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BASIC PRINCIPLES OF DRUG
DISCOVERY AND DEVELOPMENT
BASIC PRINCIPLES OF
DRUG DISCOVERY AND
DEVELOPMENT
SECOND EDITION
BENJAMIN E. BLASS
Temple University School of Pharmacy, Department of Pharmaceutical Sciences,
Moulder Center for Drug Discovery Research, Philadelphia, PA, United States
Academic Press is an imprint of Elsevier
125 London Wall, London EC2Y 5AS, United Kingdom
525 B Street, Suite 1650, San Diego, CA 92101, United States
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
Copyright © 2021 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying, recording, or any information storage and
retrieval system, without permission in writing from the publisher. Details on how to seek
permission, further information about the Publisher’s permissions policies and our arrangements
with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency,
can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the
Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience
broaden our understanding, changes in research methods, professional practices, or medical
treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in
evaluating and using any information, methods, compounds, or experiments described herein. In
using such information or methods they should be mindful of their own safety and the safety of
others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors,
assume any liability for any injury and/or damage to persons or property as a matter of products
liability, negligence or otherwise, or from any use or operation of any methods, products,
instructions, or ideas contained in the material herein.
British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
ISBN: 978-0-12-817214-8
For Information on all Academic Press publications

visit our website at https://www.elsevier.com/books-and-journals
Publisher: Susan Dennis

Acquisitions Editor: Emily McCloskey
Editorial Project Manager: Lindsay Lawrence
Production Project Manager: Paul Prasad Chandramohan
Cover Designer: Mark Rogers
Typeset by MPS Limited, Chennai, India
Dedication
Sir Isaac Newton, one of the greatest scientists of his time, wrote “If I
have seen further it is by standing on the shoulders of Giants.”
Although he was almost certainly referring to his scientific achieve-
ments, the underling concept of learning from our forbearer is true in
any endeavor. Indeed, this concept can be further extended to include
those who are there in the present day, supporting the activities of an
individual as he or she attempts to accomplish that which they view as
important. With this thought in mind, I have dedicated this book to the
scientists who came before me, those who mentored me, and those who
work with me on a daily basis. In addition, and perhaps more impor-
tantly, this text is dedicated to the loving and supportive family that
has helped me become the person that I am today. Special thanks are
offered to my mother, father, sister, brother, my three children, and of
course, my wife Kathleen. These are the giants on whose shoulders I
have stood upon.
Contents
Foreword xv
1. Drug discovery and development: An overview of modern

methods and principles 1
Drug discovery and development from 20,000 Feet 11
Target selection: the first step forward 15
Hit identification: finding a starting point 19
Identify a clinical candidate: juggling the properties 25
Questions 31
References 31
2. The drug discovery process: From ancient times to the

present day 43
The age of botanicals: pre-industrial drug discovery 44
Early biotherapeutics: before the biotechnology revolution 48
Paul Ehrlich: the father of modern drug discovery 51
Milestones in drug discovery 53
Milestones in animal models: breeding a better model 54
The Wistar rat 54
Immunocompromised mice 55
Transgenic animal models 56
Knockout animal models 58
Milestones in molecular science 60
X-ray crystallography 60
Molecular modeling and computational chemistry 63
High-throughput technology: chemical synthesis and screening science 64
Milestones in biotechnology 70
Recombinant DNA and transfection technology 72
Polymerase chain reaction technology 75
DNA Sequencing and genomics 76
Proteomics 80
Monoclonal antibody and hybridoma technology 83
The rise of biologics and macromolecular therapeutics 86
Societal and governmental impacts 87
The Pure Food and Drug Act of 1906 88
The Elixir of Sulfanilamide Disaster of 1937 89
The Thalidomide story 91
Regulatory milestones 93
vii
viii CONTENTS
Durham-Humphrey Amendment of 1951 94

Kefauver-Harris Amendment of 1962 95
Hatch-Waxman Act of 1984 96
Biologics Price Competition and Innovation Act of 2009 97
Future developments in drug discovery 99
Questions 99
References 100
3. Classical targets in drug discovery 111

Protein structure 113
Enzymes 119
Inhibition of enzymes 125
G-protein-coupled receptors 130
G-protein-dependent signaling pathways 133
Cyclic adenosine monophosphate signaling 134
IP3 signaling 136
β-Arrestin pathways 138
G-protein-coupled receptor signaling pathways 140
Modulating G-protein-coupled receptor signaling 141
Ion channels 143
Gating mechanisms 149
Ligand-gated channels 149
Voltage-gated channels 152
Other gating mechanisms 155
Membrane transport proteins (transporters) 156
Nuclear receptors 164
Nuclear receptor signaling pathways 167
Modulating nuclear receptor activity 169
Biomolecular interactions: protein/protein, protein/DNA, and protein/RNA
interfaces 170
Types of “hot spots” in biomolecular interactions 172
Stabilizing biomolecular interactions 174
Questions 176
References 176
4. In vitro screening systems 185

The language of screening: basic terms 186
Concentration response curves and IC50s 187
Dissociation constants (Kd) and inhibition constants (Ki) 188
Efficacy versus binding: EC50s 190
Agonist, partial agonist, antagonist, allosteric modulators, and inverse agonists 191
Agonists and partial agonists 192
Antagonists 193
Basal activity and inverse agonist 193
Receptor reserve 193
Allosteric modulation 194
CONTENTS ix
Streptavidin and biotin 195

Biochemical versus cellular assays 196
Assay systems and methods of detection 198
Radioligand systems 199
Scintillation proximity assay 201
Enzyme-linked immunosorbent assay 204
Fluorescence-based assay systems 206
Fluorescence polarization 207
Fluorescence resonance energy transfer 210
Time-resolved fluorescence resonance energy transfer 214
Amplified luminescent proximity homogeneous assay (AlphaScreent) 217
Fluorescent detection of calcium flux 220
Reporter gene assays 223
Chloramphenicol acetyltransferase 224
β-Lactamase reporter assays 224
Luciferase reporter assays 226
Bioluminescence resonance energy transfer assays 228
Kinetic fluorescent measurement systems 230
Label-free assay systems 231
Cellular dielectric spectroscopy 232
Optical biosensors 233
Surface plasmon resonance technology 236
Electrophysiological patch clamp 237
Thermal shift assay 240
High content screening 243
General consideration for all screening methods 245
Questions 248
References 248
5. Medicinal chemistry 257

Structure activity relationships and structure property relationships 258
The role of chirality 263
Push and pull in structure activity relationships 266
Quantitative structure activity relationships 267
The pharmacophore 272
Developing an structure activity relationship data set 276
The structure activity relationship cycle 287
Bioisosterism 288
Structure activity relationship, selectivity, and physicochemical properties 294
“Drug-like” guidelines 295
Questions 297
References 298
6. In vitro ADME and in vivo pharmacokinetics 305

Absorption 309
Solubility 310
Permeability 315
x CONTENTS
Distribution 324
Permeability 326
Transporters 328
Plasma protein binding 330
Elimination pathways 332
Metabolism 333
Excretion 345
In vitro ADME model systems 348
In Vivo pharmacokinetics 351
Volume of distribution 353
Clearance 355
Half-life 356
Bioavailability 359
Species selection 362
Questions 362
References 364
7. Animal models of disease states 371

Sources of animal models 373
Validity of animal models 376
Species selection 377
Number of animals 378
Exemplary animal models by disease category 378
Animal models in neuroscience 379
The forced swimming test: A model of depression 379
The elevated plus maze: A measure of anxiety 380
The novel object recognition test: A model of memory and cognition 381
Contextual fear conditioning model: A model of contextual learning 382
The Morris water maze: A model of spatial learning and memory 383
Animal models of neurodegeneration 384
The SOD1G93A mouse of amyotrophic lateral sclerosis (ALS) 384
The MPTP model of Parkinson’s disease 386
Animal models of cardiovascular disease 387
Models of hypertension 387
Models of hyperlipidemia and high cholesterol 389
Models of atrial fibrillation 391
Models of heart failure 393
Animal models of infectious disease 396
Murine thigh infection model 397
Murine model of systemic infection 397
Mouse model of influenza virus infection 398
Limitations of animal models of infection 399
Animal models of oncology 400
Mouse xenograft tumor model 400
Mouse allograft tumor model 401
Genetically engineered mouse models of cancer 402
CONTENTS xi
Animal models of pain 403

The Von Frey test 404
The Ramdall-Selitto test 405
Heat based models 406
Inflammation based models 408
Surgical models 409
General consideration for pain models 409
Animal models of diabetes 409
Animal models of drug addiction 411
Conclusion 413
Questions 414
References 414
8. Safety and toxicology 421

Sources of toxicity 423
Acute versus chronic toxicity 430
Cytotoxicity 430
Carcinogenicity, genotoxicity, and mutagenicity 432
Drug drug interactions 436
Cardiovascular safety and toxicology studies 438
Central nervous system safety and toxicology studies 445
Immune system-mediated safety issues 447
Teratogenicity 450
In vivo toxicity and safety studies 451
Questions 453
References 453
9. Antibody-drug discovery 459

IgG structure and function 461
Antibody therapy drug discovery 463
Hybridoma technology 463
Antibody phage display 469
Modified monoclonal antibodies 473
General considerations 476
Conclusion 478
Questions 478
References 478
10. Basics of clinical trials 483

Before the clinic 486
Drug supply 486
Delivery methods 489
Formulation 491
Investigational new drug application 502
Phase 1 clinical trials 504
xii CONTENTS

Adaptive clinical trial design 516
Meta-analysis of clinical trial data 517
Questions 519
References 519
11. Translational medicine and biomarkers 523

Definition of a biomarker and their classification 526
Characteristics and impact of biomarkers 529
Biomarkers versus surrogate endpoints 531
Imaging technologies 533
The practical application of biomarkers 540
Dipeptidyl peptidase IV inhibitors (Januvia) 541
Physiological measurements as biomarkers: Orexin antagonists 543
FDG PET imaging agent 545
The neurokinin 1 receptor, depression, and PET imaging:
The Aprepitant story 546
Cancer biomarkers 548
Conclusion 553
Questions 553
References 554
12. Organizational consideration and trends in the

pharmaceutical industry 561
Organizational structures of pharmaceutical companies 562
Business divisions interactions 562
The discovery project team evolutionary cycle 563
The business climate 567
Mergers and acquisitions 567
Contract research organizations 573
Academic drug discovery 575
Funding issues 581
Conclusion 584
Questions 584
Appendix 1 585
References 591
13. Intellectual property and patents in drug discovery 595

Patentable subject matter 597
Inherent properties and patentability 601
Novelty and the prior art 603
Obviousness and the prior art 604
Inventorship 607
CONTENTS xiii
Assignment and ownership 609

Classification of patents and patent applications 611
Impact of overlapping patents 612
Patent applications and their contents 612
Contents of a patent application 617
Conclusion 621
Questions 622
References 622
14. Case studies in drug discovery 625

Tamiflu: From mechanism of action to marketed drug 625
Histone deacylase inhibitors: Physicochemical optimization via structural change 630
HIV protease inhibitors: Chemically complex miracle drugs 632
Nitrofurantoin: A surprisingly successful drug 637
Seldanes (Terfenadine) vs Allegras (Fexofenadine): Metabolism matters: Safety 639
Claritins (Loratadine) versus Clarinexs (Desloratadine): Metabolism matters:
Pharmacokinetics 642
MPTP: Parkinson’s disease in a bottle 644
Bupropion and Methylphenidate: Improving performance via formulation
changes 647
Selective inhibition of COX-2: The impact of an inadequate written description 651
Antibiotic resistant bacteria and the development of β-lactamase inhibitors 652
Association for Molecular Pathology vs Myriad Genetics: The validity of gene
patents 655
Conclusion 658
Questions 659
References 659
Answers to questions in textbook by chapter 665
Subject Index 693

Drug Index 713
Foreword
The last several decades have witnessed a revolution in the drug dis-
covery and development process. Medicinal chemistry and in vitro
screening that were once major bottlenecks in the process of identifying
novel therapeutics have been dramatically accelerated through the
incorporation of automation and the development of enabling technolo-
gies such as recombinant DNA and transfection technology. High-
throughput screening, parallel synthesis, and combinatorial chemistry
have facilitated the synthesis and biological evaluation of large numbers
of potentially useful compounds. These activities, in turn, have gener-
ated vast amounts of data that can be analyzed to develop structure
activity relationships and structure property relationships useful for the
optimization of lead compounds. At the same time, new techniques,
technological advances, and a greater understanding of the importance
of pharmacokinetics, animal models, and safety studies have dramati-
cally altered how new molecules are selected for clinical study. Clinical
trial design strategies, biomarkers, translational medicine, the regulatory
landscape, intellectual property rights, and the business environment
have also changed dramatically over the course of the last 40 years.
The complexities of the drug discovery and development process
cannot be overstated, nor can the wide range of expertise required for
the successful development of new, marketable therapeutics. In order to
thrive in this very changing landscape, individuals interested in a career
in the pharmaceutical industry or related fields must be more than sim-
ply experts in their chosen field of study. They must also have an
understanding of the numerous, overlapping fields of their colleagues.
Basic Principles in Drug Discovery and Development has captured the criti-
cal information on the disparate processes, technologies, and expertise
required for modern drug discovery and development and presents it
in a logical and concise manner for students, practicing scientists, and
nonscientist with an interest in the pharmaceutical industry. Dr.
Benjamin E. Blass, an experienced educator and scientist with founda-
tional knowledge in medicinal chemistry, drug design, biological tar-
gets, and over 25 years of experience in industrial and academic drug
discovery and development, provides a comprehensive account of the
many functions involved in drug discovery and development, from
xv
xvi Foreword
initial medicinal chemistry conceptualization and in vitro biological

evaluation to clinical trials and beyond.
There are many aspects of this book that will help practicing scien-
tists, graduate students, and future drug researchers to develop a strong
foundation in the concepts that govern the multidisciplinary process of
drug discovery. Through this unique text, they will acquire an under-
standing of key aspects of drug discovery and development. The organi-
zation of the subject material was chosen to allow the readers to
incrementally increase their knowledge in the wide range of disciplines
required to identify new, marketable therapeutic agents. The book is
thoroughly written and includes 14 chapters with more than 300
figures and 900 references. Throughout the text the reader will become
familiar with more than 100 drugs and clinical candidates that exem-
plify important theories and practices.
Each chapter contains examples of drugs pertaining to the material in
the chapter. The opening chapter provides an overview of drug discov-
ery and development. This serves as the foundation for the following 12
chapters that describe the various functions involved in drug discovery
and development. The early phases of drug discovery are described in
detail through the discussions of important topics such as target identi-
fication, target validation, lead identification, multidimensional lead
optimization, pharmacokinetics, preclinical pharmacodynamics, and
early toxicology. This is followed by discussions of preclinical activities,
clinical trial design, biomarkers, and translational medicine. Each chap-
ter builds on the previous chapters and this approach provides the
readers with an integrated view of the various multidisciplinary func-
tions required for the drug discovery and development process.
Chapter 12, Organizational Consideration and Trends in the
Pharmaceutical Industry, and Chapter 13, Intellectual Property and
Patents in Drug Discovery, describe two important topics essential for
running an effective pharmaceutical R&D business, organizational
structure and patent protection. These chapters give the reader a true
understanding of the organizational structure required for the success-
ful management of research and development organizations and the
importance of protecting intellectual property to ensure a good return
on investment. Patent protection is the life blood of the pharmaceutical
and biotech industries, and at the same time a source of innovation for
new discoveries. Patents ensure the sharing of discoveries and innova-
tions that might otherwise be kept as trade secrets. In the final chapter,
case studies demonstrating the practical application of the concepts and
principles described in the previous chapters are provided. These vign-
ettes also describe important lessons learned in each case, some of
which changed the way modern drug-discovery research and develop-
ment programs are executed.
Foreword xvii
Although there are numerous textbooks that discuss various aspects

of the drug discovery and development process, none of them provides
a comprehensive view of the process. Basic Principles in Drug Discovery
and Development is unique in its comprehensive approach to this com-
plex endeavor. In writing this textbook, Dr. Blass has provided an
important new tool for the education of the next generation and a valu-
able resource for people with a vested interest in the identification and
commercialization of novel medications.
Magid Abou-Gharbia, PhD, FRSC

