution of the analyte peak from all eventual matrices interferences.
Additionally, stability must be evaluated again because matrices are distinct from those
analyzed in pre-clinical stages. Similarly, it is also required to evaluate the method
selectivity, also considering the metabolites formed in vivo and drugs probably co-
prescribed to the patients in order to avoid their interference with the drug candidate
under investigation. Accordingly, it is desirable to develop flexible and robust assays
that enable minor alterations in chromatographic conditions to circumvent eventual
interfering peaks if necessary.
In current practice, MS detection is employed in the analysis of the majority
of pharmacokinetic samples from clinical studies, and the issues of selectivity and
specificity should not pose a major concern for the majority of NCEs at this stage.
However, other elements associated with LC/MS assays have to be assessed, including
the matrix effect, ion suppressing effect and, in the event of selective ion monitoring
(SIM), the generation of a common m/z ion for quantification of multiple peaks
(Srinivas 2008). It is also important to emphasize that the number of samples increases
significantly in the later phases II/III and IV clinical trials and high-throughput,
although desirable, may not be the best choice as compared to improving analytical
efficiency. At these stages, advantages can be found in automating the bioanalytical
process using fast, sensitive and reproducible chromatography with detection and
data-capturing capabilities that lead with high amount of data. In the next section,
some strategies within this scope will be outlined.
2.1.4 Bioanalysis & Pharmacokinetics, a Synergistic Partnership

on DDD
In the world of DDD, speed is essential to ensure a competitive position and success
in the highly-competitive life sciences marketplace. Therefore, it is important to
employ innovative thinking in all areas of DDD and identify laboratory technologies/
methodologies that reduce the cycle time from milestone to milestone in conformation
with all the requirements laid out for a regulated environment. To address this
challenge, besides the advances observed in the HTS in vitro and in vivo methodologies
broached in section 1.2, high-throughput bioanalysis has been continuously designed
and refined to improve the speed, quality and efficiency necessary to support the
increasing number of samples from ADME/pharmacokinetic studies. Table 2.1.6
represents the flexibility in today’s number and types of hyphenated bioanalytical
techniques, providing several recent bioanalytical techniques employed to investigate
the pharmacokinetics of NCEs in all stages of DDD programs. Study objectives, sample
preparation and validation parameters evaluated are also summarized therein.
As expected, samples obtained from in vitro and in vivo are predominantly
analyzed by LC (Table 2.1.6). Indeed, chromatography is critical to the success of
any bioanalytical assay as it allows the separation of the analytes from endogenous
substances and metabolites. Resolution of the peaks correspondent to analytes
and metabolites is essential to prevent an overestimation of the concentration of
parent compound and subsequently incorrect determination of pharmacokinetic
parameters and drug exposure. Even using highly selective MS or MS/MS detection, a
prior chromatographic separation (although simpler than those required with other
detection methods) is essential because the co-elution of endogenous or exogenous
compounds may cause ion suppression or ion enhancement, both detrimental to
the development of a sensitive method. Reversed-phase LC is the chromatographic
technique most widely employed in bioanalysis of drugs and their metabolites during
DDD due to its application to small molecules, which are separated by their different

Table 2.1.6: Examples of pharmacokinetic investigations performed during DDD, the bioanalytical conditions employed and respective parameters that were
validated.
Study type Objectives Sample preparation Chromatographic conditions Parameters Ref.

validated
Acyloxy(alkyl) ester based amino acid linked prodrugs of GOCarb
In vitro metabolic Estimate the half-life time of Samples filtration before HPLC HPLC-PDA NR Gupta,
stability in the prodrugs in Caco-2 cell analysis. C18 reversed-phase column (5 μm, 4.6 × 2013
intestine homogenates 250 mm)
Verify the conversion to the parent Mobile phase: water and ACN contained
drug (GOCarb). 0.1% TFA
Flow rate: 1 mL min-1
In vitro intestinal Evaluate the epithelial transport Samples filtered in 96-well filter LC-MS NR
permeability across Caco-2 monolayers; plates before analysis. C18 column (5 μm, 2.1 × 50 mm) with guard
Identify the most permeable column.
prodrug candidate. Mobile phase: water and ACN, containing
0.1% formic acid
Flow rate: 0.2 mL min-1
Positive ESI mode.
In vivo mice Oral (10 mg kg-1) and intravenous NR LC-MS/MS NR
pharmacokinetic administration (1 mg kg-1) to Swiss C18 (5 µm, 150 × 2.1 mm) column
studies Webster mice. Mobile phase: 0.1% formic acid/ACN
Determine Cmax, tmax, AUC and F of Flow rate: 0.2 mL min-1.
the prodrug and active metabolite Data acquired by MRM in ESI positive
(GOCarb). mode.
Bioanalysis & Pharmacokinetics, a Synergistic Partnership on DDD
89
Continued
Table 2.1.6: Examples of pharmacokinetic investigations performed during DDD, the bioanalytical conditions employed and respective parameters that
90
were validated.

validated
BMS 690514
In vitro absorption Evaluation of the Papp through NR LC-UV NR Marathe,

study Caco-2 cell monolayers. PDA detector. 2010
In vitro metabolic Investigation of the oxidative NR For microsomes studies NR

studies metabolism in LC-MS/MS
- liver microsomes and ODS-AQ S-5 120 Å (2.0 × 250 mm) column
suspensions of hepatocytes Mobile phase:
isolated from mouse, rat, dog, (A) 95% water and 5% ACN (v/v)
monkey and humans. containing ammonium acetate (pH 5.0)
- specific CYP isoforms in cDNA- (B) 100% ACN.
expressed human CYP enzymes. Flow rate: 0.2 mL min-1
Hepatocytes
LC–UV-radiometric-MSn
ODS-AQ S-5 120 Å (4.6 × 150 mm) column.
Mobile phase:
(A) water, CAN, 10mM ammonium acetate,
pH 5.0
(B) 100% ACN
Flow rate of 1.0 mL min-1

Continued
were validated.

validated
In vitro equilibrium Determination of the extent of PP with acidified acetonitrile LC-MS/MS Linearity; Marathe,
dialysis plasma protein binding in several containing IS and direct analysis C18 (2.1 × 50 mm, 5µm) Accuracy. 2010
species. of the supernatant. Mobile phase:
In vivo preclinical Analysis of the systemic, urine Brain was homogenized with (A) 10 mM ammonium formate, 0.1%
pharmacokinetic and brian pharmacokinetics in water and pretreated similarly formic acid and 0.2% ACN in water
studies mice, rats, dogs and monkeys to plasma. (B) ACN with 0.1% formic acid
after administration of a single Flow rate: 0.3 mL min-1.
intravenous or oral dose. Data acquisition by SRM transition
In vivo Describe the in vivo Blood collection to K2EDTA Quantification of brivanib in plasma NR Gong,
disposition and biotransformation of brivanib after tubes and glycine buffer, LC-MS/MS 2011
biotransformation a single oral dose of [14C]brivanib followed of centrifugation to C18 HPLC column (2.1 × 150 mm, 5 µm)
studies alaninate to rats, monkeys and obtain plasma. Mobile Phase:
humans. SPE extraction with a 96-well (A) 0.1% formic acid in water;
Identification of the metabolites SPE plate. (B ) ACN
and elucidation of their structure. IS: Stable isotope-labeled Quadrupole linear ion trap mass
brivanib spectrometer operated in positive ESI
mode. Detection achieved by MRM.
αv-ß3 bone integrin antagonist
In vitro Evaluate the binding plasma Extraction with Cyclone HTLC LC-ESI/MS/MS Validation Zhang,
ultrafiltration proteins. column (50 × 0.5 mm, 60 µm) C18 column (30 × 2.1 mm, 3 µm) according to 2006
Perform a HTS ultrafiltration and the mobile phase composed Mobile phase: 80% ACN and 20% 2mM international
method employing the 96-well of ACN and 2 mM ammonium ammonium formate (pH 3.0). guidelines.
plate. formate. Triple quadrupole mass spectrometer

IS: derivative of αv-ß3 bone performed in the positive ESI mode.
integrin antagonist Detection by SRM scan mode.
91
Continued
92
were validated.

validated
Cytarabine and 5’-amino acid esters prodrugs
In vitro Caco-2 Determination of Papp through NR HPLC-UV Linearity; Sun, 2008

Caco-2 monolayer; C18 column (5 μm, 200 × 4.6 μm) Recovery,
Identify the most permeable Mobile phase: Accuracy;
prodrug candidate and evaluate if NaH2PO4 buffer solution (5 mM): Precision.
it is a substrate of the PepT1 or if it methanol:formic acid (70:30:0.1%)
inhibits PepT1. Flow rate:1.0 mL min-1
Wavelength: 272 nm.
In vivo rat Determine the absolute Blood samples were placed HPLC-MS/MS Linearity;
pharmacokinetic bioavailability in rats after oral into heparinized tubes and C18 column (50 × 2.1 mm, 1.7 μm). Recovery,
dose response administration (5, 15, 30 mg kg-1) centrifuged to obtain plasma. Quantification by MRM Accuracy;
and compare it to that of the oral IS: lamivudine Analysis of Cytarabine and prodrug: Precision.
aqueous solution of cytarabine (8 For Cytarabine and prodrug Mobile phase: Water containing 2%
mg kg-1). extraction methanol and methanol containing 0.1%
Determination of the Plasma pretreatment by SPE formic acid.
concentrations of the prodrug, cation-exchange SPE cartridges. ESI source set in positive ionization.
cytarabine and its metabolite For Ara-U extraction Analysis of Ara-U

(ara-U) in plasma. PP with ACN. Mobile phase: Water and ACN;
IS: isoniazid ESI source set in negative ionization for
Ara-U and positive mode for IS.

Continued
were validated.

validated
CYP selective inhibitors
In vitro CYP Evaluate the effect of CYP PP with ice-cold ACN and direct UHPLC-QTOF-MS Linearity; Spaggiari,
metabolism inhibitors on each isoenzyme analysis of the supernatant. C18 column (100 × 2.1 mm; 2.5 μm) Inter and intra- 2014
studies in human activity using a specific substrate Mobile phase: day precision;
liver microsomes probe cocktail in human liver (A) water with 0.1% formic acid; Matrix effect.
microsomes. (B) ACN with 0.1% formic acid
Flow rate: 400 µL min-1
Q-TOF-MS system was operated in wide-
pass quadrupole mode
In vitro direct Evaluate the inhibition potential of PP with ice-cold ACN; LC-/MS/MS Linearity; Lee, 2013
and metabolism- test compounds on 8 human CYP Supernatant was put in 96-well C18 column (150 × 4.6mm, 5 µm) protected Accuracy and
dependent CYP enzymes. plates for analysis. by a C18 guard column (2.0 × 4.0 mm) precision.
inhibition studies IS: Carbamazepine Mobile phase:
(A ) 0.1% v/v formic acid in water;
(B) 0.1% v/v formic acid in acN.
API MS/MS operated in the positive and
negative ion mode
Quantitation was performed by MRM.
93
Continued
94
were validated.

validated
CYP substrate probes
In vitro cocktail CYP Evaluate the influence of NCEs PP with ACN. HPLC-MS/MS NR Otten,
inhibition studies in several CYP isoforms, using Plates centrifugation and direct RP-Amide column (2.1 × 50 mm, 5.0 µm) 2012
specific substrates and human injection of the supernatant Mobile phase:
liver microsomes. previously diluted. (A) Aqueous 0.1% acetic acid and 25:75
Quantify the main metabolite IS: labetalol. (v/v) methanol:ACN
of each substrate probe in the (B) 0.1% acetic acid.
presence and absence of the NCE. Flow rate: 0.6 mL min-1
API Triple Quadropole LC-MS used in the
positive ESI mode.
SRM to identify each probe substrate,
their metabolites and IS.
Clinical Simultaneous quantification of Blood centrifugation to obtain HPLC-MS/MS Full validation Oh, 2012
pharmacokinetic five CYP substrate probes and their plasma. C18 column (100 × 2.1 mm, 3.5 µm) according to
study main metabolites in plasma. Dual liquid-liquid extraction Mobile phase: FDA guideline.
Administration of the CYP with ethyl acetate. (A) 0.1% formic acid in water
substrate cocktail to healthy Organic layers evaporation (B) 0.1% formic acid in acetonitrile
volunteers and quantify the parent followed of reconstitution with Flow rate: 0.2 mL min-1
drugs and main metabolites. methanol. Linear ion trap mass spectrometer triple
IS: Propranolol quadrupole mass spectrometer equipped
with a turbo ion spray source.
ESI positive ion mode
Quantification by MRM.

Continued
were validated.

validated
Didanosine and peptidomimetic prodrugs of didanosine
In vitro Caco-2 Determination of Papp through Sample direct injection HPLC-UV NR Yan, 2011
Caco-2 monolayer; No IS C18 column (5 μm, 200 × 4.6 μm)
Identify the most permeable Mobile phase: mixture (25:75) of
prodrug candidate and very if: methanol 0.05 M and KH2PO4 buffer
- it is a substrate of the solution
oligopeptide transporter (PepT1); Flow rate:1.0 mL min-1,
- influence the uptake of Wavelength: 250 nm.
glycylsarcosine.
In vivo rat Determine the absolute Blood samples were placed UHPLC-MS/MS NR
pharmacokinetic bioavailability of the previously into heparinized tubes and Hydrophilic interaction column (50 ×
dose response selected prodrug candidate in rats centrifuged to obtain plasma. 2.1 mm, 1.7 μm)
after oral administration (5, 15, Plasma pretreatment by SPE Mobile Phase: water containing 0.1%
30 mg kg-1). using HLB cartridges (1 cc, formic acid and methanol (85:15, v/v)
Similar studies performed co- 30 mg) ESI source set in positive ionization.
administering the prodrug with IS: lamivudine Quantification by MRM.
glycylsarcosine.
95
Continued
96
were validated.

validated
Eslicarbazepine acetate, carbamazepine, and other derivatives
In vitro mice Determine the Papp through Sample centrifugation prior LiChroCART Purospher Star (C18, 3 µm, 55 Partial Fortuna,
intestine CD-1 mice intestine membrane analysis. × 4 mm; Merck KGaA) column. Validation: 2012
permeability mounted in Ussing chamber; Mobile Phase: water–methanol– Linearity;
Identify substrates of acetonitrile (64:30:6, v/v/v) Intra- and
P-glycoprotein. UV: 235 nm inter-day
precision and
accuracy.
In vivo In vivo pharmacokinetic Plasma: Chiral column LiChroCART 250-4 Full validated Fortuna,
pharmacokinetic characterization of the Blood samples were collected ChiraDex. according to 2013
studies test compounds and their to heparinized tubes and UV: 235 nm International
corresponding metabolites after centrifuged to obtain plasma. Mobile phase: water and methanol guidelines.
oral administration to mice. For CBZ, OXC, ESL, S-Lic, R-Lic and CBZ-E
Calculate Cmax, tmax, AUC, t1/2 and Brain: and main metabolites:
MRT in plasma and brain. Brain tissue was homogenized in Flow rate: 0.9 mL min-1
a 0.1 M sodium phosphate buffer IS: Chloramphenicol
pH 5 (4 mL g-1) and centrifuged. For BIA 2-059, BIA2-024, BIA 2-265 and
Plasma (300 µL) and brain their main metabolites:
homogenate (1 mL) were Flow rate: 0.7 mL min-1

pretreated by SPE using HLB IS.: CBZ-10,11-E poxide
cartridges (30 mg, 1 mL). For trans-diol:
Eluents were evaporated Mobile Phase: water-methanol-ACN
to dryness and the dry Flow rate: 1.0 mL min-1
residues were analyzed after IS.: 10,11-dihydrocarbamazepine
reconstitution.

Continued
were validated.

validated
Eslicarbazepine acetate
Clinical Studies Investigate the effect of gender, Blood samples were collected LC-MS Linearity; Falcão,
food and hepatic failure in the into tubes containing lithium LichroCART 250-4 ChiraDex analytical Precision and 2007;
biodisposition of eslicarbazepine heparin and centrifuged to column (β-cyclodextrin, 5 µm), a accuracy (with Almeida,
after oral administration of obtain plasma. LichroCART 4-4 ChiraDex guard column 3 QCs) 2008
eslicarbazepine acetate. (β-cyclodextrin, 5 µm).
Plasma pretreatment with Mobile Phase:
Schleicher and C18/100 mg 96- (A) 0.2 mM sodium acetate
well SPE plate. (B) 0.2 mM sodium acetate, MeOH.
Final extract was reconstituted in ESI source set in positive ion mode.
water:methanol.
IS: 10,11-dihydrocarbamazepine
97
Continued
98
were validated.

validated
Gabapentine enacarbil
In vitro metabolic Evaluation of the rate conversion NR HPLC-MS/MS Linearity; Cundy,

stability of gabapentine enacarbil to C8 (150 × 4.6 mm; 5 µm) Intraday 2004
gabapentine, quantifying both Mobile phase: precision;
coumpds after several times of (A) 0.1% formic acid in water Intraday
incubation in plasma, intestinal (B) 0.1% formic acid in ACN accuracy.
S9, liver S9, lung S9. Flow rate: 800 µL min-1
In vitro inhibition of In a 96-well format, analyze Detector was an API MS/MS
CYP isoenzymes the influence of gabapentine Data acquisition using the MRM mode.
enacarbil on the metabolism
of CYP substrate probes using
baculosomes..
In vitro metabolism Identify the CYP enzymes involved
by CYP isoforms in the metabolism of gabapentine
in human liver S9 enacarbil using specific inhibitors
fraction of each isoform.
In vitro Determine the plasma protein
ultrafiltration binding of gabapentine enacarbil.

In vitro transcelular Determination of the Papp of
transport gabapentine enacarbil and
gabapentine through Caco-2 and
MDCK cell monolayers.

Continued
were validated.

validated
Randomized, Investigate the pharmacokinetics Blood samples were collected LC/MS/MS Full validation Cundy,
double-blind, and tolerability after immediate- into tubes containing K2EDTA C18 HPLC column (50 × 4.6 mm) according to 2004
placebo-controlled release capsules of gabapentin and centrifuged to obtain Mobile phase: international
design. enacarbil oral administration at plasma. (A) water/ACN/formic acid (95:5:1); guidelines.
5 ascending single doses and Plasma samples were quenched (B) ACN/water/formic acid (95:5:1).
compared with commercialized with methanol immediately Flow rate: 2.0 mL min-1
gabapentine capsules. before analysis. Detector was an API MS/MS and detection
Urine samples had no performed with MRM.
preparation before analysis
Randomized Evaluate the pharmacokinetics

crossover and tolerability after extended-
release tablets of gabapentin
enacarbil had been orally
administered.
99
Continued
100
were validated.

validated
Randomized- Investigate in healthy volunteers
Blood was collected into LC-MS/MS Full validation Lal, 2009
sequenced the pharmacokinetics and tubes containing dipotassium Hydro-RP, Phenomenex (50 × 4.6 mm; according to
double blind, tolerability of gabapentin ethylenediaminetetra-acetic 4 μm) international
placebo-controlled enacarbil orally administered acid and centrifuged to obtain Mobile phase: guidelines.
crossover clinical as extended release tablets as aplasma. (A) 0.1% formic acid in water
study single oral dose. Two 1-mL aliquots were (B) 0.1% formic acid in ACN
Randomized, open- Evaluate the effect of food on the immediately transferred using Flow rate: 1200 μL min-1 Lal, 2010
label, crossover tolerability and pharmacokinetics a pipette to Nalgene tubes and API MS detector with the positive-ion
study of gabapentin enacarbil quenched with 3 mL of methanol. MRM mode.
administered as extended release IS for gabapentine:
tablets. L-4-chlorophenylalanine
Open label Describe a population IS for gabapentin enacarbil: Leu- Lal, 2013
single dose pharmacokinetic analysis of gabapentin phenylacetamide.
pharmacokinetic gabapentin enacarbil in patients
study with varying degrees of renal
function.
Randomized, Quantification of plasma levels Full validation Chen,
double-Blind, of gabapentin several times after according to 2012
placebo- and administration of gabapentin international

Active-controlled, enacarbil. guidelines.
crossover

Continued
were validated.

validated
Glimepiride
Bioequivalence Quantify glimepiride in Addition of o-phosphoric acid LC-MS/MS Full validation Kundlik,
study plasma samples from ongoing (2.5%) to plasma samples which C18 column (100 × 3 mm, 3.0 µm) according to 2012
development of an immediate- are loaded onto HLB 96 well Mobile phase was ACN and 2 mM international
release formulation. cartridges. ammonium format, pH 3.5 with formic guidelines
acid
Triple quadrupole mass spectrometer
equipped with an ESI ion source in
positive ionization SRM mode.
HM781-36B, a new anticancer drug
In vitro metabolism Profile and identify the Centrifugation and supernatant LC-MS/MS NR Kim, 2013
in liver metabolites in microsomes and evaporation to dryness. C18 column (150 × 2.1 mm, 3.5 µm)
recombinant CYPs. Analysis of the residue after Mobile phase: ammonium acetate
reconstitution. (10 mM, pH 3.6)/ACN
Use of MS, MS2 and MS3.
101
Continued
102
were validated.

validated
In vivo metabolic Profile and identify the Blood samples were collected UHPLC/Q-TOF MS NR
study metabolites in plasma, urine and into heparin tubes and UHPLC analytical conditions similar to
feces after oral administration to centrifuged to obtain plasma. those used to identify the metabolites;
dogs. Feces samples were Q-TOF mass spectrometer equipped with
Determination of AUC, tmax, Cmax homogenized after freeze-drying. an ESI source working in positive mode.
and t1/2. Liquid-liquid extraction with MS/MS experiments performed using the
0.05% formic acid and ethyl MSE.
acetate.
Supernatant evaporation and
injection of the residue after
reconstitution.
In vivo systemic Determination of jugular plasma Blood samples were collected LC-MS/MS-MRM Full validated
pharmacokinetic concentrations after oral into heparin tubes and Phenylhexyl column (50 × 2.0 mm, according to
study administration to dogs. centrifuged to obtain plasma. 5.0 µm) FDA guideline.
Determination of Cmax, tmax, AUC0-t IS: HM60781 Mobile phase: 80% methanol/20%
and t1/2. ammonium acetate buffer, 10 mM, pH 3.6.
In vitro equilibrium Determination of drug binding to MRM mode used for quantification
dialysis plasma proteins and brain tissue
using a 96-well system.

In vivo cassete Evaluate systemic and brain
dosing study pharmacokinetics of the 12 test
compounds after intravenous
bolus injection to ICR mice
(2.5 mg kg-1).

Continued
were validated.

validated
In vivo Determination of Cmax, tmax, AUC, Blood was collected into EDTA LC–MS/MS-MRM Linearity
pharmacokinetic t1/2,F and Cl after oral (10 mg tubes and centrifuged to obtain NR
evaluation kg-1) or intravenous (3 mg kg-1) plasma.
administrations PP with ACN containing the IS.
Psoralenoside, isopsoralenoside, psoralen and isopsoralen from Psoralea corylifolia extract
In vivo Calculate Cmax, tmax, AUC, MRT and Blood samples collection UHPLC-MS/MS Full validation Wang,
pharmacokinetic Cl after Psoralea corylifolia extract into heparinized tubes and C18 (2.1 × 50 mm,1.7 µm) according to 2014
study or benzofuran glycoside fraction centrifuged to obtain plasma. Mobile phase: FDA guidelines
oral administration to Wistar rats. PP with methanol; water addition (A) methanol
and injection of the supernatant. (B) 0.1% formic acid aqueous
IS: sophoricoside Flow rate: 0.2 mL min-1
Triple quadrupole mass spectrometry
Data acquisition by MRM in ESI positive
mode.
103
Continued
104
were validated.

validated
Rohitukine
In vitro hepatic Stability evaluation in PP with methanol and direct HPLC-MS/MS NR Chhonker,
and intestinal rat intestinal and hepatic analysis of the supernatant. C18 column (5 μm, 4.6 × 150 mm) column, 2014
metabolism microsomes; preceded with a guard column.
Prediction of the first pass effect Mobile Phase: 10 mM ammonium acetate
on drug bioavailability; (pH 4): methanol
In vivo Calculate Cmax, tmax, AUC, Vd, Blood samples collected Flow rate: 0.6 mL min-1 Full validation,
pharmacokinetic t1/2, Cl and F after oral (50 mg into micro-centrifuge tubes ESI source set in positive ionization. according
evaluation kg-1) and intravenous (5 mg kg-1) containing heparin and Quantification by MRM to US FDA
administrations. centrifuged to obtain plasma. guideline.
Plasma pretreatment by SPE (1
mL, C18) cartridges.
Evaporation of eluents and
analysis of the dry residue
reconstituted in methanol.
IS: Phenacetin
15 derivatives of Sulfonamide with antitumor activity
In vitro metabolic All test compounds were Centrifugation and direct LC-MS/MS NR Belka,
stability and incubated for 2 h with human liver injection of the resulting C18 column (3.0 × 100 mm, 2.7 µm) 2014
elucidation of microssomes in order to: supernatant. Mobile Phase:
metabolites - quantify the remaining parent (A) 0.1% formic acid in water
structures compound; (B) 0.1% formic acid in ACN
- estimate their metabolic Flow rate: 0.25 mL min-1
stability; MS detection with ESI positive mode.
- separate and identify the MS/MS detection with Q-TOF analyzer
metabolites generated. to collect the fragmentation spectra of
biotransformation products.

Continued
were validated.

validated
Tricin-amino acid derivatives as prodrugs
In vitro cell Investigate the Papp through MDCK Direct injection of apical and UV/Vis Spectrometry at 330 nm NR Ninomiya,
permeability cell monomlayers. basolateral solutions. 2011
evaluation Quantification of tricin
concentrations initially in apical
solution and at the end in the
basolateral solution.
In vivo systemic Determination of Cmax, tmax, AUC, Blood centrifugation to obtain HPLC-UV Linearity; Ninomiya,
pharmacokinetic t1/2,Vd and Cl of tricin after a single plasma. C18 column (250 × 4.6 mm, 5 μm) Inter-and intra- 2011;
studies oral dose of prodrugs (100, 300 or Liquid-liquid extraction with Mobile phase: ammonium acetate buffer day precision Cai 2003
1000 mg kg-1) to Crl:CD rats. acetone; water addition to the (0.1 M, pH 5.1) and methanol plus EDTA. (with 3QCs);
organic layer, centrifugation and Flow-rate: 1 mL min-1. Stability.
inject ion of the supernatant. Tricin concentration in plasma was
determined by an IS method.
ACN, acetonitrile; APCI, atmospheric pressure chemical ionization; API, atmospheric pressure ionization; Ara-U, 1-ß-D-arabinofuranosyluracil; AUC, area under
the curve; BBB , blood-brain barrier; BCRP, breast cancer resistance protein; CE, collision energy; Cl, systemic clearance; DAD, diode array detection; DP,
declustering potential; F, absolute bioavailability; FDA, food and drug administration; GOCarb, guanidine oseltamivir carboxylate; HPLC, high performance
liquid chromatography; IS, internal standard; MRM, multiple reaction monitoring; K2EDTA, dipotassium salt of ethylenediaminetetraacetic acid; MRT, mean
residence time; NR, not reported; PDA, photodiode array; PP, protein precipitation; QC, quality control samples; SPE, solid phase extraction; TFA, Trifluoroacetic
acid; t1/2, half-life time; UHPLC, ultra-high-performance liquid chromatography; UV, ultraviolet; Vd, volume of distribution;
105
affinity to the hydrophobic stationary phase in relation to the mobile phase. However,
compounds with low octanol-water partition coefficients and the majority of common
metabolites may hamper the development of a reliable reversed-phase LC method,
as they present a very short or non-existent retention time. Thus, the demand for
analytical laboratories to increase sample throughput provided the impetus for HPLC
column manufacturers to introduce new stationary phases and column geometries
in order to increase speed and sensitivity. In this context, monolithic columns were
created and have been employed with fast gradients and high flow rates for the direct
analysis of several pharmaceutical compounds in biological samples (Zhou, 2005;
Huang, 2006), but these columns have not been very frequently employed during
DDD. Indeed, ultra-high pressure liquid chromatography (UHPLC) seems to be the
most important achievement in LC evolution over the last decade and its application
is increasing in the pharmaceutical field (Table 2.1.6). This technology retains the
practicality and principles of traditional HPLC system but it is capable of providing
liquid flow at pressures higher than 10,000 psi and columns packed with small
particles (< 2 µm) that can withstand these pressures (Guillarme, 2013; Rodriguez-
Aller, 2013; Fekete, 2014; Nishi, 2014). It is well-known that the theoretical plate height
is proportional to the particle size, so using particle sizes down to 1.7 µm decreases
theoretical plate height and consequently produces a significant gain in efficiency that
does not diminish at increased flow rates or linear velocities (Gilpin, 2008). UHPLC is
becoming increasingly popular (Table 2.1.6) because it enables high speed analysis,
superior resolution, increased sensitivity and reduced overall cost per analysis,
ensuring that higher quality information is gained and laboratory productivity is
optimized using sub-2 µm particle technology. However, certain practical concerns
may need improvement, including sample introduction, reproducibility and the
possible formation of temperature gradients within UHPLC columns at such high
pressures. Moreover, extremely narrow sample plugs are required to minimize sample
volume effect on peak broadening.
Currently, LC/MS and LC/MS/MS take the bulk of the work during pharmacokinetic
evaluation in DDD programs (Table 2.1.6), particularly due to the inherent specificity
and sensitivity of these techniques, which dramatically increase throughput for
the quantitative determination of drugs and metabolites in biological matrices. In
addition, these methods reduce the need of exhaustive chromatographic separations
prior to detection, reducing analysis time. Even though both LC/MS and LC/MS/MS
are very expensive and not available to all research laboratories, bioanalysts have
been devoted to improve MS and MS/MS hardware and software, not only to identify
and quantify metabolites in a variety of in vitro and in vivo assays during all stages
of DDD, but also to allow users to benefit from lower LLOQs and ease of use. In fact,
reduced LLOQ values are essential in order to guarantee strong characterization during
the elimination phase of a compound. Besides their high sensitivity and specificity,
MS and MS/MS detections are based on a combination of the unique parent and
fragment masses of each compound, eliminating the need for baseline separation
Bioanalysis & Pharmacokinetics, a Synergistic Partnership on DDD 107
and achieving fast analyses. Prior to MS detection, analyte ionization is required and
can be achieved by electrospray ionization (ESI), atmospheric pressure ionization
(API) or atmospheric pressure chemical ionization (APCI) modes. MS detection of
ionized analytes has been carried out in a linear scan mode in which a range of
m/z values is constantly monitored. In a more selective and sensitive mode called
SIM, one specific m/z value (or more) is selected for the monitoring. Single-reaction
monitoring (SRM) or multiple-reaction monitoring (MRM) modes can also be used:
in SRM mode, a specific product ion of a specific parent ion is detected, producing
very simple plots (usually containing only a single peak). MRM delivers a unique
fragment ion that can be monitored and quantified in a very complicated matrix,
making the MRM plot ideal for sensitive and specific quantifications. Commonly,
several kinds of tandem mass spectrometers including the triple stage quadrupole,
the three-dimensional ion-trap, linear ion-traps/quadrupole coupled with time-of-
flight (Q-TOF) and hybrid triple quadrupole linear ion trap mass spectrometers have
been used in DDD.
Nevertheless, classical detection modes such as UV and fluorescence, although
less employed in current practice than MS or MS/MS detections, continue to be
applied in the early in vitro bioavailability or metabolic stability screening and
remain valuable in cases where sensitivity is not paramount or where LC/MS is not
economically viable.
2.1.4.1 Bioanalytic Support of In Vitro ADME Studies
Advances in techniques for chemical synthesis allow the synthesis of hundreds to

thousands compounds every month. As described in Chapter 2.1.2, several high-
throughput in vitro assays have been extensively used in early discovery to select the
NCEs most likely to have favorable pharmacokinetic parameters. The early ADME
screening of a larger number of samples is therefore required, which in turn calls
for high-throughput bioanalytical approaches. Although most publications on in vitro
studies hardly pay attention to the bioanalytical aspects involved in obtaining data,
it becomes clear considering the examples depicted in Table 2.1.6 that bioanalytical
techniques used to support these investigations commonly employ HPLC-UV, LC-MS
or LC-MS/MS.
LC-UV techniques are mainly used to collect data from in vitro absorption
methods, which aim at investigating the in vitro transport of drugs through intestinal
or other cell layers, predicting the in vivo transport of a drug through the intestinal
membrane and the involvement of certain transporter proteins, particularly the
efflux transporter P-glycoprotein and the oligopeptide transporter (PepT1). The most
common in vitro systems are the colorectal carcinoma cell line (Caco-2) and other
cell lines, like the MDCK (Volpe, 2011). The Ussing chamber technique has also been
employed, incorporating a healthy intestinal membrane. In both techniques, after
a specific incubation time, samples are taken from both sides of the monolayers
for bioanalytical determination of the drug concentration. Most of these in vitro
techniques are calibrated within the laboratory using a set of commercial drugs with
known human absorption fraction, which are plotted against the experimentally
obtained apparent permeability (Fortuna, 2012). In our laboratory, we have developed
an Ussing chamber technique employing mouse jejunum segments in order to predict
the human intestinal absorption fraction and the involvement of P-glycoprotein in
drug absorption. The technique was initially validated with reference compounds
and then applied to compare the absorption of nine derivatives of carbamazepine, a
classical antiepileptic drug (Fortuna, 2012). An HPLC-UV technique was successfully
employed in order to quantify each compound in our samples. However, a major
drawback is one frequently ascribed to the use of HPLC-UV in these analyses, which
is that for poorly permeable drugs, the LLOQ achieved is often not compatible with
the amount of drug present in the samples. At this point, some companies are opting
to develop LC-MS techniques or automated LC-MS/MS approaches to circumvent this
limitation (Table 2.1.6).
Nevertheless, the major application of LC-MS and LC-MS/MS during in vitro
ADME analysis regards drug metabolism. In fact, due to the publication of the FDA
guidelines on metabolites in safety testing (MIST) and DDI, metabolite profiling and
screening for metabolites in plasma samples have been recently included as part of
new drug discovery stages, implying the development of bioanalytical techniques
that quickly identify and quantify the parent drug and its metabolites. Information
generated from these studies could therefore enable the scientist to predict earlier
the major metabolite exposure in animals and humans, allowing a better strategy to
be developed. For instance, for compounds with extensive metabolism, identification
of metabolites may help medicinal chemists to modify the NCE to block the sites of
metabolism. On the other hand, active metabolites may have better pharmacokinetic
profiles than the dosed NCE and may become a new lead compound.
Moreover, the DDI guidance of EMA recommends the identification of the enzymes
involved in the formation and elimination pathway of metabolites that contribute
to more than 50% of the in vivo pharmacological effect (EMA, 2012). Since CYP
enzymes are the most frequently involved in drugs metabolism, the identification of
the specific isoforms involved in the metabolism of new compounds at early in vitro
stages become crucial, specifically whether the drug candidate is metabolized by
single or multiple enzyme isoforms and whether highly polymorphic enzymes, such
as CYP2D6 and CYP2C19, contribute to their metabolic clearance. In addition, these
in vitro techniques (mainly employing microsomes and hepatocyte cell lines) can
also be useful in identifying drug candidates that are inhibitors or inducers of CYP
isoforms, and can therefore predict their DDI potential. For these investigations, CYP
probe substrates must be used and their respective metabolites must be quantified
in the presence and absence of the drug candidate. Thus, it is not surprising that
significant efforts have been devoted to develop rapid chromatographic techniques
that simultaneously quantify CYP probe substrates/metabolites. At this level, LC-MS

and LC-MS/MS are undeniably involved in the characterization and quantification of
the metabolites and have been most frequently used to support these in vitro studies
(Table 2.1.6). To increase throughput, many probes have been applied to assess multiple
CYP activities simultaneously within a single experiment. This strategy, named the
cocktail approach, is starting to be employed in vitro to predict the clinical impact of
genetic polymorphism to CYP-based DDI (Rhodes, 2011; Kozakai 2012; Pillai 2013).
Traditional analytical methods using LC coupled with UV, radiometric, fluorescence
and luminescence detections have some limitations including a lack of substrate
specificity, solvent assay sensitivity and analytical interferences. Thus, LC-MS and
LC-MS/MS offer advantages over these classical methods such as high specificity and
sensitivity to investigate several enzyme-specific substrate probes and selectively
quantify each specific metabolite. The current methodologies employing the cocktail
approach to assess in vitro the activity of human CYPs can be found in the recent review
published by Spaggiari et al. (2014).
According to Table 2.1.6, ion trap mass spectrometers have been used to a large
extent to support in vitro metabolic studies as a qualitative tool, but in comparison
with classical quadrupole MS/MS, the quantitative features of ion trap mass
spectrometers remain less used. In fact, due to its small footprint and high sensitivity,
the ion trap was considered somewhat of an alternative to the triple quadrupole MS
for quantitative high-throughput analysis. However, the generation of MS/MS data
requires a relatively long duty cycle, limiting the numbers of analytes that can be
quantified simultaneously. Furthermore, the LC-Q-TOF MS technique has been widely
applied to obtain high-resolution mass spectral data of precursor and product ions
due to its high-throughput analysis, specificity, selectivity and resolution power. The
enhancement of its selectivity is attractive to reduce the problems such as matrix
suppression and metabolite interferences. It is also of great value in the interpretation
and elucidation of the chemical structure of the metabolites of each NCE. As a result,
the best strategy uses an ion trap to obtain the structural information by sequential MSn
experiments and MRM screening could be subsequently used to perform quantitation
for the drug and metabolites at trace levels, not only in vitro but also in vivo (Table
2.1.6). For further confirmation of metabolites, the LC-Q-TOF-MS/MS technique can
be applied for accurate mass measurement of the protonated molecules and their
fragment ions.
It must be emphasized that LC-MS/MS is also frequently employed to quantify
the drug fraction unbound to plasma proteins during early screening studies (Table
2.1.6). Indeed, the plasma protein binding of a drug can dramatically affect the
circulating concentration of the compound as well as its ability to be distributed and/or
accumulated through different compartments of the body. It is well accepted that only
the unbound drug is available to cross membrane barriers, be distributed to tissues,
and exert pharmacological and/or toxicological effects. In vitro equilibrium dialysis
and ultrafiltration are the techniques most frequently employed in initial phases of
DDD (Table 2.1.2). Devices based on multi-well formats have been recently developed
(Fung 2003; Zhang 2006; Plumb 2008; van Liempd, 2011; Zamek-Gliszczynski, 2011)
and employed to investigate NCEs (Table 2.1.6). Briefly, they consist of 48 or 96-well
Teflon base plates with a semi-permeable membrane that allows the passage only
of the unbound drug. When equilibrium between both membrane sides is reached
in the equilibrium dialysis technique, the total drug concentration is determined in
the plasma compartment while the free drug concentration is determined in buffer
compartment in order to calculate the percentage of drug bound to plasma proteins.
On the other hand, ultrafiltration is currently emerging as a faster and simpler
alternative to equilibrium dialysis once the centrifugation applied (approximately
2000 × g) accelerates the passage of the unbound drug through the membrane (Fung
2003; Zhang 2006). Particularly because of the low free drug levels that can be achieved
in the buffer compartment or in the filtrate, both in vitro techniques require the
application of LC-MS/MS methods to accurately quantify the drugs (Table 2.1.6). Both
MRM (Zamek-Gliszczynski, 2011) and SRM (van Liempd, 2011) detection modes seem to
be successful. Sample preparation prior to chromatographic analysis is very simple, and
relies on protein precipitation with acetonitrile (van Liempd, 2011, Zamek-Gliszczynski,
2011) or a mixture of acetonitrile/0.1% formic acid aqueous solution (90/10, v/v) (Plumb,
2008) followed by centrifugation. It is important to highlight that the bioanalytic
methodologies employed to quantify the drugs were at least partially validated in order
to confirm their acceptability for each specific compound (Table 2.1.6).
2.1.4.2 Bioanalytic Support of In Vivo ADME/Pharmacokinetic Studies
As depicted in Table 2.1.6, most of the in vitro techniques implemented to evaluate

the ADME of NCEs are complemented with non-clinical pharmacokinetic studies
performed in rodent animals (mice or rats). As expected, the major objectives include
the determination and evaluation of the extent and rate of absorption (AUC, Cmax
and tmax), distribution (Vd), clearance and duration of exposure (t1/2) of a drug and
its metabolites after oral and/or iv administration. UV, MS and MS/MS are the major
detection methods used for the quantification of parent drug in biological matrices,
but MS and MS/MS have been more frequently used and seem to be preferable for
relating metabolite response with that of the parent drug due to higher accuracy
and sensitivity. In fact, identification and structure elucidation of the metabolites
previously obtained in vitro must be confirmed in non-clinical and then in clinical
studies. Similarly, the monitoring of metabolite exposure in non-clinical species and
humans are also mandatory and therefore widely investigated in current practice
(Table 2.1.6) (Zhu et al., 2009). These results are crucial since, according to the FDA
guidelines on MIST and DDI, the relative exposure of metabolite can be obtained from
the metabolite profiling of time-averaged AUC-pooled human plasma samples from
multiple ascending dose studies.
It is interesting to note that similar chromatographic conditions can be employed to

investigate the pharmacokinetics of NCEs both in vitro and in vivo, although detection
may vary depending on the study objectives and the DDD stage. For instance,
Fortuna et al. (2012, 2013) employed a validated HPLC-UV technique to firstly determine
the in vitro apparent permeability of nine derivatives of carbamazepine through mice
intestine and secondly characterize the plasma and brain pharmacokinetics after
oral administration to mice. To attain those objectives, a C18 column was used (Table
2.1.6) and the parent compounds and main metabolites were extracted from biological
samples by a solid-phase extraction (SPE) procedure. The same chiral column and
similar SPE protocols were employed to analyze plasma samples from clinical studies
(Falcão 2008; Almeida 2008) but with MS/MS detection instead of UV. Moreover, Lv
et al. (2013) developed an effective screening strategy to select new agents for brain
tumor chemotherapy from a series of low molecular weight anticancer agents [ON123x]
by combining several in vitro studies. These studies aimed to evaluate compound
metabolic stability in mouse and human liver microsomes, predict their BBB
permeability using MDCK-MDR1 cell monolayers and estimate their binding to plasma
proteins and brain tissue. In vivo cassette dosing studies were then conducted in mice
for the 12 compounds, permitting the examination of in vitro/in vivo relationships to
confirm the suitability of the in vitro assays. The same bioanalytical technique was
employed for the analysis of all in vitro and in vivo samples, utilizing a C18-reversed-
phase column and MS/MS detection after a simple protein precipitation to clean-up
the samples.
It is noteworthy that the majority of the in vivo studies which quantify parent
drug and/or metabolites in biological samples require more demanding and complex
sample preparation protocols than in vitro techniques, resorting to validated analytical
techniques which guarantee the acquisition of precise and accurate data (Table 2.1.6).
Indeed, it is well known that besides chromatographic separation, sample preparation
is essential to reduce the effect of the endogenous and exogenous compounds that
exist in biological samples and to concentrate the analytes to consequently enhance
the method sensitivity.
In current practice, SPE seems to be the most widespread used procedure
to extract drugs and metabolites from biological samples (Table 2.1.6), probably
because it is versatile, very efficient and easily automated. Similarly to HPLC column
packing materials, SPE cartridges utilize a wide range of silica-based and polymer-
based sorbents and the extraction procedure follows the generic chromatographic
protocol. A novel 96-well SPE plate format is being widely employed, conferring a
unique design that makes the use of the sorbents higher efficiency and allows elution
of target compounds with small quantities of solvent. This HTS sample preparation
procedure is useful in initial phases of DDD but also in clinical trials including BA/BE
studies, since high number of samples must be analyzed as fast as possible (Falcão
2007; Almeida 2008; Xie, 2010; Boer 2012; Kundlik, 2012). Particularly the 96-well HLB
µElution SPE plate is composed of a 2 mg high-capacity SPE sorbent (divinylbenzene/
N-vinylpyrrolidone polymer), exhibiting excellent wetting properties and strong

hydrophobic retention. Due to these properties, the sorbent can be run to dryness
without loss of recovery, which is a major drawback of SPE sorbents based on silica-
C18. Moreover it allows elution of target compounds with only 25 µL of solvent and the
high capacity of the sorbent also tends itself to a concentration step (up to 15 times)
with no evaporation or reconstitution, which is a potentially critical factor in low dose
scenarios (Mallet, 2003; Yang 2005; Murphy, 2007; Lindegardh, 2008). On the other
hand, the liquid-liquid extraction (LLE) is also used due to its simplicity, ability to
provide clean extracts and the lower costs required. The extraction efficiency of LLE
is related with the partition coefficient that is controlled by the characteristics of the
extraction solvent (e.g., viscosity, surface tension, solubility in water, etc). In general,
organic solvents with low miscibility in water and are preferred for LLE. However,
selecting the right solvents (and their respective quantities) for sample partitioning
makes the development of LLE methods laborious. Once optimized, the application
of liquid-handling workstations allows semi-automated operations and therefore few
LLE performed in 96-well format have been employed for high-throughput analysis
(Chapter 12). Furthermore, 96-well LLE procedures may require large volumes of
extraction solvent, limiting their application during DDD programs. Instead, the
automated LLE combined with LC-MS/MS has been increasingly used in recent
years (Eerkes, 2003; Xue 2004) for the analysis of pharmaceutical moieties based on
advantages in efficiency, cost, and throughput. It is important to bear in mind that
plasma protein precipitation, dilution and shoot approaches can also be used, but
only to prepare biological samples that are simple and mainly composed of water,
such as urine, cerebrospinal fluid, saliva and tears.
2.1.5 Conclusions
Current discovery and development programs of new drugs promote pharmacokinetic

analysis with in vitro techniques from the earliest stages and throughout preclinical
and clinical stages. Thus, the quantification of the parent compound and its main
metabolites, as well as the identification of the major metabolites in buffers and
biological samples become essential and may determine the success of a developing
drug. The introduction of advanced analytical technologies with improved sensitivity
and selectivity has opened new frontiers in obtaining pharmacokinetic information
with higher accuracy and speed. Conventional HPLC techniques particularly when
coupled to mass spectrometry detection (MS or MS/MS) have been successfully
employed over the last decade for drug metabolism and clinical investigations.
Bioanalysis should not be performed blindly and the analytical methods need
to be subject to appropriate method validation depending on the DDD stages. The
development and validation of sound bioanalytical methods to support the DDD
process is paramount to produce high-quality data, contributing for a reliable
interpretation of pharmacokinetics.
References 113
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Ma-Li Wong, Martin Lewis and Julio Licinio*4
2.2 Translational Research in Endocrinology and
Neuroimmunology Applied to Depression
2.2.1 Major Depressive Disorder
Major depressive disorder (MDD) is a complex chronic illness of gene environment-

interactions with enormous medical, social and economic impact. It affects
1.4 million Australians yearly, having the highest non-fatal disease burden and being
the nation’s leading cause of disability (Tempier, 2009). MDD is a major cause of
suicide, which accounts for the third highest fatal disease burden in Australian men
(Tempier, 2009). MDD has considerable morbidity and mortality (Wong & Licinio,
2001, 2004). Currently, the point prevalence of this disorder ranges around 4−7%
and the lifetime prevalence estimate is about 15-20% (Kessler, 2005; Kessler, 1994).
In Australia, depression is the most common mood disorder, affecting 6.2% of the
population (Lohoff, 2010), it is the largest single cause of nonfatal disease burden
and the leading cause of disability (Vos, 2004). By the year 2020, MDD will become
the second most important contributor to the global burden of disease (Lopez &
Murray, 1998). However, MDD has been one of the oldest medical mysteries, described
since Hippocrates (460-37 BC) as melancholia (μελαγχολια). It has been extensively
studied in the past forty years; however, we continue to understand little about its
fundamental biology (Kessler, 2005; Kessler, 1994; Licinio & Wong, 2011) and the
genetic factors conferring susceptibility to this disorder (Sullivan, 2012).
The following findings provide persuasive evidence for a role of the stress system
in the pathophysiology of MDD: a) antidepressants directly down regulate the
hypothalamic-pituitary-adrenal (HPA) axis function; b) antagonism of corticotropin-
releasing hormone (CRH) reduces neuroendocrine, autonomic, and behavioural
responses to stress in primates, and c) increased cerebrospinal fluid (CSF)
concentrations of noradrenaline are increased around the clock, including in sleep,
which imply that dysregulation of a stress-related system is primary and not simply a
consequence of depressed mood (Wong, 2000; Wong & Licinio, 2001). Thus, chronic
uncontrollable stress may promote the onset of MDD and a shift towards environment
withdrawal (Clark & Beck, 2010).
Ma-Li Wong, Martin Lewis, Julio Licinio: Mind and Brain Theme, South Australian Health and Medical
Research Institute, PO Box 11060 Adelaide SA 5001, South Australia, Australia; Department of
Psychiatry, Flinders University School of Medicine, Sturt Road, Bedford Park 5042, South Australia,
Australia, *Email: julio.licinio@anu.edu.au
© 2015 Ma-Li Wong, Martin Lewis and Julio Licinio

120 Translational Research in Endocrinology and Neuroimmunology Applied to Depression
2.2.2 The Stress Response
A series of physiological and behavioral stereotyped responses, which have been

evolutionary developed to promote survival, characterize our reaction to danger.
That reaction, popularly known as the “fight-or-flight response”, is better reflected
by the term “fight, flight, freeze or fawn response” and includes increased heart
rate and defensive/offensive behaviors, which are modulated by an extensive and
complex circuitry that prepares the cardiovascular, musculoskeletal, endocrine,
metabolic and immune systems to deal with threating events, and inhibits certain
physiological functions, such as reproduction, sleep, foraging/digestion and
growth, which are dispensable until the cessation of the short-term acute stress
(Cahill & McGaugh, 1998; LeDoux, 1995). The core stress system includes the CRH,
the locus ceruleus-norepinephrine (LC-NE), and the immune systems. This core
system detects and monitors the intensity and duration of the stress response,
promotes arousal, modulates the limbic system and the cortical functions in order
to favor survival.
While the stress response to acute, short-term stress is physiological and adaptive
to promote survival during threatening situations, the persistent stress response to
long-term chronic stress is generally maladaptive and harmful, as it dysregulates the
stereotyped stress response (McEwen, 1998).
2.2.2.1 The CRH System and the Stress Response
Four related ligands (CRH, urocortin I, II and III), two receptors (CRHR1 and 2),
and a binding protein (CRHBP) that acts as an endogenous antagonist have been
recognized in this system (Aubry, 2013; Heinrichs, 1997). For the purpose of this
chapter, we will focus on the CRH and the CRHR1, as both are widely distributed in
the brain, and CRH has high affinity to CRHR1 and poor affinity to CRHR2.
CRH is the key hypothalamic factor in the HPA axis, it stimulates the pituitary
gland to release adrenocorticotropin hormone (ACTH) and indirectly regulates
glucocorticoids secretion and its production in the adrenal cortex. Besides its
critical role in the HPA axis, CRH plays multiple additional roles in the stress
response; it is implicated in coordinating the behavioral, neuroendocrine,
autonomic and neurovegetative aspects of the stress response; it is relevant in
functions such as the activation the LC-NE system, the sympathetic nervous
system, catecholamine synthesis in the adrenal cortex, fear-related behavior,
and the inhibition of exploratory behavior, food consumption, and growth and
reproduction functions (Chrousos & Gold, 1992; Gold, 1988a, 1988b). CRH also
modulates anxiety behavior, as corroborated by findings in studies of mice lacking
CRHR1, which display decreased anxiety (Smith, 1998) and of CRHR1 antagonists,
such as Antalarmin, which is a non-peptide selective CRHR1 antagonist.
The Stress Response 121
Antalarmin administration results in inhibition of anxiety-like responses induced

by stress and the promotion and establishment of conditioned fear (Deak, 1999;
Habib, 2000). Antarlamin also appeared to prevent increases in circulating ACTH,
NE, epinephrine, and cortisol levels (Habib, 2000).
2.2.2.2 The Locus Ceruleus Norepinephrine (LC-NE) System and Other Central Nervous
System (CNS) Structures that Modulate the Stress System
Located in the brainstem, the LC is a homogenous nucleus with the largest

number of noradrenergic neurons in the brain that innervates the entire neuroaxis.
LC neurons have synchronous spontaneous activity that provides a mechanism for large-
scale NE release across the CNS in response to stimuli (Berridge & Waterhouse, 2003). The
LC acts as the brain’s alarm system, after a stressful challenge there is strong
activation of this system that provides a mechanism by which external and internal
stimuli induce arousal and vigilance. Multiple stressors that activate the HPA
axis, also engage the LC-NE system, and induce immediate-early gene expression
and NE release (Cullinan, 1995). The LC-NE system stimulates the HPA axis and the
sympathetic nervous system while hindering neurovegetative functions and the
parasympathetic nervous system (reviewed in Aston-Jones, 1996).
The LC promotes survival during a crisis by favoring fast and simpler
responses through inhibition of frontal cortex functions (Arnsten, 2000) and via
the amygdala and other CNS structures that encode aversive memories.
LC neurons discharge in 2 types of modes: tonic and phasic to modulate
attention and behavior (Aston-Jones & Cohen, 2005). The effects of CRH or
stressors not only increases LC activity but also modify LC discharge toward the
tonic mode; this shift in discharge style is attenuated by CRH antagonism or by
opiate antagonism and the combined removal of CRH and opioid actions rendered
the LC activity resistant to the effects of the stressor (Curtis, 2012). Stress-induced
LC activation increasing arousal but could also facilitate behavioral flexibility
during threatening situations (Snyder, 2012).
The amygdala can also modulate hypothalamic CRH release and autonomic
centers in the brainstem to respectively increase HPA axis and LC-NE system
activities while inhibiting the prefrontal cortex (Cahill & McGaugh, 1998).
Neurons in the hypothalamus, LC and amygdala contain multiple feed-forward
connections that can promote a robust and persistent stress response.
The dorsolateral prefrontal cortex is implicated in cognition and attention, and
the ventromedial prefrontal cortex regulates affect, neuroendocrine, and autonomic
activities [reviewed in (Arnsten, 2000; Fuster, 2000, 2001; Smith & Jonides, 1999)].
Cortical lesions in humans and rodents cause exaggerated autonomic and endocrine
responses; thus, there is a reciprocal inhibitory relationship between the prefrontal
cortex inhibiting the stress system, in which they can inhibit the activity of each
other (Fuster, 2000, 2001; Smith & Jonides, 1999), and activation of the prefrontal
cortex and consequent restraint of the stress and the sympathetic nervous systems
promote flexibility in cognition and affect (Smith & Jonides, 1999).
2.2.2.3 The Immune System
Stress affects many central and peripheral systems in the body, and the immune
system is critical for the promotion of survival; therefore, it has key roles during the
stress response and immune functions can be enhanced or suppressed by stressors.
Endocrine and cytokine mediators modulate the immune function during short-term
acute stress, which can modulate immune responsiveness encompassing humoral
and cellular aspects of both innate and adaptive immune responses.
The humoral changes related to stress response to psychological stressors, such
as the Trier Social Stress Test, include significant increases in the concentration of
cytokines, including interleukin-6 (IL-6) and IL-1ß (Altemus, 2001; Aschbacher,
2012; Pace, 2006; Prather, 2013; Puterman, 2014; Steptoe, 2007). Cytokines are small
proteins released by the immune system, typically under inflammatory situations.
These increased circulating cytokine levels may enhance the immune system
during acute stress, contribute to survival, and are also related to emotional states.
Peripheral IL-6 levels during stress reaction are related to the experience of anger;
it is likely that an angry individual will engage in an aggressive confrontation and
sustain injuries and an enhanced immune response will help promote wound healing
(Puterman, 2014).
It is now understood that peripheral cytokines modulate brain functions during
physiological conditions, where they can regulate neuronal processes, including
stress, inflammatory challenges, sickness behaviour, feeding, sleep, learning and
memory (Vitkovic, 2000a; 2000b), and that the communication between the brain
and the immune system is bidirectional. Our lab has contributed to the understanding
of many of the central aspects of cytokine response. We described the expression of
cytokines and immunomediators in the brain during baseline and after inflammatory
challenges (Licinio, 1991, 1992; Wong, 1997; 1996; Wong & Licinio, 1994); during
systemic inflammation, there is a high expression of central IL-1ß. Contrary to the
potent systemic counter regulatory anti-inflammatory response, in the brain the
expression of counter regulatory cytokines, such as IL-1 receptor antagonist and IL-
10, is much lower, which supports that differential regulation of anti-inflammatory
cytokines in the CNS and periphery (Wong, 1997).
Acute stress also orchestrates a massive redistribution of immune cells in the body,
which is consistent with the critical role the immune system has on survival; therefore,
functions such as wound healing and immune surveillance (Dhabhar, 2012) would be
enhanced during acute stress. Stress responsive hormones such as NE, corticosterone
and epinephrine (EPI) influence many different subsets of immune cells. NE and EPI
The Effect of Chronic Stress and MDD in Dysregulating the Core Stress System 123
are released early in the stress response; while, NE increases leucocyte numbers,
mobilizing immune cells, including neutrophils, monocytes, and lymphocytes to
enter the blood; EPI mobilizes neutrophils and monocytes into the blood, but directs
lymphocytes to leave the circulation to specific tissues, such as skin. Corticosterone is
then released and mobilizes immune cells to leave the blood towards tissues.
Acute restraint stress increases the numbers of T cells, such as memory and
effector helper cells, in sentinel lymphnodes (Dhabhar & Viswanathan, 2005). Stress-
induced increments in T cell memory may stimulate the increase of infiltrating
lymphocytes and macrophage detected on antigen re-exposure several months later
and this process is driven by increased levels of CD4+ T helper (Th) cells type 1 (Th1)
cytokines IL-2, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α).
2.2.3 The Effect of Chronic Stress and MDD in Dysregulating the

