Drug Discovery

and Development
New Advances

Edited by Vishwanath Gaitonde,

Partha Karmakar and Ashit Trivedi
 Drug Discovery and
Development - New
Advances
Edited by Vishwanath Gaitonde,
Partha Karmakar and Ashit Trivedi

Published in London, United Kingdom

Supporting open minds since 2005
Drug Discovery and Development - New Advances
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Edited by Vishwanath Gaitonde, Partha Karmakar and Ashit Trivedi

Contributors
Marta Vázquez, Natalia Guevara, Marianela Lorier, Pietro Fagiolino, Iris Feria-Romero, Sandra
Orozco-Suarez, Grace Mugumbate, Lorraine Yamurai Bishi, Dr Sundeep Chaitanya Vedithi, Tom L.
Blundell, Donato Bonifazi, Angelica Intini, Giovanni Migliaccio, Carsten Wrenger, Arne Krüger,
Thales Kronenberger, Vinicius Gonçalves Maltarollo, Thuluz Meza-Menchaca, Rossana C Zepeda, Claudia
Juárez-Portilla, Mallika Alvala, Aaftaab Sethi, Khusbhoo Joshi, Sasikala K, Mohammed Chyad Al-
Noaemi, Vishwanath Gaitonde

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Drug Discovery and Development - New Advances

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 Meet the editors

Dr Vishwanath Gaitonde is currently appointed as a Senior Re-

search Scientist at Cambrex High Point, NC, USA, where he de-
signs, develops, and optimizes chemical processes for multi-kilo
scale cGMP and nGMP production campaigns to support clinical
trials in Phase I – Phase III. Prior to joining Cambrex, Dr Gai-
tonde was engaged in optimization of a lead molecule targeting
broad spectrum viral respiratory disease in the Antiviral Me-
dicinal Chemistry Department, GlaxoSmithKline, NC, USA. Dr Gaitonde’s PhD
research was focused on the development of carbohydrate-based molecules as bio-
chemical tools to probe the Mycobacterium tuberculosis GlgE mechanistic aspect.
Additionally, his PhD research involved development of a novel amorphous polyes-
ter material based on sustainable chemistry.

Dr Partha Karmakar is currently appointed as a Research Scien-

tist at the Washington University School of Medicine where his
research involves development of a new therapeutic strategy for
cancer treatment including development of small molecules and
bioconjugates probes and an in-vitro and in-vivo efficacy study
emphasizing clinical translation. Dr Karmakar was previously
engaged in development and optimization of a lead molecule
targeting the bacterial colony growth mechanism in the Antibacterial Drug Dis-
covery Department at GlaxoSmithKline – PA, USA. Dr Karmakar’s PhD research
involved development of glycopeptide-based vaccines and their in-vivo immuno-
logical evaluation.

Dr Ashit Trivedi is currently appointed as a Scientist in the

Clinical Pharmacology Modeling and Simulations group at
Amgen, USA, where his research work is extended into different
therapeutic areas including oncology and neurosciences with a
focus in cardiovascular diseases. As a clinical pharmacologist, Dr
Trivedi participates in multifunctional study teams to design and
conduct clinical pharmacology studies to support the global reg-
istration of pipeline drugs, perform PK and exposure-response analysis to inform
dose selection, summarize and interpret PK, PK/PD, and clinical pharmacology
results for internal/external presentations, study reports, and regulatory documents
and interact with the line-management and different functions to implement strate-
gies for drug label.
Contents

Preface III

Section 1
Introduction 1

Chapter 1 3
Introductory Chapter: The Modern-Day Drug Discovery
by Partha Karmakar, Ashit Trivedi and Vishwanath Gaitonde

Section 2
Computational Chemistry Advances in Modern Day Drug Discovery 11

Chapter 2 13
Past, Present, and Future of Molecular Docking
by Thuluz Meza Menchaca, Claudia Juárez-Portilla
and Rossana C. Zepeda

Chapter 3 27
Molecular Docking in Modern Drug Discovery: Principles
and Recent Applications
by Aaftaab Sethi, Khusbhoo Joshi, K. Sasikala and Mallika Alvala

Chapter 4 49
Computational Deorphaning of Mycobacterium tuberculosis Targets
by Lorraine Yamurai Bishi, Sundeep Chaitanya Vedithi, Tom L. Blundell
and Grace Chitima Mugumbate

Section 3
Current View towards Pharmacokinetics in Drug Discovery 69

Chapter 5 71
Revisiting Pharmacokinetics and Pharmacogenetics of Methadone
in Healthy Volunteers
by Natalia Guevara, Marianela Lorier, Marta Vázquez, Pietro Fagiolino,
Iris Feria-Romero and Sandra Orozco-Suarez

Chapter 6 85
ADME Profiling in Drug Discovery and a New Path Paved on Silica
by Arne Krüger, Vinicius Gonçalves Maltarollo, Carsten Wrenger
and Thales Kronenberger
Section 4
Recent Case Studies: Advances in Drug Discovery Research 117

Chapter 7 119
Successive Drug Therapy for a Very Rare Autosomal Diseases
by Mohammed Chyad Al-Noaemi and Hassan Ali Daghriri

Chapter 8 135
Challenges and New Frontiers in the Paediatric Drug
Discovery and Development
by Angelica Intini, Donato Bonifazi and Giovanni Migliaccio

XII
 Preface

Prevention and cure cases from various diseases reach a billion individuals every
year. This is possible due to the organized efforts in the drug discovery program
across the world led by pharmaceutical companies and academic institutions.
Nevertheless, the majority of deaths globally are attributed to heart disease, stroke,
pulmonary disease, lower respiratory infections, Alzheimer’s, cancer, diabetes,
and tuberculosis. One common aspect of these disease conditions is that they are
preventable to some extent and a comparatively better quality of life can be pro-
vided to the patients with proper and timely treatment. To surmount this challenge,
the drug discovery programs have advanced significantly in the past decade. This
book, “Drug Discovery and Development – New Advances”, brings together some
of the recent progress in the field through the complex process, starting with
computational modeling right through launching a drug in the market. The book
aims to provide expert opinions and experiences from researchers encompassing
diverse key topics critical in drug discovery and development. Case studies of some
selected disease conditions will showcase the challenges and burdens faced by the
pharmaceutical industries. The book covers the latest advances in molecular dock-
ing, pharmacokinetics, and new therapeutic options of some rare diseases along
with pediatric drug discovery and development. Overall, the book represents a view
of the complex fabric of the modern-day drug discovery program.

Dr. Vishwanath Gaitonde

Senior Research Scientist,
Department of Chemical Research and Development,
Cambrex High Point, Inc.,
High Point, NC, USA

Partha Karmakar
Research Scientist,
Washington University School of Medicine,
USA

Ashit Trivedi
Clinical Pharmacology Modeling and Simulations Group at Amgen,
USA
 Section 1

Introduction

1
Chapter 1

Introductory Chapter: The

Modern-Day Drug Discovery
Partha Karmakar, Ashit Trivedi and Vishwanath Gaitonde

1. Drug discovery: a brief outline of 5000 years of history

The history of drug discovery and development is as old as some of the oldest
human civilizations. The practice of Ayurveda in India and traditional Chinese
medicines in China are over 5000-year-old therapeutic traditions that are still
in practice at large. Papyrus Ebers is evidence of medicinal practice in Egypt
about 3000 years ago [1–6]. The Greek and Roman medicines became popular
in Europe and western Asia between ~700 BC and 200 BC [7]. The ancient Arab
medicines were in practice to a great extent until 1500 AD and are still in use in the
Mediterranean gulf [8, 9]. The beginning of modern era medicine can be consid-
ered from the time when Edward Jenner discovered immunization for smallpox.
The development in the field was gradual until Sir Alexander Fleming discovered
Penicillin in 1928; since then, the field of medicinal chemistry and drug discovery
has flourished, and by the end of the twentieth century, it became a complex inter-
disciplinary platform primarily based on synthetic organic chemistry expanding
into various biological specificities [10–13]. As a result, the global pharmaceutical
market strengthened to nearly 400 billion US dollars by the year 2001 [14, 15].

2. Modern-day fabric of pharmaceutical industry

At the beginning of the twenty-first century, drug discovery research faced

new challenges transforming the classical concept of drug development that was in
practice for half a century. With advances in science and technology, the pharma-
ceutical, health care, and IT industry, accompanied by high-pace shifts in the global
economy, bolstered the process of modern-day drug discovery and development to
a large significance. Novel interdisciplinary research involving metal and polymer
nanoparticles, liposomes, antibodies, and neo-antibiotics in both academia and
industries have opened venues for precision diagnosis, targeted drug delivery,
and innovative immunotherapy [16–24]. Although the classical steps in drug
discovery (involving target validation, lead molecule design, chemical synthesis,
pre-clinical evaluation, ADME, clinical trials and development for market of the
pharmaceutical agents) are followed to date, the distribution of funding at each
stage have changed due to the changing global market and healthcare policies [25].
Even though pharmaceutical companies relatively survived the recession phase
of the early twenty-first century, a significant amount of budget cuts in R&D and
new drug development pipeline was evident [26]. Post-recession, in the course
of recovery, the collaborative efforts of the pharmaceutical and IT industry have
brought state-of-the-art analytical tools that can pull multifaceted data in large
quantities and predict the patients’ needs and market trends [27]. This has enabled

3
Drug Discovery and Development - New Advances

the pharmaceutical companies to reorganize the drug discovery and development

programs in a more efficient and cost-effective way. Furthermore, market research
has contributed to global pharmaceutical growth that is projected to reach 1.18
trillion US dollars by 2024 [28].
The main reason of this success is the data-driven integration of every major
component of pharmaceutical industries with the healthcare industries that
includes hospitals, doctors, patients, and insurance companies along with the
regular drug discovery units. This has transformed the classical linear drug discov-
ery road (Figure 1) into a complex multidimensional map (Figure 2), where the
whole industry is revolving around the power of the market analysis in a symbiotic
fashion. Though the specific needs of different companies are different depending
on their size, resources, and target market, the cumulative fabric of symbiosis is
common [29]. The existing market data has accelerated the process of “new target
identification.” It has also helped in repurposing existing and abandoned therapeu-
tics from different phases of drug development in an unprecedented way [30, 31].
Proper analysis of healthcare data and labor market research have shown positive
impact on government policies in allotting and redistributing funds for healthcare
industries and basic academic research that are closely associated to drug discovery
research, which consequently helps the pharmaceutical market to grow [32, 33].
The huge success in genomics research, high-throughput screening (HTS) robotics,
and gene sequencing technologies resulted a pull of publication that have reported
synthesis or extraction of a cumulative of over 90 million drug-like compounds
[34]. Moreover, advances in large-scale cell and tissue imaging have enabled precise
location determination of the drugs and measured variety of phenotypes in cells
and whole organism [35]. These advances in hardware instruments, research
methodologies, and data processing synergistically contribute at various stages
of drug development. The application of deep learning in leveraging these large-
scale heterogeneous database is now an integral part of industrial pharmaceutical
research [36]. Although machine learning (ML) is at its infant stage, it has already

Figure 1.
Classical components of drug discovery.

4
Introductory Chapter: The Modern-Day Drug Discovery
DOI: [Link]

Figure 2.
Modern-day symbiotic fabric of drug discovery.

reduced the library sizes for HTS and helped to understand complex multiomic data
[37, 38]. The rapid progress of different ML methods will have considerable impact
on future therapies [39].

3. Importance of PK/PD in modern-day drug discovery

The historical prototype for clinical drug development was to conduct a few
Phase 1 studies followed by a couple of Phase 2 studies consequently leading to
multiple expensive Phase 3 trials to demonstrate the efficacy of the drug candidate.
With the changing landscape and regulatory requirements, the number of clinical
studies to elucidate multiple questions related to drug properties such as the
mechanism of actions, pharmacokinetics (PK), pharmacodynamics (PD), and drug
metabolism increased overwhelmingly prior to Phase 3 studies. The increase in the
number of clinical trials has made drug development more lengthy and exorbitant.
To overcome this limitation and reach patients promptly, it is imperative to utilize
advanced technologies and approaches. One such approach is the PK/PD guided
drug development. PK/PD modeling has been extensively employed to generate
first-in-human dose predictions and selecting optimal doses for Phase 2 and Phase
3 trials. PK/PD modeling also plays an instrumental role in identifying if any dose
adjustments are needed in special populations such as pediatrics and geriatrics and
patients with hepatic or renal impairments [40, 41]. Additionally, PK/PD model-
informed drug development (MIDD) has gained increasing momentum in recent
years and is extensively used across pharmaceutical industries globally.
MIDD has become a crucial tool after receiving formal recognition in
Prescription Drug User Fee Act (PDUFA) VI, thus paving a path forward to opti-
mize drug dosing prior to approval and post-marketing and in special populations

5
Drug Discovery and Development - New Advances

in the absence of dedicated clinical trials. Dose optimization and clinical trial design
have been most established domains of MIDD; new technologies such as artificial
intelligence, ML, and real-world data (RWD), wearables along data science, have
the potential to transform MIDD.
ML approaches provide a set of tools that can improve decision-making for
well-specified questions with abundant, high-quality data. While using ML in the
early stages of drug design, target selection, and high-throughput screening is
almost standard today, the potential of ML during drug development has not been
recognized. The observed data/evidence obtained during the developmental phase
does not necessarily answer all the questions; thus the scope of MIDD is largely
expanded with analysis of RWD to generate real-world evidence (RWE) to resolve
these unanswered questions. Although RWD is obtained under less-controlled
settings requiring proper interpretation of the findings, it should be considered as
an attractive tool appealing for MIDD [42].
The emerging new techniques, such as portable devices, wearables, and applica-
tions (apps), may improve the dosing accuracy for patients and the quality of the
collected medical information in real-world medical practice. These tools may
improve the quality of electronic health records, making real-world data a reliable
source for drug development and dose optimization or individualization. All these
tools will make real-world data/real-world evidence a more appealing source for
MIDD [43].
Along with the power of data analytics, advances in computational chemis-
try, and new diagnostic techniques, PK/PD modeling tools have also influenced
the drug discovery research and development. These advances assist to build a
comprehensive protein-receptor database, thereby enabling a defined library size
for designing and optimization of a lead molecule. Along with the classical small
molecule drug discovery and development, many protein and antibody-based
pharmaceuticals have appeared as blockbuster drugs.

Author details

Partha Karmakar1, Ashit Trivedi2 and Vishwanath Gaitonde3*

1 Department of Radiology, Washington University School of Medicine, St. Louis,

MO, USA

2 Clinical Pharmacology Modeling and Simulations, Amgen, Thousand Oaks, CA,

USA

3 Chemical Research and Development, Cambrex High Point, Inc., High Point, NC,
USA

*Address all correspondence to: [Link]@[Link]

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License ([Link]
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

6
Introductory Chapter: The Modern-Day Drug Discovery
DOI: [Link]

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9
 Section 2

Computational Chemistry
Advances in Modern Day
Drug Discovery

11
Chapter 2

Past, Present, and Future of

Molecular Docking
Thuluz Meza Menchaca, Claudia Juárez-Portilla
and Rossana C. Zepeda

Abstract

The interface of any given ligand and protein—normally considered a macro-

molecule—of a known or predicted/modeled structure can be computed by deter-
mining each potential ligand position, resulting in an array of possibilities which
are finally expressed in numerical energy values based on their thermodynamic
affinity. Over the past few decades, this premier approach technique has proved to
be crucial as an automated method in drug design and discovery, as well as in other
fields. Data are retrieved from contour surface calculations for each ligand probe
and can be analyzed to delineate regions of attraction on the basis of energy levels.
Negative energy levels from contours are used to infer protein-ligand affinity clefts
and are therefore relevant to drug design. Accordingly, molecular docking, framed
as the “new microscope,” is part of a group of in silico computational techniques
that enable the behavior of molecular chemistry to be analyzed and predicted
in an inexpensive manner. From the starting point of framing the key terms in
the binomial macromolecule-ligand docking approach, this chapter presents an
introductory description of the progress made in this field of research over the past
several years, in addition to present and future perspectives. This chapter presents a
broad plethora of possibilities arising from the old docking alternatives to the cur-
rent software technology and critically dissects and discusses the emerging trends.
Despite the emergence of more degrees of freedom, a number of flexible conglom-
erates have not been well developed, and there are still computational limitations
to solve, including several features in the focused technique. The present goals,
such as molecular flexibility, binding entropy, and the presence of ions and solute
conditions, are revisited with the purpose of anticipating the challenges, goals, and
achievements in this field over the next few years or decades.

Keywords: molecules, modeling, structure, proteins

1. Introduction

In biology, dissimilar molecules dock and interact to enable the perpetuation of

the primordial logistics of living organisms. Molecular docking methodologies can
be used to identify the interaction between a small ligand and a target molecule and
to determine whether they could behave in combination as the binding site of two or
more constituent molecules with a given structure. The comparison of docking mol-
ecules for proteins, other drug-like molecules, or even fragments from the original
molecule enables a pool of prominent candidates to be calculated with listed values.

13
Drug Discovery and Development - New Advances

Interestingly, a wide spectrum of molecular binding interactions can be explored

with this technique, including lipid-protein, lipid-lipid, enzyme-substrate, drug-
enzyme, drug-nucleic acid, protein-nucleic acid, nucleic acid-nucleic acid, protein-
drug, and protein-protein potential affinities, with key functions in every molecular
biological or biochemical stage, as well as structural coupling [1–2].
The analysis of the binding scores between the constituent molecules in molecu-
lar recognition is essential to explain the constitutive processes and subsequently
suggest a possible therapy in the context of a particular disease. The molecular
docking in silico approach seeks the optimization of this process, not only in terms
of techniques but also in relation to time and economic resources. For instance,
there is no microscope with a sufficient power of resolution to capture an image at
the dynamic (real-time) molecular level, and accordingly, theoretical and com-
putational approaches can be used to predict the best binding and most probable
trajectories. Faster techniques and reduced resources are related to efficiency, in
contrast to in vitro approaches, in which the examination of every synthesized and
purified protein can have higher time and material costs. On average, traditional
in vitro research can take about a decade to complete and can cost around 800
million USD; in silico method importantly diminishes these costs [3]. As such, due
to the difficulties in determining the structures of complexes, in silico approaches,
including molecular docking, are suitable for predicting binding modes by investi-
gating thousands of ligand positions using the lowest energy score analyzed.
Since 1975, the development of high-throughput protein purification X-ray
crystallography and nuclear magnetic resonance spectroscopy has continued to
advance, predominantly contributing to a better understanding of the structural
details of macromolecules and complexes with ligands [4]. Molecular docking, as
with many other in silico tools, has become more common and easier to apply to the
field of drug discovery; however, it is not entirely dependent on molecular structure
databases. It is not impossible to work with molecules that are absent from the data-
bases, as they can be modeled by using one or multiple similar structures to build a
novel chimeric output that can mimic the original molecule. In the docking process,
the parameters can be further adjusted to test the function of the drug molecule
versus a particular target molecule.
After the molecular docking has been performed, the software executes a
systematic search on the algorithm, in which the ligand conformation is recurrently
approached until the minimum energy conformation is identified. The final result
will have a negative value of ΔG (U total in kcal/mol), in which a number of electro-
static and van der Waals energy variables will have been synthesized. These energies
are related through the interaction between two molecules. This association allows a
final scoring function to classify the candidate positions through the driving forces
of the specific interactions to be obtained. The structural shape and electrostatic
forces of both the ligand and the target molecule at specific binding-site surfaces
are key aspects in biological complementarity systems. In the drug discovery field,
several key aspects must be considered when predicting whether the molecule
will bind with the receptor target, such as the structural shape and electrostatic
interactions of the protein-ligand, ligand-ligand, or protein-protein. In this sense,
several physicochemical parameters, including the van der Waals forces, Coulombic
interactions, and the formation of hydrogen bonds, play relevant roles. The com-
bination of all these values and potential binding is predicted by a docking score.
Essentially, for drug design, it is possible to use a rigid system in which a rotational
and translational space in six dimensions is explored to fit the ligand into a specific
binding structure site [5].
The constantly growing number of biological targets for the design of rational
structure-based ligands in public databases has gained interest in the research

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DOI: [Link]

community. In the drug discovery field, the essential processes in computational

docking are the design of the ligand and the search for targets of the existing can-
didate ligands. The latter are used to predict a reliable binding affinity, in which the
best possible physicochemical prediction of how the target and ligand will interact
is made. A strategy to enhance the selection of drug candidate ligands is based on
the scores obtained from in silico approaches. These scores not only significantly
reduce the amount of inefficient compounds synthesized but also decrease the
amount of unnecessary biological tests by taking into account valuable information
about crucial binding elements in a given ligand-receptor conglomerate. Molecular
docking approaches are used to calculate the scores of ligand-binding types and
linking affinities. The estimation of reliable ligand-binding associations and modes
is a difficult challenge. During the last few decades, the scientific community has
gradually shown an increasing interest in molecular docking methods, illustrated
by the increase in references and the number of publications in the field [6].
Nevertheless, there is currently no standard consensus regarding the criteria that
should be used to classify a docking mode as correct or incorrect. Most docking
methods are based on the use of general scoring functions to predict molecular
suitability for a wide range of applications. In order to accomplish what is needed, a
reliable scoring function, reasonable protein flexibility, and a treatment for ligand
conformational changes are required.
In the context of molecular biology, the interactions between molecules are key
to understanding the mechanisms that underlie a particular biomedical event. The
latest achievements have been the improvement of computational methods essential
to the process of drug discovery, modeling in the prelaminar stage, and the actual
analysis of putative binding interactions. It is possible to conduct exploratory work
by examining the best score function values or by using a large set of multivariate
experimental data. In both cases, it is possible to analyze how changes in ligands or
macromolecules can have an effect on their interactions by validating the associated
biological processes, with the aim of gaining a better understanding of the interplay
between the biomolecular functions of the bioactive candidates through the char-
acterization of the kinetics and binding score values imperative to their molecular
recognition. In order to better understand the historical and conceptual implications
of the development of this interesting and well-established technique, past and
present achievements must be considered, as well as the current limitations with the
potential to change the course of the technological methods developed in the future.
In comparison to “wet lab” experimental procedures such as, e.g., microarray tech-
nology or even sequencing, virtual screening is inexpensive and efficient. However,
several considerations need to be taken into account [7]. Overall, computational
methods have been a recurrent option due to the focus approximation of the analysis.

2. The development of molecular docking techniques

As one of the most commonly used approaches since the 1980s, the experimen-
tal data obtained through molecular docking techniques have grown at an increas-
ing rate since the approach was first established. Programs configured through
different algorithms for molecular docking analysis have been developed on an
almost yearly basis, significantly improving pharmaceutical research [6]. The first
algorithms were designed for protein-protein interactions. Along with the scoring
function, which is used to determine the best binding poses, algorithms designed to
calculate the best geometrically complementary shapes as rigid bodies are necessary
to identify the most favorable orientations and conformational bindings with the
potential to confer a putative drug candidate.

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Drug Discovery and Development - New Advances

The gradual achievement of more powerful and complex algorithms with the
addition of further parameters has paralleled computational technological advances
over the last few decades. In order to achieve optimum flexibility, in silico methods
use different tools with different approaches. Docking software depends on the
algorithms employed, which comprise three different kinds: systematic, stochastic,
or deterministic.
In the beginning, calculation algorithms that consider docking complexes to
be rigid structures were used. In rigid docking, the objective is to match the ligand
to the protein receptor, with the main aim being the generation of as many poses
as possible in order to achieve the optimum of all poses. Through this process, all
possibilities are considered heuristically to identify a group of complementary
matches that present the most favorable van der Waals forces between the ligand
and the macromolecule receptor. Intermolecular interaction calculations avoid
any flexibility but nevertheless have a level of freedom dependent on a 3x3 matrix
plus the vector rotation. This means that three rotational and three translational
degrees of freedom cover all possible moves in three-dimensional space within the
active site. However, no binding is permitted, as the macromolecular structures are
simplistically represented as solid structures located under a center of mass and
longitude [8].
The earliest work was performed using structural shape contacts, in which the
fitting of outlines enables the best possible complementary configuration between
two proteins to be identified [9]. A little later, a shape matching strategy algorithm
was used by Kuntz and collaborators in UCSF8 to continue searching for possible
configurations using the geometric distance between the ligand atoms and the
macromolecule or receptor spheres (Figure 1).
In this method, the ideal intersection or match between the ligand and recep-
tor is viewed as a “negative image” that represents the active site. The image is
produced by covering the receptor surface region and overlapping spheres with a
solvent, in which a part of the overlapping spheres comprises the actual binding
site. This constitutes the fundamentals of the DOCK search algorithm [10]. A few
years later, Kuntz also developed a more advanced approach by conferring flex-
ibility to the ligand; however, this variant is still categorized as “flexible docking.”

Figure 1.
Top left, binding site; top right, ligand. Down below conjugate with geometrical fitness functional group related
proposed by the earliest docking algorithm model.

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Past, Present, and Future of Molecular Docking
DOI: [Link]

Subsequently, the investigation of HIV-1 protease using this approach was notable
for leading to the technique’s exponential use in drug discovery [11].
Following the pioneering work from Kuntz, a different approach was taken a
decade later in order to develop an improved new geometric recognition method,
which was developed through an algorithm called Fourier transformation [12]. For
the first time, the molecules could be described by a digital model, allowing their
interior and exterior parts to be distinguished. This novel method allows faster
calculation by determining the surface of contact, overlap, and approximation
using the six degrees of freedom. In this method, molecules are considered rigid
bodies, and the changes in structure have the degrees of freedom. This technique
makes it possible to process atomic coordinates, and Zdock represents an example
of this approach. Nevertheless, rigid-body algorithms are very erratic and ineffec-
tive in terms of any structural and conformational change arising due to the inter-
face between the ligand and the receptor. In this context, new alternatives to enable
torsions and angle movement became a matter of interest. In the same period, a new
semiflexible docking innovation was achieved using the HADDOCK protocol [13],
which involves rigid-body docking complemented by semiflexible optimization
in order to describe possible torsion angles in the main backbone and side chains.
Unlike the previous Fourier transformation method [12], which uses a grid, this
method adopts a Cartesian approach with particular coordinates, in which one of
the two molecules is flexible and the solvent can be selected. One of the two mole-
cules therefore needs to be small in order to be computationally possible in terms of
the number of conformational variations. Other methods also attempt to describe
flexible bodies undergoing rotational conformational, rotational, and translational
changes, mimicking the nature of biological molecules. In this category, both the
ligand and the receptor that are modeled by simulating protocols are flexible.
However, the flexibility needs to be lowered to make computational configuration
possible. In the end, flexible docking approaches offer a more precise technique
capable of imitating in vivo behavior of the possible structural conformations.
In flexible docking, there are two different logarithmic approaches, determinis-
tic incremental construction and stochastic. Systematic incremental construction
algorithms are most commonly used, which gradually develop binding predictions
on the basis of all possible ligand-binding poses covering all specified areas, e.g.,
DOCK [14], Glide [15], LUDI [16] FlexX [17], Hammerhead [18], and Surflex
[19], in which on-the-fly incremental ligand construction is implemented. In this
method, the number of analyses grows in line with increases in the degrees of
freedom as part of anchor-and-grow methods. In a different example, in eHiTS, the
ligand is fragmented, and each piece is tested for rigid docking, commonly based
on library screening for the best conformations to religate the fragments and test
their flexibility.
A different approach randomizes probabilistic or stochastic algorithms to
selectively reject or accept configurations through the criteria spectrum, in which
computational efforts are optimized, e.g., AutoDock [20], DARWIN [21], Monte
Carlo [22], and GOLD [23]. By the middle of the 1990s, this technique was the point
of origin of a diverse set of methods that are most commonly present in the genetic
algorithm, named after Darwin’s theory of evolution, in which the ligand is inter-
preted as a chromosome and its fragments are considered genes [24]. Every gene
exhibits conformational behavior due to its torsional/translational nature. During
computational analyses, the information is transmitted and altered through sto-
chastic crossover and mutational events evolving through specific parameters. The
changes improve the conformational binding pose from the ligand and the receptor,
e.g., Lamarckian (AutoDock). In the case of the Monte Carlo stochastic variant that
produces randomized translational conformations, the most thermodynamically

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Drug Discovery and Development - New Advances

stable potential bindings are explored by focusing on the local minimum energy
using a decision criteria parameter that is based on a temperature reaction, called
Metropolis. The flexibility also alternates with rigid rotation, displaying several
parameters at once. A more recent development is the deterministic method, which
has been used for Newton equation simulations and also employs Monte Carlo
methods that can measure trajectories, using Amber, Charm, and GROMACS;
however, this scope forms the focus of the present work, and wide reviews have
been provided by other researchers [25–27].

3. Molecular docking at present: a diverse and common approach

The drug discovery informatics market had an estimated value of 713.4 million
USD in 2016 [28]. The presence of in silico tools that can allow the computation
of data flowing from diverse methodology pathways in parsimony with medical
chemistry can be synergistic in terms of upgrading the market and are well-known
in the scientific literature. In this manner, molecular docking has been consolidated
as a useful technique among sequence analysis platforms, molecular modeling, and
clinical training management. The use of molecular docking in each of these fields
is enhancing drug discovery in the pharmaceutical and biotechnology sector. As
it comprises several stages and workflows, the discovery of new drugs relies on in
silico tools and molecular docking in particular to simplify the overall process.
A crucial factor is the steadily rising number of structures stored in the Protein
Data Bank (PDB). The PDB is the most robust, currently storing over 151,000
structures and counting. The 3D structure information bank includes a large set
of proteins, lipids, carbohydrates, and nucleic acids, in both single structures and
complexes [29]. On the other hand, nearly a hundred different forms of molecular
docking software are available, which offer analogous implementations with vari-
ous implementation options. There has been rapid progress in developing faster
architecture based on graphics processing unit clusters, more adequate algorithms
for optimized computational analysis, and the tracking of ligand-receptor binding
expressed in scoring functions.
Although there is a need to maintain computational equipment, the associated
expenses are certainly lower than the costs of “wet lab” experiments, and molecular
docking is therefore an affordable technique. One of the most challenging tasks
in bioinformatics sciences is undoubtedly the development of new and effective
drugs, which is currently an almost mandatory step before wet lab experiments. In
structure-based drug modeling, obtaining the most accurate and efficient model of
ligand-receptor binding is a crucial step and is a suitable starting point for further
evaluation to test new compounds or drug candidates, but also and no less impor-
tantly, to discard the improbable candidates. Molecular-ligand docking is a signifi-
cant tool in pharmacology at present and an important area of drug discovery that
has comprised a central node of important achievements over the current century.
As an interdisciplinary process of multiple joint efforts mainly from the pharma-
ceutical sector, biotechnological companies, and academic researchers, as well as
many other fields, the process is highly complex and requires the most accurate and
precise tools and methodologies. This has been enhanced by an increasing number
of protein coordinates and the high number of available software programs that are
constantly evolving with more sophisticated levels and a wider field of applications,
in combination with more numerous candidates. In order to discover new drugs, as
well as improve the existing ones, it is necessary to understand the targets as well
as the nature of the possible drug candidates. In silico bioinformatics approaches
have attracted increased interest due to the results of post-genomic era sequencing.

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Past, Present, and Future of Molecular Docking
DOI: [Link]

Figure 2.
Chart bar displaying paper publications per year (1982–2020) (NCBI, accessed on January 12, 2019).

Due to the limited set of protein-coding genes, the complexity is much higher due
to posttranscriptional modifications, prosthetic groups, multimeric complexes,
and other various phenomena, clearly demonstrating the need to better understand
their nature to fulfill biomedical objectives. Interestingly this year’s (2019) publica-
tions account for the first time a pause in the upper trend of docking publication
number (Figure 2). This may be symptomatic on how the future holds already
crucial challenges.