Laura H. Carnell Professor
Director Moulder Center for Drug Discovery Research
School of Pharmacy, Temple University
Philadelphia, PA, United States
C H A P T E R
1
Drug discovery and
development: An overview of
modern methods and principles
Over the course of the last two centuries, modern medicines have
improved the lives of countless patients. Diseases and conditions that
were once deemed incurable or fatal have been conquered with thera-
peutic agent designed to extend and improve quality of life. The most
recent, and perhaps most notable of these accomplishments is the transi-
tion seen in the consequences of infection with human immuno-
deficiency virus (HIV), the virus known to cause acquired
immunodeficiency syndrome (AIDS).1 When the virus was first identi-
fied by two research groups in 1983,2 there were few antiviral agents
available, none provided effective treatment for HIV infection, and
infection progressed rapidly to AIDS and death by opportunistic infec-
tion. By 1987, AZTs (Retrovirs and Azidothymidine; Fig. 1.1), the first
nucleoside reverse transcriptase inhibitor (NRTI), was approved for
clinical application for the treatment of HIV infection,3 and additional
treatment options were developed through the next three decades. New
NRTIs such as Vireads (Tenofovir)4 and Zeffixs (Lamivudine)5
(Fig. 1.1) that shared AZT’s mechanism of action (phosphorylation,
incorporation into a growing DNA chain, and chain termination)
expanded this class of HIV drugs. The development of non-NRTIs such
as Viramunes (Nevirapine)6 and Doravirines (Pifeltro)7 (Fig. 1.1) fur-
ther advanced HIV therapy and demonstrated that reversible, allosteric
inhibition (enzyme inhibition accomplished through drug binding at a
site other than the catalytic site, see Chapter 3) of reverse transcriptase
was a viable approach. HIV protease inhibitors such as Viracepts
(Nelfinavir),8 Norvirs (Ritonavir),9 and Crixivans (Indinavir)10 entered
the market at the beginning in the mid-1990s, adding a new dimension
to HIV therapy. At the same time, multidrug cocktail treatment
Basic Principles of Drug Discovery and Development

DOI: https://doi.org/10.1016/B978-0-12-817214-8.00001-4 1 © 2021 Elsevier Inc. All rights reserved.
2 1. Drug discovery and development: An overview of modern methods and principles
NH2 O
OH O
H HN
N N S N O CN
O H N NH2 N
HO OH HN
N O N P O N N
N N
N3 NH O O O N N CF3
O OH O
Cl
® ®
AZT Viread® Zeffix Viramune® Doravirine®
(Retrovir) (Tenofovir) (Lamivudine) (Nevirapine) (Pifeltro)
O
O
N N OH HN O
S HN O H O
O HO NH
O S
N N N
N N O N H
H H N OH N
OH H
S
H
OH
Viracept® Norvir® Crixivan® N
(Nelfinavir) (Ritonavir) (Indinavir)

O O O OH OH O H
N N O F F F F O
N N N N N
H H H H
O N N N N N
OH F O O
H H H
O O F O
® ®
Raltegravir Dolutegravir Bictegravir®
(Isentress) (Tivicay) (GS-9883)
FIGURE 1.1 Reverse transcriptase was the first enzyme successfully targeted in a drug
discovery program that focused on developing treatment options for HIV infection and
AIDS. AZTs (Retrovir), Vireads (Tenofovir), and Zeffixs (Lamivudine) are nucleoside
inhibitors of this important enzyme that terminate DNA chain growth, while Doravirines
(Pifeltro) is a non-nucleoside inhibitor of reverse transcriptase that reversibly binds to an
allosteric site of the target. HIV protease, another enzyme critical to the progression of
HIV and AIDS, has also been the subject of intense study. The antiviral agents Viracepts
(Nelfinavir), Norvirs (Ritonavir), and Crixivans (Indinavir) are HIV protease inhibitors
that were developed for the treatment of HIV infection and AIDS. HIV integrase inhibitors
such as Raltegravirs (Isentress), Dolutegravirs (Tivicay), and Bictegravirs (GS-9883) were
also developed for the treatment of HIV. AIDS, Acquired immunodeficiency syndrome;
HIV, human immunodeficiency virus.
regimens, also known as highly active antiretroviral therapy,11 were

introduced, further enhancing treatment options, culminating in the
development of all in one fixed combination medications such as
Compleras12 and Stribilds.13 By the turn of the 21st century, yet
another class of HIV therapies began to reach the market. HIV integrase
inhibitors such as Raltegravirs (Isentress),14 Dolutegravirs (Tivicay),15
and Bictegravirs (GS-9883)16 (Fig. 1.1) became available to patients as
individual drugs or as parts of the combination therapies Triumeqs
(Dolutegravir/Lamivudine 3TC/Abacavir)17 and Biktarvys (Bictegravir/
Emtricitabine/Tenofovir alafenamide).18 While additional progress is
clearly still required, it is clear that modern drug discovery and devel-
opment changed the course of the AIDS epidemic, allowing patients
who were once given a death sentence to lead productive lives.19
Cancer treatment has seen a similar transition, as survival rates for
many types of cancer have dramatically improved as a result of the

Drug discovery and development: An overview of modern methods and principles 3
discovery and development of novel therapeutic agents. In the

United States, overall cancer death rates have declined 11.4%
between 1950 and 2009, and progress against some specific cancer
types has been substantial. Breast cancer, prostate cancer, and mela-
noma, for example, have seen significant increases in their 5-year sur-
vival rates over the same period. The 5-year survival rates for breast
cancer increased from 60% to 91%, while the survival rates for pros-
tate cancer increased from 43% to over 99%, and melanoma survival
rose from 49% to 93%.20 A significant portion of the improved clinical
outcomes in cancer can be attributed to improved chemotherapeutic
agents. The identification of antitumor natural products and
natural product analogs such as Taxols (Paclitaxel),21 Velbans
(Vinblastine),22 Adriamycins (Doxorubicin),23 and Hycamtins
(Topotecan)24 have clearly demonstrated the importance of natural
products in modern medicine, while the development of small mole-
cule kinase inhibitors such as Gleevecs (Imatinib),25 Tasignas
(Nilotinib),26 and Tarcevas (Erlotinib)27 provide clear evidence of the
power of modern drug discovery techniques (Fig. 1.2).
Much work remains to be done; however, as statistics from the
American Cancer Society indicate that in 2018, over 609,000 people will
die from cancer in the U.S. alone, and that the 2015 U.S. direct medical
care costs exceeded $80 billion. In addition, the 5-year survival rates for
cancers of the liver (18%), esophagus (18%), lungs (18%), and pancreas
(8%) are all low.28 Newer small molecule therapeutics such as
Vizimpros (Dacomitinib),29 Zejulas (Niraparib),30 and Alunbrigs
(Brigatinib)31 (Fig. 1.3) and biotherapeutics such as Keytrudas
(Pembrolizumab) (Fig. 1.3),32 Bavencios (Avelumab),33 and Opdivos
(Nivolumab)34 will almost certainly lead to further improvements in
cancer survival rates.
The treatment of cardiovascular disease has also seen dramatic
improvements in the wake of the discovery of a multitude of therapeu-
tic agents designed to mitigate symptoms or prevent the underlying
causes of the disease. A myriad of treatments has been developed to
address hypertension, also known as “The silent killer” because of its
asymptomatic nature, leading to improvements in both the quality of
life and life expectancy of patients. Diuretics (e.g., Midamors
(Amiloride),35 Lozols (Indapamide)),36 β-blockers (e.g., Tenoretics
(Atenolol),37 Inderals (Propranolol)),38 and angiotensin-converting
enzymes (ACE) inhibitors (e.g., Capotens (Captopril),39 Vasotecs
(Enalapril))40 are just a few of the types of treatments currently available
to lower blood pressure and keep cardiovascular disease at bay.
Revolutionary changes occurred in the prevention of cardiovascular dis-
ease with the introduction of HMG-CoA reductase inhibitors, also
known as statins.41 Lipitors (Atorvastatin),42 Zocors (Simvastatin),43

O OH
O O OH O OH O N
N OH
N OH HO O
O NH O H N
O N O H
H H H O N
O O O O OH O O NH2
HO O O OH O
OH
N
O
O O O O
O OH
OH O
NH2
Taxol® Velban® Adriamycin® Hycamtin®

(Paclitaxel) (Vinblastine) (Doxorubicin) (Topotecan)
O
O
HN
NH
O N
N O
N NH
NH O N
N O
N F
N HN
N N N F
F N
N
Gleevac® Tasigna® Tarceva®

(Imatinib) (Nilotinib) (Erlotinib)
FIGURE 1.2 The natural products Taxols (Paclitaxel), Velbans (Vinblastine), Adriamycins (Doxorubicin), and Hycamtins (Topotecan) are exem-
plary natural products that have been developed for the treatment of cancer, while Gleevecs (Imatinib), Tasignas (Nilotinib), and Tarcevas
(Erlotinib) were developed for the treatment of cancer through the application of modern drug discovery programs.
O H2N O
P
H H
N N N N
N
N
N O Cl NH
N
Alunbrig® Zejula®
N (Niraparib)
(Brigatinib)
F
HN Cl
H
N
N N
O
O N
Vizimpro® Keytruda®
(Dacomitinib) (Pembrolizumab)
FIGURE 1.3 Vizimpros (Dacomitinib) was approved in 2018 for the treatment of meta-
static non-small cell lung cancer. Zejulas (Niraparib) was approved in 2017 for the treatment
of epithelial ovarian, fallopian tube, and primary peritoneal cancers. Alunbrigs (Brigatinib)
was approved in 2017 for the treatment of metastatic non-small cell lung cancer. Keytrudas
(Pembrolizumab) was approved in 2014 for the treatment of advanced melanoma.
H2N Cl OH
O N H OH
O H O N H
H2N NH2 N O O N
S N
N N H2N
O O H2N
H2N Cl
Midamor® Lozol® Tenoretic® Inderal®
(Amiloride) (Indapamide) (Atenolol) (Propranolol)
HO
HO O
O O O OH
O HO
HS O O
O
O N O N
N H O F
N H
OH OH
O
® ® ®
Capoten Vasotec Zocor Lipitor®
(Captopril) (Enalapril) (Simvastatin) (Atorvastatin)
FIGURE 1.4 The diuretics Midamors (Amiloride) and Lozols (Indapamide), the
β-blockers Tenoretics (Atenolol) and Inderals (Propranolol), the ACE inhibitors Capotens
(Captopril), Vasotecs (Enalapril), and the HMGCoA reductase inhibitors Lipitors
(Atorvastatin) and Zocors (Simvastatin) have significantly improved the treatment of car-
diovascular disease. ACE, Angiotensin-converting enzyme.
and a number of related compounds have demonstrated a remarkable

capacity to lower cholesterol levels, a major risk factor associated with
cardiovascular disease (Fig. 1.4).44 The introduction of monoclonal anti-
bodies Praluents (Alirocumab) and Repathas (Evolocumab)45 further
advanced the treatment of cardiovascular disease. These agents target
proprotein convertase subtilisin/kexin type 9 (PCSK9), a key protein in

the degradation of low-density lipoproteins (LDL) receptors, and have

been shown to significantly lower circulating cholesterol levels. The
high cost of these agents (B$14,000/year),46 however, has been a driv-
ing factor in the pharmaceutical industry’s keen interest in developing
less expensive small molecules capable of achieving the same impact.
Similar improvements in disease management, symptomatic relief,
and improvements in the quality of life through the development of
novel chemotherapeutics could be described across a wide range of
health issues. It is clear the treatment of infectious disease, pain man-
agement, respiratory disease, and many other conditions have been pro-
foundly and positively impacted by the identification of novel
therapies.47 There are, however, many challenging areas of health care
that remain in need of improved medicine and advances in current ther-
apy. Alzheimer’s disease, for example, is the most common form of
dementia, and was originally described by German psychiatrist and
neuropathologist Alois Alzheimer in 1906. Over 100 years later, treat-
ment options for this disease remain limited at best, despite the enor-
mous amount of effort and research funding dedicated to identifying
novel treatments. Potential drug targets such as β-secretase (BACE),
γ-secretase, glycogen synthase kinase 3β (GSK3β), and cyclin-dependent
kinase-5 (CDK5)48 have been extensively studied and resulting clinical
leads such as Verubecestats (MK-8931),49 Lanabecestats (AZD3293,
LY3314814),50 Semagacestats (LY-450139),51 (Fig. 1.5) Bapineuzumab52
showed great promise in animal models. Unfortunately, these com-
pounds and many others failed to produce statistically significant
improvements in patients. Clinically effective, disease-modifying agents
remain as elusive today as they did when Alzheimer’s disease was first
identified.
Additional challenges also exist in areas once thought to have been
conquered by modern science. In the 1960s and 1970s, for example,
it was widely believed that modern medicine had all but conquered
infectious disease and that the major classes of antibacterial agents,
F O
O
OH O
N NH2 N H
N N N
H N N
N N N H
F S H2N O O
O2
Verubecestat® Lanabecestat® Semagacestat®

(MK-8931) (AZD3293, LY3314814) (LY450139)
FIGURE 1.5 Verubecestats (MK-8931), Lanabecestats (AZD3293, LY3314814),
s
Semagacestat (LY-450139) were each developed as possible treatments for Alzheimer’s disease,
but none have shown efficacy in clinical trials.