Core Stress System
In chronic or long-term stress the physiological stress response continues long after
the stressor has ended, resulting in a prolonged exposure to stress hormones and
stress-related reactions, or is activated repeatedly as a result of continued exposure to
the stressor. Chronic activation of the HPA axis and sympathetic nervous system are
common in MDD, melancholic subtype; circulating levels of cortisol are elevated and
it is commonly accepted that the central CRH level is increased (or inappropriately
non-suppressed for the level of hypercortisolism). Elevations of the cerebrospinal
fluid (CSF) 24-hour levels of NE and plasma cortisol indicate that there is a persistent
stress-system dysfunction in MDD, melancholic subtype, which is not conditional
on the conscious awareness of stress (Wong, 2000). Circadian measures of CSF NE
and CSF CRH and peripheral cortisol suggest mutually reinforcing bidirectional
relationships between brain NE and CRH pathways that promotes increased central
noradrenergic, adrenomedullary, and adrenocortical secretion, which promote a state
of hyperarousal and anxiety found in MDD (Gold, 2005; Wong, 2000).
In MDD, atypical subtype, patients display fatigue, lethargy, hypersomnia,
and hyperphagia, which are related to suppression of the stress system mediators
(Chrousos & Gold, 1992; Gold, 1988a, 1988b). Patients with atypical features have
been reported to be eucortisolemic and have low CSF CRH levels (Geracioti, 1997;
Gold, 1995). However, detecting decrement in HPA axis is challenging. In response
to synthetic CRH, these patients show delays in peripheral ACTH response and very
attenuated cortisol responses (Schulte, 1984).
We have reported the involvement of the CRHR1 gene in antidepressant response;
anxious patients homozygous for the GAG haplotype of CRHR1 gene had a better
antidepressant response (Licinio, 2004); as pre-clinical studies suggested that
antidepressants suppress CRH gene expression (Brady, 1992; 1991; Reul, 1993) and
clinical studies reported that antidepressants reduce CSF CRH concentrations (De
Bellis, 1993; Heuser, 1998; Veith, 1993), downregulation of CRH activity could be a
common pathway for the therapeutic actions of antidepressants.
Chronic stress can also result in dysregulation with activation or suppression of
the immune system; clinical and pre-clinical research support a role of immune system
dysregulation in MDD with activation of the cellular and humoral immune responses
(Kronfol & Remick, 2000; Licinio & Wong, 1999). Increased circulating lymphocytes
and monocytes have been described (Herbert & Cohen, 1993; Rothermundt, 2001;
Seidel, 1996). Increased circulating IL-6 levels have been described in MDD and these
levels were significantly correlated with self-reported mood ratings (Alesci, 2005).
Pro-inflammatory cytokines, such as IL-1ß and IL-6, stimulate the HPA axis (Plata-
Salaman, 1991). Stress-induced depressive-like behaviours also increase peripheral
IL-6 levels in rodents (Himmerich, 2013), and recently brain IL-6 has been implicated
in facilitating cognitive flexibility in the orbitofrontal cortex (Donegan, 2014). A
depressive syndrome can be associated with cytokine administration, typically
during IFN-α treatment of hepatitis C or malignant melanoma (Bonaccorso, 2000;
Dieperink, 2004; Hauser, 2002; Kraus, 2003; Schaefer, 2004), the psychopathological
presentation can include changes in mood or cognition, psychomotor retardation,
fatigability, sleep disturbances and decreased appetite (Capuron & Miller, 2004;
Loftis, 2006; Raison, 2006).
Dysregulation of the T-cell arm of the immune system has corroborated the role
of immunomediators in depression (Kronfol & Remick, 2000; Pavon, 2006). Evidence
supporting both the predominance of T-cells Th1 or Th2 have increased and the
balance of these two subtypes of cytokine-producing T cells Th1/Th2 regulates the
immune response to pathogens. These two main lineages of cells differentiate from
naïve CD4+ T lymphocytes and have distinct cytokine profiles. Th1 patterns are related
to increased inflammation and autoimmune conditions, and the production of IFN-γ,
TNF-α or IL-1; conversely, Th2 patterns are related to allergic responses and asthma,
and the production of IL-6, IL-10 or IL-13 (Kimball, 2005; Pavon, 2006; Scott, 2007).
Data from our lab supports that there is an imbalance of Th1/Th2 activities with
a predominance of Th1 in MDD; we found increased chemokine CXCL10/IP10 levels,
which decreased with antidepressant treatment, and decreased IL-13 levels; although
no distinct Th1 or Th2 cytokine patterns was found in our MDD patients (Wong, 2008).
Nevertheless, different types of MDD may exhibit divergent immune profiles, and
decreased Th1 activation has been described in melancholic depressed patients, which
is accompanied by a marked HPA axis activation. Non-melancholic MDD patients show
increased inflammation features with elevated monocyte count and levels of IL-1ß and
α2-macroglobulin (Kaestner, 2005; Rothermundt, 2001). Post-partum depression also
shows a predominance of Th1 activation (Maes, 2002; Ostensen, 2005), and symptoms
of anxiety and depression in the early post-partum period are related to increased
levels of pro-inflammatory cytokine IL-8 (Maes, 2002).
In the brain, stress-induced increased glucocorticoids enhances immune function,
promotes microglia proliferation and activation, and a loss of astrocytes number and
References 125
volume (Czeh, 2006). Increased concentrations of pro-inflammatory mediators can

lead to hippocampal dendritic atrophy and neuronal death (Sapolsky, 1985; Woolley,
1990).
Overall, antidepressants reduce Th1 cytokines production in vitro, promoting
a shift towards Th2 patterns (Leonard, 2001). For instance, imipramine, clomipramine,
fluoxetine, sertraline and venlafaxine amongst other antidepressants, significantly
decrease the IFNγ/IL-10 ratio (Kubera, 2001; Maes, 1999; Szuster-Ciesielska, 2003).
Amitriptylin and nortriptylin inhibit IL-1ß and TNF-α release in in vitro cultures of
microglial and mixed glial cells (Obuchowicz, 2006). In MDD patients, decreases in
circulating IL-6 levels and TNF-α (Basterzi, 2005; Frommberger, 1997; Narita, 2006;
Sluzewska, 1995), and increases in anti-inflammatory Th3-cytokine transforming
growth factor beta (TGF-ß) (Myint, 2005) have also been reported during antidepressant
treatment.
2.2.4 Summary and Conclusions
Optimal performance of the CNS requires that the core stress system be maintained
within a particular range, and emotional responses involve the integration of several
areas of the brain. Superb collaboration of several CNS and peripheral systems in a
sequentially coordinated manner are required for the optimal response of the stress
system, which will ultimately determine the ability of an organism to survive under a
threat. Dysregulation of the stress system is implicated in MDD; melancholia involves
the hyperactivity of the stress system and atypical features are related to the down-
regulation of the stress response; likewise, dysregulation of the immune response
implicates the activation or suppression of humoral and cellular immune components.
The core stress and immune systems interact with other CNS pathways that have also
been reported to be dysregulated in MDD, including the serotonergic, glutamatergic,
and kynurenine pathways (Muller & Schwarz, 2007).
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Cidália Pereira, Rosário Monteiro, Maria João Martins*5
2.3 Understanding the Metabolic Syndrome Using
a Biomedical Chemistry Profile
2.3.1 Introduction
For quite some time, it has been identified that high blood pressure, dyslipidemia
[increased triglycerides and reduced high-density lipoprotein (HDL) cholesterol
levels], impaired glucose homeostasis and abdominal obesity take place concurrently
more than by random, supporting the existence of the metabolic syndrome (MetSyn).
Additionally, hyperuricemia, a prothrombotic state, oxidative stress, chronic low-
grade inflammation, increased levels of apolipoprotein-B and small dense low-
density lipoprotein (LDL) cholesterol (contributing to atherogenic dyslipidemia),
non-alcoholic fatty liver disease and/or non-alcoholic steatohepatitis, obstructive
sleep apnea and/or polycystic ovarian disease (Fulop, 2006; Alberti, 2009; Roberts,
2009; Ma, 2012; Matsuda, 2013; Mule, 2014; Carson, 2015) are quite often present on
the MetSyn, although not yet included in its current/actual definition. Taking this into
consideration, it is not surprising that the MetSyn associates with an increased risk
of type 2 diabetes mellitus (T2DM) and atherosclerotic cardiovascular disease (Fulop,
2006; Qiao, 2007; Carson, 2015).
Epidemiological and experimental evidence has demonstrated beneficial effects of
dietary magnesium, calcium, potassium and bicarbonate on the MetSyn or some of its
individual components (Luft, 1990; Van Leer, 1995; Schorr, 1996; Whelton, 1997; Franch,
2004; Rylander, 2004; Schoppen, 2004; Franzoni, 2005; Karppanen, 2005; Schoppen,
2005; He, 2006; Feldeisen, 2007; Schoppen, 2007; Champagne, 2008; Rylander, 2008;
Volpe, 2008; Perez-Granados, 2010; Adeva, 2011; Rice, 2011; Lee, 2013).
Natural mineral-rich waters are good sources of highly absorbable and bioavailable
minerals (such as calcium, magnesium and potassium) and bicarbonate (Heaney,
1989; Bohmer, 2000; Kessler, 2000; Sabatier, 2002; Bacciottini, 2004; Kiss, 2004;
Heaney, 2006; Karagulle, 2006; Petraccia, 2006; Karagulle, 2007; Sabatier, 2011). So,
the particular composition of natural mineral-rich waters would be responsible for
their favorable effects. In fact, the World Health Organization has recognized that
Cidália Pereira: Department of Biochemistry, Faculty of Medicine, University of Porto, Alameda Prof.
Hernâni Monteiro, 4200-319, Porto, Portugal; Escola Superior de Saúde, Instituto Politécnico de Lei-
ria, Campus 2 – Morro de Lena - Alto do Vieiro, Apartado 4137, 2411-901, Leiria, Portugal.
Rosário Monteiro, Maria João Martins: Department of Biochemistry, Faculty of Medicine, Universi-
ty of Porto, Alameda Prof. Hernâni Monteiro, 4200-319, Porto, Portugal ; Instituto de Investigação
e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal.
*Email: mmartins@med.up.pt.
© 2015 Cidália Pereira, Rosário Monteiro, Maria João Martins

Natural Mineral-rich Waters and MetSyn 133
mineral-rich drinking-waters may provide substantial contributions to total intakes

of calcium and magnesium in some populations or population subgroups (Cotruvo,
2009). In line, two recent and exhaustive studies revealed that consumption of public
drinking waters and bottled natural mineral waters is a relevant complementary
source of calcium and magnesium in Spain (Vitoria, 2014; Maraver, 2015).
Beneficial effects of natural mineral-rich waters ingestion on MetSyn features,
included or not in its definition, and MetSyn complications (Luft, 1990; Simunic, 1990;
Schorr, 1996; Polushina, 1998; Polushina, 2002; Rylander, 2004; Schoppen, 2004;
Schoppen, 2005; Schoppen, 2007; Benedetti, 2009; Botvineva, 2010; Perez-Granados,
2010; Santos, 2010; El-Seweidy, 2011) as well as on the MetSyn itself (Pereira, 2012a;
Pereira, 2013; Pereira, 2014a; Pereira, 2014b; Pereira, 2014c; Pereira, 2015) have been
published.
2.3.2 Natural Mineral-rich Waters and MetSyn
The consumption of sodium bicarbonate containing/mineral-rich waters decreased

systolic blood pressure in mildly hypertensive men (3 L day-1, 7 days) (Luft, 1990) and
mean arterial blood pressure in elderly normotensive individuals (1.5 L day-1, 4 weeks)
(Schorr, 1996). The ingestion of sulfate, calcium, magnesium and bicarbonate-rich
natural mineral water (at least 1 L day-1, 4 weeks) reduced systolic and diastolic blood
pressure in adults with borderline hypertension and low urinary magnesium and
calcium excretion levels (effect perceived after 2 weeks of consumption and sustained
until the end of the dietary protocol) (Rylander, 2004). Vaquero et al. showed that, in
moderately hypercholesterolemic young adults, the ingestion of bicarbonated natural
mineral water, rich in sodium, chloride and potassium, and with a high bicarbonate to
sodium ratio (1 L day-1, 8 weeks), reduced systolic blood pressure (an effect already seen
after 4 weeks), apolipoprotein-B, total-cholesterol and LDL-cholesterol fasting serum
levels as well as total-cholesterol and LDL-cholesterol to HDL-cholesterol ratios (Perez-
Granados, 2010). The same group showed, in healthy postmenopausal women, that
a) the ingestion of the previous water (1 L day-1, 2 months) increased HDL-cholesterol
and decreased endothelial dysfunction markers, glucose, total-cholesterol and LDL-
cholesterol fasting serum levels as well as total-cholesterol and LDL-cholesterol to
HDL-cholesterol ratios (Schoppen, 2004), and b) the consumption of sodium-rich
bicarbonated mineral waters (0.5 L each) with a standard fat-rich meal increased
insulin sensitivity [more distinctly in the women with higher homeostasis model
assessment (HOMA) index values] and decreased lipemia (Schoppen, 2005; Schoppen,
2007). Both a decrease in lipid and protein oxidation products and an increment of total
antioxidant capacity and total thiol plasma levels were observed in healthy individuals
drinking a sulfurous mineral water (0.5 L day-1, 2 weeks) (Benedetti, 2009).
Our group evaluated the effects of the ingestion of a Portuguese natural mineral-
rich water, and some of the possible mechanisms involved, on metabolic function in
134 Understanding the Metabolic Syndrome Using a Biomedical chemistry profile
a well-validated MetSyn animal model (Polizio, 2006; Rayssiguier, 2006; Oron-

Herman, 2008): male Sprague-Dawley rats treated with 10% fructose in drinking water,
for 8 weeks. Animals were randomly assigned into three groups with free access to
food and a) tap water, b) 10% fructose in tap water or c) 10% fructose in Portuguese
natural mineral-rich water. As expected, 10% fructose in tap water induced metabolic
features characteristic of the MetSyn, such as increased plasma levels of triglycerides,
insulin and leptin [with a strong tendency toward decreased insulin sensitivity index
(Cacho, 2008)] and decreased plasma levels of magnesium as well as increased systolic
blood pressure and heart rate. Fructose-induced effects in the redox state (liver),
endoplasmic reticulum homeostasis (liver), glucocorticoid and insulin signalling
pathways (liver and visceral and/or subcutaneous adipose tissue) and endothelial
dysfunction markers expression (cavernous tissue) may have contributed to explain
the induction of MetSyn; some compensatory mechanisms against fructose-ingestion
were also revealed. Importantly, the co-ingestion of the Portuguese natural mineral-
rich water reduced and/or prevented most of the changes induced by fructose and,
additionally, strengthened the compensatory mechanisms and induced per se
protective pathways in response to stress (Pereira, 2012a; Pereira, 2013; Pereira, 2014a;
Pereira, 2014b; Pereira, 2014c; Pereira, 2015). This Portuguese natural mineral-rich
water also increased hepatic catechol-O-methyltransferase activity in healthy Wistar
Han rats (Bastos, 2014). Its high-content of protective minerals, such as magnesium,
calcium and potassium, as well as bicarbonate, and low chloride content may explain
the favourable results obtained (Pereira, 2012a; Pereira, 2013; Pereira, 2014a; Pereira,
2014b; Pereira, 2014c; Pereira, 2015).
2.3.3 Magnesium and MetSyn/MetSyn Features – Associated

Mechanisms
Chronic deficiency of magnesium (in animal models with low magnesium intake) is
associated with hypertension and increased heart rate (and somewhat higher plasma
corticosterone levels) as well as dyslipidemia, insulin resistance and oxidative stress
(Caddell, 1991; Balon 1994; Laurant, 1999; Busserolles, 2003; Takaya, 2012).
Clinical and experimental studies point to magnesium intake/status being
inversely associated with the risk of hypertension, T2DM and coronary heart disease.
Additionally, magnesium intake may decrease triglycerides and increased HDL-
cholesterol circulating levels (Balon, 1994; Touyz, 2003; Takaya, 2004; Barbagallo,
2007; Belin, 2007; Abete, 2011; Heer, 2015).
Individuals with MetSyn (or with some of its individual components) frequently
show reduced magnesium status and reduced magnesium intake as compared with
non-MetSyn (or healthy) subjects (Barbagallo, 2007; Belin, 2007; Evangelopoulos,
2008; Abete, 2011; Heer, 2015). Interestingly, hypomagnesaemia has been associated
with metabolic abnormalities characteristic of MetSyn in the absence of obesity and,
Magnesium and MetSyn/MetSyn Features – Associated Mechanisms 135
conversely, normal circulating levels of magnesium seem to be protective against the

development of metabolic complications in obese individuals (Guerrero-Romero,
2013). Often, circulating and/or intracellular magnesium levels are reduced under
insulin resistance/T2DM in (obese) children, adolescents and adults (Takaya, 2004;
Huerta, 2005; Belin, 2007; Wells, 2008; Celik, 2011). Lecube et al. provided evidence
that T2DM was the main factor accounting for the hypomagnesemia found in morbidly
obese individuals. They observed that the percentage of morbidly obese individuals
with serum magnesium concentration lower than 0.75 mmol L-1 was three fold
higher in T2DM patients than in non-T2DM subjects. They also found that not only
the degree of blood glucose control (when considering fasting plasma glucose and
HbA1c levels) and serum magnesium concentration were significantly and negatively
correlated but also fasting plasma glucose and HbA1c levels were, in multiple linear
regression analysis, independently associated with serum magnesium concentration.
Additionally, in the morbidly obese patient subgroup that went through bariatric
surgery, serum magnesium levels increased in T2DM subjects in whom diabetes
resolved; serum magnesium levels lasted unchanged in whom T2DM did not resolve
(the same happened in non-T2DM obese subjects) (Lecube, 2012).
The mechanisms by which T2DM could lead to low serum magnesium levels
remain to be fully understood. However, insulin resistance, hyperinsulinemia,
hyperglycemia and/or glycosuria may negatively interfere with renal reabsorption
of magnesium, contributing to hypomagnesemia (McNair, 1982; Djurhuus, 1995;
Barbagallo, 2007; Belin, 2007; Lecube, 2012; Takaya, 2012). Insulin has a prime role
in magnesium metabolism regulation and insulin resistance/inhibition of insulin-
stimulated glucose uptake may decrease magnesium uptake by tissues and increase
magnesium efflux from tissues (Takaya, 2004; Barbagallo, 2007; Belin, 2007).
Magnesium is a cofactor of several enzymes involved in insulin and glucose
metabolism, among other processes (Takaya, 2004; Barbagallo, 2007; Belin, 2007;
Guerrera, 2009; Takaya, 2012). Briefly, insulin binds to its receptor (IR) inducing
its autophosphorylation on tyrosine residues and, subsequently, tyrosine residues
phosphorylation of its substrates (IRS) and Src homology 2 domain containing
transforming protein 1 (Shc). Phosphorylated IRS, particularly IRS1 and 2, activate
phosphatidylinositol 3-kinase (PI3K), which converts phosphatidylinositol (4,5)-
bisphosphate into phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on the plasma
membrane. PIP3 recruits, binds and activates phosphatidylinositol-dependent protein
kinase-1 (PDK1), which phosphorylates protein kinase B (Akt) contributing to its
activation. Akt activation mediates insulin-induced glycogen and protein synthesis,
gluconeogenesis inhibition and glucose transporter 4 (GLUT4) translocation to the
plasma membrane (what increases glucose uptake by insulin sensitive tissues).
Instead, phosphorylation of Shc by IR promotes a parallel signaling pathway, leading
to the activation of serine/threonine kinases, such as MAPK-kinase (MEK-1/2) and
extracellular receptor kinase (ERK), responsible for insulin-induced cell growth and
differentiation (Cohen, 2006; Tsatsoulis, 2013). Interestingly, magnesium has a positive
impact on tyrosine kinase activity at the IR level as well as on the translocation of

the GLUT4 to the cellular membrane (Takaya, 2004; Barbagallo, 2007; Belin, 2007;
Guerrera, 2009; Takaya, 2012). Moreover, magnesium deprivation, in a renal epithelial
cell line (Madin-Darby canine kidney cells), inhibited cell proliferation and decreased
ERK1/2 phosphorylation; re-addition of magnesium increased phosphorylated ERK1/2
levels. The use of a specific inhibitor of the MEK-ERK cascade inhibited this last effect,
indicating that magnesium is involved in the regulation of the MEK-ERK cascade and
cell proliferation, at least in this cell line (Ikari, 2010).
Magnesium deprivation may also increase glucocorticoid exposure, even during
fetal development (Caddell, 1991; Laurant, 1999; Takaya, 2011a; Takaya, 2012), which
is involved in insulin resistance/T2DM and dyslipidemia (Pereira, 2011; Pereira,
2012b; Pereira, 2012c; Paredes, 2014; van Raalte, 2014). Takaya et al. investigated the
effects of feeding pregnant rats a very-low magnesium diet (0.003% magnesium)
upon cytosine-guanine dinucleotides methylation in hepatic glucocorticoid genes of
neonatal offspring versus controls (0.082% magnesium). Mean methylation of the 11β-
hydroxysteroid dehydrogenase type 2 gene (Hsd11b2) promoter (11β-hydroxysteroid
dehydrogenase type 2 inactivates tissue’s glucocorticoids) in the magnesium-deficient
offspring was three times higher than in controls, predicting higher hepatic intracellular
glucocorticoid exposure (Takaya, 2011a). Additionally, in female weanling Wistar/NIN
rats, maternal and postnatal Mg deficiency played important (and different) roles in
the programming of increased body adiposity, insulin resistance and impaired glucose
tolerance and insulin secretion in rat offspring (Venu, 2005; Venu, 2008).
Magnesium deficiency is associated with vascular smooth muscle cells (SMC)
proliferation, intimal thickening, thinning and fragmentation of elastic membranes,
collagen accumulation and calcification as well as inflammation [increased
interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and C-reactive protein (PCR)
circulating levels], high rate of free-radical formation (which leads to an increased
NO degradation by superoxide anions), increased susceptibility of lipoproteins to
peroxidation and increased tissue lipid peroxidation. All these effects contribute
to/have been associated with hypertriglyceridemia, pro-atherogenic alterations in
lipoprotein metabolism, cardiovascular lipid deposition and/or the pathogenesis
of vascular lesions following magnesium deficiency (Bussière, 1995; Gueux, 1995;
Laurant, 1999; Busserolles, 2003; Rayssiguier, 2006; Rayssiguier, 2010).
Through calcium antagonism, magnesium promotes vascular relaxation and
thus plays an important role on blood pressure control (Laurant, 1999; Rice, 2011).
Conversely, magnesium deprivation activates the sympathetic nervous system which
contributes to increased blood pressure and heart rate (Laurant, 1999; Rayssiguier,
2010). Aldosterone, due to both genomic and non-genomic effects, also controls
systemic vascular resistance, blood pressure and heart rate (MacFadyen, 1997; Freel,
2004). In line, magnesium deficiency has been described to stimulate the synthesis
and secretion of aldosterone, possibly by decreasing the antagonism to calcium influx
in the zona glomerulosa of the adrenal glands (Laurant, 1999).
Calcium and MetSyn/MetSyn Features – Associated Mechanisms 137
Magnesium supplementation during 4 weeks not only decreased fasting circulating

C-peptide and insulin concentrations in healthy overweight/obese individuals but also
altered whole blood gene expression [with negative regulation of 36 genes (including
some involved in metabolic and inflammatory signaling pathways) and with positive
regulation of 24 genes (some of them involved in magnesium homeostasis)] (Chacko,
2011). Additionally, magnesium supplementation reduced liver damage induced by
ethanol ingestion both in humans (Poikolainen, 2008) and in rats (Markiewicz-Gorka,
2011). Although magnesium deprivation alone may induce metabolic abnormalities
similar to those observed in rodents under chronic high-fructose feeding (model of
diet-induced MetSyn), upon magnesium deficiency high-fructose feeding metabolic
actions are potentiated (Busserolles, 2003; Rayssiguier, 2006). In this context,
magnesium supplementation of rats submitted to high-fructose feeding prevented
and/or improved the alterations induced by fructose. In particular, it ameliorated
insulin sensitivity [evaluated as homeostasis model assessment insulin resistance
(HOMA-IR) and muscle glucose utilization] and fasting circulating lipid profile, and
decreased blood pressure as well as fasting circulating glucose, insulin and lipid
peroxidation marker levels (Balon, 1994; Olatunji, 2007).
In this regard, we have described that the Portuguese natural mineral-rich water
mentioned above partially prevented the decrease of the hepatic magnesium content
of fructose-fed Sprague-Dawley rats (Pereira, 2014a).
2.3.4 Calcium and MetSyn/MetSyn Features – Associated

Mechanisms
Serum calcium levels are increased in individuals with MetSyn and positively
correlated with serum triglycerides and blood pressure values (Park, 2012; Cho,
2011). Calcium and/or dairy products ingestion has been associated with decreased
adipose tissue mass, lower blood pressure, better circulating lipid profile (increased
HDL-cholesterol and decreased total-cholesterol and LDL-cholesterol levels as well as
decreased total-cholesterol to HDL-cholesterol ratio and increased HDL-cholesterol to
LDL-cholesterol ratio) and prevention/reduced incidence of insulin resistance, T2DM
and MetSyn (Teegarden, 2006; van Meijl, 2008; Tremblay, 2009; Abete, 2011; Rice,
2011). The inconsistency between serum calcium levels and dietary calcium intake
values with MetSyn still needs to be clarified (Park, 2012; Cho, 2011).
The increase of fecal excretion of fat and inhibition of intestinal bile salts
absorption (with the consequent increase of cholesterol conversion into bile acids
in the liver) as well as the stimulation of lipolysis and inhibition of lipogenesis
improve plasma lipid profile and decrease adipose tissue mass as a consequence of
calcium intake (Abete, 2011; Rice, 2011). Regulation of parathyroid hormone and 1,25-
dihydroxycholecalciferol levels by dietary calcium mediates its effects on fat mass
and insulin signaling/action (Teegarden, 2006). There is also evidence that adequate
calcium intake, by decreasing the concentration of 1,25-dihydroxycholecalciferol,

decreased the uptake of calcium by vascular SMC and, so, impaired contraction and
reduced peripheral resistance and blood pressure (Rice, 2011). Additionally, lowering
of 1,25-dihydroxycholecalciferol associated with a calcium-induced decrease of
reactive oxygen species, oxidative stress markers and pro-inflammatory markers
(such as TNF-α, IL-6 and PCR) and increase of anti-inflammatory markers (such as
adiponectin), in adipose tissue and/or blood in obese humans and/or obese mice
(Zemel, 2008). Dietary calcium supplementation (started 120 days after birth and
lasting for 2 months) in adult offspring rats programmed during lactation by maternal
nicotine exposure restored insulin sensitivity, reversed the concentration of serum
leptin as well as the percentage of both total body fat content and visceral fat mass,
and decreased the mRNA expression of leptin in visceral adipose tissue versus the
nicotine exposed/conditioned rat group. Dietary calcium supplementation of the
programmed rats also increased the hepatic expression of sirtuin 1 (Sirt1) versus the
control rat group (without nicotine exposure or calcium supplementation) (Nobre,
2011). Regarding Sirt1, growing evidence suggests that its deacetylase activity
regulates glucose-lipid metabolism, glucose production, inflammation, oxidative
stress, autophagy and mitochondrial function and biogenesis as well as adiponectin
and insulin secretion. Positive effects of Sirt1 overexpression and Sirt1 activators have
been described (Kitada, 2013a; Kitada, 2013b; Xu, 2013; Li, 2014).
With an opposite approach, female Wistar rats subjected to a very-low-calcium diet
[(0.008% calcium) versus regular-calcium control diet-fed rats (0,9% calcium)], for 2
weeks, showed lower fasting serum levels of adiponectin and higher HOMA-IR. Moreover,
the mRNA expression of 11β-hydroxysteroid dehydrogenase type 1 (activates tissue’s
glucocorticoids) in the liver was up-regulated (with the same tendency for the hepatic
glucocorticoid receptor), before the animals developed obesity or other evident features
of MetSyn (Takaya, 2011b), amplifying the glucocorticoid exposure in this tissue.
2.3.5 Potassium and MetSyn/MetSyn Features – Associated

Mechanisms
Dietary potassium is inversely associated with hypertension and potassium

supplementation may improve and/or prevent hypertension (Whelton, 1997; Franzoni,
2005; Rice, 2011). Inhibition of pro-inflammatory events in vascular SMC, reduction
of platelet aggregation and reduction of renal vascular resistance seem to mediate
potassium favorable effects on blood pressure (Rice, 2011).
Potassium intake and serum potassium levels have also been negatively associated
with MetSyn prevalence (Lee, 2013; Sun, 2014). Lower serum potassium levels, and to
a minor degree lower dietary potassium intake levels, have been associated with an
increased risk of diabetes (Chatterjee, 2011; Lee, 2013). Even a moderate depletion of
serum potassium (without frank hypokalemia) is associated with glucose intolerance/
insulin resistance and, hence, with an increased risk of T2DM, by reducing insulin
Bicarbonate and MetSyn/MetSyn Features – Associated Mechanisms 139
secretion (Norbiato, 1984; Lee, 2013). Serum potassium levels are closely regulated
by homeostatic mechanisms and depend on dietary potassium intake and potassium
excretion (and its regulators) as well as on partitioning between intracellular and
extracellular spaces (modulated by insulin, catecholamines and thyroid hormone)
(Chatterjee, 2011). Renal potassium excretion is primarily controlled by sodium
delivery to the distal nephron and urine flow, vasopressin levels, acid-base status
(also hormone regulated, as above) and the renin-angiotensin-aldosterone system
(Chatterjee, 2011). As a consequence of the strict control of serum potassium levels,
dietary and serum potassium levels are not inevitably associated (Chatterjee, 2011).
2.3.6 Bicarbonate and MetSyn/MetSyn Features – Associated

Mechanisms
In fact, besides calcium, magnesium and potassium content on natural mineral-

rich waters, hydrogen carbonate concentration also deserves attention. Hydrogen
carbonate-rich mineral waters may decrease calcium and magnesium renal excretion
(by increasing their renal reabsorption) and, so, contribute to minerals´ homeostasis
in the body (Brandolini, 2005; Rylander, 2008). Water pH and water hydrogen
carbonate content are particularly relevant when considering the increased acid load
of the Western diet, mainly related to the: a) high-ingestion of proteins (especially
from animal origin), since sulfur ions are formed during amino acids metabolism,
as well as high-sodium chloride consumption, and b) low-ingestion of fresh fruit,
vegetables, tubers, roots and nuts, that are net base producers. As a result, the
consumption of a Western diet induces a chronic, low-grade metabolic acidosis that
worsens with the decline of kidney function, for example with aging (Cordain, 2005;
Rylander, 2008; Zhang, 2009; Adeva, 2011). In line, it was observed that renal sulfate
excretion negatively correlates with urine pH and is higher in insulin resistance
versus normal insulin sensitivity, highlighting an association among (animal) protein
ingestion, endogenous acid production and insulin resistance. Furthermore, insulin
resistance has been linked with metabolic acidosis markers (such as low urine pH and
low serum bicarbonate levels) (Adeva, 2011).
Metabolic acidosis may induce insulin resistance by impairing the insulin signaling
pathway through inhibition of PI3K activity and, consequently, of its downstream
effectors, in the skeletal muscle (Franch, 2004; Adeva, 2011). This gives dietary acid
load a solid role in anticipating the metabolic dysfunction and the cardiovascular risk
of the healthy, overweight and obese individuals as well as diabetic, hypertensive and
chronic kidney failure patients (Zhang, 2009; Adeva, 2011; Odermatt, 2011). Moreover,
acidosis-induced inhibition of PI3K activity blocked the antiproteolytic effect of
insulin, which could be related to decreased lean body mass in chronic kidney failure
patients (Franch, 2004). Metabolic acidosis increases both glucocorticoid secretion
and plasma cortisol levels, which definitely contribute to insulin resistance, T2DM,
MetSyn, blood pressure and inflammation. Contemporary acidogenic diet associates
with cortisol excess, the latter being prevented by bicarbonate administration (Zhang,
2009; Adeva, 2011; Pereira, 2011; Pereira, 2012b; Pereira, 2012c).
In this context, once more, natural mineral-rich water consumption would be
extremely beneficial.
2.3.7 Magnesium, Calcium, Potassium and Bicarbonate versus Sodium
As highlighted above, an adequate intake of magnesium, calcium or potassium has

a favorable effect on metabolic regulation and/or blood pressure (Whelton, 1997;
Geleijnse, 2005; Karppanen, 2005; Teegarden, 2006; Feldeisen, 2007; Olatunji, 2007;
van Meijl, 2008; Cho, 2009; Tremblay, 2009; Chaudhary, 2010; Abete, 2011; Rice, 2011).
However, their association, as occurs in the dietary approaches to stop hypertension
(DASH diet), which includes, among others, magnesium, calcium and potassium-
rich foods, seems more effective, particularly in blood pressure control (Vaskonen,
2003; Al-Solaiman, 2010). Furthermore, magnesium, calcium and potassium reduce
sodium retention, which may contribute to their positive effects on blood pressure
(Vaskonen, 2003; Rice, 2011). In some studies, sodium chloride is clearly associated
with hypertension (Ziomber, 2008; Santos, 2010). This association depends on
sodium being accompanied by chloride, as sodium per se (in the drinking solution)
does not increase blood pressure (Ziomber, 2008; Santos, 2010). Possibly through
sodium potassium ATPase inhibition (Blaustein, 2006), only sodium chloride seems
to increase plasma volume and blood pressure (Kunes, 2004; Santos, 2010), although
sodium salts, with chloride or other anions (with equimolar amounts of sodium),
produce similar suppression of the renin-angiotensin axis (Luft, 1990; Santos, 2010).
In line with these findings, natural mineral-rich waters intake, with a high
content of both sodium and bicarbonate, decrease blood pressure (Schorr, 1996;
Perez-Granados, 2010) or has no effect on it (Schoppen, 2004; Santos, 2010).
2.3.8 Conclusion
As shown above, an adequate body homeostasis of magnesium, calcium, potassium

and bicarbonate has a prime role in preventing and/or improving MetSyn, or some
of its features. The ingestion of natural mineral-rich waters could contribute to an
adequate supply of those dietary micronutrients, being most relevant in the context
of the Western diet.
Acknowledgements: This work has been supported by FCT (Fundação para a Ciência
e Tecnologia, PEst-OE/SAU/UI0038/2014) and Instituto de Investigação e Inovação em
Saúde, University of Porto, Porto, Portugal. We thank Unicer Bebidas, S.A. (Leça do Balio,
Matosinhos, Portugal) the supply of the Portuguese natural mineral-rich water tested.
References 141
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Andrea Lobo & Teresa Summavielle*6
2.4 Brain Neurochemistry and Cognitive
Performance: Neurotransmitter Systems
In Biomedical Chemistry: Current Trends and Developments
Though there is no universal definition of cognition, it is widely accepted that it

may encompass many different components including: concept formation, mental
abstraction, language acquisition, text comprehension, higher linguistic abilities,
inference, learning, symbolic reasoning, planning, decision making and metacognition
(Metzger, 2011). However, this description may also extend to empathy, procedural
memory, introspection and emotional components relevant to the cognitive process.
Globally, cognition relies on a balanced function of several neural circuits and
pathways modulated primarily by a range of neurotransmitters.
The present chapter reviews how altered neurochemical function may result in
impaired cognition, with a focus on major classical neurotransmitters. Nonetheless,
several other neurotransmitters, mainly small neuropeptides, but also purinergic
elements and lipidic endocannabinoids among others, are now known to play relevant
roles in cognitive processes. At the end of the present chapter we included a short
review of two emerging areas of knowledge in the field of cognitive science: i) the role
of gliotransmitteres, referring to the cross-talk between neurons and glial cells, which
has previously been underestimated; and ii) the modulation of neurotransmission
systems to achieve “neuroenhancement“ or cognitive potentiation.
2.4.1 Monoaminergic Neurotransmission and Cognition
Dopamine appears early in the embryonic CNS, and plays a key role in neural
development. Interfering with the normal interaction between dopamine and
dopamine receptors, during developmental periods is long known to alter the standard
embryonic development, disrupting neuronal proliferation, cell migration, neuronal
maturation, pruning and proper connectivity.
*Corresponding author: Teresa Summavielle: Addiction Biology Group, Instituto de Investigação e

Inovação em Saúde (I3S), Universidade do Porto, Rua Alfredo Allen, 208 | 4200-135 Porto, Portugal
Porto, E-mail: TSummavi@ibmc.up.pt
Andrea Lobo: Addiction Biology Group, Instituto de Investigação e Inovação em Saúde (I3S), Univer-
sidade do Porto, Rua Alfredo Allen, 208 | 4200-135 Porto, Portugal
© 2015 Andrea Lobo & Teresa Summavielle

Monoaminergic Neurotransmission and Cognition 149
Dopaminergic neurons are concentrated in three major cell groups: the substantia
nigra (SN) the ventral tegmental area (VTA), and a cell group of dopamine-containing
cells in the caudal extension of the SN (Fallon, 1978; 1985a; Deutch, 1988). The SN and
VTA regions are strongly implicated in cognition, affective and behavioral disorders
(Roeper, 2013), which is partially explained by a relevant overlap with the brain
reward and limbic systems (Koob, 1988; 1992). The main efferent connections from the
SN-VTA are well described and can be summarized in three groups: the mesostriatal
or nigrostriatal projections, the mesolimbic projections and the mesocortical
projections (sometimes referred together as mesocorticolimbic). The mesostriatal
pathway originates largely from dopaminergic neurons in the ventral sheet of the
SNc projecting into the dorsal striatum (Fallon, 1985b; 1988; Voorn, 1988). In the
dorsal striatum, dopaminergic terminals synapse with GABAergic and cholinergic
neurons, resulting in either excitatory or inhibitory effects depending on the pattern
of dopaminergic receptors displayed at the synapse. Dopamine in the dorsal striatum
plays an important role in controlling patterns of motor activity, reward-based
learning and sequencing, which constitute a complete motor act and represent a
cognitive function (Calabresi, 2007; Leisman, 2014). The striatum receives major
glutamatergic inputs from the cortex and can control dopaminergic neurons in the
SN-VTA through striatal feedback projections (Voorn, 1988; Robbins, 1992; Calabresi,
2000). The mesolimbic pathway arises predominantly from the dorsal sheet of the
SNc, SNl and dorsal VTA (Fallon, 1995). These projections innervate, among other
areas, the amygdala, and the nucleus accumbens (Fallon, 1985b; 1988), which plays
a major role in the limbic-motor integration (Nicola, 2000), underlying complex
adaptive behaviors involved in brain stimulation reward and self-administration of
psychoactive drugs (Woodward, 1999; Schramm, 2002). In the nucleus accumbens,
DA seems to modulate limbic hippocampal and amygdalar inputs (Mogenson, 1988).
Dopamine in this brain region was also shown to be involved in stimulus conditioned
responsiveness (Bassareo, 2002) and locomotor activity. The mesocortical pathways
of dopaminergic projections arise mainly from the VTA and medial SN, densely
innervating the prefrontal, cingulated, suprarhinal and entorhinal cortices, and the
dorsal and ventral hippocampus and the visual cortex. Dopamine was described
to act in the prefrontal cortex (PFC), by modulating signals received from relevant
thalamic afferents (Thierry, 1988). Under stressful situations, dopamine metabolism
is increased in the PFC and, therefore, dopamine seems to be also relevant in plastic
responses to cope with the stressors (Sullivan, 1998; 2004). Dopamine is also involved
in the control of memory-guided behavior in the prefrontal cortex (Goldman-Rakic,
1992) and in cognitive tasks (Weinberger, 1988; Puig, 2014). It is noteworthy that these
pathways are under a complex net of direct stimulation and feedback regulation
within the dopaminergic system.
In the CNS, serotonin (5-hydroxytriptamine, 5-HT) is a key player in mood control.
Alterations in 5-HT function have been implicated in several clinical states, including
affective disorders, obsessive-compulsive disorders, schizophrenia, anxiety states,
150 Brain Neurochemistry and Cognitive Performance: Neurotransmitter Systems
phobic disorders, eating disorders, migraine, and sleep disorders (Stockmeier, 2003;
Albert, 2014). There is also evidence that 5-HT is involved in learning and memory
processes (Meneses, 1999; Faulkner, 2014).
The majority of serotonergic neurons are located in the midline raphe nuclei
(Tork, 1990). Serotonergic neurons in the raphe nuclei have extensive projections to
virtually all areas of the brain and spinal cord (Tork, 1990), but the brain regions
usually implicated in cognition and mood receive 5-HT projections mainly from the
dorsal raphe nucleus (DRN) and median raphe nucleus (Halliday, 1995). The majority
of the ascending 5-HT fibers originate at MRN or DRN, while a caudal cluster gives rise
to almost all the descending 5-HT fibers (Steinbusch, 1981; Halliday, 1995). The early
development of the 5-HT cell bodies and their extensive net of connections through all
the brain, are indicative of the important role that serotonin plays in the development
and functioning of the CNS. Diencephalic structures, the basal forebrain, septal
regions, hippocampal formation and the cerebellum all receive ascending projections
from the DRN and MRN (Halliday, 1995). These projections are essentially non-
overlapping and distribute to separate sites. Fibers from the DRN primarily project
to the SNc, VTA, amygdala, striatum, lateral preoptic area, substantia innominata,
nucleus accumbens and several regions of the cortex, while MRN fibers distribute
mainly to midline and para-midline structures (Vertes, 1997; 1999). Direct synaptic
connections between raphe serotonergic terminals and DA neurons were first
demonstrated in the VTA (Herve, 1987), since then, a close relationship between the
serotonergic and the dopaminergic systems has been well established.
Synthesis of dopamine in DA-neurons starts with the metabolization of L-tyrosine
into 3,4-dihydroxiphenylalanine (L-DOPA) by TH (Nagatsu, 1964; 1998), followed
by almost immediate conversion of L-DOPA into dopamine by L-aromatic amino
acid decarboxilase (L-AADC) (Deutch, 1999). Dopamine is stored into vesicles in
the presynaptic terminal of dopaminergic neurons, which not only prevents DA
from degrading, but also delays its diffusion to the extracellular space. Dopamine
is released into the synapse by calcium dependent exocytotic mechanisms. Once in
the synaptic cleft, DA can interact with presynaptic autoreceptors (D2-like receptors
that include D2, D3 and D4), regulating DA synthesis, release and neuronal firing-
rate; or with postsynaptic receptors (D1-like, that include D1 and D5), modulating the
response of the postsynaptic neuron. The D1 receptor is the most widespread receptor,
the most expressed and exclusively postsynaptic (Vallone, 2000). The D2 receptor
is found mainly expressed by GABAergic neurons (Civelli, 1991). Importantly, these
receptors are known to form heteromers, which can have relevant functions in the
dopaminergic brain (Hasbi, 2011; Perreault, 2014). The dopamine transporter protein
(DAT) seems to be targeted by a complex net of regulation mechanisms, which depend
primarily on the extracellular levels of DA. This transporter is a major target for
psychostimulant drugs, such as cocaine and amphetamines, which can easily block it
or use it to enter the dopaminergic terminal, leading to increased extracellular levels
of DA and increased oxidative stress (Reith, 1997; Chen, 2000). DAT increased levels
are potentially dangerous to the neuron (due also to its ability to transport toxins). A
positive correlation between the levels of DAT expression and neuroinjury has been
demonstrated (Miller, 1999). Storing of DA depends on a vesicular transporter protein,
the vesicular monoamine transporter (VMAT). If left free on the cytoplasm, DA is
rapidly inactivated by the mitochondrial enzyme MAO-A, simultaneously producing
H2O2.
Serotonin is synthesized from the amino acid tryptophan that is hydroxylated into
5-hydroxytryptophan (5-HTP) by the rate-limiting enzyme tryptophan hydroxylase
(TPH). The serotonin precursor, 5-HTP, is decarboxylated by the L-AADC originating
the neurotransmitter 5-HT. Synthesis of serotonin depends mainly on the availability of
tryptophan, which is present in high levels in the plasma and crosses the blood-brain
barrier by active transport. Dietary levels of tryptophan may affect substantially the
levels of serotonin in the brain. In the presynaptic terminal, 5-HT is stored in vesicles
by a process identical to the one described for dopamine. Interaction of extracellular
5-HT with 5-HT autoreceptors regulates synthesis and release of 5-HT. Inactivation
of released 5-HT is mainly attained by 5-HT reuptake through the membrane carrier-
protein 5-HT transporter (SERT). Once inside the neuron, 5-HT can be reacumulated
into vesicles, or inactivated by the mitochondrial enzyme MAO-B, that metabolizes
5-HT into 5-hydroxyindoleacetic acid (5-HIAA) simultaneously producing H2O2.
Inhibition of 5-HT neuron firing activity is mediated through the somatodendritic
5-HT1A autoreceptor, increasing the membrane hyperpolarization (Pineyro, 1999).
Serotonin receptors represent the most complex family of neurotransmitter receptors.
With the exception of the 5-HT3 receptor, which is a ligand-gated ion channel, all
5-HT receptors belong to the G-protein-coupled receptor superfamily (Hoyer, 2002).
Due to its role in antidepressant drug action, 5-HT receptors have been the target of
intense research (Pineyro, 1999). These receptors have been divided, according to
their primary function, in three main groups. The first group comprises the 5-HT1-
like receptors (5-HT1A, 5-HT1B and 5-HT1D), which couple preferentially to Gi/o proteins
(pertussis toxin-sensitive) to inhibit cAMP formation (Hoyer, 2002). In the raphe nuclei,
5-HT1A receptors are somatodendritic and act like autoreceptors inhibiting cell firing,
while postsynaptic 5-HT1A receptors, are present in a number of limbic structures,
particularly in the hippocampus (Hoyer, 2002). The 5-HT1A receptor was associated
with modulation of anxiety-related behaviors (Heisler, 1998) and also with locomotor
activity (Carey, 2001). The 5-HT1B receptors are expressed mainly in the basal ganglia,
striatum, nucleus accumbens and frontal cortex, and are thought to act as terminal
autoreceptors; in addition, they can also serve as terminal heteroreceptors, controlling
the release of other neurotransmitters (Hoyer, 2002). The 5-HT2-like receptor class
comprises the 5-HT2A, 5-HT2B and 5-HT2C (formerly 5-HT1C) receptors, which couple
preferentially to G proteins that employ protein kinase C, (Blaukat, 2000) to increase
the hydrolysis of inositol phosphates and increase cytosolic calcium (Hoyer, 2002). In
the CNS, the 5-HT2A receptors are mainly located in the cortex, claustrum and basal
ganglia, and are particularly important to the behavioral effects of drugs of abuse (Filip,
2001; Munzar, 2002; Porras, 2002). The 5-HT2B and 5-HT2C receptors are restricted to a
few brain regions and were reported to be implicated in mediating hypo/hyperphagia,
grooming frequency and anxiety behavior (Hoyer, 2002). The 5-HT3 receptors are
intrinsic ligand-gated channels, which trigger rapid depolarization due to transient
inward current. The 5-HT7 receptor, in accordance with its distribution in the limbic
system and thalamocortical regions, plays a relevant role in the pathophysiology of
affective and mood disorders (Vanhoenacker, 2000; Hoyer, 2002).
Accumulating evidence shows that optimal brain function relies on a complex
equilibrium of neurotransmitters and respective receptors in well-organized
circuitries. For most of these players, an inverted “U”-shaped curve is the best fit
to describe the relation between either sub-optimal or supra-optimal levels and a
specific behavioral performance (Cools, 2011; Yadid, 1998). For DA this seems to be
particularly relevant. Balanced DA transmission is essential for correct modulation
of the cognitive process. Deregulation of the DA function at the frontocortical level
will impact on decision-making, judgment capacity, planning, working-memory,
attention, cognitive flexibility, and impulsivity (Brozoski, 1979; Cai, 1997; Fischer,
2010). As such, the dopaminergic system is often targeted by pharmacologic
approaches meant to improve the cognitive function in several diseases (but also in
healthy individuals). A common example is the prescription of methylphenidate, a
DAT blocker, to attention-deficit hyperactivity disorder (ADHD) patients (Agay, 2010;
Gamo, 2010). Likewise, most typical and atypical antipsychotic drugs display a
significant affinity to dopaminergic (and serotonergic) receptors. Deficient decision-
making, judgment capacity and impulse control are likely to result in increased risk-
tanking and facilitate drug experimentation, binge intoxication and development of
addictive behaviors (either substance or non-substance dependent).
Addiction to drugs refers to the loss of control over drug intake or the compulsivity
to seek and consume drugs despite adverse effects. Addiction only occurs through
repeated exposure and implies adaptations of several pathways at the molecular
and cellular levels (Nestler, 2001). Behavioral adaptations in addiction result from
the dysfunctional interaction of three key neural systems: an hyperactive/impulsive
amygdala-striatum loop that promotes automatic and repetitive behaviors; an
hypoactive/reflective PFC, impairing decision-making; and the insula-dependent
decision-making processes related to uncertain risk/reward, which potentiates
impulsivity and weakens the cognitive function (Evans, 2008; Noel, 2013). In addicted
persons, the uncontrollable urge to obtain drugs and relapse is associated with a
pathological balance between excitatory and inhibitory neurotransmission circuits,
leading to persistent behavioral abnormalities (Winder, 2002).
Changes in DA balance are also implicated in the physiopathology of other
psychiatric conditions, such as schizophrenia, and anxiety and mood disorders
(Willner, 2005; Chaudhury, 2013; Tye, 2013). In schizophrenia, manifestation of
the positive symptoms (hallucinations, delusions and psychosis) is a consequence
of an hiperactive mesolimbic DA system, while hipofrontality is linked to reduced
mesocortical DA function (Abi-Dargham, 2002). Although it is now accepted that

