Basic Principles of Drug Discovery and

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Benjamin E. Blass
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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
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 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

Phase 2 clinical trials 506

Phase 3 clinical trials 510
Phase 4 clinical trials 515
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

Basic Principles of Drug Discovery and Development

 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

Basic Principles of Drug Discovery and Development

 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.
 Drug discovery and development: An overview of modern methods and principles 5

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

Basic Principles of Drug Discovery and Development

6 1. Drug discovery and development: An overview of modern methods and principles

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.

Basic Principles of Drug Discovery and Development

 Drug discovery and development: An overview of modern methods and principles 7

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

Basic Principles of Drug Discovery and Development

8 1. Drug discovery and development: An overview of modern methods and principles

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,

Basic Principles of Drug Discovery and Development

 Drug discovery and development: An overview of modern methods and principles 9

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.

Basic Principles of Drug Discovery and Development

10 1. Drug discovery and development: An overview of modern methods and principles

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.

Basic Principles of Drug Discovery and Development

 Drug discovery and development from 20,000 Feet 11

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.”

Basic Principles of Drug Discovery and Development

12 1. Drug discovery and development: An overview of modern methods and principles

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 20
100 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 100
300 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

Basic Principles of Drug Discovery and Development

 Drug discovery and development from 20,000 Feet 13

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 1000
3000 patients,
multiple clinical sites, institutional review boards, and are multiyear endea-
vors (typically 2.5
5 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

Basic Principles of Drug Discovery and Development

14 1. Drug discovery and development: An overview of modern methods and principles

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.

Basic Principles of Drug Discovery and Development

Another random document with
no related content on Scribd:
[453]

Howard, Bull. Dep. Agric. Ent. N.S. No. 4, 1896.

[454]

Schimkewitsch, Zool. Anz. vii. 1884, p. 673.

[455]

P. Boston Soc. xxvi. 1894, pp. 312-355.

[456]

Monographie der Ordnung Thysanoptera, Königgrätz, 4to, 1895.

[457]

Bull. Essex Inst. xxii. 1890, p. 24; also Amer. Natural. xxx. 1896, p.
591.

[458]

Jordan in an interesting paper, Zeitschr. wiss. Zool. xlvii. 1888, p.

573, says that in the division "Terebrantia" there are only three
pairs of stigmata.

[459]

Insect Life, i. 1888, p. 138.

[460]

See Lindeman, Bull. Soc. Moscou, lxii. 1886, No. 2, p. 296, and
Uzel, Mon. 1895, pp. 397, 398.

[461]

Entomological Magazine, iii. 1836, p. 439, and iv. 1837, p. 144.

[462]

Zeitschr. wiss. Zool. xvi. 1866, p. 389.

[463]

Arb. Inst. Wien. iv. 1882, p. 415.

[464]

Tr. Amer. Phil. Soc. xix. 1896, p. 176.

[465]

P. ent. soc. Washington, iii. 1895, p. 241.

[466]

Ent. Nachr. xxii. 1896, p. 173.

[467]

Zool. Anz. 1897, No. 527, p. 73.

[468]

Arch. Anat. Physiol. 1874, p. 313, and 1875, p. 309.

[469]

For the structure and development of the Hemipterous trophi, see

Mayer, Arch. Anat. Physiol. 1874 and 1875; Mecznikow, Zeitschr.
wiss. Zool. xvi. 1866, p. 389; Geise, Arch. Naturgesch. xlix. 1,
1883, p. 315; Wedde, op. cit. li. 1, 1885, p. 113; Mark, Arch. mikr.
Anat. xiii. 1877, p. 31: Smith, Tr. Amer. Phil. Soc. xix. 1896, p. 176.

[470]

Ent. Nachr. xix. 1893, p. 369.

[471]

Naturhist. Tidskr. (3) vi. 1896; translated in Ann. N. Hist. (4), vi.
1870, p. 225.

[472]

Ent. Zeit. Stettin, xxvii. 1866, p. 321.

[473]

Ent. Nachr. xix. 1893, p. 375.

[474]

On this subject, see Reuter, Ann. Soc. ent. France (5) v. 1875, p.
225.

[475]

Ann. Soc. ent. France (4) vii. 1867, p. 45.

[476]

The chief work on the internal anatomy of Hemiptera is still

Dufour's Recherches anatomiques et physiologiques sur les
Hémiptères, Mem. Savans Étrangers, Paris, iv. 1833, p. 129.

[477]

Künckel, Ann. Soc. ent. France (4) vii. 1867, p, 45, and C.R. Ac.
Paris, cxx. 1895, p. 1002.

[478]

In Slingerland's Cornell Univ. Bull. No. 58, 1893, p. 222.

[479]

SB. Ak. Wien. xci. 1 Abth., 1885, p. 275.

[480]

Les Insectes fossiles, etc., 1894, p. 452.

[481]

Ann. Nat. Hist. (4) vi. 1870, p. 225.