O O OH O O OH OH HO N
O OH OH
F N
H H HO H2N HO
S N HO O
N N O O O
N O O H H
O NH N OH
O O O O
OH
Staphcillin® Cipro® Vibramycin® Zithromax® O
OH
(Methicillin) (Ciprofloxacin ) (Doxycycline) (Azithromycin)
O O
F OH
H N HO Cl NH OH
OH N
N N O
H H O HO
F Cl OH
N OH O
S O
N ®
O Merrem NH2 NH2 Cl O Cl
OH (Meropenem) F O O
O HO O
®
Baxdela O OH
OH O O
H (Delafloxacin) O H H H
S N B N N N
O N N
O O N N N N
O N H H H
O HN O O
OH N H2N
HO
Vaborbactam O N
F O O
Vabomere® O Sivextro® OH Orbactiv®

HO P HO OH
(Meropenem + Vaborbactam) HO O
(Tedizolid phosphate) (Oritavancin)
FIGURE 1.6 Staphcillins (Methicillin), Cipros (Ciprofloxacin), Vibramycins

(Doxycycline), and Zithromaxs (Azithromycin) are representative examples of β-lactam,
fluoroquinolone, tetracycline, and macrolide antibiotics, respectively. Vabomeres (a com-
bination of Merrems (Meropenem) and Varobactam) combines a β-lactam antibiotic with
a β-lactamase inhibitor. Baxdelas (Delafloxacin) is a fluoroquinolone that is active
against MRSA. Sivextros (Tedizolid phosphate) is a member of the oxazolidinone class of
antibiotics, while Orbactivs (Oritavancin) is a glycopeptide antibiotic developed to
address the issue of Vancomycin-resistant bacteria. MRSA, Methicillin-resistant
Staphylococcus aureus.
β-lactams, quinolone, tetracyclines, and macrolide antibiotics (Fig. 1.6)

would provide all of the tools necessary to protect humanity. The rise of
methicillin-resistant Staphylococcus aureus (MRSA) in the 1980s and
1990s, however, has made it clear that additional tools will be required
in order to maintain the upper hand in the war against bacterial infec-
tion. Methicillin (Staphcillins) was introduced in 1959 as a means of
treating penicillin-resistant infections, but less than two years later,
resistant strains were identified in European hospitals. In the 1980s,
MRSA had spread throughout the globe, and as of 2009 MRSA infec-
tions cost the US health system $3 billion to $4 billion annually.53 In
addition, the United States Centers for Disease Control estimated that
there were .80,000 severe MRSA cases and .11,000 MRSA related
fatalities in 2013.54
Next-generation antimicrobial therapies (Fig. 1.6) have been developed
as part of the on-going efforts to address the ever-changing landscape
of human bacterial pathogens. The carbapenem β-lactam antibiotic
Merrems (Meropenem),55 for example, was approved in 1996, but
resistant organisms eventually developed. The combination therapy

Vabomeres was developed and brought to market in 2017 in response to

this growing problem. This drug combines Merrems (Meropenem) with
the β-lactamase inhibitor vaborbactam.56 The second molecule blocks a
key bacterial resistance mechanism (cleavage of β-lactams with β-lacta-
mase) and restores antibacterial potency of Merrems (Meropenem). In a
similar manner, Baxdelas (Delafloxacin)57 was also approved in 2017 as
the culmination of research focused on identifying new fluoroquinolones
capable of treating MRSA infections. The search for new classes of anti-
bacterial agents led to the identification of the oxazolidinones typified by
Sivextros (Tedizolid phosphate)58 and the development of analogs of
Vancomycin, a glycopeptide antibiotic originally introduced in 1955.59
Orbactivs (Oritavancin) and related compounds were developed to
address the growing issue of Vancomycin-resistant organisms.60
There is no doubt that the discovery of new therapeutic agents has a
positive impact on society, but to the casual observer, it is not clear how
this goal is achieved. On the surface, providing a drug necessary to
solve a medical problem would seem to be a relatively simple task;
identify the cause of the disease or malady and design a drug that will
fix or eliminate the problem. In the case of infectious disease, eliminate
the infectious agent, whether bacterial or viral, and the health problem
is solved. This is, of course a very simplistic view, as there are many
factors to consider beyond killing the offending organism. There are
millions of compounds that will kill an infectious organism, but how
many of these compounds can do so without negatively impacting the
host? How many of the remaining compounds can be delivered as safe
and effective therapeutic agents? How does one determine which of the
nearly infinite possibilities are useful and which ones are not? Of
the useful compounds, which ones will be of interest to the companies
that manufacture drugs and which ones will not be of interest? These
issues are exceptionally complex, and become even more so when the
health issue is something other than an invading organism. In consider-
ing chronic pain management, for example, a drug provided to a patient
should alleviate the chronic pain without interfering with the pain asso-
ciated with protective instincts, such as withdrawing one’s hand from a
hot stove. This added complexity is a common feature of the vast major-
ity of disease states and must be addressed in order to successfully
develop any new medication.
Given the large number of complex issues associated with drug
design and development, it should be abundantly clear that no one indi-
vidual could possibly conquer all of the tasks required to discover,
develop, and successfully bring to market a new therapeutic entity. The
process is a multidimensional one and as such, requires the coordinated
effort of individuals with a wide array of expertise such as medicinal
chemistry, in vitro biology, drug metabolism, animal pharmacology,

formulations science, process chemistry, clinical research, intellectual

property, and many other fields. Enabling technologies, such as high
throughput screening (HTS), molecular modeling, pharmaceutical pro-
filing, and biomarker studies, also play key roles in modern drug
research. It is critical that anyone interested in pursuing a career in the
development of pharmaceutically useful agents, whether in an industry
setting or an academic institution, must be willing and able to partici-
pate in collaborative research effort over a significant period of time. In
addition, it is important that any participant in this field understand the
magnitude of the risks and costs associated with the pursuit of new
drugs. The financial reward for those who are successful can be excep-
tional, as indicated by the success of compounds such as Lipitor
(Atorvastatin), which had peak annual sales of over $13 billion,61
Prozacs (Fluoxetine, peak sales $2.8 billion),62 Singulairs (Montelukast,
2011 sales $5.5 billion),63 Harvonis (Ledipasvir/Sofosbuvir, 2016 sales
$14.8 billion),64 Humiras (Adalimumab, 2017 sales $18.4 billion),65 and
Lantuss (Insulin glargine, 2017 sales $5.5 billion) (Fig. 1.7).66 The cost in
F O
OH
F
F S
HO
N O
F
Cl N
OH N O
H Singulair®
O Prozac® (Montelukast) HO
HO
Lipitor® (Fluoxetine)
(Atorvastatin)
O
NH F F
O O N
N
N N Humira® (Adalimumab)
N H N
H O FAB Fragment
Ledipasvir
HN
O O
O
NH
O
H
N O N O
P
O
O
O O Sofosbuvir
OH F Lantus®
Insulin glargine
Harvoni®
FIGURE 1.7 Lipitors (Atorvastatin), Prozacs (Fluoxetine), Singulairs (Montelukast),

Harvonis (Ledipasvir/Sofosbuvir), Humiras (Adalimumab), and Lantuss (Insulin glar-
gine) are some of the most successful drugs in the history of the drug industry. Each has
produced multibillion dollar franchises, providing their owners with ample resources for
the pursuit of additional novel therapeutic entities.

Target to hit Total cost for one marketed drug:

Success $2.87 Billion
Hit to lead Regulatory 1 Drug
rate:
80% Success Success
Programs: Lead Optimization Phase 3 rate:
rate:
24.3 75% 91%
Success Success
Programs: Preclinical Phase 2 Programs:
rate: rate: 10
19.4 1.1
85% Success Success 70% Clinical trials
Phase 1 Programs:
Programs: rate: rate:
14.6 69% Success 34% 1.6
Programs: rate: Programs:
12.4 54% 4.6 250
Programs: Preclinical
8.6 studies
Compounds Compounds Preclinical

screened screened studies Clinical studies
100,000 10,000–5000 250 250
>100,000
Screened
Discovery Development
0 2 4 6 8 10 12 14 16
FIGURE 1.8 An analysis of the various stages of the drug discovery and development
process provides an indication of the success rate of each stage of the process. Based on
these estimates, only 1 out of every 24 early stage programs (target-to-hit stage) will pro-
duce a marketed therapy. The cost to develop a single new drug must also account for the
costs associated with all of the programs that are unsuccessful. The total cost is estimated
to be $2.875 billion.
time and resource to achieve these results, however, is substantial. As

indicated in Fig. 1.8, it has been estimated that the identification of a
single marketed drug can require an initial examination of over 100,000
candidate compounds, hundreds of preclinical animal studies, and
numerous clinical trials involving thousands of patients. A recent analy-
sis of clinical trial success rates has indicated that only 1 out of every 10
clinical candidates will successfully traverse clinical trials and reach the
market. This represents a success rate of less than 0.001% if measured
by the number of compounds examined at the outset of the process. If
measured according to the number of programs required to advance a
single drug to market, program attrition rates indicate that only 1 in 24
programs are successful.
The cost associated with the identification of useful and
marketable therapeutic entities is also staggering. In 2011, it was esti-
mated that a single new drug costs over $1.75 billion to discover and
develop 67 and by 2016 the cost estimate had increased to over $2.87 bil-
lion.68 As a measure of comparison, the same amount of money could
be used to buy 33 Boeing 737-700 jet aircraft (based on 2018 prices on
Boeing’s website),69 purchase approximately 11,480 homes (assuming
$250,000 per home), 114,800 automobiles (average price $25,000), or pro-
vide for the raising 12,285 children born in 2018 to the age of 17.70
The costs and complexity of drug discovery and development is
staggering.

Drug discovery and development from 20,000 Feet (Fig. 1.9)

Fortunately, like most complex processes, drug discovery and develop-
ment can be broken down into many smaller tasks and functions. At the
highest level, the process can be divided into two major stages. The first,
referred to as drug discovery, includes all of the experimentation and stud-
ies designed to move a program from the initial identification of a biological
target and associated disease state to the identification of single compound
with the potential to be clinically relevant. The drug discovery stage may be
further broken down into three distinct phases: target discovery, lead dis-
covery, and lead optimization. Each phase of the drug discovery is designed
to establish a scientific link between a biological target (e.g., an enzyme,
G-coupled protein receptor, ion channel, etc.) and a disease state model
designed to mimic the human disease state. This process, often referred to
as target progression and target validation, is accomplished through the use
of molecular probes designed to identify multiple series of compounds that
will modulate the activity of the biological target of interest. In many cases,
known compounds are employed to facilitate target selection, and are even-
tually transitioned into novel compounds through the processes of lead
discovery and lead optimization. In lead discovery phase, sets of structurally
related compounds with the desired biological activity are identified (lead
discovery) through biological screening of large numbers of compounds.
Once a candidate series has been identified, the lead optimization phase
begins. In this phase, structural analogs within a lead series are studied
to identify a single compound that may be progressed into the drug devel-
opment stage. Typically, multiple lead series are identified in the lead
discovery and lead optimization phase through iterative rounds of experi-
mentation. In many cases, the lead discovery and lead optimization phases
overlap, as a typical drug discovery program will produce multiple sets of
related compounds with the potential for identification of candidates that
might progress into drug development. This approach is required for
Drug discovery Drug development
Target Lead Lead Proof of Full Registration/

Preclinical
discovery discovery optimization concept development launch
Target Candidate Product Market

selection selection differentiation access
Target progression/validation Indication progression
Molecular progression
FIGURE 1.9 The drug discovery and development process viewed from “20,000 Ft.”

success, as it is often difficult if not impossible to identify the lead series that
will contain the final lead candidate in the early phases of the drug discov-
ery process. Parallel operations of this type mitigate the risk of failure of any
one series of compounds. The lead discovery phase typically conclude with
the successful demonstration of in vivo efficacy in an appropriate animal
model employing a compound that possesses physical and chemical proper-
ties consistent with an eventual clinical study in the drug development
stage.
The second major stage, drug development, typically begins once a
single compound has been identified, which is then progressed through
various studies designed to support its approval for sale by the appro-
priate regulatory bodies. The first step in this process is the submission
of an investigational new drug application which requests permission
to move a clinical candidate into human study. This document provides
regulatory agencies with detailed preclinical data describing animal
pharmacology and toxicology studies, chemical manufacturing informa-
tion (including formulation, stability studies, and quality control mea-
sures), and of course, detailed clinical protocols that describe how the
clinical compounds will be studied in human populations if the studies
are approved.
While clinical trial design can vary substantially from one candidate to
another, the general goals of phase I, II, III, and IV are the same. Chapter 10
will provide a more detailed review of clinical trials, but the basic tenants of
clinical trials are as follows. In phase 1 clinical trials, safety and tolerability
of an investigational new drug is examined in a small number of healthy
individuals, typically 20100 people, with the goal of determining if safety
margins are suitable for further progression in the clinical trial process.
Pharmacokinetic (PK) and pharmacodynamic aspects of the candidate are
closely monitored, and the drug candidate is typically administered first in
a single ascending dose (SAD) study, followed by a multiple ascending dose
(MAD) study. In the SAD study, the drug is given to a group subjects once
and they are monitored to determine the impact of the drug. If there are no
adverse effects, then a second group is treated with a single higher dose of
the drug candidate and monitored as before. The cycle is repeated until
intolerable side effects appear in order to determine the maximum tolerated
dose (MTD). MAD studies are similar, but each group of subjects is pro-
vided with multiple low doses of a candidate compound over a set time. As
in the SAD studies, the manifestation of clinically intolerable side effects
defines the MTD for the MAD studies. The data developed through the
course of the phase I studies are used to determine the doses that will be
used in phases II and III clinical trials.
Phase II typically involves 100300 patients and is designed to deter-
mine whether or not the clinical candidate provides the desired biological
impact. Safety studies also continue through phase II trials. In the first part

of phase II trials, referred to as phase IIA, the goal is to determine the dose
required to provide the desired therapeutic impact or endpoint for the clin-
ical candidate. Once the proper dose levels are determined, phase IIB stud-
ies can be initiated. The goal of this portion of phase II studies is to
determine the overall efficacy of candidate compounds in a limited popu-
lation of subjects. The majority of clinical drug candidates fail in phase II
studies due to safety issues or lack of efficacy. As of 2011, only 34% of
phase II clinical candidates successfully reach phase III studies.
The effectiveness of new drug candidates in larger patient population is
determined in phase III clinical trial. These studies typically involve hun-
dreds to thousands of patients at multiple clinical trial sites, are typically
randomized, and are designed to determine the efficacy of the candidate
compound relative to the current standard of care. The cost and time associ-
ated with this phase of clinical study can vary dramatically depending on
the clinical endpoint under investigation. Clinical trials for new, acute treat-
ments, such as novel antibacterial agents, are shorter and involve far fewer
patients than clinical trials for chronic conditions such as osteoarthritis.
Patients are also closely monitored for adverse side effects, as the larger
patient pools can identify safety issues that did not become apparent in
smaller phase II trials. The number of subjects, time requirements, and com-
plex design of phase III clinical trials (especially in chronic medical condi-
tions) dictate that they are the most expensive aspect of drug discovery and
development. Phase III clinical trials typically involve 10003000 patients,
multiple clinical sites, institutional review boards, and are multiyear endea-
vors (typically 2.55 years). Upon completion of phase III trials a new drug
application is submitted to the appropriate regulatory body. This document
typically contains comprehensive details of both animal and human studies,
all safety findings (adverse effect and side effects), manufacturing proce-
dures (including methods of analysis to ensure drug quality), detailed for-
mulation information for all dosing methods studied, and storage
conditions. Regulatory reviews can lead to requests for additional informa-
tion regarding the submission, or even additional clinical trials to further
establish either safety or efficacy. Ideally, these reviews lead to regulatory
approval, including labeling requirements, and approval to market the new
drug.71
Approval of regulatory bodies does not, however, signal the end of clin-
ical trials. In many cases, regulatory agencies will require additional
follow-up studies, often referred to as phase IV trials or postmarketing sur-
veillance. In general, these studies are designed to detect rare adverse
effect across a much larger population of patients than could be supported
in phase III trials or long-term adverse effects that might be outside of the
scope of phase III trial durations. The impact of phase IV studies can
include alterations to labeling based on safety results, contraindication for
use of the new drug in combination with other medications, or even the