DA deregulation in schizofrenia is secondary to the GABAergic dysfuntion, most
therapeutic approaches target the DA and 5-HT receptors. The role of DA in anxiety
disorders has long been acknowledged. Within mood disorders, DA is strongly
implicated in depression-bipolar states, where the mania/depression cycling was
associated with hyper/hypodopaminergic states (Cousins, 2009).
Loss of DA neurons in the SNc (and also in the VTA) and consequent reduction of
dopaminergic innervation is a hallmark of Parkinson Disease (PD). As PD progresses
these patients display several primary motor symptoms characteristic of decreased
DA function within the basal ganglia, such as palsy (tremor), bradykinesia, muscular
rigidity, plus postural instability at later stages (Moore, 2003; Samii, 2004). Cognitive
decline can be seen in mild progressive stages, often accompanied by depression and
anxiety (Riedel, 2010). Apathy, anhedonia, helplessness and impaired concentration
were also reported. Loss of DA neurons results from either genetic or environmental
causes, leading to formation of cytoplasmatic insoluble protein aggregates, resulting
from mutated forms of different proteins that cannot be degraded by proteossomic
activity. Although α-synuclein is the most studied of these mutated proteins, this not
the most common form of mutation. These aggregates were associated with increased
oxidative stress, mitochondrial dysfunction and cell death. It is noteworthy that
recent studies suggest components of serotonergic, noradrenergic, cholinergic and
glutamatergic disruption in PD patients (Bohnen, 2007; Kish, 2008).
Also of relevance for the present review is the role of DA in memory formation.
Mesolimbic dopaminergic signaling was shown to act on memory encoding at the CA1
hippocampal region through D1 and D5 modulation of long term potentiation (LTP)
(Swanson-Park, 1999) These mechanisms revealed the role VTA-mediated rewarding
events in memory encoding and recalling, highlighting the relevance of DA and the
reward system in associative learning, and the role of prediction and expectation in
long-term memory (Rossato, 2009; Singer, 2009).
Serotonin also plays a key role in cognitive function. The relevance of the
serotonergic system in learning and memory formation is well documented. While
agonists on the 5-HT1 receptor were seen to impair memory retention, stimulation of
the 5-HT7 receptors or blockage of 5-HT3, 5-HT4 and 5-HT6 receptors improves memory
formation (Seyedabadi, 2014). Although, multiple serotonergic intracellular pathways
are involved in neuronal plasticity and memory formation, 5-HT seems to act through
modulation of cholinergic, dopaminergic, GABAergic or glutamatergic transmission.
In accordance, in memory impairing diseases, such as Alzheimer’s disease (AD),
where a profound alteration in the density of serotonergic receptors was shown, the
action of serotonin seems to rely on the interplay with other neurotransmitters and
second messengers relevant to memory formation.
2.4.2 Glutamate Neurotransmission and Cognition
Glutamate is a ubiquitous anionic amino acid that exists in all cell types, but
in the brain it acts as a signaling molecule, being stored and released from the
glutamatergic neurons subpopulation. Glutamate is considered the main excitatory
neurotransmitter in the CNS, used by around half of the neurons in the brain
(Fonnum, 1984; Liguz-Lecznar, 2007). Glutamate is derived from glutamine through
enzymatic conversion involving phosphate-activated glutaminase (PAG) (Albrecht,
2007), and is stored in small synaptic vesicles at nerve terminals by the action of
vesicular glutamate transporters 1 and 2 (VGLUT1 and 2). Following membrane
depolarization and Ca2+ entry into cells, synaptic vesicles fuse with the plasma
membrane and release the glutamate by exocytosis into the synaptic cleft. After
being released, glutamate is transported back to the neuron or into the glial cells by
the action of excitatory amino acid transporters (EAATs). In astrocytes, glutamate is
converted to glutamine by glutamine synthase, and glutamine is transferred back
to neurons, probably through the sequential action of amino acid system N and A
transporters (Albrecht, 2007; Liguz-Lecznar, 2007; Lee, 2010). Once released from the
presynaptic nerve terminal, glutamate binds to specific receptors in the postsynaptic
membrane to conduct excitatory transmission. Pre-synaptic glutamate receptors act
in the modulation of glutamate release. The effects of glutamate are mediated by
activation of ionotropic or metabotropic receptors, which differ in their molecular,
biochemical, physiological and pharmacological properties (Kew, 2005; Kim,
2001). The ionotropic glutamate receptors have been classified into three distinct
subgroups, α.amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA), N-methyl-D-
aspartate (NMDA) and Kainate (KA) receptors (Dingledine, 1999; Mayer, 2004). AMPA
and kainate receptors are responsible for most of the fast excitatory transmission
in the vertebrate CNS. They are voltage-independent ion channels and permeable
to Na+ and K+, leading to a net depolarizing influx of cations upon activation by
glutamate (Swanson, 1997). AMPA receptors are composed of four possible subunits,
GluA1-4, which associate in different stoichiometries to form receptors with distinct
properties (Greger, 2007). NMDA receptors are ligand-gated ion channels that exhibit
strong voltage dependence owing to the blocking of the receptor channels at negative
membrane potentials by extracellular magnesium. As a result, these receptors
contribute little to the postsynaptic response during low-frequency synaptic activity.
However, when the cell is depolarized, Mg2+ dissociates from its binding site within
the NMDAR channel, allowing Ca2+ and Na+ to enter the dendritic spine (Cull-Candy,
2001). Functional NMDA receptors are heterotrimeric complexes containing both
GluN1 and GluN2 subunits (Prybylowski, 2004). Metabotropic glutamate receptors
are coupled to G proteins (which in turn stimulate second messenger signaling
pathways), and, as such, they mediate slower synaptic responses, occurring over
seconds and minutes, rather than milliseconds as occurs for ionotropic glutamate
receptors. There are three groups of mGluR, distinguished based on sequence
Glutamate Neurotransmission and Cognition 155
homology, signal transduction mechanisms and agonist selectivity (Pin, 2002; Kim,
2008; Niswender, 2010).
Glutamatergic neurons play crucial roles in physiological mechanisms, such
as synaptic plasticity mechanism like long-term potentiation (LTP) and long-
term depression (LTD) underlying processes of learning and memory. However,
disturbances in glutamatergic signaling contribute to the pathogenesis of several
neurological disorders, such as ischemic stroke, schizophrenia, epilepsy and
neurodegenerative disorders (Dong, 2009; Szydlowska, 2010). Neuronal excitotoxicity
refers to the injury of neurons resulting from a prolonged exposure to glutamate and
consequent overactivation of both ionotropic and metabotropic glutamate receptors
(Choi, 1988; Kroemer, 2009). The associated sustained influx of ions into neurons,
and particularly, calcium overload through NMDA receptors and calcium release
from intracellular stores triggered by mGluRs is highly neurotoxic, leading to the
activation of enzymes that will degrade membranes, proteins, and nucleic acids,
which ultimately lead to cell death (Friedman, 2006; Dong, 2009).
Stroke is a complex and devastating disease, constituting the second leading
cause of death worldwide and the leading cause of acquired disability in adults
(Brouns, 2009; Lloyd-Jones, 2009; Moskowitz, 2010). Stroke can be subdivided into
ischemic and hemorrhagic (Doyle, 2008). Ischemic strokes are more frequent and
are caused by a thrombosis or an embolic occlusion of a cerebral blood vessel, more
frequently the middle and anterior cerebral arteries, or a general hypo-perfusion,
all of which result in a constraint of blood flow to the brain, reducing the delivery
of substrates, particularly oxygen and glucose, and ATP production (Dirnagl, 1999;
Doyle, 2008; Lloyd-Jones, 2009). Because of its high metabolic activity, together with
large concentrations of glutamate (Choi, 1992), the brain is particularly vulnerable to
ischemic insults. The ischemic core is the irreversibly damaged tissue characterized
by less than 20% of normal blood flow levels, reduced ATP levels and irreversible
energetic failure (Lo, 2008b). Cells in the core are killed rapidly by proteolysis, lypolysis,
bioenergetic failure and collapse of ion homeostasis (Doyle, 2008; Brouns, 2009).
In the peripheral areas of stroke, between the normal brain and the damaged core,
lies the ischemic penumbra or peri-infarct zone (Astrup, 1981). In this region, blood
flow deficits are less severe, hardly sufficient to support basal ATP levels and normal
ionic gradients (Moskowitz, 2010). Therefore, in the penumbra region the tissue is
functionally impaired but potentially salvageable, and can be rescued by enhancing
blood flow or interfering with the ischemic cascade (Lo, 2008b; 2008a; Brouns, 2009).
With time, the infarct core expands into the ischemic penumbra, so, accurate detection
of this tissue at risk can help to identify patients who might benefit from treatment
with recombinant tissue plasminogen activator (rtPA), the only approved drug to
treat acute stroke patients, by promoting clot lysis and reperfusion, therefore restore
blood flow at an early time point (NINDS, 1995; Paciaroni, 2009). The decreased ATP
production leads to the dysfunction of energy-dependent ion transport pumps, and to
the consequent depolarization of neurons and glia (Katsura, 1994; Martin, 1994). The
Na+/K+ ATPase at the plasma membrane of neurons maintains high K+ and low Na+
intracellular concentrations, that are essential for the propagation of action potentials.
After ischemia, the inhibition of ATP synthesis by mitochondria leads to a rapid ATP
consumption, which causes a neuronal membrane depolarization with the release
of K+ and Na+ entry into cells (Caplan, 2009). Energy failure also impedes the plasma
membrane Ca2+ ATPase to maintain the very low calcium concentrations usually
found within the cells (Doyle, 2008). This, together with the activation of voltage-
dependent calcium channels, leads to the release of neurotransmitters, especially
glutamate, which plays a critical role in the ischemic damage (Nicholls, 1990; Brouns,
2009). A large concentration gradient of glutamate is maintained across the plasma
membrane by sodium-dependent glutamate transporters located at the plasma
membrane, which maintain a high cytosolic glutamate concentration (approximately
10 mM) when compared to the synaptic concentration (in the micromolar range) (Hsu,
1998). Membrane depolarization during ischemia also induces a reversal of glutamate
uptake carriers and enables glutamate to exit the cells along its concentration gradient,
further increasing its accumulation in the extracellular space (Nicholls, 1990; Rossi,
2000). The extracellular accumulation of glutamate leads to excitotoxic stimulation
of synaptic and extra-synaptic ionotropic and metabotropic glutamate receptors,
which will result in neuronal dysfunction and death (Choi, 1988; Sims, 1995; Kroemer,
2009). In particular, the stimulation of AMPA and NMDA receptors causes an influx
of Na+, and NMDA receptors are also characterized by a high Ca2+ permeability and
conductance properties (Dong, 2009). Activation of group I mGluR receptors, which
includes mGluR1 and mGluR5, increases the intracellular inositol trisphospate (IP3),
thereby activating protein kinase C and releasing Ca2+ from neuronal stores (Pin, 2002;
Friedman, 2006). Therefore, overactivation of NMDA receptors and mGluR contribute
to a [Ca2+]i overload. AMPA receptors are not normally calcium permeable due to the
GluA2 subunit, but the downregulation of this subunit after ischemia increases the
calcium permeability of these receptors, and thus allows AMPA receptors to contribute
to delayed cell death (Liu, 2006; Peng, 2006).
Glutamatergic signaling and excitotoxic mechanisms play a role in chronic
neurodegenerative disorders. Alzheimer’s Disease is characterized by 3
neuropathological hallmarks: deposits of amyloid beta (Aβ) peptides that form
senile plaques, neurofibrillary tangles of hyperphosphorylated tau and neuronal
loss (Parameshwaran, 2008). Several studies have demonstrated that excitatory
synaptic transmission and plasticity are impaired in the hippocampus of AD, and
particularly the glutamatergic system is altered leading to synaptic dysfunction and
neurodegeneration (Parameshwaran, 2008). Several studies demonstrated that levels
of VGLUTs are decreased, particularly VGLUT1, in AD patient’s cortices, and that Aβ
peptides accumulate preferentially in glutamatergic neurons, expressing VGLUT1/2
(Sokolow, 2012). Also, EAAT levels are reduced, indicating that glutamate reuptake
from the synaptic cleft is compromised (Scott, 2011). Furthermore, glutamate synthase
levels are reduced, increasing glutamate levels in astrocytes, which can be released
to the synaptic cleft (Robinson, 2001). These effects may increase glutamate levels at
the synapse, triggering excitotoxic mechanisms that will contribute to cell death in
AD (Dong, 2009). It has been hypothesized that changes in AMPA receptors (AMPARs)
number and function play an important role in AD pathogenesis. Various studies
reported a downregulation of AMPARs by Aβ peptides at initial stages of the disease,
probably related to a synaptic failure underlying initial cognitive impairment, prior
to neuronal loss (Selkoe, 2002). This reduction of AMPARs levels contributes to loss
of synaptic function at initial stages of the disease, and is related to caspase cleavage
or endocytosis of the receptors triggered by Aβ peptides (Chan, 1999; Chang, 2006).
Moreover, Aβ affects proteins related to AMPARs insertion and stabilization at the
plasma membrane, such as post-synaptic density protein 95 (PSD-95) in a NMDA
receptors (NMDARs) dependent mechanism (Roselli, 2005). Excitotoxic neuronal
death triggered by excessive glutamate at the synapse and sustained Ca2+ influx
through NMDARs is believed to be one of the major causes of neurodegeneration in AD
(Harkany, 2000). Abnormal NMDARs upregulation contribute to elevated production
of Aβ, resulting in increased glutamate levels and activation of NMDARs as disease
progresses, indicating that NMDARs play a key role in Aβ induced neurotoxicity
(Miguel-Hidalgo, 2002). It is also believed that a functional downregulation of NMDARs
at initial stages of the disease is related to a compromised glutamatergic function. Aβ
peptides reduce surface NMDARs levels at the synapse through endocytosis, which
depresses the glutamatergic function (Snyder, 2005). Moreover, Aβ facilitates LTD in a
NMDA-dependent mechanism, indicating that in AD not only receptor levels, but also
their function, is impaired and may be linked to reduced glutamatergic transmission
associated with cognitive deficits (Kim, 2001).
Parkinson’s disease (PD) main pathological hallmarks are the loss of
dopaminergic neurons in the substantia nigra pars compacta (SNc) projecting to the
corpus striatum, and the presence of cytoplasmic inclusions of protein aggregates,
composed mainly of α-synuclein, and other proteins, called Lewi bodies (Betarbet,
2002). Several studies demonstrated that dopaminergic neurodegeneration in SNc
is mainly associated with mitochondrial dysfunction and oxidative stress. However,
it is widely accepted that glutamate excitotoxicity also plays an important role
in PD pathology (Blandini, 2010). The dopaminergic denervation of the striatum
associated with SNc neurodegeneration triggers changes in the basal ganglia
circuitry which are associated with the motor neurological symptoms of the disease,
namely tremors and rigidity (Blandini, 2000). Within the basal ganglia circuitry,
glutamate neurotransmission occurs in the projections from cortical areas to the
striatum and to the subthalamic nucleus (STN), and from the STN to the substantia
nigra, therefore playing an important role in the mechanisms related to PD motor
symptoms (Blandini, 2000). Also, glutamate excitotoxic effects may be relevant to
the neurodegenerative processes occurring in PD. Dopaminergic neurons from SNc
are particularly vulnerable to excitotoxicity triggered by a bioenergetic failure related
to an impairment of mitochondrial function (Erecinska, 1990). Also, the presence
of dopamine contributes to the sensititvity of SNc neurons to glutamate toxicity,

since it may amplify glutamate signaling (Shimizu, 2003). The neurodegeneration
of dopaminergic neurons in PD is associated with rearrangements in basal ganglia
circuitry. In initial phases of the disease, STN glutamatergic activity may increase
the dopaminergic activity of surviving neurons in SNc. However, through disease
progression, persistent glutamatergic stimulation of dopaminergic neurons will be
neurotoxic, further contributing to neurodegeneration (Shimo, 2009).
Schizophrenia is characterized by a dysfunction in cognitive function that
compromises daily activities, and brain regions involved in cognitive processes
show glutamatergic impairment (Miller, 2000; Falkenberg, 2014; Plitman, 2014). The
glutamatergic system is currently recognized to have a crucial role in the pathogenesis
of schizophrenia (Coyle, 2012). It is proposed that hypofuncional NMDARs in
inhibitory interneurons disinhibit pyramidal excitatory neurons, therefore increasing
glutamate release (Moghaddam, 2012). The glutamate release will overstimulate non-
NMDARs, particularly AMPARs, which will result in an excitotoxic effect related to
structural features of the disease (Deutsch, 2001). Neuroanatomical changes were
reported in patients, such as a loss of grey matter in several brain regions, like the
cortex, thalamus and basal ganglia; whole brain volume reduction and white matter
changes (Lawrie, 1998; Meyer-Lindenberg, 2011; Colibazzi, 2013). Various studies
linked these neuroanatomical changes to neurodegeneration triggered by excitotoxic
mechanisms, particularly related to decreased levels of glutamate in the thalamus,
that will hypostimulate NMDARs in interneurons, leading to increased glutamate
release in thalamocortical pathways (Theberge, 2007). Glutamate overstimulation of
AMPARs will probably lead to excitotoxic increase of intracellular Ca2+ (Deutsch, 2001).
Also, other studies implicated glutamate transporters EAATs in the glutamatergic
dysfunction occurring in schizophrenia. Particularly, both EAAT1 and 3 where shown
to be upregulated in schizophrenic patients, and the increase in EAAT3 may attenuate
NMDARs function, leading to the hipo-NMDARs stimulation (Bauer, 2008; Rao,
2012).
Ataxia is a deregulation of limb movements and poor coordination of the limbs.
The most common ataxias are cerebellar ataxias, which are caused by a dysfunction of
the cerebellum. Spinocerebellar ataxia (SCA) is caused by an abnormal function of the
part of the cerebellar cortex that receives input from the spinal cord. Usually, hereditary
dominant forms of SCA are progressive and fatal neurodegenerative disorders. Seven
SCAs are caused by expansion of CAG-repeats in particular genes, leading to long
polyglutamine sequences in the proteins translated. These polyglutamine containing
proteins aggregate and form misfolded protein deposites that constitute characteristic
neuronal cytoplasmic or nuclear inclusions, which are hallmarks of SCAs and are
associated with toxicity and eventually neuronal death (Zoghbi, 2000; Ross, 2004). In
a number of SCAs, disruption of dendritic Ca2+ spikes through stimulation of AMPARs
in Purkinje cells, and subsequent downstream signaling, is associated with disease
pathology (Carlson, 2009). For instance, in SCA1, mutant ataxin 1 disrupts motor
function by affecting synaptic plasticity in Purkinje cells, associated with an increase

in intracellular calcium levels through glutamate receptors (Clark, 1997; Serra, 2004).
In SCA5, mutations in β-III spectrin contribute to the stabilization of EAAT4 at the
cell surface, affecting glutamatergic signaling (Ikeda, 2006). In SCA6, there is an
accumulation of mutant Ca2+ channels which leads to an increase in intracellular
calcium (Gatchel, 2005).
Status epilepticus (SE) describes a persistent epileptic state during which
epileptic seizures are unceasing and self-sustaining (Chen, 2007; Meldrum, 2007).
Simultaneously with changes in inhibitory neurotransmission, AMPA and NMDARs
subunits are recruited to the plasma membrane at synaptic sites, forming additional
glutamate receptors, further increasing excitability (Wasterlain, 2009). For instance,
GluN1 subunit translocates from extrasynaptic to synaptic sites, increasing the
number of functional NMDARs at the synapse (Chen, 2007). Furthermore, changes
in synaptic enzymes may also contribute to increased excitability, such as the
autophosphorilation of Ca2+ calmodulin-dependent protein kinase II, which may
increase glutamate release (Wasterlain, 1992). Seizures can also induce rapid changes
in AMPARs subunit composition and function, particularly involving a decrease in
GluA2 subunit, with subsequent increased Ca2+ permeability. This increases AMPARs
mediated epileptogenesis in the hippocampus (Sanchez, 2001).
In addicted persons, the uncontrollable urge to obtain drugs and relapse
is associated with a pathological function of excitatory transmission (Winder,
2002). Glutamatergic interconnections occur between amygdala, NAc and PFC
(Cardinal, 2002). The NAc core receives glutamatergic input from the PFC. This
pathway is associated with learned behaviors in response to stimuli-predicting
relevant motivational events and is highly relevant for drug-seeking (Sellings,
2003). Increased glutamate release occurs in the NAc following drug reinstatement
(McFarland, 2003). This is mainly due to adaptations in glutamatergic signaling,
promoting presynaptic glutamate and altering postsynaptic response to glutamate
release. Increased release is involved in decreased presynaptic inhibitory
regulation by mGluR2/3 autoreceptors (Baker, 2003). After withdrawal there is
a reduction in extracellular glutamate due to loss of cystine-glutamate exchanger,
which is responsible for the majority of glutamate in the synaptic cleft and maintains
the tone for mGluR2/3 (Baker, 2003). Postsynaptic responses are associated with
changes in intracellular signaling and trafficking of glutamate receptors to the
membrane, such as the reduction of scaffolding proteins like PSD-95 and Homer
(Ghasemzadeh, 2003; Yao, 2004). As an example, chronic methamphetamine (METH)
involves excitotoxicity following increased glutamate release, which is thought to
create oxidative stress and DA terminal degeneration, particularly in the striatum
(Nash, 1992). The striatum receives glutamatergic input mainly from cortical terminals,
and the corticostriatal pathway is regulated by basal ganglia circuits, particularly
the GABAergic nigrothalamic and glutamatergic thalamocortical pathways (Gerfen,
1989). The striatal GABAergic projections terminate in the SN, which contains a
high density of DA neurons that project to the striatum (Trevitt, 2002). DA regulates
GABAergic signaling in the SN and to the thalamus (Aceves, 1995; Timmerman,
1997). METH increases glutamate in the striatum through a polysynaptic pathway,
characterized by an increase in striatonigral GABA transmission, which will decrease
nigrothalamic GABAergic signaling, disinhibiting thalamocortical glutamatergic
transmission, ultimately causing an increase in glutamatergic release in striatum via
the corticostriatal pathway. The increase of glutamate in the striatum will contribute
to the degeneration of DA terminals, leading to a long-term depletion of DA in this
brain region (Mark, 2004). It was also shown that regulation of VGLUT1 expression
and function via this polysynaptic pathway facilitates vesicular accumulation and
glutamate release in the striatum after METH administration, contributing to a
sustained increase in glutamatergic transmission in the corticostriatal pathway (Mark,
2007). The increase in glutamate release might lead to an overstimulation of NMDARs
and consequent oxidative stress contributing to neuronal damage (Gunasekar, 1995).
Recent evidence also shows that glutamatergic inputs, especially those contacting
GABAergic PV-positive interneurons, may be developmentally regulated by repeated
exposure to cannabinoids, which can result in disruption of the glutamatergic
facilitation of PV-positive interneuron function and underlie the cognitive impairments
seen in young adults that chronically abuse these drugs. Overstimulation of the CB1
receptor over the adolescent developmental period seems to alter frontal circuits
leading to a schizophrenic-like disorder (Caballero, 2012).
2.4.3 GABAergic Neurotransmission and Cognition
The amino acid γ-aminobutyric (GABA) is the main inhibitory neurotransmitter in the
CNS. This neurotransmitter is synthesized by the enzyme glutamic acid descarboxylase
(GAD) which catalyzes the decarboxylation of glutamate. Although the expression
of GABA in the nervous system was first described in 1950 by Eugene Roberts and
Jorge Awapara, it was only accepted as a neurotransmitter more than 10 years later
(Roberts, 1950; Del Castillo, 1964). The difficulty in the identification of GABA as a
neurotransmitter came from its enormous abundance in the vertebrate brain (which
is about 1000 fold higher than monoamine transmitters), its simple structure and
its role in the Krebs cycle, suggesting that it was likely more involved in metabolism
than in intercellular signaling (Schuske, 2004). Cellular release of GABA may be
mediated by several different mechanisms (Saransaari, 1992): (1) GABA can be released
from neurons by exocytosis through synaptic vesicles, which is the most common
mechanism of GABA release under physiological conditions; (2) it may simply leak
through plasma membranes; (3) the plasma membrane GABA transporters-GAT may
be reversed (due to changes in the electrochemical gradients); and (4) finally, ion
channels in the membranes may also mediate GABA release despite the size of this
molecule. The release of GABA in extra-synaptic sites activates non-synaptic GABAA
GABAergic Neurotransmission and Cognition 161
receptors to generate tonic inhibitory currents. These synaptic and extra-synaptic

modes of GABA action have been termed phasic and tonic effects, respectively, and
control neuronal excitability in a different manner (Mody, 2004; Farrant, 2005).
Inhibitory neurotransmission in the adult nervous system is primarily mediated by
the exocytosis of synaptic vesicles containing GABA and glycine. GABAergic inhibition
predominates in the brain, whereas both glycine and GABA act as the primary
inhibitory neurotransmitter in the spinal cord and brainstem. To date, a single vesicular
transporter was identified for the filling of synaptic vesicles at both GABAergic and
glycinergic synapses; it is referred to as vesicular GABA transporter (VGAT) (McIntire,
1997) or vesicular inhibitory amino acid transporter (VIAAT) (Sagne, 1997). GABA
exerts its effects through three types of receptors, named GABAA, GABAB and GABAC
receptors. The different GABA receptor subtypes were originally characterized based on
their pharmacological properties. GABAA receptors are ionotropic chloride channels,
the activation of GABAA receptors allows the influx of chloride, hyperpolarizing the
membrane and decreasing the excitability of the cell. The chloride homeostasis in
neurons is determined by two major transporters, the Na+-K+-Cl- co-transporter, NKCC1
(a Cl- accumulator), and the K+-Cl- co-transporter KCC2 (a Cl- exporter) (Ben-Ari, 2002;
Owens, 2002). During embryonic development and maturation, neurons downregulate
NKCC1 expression and upregulate KCC2 expression, resulting in a lower [Cl-]i in most
mature neurons. The fast inhibitory actions of GABA are mediated by the activation of
GABAA receptors in the brain. A similar role is played by GABAC receptors in the retina.
GABAB receptors are metabotropic receptors that address second messenger systems
through the binding and activation of guanine nucleotide-binding proteins (G proteins)
(Campbell, 1993). GABAB receptors predominantly couple to Giα- and Goα-type G
proteins (Pinard, 2010). It is now well established that presynaptic GABAB receptors
repress Ca2+ influx by inhibiting Ca2+ channels in a membrane-delimited manner via
the Gβγ subunits. Postsynaptic GABAB receptors trigger the opening of K+ channels,
again through the Gβγ subunits (Bettler, 2006). This results in a hyperpolarization of
the postsynaptic neuron (Luscher, 1997). Besides modulating ion channels through
Gβγ, GABAB receptors activate and inhibit adenylyl cyclase via the Giα/Goα and Gβγ
subunits. Presynaptic GABAB receptors are subdivided into those that control GABA
release (autoreceptors) and those that inhibit all other neurotransmitter release
(heteroreceptors).
The activity of GABAergic synapses is important in the regulation of the overall
activity of neuronal networks, refraining the effect of excitatory activity. In the cerebral
cortex there are two main classes of neurons, pyramidal cells and interneurons,
which use glutamate and GABA as main neurotransmitters, respectively. Interneurons
comprise 20–30% of the 15 cortical neuronal population and are locally projecting cells
that control and synchronize the output of pyramidal neurons. Increasing evidence
suggests that disruption of the excitatory-inhibitory (E/I) balance maintained by
pyramidal cells and interneurons is linked to the etiology of multiple neuropsychiatric
and ischemic conditions (Obrenovitch, 2008; Wang, 2011).
Research in cerebral ischemia and excitotoxic neuronal damage has been mainly
focused on the excitatory mediators and much less attention was given to the changes
in GABAergic activity (Schwartz-Bloom, 2001). The release of GABA in the ischemic
brain and the consequent activation of GABAA receptors may be neuroprotective
through reduction of membrane depolarization. Although Cl- entry through GABAA
receptors in association with overactivation of glutamate receptors may further
increase the influx of water and cell swelling, strategies to increase GABAergic
neurotransmission, targeting both sides of the synapse, have been quite efficient in
models of ischemia (Schwartz-Bloom, 2001). The impairment of GABAergic synaptic
transmission in brain ischemia is partly due to a down-regulation of synaptic GABAA
receptors, which may contribute to the ongoing neuronal excitability and possibly to
neuronal death (Schwartz-Bloom, 2001). Furthermore, exposure of hippocampal slices
to oxygen- and glucose-deprivation was shown to induce an early release of GABA
by exocytosis, followed by a delayed phase of neurotransmitter release mediated by
reversal of the plasma membrane transporter (Allen, 2004).
The balance between excitation and inhibition is an important mechanism
in epilepsy, with inhibitory GABAergic regulation considered the main disorder
in epilepsy (Schindler, 2008). Evidence shows that during the onset of the status
epilepticus endocytosis of GABAA receptors takes place in the hippocampus,
decreasing post-synaptic inhibitory currents (Naylor, 2005a; 2005b). Glutamate and
GABA transporters also affect the excitatory/inhibitory balance. Glutamate transporter
activity could trigger GAT reversal activity in astrocytes, by elevating intracellular Na+
levels. This glutamate/GABA exchange may act as a negative feedback to decrease
excitation during seizures (Gadea, 2001; Heja, 2012; Wu, 2014). Neuronal network
remodeling is also common in epilepsy, including loss of GABAergic interneurons and
axon growth. The formation of new excitatory circuits may increase seizure intensity
(Wu, 2014). Excitatory/inhibitory balance determination shows that inhibition
superimposes on pyramidal neurons during initiation of seizure events, shifts
towards excitation during seizures propagation, and eventually inhibition dominates
again during seizures termination (Murase, 2014). The mechanism underlying such
homeostatic changes involves increased glutamate signaling and alterations in
GABAergic transmission, related to suppressed presynaptic inhibition and GABAergic
vesicular depletion (Trevelyan, 2013).
Several evidences show that GABA plays an important role in the cognitive features
of schizophrenia (Gonzalez-Burgos, 2011). Most domains of GABAergic signaling are
altered in schizophrenic patients, such as in the expression of GAD 65 and 67, and GAT1
(Pierri, 1999; Hashimoto, 2008). Alterations in GABAA receptors were also described
in schizophrenic patients (Fatemi, 2013), depending on the targeted brain region,
GABAergic transmission can be either increased or decreased (Deidda, 2014). The
imbalance of excitatory/inhibitory stimulus has also been linked to schizophrenia-
related GABAergic dysfunction (Lewis, 2005). Hypofunction of NMDARs and increased
D2 activity leads to hypostimulation of GABAergic neurons (Lewis, 2005). GABAergic
GABAergic Neurotransmission and Cognition 163
excitatory/inhibitory imbalance and consequent discoordination between GABAergic

and glutamatergic firing are critical to the cortical abnormal function and consequent
cognitive deficits reported in schizophrenia (Sullivan, 2012).
In AD, dysfunction of the GABAergic system may also play a role in the cognitive
impairment associated with this disease. It has been shown that AD patients display
lower GABA levels in the brain and in cerebrospinal fluid (CSF) (Grouselle, 1998).
Although the AD pathogenesis is usually associated with glutamate release, altered
GABAergic transmission also contributes to disrupting the excitatory/inhibitory
balance and leads to impaired cognitive function in AD patients (Rissman, 2011). Several
studies have shown that GABAergic neurons as well as GABAergic receptors appear
to be resistant to neurodegeneration in AD, particularly in the hippocampus, despite
the mild reduction reported for GABAA receptor subunits α1, α5 and β3 (Reinikainen,
1988; Marczynski, 1998). It was then proposed that Aβ decreases pyramidal cell firing,
while in these neurons GABA receptors remain unaffected, which will decrease the
activation of downstream GABAergic neurons resulting in decreased inhibition of
excitatory neurons (Kamenetz, 2003). Increased Aβ may also elicit seizures in the
cortex and hippocampus of AD, possibly related to enhanced GABA activity in the
dentate gyrus and aberrant excitatory/inhibitory balance (Minkeviciene, 2009; Palop,
2010). It was proposed that Aβ decreases pyramidal cell firing, while in these neurons
GABA receptors remain unaffected. This will decrease the activation of downstream
GABAergic neurons, which will decrease the inhibition to the inhibitory neurons they
innervate, with a resultant decreased inhibition of excitatory neurons (Kamenetz,
Tomita et al. 2003).
In Parkinson’s disease, the degeneration of dopaminergic neurons in the
Substancia Nigra (SN) and consequent loss of dopamine (DA) leads to alteration in
the neurotransmitters in the basal ganglia circuitry, particularly an imbalance in
glutamate and GABA neurotransmission in the nigrostriatal pathway, resulting in an
increased excitation in the SN promoting excitotoxicity and cell death (Ampe, Massie
et al. 2007; Calabresi, Picconi et al. 2007; Walker, Moore et al. 2012). There are strong
evidences showing a strong interaction between GABA, glutamate and dopamine in the
basal ganglia. For instance, the DA input to the striatum affects glutamate and GABA
transmission and DA modulates glutamate costicostriatal signalling to GABAergic
medium spiny neurons (Kalivas and Duffy 1997; Surmeier, Ding et al. 2007).
Dysfunctional GABAergic transmission is also present in the altered function of
the reward system that underlies the addictive behavior. Exposure to psychoactive
substances may affect the GABAergic system either directly, as in the case of alcohol
abuse, or indirectly as in chronic exposure to psychostimulants or depressants.
Acute exposure to alcohol increases the inhibitory effect of GABAA receptors, and
enhances other inhibitory modulators, decreasing also the function of excitatory
neurotransmitters. Prolonged drinking has the opposite effect, decreasing GABAA
receptor function by decreasing receptor levels or altering protein composition.
Behaviorally, acute alcohol consumption translates into a range of dose-dependent
effects that scale from social disinhibition to impaired motor control and decision-
making, followed by mild to severe ataxia and hippocampal dysfunction, and
eventually ending in sedation, coma, cardiac arrest and possible death (Tabakoff, 1996;
2013; Gilpin, 2008; Koob, 2014). Chronic exposure to alcohol leads to compensatory
adaptations in the reward circuitry, and to the development of alcohol related
behaviors, such as tolerance, meaning that over time one will need a higher alcohol
dose to obtain the same reward effect. When alcohol consumption is discontinued,
several withdrawal effects occur, such as tremors, seizures, insomnia, and confusion.
These effects may result from the hyperactive adaptations suffered under prolonged
use that are no longer balanced by the alcohol inhibitory effects. The consequent
increase in glutamatergic activity may lead to toxicity and cell death (Becker, 2014).
Under chronic exposure to psychostimulants the GABAergic component of the reward
system is also deeply affected, reduced GABAergic inhibition of the dopaminergic
mesocorticolimbic pathway contributes to addiction by disrupting the frontal cortical
circuits that regulate motivation, drive, and self-control, increasing the motivational
salience of drug-associated stimuli (Volkow, 2002)
2.4.4 Gliotransmitters
The communication between neurons and astrocytes through the so-called

gliotransmitters (such as glutamate, ATP, and D-serine) has been shown to modulate
synaptic transmission and plasticity through several mechanisms (Araque, 2014).
Research in the field of neuron-glia interactions has gained relevance over the
last few years, revealing the involvement of astrocytes and microglia directly at
the synaptic cleft. It is now clear that astrocytes and microglia play multiple roles
in brain circuitries, modulating signaling in many pathways. While recent studies
show that some of this processes seems to be age-related and, therefore, transitory
in nature (Sun, 2013), others were shown to directly modulate learning and behavior.
Astrocyte signaling and gliotransmission were proposed to represent a highly evolved
integrative interface in brain communication, coupling slow modulatory signaling
from different sources with fast synaptic transmission, contributing to fine tuning
the synaptic circuitry according to the environmental surroundings (Araque, 2014).
Microglia, rather than just reacting to injury or infection, were shown to prune
synapses by monitoring synaptic transmission (Schafer, 2013), refining neuronal
circuitry and improving plasticity (Salter, 2014). In the hippocampus (and other brain
regions), microglia is required for proper function of the glutamatergic synapses.
Several studies have shown that local microglia-derived BDNF is essential for long
term potentiation and depression, which represents a critical mechanism for memory
and learning (Salter, 2014). In accordance, with increasing data revealing the role of
microglia in many neuronal disorders, such as AD, PD, mood disorders, neurophatic
Cognitive Enhancement 165
pain or addictive behavior. Knowledge gained in this field is expected to contribute to

the design of new therapeutic approaches in most of such conditions.
2.4.5 Cognitive Enhancement
Currently, a number of pharmacologic approaches traditionally used to treat medical

conditions are used as cognitive enhancers. Medications for ADHD and narcolepsy
targeting the monoaminergic systems, such as methylphenidate and modafinil, were
found to provide acute enhancing effects to healthy individuals (Repantis, 2010).
Other compounds, like donepezil, rivastigmine or galantamine are drugs that improve
cognition targeting the cholinergic system. As discussed for dopamine, the effect of
cholinergic enhancers is usually represented by a bell shaped curve, meaning that
excessive doses may reduce the cognitive performance (Balsters, 2011). The long-term
consequences of such drugs remain mostly unexplored and its regulation is yet to be
addressed by policymakers and other stakeholders. Their use is particularly widespread
the United States mainly on college campuses, reaching a life-time use as high as 30%.
Data regarding the prevalence of such practice in Europe is yet scarce, but seems to be
lower than in the US (Franke, 2011; Ragan, 2013). While a sporadic use of such drugs is
likely to be safe, repeated exposure may lead to deleterious consequences, possibly by
reshaping the neuronal circuits of the PFC, and affecting the control of goal-directed
behaviors, decision making and risk-assessment (Lee, 2009).
Neuromodulation methodologies employing invasive and non-invasive
procedures can also be used as possible enhancers. Deep brain stimulation (DBS) is a
reversible surgical procedure that implies the insertion of small electrodes in specific
brain regions. DBS has been reported to improve memory functions, but simultaneous
changes in personality were also reported (Glannon, 2009). Non-invasive brain
stimulation is yet mostly experimental. Transcranial stimulation (electric or magnetic)
has been shown to enhance several functions, such as sensorimotor skills, memory,
attention, and problem solving (Nitsche, 2008). While possible benefits may last up
to several months (Cohen Kadosh, 2010; Iuculano, 2014), recent evidence shows that
enhancing one ability might temporarily decrease another (Iuculano, 2013).
Biofeedback, or neurofeedback, combines readings of brain activity in the form
of EEG, with operant conditioning methodologies. This technique is based on the
specificity of EEG patterns for different emotional states or cognitive tasks. Individuals
are then trained to produce desired brain states by visual and auditory feedback,
modulating in real-time their neural activity (Dornhege, 2006; Muller, 2008; Ziemann,
2008). These brain-computer interfaces are increasingly used for a wide range of
enhancement purposes, such as meditation training, computer usability research,
improved cognitive performance, creativity and impulse control. All these tools act
through modulation of neurotransmitters’ release, leading to acute and long term
reshaping of the neural circuitries.
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Section 3: Strategies to Develop New and Better Drugs
Nuno Vale*7
3.1 Amino Acids and Peptides in Medicine: Old
or New Drugs?
3.1.1 Introduction
3.1.1.1 Amino acids: biological and chemical concepts
Amino acids (AA) are molecules containing an amine group, a carboxylic acid group
and a side-chain that varies between different amino acids. AA are an important
class of cell signalling molecules, involved in the regulation of gene expression and
the protein phosphorylation cascade, as well precursors of hormone synthesis and
low-molecular nitrogenous substances (Wu, 2009). The 20 natural AA are commonly
found in proteins and are also referred to as alpha amino acids (Fig. 3.1.1). The α-amino
acids differ in the nature of the side-chain (R group) attached to their α-carbon, which
can vary in size from just one hydrogen atom in glycine to a large heterocyclic group
in tryptophan. α-Amino acids are typically divided into three categories, based on
the properties of their side-chain. The first category contains α-amino acids with
relatively nonpolar R groups (glycine, alanine, valine, leucine, isoleucine, proline,
phenylalanine, tryptophan, and methionine), the second contains α-amino acids
with uncharged but polar R groups (serine, threonine, cysteine, tyrosine, asparagine,
glutamine), and the third contains α-amino acids with charged R groups (aspartic
acid, glutamic acid, lysine, arginine and histidine) (Vig, 2013).
Proteins are key players in many vital processes in living organisms. They
transport substances, catalyze chemical reactions, pump ions or recognize signalling
molecules. The complexity and variety of proteins is tremendous: in the human
body alone, there are more than 100,000 different proteins at work. But almost all of
them are made up of just twenty different AA. Only a few highly specialized proteins
additionally contain selenocysteine (Sec, Fig. 3.1.2), the very rare 21st amino acid
discovered in 1986. Sec is identical to cysteine (Cys), except that it contains selenium
instead of sulphur. Selenocysteine is recognized as the 21st amino acid in ribosome-
mediated protein synthesis and its specific incorporation is directed by the UGA codon
(Stadtman, 1996).
Nuno Vale: Department of Chemistry and Biochemistry, Faculty of Sciences of University of Porto, Rua
do Campo Alegre, 687, 4169-007 Porto, Portugal; Research Unit on Applied Molecular Biosciences/
UCIBIO-REQUIMTE, Faculty of Sciences of University of Porto, Rua do Campo Alegre, 687, 4169-007
Porto, Portugal, *Email: nuno.vale@fc.up.pt
© 2015 Nuno Vale

Introduction 179
Aliphatic Secondary Aromatic
OH NH
S
HN
H COOH
Glycine Alanine Valine Leucine Isoleucine Methionine Proline Phenylalanine Tyrosine Tryptophan
(Gly) (Ala) (Val) (Leu) (Ile) (Met) (Pro) (Phe) (Tyr) (Trp)
Non-polar
Charged (basic)
Non Charged
Polar
N NH2 Charged (acidic)
NH2 HN
O NH2
NH N HO O O
O
OH OH SH
NH 2
OH
Lysine Argynine Hystidine Aspartic Glutamic Asparagine Glutamine Serine Threonine Cysteine
(Lys) (Arg) (His) (Asp) (Glu) (Asn) (Gln) (Ser) (Thr) (Cys)
Figure 3.1.1: Structures of natural amino acids.
(Z)
O N O
H
N
(R)
HSe (S) OH (R) (S) OH
NH 2 O NH2
L-Selenocysteine L-Pyrrolysine
(Sec) (Pyl)
Figure 3.1.2: Structures of selenocysteine and pyrrolysine.
Selenium is in the same family below sulfur in the periodic table and the two
elements share many similar chemical properties. Sec has a distinct functional
advantage because the selenol group is more fully ionized than the thiol group of Cys
at physiological pH (biosynthesis of Sec and de novo synthesis of Cys can be observed
in Fig. 3.1.3). When Sec is replaced with Cys, the catalytic activity of a selenoenzyme
is drastically reduced (Stadtman, 2000).
A big surprise was the discovery of a 22nd amino acid in methane-producing archaea
of the family Methanosarcinaceae in 2002, pyrrolysine (Pyl, Fig. 3.1.2). It is genetically
encoded in a similar manner as that of selenocysteine and the other twenty amino
acids. The archaea use this unusual amino acid in proteins required for energy
180 Amino Acids and Peptides in Medicine: Old or New Drugs?
conversion. Pyrrolysine is located in the catalytic center of the proteins carrying it and
is essential for their function. The energy generation process of the archaebacteria
would not work without pyrrolysine (Srinivasan, 2002). Biosynthesis of this amino
acid is based on the radical S-adenosyl-L-methionine (SAM) protein PylB, which
mediates a lysine mutase reaction and produces 3-methylornithine, which is then
ligated to a second molecule of lysine by PylC before oxidation by PylD to produce
pyrrolysine (Gaston, 2011). In addition, Sec and Pyl are often referred as the natural
amino acids.
Among more than 300 AA in nature, only 20 of them (α-AA) serve as building blocks
of proteins. However, represented in Fig. 3.1.4 are non-protein α-AA (e.g. ornithine,
citrulline, and homo-cysteine) and non-α AA (e.g. taurine and β-alanine), which
also play important roles in cell metabolism (Curis, 2007; Perta-Kajan, 2007; Manna,
2009).
Figure 3.1.3: Biosynthesis of Sec and de novo synthesis of Cys. The complete synthesis of Sec on its
tRNA in eukaryotes and archaea uses selenite and ATP are substrates for SPS2, yielding selenophos-
phate. This interacts with SecS and the intermediate, likely dehydroalanine, is used to generate Sec-
tRNA[Ser]Sec (shown on the right in the upper pathway). In the lower pathway, the de novo synthesis of
Cys is shown in mammals, where sulfide and ATP are substrates for SPS2 and yield thiophosphate,
which interacts with SecS and yields Cys-tRNA[Ser]Se from the SecS intermediate (once again, likely
dehydroalanine) (adapted from Turanov, 2011).
Introduction 181
O O O
H 2N (S) OH H2 N N (S) OH
H
NH2 NH2
L-Ornithine L-Citrulline
O O O
HS HO S
(S) OH NH 2 H 2N OH
O
NH2 Taurine β-Alanine
L-Homocysteine
Figure 3.1.4: Structures of non-protein α-AA (ornithine, citrulline, homo-cysteine) and non-α-AA
(taurine, β-alanine).
The catabolism of several AA exhibit a number of common characteristics throughout

different organisms. Important metabolites of AA include ammonia, CO2, long-chain
and short-chain fatty acids, glucose, H2S, ketone bodies, nitric oxide (NO), urea,
uric acid, poly-amines, and other nitrogenous substances with enormous biological
importance. Complete oxidation of AA carbons occurs only if their carbons are
ultimately converted to acetyl-CoA, which is oxidized to CO2 and H2O via the Krebs
cycle and the mitochondrial electron transport system (Wu, 2009).
Biotransformation of amino acids into bioactive molecules can be essential
in biomedical considerations, such as the hydroxylation of Tyr into L-Dopa
(hihydroxyphenylalanine) followed by another decarboxylation to produce
dopamine in the brain (Youdin, 2006). Histamine is a small molecule derived from
the decarboxylation of the amino acid histidine (Haas, 2008). Trp is processed to form
serotonin, a neurotransmissor that is linked to depression at low levels (Oxenkrug,
2013). After the action of serine dehydratase, Ser is converted into aminoacrylate that
tautomerizes to the imine which then hydrolyzes into pyruvate and ammonia (Birolo,
1995). Arg is hydrolysed to urea and ornithine by arginase before it can be used for
the synthesis of polyamines or Pro (Grillo & Colombatto, 2004). Arg is also associated
with Glu formation, as well His. Met reacts with ATP to form S-adenosylmethionine
(Lu & Mato, 2012). Deriving from Cys, taurine (2-aminoethanesulfonic acid) is the
most abundant amino acid in mammals. Being widely distributed in the CNS,
second only to glutamate in concentration, its concentration differs depending on
the regions of the brain, brain activity and animal species studied. It also presents
different functions, which have been studied for their potential in neurology as a
trophic factor in brain development, regulating calcium transport, in the integrity of
the eardrum, as an osmoregulator, neurotransmitter and neuromodulator as well as
for its neuroprotective action (Wu & Prentice, 2010).
3.1.2 Amino Acids and Drug Development
All α-amino acids except glycine have a chiral α-carbon and can exist in two optical
isomers, L-form and D-form. The L-form of amino acids occurs naturally and prodrugs
utilizing these amino acids are generally activated by naturally occurring enzymes.
Both L- and D-amino acid prodrugs tend to have very similar physicochemical
properties but the latter is generally more stable to hydrolysis by naturally occurring
enzymes (Vig, 2003). This property of amino acids are often utilized by medicinal
chemists to develop stable amino acid prodrugs. In addition to the natural amino
acids and their D-forms, extensive arrays of synthetic amino acids and di-/tri-peptides
are commercially available for medicinal chemists as promoieties (Ma, 2003).
3.1.2.1 Rationale for Drug Design
Amino acids can be associated with drugs as a component of prodrug development.

This association depends on the purpose of the prodrug, type of functional groups
available on the parent drug, chemical and enzymatic conversion mechanisms of the
prodrug to the parent drug and safety of the promoiety. Amino acids as promoieties
ease manufacturing and offer several other advantages (Table 3.1.1; Vig, 2013).
Most amino acid prodrugs are either esters or amides, in which the α-amine
or α-carboxylic group of an amino acid is attached to parent functional groups
(hydroxyl, amine, and carboxyl). α-Amine or α-carboxylic groups of amino acid can
also be linked to the parent drug via carbonate and carbamate links. The use of amino
acid side chains, which offer wealth of functional groups (e.g. amine, carboxylic acid,
alcohol, thiol) as prodrug handles presents tremendous opportunities in prodrug
design. While in most cases amino acids are directly conjugated to the parent drugs,
bifunctional linkers have been used to further increase the structural diversity and
types of parent drugs that could be linked to amino acids (Gupta, 2009; Cao, 2012).
Amino Acids and Drug Development 183
Table 3.1.1: Advantages of using amino acids in drug development (adapted from Vig, 2013).
Advantage
• Large structural diversity
• Wide-range of functional groups for attachment to parent drug
• α-Amine and α-carboxylic group
• Well established prodrug chemistry
• Commercial availability
• Fewer safety concerns
• Substrates for various intestinal influx transporters
• Availability of commercially successful amino acid prodrugs
• Used to improve pharmaceutical properties of marketed drugs or difficult compounds

Rationale
• 20 common natural amino acids
• A number of other naturally occurring amino acids
• Extensive array of synthetic amino acids
• α-Amine and α-carboxylic groups
• Side chain functional groups e.g. amine, carboxylic acid, alcohol, and thiol
• Commonly used linkage chemistry between amino acid and parent including ester,
amide, carbonate and carbamate bonds
• Large number of amino acid suppliers available
• Amino acids are building blocks for proteins and are generally regarded as safe
• Can potentially target carrier-mediated transporters for improved delivery across cell
membranes
• Many amino acid prodrugs have been commercially developed and help patients
• Commercial and regulatory precedence in developing amino acid prodrugs
• Improved pharmaceutical properties of already marketed compounds (e.g. valacyclovir,

valganciclovir) and new chemical entities (e.g. brivanib alaninate, LY354740)
• Proven to improve solubility, permeability, sustained release, targeting transporter,

overcoming resistance.
Introducing an amino acid, either a natural or its derivative, to a parent drug usually
increases the water solubility by several orders of magnitude through an ionized
carboxylate anion or ammonium cation. Consequently, there are several amino acid
ester prodrugs that are investigated as water soluble derivatives for oral administration
(Chan, 1998; Mulholland, 2001; Gingrich, 2003; Marathe, 2009). Amino acid esters
(Bundgaard, 1984) amino acid amides (Pochopin, 1995; Bradshaw, 2002; Mittal, 2007;
Rasheed, 2011) or other amino acid examples like amidoximes (Kotthaus, 2011) or
ureas (Hemenway, 2010) have been investigated for parenteral use, albeit to a lesser
extent. In proline, the side-chain links to the α-amino group, meaning it is the only
amino acid containing a secondary amine at this position. The pKa of the α-amino
group is typically in the range of 8.8 to 10.6 and the acidic α-carboxyl group has a
pKa typically between 1.7 and 2.6. The dissociation constant of both the α-amino and
α-carboxyl groups are affected by each other and the side chain. The α-amino group
in its charged state is strongly electron withdrawing and makes the α-carboxyl group
more acidic, thereby lowering its pKa as compared with the typical carboxylic acid
pKa of approximately 4.5. At physiological pH, the predominant form of α-amino
acids contains both a negative carboxylate and a positive ammonium group. This
molecular state is known as a zwitterion and has minimum solubility at the isoelectric
point (mid-point between 2 pKa). However, in most α-amino acid prodrugs either the
carboxylic acid or amine group is linked to a parent drug, abolishing the zwitterionic
form. Exceptions are α-amino acids that have an additional functional group in the
side chain amenable to prodrug formation (e.g. -SH in Cys, -OH in Tyr, -NH2 in Lys,
-COOH in Asp and Glu) which consequently can maintain a double charge. Water
solubility of α-amino acids themselves is largely a function of the polar or nonpolar
nature of the side chain. An increase in hydrocarbon content of the side chain (R
group) from Gly to Val or Leu decreases water solubility. Consequently, amino acids
with shorter hydrocarbon side chains resulted in higher aqueous solubility versus
amino acids with longer hydrocarbon side chains for a series of α-amino acid amide
prodrugs of dapsone (Fig. 3.1.5, Pochopin, 1995).
Moreover, α-amino acids with uncharged but polar R groups are generally more
soluble than the corresponding α-amino acids having nonpolar R groups. This trend is
in line with the observations from several studies, where phenylalanine as a promoiety
resulted in the least soluble prodrug in a series of amino acid prodrugs (Pochopin,
1995; Rautio, 1999). Among the α-amino acids with ionizable R groups, Lys renders
relatively high water solubility over the wide pH range due to the ionized ε-amine
regardless of prodrug type (e.g. ester or amide) (Pochopin, 1995; Nam, 2003).
H
H2 N S N R
L-Ala L-Leu L-Phe Gly

O
L-Lys D-Ala D-Leu D-Phe
D,L-Ala D,L-Leu D,L-Phe
Figure 3.1.5: Structures of dapsone (R=H) and prodrugs with amino acids.
Amino acid prodrugs are expected to pose synthetic challenges similar to other
prodrugs. Amino acid prodrug linkage chemistry is well-established, therefore many
of the synthetic challenges will depend on the number and type of protecting groups
required to mask the active groups on the amino acid and parent drug. Furthermore,
all amino acids except glycine introduce a stereocenter to the prodrug. Like other
prodrugs, amino acid prodrugs increase the molecular weight of the parent, requiring
mindfulness of the increase in dose due to the “non-active” contribution of the
prodrug relative to the bioavailability enhancement with prodrug (Vig, 2013).
3.1.2.2 Amino Acid Prodrug in Drug Delivery
Many drugs suffer from an extensive first-pass metabolism leading to drug inactivation
and/or production of toxic metabolites, which makes them attractive targets for prodrug
design. The classical prodrug approach, which involves enzyme-sensitive covalent
linkage between the parent drug and a carrier moiety, is a well-established strategy to
overcome bioavailability/toxicity issues. However, the development of prodrugs that
can regenerate the parent drug through non-enzymatic pathways has emerged as an
alternative approach in which prodrug activation is not influenced by inter- and intra-
individual variability that affects enzymatic activity. As discussed above, amino acids
are excellent promoieties to increase the aqueous solubility of the parent drug. To
increase the dissolution rate further, amino acid prodrugs can be converted to their
salt forms. In the following sections, we will present some examples of commercial
as well as investigational amino acid prodrugs that have been designed to improve
1) oral bioavailability, 2) sustained drug delivery, 3) intravenous drug delivery, 4) to
target drugs to their site of action and 5) improve enzymatic stability.
Prodrug strategies have arguably been successful for a number of clinically-
used therapeutic agents. However, prodrug research encounters various challenges
and requires additional work in preclinical and clinical settings, much of which can
be attributed to understanding the bioconversion mechanisms of prodrugs. Many
enzymes involved in prodrug activation are subject to interindividual variabilities in
their activities. The main factors contributing to this variability are intrinsic, especially
polymorphisms in the genes encoding the enzymes, but can also be extrinsic (i.e.
interactions caused by other drugs and xenobiotics). Both intrinsic and extrinsic
factors may cause insufficient or excessive conversion of the prodrugs into their active
forms. Moreover, interspecies differences in enzyme activation represent another
hurdle to the prediction of human disposition of certain prodrugs (Hutunen, 2011).
The enzymatic hydrolysis of amino acid esters and amides by various hydrolases
(e.g. esterases and/or peptidases) is far more effective than chemical hydrolysis.
The half-lives of prodrugs are usually several orders of magnitude shorter in blood
or tissue homogenates than in aqueous solutions. Often the activating enzymes are
unidentified. However, it has been suggested that the biphenyl hydrolase-like protein
human valacyclovirase (VACVase) is at least partly responsible for hydrolysis of the
prodrugs valacyclovir and valganciclovir (Fig. 3.1.6) and might be involved in the
activation of other amino acid prodrugs as well (Kim, 2003; 2004; Lai, 2004). As the
specificity of this enzyme resides mainly in the amino acid acyl promoiety (and to a
lesser extent in the alcohol moiety of a parent drug) and that the α-amino group in
the substrate is important for activity, the enzyme can be better defined as an α-amino
acid ester prodrug-activating enzyme (Burnette, 1995; Lai, 2004). VACVase prefers
small, hydrophobic (valine, proline) or aromatic side chains (phenylalanine) over
the charged amino acids (lysine, aspartic acid). It has shown clear stereoselectivity
for L-valine over D-valine prodrugs irrespective of the parent drug while exhibiting
comparable hydrolytic activity toward D-phenylalanine and L-phenylalanine esters,
as indicated in a study of various nucleoside analogue prodrugs (Kim, 2004). The
success of valacyclovir and valganciclovir has proved that an L-valine prodrug
approach is a very efficient option to improve the oral bioavailability by utilizing
amino acid transporters. Therefore, there are several other L-valyl ester prodrugs,
including nucleoside analogues levovirin valinate, valopicitabine, and valtorcitabine
currently undergoing clinical evaluation.
L-Valyl ester prodrugs are probably the first commercial examples of amino
acid prodrugs that utilize intestinal transporters to increase the permeability and
consequently poor oral bioavailability of their parent drugs. Valacyclovir (Valtrex®)
was the pioneer of L-valyl ester prodrugs, which achieved 3-5-times higher oral
bioavailability (> 60%) (Soul-Lawton, 1995) than that of its parent drug, acyclovir
(10−20%) (de Miranda and Blum, 1983). Valacyclovir is absorbed by the PepT1 and
ATB0,+ transporters and is then rapidly hydrolyzed to acyclovir, predominantly by
biphenyl hydrolase-like protein (valacyclovirase) (Kim, 2003; Hatanaka, 2004).
Finally, acyclovir triphosphate acts as an antiherpetic agent by inhibiting viral DNA
replication. Soon, after the discovery of valacyclovir, valganciclovir (Valcyte®) was
designed. Valganciclovir is also absorbed by the PepT1 and ATB0,+ transporters then
bioactivated by valacyclovirase (Kim, 2003; Umapathy, 2004). The oral bioavailability
of ganciclovir is approximately 60%, which is almost 10-times higher than that from
oral ganciclovir (6−8%) (Jung & Dorr, 1999).
O O
N H 2N N H2N
HN HN OH
N O N O
H 2N N H2N N
O O O
O
Valacyclovir Valganciclovir
valacyclovirase
O O
N N
HN HN OH
OH N OH
N H 2N N
H 2N N
O O
Acyclovir Ganciclovir
1. thymidine kinase 2. cellular kinases
O O
N N
HN HN OH
O O O
N N O P O P O P OH
H 2N N H 2N N
OH OH OH
O O
O O O Ganciclovir triphosphate
Acyclovir triphosphate O P O P O P OH
OH OH OH
Figure 3.1.6: Bioconversion of valacyclovir and valganciclovir to their parent drugs by valacyclovi-
rase and to their corresponding triphosphates, firstly by thymidine kinase and secondly by cellular
kinases.
The successful results obtained for valacyclovir and valganciclovir, amino acid ester
prodrugs of acyclovir and ganciclovir, respectively, has prompted the investigations
of amino acids as promoieties for other agents. This success has been attributed to
their enhanced intestinal transport via oligopeptide transporters. The work of Han
prove that amino acid ester prodrugs significantly improve the cellular uptake of the
parent drugs via peptide transport mechanism, though there is no peptide bond in
their structures (Han, 1998). The fact that some cancer epithelial cells are rich in this
type of transporter suggests their use for the delivery of peptidomimetic anticancer
agents (Gonzalez, 1998; Nakanishi, 2000; Vig, 2003). Anticancer drugs are primarily
cytotoxic agents and exert their antitumor activity by interfering with some aspects
of DNA replication, repair, translation or cell division. However, these agents are not
“magic bullets”, as they do not destroy tumor cells while sparing the normal cells
(Sinhababu, 1996).
The prodrug strategy is once again used with anticancer agents as a way to
alleviate their toxicity. Different prodrug strategies have been employed to not only
to improve solubility, transport and pharmacokinetic properties, but also to enable
selective activation in target tissues. As a means to reduce the toxic effects of these
agents, prodrugs designed for selective activation in target tissues are by far the most
efficient and attractive option. However, an enzyme or transporter that is exclusively
or preferentially expressed in these tissues is a prerequisite for selective targeting
(Landowski, 2006; Sinhababu, 1996).
There are numerous studies of amino acid derivatives of the clinically-effective
anticancer agents floxuridine (Vig, 2003) and gemcitabine (Song, 2005) concerning the
activation of the prodrug. Unlike the desired rapid activation required for valacyclovir,
extensive intestinal activation of floxuridine and gemcitabine prodrugs would lead
to severe intestinal toxicity. In this regard, Song and Landowski demonstrated that
amino acid ester prodrugs provided resistance to deamination of gemcitabine (Song,
2005) and to floxuridine cleavage (Landowski, 2005a), respectively.
The studies developed with the amino acid esters of fluxoridine were consistent
with previous findings: 5’-L-valyl floxuridine was the most efficiently transported
floxuridine prodrug, exhibiting the highest PEPT1-mediated transport and permeability
across Caco-2 monolayers. The length and stereochemistry of the amino acid moiety
side chain influences the transport efficiency of floxuridine prodrugs. The slightly
more branched isoleucyl side chain reduced the transport of these prodrugs to half,
but the branching at γ carbon (as in leucine) side chain decreases this transport
even further. The permeability of 5’-monoester prodrugs of floxuridine across Caco-2
monolayers was significantly higher than that of the parent drug and also reflected
a profound promoiety dependency. Thus, the permeability of the 5’-L-valyl prodrug
was roughly 2- and 5-fold higher than the permeability of 5’-L-isoleucyl and 5’-leucyl
floxuridine prodrugs, respectively (Landowski, 2005a).
The metabolic conversion of floxuridine to 5-fluorouacil (5-FU) following systemic
delivery has been shown to be detrimental to the therapeutic efficacy of floxuridine.
The mechanism of action of these two drugs are well understood, where the toxicity
of 5-FU is predominantly caused by 5-FU incorporation into RNA. Unlike 5-FU,
floxuridine is specifically incorporated into DNA, which leads to the minimization of
adverse effects. It is important to highlight that floxuridine has shown to inhibit cell
proliferation 10- to 100-fold more than 5-FU. However, floxuridine is rapidly converted
to 5-FU in many tissues (including the liver) by the enzyme thymidine phosphorylase
(Tsume, 2008). As a consequence, higher doses of floxuridine are required to maintain
clinical efficacy, which leads to greater toxicity. Therefore, protection of floxuridine
prodrugs against this enzyme is essential to enhance therapeutic efficacy at low doses
and obviate toxicity.
All amino acid ester prodrugs examined by Landowski and co-workers were stable
to glycosidic bond cleavage by thymidine phosphorylase. It is therefore possible to
conclude that modification of one or both of the free hydroxyl groups on the sugar
moiety provide protection from glycosidic bond cleavages. The rate of conversion of the
prodrugs to the parent drug after transport would determine floxuridine disposition
and therapeutic action (Landowski, 2005a). As previously reported to structure,
stereochemistry and the site of esterification of the amino acid promoiety affect the
rates of activation of floxuridine prodrugs. Therefore, the wide range of variations
for prodrug structure suggests that the hydrolysis rate can be tailored to produce a
prodrug with the desired half-life (Landowski, 2005a; Vig, 2003).
The roughly 5- to 12-fold higher activity in Caco-2 cell homogenates compared
with pH 7.4 buffer suggests the predominance of enzymatic bioconversion of the
prodrugs. The results obtained for the leucyl ester prodrugs indicate that they would
not be suitable candidates. In comparison, isoleucyl ester prodrugs of floxuridine
are enzymatically more stable than the valyl ester floxuridine prodrugs and the
reference drug valacyclovir (Landowsky, 2005b). The combined results of the in vitro
studies suggest that isoleucyl monoesters of floxuridine may be potentially promising
candidates for improving oral bioavailability of floxuridine in vivo. The prodrugs
given orally could improve the intestinal uptake of floxuridine as well as shield it from
unwanted degradation (Landowsky, 2005b). Various dipeptides and peptidomimetics
have also been tested to characterize the hPEPT1 transporter and improve its affinity,
and mono amino acid ester prodrugs have been evaluated as hPEPT1 substrates
(Landowsky, 2005a; 2005b; Song, 2005). Based on those reports, six amino acids were
chosen to be N-terminal amino acids of the dipeptide and paired with three others to
test the hypothesis that molecular sizes may structurally affect its ester bond stability
(Tsume, 2008). In this study, dipeptide prodrugs appeared to be less stable in pH
7.4 buffers than the corresponding mono amino acid ester prodrugs. Since no mono
amino acid ester prodrug degradation products were detected, it is quite likely that
the dipeptide monoester prodrugs degrade through parallel pathways, as previously
suggested for some anti-viral dipeptide prodrugs.
Although gemcitabine is clinically effective in the treatment of advanced or
metastatic pancreatic cancer, it also exhibits various side effects which are attributed
to its inability to distinguish between normal or target cells. It is known that
gemcitabine exerts its antiproliferative activity via multiple mechanisms of action. It
is initially phosphorylated intracellularly by deoxycytidine kinase and subsequently
by nucleotide kinases to its active metabolites, gemcitabine diphosphate and
gemcitabine triphosphate. The influence of gemcitabine on DNA synthesis has been

strongly correlated with the gemcitabine triphospate intracellular concentration.
However, extensive degradation of gemcitabine by cytidine deaminases to an inactive
metabolite adversely affects gemcitabine activity (Huang, 1991; Song, 2005). To
overcome this disadvantage, Song and co-workers reported the synthesis of amino acid
ester prodrugs of gemcitabine and evaluated their affinity to oligopeptide transporterts
(hPEPT1), which are overexpressed in the gastrointestinal tract and recently known
to be expressed in tumor cells (Song, 2005; Vig, 2003). The promoieties used in this
study were the aliphatic amino acids L-valine, D-valine, and L-isoleucine, as well as
the aromatic amino acids L-phenylalanine and D-phenylalanine. The results obtained
were once again consistent with the previously mentioned findings with amino
acid prodrugs. All gemcitabine prodrugs (Fig. 3.1.7) showed a greater affinity to the
oligopeptide transporter compared with the parent drug and the preference for the
5’-monoester and L-configuration persist. The prodrugs rates of hydrolysis were also
observed to be affected by the structure, stereochemistry and site of esterification of
the promoiety (Song; 2005).
NH2 NH2
HN HN
O N O N
HO O O
O O
F F
R NH 2
OH F OH F
Figure 3.1.7: Amino acid-gemcitabine prodrug (R = L-Val, L-Ile, L-Phe, D-Val, D-Phe).
The chemical stability and rapid enzymatic bioconversion characteristic of the 5’-
L-valyl-gemcitabine derivative suggests it potential in enhancing oral absorption
of gemcitabine. On the other hand, the 5’-L-isoleucyl-gemcitabine showed a slow
bioconversion in Caco-2 cells and in human plasma, as well as an unusual resistance
to cytidine deaminase deactivation. This way a longer systemic circulation half-life is
possible which may facilitate the targeting of cells over expressing hPEPT-1 transporter
(Song, 2005).
Spontaneous cyclization of oligopeptides and prodrugs with amino acids in
solution does not easily occur without the intervention of specific enzymes, with the
exception of dipeptides that easily cyclize to piperazine-2,5-diones or diketopiperazines
(DKP, 1). The DKP scaffold is widely found in compounds of biological interest
and could serve as a drug template with appropriately arrayed pharmacophores
(Gomes, 2007). The major role of DKP in prodrug design falls in the domain of approach
(ii): by linking adequate dipeptide carriers to a drug, a prodrug can be created which
undergoes a strictly chemical cyclization-elimination process via intramolecular
aminolysis of the dipeptide moiety to a DKP, with simultaneous departure of the free
parent drug (Fig. 3.1.8, Gomes, 2003).
Peptide derivatives of some drugs have been prepared and evaluated as prodrug
candidates where prodrug activation processes involved DKP formation. One example
are the peptide conjugates of the cytotoxic agent vinblastine, designed as potential
prodrugs targeted at prostate cancer cells. In vitro and in vivo evaluation showed
the best derivative was a conjugate bearing an octapeptide segment attached by an
ester linkage to position 4 of vinblastine. This conjugate was found to undergo fast
(t1/2 = 12 min) and specific cleavage of the Gln-Ser peptide bond by prostate enzymes,
and additional data from metabolism studies supported that the final spontaneous
vinblastine release was driven by DKP formation from a dipeptidyl intermediate
(Brady, 2002).
O R2
H2 N Parent Drug
N
H
R1 O
R1
NH
HN
R2
Parent Drug
Figure 3.1.8: Prodrug intramolecular activation via DKP formation.