4. Future challenges, endeavors, and perspectives

The drug discovery informatics market is estimated to grow from 1.5 billion
in 2016 to 2.84 billion by 2022 and may continue expanding. Accordingly, there is
currently a rising demand for the discovery and implementation of novel infor-
matics solutions. The major factors driving the expansion of the global market
include the transition from pure research to clinical treatment. More skilled
professionals, interdisciplinary backgrounds, and the high pricing of informatics
software may have a crucial impact on the growing market. At present, a number of
well-established applications have been made available for free or as paid software
or services. However, many challenges remain to be addressed to enable the full
potential of this powerful technique to be realized.
Nevertheless, in the case of pharmacology, the synergistic aspect is an important
chemical phenomenon in which two different biomolecules with different origins
can have an exponential effect in combination that is greater than their separate
effects. If it is determined that a particular structure is more favorable [30] in terms
of the docking score and it may be correlated with synergism, this can be second-
ary, due to the fact that a molecular docking procedure has not been developed to
examine it in a particular scoring function. A linear/quadratic formula could be
developed to measure synergy by discriminating between synergistic, additive, or
antagonistic effects, which can be expressed both qualitatively and quantitatively.
In this sense, further work is needed to investigate how the chemosensitivity
between a macromolecule and ligand could be detected once more than one ligand

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Drug Discovery and Development - New Advances

is included. Although unmanageable amounts of data make this process difficult,

it is possible to analyze the small targets that are the most restricted to the binding
site being examined, especially in drug-protein analysis. System biology models that
depend on a drug synergy test need to be developed in a more comprehensive man-
ner, perhaps by including qualitative features in combination with the quantitative.
In this sense, a novel input could be developed in computational docking analysis to
enable, e.g., the measurement of molecular signaling that has been established to be
part of several components, ligands, or targets. These systematic synergy model-
ing methods could support drug synergy research with the aim of improving the
accuracy of experimental results.
An improvement of the molecular structure databases is necessary for further
development. Filters are needed to ensure the structural models they contain are
of a better quality, as this will influence the reliability of the results. The PDB
was established in 1971 as a pioneer crystal structure database, and today it is
the most common source for molecular in silico modeling, harboring more than
150,000 experimentally proven 3D models. However, there is no guarantee that the
chosen structures are error-free, including even those with excellent geometrical
parameters, and this must be taken into account. High-quality statistics are not an
indication that the structure is perfect. Therefore, an improvement of their quality,
protocols, and validation would allow the construction of better models that could
be valuable in the inevitable task of structure refinement. However, a better model
will not be more informative in terms of more detailed biological information,
which means that the interpretation of a scientist will be necessary. However, the
confirmation of outcomes and the precision of the docking tool in a certain interac-
tion can be tested. Although docking strategies have become more complex, false
positives are a recurrent issue with this technique, and as such, refining the struc-
tures stored in the PDB will undoubtedly lead to an improvement and better results
from pharmacodynamics studies [31].
Those who devote their time to molecular docking are well aware of the large
number of docking techniques. In the years to come, docking experiments will need
to be more consistent in terms of the outputs generated by different docking meth-
ods. Using meta-experimental databases, including a large-scale and diverse variety
of targets and ligands, comparisons of scoring functions have shown that accuracy
and reportability are far from being reached. A standardized common workflow
that follows the same procedures and is associated with the same advantages and
issues is therefore necessary. A streamlined validation process to define standard
test protocols needs to be agreed for every aspect of the docking method; otherwise
there will be a lack of reproducibility in the output process used by each research
group and for each given software [32].
The interaction model of the ligand and the active site must achieve the most
optimum site of recognition. Docking ensembles using rigid proteins can be
slightly inaccurate. Through the ensemble, the protein can fluctuate according to
the relative energy, with more time spent in the lowered energy structure. On the
other hand, the conformations of ligands fluctuate partially, making the whole
ensemble more stable. This can be misleading for dockings that are not flexible,
due to the fact that a given conformation may not be the most stable choice in the
structure. Up-to-date docking scores have been oriented for machine learning
scoring and mainly consist of four building blocks: descriptors, a model, a training
set, and a test set. Currently, SFCscore, NNscore, or RFscore represents promi-
nent examples of nonlinear and nontrivial correlations of data in order to avoid
obstacles to interpretation [33]. Techniques that provide free access to the scoring
function are still a minority and more options are needed, particularly those with
open access. The number of poses needs to be exhaustive; however, this has not

20
Past, Present, and Future of Molecular Docking
DOI: [Link]

been well-established. In this sense, we can state that the sensitivity of the original
conformation of the ligands remains unanswered. Furthermore, in the case of mul-
tidomain proteins, proteins are frequently composed of more than a single effector
domain, and this should be taken into consideration.
With regard to a different aspect, how water is placed around the binding site
is not a straightforward problem to solve, although recent studies have proposed
the use of this parameter as functionally valid in specific contexts [34] within and
around the conglomerate binding site. X-ray crystallography is the most extensively
used tool for predicting 3D conformational structure; however, the actual output is
only partially informative, due to the fact that the density limits are out of resolu-
tion and, on occasion, the electron density can be of insufficient quality. Future
efforts need to endorse novel alternatives to increase the capacity and parameters
that can be used in every aspect of a given analysis, not only in terms of water but
also the physiological solutes found in nature and even protonation, in addition to
the pH potency spectra.
An understanding of the biological functions and roles of a protein in a particu-
lar cell or tissue is highly relevant in determining the role of a protein’s structure,
including all of its functional domains. Genome-wide studies have demonstrated
that multidomains are present in over 70% of eukaryotic proteins. Nevertheless,
protein-folding studies usually consider only single domains and are therefore not
focused on the mechanisms in multidomains that can even influence the folding
structure [34]. Very crucial obstacles are involved in multidomain docking analyses.
In some examples, the understanding of intermolecular movement can be restricted
by rigid docking methodologies that lack the ability to consider the effect of mul-
tiple domains in a single macromolecule. A given protein is not always present in a
static and simplistic single conformational shape but can be present in a collection
of scaffolds, stages, and intersections of conformational shapes. As a consequence,
the free energy landscape can be profoundly affected, distinctively changing the
scoring function’s output. This continues to present a major issue [35].
To improve modeling, the role played by multiple molecules in the context of
a certain reaction is an indispensable step that must be considered. At the current
stage of technology, this does not fall under the current scope of molecular docking,
due to the fact that the processes are far too complex and it is difficult to manage
all of the interactions that occur during a molecular binding and reaction. In order
to mimic how chemistry works in nature, the inclusion of more than two factors
(ligand/macromolecule) where methodologically possible would be a priority to
enable the possible interactions in a molecular group to be predicted. Although a
few software packages use this approach, in the future, it needs to become more
common in other methods to address the binding modes of ligands in assessments
with higher stoichiometry using multiple ligand complexes against the molecular
target. Additionally, as stated earlier in this work, it would be of great interest to
evaluate the synergy of ligand combination conjugates.

5. Conclusion

Over the last four decades, molecular docking has improved quite remarkably,
contributing to the enhancement and improvement of pharmacology in addition
to many different areas of applied and molecular biology. After the first complete
draft of the Human Genome Project was announced in 2003, the scientific com-
munity concluded that there are far fewer protein-coding genes than expected and
it has therefore been swift to study how molecules interact by investigating more
possible target bindings of a given molecule. The increasing demand for molecular

21
Drug Discovery and Development - New Advances

docking has paralleled the revolutionary advancement of its technological back-

ground. Nevertheless, several biochemical and physical properties of proteins,
particularly at the surface of contact, need to be included in docking algorithms in
conjunction with those already present. On the other hand, the question of how
to diminish unnecessary calculations and outputs from undesirable rotations and
therefore translations is a big challenge to be considered in the near future, espe-
cially in virtual screening. The right implementation needs to be standardized, and
closer multi- and interdisciplinary teams must overcome this challenge in order to
fine-tune this already widely explored technique.

Author details

Thuluz Meza Menchaca1*, Claudia Juárez-Portilla2 and Rossana C. Zepeda2

1 Laboratorio de Genómica Humana, Facultad de Medicina, Universidad

Veracruzana, Veracruz, Mexico

2 Centro de Investigaciones Biomédicas, Universidad Veracruzana, Veracruz,

Mexico

*Address all correspondence to: thuluz@[Link]

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License ([Link]
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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Past, Present, and Future of Molecular Docking
DOI: [Link]

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Sanner MF, Belew RK, Goodsell DS,
(In-House, Outsourced), by Services,
et al. AutoDock4 and AutoDockTools4:
by Region, Vendor Landscape, and
Automated docking with selective
Segment Forecasts 2018-2025. 2016.
receptor flexibility. Journal
978-1- 68038-749-0
of Computational Chemistry.
2009;30(16):2785-2791 [29] Berman HM, Bhat TN, Bourne PE,
Feng Z, Gilliland G, Weissig H, et al.
[21] Taylor JS, Burnett RM. DARWIN: A The protein data bank and the challenge
program for docking flexible molecules. of structural genomics. Nature
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[22] Liu M, Wang S. MCDOCK: A [30] Wang X, Song K, Li L, Chen L.

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[23] Jones G, Willett P, Glen RC, [31] Markosian C, Di Costanzo L,

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[32] Rentzsch R, Renard BY. Docking

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25
Chapter 3

Molecular Docking in Modern

Drug Discovery: Principles and
Recent Applications
Aaftaab Sethi, Khusbhoo Joshi, K. Sasikala
and Mallika Alvala

Abstract

The process of hunt of a lead molecule is a long and a tedious process and one is
often demoralized by the endless possibilities one has to search through. Fortu-
nately, computational tools have come to the rescue and have undoubtedly played a
pivotal role in rationalizing the path to drug discovery. Of all techniques, molecular
docking has played a crucial role in computer aided drug design and has swiftly
gained ranks to secure a valuable position in the modern scenario of structure-based
drug design. In this chapter, the principle, sampling algorithms, scoring functions
and diverse available software’s for molecular docking have been summarized. We
demonstrate the interplay of docking, classical techniques of structure-based design
and X-ray crystallography in the process of drug discovery. In addition, we dwell
upon some of the limitations faced in docking studies. Finally, several success
stories of molecular docking approaches in drug discovery have been highlighted,
concluding with remarks on molecular docking for the future.

Keywords: molecular docking, virtual screening, drug discovery, computer aided

drug design, conformational sampling, scoring functions

1. Introduction

The path to drug discovery is a long, challenging & arduous task not to mention
the overburdening finances it demands. As of 2014, the average cost of developing a
new drug from scratch was found to be an estimated $2.5 billion, an increase of
145% from the previous study done by the same organization in 2003. The major
reasons for this drastic increase in the cost is mainly attributed to high failure rate of
drugs among others [1]. Understanding of the drug discovery process is important
to handle the challenges faced by the pharma companies in terms of cost and
innovation.
The process of identifying a target, synthesizing an active compound with
suitable characteristics like minimal toxicity, high bioavailability, cost-effective
synthesis, etc., and finally developing it to introduce in the market is a time-
consuming, extremely complex and risky endeavor [2]. Initially, a target is identi-
fied which plays a key role in progress of the disease. Once a link between the target
and the disease has been established, the next step is to identify potential candidates
which can stop or reverse the progress of the disease [3]. This process starts with the

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Drug Discovery and Development - New Advances

discovery of molecules that show efficacy in a simple screen, called “hits.” Screen-
ing is a process in which normally a large number of compounds from natural
products and online databases are examined for biological activity in high-
throughput assays. This step in the drug discovery process is very crucial and
demands maintaining huge molecular libraries and carrying out thousands or mil-
lions of assays, which leaves the academicians and small pharmaceutical companies
at a disadvantage and also shoots up the cost for larger industries. Next, the “hits”
found are chemically modified to give improved pharmaceutical properties, such
compounds are often called “leads.” But, it is quite apparent that the method stated
above for discovery of a drug has a number of pitfalls. From an academic point of
view, carrying out high throughput screens (HTS) is costly, time-consuming and
not feasible; while, from an industrial perspective, it does nothing to improve the
eminent danger of market saturation.
Truly innovative and blockbuster drugs are what drive the pharmaceutical
industry forward but, over the past few years introduction of new molecular enti-
ties (NMEs) has vastly reduced. For example, in 2007 only 19 NMEs were approved
by the US Food and Drug Administration (FDA), the least since 1983 [4]. Cur-
rently, and even in the future it is expected that only slight modifications of the
existing blockbuster drugs would be carried out which would further aggravate this
problem [5]. HTS would not help in either curbing the rising costs of discovering
hits or the problem of finding truly innovative and blockbuster NMEs, the two
major hurdles that the pharmaceutical industry faces now-a-days.
To overcome these challenges, molecular docking is an exemplary tool. During
the first step to find hits from existing chemicals for a drug discovery and develop-
ment project, virtual screening (VS) is a perfectly viable and an alternative or
complementary approach to HTS for fulfilling the screening of thousands or mil-
lions of compounds within a few days. In addition, the speed of VS helps in kick-
starting projects for newer targets for which no leads are available [6]. Molecular
docking is one of the most applied virtual screening methods and has become
increasingly useful overtime on account of immense growth in 3D X-ray and NMR
structures and their improved resolution (physics and knowledge based docking
algorithms depend on it) reported in the Protein Data Bank (PDB). As an example,
in total 46,541 X-ray structures were reported at the end of 2008 in PDB, but by the
end of 2018 it had grown to a staggering figure of 131,993 [7]. In addition, it is a
resource saving technique which provides accessibility of screening to academia and
small industries which were earlier limited to large pharmaceutical giants.
In this chapter, we will discuss a particular class of molecular design, i.e.,
“Docking” along with the various algorithms, techniques, success stories and limi-
tations related to it. In the end, we will conclude with its scope in the near future.

2. Molecular docking

Two molecules can interact in a number of ways let alone the interaction of a
protein and protein or a protein and small molecule. Molecular docking helps us in
predicting the intermolecular framework formed between a protein and a small
molecule or a protein and protein and suggest the binding modes responsible for
inhibition of the protein. To accurately carry out docking studies one requires the
high-resolution X-ray, NMR or homology-modeled structure with known/predicted
binding site in the biomolecule. To date, 148,827 are available in the database (PDB)
[3]. Docking methods fit a ligand into a binding site by combining and optimizing
variables like steric, hydrophobic and electrostatic complementarity and also
estimating the free energy of binding (scoring) [8].

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Molecular Docking in Modern Drug Discovery: Principles and Recent Applications
DOI: [Link]

There are two basic components which distinguish the variety of docking soft-
wares available to choose from—One is sampling algorithm and the other is scoring
function, these are discussed in detail.

2.1 Sampling algorithm

As pointed out earlier, there are a huge number of modes of binding between
two molecules and even with advances in parallel computing and higher clock
speeds of modern computers it would be expensive and time-consuming to generate
all the possible modes. Therefore, algorithms were needed which could fish out
valuable conformations from the fruitless ones.
Various algorithms were developed in this regard and can be classified by the
number of degrees of freedom they ignore. The simplest of the algorithms intro-
duced treated the molecules as two rigid bodies thereby reducing the degree of
freedom to just six (three translational and three rotational). A very well cited
example of a program using this algorithm is DOCK [9]. This program was designed
to find molecules which had a huge extent of shape similarity to the pockets/
grooves or binding sites. It derives an image of suspected binding sites present on
the surface of the protein. This image consists of several overlapping spheres of
varying radii which touch the molecular surface of the macromolecule at just two
points. The ligand molecule is also considered as a set of spheres which approxi-
mately fill the space occupied by the ligand. Once the respective representations of
the protein surface and the ligand in terms of sphere are complete, the pairing rule
is applied. The pairing rule is based on the principle that ligand sphere can be paired
with a protein sphere if the internal distances of all the spheres in the ligand set
match all the internal distances within the protein set, allowing some user specified
tolerance. Thus, it allows the program to identify geometrically similar cluster of
spheres on the protein site and the ligand. Many other programs were developed
later which make use of such matching algorithm (MA) which include LibDock [8],
LIDAEUS [10], PhDOCK [11], Ph4DOCK [12], Q-fit [13], SANDOCK [14], etc.
All these programs based on MA have the advantage of speed but have several
limitations such as prior need for detailed receptor geometry and lack of molecular
flexibility which does not accurately define many aspects of ligand-protein
interactions.
The second algorithm is that of incremental construction (IC), wherein the
ligand is fragmented from rotatable bonds into various segments. One of the seg-
ments is anchored to the receptor surface. The anchor is generally considered to be
the fragment which shows maximum interactions with the receptor surface, has
minimum number of alternate conformations and fairly rigid such as the ring
system. Once the base/anchor has been established, the next step is to add each of
the fragments step by step. Ideally, those fragments are added first which have a
greater chance of showing interactions like hydrogen bonding since they are direc-
tional in nature and are responsible for specificity of the ligand. In addition, hydro-
gen bonds lead to more accurate prediction of geometry. Once a particular fragment
is added, the poses with the least energies are considered for the next iteration,
making the algorithm extremely fast and robust [15]. IC has been used in programs
like DOCK 4.0 [16], FlexX [15], Hammerhead [17], SLIDE [18] and eHiTS [19],
SKELGEN [20], ProPose [21], PatchDock [22], MacDock [23], FLOG [24], etc. One
major limitation of this program is that it is restricted to medium sized ligands and
is not feasible for large size ligands where the number of fragments generated pose a
problem.
Another useful algorithm is the Monte Carlo (MC) technique. In this approach, a
ligand is modified gradually using bond rotation and translation or rotation of the

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Drug Discovery and Development - New Advances

entire ligand. More than one parameter can also be changed at a time to get a
particular conformation. That conformation is then evaluated at the binding site
based on energy calculation using molecular mechanics and is then rejected or
accepted for the next iteration based on Boltzmann’s probability constant. Accep-
tance or rejection of the conformation is a function of the change in energy with
respect to a parameter T, which can be physically interpreted as temperature (sim-
ulated annealing). This criterion of acceptance or rejection makes this method
strikingly different than the others. Whereas the other algorithm favor decrease in
energy, in MC method increases are also possible. For higher values of T increases
are likely. If one starts at a high value of T, then small energy barriers can be jumped
and the configuration can move beyond local minima and is therefore particularly
useful in situations where a global minimum is sought among many local minima
[25]. An interesting spin-off of the MC approach is the Tabu search, which main-
tains a record of the search space of the binding site which has already been visited
and thus ensures that the binding site is explored to the maximum [26]. MC
approach has been made use of in programs like DockVision 1.0.3 [25], FDS [27],
GlamDock [28], ICM [29], MCDOCK [30], PRODOCK [31], QXP [32],
ROSETTALIGAND [33], RiboDock [34], Yucca [35], AutoDock [36], etc. One of
the major concerns with MC approach is the uncertainty of convergence, which can
be improved by performing multiple independent runs.
Genetic algorithm (GA) is quite similar to MC method and is basically used to
find the global minima [37]. These are much inspired by the Darwin’s Theory of
Evolution [38]. GA maintains a population of ligands with an associated fitness
determined by the scoring function. Each ligand represents a potential hit. The GA
alters the ligands of the population by mutation or crossover. In the first stage, a
new population is created by accessing and then selecting the more fit ligands from
the previous step. The members of the populations are then transformed in the
alteration step. The mutation operator creates new ligands from a single ligand by
randomly changing a fragment in its representation while the crossover operator
exchanges information between two (occasionally more) members of the popula-
tion [39–41]. GA has been incorporated in programs like Autodock 4.0 [42], DAR-
WIN [43], DIVALI [39], FITTED [44], FLIPDock [45], GAMBLER [46], GAsDock
[47], GOLD 3.1 [48], PSI-DOCK [49]. GA has a similar limitation like that of MC
method wherein the uncertainty of convergence is a major drawback.
Another approach is the hierarchical method. In this approach, the low energy
conformations of the ligand are pre-computed and aligned. The populations of the
pre-generated ligand conformations are merged into a hierarchy such that similar
conformations are positioned adjacent to each other within the hierarchy. After-
wards, on carrying out rotation or translation of the ligand, the docking program
will make use of this hierarchical data structure and thus minimize the outcomes.
Let us understand with a simple example—if an atom near the rigid center of the
ligand is found to clash with the protein in a given rotation/translation, then this
approach can reject all of the conformations lying below in the hierarchy to that of
the conformation under scrutiny, because the descendants must contain the same
clash as well [50]. GLIDE software makes use of the hierarchical method [51, 52].

2.2 Scoring functions

Sampling changes among varying degrees of freedom must be performed with

sufficient accuracy to identify a conformation that best matches the receptor struc-
ture, and also must be fast enough to permit the evaluation of millions of com-
pounds in a set computational time. This is taken care by the variety of algorithms
discussed above. Algorithms are further complemented by scoring functions.

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Molecular Docking in Modern Drug Discovery: Principles and Recent Applications
DOI: [Link]

The evaluation and ranking of predicted ligand conformations is a crucial aspect

of VS. When we are interested in only how a single ligand binds to a biomolecule,
then the scoring function needs to predict the docked orientation which most accu-
rately represents the “true” structure of the intermolecular complex. On the other
hand, if we are interested to evaluate multiple ligands, in that scenario the scoring
function should not only identify the “true” docking pose but also be able to rank one
ligand relative to another. Therefore, the design of reliable scoring functions and
schemes which can rank different poses is of fundamental importance [53].
The scoring functions usually estimate binding energy of complex using many
assumptions and simplifications to arrive as close as possible to actual binding
energy in minimum time. Popular scoring functions have an adequate balance
between accurate estimation of binding energy and computational cost in terms of
time. There have been a number of scoring functions developed over the past many
years and can be classified into three main categories—force field, empirical and
knowledge based [54].
Force field functions: force field (FF) scoring functions are developed based on
physical atomic interactions like van der Waals interactions, electrostatic interac-
tions and bond lengths, bond angles and torsions [55]. Force field functions and
parameters are usually derived from both experimental data and ab initio quantum
mechanical calculations according to the principles of physics.
!
Aij Bij qi qj
E ¼ ∑ ∑ 12 � 6 þ � � (1)
i j rij rij ε rij rij

Here, rij stands for the distance between protein atom i and ligand atom j, Aij and
Bij are the van der Waal parameters, qi and qj are the atomic charges and ε(rij) is the
distance dependent dielectric constant.
One common example of a FF scoring function is that of the program DOCK
[56] represented in Eq. (1), where, the effect of solvent is indirectly considered by
the distance dependent dielectric constant e(rij) seen in the Coulombic potential.
One major drawback of this function is that it does not consider an important
solvent effect that charged groups favor aqueous environments whereas non-polar
groups tend to stay in non-aqueous environments, commonly referred to as the
desolvation effect [57]. Ignorance could lead to biased results as the function would
now be totally dependent on the coulombic interactions and would thus favor
highly charged ligands. In other words, it only takes into account the interaction of
protein and ligand, which is inadequate. To build a more robust function one needs
to also evaluate how both interact with water before the formation of the complex
and how water mediates this process.
Later the Shoichet group [58] improved upon the existing function by adding the
effects of the solvent on protein-ligand interactions using implicit solvent models.
They employed the Poisson-Boltzmann approach to model the electrostatic potential
of the protein. The van der Waals interactions were calculated using the Lennard-
Jones potential; the electrostatic interaction between the ligand and the protein was
estimated using a precomputed receptor potential determined with DelPhi [59].
Ligand desolvation penalties were calculated with HYDREN [60]. The solvent-
corrected scores were found to be closer to experimental binding free energies than
the DOCK program scores, but were still too favorable. The overestimation of
complex stability could be due to the neglect of solute entropic terms [58].
There a few scoring functions which be classified in this category such
as DockScore [56], GoldScore [61], HADDOCK Score [62], ICM SF [29],
QXP SF [32], etc.

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Drug Discovery and Development - New Advances

Empirical scoring functions: the basis of this scoring function is that the binding
energies of a complex can be approximated by a sum of individual uncorrelated
terms. The coefficients of the various terms involved in calculation of binding
energy are obtained from regression analysis using experimentally determined
binding energies or potentially from X-ray structural information. Empirical func-
tions have simpler energy terms to evaluate when compared to force field scoring
functions and thus are much faster in binding score calculations.
The first empirical scoring function developed to predict binding free energies
was implemented in LUDI, credited to the pioneering work of Bohm [63]. The
energy was derived using experimental binding free energies and protein-ligand
crystal structures for 45 complexes.
 
ΔGbind ¼ ΔGO þ ΔGhb ∑ f ðΔR; ΔαÞ þ ΔGionic ∑ f ðΔR; ΔαÞ þ ΔGlipo Alipo 
h�bonds ionic int:

þ ΔGrot NROT:
(2)

Here, ΔGo is the binding energy independent of protein interactions, ΔGhb

describes contribution to binding energy from hydrogen bonds, ΔGionic denotes con-
tribution to binding energy from unperturbed ionic interactions, ΔGlipo considers
contribution to binding energy through lipophilic interactions while Alipo is the lipo-
philic contact surface between the protein and the ligand, ΔGrot describes the loss of
binding energy due to freezing of internal degrees of freedom in the ligand while
NROT represents number of rotatable bonds and f(ΔR, Δα) is a penalty function that
accounts for large deviations from ideal hydrogen bond and salt bridge geometry.
As shown in Eq. (2), the binding free energy is modeled using hydrogen bonds,
salt bridges, the hydrophobic effect, and solute entropy terms. The hydrogen bond
and salt bridge terms are modified by a penalty function which accounts for devia-
tion from ideal geometry. Entropy loss of the ligand upon complex formation is
based on the Number of ROTatable bonds (NROT) in the ligand [64, 65]. Eldridge
et al. presented an empirical scoring function referred to as ChemScore by taking
into account different energetic parameters like hydrogen bonds, the lipophilic
effects of atoms, the effective number of rotatable bonds in the ligand among
others. The scoring function was calibrated using 82 ligand-receptor complexes
with known binding affinities [66].
By including different empirical energy terms, many different empirical scoring
functions have been developed such as SCORE2 [67], ChemScore [66], RankScore
[68], LigScore [69], GlideScore [51], HINT [70], etc. The empirical scoring func-
tions take into account many different energy terms and thus the problem of
unknowingly double-counting of certain energy terms difficult issue to tackle.
Knowledge based scoring functions: these are derived from the structural infor-
mation embedded in experimentally determined atomic structures. The functions
use statistical analysis on crystal structures of complexes to obtain the interatomic
contact frequencies between the protein and the ligand based on the presumption
that stronger an interaction is, the greater the frequency of its occurrence will be.
The overall score is calculated with the help of Eq. (3) by accounting for favorable
contacts and repulsive interactions between each atom in the ligand and protein
lying within a sphere with a specified cutoff [71–78].

wðrÞ ¼ �kB Tln ½ g ðrÞ�, g ðrÞ ¼ ðrÞρðrÞ=ρ ∗ ðrÞ (3)

Here, kB is the Boltzmann constant, T is the absolute temperature of the system,

ρ(r) is the number density of the protein-ligand atom at distance r, ρ*(r) is the pair

32
Molecular Docking in Modern Drug Discovery: Principles and Recent Applications
DOI: [Link]

density in the reference state where interatomic interactions are zero and g(r) is
pair distribution function.
Popular knowledge based functions include DrugScore [79], PMF [72, 80],
MScore [81], SMoG [71], BLEEP [74], ITScore/SE [75], etc. The computational
simplicity of such functions is a major advantage especially when one has large
databases at hand however, the accuracy of predicting the reference state and
underrepresentation of interactions with halogens and metals are the major hurdles.
Each of the above classified have their inherent drawbacks, in this regard,
combination of more than one scoring functions has given improved results. This
approach has been widely regarded as “Consensus Scoring” [46].
Another set of scoring functions which have recently started to attract attention
are based on machine learning. One of the programs based on functions incorpo-
rating machine learning was able to achieve an astounding hit rate of 88.6% [82].
The nexus of machine learning and scoring functions is promising but the develop-
ment of such a tool is slow owing to its complexity.
In order to compare the variety of scoring functions that have been developed
up until now, comparative assessment of scoring functions (CASF) is an incredible
platform to begin with [83].
There is another set of classification proposed for the scoring functions namely
physics-based methods, empirical scoring functions, knowledge-based potentials,
and descriptor-based scoring functions but there is still no clear consensus on which
classification of scoring functions would be appropriate [84].

3. Applications

Molecular docking has been developed and improving for many years, but its
ability to generate a viable drug is still generally questioned. In the section below,
you will find examples where docking approach lead to recognition of active hits for
a variety of different receptors/targets.
HIV 1 Integrase—a new binding site for drugs treating AIDS was discovered
by Schames et al. using docking while considering the flexibility of the receptor
through molecular dynamics. The group used AutoDock in conjunction with
the relaxed-complex method to discover novel mode of inhibition of HIV
integrase [85].
α1A Adrenergic receptor—Evers et al. generated a model of the receptor using
homology modeling based on the X-ray crystallographic structure of bovine rho-
dopsin. Hierarchical virtual screening method was performed by them on the
Aventis in-house compound repository in a stepwise manner. 22,950 filtered com-
pounds were then docked into the α1A receptor homology model with the program
GOLD and scored with PMF. The top scoring compounds were finally clustered
according to their unity fingerprint similarity, and a diverse set of 80 compounds
was tested in a radio ligand displacement assay. Thirty-seven compounds displayed
a Ki < 10 μM with the most active having Ki = 1.4 nM [86].
Type I TGF-beta receptor kinase—A striking example and a proof of the benefit
of in silico approach over classical high-throughput screening involves the discov-
ery of novel Type I TGF-beta receptor kinase inhibitor. The same molecule (HTS-
466284); Figure 1, a 27 nM inhibitor, was discovered independently using virtual
screening [87] and also by traditional enzyme and cell-based high-throughput
screening in the same year [88]. The compound discovered experimentally required
in vitro screening of a large library of compounds in a TGF-β-dependent cell-based
assay which required more time, proved to be costlier and required usage of a
variety of chemicals when compared to its computational counterpart.

33
Drug Discovery and Development - New Advances

Figure 1.
Structure of HTS-46628, type I TGF-beta receptor kinase inhibitor.

Figure 2.
Structures for Aurora Kinase A inhibitor with IC50 12 and 43 pM respectively.

Aurora Kinase A—A major improvement was seen in the inhibitory activity of
Aurora Kinase A inhibitors which were designed using in silico techniques by Park
et al. [89]. This research group made use of a genetic algorithm to carry out the
sampling while the scoring function involved the energy terms from the AutoDock
program with a slight modification of the dehydration energy term. The design
strategy and tools used to carry out the study proved to be immensely successful
with some inhibitors revealing exceptionally high potency at low picomolar levels;
Figure 2 [89].
Dopamine D3 receptor—The 3D structure of the Dopamine 3 (D3) subtype
receptor was modeled by Varady et al. from the X-ray crystallographic structure of
rhodopsin and validated using experimental data. A D3 pharmacophore model was
devised by them from 10 selective and potent known D3 receptor ligands. Using
their model, 250,251 compound were screened from the National Cancer Institute
(NCI) 3D database. The hit list of 2478 potential ligands was then filtered for known
chemotypes. After removal of all compounds that were structurally similar to
known D3 receptor ligands, 1314 candidates remained. At the end, 20 compounds
supplied by NCI to the group were tested, out of which eight had Ki values below
500 nM, among which one of the compounds had Ki = 11 nM; Figure 3 [90].
Serotonin receptor (5HT1A)—Due to lack of structural information available for
the receptor, Becker et al. made use of PREDICT, to develop a unique non-
homology model for building a virtual 3D structure of the receptor. Using the
model, 40,000 compounds from Predix’s compound library were screened for
molecular docking and 78 virtual hits were discovered and then purchased by them
from respective vendors. The in vitro 5-HT1A binding assays elucidated that 16 of
the 78 compounds tested by the group were found to be hits with Ki < 5 μM,
reflecting a 21% hit rate, 9 of which had a Ki < 1 μM. The most potent molecule had
Ki = 1 nM (Figure 4) and was selected as a lead molecule for further optimization.
One significant feature of the study which highlights the utility of docking was that

34
Molecular Docking in Modern Drug Discovery: Principles and Recent Applications
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Figure 3.
Structure of dopamine D3 receptor inhibitor with Ki = 11 nM.