O
S N
O
O Cl
O
Vioxx® Meridia®
(Rofecoxib) (Sibutramine)
O OH OH
HO O
Baycol®
(Cerivastatin) N
FIGURE 1.10 Vioxxs (Rofecoxib) and Meridias (Sibutramine) were removed from the
market as a result of an increased risk of ischemic events, while and Baycols (cerivastatin)
was withdrawn from the market based on increased occurrence of fatal rhabdomyolysis
versus similar drugs in its class.
withdrawal of marketing approval if the findings are severe enough. The

COX-2 selective non-steriodal antiinflammatory agent Vioxxs (Rofecoxib),72
for example, was removed from the market after phase IV studies indi-
cated that it increased the risk of ischemic events in patients (Fig. 1.10).
In a similar fashion, Meridias (Sibutramine), a monoamine oxidase
inhibitor approved in 1997 for the treatment of obesity, was voluntarily
removed from the market by its manufacturer in 2010 after follow-up
studies demonstrated increased risks of cardiovascular events and lower
than anticipated efficacy (Fig. 1.10).73 Finally, Baycols (cerivastatin), an
3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibi-
tor marketed by Bayer AG for the treatment of high cholesterol and car-
diovascular disease, was voluntarily removed from the market after
reports of fatal rhabdomyolysis (Fig. 1.10).74
It is should be noted that safety studies are not the only reason for
phase IV clinical trials. Companies often use the data provided in post-
marketing surveillance and additional clinical studies to identify com-
petitive advantages, new markets, and new indication for their
products. There is some level of risk associated with conducting trials
designed to identify clinical superiority, as the results of competitive
trials are often difficult to predict. In some cases, a company’s plan to
demonstrate that their compound is superior to a competitor’s drugs
backfires, and they prove the opposite.

Target selection: the first step forward

The process of identifying a new drug candidate begins with identifying
a disease state or condition that can be addressed or modified through the
application of a suitable chemotherapeutic intervention. In theory, the
most pressing medical needs would have the highest priority in order to
ensure the improvement of the overall quality of life for patients. In prac-
tice, however, there are many factors that contribute to the decision of
which disease or condition to attempt to address through drug discovery
programs. First, the pathway to develop a therapeutic intervention may
not be clear for a particular disease or condition even though the medical
need is urgent. For example, while it is clear that Alzheimer’s disease is
pressing medical need,75 to date there are no disease-modifying therapies
available, despite the extraordinary amount of capital expended in an
effort to identify useful therapies. This is in part due to the lack of proven
targets for Alzheimer’s disease. Similarly, while there is a clear and press-
ing need for additional treatments for schizophrenia,76 the current level of
understanding of the disease state and lack of sufficient animal models77 is
a hindrance to progress in this important area.
There are also drug targets that have a theoretical connection to a particu-
lar disease states, but have as yet to be proven relevant to the human condi-
tion through the application of an appropriate chemotherapeutic agent.
Cholesteryl ester transfer protein (CETP), for example, plays a key role in
the interconversion of high-density lipoproteins (HDL) and LDL, and it has
been suggested that inhibitors of this enzyme would have a positive impact
on patients suffering from hypercholesterolemia.78 The potent CETP inhibi-
tors Torcetrapib (CP-529,414)79 and Dalcetrapib (JTT-705)80 (Fig. 1.11) were
O O F
N
O O
O
CF3 O N
NH
N O
O S F3C
F3C
O
F3C CF3 CF3
Torcetrapib® Dalcetrapib® Anacetrapib®

(CP-529,414) (JTT-705) (MK-0859)
FIGURE 1.11 Torcetrapib (CP-529,414), Dalcetrapib (JTT-705), and Anacetrapib (MK-

0859) are CETP inhibitors that have been clinically studied as potential treatments for
hypercholesterolemia. Torcetrapib increased HDL levels and decreased LDL levels but
increased mortality rates while Dalcetrapib was not efficacious in clinical trials.
Anacetrapib increased HDL levels and decreased LDL levels and did not negatively
impact mortality rates. CETP, Cholesteryl ester transfer protein; HDL, high-density lipo-
proteins; LDL, low-density lipoproteins.

identified as viable clinical leads, but neither was approved for marketing as
these clinical candidates failed to demonstrate statistically significant benefi-
cial effects in patients. It is possible that these results are an indication that
CETP inhibition is not a viable drug target for the treatment of cardiovascu-
lar disease. It is also possible, however, that these clinical candidates are
flawed in ways unrelated to CETP that prevented them from functioning in
the desired manner (e.g., off-target effects, pharmacokinetic issues).
In the case of Torcetrapib (CP-529,414), clinical trial data demon-
strated that the drug candidate increased HDL levels and decreased
LDL levels, indicating that clinical efficacy could be achieved.81
Unfortunately, the clinical candidate also caused increased blood pres-
sure and increased mortality rates, leading to the termination of clinical
development in 2006.82 Dalcetrapib (JTT-705) clinical studies were ter-
minated by Roche in 2012 due to lack of efficacy.83 On the other hand,
clinical trials with Anacetrapib (MK-0859) (Fig. 1.11),84 which also tar-
gets CETP, have successfully demonstrated that this compound can
increase HDL levels and decrease LDL levels without increases in blood
pressure or increased risk of cardiovascular disease-related deaths or
events.85 Longer term clinical trials demonstrated that the combination
of this drug with an HMGCoA reductase inhibitor produced a statisti-
cally significant reduction in major coronary events.86 These findings
demonstrate the validity of CETP as a therapeutic target and illustrate
the risk of overly generalizing the meaning of the clinical failure of indi-
vidual compounds. Despite these results, Merck elected not to pursue
regulatory approval for marketing Anacetrapib (MK-0859)87 for reasons
that remain undisclosed as of the writing of this text.
A similar scenario has surrounded γ-secretase inhibition. While it is
known that γ-secretase plays a key role in the formation and deposition
of amyloid plaques during the progression of Alzheimer’s,88 inhibitors
of this key enzyme have failed to provide the clinically beneficial results
expected. Semagacestats (LY450139) (Fig. 1.12), a compound developed
NH2
O CF3
OH O
H O2S N N O
N N
N
H N
O O
F
Cl
Semagacestat® Avagacestat®
(LY450139) (BMS-708163)
FIGURE 1.12 The γ-secretase inhibitor Semagacestat (LY450139) failed to improve cog-
nitive function in Alzheimer’s patients, despite the fact that it lowered amyloid plaque for-
mation. It was theorized that this failure was the result of its interference with notch
signaling, which plays a key role in cognitive function. Avagacestats (BMS-708163) is notch
sparring γ-secretase inhibitor that was developed to test this hypothesis in clinical trials.

by Eli Lilly inhibits γ-secretase, shows a dose-dependent lowering of

amyloid plaque formation in humans, but did not improve cognitive
function in patients. In fact, Semagacestats (LY450139) produced statis-
tically significant declines in cognitive function compared to the placebo
group in clinical trials.89 Once again, this raises the question as to
whether the target pathway is a dead end for treatment of the disease in
question or if the compound in question is flawed in some unforeseen
manner. In the case of Semagacestats (LY450139), it is possible that
unexpected off-target activity may be clouding the clinical results.
Semagacestats (LY450139) also interferes with Notch signaling, which
plays a key role in cognitive function,51 and it is not unreasonable to
suggest that Notch signaling modulation masked potential positive
effects that might have been observed if this off-target activity was
absent. This theory was put to the test by the next generation of
γ-secretase inhibitors that were specifically designed to avoid interac-
tions with the Notch signaling pathway. The clinical candidate
Avagacestats (BMS-708163) (Fig. 1.12), for example, was identified by
Bristol-Myers Squibb,90 but human trials were terminated when phase 2
studies demonstrated worsening cognition and greater adverse effects
in the treated patients.91 These findings contradicted the Notch signal-
ing hypothesis and lead to further questioning of the value of
γ-secretase as a therapeutic target for Alzheimer’s disease.
The question raised by the failures of clinical candidates such as
Torcetrapib (CP-529,414), Dalcetrapib (JTT-705), Semagacestats (LY450139),
and Avagacestats (BMS-708163) highlights the risks associated with choos-
ing a target that is not clinically proven, as well as the potential for negative
clinical results to be clouded by factors not related to the targeted mecha-
nism. There are, however, substantial financial incentives to attempt to
develop a “first in class” therapeutic agent. Prior to the introduction of the
statins (or HMG-CoA reductase inhibitors),92 there was no clear pathway
forward to inhibit cholesterol synthesis. The companies that took the leap of
faith that inhibition of HMGCoA reductase would provide therapeutic relief
for the prevention of cardiovascular disease received significant financial
rewards in the marketplace as indicated by the success of drugs like
Mevacors (Lovastatin)93 and Lipotors (Atorvastatin).94
There are, of course, many biological targets with proven clinical
utility that an organization might choose to focus on, such as
phosphodiesterase-5 (PDE-5),95 β-adrenergic receptors,96 and 5-
hydroxytryptamine receptors.97 In considering whether or not to pursue
known drug targets, one must keep in mind both the benefits and
potential pitfalls related to previously defined targets. On the positive
sides, a wealth of research and development information will be avail-
able in the literature, as companies and research institutions (universi-
ties, non-profit research institutions, etc.) patent and publish their

CO2H
OH OH
N N
OH OH
Seldane® Allegra®
(Terfenadine) (Fexofenadine)
FIGURE 1.13 Seldanes (Terfenadine), the first non-sedating antihistamine, dominated
the market until serious safety issues were identified. It was replaced by Allegras
(Fexofenadine), an active metabolite that is safer than the original.
research in order to garner support for their marketed products and

research programs. The availability of research tools such as biological
assays, reference compounds, and clinical trial data can provide an
excellent springboard for a drug discovery program. On the other hand,
the availability of this kind of information presents a significant hurdle
to the development of new therapeutic agents, as any new compound
or biological agent will be required to demonstrate clinical superiority
to the current standard of care. Also, scientific disclosures in the
literature will be available as prior art and could prevent an organiza-
tion from gaining patent protection for their research (this topic
will be covered in more detail in Chapter 13). If, however, one is
successful in developing a new therapeutic entity based on clinically
proven targets, substantial benefits can be available. Sepracor, now a
division of Sunovia, for example, took the risk of developing a new
antihistamine at a time when the market was dominated by Seldanes
(Terfenadine).98 They were able to demonstrate that Allegras
(Fexofenadine), a metabolite of Seldanes (Terfenadine), is significantly
safer than its predecessor and quickly took over the antihistamine mar-
ket (Fig. 1.13).99
Financial considerations also play a major factor in the determina-
tion of which diseases and potential drug targets are examined and
which are not. Clearly, the amount of money and time available to
pursue new therapeutic entities is limited, so not every target or dis-
ease state can be addressed. In the corporate world, disease state and
target selection are generally driven by the ability to generate
profitable products whose sale will support future research pro-
grams. On the surface, this would seem to dictate that only diseases
or conditions with large numbers of patients would be of interest to
corporate entities, but this is not the case. Chronic diseases such
as osteoporosis, hypertension, hypercholesterolemia, and arthritis
clearly have a large patient population that creates opportunities for

corporations. Rare diseases, however, also present significant oppor-

tunities and a pathway for growth. Amyotrophic lateral sclerosis
(ALS), for example, is a disease with a small, but consistent patient
population with significant unmet medical needs. At any given time,
there are only 20,00030,000 ALS patients in the U.S. whose life expec-
tancy is only 35 years, and there are no life extending therapies currently
available.100 This would appear to be a very small market that is unlikely
to provide the kind of profitability required to sustain a corporation.
However, it is important to realize that if a suitable treatment where avail-
able, this terminal condition would become a chronic condition wherein
patients would be treated for the disease throughout the course of an oth-
erwise normal life span. In addition, increased survival time for ALS
patients would lead to a larger patient pool, providing additional revenue
to a company that develops a life-extending treatment for ALS.
The selection of targets and disease states of interest sets the course
for all future aspects of a research program, so the importance of this
decision cannot be understated. Once the biological target is selected,
the process of identifying a clinical candidate can begin.
Hit identification: finding a starting point

Once a target of interest has been identified, the remainder of the
research program is essentially a quest to identify a single compound
that is suitable for use in a clinical setting. Of course, this relatively sim-
ple statement is actually a representation of an exceptionally complex
and multifaceted problem. Currently, there are over 144 million com-
pounds registered in the Chemical Abstract database,101 and the total
number of possible compounds to consider as drug candidates is nearly
infinite, so the question of where to start the process is significant.
Fortunately, there are some guidelines that have been developed in
order to provide some guidance as to where one might begin to look for
biologically useful molecules. Lipinski’s rule of 5,102 for example, sug-
gests that the majority of drug-like compounds exist within a limited
portion of chemical space. This concept will be discussed in greater
detail in Chapter 5, but for the purposes of this discussion, Lipinski’s
rules suggest that “drug-like” compounds will (1) have a molecular
weight lower than 500, (2) a logP below 5, (3) less than 5 hydrogen
bond donors, (4) less than 10 hydrogen bond acceptors, and (5) less
than 10 rotatable bonds. While there are exceptions to these rules (most
notably in the natural products arena), their application to chemical
space can be useful in that it provides a framework for
further movement toward a manageable number of compounds for
consideration.