The formation of DKPs was reported as a major degradation pathway for simple alkyl
esters (Larsen, 2004) and dipeptide p-nitroanilides (Goolcharran, 1998). Dipeptides
have been thus proposed as drug carriers to deliver the parent drug through enzyme-
independent processes, namely via DKP formation. Dipeptides are also readily
accessible carriers that can be easily modified to optimize the rate of release of the
parent drug (Shan, 1997). Some authors have therefore considered that dipeptides
might play a crucial role as carriers for hydroxyl-containing drugs. This hypothesis
was developed in a systematic study of the reactivity of paracetamol dipeptide
esters, as this phenolic drug represents an excellent leaving group in the course of
intramolecular dipeptide ester aminolysis (Santos, 2005) and is known to originate
hepatotoxic metabolites (Bertoloni, 2006). Dipeptide esters of paracetamol were
found to be quantitatively hydrolysed to the parent drug and corresponding DKPs at
physiological pH and temperature. Additional evidence that the rate of paracetamol
release depended on the structure of the dipeptide carrier also supported an
intramolecular pathway. Paracetamol esterification with dipeptides also led to
significant decrease or even elimination of the drug’s hepatotoxic effects on mice,
reinforcing the great potential of dipeptide carriers in prodrug design (Santos, 2005).
From what has been described, it is clear that a major drawback of dipeptide-based
prodrugs is their susceptibility to non-specific peptidases. However, this problem can
be easily solved by incorporating at least one non-natural amino acid. For instance,
enzymatically stable dipeptides (e.g. containing α-aminoisobutyric acid or Sar) have
been successfully employed in prodrugs of cytarabine (Wipf, 1996) and cyclosporine
A (CsA) (Hamel, 2004). Dipeptide esters of CsA showed high thermodynamic stability,
differential conversion rates under physiological conditions and strongly increased
water solubility, offering a novel route for the design of CsA prodrugs (Hamel, 2004).
Unnatural amino acids were also used to construct prodrugs of 5-fluorodeoxyuridine
(FdU) to improve prodrug stability against protease/esterase action. Interestingly, these
compounds were designed to be resistant to certain enzymes but to be susceptible to
others. In fact, the antibacterial prodrugs of FdU developed by Wei and Pei are first
activated in vivo by peptide deformylase, after which spontaneous intramolecular
aminolysis of the resulting dipeptide carrier forms a DKP, with simultaneous release
of FdU (Wei, 2000).
3.1.2.3. L-type Amino Acid Transporter
Although the classical approach to improve membrane permeability of polar drugs

uses lipophilic derivatives to increase passive membrane penetration, the targeted
prodrug approach uses transporters designed for facilitating membrane transport
of polar nutrients such as amino acids and peptides. There is direct and indirect
evidence for the participation of carrier-mediated membrane transport mechanisms,
where several hydrophilic compounds seem to be absorbed efficiently via specific
transporters (Mizuma, 1992). Therefore, targeting specific membrane transporters is

particularly important when prodrugs are polar or charged. From this point of view,
use of intestinal epithelial transporters to facilitate the absorption of appropriately
modified drugs seems to be an attractive strategy for improving the bioavailability
of poorly absorbed drug molecules. Prodrugs can be designed to structurally
resemble intestinal nutrients and to be absorbed by specific carrier proteins. In this
case, prodrugs may have the additional advantage of producing nontoxic nutrient
by products during conversion to the parent drug molecules. There have been many
attempts to improve drug absorption targeting specific membrane transporters,
including amino acid, peptide, and glucose transporters (Han & Amidon, 2000).
To exemplify the utility of amino acid prodrugs targeted to transporters expressed
in the gastrointestinal tract, gabapentin and baclofen are structural analogs of GABA
(gamma amino butyric acid) used for the treatment of various neurological disorders.
Both gabapentin and baclofen have structural features of amino acids. They are both
absorbed in the upper small intestine by a low-capacity solute transporter localized
in the upper small intestine, possibly an L-type amino acid transporter (Vig, 2013).
XP13512 and XP19986 are novel prodrugs of gabapentin and baclofen, respectively,
designed to overcome pharmacokinetic limitations of the parent drugs. Transport
of these prodrugs is mediated by monocarboxylate transporter type 1 (MCT-1) and
sodium-dependentmultivitamin transporter (SMVT) (Cundy, 2004; Lal, 2009).
L-type amino acid transporter 1 (LAT1) is a sodium-independent heterodimeric
transmembrane protein found in brain, testis and placenta. The levels of functional
LAT1 are also significantly up-regulated in the surface of several human tumour cells,
highlighting its essential role in cell growth and proliferation. LAT1 is responsible
for transporting large neutral amino acids such as L-Leu, L-Trp, L-Ile and L-Phe into
cells (Kanai, 1998) but can also carry amino acid-derived drugs such as levodopa,
gabapentin, melphalan and baclofen (Cornford, 1992; Kageyama, 2000; Wang & Welty,
1996). Furthermore, LAT1 has been demonstrated to be able to transport amino acid
prodrugs, where the amino acids have been conjugated with drug molecules which
are not LAT1 substrates as such (Killian, 2007; Walker, 1994). Recently, Gynther and
co-workers described a feasible means to achieve carrier-mediated drug transport into
the rat brain via the specific transporter by conjugating a model compound to L-Tyr
(2) or L-Lys (3) with the hydrophilic drug ketoprofen, which is not a substrate for LAT1.
The mechanism and kinetics of the brain uptake of the prodrug were determined with
an in situ rat brain perfusion technique. The brain uptake of the prodrug was found
to be concentration-dependent. These results showed that a prodrug approach can
achieve uptake of drugs via LAT1 into the brain intracellular fluid. The distribution of
the prodrug in the brain parenchyma and the site of parent drug release in the brain
were also shown with in vivo and in vitro studies (Gynther, 2008; 2010).
O
O-Tyr (L) (2)
R
NH-Lys (L) (3)

O
Peptide transporters appear to be attractive targets in prodrug design for their high
capacity, broad substrate specificity, strong expression in the intestinal epithelium and
low occurrence of functional polymorphisms. Conjugating a specific amino acid with
a drug can make it a substrate for PepT1 to enhance the absorption of the parent drug.
Two excellent examples of marketed prodrugs that exploit carrier-mediated transport
are valacyclovir (Valtrex; GlaxoSmithKline) and valganciclovir (Valcyte; Roche) (Cao,
2012). They are L-Val esters (i.e. valine as the promoiety) of acyclovir and ganciclovir,
which both have limited and variable oral bioavailability owing to their high polarity.
These amino acid prodrugs increased the intestinal permeation of their parent drugs
by 3−10-fold, and their membrane transport was mediated predominantly through
the di- and tripeptide transporter (hPepT1) expressed in intestinal epithelial cells
(Han, 1998; Sugawara, 2000).
3.1.2.4. Variability of Amino Acid Application to Exclusive Drugs
Primaquine (PQ, 4, Fig. 3.1.9) is the only generally available anti-malarial that
prevents relapse in vivax and ovale malaria, and is the only potent gametocytocide in
falciparum malaria. Primaquine becomes increasingly important as malaria-endemic
countries move towards elimination, and although it is widely recommended, it is
commonly not given to malaria patients because of haemolytic toxicity in subjects who
are glucose-6-phosphate dehydrogenase (G6PD) deficient (gene frequency typically
3−30% in malaria endemic areas; > 180 different genetic variants) (Ashley, 2014). PQ
is often associated with serious adverse effects because of its toxic metabolites (Vale,
2009). Blocking the terminal primary amine in PQ may represent a huge improvement
in terms of PQ bioavailability, as it can significantly reduce the extent of PQ conversion
into carboxyPQ, the principal metabolite. N-Acylation of anti-malarials with amino
acids and oligopeptides has been used in several works aimed at improving drug
transport into malaria-infected erythrocytes and over the last two decades, these
modifications started to be also seen as a means to avoid premature PQ inactivation
by oxidative deamination to carboxyPQ (Kirk, 2001). An earlier work describes the
synthesis of N-cysteinyl-PQ that was subsequently coupled to carrier proteins, and
both the anti-malarial activity in vivo and the acute lethal toxicity of the protein-drug
conjugates were evaluated. The causal prophylactic activity of the lactosaminated
serum albumin conjugate was two times higher than that of the free drug, whereas
its acute lethal toxicity was at least 6.5-fold lower than that of PQ [mean lethal dose
(LD50) > 85 mg of PQ base kg-1]. This yielded a therapeutic index for the conjugate at
least 12 times higher than that of the free parent drug (Hofsteenge, 1986).
Oligopeptide derivatives of PQ such as PQ-Lys-Leu-d-Val, PQ-Lys-Leu-Ala and
PQ-Lys-Leu-l-Val have been prepared and tested for radical curative anti-malarial
activity against P. cynomolgi in rhesus monkeys and blood-schizontocidal activity
against P. berghei in mice. The d-Val-containing derivative was found to be less
toxic and more active than both its l-Val counterpart and PQ, whereas the activity
of the Ala-bearing compound was comparable to that of the l-Val derivative (Philip,
1988). PQ N-acylation with amino acids or oligopeptides yields structures that are
amenable to proteolytic degradation. Despite this bioreversibility being desirable in
a prodrug, fast conversion into PQ will not overcome the problems associated with
PQ-based therapies. Thus, additional protection of the peptide moiety in peptidyl-
PQ compounds became a new goal so structures could be obtained with resistance
to early proteolytic cleavage and bioreversibility at target tissues or cells to release
the active molecule (Bundgaard and Moss, 1989). Two groups of tetrapeptides with
general formula PQ-Y-Ala-Leu-X-NH2 were also synthesized. In the first group, Leu,
Tyr, Lys and Asp were used in the Y positions and Ala at X. In the second group, Ala,
Tyr, Lys and Asp were used in X positions, while Y was Leu. The peptide derivatives
were then coupled to polyacryl starch microparticles (via the N-terminal amino acid
α-amino group), forming lysosomotropic drug carriers (Borissova, 1995).
Dipeptide derivatives of PQ (PQ-Arg-Phe, PQ-Arg-Lys and PQ-Ala-Phe) were also
synthesized and tested against Chagas disease. PQ-Arg-Lys was seen to be active
against T. cruzi development inside host cells, probably by interfering with the initial
steps of trypomastigote-amastigote transformation. This derivative was more active
than the other two, so it seems that specific cleavage has an important role in PQ release
(Chung, 1997). Portela have also evaluated the dipeptide derivatives of PQ as novel
transmission-blocking anti-malarials. In contrast with PQ, none of the compounds
were able to block oocyst production in mosquitoes’ midguts at 3.75 mg kg-1 but all
were found to completely inhibit the formation of oocysts at 15 mg kg-1, where
N-acetylPQ is not active at this dose. As a whole, this work has demonstrated that
acylation of the PQ’s primary amino group effectively prevents the early conversion of
PQ into carboxyPQ while confirming that the presence of a terminal amino group, as in
the dipeptide derivatives of PQ, is essential for gametocytocidal activity (Portela, 1999).
Gomes worked on the further transformation of the amino acid moiety linked to the PQ
primary amine with the introduction of an imidazolidin-4-one ring at the amino acid’s
α-amino group (5, Fig. 3.1.9). Condensation of the N-aminoacyl derivatives of PQ with
carbonyl compounds (ketones or aldehydes) was suggested by these authors as a means
to protect the amino acid moiety against premature proteolytic degradation (Gomes,
2004). Imidazolidin-4-ones were highly stable in human plasma, with a weak or null
degree of PQ release after 3 days of incubation. Moreover, compound 5 hydrolyzed 50-
100 times slower in aqueous buffer at physiological pH and T than the corresponding
imidazolidin-4-ones derived from di- and penta-peptides (Araújo, 2005; Chambel,

2006). These imidazolidin-4-ones (5) were effective in preventing P. berghei malaria
transmission from BalbC mice to Anopheles stephensi mosquitoes (Araújo, 2005; Vale,
2005). Most of these compounds were also active against Pneumocystis carinii above
10 μg mL-1, which was correlated to their anti-P. falciparum blood-schizontocidal
activity in vitro (Vale, 2008a).
Later, PQ dipeptide derivatives bearing an imidazolidin-4-one moiety at the
N-terminus (6, Fig. 3.1.9) were synthesized and evaluated as potential transmission-
blocking antimalarial prodrugs. All compounds were hydrolyzed to the parent
dipeptide derivative of PQ in neutral and basic solutions, with half-lives ranging
from 0.7 to 31 h at 37°C, depending on the nature of the substituents present in the
imidazolidin-4-one moiety and in the C-terminal amino acid directly coupled to PQ.
The imidazolidin-4-one derived from Ala-Ala-PQ and acetone reduced the transmission
of the infection to mosquitoes more efficiently than PQ, as shown by the significant
decrease in the number of oocysts in the midguts of the mosquitoes at 10 and
50 mmol kg-1 when compared to the control (Vale, 2009a).
A recent report from the same authors provides the synthesis and biological
activity of N1-aminoacyl derivatives of 5 (7, Fig. 3.1.9) as mimetics of PQProXaa
structures (where Xaa represents a general amino acid). Such structures were again
found to be remarkably stable, modestly active as a blood-schizontocidal against a
CQ-resistant strain of P. falciparum and effective inhibitors of the sporogonic cycle
of P. berghei in A. stephensi mosquitoes (Vale, 2008b).
O O
R4
O
N N O
NH
HN HN N
NH 2 N
H R3
R2
R1
4 6
O
O
N O
N O
HN
N HN
R1
N
R1
R2
NH
R2
N
5 R3 NH 2
R3
7
O
R4
Figure 3.1.9: Structures do PQ and its imidazolidin-4-ones derived from L-amino acids and carbonyl
compounds.
Peptides for Biomedicine 197
This research group incorporated the imidazolidin-4-one moiety into dipeptide

derivatives of PQ (7, Fig. 3.1.9), both to introduce a terminal basic amino group reported
as relevant for activity, and effectively suppress hydrolysis of the imidazolidin-4-one
through acylation of the N1 nitrogen atom (Vale, 2008b; 2008c). These peptidomimetic
derivatives were active against a chloroquine-resistant P. falciparum strain and
inhibited the development of the sporogonic cycle of P. berghei, affecting the
appearance of oocysts in the midguts of the mosquitoes and were extremely stable,
both in human plasma and in pH 7.4 buffer as a result of N1-acylation (Vale, 2008b). All
compounds were also active in many biological assays (in vivo transmission-blocking
activity, in vitro tissue-schizontocidal activity on P. berghei infected hepatocytes
and in vitro anti-P. carinii activity), with generally lower activity than the parent
drug. However, these imidazoquines are stable compounds because of blockage of
the aliphatic amine of PQ by insertion of the peptidomimetic carrier. Structure 7
compounds are not vulnerable to oxidative deamination, which is the main metabolic
process behind the low oral bioavailability of PQ. On the other hand, the use of a
peptidomimetic instead of a dipeptide carrier makes compound 7 stable to proteolytic
degradation by action of amino- or endopeptidases (Vale, 2009b).
3.1.3 Peptides for Biomedicine
To fulfil their therapeutic function, most drug-like compounds must pass through the
cellular membrane to enter a cell. Small molecules that obey Lipinski’s rule of five
(no more than 5 hydrogen bond donors, not more than 10 hydrogen bond acceptors,
a molecular mass less than 500 Da and an octanol-water partition coefficient not
greater than 5) have a reasonable chance of penetrating the cell membrane. However,
an increasing number of therapeutics, such as biologics, cannot be considered
‘small molecules’ and these therapeutic are often unable to enter the cell unassisted
(Liskamp, 2014). One of the vehicles used to facilitate the passage of these compounds
into cells are peptides.
Peptides and proteins play a central role in numerous biological and physiological
processes in living organisms: they are involved as hormones and neurotransmitters
in intercellular communication, act as antibodies in the immune system to protect
organisms against foreign invaders, and are also involved in the transport of various
substances through biological membranes. Peptides have significant advantages over
other small molecules in terms of specificity/affinity for targets and toxicity profiles,
and over antibodies in terms of tissue penetration owing to their smaller size. A large
number of peptide-based drugs are now being marketed and the number of candidates
entering clinical evaluation in recent years is steadily increasing. Therefore, the
synthesis of such structures has been a major focus of organic chemistry for over a
century in order to improve the prospects for synthetic therapeutic peptides. In this
section, the relevance of peptides in biomedicine will be discussed.
3.1.3.1 Antimicrobial Peptides (AMPs)
Parasitic diseases are most common in tropical regions with poverty and undeveloped
health care systems. Many parasitic diseases have been out of focus on national and
international agendas and increasing drug resistance combined a lack of vaccines in
several countries make this very alarming. To combat this reality, it is imperative to
understand the relationship between insect and host associated to parasitic disease.
Insects are widely distributed and developed in various ecological niches and serve
as vectors of many parasitic diseases in humans and animals, suggesting outstanding
strategies for defending against pathogens such as microorganisms by overcoming and
adapting to different environmental conditions (Lowenberger, 2001). After infection,
the parasites are presented to the host immune system and induce the production of
defence compounds such as proteins and peptides. Several of these humoral response
peptides exert antibacterial, antifungal, or antiviral properties (Bulet, 1999) and are
known as small cationic peptides, host defense peptides or antimicrobial peptides
(AMPs) (Bell, 2011).
AMPs mostly contain 15−45 amino acid residues and are generally cationic at
physiological pH, often with an amphipathic character and encoded by separated
genes. They form the first line of host defence against pathogenic infections and are
a key component of the ancient innate immune system. AMPs have been identified in
various species ranging from bacteria and frogs to mammals, including humans. In
insects, AMPs are synthesized in the fat body, blood cells (hemocytes) or epithelia and
are released into the hemolymph, the insect blood. In vertebrates, AMPs are present
in amphibian skin secretions (Simmaco, 1999) and epithelia (Ganz & Weiss, 1997;
Bals, 1998). In mammals, they were also observed in lymphocytes (Agerberth, 2000)
and leukocytes (Sorensen, 1997). Because of their broad activity against microbes and
expression triggered by various infections, AMPs are currently intensely examined as
potential antiparasitic compounds (Vale, 2014).
In 2004, the antimicrobial peptide database (APD, http://aps.unmc.edu/AP/
main.php) reported a significant number of AMPs that have been discovered at both
the gene and protein levels (Wang Z & Wang G, 2003). The APD was later updated
and expanded to allow users to search peptide families (bacteriocins, cyclotides, or
defensins), peptide sources (fish, frogs or chicken), post-translationally modified
peptides (amidation, oxidation, lipidation, glycosylation or D- amino acids), and
peptide binding targets (membranes, proteins, DNA/RNA, LPS or sugars) (Wang,
2009).
3.1.3.1.1 AMPs: Mechanism of Action and Peptide Families

Several models have been proposed for the mechanism of action of AMPs on
membrane permeabilization or insertion, and this mechanism depends on a
combination of hydrophobic and electrostatic effects (Shai, 1999; Sanderson, 2005).
The most prominent models are the carpet mechanism, the barrel-stave model, and the
toroidal pore model. Overall, the mechanism of interaction of AMPs with membranes
results either in the translocation of peptides into the cell, the formation of pores
embedded in the membrane to enable a flux of ions and molecules, or in membrane
permeabilization/disruption (Fig. 3.1.10).
A
D
Figure 3.1.10: Proposed mechanisms of AMP‑mediated membrane disruption. A) Barrel-stave pore.

Peptides insert perpendicularly in the bilayer, associate and form a pore. The peptides line the pore
lumen in a parallel direction relative to the phospholipid chains, which remain perpendicular to
the bilayer plane. B) Carpet mechanism. Peptides adsorb parallel to the bilayer and, after reaching
sufficient coverage, produce a detergent-like effect that disintegrates the membrane. C) Toroidal
pore. As with the barrel-stave pore, peptides insert perpendicularly in the bilayer, but instead of
packing parallel to the phospholipid chains, the peptides induce a local membrane curvature in such
a way that the pore lumen is lined partly by peptides and partly by phospholipid head groups. D)
Disordered toroidal pore. A recent modification to the toroidal pore model proposes that less-rigid
peptide conformations and orientations are formed, where the pore lumen is lined by the phospholi-
pid head groups (adapted from Melo, 2009).
The potency and range of the antimicrobial activity of AMPs vary depending on
various parameters, such as the amino acid composition of the structure and its
stability under different environmental conditions, as well as on parameters of the
target cell such as membrane composition and structural features (Fig. 3.1.11). As the
factors that contribute to peptide-lipid interactions are beginning to be understood,

many opportunities remain for the organic chemist to design new systems that address
some of these issues.
Eukaryotic plasma membranes contain phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine and sphingomyelin, and are enriched in cholesterol in
contrast to bacterial plasma membranes, which are often composed of one major
lipid (Pretzel, 2013). Besides the negatively-charged phosphatidylserine, none of the
aforementioned phospholipids has a net charge, meaning eukaryotic membranes
are generally neutral. In contrast, prokaryotic membranes are enriched in negatively
charged lipids such as in Gram-positive bacteria, which contain the negatively
charged teichoic and teichuronic acids in their envelope (Yeaman & Yount, 2003).
Combined with differences in the electrochemical gradients of cells from different
organisms, differences in the membrane environment and the remodeling of the host
cell membrane by intracellular parasites (Hsiao, 1991), these factors could explain
why AMPs discriminate between microbial cells and host cells, or between parasites
and mammalian cells (Pretzel, 2013; Yeaman & Yount, 2003).
Figure 3.1.11: Interrelationship among structural determinants in antimicrobial peptides. Funda-

mental composition and amino acid sequence influence not only the biochemical properties of the
peptide [e.g. charge (Q), amphipathicity (A), and hydrophobicity (H)], but also govern their three-
dimensional configuration [e.g. conformation (χ), polar angle (θ), and overall stereo geometry].
Changes in composition, sequence, and intramolecular bonds may profoundly affect the structure-
activity relationships of antimicrobial peptides in solution, upon binding to target membranes, or
during conformational phase transition to activated states. Therefore, optimal antimicrobial peptide
efficacy lies in the relevant coordination of these relationships (shaded area) as they relate to
microbial target versus host cells in a particular context of infection (adapted from Yeaman & Yount,
2003).
In current practice, it is known that AMPs not only target cell membranes but
may have intracellular targets. The AMP buforin II kills E. coli without lysis of the
cell membrane. Buforin II penetrates the cell membrane, accumulates inside the
bacterial cell and binds to the DNA and RNA, which leads to cell death by inhibiting
cellular functions (Park, 1998). Pleurocidin inhibits DNA and protein synthesis in
E. coli without damaging the cytoplasmic membrane (Patrzykat, 2002). Other AMPs
inhibit enzymatic activities inside the target cell such as phyrrhocoricin, apidaecin,
and drosocin, which interact with the E. coli heat shock protein DnaK and inhibit
protein folding (Kragol, 2001). Some AMPs are capable of inducing apoptotic cell
death, including the breakdown of mitochondrial membrane potential and activation
of caspase 3-like activity, such as in Leishmania (Kulkarni, 2006). AMPs may have
different target sites and act in more than one mechanism to kill the same species, and
different AMPs may act synergistically (Fieck, 2010).
According Boman, AMPs can be classified into three groups: a) linear, often
α-helical peptides free of cysteine residues; b) peptides containing disulfide bridges,
giving peptides a β-sheet structure; and c) peptides with an overrepresentation in
certain amino acids, such as proline, arginine, tryptophan, or histidine (Boman,
2003).
3.1.3.1.2 α-Helical Peptides without Cys Residues

Cecropin-AMP families share a similar structure containing two α-helical domains
linked by a flexible region, and the different cecropins from different organisms vary
in their range of antimicrobial activity. The antibacterial activity of insect cecropins
is based on the pore formation in bacterial membranes (Ekengen & Hultmark, 1999;
Tanaka, 2008). A positive surface charge of cholesterol present in the membrane
bilayer decreases the channel formation potency of cecropins (Christensen, 1988).
Cholesterol increases the thickness and condensation of membranes, so cecropins
have little to no effect on eukaryotic cells, which contain a high amount of cholesterol
in contrast to bacteria (Beevers & Dixon, 2010).
A second class of linear, cysteine-free peptides, the magainins, is exclusively
found in amphibians. Magainin 1 and magainin 2 adopt an α-helical conformation in
solution (Zasloff, 1987). Magainins are proposed to induce toroidal pores in bacterial
membranes (Ludtke, 1996). The nonhemolytic feature of magainin 2 and its protocidal
activity make it highly interesting for the examination of its activity against human
parasites. Another family of amphibian AMPs is the dermaseptin superfamily.
These AMPs exhibit a broad range of antimicrobial activity and some aggregate
on the membrane surface in a carpet-like manner (Pouny, 1992; Raghuraman &
Chattopadhyay, 2007). Melittin is an α-helical cationic peptide, composed of 26 amino
acid residues in which the amino-terminal region is predominantly hydrophobic and
the carboxy-terminal region is hydrophilic due to the presence of a stretch of positively
charged amino acids (Raghuraman & Chattopadhyay, 2007), and the membrane
permeabilization mechanism is proposed to result from pore formation according

to the toroidal model (Yang, 2001). Cationic antimicrobial peptides are a class of
small charged peptides, imparted by the presence of multiple Lys and Arg but with a
substantial portion (50% or more) of hydrophobic residues. They are known for their
broad-spectrum antimicrobial activity and, most recently, the ability to modulate the
innate immune response (Powers & Hancock, 2003).
3.1.3.1.3 Peptides Containing Disulfide Bridges

Defensins are peptides from mammalian phagocytes that contain 6 Cys residues
(or for some insect defensins, eight Cys) that stabilize the peptide structure by
forming three intramolecular disulphide bridges (Selsted, 1985). The mechanism of
action of these peptides is based on membrane permeabilization, probably by pore
formation, and defensins are more active against negatively charged phospholipids
(Lehrer, 1989; Wimley, 1994). The penaeidin class of peptides consist of a proline-rich
N-terminus and a C-terminus containing six Cys residues engaged in three disulfide
bridges (Destoumieux, 2000). The Pro-rich domain of penaeidin class AMPs confers
the target specificity and antimicrobial activity of penaeidin (Cuthbertson, 2004). The
carboxyl Cys-rich domain consists of an amphipathic helix linked to the upstream
and downstream coils by two disulfide bonds.
3.1.3.1.4 Peptides Rich in Pro, Gly, His, Arg and Trp Residues
This group includes AMPs such as apidaecins, short-chain Pro-rich peptides that
may adopt a polyPro helical type II structure, possibly forming the structural basis
to bind specific targets and confer antibacterial activity (Li, 2006). The mechanism
of membrane action does not include the formation of pores but is energy-driven,
resulting in a transporter-mediated model (Castle, 1999).
As for the Pro-rich antimicrobial peptide class, the Gly rich peptides have variable
sizes and do not show clear sequence consensus, apart from the high proportion of
Gly in their primary sequence. These peptides are in general longer than AMP from
other classes and between 25 to 50% of their amino acids are glycine. They have
disordered structure in water but tend to self-order when in contact with artificial
membranes (Bruston, 2007). Attacins are a group of six glycine-rich AMPs and can
be grouped into four basic (A-D) and two acidic (E-F) peptides, probably derived
from two attacin genes (Yi, 2014). Attacin inhibits the synthesis of outer membrane
proteins of E. coli by blocking the transcription of the respective genes (Carlsson,
1991). This is presumably achieved by an indirect mechanism, where attacin binds
to lipopolysaccharides (which serve as a receptor for attacin) but does not enter the
bacterial cell (Carlsson, 1998).
The archetypical Trp rich peptide is indolicin, which unlike the amphipathic alpha
helical structure of the cecropin class of peptides, has a linear structure (no disulfide
bridges) and no particular secondary structure in water. Some authors suggest that
structural changes and strong membrane affinity are key to the antimicrobial activity
of indolicin (Ladokhin & White, 2001). Trp-rich AMP sequences contain more than
25% of Trp. This peptide has the ability to permeate bacterial membranes and,
depending on its three dimensional shape, inhibits DNA synthesis by binding to its
strand (Hsu, 2005).
His-rich amphipathic cationic peptides are peptides with 25% of their amino acids
represented by His. They show a global cationic amphipathic helical structure. They
trigger microorganism membrane disruption when the peptide adopts an alignment
parallel to the membrane surface. However, pore formation is not essential for their
high antimicrobial activity (Mason, 2009). Clavinin (van Kan, 2002) and daptomycin
(Jeu & Fung, 2004) are two studied members of this antimicrobial peptide class.
Cell-penetrating Peptides: A Tool for Effective Delivery

The hydrophobic nature of cellular membranes protects cells from an influx of
exogenous molecules, including bioactive molecules such as peptides, proteins, and
oligonucleotides. New strategies have been developed to overcome these aspects,
as microinjection, electroporation, and liposome and viral-based vectors. However,
these methods have various drawbacks, including low efficiency, high toxicity, low
bioavailability and poor specificity. An alternative strategy to traverse the phospholipid
bilayer of the cell membrane emerged from two unexpected findings. In 1988, the
HIV TAT transactivating factor was discovered (Frankel & Pabo, 1988) and a few years
later, the Drosophila Antennapedia transcription factor (later named penetratin) was
also discovered. TAT and penetratin served as the foundation for the development
of a new type of molecular vector able to promote the delivery of a variety of cargos:
cell-penetrating peptides (CPPs). A vast number of interdisciplinary studies report
numerous applications for CPPs in the delivery of various cargos such as nucleic
acids, polymers, liposomes, nanoparticles and low molecular weight drugs. The main
characteristics of CPPs are low cytotoxicity, the ability to be taken up by a variety of
cell types, dose-dependent efficiency and no restriction with respect to the size or type
of cargo (Heitz, 2009). With a broad sequence variety and large differences in terms
of physical chemical properties, CPPs can be linear, cyclical, cationic, hydrophobic,
hydrophilic, amphipathic, non-amphipathic, random coiled, α-helical or β-sheets.
CPPs differ from most other peptides with respect to specific features that reflect
various mechanisms used to enter the cell (Milletti, 2012).
Cell-penetrating peptides (CPPs) are relatively short peptides that consist of
less than 40 amino acids, are able to enter cells by means of various mechanisms,
including endocytosis, and are able to assist in the further intracellular delivery of
covalently or noncovalently conjugated bioactive cargos (Heitz, 2009; Vale, 2016), as
illustrated in Fig. 3.1.12.
X Y
X Y
Drug Linker CPP
X and Y can be similar or different

functionalities. Some possibilities can be:
O
H H2
a) S S b) C N c) C S
O O
H H H
d) N C N e) C O f) C N N
H
Figure 3.1.12: The structure of a drug-linker-peptide (CPP) conjugate developed in our research
group. X and Y represent the common functional groups used to connect either the drug or the
peptide to the linker. X may be similar to or different than Y. Here, the nature of the X bond and the
drug peptide conjugation chemistry has been classified according to the nature of the X bond: a)
disulfide bond, b) amide, c) thioether, d) carbamate ester, e) carboxylic acid ester or f) hydrazone
bond.
Sequences of common CPPs are provided in Table 3.1.2. The common virtue of all CPPs
is the ability to efficiently pass through cell membranes while carrying a wide variety
of cargos inside cells (Fig. 3.1.13) (Capolovici, 2014). Interestingly, CPP sequences are
known to vary considerably as seen by examining Table 3.1.2. There are, however,
several similarities between the structural natures of these short peptides. Almost
every CPP sequence involves positively charged amino acids. In fact, a chain of
arginines form one of the most widely used CPPs (Myrberg, 2008). The membranolytic
properties of a given CPP can also be governed by its secondary structure, specifically
its helicity. It has been shown that peptides with an α-helical region can enter cells
more efficiently.
Table 3.1.2: Sequences of common CPPs.
Name Sequence / (Refs) Origin

TAT (48-60) GRKKRRQRRRPPQ Human immunodeficiency virus type 1
(HIV-1) TAT
(Frankel and Pabo, 1988; Green and
Loewentein, 1988)
Penetratin RQIKIWFQNRRMKWKK Drosophila Antennapedia homeodomain
(Derossi, 1994)
MAP KLALKLALKALKAALKLAa Amphipathic model peptide
(Oehlke, 1998)
Transportan/ GWTLNS/ Galanin-Lys-mastoparan
TP10 AGYLLGKINLKALAALAKKILa
(Pooga, 1998; Soomets, 2000)

VP22 NAKTRRHERRRKLAIER Herpes simplex virus
(Eliott & O’Hare, 1997)

Polyarginine Rn,a n = 8,9 Positively charged sequence
(Futaki, 2001)
MPG GALFLGFLGAAGSTMGAb Hydrophobic domain from the fusion
sequence of HIV gp41 and NLS of SV40
(Morris, 1997)
T-antigen
Pep1 KETWWETWWTEWSQPKKKRKVb NLS from Simian Virus 40 large T antigen
and reverse transcriptase of HIV-1
(Chaloin, 1998)
pVEC LLIILRRRIRKQAHAHSKa VE-cadherin
(Säälik, 2004)
YTA2 YTAIAWVKAFIRKLRKa MMP cleavage site as seeding sequence
(Lindgreen, 2006)
YTA4 IAWVKAFIRKLRKGPLGa MMP cleavage site as seeding sequence
(Lindgreen, 2006)
M918 MVTVLFRRLRIRRACGPPRVRVa Tumor suppressor protein p14ARF
(El-Andaloussi, 2007)
CADY GLWRALWRLLRSLWRLLWRAb Derived from PPTG1 peptide, W and
charged amino acids
(Crombez, 2007)
a
C-terminal amide. b C-terminal cysteamide.
Initially, CPPs were composed of only natural amino acids but recently have included
non-primary and even unnatural amino acids (Farrera-Sinfreu, 2005). Furthermore,
when lysine residues are replaced with ornithine residues, the peptide becomes more
resistant to cellular degradation (Ezzat, 2011; Angeles-Boza, 2010). Cargo delivery
efficiency can also be improved by changing the structure of the peptides, such as
modification into dendrimers (Angeles-Boza, 2010; Bode, 2014), cyclic peptides
(Mandal, 2011; Northfield, 2014), and the often-used strategy of modifying the side
chains of CPPs, such as in TP10 derivatives (Andaloussi, 2011; Oskolkov, 2011).
Recently, Vale’s research group developed new CPP-Gemcitabine conjugates for drug
delivery in cancer therapy (Vale, 2016).
Further investigations into the structure and mechanisms of uptake of CPP-cargo
complexes will be required to evaluate CPPs as potential delivery tools for biomolecules
in vitro and in vivo models. However, considering evidence from numerous studies,
CPPs have the potential to become a universal tool to carry therapeutic molecules
across cellular membranes without a risk of toxicity or inflammatory reactions.
A B
Figure 3.1.13: A) Applications of cell-penetrating peptides as molecular delivery vehicles. B) Intracel-

lular delivery of CPP-cargo complexes (specific internalization pathways have not been fully elucida-
ted and seem to depend on the specific nature of the CPP and the cargo attached to it). Adapted from
(Capolovici, 2014; Wang, 2014).
3.1.3.3 Peptides: Scaffolding Materials in Tissue Engineering
3.1.3.3.1 Peptide-based Biopolymers

Peptides provide an extensive repertoire of signals that can be used to synthetically
recreate complex biological processes required for tissue regeneration, such as
enabling naturally occurring enzyme-mediated degradation, specific binding of
biomolecules, or directing cell adhesion, proliferation and differentiation. Therefore,
peptides represent attractive functional and structural building blocks to create
versatile materials (Chan & Mooney, 2008; Smith, 2011).
The use of peptide therapeutics is expected to increase in the near future not only
as the active ingredient, but also as new conjugated forms with polysaccharides or
synthetic polymers to improve potency and specificity (Vlieghe, 2010). In this context,
peptides can be used as targeting moieties, as carriers to provide transport across cellular
membranes, and to modify the bioactivity of the original compound/material. In the field
of biomaterials, peptides have also been extensively used as cell-instructive motifs with
different roles, namely to promote cell-adhesion to otherwise non-adhesive polymers,
as well as proliferation and differentiation (Collier & Segma, 2011; Wojtowicz, 2010).
Finally, peptides by themselves are paving the way as new biomaterials. This is the case
for a new class of materials named self-assembling peptides (SAPs), which self-assemble
into nanofiber-like hydrogen bond networks under physiological concentrations of salt
solutions and have found numerous applications as three-dimensional matrices in the
biomedical field (Collier & Segma, 2011; Matson & Stupp, 2012).
3.1.3.3.2 Strategies to Create Scaffolds as Instructive Extracellular Microenvironments

for Tissue Engineering - Peptide-conjugated Polymers to Mimic Natural ECM
New generations of synthetic biomaterials are being developed not only to provide
structural support for damaged tissues, but also to integrate with these tissues and
ideally promote regeneration. The surface of biomaterials can be functionalized with
specific factors that are capable of modulating cell behaviour to promote wound
healing and tissue regeneration (Bellis, 2011). Synthetic ECMs replace many functions
of the native ECM: organizing cells into a three-dimensional architecture, providing
mechanical integrity to the new tissue and providing a hydrated space for the diffusion
of nutrients and metabolites to and from the cell (Rowley, 1999).
Cell adhesion is not only critical for stimulating proper tissue development at
implant/tissue interfaces, but also necessary for materials that serve as carriers for
the delivery of reparative cells to wound sites. Furthermore, cell attachment to a
biomaterial scaffold is an important early step in the generation of in vitro-engineered
tissue substitutes. There are several molecular interactions that can mediate cell
attachment, however much of the research in this area has focussed on utilizing
pro-adhesive factors, such as adhesive peptides, that engage and activate integrin
adhesion receptors on the cell surface. Integrins are heterodimeric transmembrane
receptors that bind to proteins within the ECM including fibronectin, laminin, various
collagens, and many other molecules (Bellis, 2011).
The most widely studied cell-adhesive peptide in the biomaterials field is
the oligopeptide Arg-Gly-Asp (RGD), identified as the minimal essential cell-
adhesion sequence in fibronectin (Ruoslahti & Pierschbacher, 1987; Cha, 2012). The
incorporation of this motif as described initially by Rowley is highly effective at
promoting the attachment of numerous cell types to a plethora of diverse materials
(Rowley, 1999; Bellis 2011).
The RGD sequence can bind to multiple integrin species, and synthetic RGD peptides
offer several advantages for biomaterials applications. Because integrin receptors
recognize RGD as a primary sequence (although conformation of the peptide can
modulate affinity), the functionality of RGD is usually maintained throughout the
processing and sterilization steps required for biomaterials synthesis, many of which
cause protein denaturation. The use of RGD, as compared with native ECM proteins,
also minimizes the risk of immune reactivity or pathogen transfer. Another benefit
is that the synthesis of RGD peptides is relatively simple and inexpensive, which
facilitates translation into the clinic (Bellis, 2011).
3.1.3.3.3 Peptide-based Biomaterials Responsive to Environmental Cues

The design and development of materials that react accordingly to the surrounding
environment is expected to open up the potential of peptide-based materials (Mart,
2006). Peptides are ideally suited for this purpose because of the range of distinct
physical properties available from the naturally occurring amino acids. This diversity
allows for rational incorporation of non-covalent interactions including electrostatic
(acidic and basic amino acids), hydrophobic, pp-stacking (aromatic amino acids),
hydrogen bonding (polar amino acids) as well as covalent (disulfide) bonds and steric
contributions (strand directing amino acids). While individually these interactions are
quite weak, collectively they can give rise to very stable structures. These interactions
depend significantly in different ways on environmental conditions such as ionic
strength, pH and temperature (Mart, 2006).
Enzyme responsiveness can be programmed into these materials by
incorporation of peptide sequences that are known substrates for proteases, kinases,
or phosphatases. The dynamic nature of these interactions allows the protein
molecular organization to be altered in response to changes in the direct environment.
The degradation rate of the scaffold is another critical factor that can control tissue
morphogenesis. In this context, enzyme-sensitive hybrid hydrogels composed of
synthetic or natural polymers and peptide/protein domains, which respond to
specific cell-secreted proteases (particularly proteases) have been prepared using
genetic engineering and/or chemical approaches (Lin & Anseth, 2009). The action
of enzymes can be used as stimuli for triggering drug release and also to facilitate
scaffold remodelling and replacement by resident and host cells, enabling the
infiltration of blood vessels as well as controlling the release of matrix-associated
growth factors and morphogens to enhance tissue regeneration (Lin & Anseth, 2009;
Chan & Mooney, 2008).
Most ECM proteins, including collagen, fibrin, fibronectin, and laminin have
specific cleavage sites for degradation by enzymes, such as matrix metalloproteinases
(MMPs), plasmin, and elastase. In this perspective, MMPs and plasmin are particularly
interesting as they participate in ECM remodelling and degradation, having a key role
in wound healing and tissue regeneration. In particular, plasmin can degrade and
remodel the provisional fibrin matrix act generated at the onset of clot formation
and can also activate latent growth factors or morphogens by specific cleavage
events (Page-Mc Caw, 2007; Patterson & Hubbell, 2011; Aizawa, 2012; Fonseca, 2013).
The cleavage site specificity of proteolytic enzymes can be determined by means
of combinatorial libraries which provide information applicable to the design of

inhibitors and to the identification of protein substrates (Turk, 2001; Chau, 2004).
The development of efficient drug delivery systems with enhanced therapeutic
efficiency is relevant in cases where a prolonged treatment is required. Biodegradable
enzyme-sensitive hybrid polymers are susceptible to the action of specific proteases
yielding small nontoxic metabolites that can be easily excreted through natural body
mechanisms. Their biocompatibility and low immunogenicity make them suitable
for repeated parenteral administration and allow the use of high molecular weight
carriers to optimise pharmacokinetics and high polymer doses (Duro-Castano, 2014).
3.1.3.3.4 Self-assembling Peptides as Biomaterials

Biocompatible and bioactive small molecules, capable of self-assembly and
degradation into predictable metabolites over time are ideal building blocks for
scaffolds to regenerate tissues and organs. The high signaling capacity and therapeutic
efficacy of peptide-based scaffolds is expected to open up the potential of self-assembling
peptides as new therapeutic agents in regenerative medicine. Furthermore, these
hydrogels are biocompatible since they are composed of simple naturally-occurring
amino acids. There are several classes of self-assembling peptide-based materials,
such as peptide amphiphiles, Fmoc-peptides, self-complementary ionic peptides and
hairpin peptides, among others (Matson & Stupp, 2012; Ischakov, 2013).
The peptide-based hydrogels are developed through molecular self-assembly of
peptides and their derivatives present several advantages over other polymeric gelators.
These hydrogels are formed without the use of potentially hazardous chemicals that
could affect their biocompatibility. They also comprise nanofibrous networks which
imitate the structural architecture of natural ECM. Finally, molecular self-assembling
systems can also incorporate features that enhance the overall hydrogel properties
(Johnstone, 2013). Short peptide building blocks are easy to manufacture in large
quantities and can also be chemically decorated with biologically-active components,
facilitating the design of hydrogels with improved targeting and in vivo stability (Zhou,
2009; Ischakov, 2013).
A large number of self-assembling materials responsive to biologically relevant
stimuli, such as pH or temperature, are being developed based on small molecular
building-blocks (Table 3.1.3). Aggeli and colleagues have designed short β-sheet
forming peptides that self-assemble into a variety of nanostructures with tunable
physical and chemical properties (Aggeli, 2003). Pochan and colleagues have
developed β-hairpin peptides that fold to acquire an intramolecular amphiphilic
structure to form self-assembled hydrogel networks (Pochan, 2003). Recent studies
demonstrated that self-assembled peptides can be used to mimic the structure of
natural ECM fibers and to trigger physiological processes in cells. The design of self-
assembled scaffolds that can act as anchors to the cultured cells can be achieved by
incorporation of short adhesive motifs into the fibers of the backbone of a synthetic
ECM-like matrix (O’Leary, 2011; Fleischer & Dvir, 2013).
Table 3.1.3: Examples of self-assembling peptides.
Peptide Sequence / Activity

References
β-Hairpin peptides β-Hairpin secondary structures (two β-strands linked by a kink) can
(Loo, 2013) be rationally designed to self-assemble into ibrillary macromolecular
scaffolds
Fmoc-peptides Fluorenylmethoxy-carbonyl (Fmoc)-protected amino acids and dipeptides

(Orbach, 2009) can form hydrogels. The Fmoc moiety is widely used as a protecting group
in peptide synthesis and it was even reported that a number of Fmoc-
amino acids show anti-inflammatory properties
β-sheet β-sheet peptides are ionic self-complementary, as a result of positive and

(Loo, 2013; Hauser & negative side chains on one side of the β –sheet and hydrophobic side
Zhang, 2010) chains on the other. This motif causes the peptides to fold into β-sheet
secondary structures with distinct hydrophobic and hydrophilic surfaces.
The hydrophobic side forms a double sheet inside of a nanofiber and
the hydrophilic side becomes the exterior of the nanofibers, which
interacts with water molecules to form an extremely high water content
hydrogel. The 4 ionic self-complementary peptides RADA16-I, RAD16-II,
EAK-I and EAK16-II form stable β –sheet structures in water and undergo
spontaneous assembly to form nanofiber scaffolds
α-helical coiled coil α-helices result from the hydrogen bonding of a peptide backbone amide
(Stephanopoulos, 2013) hydrogen to a backbone carbonyl four residues away. This motif results
in a right-handed helical twist configuration. The formation of α-helices is
determined by the amino acid sequence and is dependent on hydrophilic
and hydrophobic amino acid residues organizing into a hydrophobic and
a hydrophilic face. The gelation can be controlled using heat to provide
a versatile scaffold for cell culture, especially when gel dissolution at
elevated temperatures is desired. The α-helical peptide gels proved
highly cytocompatible, and PC12 cells cultured in them were able to
proliferate and differentiate
Peptide amphiphiles Peptide amphiphiles are short peptide sequences attached to a

(Cui, 2010; Matson, 2011; hydrophobic tail, usually an alkyl chain. Peptide amphiphiles combine
Matson & Stupp, 2012) the structural features of amphiphilic surfactants with the functions
of bioactive peptides and are known to assemble into a variety of
nanostructures
3.1.3.4 Therapeutic Peptides and Market
The obstacles that remain for the development of peptides as therapeutics are
significant. As in all drug development, in vivo efficacy might differ from promising
in vitro models. The reason can be the result of interactions with many in vivo
components in the circulation, gastrointestinal tract, dermis, relative clearance and
affinity to target receptors (Lin & Lowman, 2003).
The main limitations generally attributed to therapeutic peptides are (i) oral
bioavailability (injection is normally required), (ii) short half-life because of rapid
degradation by proteolytic enzymes of the digestive system and blood plasma, (iii)
rapid removal from the circulation by the liver or kidneys, and (iv) high synthetic and
production costs (Bray, 2003; Picherean & Allary, 2005).
However, therapeutic peptides are becoming increasingly attractive for the
discovery and development of new generations of drugs. With the recent addition
of new methodologies, peptides can be engineered to have long plasma half-
lives and low immunogenicity (Salo, 2006), along with alternative routes of
administration. Production of synthetic therapeutic peptides has become possible for
the pharmaceutical industry with recent developments of SPPS, initially developed
by Merrifield. SPPS is crucial in the early steps of preclinical research and in the
production of peptide-based active pharmaceutical ingredients (APIs) (Bruckdorfer,
2004). SPPS is especially suited for medium-sized peptides (up to 80 amino acids
residues), which comprise the majority of therapeutically-relevant peptides.
3.1.3.4.1 Chemical Strategies to Improve Peptide Biological Activity

To develop a peptide as a therapeutic agent, the crucial parameters to consider are its
biological effect, pharmacokinetic profile and low immunogenicity. Various chemical
strategies have been developed to try and overcome the limitations of peptides by
increasing in vivo plasma residence time (Table 3.1.4) (Vlieghe, 2010).
The chemical optimization strategy of a therapeutic peptide is based on structure-
activity relationship (Witt, 2001; Ladner, 2004; Wittand & Davis, 2006) with the aim
of improving bioavailability, reducing elimination and biodegradation as well as
increasing selectivity or affinity to its target or receptor. According to Lipinski’s rule of
five (Lipinski, 1997; 2000; 2004), completed by Veber analysis (Veber, 2002), peptides
are poor candidates to move from the digestive tract to the circulatory system based
on their physicochemical properties. Until a few years ago, therapeutic peptides were
generally administered by subcutaneous, intramuscular or intravenous routes to
circumvent the gut barrier. When administered orally, peptides have to face a strongly
acidic gastric environment, high levels of intestinal proteolytic activity and a high
intestinal permeability barrier (Woodley, 1994).
Table 3.1.4: Lead peptide chemical optimization (adapted from Vlieghe, 2010).
Lead Attribute
1 Blocking N- or C-terminal ends by N-acylation, N-pyroglutamate, C-amidation and so on,

or addition of carbohydrate chains (glycosylation: glucose, xylose, hexose and so on) to
increase plasma stability (notably, resistance towards exopeptidases)
2 Search for the minimum active sequence (MAS) from N- and/or C-terminal truncated
analogues
3 Simplification and/or optimization of the structure after alanine scanning (Ala-scan) and/
or D-scanning (D-scan) to eliminate potential sites of cleavage (notably by endopeptidases)
and to determine important functional groups involved in the interaction with the target of
interest
4 Isosteric, or not, amide bond replacement between two amino acids: NH-amide alkylation,
the carbonyl function of the peptide bond can be replaced by CH2 (reduced bond: -CH2-NH-),
C(S) (endothio-peptide, -C(S)-NH-) or PO2H (phosphonamide, -P(O)OH-NH-). NH-amide bond
can be exchanged by O (depsipeptide, -CO-O-), S (thioester, -CO-S-) or CH2 (ketomethylene,
-CO-CH2-). The peptide bond can also be modified: retro-inverso bond (-NH-CO-), methylene-
oxy bond (-CH2-), thiomethylene bond (-CH2-S-), carba bond (-CH2-CH2-), hydroxyethylene
bond (-CHOH-CH2-) and so on, to increase plasma stability of the peptide sequence (notably
towards endopeptidases)
5 Cyclization of the peptide sequence (between side chains or ends of the peptide sequence:
head to tail, N-backbone to N-backbone, end to N-backbone, end to side chain, side chain to
N-backbone, side chain to side chain) through disulfides (disulfide-bond cyclization scan),
lanthionine, dicarba, hydrazine or lactam bridges, to decrease the conformational flexibility
of linear peptides and reduce hydrogen bonding, enhance membrane permeability, and
most importantly to increase their stability against proteolysis by endo- and exopeptidases
6 Substitution of a natural amino acid residue by an unnatural amino acid (D-configuration),
an N-methyl-α-amino acid, a non-proteogenic constrained amino acid or a β-amino acid, to
increase plasma stability (e.g. resistance to endopeptidases) of the peptide and/or affinity
(activity) for its target
7 Deletion of one or more consecutive amino acid(s) and combinatorial deletion with two or
more positions omitted independently throughout the sequence
8 Significance of the N- and C-terminus

9 N-terminal esterification (phosphoester) or pegylation modifications to enhance plasma
stability (e.g. resistance to exopeptidases) and to reduce immunogenicity. Pegylation is also
designed to make the peptide larger (generally > 50 kDa) to retard excretion through the
kidneys (renal clearance)
Alternative routes for the administration of peptide-based drugs have been improved
in recent years and novel peptide delivery technologies have emerged, including
controlled-release parental drug, mucosal route, oral route and transdermal route
(Pettit & Gombotz, 1998). In spite of some limitations, synthetic therapeutic peptides
present numerous advantages compared with their homologous compounds (proteins
and antibodies) and with small organic molecules, thanks to progress in chemical
synthesis and routes of administration (Vlieghe, 2010). In Table 3.1.5, the principal
advantages of peptides over proteins/antibodies or over small organic molecules that
make up traditional medicines are presented.
Table 3.1.5: Advantages of peptides over other drug candidates as proteins/antibodies or organic
molecules (Loffet, 2002; Lien & Lowman, 2003; Witt & Davis, 2006; Vlieghe, 2010; McGregor, 2008;
Hummel, 2006).
Proteins and antibodies Organic molecules

 Peptides have the potential to penetrate  Organic molecules often represent the
further into tissues owing to their smaller smallest functional part of a protein,
size and charge offering greater efficacy, selectivity and
specificity
 Therapeutic peptides are generally less
immunogenic than recombinant proteins  The degradation products of peptides
and antibodies are amino acids (minimizing the risks of
systemic toxicity or drug-drug interactions
 Lower manufacturing costs (synthetic vs.
recombinant production)  Because of their short half-life, few
peptides accumulate in tissues (reduction
 Higher activity per unit mass
of risks of complications caused by their
 Lower royalty stack metabolites)
 Greater stability  Peptides derived from natural source are

receptor agonists, and generally, small
 Produced potential for interaction with the quantities of these peptides are necessary
immune system to activate the targeted receptors
 Better organ or tumor penetration.
3.1.3.4.2 Market
The USA and EU are the major markets for drugs of all kinds, so first approvals for
peptide therapeutics have occurred primarily in one of these two regions. All 19
peptides approved in the USA during the period 2001 to 2012 were first approved in
either the USA or EU. The notable expansion of peptide therapeutic development in
the late 1990s and 2000s led to an unprecedented number of marketing approvals
in 2012, and has provided a robust pipeline that should deliver numerous approvals
during the remainder of the 2010 (Kaspar & Reichert, 2013).
In the US, annual sales of peptide drugs exceed 13 billion, representing 1.5% of
drug sales globally. Protein drugs such as therapeutic antibodies also represent a
larger share with the combined biopharmaceutical market valued at over 70 billion. In
Europe, Germany and the UK account for 63% of the peptide therapeutic market with
France, Italy, Scandinavia and Spain making up the rest of the major users (Hervé,
2008).
According to a review by Vlieghe, more than 60 synthetic therapeutic peptides