Figure 4.
Structure of serotonin receptor inhibitor with Ki = 1 nM.

the complete discovery process, i.e., from in silico screening through lead optimi-
zation, preclinical, and into clinical studies, was very rapid, requiring less than a
couple of years from program initiation to Phase I clinical trial [91].
Crystal structure prediction challenge—The International Blind Test is a chal-
lenge organized by the Cambridge Crystallographic Data Center wherein a previ-
ously determined crystal structure is only revealed once all the participants submit
their respective structures. In the Fifth International Blind Test, the challenge was
toughened by including flexible molecules with 50–60 atoms. The successful pre-
diction by two participants of the crystal structure of molecule XX in the blind test
indicated that search methods and models for lattice energy are capable of provid-
ing worthwhile results, both in terms of the range of structures considered in the
search and relative energies of the structures and thus can act as efficient ranking
systems [92].
Muscarinic M3 receptor—A pharmacophore model was constructed by Marriot
et al. from the known molecules showing significant M3 potency [93]. The research
group utilized the program DISCO, which generated five models. Three models
were rejected based on structural overlay. 3D screening was performed by Unity 3D
of the Astra compound database. The first model developed by them gave 176 hits
while the second model gave 173 hits; 172 compounds were common to the two sets
and were tested for their M3-antagonistic potency. Several compounds with

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Drug Discovery and Development - New Advances

micromolar and even submicromolar activities resulted, for example, compound

below had A50 M3 antagonism ≈ 0.2 μM; pA2 = 6.67; Figure 5 [93].
Checkpoint Kinase 1—Lyne et al. utilized virtual screening to discover Check-
point Kinase 1 (Chk-1) inhibitors [94]. Compounds with molecular weight > 600 or
with more than 10 rotatable bonds were excluded from the database. Then 3D
structures of the ligands were generated using Corina and a maximum of 8 stereo-
isomers were generated for each molecule. A 3D pharmacophore search was
performed with their in-house program Plurality to eliminate compounds that do
not have the typical binding motif for the kinase region. The remainder of the
compounds were docked into the ATP binding site of Chk-1, using the program
FlexX-Pharm, which considers full flexibility of the ligand but treats the protein as a
rigid structure. The research group then utilized consensus scoring to identify
molecules which were consistently giving good score with different scoring func-
tions. Finally, visual inspection by the group of the 250 highest scoring hits for
unfavorable interactions with the binding site or compounds with unrealistic con-
formations resulted in a list of 103 compounds for biological testing. Thirty-six hits
were identified with IC50 ranging from 110 nM to 68 μM; Figure 6 [94].
Human Cathepsin K—Schröder et al. presented the implementation of a
docking-based virtual screening workflow for the retrieval of covalent binders,
human cathepsin K was utilized as a test case [95]. By using the filter of electrophilic
war heads, a database with two million structurally diverse compounds with a

Figure 5.
Structure of muscarinic M3 receptor antagonist.

Figure 6.
Structures of checkpoint kinase 1 inhibitor with IC50 450 nM and 4 μM respectively.

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Molecular Docking in Modern Drug Discovery: Principles and Recent Applications
DOI: [Link]

variety of functional groups was reduced to a data set of just 343 test compounds.
Molecular docking was performed by them and the top scoring poses of the
GoldScore ranking list were taken into account for the manual selection of the
virtual hits based on visual inspection of the appropriate fit of the molecule in the
active site. A data set of 44 compounds including the five low scoring compounds
were finally selected for experimental evaluation. The activity of 21 out of the
selected 39 in silico hits was experimentally confirmed and four out of the five
structures predicted as inactive showed no activity on cathepsin K. This study
demonstrated to a huge extent the ability of docking to generate positive outcomes
(Figure 7) [95].
Human aldose reductase (ALR2)—ALR2 catalyzes a key reaction in the polyol
pathway of glucose metabolism, a process implicated in the long-term complica-
tions of diabetes. Its inhibitors were designed by Wang et al. using molecular
dynamic (MD) simulations and virtual screening [96]. A major challenge encoun-
tered by them in the in silico studies was that the binding site of the enzyme
underwent large conformational changes and adopted distinct configurations upon
binding different classes of ligands. To address this issue, the group sampled poten-
tially accessible binding site conformations by MD simulations based on the avail-
able crystallographic structures of ALR2. After this procedure, three average
conformations were selected for the docking. FlexX was utilized to carry out
docking of 7200 compounds of which 128 compounds were selected by them for
further screening. Out of these 72 molecules were selected which had RMSD < 3.00
A for experimental assay, of which 15 novel ALR2 inhibitors hits were discovered.
The most potent inhibitor had an IC50 = 0.24 μM; Figure 8 [96].
Cyclooxygenase-2 (COX-2) and β-amyloid aggregation inhibitors—Dadashpour
et al. made use of AutoDock4.2 to carry out docking studies of designed molecules

Figure 7.
Respective structures for active and inactive covalent binders of human cathepsin K.

Figure 8.
Structure of human aldose reductase inhibitor with IC50 = 0.24 μM.

37
Drug Discovery and Development - New Advances

Figure 9.
Structure of cyclooxygenase-2 inhibitor with IC50 = 10.1 μM.

based on diaryltriazine as lead. To validate the enzyme-inhibitor complex, the key

molecular interactions and calculated binding energy were considered by them.
Among the designed molecules, one of the compounds (Figure 9) showed an IC50
of 10.1 μM in experimental COX-2 assay. In addition, it showed potent anti-
aggregation activity on β peptides [97].

4. Limitations

The major limitation of molecular docking is due to the lack of confidence on the
ability of scoring functions to give accurate binding energies. This stems from the
fact that some intermolecular interaction terms are hardly predicted accurately,
such as solvation effect and entropy change [98]. In addition, some intermolecular
interactions are rarely considered in scoring functions which have been proven to
be of significance. For instance, halogen bonding is verified to make a contribution
to protein-ligand binding affinity [99] and so do guanidine-arginine interactions
[100], but are not considered.
Transthyretin-thyroxine complex—One critical example wherein energy func-
tions failed is that of transthyretin-thyroxine complex. The docking simulations
with energy functions resulted in generation of two binding modes, one similar to
the native binding mode of thyroxine and the other belonging to an alternate
binding domain with a root mean square deviation (RMSD) of 8.97 Å from native
binding state. The energy simulation was carried out and the lower energy solution
picked by the docking program was the one with higher RMSD. Thus, in this case
molecular docking failed to make the correct prediction of binding mode. Thereby,
it would be fair to conclude that we might get many false negatives during the
process of VS. [101].
It is still an unsolved problem to accurately deal with the water molecules in
binding pocket during docking process, which is tough task and needs a lot of
attention in the near future due to two reasons. Firstly, the x-ray crystal structures
lack the coordinate information of hydrogen, due to inefficient scattering by
smaller atoms. Not knowing the exact position of hydrogen leads to inaccuracies in
identifying water molecules which might be acting as a bridging molecule between
the ligand and the receptor. Secondly, no reliable theoretical approach is available
to accurately predict how water molecules are affected by ligands and how strong
the effect is. On top of that, it impossible with our current knowledge to predict
how many water molecules in the binding pocket would be replaced by potential
ligands and how the hydrogen bonding network would be disturbed by ligand
binding [102].

38
Molecular Docking in Modern Drug Discovery: Principles and Recent Applications
DOI: [Link]

One of the major challenges faced in the field of docking is that of rigid receptor.
A protein can adopt many different conformations depending upon the ligand to
which it binds. As a result, docking performed using a rigid receptor will corre-
spond to a single receptor conformation, which leads to false negatives in many
cases where later the ligand was found to be active. This happens because a protein
can exist in constant motion between different conformational states having similar
energies, which is usually neglected in docking [58].
Finally, the spectrum of activity against off-target proteins is something rarely
seen even in computational screens and is only dealt by animal and human trials.

5. Conclusion

Thus, it is quite evident from the case studies highlighted above and many more
success stories that one can find in literature related to computer aided drug design,
that in silico approaches in combination with biophysical data, experimental high
throughput screening and biology/toxicology/clinical studies are an indispensable
tool in the process of drug discovery. It assists in decision making, conceptualizing
new ideas and exploring them in a rapid manner to test a hypothesis, bringing
solutions to problems that cannot be assessed experimentally either because the
experiments is too difficult to design or because it would cost too much.
Undoubtedly, many challenges still remain to be addressed such as role of water
molecules, solvent effects, entropic effects, and receptor flexibility.
There is more than sufficient information now that proves the utility of compu-
tational tools in drug design and there is no scope for any debate regarding the
effectiveness and advantage of computational tools in the process of drug discovery.

Author details

Aaftaab Sethi, Khusbhoo Joshi, K. Sasikala and Mallika Alvala*

National Institute of Pharmaceutical Education and Research, Hyderabad,
Telangana, India

*Address all correspondence to: mallikaalvala@[Link]

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License ([Link]
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

39
Drug Discovery and Development - New Advances

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47
Chapter 4

Computational Deorphaning of
Mycobacterium tuberculosis Targets
Lorraine Yamurai Bishi, Sundeep Chaitanya Vedithi,
Tom L. Blundell and Grace Chitima Mugumbate

Abstract

Tuberculosis (TB) continues to be a major health hazard worldwide due to

the resurgence of drug discovery strains of Mycobacterium tuberculosis (Mtb) and
co-infection. For decades drug discovery has concentrated on identifying ligands
for ~10 Mtb targets, hence most of the identified essential proteins are not utilised
in TB chemotherapy. Here computational techniques were used to identify ligands
for the orphan Mtb proteins. These range from ligand-based and structure-based
virtual screening modelling the proteome of the bacterium. Identification of
ligands for most of the Mtb proteins will provide novel TB drugs and targets and
hence address drug resistance, toxicity and the duration of TB treatment.

Keywords: Mycobacterium tuberculosis, target deorphaning, target deconvolution,

proteome modelling, virtual screening

1. Introduction

Tuberculosis (TB) continues to be a major public health concern with over

2 billion people currently infected, 8.6 million new cases per year, and more than
1.3 million deaths annually [1]. The current drug-regimen combination for drug
sensitive TB consists of isoniazid, rifampicin, ethambutol and pyrazinamide,
administered over 6 months [2]. If this treatment fails, second-line drugs are used,
such as para-aminosalicylate (PAS) and fluoroquinolones, which are usually either
less effective or more toxic with serious side effects. Although this regimen has a
high success rate, it is marred by compliance issues, which have resulted in the rise
of multidrug resistant (MDR), extensively drug resistant (XDR) and totally drug
resistant (TDR) strains of the causative agent, Mycobacterium tuberculosis (Mtb)
[3, 4], in both immunocompetent and immunocompromised patients worldwide
[5]. However, it took about 40 years for a new TB drug to be discovered and most
of the current TB drugs target a total of only ~10 proteins, even though the com-
plete genome of Mtb was published nearly 20 years ago [6]. Consequently, most
of the essential proteins are orphans since their ligands are still to be identified. In
our context, target deorphaning or deconvolution encompasses identification of
ligands for Mtb proteins not currently exploited in TB chemotherapy and those of
old TB targets. Targeting further essential proteins should allow the fight against
drug resistance to be enhanced, and possibly lead to a reduction in the duration of
TB treatment.

49
Drug Discovery and Development - New Advances

The conventional target deorphaning process involves experimental work,

which characteristically includes genetic, proteomics and transcriptional profiling
and then identification of the ligands for the proteins using many more chemical-
proteomic approaches [7]. This approach is usually long, expensive and time
consuming. However, developments in bioinformatics and chemoinformatics,
together with advances in computer tools and resources, have fortunately revolu-
tionised target deorphaning. Bioinformatics describes the target space in Mtb from
the genome to the proteome, whilst chemoinformatics provides information about
the available chemical space and tools for navigation of the space. Together these
developments have led to a mushrooming of computer-based target deorphaning
methods ranging from modelling proteomes, virtual screening, machine and deep
learning, and chemogenomics [8–10]. When used effectively in conjunction with
experimental work, computational methods can facilitate identification of new TB
targets and drugs [11–13].
Therefore, in this chapter we present an overview of the genome of Mtb, giving a
detailed account on how the computational techniques have been used to de-orphan
Mtb targets including case studies, the current and proposed future impacts of these
techniques on the number of de-orphaned Mtb targets and their impacts in boosting
the biomedical efficacy of TB drugs. The collated data will provide researchers in
academia and industry with knowledge of target-ligand pairs and interactions, infor-
mation crucial for the design of novel drugs with known targets that are less prone to
resistance, with minimal side effects and interactions with e.g. anti-HIV drugs.

2. Method

An extensive literature search was performed to give an overview of the genome of

the Mtb and status of the currently used tuberculosis drugs and their targets. An analy-
sis of the essential proteins in Mtb and the number of proteins targeted by the current
TB drugs was performed. To boost this data Mtb target-ligand data was extracted from
the ChEMBL database version 24 ([Link]
targets), which was used to determine the number of the proposed new targets.
An overview of computational deorphaning of Mtb targets is provided, using data
extracted from literature and a description of the efforts made from our laboratory.
To sum this up, a detailed account of modelling the proteome for Mycobacteria, and
identification of the hotspots and druggability of the proteins is given.

3. Genome sequence of Mycobacterium tuberculosis

Cole and co-workers [14] in 1998 reported the complete sequence of Mtb, which
comprises of 4,411,529 base pairs. The genome has an evenly distributed guanine-
cysteine content of 65.6% and represents the second-largest bacterial genome
sequence currently available. Additionally, the genome is rich in repetitive DNA,
particularly insertion sequences, and in new multi-gene families and duplicated
housekeeping genes, providing evidence for horizontally-transferred pathogenicity
islands of a particular base composition [14].
The genome of Mtb has some exceptional features, for example there are over
200 genes that encode enzymes for the metabolism of fatty acids, comprising 6% of
the total (Table 1). Among these, about 100 are predicted to function in the oxida-
tion of fatty acids. This large number of Mtb enzymes that putatively have fatty
acids as substrates may be linked to the ability of this pathogen to grow in the tissues
of the infected host, where fatty acids maybe the major carbon source. Another

50
Computational Deorphaning of Mycobacterium tuberculosis Targets
DOI: [Link]

Function No. of % of total % of total coding

genes genes capacity

Lipid metabolism 225 5.7 9.3

Information pathways 207 5.2 6.1

Cell wall and cell processes 517 13.0 13.5

Stable RNAs 50 1.3 0.2

IS elements and bacteriophages 137 3.4 2.5

PE and PPE Proteins 167 4.2 7.1

Intermediary metabolism and respiration 877 22.0 24.6

Regulatory proteins 188 4.7 4.0

Virulence, detoxification and adaptation 91 2.3 2.4

Conserved hypothetical function 911 22.9 18.4

Proteins of unknown function 607 15.3 9.9

Table 1.
General classification of Mtb genes. Adopted from [15].

unusual feature of the Mtb genome is the presence of the unrelated Pro-Glu (PE)
and Pro-Pro-Glu (PPE) families of proteins that have conserved N-terminal
domains of 100 and 180 amino acids respectively. The antigenicity of these proteins
has led to the assumption that at least some of these proteins may be involved in
antigenic variation of Mtb during infection [15].

3.1 Current status of tuberculosis drugs and targets

3.1.1 Tuberculosis drugs

The success of TB chemotherapy derives from an “intensive” phase involving

a cocktail of four first-line drugs, comprising, rifampicin (RIF), isoniazid (INH),
pyrazinamide (PZA), and ethambutol (EMB). A threatening global issue of this
epidemic is the emergence of drug-resistant bacteria, a trend that is on the rise, as
such strains are easily spread with low fitness costs associated with transmission
[16]. The World Health Organisation (WHO) reported that globally 3.5% of naive
infections already expressed resistance to the two most efficacious frontline agents
used to treat the disease, RIF and INH, thereby classifying the infection as multi-
drug resistant tuberculosis (MDR-TB) [17]. Treatment of drug-resistant Mtb is dif-
ficult already, requiring 6–9 months of combination therapy of second-line drugs,
such as PAS, fluoroquinolones e.g. levofloxacin, and aminoglycosides e.g. kanamy-
cin, capreomycin, ethionamide and cycloserine. Complicating the issue is the fact
that TB is endemic to the developing world; thus, access to adequate healthcare
facilities and drugs can be limited for those patients. This leads to non-compliance
by most patients, relapse of the disease and severe side-effects especially of second-
line drugs [18]. Treatment for MDR-TB can extend upwards of 2 years and relies on
more toxic, less efficacious second-line drugs, many of which are even more scarce
than frontline drugs in affected areas [16].
In addition, comorbidity with HIV causes massive diagnostic and therapeutic
challenges and results in adverse drug interactions [19]. This is because RIF is
a potent inducer of drug-metabolising enzymes, including cytochrome P450
(CYP) 3A4. This induction dramatically reduces plasma levels of several highly
active antiretroviral therapy drugs; thus, patients are often forced to complete

51
Drug Discovery and Development - New Advances

their TB treatment before beginning HIV treatment [20]. Patients who contract
MDR-TB with HIV have a very poor prognosis due to the duration of treatment;
these individuals frequently succumb within a few months. Therefore, there is an
urgent need to develop continually new active agents to combat MDR-TB which has
been compounded by the emergence of XDR-TB. Furthermore, cases of TDR-TB
have been noted in China, India, Africa, and Eastern Europe. In TDR-TB, the
Mycobacterium are resistant to all available therapeutics [19]. To address this, in
2012 the U.S. Food and Drug Agency (FDA) approved bedaquiline for MDR-TB [21]
and later delamanid was approved as a compassionate care option for XDR-TB and
TDR-TB infections, nonetheless the EMA approved both agents for MDR-TB [22].
The biggest challenge is that these drugs have reported human ether-a-go-go related
gene (hERG) toxicity, as well as multiple absorption, distribution, metabolism and
excretion (ADME) issues due to their high lipophilicity [21]. This leads to an urgent
need for development of new agents that have successful therapeutic effects.

3.1.2 Mycobacterium tuberculosis drug targets

To date the number of essential Mtb proteins encoded by approximately 4000

genes is just over 500 (Figure 1), and this provides a rich source for novel targets
for new and current TB drugs. However, Lamichhane et al. [23] reported that TB
chemotherapy exploited only 10 of these proteins; Table 2, gives a summary of
the targets, and their current and/or new drug ligands. The most popular target is
enoyl[acyl-carrier protein] reductase, important for the biosynthesis of mycolic acid.
Efforts to identify genes that code for new potential drugs are underway, as evidenced
by 76 TB data points recorded in the ChEMBL database version 24 ([Link]
[Link]/chembl/beta/g/#browse/targets), consisting of small bioactive compounds,
their targets and bioassay data. There are 73 single proteins, including the 10 proteins
already targeted by both first-line and second-line drugs during TB chemotherapy.
Thus, 63 new drug targets are being explored in a plethora of bioassays.
This is of paramount importance because Mtb secreted proteins play a vital
role in host-pathogen interactions and facilitate nutrient acquisition, pilot the host
immune response and interfere with therapeutic intervention. Therefore, the Mtb
secretome consists of proteins essential for successful invasion and in vivo growth
during host infection. The essential proteins are the most suitable drug targets for
the development of diagnostic tools and new drugs, because of their key role in in
vivo bacterial survival and growth. Identifying ligands for these proteins required
for growth and survival in the infected host could lead to the discovery of poten-
tially useful biomarkers to add on the above mentioned drug targets [27].

Figure 1.
Circular diagram of the genome of Mtb genes, essential proteins and the number of proteins that are drug
targets.

52
Computational Deorphaning of Mycobacterium tuberculosis Targets
DOI: [Link]

Targets Function Conventional New ligands

drugs
Enoyl-(acyl-carrier- Biosynthesis of mycolic Isoniazid Tetrahydropyrans
protein) reductase (InhA), acids, that is essential Ethambutol (PT070)
Fatty acid synthase for growth and Pyrazinamide Methylthiazoles
virulence Delamanid Diazaborines
Pyrrolidine-carboxamide
Piperazine
indoleformamides
Aminoproline
Arylamides
Imidazopiperidines
DNA gyrase An ATP-dependent Fluoroquinolones Clinafloxacin
enzyme that acts by
creating a transient
double-stranded DNA
break
Ubiquinol-cytochrome Electron carriers of the Pyrrolo[3,4-c]pyridine-
C-reductase (QCrB) respiratory chain 1,3(2H)diones
Lansoprazole
Transmembrane transport Responsible for heme SQ109
protein large (MmpL3) uptake into the cell. Adamantyl ureas
Responsible for the Phenylpyrroles
transport of ions, Benzimidazoles
drugs, fatty acids and Tetrahydropyrazolo
bile salts [1,5-a]pyrimidine-3-
carboxamide
Spiropiperidines
Decaprenylphospo-β-D- Cell wall synthesis Benzothiazinones
ribofuranose-2-oxidase (BTZ043)
(DprE1) Benzothiazole (TCA1)
4-aminoquinolone
piperidine amides
2-carboxyquinoxalines
Oxadiazoles
Benzo [b]thiophenes
Pyrazolopyridones
RNA polymerase Responsible for Rifampicin
transcription Rifapentine
Rifabutin
Protein synthase Protein synthesis Linezolid PNU100480
([Link] AZD5847
[Link]/
drugs/DB00601)
ATP Synthase ATP synthesis Bedaquiline D-Dethiobiotin
Cytidine triphosphate Catalysis of amination Thiophenecarboxamide
(CTP) synthetase of uridine triphosphate 4-(pyridine 2-yl) thiazole
(UTP) into CTP
Transcription factor Regulating the Benzo-thiazol benzene
(IdeR) intracellular levels of sulfonic acid
iron
Lysine-ε-amino Catalysing reversibly Benzothiazole
transferase (LAT) the transamination
of lysine into
α-ketoglutaric acid

Table 2.
Mtb drug targets and the current used drugs [24–26].

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Drug Discovery and Development - New Advances

4. Computer resources and tools for tuberculosis drug targets

The development in genomics, coupled with advances in high performance

computing and validation of molecular targets, has introduced new approaches
to drug discovery that provide a shift from the historical pipeline that focuses on
target identification and in most cases involves single targets. In this era of extensive
discovery of new chemical entities for treatment of TB and other infectious diseases
like HIV/AIDs, a number of research institutes as well as pharmaceutical companies
are eagerly developing computational tools and protocols to facilitate drug discovery
and development [28]. Genomics provide DNA, RNA, transcriptomic and proteomic
data that is housed in a variety of databases and provide resources e.g. from the
European Bioinformatics Institute (EBI) [Link] and the National
Centre for Biotechnology Information (NCBI) [Link]
which can be easily retrieved and analysed, thereby shifting the drug discovery focus
from a single to a multi-protein target approach. In this approach Mtb genomic data
are analysed for network, structure and function of a number of essential proteins
that are druggable and validated as potential targets for a number of bactericidal or
bacteriostatic chemical compounds. In this section, different databases, resources
and tools for target deorphaning are discussed with a particular focus on Mtb targets.
The revolution in genomics led to the availability of a number of mycobacterial
genomes and the development of a variety of databases consisting of Mtb genomic
and transcriptomic data. The genomic databases provide information about the
structure, function and evolution of Mtb genes, whilst the transcriptomics provide
information crucial for analysis of gene expression using large scale RNA sequences
[29]. On the other hand proteomics provides information about the function,
networks and structure of proteins. In their paper, Machado et al. [29] give a detailed
summary of most computational resources for TB and we encourage readers to con-
sult the article for more information. Similarly a number of chemogenomic resources
and database containing data for Mtb ligand annotated targets have been developed.
Examples of such databases include the ChEMBL database [30], a database of small
bioactive molecules and their targets, TIBLE [31] a database containing MIC and tar-
get data for mycobacterial species and TDR targets containing target-ligand informa-
tion for neglected tropical diseases including TB. The databases are freely available
and provide easy access to target-ligand data for Mtb. In these databases each target is
associated to ligand(s) obtained from bioassays and vice versa.

5. Computational target deorphaning techniques

A number of computational methods are being explored in order to identify

ligands for both host and pathogen targets and for targets from other organ-
isms like Plasmodium falciparum [32]. In most cases two or more complementary
ligand-based and structure-based deorphaning approaches are used; statistical
methods involving machine learning [8] and deep learning strategies are applied in
conjunction with biological and/or biophysical methods to validate the computa-
tional results or the computational methods are used to provide the protein-ligand
binding information in the absence of X-ray co-crystallised structures of the ligand
[12, 13]. In their work, Mendes and Blundell [13] applied cheminformatics to
complement current efforts for target identification of fragment-sized molecules
that target e.g. the PanC that synthesises pantothenate important for generation of
the Mtb co-enzyme A. This has led to the identification of ‘hotspots’ in the binding
pockets of a number of proteins, which highlight the most favoured binding spots
for the protein. Hotspots and druggability will be discussed in detail in Section 6.

54
Computational Deorphaning of Mycobacterium tuberculosis Targets
DOI: [Link]

5.1 Ligand-based and structure-based virtual screening methods

Structure-based virtual screening is an approach used in drug discovery to com-

putationally screen small molecule databases for compounds that target proteins of
known 3D structure that are experimentally validated. Brain Shoichet [33] has pointed
out that this approach was first published in the 1970s, however most new ligands and
their targets were not identified until the early 2000. The method offers the opportu-
nity to access a large number of potential new chemical ligands for old and new targets.
In the presence of available ligands for named biological targets, ligand-based virtual
screening may be used using a variety of techniques ranging from molecular similarity,
pharmacophoric search, to machine learning and most recently deep learning.

5.1.1 Structure-based techniques

Structure-based virtual screening plays a significant role in drug discovery in

that it is used to identify ligands for biological targets when the 3D structures of
the Mtb targets from X-ray crystallography, nuclear magnetic resonance (NMR)
or cryoelectron microscopy are available in the Protein Data Bank, or homol-
ogy models available in the CHOPIN database and/or generated in house. This
method applies structural data of proteins/receptors to provide small molecules
with specific structural attributes for good binding affinity [34]. Generally, the
process involves three crucial steps, namely preparation of 3D crystal structures
of proteins obtained from the Protein Data Bank (PDB) and the ligand structures,
docking calculation and data analysis. Protein structure preparation involves adding
hydrogen atoms that are normally missing in the coordinate files, adding missing
residues, optimising hydrogen bonds, removing atomic clashes, as well as sampling
the degrees of freedom such as flip that are not clear in standard resolution crystal
structures, for example the 180o flips of chain terminal rotatable side-chain groups
e.g. in shape-symmetric amino acids Asn and Gln, tautomer and/or ionisation state
and relaxation of the target and ligand structure [35]. Most docking software is
associated with protein and ligand preparation tools, for example Autodock4 or
VINA require structures prepared using AutoDockTools (ADT) and the protein
preparation script to generate Autodock-type atoms containing Gasteiger charges,
and produce the pdbqt files that are compatible with the tool [36]. Similarly, the
Primex and Ligprep tools are used to prepare the protein and ligand structures
respectively before docking with GLIDE [37]. The quality of input structure files
contribute to the quality of the docking results, and the importance of protein and
ligand preparation have been highlighted by Sastry [35].

[Link] Molecular docking

Molecular docking calculations are capable of predicting the binding conforma-

tion of ligands inside the binding pocket of a target, as such they are used to map
small molecules onto targets and hence provide essential binding information for
structure-based drug design. To achieve this, a number of docking algorithms like
Autodock [36], perform a stochastic conformational search or e.g. in GLIDE, a [37]
that perform a systematic search [34]. In a stochastic search structural parameters,
such as torsional, translational and rotational degrees of freedom of the ligand,
are randomly modified to generate an ensemble of molecular conformations and
increase the chances of finding the energy global minimum, whilst in a systematic
conformational search structural features are gradually changed until a local or
global minimum is reached [34]. During the search, conformations of a number of
potential binding compounds are explored and evaluated using a specific scoring

55
Drug Discovery and Development - New Advances

function. In addition, the conformations are ranked based on their calculated bind-
ing energy. Highly ranked compounds are selected as ligands for the target. On the
other hand, reverse or inverse docking is used for identifying targets of drug pheno-
typic hits from a sea of targets. In this way, structure-based screening helps to iden-
tify and explain polypharmacology, molecular mechanism of action of substances,
facilitate drug repurposing, detect adverse drug reactions and hence toxicity.

[Link] Deorphaning the HTH transcription regulator, EthR

In an effort to de-orphan the HTH transcription regulator, EthR, and identify

the binding mode of the ligand, we docked 200 fragment-like compounds from the
Maybridge database to the highest quality crystal structure of the 23 PDB entries
using the GOLD algorithm (unpublished work). We used Arpeggio [38], an online
tool that identifies non-covalent interactions in protein-structures, to assess the role
of each EthR binding site residue and each small-molecule ligand moiety in con-
tributing to protein-ligand interactions. Visual assessment of interactions involved
calculating interactions using the Arpeggio web server ([Link]
[Link]/arpeggio) and downloading the results as PyMOL session files, to analyse the
non-covalent interactions of each residue. We found that in addition to using polar
contacts, most ligands are stabilised by a cascade of pi-interactions starting from
Tyr103 close to the entrance of the allosteric pocket to Phe114 located close to the
HTH-domain and beyond (Figure 2). Furthermore, potential ligands for the protein
were identified. Information obtained from these results is vital identify ligands with
a higher probability of binding to EthR, and so improve the potency and safety of
ethionamide (ETH).

Figure 2.
(A) Binding modes of two fragment-like molecules inside the long cylindrical allosteric binding pocket of
EthR defined by five helices. Yellow sticks depict the molecule occupying the upper binding site close to the
entrance of the pocket and cyan sticks represent a molecule occupying the inner binding site close to the HTH
domain. (B) EthR-ligand interactions involving Trp103 (yellow) at the entrance of the binding pocket of the
protein. Ligand atoms and bonds are in pink, grey rings are hydrophobic interactions, red rings show hydrogen
bonds. (C) EthR-ligand (pink) interactions involving Phe110 located at the center of the binding pocket of
EthR.

56
Computational Deorphaning of Mycobacterium tuberculosis Targets
DOI: [Link]

Similarly, docking calculations were used to assess binding of ligands identified

from for a novel TB drug target, inosine monophosphate dehydrogenase (IMPDH)
protein Guab2 that is responsible for the synthesis of xanthosine monophosphate
(XMP) from IMP, identified from high throughput screening [12]. Hit compounds
were identified in a single shot high-throughput screen, validated by dose response
and subjected to further biochemical analysis. The compounds were also assessed
using molecular docking experiments, providing a platform for their further opti-
misation using medicinal chemistry. From the results, it was observed that occupa-
tion of the nicotinamide sub-site was correlated with interactions of the ligands
with the purine ring of IMP.

[Link] Applying concerted computational and experimental approaches

Likewise, we used a combination of ligand-based and structure-based chemoge-

nomic approaches, followed by biophysical and biochemical methods, to identify
targets for Mtb phenotypic hits deposited in the ChEMBL database [11]. In this
work, EthR and InhA emerged as potential targets for many of the hits, and some of
them displayed activity through both targets. From the 35 predicted EthR inhibitors
25 displayed an inhibition of better than 50%, of which eight showed an IC50 better
than 50 μM against Mtb EthR and three were confirmed to be also active against
InhA. Further the EthR-ligand complexes were validated using X-ray crystallogra-
phy in the Blundell laboratory to give new crystal structures which were deposited
in the Protein Data Bank. These results provide new lead compounds that could be
further developed into highly active ligands of EthR and InhA and enhance treat-
ment of drug-resistant TB.