HO
HO O
O OH
O HO
O
N
O F
H
Mevacor® Lipitor®
(Lovastatin) (Atorvastatin)
HO
OH OH O
O
HO OH N OH
N N N
O S
F F
O
Lescol® Crestor®
(Fluvastatin) (Rosuvastatin)
FIGURE 1.14 The HMGCoA reductase inhibitors Mevacors (lovastatin), Lipitors
(atorvastatin), Lescols (Fluvastatin), and Crestors (Rosuvastatin) have some structural
similarities, but there are a number of differences that make each unique.
However, these limitations still leave an enormous expanse of

chemical space that could be mined in an effort to identify com-
pounds that interact with the biological target of interest. This issue
is further complicated by the fact that drugs interacting at the same
target may have very little structural overlap. There are, for example,
clear similarities between the HMGCoA reductase inhibitors
Lipitors (atorvastatin),103 Lescols (Fluvastatin),104 and Crestors
(Rosuvastatin)105. They each contain a para-fluorobenzene ring and
1,3-diol-carboxylic acid side chain displayed in a similar orientation,
but the remainder of the three compounds are substantially different
from each other. Mevacors (lovastatin),106 which also inhibits
HMGCoA reductase (Fig. 1.14), is from an entirely separate structural
class, and it is not clear to the naked eye how this compound is

O O
H
N N
OO HN
N
N S
N N
H
O N
O
Cialis® Viagra®
(Tadalafil) O (Sildenafil)
O
FIGURE 1.15 The PDE-5 inhibitors Cialiss (tadalafil) and Viagras (sildenafil) are
structurally dissimilar even though they interact with the same macromolecular target.
N O
H
O O
N N
OH
O N
OH Demerol® Duragesic®
Morphine (Meperidine) (Fentanyl)
FIGURE 1.16 Morphine, Demerols (Meperidine), and Duragesics (Fentanyl) are all
μ-opioid receptor agonists.
related to the previously mentioned drugs. Similarly, Viagras (sil-

denafil)107 and Cialiss (tadalafil)108 are both PDE-5 inhibitors, but
structurally, they are quite different (Fig. 1.15). It is not immediately
clear how these two compounds are related or why they would serve
the same biological function. The same is true of Morphine,109
Demerols (Meperidine),110 and Duragesics (Fentanyl) (Fig. 1.16).111
While all of these compounds are μ-opioid receptor agonists, it would
not be obvious to a casual observer that they share a common
biological target. The selective serotonin reuptake inhibitors Zolofts
(Sertraline),112 Zelmids (Zimeldine),113 Celexas (Citalopram),114 Prozacs
(Fluoxetine),115 and Paxils (Paroxetine)116 represent distinct chemical
classes that are not clearly related to each other (Fig. 1.17). Given the
breadth of structural diversity that can be employed for any single molec-
ular target, it is clear that even the process of finding an initial chemical
lead for a drug discovery program can be challenging.
Fortunately, a number of tools and methods have been developed
to address the simple and yet very complex question of identifying a
molecular starting point for a drug discovery program. Essentially,

H
F N
N N
HN
Cl
H H H
O
H O
Cl F3C O NH
N Br N O
F O
Zoloft® Zelmid® Celexa® Prozac® Paxil®
(Sertraline) (Zimeldine) (Citalopram) (Fluoxetine) (Paroxetine)
FIGURE 1.17 Zolofts (Sertraline), Zelmids (Zimeldine), Celexas (Citalopram),

Prozacs (Fluoxetine), and Paxils (Paroxetine) are all selective serotonin reuptake inhibi-
tors (SSRIs) useful for the treatment of depression, but structural similarities are limited.
there are two general methods utilized in modern drug discovery

programs, physical HTS methods117 and virtual HTS methods.118
There is some degree of overlap between the two categories, and the
use of one set of tools does not preclude the use of the other. In point
of fact, they are often employed in tandem in order to improve the
likelihood of success. Physical HTS approaches (also referred to as
database mining) depend on the ability to screen large compound
libraries containing hundreds, thousands, or even millions of sam-
ples. Larger libraries are often designed to be chemically diverse in
order to cover as much of the “drug-like” chemical space as possible,
but focused libraries designed to target specific types of biological
targets (e.g., kinases, phosphatases) have also been employed.
Physical samples for screening are available from commercial sources
(e.g., Maybridge, Enamine, Aldrich, etc.) and pharmaceutical compa-
nies generally maintain an internal compound collection of proprie-
tary compounds.
Physical HTS techniques also require sophisticated, fully auto-
mated systems capable of manipulating reagents in 96, 384, or even
1496 well microtiter plates, as well as reagent distribution, data
acquisition, and waste disposal for thousands of samples per hour.
The cost of consumables is also significant. Consider, for example, an
HTS screening assay designed to identify compounds that inhibit
50% of an enzyme’s activity at 1.0 μM. If this assay were executed on
a library of 100,000 compounds in a 384 well plate format, it would
require nearly 800 assay plates to run the experiment in triplicate,
100,000 pipet tips to distribute the test compound, additional pipet
tips to add required reagents (enzyme, substrate, etc.), and assay sol-
vents. Automated data analysis is also required in order to handle
the volume of information generated in a typical HTS run. The afore-
mentioned assay of 100,000 compounds would produce 300,000 data
points if run in triplicate, while an HTS assay designed to generate
an 8-point IC50 curve for each compound would produce 2,400,000
data points for evaluation.

There are some key points that one must consider in evaluating
the data provided by an HTS screen. First and foremost is the possi-
bility of false-positive and false-negative results. In physical screen-
ing methods, the sheer number of manipulations involved leaves
open the possibility that an error may occur during some facet of
reagent handling (such as a clogged pipette tip). There is also the
possibility that the screening sample may have degraded over time,
creating “ghost samples” within the chemical library (in other words,
a sample whose structure no longer matches the material originally
entered into the library). In order to ensure that programs are
directed towards authentically active compounds, the chemical integ-
rity of “hit” samples is generally assessed using HPLC/MS methods.
In addition, biological screening is often repeated with the “hit” com-
pounds in order to validate the HTS results.
As an alternative to physical HTS methods, it is also possible to per-
form virtual HTS (also referred to as in silico screening). In this scenario,
advanced molecular modeling techniques are combined with virtual
chemical libraries (data files containing detailed structural information
on millions of compounds) and structural data on the biological target
in order to assess a compound’s ability to interact with the target of
interest. Virtual chemical libraries are often freely available from com-
mercial vendors (the largest of which is the Zinc database, which con-
tains over 35 million commercially available compounds; http://zinc.
docking.org/) and, as with physical samples, pharmaceutical companies
generally maintain virtual libraries of their proprietary compounds for
internal use. Structural information on biological targets may be avail-
able through X-ray crystallography, as a large number of protein crystal
structures are available through the Research Collaboratory for
Structural Bioinformatics (RCSB) Protein Data Bank (in 2018, the RCSB
database included over 145,000 structures https://www.rcsb.org/). If a
structure is not readily available, it may be possible to create a homol-
ogy model of the biological target using crystal structure data on closely
related macromolecules.119 In either case, the individual compounds of
the chemical libraries can then be “docked” in a hypothetical binding
site in the target of interest to determine a relative rank order for the
entire set of compounds. Automated data analysis tools are then
employed to organize the predictions provided by the “docking” of the
chemical libraries to the hypothetical binding sites of the biological tar-
gets. The predictions can then be used to select a smaller subset of a
large library for physicalbiological screening as potential starting
points.
Much like physical HTS, there are some important limitations that
must be considered in evaluating virtual screening data. First and
foremost, virtual screening results are predictions based on the model

system and not actual data on physical compounds. As such, the

quality of the results will depend on the quality of the model. In silico
models based on X-ray crystal structures tend to be stronger models
than homology models built on related biological structures, but it is
important to realize that there are limitations associated with X-ray
crystal-based models as well. Crystal structures can provide excep-
tionally detailed structural information, with resolution as low as
1.5 angstroms, but by definition, X-ray crystal structures are solid
state version of the desired target. It is possible that the structure
provided by X-ray crystallography matches the biologically active
form of the biological target of interest, but it may not. In “real life”
situations, biological targets are either dissolved in water or
membrane bound, and it is possible that they may have a different
configuration in these environments when compared to the closely
packed structure of a crystal form. Also, in many cases, sections of a
macromolecule must be altered or removed in order to generate a
crystallizable form of the biological target (with or without a ligand).
Given these limitations, virtual “hits” should also be physically
validated in biological screening efforts in order to confirm that the
predictions provided by molecular modeling are representative of the
real system.
Irrespective of the initial screening method employed (physical or
virtual), a successful screening effort will produce a subset of potential
“hit” compounds that will need to be examined in order to determine
whether or not follow-up efforts are warranted. This determination, also
referred to as “lead discovery” (Fig. 1.9), can be quite complex in itself
depending on the number and nature of the “hits” identified. If 500,000
compounds are screened and only 0.1% of the compounds in the library
provide interesting biological results, this still leaves some 500 com-
pounds to evaluate. Ideally, the initial “hits” will belong to a relatively
small number of structural classes, and then each structural class can be
independently analyzed to determine if further effort in the class is war-
ranted. Small groups of related compounds demonstrating the desired
biological activity can provide a significant advantage in further efforts,
as structureactivity relationship data may become apparent at an early
stage (the concept of structureactivity relationships will be covered in
more detail in Chapter 5). Also, the preparation of additional analogs
may be simplified, as synthetic methods may already be available. On
the other hand, the presence of set of related compounds within a
library suggests that they may have been prepared for a project with a
different biological target. Intellectual property issues may also exist, as
patent rights and ownership could become a serious question, especially
if the compounds were part of a set that has been previously patented,
previously published, or purchased from a commercial vendor.

New structural change

Yes
Keep structure change
Lead structure Structural New Biological Improved
“Hit” change analog screening potency?
No
Discard structure change
New structural change
FIGURE 1.18 The lead optimization cycle begins with the identification of a lead struc-
ture (hit) in a relevant biological assay. New analogs with structural modifications are pre-
pared and screened in the biological assay. If the assay results improve, then the changes
are kept and the cycle is repeated. If the changes are detrimental, then the changes are dis-
carded and the cycle is repeated. This process continues until a candidate compound with
the desired properties is identified.
Intellectual property consideration will be explored in more detail in

Chapter 13.
In some instances, “hit” compounds may be singletons. Isolated com-
pounds can be more difficult to follow-up on, as the original HTS data
set will not provide any additional guidance on how to proceed. It is,
however, still possible to generate more data on related compounds that
may be available from outside of the original compound library through
either commercial sources or additional synthetic efforts.
Once the initial “hit” compounds have been identified, confirmed, and a
compound class (or perhaps more than one compound class, depending on
the available resources) has been selected for further study, an iterative pro-
cess of compound acquisition/synthesis, biological screening, and data eval-
uation begins with the goal of improving the potency of the compounds
(Fig. 1.18). In each cycle of the “lead optimization” process, new data is pro-
duced as changes in the molecular structure are made to the “lead” com-
pounds, and this data is used to design the next generation of compounds.
This cycle of generating structureactivity relationship data continues until
a compound suitable for clinical evaluation has been identified. The nature
of this process and the associated medicinal chemistry will be discussed in
greater detail in Chapter 5.
Identify a clinical candidate: juggling the properties

Simply identifying a compound that is potent at the target of interest is a
difficult task to begin with, but shear potency is not enough to allow a com-
pound to be considered for clinical development and ultimately commercial
deployment. The process of discovering a suitable drug candidate is, in
many ways, a juggling act performed by drug discovery scientists
(Fig. 1.19). As programs progress from hit and lead identification to lead

Active Stable
Novel Selective Safe
PK Soluble
FIGURE 1.19 The identification of a clinical candidate requires the consideration of a

variety of properties beyond activity at the biological target of interest. Drug discovery
and development programs seek to optimize as many of these properties as possible in
order to identify the best opportunity for success.
optimization and an eventual clinical study, hundreds if not thousands of

compounds will be examined. It is the drug discovery scientist’s responsibil-
ity to identify a compound that will not only modulate the target of interest,
but also possesses the correct balance of properties required to create a use-
able drug. Potency at a biological target is only the beginning of a long series
of screening processes that must be performed in order to demonstrate that
a compound will survive the rigors of a discovery program. The specific
strategies employed are different for each program, but, in general, they can
be mapped in a screening cascade (Fig. 1.20) that sets gating guidelines for
each level of the screening process, from initial activity screening through
in vivo animal efficacy studies. The screening cascade is designed to decrease
the number of compounds examined at each level in order to ensure that
compounds with flaws are removed as early as possible. The cascade, also
referred to as a screening tree, begins with in vitro profiling and then transi-
tions into in vivo studies designed to determine a compound PK profile and
demonstrate efficacy in an appropriate animal model.
At the top of the cascade, compounds are screened for activity
against the biological target and a threshold of interest is generally set
to determine if compounds are active enough to warrant further investi-
gation. Potency is, of course, an important issue, as dosing requirements
are lower for compounds that are more potent. All other things being
equal, compounds with higher potency can be dosed at lower levels,
decreasing the likelihood of side effects. A compound with target
potency of 5 nM in theory could be provided to a patient at a signifi-
cantly lower dose than a compound serving the same function but with
a potency of 5 μM.
Once a compound has satisfied the potency criteria, selectivity and
physicochemical properties criteria are typically examined. Nature has
developed exquisite systems to accomplish very specific tasks with

Primary screening assay FIGURE 1.20 A screening tree is

designed to identify lead compounds
Biological activity by establishing a series of qualifica-
cutoff tions or “gates” that a compound must
surpass in order to advance through
the process.
Selectivity/off-Target Physicochemical
screening properties assessment
Biological activity Physicochemical

cutoff properties cutoff
Secondary screening
assay
Biological activity cutoff
Pharmacokinetic studies
Pharmacokinetic cutoff
In Vivo efficacy Studies
In vitro In vivo Gate
highly selective systems, but many of these systems overlap structur-

ally, and this can have a significant impact on the biological properties
of a given test compound. Thus the next biological screening step in a
typical screening cascade is often an assessment of a compound’s
potency at biological systems that are closely related to the target of
interest. The Kv1.5 channel, for example, is a voltage-gated potassium
channel that has been the target of research programs aimed at atrial
arrhythmia, and many compounds have been identified that can block
this channel with a high level of potency.120 There are, however, over 70
other potassium channels with varying degrees of similarity to the
Kv1.5 channel, and undesired activity at any of these related channels
could create unwanted side effects in human or animal studies. For
example, the Kv1.5 channel is closely related to the hERG channel.
Blockade of the hERG channel has been linked to torsade de pointes
and sudden cardiac death,121 so any program moving forward in this
area would need to counter screen compounds of interest for hERG
activity in order to ensure that advancing compounds do not present a
risk of sudden cardiac death in a clinical setting. This is a rather extreme
example of the importance of proper selectivity, but it should be clear
that failure to achieve proper target selectivity in this area represents a
significant barrier to moving a program forward.