(including those used for medical diagnostics or imaging) with a size < 50 amino
acids, have reached the American, European and /or Japanese pharmaceutical
markets through marketing authorization as APIs (Vlieghe, 2010).
The peptides at each phase of development are undergoing evaluation for a wide
variety of indications (Fig. 3.1.14). The diversity of therapeutic areas (TAs) represented
is substantially higher in Phase I and II compared with Phase III, which is at least
in part because of the greater number of peptides in early-stage compared with
pivotal studies. The top two are metabolic disease and oncology for the Phase I and II
peptides, whereas oncology and infectious disease are the top two TAs for peptides in
Phase III (Kaspar & Reichert, 2013).
Figure 3.1.14: Therapeutic areas for peptides in clinical studies (on 15 February 2013; CNS-central
nervous system; CV-cardiovascular; adapted from (Kaspar & Reichert, 2013).
The commercial value of peptide therapeutics that have been marketed for years is
well-established, but could substantially increase as recently approved products and
those on the horizon gain market share. Peptides will probably be used intensively
in the near future for various applications in the treatment of CNS diseases, notably
in the design of peptide regulators of protein activity, if they are able across the BBB
by absorptive-mediated transport (Hervé, 2008; Pardridge, 2003). CMT- or RMT-based
peptides (de Boer & Gaillard, 2007a; 2007b) developed for CNS drug targeting will also
certainly contribute to the development of peptide-based prodrugs with facilitated
Conclusions 215
access to the CNS. Also, in current trials conducted to treat cancer, gene disorders, and
autoimmune diseases, CPP-based therapies are being used. Gene-targeted therapies
have a huge potential to fight rare genomic diseases and will be associated to the
usage of CPP to deliver therapeutic agents into cells (Figueiredo, 2014).
3.1.4 Conclusions
Many drugs see medicinal application denied due to physical, pharmacokinetic or

pharmacodynamic properties. The use of different strategies to improve solubility,
stability, permeability and targeting problems in drug discovery and development
is crucial. The implementation of a prodrug approach in the early stages of drug
discovery is a growing trend. Prodrugs are bioreversible derivatives of drug molecules
that undergo an enzymatic and/or chemical transformation to release the parent drug.
There are several promoieties that can be use in prodrug design, where the selection
of a specific promoiety is essential. The ultimate goal is a chemically stable prodrug
that does not generate toxic metabolites after bioconversion. Some of these works
were based on amino acid applications. Amino acids are basic constituents of a cell
structure and require specialized transport systems to cross the plasma membrane.
Over the past few years, several amino acid transport systems have been identified
and classified on the basis of their substrate affinity, dependence on sodium ions,
energy and pH. The low toxicity of amino acids makes them attractive carriers for
the development of prodrugs for poorly absorbed therapeutic agents. To make the
prodrug a substrate for carrier mediated transport, an amino acid promoiety also
balances the water solubility of the prodrug.
New synthetic strategies for limiting metabolism and alternative routes of
administration have emerged in recent years and resulted in a large number of peptide-
based drugs now being marketed. Peptide-based drug discovery could be a serious
option for addressing as-of-yet unresolved problems. New peptide therapeutics are
delivering significant commercial value in a broad range of indications including
expanding markets such as diabetes and oncology, as well as orphan indications.
Developments in solid-phase peptide synthesis (SPPS) using Fmoc chemistry
have led to larger peptide molecules and small proteins being accessible by chemical
synthesis on scales of multiple kg. The great advantage of SPPS over solution-phase
synthesis is the simplicity, readiness and efficiency of the synthetic process. In
solution-phase synthesis, each synthetic intermediate has to be isolated and purified
after every reactional step, which leads to a very expensive and time consuming
process. Furthermore, it is not convenient to use a large excess of reagents, since
it significantly complicates the necessary purifications. Following a SPPS strategy,
the synthesis is significantly less time consuming as there is no need to isolate the
synthetic intermediates. The global yield also increases, since the whole process is
performed in the same reaction vessel and the use of surplus reagents is admissible
as they are later removed by washing and filtration of the peptidyl-resin contained in
the vessel, which is usually attached to a vacuum filtration system.
The progression of peptide compounds into clinical therapy goes along with
the increasing need for new therapeutic strategies due to the limitations of current
biomaterials for engineering complex tissues. The chemical synthesis of peptides
enables the conjugation of other small molecules or incorporation of non-natural
amino acids by design. Conjugation of small molecules to a peptide opens up the
possibility for greater chemical diversity, analogous to small-molecule medicinal
chemistry approaches for developing high-affinity, high-specificity molecular
recognition increasing the range of potential applications of these molecules in the
growing area of tissue engineering.
Although the interaction of CPPs with cellular membranes often determines their
uptake efficiency, CPPs are first and foremost delivery vectors. This means that they
must efficiently deliver various cargo molecules to their designated location, whether
it is in the cytoplasm or nucleus. CPPs were discovered 20 years ago based on the
potency of several proteins to enter cells. In recent years CPPs have been associated
with biodistribution and efficacy of oral biodrug delivery. Nevertheless, the recent
progress of CPPs as new carriers for intracellular cargo delivery were focused in
tumor cells, where only small quantity of drugs were used as cargo. We propose that
new methodologies will be used to design and develop CPP-drug complexes, in that
CPPs transport their cargo inside cells using endocytosis. Each modification will be
carefully designed to avoid problems that could lead to low synthetic yields, poor
solubility, aggregation or toxicity. The in vivo delivery of drugs (as cargo) requires a
longer drug circulation time, which in turn requires a more stable complex between
the CPP and its drug, as it introduces different hydrophilic groups to an already cell-
permeable peptide. When using CPPs as a delivery system, one must consider that
drug delivery must often be highly specific.
Chemical strategies can improve the in vivo plasma residence time and, after that,
introduce new peptides as drugs in the market. This way, the commercial value of
peptides therapeutic will be well-established.
Acknowledgments: Fundação para a Ciência e Tecnolgia (FCT, Portugal) for financial

support through IF00092/2014, UID/Multi/04378/2013 projects, and for holding an
IF position 00092/2014. Thanks are further due to ON.2 and FCUP for co-funding
refurbishment of the Porto Peptide Synthesis Facility (POP-UP) through operation
NORTE-07-0162-FEDER-000111. NV is also grateful to all authors that sharing their
works and Dr. Mariana Barbosa (U. Porto) by it’s participation in “Peptides: Scaffolding
materials in Tissue Engineering”.
References 217
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James J. Chambers*8
3.2 Targeting Calcium-mediated Excitotoxicity in the
CNS
3.2.1 Introduction
At the heart of our ability to recognize, remember, and perform basic motor functions,
is the highly coordinated placement of neuronal receptors at the interface between
adjacent cells that control the flux of metal ions across the membrane (Kerchner &
Nicoll, 2008; Ribrault, 2011; Wang, 2012). Glutamate is the neurotransmitter that
mediates the majority of fast, excitatory neurotransmission in the central nervous
system (Cotman & Monaghan, 1986). Vesicles of glutamate are released from the
presynaptic cell when action potentials depolarize the membrane of synaptic boutons
(terminals) and vesicles fuse with the membrane (Debelleroche & Bradford, 1977).
Glutamate then diffuses across the synapse and activates postsynaptic glutamate
receptors, initiating postsynaptic current flow via ion entry and depolarizing that
cell, thus completing chemical synaptic communication (Moore & Buchanan, 1993).
Despite decades of inquiry, we still do not fully understand the elegant electrical and
chemical neuronal signals that are generated in a highly complex tangle of minuscule
structures.
Communication between cells in the nervous system requires neurons to quickly
alter their membrane potential, transiently entering a depolarized, excited state to
pass action potentials along to neighboring neurons, and then rapid repolarization
to be prepared for new incoming information. Over-excitation of neurons by various
means maintains a depolarized state for longer than usual and can contribute to
neurological disease. Some of these diseases, which are linked to neuronal over-
activity, can also cause cell damage and death via a process called excitotoxicity.
Excitotoxicity is thought to play a role in neuronal cell and network damage following
stroke and traumatic brain injury and, quite possibly, in the progressive damage
associated with a number of congenital and sporadic neurodegenerative disorders
such as Alzheimer’s, stroke, Huntington’s, Parkinson’s, Amyotrophic Lateral Sclerosis
(ALS), and Multiple Sclerosis (MS).
Receptors located at the neuronal membrane help regulate these fluctuations
in membrane potential by controlling the opening and closing of transmembrane
ion channels, which variously allow cations and anions to flow into and out of the
James J. Chambers: Department of Chemistry, University of Massachusetts, Amherst, Amherst, MA,

01003, USA; Neuroscience and Behavior Graduate Program, University of Massachusetts, Amherst,
Amherst, MA, 01003, USA, *Email: jjchambe@umass.edu
© 2015 James J. Chambers

230 Targeting Calcium-mediated Excitotoxicity in the CNS
neuron down their electrical and chemical concentration gradient. During typical
communication at an excitatory synapse, the presynaptic cell releases glutamate, the
primary excitatory neurotransmitter, after an influx of calcium that enters through
voltage-gated calcium channels. Glutamate crosses the synaptic cleft by diffusion and
binds to receptors on the postsynaptic cell. Some glutamate receptors are ionotropic
ligand-gated channels that are found on the postsynaptic neuron. When an agonist
such as glutamate binds, the associated ion channel opens, allowing monovalent
cations, primarily sodium, to flow into the neuron. As the positive charge flows into
the neuron, the membrane potential of that postsynaptic cell depolarizes, and if it is
sufficiently depolarized, other receptors located nearby on the membrane will open
as well, eventually allowing calcium to enter the postsynaptic cell.
Glutamate is the most prevalent excitatory neurotransmitter in the central
nervous system (CNS) and glutamate receptors (GluRs) play a key role in both normal
excitatory neurotransmission, and in modulating both normal and constructive
plasticity as well as excitotoxic biochemistry, the main topic of this chapter (Cotman
& Monaghan, 1986; Debelleroche & Bradford, 1977; Moore & Buchanan, 1993). There
are two major types of GluRs that differ in the way they influence neuronal response
to excitatory neurotransmission. The first are the metabotropic glutamate receptors
(mGluRs) which, upon binding to glutamate, set off an intracellular biochemical
cascade via second messenger signaling. While mGluRs are certainly targets of interest
for negating the excitotoxic effects of over-stimulation, they are not the focus of this
work, however, a well-written and expansive review of mGluRs and pharmacological
agents was published recently (Williams & Dexter, 2014). Of more importance here,
ionotropic glutamate receptors (iGluRs) mediate fast responses to glutamate release
and also, as their name implies, allow ions to enter or leave the cell via an intrinsic ion
channel. iGluRs are typically heteromultimeric, integral membrane proteins that are
composed of four subunits that form a transmembrane ion channel. This channel is
allosterically connected to the agonist-binding site and upon binding to glutamate or
an exogenous agonist, the channel portion opens and allows cations to pass into the
cell which directly depolarize the membrane potential.
3.2.2 Glutamate and Glutamate Receptors
The postsynaptic iGluR composition has been found to mediate the ionic makeup
of the current that results from glutamate binding. The iGluRs are subdivided into
NMDA receptors and non-NMDA receptors with the latter being further divided into
AMPA receptors and kainate receptors. The AMPA receptors, named after the synthetic
agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, are located on the
acceptor, or postsynaptic, dendrite where they bind neurotransmitters, causing a
conformational change in the receptor protein structure that opens intrinsic sodium
ion channels. AMPA receptors are responsible for the vast majority of excitatory
Glutamate and Glutamate Receptors 231
neurotransmission and they open quickly in response to glutamate and close fast as
well. The influx of sodium ions alters the electrical potential of the postsynaptic cell,
producing a local signal that, after summation and integration at the neuron and then
circuit level, ultimately results in cognition or action. Another family of iGluRs is
the kainate receptors. These appear to be more related to AMPA receptors, but they
are still the subject of ongoing research and their roles in either normal signaling
or excitotoxicity has not been fully explored. If the cellular polarization is above a
certain threshold, neighboring NMDA receptors, named after the synthetic agonist
N-methyl-D-aspartate, fully or partially eject an ion channel-bound magnesium
ion allowing calcium to flow into the cell. The entry of calcium then sets off myriad
intracellular events, not least of which is activation of calcium-activated kinases and,
eventually, synaptic potentiation (Lisman, 2012). A change in the communication
strength between adjacent neurons arises from trafficking of receptors to or from the
synaptic zone and it is presently thought that this molecular trafficking plays a crucial
role in synaptic plasticity, which, in turn, is believed to underlie memory formation
and loss.
3.2.2.1 AMPA Receptors
AMPA receptors are assembled from subunits GluA1 through GluA4 and are typically
comprised of heterotetramers, quite often containing GluA2 subunits. These
receptors are known to play a critical role in long-term potentiation (LTP), one of the
mechanisms thought to underlie memory formation as well as maladaptive and even
neurodegenerative plasticity (Kang, 2009). Most AMPA receptors are not permeable to
calcium ions, due to the presence of the GluA2 subunit. In the GluA2 subunit RNA, a
key modification is made to translate a codon to arginine instead of a glutamine in the
ion channel pore. The incorporation of arginine in the ion conduction pore results in a
channel that does not allow divalent cations, such as calcium, to pass.
A subset of AMPA receptors that are currently of considerable interest in the
neurobiology field and in particular the field of neurodegeneration are those that lack
the GluA2 subunit. GluA2-containing channels are quite often calcium-impermeable
due to an mRNA codon editing event (Araki, 2010; Isaac, 2007). The precise role of
GluA2-lacking receptors in synaptic plasticity or, for that matter, normal synaptic
signaling, is still the subject of ongoing debate in the field (Henley, 2011). While it
is thought that most functional AMPA receptors contain an edited GluA2 subunit,
there is now considerable evidence for a subpopulation of GluA2-lacking receptors
that result in calcium-permeable AMPA receptors (Keller, 1992; McDermott, 2003;
Szabo, 2012; Wright & Vissel, 2012). The enzyme Adenosine deaminase acting on
RNA 2 (ADAR2) specifically mediates RNA editing of the glutamine/arginine (Q/R) site
of GluA2 subunit of the AMPA receptors and it has been found that motor neurons
expressing Q/R site-unedited GluA2 undergo slow death in conditional ADAR2
knockout mice (Hideyama, 2012). This line of research also found that unedited
GluA2 mRNA was expressed in a large proportion of motor neurons from ALS patients
while motor neurons from normal and disease control subjects expressed only edited
GluA2 mRNA. ADAR2 was significantly down-regulated in all the motor neurons of
ALS patients, more extensively in those expressing Q/R site-unedited GluA2 mRNA
than those expressing only Q/R site-edited GluA2 mRNA. These findings suggest that
antagonism of calcium-permeable AMPA receptors could slow disease progression, a
topic we will discuss later in this manuscript.
3.2.2.2 Kainate Receptors
Kainate receptors are also heterotetrameric ion channels assembled from subunits
GluA5-7 and KA1-2 (also known as GluK1-2). The properties of kainate channels are
similar to AMPARs in that they allow ion flux if glutamate activates the channel.
Further, kainate receptors are mostly impermeable to calcium. One main difference
between AMPA and kainate receptors is their subcellular location. While most
AMPA receptors are located at the postsynaptic membrane, kainate receptors have
been found on the presynaptic side as well as the postsynaptic side. It remains
unclear if presynaptic kainate receptor stimulation by glutamate results in more or
less vesicular release of glutamate into the synapse, though in the case of neuronal
necrosis and ischemia, it very likely does. These are discussed later in this chapter.
The function of postsynaptic kainate receptors is similar to that of AMPA receptors
in that depolarization via glutamate-evoked kainate channel current can unplug the
magnesium ion blocking NMDA receptors, thus leading to calcium entry into the
postsynaptic spine.
3.2.2.3 NMDA Receptors
NMDA receptors are heterotetramers and are assembled from three major families
of subunits. The NR1 subunit is ubiquitously expressed, there are four NR2 subunits
(A-D), and two NR3 members (A and B). Most NMDA receptors that have been studied
to date contain both NR1 and NR2 subunits and they share the common attributes of
being activated by glutamate but normally being blocked by a magnesium ion that is
removed upon strong stimulation of the postsynaptic spine. Besides glutamate, it was
found that glycine was a required co-agonist to elicit NMDA channel opening.
NMDA receptors have slow gating kinetics, but once open, are highly permeable to
both calcium and sodium. Because of the large ion permeability to calcium, this influx
generates intracellular calcium waves or transients (also the term “calcium spikes” is
popular in the literature) that set off myriad calcium-mediated biochemical cascades.
The NMDA receptors are bound to postsynaptic cytoskeletal scaffolding proteins that
The Role of Calcium in Normal Neuronal Biochemistry 233
are collectively known as the postsynaptic density. This scaffolding also supports
kinases and other proteins that have downstream signaling roles, likely to receive the
calcium transient as efficiently as possible. Under normal circumstances, glutamate is
quickly removed from the synaptic cleft by excitatory amino acid transporters (EAAT)
located on nearby glial cells. This ensures that only a limited amount of calcium will
be able to enter the postsynaptic neuron.
3.2.3 The Role of Calcium in Normal Neuronal Biochemistry
NMDA receptors are opened when the neuronal membrane potential is depolarized
to a sufficient level to expel the bound magnesium ion. Opening of these ionotropic
receptors leads to a rise in postsynaptic calcium concentration, which has been linked
to long-term potentiation (LTP) via protein kinase activation. It is thought that strong
depolarization fully expels the magnesium plug, whereas milder depolarization
partially displaces the magnesium, resulting in less access to calcium through NMDA
receptors. This latter process is thought to underlie long-term depression (LTD), in
which lower concentrations of postsynaptic calcium activates protein phosphatases
and initiates partial dismantling of the synaptic connection.
The activated protein kinases that arise from the high calcium concentration
phosphorylate a variety of postsynaptic targets including some AMPA receptors.
These post-translational modifications have been shown to enhance single channel
conduction through these channels, thereby potentiating the connection between
these two neurons. Further, these kinases can phosphorylate the protein machinery
that determines the number of postsynaptic AMPA receptors, thus opening more
“docking” sites for new AMPA receptors from nearby locations. In the end, the result
of controlled calcium entry into a synaptic spine is more AMPA receptors that are
more permeable to cations. Of course, protein phosphatases can act to reverse these
potentiation modifications to return the synapses to the pre-LTP levels.
Besides direct activation of postsynaptic kinases, calcium entry can activate a
second messenger cascade to turn on gene transcription of proteins such as CaMKII
and PKAII leading to relatively high local concentrations of these kinases in the
synaptic spine. Both of these kinases have been found to play a role in LTP processes,
such as spine volume, addition of new AMPA receptors, and direct phosphorylation of
ion channels. It is this local effect of LTP that allows for selective potentiation of single
synapses and, because of this, each synaptic spine could be considered isolated from
other postsynaptic zones as all of the changes in kinase activity are highly restricted
to that local spine. This addressable and highly spatial system is likely related to long-
term memory storage.
3.2.4 Excitotoxicity
A common molecular mechanism in both acute and chronic neurodegenerative

diseases including stroke, Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis,
multiple sclerosis, and others is the excitotoxic loss of dendritic synaptic spines and
eventual neuronal death. Dendritic synaptic spines are the postsynaptic regions of
a neuron that receive the chemical signal from the presynaptic cell, in this case via
glutamate diffusion. These spines are essential to brain function, such as memory
formation and related neural plasticity mechanisms. Pruning or loss of synaptic spines
may occur through misdirected voltage- or ligand-gated ion channels. The excitotoxic
effects of calcium have long been thought to be due to excess quantities of calcium,
but evidence is accumulating that the route by which calcium enters the cell may
be at least as important (Szydlowska & Tymianski, 2010). Recent research suggests
that in Huntington’s disease, a neurodegenerative disorder, calcium entering neurons
via NMDA receptors located outside the synapse (extrasynaptic NMDA receptors)
may initiate different and more damaging molecular cascades than calcium entering
neurons via synaptically-located NMDA receptors (Hardingham & Bading, 2010).
3.2.5 The Role of Calcium in Excitotoxic Neuronal Biochemistry
Controlled increases in glutamatergic activity can result in increased postsynaptic

calcium, which plays a critical role in neuronal processes supporting learning and
memory. Over-activation of glutamate receptors, however, can lead to excitotoxic
concentrations of calcium. This can lead to neuronal death and degeneration through
a variety of molecular mechanisms. Intracellular calcium activates a number of
enzymes that can have deleterious effects including phosphatases, proteases, lipases,
caspases, and endonucleases and eventually the cell can undergo apoptosis through
activation of caspases. Additionally, the NMDA receptor interacts directly with nitric
oxide synthase, an enzyme sensitive to calcium influx that induces the production
of toxic nitric oxide along with reactive oxygen species (Szydlowska & Tymianski,
2010).
One of the earliest findings of pathological excitotoxicity was the release of
glutamate into extracellular space minutes after spinal cord injury by neurons that
were at the site of injury. This non-controlled glutamate release can stimulate glutamate
receptors, further stimulating the local cohort of neurons. In stroke patients, blood
flow is reduced to inadequate levels and the concentration of extracellular glutamate
rises, causing uncontrolled excitation of glutamate receptors. This excitatory insult is
thought to be exacerbated by a lack of oxygen and glucose to power the machinery to
remove the glutamate from the effected area.
The effect of the increased concentrations of glutamate in the extracellular
space is that neurons are strongly depolarized to the point where the removal of the
Diseases that are Potentially Exacerbated by Calcium-mediated Excitotoxicity 235
magnesium ion that normally blocks NMDA receptors occurs. This, of course, then
results in uncontrolled calcium entry through the NMDA receptors into a previously
unaffected neuron, thus propagating the cellular insult. In addition, it has been
shown that the NMDA receptors found on oligodendrocytes, the cells that provide
myelin sheaths the axons in the CNS, are activated as well, thereby exacerbating
excitotoxicity. Once in the cell, the increased calcium activates kinases, but also has an
effect on mitochondria. In response to high calcium, the mitochondrial permeability
transition pore is opened and it is presently thought that pore opening may allow the
release of reactive oxygen species, further contributing to apoptosis and slowing or
halting the local production of adenosine triphosphate (ATP). Lack of ATP quickly
eliminates the electrochemical gradient across the membrane of some ions, which
in turn shuts down glutamate transporters, the proteins that remove glutamate from
extracellular space, to terminate glutamatergic signaling. The result of this excitotoxic
cascade is the accumulation of glutamate in the extracellular milieu which can spread
to neighboring neurons.
3.2.6 Diseases that are Potentially Exacerbated by Calcium-media-

ted Excitotoxicity
3.2.6.1 Amyotrophic Lateral Sclerosis (ALS)
Calcium dysregulation is a central factor in the incurable neurodegenerative disorder

ALS (Grosskreutz, 2010). This disorder results in muscle atrophy followed by paralysis
and eventual death of patients 3-5 years after the presentation of symptoms. The
underlying cause of ALS is the selective degeneration of motor neurons in the spinal
cord and brain stem as well as the motor cortex. Degeneration of these cells results
in difficulty of movement, speech, and swallowing and progresses to problems with
respiratory function. A dismaying psychological factor is that patients with ALS
typically suffer no cognitive deficit and are thus fully cognizant of their own demise as
the disease progresses inexorably toward death. Present numbers on the prevalence
of ALS indicate that the adult lifetime risk for ALS is near 1 in 400, a number which is
similar to that for multiple sclerosis (Benatar & Wuu, 2012).
The best known genetic factor for familial ALS, occurring in about 20% of the
cases, are mutations in a gene that encodes the copper/zinc-dependent superoxide
dismutase protein (SOD1). In more recent studies, further mutations have been found
by using genome wide association studies of patients afflicted with ALS, however,
in many of these cases, the underlying molecular pathology is not yet known
(Renton, 2014). Sporadic ALS does not have such target mutations, however, there
are a few examples of environmental exposures that can result in ALS or ALS-like
neurodegeneration and new gene targets with incomplete penetrance (Turner, 2013).
One of the striking characteristics of familial ALS as well as SOD1 mutant mouse models
is the high degree of selectivity that the disease shows for motor neurons over all other
cells in the body, even though the mutant form of SOD1 protein is in many other cell
types in the patient. A possible mechanism for this selectivity is based on excitatory
neurotransmitter-mediated neuropathology, in particular glutamate. It is presently
believed that motor neurons are especially susceptible to excitotoxicity because
they are known to receive strong glutamatergic input. Additionally, spinal motor
neurons have recently been found to express calcium-permeable AMPA receptors on
their surface. Multiple lines of neurobiological research have resulted in the current
understanding that calcium-permeable AMPA receptors play crucial roles in synaptic
signaling and plasticity in the CNS. An overabundance of these receptors, coupled
with glutamatergic excitation, could overwhelm the calcium buffering capacity of a
cell, resulting in metabolic/mitochondrial breakdown followed by cell death.
Presently, riluzole (Rilutek) is the only United States Food and Drug
Administration approved therapeutic to treat ALS. The specific biological target of
riluzole is controversial, but it is thought that the drug acts by reducing excitatory
neurotransmission, resulting in the influx of less calcium into motor neurons slowing
the progression of the disease. Unfortunately, riluzole is not a cure and only offers
some of those suffering with ALS around 3 additional months of life.
3.2.6.2 Multiple Sclerosis (MS)
MS is an inflammatory disease of the myelin sheath that insulates neurons in the

brain and spinal cord. The breakdown of this insulator erodes the ability for neurons
to communicate and results in a wide variety of symptoms in patients suffering
from MS, ranging from physical disability to psychiatric and cognitive deficits. The
progression of MS has been observed to occur either in isolated “attacks” and a slower
degenerative progression. In each case, permanent damage is done as the disease
advances, however, it appears that some functions can be relearned likely through
network plasticity.
Presently, the molecular cause of MS is not fully elucidated but much evidence
points to a failure of the immune system in which the myelin-producing cells are
attacked. Glutamate-mediated excitotoxicity has also been implicated as a root
cause. As discussed previously, the oligodendrocytes, the cells that provide the
myelin sheath for neurons in the CNS, are also susceptible to excitotoxicity. In a recent
study in an animal model of MS, glutamate receptor blockers were found to increase
the survival of oligodendrocytes and prevent some of the molecular mechanism from
over-stimulation (Suhs, 2014).
3.2.6.3 Alzheimer’s Disease (AD)
Characterized by progressive death and degeneration of neurons, Alzheimer’s disease

leads to increasing memory loss and eventual dementia. A small percentage of cases
(Ballard, 2011) have been found to be caused by a congenital defect and these cases are
classified as Familial Alzheimer’s Disease (FAD). The majority of Alzheimer’s disease
cases are classified as sporadic Alzheimer’s and there is presently no known direct
cause. Two classic indications of the disease that can be observed in brain tissue of
both FAD and sporadic Alzheimer’s sufferers are the presence of neurofibrillary tangles
of hyperphosphorylated tau inside of neurons and plaques consisting primarily of
extracellular aggregations of beta-amyloid (Aβ) peptide fragments.
Aβ clusters have been found to influence glutamate transmission in several
ways, some of which could lead to excessively high concentrations of calcium
and thus excitotoxicity. It was shown that the presence of Aβ impairs the ability of
glutamate transporters to help remove glutamate from the extracellular area around
the synapse, potentially resulting in excitotoxic effects (Li, 2009). Aβ clusters also
appear to increase the population of expressed NMDA receptors containing the NR2B
subunit and the number of metabotropic mGluR5 receptors present at the synapse,
which could also lead to excitotoxicity. Recent research has also suggested that
extrasynaptic NMDA receptor activation could lead to an increase in Aβ production,
unlike synaptic NMDA receptor activity. Further complicating matters is the fact
that the different groups (group I and group II) of metabotropic glutamate receptors
(mGluRs) have differing effects on Aβ production by neurons (Hu, 2012). Further, two
observed effects of Aβ are not directly linked to excitotoxicity, but further implicate
glutamatergic transmission in AD. When cultures of neurons that were collected from
the hippocampus, an area of the brain critical to memory formation, were exposed to
sub-nanomolar concentrations of Aβ, it was found to promote glutamate release in an
activity dependent manner. While this may possibly be a normal function as it would
support the cellular and molecular processes underlying learning and memory, other
experiments on hippocampal neurons found that high nanomolar concentrations of
Aβ clusters bind to the postsynaptic spines of glutamatergic synapses. This binding
is associated with the internalization of AMPA receptors, which can lead to the loss
of the synaptic spine, and thus that synaptic connection. This synaptic loss would
reduce the ability of affected neurons to communicate with each other, and could
impair the neuronal connections that support memory formation and maintenance.
There is also some evidence that hyperphosphorylated tau may interfere with iGluR
trafficking, which could also impair synaptic formation and maintenance (Hu, 2012).
3.2.6.4 Huntington’s Disease (HD)
Huntington’s disease is an inherited degenerative disorder that results in the death

of cortical and striatal neurons, specifically certin GABA-releasing neurons known
as GABAergic medium-sized spiny neurons (Mony, 2009). The loss of these neurons
results in a range of physical, psychological, and cognitive symptoms. Involuntary
spastic movement and difficulty initiating and controlling voluntary movement
are common in the early stages of this progressive disorder. These symptoms are
often accompanied by cognitive decline. The cause of the disease is a mutation in
the gene that encodes the huntington protein, which results in the production of a
longer than normal sequence of glutamine residues at the N-terminus of the protein
(Okamoto, 2009). Although the exact mechanism by which the mutation causes the
progressive death of these neurons remains unknown, some evidence suggests that
the mechanism may be a dysregulation in expression of NMDA receptors at synapses
and, more importantly, expression of these receptors at extrasynaptic locations.
When intracellular calcium concentrations are increased due to extrasynaptic NMDA
receptor activity, the calcium appears to trigger different downstream cascades
than calcium ions that enter the neuron through synaptic NMDA receptors. As
discussed previously, controlled increases in calcium that result from synaptic
activity can activate beneficial, synapse building pathways; equivalent increases
through extrasynaptic NMDA receptor activity were shown to cause disruptions to
mitochondrial membrane potential and cell death (Hardingham & Bading, 2010;
Milnerwood, 2010). It has been suggested that weak glutamate antagonists such as
memantine, or NR2B subunit specific antagonists, may provide a clinically relevant
outcome if they can be designed to block extrasynaptic NMDA receptors without
preventing normal synaptic NMDA receptor activity (Mony, 2009).
3.2.6.5 Stroke
During a stroke, the blood flow to part of the brain is disrupted either due to a blockage
in a blood vessel feeding the brain (ischemic stroke) or due to a rupture in one of these
vessels (hemorrhagic stroke). Neurons in the area of the brain that are supplied by
the affected vessel are often deprived of the required supply of oxygen and glucose
and are thus unable to continue operating the energy-demanding ion transporters
that establish the negative membrane potential. With a loss of these pumps, the
membrane begins to depolarize and when it has sufficiently depolarized to the point
that magnesium plugs have been ejected from the NMDA receptors, calcium ions enter
the neuron at the slightest exposure to glutamate. As already discussed, this calcium
entry then initiates excitotoxic molecular mechanisms (Lipton, 2006).
After a stroke, excess calcium is also able to enter neurons by a non-glutamate
receptor pathway. The Transient Receptor Potential (TRP) channels allow calcium
to enter neurons in response to ischemic-linked changes in extracellular levels of

divalent cations, pH, and levels of reactive oxygen species. Further, the stored calcium
that is normally localized to the endoplasmic reticulum or mitochondria may also be
released into the cytoplasm following ischemia, further exacerbating the condition
by increasing the concentration of calcium in the neuronal cytoplasm (Szydlowska &
Tymianski, 2010).
A number of drugs have been developed specifically to treat excitotoxicity in
the aftermath of stroke and, for the most part, they are aimed at preventing calcium
entry into neurons or by targeting downstream molecules involved in the excitotoxic
cascade, but clinical trials have been largely unsuccessful. These compounds include
glutamate receptor antagonists, glutamate release blockers, nitric oxide synthase
inhibitors, and free radical scavengers that are designed to prevent oxidative damage.
Most of these agents that have been tested unfortunately have been ineffective or
have had intolerable side effects precluding their use in the clinic (Lau & Tymianski,
2010).
An additional molecular factor that may increase the excitotoxic effects of
stroke is an upregulation of calcium-permeable AMPA receptors. As described in
the introduction to glutamate receptors previously, most AMPA receptors contain a
GluA2 subunit that prevents calcium from passing through the ion channel. However,
following some types of ischemic events, it has been found that the expression of
GluA2 mRNA is reduced; suggesting that there could be an increased expression of
calcium-permeable AMPA receptors (i.e. those that lack the GluA2 subunit) would
result. An increase in these receptors offers yet another possible means for calcium to
enter into neurons already suffering from hyperactivation, leading to abnormally long
periods of depolarization and excessive calcium influx (Lau & Tymianski, 2010).
3.2.6.6 Parkinson’s Disease
The progression of Parkinson’s disease involves the loss of the dopaminergic neurons
in the substantia nigra. These neurons help control muscle movement and as they
degenerate, patients lose control of voluntary and spontaneous muscle movement
presenting symptoms that include resting tremors, slowed movement, difficulty with
movement, and balance problems. In addition, a number of cognitive symptoms
typically develop as the disease progresses, including memory loss, depression, and
eventually dementia. The mechanism behind the initial neuronal degeneration is
only known in a small percentage of cases, termed familial Parkinson’s, which has
been linked to an inherited genetic mutation. However, like AD, most other cases
are sporadic Parkinson’s and there is no known cause. However, recent research
has implicated glutamate excitotoxicity as a factor as the reduction in dopaminergic
transmission leads to a loss of regulation of striatal neurons, resulting in an in increase
in glutamatergic activity (Koutsilieri & Riederer, 2007; Meissner, 2011; Mony, 2009).
A logical method to prevent glutamate-mediated excitotoxicity is to prevent glutamate

release and/or binding to the iGluRs. Recent experimental treatments have included
drugs that do just this (Meissner, 2011). The results have interestingly demonstrated
that, in animal models of Parkinson’s, a shift in the subunit composition of the
NMDA receptor is found, which is thought to be a potential excitotoxic mechanism.
L-DOPA treatment was shown to alter NMDA receptor subunit composition as
well, leading to L-DOPA induced dyskinesia. Unfortunately, though general NMDA
receptor antagonists have been found to be successful in treating both Parkinsonian
and dyskinesia symptoms in animal models of the disease, the side effects of these
agents in humans preclude their use in the clinic. In an effort to circumvent these side
effects, drug developers have focused on compounds targeted to the NR2B subunit of
the NMDA receptor. One such compound, CP-101,606 (Traxoprodil) worked well for
the treatment of both Parkinsonian and dyskinetic symptoms in animal models, but
in human trials only appeared to treat dyskinetic symptoms (Koutsilieri & Riederer,
2007; Mony, 2009).
3.2.6.7 Traumatic Brain or Spinal Cord Injury
Intracranial injury, more commonly know as traumatic brain injury (TBI), occurs when
a head injury results in damage to neuronal structure and integrity. There are two
phases of TBI; the acute initial damage from the insult and the secondary injury that
are caused by excitotoxicity. Much like the progressive neurodegenerative diseases
already discussed and acute stroke, glutamate dysregulation leads to calcium influx
into neurons close to the region of initial insult, which results in necrosis of neurons.
TBI is complicated as there may or may not be blood flow alterations, hypoxia,
swelling, and intracranial pressure. Presently, no pharmacological intervention is
available to treat this secondary, excitotoxic cascade that results from TBI. Like other
excitotoxic diseases, NMDA receptor antagonists showed promise in animal studies
but failed to show efficacy in human clinical trials.
3.2.7 Why not Fully Block Calcium Entry via Pharmacological

Agents?
Could it be as simple as blocking the glutamatergic transmission so as to cut off

the link between over-stimulation by extracellular glutamate and calcium-induced
excitotoxicity inside of downstream neurons? Based on the studies that have been
performed, it appears that is not the case. One very possible problem that is presented,
though likely underappreciated, when fully antagonizing these receptors is the self-
regulating mechanism that synapses use, called homeostatic plasticity.
Emerging Targets for Reducing Calcium-mediated Excitotoxicity 241
Homeostatic plasticity is the broad term used to describe all of the molecular plastic
changes that a neuron uses to govern and adjust its own intrinsic excitability. One
common underlying method that neurons use to make these adjustments is synaptic
scaling (Thalhammer & Cingolani, 2014). That is, the neuron will adjust the properties
of ion channels to meet a certain set point. This is a process that was first observed
in neurons adjusting their excitatory response to glutamate release after chronic
manipulations of their activity. In vitro experiments with neurons have demonstrated
that chronic blockage of either NMDA receptors or AMPA receptors results in a
process that looks like LTP (Lee & Chung, 2014). There have been reports of enhanced
trafficking of iGluRs to the surface of the cell and delivery to synaptic locations after
treatments. Thus, we draw a parallel to these findings and suggest that full blockage
of iGluRs could lead to homeostatic plasticity induction and this would be counter-
productive to reducing excitotoxicity, since it would result in an increased expression
of just the channels that clinicians are trying to modulate. We hypothesize that a
partial antagonist will be effective in reducing spine loss and neuron death due to
excitotoxic calcium influx. In fact, one of the few pharmacological agents that actually
demonstrates some efficacy on slowing excitotoxic effects is memantine, a partial and
weak antagonist of NMDA receptors (Gardoni & Di Luca, 2006).
3.2.8 Emerging Targets for Reducing Calcium-mediated

Excitotoxicity
As described earlier in this chapter, calcium-permeable AMPA receptors are garnering

much attention as they have been found to play a role now in both normal plasticity as
well as neurodegenerative. Interestingly, in almost all studies of calcium-permeable
AMPA receptors, one common ligand has been employed in many experiments
(Meyer, 2012; Wen & Barth, 2012). This molecule is naphthylacetylspermine (NAS)
and is a synthetic agent that was designed based on a number of natural toxins
biosynthesized by certain spiders and wasps. All of these molecules are use-
dependent, full antagonist, pore blockers of calcium-permeable AMPA receptors.
Calcium-permeable AMPA receptors are inserted into the membrane after broad AMPA
receptor antagonism in cultured neurons, suggesting a role for them in homeostatic
plasticity and maintenance of synaptic communication (Beique, 2011). This latter
point leads us to believe that partial antagonism may be more fruitful than simply
blocking all CP-AMPARs for ALS treatment.
There is much experimental evidence that suggests CP-AMPARs are expressed on
cultured motor neurons (Carriedo, 1996; Van Den Bosch & Robberecht, 2000; Van den
Bosch, 2002). These specific receptors have been shown to be a calcium conduit that
could be responsible for excitotoxic levels of calcium entering motor neurons and it
is known that blockage of these channels can prevent or delay cell death (Corona &
Tapia, 2007; Van Damme, 2002; Van Den Bosch & Robberecht, 2000). In addition,
the calcium-permeable AMPA receptor-mediated excitotoxicity can be delayed by

chelation of calcium in models of ALS by application of BAPTA (Corona, 2007). Our
present understanding of the molecular determinants of ALS progression all point to
the involvement of calcium-permeable AMPA receptors and thus these ion channels
warrant further study and targeting for new pharmacological agents. Of course, an
alternative method to potentially reduce the expression of calcium-permeable AMPA
receptors would be to restore the enzymatic activity of ADAR2, the enzyme responsible
for editing the GluA2 RNA to make calcium-impermeable AMPA receptors.
3.2.9 Conclusions and Outlook
With an increasingly large aging population, the numbers of people afflicted with the
diseases of excitotoxicity will continue to grow. Treatment of the effects of calcium-
mediated excitotoxicity should be at the forefront of our collective efforts to treat
these diseases. In too few cases, there are obvious genetic targets that can potentially
be addressed with the new genome editing technology that is currently maturing.
However, for the vast majority of sporadic cases of disease, the root cause is either
undetected genomic mutations or environmental or developmental insults. For these
categories, genome editing has no known target and thus, must be tackled at the site
of molecular damage. With calcium-mediated excitotoxicity implicated in so many
neurological diseases, the prize for the scientist who solves this problem will likely be
substantial and society will be indebted to them.
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Rebecca Fransson, Christian Sköld and Anja Sandström*9
3.3 Strategies for Conversion of Peptides to Peptido-
mimetic Drugs
3.3.1 Peptides as Starting Points in Drug Discovery
Due to their importance in many biological functions, bioactive peptides are

interesting as starting points in drug discovery and as valuable research tools in
initial investigations of the biological mechanisms of various diseases (Sewald,
2002). A compound intended as an oral therapeutic agent should display not only
the desired effect (pharmacodynamics), but also satisfying pharmacokinetic criteria
(Fig 3.3.1). Thus, adsorption, distribution, metabolism and excretion (ADME)
properties play a critical role in defining a good drug candidate. However, due to the
structural characteristics of peptides, they suffer from inherent drawbacks, such as
low bioavailability, low metabolic stability, poor absorption from the gastrointestinal
tract (GI), and poor brain exposure as a result of low permeability over the blood-
brain barrier (BBB) (Humphrey, 1986; Veber, 1985).
Figure 3.3.1: Drug distribution in the human body is determined by its ADME properties.
Rebecca Fransson, Christian Sköld and Anja Sandström: Division of Organic Pharmaceutical
Chemistry, Department of Medicinal Chemistry, BMC, Box 574, Uppsala University, SE-751 23
Uppsala, Sweden, *Email: anja.sandstrom@orgfarm.uu.se
© 2015 Rebecca Fransson, Christian Sköld and Anja Sandström

246 Strategies for Conversion of Peptides to Peptidomimetic Drugs
Furthermore, peptides have a large degree of conformational flexibility, and can fold
into complex tertiary structures crucial for their molecular recognition and their
ability to produce a biological response. In order to fully benefit from the potential
of biologically active peptides in chemical biology and in drug discovery, different
approaches to overcome their limitations are needed.
There are several strategies available that can be utilized for the development
of drugs that modulates the physiological events that are triggered by peptides. One
common approach is high-throughput screening (HTS) of libraries of small molecules.
This approach only considers the macromolecular target and can be utilized if the target
is known and a suitable biochemical assay is available. Peptide-based therapeutics
is another, currently growing, area that instead focuses on the biologically active
peptide and aims to circumvent the limitation of peptides by introducing various
modifications (e.g. PEG and phosphoesters linkages) or by employing different
ways of administration (e.g. parenteral, mucosal, oral and transdermal routes). As
a complement to HTS and peptide modifications, rational design based on step-wise
transformation of peptides into low-molecular-weight and bioavailable drug-like
molecules that mimic the action of peptides, i.e. peptidomimetics, is a viable way to
overcome the problems associated with peptides (Hruby, 2002; Olson, 1993; Ripka,
1998; Vagner, 2008). Peptidomimetics are molecules with significantly reduced
peptide character that mimic the bioactive conformation of peptides, and thus retain
the ability to interact with the biological target and cause the same biological effect
(Grauer, 2009). As these compounds are non-peptides which often possess desired
improved pharmacokinetic properties, such as better absorption, metabolic stability
and/or bioavailability. There are several successful examples where a rational design
approach has resulted in approved drugs, e.g. the development of protease inhibitors
such as angiotensin-converting enzyme (ACE) inhibitors, HIV protease inhibitors,
and hepatitis C virus (HCV) inhibitors.
3.3.1.1 Strategy for the Development of Peptidomimetics
To transform biologically active peptides into small drug-like pseudopeptides or

peptidomimetics in a rational way, a stepwise procedure, similar to the one outlined in
Fig. 3.3.2, can be employed. Biological evaluation (both in vitro and in vivo), evaluation
of physicochemical and pharmacokinetic properties, and computational modeling
guide the stepwise structural modifications and should be run in parallel with the
different steps. The strategy will be described in the following section, and further
exemplified in a case study related to a metabolite to the neuropeptide Substance P
(Fransson, 2008; 2010; 2013) .
Peptides as Starting Points in Drug Discovery 247
Figure 3.3.2: A step-wise strategy for development of drug-like peptidomimetics.
3.3.1.1.1 Property Elucidation
The activity at the target and the exposure (e.g. concentration and duration)
determine the efficacy of a drug. In the body, several barriers to drug exposure can
be found, for example cell membranes, metabolic enzymes, efflux transporters,
and binding proteins. How a compound performs at a specific barrier is connected
to its drug properties. In the GI tract, compounds can cross the cellular membrane
barrier by three major mechanisms (van de Waterbeemd, 2001). The two most
common are transcellular absorption, i.e. passive transfer by diffusion across the
lipid membranes, and paracellular absorption, which proceeds through aqueous
pores at the tight junctions between the cells. The third mechanism is active uptake
by transport proteins that usually transport nutrients across the membrane (Kerns,
2008; van de Waterbeemd, 2001). The most important mechanism for drug absorption
is passive diffusion, and about 95% of all commercial drugs are absorbed by this route
(Kerns, 2008). Metabolizing enzymes in the GI tract, e.g. the cytochrome P450 (CYP)
enzymes and proteolytic enzymes as well as efflux transporters, e.g. P-glycoprotein
(PgP), are expressed, which limit the oral absorption of compounds (Fig. 3.3.3) (van
de Waterbeemd, 2001). In addition to avoiding the efflux of PgP and metabolism by
gut wall enzymes, good permeability is important to maximize the oral absorption
of a compound. In general, therapeutic peptides suffer from short half-lives due to
rapid degradation by proteolytic enzymes of the GI tract and blood plasma, rapid
clearance from the circulation by liver and kidneys, as well as limited permeability
across physiological barriers because of their hydrophilic structure (Vlieghe, 2010).
Especially CYP3A4 and PgP have been shown to have a significant impact on the
bioavailability of peptidic and peptidomimetic drugs (Wacher, 1998). One means

of enhancing oral bioavailability is to increase passive diffusion by changing the
physiochemical properties (i.e. decreasing the hydrophilicity) of the compound
(Wang, 1999). It should be noted that increasing the lipophilicity, in order to improve
membrane permeability, can also lead to increased efflux and metabolism. Two other
strategies that can be used to improve permeability are reducing hydrogen bonding
and decreasing the polarity (Kerns, 2008).
Figure 3.3.3: Illustration of the barriers to drug absorption in the GI tract.
In the bloodstream, enzymatic hydrolysis by proteolytic enzymes and plasma protein

binding (PPB) constitute barriers preventing drugs from penetrating into the tissues.
The affinity of a compound to plasma proteins determines the ratio of bound to
unbound (“free”) drug in solution, and only the unbound drug can enter the tissues.
If a compound has a high binding affinity it can be difficult to achieve concentrations
in the tissue sufficient to produce the desired pharmacological effect. High PPB also
reduces the clearance of a compound and thus increases the PK half-life, since it
prevents the drug from permeating into the liver and kidneys (Kerns, 2008).
A further dimension that must be considered when working with central nervous
system (CNS) active agents is the uptake in the brain. Here, the blood–brain barrier
(BBB) constitutes an additional barrier to absorption. It has been shown that the
penetration of a compound into the brain decreases with increasing polar surface area
(Van de Waterbeemd, 1998). However, it is not only the physiochemical properties
that influence brain uptake. Transporters, especially PgP, and metabolizing enzymes
are also limiting factors. Several structure modification strategies to reduce PgP efflux
have been described (Kerns, 2008). These include reducing the number of hydrogen
bond donors, decreasing the hydrogen bond acceptor potential of a compound, and
decreasing the overall lipophilicity of the structure.
During rational peptide lead optimization various in vitro property assays (e.g.
solubility, permeability, chemical stability, metabolism, protein binding and transport)
are used to assess the drug-like properties of the generated peptide analogues (Kerns,
2008). Extensive in vitro profiling regarding the PK data of lead compounds in the
early stages of development can thus provide the medicinal chemist with information
on the structure-property relationship important for the further development of orally
active compounds.
3.3.1.1.2 Structure–Activity Relationship

In order to develop peptidomimetics with retained affinity to the biological target
it is crucial to investigate what parts of the peptide are directly involved in target
recognition. Thus, attainment of the structure–activity relationships (SARs) and
identification of the minimal active sequence of the peptide required for biological
activity are important first steps in the process. This is normally achieved by the
evaluation of binding affinities of peptide analogs to the target protein. In practice,
such information is normally gathered through amino acid scans, truncations and
N- and C-terminal modifications (Grauer, 2009; Hruby, 2002; Wiley, 1993; Vlieghe,
2010). Amino acid scans determine the importance of amino acid side chains by
systematically replacing each residue within the peptide with alanine, glycine or
the corresponding d-amino acid. Alanine and glycine are the smallest amino acids
available, having a methyl and a hydrogen side chain, respectively. Thus, they should
have only a small impact on the overall binding affinity, unless they substitute a crucial
amino acid in the original peptide. N- or C-terminal truncation removing one amino
acid at a time provides information about the minimal sequence needed for biological
activity. The importance of a basic N-terminal or an acidic C-terminal is determined
by the introduction of capping groups. These initial investigations will lead to the
identification of the essential residues in the peptide and potential pharmacophoric
groups, i.e. structural features that are required for the biological activity. Based
on the information gained from these studies, further structural modifications are
undertaken to improve stability, potency and selectivity.
3.3.1.1.3 Bioactive Conformation

One of the central steps in peptidomimetic design is to elucidate the bioactive
conformation of the peptide, i.e. the conformation adopted when it is bound to the
macromolecular target. Conformational restrictions are frequently used to explore
the bioactive conformation and to enhance bioavailability by improving enzymatic
stability, (Grauer, 2009; Veber, 1985). A constraint that leads to a reduction in the
loss of conformational entropy upon interaction with the target can also increase
the binding affinity (Rizo, 1992). Global and local constraints can be achieved by
cyclization (Hruby, 2002), N-methylation (Rizo, 1992), isosteric substitution (Rizo,
1992; Sewald, 2002) or by secondary structure replacement (which will be discussed

in more detail below) (Kim, 2000) as exemplified in Fig. 3.3.4.
A B
i. N- to C-terminal Internal N-methylation
i
α-Carbon methylation C-terminal methylation
ii
R O
H
R O R N
H H2 N OH
N OH
H2 N N O R
H iv ii. Side chain to
O R O
side chain N-terminal methylation Side chain methylation
iii
iii. Backbone to backbone iv. Backbone to side chain
C D
H
O S
S
N N H 2N
H H 2N O
HO
O OH
H O
N OH β-turn mimetic β-turn mimetic
O R OH
H R
Retro-inversion Hydroxyethylene (E)-alkene N
O S O
H 2N N
N N
H H OH
O N3
Reduced Reduced Thioamide OH
O
β-turn mimetic γ-turn mimetic
Figure 3.3.4: A) Cyclization strategies that can be performed in a peptide sequence to introduce
global constraints (Rizo & Gierasch, 1992). B) Positions in a peptide that can be methylated.
Methylation of both the backbone atoms and the side chains can introduce local constraints (Rizo,
1992). C) Isosteric replacement of the peptide bond can introduce local constraints. It should be
noted that such replacements are not always real constraints, but alter the overall conformational
behavior of the peptide backbone to varying degrees. In some cases the flexibility is increased (Rizo,
1992; Sewald, 2002). D) Secondary structure mimetics can induce a desired conformation when
introduced into the peptide backbone (Rosenström, 2006; Schmidt, 1998; Sewald, 2002).
Many high-quality 3D structures of ligands, when bound to their macromolecular

target, have been obtained using X-ray crystallography. However, this technique is
not readily applicable to all types of systems. For example, enzymes have been easier
to crystalize as compared to membrane bound G-protein coupled receptors (GPCRs),
although the number of crystalized receptors are increasing. To model the receptor-
bound conformation when the 3D structure of the target is unknown, ligand-based
experimental or theoretical studies have to be performed. Conformations adopted
by flexible linear peptides are numerous, and are strongly influenced by interactions
with the environment (Rose, 1985). Therefore, to limit the conformational flexibility,
conformational constraints can be introduced into the peptide to provide information
about the bioactive conformation. In many cases the constrained part of the peptide is
not completely rigid (e.g., using side chain cyclizations as constraints) and therefore
several differently constrained analogues of the peptide may be required in order to
derive a putative receptor-bound conformation. When peptides bind to their receptors
they become an integral part of the protein structure and thus can be predicted to
adopt secondary structure motifs as part of the bioactive conformation (Hruby, 2002).
Therefore, conformational constraints or organic scaffolds that induce or mimic
secondary structures can give valuable information when searching for the bioactive
conformation and may provide a valuable starting point for the development of
peptidomimetic drugs.
3.3.1.1.3.1 Secondary Structure Mimetics

The main secondary structures found in proteins and peptides are the α-helix, β-sheet
and turns. For peptide ligands, secondary structures correspond to local rigidified
structure motifs with a specific arrangement of the residue side chains, which could
provide important recognition elements during receptor binding and activation
(Rose, 1985). Thus, mimicking such structural elements of the peptide with organic
scaffolds is a rational approach in the development of peptidomimetic compounds.
In addition, such an approach may also give compounds with increased metabolic
stability and higher receptor specificity, and provide a rational basis for exploring
the conformational effect on activity (Hruby, 1982). There are indications that turn
structures are present in several peptide ligands when bound to their receptors, and
this makes it appealing to mimic turns (Rose, 1985). The major types of turns present
in proteins and peptides are the β-turn and the γ-turn, depicted in Fig. 3.3.5, where
turn mimetic examples also can be found.
Venkatachalam first suggested the presence of β-turns in proteins based on
modeling studies in 1968 (Venkatachalam, 1968). The β-turn causes reversal of the
peptide backbone and is defined as a sequence of four amino acid residues in a
non-helical segment with a distance of less than 7 Å between C α ,i and C α,i+3 (Lewis,
1973). The β-turns are often, but not necessarily, stabilized by a COi–NHi+3 hydrogen
bond. The β-turn has been divided into several types, depending on the values of the
backbone torsion angles of residues i+1 and i+2. The most common β-turns found in
proteins are types I, I′, II, II′, and VIII (Table 3.3.1) (Hutchinson, 1994; Richardson,
1981; Venkatachalam, 1968; Wilmot , 1988). In addition to these, other classes such
as III, III′, VIa, VIb and VII have been proposed (Lewis, 1973; Venkatachalam, 1968).
The miscellaneous category IV has been introduced to accommodate β-turns that do
not fit any specific type.
ψi+ 1
Ri+1 O φ Ri+2
i+2
ψi+2 R i+1 ψi+1
φi+1 φi+1
N O O
HN H HN
H R i+2
N R i+3 N N
O H
Ri
O H
NH O Ri O
A B
HO
OH
H
N
O
O H HN
N
HN N N
O O
C D
Figure 3.3.5: A β-turn (A) and a γ-turn (B) and examples of a β-turn mimetic (C) (Rosenström, 2006)
and a γ-turn mimetic (D) (Rosenström, 2005). The backbone torsion angles phi (φ) and psi (ψ) are
indicated for the relevant residues in the peptide turns.
Table 3.3.1 Idealized backbone torsion angles for the most common β-turn types (Venkatachalam,
1968; Wilmot, 1988) and the backbone torsion angles for the classic and inverse γ-turn (Matthews,
1972; Nemethy, 1972).
Residue i+1 Residue i+2
β-Turn Phi (φ) Psi (ψ) Phi (φ) Psi (ψ)

I -60 -30 -90 0
I′ 60 30 90 0
II -60 120 80 0
II′ 60 -120 -80 0
VIII -60 -30 -120 120
γ-Turn (classic) 70 to 85 -60 to -70
γ-Turn (inverse) -70 to -85 60 to 70
The γ-turn is not as common as the β-turn but has been found to be adopted by
peptides as short as tripeptides (Motta, 2005). A γ-turn spans over three amino acid
residues with a hydrogen bond between COi and NHi+2, resulting in the shape of a
seven-membered ring. There are two types of γ-turns: the classic γ-turn with the i+1
residue side chain in an axial geometry and the inverse γ-turn with the side chain in
an equatorial geometry (Table 3.3.1) (Matthews, 1972; Nemethy, 1972). Although the
classic γ-turn was proposed first, it has been shown to be very rare and the inverse
γ-turn is the significantly more common of the two (Milnerwhite, 1988). The classic
γ-turn conformation causes reversal of the backbone direction and is mainly found
in β-hairpin structures, as a tight turn, forming an antiparallel β-sheet structure.
The inverse γ-turn can be found as backbone kinks and rarely causes reversal of the
backbone direction (Milnerwhite, 1988).
The information gained from the investigation of the bioactive conformation
can provide indications regarding the presence of a turn structure in the bioactive
conformation. Attempts to replace this part of the peptide with a turn mimetic would
then be a rational step towards a peptide mimicking compound. However, how does
one know if a particular organic scaffold indeed does mimic a turn, and in that case,
which specific class of turns? One way of characterizing a potential turn mimicking
moiety is to compare the geometry with experimental turn structures found in proteins
(Claerhout, 2012; Rosenström, 2006; Whitby, 2011). The great abundance of protein
3D structures determined by X-ray crystallography in the Protein Data Bank (Berman,
2000) provides a good source of experimental protein secondary structures that can
be used for this purpose. Using this method, both β- and γ-turn mimicking scaffolds
have been characterized, which are exemplified below.
β-Turn Mimicking Scaffold C

Since turn mimicking moieties usually have reduced peptidic character and thus
do not have a straightforward atom-to-atom match to the peptide, the phi and psi
dihedral angles used to classify β-turns can be difficult to use for classifying β-turn
mimetics. Instead, another rational classification approach is to compare the
geometries of the mimicking moiety and the different β-turn types by superimposing
important structural features. The main features in a protein turn are the incoming and
outgoing directions of the protein backbone and the side chains. If the corresponding
directions can be identified in the peptidomimetic moiety, the characterization can
be done by superimposing the relevant atoms to the corresponding protein turn
structures. The quality of the match can be quantified by the root-mean-square atom
pair distance between the two structures. In the case of scaffold C (Fig. 3.3.6) low
energy conformations of the scaffold were superimposed to more than 13,000 β-turn
structures collected from protein 3D structures in PDB. Out of the well-defined β-turn
types, scaffold C had best match to type II β-turns (Fig. 3.3.6) and thus may be suited
as a mimetic of this class of β-turns. In angiotensin II (Ang II, Table 3.3.2, entry 1) there
have been indications of a turn structure present in the central part of the peptide and
scaffold C has been used to replace amino acids in the center of Ang II to yield high
affinity Ang II type 1 (AT1) and Ang II type 2 (AT2) receptor ligands (Table 3.3.2, entries
2 and 3).
Figure 3.3.6: Low energy conformation of β-turn mimetic scaffold C (dark grey carbons) superimpo-
sed to a type II β-turn (light grey carbons; PDB ID 1H2C, chain A, sequence Ile142-Pro143-Asp144-His145).
For clarity, only Cβ is shown for the side chains and non-polar hydrogen atoms are omitted.
Table 3.3.2: AT1- and AT2 receptor affinity of Ang II and Ang II mimicking pseudopeptides.
Entry AT1a AT2b Reference

Ki (nM) ± SEM Ki (nM) ± SEM
1 H 2N NH OH 0.24 ± 0.07 0.23 ± 0.03 Rosenström,
NH HN
N
2005
O H O H O H O
H 2N N N N N
N N N OH
HO H O H O H O O
2 HO 1,668 ± 20 4.7 ± 0.3 Rosenström,

O
H
2006
N N
HN
HO O
H O
N O O
H 2N N N H
H N N
O N OH
H
O O
HN
H2 N NH
3 H 2N NH HO 14.9 ± 0.4 1.8 ± 0.04 Rosenström,

HN
H
N
2006
N HN
O H O
O
H 2N N O O
N N N H
H H N N
HO O N OH
H
O O O
Continued
Table 3.3.2: AT1- and AT2 receptor affinity of Ang II and Ang II mimicking pseudopeptides.
Entry AT1a AT2b Reference

Ki (nM) ± SEM Ki (nM) ± SEM
4 H 2N NH
OH
> 10,000 2.8 ± 0.2 Rosenström,
HN 2005
N
O HN
H O
H2 N N HN O
N H O
H N N N
HO O N OH
H
O O O
5 OH > 10,000 0.8 ± 0.1 Rosenström,