6. Modelling proteomes for mycobacteria, hotspots and druggability

A comprehensive understanding of the structural proteomes of mycobacteria

is essential for novel drug discovery and elucidating the roles of mutations in drug
resistance. Most researchers begin by defining the 3D-structure using X-ray crystal-
lography, NMR or increasingly cryo-EM. For phenotypic screening and under-
standing off-target hits, where the target is not identified, prior knowledge of the
structures of all gene products in the target organism is helpful. This has stimulated
the establishment of several consortia in what is usually known as structural
genomics, but might more appropriately termed “structural proteomics”.

6.1 Evolution of structural genomics consortia

The Structural Genomics Consortium (SGC) [39] which has focused on proteins
of interest to medicine, has impressive achievements, in 2011 defining ~40% of
the structures of proteins from human parasites deposited in the PDB [40]. The
Tuberculosis Structural Genomics Consortium (TBSGC), an international col-
laboration involving 53 countries, has focused on 3D structures of Mtb [40]. This
activity and others working on Mtb proteomes have deposited 2274 structures in
the PDB, but still representing less than 583 gene products, only 13.97% of genome.
Although this is a small percentage, it compares impressively with knowledge
of protein structures of two other mycobacterial pathogens where there is great
clinical interest: for M. leprae causing leprosy there are experimentally-defined 3D
structures for 15 gene products and for M. abscessus, a free living Mycobacterium,
which is a growing challenge for cystic fibrosis patients, there are 53 experimen-
tally-defined 3D structures in the PDB.

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6.2 Comparative 3D modelling of proteins

Comparative modelling proteins, based on the fold recognition and structural

alignment with the closest homologues that have experimentally solved structures,
began using interactive graphics in the 1970s [41–43]. The development of auto-
mated modelling software began in the 1980s, initially with Composer [44] and
later developed with Comparer [45] and Modeller [46], based on satisfaction of 3D
restraints derived from structurally aligned homologues. Modeller has now been
cited ~10,500 times in the literature!

6.2.1 Computational modelling pipelines and structural proteome databases

Rapid progress in this and other related software coupled with increasing com-
puting power has enabled genome scale prediction of protein structures, as a viable
alternative to experimental determination. In order to construct computational models
of all gene products, which we here refer to as the structural proteome, we identify
templates by a sequence-structure homology search using Fugue [47], which uses
local-structural-environment-specific substitution tables to predict the likelihood of a
common 3D structure. We have incorporated Fugue into a pipeline (Vivace), in which
templates are selected from TOCCATA (Ochoa Montaño and Blundell, unpublished),
a database of consensus profiles built from CATH 3.5 [48] and SCOP 1.75A [49] based
classification of proteins structures (PDB files). PDBs within each profile are clustered
based on sequence similarity using CD-HIT [50] and structures are aligned using
BATON, a modified version of COMPARER [45]. After further optimization of the
clusters by discarding templates with more than 20% difference in sequence identity to
the maximum hit, remaining templates are classified into states based on ligand binding
and oligomerization. Five different states, known as “liganded-monomeric,” “liganded-
complexed,” “apo-monomeric,” “apo-complexed” and “any,” are generated in each
profile hit. Models are built in each of these states using Modeller 9.10 [46] and refined.
Later NDOPE, GA341 [51] Molprobity [52] and SSAG [53] are used to determine the
quality of the models.

6.2.2 Mycobacterial proteome databases

The first application of this approach was to construct the Chopin Database
([Link] a database of protein structures for
H37Rv strain of Mtb. This has provided structures that are reasonably certain for around
65% of gene products. These have proved reliable indicators of the overall structures but
may have some uncertainties especially in loop regions and domain-domain relation-
ships. A further ~19% probably have correct folds while the remaining would unlikely
to be correct. Nevertheless, compared to those structures defined experimentally by
X-ray analysis, this represents a 6-fold increase of structural information available that
might be useful in assessing druggability and the impacts of mutations.
Similar models of the structural proteome for M. abscessus (Skwark et al.,
unpublished) and M. leprae (Vedithi et al., unpublished) have been developed in
the group. In M. leprae, of the 1615 gene products, templates were identified for
1429 gene products and we were able to model 1161 proteins with high confidence.
A total of 36,408 models were built in different ligand bound and oligomeric states
for the 1161 proteins. The distribution of Fugue Z score across models indicates
that only 4% of the proteome has no hits and 15% has poor scores. ~80% of the
proteome has acceptable and good hits, and the corresponding Z scores. Around
47% of the protein queries identified templates with identity and coverage greater
than 40 and 67% of the models in the proteome are of best quality as estimated by
NDOPE, GA341, Molprobity and Secondary Structure Agreement (SSAG).

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6.2.3 Oligomeric protein models

Current work on structural proteomes includes efforts to extend the modelling

pipeline to homo-oligomeric (and eventually hetero-oligomeric) structures using com-
parative approaches (Malhotra et al., unpublished), extending models and improving
models of small molecule complexes, and linking individual protein structures into
the metabolic networks and interactions in the cell (Bannerman et al., unpublished).
An example of an oligomeric structure is CTP-synthase, encoded by PyrG, which is
an essential gene in Mtb identified by transposon saturation mutagenesis [54] and
catalyses ATP-dependent amination of UTP to CTP with either L-glutamine or ammo-
nia. The allosteric effector GTP functions by stabilising the protein conformation that
binds to the tetrahedral intermediates formed during glutamine hydrolysis. Its closest
homologue in M. leprae ML1363 is a target of choice and was modelled using Vivace
during the proteome modelling exercise. We modelled the apomeric and ligand bound
states of the model and oligomerized the protomer using our inhouse oligomerization
pipeline. The protomeric and oligomeric states are depicted in Figure 3A and B.
The models were built by using templates PDB-IDs: 4zdI and 4zdK for PyrG
of Mtb [55]. Both the templates are 89% identical and 100% coverage to the query
sequence. The superposition of the models with the templates indicated a root mean
square deviation (RMSD) of 0.758.

6.3 Structural implications of mutations

We have also spent time over 2 decades analysing the impacts of mutations
evident in the increasing wealth of available genome sequences for pathogenic myco-
bacteria and cancers. We originally developed SDM [56] in 1997, a method depend-
ing on statistical analysis of environment-dependent amino-acid substitution tables
[57, 58]. In 2013 machine learning was introduced with the arrival of Douglas Pires in
Cambridge, developing first mCSM for stability [59] followed by several “flavours”
including mCSM-PPI for impacts on protein-protein interactions, mCSM-NA [60]
for nucleic acid interactions and mCSM-lig for impacts on small-molecule ligand
interactions useful for understanding drug resistance [61]. A critical part of using
machine learning is to have an extensive database of experimentally-defined impacts
of mutations on stability and interactions, such as Platinum by David Ascher when
in Cambridge [62], a database of experimentally measured effects of mutations on
structurally defined protein-ligand complexes that was developed for mCSM-lig.
These two structural approaches to predicting the impacts of mutations (SDM &
mCSM) have proved complementary and more reliable than most sequence-only

Figure 3.
(A) Protomeric model of PyrG (CTP-Synthase) of M. leprae modelled with a quality of 4.25 (best).
(B) Homo-8-mer of PyrG of M. leprae modelled with a quality of 4.25 (best).

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Drug Discovery and Development - New Advances

Figure 4.
Indicates the maximum destabilising effect a mutation can induce on the stability of RNA-polymerase
β-subunit of M. leprae (target for rifampin) measured by mCSM-stability.

methods. They also allow the application of saturation mutagenesis, facilitating

in silico systematic analysis of mutations [63], an approach now being adopted to
whole proteomes where every residue in each of the proteins in the proteome is
mutated to all the other 19 amino acids and the effects of the mutations are measured
using various methods mentioned above. In structure-guided fragment-based drug
discovery, this provides comprehensive information on the regions of the protein
that are less likely to lead to drug resistance and therefore can be probed by elabora-
tion of fragments/small molecules. We performed saturation mutagenesis on the
drug targets in M. leprae for leprosy and the average or highest impact a mutation
can induce in each residue position is depicted on the structure (Figure 4).

6.4 Active sites, cavities and fragment hotspot maps

Although comparative modelling of homologues in complex with ligands can

often give clues about active sites, cofactor binding and substrate or other ligand
binding sites, this is not always possible. In order to indicate putative binding
sites in the absence of appropriate experimental data, we have exploited cavity-
defining software such as VolSite [64] for novel binding site description together
with an alignment and comparison tool (Shaper) [65]. We have used FuzCav, a
novel alignment-free high-throughput algorithm to compute pairwise similarities
between protein-ligand binding sites [66] and GHECOM [67], to study the small
pockets that often characterise protein-protein and protein-peptide interactions.
Further to the identification of cavities and pockets, it is also useful to be able to
identify hotspots, region(s) of the binding site defined as a major contributor to the
binding free energy, and often characterised by their ability to bind fragment-sized
organic molecules in well-defined orientations. The usual understanding is that the
fragment, with a mixed polar and hydrophobic character, can displace an “unhappy
water.” We have tried to mimic this in silico by using SuperStar [68] to generate atomic
interaction propensities on a grid. We then carry out a search with three fragments,
each having a six-membered carbon ring, but having a donor, acceptor or a non-polar
substituent. The resulting map is convoluted with an estimate of the depth below the
surface, which generally appears to correlate with favourable entropic gain on water
release on binding of a ligand [69]. The hotspot maps, computed in this way and

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DOI: [Link]

indicating donor, acceptor and lipophilic interactions correlate well with experimental
binding sites of fragments that can be elaborated in fragment-based discovery. For the
ligand bound structures, lower contouring can provide “warm spots” for the binding
sites, indicating possibilities for elaborating the fragment in the binding pocket.
The models of individual molecules of the modelled proteome can be individually
decorated with the hotspot maps. They give a good indication of the known func-
tional sites on experimentally defined structures of proteins, often demonstrating
that a functional site comprises several hotspots involved in binding substrates and
cofactors. They also provide a good indication of the location of allosteric sites [70].

7. Conclusion

In summary we can move from the study of individual targets to an understanding

of the majority of targets coded by the genome. Indeed, we can build 3D structures
for a majority of the genes, so providing a model of the “structural proteome”.
Hotspots and cavities provide a basis for identification of the ligandability of putative
binding sites and have been used in our group to predict pharmacophores that can be
used in docking and virtual screening and so deorphaning of mycobacterial proteins.
To identify druggable proteins from the structural proteome, we have adopted
a hierarchal selection process wherein chokepoint analysis is initially performed to
identify metabolic reactions that are critical to cell survival. Gene products identi-
fied in this screen are later subjected to essentiality analysis using either flux balance
analysis (FBA) based models or by data from the transposon saturation mutagenesis
experiments in the literature. Genes that are essential are chosen at this stage and
understanding of the gene expression profiles in different growth conditions is anal-
ysed. Genes whose expression is condition specific are excluded. Later for the selected
genes, the structural information of the corresponding proteins is analysed in the
context of prior knowledge and attempts in drug discovery, druggable pockets and
fragment hotspots maps, small molecule bound states, non-human homologue, non-
homologous to human microbiome, cellular localization and biochemical properties
of the proteins. Structure-guided virtual screening is performed on the selected drug
targets with a choice of fragment and compound libraries using CCDC Gold (The
Cambridge Crystallographic Data Centre) [71]. Best poses with good scores lead the
experimental process of structure-guided fragment-based drug discovery.
The challenge now is to test the computational methods outlined here for
identifying ligands and understanding the druggability of the proteome—several
thousand gene products from the whole genome of Mtb. We can then begin to assess
the degree to which we can de-orphan the many Mtb proteins that have until now
not featured as targets in the worldwide efforts to combat the global challenge of TB
to the health and well-being of human kind.

Acknowledgements

LYB and GCM are grateful to Chinhoyi University of Technology for their
support in introducing computational drug discovery and development research
work at the University and all our collaborators. TLB and SCV thank the Gates
Foundation, the Cystic Fibrosis Trust and the American Leprosy Mission for their
funding of computational and experimental work on approaches to combating
disease from mycobacterial infections. They also thank colleagues in Cambridge
and elsewhere who have contributed over the years to our efforts to develop new
approaches to structural biology, computational bioinformatics and drug discovery.

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Author details

Lorraine Yamurai Bishi1, Sundeep Chaitanya Vedithi2, Tom L. Blundell2

and Grace Chitima Mugumbate1*

1 Department of Chemistry, School of Natural Sciences and Mathematics,

Chinhoyi University of Technology, Chinhoyi, Zimbabwe

2 Department of Biochemistry, University of Cambridge, Cambridge,

United Kingdom

*Address all correspondence to: gmugumbate@[Link]

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License ([Link]
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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Computational Deorphaning of Mycobacterium tuberculosis Targets
DOI: [Link]

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[64] Desaphy J, Azdimousa K,
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[65] Ehrt C, Brinkjost T, Koch O. Impact

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[67] Kawabata T. Detection of

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10.1002/prot.22639

67
 Section 3

Current View towards

Pharmacokinetics in Drug
Discovery

69
Chapter 5

Revisiting Pharmacokinetics and

Pharmacogenetics of Methadone
in Healthy Volunteers
Natalia Guevara, Marianela Lorier, Marta Vázquez,
Pietro Fagiolino, Iris Feria-Romero and Sandra Orozco-Suarez

Abstract

Methadone acts as a μ opioid agonist, a serotonin and norepinephrine reuptake

inhibitor, and a noncompetitive N-methyl-D-aspartate receptor antagonist. These
actions altogether are responsible for its efficacy in the management of chronic
pain. It is available as a racemic mixture of (R)- and (S)-methadone, both being
stereoisomers responsible for its analgesic effect. Methadone elimination occurs
mainly through metabolism in the liver by CYP3A4, CYP2B6, and CY2C19 and to
a lesser extent by CYP2D6 and in the intestine by CYP3A4. The relative intestinal
content of CYP2B6 and CY2C19 is unknown but it seems that CYP2B6 is not pres-
ent at the intestine. CYP3A4, CYP2B6, and CYP2C19 convert methadone mainly
into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine(EDDP). CYP2B6 and
CYP2C19 are stereoselective to S- and R-enantiomer, respectively. The pharmacoki-
netic study carried out in healthy volunteers by our research group confirmed that
MTD undergoes recirculation via gastric secretion and intestinal reabsorption and
revealed that the drug is extensively metabolized in the liver but intestinal metabo-
lism is not only relevant but also stereoselective. Polymorphisms of the CYP2B6 and
CYP2C19 isoenzymes and their relationship with the pharmacokinetics of MTD
were also assessed.

Keywords: methadone stereoisomers, EDDP stereoisomers, pharmacokinetics,

pharmacogenetics, stereoselectivity

1. Introduction

Methadone (MTD) is a synthetic opioid with primarily a μ and δ opioid agonist

action, but some other novel mechanisms implied in pain relief such as antagonism
of the N-methyl-D-aspartate (NMDA) receptor, and inhibition of serotonin and
norepinephrine reuptake are also reported in the literature [1–5]. These multiple
receptor activities make it an attractive choice for analgesia. It is increasingly used
to manage cancer and chronic nonmalignant pain [6, 7] and although some authors
stated its use in neuropathic pain as well, [3, 8, 9] good evidence for this use is still
lacking [10]. NMDA antagonism has an important role in attenuating tolerance [11].
In comparison to oral morphine and other opioids, MTD has a higher bioavail-
ability and initial rapid and extensive distribution and a slower elimination rate.

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Drug Discovery and Development - New Advances

Unfortunately, it is the unique pharmacokinetics and pharmacodynamics of MTD

that render its somewhat unpredictable effects.
This chapter focuses on revising plasma-gastrointestinal-plasma recirculation
of MTD, evaluating the relative importance of CYP3A4, CYP2B6, and CYP2C19
isozymes in the metabolism of the drug, and assessing the possibility of attenuating
the metabolism mediated by localized isoenzymes, mainly in the liver to favor the
recirculation of the drug. Polymorphisms of the CYP2B6 and CYP2C19 isoenzymes
and their relationship with the pharmacokinetics of MTD in healthy volunteers are
also dealt with.

2. Pharmacokinetic study

MTD is a racemic mixture of two enantiomers: (S)-methadone and

(R)-methadone. (R)-methadone accounts for its opioid effect with a minor antago-
nism on NMDA-receptors, whereas (S)-methadone is responsible for serotonin and
norepinephrine reuptake inhibition and NMDA-receptor antagonism [4, 5, 12–14].
The mean bioavailability of MTD is around 75% (range 36–100%). MTD
undergoes first-pass metabolism and is detected in plasma 30 minutes after intake.
The time needed to reach peak concentration in plasma (Tmax) in patients is
4.4–6 hours and 2.8 hours in healthy volunteers [15, 16]. It is also an efflux trans-
porter (P-glycoprotein) substrate [17]. MTD is a highly lipophilic drug with basic
properties (pKa = 8.3) [18]. Following absorption, it is distributed to the brain,
liver, kidneys, lungs, and muscles. It binds to alpha-1-acid glycoprotein (60–90%)
[19]. The fluctuation in the levels of this protein with physiological and pathological
changes and with age and sex explains the variability in plasma protein binding of
basic drugs both between individuals and within individuals [20]. (R)-MTD has
lower plasma protein binding in comparison with the (S)-enantiomer [21].
The metabolism of MTD is thought to occur mainly in the liver by the cyto-
chrome P450 (CYP450) enzyme system, primarily by CYP3A4 also located in the
intestine, but human drug-drug interaction studies are not consistent with this and
other enzymes are thought to be more involved in its hepatic metabolism such as
CYP2B6 and CYP2C19. Excretion through the kidneys and feces is not negligible
and since MTD is a basic drug, if urinary pH increases, MTD clearance in urine
decreases [22]. Its principal metabolite is N-demethyl MTD which rapidly converts
into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The hierarchy
of transforming MTD into EDDP is CYP2B6 > CYP2C19 ≥ CYP3A4. CYP2B6 is
responsible mainly for metabolizing the (S)-enantiomer, while CYP2C19 shows
preference for the (R)- enantiomer. CYP3A4 shows no enantioselectivity [23–25].
The isoenzyme CYP2D6 is also implicated in metabolizing MTD but through a dif-
ferent pathway and to a lesser extent [26]. The unbound MTD clearance is stereose-
lective, being the S-enantiomer cleared faster [21].
The elimination half-life after the first dose is longer than at steady state due to
induction of CYP3A4 and P-glycoprotein by MTD [15, 16, 27]. Our research group
found a nonlinear relationship between steady-state MTD plasma concentrations
and daily dose [28].
Due to its basic properties, MTD can be recovered in gastric juice [29] and
subsequently reabsorbed after the gastric content is emptied into the duodenum
completing a blood-gastrointestinal-blood recycling.
Although venous plasma drug concentrations are the ones used in pharmaco-
kinetics studies, vein and artery drug concentrations are not the same throughout
time. Arterial drug concentrations are higher than the respective venous concen-
trations during drug input. For highly lipophilic drugs rapidly distributed from

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DOI: [Link]

arterial blood to tissues such as MTD, such increased tissue/venous plasma ratio
would explain the toxicity of MTD in certain tissues and the lack of correlation
between venous MTD concentrations and adverse effects [15]. When elimination
predominates, the opposite is observed [30]. However, if MTD recycling is operat-
ing at the monoexponential decay of levels, increased arterial/venous plasma drug
concentration ratios will also be observed due to drug reabsorptions.
So its storage in body tissues and the slow release to plasma as well as its
recycling process could be responsible for its prolonged elimination half-life.
This last fact is exploited in preventing withdrawal symptoms. However, the long
half-life does not seem to correlate with the observed shorter duration of analgesia
(6–12 hours) after steady state is reached [31].
As measuring drug levels in arteries is an uncommon practice, our group has
been working for a long time [32–35] using saliva in order to surrogate arterial free
plasma drug concentrations as this biological fluid highly correlates with arte-
rial plasma due to the fact that it is produced by ultrafiltration of the latter [36].
Salivary peaks during the elimination phase would be indicating reentry processes
as it was observed in a study carried out with patients [35].
It is important to study the stereoselectivity of MTD metabolism once the blood-
gastrointestinal tract cycling is operating, and to investigate whether the intestinal
metabolism of MTD could be assessed as relevant in relation with the hepatic one.
For this purpose, our research group has carried out an in vivo study.

2.1 Subjects and study design

An in vivo randomized, single-dose, crossover, and compensated study with two

periods and two treatments (A and B) was carried out. A single dose (10 mg) of
MTD was administered to 12 healthy volunteers (six women and six men between
18 and 42 years old) under fasting conditions. Blood, saliva, and urine samples were
taken to determine pharmacokinetic and exposure parameters for both enantiomers
of MTD and of its main metabolite (EDDP), as well as for the genotyping studies.
The previous night and 30 minutes before the administration of MTD, the subjects
received a dose of 10 mg of metoclopramide in order to avoid nauseas and vomits.
Part of these results has already been published [37].
Food intake was standardized in the study protocol and was different for treat-
ments A and B. There was a higher frequency of food intake in the latter in order to
investigate the impact of blood-gastrointestinal tract-blood recirculation processes on
MTD metabolism. In treatment A, volunteers received lunch, dinner, and breakfast
at 4, 13, and 24 hours post dose, while during treatment B, the volunteers received
lunch, a light meal, a snack, dinner, and breakfast at 4, 7, 10, 13, and 24 hours post
dose. Only frequency of food intake differs between treatments A and B.
The study conformed to standards indicated by the Declaration of Helsinki
and its later amendments, approval was provided by the Ethics Committee of the
Faculty of Chemistry (Uruguay), and all healthy volunteers in the study gave writ-
ten informed consent prior to participation.

2.2 Sampling and MTD and EDDP determination

Blood samples were withdrawn from the antecubital vein through cannula-
tion and saliva samples were collected in Salivette® tubes at the following times:
0–0.5–1–2–3–4–6–8–10–12–16–24–36–48–72 and 96 hours post dose. Urine was col-
lected at 0 (before dose intake) and at the end of the following intervals: 0–2, 2–4,
4–7, 7–8.5, 8.5–10, 10–11.5, 11.5–13, 13–14.5, 14.5–16, and 16–24 hours after dosing
and sample volumes were recorded. Aliquots of urine samples were kept in order to

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Drug Discovery and Development - New Advances

measure the analyte content. Immediately after sampling, pH was measured using
a portable pH meter for urine samples. All samples were kept in a freezer at −25°C
until the time of analysis.
When the pre-dose blood sample was taken, another blood sample was taken
to obtain genomic DNA in order to determine the genotype of the CYP2B6 and
CY2C19 isoenzymes of the subjects.
MTD enantiomers in plasma, saliva, and urine were quantified. EDDP enantiomer
quantification was performed in urine. MTD and EDDP were extracted with a mixture
of hexane and isoamyl alcohol from 2.0 mL of plasma or 1.0 mL of urine or saliva
samples that were previously alkalinized. Then, the organic phase was evaporated
under a stream of nitrogen, and the residue was reconstituted with the mobile phase.
Imipramine (10.00 μg/mL) was used as the internal standard and 50 mL was added
to plasma or urine or saliva. MTD (in all the three fluids) and EDDP (only in urine)
quantification was performed using a validated HPLC-UV chiral method, which was
an adaptation of a previously published methodology [38]. The mobile phases con-
sisted of phosphate buffer 20 mM pH 6.0 + 2 mM diisopropylamine: acetonitrile (92:8)
for urine analysis and phosphate buffer 20 mM pH 7.0 + 2 mM diisopropylamine:
acetonitrile (82:18) for plasma and saliva analysis. The flow rate was 0.7 mL/min. The
separation of the compounds was performed on a CHIRALPACK AGP™ (100 × 4 mm;
5 μm) column with a silica guard column. Detection was performed at a wavelength of
215 nm. The analysis was carried out at 25°C and the injection volume was 80 μL.
The HPLC method was linear for MTD between 4.0 and 160 ng/mL and
between 19.0 and 3280 ng/mL for plasma or saliva and urine samples, respectively.
The linearity for EDDP in urine was proven from 52.0 to 4200 ng/mL. Inter- and
intra-day precision and accuracy were below 14% for both compounds.

2.3 Pharmacokinetic and statistical analysis

The following pharmacokinetic parameters were obtained from the MTD plasma
and saliva concentration versus time curves for both enantiomers of MTD:

• Cmax: Maximum concentration.

• Tmax: Time to maximum concentration.

• AUC [0–96]: Area under the concentration-time curve from 0 to 96 hours.

• AUC [0–24]: Area under the concentration-time curve from 0 to 24 hours.

• R/S: Concentration ratio of the enantiomers.

Experimental Cmax and Tmax were computed and the AUC was estimated by
the trapezoid method up to 96 hours, or until the last quantifiable concentration
time. As for most of the subjects, the concentrations were not quantifiable for times
longer than 24 h and AUC was determined up to 24 h. The R/S concentration ratio
was computed as an indicator of possible stereoselective metabolic changes because
of drug recycling.
From the urinary concentrations of MTD and EDDP and the volumes of urine
recorded, the amounts excreted in the time interval between two consecutive
micturitions were calculated. Excretion rates versus time were plotted and the R/S
ratios of MTD and EDDP were calculated for this parameter.
Statistical significances between means were assessed by a nonpaired (between
sexes) and a paired (between enantiomers) t-student test.

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DOI: [Link]

2.4 Results and discussion

Mean R- and S-MTD plasma concentration-time profiles for treatments A and B

in women and men are shown in Figure 1. As it is shown in this figure and in Table 1,
a higher exposure of S-MTD for both treatments can be observed due to its higher
plasma protein binding.
Figure 1 also shows a secondary peak 8 hours post dose (4 hours post lunch).
This means a re-entry of the drug into the bloodstream, as a consequence of a
plasma-gastrointestinal-plasma recirculation process of MTD.
The feasibility of MTD to follow this recirculation process is due to its basic
nature previously mentioned. MTD can be secreted into the gastric juice as
a consequence of the pH gradient between plasma (pH = 7) and gastric juice
(pH = 1.2). In addition, after food intake there is an increase in blood flow and in
the fraction of cardiac output destined to the gastric area, which would favor the
secretion of MTD to the gastric juice. When food reaches the stomach, several
milliliters of gastric juice are poured into the gastrointestinal tract, so molecules
of MTD that could have been secreted into the gastric juice from the blood
would pass into the intestinal lumen and could be re-absorbed from there again,
re-entering the bloodstream. This secondary peak was evidenced in the sample
obtained 8 hours post dose for both treatments, but the process could have begun
sometime before as a result of food intake and depending on the gastric emptying
of each volunteer. No differences were observed in the appearance of secondary
peaks between treatments A and B, so a higher frequency of food intake does
not add more mass of recirculating molecules, but perhaps a prolongation of the
recirculation process.
Table 1 summarizes the results obtained from the plasma samples.

Figure 1.
Mean R- and S-MTD plasma concentration-time profiles for treatments A and B in women and men.

t1/2 AUC (0–24) CMAX TMAX

± SD (h) ± SD (ng.h/mL) ± SD (ng/mL) (range)a (h)

R S R S R S R S
b b b b
Plasma Women 27.3 ± 12.6 22.1 ± 7.8 190 ± 61 302 ± 89 18.6 ± 10.3 32.2 ± 8.4 3.5 2.0
(2.0–8.0) (1.0–8.0)

Men 25.0 ± 4.7 24.6 ± 6.8 192b ± 101 304b ± 118 17.5b ± 7.9 31.0b ± 6.3 3.0 2.0
(1.0–10) (1.0–8.0)
a
median (range).
b
p < 0.01, paired t-student test between R and S.

Table 1.
Mean (± standard deviation) pharmacokinetic parameters of MTD obtained in women and in men.

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Drug Discovery and Development - New Advances

The value of Tmax obtained in healthy volunteers is in agreement with the

literature [16, 27]. In patients, the value of Tmax is higher in comparison to healthy
volunteers as chronic use of MTD delays gastric emptying and gastric motility and
hence absorption.
Urinary exposure, as can be seen in Figure 2, showed an inverse relationship
between isomers. Bearing in mind that the rate of urinary excretion of MTD could sub-
rogate its free plasma concentration, a lower intrinsic clearance of the R-isomer could
be evidenced and therefore a stereoselective biotransformation in favor of the S-MTD.
Volunteers excreted significantly (p < 0.01) more (R)-methadone and (S)-EDDP
(p < 0.001) than the corresponding enantiomers as is shown in Figures 2 and 3,
respectively. However, as information about the stereoselectivity of the metabolite
clearance is lacking, no conclusion can be drawn about its bioavailability.
The profile of the urinary excretion rate of MTD did not show the same pattern
of secondary peaks as the profile of MTD plasma concentrations did. This could be
explained by a significant drop in the rate of excretion after lunch, which can be
attributed to the well-known increase in urinary pH after food intake (postprandial
alkaline tide), causing a decrease in urinary MTD excretion.
A higher incidence of nausea was detected in women than in men during the
experimental phase of the study; in fact, this adverse effect was not observed in
men. This motivated a differentiated analysis of the results according to the sex of
the subjects, as differences in the pharmacokinetics of opioids between the sexes
can affect the safety and efficacy of the treatments. The pharmacological activity
can be better predicted from free plasma concentrations than from the total ones,
and as mentioned above, the rate of urinary excretion of MTD could subrogate the
free plasma concentration. Women presented a higher urinary exposure of R-MTD
(mainly responsible for the μ effect), which correlates with the greater intensity of
adverse effects that they presented around Tmax in comparison to men. This is also
shown in the profiles of saliva concentrations of MTD (Figure 4), which are also
related to free plasma concentrations.
To assess stereoselectivity in MTD metabolism, R/S ratios were studied through-
out time as is shown in Figure 5. R/S ratios of MTD were constant once absorption

Figure 2.
Mean urinary excretion rates of R- and S-MTD versus time in men and women.

Figure 3.
Mean urinary excretion rates of R- and S-EDDP versus time in men and women.

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DOI: [Link]

Figure 4.
Mean R- and S-MTD saliva concentration-time profiles for treatments A and B in women and men.

Figure 5.
Mean (± 95%CI) R-to-S MTD and EDDP urinary excretion rate ratios after oral administration of MTD.

had finished. During the absorption and rapid disposition phase, this ratio is
increasing. However, R/S ratios of EDDP were constant from the beginning, except
after food intake (mainly between 3 and 7 hours post lunch intake when MTD recir-
culation is taking place) when the ratio decreased and this might explain differences
in EDDP systemic formation.
The molecules of MTD present in the systemic circulation undergo both intestinal
and hepatic stereoselective metabolism by CYP2C19 and CYP2B6 enzymes. CYP2C19
is stereoselective towards the R-isomer while CYP2B6 towards the S-isomer. After food
intake, when a process of drug reentry is operating, the molecules of MTD that had
been secreted into the gastric juice can be reabsorbed in the intestine. Consequently,
a greater number of molecules enter the enterocyte. The change observed in the R/S
ratio of EDDP after the ingestion of meals evidences a different stereoselectivity
between intestinal and hepatic metabolism, possibly due to a relative differential
content of CYP3A4 and CYP2C19 in enterocytes and hepatocytes, being the relative
presence of CYP3A4 greater at the intestine. In the case of MTD, the metabolism of the
S-enantiomer is favored after the passage of MTD through the intestine compared to
its passage through the liver. Although during food intake there is an increased blood
flow to the splanchnic area, and the liver and the other organs in this region receive a
greater number of molecules from the blood coming from areas that do not belong to
the splanchnic region, for drugs secreted in the gastric juice, the fraction of molecules
that the intestine receives is even greater because there is a supplementary quantity of
molecules that enter the intestine coming from the gastric juice. If no secretion was
taking place, the molecules would be transferred from the stomach directly to the liver
through the portal bloodstream without passing through the enterocytes.
Therefore, by favoring recirculation rather than bypassing hepatic metabolism,
the intestinal metabolism would be increasing. Our research reveals an important
role of the intestine in the systemic (and pre-systemic) metabolism of MTD,
presenting a greater stereoselectivity towards the S-isomer. Although this isomer
has little or no activity as an opioid agonist, it is able to inhibit the reuptake of

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Drug Discovery and Development - New Advances

serotonin and noradrenaline, in addition to acting as a noncompetitive antagonist of

NMDA receptors, actions that enhance the opioid analgesic effect of the R- isomer.
As a result, by favoring recirculation, the analgesic potency of MTD would not be
increasing but decreasing instead. This could explain the shorter duration of the
analgesic effect of MTD in view of the reported long elimination half-life.