Similarly, there are over 500 known kinase enzymes,122 and any drug
discovery program designed to target a single kinase, or even a family
of kinases, has an associated risk of identifying compounds that are
active at multiple members of this large family of related enzymes. In
order to mitigate this risk, kinase programs routinely screen test com-
pounds against panels of related kinases in order to understand the
risks associated with off-target activity.
In general, compounds that are potent not only at the target of inter-
est, but also at a variety of other targets (promiscuous compounds) do
not move forward in a drug discovery program, as the risk of undesired
(or unpredicted) side effects is too high. The level of selectivity required,
however, depends on the program, the nature of the potential side effect
presented by off-target activity (Off-target activity leading to excessive
hair growth might be tolerable, whereas sudden cardiac death through
poor hERG selectivity is not.), the target patient population, duration of
treatment (Some side effects only appear upon extended exposure to a
drug.) and a variety of other factors. Overall, target selectivity is a major
factor to consider.
An active and suitably selective compound, however, is not necessar-
ily a good drug candidate. Physicochemical properties also play a major
role in determining whether or not a compound is suitable for further
investigation. In vitro screening assays designed to predict absorption,
distribution, metabolism, and excretion (in vitro ADME) are generally
performed early in a program in order to ensure that candidates reach-
ing the drug development pipeline are “drug-like” in nature (Fig. 1.21).
Compounds that have poor aqueous solubility, for instance, are often
difficult to develop as drugs. In order for a drug to exert an influence
on a biological target, it must be soluble in biological fluid at a level
consistent with its potency. Thus the level of solubility required for a
given compound is directly linked to its potency. As potency increases,
the requisite solubility decreases, as less drug is required to provide the
intended effect. Solubility also has a direct impact on absorption, as a
compound must be soluble in biological fluids in order for a compound
to successfully pass through a biological membrane and reach its
intended target.
The ability of a compound to penetrate cellular membrane (its perme-
ability) can also be a determining factor in the success or failure of a
given candidate compound. If a compound is potent, selective, and sol-
uble but unable to pass through a biological membrane, it may not be
able to reach the target of interest and fail to demonstrate the desired
efficacy. Orally active drugs must be absorbed in the gastrointestinal
tract, and drugs that target intracellular system must also pass through
the cell membrane in order to reach their intended targets. Extracellular
targets, of course, do not face this added issue, but CNS drug

FIGURE 1.21 An in vitro

ADME profile can be used to
Metabolic
Permeability identify compounds that have
stability
“drug-like” properties. Assays
are available to determine meta-
BBB Plasma bolic stability, plasma stability,
penetration stability
aqueous solubility, Pgp efflux
In vitro susceptibility, solution stability,
ADME Cyp450 inhibition, bioassay solu-
Bioassay profile Aqueous bility, bloodbrain barrier pene-
solubility solubility tration, and permeability. ADME,
Absorption, distribution, metabo-
lism, and excretion.
Cyp450 Pgp
inhibition Solution efflux
stability
candidates face the added complexity of required permeability through

the bloodbrain barrier. In addition, there are efflux pumps (e.g.,
P-glycoproteins (Pgps))123 designed to remove xenobiotic material that
can limit permeability, preventing efficacy. The inability of a compound
to penetrate a cell membrane represents a significant issue that could
prevent further investigation of the candidate compound.
Metabolic and chemical stability are also important considerations. If
all of the previously mentioned criteria are met, and a compound is
able to enter the body, but is immediately metabolized, efficacy studies
will fail to show the desired results. However, the relative rate of
metabolism that can be allowed for a successful compound depends on
the goals of the project. If, for example, the goal is to develop a new
antibacterial agent, then high metabolic stability will likely be desired
so that the potential drug candidate will be available in the circulation
long enough to kill the invading organism. If, however, the goal is to
develop a new surgical anesthetic, metabolic stability may be less of an
issue, as it may be desirable for drug efficacy to fade rapidly upon the
termination of dosing regimens.
In a related sense, compounds that have chemical stability issue may
be problematic as drugs. Special packaging systems, some as simple as
amber bottles for light-sensitive compounds or cold storage, may be
required in order for the drug to be available commercially when a
patient is in need. While these kinds of issues are not insurmountable,
generally speaking, chemically stable compounds are preferred.
It is also important to consider how candidate compounds may
impact the normal metabolic processes, potentially altering the

metabolism of drug products used in tandem with the candidate com-

pound. Inhibition of key metabolic enzymes in the liver such as
Cyp3A4, Cyp2D6, and Cyp2C9, members of the cytochrome P450
(Cyp450) family of metabolic enzymes, are often studied using in vitro
screening methods (liver microsomes) in order to determine the risk of
drug/drug interactions.124 A compound that meets all other in vitro cri-
teria and demonstrates efficacy may still fail as a drug candidate if it is
determined that there is significant risk of drug/drug interactions. The
withdrawal of Seldane98 from market is a classic example of the risks
associated with unintended inhibition of the normal metabolic processes
and is discussed in greater detail in Chapter 14.
Positive results through the in vitro screening portion of a discovery
program represent a significant accomplishment but are still not neces-
sarily indicative of success. The PK properties of a candidate compound
must be determined in order to answer key questions about the in vivo
fate of the potential drug candidate. For example, if the candidate com-
pound is dosed orally, what percentage of the oral dose actually reaches
the systemic circulation? How rapidly is the candidate compound
excreted or metabolized? Does the compound reach systemic concentra-
tions high enough to suggest that in vivo efficacy should be expected in
an animal model? Is the compound freely distributed through the body,
or does it concentrate in a particular organ or tissue type? The answers
to these and a number of similar questions will have a significant
impact on the ability of any given compound to provide the desired
in vivo efficacy in a given animal model. Irrespective of the positive
results of in vitro screening, compounds with poor PK profiles are not
likely to be successful drugs.
Compounds found to possess suitable PK profiles must, of course,
demonstrate activity in key animal efficacy trials before they can be con-
sidered for clinical study. The type of efficacy studies required is based
on the desired biological endpoint (disease state), and a full discussion
of in vivo efficacy models is well beyond the scope of this text. Some
examples are given in Chapter 7, but it should be clear that the ultimate
goal of a discovery program is to identify compounds that meet all of
the aforementioned in vitro criteria, demonstrate efficacy in the appro-
priate animal model, and have PK properties consistent with the desired
dosing regimen.
Safety and side effect profiles are also major concerns, and there are a
number of in vitro and in vivo screens that can be used to assess the risks
associated with a compound (e.g., in vitro hERG screening,125 Ames
mutagenicity screening,126 and dog cardiovascular safety assessment).127
The nature and scope of safety studies is well beyond the scope of this
text, but should always be a major concern in the minds of drug discov-
ery scientist as any project moves forward. It is also important to realize

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It was an impossible room—I may say it at once—quite the typical
tawdry boudoir of an ex-coryphée. She was not there, I was relieved
to find, in person; but her multiplied presentment simpered and
abashed one from a dozen places on walls and mantelpiece.
“Claudine” (she might have been a hair-wash, and enjoyed the same
sort of popularity) posed, for all the blind purposes of vanity, in the
tights and kid boots of a past generation. Looking from queer old
daguerreotypes, in skirts like curtailed crinolines; ogling from
wreaths, her calves, crossed to display their strength, in disfiguring
proportion with the thin bosom above, she seemed to make an
outrage of the dear ungainly sanctities which appeal to us, in
pegtops and voluminous skirts, from the back parts of our albums.
There are certain people who, with the best intention in the world to
be held sweet, are unsavoury; and Aunt Mim was one of them. All
the more wonder that such fruit could be born of her stock.
For she was certainly attractive, was the girl—pure and pretty and
unaffected. I had to own it grudgingly to myself, as I bowed to her,
and turned interrogatively to my friend.
He had gone to the back of her chair, where she sat away from the
open window. There was some discarded work in her lap, and in her
eyes some look of a vague sadness and bewilderment.
“Nanny,” he said softly, “this, Mr. Verender, is my great friend—my
counsel out of Court. Will you just do this for me—make him yours,
too? Will you try to explain to him, while I go away, what you find it
so hard to explain to me—your sense of the something that keeps us
apart?”
I made an instant but faint demur. Nanny, as faintly, shook her
head.
“O,” he said, “but he will listen to you, I know! Because I am
unhappy, and you are unhappy, and I love you so, Nanny, and he is
my best friend. Try to explain to him, dear, the difficulty of your case.”
This novel enlargement on our relations, his and mine, vaguely
annoyed me.
“Why should there be any?” I put in impatiently. “Our friend can
give you great social and other advantages, Miss Nolan. If he is
decided on this course—you don’t dislike him, I think—forgive me, I
can see no reason for objection on your part.”
She rose, as if scared, to her feet. He put a hand on her shoulder.
“Hush!” he said. “Be just to me, and try to tell him.”
He left the room and the house; and I was in two minds about
following him. Was ever man put in a more ridiculous position? Yet
the look of the girl gave me pause. She seemed to me to be yet only
half awake; and indeed, I think, that is something to understate the
case.
“Well,” I said stumblingly, as she stood before me. “You heard
what he said, Miss Nolan?”
I was not sympathetic. I knew it. Perhaps, having once asserted
myself, I might have grown so. But she would not give me the
opportunity. In the meantime, I did not feel the less the full force of
this mismatch.
She put her hand in a lost way to her forehead.
“I will try,” she said, in a low voice, “because he asked me. There
—there was a great trouble—O! it was so far back. I can’t remember
it—and then everything went.”
“He is willing, it appears, to take that interval, that trouble, on
trust,” I said. “He only asks you, it seems, to repay his confidence.
What you are is what he desires. Cannot you consider yourself new-
born into his love?” (I positively sneered the word to myself.)
“There is something stands between us,” she only murmured
helplessly.
“He doesn’t admit it for himself,” I insisted irritably. “It might be the
ruin of his career, of his position, as foreseen by his friends. I
suppose he wishes to assure you that that counts for nothing with
him, if by any chance the bar between you lies in your dim
consciousness of such a sentiment.”
I had been brutal, I admit it. I can only palliate my behaviour by
confessing that it was intended to sound the first note of my moral
surrender to the appeal of those poor, pain-troubled eyes. Now, at
least, I had got my shaft home. She looked up at me with a light of
amazed knowledge in her face.
“Thank you,” she said. “I knew there was a right reason; and all
the time I have been hunting for a fancied one.”
I suffered an instant reaction to dismay. I had had no right
whatever to make this point. Whatever my private opinion of
Valentine’s folly, I had allowed myself to be accredited his
ambassador.
“Come; it is no reason at all,” I said. “There is no such thing as a
misalliance in love” (I threw this atrocious sop to my own panic). “If
only the practical bar between you could be as easily disposed of.”
“The practical bar?”
She turned upon me with a piteous pain in her voice. I had opened
a door of release to her, I suppose, and before she could escape
shut it again in her face. I was stumbling weakly on an explanation,
when suddenly from somewhere above the baby began to wail.
Instantly her face assumed the strangest expression—a sort of
exalted hardness. She put up her hand, listened a moment, then,
without another word, glided from the room. I am ashamed to say
that I seized the opportunity to put an instant period to my visit.
I expected to meet Valentine loitering without; but to my relief he
did not appear. So I went on my way fuming. What right had the man
to try to inveigle me into seeming to sanction his idiotcy by claiming
me its advocate? He wanted to buy justification, I suppose. There
are certain natures which cannot properly relish their own grief or
happiness unless a witness be by to report upon them. Such was
Valentine’s, I thought; and the thought did not increase my respect
for my friend. I fancied I had already plumbed the shallows of that
pretentious reserve, and was angry and half contemptuous that he
had so soon revealed himself to me. There was certainly something
attractive about the girl; but—well, he had not been the first to
discover the fact, and, when all was said, his infatuation showed him
a fool in my eyes.
That evening, when I was sitting alone writing, she suddenly stood
before me. My first shock of amazement was followed by a glow of
fury. I felt that I was being persecuted.
“Well, what is it?” I said harshly.
“I only wanted to tell you,” she said low, panting as if she had run;
“I wanted you to tell him that—that I know now what it is. I found out
the moment I left you; and I came to say—but you were gone.”
“Well?”
“It is the child, sir.”
“Yes, you are quite right—it is the child.”
No sooner had I said it, than I felt the weight of my self-
commitment. Had she discovered—remembered all? Did she
conceive the impediment as associated with some scandal attaching
to the ineffable Aunt Mim? or was the baby, in her clouded soul, but
an unattachable changeling, which had come to disrupt the kind
order of things and brand their household with a curse?
“Yes, it is the child,” I said, and leaned my forehead into my hand
while I frowned over the problem.
She made no answer. When I looked up at the end of a minute,
she was gone.
I started to my feet, and went up and down. I made no attempt to
follow. “It is better,” I thought angrily, “to let this stuff ferment in its
own way. I could have given no other answer.”
At the twentieth turn I saw Valentine before me, and stopped
abruptly.
“Well,” he said; “were you able to get it out of her?”
“What?” I asked defiantly.
“The reason—the impediment, you know?” he answered.
“Sit down, Valentine,” I said. “I will tell you the truth. I hinted that
the mésalliance might be her unconscious consideration.”
“She is not so proud,” he said quietly; “though I’m unworthy to
buckle her little shoe for her.”
I positively gasped.
“O! if that’s your view! But, anyhow, she was seeming to accept
mine, when the infant hailed her, and she left me, and I bolted. You
put too much upon me—really you do, Val; and here’s the sequel.
Ten minutes ago she appeared in this room and told me that she had
discovered the reason—the real one this time.”
“And it was?”
“The baby—no less.”
“What! Does she——?”
“I don’t know from Adam. I was thinking over my answer; and
when I looked up, she was gone.”
“And you gave her no reply?”
“O yes! I told her I entirely agreed with her. I had to be honest.”
“Verender! You must come with me!”
“Go with you!”
“You’ve called the tune; you must pay the piper.”
“I don’t know what you mean. I’ll see you—cremated first!”
He stared at me a moment, his teeth showing, his eyes rounding
in the dusk, his fists clinching and unclinching; then he, too, was
gone. And I went and stood at the window, slinking into the curtains,
and feeling myself the most abused cur in all London.
For a fortnight this state continued in me, through alternations of