O
N 2005
HN
HO H O
H O
N N HN O
H 2N N H O
H N N N
O O N OH
H
O O
NH
H 2N NH
a
Rat liver membranes
b
Pig uterus myometrium
γ-Turn Mimicking Scaffold D

In the case of the γ-turn mimicking scaffold D (Fig. 3.3.7) an idealized γ-turn was first
used to perform geometry comparison with respect to distances and angles in the turn
structure. In scaffold D two amino acid residues corresponding to i+1 and i+2 can be
directly mapped onto a peptide as seen in Fig. 3.3.7. The third atom building up the Cα
atom triangle is part of a benzene ring, but is easily mapped to a peptide backbone via
atom count either from the N-terminal or from residue i+1. As seen in Fig. 3.3.7 the Cα
–Cα distances and angles match very well between scaffold D and an idealized peptide
γ-turn, which indicate that this scaffold is a promising γ-turn mimetic. Scaffold D has
been experimentally evaluated and has successfully been used to replace two or three
amino acids in Ang II, which furnished selective, high affinity pseudopeptides (Table
3.3.2, entries 4 and 5) that act as agonists at the AT2 receptor.
OH
O
HN Cα 3.8 Å 3.8-3.9 Å O
H i+1 92 o H HN
N N N
Cα i+2 44o 43-44 o N
O Cα i O H O
NH 5.5 Å
O NH
O
Figure 3.3.7: Comparison of Cα distances and angles in an ideal γ-turn model peptide (left) and
corresponding atoms in the γ-turn mimicking scaffold D (right).
Although the distances and angles correspond well between an idealized γ-turn and
scaffold D, such an analysis does not include the incoming and outgoing directions of
the peptide backbone or the direction of the side chain of residue i+1. As in the case
of the β-turn mimetic classification, one way of characterizing the γ-turn mimetic is to
compare relevant parts to a determined 3D structure of a γ-turn. In Fig. 3.3.8, an energy-
minimized conformation of scaffold D can be seen superimposed on a γ-turn found in
a protein 3D structure, which shows the good match between the structures.
Figure 3.3.8: Low energy conformation of scaffold D superimposed on an inverse γ-turn (light grey
carbons, PDB ID 1NNF, chain A, sequence Thr82-Ala83-Gln84). For clarity, only Cβ is shown for the side
chains and non-polar hydrogen atoms are omitted.
3.3.2 A Case study of Rational Peptide Lead Optimization:

Development of Small and Constrained Peptides Targeting the
Substance P 1-7 Binding site
Substance P (SP) was the first neuropeptide to be identified (Von Euler, 1931). In the
beginning of the 1970s the peptide sequence, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-
Gly-Leu-Met-NH2, was disclosed (Chang, 1971) and a decade later its belonging to the
tachykinin family was affirmed (Erspamer, 1981). Together with neurokinin A (NKA)
and neurokinin B (NKB), SP is the most well-known member of this family (Harrison,
2001). Three mammalian tachykinin receptors are known today: the neurokinin (NK)
receptors NK1, NK2 and NK3 (Otsuka, 1993). SP is the preferred endogenous ligand for
the NK1 receptor, where it acts as a neurotransmitter and a neuromodulator in both
the central and peripheral nervous system. In the brain, SP and its corresponding
receptor are expressed in areas related to depression (Kramer, 1998), anxiety (De
Araujo, 2001) and stress (Culman, 1995; Ebner, 2008) as well as in areas involved
in motivation and reward (Hasenohrl, 1991; Huston, 1993). In the spinal cord, SP is
expressed in pain processing pathways (Zubrzycka, 2000).
A Case study of Rational Peptide Lead Optimization: Development of Small and Constrained... 257
3.3.2.1 SP1-7 and its Binding Site
Several of the degradation products of SP have been found to be bioactive, and

especially the C-terminal fragments can mimic the effects of the mother peptide. For
example, infusion of the C-terminal metabolite SP5–11 into the spinal cord induces
nociceptive reactions (Skilling, 1990) and when injected into the dorsal periaqueductal
gray matter in rats the C-terminal fragment SP6–11 was shown to produce anxiogenic
effects mediated via the NK1 receptor (De Araujo, 2001). The N-terminal fragment SP1–7
is one of the major metabolites of SP. In contrast to the C-terminal metabolites of SP, SP1–7
has been shown to oppose several effects of SP, e.g. the nociceptive effect (Sakurada,
2004), the inflammatory effect (Wiktelius, 2006) and the potentiating effect on opioid
withdrawal symptoms (Kreeger, 1993; Zhou, 2003). Interestingly, these effects were
not found to be mediated through the NK1 receptor and although the actions of this
heptapeptide are well-known, no explicit receptor has yet been identified. However,
the potential existence of a specific receptor for the N-terminal partial sequences of SP
in mouse spinal cord was discussed at the beginning of the 1980s (Piercey, 1982) and
in 1990 Igwe et al. characterized the specific binding site of SP1–7 in mouse brain and
spinal cord (Igwe, 1990). Binding was found to be specific, saturable and reversible,
which strongly supported the existence of an N-terminal-directed SP receptor. In
2006, Nyberg and coworkers demonstrated the presence of specific binding sites for
SP1–7 in rat spinal cord in accordance with previously reported results from the mouse
spinal cord and brain (Botros, 2006; Igwe, 1990). A binding assay was developed using
spinal cord tissue homogenate and measurements of the binding affinity for various
compounds by displacement of tritiated SP1–7 ([3H]-SP1–7), which was a prerequisite for
the medicinal chemistry program which will be described below.
Initially, ligands for opioid and neurokinin receptors, as well as various N-terminal
SP fragments, were screened against the SP1–7 binding site. In short, none of these
ligands did bind efficiently to this binding site. However, interestingly, the high-
affinity µ-opioid receptor agonists endomorphin-2 (EM-2) and endomorphin-1 (EM-1)
were shown to interact differentially with the binding site of SP1–7. Thus, EM-2 had only
a 10-fold lower affinity than SP1–7, whereas EM-1 had a 1,400-fold lower affinity. To rule
out involvement of the µ-opioid receptor, the binding affinity of SP1–7 to the µ-opioid
receptor and the ability of the heptapeptide to activate it were further investigated
(Botros, 2006). However, no specific binding or any activation of the µ-opioid receptor
was observed, which is indicative of a specific target protein for SP1–7, identical to
neither the tachykinin receptors nor the µ-opioid receptor.
3.3.2.2 SAR and Truncation Studies of SP1–7 and EM-2
3.3.2.2.1 Strategy
A SAR study of the two peptide leads, SP1–7 and EM-2, was initiated, with the intention
of identifying the pharmacophoric groups. This design strategy included Ala scans,
truncations and C- and N-terminal modifications of the two target peptides (Fig.
3.3.9). Thus, a series of peptide analogs, in which each amino acid residue of the two
target peptides was replaced sequentially with an alanine, was synthesized. In the
truncation studies, one amino acid at a time was removed from the N-terminal. Both
C-terminal carboxylic acids and carboxamides were included.
Alanine substitution of each amino acid residue

H 2N NH
NH NH 2
N-terminal acylation
NH 2
O
N
H2 N O OH
O N H
N N N
O H H
O O
N
O H O
N-terminal truncations Substitution with a primary amide
O
H 2N
Alanine substitution of
each amino acid residue
HO
Substitution with a carboxylic acid
N O
H2 N H
O N NH 2
N
O H
O
N-terminal truncations
Figure 3.3.9: Illustration of the modifications of A) SP1–7 and B) EM-2, used in the SAR study.
3.3.2.2.2 Structure–activity Relationship

The two lead peptides, SP1–7 (1) and EM-2 (2), the Ala-substituted peptides 3–13, the
N- and C-terminally modified analogs 14–16 and 28–29, the truncated analogs 17–
27, and the (d) and (l) variants 30–32 were prepared and biochemically evaluated
(Tables 3.3.3 and 3.3.4).
Table 3.3.3: Ki values of SP1–7 and EM-2 analogs for inhibition of [3H]-SP1–7 binding to rat spinal cord
membrane.
H 2N NH
NH NH 2 HO
NH 2
O
N N O
H2 N O OH H2 N H
O N H O N NH 2
N N N N
O H O H
O H O O
SP1-7 N EM-2
O H O
O
H 2N
Compound Sequence Ki ± SEM (nM)
1 (SP1–7) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH 1.6 ± 0.1
2 (EM-2) H-Tyr-Pro-Phe-Phe-NH2 8.7 ± 0.1
Alanine-substituted SP1–7
3 H-Ala-Pro-Lys-Pro-Gln-Gln-Phe-OH 12.3 ± 0.4
4 H-Arg-Ala-Lys-Pro-Gln-Gln-Phe-OH 1.7 ± 0.1
5 H-Arg-Pro-Ala-Pro-Gln-Gln-Phe-OH 2.8 ± 0.1
6 H-Arg-Pro-Lys-Ala-Gln-Gln-Phe-OH 2.8 ± 0.1
7 H-Arg-Pro-Lys-Pro-Ala-Gln-Phe-OH 78.6 ± 5.1
8 H-Arg-Pro-Lys-Pro-Gln-Ala-Phe-OH 365 ± 3
9 H-Arg-Pro-Lys-Pro-Gln-Gln-Ala-OH > 10 000
Alanine-substituted EM-2
10 H-Ala-Pro-Phe-Phe-NH2 11.5 ± 0.1
11 H-Tyr-Ala-Phe-Phe-NH2 10.2 ± 0.3
12 H-Tyr-Pro-Ala-Phe-NH2 9.4 ± 0.1
13 H-Tyr-Pro-Phe-Ala-NH2 1460 ± 15
Terminally Modified SP1–7 and EM-2
14 Ac-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH 7.1 ± 0.0
15 H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-NH2 0.3 ± 0.0
16 H-Tyr-Pro-Phe-Phe-OH 30.2 ± 1.7

Table 3.3.3: Ki values of SP1–7 and EM-2 analogs for inhibition of [3H]-SP1–7 binding to rat spinal
Continued
cord membrane.
Compound Sequence Ki ± SEM (nM)
Truncated SP1–7 Peptides
17 H-Pro-Lys-Pro-Gln-Gln-Phe-OH 29.6 ± 0.8
18 H-Pro-Lys-Pro-Gln-Gln-Phe-NH2 2.8 ± 0.25
19 H-Lys-Pro-Gln-Gln-Phe-OH 30.9 ± 0.4
20 H-Lys-Pro-Gln-Gln-Phe-NH2 4.4 ± 0.1
21 H-Pro-Gln-Gln-Phe-OH 26.2 ± 0.7
22 H-Pro-Gln-Gln-Phe-NH2 4.5 ± 0.3
23 H-Gln-Gln-Phe-OH 20.4 ± 0.8
24 H-Gln-Gln-Phe-NH2 1.9 ± 0.05
Truncated EM-2 Peptides
25 H-Pro-Phe-Phe-NH2 10.9 ± 0.7
26 H-Phe-Phe-NH2 1.5 ± 0.1
27 H-Phe-NH2 5028 ± 31
The results were remarkably consistent for SP1–7 and EM-2 peptides (Table 3.3.3).
Substitution with alanine in the N-terminal part of SP1–7 was well tolerated without
affecting the binding affinity significantly (cf. 1 with 4, 5 and 6), although replacement
of the basic amino acid arginine rendered a 10-fold lower affinity (cf. 1 and 3). Likewise,
the three N-terminal amino acid residues in EM-2 (tyrosine, proline and the internal
phenylalanine) could be substituted with an alanine and still retain binding affinity
(cf. 2 with 10, 11 and 12). However, removal of the two primary amide functions of
the side chains of the glutamine in the C-terminal of SP1–7 resulted in a considerable
decrease in affinity (7 and 8). The C-terminal phenylalanine was absolutely crucial for
strong affinity in both SP1–7 and EM-2. Replacement of the C-terminal phenylalanine in
SP1–7 gave analog 9, which was devoid of affinity. Making the same substitution in EM-2
resulted in compound 13 with a Ki value of 1460 nM. The finding that the C-terminal
phenylalanine plays such an important role in binding affinity is in line with the
peptide scan discussed above, where the C-terminal fragment SP1–6 possessed very
weak binding affinity to the SP1–7 binding site (Botros, 2006; Igwe, 1990). Moreover, the
potency of SP1–7 showed a five-fold increase upon amidation of the terminal carboxyl
group (15). Similarly, the binding affinity of EM-2 was reduced by a factor of four upon
removal of the amide function (16). Since SP1–7 is a proteolytic product of SP resulting
in a C-terminal carboxylic acid, it was surprising that the amidated analog 15 led to
improved binding affinity.
As deduced from the two Ala scans, the N-terminal parts of SP1–7 and EM-2 does
not seem to be engaged in molecular recognition or binding to the target protein, a fact
that prompted synthesis of the truncated analogs 17–24 and 25–27. For SP1–7, removal
of the N-terminal arginine rendered the hexapeptide 17 with a 20-fold reduction
in affinity compared to SP1–7 and a little more than 2-times lower affinity than the
alanine derivative 3. The affinity could, however, be recovered by amidation of 17,
resulting in peptide 18, which was 10 times more potent. Further truncation down to
the tripeptide level was possible without loss of affinity, and C-terminal amidation of
all the truncated SP1–7 analogs improved the affinity 5–10-fold. Hence, the tripeptide
H-Gln-Gln-Phe-NH2 (24) exhibited a Ki of 1.9 nM.
For EM-2, simultaneous removal of the two N-terminal amino acids tyrosine and
proline improved the binding affinity six times, resulting in the notable discovery of
the dipeptide H-Phe-Phe-NH2 (26) with a Ki value similar to that of SP1–7 itself. It should
be emphasized that the Tyr-Pro sequence is the critical fragment for binding to the
µ-receptor, whereas the two C-terminal phenylalanines are not, facts that highlight
the double nature of EM-2 (Fichna, 2007; Kruszynski, 2005; Okada, 2003). In the
Ala scan of EM-2, substitution of the internal phenylalanine was accepted (12), but
truncation down to a single phenylalanine resulted in 27 with no binding affinity (Ki
of 5028 nM).
The binding features of the new dipeptide lead compound H-Phe-Phe-NH2 were
further explored via the synthesis of peptides 28–32 (Table 3.3.4). As observed for SP1–7
and EM-2, the C-terminal function should be a primary amide. The corresponding
carboxylic acid was devoid of activity (cf. 26 with 28). All four stereoisomers of H-Phe-
Phe-NH2 (26, 30, 31 and 32) were synthesized and evaluated. The natural l-Phe-l-Phe
isomer (26) was found to be preferred, followed by the d,d compound (31), although
with a 40-fold lower affinity. As mentioned above, incorporation of d-amino acids into
neuropeptides can change their biological function from an agonist to an antagonist,
e.g. d-SP1–7, which makes it interesting to evaluate the analogs 30, 31 and 32 in animal
studies concerning their functional activities.
Due to the smaller size of 26 in comparison to the heptapeptide SP1–7, lower
selectivity can be expected. Moreover, 26 resembles ligands for the NK3 receptor
(Boden, 1995; 1994). Hence, the possible binding affinity of 26 to the human neurokinin
receptors NK1 and NK3 was studied. Binding was evaluated in agonist radioligand
binding assays relying on the displacement of [Sar9, Met(O2)11]-SP from NK-1 receptors,
and [MePhe7]-NKB from NK-3 receptors (Anthes, 2002; Heuillet, 1993), 26 was tested at
a concentration of 10 µM, but showed no affinity for any of the receptors.
Table 3.3.4: Ki values of Phe-Phe analogs for inhibition of [3H]-SP1–7 binding to rat spinal cord
membrane.
Compound Sequencea Ki ± SEM (nM)

26 1.5 ± 0.1
O
H
N (S)
H2 N (S) NH 2
O
Terminally Modified Phe-Phe Peptides

28 > 10 000
O
H
N (S)
H2 N (S) OH
O
29 18.5 ± 1.7
O O
H
N (S)
N (S) NH 2
H
O
Phe-Phe Analogs
30 540 ± 20
O
H
N (R)
H2 N (S) NH 2
O
31 64 ± 2
O
H
N (R)
H2 N (R) NH 2
O
32 175 ± 13
O
H
N (S)
H2 N (R) NH 2
O
a
S configuration = l configuration and R configuration = d configuration.
3.3.2.2.3 Effects of SP1–7 and its Analogs

As mentioned in the introduction, SP1–7 has been shown to influence opioid withdrawal
symptoms and possess antinociceptive properties. Consequently, the synthesized
compounds 15 and 26 were evaluated in different in vivo models. The amidated
C-terminal analog SP1–7-NH2 (15) was demonstrated to attenuate the expression of
naloxone-precipitated withdrawal in morphine-dependent rats when administered
intracerebroventricularly (Zhou, 2009; Zhou, 2003). In agreement with the binding
affinities obtained in the SAR study, the C-terminal amide analog is more efficient in
reducing opioid withdrawal symptoms than SP1–7.
SP1–7-NH2 (15) and also 26 have been further tested regarding their potential
antinociceptive effect in both non-diabetic and diabetic mice after intrathecal
administration (Fig. 3.3.10) (Carlsson, 2010; Fransson, 2010b; Nyberg, 2010;
Ohsawa, 2010; 2011). The use of diabetic mice for evaluation is due to their reduced
pain threshold compared to non-diabetic mice, a reduction thought to arise from
hyperalgesia caused by neuropathy, which makes them a good model for studying
neuropathic pain. Interestingly, morphine was unable to induce any antinociceptive
effect in the diabetic mice, whereas SP1–7 showed a dose-dependent antinociceptive
effect in both diabetic and non-diabetic mice (Carlsson, 2010). The effect was higher
in diabetic mice, which suggests that the compound is more effective on neuropathic
pain and that SP1–7 ameliorates signs of hyperalgesia (Fig. 3.3.10). In agreement with the
results obtained from the opioid withdrawal test, SP1–7-NH2 proved to be more efficient
in reducing pain as compared to the native heptapeptide. It was also demonstrated
that the dipeptide 26, which possessed the same binding affinity as SP1–7 (1), showed
greater antinociceptive potency in diabetic mice than SP1–7 (Fransson, 2010b; Nyberg,
2010; Ohsawa, 2010).
Non-diabetic mice Diabetic mice

SP1-7 –NH2
H-Phe-Phe-NH2
SP1-7
Figure 3.3.10: The antinociceptive effect of SP1–7 (1), SP1–7-NH2 (15) and H-Phe-Phe-NH2 (26) in
non-diabetic (left) and diabetic (right) mice. The antinociceptive effect was evaluated by the AUC
calculated from the time-response curve of tail-flick latency. Each column represents the mean with
S.E.M. (n = 6).
3.3.2.3 Design and Synthesis of Small Constrained H-Phe-Phe-NH2 Analogs
3.3.2.3.1 Strategy
The potent dipeptide lead H-Phe-Phe-NH2 (26), discussed above, was chosen for
further optimization studies with the overall aim of developing metabolically stable
and selective SP1–7 analogs. The introduction of local constraints can enhance stability,
selectivity and bioavailability. The intestinal permeability is an important factor in the
development of orally bioavailable drugs. In the intestine, the di/tri-peptide transporter
PepT1 enables the absorption of small peptides from the digestion of dietary proteins.
This transport system has also been shown to transport a variety of peptidomimetic
drugs, such as β-lactam antibiotics and ACE inhibitors and might be exploited in
order to increase the absorption of our small compounds (Brandsch, 2009; Brodin,
2002; Rubio-Aliaga, 2002). A known problem with peptides is their susceptibility to
efflux. For peptides targeting functions in the CNS, uptake in the brain, i.e. crossing
the BBB, is a crucial factor. As a defense mechanism preventing harmful substances
from entering the brain, the BBB is equipped with efflux transporters (Witt, 2001). PgP
is one of the most important, and can actively transport substances out of the brain
(Giacomini, 2010). Such transporters can be an obstacle to entering the CNS.
A series of H-Phe-Phe-NH2 analogs (33–43, Tables 3.3.5 and 3.3.6) incorporating
different types of constraints were designed, synthesized and evaluated regarding
their binding affinity, stability, uptake and permeability (Fig. 3.3.11). N-methyl
and α-methyl amino acids were incorporated, substituting one residue at a time.
Furthermore, β-methylation of the phenylalanine side chain was used to reduce the
conformational flexibility, which can be advantageous upon binding. This approach
has been successful in other projects in obtaining neuropeptide analogs resistant to
metabolism while still retaining their biological activity (Veber, 1985). Both N- and
C-terminal rigidifications were accomplished by the introduction of a 3-phenylproline
derivative (Sewald, 2002).
Figure 3.3.11: Overview of different modification strategies.

3.3.2.3.2 Structure–activity relationship and ADME properties

The binding affinities of the dipeptides were evaluated as described previously.
The dipeptide lead H-Phe-Phe-NH2 was re-tested with the new peptides for a more
accurate comparison of the Ki values. The metabolic stability was evaluated by
incubating the peptides with pooled human liver microsomes. In vitro half-life (t1/2)
and in vitro intrinsic clearance (Clint) were calculated using previously reported
models (Houston, 1994; Obach, 1999). The Ki values and metabolic stabilities (t1/2 and
Clint) of the methylated analogs 33–37 are presented in Table 3.3.5, while the results of
the rigidified and C-terminal-phenylalanine-modified analogs 38–43 are presented in
Table 3.3.6.
Table 3.3.5: Binding affinity (Ki values) and metabolic stability (Clint and t1/2) of the methylated H-Phe-
Phe-NH2 analogs
Compound Structure Binding affinity Clearancec Half-lifec

Ki ± SEM (nM) Clint (µL min mg ) t1/2e (min)
d -1 -1
26 8.4 ± 0.4a 121 ± 39 12 ± 4

H
N (S)
O
(1.5 ± 0.1)b
H2N (S) NH 2
O
33 189 ± 3 2.7 ± 1.5 597 ± 40

O
H
N (S)
N (S) NH 2
H
O
34 70 ± 3 64 ± 11 22 ± 4
O
H
N (S)
H 2N (S) NH 2
O
35 9.4 ± 0.1 92 ± 0 15 ± 1
O
N (S)
H 2N (S) NH 2
O
36 26 ± 1 38 ± 15 40 ± 16
O
H
N (S)
H 2N (S) NH 2
O
37 136 ± 2 175 ± 5 7.9 ± 0.2

O
H
N (S)
H 2N (S) N
H
O
a
Ki value determined on the same occasion as for 33–46. b Previously determined Ki value c The meta-
bolic stability data are expressed as mean ± SD.
d
Clint = in vitro intrinsic clearance. e t1/2 = in vitro half-life.
In the methylated series, only compound 35 with internal N-methylation, retained

binding affinity comparable to that of H-Phe-Phe-NH2 (26); unfortunately, no significant
improvement in the metabolic stability was achieved. Methylation of the N-terminal
resulted in 22-fold lower affinity, but was found to have a pronounced impact on the
stability, which increased the half-life 50 times (cf. 26 and 33). C-terminal methylation
(37) was accompanied by a roughly 20-fold decrease in binding affinity, but without
improvement of the stability. A potential reason for the reduced binding affinity of
37 is that the secondary amide loses a hydrogen bond donor feature compared to 26,
which may be important for the affinity.
Incorporation of an α-methyl amino acid (34 and 36) also reduced the binding
affinity, but less than for the N-methyl amino acids in the terminal parts of H-Phe-
Phe-NH2. A 2- to 3-fold increase in the half-life was thus observed for the α-carbon
methylated analogs.
When the cis 3-phenylproline derivative was incorporated into the N-terminal part
of H-Phe-Phe-NH2 replacing phenylalanine (38 and 39), the binding affinity decreased
4 and 6 times, respectively. A possible reason for this loss in affinity is that these
compounds may have problems adopting an optimal binding conformation because
of the introduced rigidification provided by the proline analogue. Replacement of the
C-terminal phenylalanine by the cis 3-phenylproline moiety in the S,S,S configuration
(40) resulted in a more potent ligand than H-Phe-Phe-NH2. This rigidification also
increased the half-life by 7-fold (cf. 26 and 40).
β-Methylation of the C-terminal phenylalanine gave compounds 42 and 43, with
lower binding affinity than H-Phe-Phe-NH2. When comparing the metabolic stability
in all the diastereomeric pairs (38 vs. 39, 40 vs. 41, and 42 vs. 43), the natural S,S,S
configuration was more easily metabolized. Incorporation of d-amino acids is a known
strategy to improve metabolic stabilization (Humphrey, 1986; Veber, 1985).
The physiochemical properties of all the synthesized compounds were further
explored by evaluation of their intestinal epithelial permeability. This was determined
from transport rates across a Caco-2 cell monolayer, and is expressed as the apparent
permeability coefficient (Papp) (Hubatsch, 2007). A good relationship between the
permeability across the Caco-2 monolayer and the extent of absorption in vivo has been
reported (Stewart, 1995). Each compound was investigated in the apical to basolateral
(a–b) and basolateral to apical (b–a) direction. One of several efflux transporters
present in the Caco-2 cells is PgP. Measurement of the efflux (the ba/ab ratio) can thus
indicate whether or not the compounds are substrates for PgP.
Since uptake transporters can enhance absorption, the possibility of the peptides
being actively transported was studied in Chinese hamster ovary (CHO) cells stably
transfected with the PepT1 transporter (CHO)-PepT1, using CHO-K1 cells as the control.
The results are expressed as pmol mg-1 of protein/min. For peptides being actively
transported the PeptT1/K1 ratio should be greater than one. Uptake and permeability
are reported in Tables 3.3.7 and 3.3.8.
Table 3.3.6: Binding affinity (Ki values) and metabolic stability (Clint and t1/2) of the rigidified and
C-terminal-modified H-Phe-Phe-NH2 analogs
Compound Structure Binding affinity Clearancec Half-lifec

Ki ± SEM (nM) Clint (µL min mg ) t1/2e (min)
d -1 -1
38a 34 ± 3 28 ± 3 50 ± 6
(S) O
(S)
H
N (S)
N NH 2
H
O
39a 51 ± 2 16 ± 9 103 ± 6
(R)
O
(R) H
N (S)
N NH 2
H
O
40a 2.4 ± 0.6b 16 ± 3 88 ± 15

(S)
N
H 2N (S) (S)
O NH 2
O
41a 93 ± 0 7.6 ± 1.9 187 ± 5

(R)
N
H 2N (S) (R)
O NH 2
O
42a 18 ± 1 39 ± 4 36 ± 3
O
H 2N (S)
HN (S)
(S)
O NH 2
43 a
68 ± 1 16 ± 7 98 ± 4
O
H2N (S)
HN (R)
(R)
O NH 2
a
The stereochemistry of each diastereomer pair was estimated from the pharmacophore model. b
IC50 value. Analog 40 was tested at a 2:1 ratio with analog 41. It was tested once, at six different con-
centrations, in triplicate. c The metabolic stability data are expressed as mean ± SD. d Clint = in vitro
intrinsic clearance. e t1/2 = in vitro half-life.
Table 3.3.7: Active uptake and permeability data for the methylated H-Phe-Phe-NH2 analogs.
Compd. Uptakea Caco-2 permeabilitya

(pmol mg-1 protein min-1) Pappb (10-6 cm s-1)
Structure CHO-PepT1 CHO-K1 Ratio a–bc b–ad Ratio Ratio
PepT1/ ab/ba ba/ab
K1
26 0.3 ± 0.0 0.3 ± 0.0 1.0 0.02 ± 0.00 0.2 ± 0.0 0.1 11
O
H
N (S)
H2N (S) NH 2
O
33 2.2 ± 0.7 2.4 ± 0.5 0.9 14 ± 1 124 ± 21 0.1 9

O
H
N (S)
N (S) NH 2
H
O
34 1.1 ± 0.2 1.5 ± 0.2 0.7 18 ± 0 87 ± 0 0.2 5

O
H
N (S)
H 2N (S) NH 2
O
35 19 ± 0 23 ± 3 0.8 9.0 ± 2.2 124 ± 19 0.1 14

O
N (S)
H 2N (S) NH 2
O
36 33 ± 2 32 ± 9 1.0 20 ± 2 224 ± 5 0.1 11

O
H
N (S)
H 2N (S) NH 2
O
37 0.3 ± 0.1 0.4 ± 0.1 0.7 0.3 ± 0.1 0.9 ± 0.2 0.3 3
O
H
N (S)
H 2N (S) N
H
O
a
The results are expressed as mean ± SD. b Papp = apparent permeability coefficient. c a–b = apical to
basolateral. d b–a = basolateral to apical.
Conclusion 269
A Papp value below 0.2 × 10-6 cm s-1 indicates low permeability, a Papp value ranging from
0.2 × 10-6 cm s-1 to 1.6 × 10-6 cm s-1 indicates moderate permeability and a Papp value above
1.6 × 10-6 cm s-1 indicates high permeability, in this particular setting (Bergström, 2003).
The permeability in the a–b direction increased substantially for all the methylated
analogs (ranging from 0.3 × 10-6 cm s-1 to 20 × 10-6 cm s-1) compared to H-Phe-Phe-NH2
(26, 0.02 × 10-6 cm s-1). All the compounds, except 37, were classified as having high
permeability, with the highest permeability observed for the α-methylated analogs
34 and 36, 18 × 10-6 cm s-1 and 20 × 10-6 cm s-1, respectively. However, the methylated
compounds were not actively transported, as can be seen from the PepT1/K1 ratio.
Furthermore, the peptides also displayed efflux (the ba/ab ratio ranging from 3 to 14),
where compounds 35 and 36 showed the highest tendency towards efflux and were
equal to 26.
Compared to the methylated analogs, the rigidified and the C-terminal-
phenylalanine-modified analogs possessed much lower permeability (ranging from
0.02 × 10-6 cm s-1 to 4.4 × 10-6 cm s-1). Satisfyingly, the high affinity analog 40 turned out
to have high permeability (3.6 × 10-6 cm s-1). In this series, the efflux was much higher;
8−95 times, in the b–a direction than in the a–b direction. The stereochemistry of
the compounds also seemed to influence the predisposition for efflux. Thus, the
compounds 38, 40 and 42 all showed lower efflux ratios than their diastereomeric
counterparts 39, 41 and 43. The replacement of the phenylalanine in the N- or
C-terminal by the 3-phenylproline moiety seemed advantageous in improving the
active uptake of these compounds, since a slight increase in the PepT1/K1 ratio was
observed (cf. 26 vs. 38, 39, 40 and 41).
3.3.3 Conclusion
The case study presented herein is an illustrative example of rational design of drug-like
molecules mimicking the actions of a bioactive peptide. It highlights the importance
working in an iterative manner, where structural modifications of compounds are
evaluated both from a pharmacodynamic and pharmacokinetic perspective.
The optimization process, starting with the bioactive heptapeptide SP1-7 and the
tetrapeptide EM-2, resulted in the remarkable discovery of the dipeptide H-Phe-Phe-
NH2, which was equipotent to endogenous SP1-7 and had a higher binding affinity
than EM-2. However, in the ADME assessment, the dipeptide showed poor metabolic
stability and permeability, and suffered from high efflux rates. By introducing local
constraints in the C-terminal of H-Phe-Phe-NH2, via the introduction of the cis
3-phenyl-pyrrolidine moiety, the pharmacokinetic properties could be substantially
improved and the binding affinity retained.
It should be emphasized that although numerous strategies and attempts for
transforming a bioactive peptide into small peptidomimetics have been reported, it
is not straightforward, it is case dependent, and there is no guarantee of success.
The decreasing number of approved drugs during recent years has put enormous
pressure on the pharmaceutical industry, resulting in a revival of interest in peptides
as potential drug candidates (Vlieghe, 2010). By using synthetic strategies to limit
metabolism and exploring alternative routes of administration, a number of peptidic
drugs have been brought to the market (Vlieghe, 2010), showing that it is not necessary
to remove the peptide character completely, and that small pseudopeptides can be
useful as drugs.
Table 3.3.8: Active uptake and permeability data for the rigidified and C-terminal-modified H-Phe-
Phe-NH2 analogs.
Compd. Uptakea Caco-2 permeabilitya

(pmol mg-1 / protein min-1) Pappb (10-6 cm s-1)
Structure CHO-PepT1 CHO-K1 Ratio a–bc b–ad Ratio Ratio
PepT1/ ab/ba ba/ab
K1
38 31 ± 4 22 ± 2 1.4 0.5 ± 0.0 26 ± 1 0.0 53
(S) O
(S)
H
N (S)
N NH 2
H
O
39 15 ± 2 11 ± 1 1.4 0.8 ± 0.1 76 ± 1 0.0 95

(R)
O
(R) H
N (S)
N NH 2
H
O
40 13 ± 2 11 ± 1 1.2 3.6 ± 0.4 75 ± 2 0.1 21

(S)
N
H 2N (S) (S)
O NH 2
O
41 11 ± 1 8.7 ± 0.8 1.3 0.6 ± 0.0 51 ± 2 0.0 86

(R)
N
H 2N (S) (R)
O NH 2
O
42 0.6 ± 0.1 0.7 ± 0.0 0.9 0.7 ± 0.0 5.4 ± 0.1 8

0.4
O
H 2N (S)
HN (S)
(S)
O NH 2
43 25 ± 4 22 ± 3 1.1 4.4 ± 0.1 171 ± 6 0.0 39
O
H2N (S)
HN (R)
(R)
O NH 2
a
The results are expressed as mean ± SD. b Papp = apparent permeability coefficient. c a–b = apical to
basolateral. d b–a = basolateral to apical.
References 271
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Brandon S. Pybus*10
3.4 Synthetic Vs. Natural Bioactive Compounds
Against Tropical Disease
3.4.1 Introduction
Tropical diseases, although largely neglected by the commercial pharmaceutical

industry, are a major problem for people in poor and underdeveloped nations.
Perhaps the most significant of these threats comes from malaria. Roughly half of
the world’s population live in malaria endemic regions, and the WHO estimates that
it caused 627,000 deaths in 2012 (CDC, August 18, 2014). Nearly 500 million cases of
malaria are confirmed worldwide annually, with roughly 80% of those caused by
Plasmodium vivax, a persistent strain of the parasite (Goncalves, 2014). Due to the
constant emergence of resistance, there is an ongoing need for the development of new
drugs for malaria (Rosenthal, 2003). For this reason, and in the interest of outlining
a generic drug development paradigm in limited space, we will focus the topic of the
current discussion on the historical successes and failures of natural products in
treating malaria and the efforts in the modern era to combat drug resistance and poor
drug tolerance with synthetic drugs.
3.4.2 Early History of Malaria Treatment; Quinine and Artemisinin
“The angel of disease and death, ascending from his oozy bed, along the marshy
margins of the bottom grounds […] floats in his aerial chariot, and in seasons favorable
to his progress, spreads mortal desolation as he flies”, so it was written in an Ohio
newspaper article from 1820 (Findley, 1968). From this quote one can see how parasitic
disease plagued the early frontiersmen of the 19th century. However, the unhappy
coexistence of humans and parasitic disease goes back much further into the past.
In fact, the earliest recorded descriptions of a disease with symptoms consistent
with those of malaria date back to 2700 BC from imperial China. During this period
traditional Chinese medical practitioners discovered the use of sweet wormwood
(Artemesia annua) to treat this mysterious fever. Meanwhile, on the other side of the
planet Quechuas from South America used the bark of the cinchona tree in the treatment
of similar fevers. Jesuit monks brought this bark from the new world and introduced
Brandon S. Pybus: Division of Experimental Therapeutics, Military Malaria Research Program, Walter
Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910, USA,
*Email: brandon.s.pybus.mil@mail.mil
© 2015 Brandon S. Pybus

276 Synthetic Vs. Natural Bioactive Compounds Against Tropical Disease
its powerful medicinal properties to Europe during the 17th century, however, the active
component, quinine, was not isolated until 1820 (Faurant, 2011). Quinine is known
for several unpleasant and potentially severe adverse reactions including cinchonism,
hearing impairment, increased risk of hemolysis in G6PD deficient patients, and is
linked to a severe syndrome in some patients, dubbed blackwater fever. Modern efforts
suggest the latter may be the result of redox active metabolites (Marcsisin, 2013). As we
will explore, many of the ongoing efforts in anti-malarial therapy revolve around not
only circumventing resistance, but in mitigating many of the safety risks associated
with historically used drugs. These reasons and the outbreak of war in malaria endemic
regions of the world led to a surge of interest in the development of safe and effective
drugs to treat malaria that we will explore further.
3.4.3 Post World War II and the Development of Synthetic Anti-

malarials
“Doctor […] this shall be a long war if for every division I have facing the enemy I
must count on a second division in the hospital with malaria and a third division
convalescing from this debilitating disease!” Thus opined Gen. Douglas MacArthur
concerning the ravages of malaria during the second world war (Coates, 1963). This
constant struggle with non-combat related injury from disease was a tremendous
burden on medical logistical chains, morale, and overall readiness on fronts across
Africa, Asia, and even in parts of Europe. This reality was a major shaping force for a
massive effort to prevent and treat malaria.
During WWI, Germany had no access to quinine. As a result, a large-scale effort
ensued in the intervening years between WWI and WWII in which the Germans
synthesized and screened thousands of compounds for anti-malarial activity.
Among these were several 8-aminoquinolines, atabrine (quinacrine), and a drug
they discarded for toxicity called Resochin (chloroquine) (Coates, 1963; Vale, 2009).
After access to quinine from Indonesia was cut to Allied forces by the entry of Japan
into WWII, US and British chemists began a similar push resulting in the adoption of
atabrine as the drug of choice for malaria treatment and prophylaxis during the later
years of the war. Post-war, powerful new anti-malarial drugs began to emerge as the
culmination of follow-on efforts by US and British chemists to exploit the original
efforts by Germany. Many of these drugs are still in use today, with robust analog
efforts being employed around them to overcome developing resistance.
3.4.4 Modern Efforts in Antimalarial Drug Development
Modern efforts in anti-malarial drug discovery in general follow six separate

approaches including optimization of therapy with existing agents, development
Modern Efforts in Antimalarial Drug Development 277
of analogs of existing agents, natural products, repurposing of drugs from other

therapeutic areas, reversal of resistance, and discovery of compounds active against
novel targets (Rosenthal, 2003). Table 3.4.1 illustrates examples from current literature
from each approach. It is interesting to note that many of these approaches revolve
around existing agents and their analogs, many of which were known since the 1920s,
30s, or 40s. For the purposes of this discussion, we will focus on ongoing work in
existing classes and development of novel classes of anti-malarials, as discussions of
combinations of existing drugs can become lengthy and are better suited in a clinical
pharmacology text.
3.4.4.1 Quinine, 4-Aminoquinolines, and Quinoline Methanols
For natural products, quinine is a success story that has endured for centuries.
Quinine is an aryl amino alchohol derived from the bark of the cinchona tree (Fig.
3.4.1). Despite its discovery over 400 years ago, it remains an enormously important
drug in the treatment of malaria in the developing world. Quinine is rapidly absorbed,
both orally and parenterally, broadly distributed throughout the body, and has proven
highly effective for the treatment of uncomplicated or severe malaria (Achan, 2011).
In the age of resistance, quinine has made a resurgence in usage. Although quinine
resistance has been reported, it is generally low-grade, and is largely found in Asia
and South America (Noedl, 2006; Parola, 2001).
Perhaps a larger issue still in play for quinine is tolerance. Quinine has several
notable and potential severe adverse events associated with usage. Most common side-
effects include tinnitus and hearing impairment, but more severe effects can include
vertigo, vomiting, abdominal pain, or hypotension (Achan, 2011). The most severe
and potentially least understood is a syndrome called blackwater fever characterized
by hemolysis and hemoglobinuria (George, 2009). Recent studies suggest a potential
link between CYP mediated oxidative metabolites of quinine and this potentially fatal
reaction (Marcsisin, 2013). It should also be noted that while clinically different from
the hemolytic events observed with the 8-aminoquinolines (which we will discuss
in a later section), as with the 8-aminoquinolines a link may exist between glucose-
6-phosphate (G6PD) deficiency and hemolysis (Hue, 2009). This is significant as it
suggests common metabolic pathways for some quinoline drugs that should be avoided
or at least considered when developing new drugs in these classes. For quinine, the
formation of redox active quinones may lead to increased oxidative stress under
conditions of high parasitemia or in G6PD deficient individuals that ultimately results
in the destruction of red-cells through mechanisms which are not fully understood
(Fig. 3.4.2) (Marcsisin, 2013).
Arguably one of the most successful classes of drugs to arise from WWII efforts is
the 4-aminoquinolines. Fig. 3.4.3 illustrates two members of the class, chloroquine and
amodiaquine, and can serve as a generic paradigm for its structure. Chloroquine was
originally discovered by Hans Andersag in 1934 while working for Bayer, however, the
drug was discarded for perceived toxicity. After its re-discovery by British and American
chemists, it quickly won favor as a safe and effective anti-malarial (CDC, August 18,
2014). In fact, chloroquine was used extensively in post-war eradication efforts. While
the mechanism of action for all 4-aminoquinolines is not exactly known, they are
known to be effective in treating only erythrocytic stages of infection. Poor compliance
or poor management of care with such drugs can rapidly lead to resistance. This was
exactly the case with chloroquine. By the early 1950’s, chloroquine resistance was
identified along the Thai-Cambodian border and Colombia. Within two decades it had
spread to every malaria endemic region of the world (Farooq & Mahajan, 2004).
While the mechanism of action for the 4-aminoquinolines is not completely
understood, the most widely cited hypothesis is that accumulation in the digestive
vacuole of the parasite interferes with hemoglobin digestion. Resistance is believed
to be conferred by the presence of a chloroquine specific P-glycoprotein pump (Foley
& Tilley, 1998). Several loci in the P. falciparum genome have been implicated in this
resistance (Farooq & Mahajan, 2004). This mechanism, or a variant thereof may well
be important in quinine, mefloquine, or other quinoline resistance mechanisms,
however, it should be noted that more lipophilic quinolines do not concentrate in the
food vacuole to the same extent as chloroquine, and therefore other mechanisms need
to be explored (including potential alternative targets of efficacy) when considering
these drugs.
A considerable effort is still ongoing to circumvent resistance through analog
campaigns or co-administration with other drugs (Hanboonkunupakarn, 2014; Le
Garlantezec, 2014; I. Opsenica, 2011; I. M. Opsenica, 2013; Thriemer, 2014). While
it is tempting to synthesize analogs of a compound with emerging or established
resistance, in the author’s opinion, such efforts should be entered into with full
knowledge that selection pressures in malaria endemic areas quickly erode the
efficacy of new drugs from old classes. It is advisable to pursue a combination
approach wherein fast acting short half-life drugs (e.g. artemesinins) are combined
with long half-life (longer exposure of parasite to drug) quinolines prone to emergence
of resistance. Further, future efforts in this class should pay careful attention to early
signs of cross resistance in chloroquine resistant strains. While small jumps in IC50
in a resistant strain may not raise alarms if those IC50s are still significantly less than
the anticipated maximum concentrations achieved in plasma post-exposure, they
could be indicative of shared mechanisms of resistance that might worsen over time
with large exposures in environments with poorly controlled administration or where
monotherapy is used.
Mefloquine, a quinoline methanol, was developed by the US Army in the 1970s,
and was considered very desirable for both treatment and prophylaxis due to its
long half-life (Croft, 2007). In recent years mefloquine usage has decreased due to
emerging resistance, and more significantly, poor tolerance (Farooq & Mahajan,
2004; Milner, 2010). Mefloquine has been linked to neurological side-effects including
Modern Efforts in Antimalarial Drug Development 279
vertigo, loss of balance, and polyneuropathy, which have recently been labeled as
potentially irreversible by the FDA (Nevin, 2014). Extensive efforts have shown
promise in improving the therapeutic index of mefloquine by opening or removing
the piperadine side-chain (Dow, 2006; 2011; Milner, 2011). With the notoriously poor
predictive power of models of CNS toxicity and the stigma attached to the quinoline
methanol class, these efforts have largely been abandoned. Further, recent trials with
enantiomerically pure mefloquine (currently marketed as a racemic mixture) have
shown little promise in improving tolerability (Nevin, 2014).
3.4.4.2 8-Aminoquinolines
Another powerful class of anti-malarial drugs to emerge from war efforts is the
8-aminoquinolines. Although still quinoline based, this class has several key features
which make it unique. One primary feature of this class is its ability to act as a causal
prophylactic, or to prevent the initial infection of parasites in the liver. Further, the
class has powerful anti-hypnozoite activity. Hypnozoites being the dormant liver stage
of P. vivax and P. ovale, this imparts a separate prophylactic use, namely presumptive
anti-relapse therapy or PART. Combined with this unique exoerythrocytic activity
is the gametocytocidal activity, whereby 8-aminoquinolines kill the sexual stages
of the parasite blocking further transmission of infection. This combination of
activities makes this class highly attractive for prophylaxis, treatment of relapsing
strains of malaria, and/or elimination efforts. The class has also shown in vitro anti-
leishmanicidal activity and has clinical utility in the treatment of pneumocystic
pneumonia, and trypanosomiasis (Vale, 2009). However, as with other classes that
we have reviewed here tolerability is a substantial problem for the 8-aminoquinolines.
From this class, currently only primaquine is clinically available for these uses.
Administration of 8-aminoquinolines is known to cause methemoglobinemia
and hemolysis in G6PD deficient individuals. Both of these effects are linked to
oxidative metabolites rather than the parent drug itself (Ganesan, 2009; Ganesan,
2012). Interestingly, these same metabolites were recently shown to play a crucial
role in activity (Bennett, 2013; Marcsisin, 2014; Pybus, 2013). For both primaquine
and tafenoquine (8-aminoquinoline, currently in clinical development), activity is
mediated by CYP 2D6 (Fig. 3.4.2). The mechanism of activity for the 8-aminoquinolines
is not fully understood, however, contemporary thought is that redox active metabolites
produce peroxides and superoxides (oxidative stress), which ultimately interferes
with electron transport in the parasite. Not surprisingly, what is also unknown at
present is whether the toxicity can be mitigated while preserving efficacy. Work in
this area is of paramount importance, as new 8-aminoquinolines with similar profiles
to primaquine are of little clinical utility.
The WHO currently calls for a 30 mg dose of primaquine as a chemoprophylaxis
in areas where P. vivax is the predominant risk, however significant fractions of the
population (10% in Europeans and as much as 50% in some Asians) possess 2D6 alleles
with reduced function (Deye & Magill, 2014). It is therefore likely that primaquine
prophylaxis and even anti-relapse therapy will fail in many of these individuals.
All things considered, the importance of this class cannot be overemphasized and
it cannot be abandoned without more study. As stated previously, primaquine is
currently the only clinically available drug for the treatment of relapsing malaria.
As such, the challenges posed for future 8-aminoquinoline development include
re-routing metabolism through a non-CYP 2D6 pathway, circumventing hemolytic
toxicity, or both. The primaquine enamine bulaquine showed some promise in recent
animal studies at improving the therapeutic index (Lal, 2003; Mehrotra, 2007). This
appears to be due to differential pharmacokinetic exposure patterns as compared
to parent primaquine. The addition of the enamine group on the side-chain creates
an almost time-release effect as it is hydrolyzed to release primaquine. This leads
to an interesting hypothesis that perhaps while the same metabolites may well be
responsible for efficacy and toxicity, efficacy could be exposure driven while toxicity
is concentration dependent, providing an in-road to improvements in therapeutic
index. Although in the author’s opinion, circumstantial evidence overwhelmingly
points to an inextricable link between efficacy and toxicity in this class that may
make it difficult, if not impossible, to support the large-scale development of a new
candidate.
3.4.4.3 Artemisinins and other Endoperoxides
Although sweet wormwood was used for over 2000 years in China to treat malaria, the
active ingredient, artemisinin, was not identified until the 1970’s (Fig. 3.4.5) (Faurant,
2011). At present, several artemisinin compounds are clinically available for use
including artemisinin, artesunate, dihydroartemisinin (DHA), and artemether. Other
experimental drugs exist but are not clinically available. Due to poor tolerability
and hence compliance with quinine, artemisinin compounds have drastically risen
in popularity for the treatment of falciparum malaria (Shanks, 2006). Artemisinin
resistance was first reported in Southeast Asia in 2008, and evidence suggests that
it is spreading (Ashley, 2014; Dondorp, 2009; Thriemer, 2014). Widespread resistance
to the most powerful new weapon in the fight against malaria could de-rail many
ongoing efforts to eradicate the disease in the endemic world. As such, it is widely
acknowledged that combination therapy should be the standard of care. The WHO
currently recommends artemisinin combination therapy (ACT) consisting of DHA-
piperaquine, followed with a 0.75 mg kg-1 single dose of primaquine for the treatment
of uncomplicated malaria (WHO, 2010).
At present, research is still ongoing as to the exact killing mechanism for the
functional endoperoxide bridge contained in the artemisinins, however, it is thought
to be a result of membrane depolarization and subsequent interference with electron
Natural Products in the Treatment of Malaria 281
transport (Antoine, 2014). Further development of synthetic and semi-synthetic

artemisinins is still ongoing as well as the development of novel endoperoxide
containing compounds which have proven effective as anti-malarial agents in pre-
clinical studies (Lanteri, 2014; Oliveira, 2014).
3.4.4.4 Repurposed Drugs
For the treatment of malaria and many other tropical diseases, limitations in funding
have led to discoveries in the area of repurposing of drugs commonly used for
other indications. Broad spectrum antibiotics have proven highly successful in the
treatment of malaria. Doxycycline, a synthetic tetracycline, was shown to have partial
prophylactic efficacy against malaria in the 1970’s (Andrews, 2014). In fact, despite
its own set of tolerance issues, doxycycline is now the prophylactic drug of choice
for the US Army due to more serious concerns with mefloquine (Kime, 2012). It is also
effective for use in treatment when partnered with a fast acting anti-malarial like
quinine or quinidine (Tan & Centers for Disease Control and Prevention, 2011). Other
antibiotics including clindamycin and sulfonamide antibiotics like sulphadiazine and
sulphadoxine have also proven effective in the treatment of malaria (Andrews, 2014).
Many of these target folate synthesis, which is a common pathway for other classical
anti-malarials. Antifolate drugs block the synthesis of tetrahydrofolate, which in
turn shuts down nucleic acid synthesis (Shanks, 2006). Among these is dapsone.
Dapsone, a sulfone, was first used to treat leprosy but was introduced by GSK as
Lapdap (dapsone/chloroproguanil) in the late 1990s. This drug was removed from
the market in 2008 due to hemolytic anemia similar to that of the 8-aminoquinolines.
Exploration of this strategy still continues with significant effort. Several examples can
be found in the literature with protease inhibitors, antibiotics, and quinolones found
to act against various targets in the parasite (Rosenthal, 2003). With pre-existing and
well established safety profiles, re-purposing of old drugs is an attractive strategy if
for no other reason than bypassing several regulatory hurdles and saving money in
development. Another potential benefit for the developing world is often the cost of
these drugs once marketed. Doxycycline, for example, is pennies per dose compared
to Malarone (atovaquone/proguanil) and inexpensive drugs are far more likely in
that regard to make a meaningful impact on the fight against parasitic disease world-
wide.
3.4.5 Natural Products in the Treatment of Malaria
As we have noted many of the progenitive compounds (quinine, artemisinin) for

the treatment of malaria are naturally derived. Efforts continue to isolate natural
compounds with anti-protozoal activity (Mohammed, 2014; Singh, 2014; Traore,
2014). Natural sources including plants, microbes, and animals provide libraries with
rich chemical diversity, however, isolation and purification of active components
presents a significant challenge in natural product development. The complexity of
isolation is highly dependent on structure, which can often be quite complex with
compounds isolated from natural sources. This of course leads to a second challenge
for the development of natural products, namely synthesis. Once a hit is identified
from a mixture, large quantities are often needed for further pre-clinical or clinical
testing. The likelihood of identifying a clinic-ready compound from a natural source
in today’s regulatory environment is small. The more likely scenario involves the
identification of a hit compound, which can be potentially modified to improve drug-
like characteristics or decrease potential liabilities from toxicity. As we will discuss
in the next section, the source of starting material for a drug development project is
largely irrelevant once a hit is identified, as all compounds should be gated through the
same testing scheme to ensure the end product matches the target product profile.
3.4.6 Considerations for Anti-parasitic Drug Development
In any drug candidate’s journey down a development pipeline there are many
pitfalls. Sadly, activity against a biological target or even in vivo efficacy does not
make a successful drug. Careful consideration should be given early to liabilities
that might lead to late stage failure, which could be costly and drain resources vital
to the discovery of a potential candidate. In 1991, the leading cause of attrition for
drug candidates was poor pharmacokinetics and/or bioavailability (40%), yet by
2000 this had decreased to less than 10% (Kola & Landis, 2004). This is largely
attributable to the advent of in vitro screens for physiochemical properties likely to
create downstream liabilities. The placement of these screens early in the pipeline
forces compounds with poor solubility, bioavailability, and metabolic stability to fail
during far cheaper stages of development and spares the opportunity cost otherwise
missed in development of these compounds. Fig. 3.4.6 outlines a generic paradigm
for screening compounds. In this paradigm early hits from in vitro activity screens are
passed through an absorption, distribution, metabolism, and excretion (ADME) gate
before passing into more expensive animal models. The source of compounds could
be from commercially available large libraries, libraries of natural product extracts,
small scale synthesis, etc. The process remains unchanged. However, feedback
from all stages should be iteratively incorporated into the next round of screening.
Acceptable physiochemical properties for a drug ultimately depend on the target
product profile for intended use; however, it is reasonable to assume that compounds
with low solubility or poor permeability may not fit a profile for oral prophylaxis, for
example. It should also be noted that many of these properties go hand-in-hand, and
that it is often necessary to strike a balance. For example, lipophilicity, permeability,
and metabolic instability tend to correlate since CYPs tend to favor lipophilic drugs.
Considerations for Anti-parasitic Drug Development 283
From this example, if one were trying to develop a long half-life lipophilic drug to
concentrate in the food vacuole of a parasite, it might be necessary to trade off some
lipophilicity (and with it potentially some biological activity) to enhance half-life. The
subtleties of this interplay of course depend again on the intended use of the drug.
Feedback from all stages of screening should be used to inform later rounds. In such
a manner, successive rounds of screening should get closer and closer to the desired
chemical space with optimum physiochemical properties and biological activity for
intended use.
From the example of the 8-aminoquinolines, a very modern consideration for drug
development arises. Pro-drugs should be fully metabolically characterized, with all
pertinent pathways identified. Consideration should be given to the pharmacogenomic
make-up of the target population. Pro-drugs activated by CYPs 3A4, 2D6, or 2C19 may
lack efficacy in some populations due to high polymorphism. If at all possible, non-
CYP mediated conversion is desirable to avoid such liabilities in later development.
Likewise, toxicity can be affected in a similar manner if, for example, the formation of
a reactive metabolite is mediated by a highly polymorphic CYP.
Table 3.4.1: Approaches to antimalarial drug discovery and development. Adapted from Philip J.
Rosenthal’s review on Antimalarial drug discovery: old and new approaches (Rosenthal, 2003).
Approach Examples
Optimize therapy with existing drugs Amodiaquine/sulfadoxine/pyrimethamine
(combinations) Amodiaquine/artesunate
Artesunate/sulfadoxine/pyrimethamine
Artesunate/mefloquine
Artemether/lumefantrine
Chlorproguanil/dapsone
Chlorproguanil/dapsone/artesunate
Atovaquone/proguanil
Develop analogs of existing drugs New aminoquinolines
New endoperoxides
New folate antagonists
Natural Products New natural products
Repurposing of drugs from other Folate antagonists
therapeutic areas Antibiotics
Atovaquone
Iron chelators
Reversing drug resistance Verapamil, desipramine, trifluoperazine, chlorpheniramine
Discovery of compounds active Antibiotics, Quinolones, etc.
against novel targets
Figure 3.4.1: The structure of quinine.
Figure 3.4.2: Metabolism of quinine leads to the formation of redox active quinones. Adapted from
Marcsisin 2013 (Marcsisin, 2013).
Figure 3.4.3: Two important members of the 4-aminoquinoline class: A. Amodiaquine B. Chloroquine
Considerations for Anti-parasitic Drug Development 285
Figure 3.4.4: CYP 2D6 mediated metabolism of primaquine (Pybus, 2013). Several potentially oxi-
dative metabolites formed by CYP 2D6 may be key in primaquine activity. Carboxyprimaquine is the
primary metabolite found is plasma. Its production is likely MAO mediated and the pathway is not
shown.
Figure 3.4.5: Artemisinin was identified as the active ingredient in sweet wormwood in the 1970s.
Several synthetic artemisinins and other endoperoxides now exist for the treatment of malaria.
Figure 3.4.6: Generic paradigm for drug discovery that uses ADME properties to down-select poten-
tial candidates prior to expensive and labor intensive animal modeling. Dashed lines indicate a
screen that is not necessarily a mandatory gate, but which can aid in the interpretation of potential
later stage failure.
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Mónica Ferreira, Patrick Almeida, Mohammad-Ali Shahbazi,
Alexandra Correia and Hélder A. Santos*11
3.5 Current Trends and Developments for

Nanotechnology in Cancer
Abstract: In spite of the incessant development in medicine and technology, cancer
continues to be one of the leading causes of death worldwide. Conventional systems
for cancer therapy that are available in the market have limited and unspecific access
to tumor sites. Thus, in the recent years, nanotechnology has been applied to the field
of medicine, opening new avenues to the treatment, diagnosis, and monitoring of
cancer diseases. This horizon has become closer with a considerable number of nano-
formulations being recently approved for commercialization or reaching preclinical
and clinical stages. In this context, remarkable advances in nanotechnology led to the
emergence of nanodelivery systems that can specifically target an extensive variety of
malignant tissues, control precisely the release of the cargos, as well as to improve the
biological effects of the immunostimulatory molecules via different mechanisms for
cancer immunotherapy. In addition, imaging techniques combined with nanotechnol-
ogy render extraordinary sensitive and powerful diagnostic and imaging tools. Multi-
functional systems encompassing both therapeutic and diagnostic functions provide
great advantage for tracking, in real time, the drug payloads delivered to the tumour
site. The bench-to-bedside translation of the nanomedicines and technologies have
introduced a new era in the design and development of innovative, but simultane-
ously complex targeting nanoparticles for delivery of both therapeutic and diagnostic
agents to tumors. In this chapter, we focus on the current trends and developments
of nanotechnology for cancer, highlighting some of the most advanced drug delivery
nanosystems, targeting strategies, the nanotools used for cancer imaging and diag-
nostics, as well as the recent nanotechnological approaches used for cancer immu-
notherapy.
Keywords: Nanotechnology, nanomedicine, drug delivery, targeting, cancer

immunotherapy, diagnostics, imaging
*Corresponding author: Hélder A. Santos: Division of Pharmaceutical Chemistry and Technology,

Faculty of Pharmacy, FI-00014 University of Helsinki, Finland, *E-mail: helder.santos@helsinki.fi
Mónica Ferreira, Patrick Almeida, Mohammad-Ali Shahbazi, Alexandra Correia: Division of Pharma-
ceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014 University of Helsinki, Finland
© 2015 Mónica Ferreira et al.