3. Pharmacogenetic study

CYP2B6 and not CYP3A4 is the principle determinant of clinical MTD elimina-
tion and is one of the most polymorphic cytochrome P450 (P450) genes in humans
and, currently, it has 30 defined alleles with over 100 described polymorphisms
[39]. According to Kharasch et al. [40], CYP2B6*6 allele carriers showed higher
MTD concentrations and slower elimination, whereas CYP2B6*4 carriers had lower
concentrations and faster elimination.
CYP2C19 plays an important role in MTD metabolism and CYP2C19 gene is highly
polymorphic as well. Loss of enzyme activity results from the CYP2C19*2 allele and
the CYP2C19*17 allele is associated with increased enzymatic activity [41, 42].

3.1 Methodology

Once the genomic DNA was obtained from the leukocyte fraction, the individu-
als were genotyped for the CYP2B6 and CYP2C19 genes by massive sequencing,
which was carried out at the Institute of Genomic Medicine (INMEGEN) in Mexico.
In order to be processed by massive sequencing, genomic DNA samples should
have a concentration higher than 10 ng/μL, and the ratio of absorbances 260/280
and 260/230 should be approximately 2 to be able to consider that the DNA
obtained was of good quality. In cases in which the sample did not meet these
requirements, purification was performed using the Mag Jet Genomic DNA Kit
(Thermo Scientific) which includes incubation with proteinase and RNAse and
purification with magnetic beads.
As a result of this processing, the genotype of the 12 volunteers was obtained for
CYP2B6 and CYP2C19 enzymes. Considering the polymorphisms found and based
on the literature, we determined the phenotype that would be expected, that is,
increased, normal, or decreased enzyme activity.

3.2 Results and discussion

Regarding the polymorphisms in the gene that encodes CYP2C19, 5 of the volun-
teers in our study presented the allelic variant * 2 (rs4244285), which is associated with
a decrease in the activity of the enzyme, whereas 2 volunteers presented the allelic
variant * 17 (rs3758581), which is associated with an increase in the activity. Regarding
the polymorphisms in the gene that encodes CYP2B6, 6 volunteers presented the allelic
variant * 4 (rs2279343), which determines an increased enzymatic activity.
S/R ratios for MTD in plasma and urine and the S/R ratios for EDDP in urine
were calculated. The individuals were grouped into two. Group 1 included those
volunteers in whom the activity of CYP2B6 was increased and CYP2C19 activity
was normal or decreased as well as those volunteers in whom CYP2B6 activ-
ity was normal but CYP2C19 activity was diminished. Group 2 included those
individuals with normal activity of both enzymes as well as those in which the
activity of CYP2B6 was normal but that of CYP2C19 was increased and a case

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DOI: [Link]

Group Volunteer CYP2B6 CYP2C19 S/R S/R S/R

activity activity MTD in MTD EDDP
plasma in in urine
urine
Group 1 Vol. 1 Increased Decreased 1.63 0.840 1.757

Vol. 2 Increased Decreased 1.09 0.684 1.992

Vol. 4 Increased Normal 1.80 0.949 1.705

Vol. 5 Increased Decreased 1.65 0.818 2.019

Vol. 8 Normal Decreased 1.84 0.732 1.751

Vol. 9 Normal Decreased 1.61 0.782 2.050

Vol. 11 Increased Normal 1.55 0.567 1.707

Group 2 Vol. 3 Increased Increased 1.55 0.703 2.071

Vol. 6 Normal Normal 1.43 0.869 1.729

Vol. 7 Normal Increased 1.89 0.846 2.011

Vol. 10 Normal Normal 1.86 0.743 2.277

Vol. 12 Normal Normal 1.51 0.528

Average of the total 1.62 0.76 1.92

number of volunteers

Standard error 0.065 0.032 0.058

Average Group 1 1.60 0.77 1.85

Standard error Group 1 0.093 0.046 0.060

Average Group 2 1.65 0.79 2.02

Standard error Group 2 0.094 0.036 0.113

Table 2.
S/R ratios for MTD in plasma and urine and for EDDP in urine obtained in treatment A and the activity of
CYP2B6 and CYP2C19.

in which the activity of both enzymes was increased. This classification allowed
grouping those individuals, in whom a preferential biotransformation was
expected on the S isomer, considering the activity of the enzyme together with
its stereoselectivity. The averages of the S/R ratios for each group were calculated,
both for treatment A and for treatment B, and the results are shown in Tables 2
and 3, respectively.
The three average S/R ratios were compared by a t-student test, and no signifi-
cant differences were obtained in any of the cases. However, the S/R ratios of MTD
either in plasma or in urine are lower in Group 1 compared to Group 2, which is
in agreement with the stereoselectivity of CYP2B6 towards the S-MTD since the
metabolism of the S-isomer is greater compared to the R-isomer when the activity
of CYP2B6 is increased and the activity of CYP2C19 decreased. The results obtained
for the S/R ratios of EDDP are different, probably because the biotransformation of
MTD mediated by these enzymes also leads to the formation of other metabolites.
Moreover, there is a lack of information in the literature about the stereoselectivity
of EDDP clearance.
Genetic variation of CYP2C19 mainly affects MTD metabolism, and it has a
minor effect on the metabolite, maybe because it contributes very little to EDDP
formation (1/10 compared to CYP2B6 contribution).

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Group Volunteer CYP2B6 CYP2C19 S/R S/R S/R

activity activity MTD in MTD EDDP
plasma in in urine
urine
Group 1 Vol. 1 Increased Decreased 1.74 0.874 1.534

Vol. 2 Increased Decreased 1.26 0.716 1.848

Vol. 4 Increased Normal 1.41 0.958 1.565

Vol. 5 Increased Decreased 1.83 0.776 1.993

Vol. 8 Normal Decreased 1.63 0.717 1.657

Vol. 9 Normal Decreased 1.53 0.749 2.397

Vol. 11 Increased Normal 1.77 0.638 1.923

Group 2 Vol. 3 Increased Increased 1.37 0.820 2.126

Vol. 6 Normal Normal 1.86 0.866 1.871

Vol. 7 Normal Increased 1.71 0.781 1.635

Vol. 10 Normal Normal 1.65 0.766 1.961

Vol. 12 Normal Normal 1.95 0.580

Average of the total number of volunteers 1.64 0.77 1.86

Standard error 0.061 0.027 0.078

Average Group 1 1.60 0.78 1.85

Standard error Group 1 0.078 0.041 0.114

Average Group 2 1.71 0.81 1.90

Standard error Group 2 0.100 0.020 0.102

Table 3.
S/R ratios for MTD in plasma and urine and for EDDP in urine obtained in treatment B and activity of
CYP2B6 and CYP2C19.

4. Conclusions

Our results confirm MTD recirculation via gastric secretion and subsequent
intestinal reabsorption. MTD is extensively metabolized in the liver but intestinal
metabolism is not only relevant but also stereoselective.
Although the opioid effect of MTD is mainly due to the R-isomer, the S-isomer
also has an analgesic action by inhibiting the reuptake of serotonin and nor-
adrenaline and by exhibiting a noncompetitive antagonism of the NMDA receptor.
The latter action is also responsible for preventing or attenuating tolerance and
withdrawal syndrome. Therefore, in those patients who have an increased activity
of the CYP2B6 enzyme or a normal activity of this enzyme in combination with a
decreased activity of CYP2C19, (situations that favor the S-isomer metabolism), the
analgesic effect could be diminished and the development of tolerance as well as the
withdrawal symptoms could be exacerbated.
Despite the fact that blood-gastrointestinal-blood recycling extends the resi-
dence of a drug in the body, in this case, the elimination of the S-isomer is increased
with each passage through the enterocyte. Consequently, the recycling process of
MTD would not be favoring an increased analgesic effect as it would be expected.
This is in agreement with the shorter duration of analgesia observed in the clinical
setting after steady state is reached.

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DOI: [Link]

The occurrence of frequent adverse effects such as nausea was observed only
in women, even after receiving two doses of metoclopramide prior to the dose of
MTD. Although tolerance to nausea and vomits develop with chronic use, the physi-
cian should consider a lower starting dose of 5 mg/day for women. Apparently, an
initial dose of 10 mg/day for men could be appropriate.

Acknowledgements

The genetic part of this work was supported by Consejo Nacional de Ciencia
y Tecnología (Conacyt, México) Research [grant number 248513]. Natalia
Guevara received fundings to perform this work from the Comisión Sectorial
de Investigación Científica (CSIC), Programa de Iniciación a la Investigación,
Universidad de la República (Uruguay).

Conflict of interest

The authors declare no conflict of interest.

Author details

Natalia Guevara1, Marianela Lorier1, Marta Vázquez1*, Pietro Fagiolino1,

Iris Feria-Romero2 and Sandra Orozco-Suarez2

1 Pharmaceutical Sciences Department, Faculty of Chemistry, Universidad de la

República, Montevideo, Uruguay

2 Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de

Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro
Social, Ciudad de México, México

*Address all correspondence to: mvazquez@[Link]

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License ([Link]
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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Drug Discovery and Development - New Advances

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Chapter 6

ADME Profiling in Drug

Discovery and a New Path Paved
on Silica
Arne Krüger, Vinicius Gonçalves Maltarollo, Carsten Wrenger
and Thales Kronenberger

Abstract

The drug discovery and development pipeline have more and more relied on
in vitro testing and in silico predictions to reduce investments and optimize lead
compounds. A comprehensive set of in vitro assays is available to determine key
parameters of absorption, distribution, metabolism, and excretion, for example,
lipophilicity, solubility, and plasma stability. Such test systems aid the evaluation
of the pharmacological properties of a compound and serve as surrogates before
entering in vivo testing and clinical trials. Nowadays, computer-aided techniques
are employed not just in the discovery of new lead compounds but embedded as
part of the entire drug development process where the ADME profiling and big data
analyses add a new layer of complexity to those systems. Herein, we give a short
overview of the history of the drug development pipeline presenting state-of-the-
art ADME in vitro assays as established in academia and industry. We will further
introduce the underlying good practices and give an example of the compound
development pipeline. In the next step, recent advances at in silico techniques will
be highlighted with special emphasis on how pharmacogenomics and in silico PK
profiling can enhance drug monitoring and individualization of drug therapy.

Keywords: ADME, drug discovery, in silico prediction, pharmacokinetics prediction,

QSAR

1. Introduction

Drug discovery and development grew into a wide interdisciplinary field

during the last decades and many factors played and play an important role in
the successful evolution from a bioactive compound, or so-called new molecular
entity (NME), into a potential drug [1]. Herein, we discuss the drug discovery
and development (DDD) process where the pharmacokinetic profiling in terms of
ADME assessment is concerned. Therefore, we provide a short overview of the in
vitro, ex vivo, and in vivo state-of-the-art techniques used in academy and industry
with special emphasis on how recent advances in computer science paved the
path for in silico prediction in the DDD process for small molecules. However, the
discussion of the whole topic is out of the scope of this review, which only aims to
give insights into the principle process of (computer-aided) drug discovery and
development.

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Drug Discovery and Development - New Advances

The current state of pharmaceutical DDD estimates that only up to ten com-
pounds out of thousand screened hits would result in optimized leads and enter
preclinical testing, with a chance of 9.6% to pass the clinical testing phase [1, 2].
Additionally, the drug approval process is estimated to last in average 15 years, with
major expenses in phases II and III of clinical trials, which highlights the drawback
a failure in (pre-) clinical testing causes [3–6], where the overall DDD cost for each
drug can reach as high as ~$2.56 billion preapproval rising to $2.87 billion including
postapproval investments [6–8]. From the initial small molecule screened as hit to
the optimized lead, a variety of in vitro tests are performed to guarantee efficacy
and safety, but also to find structure-activity relationships (SAR), which can then
be connected to specific physicochemical properties of the compound and further
aid in the lead optimization phase [8–10].
The drug development phase starts with preclinical testing followed by the clini-
cal stage comprising phase I–III human trials. Each of the phases aims to answer
a specific question. Initially, preclinical trials are conducted in animals and can
provide information about whether a drug is toxic or not. Compounds that show no
toxicity in animals then advance to phase I trials, which will study whether the drug
is also safe in healthy humans and provide an initial idea for appropriate dosage.
In phase II, the efficacy of the drug is examined in parallel to potential side effects
to answer the question if it principally meets the expected performance. Phase II
presents the biggest hurdle with a transition success rate as low as 30%. Ultimately,
drug candidates enter clinical phase III in which the preliminary results found so far
need to be proofed and any adverse reactions monitored to make sure that the drug
really helps treating the disease [2, 11].
Starting from the generation of a lead compound assessment, and optimiza-
tion of pharmacokinetic properties and correlation to pharmacodynamic effects
increases in importance as one of the three major attrition causes among toxicity
and efficacy [8, 12]. In this sense, it is not surprising that the period between lead
and the clinical candidate is sometimes referred to as “valley of death” due to the
often occurring failures and dead ends during this time of the DDD process, which
results in high costs and missing deadlines [13].

2. Role of computer-aided techniques in drug discovery

In a long ongoing effort, more and more in silico techniques are being integrated
into several points of DDD with different purposes. In silico techniques can ease the
process of SAR assessment as well as the generation of compound series by guiding
combinatorial chemistry since they allow fast and easy evaluation of compounds
prior to synthesis from big libraries. For instance, combinatorial chemistry offered
an option to readily produce a broad range of potentially pharmaceutical active
small molecules in a short time, while SAR data in combination with complex math-
ematical algorithms, such as regression analyses based or machine-learning–based
approaches, allow to determine the potential effects of the analogues and deriva-
tive’s structures a priori [8].
Latter approach can save time and resources by eliminating in early stages
molecules that have predicted low efficacy against the target or to suggest the
next round of chemical modifications [14, 15]. Still, lead generation and/or
optimization will eventually also include in vivo testing after no toxic side effect
was shown in vitro. In vivo efficacy testing will be carried out as proof of concept
followed by PK assessment and ultimately animal models of human disease to
find correlations between preliminary data and potential performance later on in
humans [12].

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ADME Profiling in Drug Discovery and a New Path Paved on Silica
DOI: [Link]

In silico ADME prediction aims to generate tools and models based on experi-
mental data to calculate in vivo behavior of compounds by finding quantitative
structure-property relationships (QSPRs), which connect structural information
to physical and chemical characteristics or even biological behavior (quantita-
tive structure-activity relationship; QSAR). Gained empirical data are then
related to descriptors/properties thereby supporting the process of hit-to-lead
optimization [10, 16].
When using in silico methods for prediction, it is important to keep in mind that
algorithms and tools applied are only models thus being only as good as the data
and idea they are based on. That implies a continuous experimental validation and
improvement as a basic principle that is supported by an interdisciplinary team. In
this sense, frequently used models include QSPR predictors, matched molecular
pair (MMP), and data trend analysis since they allow comparably easy application
and are based on a high amount of (end) point data. For instance, some experi-
ments offer highly convenient data but do not contribute much to model design,
whereas others show high variability but lead to impactful models. Considering
the nature of data, it is important to know which type can be used as input from
different sources (low variability biological and activity data or homogeneously cal-
culated chemical descriptors) in contrast to data that should only be used from one
source (Caco-2, MDCK). A sophisticated approach to generate reliable data or to
determine differences between individual experiments is to use assays with control
compounds [10]. The target property must be obtained under the same experimen-
tal condition and, in the best scenario, obtained from the same laboratory aiming to
avoid interlaboratory and interpersonal data noise [17].
Furthermore, the choice of the number and type of molecular descriptors has a
high impact, since it influences the accuracy and interpretability of the model. One
would expect that using the maximum number of descriptors would be beneficial,
but in reality, the risk of overfitting the data or losing the interpretability is a trade-
off. This leads to the point that it is fundamental for a “good” model to find the per-
fect compromise between quality and quantity. Nevertheless, it is crucial to test and
train a model and to evaluate its predictability by different means, such as statistical
measures and internal and external validation as recommended by organizations as
OECD [18], and also includes outlier analysis to reduce the noise in the model. An
extensive review of different adequate validation methods is discussed in [19].
As a result of newly achieved advances in computational capability, more com-
plex models and algorithms can now be applied. Despite this, it is still a challenge
to create a model for the pharmacokinetic and pharmacodynamic phenomena and
interactions within an organism as complex as a mammal, let alone humans [10].
Finally, notwithstanding the apparent linearity, the development of a new chemical
entity into a drug is an iterative process, even more, where modeling is concerned,
with data from failed attempts being integrated into the new predictions [13].

3. How specific parameters shape the pharmacology studies

Pharmacology is a major part of the DDD process and describes the interaction
of an organism and the drug. It can be divided into two main branches: while phar-
macodynamics (PD) describes what the drug does to the body, pharmacokinetics
(PK) is interested in what the body does to the drug [20]. The main processes of PK
are absorption, distribution, metabolism, and excretion (ADME), finally comple-
mented by toxicity (ADMET). While ADME tries to maximize the pharmacological
performance of a small molecule, toxicology aims to ensure that it causes no harm
in any kind of side effect [21].

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Drug Discovery and Development - New Advances

The big hurdle to overcome is to combine appropriate physicochemical proper-

ties of the drug, which would drive its interaction with the organism and show
biological activity [22]. Or according to Hodgson: “A chemical cannot be a drug, no
matter how active nor how specific its action, unless it is also taken appropriately
into the body (absorption), distributed to the right parts of the body, metabolized
in a way that does not instantly remove its activity, and eliminated in a suitable
manner—a drug must get in, move about, hang around, and then get out” [21].
As already reviewed [23] and suggested by the FDA, PK/PD assessment is
one of the main focuses for optimization in the drug development process. This
is apparently an idea that was shared among many: whereas ADME evaluation
was previously addressed in the late stages of preclinical development, currently
it became a major concern throughout the whole DDD process, starting from
the very beginnings in drug discovery approaches until the very last steps in lead
optimization [21, 24].
For each step in the drug’s path through the body, several parameters determine
the destination of the drug. In respect to this, each of those parameters would be
addressed directly and individually. Unfortunately, to address experimentally each
potential parameter is timely unviable, due to the complexity of the human body
where all those parameters influence each other. This is not only restricted to mecha-
nisms within the body between different compartments but also extends to interper-
sonal variations introduced through gender, age, genetic state, disease, etc. To find
an approximation, most of the important variables are indirectly evaluated by either
models or surrogates (Table 1). In an approach to characterize the properties of
compounds, facilitate calculations, and allow standardization between experiments,
descriptors are introduced as numerical representations encoding aspects of the
chemical information of a molecule. Examples of descriptors and properties include
molecular weight and H-bond donors/acceptors and they can be directly obtained
from experimental or generated by computational techniques [25].

In vitro Ex vivo/cells In vivo In silico

Absorption Physicochemical MDCK Mouse model QSPR/QSAR

properties Caco-2 Knock-out/ pKa
Dissolution and Transfected cells down mice logP, logD
solubility Humanized Binding and
Cell monolayers mice expression of
Artificial membranes transporters
Inhibition of efflux
pumps

Distribution PAMPA Plasma/tissue Vd Vd

IAM binding Plasma Plasma protein
HAS-coupled (RP-) protein binding
HPLC binding Activity and
expression of
transporters

Metabolism S9 fraction Hepatocytes Humanized Half-life

Liver microsomes (HepG2) animals Activity and
Recombinant enzymes Isolated tissue expression of
Isolated organ transporters

Excretion Isolated tissue Urine analysis Half-life prediction

Isolated organ
Transfected cells

Table 1.
Tools for ADME evaluation.

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ADME Profiling in Drug Discovery and a New Path Paved on Silica
DOI: [Link]

For instance, although the perfect approach of PK profiling would also reflect
the kinetics of drug administration and concentration at the site of action, most in
vivo systems rely on plasma sampling as a medium of drug equilibrium since it is
easily accessible. As a consequence, results are highly influenced by intrinsic and
extrinsic factors such as interpersonal variances as already stated above [20].
Each compound possesses individual physicochemical properties, such as solu-
bility or lipophilicity, which are influenced by biochemical properties of the body
as the different pH of tissues. Although they can be similar, each compound will
behave differently, and it is futile to address in vivo behavior without any prelimi-
nary knowledge of the basic PK parameters in vitro [26].
Furthermore, every PK assessment varies depending on the route of admin-
istration and requires different models and assays. While some routes depend on
absorption mechanisms like oral and transdermal administration, others (i.e.,
intravenous) directly target the bloodstream and the bioavailability is essentially
equal to 100%. Hereafter, we will discuss oral administration parameters of small
molecules as the most common form due to many advantages like reliability, safety,
price, their experimental approaches, and most common prediction modes [27, 28].
Passive transport across membranes is defined as permeability, which is depen-
dent on lipophilicity, since biological membranes are virtually lipid bilayers, and is
by far the most important transport for small molecules, especially in oral absorp-
tion [8, 24, 29]. Nonlipophilic compounds normally do not traverse membranes
passively, while highly lipophilic molecules run the risk to get stuck within the
membranes [30].
Properties utilized for measuring lipophilicity are the logarithm of the partition
coefficient (logP) and the distribution coefficient (logD) with the first not dif-
ferentiating between ionized and nonionized species. Both are normally applied for
n-octanol/water representing an organic and aqueous phase, respectively [21, 26].
Ionizability and lipophilicity provide a strong indication if a compound is likely
to be orally absorbed or not [21].
Ultimately, also the molecular size of the compound is involved in successful
absorption due to the aforementioned effects on permeability and solubility [31].
Usually, increasing molecular weight by adding new chemical moieties leads to
decreased solubility in aqueous solutions [32] and while big lipophilic compounds
partition passively along membranes (transcellular), small charged molecules can
also cross membranes via tight junctions (paracellular) [26]. For oral absorption in
terms of permeability, Lipinski and collaborators already proposed in 1997 [33, 34]
that orally active compounds should fit at least three of observed four parameters:
molecular weight < 500 g mol−1, logP < 5; number of hydrogen bond acceptors <10;
number of hydrogen bond donors <10; the well-known Lipinski’s rule of 5 (Ro5).
In other words, Ro5 stated a physicochemical space in which molecules outside its
domain has a low probability to become orally active. Other rules, as Veber rules
[35], Daina and Zoete [36], Egan and collaborators [37], Lovering et al. [38], and
Ritchie and colleagues’ [39] works for example, also included other properties as the
sum of hydrogen bond acceptor and donors, rotatable bonds count, polar surface
area, number of aromatic rings, and fraction of sp3 carbon atoms.
Despite the criticism and overinterpretation of Lipinski and derived rules, the
influence of physicochemical parameters on oral bioavailability and related param-
eters (as logP and aqueous solubility) is notable. Moreover, these rules are still being
employed nowadays in virtual screening campaigns aiming to reduce the number
of compounds from massively large available libraries (e.g., ZINC, which contains
more than 750 millions of compounds) [40–42]. Furthermore, those initial steps
instigate the generation of more complex models to predict not just oral bioavail-
ability but other PK-related parameters as Caco-2 permeability, aqueous solubility,

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Drug Discovery and Development - New Advances

and logP as indirectly related properties as well as other direct parameters as

intestinal absorption, metabolism, clearance, etc.

3.1 Aqueous solubility and lipophilicity

As already mentioned, ionizability is one of the most important properties in

PK, thus making pKa the physicochemical property with the highest impact.
Early attempts to increase the efficiency of pKa evaluation were reported by
Morgan and colleagues by scaling down the classical titration and spectrophotomet-
ric methods introducing microscale versions [43].
These alterations, however, could not overcome the principle demands of each
technique, which are moderate precision and frequent calibration (potentiometric),
and the need for a chromophore within the analyte (spectrophotometric) [44].
Starting in 1998, capillary electrophoresis (CE) was effectively used to determine
pKa of many compounds and was further upgraded from Pfizer by implementing
pressure-assisted capillary electrophoresis (PACE) as a standard method, which is
nowadays readily applied in industry settings showing superior features compared
to the aforementioned methods [44, 45]. Other variants such as vacuum-assisted
multiplexed capillary electrophoresis also exist (VAMCE) [46]. A different approach
better suited for HTS is called pH gradient titration offered from Sirius Analytical
Instruments but is still limited due to the UV spectroscopy technology [30].
It is well established that solubility in aqueous media is one of the most impor-
tant physicochemical properties to be evaluated in oral administration. It is not only
necessary for absorption in the GI tract but also a requirement for almost all in vitro
and in vivo assays, which depend on a solved compound. Poor solubility can affect
the reproducibility of assay results by introducing high variability and further
increase development costs of leads with low solubility [26, 47]. Traditionally,
solubility measurements were conducted via labor-intensive potentiometric tech-
niques [48] or equilibrium solubility (thermodynamic; e.g., shake flask) [26]. HTS
alternatives comprise laser nephelometric scans (kinetic) [47] and LC-MS/HPLC
techniques, which can also be performed with DMSO solutions of the compound—
the standard for HTS applications [47, 49]. It should be noted, though, that aqueous
solubility, as described above, is not an optimal model for GI solubility since it does
not consider the composition of the GI fluids [49].
On the other hand, generally speaking, lipophilicity is the ability of a compound
to dissolve in lipids and/or organic solvents thus being able to pass biological
membranes. Descriptors for lipophilicity are the logarithm of the partition coef-
ficient (logP) or distribution coefficient (logD). Classically, logP was determined
using the shake flask method applying n-octanol/water phases. Later, UV spectros-
copy became the standard, which unfortunately is not applicable for compounds
without absorption in the UV range [50]. Today, RP-HPLC methods are frequently
in use due to superior properties [25, 51]. As with many methods, comparison of
results obtained under different conditions and in different laboratories proves to
be difficult with RP-HPLC. A solution offers the implementation of a standardized
lipophilicity value, for example, the chromatographic hydrophobicity index (CHI).
In recent years, a great effort has been made to improve the ability of in silico
models to accurately predict aqueous solubility. One of the most developed model is
Yalkowsky and Jain’s [52] general solubility equation (GSE), which is based on the
melting point (m.p. °C − 25) and logP (the octanol-water partition coefficient of
the un-ionized molecule) of a chemical substance (Eq. (1)), with a relevant predic-
tion power as represented by the coefficient of determination (R2) = 0.96 and root-
mean-square error (RMSE) = 0.53 in a dataset of 1026 organic compounds [53].
General solubility equation as proposed by Yalkowsky and Jain’s [52]:

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logS = 0.5 − 0.01(m.p.°C − 25)–logP (1)

Modifications in terms of the GSE have been proposed, for instance with the
SCRATCH model, which replaces the melting point by molar aqueous activity
coefficient, with comparable accuracy (R2 = 0.956, RMSE = 0.859 in a dataset of
883 compounds) [54]. Ali and collaborators suggested replacing the melting point
descriptor of the GSE with TPSA, aiming to overcome the issues with compounds
with high melting points and also to explicitly take into account the effect of polar
and polarizable atoms on the aqueous solubility [55].
The argument that real drugs are actually more soluble than drug-like molecules,
filtered by Lipinski’s rule of five [56], pointed out the studies in the direction of more
complex models. Indeed, nowadays, the quantitative structure-property relationship
(QSPR) models correlating the aqueous solubility with various molecular descriptors
are often employed. As an example, Chevillard et al. reported the use of a random
forest protocol to select the most accurate model among several available, both in
commercial or free software packages, for each compound [57]. They report that the
multimodel approach can enlarge the applicability domain given that more accurate
results for solubility prediction were obtained in comparison to using individual
models. This approach agrees with other reports that consensus of local QSAR
models can generate predictive workflows, especially for datasets with large struc-
tural diversity [58, 59]. It is worth noting that Lipinski himself recently revisited his
own rules [60], in vision of new potential classes of drugs, such as natural products,
peptide-like, and fragments, which, despite the validated effect, would defy the
original Ro5 limits.

3.2 Ionization state and pKa prediction

Early pKa measurement proves beneficial in lipophilicity assessment since logD

values at any pH can be calculated from the pKa and logP values [25, 50]. Although
octanol/water logP is similar to most components in the body, not all biological
partition processes (i.e., blood-brain barrier and gastrointestinal absorption) can be
easily modeled by it [25].
The prediction of ionization state of compounds, which is indicated by the pKa
value, is relevant to derive several other physicochemical and ADME properties of
drugs, including solubility, lipophilicity, and pharmacokinetic profile. The use of
pKa prediction can be placed in two different stages along the DDD, in the begin-
ning with fast models for larger libraries, intending to generate all possible state
populations of particular compounds, and/or later on with more refined semiem-
pirical and, computationally expensive, the density functional theory (DFT), in
which more accurate ionization states can be accessed. Examples of fast prediction
methods for ionization states, which are available as computer programs, are
SPARC [61], MoKa [62], and Epik, which use the Hammett and Taft approaches for
the pKa prediction [63]. On the other hand, once smaller subsets of molecules are
being addressed, the use of semiempirical or density functional theory (DFT) with
more computationally expensive models was reported to accurately incorporate the
structural features and diversity into the pKa prediction [64, 65].

3.3 Permeability and the use of cellular and noncellular models

As already seen, lipophilicity (logP, logD) is highly involved in membrane

permeability. Apart from the already described in vitro methods for logP and logD
determination, systems for ex vivo/in situ but also in vivo assessment exist as “direct
empirical” determination of permeability. When talking about permeability, the

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difference between passive diffusion and transporter-mediated active transport

needs to be considered.
Cell culture methods have been applied to study intestinal absorption for several
decades already [66]. Finding the correct model or cell line is crucial to assess the
desired parameters such as passive or active transport. In general, it cannot be
distinguished between the different transport mechanisms when using cell culture
approaches, but several models exist to shift the focus on one of the parameters.
Two main cell lines are in use as models for intestinal absorption: Caco-2 and
MDCK cells. Caco-2 cells are derived from a human colorectal carcinoma and
possess many of the typical properties of the small intestine, therefore represent-
ing a well-established and validated assay system for absorption, permeability,
and secretion studies [21, 67]. This assay is mainly used for rank ordering of
compounds in terms of oral absorption and permeability in early phases of drug
design. Unfortunately, results obtained in different batches and laboratories vary
heavily due to several reasons, which make control compound usage necessary and
represent a drawback of the technique [26]. Additional disadvantages include long
preparation times (about 3 weeks) and no specific permeation mechanism evalua-
tion. Caco-2 assays are usually used as a primary assay followed and complemented
by other in vitro and ex vivo methods [68]. Recently, a 3D version of the Caco-2
assay, “Caco-2 3D spheroid permeability assay” was reported, increasing the overall
performance and correlation to a human in vivo data [69].
As already stated above, transcellular permeation either occurs passively via
diffusion of lipophilic molecules or is driven by membrane transporters. Important
transporter includes ATP-dependent efflux transporter such as MRP2, BCRP, and
P-gp and the organic solute transporter and the multidrug resistance protein 3
(MRP3) on the luminal and basolateral membranes, respectively [26].
Madin-Darby canine kidney (MDCK) cells are an alternative to Caco-2
cell-based assays and the next most common cell line for passive permeability
assessment as well as drug-receptor interaction [70]. MDCK cells also are ideal for
transfection and overexpression experiments with human transporters and recep-
tors due to the lack of P-glycoprotein [68, 71]. For instance, the MDCK-MDR1 cell
line overexpresses the multidrug resistance protein 1 (MDR1, P-glycoprotein) and
can be used in concert with other cell-based assays to specifically address the influ-
ence of MDR1 in drug efflux [72].
Immobilized artificial membranes (IAMs) were already used very early on for lipo-
philicity determination and are gaining interest again in recent years for direct perme-
ability measures [25]. IAMs are also intensively used in the measurement of the volume
of distribution to mimic in vivo binding to phospholipids and phospholipid bilayers
(membranes). Therefore, IAMs are discussed in more detail in the following section.
The parallel artificial membrane permeability assay (PAMPA) [73] is a cheap
and fast in vitro alternative to cellular-based assay systems. A very comprehensive
review of recent PAMPA methodologies and applications is available [74]. In prin-
ciple, PAMPA was developed to overcome cellular-based systems (Caco-2, MDCK,
etc.) for passive permeability evaluation, which are error-prone, more difficult,
and labor and time intensive and tend to report false negatives. Another advantage
of PAMPA over conventional cell-based assays is the ability to selectively measure
passive permeability, while in cell-based systems influence of membrane transport-
ers cannot be left out. PAMPA assays can be readily applied in high throughput
processes or scales and different variants exist to address ionic and H-bonding with
membranes that influence permeability and complement the use of Caco-2 and
other cell assays [73]. Bermejo and colleagues also showed a significant correlation
between Caco-2, in situ rat perfusion, and PAMPA assay data underlining applica-
bility of the method for ADME assessment [75].