depression, self-accusation, and savage bursts of rebellion. On the
sixteenth day a brief note, begging me to call at his rooms, reached
me from Valentine.
“I won’t go,” I swore through my teeth, feeling an inclination to tear
the paper in them; and five minutes later was on my way.
“He shall justify me to myself,” I had thought. “I’ll let my conscience
be his footstool no longer.”
The fellow lived en prince in Piccadilly. I found him in the midst of
a litter—boxes and packages and strewed floors—evidently on the
eve of a journey. He greeted me, twinkling, in high excitement—not a
trace of grievance or embarrassment in his manner.
“Leave those things, Phillips,” he said to his staid valet. “We’ll
finish by and by.”
The man left the room; and his master took me by the sleeve,
while I held myself in reserve—unconsciously, at the same time,
softening to his geniality.
“We’re off to Capri—Egypt,” he said, “following our late summer
with the swallows.”
“You and—Phillips?” I asked.
“I and my wife,” said he, with a laugh. “Hush! She’s seeing the
baby to sleep.”
He could say it without a blush; and they had been married, as I
came to learn, just a week! He led me on tiptoe to a distant room,
and bade me look through the opened door. Nanny, crowned, proud
as any young queen, with conscious maternity, was bent, singing
softly, above a little cot. The sight of her—Val’s wife—restored me at
once to my self-respect. I had done nothing after all, it seemed, but
help to precipitate an event I deplored. My shout had brought down
the avalanche. Henceforth my position was to be that of the amused
onlooker.
He let me stare; then led me away with all his old affectation of
pregnant mystery. We went out together—I don’t know why—into the
Green Park. It seemed remote and silent, and the better shadows of
night were beginning to troop under its trees. Then he spoke to me,
as follows:
“Verender, you have a right to know. You remember what you told
me that evening? I wasn’t just to you, perhaps. I foresaw issues to
which you must necessarily be blind. The baby stood between us,
you said. It did, but not in the way you meant to imply. I am its
father.”
I listened perfectly silent, and very grave, as we stepped on
together.
“I will say of, not for, myself,” he continued, “that I had known
nothing of the fruits of a little moonlight idyll out in that Kent village. I
was hop-picking, as she was, but for a worser reason. Our encounter
in B—— Hospital was my first intimation of the truth. Till that moment
I had never considered, at least had been careless of, a sequel; had
never, of course, had a shadow of thought to identify the patient with
my victim. Then in a moment—Verender, her helplessness found all
that wasn’t bad in me. She didn’t know me—the curtain was too
thick. I determined to woo and win, as a stranger, what was already
my own. Was I right?”
I nodded. “Yes, you were right.”
“Then came the strange part,” he said—“a sort of
subconsciousness of an impediment she could not define. It was her
dishonour, Verender—my God! Verender, her dishonour!—that found
some subtle expression in the little life introduced into her home. She
always feared and distrusted the child; and I tell you I lived in horror
that some day her witlessness would arm her gentle hand to do it a
hurt. For she wanted to come to me, Verender, she wanted to come,
and it was as if she couldn’t, and nobody would tell her why.
“You told her, you old rascal! And with what result, do you think?
When I followed her, I found her gone—she had taken the baby from
its cot, and hurried out. The old harpy was there, raving and gobbling
beyond reason. I had her down on her knees to confess. She
admitted that the girl had come in, in a fever to proclaim her
knowledge of the bar which separated us. Nanny had rounded upon
her, it appeared, and accused her, Aunt Mim, of wantonly causing
the scandal which had brought this shadow into her life. And then—
perhaps it wasn’t to be wondered at—Auntie exploded, and gave up
all.”
“The truth?”
“All of it but what the old hag herself didn’t know—the name of the
villain. That, circumstances had kept from her, you see. She let
loose, did Auntie—we’ll allow her a grain of justification; to have her
forbearance turned upon herself like that, you know!—and screamed
to the girl to pack, and dispose of her rubbish somewhere else. And
Nanny understood at last, and went.”
“Where?”
“Ah, where? That was the question. I’d only one clue—Skene and
the river; but I seized upon it, and my inspiration proved right. She’d
gone instinctively to the only place where, it seemed, her trouble
could be resolved. You see, she hadn’t yet come to identify me with
it. But I followed, and I caught her in time.”
He hung his head, and spoke very low.
“I took the next train possible to Skene. Verender, I’m not going to
talk. It was one of those fainting, indescribable experiences, like the
voice in the burning bush. Cold dawn it was, with a white bubbling
river and the ghost of an old moon. She had intended to commit it to
the water—the fog wasn’t yet out of her brain—and then, all in an
instant, the mother came upon her, and the memory of me; and she
ran to cast herself in instead, and saw me coming.”
There followed a long interval of silence.
“And Aunt Mim?” I asked drily, chiefly to keep up my character.
He laughed.
“O! we’ve added an honourable moiety to a dishonourable
pension, and settled her,” he said.
Another silence followed.
“Well, I apologize,” I said grumpily.
THE SOUL OF THE PROFESSOR
John Stannary hungrily paced his laboratory, awaiting an expected
advent. A brilliant coronal of candles, concentrated within a shade
and pendent from a black beam over the dissecting table, regularly
identified him as he came within its radiance, and as regularly, when
he had passed without it, returned to its scrutiny of the empty slab
beneath, as if it were trying to trace on that blank surface the
unwritten hieroglyphics of his development. Yet, if each of its half-
score fiery tongues had been as polyglot as the Apostles’ tongues of
flame, it could have found among them all no voice to dispute his
lifelong consistency with himself. From the hard, ungracious child,
who had rejoiced to discomfit the love which sought to hedge him;
from the cynic schoolboy, to whom to awaken and analyze pain in
the living had been the only absorbing sport; from the
unimpassioned student, who had walked the hospitals like a very
spectre of moral insensibility; from the calculating libertine, whose
experimental phase of animalism had been as brief as it was
savage; from the lust of life, soon spent, to the bloodless analysis of
its organic motives; from the soulless child to the virile monster of
science; from nothingness to a great early reputation, to honours, to
a fine house, to his present self and condition, in short, Professor
Stannary’s progress had been entirely and unerringly consistent. He
was one of those born to account for results, not by any means with
a view to clearing the stream of tendency by cleansing its source. On
the contrary, he never would have hesitated further to contaminate it,
could he by so doing have evolved some novel epidemic. His fight
was not to win Nature to God, but to the laboratory; and, if he had a
conqueror’s ambition, it was to die gloriously upon a protoplast, at
that beginning of things which his fathers had struggled through
æons to forget. To have called him a dog nosing back for a scent,
would have been to libel the sorriest of mongrels with an inch of tail
to wag at a kind word. Yet he had routled so much, nevertheless,
that his eyes were inflamed, and his features pimpled, and his nose
itself sharpened as with much whetting on carrion. And, still
unappeased, he paced his shambles that evening like a caged
ravening jackal.
In those early decades of the nineteenth century,
anthropophagous science, especially when non-official, was often
hard put to it for a meal. The “ringing grooves of change” were
sounding; discovery was a new-risen star; ghoulish explorers
shouldered one another in their struggle for the scientific pabulum
which the grave afforded. But the supply, die as men would, was
unequal to the demand. The hospitals kept their own; the others, à
contre-cœur, must keep the resurrection-men. They pulled the blinds
down on their consciences; they were willing to accept the least
plausible of explanations, for the Cause was paramount. But, indeed,
we are all casuists when we want to justify our lusts to ourselves.
Still the material lacked, only, according to the universal law of
necessity, to evolve its more desperate instruments of supply. And
then at last, hard-driven, first one, and after him another and another,
had the panic courage to pull up those blinds, and let in the light on
some very shocking suspicions—with the result that Burke was
hanged in Edinburgh, and Bishop and Williams in London.
Professor Stannary was not of these white-livers. He pulled up no
blind, for the simple reason that he had let none down. He would
have diagnosed conscience as a morbid disease characterized by a
diathetic condition, and peculiar to fools of both sexes and all ages.
He would have said that to ask any question in this world was to
invite a lie, and that in all his thirty years’ experience he had found
none but the dead to answer back the unswerving truth. Well, if they
did? Did it matter to them, being dead? But it mattered greatly to the
cause of science to get at the truth; and he, for one, was certainly
not going to question the means so long as the results came to
justify them.
While yet, however, the blinds were down and the pitch-plaster but
an ugly suspicion, the competition for material had not so ceased of
its keenness but that Professor Stannary had found himself checked,
one day, under the very near-conquered crest of a physiological
peak, for want of the final clue to that crowning achievement—a clue
which, like Bluebeard’s key, turned upon nothing so intimately as
dead bodies. Step by step he had reasoned his way to this position,
only, when within touch of the end, to find himself held up, tantalized,
irritated by an inability to proceed farther until a nice necessity
should be provided. His material, in fact and in short, had given out
at the psychologic moment. His laboratory was already a museum of
shredded particles—the pickings from much inevitable waste of dead
humanity. There needed only the retraversing of certain nerves, or
ducts, or canals to put the finishing touches to a great discovery. And
then—the summit, the tribune, as it were, from which he was
engaged to announce on the morrow to the Royal Society the
triumphant term to his investigations.
Already, pacing to and fro, eager and impatient, in the silent room,
he foresaw himself the recipient of the highest honour it was in the
power of that body to bestow. He was not above desiring it. He was
not himself so superlatively rational but he could covet applause for
knocking yet another long nail into the coffin of irrationality; could
expect the recognition of the world for his services in helping to
reduce it, its passions and its hopes and its pathetic fallacies, to
some mathematical formulæ. There is nothing so incomprehensible
to the men of science as the reluctance of the unscientific to part
with their doting illusions. To be content to rejoice or sorrow in things
as they seem, and not to wish to know them as they are—that, they
think, is so foolish as to justify their hardest castigation of the folly. At
least so thought John Stannary, as, with his lips set sourly, he
paused, and consulted his watch, and listened for a sound of
expected footsteps shuffling down the hang-dog passage which
skirted that wall of the house, and into which his laboratory door
conveniently opened.
Not a whisper, however, rewarded him. He put his ear to the
panels. Even then, the surf-like murmur of distant traffic—or the thud
of his own excited heart, he could not tell which—was the only
articulate sound. He glanced up angrily at the shortening candles
before resuming his tramp. As he passed to and fro, from dusk to
light, and into dusk again, he seemed to be demonstrating to a
theatre of spectral monstrosities the hieroglyphics of that same
empty slab. For the central core of radiance, concentrating itself with
deadly expectancy upon its surface, had, nevertheless, its own
ghostly halo—a dim auditorium, tier over tier, peopled with shadows
of misbegotten horrors. Sets of surgical steel in the pit, arrayed
symmetrically on a table as if for a dinner party of vampires;
nameless writhed specimens on cards or in bottles, standing higher
behind the dry sleek of glass; over all, murderous busts in the
gallery, the dust on their heads and upper features giving them the
appearance of standing above some infernal sort of footlights—with
such shapes, watchful and gloating in suggestion, was the man
hemmed in. They touched his nerves with just such an emotion as
the ordinary citizen feels towards his domestic lares; they affected
him in just such proportion as he was moved by the thought of the
possible manner in which an order he had given to some friends of
his that morning might be executed. That is to say, his feeling
towards these dead members, as towards the means taken by
others to procure him the use of them, was utterly impersonal. He
had had at this pass a great truth to demonstrate. “A body, this night,
at any cost,” he had simply ordered, and had straightway shut the
door on his caterers. He had had no thought of scruple. His
responsibility in these matters was to the ages; never to the
individual.
A low tap sounded at the door. He was there in three strides, and
opening swiftly, let in two men, the one shouldering a sack, the other
hovering about his comrade in a sort of anxious moral support.
Professor Stannary, without a word, pointed to the table. The
laden one, as mutely, shuffled across, backed, heaved his burden
down, and stood to take off his hat and mop his brow. He was a
burly, humorous-looking fellow, with a sort of cheerful popularity
written across his face—an expression in strong contrast with that of
the other, who, tall and stealthy, stood lank behind him, like his
shadow at a distance, watching the persuasive effect of his principal
on the customer. It was he who had closed the door gently upon
them all as soon as they were in, and now stood, his teeth and
eyeballs the prominent things in him, softly twirling his hat in his
hand.
“Take away the sack,” said the Professor quietly.
The burly man obeyed, sliding it off like a petticoat; and revealed
the body of a young woman. Lowering and rubbing his jaw, the
Professor stood some moments pondering the vision. Then he
turned sharply.
“You are late. I expected you sooner.”
“The notice was short, sir,” answered the man coolly. “These ’ere
matters can’t be accommodated in a moment. As it is she’s warm. I
bought the body off of——”
The other interrupted him—
“I don’t want to know. You can hold your tongue, and take your
price, and go.”
“Short and sweet,” said the man.
He laughed, and his friend laughed in echo, putting his long hand
to his mouth as if in apology for such an unpardonable ebullition of
nature.
“As to the price,” said the former, “taking into consideration the
urgency, and the special providence, so to speak, in purwiding, at a
moment’s notice too, this ’ere comely young——”
A certain full chink of money stopped him.
“Thankee,” he said, after a short negotiation. “I’ll own you’ve done
the handsome, sir, and we’ve no cause to complain. Not but what I
had to give——”
“Good night!” said the Professor.
Not till they were gone, locked out, and the very trail of their filthy
footsteps hidden under the black droppings of the night, did he turn,
for all his impatience, and regard the body again.
“Nancy,” he murmured to himself; “yes, it’s Nancy.”
Into the vast study of biology it is perfectly certain that a personal
knowledge of biogenesis must enter. Here, too, the individual must
be sacrificed to the cause. Well, by virtue of that phase in his career
before-mentioned, he had already once made of Nancy a holocaust
to science. If the fruits of that sacrifice had been to be found in a
ruined life, a social degradation, a gradual decline upon infamy, what
was it all to him? As german to the general subject as the dead
specimens on his walls was the living specimen of his passion. Ex
abusu non arguitur ad usum. Still, it was a strange coincidence that
she should come thus to consummate his work.
Looking upon her frozen face, he was aware of certain tell-tale,
rudely-erased tokens about the mouth. He never had a doubt as to
what they signified. It was a rude kiss that of the pitch-plaster, more
close and savage than any he himself had once pressed upon those
blistered lips. So, the murderous beasts had gone the short way to
supply his urgency! Would they have taken it none the less, he
wondered, if they could have known in what relation their victim once
stood towards their employer? Very likely. Very justly, too, could they
believe him consistent with himself.
Nevertheless, though he had no conscience, though he had too
often scored that fact on human flesh with a knife to be in any doubt
about it, it was notable that, as he moved now, perfectly cold and
collected, to make some selection of tools from the table hard by, he
was registering to himself a vow, mortal to some folks, that no effort
of his should be lacking to help bring certain vile instruments to their
judgment—so soon as his disuse of them should find warrant in a
fuller supply of the legitimate material.
As he groped for what he wanted, a sparrow twittered somewhere
in the dark outside. He started, and dwelt a moment listening. Birds!
the little false priests of haunted woods, who sang their lying
benedictions over every folly perpetrated in their green shades! Why,
he had loathed them, even while he had been making their loves the
text for this early experiment of his in rustic nature. They had been
singing when——grasping his knife firmly, he returned to the table.
Something had happened there. For one moment the blade shook
in his hand. To his practised eye there were signs—the ghostliest,
the most remote—but signs still. A movement—a tremor—the
faintest, faintest vibration of a soul, unreleased, struggling to return
to the surface—that was what he felt rather than saw. He recalled
the hasty character of the deed; he thought of the shock, of the
suspended trance into which such a deed might cast a sensitive
subject. Mastering himself, with the dry firm will of an operator, he
walked once more unhurriedly to the instrument table, and made a
further selection.
The sparrow twittered again. Birds in the wood—small
procuresses to Sentiment! What a trollop she was, that Sentiment!
He had known ethereal creatures go from picking the bones of
stuffed larks, to moralize sweetly on the song of nightingales under
the moon. For himself, barring his natural asceticism, he would have
no remorse whatever in devouring nightingales. Such emotions were
born of surfeit, and the moral of them all was that a bird in the
stomach was worth two, or two hundred, in the bush. The one was
the decoy which brought the many into notice. Why, he himself,
when flushed with passion——
Harder than steel, hard as flesh can be, he stepped back to the
table.
Yes, there was no longer doubt about it. He must decide quickly.
Decide! What was there in all his life to warrant in him a moment’s
indecision? His pledge to to-morrow was paramount over his pledge
to yesterday. It was by very virtue of the past that he owed
everything to the future. The Cause was himself, bone of his bone,
flesh of his flesh. As she had made herself one with him, so must
she consummate the gift. He chose to believe, even, that she would
not hesitate could she know. He grasped his knife.
Her hair! He had said some foolish things about it once. In a
sudden fury he seized it, and sliced it off close by her head, and
flung it aside. She looked strangely innocent and boyish thus shorn.
He had a momentary grotesque thought that he would excuse
himself to himself by pretending that she was a boy. It passed on the
instant. What excuse was necessary? He remembered how once, to
his idle amusement, when she had fancied herself secure of him,
she had coveted greatness for his future. Well, it was within his
grasp at length, and by her final means.
Damn the sparrow! What was there in all the murky town to tempt
his twittering? He had blunted his knife’s edge on the hair. He must
fetch another.
As he came back with it, the bird seemed to flutter and cry out
against his very door. In a swift access of passion he strode to it, and
opened. Whether old, or wounded, or poisoned in the drooping fog,
there lay the little thing, gasping, with outspread wings, upon the
pavement. One moment the Professor hesitated; then, crushing out
the tiny shrieking life under his foot, he relocked the door and
returned with a firm step to the table.
*****
His treatise, read the next day before an august body, was said
masterly to resolve an intricate and long obscure physiologic
problem.
It brought him additional and great honour, and, what he prized
above all, that gift of the Society’s gold medal, which is only granted
to discoveries of the first importance. But then, it must be
remembered, he had given his soul to the Cause. The stain of its
sacrifice was yet red on the stones outside his door.
A GHOST-CHILD
In making this confession public, I am aware that I am giving a
butterfly to be broken on a wheel. There is so much of delicacy in its
subject, that the mere resolve to handle it at all might seem to imply
a lack of the sensitiveness necessary to its understanding; and it is
certain that the more reverent the touch, the more irresistible will
figure its opportunity to the common scepticism which is bondslave
to its five senses. Moreover one cannot, in the reason of things, write
to publish for Aristarchus alone; but the gauntlet of Grub Street must
be run in any bid for truth and sincerity.
On the other hand, to withhold from evidence, in these days of
what one may call a zetetic psychology, anything which may appear
elucidatory, however exquisitely and rarely, of our spiritual
relationships, must be pronounced, I think, a sin against the Holy
Ghost.
All in all, therefore, I decide to give, with every passage to
personal identification safeguarded, the story of a possession, or
visitation, which is signified in the title to my narrative.
Tryphena was the sole orphaned representative of an obscure but