Introduction 291
3.5.1 Introduction
Cancer is one of the leading causes of death in the present days, killing millions of
people every year (Sutradhar, 2014). Cancer is a pathological problem derived from
genetic instability and multiple molecular alterations, caused by uncontrollable cell
division which invade the surrounding tissue and destroy it (Mody, 2011). More than
8.2 million people died from cancer in 2012, and around 60% of new cases occurred in
Africa and Asia, but 30% of cancers were prevented (WHO, 2014). In 2014, more than
1.6 million new cancer cases and more than half million cancer deaths are estimated to
occur in the United States (ACS, 2014). During the most recent 5 years of existing data
(2006−2010), delay-adjusted cancer incidence rates declined slightly in men (by 0.6%
per year) and were stable in women, while cancer death rates decreased by 1.8% per
year in men and by 1.4% per year in women. The combined cancer death rates (deaths
per 100,000 inhabitants) have been continuously declining for 2 decades, from 215.1
in 1991 to 171.8 in 2010. This 20% decline translates to the avoidance of more than 1.3
million cancer deaths (almost 1 million among men and more than 0.3 million among
women) during this time period (Siegel, 2014). In Europe, around one quarter of the
total population is affected by new cancer cases, with about 3.2 million new patients
every year (WHO, 2014). Breast, colorectal, lung, liver and stomach cancers are some
of the most common cases of cancers.
While there have been many advances in cancer treatment, such as chemotherapy
and radiotherapy for cancer, they are still far from being ideal. The problematic issue
is to achieve the desired concentration of therapeutic agents at the tumor site, thereby
destroying cancer cells, while minimizing the damage to normal cells. In order to
overcome some of the problems of conventional anticancer therapeutics, cancer
nanotechnology has been implemented, which indicates a major breakthrough in
cancer detection, diagnosis and treatment of cancer (Misra, 2010).
Nanotechnology is one of the fastest areas of growth and development in the 21st
century, where several material types (e.g., organic, inorganic, and polymeric based),
medicines and devices are used to manipulate matter with size in the range of 1−100
nm (Reddy, 2011) (Fig. 3.5.1). Some of the major challenges in nanotechnology are a
proper identification of neoplasic early biomarkers, as well as the understanding of
their evolution, screening and early detection, development of technology to provide
different selective therapeutic nanoplatforms and the ability to pass through biological
and physical barriers (Pinheiro AV1, 2011). Some nanosystems are developed to overcome
drug resistance, since they control the efflux of P-glycoprotein, the major mechanism
of drug resistance (Dong, 2010). Therefore, nanotechnology has brought new hope for
cancer detection due to the development of nanozised probes (Mody, 2011).
292 Current Trends and Developments for Nanotechnology in Cancer
Figure 3.5.1: A summary of nanoparticles that have been explored as carriers for drug delivery in
cancer therapy, together with illustrations of their biophysicochemical properties. Reprinted with
permission from (Sun, 2014).
Nanotechnology applied to the field of medicine or “nanomedicine”, entails strong

advantages in the treatment, diagnosis, monitoring, and control of biological
systems, providing several innovative tools, some of them taking the form of
nanoparticles, polymeric nanoconstructs, nanofibers, nanoscale microfabrication-
based devices and sensors. (Moghimi, 2005). These nanotools aid in the design of
imaging agents and diagnostics, allowing easy treatment and early detection of cancer
(Table 3.5.1).
Nanomedicines are being used as vehicles to deliver therapeutic agents to a
specific location in the body (Markman, 2013). The selective nanomedicines for
cancer treatment have revolutionized the research field of cancer and technology due
to the possibility to precisely synthetize nanoparticulate systems with variable size,
shape and physicochemical properties (Paulo, 2011). Nanomedicine can be applied to
actively or passively target the tumor tissue (Krishnan, 2014). In passive targeting, the
physicochemical properties of the drug nanocarrier are molded so that it escapes from
the immune system and accumulates in the target tissue. Active targeting involves the
attachment of an antibody, carrier protein or other ligand to the nanovector, so that it
has affinity to a cell receptor or biomarker (Danhier, 2010). Targeted nanoparticulate
imaging agents offer new opportunities for accurate cancer diagnosis. Due to the
highly engineerable nanomaterials, nanoparticles offer great advantages such as
increased sensitivity to contrast and the avidity of binding and cleavage specificity
(Gautier, 2013). Considering the nanoparticles and their ability to reach a specific
location and generate detectable signals, these nanoparticles can also be used to
Introduction 293
study the biophysical interactions (Sumer, 2008). Drug development and imaging
nanomedicines are thus interconnected, due to the advantages of the application
and non-invasiveness in high yield imaging studies. All imaging modalities can be
used in drug studies to provide anatomical, pharmacokinetic and pharmacodynamic
information (Wang, 2010).
Table 3.5.1: Nanomedicines approved by one or more regulatory bodies. Reprinted from (Wang, 2013).
Product Nanoplatform/agent Indication Status Company
Doxil PEGylated liposome/ Ovarian cancer Approved Ortho Biotech

doxorubicin 11/17/1995 (acquired by JNJ)
hydrochloride FDA50718
Myocet Non-PEGylated Metastatic breast Approved in Europe Sopherion
liposomal doxorubicin cancer and Canada, in Therapeutics, LLC
nanomedicine combination with in North America
cyclophosphamide and Cephalon, Inc.
in Europe
DaunoXome Lipid encapsulation of First-line treatment Approved in the USA Galen Ltd.
daunorubicin for patients
with advanced
HIV-associated
Kaposi‘s sarcoma
ThermoDox Heat-activated Breast cancer, Received Fast Celsion
liposomal encapsulation primary liver Track Designation,
of doxorubicin cancer approval expected
by 2013
Abraxane Nanoparticulate Various cancers Approved 1/7/2005 Celgene
albumin/paclitaxel FDA21660
Rexin-G Targeting protein Sarcoma, Fully approved in Epeius
tagged phospholipid/ osteosarcoma, Philippine Biotechnologies
microRNA-122 pancreatic cancer, Phase II/III (Fast Corp.
and other solid Track Designation,
tumor Orphan Drug Status
Acquired) in USA
Oncaspar PEGylated asparaginase Acute Approved Enzon
lymphoblastic 24/06/2006 Pharmaceuticals,
leukemia Inc.
Resovist Iron oxide nanoparticles Liver/spleen lesion In 2001, approved Bayer Schering
coated with imaging for the European Pharma AG
carboxydextran market
Feridex Iron oxide nanoparticles Liver/spleen lesion Approved by US-FDA Berlex
coated with dextran imaging in 1996 Laboratories
Endorem Iron oxide nanoparticles Liver/spleen lesion Approved in Europe Guerbet
coated with dextran imaging
The current nanoplatforms used for the purpose of targeting drug delivery to cancer
tissues can also be addressed to selectively deliver imaging agents (Chen, 2014).
For example, the first evaluation of the effectiveness of a particular formulation to
a patient can be performed with imaging agents, in order to verify that the delivery
system will primarily target the cancerous tissues, even before any drug regimen test
is initiated (Toy, 2014). However, imaging is not only used for detection, it is also
important to determine the stage and the precise locations of cancer. Distribution of
multistage systems have shown potential to meet the challenges of drug targeting and
overcome biological barriers (Miele, 2012). Image-based nanomedicines have also a
crucial importance in cancer diagnostics due to their highly sensitive detection probes
(Pan, 2010). Imaging techniques, which are methods of producing images of the body,
are an important element of early detection of cancer diseases (Shukla-Dave, 2014).
Transformation procedures leading to malignancy can be detected by a matter of
routine screening, non-invasive means, such as standard proteomic analysis of blood
samples or in vivo imaging of molecular profiles (Bellisola, 2012). Some of the main
challenges for nanomedicines are the development for the detection and monitoring
of cancer markers in vivo, creation of technological platforms for early detection of
cancer biomarkers ex vivo, and engineering of nanoparticles to prevent biophysical
and biological barriers (Ferrari, 2005).
In this chapter we focus on the current trends and developments of nanotechnology
for cancer, highlighting some of the most advanced drug delivery nanosystems,
targeting strategies, nanotools used for cancer imaging and diagnostics, as well as
recent nanotechnological approaches used for cancer immunotherapy.
3.5.2 Drug Delivery Nanosystems in Cancer Therapy
Cancer drug delivery is a demanding subject that requires deep and comprehensive
research. Thus, this area of research has been under intensive investigation during the
last few years. For example, problems associated with selective targeting, improved
penetration through biological barriers and a controlled release of therapeutic cargos
over time are details that influence the design of nanocarriers for drug delivery.
Therefore, recently, sophisticated nanosystems have been developed in order to
provide delivery of one or more therapeutic molecules in a controlled, selective and
“smart” way to the desired cancer cells/tissues.
3.5.2.1 Controlled Drug Delivery
Conventional systems for cancer therapy that are available in the market have limited
and unspecific access to tumor sites, and thus, it is well-known that patients experience
serious side effects when treated with anticancer drugs, frequently associated to low
Drug Delivery Nanosystems in Cancer Therapy 295
therapeutic efficacy (Siegel, 2011). Another factor that needs to be considered for
drug delivery is the physicochemical properties of the drugs themselves. Many of
the anticancer drugs used in the clinic at the present are rather hydrophobic, and
thus, their poor solubility may lead to local toxicity associated with the fact that they
may not be soluble enough to go through the aqueous environment surrounding the
tumor cells and cross the cell membrane to ultimately reach the intracellular targets
(Owen, 2012). On the other hand, successful utilization of hydrophilic drugs (e.g.,
macromolecules such as proteins or nucleic acids) has been stalled by a number of
obstacles, such as poor cell internalization, because of their inability to cross the lipid
bilayer of the cell membrane, as well as short half-life in the bloodstream, due to poor
stability against proteolytic and hydrolytic degradation (Fattal, 2009; Ishihara, 2010;
Sun, 2014).
Therefore, by applying nanotechnology, many scientists have focused on
investigating new ways to develop novel drug delivery systems with the aim to
maintain high therapeutic drug levels at the malignant cell sites and as low as
possible in healthy cells, hence overcoming and improving the poor physicochemical
properties of the drug (Grinberg, 2014; Sun, 2014). For this purpose, several strategies
to both target the tumor site and release the drug(s) in a controlled fashion have been
developed in order to selectively deliver therapeutic cargos over time. In this section,
we will briefly focus on the most recent controlled release strategies and give some
examples of the nanosystems used to achieve the abovementioned aims.
It is possible to fine-tune and control the release of payloads from the nanocarrier
through diverse mechanisms. For example, for porous materials, controlling the pore
size and the surface chemistry of the pore walls can lead to different diffusion release
profiles, either improving or sustaining the release of the loaded cargos. Porous hollow
Fe3O4 nanoparticles were able to provide the delivery of cisplatin via a slow diffusion-
controlled process, exhibiting different kinetic profiles of cisplatin depending of the
pore gap sizes (Cheng, 2009). Porous silicon (PSi) materials are another good example
of a biomaterial widely used for biomedical applications (Santos, 2014). The small
and tunable size of the pores, high surface area and large pore volume enables high-
drug-loading capacity. The confinement of large amounts of drug molecules, together
with their interaction with the nanocarriers, and depending on the physicochemical
properties of the PSi materials, leads to a tunable and controllable release of cargos
(Mäkilä, 2014; Salonen, 2008). Controlled release of drug molecules can be also
achieved through the uniform incorporation of the drug into matrix-based systems
(water-insoluble polymer carriers). These systems enable sustained drug release
because the diffusion of drug molecules from the inner matrix of the polymers to the
surface takes time and may depend on the physicochemical properties of the drug,
on the interactions between the drug and the polymeric matrix, and on the density
of the matrix itself (Sun, 2014). Ma have synthesized a PCL-Tween 80 copolymer
nanoparticulate matrix from ε-caprolactone and Tween 80 to maintain the release
of cargos through diffusion for as long as 28 days, with a better in vitro cytotoxicity
towards C6 glioma cells compared to the commercial docetaxel (Taxotere) at the same
drug concentration (Ma, 2011). Sustained release can similarly be achieved by using
nanocarriers made of erodible or degradable materials. The release can be tailored by
tuning the erosion kinetics of nanoparticles through the selection of biodegradable
polymers, such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA),
polyethylene glycol (PEG), polycaprolactone (PCL) among others (Sun, 2014), and
through different encapsulation methods (Zilberman, 2008). Sun have successfully
constructed a biodegradable nanoparticle based on a triblock copolymer PEG-b-PCL-
b-poly (2-aminoethylethylene phosphate) (Fig. 3.5.2) with a proven fruitful delivery of
small interfering RNA (siRNA) and paclitaxel for synergistic tumor suppression (Sun,
2011).
Figure 3.5.2: Chemical structure of the triblock copolymer PEG-b-PCL-b-poly (2-aminoethylethylene

phosphate) and schematic illustration of the micellar nanoparticle formation and loading of paclita-
xel and siRNA. Reprinted with permission from (Sun, 2011).
Nowadays, the most advanced systems are not so simple and combine a series of
different functionalities that render tunable release of cargos, triggered release
properties, selectivity, and the possibility of tracking the nanocarriers by imaging
and even combined therapy of several active substances. Such systems are further
described in the sections below.
3.5.2.2 Stimuli-responsive Controlled Drug Delivery Systems
Nanoparticulate systems have been widely used for drug delivery when combined to
molecules that have the particularity to react to certain physiological or pathological
changes in the surrounding environment (Jhaveri, 2014). This is an approach that
offers additional opportunities to enhance tumor accumulation of drugs that are

administered systemically, increasing the chances of a more successful therapeutic
outcome when treating solid tumors and avoiding unnecessary drug distribution
in healthy tissues, thus minimizing the toxic side effects of the drugs (Nakayama,
2014). Different strategies have been investigated and nowadays there are different
nanosized platforms capable of responding to different stimuli (Fig. 3.5.3) that can be
tumor extrinsic, such as application of light, magnetic field, heat or ultrasounds; or
intrinsic to the tumor-like pH, reductive potential changes or up-regulation of proteins
or enzyme expression (MacEwan, 2010; Nakayama, 2014; Yang, 2013; Zhu, 2013). In
this section we will explain different tumor-intrinsic stimuli-responsive strategies
recently applied in the research field.
Figure 3.5.3: Stimulus-responsive delivery strategies for tumor targeting. Abbreviations: EPR, enhan-
ced permeability and retention effect; GSH, glutathione. Reprinted with permission from (Zhu, 2013).
Among the nanosystems generated to trigger the release of cargos in a pH-dependent

manner, many systems have been developed taking into account the difference
in pH values existing between the healthy tissues (pH = 7.4) and the extracellular
environment of solid tumors (pH = 6.5–6.8) (Helmlinger, 2002). Other systems exploit
the pH values of the internal cellular organelles, such as lysosomes and endosomes
(Sorkin, 2002). It is well-known that, environmentally, these cellular compartments
have characteristic features, such as lower pH and reductive potential, which are
divergent from the extracellular surroundings, offering a chance to exploit these
special properties to release actively the cargos intracellularly, inside endosomal
vesicles and/or lysosomal organelles (Elzoghby, 2012; MacEwan, 2010). For example,
mesoporous silica nanoparticles (MSN) have been widely used for stimuli-responsive
drug delivery in cancer therapeutics and were proved to be a versatile, easily
functionalized platform, enabling an easy manipulation of the nanopore openings,
with noncytotoxic solid supports for nanovalve-controlled drug delivery (Fig. 3.5.4)
(Coti, 2009; Tarn, 2013; Xia, 2009).
Figure 3.5.4: Schematic of a multifunctional mesoporous silica nanoparticle showing a core/shell

design, surface modifications, and multiple types of cargos. Reprinted with permission from (Tarn,
2013).
It is also known that MSNs are endocytosed by cells in an energy-dependent manner

and colocalize to the lysosomes (Yanes, 2012). Thus, many researchers have reported
pH-responsive MSNs modified with different kinds of gatekeepers. The activated
release of anti-cancer drugs from mesoporous materials due to a pH change in the
environment has mainly been achieved by using polyelectrolytes, supramolecular
nanovalves, pH-sensitive linkers, and acid-decomposable inorganic materials (Yang,
2014; Zhang, 2014b). In a study reported by Meng, (Meng, 2010), a nanovalve-based
delivery system was designed to meet the pH features of the acidic compartments of
lysosomal organelles. The nanosystem had the ability to have the nanovalves closed
at physiologic pH by non-covalent interactions, but open in response to changes
in pH acidic conditions inside endosomal compartments by dissociation at pH 6 or
lower after cellular uptake. The principle used here was based on the construction of
stalks that were covalently attached to the nanopore openings, and the same stalks
were bonded to a capping agent, in this case β-cyclodextrin (β-CD), that responded to
the changes in the pH environment by opening or closing the nanopore accordingly
in a reversible manner. In addition, the authors were able to demonstrate that this
nanosystem was taken-up into the acidic endosomal compartments in THP-1 and KB-
31 cells, showing also an efficient entrapment of dye molecules and anticancer drug
doxorubicin inside the nanopores at physiological pH. In contrast, when the pH was
decreased to values below 6, it caused dissociation of the β-CD caps and released the
entrapped cargos, leading to apoptotic events of the cells. The opposite systems was
also designed, having the β-CD rings immobilized and the stalks movable, making
also possible the storage and delivery of different payloads (Zhao, 2010).
Other stimuli-responsive approaches are related to the use of pH-sensitive linkers
that connect the drug molecule with the nanoparticle itself. Lee created a delivery
system that linked doxorubicin to the MSN surface by hydrazone bonds, which were
sensitive to the pH environmental changes, cleaving at low pH values from 4−6
(compatible with pH in the lysosome) and being intact at physiological pH, thus
maintaining the drug molecules safely connected to the carrier during circulation
in the bloodstream (Lee, 2010). Polymer coatings have also been used as coating
agents to prevent the fast release of drug payloads. For example, Liu used poly(4-
vinyl pyridine) (PVP) as a pH-responsive capping nanoshell to coat MSNs, where the
releasing kinetics of the molecules was dependent on the protonation degree of PVP
at different pH values. As the pH decreased, the drug release rate increased, since the
protonated polymer became swollen and permeable to the trapped molecule. This
approach may be useful to deliver drug cargos into microenvironments with lower pH,
such as the tumor region and/or even the endosomal organelles (Liu, 2011).
Redox-responsive drug delivery approaches have also been recently exploited
for cancer therapy applications (Remant, 2014; Shi, 2014), taking advantage of the
fact that tumor cells have greater reducing features compared to healthy tissues,
thus showing a much higher concentration of glutathione in the cytosol of tumor
cells (Kuppusamy, 2002). Chuan have developed a novel nanosystem consisting
of a self-assembling poly(ethyleneglycol)-disulfide-paclitaxel (PEG-SS-PTX/PTX)

delivery vector containing free paclitaxel (PTX) and conjugated PTX in the same
nanoparticulate system with a redox-triggered drug-release mechanism (Fig. 3.5.5).
The principle is based on the instability of disulfide bonds in a reductive environment.
The authors were able to improve the drug loading method, obtain programmed drug
release, reduce the drug toxicity, and enhance the antitumor efficacy (Chuan, 2014).
Figure 3.5.5: Schematic representation of the self-assembly, accumulation at the tumor site, uptake
by tumor cells, and triggered intracellular drug release of redox-responsive PEG-SS-PTX/PTX nano-
particles. The programmed drug release undergoes two phases: release of loaded PTX and conjuga-
ted PTX. Abbreviations: PTX, paclitaxel, GSH, glutathione, EPR, enhanced permeability and retention
effect. Reprinted with permission from (Chuan, 2014).
Variations in the composition and expression of local enzymes are other potential
factors that may provide an opportunity to selectively deliver drugs efficiently in the
tumor sites via an enzyme-triggered mechanism. One of the most popular approaches
is based on the altered expression of matrix metalloproteinases (MMPs), since in the
MMP family, the up-regulation of MMP2 and MMP9 is generally recognized as involved
in the invasion, progression and metastasis of most human tumors (Mansour, 2003).
Zhu developed a multifunctional complex drug delivery system that has prolonged
blood circulation time, targets specifically to tumor cells, responds to the up-regulated
matrix metalloprotease 2 (MMP2) in the tumor microenvironment and has enhanced
intracellular delivery by coupling specific moieties to the surface of a liposomal
nanovector (Zhu, 2012). They were able to show a significant increase in the tumor
targetability, as well as tumor cell internalization of the nanocarrier, and thus, this
approach may be useful for selective drug delivery of cargos in the tumor sites. Other
up-regulated enzymes, namely cancer-associated enzymes, have been utilized for
the same purpose in liposomal formulations, yielding positive results of fast release
kinetics in the specific tumor site (Basel, 2011).
3.5.2.3 Combination Therapy
Cancer is a multifactorial disease and often the clinical treatment requires the
administration of more than one drug molecule in order to achieve successful
therapeutic outcomes. In addition, cancer cells frequently become multidrug resistant,
by developing defense mechanisms against the therapeutic approach, like expression
of different receptors/target proteins, self-repairing mechanisms or/and alterations in
their own metabolism, evolving towards a weaker response to the treatment (Holohan,
2013). The combination of different therapeutics can lead to reduced drug resistance
and synergize the therapeutic effect of different active substances, and thus, a dual
positive outcome on the improvement of therapeutic effectiveness and decrease of
deleterious effects (Hu, 2010). Despite the positive advantages of the association of
multidrugs in a delivery system, it is rather challenging to efficiently co-load different
drug molecules, often with distinct physicochemical properties, in the same ordinary
nanocarrier (Hu, 2010; Kratz, 2012; Zhang, 2011), and even more challenging to deliver
such cargos in vivo, where it is absolutely necessary to supply the proper drug ratio at
the tumor level (Mayer, 2006). Several nanoparticulate systems such as lipid-based,
inorganic and polymeric nanoparticles, among other types, have been used to co-
load two or more different payloads adopting different strategies (Hu, 2010). Some
include the physical adsorption of drugs to the nanovector, with the disadvantage
that only drugs with similar physicochemical properties can be loaded simultaneously
(Herranz-Blanco, 2014; Liu, 2012; 2014a). It is also possible to co-load molecules with
different physicochemical properties via a sequential adsorption procedure (Liu,
2013a). Others have followed the incorporation of therapeutics into the particle matrix
during particle preparation (Doane, 2013), and covalently bonded the drug molecules
to the polymeric backbone after particle preparation (Aryal, 2011).
Despite the obstacles encountered in cancer therapy, extensive progress has been
made so far and nowadays there are several nanoformulations that combine two or
more different anticancer drugs (Liao, 2014; Tardi, 2009) or even two different classes
of therapeutics (Deng, 2013; Liu, 2014b; Saad, 2008). For example, a nanoparticulate
system was developed by Deng using a layer-by-layer deposition approach (Deng,
2013). Liposomes were chosen to be encapsulated by polycationic PLA through the
layer-by-layer deposition, and finally coated by a layer of hyaluronic acid that enhances
the in vivo stability of the nanosystem (Poon, 2011). This system co-delivered siRNA
molecules and doxorubicin loaded in the core of the liposomes, which were able to
knockdown a drug resistance pathway in tumor cells (Whitehead, 2009). This means
they can be applied in the treatment of triple-negative breast cancer form, which is
an estrogen, progesterone and human epidermal growth factor receptor 2 (HER2)-
negative cancer type with a more aggressive effect than other breast cancers (Kassam,
2009; Livasy, 2006). The successful co-delivery of both siRNA and doxorubicin was
achieved, as well as significantly enhanced DXR efficacy by 4-fold in vitro (Fig. 3.5.6).
The in vivo results showed an 8-fold decrease in tumor volume compared to the
control treatments, with no observed toxicity to the animals. An ultimate outer layer
of hyaluronic acid (HA) was deposited to increase the stability of the system (Deng,
2013).
Figure 3.5.6: The co-delivery of siRNA and doxorubicin using the modular doxorubicin-liposome/
PLA/siRNA/PLA/HA LbL nanoparticle platform. (A) Schematics of the siRNA-doxorubicin LbL liposo-
mes. (B) Release profile of the two therapeutics components: siRNA and doxorubicin from the LbL
liposomal nanoparticles in tissue culture medium over 72 h (n = 3). (C) Evaluation of the siRNA-
enhanced cytotoxicity of the combo therapy in MDA-MB-468 cells. The results represent mean ±
standard deviation (n = 3; P < 0.05). Reprinted with permission from (Deng, 2013).
Cancer Targeting 303
Another study that combines siRNA and the co-delivery of paclitaxel in a two-in-one
micelleplex system showed that it is possible to reduce by hundreds of times the dose
of paclitaxel given by associating it with siRNA, and equally achieve a synergistic
therapeutic effect (Sun, 2011). This and other systems are able to provide the co-
delivery of different drugs and therapeutic classes to the tumor sites in vivo, giving
hope to the decrease of harmful side effects of the anticancer drugs.
As described before, there are a variety of strategies that can be applied to
combine different molecules for treatment of tumor diseases. In addition, there is
the possibility that the nanocarrier itself is modified in such a way that it also has
some therapeutic activity. Interestingly, the following study combined doxorubicin
to the pro-apoptotic effect of the nanocarrier itself by means of incorporation of a
sphingolipid C6-ceramide into the liposomal formulation, targeting at the same time
nucleolin, a protein that is greatly expressed in cancer and endothelial cells of tumor
angiogenic blood vessels (Shi, 2007). In this work, the sinergistic combination of
DXR:C6-Cer in a 1:2 molar ratio was identified and tested, which increased the cell
toxicity potential, and thus, enabled at least a 4–6-fold dose reduction of doxorubicin,
independently of the cell line tested. Hence, this approach might be valid to overcome
drug resistance and, at the same time, decrease the deleterious effects of doxorubicin
(Fonseca, 2014).
3.5.3 Cancer Targeting
Recent developments in nanotechnology are expected to revolutionize the

current scenario of cancer therapy and diagnosis. This horizon has become closer
with a considerable number of nano-formulations being recently approved for
commercialization or reaching preclinical and clinical stages. The unique features
of engineered nanotherapeutics can be translated into potential and attractive
advantages over the conventionally practiced chemotherapeutic modalities,
including: the reversion of unfavorable physicochemical properties of cytostatic
drug molecules, resulting in improved pharmacokinetic and pharmacodynamics
profiles (Alexis, 2008; Bertrand, 2012; Bertrand, 2014; Kipp, 2004); improvement of
drug stability by confining the therapeutic agents inside the nanocarriers, preventing
their degradation (Whitehead, 2009); delivery of the bioactive payload to the tumor
site through passive and/or active targeting strategies, consequently improving its
therapeutic efficacy and reducing adverse side effects by hindering the drug release
during systemic circulation; and designing multifunctional nanoplatforms for
combined therapy and/or theranostic applications, encompassing both therapeutic
and diagnostic functions (Fernandez-Fernandez, 2011; Janib, 2010; Sanna, 2014).
Since the concept of the “magic bullet” was introduced, nearly one hundred years
ago (Strebhardt, 2008), significant efforts have been focused on a deeper understanding
of the tumor biology and microenvironment, as well as on the screening of receptors
overexpressed by the cancer cells, with the ultimate goal of defining strategies for
specifically targeting the tumor tissues. In this context, remarkable advances in
nanotechnology led to the emergence of targeting nanodelivery systems as innovative
as complex, which exploit both passive and active mechanisms for targeting an
extensive variety of malignant tissues (Fig. 3.5.7) (Davis, 2008; Farokhzad, 2009;
Ferrari, 2005; Nicolas, 2013). This section briefly describes the fundamentals behind
tumor targeting, scrutinizes different strategies that have been employed for targeting
cancer tissues, and highlights the most recent breakthroughs on designing and
developing targeting drug delivery nanoplatforms for application in cancer therapy.
Figure 3.5.7: Strategies adopted for drug targeting and localization of nanosystems to tumor cells and
tissues. (A) Passive targeting. Circulating nanoparticles passively extravasate in solid tumor tissue
via the enhanced permeability of blood vessels, i.e., through the disorganized and leaky vasculature
surrounding the solid tumor coupled with the absence of lymphatic drainage, and preferentially
accumulate in tumor cells (the EPR effect). (a) The drug is released into the extracellular matrix and
diffuses through the cells and tissue. (B) Active targeting. Once nanoparticles passively extravasate
and concentrate in the target tissue via the EPR effect, the presence of ligands grafted onto the
nanoparticle surface enable active targeting of xthe nanoparticles to receptors that are overexpressed
on tumor cells or tissue, resulting in enhanced uptake and internalization via receptor-mediated
endocytosis. (b) Tumor-specific ligands on the nanoparticles bind to cell surface receptors, triggering
internalization of the nanoparticles into the cell through endosomes on which, due to an internal
acidic pH, the drug is released from the nanoparticles and diffuses into the cytoplasm. (C) Active
targeting to endothelial cells. Nanoparticles can be targeted to bind to angiogenic endothelial cell
surface receptors with the aims of enhancing drug accumulation in the tumor endothelium, thereby
inhibiting growth of blood vessels supplying the tumor rather than inhibiting tumor cells per se (c);
and improving delivery of chemotherapeutic agents to tumor cells via the EPR effect with the potential
to act synergistically in targeting both the vascular tissue and tumor cells. Adapted and reproduced
with permission from (Lammers, 2012). Abbreviation: EPR, enhanced permeability and retention.
3.5.3.1 Passive Targeting
3.5.3.1.1 The Fundamentals of Passive Targeting and the EPR Effect

The accumulation of drug molecules and nanosystems in certain target tissues,
relying exclusively on their pathophysiological features (i.e., not involving any ligand-
driven mechanism), can be referred as “passive targeting” (Arias, 2011; Jhaveri, 2014).
The majority of nanoparticulate systems with the size ranging between ~10–500 nm
present the capability of exploiting the unique intrinsic characteristics of the tumor
microenvironment, and eventually accumulating at the tumor (Torchilin, 2011). The
rationale behind the preferential distribution of nanoparticles to malignant tissues,
after intravenous administration, has been intensively investigated and, nowadays,
it is well recognized that such behavior relies on the EPR effect (Fang, 2011; Maeda,
2001; Maeda, 2000; Matsumura, 1986; Torchilin, 2011).
Contrarily to what happens in normal tissues, the microvasculature of solid
tumors is characterized by a low degree of differentiation, a discontinuous highly-
fenestrated endothelium and a disrupted basal membrane, essentially resulting from
an active angiogenesis stimulated by a frenetic and metabolically demanding tumor
growth (Carmeliet, 2000; Jain, 1998; 2010). The interendothelial fenestrations of this
imperfect vascular architecture result in an increased permeability and consequent
lower resistance to the extravasation selective accumulation of nanoparticles and
active macromolecules from the blood vessels lumen to the tumor extravascular space
(Fig. 3.5.7A) (Danquah, 2011). Additionally, the tumor tissues exhibit a compromised
lymphatic drainage function, hindering the renewal of the interstitial fluid and,
consequently, an effective clearance of the extravased nanoparticles leading to
their accumulation in the tumor interstitium (Padera, 2004). The aforementioned
pathophysiological phenomena occurring in the tumor milieu represent the
fundamentals of the EPR effect, and consequently, of the EPR effect-driven passive
targeting of nanosystems to tumors.
3.5.3.1.2 Physicochemical Properties of Nanoparticles Affecting the Passive Targeting

The physicochemical properties of the nanocarriers, namely the size, surface
charge, shape and hydrophobicity, play a major role on exploiting the EPR effect
and, consequently, the passive mechanism as a strategy to target tumors, not only
for influencing the extravasation capacity of the nanoparticles from the tumor
surrounding capillaries and their accumulation in the tumor site, but also, and equally
relevant, for influencing their stability in blood circulation, which significantly
impacts the clearance by the reticuloendothelial system (RES), and ultimately the
blood circulation kinetic profiles (Alexis, 2008; Bertrand, 2012; Dreher, 2006; Maeda,
2001). The meticulous and rational design of the nanoparticles intended for cancer
therapeutic and diagnostic applications is, therefore, of utmost importance when
taking a passive targeting strategy into consideration.
The size of the nanocarriers represents a crucial parameter that significantly

influences the extent and kinetics of accumulation in the tumor tissues. In order
for the nanoplatforms to extravasate from the tumor microvasculature to the tumor
interstitial fluid, their hydrodynamic radius needs to be lower than the threshold of
the capillary interendothelial gaps. In contrary, nanoparticles sized over the referred
limit are more likely to be engulfed by the RES (Arias, 2011). Recent works have been
shown the impact of the nanocarriers size on the efficiency of tumor passive targeting
(Cabral, 2011; Huo, 2013; Lee, 2013b; Perrault, 2009; Wong, 2011). Chan evaluated
the influence of the nanoparticles’ size and surface chemistry on the in vivo passive
targeting of tumors, and it was observed that nanoparticles of reduced size are capable
of extravasate to the tumor interstitium, contrarily to their larger counterpartes, which
were confined inside the neovasculature (Perrault, 2009). The histological data (Fig.
3.5.8) revealed that, although no difference was observed on the extension of tumor
penetration by nanoparticles with variable sizes (20, 60 and 100 nm) after 1 h, the
smaller particles diffused inside the tumor mass throughout time, while the larger
ones were mostly restrained inside the blood vessels.
Figure 3.5.8: Histological data of the nanoparticle uptake in tumors when different sized nanopartic-
les were used in vivo. Reproduced with permission from (Perrault, 2009).
In a study conducted by Tseng, magnetothermally-responsive doxorubicin loaded

supramolecular magnetic nanoparticles (Dox-SMNPs) were synthesized with the
sizes of 70, 100 and 160 nm, and subsequently injected intravenously into DLD-1
colorectal adenocarcinoma tumor bearing mice, aiming to examine the influence
of the nanoparticles’ size on their retention at the tumor site (Jain, 1987). The two-
dimension (2D) micro-positron emission tomography (PET) cross sectional images
displayed a considerably higher accumulation of the 70 nm nanoparticles at the
malignant tissue, comparatively to their larger counterparts (Fig. 3.5.9).
Figure 3.5.9: 2D micro-PET cross-sections images, created using a filtered back-projection, of mice
bearing a DLD-1 tumor (n = 3) at 36 h post-injection of 70 (A), 100 (B), and 160 nm (C) 64Cu-labeled
Dox-SMNPs. Each image was scaled to its own maximum. The 70 nm Dox-SMNPs show the highest
tumor-specific uptake among the three studies. Abbreviations: Dox-SMNPs, doxorubicin-loaded
supramolecular magnetic nanoparticles. Adapted and reproduced with permission from (Lee,
2013b).
Similarly to the size, the surface charge of nanoparticles exert an effect on their
behavior in the systemic circulation, including the circulation time, interaction
with elements of the RES and predisposition for interacting with the tumor cells,
affecting cellular association and internalization (Alexis, 2008; He, 2010). In this
regard, positively charged nanocarriers are recognized as owing more propensity for
interacting and, consequently, accumulating at the tumor tissues, as suggested by
studies performed in animal models (He, 2010; Hu-Lieskovan, 2005). An enhanced
interaction between positively charged nanoparticles and the endothelial cells of the
tumor microvasculature is suspected to be one of the mechanisms involved on the

referred predisposition, and the application of this phenomenon on the targeting of
the endothelium of the tumor vasculature for delivery of antiangiogenic agents has
already been explored (Fasol, 2012; Lohr, 2012; Schmitt-Sody, 2003; Strieth, 2008).
The coating of nanodelivery systems with hydrophilic polymers, such as PEG, has
been widely used for improving biocompatibility, as well as for sterically stabilizing the
nanocarriers (Alexis, 2008; Otsuka, 2003; Torchilin, 2006). The so-called PEGylation
of the nanoparticles’ surface renders the nanocarriers capable of evading the
recognition and opsonization by the RES, consequently reducing their clearance from
the systemic circulation, prolonging the circulation time, and ultimately promoting
the accumulation in the tumor site through the EPR effect (Arias, 2011; Danhier, 2010;
Maruyama, 2011; Sawant, 2012; Sultana, 2013). The aforementioned formulations
strategies have been explored on the engineering of nanoparticulate drug delivery
platforms that exclusively exploit a passive mechanism for targeting cancer and,
in fact, a bench-to-bedside translation can already be considered as a reality with
many of those being approved for commercialization or undergoing clinical trials for
treating a wide range of cancers (Table 3.5.2).
3.5.3.1.3 Challenges and Future Prospects of Passive Targeting

Despite the potential of the passive targeting that has been discussed above, this
mechanism presents some limitations and faces some challenges, being now
considered as a more complex and heterogeneous phenomenon than previously
assumed (Prabhakar, 2013). The inter- and intratumoral variability of the tumors
result in significant differences on the physiology of the tumor microvasculature and
microenvironment and, consequently, on the therapeutic response to passive targeted
nanomedicines (Jain, 2010).
In addition, the capability of the loaded therapeutic agents to succesfully reach
their pharmacological target, thus exerting their therapeutic effect, does not solely
depend on the biodistribution of the nanocarrier and its accumulation in the tumor
site. An effective drug delivery might be hindered by an increased interstitial fluid
pressure which induces the eflux of therapeutics back in the systemic circulation,
as well as by the inability of the drug molecules to homogeneously diffuse inside the
tumor mass (Dreher, 2006; Sawant, 2012).
Therefore, the screening of individual tumor genetic profiles and their particular
predisposition for the EPR effect, paralellely to the specific fine-tuning of the drug
release profiles based on the bahavior of the drugs in the tumor microenvironment,
may become crucial when designing and engineering personalized and optimized
nanotherapeutics exploiting cancer passive targeting.
Table 3.5.2: Examples of non-targeted nanosystems in clinical use for anticancer therapy. Adapted
with permission from (Sanna, 2014).
Name Formulation Bioactive Indication Status

compound
Liposomes
DaunoXome® Non-PEGylated Daunorubicin Kaposi’s sarcoma Approved
liposomes
Myocet® Non-PEGylated Doxorubicin Breast cancer Approved
liposomes
Onco TCS® Non-PEGylated Vincristine Non-Hodgkin’s Approved
liposomes lymphoma
Depocyt® Non-PEGylated Cytarabine Leukemia Phase III
liposomes Glioblastoma Phase I/II
Doxil®/Caelyx® PEGylated liposomes Doxorubicin Breast cancer, ovarian Approved
cancer, multiple
myeloma,Kaposi’s
sarcoma
Thermodox® PEGylated liposomes Doxorubicin Liver cancer, breast Phase III
cancer
SPI-77 PEGylated liposomes Cisplatin Ovarian cancer Phase II
NL CPT PEGylated liposomes Irinotecan Glioma Phase I
Polymeric nanoparticles
Genexol-PM® PEG-PLA Paclitaxel Breast cancer, lung Phase II

cancer, ovarian cancer
NK105 PEG-poly(aspartic acid) Paclitaxel Gastric cancer Phase I
Breast cancer Phase III
NK911 PEG-poly(aspartic acid) Doxorubicin Various solid tumors Phase II
Opaxio™ PGA-paclitaxel Paclitaxel Lung cancer, ovarian Phase III
cancer
CRLX101 PEG-cyclodextrin Camptothecin Non-small-cell lung Phase II
cancer
NC-6004 PEG-poly(glutamic acid) Cisplatin Pancreatic cancer Phase II
ProLindac™ HPMA DACH-Pt Ovarian cancer Phase II
Others
Abraxane® Albumin-based Paclitaxel Breast cancer Approved
Paclical® Micellar retinoid-derived Paclitaxel Ovarian cancer Phase III
NC-4016 Micellar PEG/polyamino Oxaliplatin Various solid tumors Phase I/II
acid
Oncaspar® PEG-L-asparaginase Asparagine Acute lymphoblastic Approved
specific enzyme leukemia
Note: DaunoXome® (Galen US Inc., Souderton, PA, USA); Myocet® (Sopherion Therapeutics Inc.,
Princeton, NJ, USA); Onco TCS® (Inex Pharmaceuticals Corp., Burnay, BC, Canada, and Enzon
Pharmaceuticals Inc., Bridgewater, NJ, USA); Depocyt® (Pacira Pharmaceuticals Inc., San Diego, CA,
USA); Doxil®/Caelyx® (Janssen Biotech Inc., Horsham, PA, USA / Janssen-Cilag Pty Ltd, Macquarie
Park, NSW, Australia); Thermodox® (Celsion Corporation, Lawrenceville, NJ, USA); Genexol-PM®
(Samyang Biopharmaceuticals Corporation, Jongno-gu, Seoul, Korea); Opaxio™ (Cell Therapeutics,
Inc., Seattle, WA, USA); ProLindac™ (Access Pharmaceuticals Inc., Dallas, TX, USA); Abraxane®
(Celgene Corporation, Inc., Berkeley Heights, NJ, USA); Paclical® (Oasmia Pharmaceutical AB, Uppsala,
Sweden); Oncaspar® (Enzon Pharmaceuticals Inc., Bridgewater, NJ, USA).
Abbreviations: PEG, poly(ethylene glycol); HPMA, hydroxypropylmethacrylamide; DACH-Pt,
diaminocyclohexane-platinum.
3.5.3.2 Active Targeting
3.5.3.2.1 The Fundamentals of Active Targeting

The active targeting of nanoparticles, also denominated ligand-mediated targeting,
involves the surface-functionalization of the nanoparticles with active moieties
possessing intrinsic affinity to specific receptors/antigens overexpressed in the
diseased tissues or cells, functioning not only as driven-force for the nanocarries’
accumulation at the target site, but also as a mechanism for enhanced cellular
association and internalization by receptor-mediated endocytosis (Fig. 3.5.7B) (ACS,
2014; Allen, 2002; Arias, 2011; Cho, 2008; Couvreur, 2006; Danhier, 2010; Kamaly,
2012; Lammers, 2012; Peer, 2007; Shi, 2011; Torchilin, 2006). Considering the specific
cellular recognition and consequent enhancement of cellular uptake as the main
achievement of the active targeting, this strategy is assumed to ultimately result in an
improved therapeutic efficacy of the targeted nanoparticulate systems, comparatively
to their non-targeted counterparts (Kamaly, 2012). The active targeting mechanism
requires the proximity of the ligand-anchored nanoparticles to the targeted antigen,
enabling their recognition and the interaction between the two components.
Therefore, the intrinsic characteristics of the nanocarriers influencing the EPR effect
and the blood circulation kinetics also play an important role on the delivery and
therapeutic and efficiency of active-targeted nanoparticles, since those primarily need
to extravasate from the tumor microvasculature in order to interact with their specific
targets located in the extravascular tissue (Lammers, 2012; Taurin, 2012). Hence, the
active and passive strategies for targeting of nanoparticles to tumors are currently
considered as complementary to ultimately improve the therapeutic efficiency of
anticancer nanodelivery systems.
3.5.3.2.2 Factors Affecting Tumor Active Targeting

The meticulous and rational design of actively targeted nanocarriers is of utmost
importance for the efficient targeting and recognition of the targeted receptor, both in
vitro and in vivo. This optimal engineering of the nanoparticles’ architecture should
encompass not only the physicochemical properties of the nanoplatform, including
its size, shape and surface properties (i.e., charge and hydrophobicity), but also
parameters associated with the targeting ligand, such as the affinity of the ligand to the
targeting receptor, the ligand density and the conjugation chemistry (Berretta, 2011;
Chen, 2012; Humrich, 2010; Lesterhuis, 2004; Signori, 2010). The combination of these
factors can synergistically impact the systemic circulation kinetics, biodistribution
profiles and targeting efficiency of actively-targeted nanodelivery systems, therefore
dictating their performance in vivo (Fig. 3.5.10).
Figure 3.5.10: The physicochemical properties of the ligand and the nanoparticles affect their blood
circulation profiles, their biodistribution and their ability to be internalized by cancer cells. Reprodu-
ced with permission from (Bertrand, 2014).
The optimization of the targeted nanoparticles’ size is therefore crucial for their
extravasation from the tumor vasculature and accumulation at the tumor size.
Rationally, when a ligand functionalization is intended, the increase on the
hydrodynamic radius after conjugation of the targeting moiety must be taken into
consideration, particularly in the case of large ligands, such as antibodies, proteins or
aptamers, under penalty of negatively influencing the targeting efficiency by hindering
the accumulation of the nanocarrier in the tumor (Humrich, 2010). The size of targeted
nanoparticles was also revealed to impact their intracellular trafficking, with smaller
particles being increasingly found deposited in the cytoplasm and nucleus of cancer
cells, in a study conducted by Lee (Kapsenberg, 2003). In parallel, the influence of the
shape of actively targeted nanoparticles on their cell internalization by cancer cells
has also been recently investigated (Coosemans, 2014; Hanke, 2013; Subbotin, 2014).
The surface of the actively targeted nanoparticles also impact on the interaction
between those and the targeted cells’ surface, therefore contributing for the efficacy
of the targeting. Cationic nanoparticulate systems are acknowledged for more
extensively interacting with negatively-charged cell membranes, resulting in a non-
specific increased cellular association and internalization (Offringa, 2009). However,
an excess in the positive charge of the nanoparticles might increase toxicity and
stimulate immunologic response (Moghimi, 2005). When the nanoparticles’ surface
is functionalized with a targeting moiety, the surface charge is not only influenced
by the intrinsic charge of the ligand and the nanoplatform, but also by the ligand
density.
Besides the surface charge, the hydrophobicity of the nanocarriers can also
interfere with the way they interact with targeted cells, since the adsorption of plasma
proteins onto the surface of non-sterically stabilized nanoparticles may decrease
the capacity of those to bind to the targeted receptor (Homma, 2005). Contrarily,
the nanoparticles’ surface functionalization with high molecular weight polymers,
such as PEG, may hinder the targeting moieties to efficiently bind to their antigen
(Lowin, 1995; Peters, 1991). The development of a PEG-shielded actively-targeted
nanoplatform with prolonged circulation time and capable of undergoing cleavage
of the steric protection in the tumor interstitium, consequently exposing the targeting
moiety to the cancer cells, might constitute an interesting approach demanding
further investigation (Arroyo, 2002).
Finally, the density of the targeting molecules functionalizing the nanoparticles’
surface significantly influences the avidity of targeting nanotherapeutics to the
complementary receptor. Theoretically, a higher density of the targeting moieties
generates a favorable binding enthalpy in the system, consequently inducing the
interaction between the ligand and the antigen (Callard, 1977; Liu, 2007), and also
stimulates the overexpression of the receptor at the cells’ surface, therefore resulting
in an enhanced internalization of the actively targeted nanoparticles by the tumor
cells. Nevertheless, various aspects, including the inappropriate orientation of the
targeting ligand, the steric hindrance of subjacent molecules and a competitive
behavior towards the receptor binding may revert this relation between the ligand
density and the efficiency of cell association (James, 1981; Valdez, 2000).
3.5.3.2.3 Ligands for Tumor Active Targeting

The adequate selection of the most suitable targeting moiety to functionalize the
surface of nanoparticles is absolutely imperative for the efficiency of their targeting to
specific receptors overexpressed by the tumor tissues, therefore representing one of the
major aspects to be considered during the design of actively-targeted nanomedicines.
A variety of targeting moieties are available to functionalize the surface of carriers
through different approaches, and those encompass monoclonal antibodies and their
fragments, proteins and peptides, aptamers and other nucleic acids, as well as small
molecules (Yu, 2010).
3.5.3.2.3.1 Monoclonal Antibodies

In the past few years, monoclonal antibodies (mAb) have remained the preferred and
most extensively used and investigated type of targeting moieties, due to the high
affinity and specificity they render to the nanoparticles. This potential of the application
of mAb in targeted cancer therapy led to the approval of a large number of these
macromolecules for clinical use, including rituximab, cetuximab and trastuzumab,

as well as to the recent development of chimeric and humanized derivatives with
modulated immunogenicity (Kamaly, 2012; Ndungu, 2012; Yoshida, 2010). However,
despite the aforementioned attractive features of mAb for targeting anti-tumor
nanotherapy, the performance of these molecules in vivo still present some limitations
and challenges, including their large size and molecular weight, their immunogenicity
resulting in the prompt recognition by the RES and rapid nanoparticle clearance
from the systemic circulation (Taniguchi, 2012; Vivek, 2014), as well as their liability
to environmental alterations and organic solvents, which challenges the processes of
scale-up and manufacturing, impacts the cost/effectiveness of the formulations and
compromises their stability. In this regard, there is an emerging interest on engineering
antibody fragments, including Fab fragments, single chain variable fragments (scFv)
minibodies, diabodies and nanobodies, for functionalizing the surface of nanodelivery
systems, maintaining their targeting specificity and considerably reducing their size
and immunogenicity (Peer, 2007; Scott, 2012; Weiner, 2000). Antibody fragment-
targeted nanomedicines have already reached clinical trials, as for example MCC-465
and SGT-53 (Table 3.5.3). MCC-465 exhibited an adequate biodistribution and highly
efficient delivery of doxorubicin for the treatment of stomach cancer in preclinical
studies (Baxevanis, 2008), and SGT-53 sustained the tumor growth in different
cancers, including head and neck, breast and prostate, by delivering the p53 supressor
protein after targeting the transferrin-receptor (Tf-R) overexpressed by the tumor cells
(Bonsignori, 2012; Le Garff-Tavernier, 2014).
3.5.3.2.3.2 Proteins and Peptides

Recent advances in bioinformatics and cancer proteomics enabled the high-
throughput screening of protein antigens overexpressed in tumors. These biomarkers
can be selectively recognized by targeting proteins and peptides that functionalize the
nanoparticles’ surface, resulting in their receptor-mediated endocytosis (Mody, 2011).
Transferrin (Tf), a serum glycoprotein involved in the iron homeostasis and regulation
of cell growth has focused attention for specifically binding to the internalizing
transferrin-receptor (Tf-R), approximately 100-fold overexpressed in various tumors
comparatively to healthy cells (Chandrasekhar, 2013). Therefore, transferrin has been
exploited as a potential ligand for decorating nanoparticulate drug delivery systems
intended to target Tf-R and some nano-formulations are presently under clinical
evaluation (Prang, 2005). However, the feasibility of using proteins for targeting
purposes has been limited by the immunogenicity of these molecules, resulting in
their increased predisposition for opsonization by the RES. In addition, the expression
of the targeted receptors by nonmalignant tissues may originate unwanted off-target
effects, compromising one of the main advantages on using targeting therapy.
In order to circumvent the aforementioned bottlenecks, attention has been
focused on the synthesis of peptide-based ligands exhibiting smaller molecular
sizes and simpler 3D conformation, consequently resulting in higher stability and

relatively lower immunogenicity when compared with the majority of the proteins.
The identification of new ligand-receptor combinations normally derive from the
application of recently developed high-throughput screening techniques and phage/
plasmid/bacterial peptide display libraries (Paulo, 2011). The RGD sequence (i.e.,
arginine-glycine-aspartic acid) has been extensively included in an immense variety
of peptides sharing the capability to specifically target the αvβ3 integrin receptors, an
endothelial cell surface receptor overexpressed in neovascular endothelial cells (Fass,
2008; Kamaly, 2012; Miele, 2012). Despite the potential and promising application
of RGD peptides on the targeting of endothelium of the tumor microvasculature,
the expression of the αvβ3 integrin receptor by normal and inflamed tissues limits
its clinical translation. In that sense, investigations have been undergoing with the
purpose of generating new RGD analogs with improved targeting specificity (Kamaly,
2012; Nembrini, 2011).
3.5.3.2.3.3 Aptamers
Nucleic acid-based aptamers are small, single-stranded RNA or DNA oligonucleotides,
where the conformational structure can be designed, rendering them the capability
of binding antigens with high affinity and specificity (Bellisola, 2012; Pan, 2010).
Candidates for targeting a specific receptor have been screened with oligonucleotide
libraries using high throughput screening methodologies. The Promising aptamers are
subsequently selected and amplified in detriment of the remaining ones. Recently, an
in vitro chemical technique denominated “systemic evolution of ligands by exponential
enrichment” (SELEX) has been explored for identification of the referred candidates
(Wang, 2013). This technique has been used by Langer and Farokhzad for developing
customized controlled-release nanoplatforms capable of specifically targeting the
prostate-specific membrane antigen (PMSA) (Ferris, 2010; Ma, 2013; Monjanel, 2014;
Nembrini, 2011; Tao, 2012). Aptamers present attractive intrinsic features to be used as
targeting ligands, such as smaller size and reduced immugenicity, in comparison with
mAb or other macromolecules, resulting in improved stability and biodistribution
profiles (Oki, 2012; Pawelec, 1999). Similarly to the other classes of targeting moieties,
the use of aptamers presents some limitations, which hinder their clinical success.
First of all, the susceptibility of nucleic acids to the action of nucleases, the stability
of the aptamer conjugation onto the nanoparticles’ surface becomes a major concern.
Second of all, the negative charge of the aptamers attributed to the phosphodiester
backbone is suspected to impact the systemic circulation kinetics of the actively-
targeted nanosystem. Finally, although presenting a relatively small molecular size,
the stiffness of the nucleic acid conformation may be translated into an increased
hydrodynamic radius of the targeting nanosystem after ligand conjugation, hence
influencing its accumulation at the targeted site, as previously discussed in this
section.
3.5.3.2.3.4 Small Molecules

Small molecules, usually defined as possessing a molecular weight < 500 Da, are
currently considered as a potential group of targeting moieties, due to the attractiveness
of their properties. These targeting molecules present a small size, improved stability,
and almost no immunogenicity. In addition, the availability of simple coupling
chemistries for their conjugation onto the nanoparticles’ surface enable the control
of the functionalization process and, consequently, the fine-tuning of the ligand
density and charge on the nanosystems’ surface, which play a significant role in the
stability, blood circulation kinetics and targeting efficiency, as previously discussed.
Furthermore, a wide range of ligands with different physiochemical properties and
chemical functionalities are available and the low production costs, reproducible
scale-up and simple manufacturing render these small molecular weight compounds
very promising targeting ligands for tumor targeted therapy (ACS, 2014; Kamaly, 2012;
Moghimi, 2005).
Folic acid has been one of the most employed and studied molecules among
the numerous targeting ligands used in cancer targeted therapy, fundamentally due
to its high affinity to the folate receptor (FR), which was found of being frequently
overexpressed on many tumors, including ovarian, breast, brain, renal, colon and
lung cancers (ACS, 2014; Annabi, 2014; Bachmann, 2010; Lee, 2011). Unfortunately,
the FR expression seems to rely on inter-tumor variability and, therefore, the
tumor predisposition for accumulating folate-targeted nanomedicines needs to be
individually evaluated (Bachmann, 2010; Kamaly, 2012). Recently, folate-targeted
imaging strategies have emerged for identifying FR-positive patients, permitting the
selection of those that would in fact benefit from the administration of the folate-
targeted nanoparticles (Bimbo, 2013). Interestingly, these images would possibly
enable the quantification of the receptor displayed in the cellular surface and readily
available to interact with the targeting ligand. Additionally to the tumor inter-
variability of the FR overexpression, this receptor is also expressed in healthy tissues,
a fact that might constitute a major downside on the administration of folate-targeted
nanosystems for specific drug delivery to cancer.
Definitively, a successful case on the field of tumor targeted therapy, particularly
when a small molecule is used as a targeting moiety, is the one of BIND-014 (Table
3.5.3). This nanomedicine relies on the targeting affinity of a urea-based small molecule
denominated S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (ACUPA)
to PSMA (Binjawadagi, 2014; Nembrini, 2011). Docetaxel-loaded nanoparticles with
a ligand density of approximately 200 ACUPA molecules per particle exhibited an
optimized targeting of PSMA-positive prostate tumors, with no visible change in the
systemic circulation kinetics (Binjawadagi, 2014; Chen, 2012).
Table 3.5.3: Tumor-targeted nanomedicines in clinical development. Adapted and reprinted with
permission from (Bertrand, 2014).
Name Formulation Targeting ligand Bioactive Indication Status

compound
Liposomes
CALAA-01 Cyclodextrin- Transferrin siRNA Various solid Phase I
based NP tumors
containing anti-
RRM2
MBP-426 Liposomes Transferrin Oxaliplatin Gastro- Phase II
esophageal
adenocarcinoma
MCC-465 PEGylated F(ab‘) 2 fragment Doxorubicin Metastatic Phase I
liposomes of human Ab GAH stomach cancer
SGT53 Liposomes Anti-transferrin p53 gene Solid tumors Phase I
receptor
single-chain
Ab fragment
(TfRscFv)
C225-ILS-DOX PEGylated Antigen-binding Doxorubicin Advanced solid Phase I
liposomes fragments (Fab) tumors
of cetuximab
Polymeric nanoparticles
BIND-014 PEGylated PL(G)A PSMA (Small Docetaxel Solid tumors Phase II
(Accurins™) molecule)
Atu027 Liposomes Protein kinase N3 siRNA Solid tumors Phase I
C-VISA-BikDD Liposomes Proapoptotic BikDD Pancreatic cancer Phase I
gene plasmid DNA
Note: AccurinsTM (BIND Therapeutics, Inc., Cambridge, MA, USA).
Abbreviations: NP, nanoparticles; PEG, poly(ethylene glycol).
3.5.4 Nanotechnology and Immunotherapy
Cancer immunotherapy is a therapeutic strategy in which the body’s own defense

mechanisms can become activated to fight against the abnormally fast growing
cells by reinforcing the immune system. Nowadays, this method has attracted a
lot of attention because of the discovery and application of different synthetic and
natural immunogenic materials (Egilmez, 2012; Gajewski, 2013; King, 2008). The
three most common methods for cancer immunotherapy include the application of
immunoadjuvant materials for non-specific treatment, vaccination using antigens
and the application of monoclonal antibodies (Chandramohan, 2013; Gedeon,
2014; Monjazeb, 2012; Nembrini, 2011). Non-specific immunotherapies can be given
as a single therapy or at the same time with another treatment, such as chemo- or
Nanotechnology and Immunotherapy 317
radiotherapy for improving the therapeutic responses. Interferons and interleukins