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Although high-throughput applications of newly developed and standardized

techniques allow gathering of an exorbitant amount of data, it is crucial to also
(cor)relate the physicochemical and biomimetic properties to structural features
of the compound. This will facilitate the development of QSPRs and allows the
construction of in silico models ultimately guiding the medicinal chemistry
efforts [25].
When dealing with oral administration, it is important to note that the drug is
not only confronted with the hurdles of solubility and permeability in the absorp-
tion process but is also facing metabolizing mechanisms (i.e., enzymes) in the
gastrointestinal tract, which are referred to as first-pass metabolism [24, 76]. These
include but are not limited to P-glycoproteins, uridine diphosphate glucuronosyl-
transferase, and mainly cytochrome P450s (CYP450) [24]. This will be discussed
more deeply in the Metabolism section.
Permeability has a direct influence on the drug absorption rate and, as dis-
cussed, despite the several in vitro cellular models available (e.g., Caco-2, PAMPA,
and MDCK), the high costs justify the use of in silico prediction. Further, QSPR
study developed using a large compound dataset of Caco-2 permeability data (1272
compounds) presented good apparent permeability prediction accuracy (R2 = 0.81
for the test set) using the polar volume, number hydrogen bond donors, and the
surface area as main descriptors [77].
However, we are far from a model that can predict overall permeability and, the
current status, rather focuses on individual compartments and tissues, such as the
gastrointestinal (GI) tract, skin, buccal membrane, and the blood-brain barrier
(BBB). Since the first BBB permeability correlations with logP in 1977 [78], models
to predict BBB permeability, particularly logBB (Eq. (2)), have greatly advanced.
Current models using an array of machine-learning methods such as multilinear
regression, support vector machine (SVM), and artificial neural network (ANN)
against a dataset of 320 unique compounds had good predictive power (R2 = 0.89)
[79]. The work of Shen et al. developed SVM models using 1593 compounds (1283
BBB+ and 310 BBB−) by using different pattern selection methods and obtained the
overall accuracy of 98.2% [80]. Both methods have the limitation of unbalanced
datasets (where the number of BBB+ is higher than the BBB− within the training
set), which was addressed on the work of Wang et al. by using resampling meth-
ods coupled with the machine-learning techniques, to achieve accuracy rates of
0.919 in external test data [81]. Wang and collaborators compiled a dataset of 439
unique molecules, which were employed to generate a diverse set of QSAR models
and consensus (R2 = 0.504 for external dataset prediction). They also reported the
use of transporter profiles as additional biological descriptors to develop hybrid
QSAR BBB models, with an improved correlation coefficient R2 = 0.526 for external
validation [82].
LogBB can be calculated by the log of the ratio between the concentration within
the brain (Cbrain) by the bloodstream concentration (Cblood) of the determined
chemical entitiy

logBB = log (_____ )

C
brain
(2)
Cblood

Finally, beyond the usual ADME parameters of interest in DDD, there are
several other unusual ones that also can be predicted; as examples, we here point
the permeability of the models for skin permeability, which evolved from simple
diffusion models based on molecular weight and n-octanol/water partition coef-
ficient [83, 84], until more sophisticated models, such as (non)-linear QSPR models
and even molecular dynamics simulation (as extensively reviewed by [85, 86]).

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4. ADME properties: experimental approaches and in silico models

4.1 Absorption

Oral bioavailability is defined as the amount of drug that reaches the site of
action after oral administration and is influenced by factors like drug solubility and
dissolution, chemical and enzymatic stability in the gastric and intestinal lumen,
interacting luminal contents (food), gastrointestinal transit time, enterocyte per-
meability, and intestinal and hepatic metabolism [24]. Recently, bioavailability has
been also described as the rate and speed of the drug to reach systemic bloodstream,
considering the initial formulation as the starting point.
Oral administration includes a pharmaceutical phase—prior to PK and PD
phases—that comprises disintegration and dissolution of the dosage form. When
using oral dosage forms, the shape and chemical composition (e.g., tablets) play
an important role since they contribute to the time needed for disintegration and
dissolution.
Following the pharmaceutical phase, absorption is the first step in the phar-
macokinetic phase and is defined as the movement of the drug from the site of
administration to the bloodstream. The main properties determining the rate of oral
absorption for small molecules are permeability and solubility [87].
As such, the rate of dissolution and ionization, which are described by the
Noyes-Whitney and Henderson-Hasselbalch equation, respectively, is the key fac-
tors in lead optimization for oral administration and is complemented by lipophilic-
ity as an additional factor influencing membrane permeation and solubility of the
compound [31].
Dissolution can be expressed by a function of the aqueous solubility of a com-
pound, the surface area of the administered tablet (or the particles in other solid
formulation), and a specific dissolution rate constant. Altering any of these param-
eters directly affects the dissolution profile [26]. While solubility is an endpoint
value indicating the amount of a compound that is soluble in a solvent, dissolution
describes the kinetic process of a compound being solved in a solvent [88].
On the other hand, ionization reflects if a compound is present in the charged
or uncharged state and is at least influenced by two major parameters. The physico-
chemical property responsible for ionization is the pKa and describes the ionization
state of that entity at a given pH. It is also referred to as aqueous ionization constant
[30]. Thereby, it is directly influenced by the pH of the environment, the second
parameter, which drastically changes on the way through the GI tract, from about
pH 1 to 8 in the stomach and ileum, respectively.
The determination of the ionization state of a compound in the gastrointestinal
system (stomach, jejunum, ileum, and colon) is crucial for absorption since it
not only influences the solubility of a compound but also the lipophilicity and
permeability [26, 30, 89]. About 60–70% of all drugs (effective 1999) are ioniz-
able, which underlines the role that ionization plays in ADME assessment [30, 90].
While charged molecules easily dissolve in aqueous systems (GI tract), they do
not permeate membranes via passive diffusion and are reliant on active transport.
The contrary is true for uncharged molecules, which pass biological membranes
passively but show low solubility in aqueous solutions. Mechanisms of drug absorp-
tion include passive diffusion, active transport, and receptor-mediated endocy-
tosis, which are influenced by different factors and can themselves influence the
bioavailability.
Similar to model and prediction, the absorption of a drug is a complex process,
which is influenced not only by the physicochemical properties of drugs themselves
but also by the physiological state of the tissue in question. As such, there are a large

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number of prediction models available, which were generated based on the physico-
chemical properties involved in the absorption process, such as membrane perme-
ability and drug solubility. These models can help formulation scientists to optimize
drugs with poor absorption due to low aqueous solubility.
Initial absorption models can be separated into dispersion and compartmental
models [91]. While dispersion models treat the GI as a continuous system, with
variable pH and surface area, compartmental models take into account physi-
ological factors such as transporters. The compartmental absorption transit (CAT)
was one of the first models to regard distinct physiological properties, such as the
minimal absorption in the stomach and colon, while assuming some mathemati-
cal simplifications, such as the instant dissolution of the drug and linear kinetics
[92]. CAT was further modified as advanced CAT (ACAT), by including nonlinear
absorption kinetics and the effects of the first-pass metabolism. ACAT also consid-
ers the gastrointestinal tract as nine subsections, each with unique physicochemical
properties, such as pH, allowed solubility, particle size, and permeability [93].
Novel developments have included other absorption routes other than the GI,
which have been recently included in commercially available software, such as oral
absorption for the development of sublingual zolpidem tablets [94]. The absorption
constant (Ka, expressed in terms of h−1 min−1), or also called first-order absorp-
tion rate constant (to not be confounded with pKa), is employed in most of the
aforementioned models and is determined as a result from the changes in mass of
absorbable drug over time at the site of administration. Ka can be derived from the
decrease in the drug amount of absorbable present at the site of administration over
time; however, it is often indirectly determined by the drug amounts measured in
the blood and/or urine.
Along physicochemical models, which have a global application, machine-learning
techniques were extensively employed to model absorption (as comprehensively
reviewed by Kumar et al. [95]) and are inclined to be local models, since they are
mostly based on a small, homogeneous dataset that influences their applicability
domain.

4.2 Distribution and the role of plasma-binding proteins

After being absorbed and entering the circulatory system, the drug moves
reversibly between different compartments within the body, which is described
as distribution and influenced by several physicochemical properties of the
drug and biological factors of the body. One of the most important properties is
lipophilicity, and as such logP/logD, since it reflects the ability of the compound
to pass biological membranes to reach other sites, tissues, and organs within the
body [25]. Additional factors include phospholipid and (plasma) protein binding,
which reduces the free drug concentration within the body, can prevent the migra-
tion to the receptor side/side of action, and causes drug-drug interactions [25,
96]. Interestingly, binding to plasma proteins can also prolong the drug action by
releasing the drug over a longer period of time. It is also important to note that the
influence of lipophilicity on plasma protein binding is hypothesized to be higher for
acidic compounds than for bases, meaning that negative charges contribute highly
to plasma protein binding and prevent tissue binding, which leads to diminished
volumes of distribution (Vd, Eq. (3)). The Vd is the amount of drug that is freely
available in the blood, thus not bound to plasma proteins or other components [25,
97, 98]. Vd is an apparent volume that increases proportionally to the extravascular
drug binding and not an anatomically defined volume. Consequently, exten-
sive drug binding outside the bloodstream leads to increasing values of volume
distribution.

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Volume distribution (Vd) is defined by the ratio between the amount of drug in
the body (A) and the drug concentration in plasma (C, comprising both free drug
and protein-bound drug):

A
Vd = __ (3)
C

The parameter describing protein binding is the plasma protein affinity con-
stant Ki. Many efforts to determine distribution led to chromatography-based meth-
ods, such as (RP-)HPLC to mimic n-octanol/water logP or lipophilicity to measure
distribution. In general, chromatographic methods are believed to resemble biologi-
cal partition processes more than octanol/water partition [25]. In the beginning,
stationary phases in (RP-)HPLC were either silica-based or polymer-based but both
had difficulties to reproduce logP and logD values despite several additives in the
mobile phases [99]. The introduction of biomimetic (stationary) phases coated with
human serum albumin (HAS), α1 acid glycoprotein (AGP), or immobilized artificial
membranes (IAM) revolutionized the methodology since they allowed a better
approximation of the biological system [25, 100].
A method to address plasma protein binding is the use of HSA and other plasma
proteins (e.g., α1 acid glycoprotein) coupled with RP-HPLC [25, 101]. On the other
hand, HPLC combined with IAMs is a popularly accepted technique for phospho-
lipid interaction and partition and several IAM columns are commercially available
for DDD projects. Both techniques represent good assay systems to model in vivo Vd
in high-throughput scale [98]. Problems with HPLC techniques, which are also true
for biomimetic phases, include the lack of a gold standard that is needed to calibrate
and later standardize results to make a comparison possible [25].
In vitro standard methods for unbound plasma fraction calculation include equi-
librium dialysis and ultrafiltration among several others as the two most commonly
used methods and are considered the gold standard for binding assessment [26].
To calculate the Vd “a priori”/nonexperimentally, plasma protein binding,
experimental logD and pKa are necessary. Then again, based on the Vd, the half-life
(t1/2) of a compound can be calculated [102]. Apart from protein binding, tissue
binding is also involved in the distribution of the compound. Generally, “tissue”
here comprises several components of the human body such as lipids, DNA, or RNA
and is also referred to as nonspecific binding [26].
In silico models to predict the Vd are often based on lipophilicity and solubility
descriptors, which correlate with the fractions of the drugs that are either bound to
plasma proteins or freely available. The work of del Amo et al. not just accurately
predicts Vd and unbound drug fraction but also compares the model’s performance
against the commercially available software VolSurf+ with comparable accuracy
(R2 = 0.70 and 0.71, respectively) [103].
Expanding these studies, the work of Lombardo and Jing generated a set of
models to predict the Vd in the steady state (Vss), using a dataset of 1096 diverse
compounds [104]. They compared models generated by linear (PLS) with nonlinear
(Random Forest) models, recommending the latter, with 33 descriptors, as the
optimal method for Vss prediction.
The Vd of drugs is greatly influenced by binding to plasma proteins with
several machine-learning–based models generated to predict this interaction.
Protein-protein interaction (PPI) information derived from molecular docking
was employed to derive a PPI-QSAR model for a small dataset of antibiotics (65
unique compounds), which resulted in an accurate model (R2 = 0.86 for the test set)
[105]. Additionally, global quantitative models using an array of classification and
regression models using physicochemical and molecular descriptors derived from

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a dataset of 794 compounds were shown to correctly classify the binding status of
the test set compounds and could be used as a prescreening [106]. Another recent
QSAR study using an extensively curated training set of 967 diverse pharmaceuti-
cals aimed to predict plasma protein-bound fractions (fb) using models generated
by six machine-learning algorithms with 26 molecular descriptors [107]. This study
is particularly interesting where the applicability domain is concerned allowing to
differentiate whether the classification derives from (un-)favorable regions.
del Amo et al. recently reported one of the first QSPR models to predict intra-
vitreal volume of distribution and clearance of small molecules [108]; the model
relies on the LogD and hydrogen bond capacity to understand phenomena such as
intraocular pressure and guide drug discovery. Complementarily, the prediction of
the drug passage through the blood-ocular barrier was described to be an important
factor to evaluate volume distribution in this organ [109].
Recently, as a novel approach bridging the animal experiments with human
results, it was shown that in PXB mice, a chimeric mice linage with a humanized
liver, plasma concentration-time profiles could be used to infer human’s compound
half-life [110].
Volume of distribution is also closely related to half-life and clearance param-
eters. As the Vd is a relative measurement of the free concentration of drug in the
blood, this same amount could be excreted by kidneys in the glomerular filtration
(clearance). Consecutively, the rate of clearance (discussed below in Excretion sec-
tion) directly influences the amount of available drug. Naturally, the concentration
of free drug that can bind its molecular target is related to the therapeutic dosage
and the half-life of the administered drug (as seen in Eq. (4)).
Half-life definition. Half-life is calculated by a ratio between the Napierian
logarithm multiplied by the volume of distribution (Vd) and renal clearance (CL):

[Link]
t1 / 2 = _______ (4)
CL

4.3 Metabolism

Drug metabolism normally involves enzymatic modification or degradation of

the compound to facilitate excretion via one of the major clearance organs: liver,
kidney, spleen, or bile. While phase I enzymatic reactions include modifications
such as oxidation, hydrolysis, and reduction to either introduce a functional group
to the molecule or make it accessible, phase II reactions are conjugation mechanisms
(e.g., methylation, acetylation, glutathione conjugation, amino acid conjugation,
and others) that result in polar products that can be actively effluxed [26]. Thus,
isozymes of the CYP450 family and efflux transporters such as P-glycoprotein and
members of the multidrug resistance transporter MRP family are highly involved
in the metabolism of drugs as well as drug-drug interactions, which are a major
attrition cause. For instance, CYP3A4, CYP2C9, and CYP2D6 together catalyze the
hepatic metabolism of about 50% of drugs, which underlines the importance of the
superfamily. Interestingly, when CYP3A4 is expressed, usually P-glycoprotein is as
well [8, 10, 14, 24, 111]. An approximation for metabolic behavior analysis is the use
of either liver microsomes or S9 fractions although also recombinantly expressed
proteins are partially in use [24, 26].
When available, the 3D structure of those proteins could be employed in
molecular docking and molecular dynamics simulations aiming to predict the bind-
ing affinity of drugs or drug candidates aiming the estimation of a PK profile [112].
The metabolism prediction combines mathematical models to predict whether

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the target compound could be a substrate of a specific enzyme in combination

with metabolism site predictions. Usually, those initial predictions are followed
by molecular docking simulations and quantum mechanics simulations due to the
dependency of electronics structure from both substrate and enzyme in catalyzed
reaction [113, 114].
Nowadays, several attempts have been made to develop in silico models for
predicting drug metabolism, specifically site-of-metabolism (SOM), and quite
often are also converted into online server prediction tools for general use, for
instance, the FAst MEtabolizer (FAME) model, which was generated from a diverse
chemical datasets of more than 20,000 molecules and their respective experimen-
tally determined metabolism sites. FAME prediction rates were comparable to other
metabolism site predictors focused on specific enzyme families, despite using only
seven chemical descriptors [115]. Similarly, SMARTCyp server, which initially
relied on the 2D structure of the molecule, without considering electronic proper-
ties or generating 3D structures, to predict CYP2D6 [116], was later expanded for
other CYP isoforms. A more refined version was later updated to include the atomic
solvent accessible surface area, which is independent of 3D coordinates, slightingly
improving the overall prediction accuracy for different CYP isoforms [117]. The
newest SMARTCyp version (3.0) uses the activation energies calculated by
the density functional theory (DFT), meaning the difference between the energy
of the transition state and the reactant complex, to predict SOMs of molecular frag-
ments of the query in an unsupervised fashion. SMARTCyp 3.0 also calculates the
similarity between the query molecule and the model fragment [118].
IDSite approach aims to overcome the ligand-based bias of SOM prediction by
using it as a part of a large framework, more precisely by combining it with molecu-
lar docking, where an atom can be considered a significant SOM by a P450 enzyme
when accessible to the reactive heme iron center, and/or quantum calculations,
where the candidate atom must have some degree of reactivity in the absence of the
enzyme [119]. Similarly, the work of Kingsley et al. combined different approaches
into a framework to predict CYP2C9 substrates. They validated the predictions
from SMARTCyp in an ensemble docking, followed by a QSAR model to account for
influences of both the inherent reactivity of each atom and the physical structure
of the CYP2C9 binding site [120]. This combined approach resulted in 88% of true
SOMs accurately predicted among the top ranked sites.

4.4 Excretion

Excretion is guided by one of the major clearance organs, and the assessment
of clearance behavior sometimes involves isolated organs or tissues [24]. Humans
rely on the kidney clearance as a major route for xenobiotic excretion, despite other
available routes such as feces, bile, sweat, and breath. The excretion pathways
directly impact the concentration of available drugs and are often measured in
terms of half-life and the initial administered dose.
The renal clearance of a drug is another important parameter, which is usually
employed to predict drug excretion. Experimentally, clearance is defined by the
drug concentration drug along a defined time of renal excretion by a linear equa-
tion (Eq. (5)).
Equation for renal clearance. m is the substance’s mass generation rate, K is the
clearance and C is the concentration at the time, and V is the volume where the drug
is distributed, or for systemic approaches the total body water.
dC = − K.C + m
V.___ (5)
dt

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Gombar et al. developed SVM- and MLR-based QSAR models to predict both
systemic clearance and apparent volume of distribution from intravenous data
[121] using as input structural fingerprints and electro-topological states (so-called
E-states), respectively. The model performed with high accuracy, despite the highly
diverse initial dataset employed for its generation, which points the importance of
those models in early steps of the drug-discovery pipeline.
Also, the work of Kusama et al. established a chemoinformatic-based classifica-
tion model to predict the major clearance pathways of 141 approved drugs based
on four physicochemical parameters: charge, molecular weight, lipophilicity, and
protein unbound fraction in plasma, resulting in a final model with an accuracy
of 88% [122]. This model approach was further refined by using support vector
machine and increasing the number of relevant descriptors [123]. In order to better
model the biotransformation processes, often the major triggers of excretion,
the work of Berellini et al. used ELASTICO (Enhanced Leave Analog-Structural,
Therapeutic, Ionization Class Out) to provide an appropriate sampling during the
validation process. Their partial least-square models resulted in a highly accurate
model derived from 754 compounds [124].
On another topic, ABCB1, also known as P-glycoprotein (P-gp or MDR1), is
a membrane protein member of the ATP-binding cassette (ABC) transporters
superfamily. Together with the hERG channel and CYP3A4, P-gp is one of the most
widely studied antitarget, where its inhibition could bring consequences for several
processes, such as the absorption, distribution, and excretion of drugs. Classical
studies used chemometric methods to describe bioavailability in terms of P-gp
and CYP enzyme activities, generating QSAR models based on 805 unique drug
molecules with high accuracy (R2 = 0.80 for the test set) [125]. Alternatively, an
approach to predict P-glycoprotein inhibition using molecular interaction fields,
derived from a literature collection of more than 1200 structures, generated a
pharmacophore model for competitive P-gp inhibition [126].
The most recent reported studies involving prediction of drug clearance, both
from human and rat hepatic in vitro systems, were based on microsomes, with a
recent emphasis on the use of hepatocytes. Wood et al. discuss the inherent limita-
tion of using human hepatocyte predictions, due to underprediction when com-
pared to in vivo clearance data, and the comments on the potential causes for those
divergences [127].
As the pinnacle of ADME in silico approaches, the holistic physiologically based
pharmacokinetic (PBPK) modeling was initially conceptualized by Teorell [128],
aiming to enable the prediction of drugs’ pharmacokinetic behavior in humans using
preclinical data. Recent PBPK models benefit from the large amount of available
ADME data not only to aid the drug discovery process and dose regiment selection
but also to guide the risk assessment for regulatory reviews [129]. PBPK models are
compartmentalized representations of the different organs, and each compartment
can be described by a specific tissue volume and blood flow rate, which communi-
cates with the blood (venous and arterial). Each organ/tissue has a unique volume,
permeability, and eliminating anatomical constants and terms, which are deter-
mined independently from the studied drug, while other physiological drug-specific
parameters are later incorporated, such as affinity toward plasma proteins, tissue-to-
plasma distribution rate, and even on target activity (Km, Vmax, or binding kinetics).
One of PBPK models’ important features is the perspective for the mechanistic
and prospective prediction of a drug’s pharmacokinetic profiles. The use of drug-
dependent parameters includes, but is not limited to, physicochemical properties,
solubility and permeability values, and also the role of individual enzymes and
transporters in the metabolism. Those parameters can be determined in vitro

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Name Description Link/reference

ADME prediction
vNN-ADMET Public web server for ADMET property [Link]
prediction based on 15 nearest neighbor [132]
models.
Swiss-ADME Public web server for ADME property [Link]
prediction. It has a very unique LogP [133]
calculation ([Link]) based on free energy.
pkCSM ADME web server based on chemical [Link]
fragment similarity (the so-called [134]
graph-based signatures).
ADMETlab Web server using similarity-based [Link]
ADME calculator models and drug- [135]
likeness analyses.
Schrodinger— Calculates pKa; LogP; water [Link]
QikProp solubility—Schrodinger also offers QikProp, Schrödinger, LLC, NY, 2019
other tools for property calculation.
DDI-Predictor DDI-Predictor is able to make [Link]
quantitative predictions of drug
exposure even in cases where the
interaction has not been studied yet.
PBPK models and platforms
GastroPlus Comprises 10 different modules [Link]
including PBPK modeling and in
vitro vs. in vivo correlation, can be
parameterized for different disease
states and age groups.
PKSIM PBK modeling tool with integrated [Link] [Link]/products/
database of anatomical and [Link]
physiological parameters for humans,
mouse, rat, dog, and monkey. Can
model different scenarios depending on
the chosen building blocks.
Simcyp Incorporates databases of genetic, [Link]
physiological, and epidemiological
information to enable simulation of
different populations and species,
ultimately is able to predict ADME
parameters.
ADMEWORKS As a differential is able to predict [Link]
DDI Simulator drug-drug interactions using nonlinear materials_life_science/products/
models. ddi_simulator

Table 2.
Tools for ADME prediction and PBPK modeling.

or calculated from the compound structure with other in silico approaches,

which allows the early use of PBPK in the DDD (the bottom-up approach).
Concurrently, it is also noteworthy that the model construction and parameter
fine-tuning are a source of knowledge for the hit development, where the predic-
tions from the ongoing model can help to understand the model’s accuracy itself
along the way (called as middle-out approach) and then prospectively be applied
to simulate unstudied scenarios. Currently, there are several free-to-use and com-
mercially available PBPK and ADME prediction options (Table 2), which are also
extensively reviewed and discussed by the works of Madden et al. [130].

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Early PBPK models, such as the work of Varma et al., described another layer of
complexity by including drug-drug interactions (DDI). The dosing time-dependent
model considering the interaction between repaglinide with rifampicin was able to pre-
dict repaglinide plasma concentrations along a day. The model also predicted the drug
interaction with other CYP3A4 and OATP1B1 inhibitors, which could result in further
DDIs. Reports of DDI leading to complications in patients with particular genotype
stimulated studies such as the one performed by Fermier et al. [131], where the effects
of polymorphic cytochromes provided the basis for a more accurate DDI prediction.

5. Biological (large) molecules

During recent years, larger molecules (LM) have gained in significance and
popularity, due to achievements and approvals, as new molecular entities. These
“biologics” are normally biotechnologically synthesized or recombinantly pro-
duced compounds of biological origin such as peptides, antibodies, and nucleic
acids [136]. From a historical perspective, drug discovery and development of
LMs are heavily delayed in comparison to SMs with their first approved entity
happening in the 1980s [137]. At about the same time, two major inventions
allowed huge progress in pharmacokinetics assessment of small molecules,
contributing to smaller drop-out rates in later DDD stages [136]. One of them was
the improved understanding of CYP450 mechanism and the other, the invention
of (HP)LC-MS technology, fueled the assessment of the ADME parameters. LMs’
discovery and development face many challenges, which demand high efforts
to overcome but also offer unique opportunities in comparison to those of small
molecules [138, 139].
The main differences between small and large molecules, despite the molecular
weight, the number of heavy atoms, and torsions, can be found in the physicochem-
ical properties, such as permeability, oral bioavailability, stability, specificity, and
immunogenicity [138, 139]. New parameters, unique for large molecules, are also of
interest, such as the physical particle size and the hydrodynamic radius, which has
a dramatic effect on the absorption. Both parameters are related to the overall shape
and correlate well with MW for globular proteins, but not necessarily for unstruc-
tured or highly modified entities. As a result, biologics are normally administered
parenterally, only targeting extracellular structures; they are also more likely to trig-
ger an immune response; and their production costs are considerably higher [139].
Interestingly, with the exception of the costs, these disadvantages can potentially
be circumvented by appropriate delivery systems, for example, nanoparticle-based
delivery to facilitate membrane permeation.
Other parameters, such as charges, which were previously modeled by pKa
in case of small molecules, are heavily heterogeneous in LMs. The charge can be
represented by the use of isoelectric points (pI), which are calculated from the
available amino-acid sequence, and surface charge, which can use individual pKa’s
and structural information to be inferred. Overall protein charge often influences
the biologic excretion [140], since negatively charged molecules undergo less renal
filtration disregarding size effect [141].
While representing difficulties in the development of new molecular entities,
the aforementioned properties also offer special advantages that small molecules
cannot cover. As such, LMs normally have longer t1/2, slower clearance, and higher
selectivity; are multifunctional; and rarely expose drug interactions [139]. Apart
from those, it was suggested that only 2–5% of the human genes can be targeted by
small molecules, offering a niche for LMs’ application against several diseases [138].

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The increasing effort and development of new technologies, driven by the belief
in higher success rates, enabled the latest advances in the field [138]. For instance,
currently, peptide drugs only account for ~2% of the drug market but are in use in
a wide range of diseases such as acromegaly and multiple sclerosis, together with
different cancer types such as prostate and breast cancer.
Several other biologics are currently in use, namely monoclonal antibodies
(mAbs) and bispecific antibodies (bsAbs), as example agents that activate or
enhance the immunologic response. Of special interest in cancer therapy is a sub-
class of bsAbs, so-called bispecific T-cell engager (BiTEs), which can recruit CD3
cells at the tumor site by binding to both cell types thereby directing the immuno-
logical response [142].
Other interesting examples for biologics comprise hormones (e.g., insulin),
cytokines (such as erythropoietin, EPO; IL-1; IL-2; IL-6) [143], nucleic acids such
as siRNA (ONPATTRO) [144], and aptamers (Pegaptanib) [145]. While such a
broad spectrum of molecule classes offers also a wide range of treatments, at the
same time, it exacerbates the need for new developments since every molecule type
exhibits different properties. In the field of predicting the biologics activity against
specific targets, classical modeling tools, such as Monte Carlo sampling, genetic algo-
rithms, docking, and molecular dynamics simulation, were adapted or even devel-
oped anew to accommodate the specifics (as extensively reviewed by [146, 147]).
On the other hand, the absence of standard techniques to assess ADME proper-
ties hampers the PK profiling and thus further development [136]. In fact, the
current knowledge of LM pharmacokinetics is even impaired compared to the basic
knowledge of ADME principles for small molecules in the 1980s [136]. Although
the basic PK principles are similar between SMs and LMs, the specific mechanisms
influencing each step of ADME are different. To begin with, the route of adminis-
tration between them can differ, which leads to different mechanisms of absorption
and first-pass metabolism. Furthermore, LMs are not metabolized by CYPs but can
still trigger the release of pro-inflammatory cytokines leading to heavy side effects
known as cytokine storm [136, 139]. Also, other modifications play a role in biolog-
ics ADME, namely glycosylation, PEGylation, and neonatal Fc receptor (FcRn)
interactions [139, 148]. Unfortunately, up until now, most of the evaluation of those
factors is only addressed on in vivo level systems, which are not suited for HTS, are
expensive and labor intensive, and require longer bioethics evaluation.
In this regard, the development of in vitro and in silico methods to evaluate
ADME should be a high-profile goal. One of the main challenges will be to find
a way to integrate as many of the biologics into the process in order to facilitate
ADME assessment and guide large molecules’ DDD as already implemented for
their smaller counterparts.