gentle family which had lived for generations in the east of England.
The spirit of the fens, of the long grey marshes, whose shores are
the neutral ground of two elements, slumbered in her eyes. Looking
into them, one seemed to see little beds of tiny green mosses
luminous under water, or stirred by the movement of microscopic life
in their midst. Secrets, one felt, were shadowed in their depths, too
frail and sweet for understanding. The pretty love-fancy of babies
seen in the eyes of maidens, was in hers to be interpreted into the
very cosmic dust of sea-urchins, sparkling like chrysoberyls. Her soul
looked out through them, as if they were the windows of a water-
nursery.
She was always a child among children, in heart and knowledge
most innocent, until Jason came and stood in her field of vision.
Then, spirit of the neutral ground as she was, inclining to earth or
water with the sway of the tides, she came wondering and dripping,
as it were, to land, and took up her abode for final choice among the
daughters of the earth. She knew her woman’s estate, in fact, and
the irresistible attraction of all completed perfections to the light that
burns to destroy them.
Tryphena was not only an orphan, but an heiress. Her
considerable estate was administered by her guardian, Jason’s
father, a widower, who was possessed of this single adored child.
The fruits of parental infatuation had come early to ripen on the
seedling. The boy was self-willed and perverse, the more so as he
was naturally of a hot-hearted disposition. Violence and remorse
would sway him in alternate moods, and be made, each in its turn, a
self-indulgence. He took a delight in crossing his father’s wishes, and
no less in atoning for his gracelessness with moving demonstrations
of affection.
Foremost of the old man’s most cherished projects was, very
naturally, a union between the two young people. He planned,
manœuvred, spoke for it with all his heart of love and eloquence.
And, indeed, it seemed at last as if his hopes were to be crowned.
Jason, returning from a lengthy voyage (for his enterprising spirit had
early decided for the sea, and he was a naval officer), saw, and was
struck amazed before, the transformed vision of his old child-
playfellow. She was an opened flower whom he had left a green bud
—a thing so rare and flawless that it seemed a sacrilege for earthly
passions to converse of her. Familiarity, however, and some sense of
reciprocal attraction, quickly dethroned that eucharist. Tryphena
could blush, could thrill, could solicit, in the sweet ways of innocent
womanhood. She loved him dearly, wholly, it was plain—had found
the realization of all her old formless dreams in this wondrous birth of
a desire for one, in whose new-impassioned eyes she had known
herself reflected hitherto only for the most patronized of small
gossips. And, for her part, fearless as nature, she made no secret of
her love. She was absorbed in, a captive to, Jason from that moment
and for ever.
He responded. What man, however perverse, could have resisted,
on first appeal, the attraction of such beauty, the flower of a radiant
soul? The two were betrothed; the old man’s cup of happiness was
brimmed.
Then came clouds and a cold wind, chilling the garden of
Hesperis. Jason was always one of those who, possessing classic
noses, will cut them off, on easy provocation, to spite their faces. He
was so proudly independent, to himself, that he resented the least
assumption of proprietorship in him on the part of other people—
even of those who had the best claim to his love and submission.
This pride was an obsession. It stultified the real good in him, which
was considerable. Apart from it, he was a good, warm-tempered
fellow, hasty but affectionate. Under its dominion, he would have
broken his own heart on an imaginary grievance.
He found one, it is to be supposed, in the privileges assumed by
love; in its exacting claims upon him; perhaps in its little unreasoning
jealousies. He distorted these into an implied conceit of authority
over him on the part of an heiress who was condescending to his
meaner fortunes. The suggestion was quite base and without
warrant; but pride has no balance. No doubt, moreover, the rather
childish self-depreciations of the old man, his father, in his attitude
towards a match he had so fondly desired, helped to aggravate this
feeling. The upshot was that, when within a few months of the date
which was to make his union with Tryphena eternal, Jason broke
away from a restraint which his pride pictured to him as intolerable,
and went on a yachting expedition with a friend.
Then, at once, and with characteristic violence, came the reaction.
He wrote, impetuously, frenziedly, from a distant port, claiming
himself Tryphena’s, and Tryphena his, for ever and ever and ever.
They were man and wife before God. He had behaved like an
insensate brute, and he was at that moment starting to speed to her
side, to beg her forgiveness and the return of her love.
He had no need to play the suitor afresh. She had never doubted
or questioned their mutual bondage, and would have died a maid for
his sake. Something of sweet exultation only seemed to quicken and
leap in her body, that her faith in her dear love was vindicated.
But the joy came near to upset the reason of the old man, already
tottering to its dotage; and what followed destroyed it utterly.
The yacht, flying home, was lost at sea, and Jason was drowned.
I once saw Tryphena about this time. She lived with her near
mindless charge, lonely, in an old grey house upon the borders of a
salt mere, and had little but the unearthly cries of seabirds to answer
to the questions of her widowed heart. She worked, sweet in charity,
among the marsh folk, a beautiful unearthly presence; and was
especially to be found where infants and the troubles of child-bearing
women called for her help and sympathy. She was a wife herself,
she would say quaintly; and some day perhaps, by grace of the good
spirits of the sea, would be a mother. None thought to cross her
statement, put with so sweet a sanity; and, indeed, I have often
noticed that the neighbourhood of great waters breeds in souls a
mysticism which is remote from the very understanding of land-
dwellers.
How I saw her was thus:—
I was fishing, on a day of chill calm, in a dinghy off the flat coast.
The stillness of the morning had tempted me some distance from the
village where I was staying. Presently a sense of bad sport and
healthy famine “plumped” in me, so to speak, for luncheon, and I
looked about for a spot picturesque enough to add a zest to
sandwiches, whisky, and tobacco. Close by, a little creek or estuary
ran up into a mere, between which and the sea lay a cluster of low
sand-hills; and thither I pulled. The spot, when I reached it, was
calm, chill desolation manifest—lifeless water and lifeless sand, with
no traffic between them but the dead interchange of salt. Low
sedges, at first, and behind them low woods were mirrored in the
water at a distance, with an interval between me and them of
sheeted glass; and right across this shining pool ran a dim, half-
drowned causeway—the sea-path, it appeared, to and from a lonely
house which I could just distinguish squatting among trees. It was
Tryphena’s home.
Now, paddling dispiritedly, I turned a cold dune, and saw a
mermaid before me. At least, that was my instant impression. The
creature sat coiled on the strand, combing her hair—that was
certain, for I saw the gold-green tresses of it whisked by her action
into rainbow threads. It appeared as certain that her upper half was
flesh and her lower fish; and it was only on my nearer approach that
this latter resolved itself into a pale green skirt, roped, owing to her
posture, about her limbs, and the hem fanned out at her feet into a
tail fin. Thus also her bosom, which had appeared naked, became a
bodice, as near to her flesh in colour and texture as a smock is to a
lady’s-smock, which some call a cuckoo-flower.
It was plain enough now; yet the illusion for the moment had quite
startled me.
As I came near, she paused in her strange business to canvass
me. It was Tryphena herself, as after-inquiry informed me. I have
never seen so lovely a creature. Her eyes, as they regarded me
passing, were something to haunt a dream: so great in tragedy—not
fathomless, but all in motion near their surfaces, it seemed, with
green and rooted sorrows. They were the eyes, I thought, of an
Undine late-humanized, late awakened to the rapturous and troubled
knowledge of the woman’s burden. Her forehead was most fair, and
the glistening thatch divided on it like a golden cloud revealing the
face of a wondering angel.
I passed, and a sand-heap stole my vision foot by foot. The vision
was gone when I returned. I have reason to believe it was
vouchsafed me within a few months of the coming of the ghost-child.
On the morning succeeding the night of the day on which Jason
and Tryphena were to have been married, the girl came down from
her bedroom with an extraordinary expression of still rapture on her
face. After breakfast she took the old man into her confidence. She
was childish still; her manner quite youthfully thrilling; but now there
was a new-born wonder in it that hovered on the pink of shame.
“Father! I have been under the deep waters and found him. He
came to me last night in my dreams—so sobbing, so impassioned—
to assure me that he had never really ceased to love me, though he
had near broken his own heart pretending it. Poor boy! poor ghost!
What could I do but take him to my arms? And all night he lay there,
blest and forgiven, till in the morning he melted away with a sigh that
woke me; and it seemed to me that I came up dripping from the sea.”
“My boy! He has come back!” chuckled the old man. “What have
you done with him, Tryphena?”
“I will hold him tighter the next time,” she said.
But the spirit of Jason visited her dreams no more.

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Basic Principles of Drug Discovery and

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British Library Cataloguing-in-Publication Data

For Information on all Academic Press publications

Publisher: Susan Dennis

1. Drug discovery and development: An overview of modern

2. The drug discovery process: From ancient times to the

Durham-Humphrey Amendment of 1951 94

3. Classical targets in drug discovery 111

4. In vitro screening systems 185

Streptavidin and biotin 195

5. Medicinal chemistry 257

6. In vitro ADME and in vivo pharmacokinetics 305

7. Animal models of disease states 371

Animal models of pain 403

8. Safety and toxicology 421

9. Antibody-drug discovery 459

10. Basics of clinical trials 483

Phase 2 clinical trials 506

11. Translational medicine and biomarkers 523

12. Organizational consideration and trends in the

13. Intellectual property and patents in drug discovery 595

Assignment and ownership 609

14. Case studies in drug discovery 625

Answers to questions in textbook by chapter 665

Subject Index 693

initial medicinal chemistry conceptualization and in vitro biological

Although there are numerous textbooks that discuss various aspects

Magid Abou-Gharbia, PhD, FRSC

Basic Principles of Drug Discovery and Development

(Nelfinavir) (Ritonavir) (Indinavir)

regimens, also known as highly active antiretroviral therapy,11 were

Basic Principles of Drug Discovery and Development

discovery and development of novel therapeutic agents. In the

Basic Principles of Drug Discovery and Development

Taxol® Velban® Adriamycin® Hycamtin®

Gleevac® Tasigna® Tarceva®

and a number of related compounds have demonstrated a remarkable

Basic Principles of Drug Discovery and Development

the degradation of low-density lipoproteins (LDL) receptors, and have

Verubecestat® Lanabecestat® Semagacestat®

Basic Principles of Drug Discovery and Development

Vabomere® O Sivextro® OH Orbactiv®

FIGURE 1.6 Staphcillins (Methicillin), Cipros (Ciprofloxacin), Vibramycins

β-lactams, quinolone, tetracyclines, and macrolide antibiotics (Fig. 1.6)

Basic Principles of Drug Discovery and Development

Vabomeres was developed and brought to market in 2017 in response to

Basic Principles of Drug Discovery and Development

formulations science, process chemistry, clinical research, intellectual

FIGURE 1.7 Lipitors (Atorvastatin), Prozacs (Fluoxetine), Singulairs (Montelukast),

Basic Principles of Drug Discovery and Development

Target to hit Total cost for one marketed drug:

Compounds Compounds Preclinical

time and resource to achieve these results, however, is substantial. As

Basic Principles of Drug Discovery and Development