(ILs) are the most common non-specific anticancer immunotherapies loaded inside
nanoparticles for activating different immunological pathways (Rosenberg, 1988).
Cancer vaccination is another approach to help the body fighting against cancer
(Nembrini, 2011). Vaccines contain an agent that resembles a disease-causing
environment and are able to improve the immunity against a particular disease. While
traditional vaccines are often made from dead or weakened forms of the microbe or
their toxins, nowadays the investigation is more focused on the development of new
vaccines made from surface proteins and DNA of the immunogenic cells (Kalkanidis,
2006; Silva, 2013; Singh, 2007). The immune system can recognize the immunogenic
molecule, keep a record of it, and more easily recognize and destroy any of the cells
containing the immunogenic molecule (Signori, 2010).
Monoclonal antibodies (mAb) can be used for immunotherapy through a number
of mechanisms, such as blocking the function of the targeted molecules, antibody
dependent cellular cytotoxicity effect, inducing apoptosis in the cells expressing the
target antigen, and increasing the phagocytosis of the target cells by macrophages
or by modulating the signaling pathways of the target cells (Chandramohan, 2013;
Ferris, 2010; Gedeon, 2014). In addition to immunotherapy, mAb can be applied for
cancer targeting and diagnosis (Ma, 2013; Tao, 2012).
3.5.4.1 Nano-based Cancer Immunotherapy
One of the main shortcomings of the current immunostimulatory materials is the

lack of dendritic cell (DC) targeting and short time immunostimulative responses, as
the concentration of the immunogenic molecules declines in the body over a short
period of time. In addition, existing immunoactivating compounds mostly elicit
Immunoglobulin G (IgG) isotype Ab and are not able to induce the secretion of various
Ab isotypes (Mallapragada, 2008). Thereby, induced immunotherapeutic protections
are not long-lasting. Accordingly, new strategies are essential to be developed for
more efficient activation of the immune system against cancer.
In general, the ideal properties of a good anticancer immunotherapeutic
formulation are high safety, the capability of eliciting the desired immune responses
after a single dose, absence of a booster dose, absence of the premature drug release,
simple and affordable preparation process, easy administration and scaling-up
process, and high physicochemical stability of the immunogenic agents and excipients
throughout the process, storage and administration (Oyewumi, 2010). To combine all
these properties in one formulation, nanoparticles have been suggested as versatile
systems capable of improving the biological effects of the immunostimulatory
molecules via different mechanisms. One of the benefits of nanovaccines is that
the morphology, size distribution, entrapment efficiency, release kinetics and other
physicochemical properties, which affect the obtained immune responses can be
controlled, leading to the successful development of promising vaccines (Kendall,

2006). In addition, the systemic severe side effects of high dose administration of the
immunostimulants, such as toll-like receptor ligands (Heikenwalder, 2004), can be
minimized using nanoparticles. Nanoparticles can also reduce the needed dose and
limit the non-specific immune responses (Diwan, 2004).
Nanomaterial-based immunotherapy is a relatively new interdisciplinary field
holding great promise by combining materials science, chemistry and immunology.
The immunostimulative biomolecules can be either captured within or conjugated
on the surface of the nanoparticles (Nembrini, 2011; Xiang, 2013). The former method
offers distinct advantages, such as reduced dose of antigen, efficient uptake and
processing by antigen presenting cells (APCs), increased stability during storage and
long-term immune response to the therapy (Foster, 2010; Katare, 2003). Although the
entrapment of immunogenic biomolecules within the particles is offered as the best
possible protective strategy, the main drawback of this method is the unavailability
of the loaded antigens upon administration because of the slow drug release profiles
(Kazzaz, 2000). In addition, the loaded bio-immunogenics can physically or chemically
degrade during the loading process (Jung, 2001). This method also causes a lower
extent of immunity compared to the nanoparticles that have immunostimulative
molecules chemically conjugated or physically adsorbed on their surface with the aim
to induce rapid and short immune responses. However, this method also suffers from
low stability and rapid degradation (Kalkanidis, 2006; Sloat, 2010).
It is currently known that the immunostimulatory products with large hydrophobic
structures are more immunostimulative than hydrophilic compounds. Therefore, it
is proposed that microbially derived adjuvants can be replaced with hydrophobic
nanomaterials. These nanosystems can also simultaneously serve as delivery vehicles
for the immunostimulative molecules (Kipper, 2002), with the aim of facilitating
single-dose vaccination and eliminate the need for booster shots through sustaining
the release of immunotherapeutic payloads and potentiating their effect via the
intrinsic adjuvanticity of the nanoparticles. In addition, most of the nanoparticles
applied for cancer immunotherapy possess high safety, and controllable rate of
degradation for the antigen release (Kersten, 2004). Owing to these benefits, it can
be possible to avoid the need for surgical removal of cancer tissues and circumvent
the disadvantages of conventional anticancer formulations by combining chemo- and
immunotherapeutic approaches using such nanostructures.
Below we discuss the adjuvanticity of nanoparticles as well as the current
progresses in the development of nanovaccines, particularly those with high potential
to be used for cancer therapy.
3.5.4.2 Nanoparticulate Adjuvants for Cancer Immunotherapy
Adjuvants are immunogenic compounds capable of accelerating and extending the

immunostimulative response of biomolecules. Currently, alum salts are the most widely
used immune adjuvants (Correia-Pinto, 2013), owing to their potential in triggering
the so-called “inflammasome” mechanism in the cells, that leads to the release of
danger signals and subsequent secretion of pro-inflammatory biomolecules, resulting
in the activation of the immune system (Marrack, 2009). Despite the popularity of
immunogenic alum salts and other conventional adjuvants over the last few decades,
they suffer from major limitations, such as adverse local reactions, degradation
during freeze-drying, lack of inducing cellular immune responses and necessity of
multi-dosing to reach long lasting protection (Correia-Pinto, 2013). Hence, for cancer
immunotherapy, the scientific community has been developing nanoparticulate
adjuvants that are able to show intrinsic immuno-adjuvanticity and also to act as
vehicles for the delivery of antigens and immunotherapeutic biomolecules (Park, 2013).
This strategy can provide an opportunity for simultaneous humoral and cell-mediated
immunity induction, which can lead to improved therapeutic effects (Rappuoli, 2011;
Wu, 2006). Nanoparticles may also assist the interaction of the delivered antigens with
APCs, enhancing the antigen-based immune responses (Hamdy, 2011; Park, 2013).
Moreover, co-encapsulation of anticancer drug molecules with immunostimulative
biomolecules can be obtained for synergised multifunctional purposes (Roy, 2013).
Accordingly, cancer nanovaccines have recently attracted a lot of interest owing
to their unique properties to overcome the limitations of immuno-therapeutics,
including low interaction with APCs, inherent instability of biomacromolecules and
lack of cross-presentation to T lymphocytes (Hamdy, 2011; Park, 2013).
The impact of nanoparticles on the maturation of DCs for cancer immunotherapy
is usually evaluated by studying the expression of co-stimulatory molecules and the
major histocompatibility complex (MHC) classes I and II bioreceptor molecules, the
production of cytokines, and the activation of signalling pathways (Almeida, 2014;
Klippstein, 2010). For example, the effects of poly(lactic-co-glycolic acid) (PLGA)
nanoparticles on the maturation of DCs were studied in mouse spleens (Reischl,
2009). The results showed a dose-dependent expression of co-stimulatory molecules,
such as CD80, CD86, CD40, and MHC class I, as well as enhanced secretion of
inflammatory cytokines and chemokines, such as tumor necrosis factor-α (TNF-α)
and interleukin-6 (IL-6). In addition, increased activity of mitogen-activated protein
kinase and nuclear factor–κB (NF-κB) signalling pathways was observed. Joshi
(Joshi, 2013) have also demonstrated that the delivery of vaccine antigens with an
appropriate nanoparticulate adjuvant can stimulate the immune responses against
cancer, reducing tumor growth and improving the survival rate in mice. Accordingly,
the authors developed an amphiphilic polyanhydride copolymer based on 1,8-bis(p-
carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane
(CPH) with inherent adjuvant properties for immunogenicity, antigen-loading
properties and antitumor activity. It was shown that mice treated with ovalbumin-
encapsulated CPTEG:CPH particles elicited very high CD8+ T cell responses on days 14
and 20, leading to a delay of the tumor progression and extended survival time. These
results suggest that the immunoadjuvant properties of the nanoparticles can enhance
antigen-specific immuno-cellular responses, and thus, they can be potentially applied
for the promotion of antitumor responses in cancer patients.
3.5.4.3 Nanoparticle Based DC Targeting for Cancer Immunotherapy
Nanoparticles have attracted more attention than microparticles for DC targeting

because of the inverse relationship between the efficiency of the uptake by DCs and
the particle size (Hamdy, 2011). Moreover, positively charged nanoparticles are more
preferred since they can highly interact with the immune cells via binding to the
negatively charged surface of the cells (Dobrovolskaia, 2012; Gjetting, 2014). After
the internalization of nanoparticles into the DCs, loaded cancer cell antigens can
be released in endosomes, degrade, and eventually bind to the MHC II molecules
on the surface of the DCs to present the antigenic molecules to the CD4+ T cells
(Trombetta, 2005). If the nanoparticles escape from the endosome and release their
immunostimulative antigens in the cytoplasm, antigens degrade to small peptides by
the proteosomes and, finally, form a complex with the MHC I in order to be recognized
by the CD8+ T cells (Davis, 2004; Trombetta, 2005). Finally, activated T cells can
recognize cancer cells expressing antigens on their surface and destroy them. These
explanations show the substantial role of the nanoparticle uptake by DCs for the
subsequent immune cell activation. For example, Ma (Ma, 2012) have shown that
tumor antigenic peptides loaded in PLGA nanoparticles can be highly colocalized in
the DCs in 30 min (Fig. 3.5.11) and induce significantly stronger antitumor cytotoxicity
of T lymphocytes than the free antigen peptide.
DC targeting can be achieved via both passive and active processes. The efficiency
of the anticancer nanovaccines to passively target DCs is strongly dependent on
the size, surface charge, hydrophobicity/hydrophilicity, and the interaction of
nanoparticle with the plasma proteins and cell-surface receptors (Bachmann, 2010).
It has been reported that nanoparticles with a size smaller than 500 nm are taken-up
more efficiently by the DCs (Foged, 2005). In addition, a pre-clinical study performed in
mice suggested that the nanoparticle size of 40–50 nm was optimal for nanovaccines
(Fifis, 2004). However, nanoparticles up to 300 nm have already demonstrated
effective induction of the CD4+ and CD8+ T cell responses (Fifis, 2004; Kasturi, 2011;
Reichert, 2011; Shima, 2013; Tomic, 2014). Positively charged particles are taken-
up more efficiently than those with a negative or neutral charge by the DCs in vitro
(Foged, 2005). However, some in vivo studies have revealed no significant difference
in the immunostimulatory effect between positively and negatively charged particles
(Yotsumoto, 2004). Some studies have also shown that the particles with positive
surface charge can immobilize the nanovaccines in negatively charged components

presented in the extracellular matrix of the cells through electrostatic interactions
(van den Berg, 2010), resulting in the inhibition of the immunostimulative responses,
owing to the reduced tissue penetration.
Figure 3.5.11: Confocal microscopy imaging of the PLGA nanoparticles in human DCs. (A) 4′,6-
Diamidino-2-phenylindole channel. (B) Fluorescein isothiocyanate channel. (C) 4′,6-Diamidino-2-
phenylindole and fluorescein isothiocyanate channels overlaid. (D) 4′,6-Diamidino-2-phenylindole,
fluorescein isothiocyanate, and reflection channels overlaid. Reprinted with permission from (Ma,
2012).
Since DCs have the intrinsic ability of phagocyting foreign material, passive DC
targeting for cancer immunotherapy is possible by directing the nanoparticles to sites
rich in DCs. For this purpose, the route of administration of the nanoparticles is very
important. For example, while subcutaneously administered small nanoparticles (<
100 nm) can be quickly transported into the lymphatic system to interact with the
DC localized in the lymph node (Cubas, 2009; Manolova, 2008; Reddy, 2007), larger
nanoparticles (> 500 nm) stay for a longer time in the injection site of the skin and
predominantly interact with the DCs or monocytes available in the skin (Manolova,
2008; Moon, 2012). To avoid the size dependency of passive DC targeting to the lymph
node, direct injection into the blood is suggested as an efficient way to reach both
blood and splenic DCs and induce strong immune responses by a variety of particles
(Perche, 2011; Tacken, 2011). However, the macrophage uptake is still one of the main
drawbacks of the cancer immunotherapy using this strategy.
DCs also express many cell surface receptors, such as mannose (Chenevier, 2002;
Ramakrishna, 2004), CD11c (Cruz, 2014), DEC-205 (Cruz, 2014), Langerin (Flacher,
2010), DC-SIGN (Cruz, 2010; Varga, 2013), clec9A (Caminschi, 2008), and DCIR2
(Dudziak, 2007), which can facilitate actively targeted cellular endocytosis of the
nanoparticles via appropriate surface modification with the ligands that target these
receptors on the surface of the DCs (Foged, 2002). It has already been reported that the
conjugation of such targeting moieties on the surface of the nanoparticles increases
their uptake by the DCs and enhances the DC maturation, as evidenced by the high
secretion of cytokine and up-regulation of CD83 and CD86 surface maturation markers
(Kempf, 2003). The type of the target receptor can have a substantial effect on the
immunological response of nano-immunotherapeutics. A study by Dudziak showed
that the targeting antigens to DEC-205 results mainly in cross-presentation of antigens
to the CD8 T cells, whereas antigens targeted to DCIR2 are preferentially presented via
the MHC class II molecules to the CD4 T cells (Dudziak, 2007). In addition, Idoyaga
(Idoyaga, 2011) have shown that, while antigens can be targeted to the splenic CD8α+
DC subset by antigen conjugation to antibodies against Langerin and DEC205, DCIR2
antibodies can specifically target CD8α‒ DCs. In addition, it has been proved that
effective active DC targeting can be achieved by reducing the nonspecific interaction
of the nanoparticles with plasma constituents and other cells in the bloodstream,
by introducing a hydrophilic biomaterial consisting of PEG onto the surface of the
particles (Hillaireau, 2009).
Polysaccharides (Weber, 2010), PLA (Primard, 2010), PLGA (Binjawadagi, 2014),
polyanhydrides (Camacho, 2011), and polyphosphazene (Garlapati, 2012) are among
the most widely studied nanomaterials prepared for immunostimulative purposes.
Different techniques have been employed to prepare the nano-immunotherapeutics
using the abovementioned polymers, including spray drying, emulsification/
solvent evaporation, and coacervation (Kalkanidis, 2006; Oyewumi, 2010). All
of these methods can produce polymeric nanoparticles with a large surface area
that improve the interaction between immune cells and the immunostimulative
payload (Bachmann, 2010), leading to enhanced uptake of antigens by DCs and
improved immune responses (Silva, 2013). The main reason for the great interest in
immunostimulatory potential of the abovementioned nanoparticles is the superior
biocompatibility, versatile chemistry, high protection of the loaded biomolecules, high
loading efficiency, efficient endocytosis and high presentation of the immunogenic
molecules (Binjawadagi, 2014; Camacho, 2011; Garlapati, 2012; Jiang, 2005; Primard,
2010; Weber, 2010). For example, some of these nanocarriers have shown endosomal
escape properties, potentiating the proteosome-dependent processing of the
immunogenic payload and cross-presentation through MHC class I (Jia, 2013; Silva,
2013). Another benefit of these nanoparticles is the ability to control the release pattern
of the loaded immunostimulative compound and to act as an adjuvant and increase
the therapeutic effect of the formulation (Cohen, 1994; Nandakumar, 2012; Oyewumi,
2010). For example, Gómez (Gomez, 2006) have reported the ability of poly(methyl
vinyl ether-alt-maleic anhydrate) (PMVE-MAh) nanocarriers for vaccination using

OVA as a model antigen. The results showed higher humoral immunity (IgG titers)
in response to the OVA-loaded nanoparticles compared to the alum, indicating the
potential of PMVE-MAh for antigen delivery and improving immune responses
(Fig. 3.5.12). Despite all the advantages nanomaterials render to immunostimulative
formulations, several hurdles, such as reproducibility, stability during production,
and appropriate methods for non-thermal sterilization, need to be taken into account
for developing proper immunotherapeutic approaches (Nandedkar, 2009; Sharma,
2009). For example, the reproducibility of the formulation can be affected by the
variation in the size of the nanoparticles, which in turn can modify the immunogenic
property of the final product (Fifis, 2004).
Figure 3.5.12: Anti-OVA IgG1 and IgG2a titers in sera of female BALB/c mice after intradermal immu-
nisation with 10 μg of ovalbumin as follows: ovalbumin solution (OVA), ovalbumin adsorbed to Alum
(OVA-Alum), blank nanoparticles (NP), ovalbumin coated nanoparticles (NP I), ovalbumin encapsu-
lated nanoparticles (NP II) and 1,3-diaminopropane cross-linked ovalbumin encapsulated nano-
particles (NP III and NP IV). The antibody titer is defined as the reciprocal dilution giving an optical
density. Reprinted with permission from (Gomez, 2006).
All the above-mentioned examples demonstrate the high potential of nanoparticles

to synergistically improve the immunostimulative effect of antigens and
other immunogenic molecules. However, using nanoparticles as anticancer
immunoadjuvants and the delivery of immunostimulative biomolecules still suffers
from several important drawbacks, including the problems associated with the scaling-
up of the system, expensive preparation processes, and limitations for producing
sterile products (Sangha, 2007). Accordingly, despite the great versatility and
promising features observed for the potential of such anticancer therapeutic systems,
intensive research studies are still needed in order to develop nano-formulations that
can be produced in large scale and applied in the clinic.
3.5.5 Cancer Imaging, Diagnostics and Multifuctional Nanosystems
The early diagnosis of tumors through screening based on imaging might contribute
to the reduction in the mortality for certain types of cancers. Real-time imaging is a
most valuable technique that enables visualization of the tissue morphology and cell
function, thus allowing the identification of pathological situations that generally
cannot be aquired in in vitro analysis (Fass, 2008). Imaging techniques are being
combined with nanotechnology, rendering, in many cases, extraordinarily sensitive
and powerful diagnostic and imaging tools that, by other means, were not available
with small or microscale tools, thus enhancing the tissue contrast or allowing
the identification of specific biological changes (Toy, 2014). As it is necessary to
specifically target the tumor sites in order to achieve a more successful outcome out
of the treatment, it is also of utmost importance that the diagnostic and imaging tools,
such as those extensively used in the medical practice: Magnetic Resonance Imaging
(MRI), Computed Tomography (CT) and Positron Emission Topography (PET), are
reliable enough to facilitate the right medical decisions, which can be improved by
alliyng them to nanotechnology (Lee, 2013a; Perez-Medina, 2014).
Contrast agents are frequently employed to assist and complement the
visualization of abnormalities in a diagnostic image, interacting with the incident
radiation to yield noticeable alterations in the final aquired images. They increase
the power of the imaging techniques since there is an increased sensitivity, allowing
the diagnosis of many diseases that are not possible to detect using conventional
methods (Power, 2011). However, nanotechnology is impacting this area of diagnostic
imaging by opening new avenues of novel imaging techniques, and by allowing
the enhancement of the sensitivity, pharmacokinetics, pharmacodinamics and
biocompatibility features of several contrast agents, as well as providing a high signal-
to-noise ratio (Rosen JE, 2011). It is becoming inevitable to not associate imaging and
therapeutic properties in a single nanoplatform. Multifunctional nanocarriers are
becoming more and more fashionable and many studies have been focusing on the
production of multistage nanoparticulate systems.
The employment of nanoparticulate systems to image and diagnose cancer has
been extensively applied and new approaches have been developed (Choi, 2012).
Some advantages of using nanoparticulate-based probes for imaging purposes is
attained to their low cytotoxicity profiles and physicochemical properties (Santos,
2013a). In general, a surface area-to-volume ratio and effective surface functionality,
are particularities used as tools to tune the nanoparticle properties for tissue or
cell targeting in vivo through high affinity to certain cell biomarkers (Davis, 2008).
Biochemical changes in vivo, such as enhanced receptor density in a certain tumor
Cancer Imaging, Diagnostics and Multifuctional Nanosystems 325
type could be imaged and measured by probing with specific nanoparticulate systems
avid to bind to it (Jin, 2014; Satpathy, 2015). These probes shall attain certain features
that allow them to be detected and promote their accumulation at the site of interest,
be safe and biocompatible, as well as to have limited side effects and to avoid the
hostile environments encountered in vivo, such as avoidance of interactions with
plasma proteins and the recognition by the phagocytic cells and consequent removal
from the systemic circulation by the mononuclear phagocyte system (Santos, 2013a).
For example, superparamagnetic iron oxide nanoparticles (SPIONs), along with many
other clinical applications, are some of the nanoparticulate systems that are most
used as highly effective contrast agents for MRI diagnosis of solid tumors (Ittrich,
2013; Rosen, 2012). Compared to other traditional contrast agents, SPIONs exhibit
several advantageous properties, among them the greater magnetic signal strength
and longer lasting contrast enhancement, low cytotoxic effects, biodegradability and
improved delineation of tumor margins (Corot, 2006; Varallyay, 2002; Wang, 2001).
Such nanoparticulate systems have been used to target solid tumors, either in a passive
way, (Zolata, 2014), taking advantage of the leaky and damaged tumor vasculature
through the enhanced permeability and retention effect (Brannon-Peppas, 2004;
Rosen, 2012), by targeting actively the tumor sites by attaching a targeting ligand to
the surface of the SPIONs, or by taking into account the tumor pathological features,
such its lower pH microenvironment. The internalization of SPIONs enables a longer
and effective imaging time by improvement of the contrast-enhancing effect of the
particles through the accumulation of a high number of SPIONs in the tumor tissue,
therefore being a more promissing and sensitive molecular diagnostics approach than
passive targeting imaging. To make it possible to biofunctionalize the SPIONs, it is
necessary to provide them with a surface coating system in order to create a desirable
platform for conjugation of targeting ligands or to make the SPIONs sensitive to tumor
characteristic microenvironment (Fig. 3.5.13) (Jin, 2014; Xie, 2010).
Figure 3.5.13: Schematic representation illustration of the polymer micelle composite system of
surface-engineered SPION for imaging and therapy applications. Adapted from (Jin, 2014).
Recently, several studies utilizing SPIONs for the active targeting of several types
of cancer were conducted and showed promising results (Kievit, 2012; Ling, 2014;
Melancon, 2011; Zhang, 2014a; Zhang, 2014c). “CLIO” nanoparticles, which are
SPIONs cross-linked with a dextran coating, have appeared as a powerful multistage
nanovector for targeted imaging and diagnostic applications, allowing a wide and
versatile conjugation of targeting moieties and compatible with diagnostic imaging
MRI, optical and PET techniques (Tassa, 2011). Imaging and diagnosis can be achieved
with this nanoscaled platform, as well as the delivery of therapeutic cargos to the tumor
sites, making multifunctional systems or “theranostic” (Choi, 2012). For example, in
a recent study, CLIO nanoparticles were developed with the purpose of producing a
multifunctional platform. Taking advantage of the eminent levels of MMPs in the tumor
microenvironment, in particular MMP-14, which has an important role in the tumor
invasion features and provides a direct cellular target for prodrug activation (Devy,
2009; Landry, 2005), it was found that functionalization of the CLIO nanoparticulate
system with the MMP-14 cleavable peptide-conjugate of azademethylcolchicine leads
to vascular collapse of the tumor (Reyes-Aldasoro, 2008). This theranostic nanovector
provided an opportunity to image selectively the tumor site and, at the same time,
track the drug payloads delivered to the tumour site. In addition, the conjugation of
a drug molecule to nanoparticles allowed for a higher amount of drug cargo to be
delivered, leading to a more effective therapeutic outcome and, again, decreasing the
associated toxicity of the anticancer drug to healthy tissues (Ansari, 2014).
PSi nanomaterials are other example of materials that have been proven to be
biocompatible and possess remarkable features for biomedical applications, including
imaging and drug delivery (Liu, 2013b; Santos, 2013b; Shahbazi, 2012). Besides the
vast medical applications (Park, 2009; Santos, 2014; Shahbazi, 2012), and taking into
account a special property of this material, its photoluminescence (Anglin, 2008;
Hua, 2005), PSi has been explored for clinical diagnosis and imaging applications
(Godin, 2011; Santos, 2013a), such as acting as a self-reporting drug delivery systems
using optical imaging in living animals (Cheng, 2008), multicolor near infrared
imaging in live mice (Erogbogbo, 2010), incorporation of gadolidium (Ananta, 2010)
and SPIONs inside the nanoporous structure for MRI applications (Kinsella, 2011), as
well as radiolabeling of PSi with radioisotopes for imaging purposes (Bimbo, 2010;
Sarparanta, 2012).
Liposomal formulations have also been used as theranostic nanoplatforms for
both cancer therapeutics and imaging, considering its safety and wide clinical use.
Recent studies included the encapsulation of Magnevist, a contrast agent for MRI
with in vivo contrast-enhancing effects into liposomes, which showed, together
with doxorubicin, an improved biocompatibility when coated with hyaluronic acid
ceramide polymer, aimed for targeted imaging and drug delivery (Park, 2014). SPIONs
have been successfully encapsulated inside immuno-liposomes targeting endothelial
cells in tumor vasculature and applied in MRI imaging (Zhang, 2014a). Multimodal
fluorescent labeled liposomes with thermo-responsive release properties of the
Conclusions and Future Prospects 327
contrast agents are some of the more advanced imaging platforms fabricated so far
(Kokuryo, 2014), and together with near infrared (NIR) fluorescent dyes, have been
similarly loaded inside theranostic liposomes with improved label efficiency (Xie,
2014).
The abovementioned nanoparticulate systems are revolutionary for the diagnostic
and imaging fields, but other materials such as gold nanomaterials, carbon nanotubes
and silica nanoparticles have also shown light absorbing properties, that when
scattered or emited, yield specific types of diagnostic/therapeutic signals, e.g. under
ultrasound, heat, Raman or fluorescence signals. These types of nanoparticles may
function as theranostic nanoparticles on their own and/or can be associated with
other molecules for increases in targeting and selectivity (Choi, 2012).
3.5.6 Conclusions and Future Prospects
Nanotechnology has provided the opportunity to explore new clues for the diagnosis
and treatment of various diseases, in particular, cancer. With the advances of
nanomedicine, the direct use of nanoparticles in prevention, diagnostic and treatment
of cancer, has become a reality. In the past decades, the use of nanotechnology
for simultaneous drug delivery, imaging and diagnostics has grown exponentially.
There is a great evolution of the biological and physical resources to improve the use
of nanoparticles for finding the best treatment for cancer. The controlled size and
multifunctionality are the main reasons for the growing applications of nanomedicines
as anticancer agents. Nanoparticles can synergistically improve the immunostimulative
effect of antigens and other immunogenic molecules. However, using nanoparticles
as anticancer immune adjuvants and delivery of immunostimulative biomolecules
still suffers from several important drawbacks, including the problems associated
with the scaling-up of the system, expensive preparation processes, and limitations
for producing sterile products
Tremendous and fast advancements are being achieved to produce better and
improved nanosystems able to provide precise information for a better understanding
of the tumor diseases, as well as for facilitating medical decisions in the clinic,
adding therapeutic functions to the novel nanoplatforms that can, in the near future,
change the practical course of the treatment and diagnosis of cancer. The bench-
to-bedside translation of the nanomedicines and technologies have introduced a
new era in the design and development of innovative but simultaneously complex
targeting nanoparticles for delivery of therapeutic and diagnostic agents to tumors.
Accordingly, despite the great versatility and promising features observed for such
anticancer therapeutic systems, intensive research studies are still needed at the pre-
clinical level in order to develop nano-formulations that can be produced in large
scale and applied in the clinic.
Acknowledgments: The Academy of Finland (projects numbers 252215 and 281300),

The University of Helsinki Research Funds, The Biomedicum Centrum, The European
Research Council under the European Union’s Seventh Framework Programme
(FP7/2007-2013) ERC Starting Grant no. 310892, The Drug Research Doctoral
Programme of the Faculty of Pharmacy, University of Helsinki, and The Finnish
Cultural Foundation (grant no. 00130129) are acknowledge for financial support.
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Index
absorption 32, 35-36, 62, 65-66, 69-76, 79, aromatic ring 17

107-108, 110, 115, 117, 137, 143, 190, 193- Aromatic Substitutions V, 15, 17
194, 218, 222, 224, 245-248, 264, 266, Aspirin V, 24-25, 44
271-274, 282 Astrocyte 164
accuracy 65, 75, 81, 84, 110, 112 ATP 12, 33, 42, 155-156, 164, 180-181, 235
acetylation 24 4-aminoquinoline 284
acid VII, 5-8, 10, 14, 16, 24-25, 27, 29, 31, 33- 8-aminoquinoline 279-280, 288
34, 40, 44, 58, 105, 110, 114, 116-118, 139, ADME VI, IX, 62, 65-66, 69, 75-76, 80-81, 88,
147, 150-151, 154, 160-161, 166, 168, 173- 107-108, 110, 113, 115, 117, 245, 265, 269,
175, 178-182, 184-190, 192-196, 198, 200- 282, 286
201, 204, 215, 217-222, 225-227, 230, 233, adolescent 160, 169, 173
242, 244, 249, 251, 253, 255, 258, 260-261, adult 131, 138, 144, 161, 175, 235, 287-288
266, 274, 281, 296, 302, 314-315, 319, 326, Alzheimer’s disease VIII, 153, 156, 167, 171-174,
329, 332-333, 337-339 237, 242-243
Acidity constant 6 Amino-Acids 59, 242
acids V, VII, 2, 5-7, 23-24, 33, 35, 37, 137, 139, AMPA receptor 167, 169, 173, 241-244
155, 172, 178-196, 198, 200-204, 206, 208- arginine 126, 178, 201, 220, 231, 260-261
212, 214-228, 249, 254-255, 258, 261, 264, atherogenic 132
266, 271, 273, 295, 312, 314 base V, 5-7, 13, 18-20, 26-27, 30, 32, 110, 139,
Active Targeting X, 292, 303-304, 310, 312, 195
326, 329 bases V, 5-7, 19, 24
acyl 5, 12, 23-24, 27, 31, 33, 35-36, 42, 186 behaviour 122, 145, 175, 207
acyl-CoA 33-34 bicarbonate VII, 132-134, 139-140, 143-144
adiponectin 129, 137-138 Bimolecular Elimination V, 19
adipose tissue 134, 137-138, 147 Bimolecular Nucleophilic Substitution V, 10
ADME VI, IX, 62, 65-66, 69, 75-76, 80-81, 88, binding site IX, 154, 256-257, 260, 271
107-108, 110, 113, 115, 117, 245, 265, 269, Bioanalysis VI, 62, 64-66, 68, 70, 72, 74, 76,
282, 286 78, 80-112, 114, 116-118
adrenocorticotropin hormone (ACTH) 120 Biomaterials VIII, 207-209, 216-219, 223-226,
alcohols 5 228, 332, 334-336, 338-339, 342
aldehydes 5, 20, 22, 25, 52, 55, 195 Biomedicine VIII, 142, 197, 199, 201, 203, 205,
aldol reaction V, 25, 27 207, 209, 211, 213, 223, 333, 339
alkenes 8, 13 Biopolymers VIII, 206, 218, 226, 274
allylic carbocation 12, 14 biotin VI, 41-43
amides 5, 182, 184, 186 blood-brain barrier (BBB) 245
amines 5, 22 blood pressure 132-134, 136-141, 144, 146-147
amino VII, 17, 22-23, 37-38, 40, 44, 139, 150- brain VII, VIII, 32, 76, 111, 115-116, 119-131,
151, 154, 160-161, 172, 178-198, 200-204, 148-152, 154-156, 158, 160-176, 181-182,
206, 208-212, 214-228, 233, 242, 249, 251, 193, 217, 219-222, 224, 229, 234-238, 240,
253-255, 258, 260-261, 264, 266, 277 244-245, 248, 256-257, 264, 271-272, 274,
amygdala 121, 149-150, 159, 167 315, 329
anhydrides 24 brainstem 121, 161
antalarmin 120, 126 Brønsted-Lowry 5
antidepressants 119, 123-125, 128-130 Cahn, Ingold and Prelog 8
anti-inflammatory response 122 carbamic acid 29
aromaticity 15-16 carbanion 17, 20
344 Index
carbinolamine 27 CRH System VI, 120

carbocation 11-14, 18-19 cyclooxygenases 24
carbon atom 2, 4, 11, 28 cysteine 31-32, 37, 44-46, 48-55, 57-59, 178,
carbonyl V, 5, 17, 20, 22-27, 29-31, 33-34, 49, 201, 226
51, 54, 57, 114, 195-196, 219 cytochrome P450 (CYP) 247
carboxybiotin 42 cytokine 122, 124, 128-129, 131, 322
carboxyl 29, 42, 182, 202, 260 cytokine response 122, 129
carboxylation VI, 41-42 1,25-dihydroxycholecalciferol 137
carboxylic acids 5, 23, 258 calcium VII, VIII, 132-134, 136-147, 150-151, 155-
catalysis 5, 24, 27, 35 156, 159, 169, 175, 182, 230-244
Chirality V, 8-9 cargo 203, 206, 216, 225, 326
chiral molecules 9 covalent VI, 44-48, 50-56, 58-59, 185, 208
calcium VII, VIII, 132-134, 136-147, 150-151, 155- cyclization 190, 225, 249-250
156, 159, 169, 175, 182, 230-244 cytotoxicity 53-54, 203, 220, 295, 302, 317,
cancer IX, X, 62, 74, 105, 143-145, 188-189, 191, 320, 324, 334-335, 338
215, 217, 219-224, 226, 290-292, 294-322, dapsone 184-185, 225, 281
324-342 decarboxylations 18
carrier 31, 185, 192-194, 197, 215, 218, 228, defensins 198, 202, 222, 225, 227
292, 299 deficiency 14, 134, 136-137, 142-143, 146, 277
Catecholamine 120, 168, 172 dehydration 23, 25-26
catechol-O-methyltransferase 134, 141 dehydrogenation 18, 33
central nervous system (CNS) VI, 121, 230, 248 delocalization of electrons 15
cerebrospinal fluid (CSF) 119, 123, 163 deprotonation 6, 20, 23, 37
children 135, 143, 336 diagnostics X, 214, 290, 292, 294, 324-325,
chloride 18, 133-134, 139-140, 143-144, 161 327, 333, 338, 340
chloroquine 52, 276-278, 284, 287 Diastereomeric resolution 10
cholesterol 132, 137, 200-201 diastereomers 9-10
Circadian measures 123 dimethylallyl tryptophan 17
cis V, 8, 32, 217, 266, 269, 273 dipeptide 50, 189, 191-192, 195-197, 219, 222,
cis-configuration 8 225-227, 261, 263-265, 269, 271
Claisen condensation V, 26 diphosphate ion 14
clearance 62, 76, 78, 105, 108, 110, 211, 247- dipole moment 37
248, 265, 267, 273, 289, 305, 308, 313, 328 distribution 4, 62-63, 65, 69-74, 76, 78-79, 105,
clinical development 64-66, 79-80, 118, 279, 110, 115, 152, 173, 175, 193, 222, 245, 282,
316 294, 297, 305, 317
clinical trial 145, 288 disulfide V, 31, 44, 202, 204, 208, 217, 300
coefficient of variation (CV) 84 Disulfide Bridges V, 31, 202
coenzyme VI, 29, 33-34, 39-40 dopamine 148-151, 157, 165-176, 181, 272, 274
combination therapy X, 280, 288, 301, 328, double bond 4, 8, 14-15, 19, 33-34
342 Doxycycline 281, 289
concerted step 11 drug absorption 75, 108, 193, 247-248, 272,
condensations 6, 18, 23, 25 274
configurations 8 drug delivery VII, IX, X, 185, 209, 216-218,
conjugate base V, 6, 19-20 220-227, 273, 290, 292, 294-299, 301, 304,
conjugated unsaturation 15 308, 313, 315, 326-342
core stress system VII, 120, 123, 125 drug design V, VII, 1, 58, 63, 182, 224, 271-272
corticosterone 122-123, 134, 142, 175 drug discovery and development (DDD) 63
corticotropin-releasing hormone (CRH) 119 drug-drug interaction 74, 78
cortisol 121, 123, 139, 145 drug stability 303
CRHR1 gene 123 dyslipidemia 132, 134, 136, 144
Index 345
E V, 5, 8, 10, 13, 17-20, 25, 29, 32, 43-44, 46, formal charge 13
48, 53, 56-59, 112-116, 118, 125-132, 140- fructose 27, 134, 137, 141, 146
148, 161, 166-169, 171-176, 180, 182, 184, fumaric acid 8
186, 192, 194, 200-204, 216-228, 239, 243- Functional Groups V, 2-8, 10, 12, 14, 16, 18, 20,
244, 246-249, 251, 257, 261, 263-265, 267, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
271-272, 278, 286-289, 291, 295, 304-305, 182, 204
310, 314, 327-332, 334-341 GABAergic VII, 149-150, 153, 159-164, 168, 171,
E-isomer 8 173, 238
electron density 5, 13, 17 gas chromatography (GC) 64
electron donating 17 gemcitabine 188-190, 226, 334-335
electronegativity 5 geometry 4, 32, 200, 217, 253, 255, 328
electron-poor 7 Gliotransmitters VII, 164, 166
electron rich 13 global burden of disease 119, 129
electrons 5-7, 13-17, 20 glucocorticoid 130, 136, 138-139
electron withdrawing 17, 184 glucocorticoids 120, 124, 131, 134, 136, 138
electrophile species 10 glutamate VII, VIII, 129, 154-164, 166, 168-174,
elderly 80, 133, 142, 146, 166 181, 229-243
Electrophiles V, 5-7, 59 guidelines for validation of bioanalytical
electrophilic V, VI, 13-14, 16-17, 20, 22-23, 42, methods 84
44-45, 48, 52-56, 58 11β-hydroxysteroid dehydrogenase 136, 138,
Electrophilic Addition Reactions V, 13, 22 145
electrophilic aromatic substitution V, 16-17, 42 heart rate 120, 134, 136, 143-144
Eliminations Reactions V, 18 hepatic function 114
empty orbitals 15 hepatitis C 124, 127-128, 246
Enantiomerism V, 9 heptapeptide 257, 261, 263, 269, 272
enantiomers 9-11 heteroaromatic 16
endoperoxide 52-54, 57-58, 280-281, 287 heterolytically 11
endoperoxides IX, 52, 280, 285-286 high performance liquid chromatography (HPLC)
endothelial dysfunction 133-134 64
enolate 26-27 high-throughput screening (HTS) 66, 246
Enzymatic resolution 10 Hippocrates 119
enzyme 10, 13-14, 24-25, 27, 30, 33, 35-39, homeostasis 126, 132-134, 137-140, 143-145,
44-46, 48, 52-53, 56, 69, 108, 115, 151, 160, 147, 155, 161, 170, 313
186, 188-189, 208, 218, 222, 224-225, 231, Huckel rule 15
234, 242-243, 246, 297, 335 Huntington’s disease VIII, 234, 238, 243
epinephrine (EPI) 122 Hybrid Drugs VI, 51, 53, 55
ester 10, 24, 26-27, 117, 184, 186-192, 204, 218, hybrid orbitals 4
220, 222-223, 226-227 hydride 18, 21, 30, 39
esterification 24, 33, 189-190, 192, 227 hydrogen carbonate 6-7, 139
ethers 5 hydrogen ions 5
European Medicine Agency 62, 79 hydroxyl 17, 23-24, 29-30, 33, 35-36, 182, 189
excitotoxic VIII, 156-158, 162-163, 167-169, 230, hypercortisolism 123, 131
234-235, 237-241 hypertension 133-134, 138, 140-144, 146-147
excretion 62, 65, 69, 79, 133, 137-139, 142, imines 20
245, 272, 282 immediate-early gene 121
FAD VI, 33, 40-41, 237 immune cells 122-123, 320, 322
fatty acids V, 2, 33, 35, 181 immune system VII, 122, 124, 128, 197-198,
fear-related behavior 120 236, 292, 316-317, 319, 329, 339
fight, flight, freeze or fawn response 120 induction 69, 75, 115, 117, 134, 145, 241, 319-
Food and Drug Administration 62, 75, 105, 236 320, 333, 336, 341
346 Index
inductive effect 11 lipid 133, 136-137, 141, 144, 200, 218, 221, 227,
inhibition VI, 36, 43, 45, 50, 52-53, 57-59, 69, 247, 295, 335
115-117, 120-121, 129, 135, 137-140, 151, liquid chromatography (LC) 64
156, 161-164, 169-170, 172, 175, 218, 225- liquid-liquid extraction (LLE) 112
226, 244, 259-260, 262, 321, 329-330 liver 44, 53, 66, 111, 132, 134, 137-138, 144,
in silico 64, 66, 115 189, 211, 218, 225, 247-248, 255, 265, 279,
intermediate 11, 13, 15-16, 19-20, 27, 53, 84, 291, 331
180, 191, 215 lone pair electrons 15
internal standard (IS) 87 lower limit of quantification (LLOQ) 84
International Conference on Harmonisation lymphocyte 216, 335, 337
(ICH) 79 macrophage 123, 321
Intramolecular Addition V, 14 magnesium VII, 132-137, 139-147, 154, 231-233,
in vitro VI, 38, 51-53, 55, 64, 66, 70-76, 80-81, 235, 238
84, 88, 106-113, 116-117, 125, 130, 189, 191, Major depressive disorder (MDD) 119
193, 196-197, 206, 211, 219, 223, 225, 241- marker 134
242, 246, 249, 265, 267, 273, 279, 282, market VIII, 63-64, 80, 211, 213-214, 216, 223,
288-289, 295, 302, 310, 314, 320, 324, 227, 270, 274, 281, 290, 294, 341
329-331, 336, 340 mass spectrometry (MS) 64
in vivo VI, 53, 64, 66, 75-78, 81, 84, 87-88, mefloquine 278-279, 281, 287-288
106-111, 113-114, 116-118, 170, 173, 189, melanoma 124, 130, 224, 336-337
191-194, 197, 206, 209, 211, 216-217, 242, membrane 63, 66, 74, 83, 107, 109-110, 135,
246, 263, 266, 282, 288, 294, 301-303, 151, 154, 156-157, 159-163, 192-194, 197,
306, 310, 313, 320, 324-326, 329-331, 334, 199-203, 215, 217-218, 221-224, 229-230,
337-342 232-233, 235, 238, 241-243, 247-248, 250,
isopenicillin-N synthase 37-38 259-260, 262, 272, 280, 286, 295, 305,
IUPAC 2, 42 314, 341
imaging X, 170, 175-176, 214, 290, 292-294, meso structures 9
296, 315, 321, 324-335, 337-342 metabolic acidosis 139, 141
imidazolidin-4-one 195-197, 217-218, 220, 227 metabolic stability 107, 111, 113, 226, 245-246,
immune surveillance 122 251, 265-267, 269, 271, 282
immunomediators 122, 124 metabolism V, 2, 14, 33, 39, 62, 65, 69-74, 79,
immunotherapy X, 290, 294, 316-321, 323, 108, 112-113, 115-118, 125, 130, 135-136,
328-332, 334-337, 339-340 138-139, 141-144, 146-147, 149, 160, 168,
inflammation 122, 124, 130-132, 136, 138-139, 170, 180, 185, 191, 215, 222-223, 228, 245,
146, 274 247-249, 264, 270-273, 280, 282, 284-285,
insulin 133-139, 141-147 288, 301, 332
interleukin-6 (IL-6) 122, 136, 319 metabolites 63-64, 66, 76, 79-81, 84, 87-88,
kainate receptors VIII, 154, 230-232 106, 108-116, 181, 185, 189, 192, 194, 207,
β-keto ester 27 209, 215, 257, 273, 276-277, 279-280, 285,
ketones 5, 20, 22, 25, 52, 55, 195 288
kynurenine pathways 125 metabolites in safety testing (MIST) 108
β-lactam 35-36, 264 mice 50, 58, 76, 110-111, 120, 130, 137, 144,
leaving group 11, 17-19, 23-24, 29, 33, 35, 192 147, 167, 170, 174, 192, 195-196, 232, 243,
leptin 134, 138, 144 263, 271-273, 287-288, 307, 319-320, 323,
leucocyte 123 326, 329, 333, 336, 341-342
Lewis acid 6-7 Michael acceptors VI, 45-46, 48-49, 53, 57-59
Lewis base 6-7 microglia 124, 164, 174
limbic system 120, 152 microsomal stability 66, 76, 114
linalyl diphosphate 14 microsomes 108, 111, 265, 273
linear 106-107, 135, 201-203, 250 molecular orbitals 4
Index 347
molecule 2, 5-6, 9, 13, 18, 20, 23, 26, 41, 54, 198-204, 206-209, 211-219, 221-228, 237,
87, 154, 160, 180-181, 195, 241, 271, 299, 246, 249-261, 263, 265, 267, 269-274, 314,
301, 315, 317, 326, 335, 340 320, 331, 335, 338
Monoaminergic VII, 148-149, 151, 153, 165 Peptidomimetic IX, 53-54, 118, 188, 197, 227,
monocyte 124, 130, 216 245-246, 248-254, 256, 258, 260, 262,
mood disorder 119 264, 266, 268, 270-274
mood ratings 124 P-glycoprotein 107-108, 115, 247, 273, 278, 291
multifunctional 290, 298, 301, 303, 319, 324, Pharmacokinetics VI, 62, 64-70, 72, 74-76, 78,
326, 331, 333-336, 340-342 80-82, 84, 86, 88-118, 209, 217-218, 221,
multiple-reaction monitoring (MRM) 107 223, 274, 282, 287-288, 324
NAD VI, 29-31, 34, 39-40, 42 phosphates 5, 11, 23-24, 151
neuroendocrine 119-121, 127, 130 plasma 64, 66, 69, 76, 79-81, 108-118, 123,
neurovegetative functions 121 125, 127, 129, 133-137, 139-142, 151, 154,
neutrophil 58, 225 156-157, 159-160, 162, 175, 190, 195, 197,
noradrenergic neurons 121 200, 211, 215-216, 226, 242, 247-248, 278,
nucleophiles V, 5-7, 20, 44-45 285, 312, 320, 322, 325, 336, 341
nucleophilic addition reactions 20-21, 23 plasmodium 52, 57, 59, 221, 275, 286-289
Nucleophilic Aromatic Substitutions V, 17 polarised 7
Nucleophilic Substitution Reactions V, 10-12 β–position 20
Nanomedicine 290, 292, 315, 327-330, 333- potassium VII, 132-134, 138-144, 146-147, 168
338, 340-341 precision 65, 75, 81, 84
nanoparticles X, 203, 221, 290-292, 294-296, preclinical 50, 112, 115-116, 118, 185, 211, 217,
298, 300-302, 304-308, 310-323, 325-342 223, 243, 290, 303, 313, 338
Nanotechnology IX, X, 290-292, 294-296, 298, pre-clinical development 87
300, 302-304, 306, 308, 310, 312, 314, primaquine 194, 217-221, 225, 227, 279-280,
316-324, 326-332, 334-338, 340, 342 285-289
nanovaccines 317-321, 336 prodrug VII, 54, 58, 114-116, 182, 184-186, 188-
natural mineral-rich waters VII, 132-133, 139 195, 215, 218-227, 326, 336
neurodegenerative diseases 168, 234, 240 protonation 20, 23, 38, 299
neuromodulation 165, 272 protons 5-6, 33
neuropeptide 246, 256, 264, 272 proline 178, 184, 186, 201, 224, 260-261, 266
neurotransmissor 181 pseudopeptides 246, 254-255, 270, 273
nicotine 138, 144 quality control 63, 81, 105
obese 135-137, 139, 141, 143-144 quinidine 281
obesity 132, 134, 138, 141-142, 144-145, 147 quinine IX, 275-278, 280-281, 284, 286, 288
oligopeptide 63, 107, 187, 190, 195, 207, 220, quinoline IX, 277-279, 287-288
224 racemates 10
optical rotation 9-10 Racemic mixtures 10
overlapping 13 radical 18, 37, 41, 55-56, 180, 195, 239
overweight 136, 139, 142 rats 76, 110, 113, 115, 128, 130, 134, 136-138,
β-Oxidation Pathway V, 14, 33 141-142, 144-147, 175, 257, 263, 274, 329
Oxidations V, 28 reactive oxygen species 137, 169, 234-235, 239
parasympathetic nervous system 121 redox reactions 28-29
Parkinson VIII, 153, 157, 166-167, 170, 172-174, Reductions V, 28, 126
229, 234, 239-240, 243 relative standard deviation (RSD) 84
particle size 106, 320, 331-332 renal function 114
passive targeting X, 292, 304-306, 308, 325 resistance 35, 51, 59, 62, 74, 105, 134-139, 141-
patient 81, 135, 156, 236, 294 144, 146-147, 188, 190, 195, 198, 222-223,
penicillin V, VI, 2, 35-38 228, 275-278, 280, 286-288, 291, 301-303,
peptide VIII, IX, 35-36, 48, 58, 188, 191-195, 305, 329, 332, 335-336
348 Index
resonance 5, 12-16, 20, 27, 36, 170, 324, 328, Th1 123-125, 129, 332
334 Th3 129
resonance structures 5, 27 Th1 cytokines 125
retinol 32 Th1 patterns 124
reversed-phase 88, 106 Th2 patterns 124-125
Rupintrivir VI, 50-51 thiazolidine 35-37
safety 51, 63, 65, 75, 79-80, 108, 114-115, 182, thioesters 5, 24, 34
276, 281, 317-318, 326 thiols V, 5, 31, 46
saturated fatty acids 35 toxicokinetics 79, 114
Schiff base 32 trans V, 8, 32, 34, 168, 170, 217
Semicarbazone V, 22 trans-configuration 8, 35
simple bond 4 transesterification 24-25
Single-reaction monitoring (SRM) 107 transforming growth factor beta (TGF-ß) 125
solid-phase extraction (SPE) 111 transpeptidase VI, 35-36
stable anion 14, 18, 23 Trier Social Stress Test 122
stereocenters 9 triple bond 4
stereogenic centers 9 targeting VIII, IX, X, 50-51, 58-59, 162-163, 165,
Stereoisomerism V, 8-9 188, 190, 193, 207, 209, 214-215, 217-218,
stereospecific 10, 27, 33, 35 222, 224, 229-230, 232, 234, 236, 238-
steric hindrance 19, 312 240, 242, 244, 256, 264, 274, 290, 292,
stress, chronic 132 294, 297, 303-315, 317, 320-322, 324-332,
stress system VI, VII, 119-121, 123, 125-126 334-342
stronger acid 6 tetraoxane 53-54
substrate 10-11, 36, 39, 42, 45, 109, 128, 186, therapeutic VIII, 44, 48, 58-59, 63-64, 80-81,
193-194, 215, 222 124, 126, 143, 153, 164, 168, 172, 185, 188-
sulfide 180 189, 195, 197, 206, 209, 211-216, 218, 222-
schizophrenia 127, 149, 152, 155, 158, 162-163, 223, 225, 227-228, 236, 242-243, 245, 247,
166-173, 175 274, 277, 279-280, 290-292, 294-295, 297,
sclerosis VIII, 229, 234-236, 242-244 301, 303, 305, 308, 310, 316-317, 319, 322-
Secondary Structure Mimetics IX, 250-251, 272 324, 326-327, 329, 331-333, 336, 339-341
self-assembling peptides VIII, 207, 209-210 thiol 33, 37, 44, 47-48, 50, 57, 133, 179, 182
serotonin 129, 145, 149-151, 153, 167, 169-170, thyroid 138
173-175, 181 tryptophan 17, 129, 151, 169, 178, 201
serum 79, 81, 116, 130, 133, 135, 137-139, 142- ultra-high pressure liquid chromatography
146, 194, 313 (UHPLC) 106
sodium VII, 114, 133, 138, 140, 142-144, 146, Unimolecular Elimination V, 18-19
168, 215, 230-232 Unimolecular Nucleophilic Substitution V, 11
Sprague-Dawley 134, 137, 142, 145 α,β-unsaturated carbonyl system 20
Stress-induced LC activation 121 unshared electrons 6
stroke VIII, 155, 166-168, 170-172, 229, 234, urocortin 120
238-240 Ussing chamber 107-108
structure-activity relationships (SARs) 249 vacant low-energy orbital 6
sulphadiazine 281 valacyclovir 186-189, 194, 222
sulphadoxine 281 valganciclovir 186-187, 194, 221, 226
sympathetic nervous system 120-121, 123, 136 Validation VI, 65, 75, 81-88, 112-115, 117-118,
tandem mass spectrometry (MS 64 142, 274
t cell 123, 320, 330, 338 vascular 79, 136-138, 141, 144-145, 220, 304-
α-terpineol V, 14 305, 326, 330-331, 335, 338, 340
tertiary carbons 11 vicinal carbons 11, 18
tetrahedral 13 vinylic systems 8
Index 349
Vision Pathway V, 32
vitamin A 32
volume of distribution 63, 76, 78, 105
weaker acid 6
Western diet 139-140, 142
Wistar 134, 136, 138, 142, 144
World Health Organization 22, 132, 142, 289,
341
wound healing 122, 207-208
Z 8, 57-58, 88, 107, 114, 116-118, 128-129, 141,
144, 168, 172, 198, 218, 220, 222, 224, 227,
242, 271, 274, 329-330, 333-334, 337, 339,
342
Z-isomer 8

Biomedical Chemistry - Current Trends and Developments

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Biomedical Chemistry - Current Trends and Developments

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Nuno Vale

Biomedical Chemistry: Current Trends and Developments

Managing Editor: Anna Rulka

Language Editor: Reuben Hudson & Michael Jones

Copyright © 2015 Nuno Vale and chapters’ contributors

Bibliographic information published by the Deutsche Nationalbibliothek

Managing Editor: Anna Rulka

Cover illustration: © Thinkstock / Garsya

Section 1: Chemical Principles in Drug Design and Discovery

1.1 Functional Groups of Biomolecules and their Reactions 2

1.2 Designing Covalent Inhibitors: A Medicinal Chemistry Challenge 44

Section 2: Chemical Basis of Drug Action and Diseases

2.1 Pharmacokinetics and Bioanalysis to Improve Drug Development 62

2.2 Translational Research in Endocrinology and Neuroimmunology Applied to

2.3 Understanding the Metabolic Syndrome Using a Biomedical Chemistry

2.4 Brain Neurochemistry and Cognitive Performance: Neurotransmitter

Section 3: Strategies to Develop New and Better Drugs

3.2 Targeting Calcium-mediated Excitotoxicity in the CNS 229

3.4 Synthetic Vs. Natural Bioactive Compounds Against Tropical Disease

3.5 Current Trends and Developments for Nanotechnology in Cancer 290

1.1.1 Functional Groups in Biological Systems

The main definition of a functional group in organic chemistry books is as a chemically

Diana C. G. A. Pinto, João M. P. Pereira, Artur M. S. Silva: Departamento de Química da Universidade

© 2015 Diana C. G. A. Pinto, João M. P. Pereira, Artur M. S. Silva

C=C double bond (alkenes)

aromatic ring (arenes)

carboxyl (carboxylic acids) R-oxycarbonyl (ester) aminocarbonyl/carboxamide

Combination Hybrid result Geometry

These hybridisations have several consequences, such as the electron density of a

The electronegativity of an element can also be an important factor to explain some

1.1.2 Acids and Bases Versus Electrophiles and Nucleophiles

Scheme 1.1.3: Acidity constant and pKa.

in organic and bioorganic transformations. Electrophiles are either positively charged

Functional group Example pKa

alkylammonium ion 10.66

alcohol CH3CH2OH 16.0

1.1.3 Stereoisomerism and Chirality

Stereoisomers having the spatial orientation of bonds as their unique difference,

1.1.3.1 Cis/trans Isomerism

Cis/trans stereoisomerism exists in alkenes (double bonds) and saturated rings

1.1.3.2 Chirality and Enantiomerism

Figure 1.1.3: trans-1,2-Dimethylcyclopropane is an example of a molecule with enantiomers. The 3D

It is worthy to note that enantiomers only differ by reflection, so their physical

1.1.4 Common Mechanisms in Biological Chemistry

1.1.4.1 Nucleophilic Substitution Reactions

Nucleophilic substitution reactions occur when a group attached to an sp3 carbon

1.1.4.1.1 SN2 – Bimolecular Nucleophilic Substitution

This type of mechanism (Scheme 1.1.5) is called a bimolecular nucleophilic substitution

Scheme 1.1.5: General reaction for an SN2 reaction mechanism.

1.1.4.1.2 SN1 ‒ Unimolecular Nucleophilic Substitution Reactions

Scheme 1.1.6: General reaction for a SN1 reaction mechanism.

A carbocation may be easily formed on tertiary carbons because the carbocation is

Scheme 1.1.7: SN1 reaction mechanism of geranyl diphosphate forming geraniol.

1.1.4.1.3 Phosphate Group Transfer – the Grey Area of Nucleophilic Substitutions in

Scheme 1.1.8: Glucose phosphorylation.

In this reaction, the phosphorus electrophilicity is reinforced trough chelation with

1.1.4.2 Electrophilic Addition Reactions

Scheme 1.1.10: Electrophilic addition reaction mechanism.

1.1.4.2.1 Synthesis of α-Terpineol ‒ Intramolecular Addition

Scheme 1.1.11: Synthesis of α-terpineol from linalyl diphosphate.