6. Conclusions

The main difficulties in PK profiling lie in the high costs and comparable low
throughput of in vivo models. The extensive use of animals in DDD also raises ethi-
cal issues and is further affected since animal models not always translate readily to
the humans, especially in terms of metabolism [149, 150]. Furthermore, the advent
of combinatorial chemistry coupled with HTS for efficacy evaluation leads to an
explosion in the number to an extent that the classical PK assays could not compen-
sate [29, 47]. In vitro PK screens are supposed to offer a solution to the problem by
complementing in vivo assessment to reduce costs while increasing efficiency, but
they also suffer from shortcomings. In general, one must distinguish between two
main forms of in vitro systems: static and dynamic models. Only dynamic models

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are suited for PK evaluation because they allow variation of compound concentra-
tions, a key factor in pharmacokinetics. In this sense, diffusion-based dynamic in
vitro models offer a solution but still are quite limited in terms of high throughput
and costs. An alternative was presented by Lockwood and colleagues in the form
of a 3D-printed fluidic device utilizing trans-well technique generating dynamic in
vitro PK profiles also applicable for HTS infrastructure [149].
What distinguishes the DDD “then” and “now” is principally two main changes.
First, in the past, pharmaceutical companies as well as academic laboratories were not
that concerned with ADMET assessment in the early stages of drug discovery (hit and
lead generation) and only addressed PK from preclinical stages on forward. Instead,
HTS/HCS, genomics, and computational chemistry were high-profile areas. Today,
almost all pharmaceutical big-players have shifted pharmacokinetic profiling to dis-
covery phases. However, only the future will tell whether those changes will yield fruit.
Second, CADD became more and more part of the DDD pipeline in dif-
ferent stages facilitating fast screening of compounds in silico and supporting
QSAR. Although bioinformatics techniques already substituted many in vitro tests,
basically all of them require in vitro and/or in vivo validation and standardization
to guarantee trustable predictions. Another important aspect, recently addressed
by the work of Ferreria and Andricopulo [151], is the importance of translating
those models into well-structured and user-friendly (online) platforms that can be
accessed and used by the drug discovery community. Still, the efficacy and reli-
ability of computer simulations increase permanently and drastically, and many see
a future of solely virtual drug discovery. Thankfully, these failures resulted in the
consequence of addressing safety and efficacy concern earlier in the drug discovery
process, for instance, via in vitro screens to assess metabolic stability and absorption
properties and diminish failure rates later on [13].

Acknowledgements

The authors would like to acknowledge the Fundação de Amparo à Pesquisa do

Estado de São Paulo (FAPESP grants 2018/08820-0, 2017/03966-4, and 2015/26722-8).
The authors would like to thank Prof Dr. José Eduardo Gonçalves for his valuable com-
ments on the manuscript.

Notes

ORCID numbers of all authors:

Thales Kronenberger: 0000-0001-6933-7590

Carsten Wrenger: 0000-0001-5987-1749
Vinicius Goncalves Maltarollo: 0000-0001-9675-5907
Arne Krüger: 0000-0002-5531-9508

Conflict of interest

The authors declare no conflict of interest.

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Drug Discovery and Development - New Advances

Author details

Arne Krüger1, Vinicius Gonçalves Maltarollo2, Carsten Wrenger1*

and Thales Kronenberger3

1 Unit for Drug Discovery, Department of Parasitology, Institute of Biomedical

Sciences, University of São Paulo, São Paulo, Brazil

2 Department of Pharmaceutical Products, Pharmacy Faculty, Federal University of

Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

3 Department of Internal Medicine VIII, University Hospital of Tübingen,

Tübingen, Germany

*Address all correspondence to: cwrenger@[Link]

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License ([Link]
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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ADME Profiling in Drug Discovery and a New Path Paved on Silica
DOI: [Link]

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 Section 4

Recent Case Studies:

Advances in Drug
Discovery Research

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Chapter 7

Successive Drug Therapy for a Very

Rare Autosomal Diseases
Mohammed Chyad Al-Noaemi and Hassan Ali Daghriri

Abstract

It is very rare to find reports concerning a drug therapy successively treating

chromosomal abnormalities. In this paper, we are reporting a successive use of
nitisinone in treating a fatal and very rare autosomal disease called hereditary tyro-
sinemia type-1 [HT-1]. HT-1 is affecting about one person in 100,000 to 120,000
births worldwide. It is due to a genetic defect in the enzyme fumarylacetoacetate
hydroxylase (FAH), which is responsible for the final degradation of tyrosine.
Accumulation of tyrosine metabolites is responsible for tissue damage such as
liver, kidney, and neural tissues, finally causing the death of the newborn babies
in their early months of life if not treated. Fumarylacetoacetate hydrolase gen has
mapped on chromosome 15q23-15q25. Since 1992, the initiation of treating HT-1
with nitisinone (NTBC) has become the medical therapy of choice in combination
with diet. NTBC therapy has shown a direct correlation between age of initiation
and subsequent clinical course. We are reporting three brothers treated safely and
successively with NTBC in combination with diet. All of them are in very good
conditions. The elder brother is on NTBC since 27 years ago.

Keywords: autosomal diseases, hepatocellular tyrosinemia, nitisinone, NTBC,

hepatocellular carcinoma, newborn screening

1. Introduction

1.1 Tyrosine

Tyrosine (4-hydroxyphenylalanine) is a nonessential amino acid with a polar

side group, 1 of the 22 amino acids that are used by cells to synthesize proteins.
Tyrosine is also a precursor to neurotransmitters (catecholamines) and hormones
(thyroxine and melatonin) [1–3]. In humans, tyrosine is obtained from two sources,
dietary intake and hydroxylation of phenylalanine [4, 5]. Tyrosine degradation
as shown in Figure 1 is catalyzed by a series of five enzymatic reactions that yield
acetoacetate, which is ketogenic, and the Krebs cycle intermediate fumarate, which
is glucogenic [4, 6, 7]. Although tyrosine degradation occurs mainly in the liver
but to a lesser extent, it occurs in the proximal renal tubules [5, 8, 9]. Impaired
catabolism of tyrosine is a feature of several acquired and genetic disorders. Four
autosomal-recessive disorders result from deficiencies in specific enzymes in the
tyrosine catabolic pathway: hereditary tyrosinemia (HT) types 1, 2, and 3, and
alkaptonuria (AKU). These disorders result in elevated blood tyrosine levels except
for AKU [4, 7, 9–11].

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Drug Discovery and Development - New Advances

Figure 1.
The steps of tyrosine degradation.

2. Hereditary tyrosinemia type 1 (HT-1)

Synonyms: hepatorenal tyrosinemia (HRT), tyrosinemia type-1, hereditary

infantile tyrosinemia, congenital tyrosinosis, and fumarylacetoacetate hydrolase
(FAH) deficiency (FAHD), and is assigned OMIM 276700.

2.1 History

Inborn errors of metabolism (IEMs) are a group of diseases involving a genetic

defect that alters a metabolic pathway and that presents during infancy.
The tyrosine degradation pathway contains five enzymes, four of which are
associated with IEMs. The most severe metabolic disorder associated with this
catabolic pathway is hereditary tyrosinemia type 1 (HT-1; OMIM 276700) [10].
In 1932, American biochemist Grace Medes, at the University of Minnesota
Medical School in Minneapolis, first described “a new disorder of tyrosine metabo-
lism” and called it “tyrosinosis” after observing 4-hydroxyphenylpyruvate in the
urine of a 49-year-old man with myasthenia gravis [12]. She proposed that the
metabolic defect in this patient was a deficiency of 4-hydroxyphenylpyruvate
dioxygenase.
In 1957, Japanese scientists, Kiyoshi Sakai and colleagues, published three
reports describing the clinical, biochemical, and pathological findings of a 2-year-
old boy with hepatorenal tyrosinemia who was then thought to have an “atypical”
case of tyrosinosis (“atypical” because it differed from the supposedly prototypical
case reported by Medes) [13–16].
Then, between 1963 and 1965, Swedish pediatrician Rolf Zetterström and
associates published the first detailed descriptions of hepatorenal tyrosinemia
and its variants, a disorder then hypothesized to be caused by a defective
4-hydroxyphenylpyruvate dioxygenase enzyme [17–20]. Furthermore, in 1964
several pediatricians in Chicoutimi (Quebec-Canada) became aware of an
increased incidence of infantile liver cirrhosis that was later recognized to be due
to hereditary tyrosinemia type [21, 22]. Both the Scandinavian and Canadian
groups suggested that the Japanese patients described earlier by Sakai and col-
leagues had the same disorder, that is, HT-1 [16]. Therefore, it has been consid-
ered that the first definite case report in the world of HT-1 was in Japan by Sakai
and colleagues in 1957 [23].

2.2 Pathophysiology

Hereditary tyrosinemia type 1 is an inborn error of metabolism, inherited

as an autosomal recessive disorder. The biochemical defect was shown to be

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DOI: [Link]

due to a genetic defect causing a deficiency (weak activity) or absence in

the enzyme fumarylacetoacetate hydrolase (FAH), the enzyme catalyzing
the final step of tyrosine catabolism pathway as shown in Figure 2 [24, 25].
Fumarylacetoacetate hydrolase gen has located on chromosome 15q23-
15q2515q23 and is composed of 14 exons [26]. This enzyme defect leads to
subsequent accumulation of the amino acid tyrosine and its toxic metabolites
such as succinylacetone, maleylacetoacetate, and Fumarylacetoacetate in the
blood and tissues such as the liver, kidney, heart, and peripheral nerves, leading
to dysfunction of these organs [24, 27–30]. The patient may develop acute and
severe liver failure that is life-threatening in early infancy (<6 months of age).
The survivors of the acute failure show before two years of life liver cirrhosis,
complex renal tubulopathy, rickets, cardiomyopathy, and hemorrhagic syn-
drome. Hepatocellular carcinoma (HCC) is a frequent complication of this form
of HT1, which is often the cause of death in early life in an untreated individual
[22, 25, 31, 32].

2.3 Prevalence of HT-1

In general, hereditary tyrosinemia type-1 is a very rare inborn genetic disease

affecting about one person in 100,000–120,000 live births worldwide [29, 31, 33,
34]. In some areas, the incidence of HT1 is noticeably higher. In Norway, Finland,
and Tunisia, the frequency of HT1 is 1:74,800, 1:60,000, and 1:14,804, respectively
[35–37]. The highest prevalence of the disorder is observed in Canada (the Province
of Quebec), which is about 1 in 16,000 live births [32, 33, 38, 39], and even in a
certain region of Quebec near Saguenay-Lac Saint-Jean Jean, it is estimated to be
1:1846 live births [32], and the carrier rate has been estimated to be between 1 in 20
and 1 in 31 [39].
The estimated incidence of tyrosinemia in the Eastern Province of Saudi Arabia
is 3 in 100,000 live births, although the authors concluded that data obtained from
their study underestimate the true number [40].

Figure 2.
Steps of tyrosine and its inborn enzymatic error of metabolism causing tyrosinemia type I, II, and III.

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2.4 Diagnosis

A diagnosis of HT-1 is made based upon thorough clinical evaluation, a detailed

patient history, and specialized tests.
Elevated blood tyrosine level in newborns should be seen as soon as possible
for clinical and laboratory evaluations for the possibility of HT-1. The diagnosis of
HT-1 is based on elevated succinylacetone (SA) levels in the blood and or urine, as
tyrosine elevation is an unreliable marker. There are many false-positive and false-
negative results when tyrosine is used as the only diagnostic parameter [41]. In the
US, Canada, and some of the European countries, they use the detection of plasma
SA as a newborn screening test for the detection of HT-1 [42].
If there is a high suspicion for HT-1, plasma amino acids (PAA) and liver func-
tion tests including prothrombin time (PT), international normalized ratio (INR),
partial thromboplastin time (PTT), and α-fetoprotein (AFP) should be evaluated at
the first visit. [43].
Clinical symptoms typically begin before two years of age, with the majority
of children presenting before the age of 6 months with hepatosplenomegaly and
evidence of acute liver failure and renal dysfunction. A few affected children may
present over the age of 2 years with isolated coagulopathy or other signs of liver
dysfunction, renal tubular disease, hypophosphatemic rickets, and failure to thrive.
All children with HT-1 are at high risk for hepatocellular carcinoma (HCC), and this
also may be the first recognized clinical event [44].
Molecular genetic testing for FAH gene mutations is available to confirm the
diagnosis [45, 46].

2.5 Differential diagnosis

HT-1 should be differentiated from another inherited inborn autosomal reces-

sive disorders with dramatically elevated blood tyrosine levels such as:
Tyrosinemia type II, which is due to tyrosine aminotransferase (TAT) defi-
ciency as shown in Figure 2, causing accumulation of tyrosine that produces a
severe dermatologic and ophthalmologic abnormalities. Type II tyrosinemia occurs
in less than 1 in 250,000 individuals [47–49].
Tyrosinemia type III is due to 4-hydroxyphenylpyruvate dioxygenase (HPD)
deficiency as shown in Figure 2. It is the rarest of the three conditions, with only
a few cases ever reported. Most of those cases have included intellectual disability
and neurologic dysfunction. It also has highly elevated blood tyrosine levels but
does not manifest liver disease or renal tubular disease [50–53].
Tyrosinemia types II and III variably respond to phenylalanine and tyrosine
dietary restriction therapies, unlike HT-I, the dietary restriction, even if begun
within the first month of life, did not eliminate the development of hepatic, renal,
or neurological complications.
Plasma amino acids (PAA) will help to differentiate tyrosinemia types II and III
from HT-1 in those cases where the children are detected by an elevated tyrosine
level but do not have detectable succinylacetone (SA).

2.6 Management of HT-1

2.6.1 Nitisinone drug therapy

Nitisinone, orfadin, or 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-

1,3-dione (NTBC). Its structure is shown in Figure 3.

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Figure 3.
The structure of nitisinone (NTBC).

2.6.2 History of nitisinone discovery

Nitisinone is a member of the benzoylcyclohexane-1,3-dione family of herbi-

cides, which are chemically derived from a natural phytotoxin, leptospermone,
obtained from the Australian bottlebrush plant (Callistemon citrinus) [29].
Nitisinone was discovered as part of a program to develop a class of herbicides
called 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors. HPPD is essential
in plants and animals for catabolism, or breaking apart, of tyrosine. In plants,
preventing this process leads to the destruction of chlorophyll and the death of the
plant [54].
In normal humans, fumarylacetoacetate hydrolase acts on the final step of
tyrosine metabolism after HPPD does. The absence or weak activity of fumaryl-
acetoacetate hydrolase as in HT-1 leads to very harmful products building up in the
body [24, 27–30]. So scientists working on making herbicides in the class of HPPD
inhibitors hypothesized that inhibiting HPPD and controlling tyrosine in the diet
could treat this disease. A series of small clinical trials attempted with one of their
compounds, nitisinone, were conducted and were successful, leading to nitisinone
brought to market as an orphan drug Swedish Orphan International, which was
later acquired by Swedish Orphan Biovitrum (Sobi). [55, 56]. Therefore, in HT-1,
the mechanism of nitisinone action will involve reversible inhibition of HPPD pre-
venting the formation of maleylacetoacetic acid and fumarylacetoacetic acid, which
have the potential to be converted to succinylacetone, a toxin that damages the liver
and kidneys. This causes the symptoms of HT-1 experienced by untreated patients.
Lock described nitisinone by a nice statement “From Weed Killer to Wonder
Drug” [57].

2.6.3 The clinical trial of nitisinone

Sven Lindstedt recognized the potential value of NTBC for the treatment of
HT-1. By blocking the proximal tyrosinemia pathway, NTBC minimizes the forma-
tion of FAA and maleylacetoacetic. It was this keen insight that led to the original
clinical trial with five patients, which documented the rapid reversal of clinical
symptoms [58].
NTBC dosing should be sufficient to completely suppress plasma and urine SA
detection and normalize liver and renal function. SA, in either plasma or urine,
should be below detectable limits (or within the limits of normal established by

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the reference laboratory). The dose of NTBC should be increased if the SA level
increases once patient adherence has been confirmed [43].
The standard recommended dosage of NTBC is 1 mg/kg body weight [43, 59, 60].
The half-life of NTBC has been measured in healthy human subjects and found
to be approximately 54 h [61]. Because of this long half-life, a single daily dose of
NTBC is satisfactory for maintaining inhibition of HPD [62, 63].
In the evaluation of its safety profile, rats and dogs exposed to NTBC developed
elevated plasma tyrosine levels and ocular lesions. The ocular lesions (keratopathy)
were caused by tyrosine crystals within the cornea, which on cessation of the diet
recovered [43, 57].
The FDA approved NTBC in January 2002 [64].

2.6.4 Nutritional therapy

The combined nitisinone and low phenylalanine and tyrosine diet treatment
should be initiated as soon as possible following the diagnosis of HT-1, to maintain
PAA concentrations within the treatment range. Phenylalanine must be restricted
in the diets of affected patients since approximately 75% of dietary phenylalanine
is hydroxylated to form tyrosine [65, 66]. The combined diet restriction and NTBC
treatment resulted in a greater than 90% survival rate, normal growth, improved
liver function, prevention of cirrhosis, correction of renal tubular acidosis, and
improvement in secondary rickets [67–69].

2.6.5 Liver transplantation

Before the availability of nitisinone for the treatment of tyrosinemia type I, the
only definitive therapy was liver transplantation. The first case of HT-1 treated with
liver transplant was in 1978 performed by Fisch and his colleagues [70]. The patient
died 3 months later, but the biochemical derangements improved. Subsequently,
the use of liver transplants in HRT cases has increased, and the benefits appear to be
confirmed [71].
Liver transplantation should be reserved for those children who (1) have a severe
liver failure at clinical presentation and fail to respond to nitisinone therapy or (2)
have documented evidence of malignant changes in hepatic tissue [72]. Transplant
recipients may also benefit from low-dose (0.1 mg/kg/day) nitisinone therapy to
prevent continued renal tubular and glomerular dysfunction resulting from the
persistence of succinylacetone in the plasma and urine [73].

2.6.6 Low phenylalanine and tyrosine diet restriction

Diet restriction for the treatment of HT-1 patients was introduced by Halvorsen
and Gjessing in 1964 [19] and for a long time was the only treatment available. It
had a beneficial effect on the renal tubular defects but did not cure the liver disease.
A girl with HT-1, diagnosed at 6 months of age, was treated with a diet restricted in
phenylalanine and tyrosine. At 9½ years of age, she developed an acutely enlarged
liver and spleen, and the diagnosis of hepatocarcinoma was made [70].

2.6.7 Genetic therapy

Gene therapy is a promising means to cure many monogenic diseases.

However, traditional gene therapies are best suited to treat diseases of deficient or
absent gene products rather than those diseases caused by aberrantly functioning
proteins [74].

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Adeno-associated virus (AAV)-mediated gene repair is feasible in vivo and can

functionally correct a mouse model [74] and pig model of HT-1 [75] and con-
cluded that further exploration of ex vivo hepatocyte genetic correction is war-
ranted for clinical use. Although AAV-mediated gene therapy in a mouse model
of HT-1 was successful as it has shown that none-treated FAH mutant control
mice died within six weeks from fulminant liver failure, FAH adenovirus-infected
animals survived 2–9 months. But this gene therapy does not obviate the tumor
risk inherent in HT-1 as nine of 13 virus-treated animals developed hepatocellular
cancer [76].

2.6.8 Family cases of HT-1 treated successively with nitisinone

We are reporting three Saudi siblings who have diagnosed as patients with HT-1.
They are living in Najran city, the southern province of Saudi Arabia.
The first case is 27-year-old male patient. He has been diagnosed at the age of
4 months in Great Ormond Street Hospital (London-UK) and treated by nitisinone
in combination with tyrosine and phenylalanine-free diet. For the next 15 years,
he used to visit the clinic for regular checking. The final report showed that he
has good general health, with normal liver and renal function test and normal
alpha-fetoprotein. Since then and until now, he attends the National Laboratory for
Newborn Screening, Department of Genetics, King Faisal Specialist Hospital and
Research Centre for a routine checkup. Still, he is on nitisinone and diet restriction.
He has graduated from Najran Technical College, and now he is doing very well
in his job as the vice director of staff affairs at Al-Ghad International College for
Applied Medical Sciences in Najran-KSA.
The second case is his brother, who is 20 years old. He has been diagnosed in
the first few days after birth by the National Laboratory for Newborn Screening,
Department of Genetics, King Faisal Specialist Hospital and Research Centre,
Riyadh, Saudi Arabia. Since then he has been treated by nitisinone in combination
with tyrosine and phenylalanine-free diet. He is having good general and mental
conditions. He is now a 2nd-year university student and doing well in his study.
The third case is their younger brother, who is 15 years old. Diagnosed since
birth by the National Laboratory for Newborn Screening, Department of Genetics,
King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. Since
then, he has also been put on nitisinone in combination with tyrosine and phenyl-
alanine-free diet. He is also having a good general and mental conditions. He is a
secondary school student, and he is also doing very well in his study.
The three brothers are now on nitisinone in a dose of 1 mg/kg/day. It is marketed
under the brand name Orfadin by the company Swedish Orphan Biovitrum (Sobi).
Also, they are on tyrosine and a phenylalanine-free diet supplemented with HCU
Anamix Junior LQ [Nutricia Advanced Medical Nutrition Company].
They have a regular visit to the National Laboratory for Newborn Screening,
Department of Genetics, King Faisal Specialist Hospital, and Research Centre,
Riyadh, Saudi Arabia for regular checking, and the last visit was a few weeks ago.
Their elder two siblings (sister and brother) died in their early life of unknown
cause. The sister died at the age of 26 months. From the history of her mother, it
seems that the daughter developed abdominal distension, and their doctors told
her that the baby has hepatosplenomegaly. She also developed jaundice and became
reluctant to milk or food. Until the day of death at the age of 26 months, she could
not sit or walk; she was very week as her mother described.
Then after, they have a boy who developed after birth hepatomegaly, and
jaundice, abdominal distension with very thin extremities and died early after birth
at the age of 4 months, as his mother said.

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After the birth of the third baby (our first case), they went to Great Ormond
Street Hospital and diagnosed as having tyrosinemia-1. At the same time, the
parents diagnosed as a carrier of the disease.
The clinical history of their previous dead siblings suggested that they did have
undiagnosed HT-1. Furthermore, the diagnosis of the parents that they are a carrier
of the disease is highly supporting that the death of their two children early life was
due to the lack of diagnosis and treatment of HT-1.

3. Discussion

HT-1 is a rare but clinically severe and fatal inborn error that principally affects
the liver, kidney, and peripheral nerve [32]. In general, the most diagnosed patients
of inborn errors of metabolism (IEMs) including HT-1, were born from consan-
guineous married parents. As HT-1 is a rare inherited autosomal recessive disorder,
it explains why it is more common in population with a high rate of consanguineous
marriages, such as in United Arab Emirates, Oman, Kuwait, and Saudi Arabia,
in which the rate of consanguineous marriages reaches up to approximately 60%
[33, 77–80], and even the first reported case of HT-1 in Japan (1957) was a child
from parents of consanguineous marriage [13, 14]. Furthermore, in our study, the
reported three family cases are also born from a consanguineous married parent.
In the sixties and early seventies HT-1 patients were treated by phenylalanine-
and tyrosine-free died; it delayed the mortality and morbidity for a few years but
it did not prevent the development of hepatic failure, renal complications and
hepatocellular carcinoma (HCC) even if begun within the first month of life [43].
Then after, treatment with hepatic transplantation was the only option for survival.
But it has been reported that the development of HCC was observed in 17–37% of
affected children [81, 82]. Furthermore, after transplantation, urine and plasma
SA decreases but is not completely suppressed [43, 63], and even other scientists
reported that plasma succninylacetone is persistently raised after liver transplanta-
tion [73] presumably because of continued production in the kidneys which could
cause damage to the liver and the kidney. Also, some of the patients died from
the complications of hepatic transplant, whether the surgical or the immuno-
suppressive drug complications [43, 63, 69]. Therefore, in 1992, the introduction
of nitisinone in combination with diet restriction was the ideal therapy for HT-1
patients especially if started in their early neonatal days [58].
The three HT-1 patients in our study used nitisinone in combination with
phenylalanine and tyrosine diet restriction from early days of their neonatal life and
till now, which is 27, 20, and 15 years, respectively. They are not only still alive but
also doing very well in their living.
These results justify implementing prevention programs that incorporate
genetic counseling and neonatal diagnostic screening tests, especially in the sus-
pected families of consanguineous marriages to detect the neonatal patients with
HT-1 as early as possible and then to start treatment which will minimize the lethal
consequences of the disease.
All subsequent children of the parents of a child with HT-1 should have urine
and blood succinylacetone analyzed as soon as possible after birth to enable the ear-
liest possible diagnosis and initiation of therapy. Early detection of newborn babies
with HT-1, followed by prompt treatment with nitisinone in combination with a low
phenylalanine and tyrosine diet has improved the survival to over 90% and resulted
in normal growth, improved liver function, prevention of cirrhosis, correction of
kidney disease, and improvement in rickets [41, 42, 83, 84]. In 2012, Larochelle

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et al. reported that patients who receive nitisinone treatment before 1 month had no
detectable liver disease after more than 5 years [69].
These data suggested that early neonatal diagnosis of HT-1 and treatment with
nitisinone and diet restriction not only keep the survival of the patients but also
keep them in good general, physical, and mental conditions.

4. Conclusion

Nitisinone (NTBC) has been used since 1992 and proves to be an effective and
safe pharmacological treatment for HT-1 in combination with phenylalanine- and
tyrosine-free diet.
In this paper, we are reporting three cases (brothers) treated safely and succes-
sively with NTBC in combination with diet. All of them are in very good condi-
tions. The elder brother is on NTBC since 27 years ago. He is one of the few cases
worldwide treated since 1992 and till now, and he is living with a very good general
health.
HT-1 is not only a rare and fatal autosomal disease, but it is a very rare genetic
disease that can be successfully, effectively, and safely treated by drug therapy,
which is nitisinone (NTBC).

4.1 Recommendation

We highly recommend establishing a national Newborn Screening Center,

which provides newborn screening test for the diagnosis of HT-1, especially for the
high-risk neonates in the suspected families. The use of tandem-mass-spectrometry
could make an early diagnosis of HT-1 by measurement of succinylacetone in blood
spot specimens.
Prenatal diagnosis is also possible by doing DNA analysis in addition to the
detection of succinylacetone in the amniotic fluid of the suspected pregnancies.
Early diagnosis and treatment of this life-threatening disease provide an oppor-
tunity to intervene before symptom onset.
Furthermore, this report justifies implementing prevention program by doing
genetic counseling and DNA analysis in the suspected families, where consanguine-
ous marriages are prevalent.

Disclosure of conflicts of interest

None.

Ethical consideration

The protocol was submitted and approved by the Research Ethical Committee of
Al-Ghad International College for Applied Medical Sciences-Najran, KSA.

Consent

Written informed consent was obtained from the family for the anonymized
information to be published in this article.

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Author details

Mohammed Chyad Al-Noaemi1* and Hassan Ali Daghriri2

1 Department of Emergency Medical Services, Al-Ghad International College for

Applied Medical Sciences in Najran, KSA

2 Al-Ghad International College for Applied Medical Sciences in Najran, KSA

*Address all correspondence to: mohammedalnoaemi@[Link]

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License ([Link]
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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Chapter 8

Challenges and New Frontiers in

the Paediatric Drug Discovery and
Development
Angelica Intini, Donato Bonifazi and Giovanni Migliaccio

Abstract

Drug discovery and development advances in the last decades allowed to find
a treatment for many preventable diseases. However, all too often, children are
excluded from these progresses since most of the new medicines have been discov-
ered and developed for the adult population. Even if paediatricians routinely give
drugs to children ‘off-label’, researchers have demonstrated that children do not
respond to medications in the same way as adults. Furthermore, certain specific
disorders are unique to children or occur in children differently than in adults.
Besides specifically testing medicines in children in proper clinical studies taking
into due account the peculiarity of this population, there is a growing recognition
of the need to develop paediatric medicines having in mind the specificities of this
vulnerable population. In this chapter, we will provide an overview on the drug
discovery and development path for children highlighting challenges and new fron-
tiers of each phase from the discovery to the preclinical and clinical development
as well as we will provide a slightest hint about paediatric biomarkers discovery,
age-appropriate formulation, pregnancy, and perinatal pharmacology and in silico
pharmacology. Finally, pricing and reimbursement policies for medicines and new
and existing research initiatives in the field will be discussed.

Keywords: human development research, paediatric drug discovery, preclinical

research, juvenile animal models, paediatric pharmacology, paediatric biomarkers,
age-appropriate formulations, perinatal pharmacology, physiologically-based
pharmacokinetic (PBPK)

1. Introduction

Even if paediatricians routinely give drugs to children ‘off-label’ (drug not

specifically approved for use in children), it is known that children respond to drugs
in a very different way than adults in terms of safety and efficacy [1]. Anatomical,
physiological and developmental differences between children and adults and
among children of different ages reflect in changes in absorption, distribution,
metabolism and excretion (ADME). Moreover, less information is available in
younger age groups and neonates. Furthermore, while certain specific disorders
are unique to children, others could be more common in children than adults or
infrequent in children compared to adults. Notwithstanding, children have been
excluded from testing of new drugs for many years and for this reason have been
defined as ‘Therapeutic Orphans’ by Shirkey in 1969 [2].

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The lack of a regulatory framework that obliged to test medications in the pae-
diatric population taking into account the specificities of children and the ethical
concerns behind resulted in several examples of therapeutic tragedies in paediatric
patients. A new liquid formulation of the antibiotic sulphanilamide was developed
in 1938 to allow oral dosing for paediatric patients who could not swallow the tablet
form. Unluckily, the solvent used to dissolve the active substance was a toxin that
caused many adverse events with a 30% mortality rate [3]. And again, Thalidomide
was marketed in Europe in the late 1950s for the treatment of nausea in pregnant
women causing severe birth defects in thousands of children including severe
shortening of the extremities, malformations of ears, heart, intestines and other
structures, depending on the embryologic stage at the time of exposure [4].
These tragedies are just an example of the high risk to which children have been
exposed for years and have led to the increasing awareness that new medications
for children should be carefully studied before they could be approved, defining
the proper requirements and ethical issues to guarantee efficacious and safer drugs
for children. As a consequence, regulations have been adopted independently in
the most developed countries, but in accordance with unified guidelines suggested
by the International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH), an organisation working on
the harmonisation of pharmaceutical regulatory requirements within the European
Union (EU), Japan and the United States (US) [5].
The European Paediatric Regulation was adopted in 2006 and entered into force
in 2007 [6] imposing to pharmaceutical companies developing drugs of potential
interest for children to prepare a paediatric investigational plan (PIP) to obtain a mar-
keting authorisation for an indication in adults, unless they were granted a product-
specific waiver by the Paediatric Committee of the European Medicines Agency
(EMA), for example if the indication does not occur in children [7]. The paediatric
regulation has defined rules concerning the development of medicinal products for
paediatric use and introduced rewards and incentives for the development of paediat-
ric drugs (i.e. the paediatric-use marketing authorization—PUMA) [5].
In the US, two main acts have complemented each other ruling the evaluation of
drugs in infants and children and increasing the paediatric clinical studies and drug
labelling for children: the PREA of 2003 [8] and the BPCA of 2002 [9], both amended
in the FDAAA of 2007 [10]. A different approach has been taken in Japan, more
focused on premiums granted to pharmaceutical companies as rewards for developing
paediatric medicines without a regulatory framework specifically addressing paedi-
atric clinical research. As an effect of these premiums, the price of those drugs is not
reduced as normally occurred every 2 years in the Japanese system [11].

2. Paediatric diseases

A major challenge in studying paediatric diseases is the relatively low incidence

rate or uniqueness of some disorders in children. Paediatric diseases may resemble
those in adults, but considerable differences may also exist with regards to aetiol-
ogy, progression, comorbidities and prognosis [12].
Several cancer types are genetically different in children compared to adults as
demonstrated by a comprehensive analysis of genetic alterations in a pan-cancer
cohort including 961 tumours from children, adolescents, and young adults, and
comprising 24 distinct molecular types of cancer [13]. Epilepsy in children is
associated with a wide range of congenital or hereditary diseases, while in adults,
it is associated mainly with strokes and brain tumours [14]. The onset of systemic
lupus erythematosus (SLE) during childhood is associated with different clinical

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DOI: [Link]

manifestations and two to three times higher mortality compared to adult-onset

SLE [15]. Moreover, frequent comorbidities are specific of premature neonates
including persistent ductus arteriosus, sepsis, intra-ventricular haemorrhage and
necrotising enterocolitis, and mortality is the highest in premature neonates born
<28 weeks’ gestation [16, 17].
The above studies provide just some examples of how children and adults can
be differently affected by similar diseases underling the importance to address
the drug discovery and development process starting from the specificities of the
paediatric population.

2.1 Changes occurring during development and age groups

Obviously, childhood is the period of life when the physiological and physical
changes are the most important and the fastest. Physiological systems and functions
are immature in neonates at birth with the degree of immaturity depending on
gestational age. These systems develop progressively and changes can be observed,
for example, in gastrointestinal motility and function, body composition and size,
activities of transporters and metabolism enzymes, and renal function. The process
is dynamic and nonlinear with progressive rapid growth and maturation in the first
weeks/months of life, and slower thereafter. These developmental changes affect
drug disposition, as discussed later, with differences among neonates, children,
adolescents and adults [18, 19].
Therefore, defining the paediatric population is a very complex issue since it rep-
resents an extremely heterogeneous population. To address the peculiarity of each
age group and to provide guidance for regulatory and clinical matters, the interna-
tional regulation on paediatric clinical trials [20] has described four subsets: pre-
term and term neonates (0–27 days), infants (1–23 months), children (2–11 years)
and adolescents (12–18 years). In addition, the recently revised EMA guideline
‘Ethical considerations for clinical trials on medicinal products conducted with
minors’ issued on September 2017 has further sub-divided the age group 2–18 years
into pre-schoolers (2–5 years), schoolers (6–9 years) and adolescents (10–18 years)
[5, 21]. It has to be underlined that this definition, although useful to unify the
system of rules and law in this field, does not always reflect the maturity of the
child, which is something that is generally recognised as crucial aspect to be taken
into account during the conduct of paediatric clinical trials [5].

2.2 Rare diseases

When it comes to talking about paediatric diseases, we cannot exclude the rare
disease field since many rare diseases are diagnosed during childhood. Rare diseases
include a very heterogeneous group of disorders, affecting any body system. A
disease is defined rare if it affects fewer than 1 in 2000 people in Europe and fewer
than 200,000 people in the United State. A high percentage of rare diseases (about
80%) affect children, and in 50% of cases, all rare diseases are characterised by
a childhood-onset with a significant impact on the well-being of the patients and
families [22–24].
Although rare diseases have by definition a low prevalence, with some having a
single identified case worldwide, collectively they affect about 6–8% of the human
population with a number of diseases recognised as rare comprised between 6000
and 8000 diseases [25].
Despite the high impact they have on the worldwide population, few treatments
are available on the market. Drug development for rare diseases poses unique scien-
tific and ethical challenges, most of which in common with the obstacles described

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in this chapter for the paediatric population. Since they affect a small population,
heterogeneous and widely dispersed, it is more difficult to enrol enough patients in
clinical studies and pharmaceutical company shows a scarce interest in this field for
the low return they may have.
Moreover, considering the high incidence and prevalence during the childhood, the
ethical issue is predominant in this field. And additional challenges may result from
the frequently progressive, life-limiting or life-threatening nature of these diseases.
As described below, new approaches in all the phases of the drug development
process may offer valuable solutions to overcome these difficulties in the rare
diseases as well as paediatric diseases field.

3. Tailored drugs for children

Drug discovery and development path represents the long process starting with
the identification of new target molecules (discovery phase), going through stud-
ies on microorganisms and animals (preclinical development) and finally testing
the new medicines in the target population (clinical development) to bring them
to the market (authorization and commercialization). Considering the differences
between children and adults above mentioned, a new drug to be used in children
should be specifically tested in children themselves in controlled clinical studies.
At the same extent, medicines for children should be developed having in mind the
specificities of this vulnerable population starting from the very initial phase of
discovery.

3.1 Drug discovery

In order to make available better medicines for children, it is mandatory to start

thinking differently from the beginnings of the long process of drug development
and stop the habit to translate results from adult to children. Even if we cannot
deny the potentialities and advantages of using existing drugs in alternative ways or
populations, as the case of repurposed drugs or the use of extrapolation in paediat-
ric drug development, these approaches should be considered complementary to a
drug discovery tailored to children and not the only way to go.
Drug discovery for children should be focused on specific targets for paediatric
indications and should not be influenced by the existing knowledge for adults.
Appropriate preclinical animal and cellular models should be used, and new emer-
gent technologies should be implemented.
The main challenges in the research of novel medications for children come
from a range of unique characteristics of this population. As highlighted before,
several paediatric diseases are unique of childhood or differ in children compared
to adult. Therefore, it is of major importance to increase our understanding of the
disease mechanism in children and of the human development mechanism relevant
for paediatric diseases and use this knowledge to favour a proper drug target selec-
tion and validation. For this aim, the availability of adequate disease models, both
at in vitro and in vivo experimentation level, is a critical factor.
The existing human cell lines are frequently derived from adult sources, making
them inappropriate as in vitro model of paediatric diseases. Indeed, several studies
have highlighted the differences existing between adult and foetal/neonatal cells.
Differences in platelet transcriptome [26] and proteome [27] have been described
between platelets derived from healthy adults and full-term neonates. Variations
between neonatal and adult fibroblasts and keratinocytes have been described as prob-
ably associated with improved wound healing during the early neonatal period [28].

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Challenges and New Frontiers in the Paediatric Drug Discovery and Development
DOI: [Link]

Xu et al. provided an overview on the in vitro models used to study paediatric brain
tumours underlined that in the initial drug screening for new therapies, it is critical
to use cell lines more closely related to the tumour and organism being studied. The
authors listed 60 paediatric brain tumour cell lines reported in the literature, of which
only a small number can be obtained from central repositories such as ATCC [29] or
Children’s Oncology Group (COG) [30], thus rendering more difficult for the research
community to have access to the most adequate cell lines [31].
Considering these findings, novel preclinical models should be evaluated as plat-
form for drug discovery for paediatric diseases, such as induced pluripotent stem
cells (iPSCs) or innovative techniques including organoids and organs-on-a-chip.
Disease-specific iPSCs represent a promising platform to understand pathologi-
cal progression in patient-derived cells presenting many advantages: iPSCs are an
unlimited source of patient-specific cells for drug testing and for the development
of personalised medicine [32]. Advances in human pluripotent stem cell (hPSC) or
tissue-resident adult stem cell (AdSC) research have led to the possibility to mimic
any tissue in the human body through three-dimensional (3D) model including
organoids and organs-on-a-chip that can be used as in vitro screening models [33].
However, to confirm the adherence of these in vitro models with their normal coun-
terparts in vivo, we need a much deeper understanding of the physiology of human
development than what is currently available.
In addition, as regarding the animal models, the number of comprehensive
studies describing the normal development of different physiological systems and
processes in laboratory animals from molecular to system levels is very limited,
and such studies usually do not exist in animal models of paediatric diseases. Thus,
questions of comparability of developmental stages across species continue to
create debate. The need to use juvenile animal models will be better discussed in the
following section.
In addition to the need of developing cellular and animal models more suitable
to study paediatric diseases and the instruments to work with immature animals,
all the new emergent technologies should be timely applied to the paediatric drug
discovery in order to speed up the pharmacological research, including pluripotent
stem cell, 3D cell cultures, target validation, patient-derived cell assays, micro-
fluidics, high-throughput cell image analysis, non-invasive drug delivery systems
and devices to measure drug safety or efficacy non-invasively.

3.2 Preclinical development

Commonly, only a small number of compounds identified in the initial discov-

ery phase will pass through to more rigorous preclinical development. Pre-clinical
studies—in vitro, in vivo, and ex vivo—are essential steps in the drug development
path to provide detailed information about the pharmacokinetic (PK) and phar-
macodynamics (PD) properties of the selected molecules. The main goal of this
phase is to improve the understanding of the drug properties in vivo, evaluating
their efficacy, biodistribution, toxicity involving multiple experts, and competences
from pharmacologists, drug metabolism specialists, chemists, toxicologists and
formulation experts.
Drug dosing and response may differ markedly between adults and children for
many reasons: anatomical and physiological differences between paediatric and
adult population [34, 35], different diseases or presentation of diseases [36], differ-
ences in PK and/or PD profiles [37], different ‘host’ responses [38] different adverse
drug reactions [39] and drug formulation.
There are many examples of drugs with a diverse PK profile in children com-
pared to adults as a consequence of a different absorption, distribution, metabolism

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Drug Discovery and Development - New Advances

and excretion (ADME) [40]. The rate and extent of the bioavailability of a drug
may vary as a consequence of the development changes that occur in absorptive sur-
faces, especially the gastrointestinal tract. Dissimilarities have also been reported
in drug metabolism, transporters expression, biliary function and renal clearance,
resulting in differences in drug disposition and elimination [41].
Similarly to PK profile, PD profile is also affected by human development and
drug targets may vary under developmental control: their level of expression,
affinity or activity may diverge according to the patient’s age, resulting in variable
drug responses depending on patients’ age group. This is particularly important
in younger infants, more vulnerable to drug toxicity and related adverse events by
modifying drug therapeutic windows [42].
Another aspect to be taken into due account is represented by the effect of the
ontogeny and genetic variation interactions on drug response, known as pharma-
cogenetics [43]. Several pharmacogenetics studies have indeed demonstrated the
differences in response to drugs between children and adults [44].
To take into consideration these aspects, age-appropriated technologies and
models in paediatric drug development should be applied: appropriate cellular
models, juvenile animal model, administration of sub-pharmacologic doses (micro-
dosing) to evaluate PK in a first-in-paediatric study, modelling and simulations and
pharmacogenetics biomarkers.
Juvenile animal models should be used to take into due account the specificities
of the paediatric population as described above and to fill the gap between devel-
opmental and mature toxicity. Indeed, the same drugs can have a different safety
profile in children compared to adults due to many aspects such as body weight,
developmental differences in growth and function of target organs, immune system
maturation and different expression of receptors system. For example, adult models
of epilepsy cannot be simply applied to the study of paediatric epilepsy and key
differences exist in human and rodent brain maturation process [45].
Extrapolation of data from adults or studies using adult animals is not always
adequate to predict these differences in safety profile for paediatric age groups.
For this aim, ‘Guideline on the need for non-clinical testing in juvenile animals of
pharmaceuticals for paediatric indications’ has been adopted in January 2008 by
the EMA. The guidelines recommend the ‘use of juvenile animal models when a
drug safety cannot be appropriately defined in the intended paediatric age group
on the basis of human data or previous animal studies’ and provide recommenda-
tion on the ‘timing and utility of juvenile animal studies in relation to phases of
drug development process’. In particular, the document points out that studies in
juvenile animals should be performed on a case-by-case basis rather than using
standardised study protocols and describes the key aspects to take into consid-
eration in the study’s design: age of the animal and duration of the studies, route
of administration, selection of species, PK and toxicokinetics, dose selection,
endpoint [46]. Juvenile studies are especially recommended when it has been
demonstrated that a medicine causes toxicity in adult at the target organ level and/
or to tissues that undergo significant post-natal development (CNS, immune, or
reproductive systems). As also underlined by Anderson et al., it is important to
conduct the preclinical experiments in the most appropriate species at the most
relevant age on the basis of comparability of the specific organ system develop-
ment in question [47]. And many issues have to be considered in juvenile toxicol-
ogy studies: difficulties in the dose administration due to the small size of the
animals, in blood and tissue sample collection, and in distinguishing direct versus
latent effects [48].
Therefore, proper animal models should be developed. As an example, Lohi
et al. described the zebrafish as a model for paediatric diseases, with particular

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DOI: [Link]

emphasis on haematopoietic and infectious diseases [49]. In this direction, several

zebrafish models for the study of leukaemia have been developed [50–52].
Preclinical data obtained from juvenile studies, extrapolated assuming a cor-
relation between developmental growth in animals and children, can be linked
to different information from a variety of data sources using the modelling and
simulation (M&S) approach.
M&S is a multidisciplinary science, which integrates knowledge about diseases,
drug characteristics, in vitro, in vivo, and ex vivo data, patient populations and
clinical trial parameters in order to optimise study design and drug labelling [53].
Modelling and simulation tools have long been used in drug development to allow
a quantitative assessment of age- or growth-related differences in drug effects and
consequently the potential implications for different paediatric age groups [54].
On this basis, software has been created to link in vitro data to in vivo ADME and
pharmacokinetic/pharmacodynamic (PK/PD) outcomes in order to predict the
potential clinical complexities prior to human studies [55].
The use of a model-based approach in the paediatric context provides several
advantages allowing the integration of prior in vitro data and physiologically based
pharmacokinetic (PBPK) models with pharmacodynamics (PD) models (PBPK-PD
models) and the optimization of experimental protocols. Finally, this approach
improves the accuracy and efficiency of data extrapolation and allows the reduction
in the number of animals per experiment that sometimes may also be replaced by in
silico experiments.

3.3 Clinical development

Finally, efficacy and safety of the new medicine should be tested in appropriate
clinical trials. When it comes to the clinical development of a drug, several issues
related to the peculiarity of the paediatric population have to be faced. Conducting
a paediatric clinical trial raises several scientific and operational challenges.
First of all, the low prevalence of many paediatric diseases leads to a limited
number of children affected by each condition. In addition, the ethical issues are
also to be considered to obtain clinical benefits for children assuring the best pos-
sible protection for these vulnerable subjects. Moreover, considering the heteroge-
neous nature of the paediatric population, the population subsets to be included in a
study should be chosen with great attention in order to be sure to consider the most
likely target population for the medicine being tested.
Another issue to be considered in the design of a paediatric clinical trial is the
lack of tools and/or methods for quantitative and qualitative assessment tailored for
the paediatric population and its sub-groups (study endpoints, questionnaires and
scales for the measurement of psychophysical parameters and tools for the assess-
ment of adverse reactions).
The difficulties described above, in testing appropriate drugs in children,
have brought to an increased use of off-label drugs with high risks for adverse
safety events and efficacy failures and to a general knowledge gap in paediatric
research [1].
The US and EU Regulatory agencies foster the drug clinical development
through regulations and incentives and the increasing number of paediatric trials
and specific label changes and dosing recommendations.
Ground-breaking methodologies such as innovative trial design, application of
modelling and simulation and other tools supporting paediatric trials (such as spe-
cific outcomes measures, biomarkers, statistical methods, etc.) can help researchers
to overcome obstacles faced in planning, initiating and conducting a clinical trial
involving children.

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For example, to reduce the number of samples required for a study, sparse and
scavenged sampling approach can be used. Sparse sampling uses a lower number of
samples per patient compared with traditional PK sampling methods. Scavenged
sampling consists in the use of residual blood/plasma samples remaining after the
laboratory tests obtained in the course of medical care. These approaches reduce
the risk for the child and eliminate the need for vascular punctures specifically for
the study and, as a consequence, increase the rate of parental consent and the avail-
ability of several samples per infant [56].
Statistical methods, such as the Bayesian design, allow the extrapolation of
results out of fewer children than in the conventional, fixed-number design, also
considering evidences in adults [5].
Modelling and simulation approaches allow to successfully predict the optimal
dosing regimens from the preclinical to the clinical phase [57].
More innovative trial design methods are being developed to overcome the
limits related to small samples and to the acceptability of the trial. These alterna-
tive approaches, limiting the amount of experimentation in children, represent a
promising way of ultimately improving paediatric care [58].

3.4 Paediatric biomarkers

A biomarker can be defined as ‘a characteristic that is objectively measured and

evaluated as an indicator of normal biological processes, pathogenic processes, or
pharmacologic responses to a therapeutic intervention’ [59].
Biomarkers can be influential in every phase of drug development, from drug
discovery and preclinical development, through each phase of clinical trials and
into post-marketing studies. Evaluation and application of biomarkers can be useful
to refine a drug dose or dose interval, or to select the appropriate population during
early-phase clinical development of a product [60].
Despite this, the discovery of paediatric biomarkers has been limited and to
cover the resultant gap, extrapolation, in children, of biomarkers identified and
employed successfully in adults has become a common practice. However, human
development impacts almost all factors and systems from organ function to drug
disposition including the commonly utilised biomarkers that are influenced by
changes occurring from birth onwards [61]. Therefore, adult biomarkers are not
always appropriate to a paediatric setting.
A major challenge in the paediatric biomarkers discovery path is the sample
availability due to the low prevalence of many paediatric diseases. Moreover,
compared to adults, the paediatric populations is more heterogeneous making
more difficult to obtain samples for biomarker discovery and validation, with
the patients often distributed among several centres. Consequently, multicentre
collaborations are often necessary in order to access sufficiently large study popu-
lations of affected children to generate big enough datasets to adequately power
research studies [62].
Additional obstacles in children are represented by the difficulty to obtain
appropriate age-matching control samples in order to minimise the influence that
age-related changes may have on biomarker discovery and validation. Research on
healthy children is generally restricted to minimal risk procedures, so although bio-
logical samples like saliva and urine can be relatively easy to obtain, blood samples
are difficult to obtain in healthy children, particularly in neonates [61].
Moreover, several ethical considerations have to be taken into due account to
enrol children in a biomarkers study: an effective and simplified consent process,
long-term retention of samples for future research, the impact of ancillary genetic
information on family members and predisposition to adult-onset disease [61].

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Current advances in molecular techniques and the speed up of the ‘-omics’

technologies (i.e. genomics, transcriptomics, metabolomics and proteomics) have
provided new tools facilitating the discovery of new biomarkers. The promise of
omics technologies is considered huge, but translation of these technologies into
clinical setting has been quite slow especially in the paediatric field.

3.5 Age-appropriate formulation

The effect of age on PK profile, as discussed above, leads to different dosing

requirements for different age groups. The proper dose administered may vary nearly
100-fold during childhood as a consequence of the body size and weight increase from
birth to adulthood [63]. Premature neonates admitted to the hospital can weigh as little
as 500 g. Moreover, since the maturation process in children is not linear, not always a
linear relationship exists between a medication dose and body size and/or weight.
The need to have safe and suitable drugs for children has led to the awareness
that drug formulations tailored to children in all the target age groups is essential.
Formulation acceptability differs across age groups as children gradually develop their
cognitive and motor skills, and improve their ability to swallow medications. And taste
of a drug may be critical to ensure acceptable adherence to paediatric oral formulations.
The ideal formulation for children should have flexible dosage increments and
minimal excipients, be palatable if given oral, easy and safe to administer, and be
stable with regard to light, humidity and heat.
Continuous effort in formulation science by academic and paediatric researchers
and commitment of policy makers and regulators should promote the preparation
of pharmaceutical formulations for paediatric use, focusing on age tailored forms,
excipient-related toxicity and safety risks in order to improve acceptability and
facilitate medication adherence in children.

3.6 Pregnancy and perinatal pharmacology

Up to 80% of women receive at least one medication, over-the-counter (OTC)

or prescribed, during pregnancy in Europe [64]. The most common drugs used dur-
ing pregnancy are anti-infectives and respiratory drugs [65]. It is recognised that
medications assumption during pregnancy can represent a risk for the foetus, and
therefore, medication use is approached with caution by pregnant women and their
health care providers [66]. Nevertheless, the majority of current therapeutics used
were never being studied in pregnancy for many reasons. Traditionally, pregnancy
usually represents an exclusion criterion for phase I testing studies and women of
childbearing age are usually excluded from clinical trials. Moreover, pharmaceutical
companies manifest a low interest in the pregnant population since this population
has more medico-legal risks and ethical concerns and represents a small percentage
of the patient population that these companies target [66].
Due to the lack of studies involving pregnant women, safety drugs profile is
usually obtained from either post-marketing surveillance or late-stage retrospective
studies and efficacy and dosing data can be extrapolated from studies conducted in
men or non-pregnant women [66].
To foster the availability of more effective and safer obstetrical drugs, a better
understanding of the changes that occur in the mother, placenta and foetus is essen-
tial and strategies to monitor the therapeutic progress have to be improved [65].
The placenta represents a maternal-foetal interface between the mother and
baby’s blood and controls exchanges of nutrients, oxygen, wastes and drug transport.
The process regulating molecular transfer across the placental barrier is poorly under-
stood leading to a lack of precious information for the drug development process.

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Most studies on human placental biology have been conducted on tissue obtained
after term delivery, or earlier, often from pathological pregnancies at various stages
of disease, or from ex vivo model system. Less information can be obtained about the
earlier phases of gestation and the normal development and functions of human pla-
centa [67]. Behind these difficulties in obtaining the tissue, the studies on placenta
require high level of expertise. To overcome these limitations, some initiatives have
been undertaken. A 3D in vitro model of human placenta has been developed by a
research group at the University of Vienna. The 3D model shows self-organisation,
self-renewal and constant growth capacity and can be also pharmacologically and
genetically manipulated allowing to study the physiological and pathophysiological
processes of human placenta [68]. Another attempt to develop a model of human
placenta has been carried out by the Huh Lab at the University of Pennsylvania,
which developed the first placenta-on-a-chip to study drug delivery to the placenta
and preterm birth. It consists of a small block of silicone that contains two overlap-
ping layers of microchannels that are lined with trophoblast cells isolated from the
outer surface of the placental barrier and separated by a porous membrane [69].
These advancements will allow a better understanding of the transport processes
through the placenta and a better designing of new obstetrician drug.

3.7 In silico pharmacology

In the last decades, advances in computer technology has led to an increase in

the use of informatics and bioinformatics in biomedical research, moving into an in
silico era. The introduction of the in silico methods in the drug discovery and devel-
opment has provided the opportunity to simulate every stage of the process, from
preclinical to clinical, allowing to combine various heterogeneous types of data into
computer-based pharmacological model.
As an example, in silico methods have been applied successfully in 2003 to drug
screening when two different research groups found an identical molecule as inhibi-
tor for the TGFb-1 receptor kinase: one using conventional ‘wet-lab’ assays and the
other using an in silico approach [70]. In parallel, computational methods for drug
development began to emerge, in order to model the interactions between drugs
and biological systems [70].
This approach has been translated in paediatrics as a promising method to sup-
port the design of in vivo studies in the early phase of drug development. Johnson
et al. predict with reasonable accuracy the in vivo drug clearance of 11 drugs that are
commonly used in neonates, infants and children using in silico prediction methods
and in particular the Simcyp® software [71]. Using a similar in silico approach, a
physiologically-based pharmacokinetic model (PBPK) was developed in PK-Sim
v4.2® to predict lorazepam PK in children as a function of age [72].
The introduction of PBPK modelling software in the field of paediatric drug
development presents many advantages considering the peculiarities of this popu-
lation. Notwithstanding these approaches could not replace totally the need for
clinical trials, but they could reduce the amount of clinical trials required in chil-
dren providing a primary exploratory investigation of drug PK, first-time dosing in
children and study design [71, 72].

4. Pricing and reimbursement policies

The issues linked to the pricing and reimbursement of drugs administered to

paediatric population are strictly linked to the mechanisms of drug marketing.
Multiple factors are involved, and alteration of the regulatory environment can

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DOI: [Link]

rapidly change the drug development pathway chosen by pharmaceutical compa-

nies. At the moment, most of the drugs used for children have a marketing autho-
rization for adults and are used ‘off-label’. No incentives are present for a company
to perform further studies in a paediatric population if the drug is used and reim-
bursed all the same.
The introduction of regulatory requirements for clinical studies in paediatric
populations [6, 8, 9] has altered this paradigm for the newest drugs but has not
changed the situation for the already used ones.
A basic principle for price calculation is the pay for quality-adjusted life years.
Theoretically, this approach should increase the value of a new paediatric drug,
but if the same drug is also used for an adult population, the payer would limit the
price as a larger population is involved. In fact, due to the age stratification of the
paediatric population, many paediatric pathologies might be considered as a rare
disease. In fact, due to the facilitation linked to the development of a drug for a rare
disease, an emerging approach from commercial entities is to develop drugs for the
smaller paediatric population and to ask for an extension of the marketing licence
to the adult group only when the licence is going to expire in a reverse approach to
maximising the revenues for each new drug.
As this is applicable to all small populations, the regulatory agencies are already
eliminating the rare diseases from the groups receiving extra benefit during the
marketing authorization process, further complicating the issue.
Overall, due to the personalised medicine approach stratification, there is a
strong need to increase the public funding during the early stages of drug develop-
ment in order to not only reduce and control the cost of new drug but also encour-
age the development of new class of drugs based on the increased knowledge of the
human normal and pathological development.

5. New and existing research initiatives in the field

The advancement of innovative technologies in the paediatric pharmacology and

preclinical phase of drug development will contribute to speed up both the develop-
ment of new medicines for children and the paediatric clinical research. The aware-
ness about the limited application of the innovative technologies in the paediatric
drug development process and the scarce availability of safer and efficacious drugs
for children has led, over the last years, to the onset of initiatives and collaborative
efforts in this field.
At European level, we can cite EnprEMA [73], a network of research networks,
investigators and centres with recognised expertise in performing paediatric
clinical studies, which have greatly contributed to increase availability of medi-
cines authorised for use in the paediatric population, according to what foreseen
in the Paediatric Regulation. The TEDDY Network of Excellence (European
Network of Excellence for Paediatric Clinical Research) [74], funded within the Sixth
Framework Programme of the European Commission as Task-force in Europe for
Drug Development for the Young and recognised as category 1 network member of
Enpr-EMA, aims to favour adequate health policies and a social awareness on the
importance of the paediatric medicines across Europe. TEDDY network goal is to
support the paediatric clinical pharmacology and reduce the current fragmentation
in the development of medicine in children. In line with this goal, TEDDY set up
the European Paediatric Medicines Database as a pan-European source of informa-
tion that includes data on paediatric medicines authorised by EMA collected by
several sources (national authorities, regulatory bodies and pharmaceutical com-
panies). Among other initiatives, we can mention the Conect4Children (C4C) IMI2

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(Innovative Medicines Initiative 2) project [75] and the PedCRIN project [76]. C4C
is a European project aimed to implement an infrastructure of clinical sites organ-
ised as a pan-European network to test medicines through well-organised, moni-
tored and evaluated profit and non-profit paediatric clinical trials. PedCRIN project
is intended to develop tools and actions for paediatric and neonatal trials in order to
better address the real needs and gaps of the paediatric research community.
Behind the initiatives mentioned above, other actions have been taken to foster
the early drug discovery and preclinical development phases. In this field, we can
mention the European Paediatric Translational Research Infrastructures (EPTRI)
project [77], aimed to design a research infrastructure (RI) completely dedicated
to paediatrics to be included in the landscape of the ESFRI RIs. EPTRI aims to be
complementary and fully integrated in the context of the existing RIs providing
services, competences, expertise in the paediatric drug discovery and develop-
ment. EPTRI will provide support to the paediatric research community through
its thematic platform: Human Development and Paediatric Medicines Discovery,
Paediatric Biomarkers and Biosamples, Paediatric Pharmacology, and Paediatric
Medicines Formulations and Medical Devices. Through them, EPTRI will promote
a translational approach from the bedside to the bench side, to make available
more efficacious and safer drugs for children. In the formulation field, it has to be
mentioned that the European Paediatric Formulation Initiative (EuPFI) [78] is a
consortium working in a pre-competitive way on paediatric drug formulations and
aimed to speed up the development of better and safer medicines for children by
identifying issues and challenges in paediatric formulation development. EuPFI has
set up the database Safety and Toxicity of Excipients for Paediatrics (STEP) that
provides updated information on excipients safety and toxicity in children.
To address specifically the rare diseases, the European Joint Programme Rare
Disease (EJP RD) [79], recently founded by the European Commission, brings over
130 institutions from 35 countries to create virtuous circle among research, care and
medical innovation in the rare disease landscape. In particular, the project will improve
the integration, the efficacy, the development and the social impact of research on
rare disease and will implement an efficient model of financial support for all types
of research on RD (fundamental, clinical, epidemiological, social, economic and
health service), providing support to accelerate the exploitation of research results
for the benefit of patients. To more specifically focus on the drug development in rare
diseases, a task force has been created within International Rare Diseases Research
Consortium(IRDiRC) [80], the Orphan Drug Development Guidebook Taskforce,
aimed at providing support to academic and industrial drug developers and describing
the available tools and initiatives specific for rare disease drug development [81].
As described, many initiatives exist as a result of the growing understanding
that children cannot be considered as small adults, but need to be addressed specifi-
cally in the drug development path. But more efforts and the involvement of the
national and international policy bodies are still needed to make the development
of medicines for children a priority.

6. Conclusion

Children represent particular vulnerable subjects and therefore should be pro-

tected and preserved by the risks that a clinical research can entail. However, at the
same time, higher risks in term of major toxicity and/or reduced efficacy can result
by the administration of drugs not properly tested and developed for them. Despite
this, the off-label drug administration is still common in the paediatric population
and children have been considered for year as the therapeutic orphans due to the

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DOI: [Link]

recognised lack of medicines specifically targeted for them. Moreover, the enor-
mous progresses and advancements reached in the pharmaceutical field have not
been applied to the paediatric population at the same extent of the adults.
The gap in the availability of proper medicines for children can be traced back to
ethical, practical and economic reasons. As discussed in the chapter, the main prac-
tical reasons can be associated with the differences existing in the diseases affecting
children compared to adults, as well as in the different physiology itself of the chil-
dren compared to adults, the low number of patients affected, the need to take into
account different age groups and the need to make available appropriate formula-
tions. Moreover, the ethical concerns make more difficult to obtain the parents’
consent. In addition, the pharmaceutical companies are not interested in this niche
market, since they cannot foresee an adequate economic return. Furthermore, more
challenges have to be faced when considering paediatric rare diseases. Complex
aetiology, small affected population and subsequently small market size, high cost,
and possibly low return on investment led to a large gap between basic research and
patient unmet needs for rare disease drug discovery.
Many initiatives have been taken over the years, also at institutional levels, to
promote a ‘good research’ in the paediatric field, in order to involve children and at
the same time preserve them by unnecessary risks. Only increasing our understand-
ing about human development processes and about how these processes impact
on the onset and progression of diseases will able us to develop specific medicines
targeted for children. The knowledge of these processes will allow us to transfer in
the paediatrics all the advancements and innovative technologies nowadays avail-
able in the adults’ pharmacological research. Thus, more efforts are needed in terms
of capitals, human resources, and technological expertise to speed up both the
preclinical and clinical drug development in children and make available to children
new medicines and appropriate treatments.

Conflict of interest

The authors declare that they have no conflict of interest.

Author details

Angelica Intini1,2, Donato Bonifazi1,2* and Giovanni Migliaccio1,2

1 CVBF—Consorzio per Valutazioni Biologiche e Farmacologiche, Bari, Italy

2 TEDDY—European Network of Excellence for Paediatric Clinical Research, Pavia,

Italy

*Address all correspondence to: ceo@[Link]

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License ([Link]
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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Drug Discovery and Development - New Advances

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152
 Edited by Vishwanath Gaitonde,
Partha Karmakar and Ashit Trivedi

The process of drug discovery and development is a complex multistage logistics

project spanned over 10-15 years with an average budget exceeding 1 billion USD.
Starting with target identification and synthesizing anywhere between 10k to 15k
synthetic compounds to potentially obtain the final drug that reaches the market
involves a complicated maze with multiple inter- and intra-operative fields. Topics
described in this book emphasize the progresses in computational applications,
pharmacokinetics advances, and molecular modeling developments. In addition the
book also contains special topics describing target deorphaning in Mycobacterium
tuberculosis, therapy treatment of some rare diseases, and developments in the
pediatric drug discovery process.

ISBN
ISBN 978-1-78985-219-6
978-1-78923-975-1

Published in London, UK
© 2020 IntechOpen
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