[482]

A table of the families is given by Ashmead, but does not work out
quite satisfactorily, Entom. Americana, iv. 1888, p. 65; a brief table
of the characters of the British families is given by Saunders,
Hemiptera-Heteroptera of the British Islands, 1892, p. 12.

[483]

Those who wish to see tables of the families are referred to

Ashmead, loc. cit.; to Pascoe, Ann. Nat. Hist. (5) ix. 1882, p. 424;
to Stål's Hemiptera Africana, vol. iv. 1866; and for the families
found in Britain to Edwards, Hemiptera-Homoptera of the British
Islands. For a discussion in Danish on the value of the characters
used, cf. Hansen, Ent. Tidskr. xi. 1890, pp. 19-76.

[484]

Ent. Mag. vii. 1870, p. 53.

[485]

Insect Life, i. 1889, p. 234.

[486]

C.R. Ac. Sci. Paris, cxviii. 1894, p. 1282.

[487]

Verh. Ges. Wien. iii. 1858, p. 157.

[488]

Ent. Mag. xxix. 1893, p. 227.

[489]

Wien. ent. Zeit. xi. 1892, p. 169.

[490]

Monograph of Phymatidae: Handlirsch, Ann. Hofmus. Wien, xii.

1897, p. 127.

[491]
Ent. Zeit. Stettin, li. 1890, p. 281.

[492]

Naturalist's Voyage, ed. 1884, p. 330; chap. xv.

[493]

Thesaurus ent. Oxoniensis, 1874, p. 197.

[494]

Ind. Mus. Notes, iii. No. 5, 1894, p. 53.

[495]

Ferrari, Monograph of Nepa, Ann. Hofmus. Wien, iii. 1888, p. 171.

[496]

Bull. Soc. Philomat. (8) v. 1893, p. 57. There is some diversity of

opinion as to the respiratory orifices, and some authorities say that
thoracic stigmata exist even in the imago.

[497]

Acta Ac. German. li. 1887, p. 224, and Zeitschr. wiss. Zool. xliii.
1886, p. 537.

[498]

Korschelt, Acta. t.c. p. 245. Compare the remarks we have made

on p. 559 as to the peculiarities of eggs of many other Hemiptera.

[499]

Bull. Mus. Paris, 1896, p. 238.

[500]

See Carpenter, Irish Naturalist, iv. 1895, p. 59.

[501]
See remarks on pp. 543, 544.

[502]

We must refer those who may wish for further information as to

this complex and difficult question to the writings of the late
Professor Riley, especially to Bulletin No. 8, 1885, U.S.
Department of Agriculture, division of entomology; and to the more
recent report by Marlatt, Bull. Dep. Agric. Ent., N.S. No. 14, 1898.

[503]

Some entomologists consider that this "railway-whistle" note is the

result of the combined efforts of several individuals. Cf. Mathew,
Ent. Mag. xi. 1875, p. 175.

[504]

It is unnecessary to say that the poet was not Sappho, but one of
the baser sex, named Xenarchus.

[505]

Swinton claims that one of the membranes in the vocal apparatus

is an auditory organ; if so, the male would be deafened by his own
noise, while the females, not possessing the organ, should not
hear the song.

[506]

P. ent. Soc. London, 1883, p. 20.

[507]

A considerable variety of these extraordinary creatures are figured

in Biol. Centr. Amer. Rhynch. Homopt. ii.

[508]

Riley, P. ent. Soc. Washington, iii. 1895, p. 88. For the younger
stages of Membracis foliata, see Tijdschr. Ent. (2) iv. 1869, pl. viii.
[509]

Tr. ent. Soc. London, 1886, p. 329.

[510]

Verh. z.-b. Ges. Wien, xxvi. 1876, p. 167.

[511]

Cornell Univ. Agric. exp. station Bulletin, 44, 1892, and Bull. 108,
1896.

[512]

Zeitschr. wiss. Zool. xlii. 1885, pp. 569-638.

[513]

Zeitschr. Naturw. (2) xii. 1875, p. 438.

[514]

Réaumur, Mém. iii. 1737, Dixiéme Mémoire.

[515]

P. ent. soc. Washington, iv. 1897, p. 66.

[516]

For list see Scott, Ent. Mag. xviii. 1882, p. 253.

[517]

There is some doubt on this point, as the earlier observers seem

to have supposed that a winged individual appearing in a
generation chiefly apterous was ipso facto, a male; it seems,
however, to be certain that perfect winged males appear in some
species in generations producing no perfect sexual females.
Speaking generally, the course of events seems to be that in
summer there exist only wingless and winged parthenogenetic
females, and that the sexually perfect forms appear for the first
time in autumn.

[518]

Mitt. Schweiz. ent. Ges. iv. 1876, p. 529.

[519]

The term pseudovum is applied, as a matter of convenience, to the

earlier condition of the viviparously-produced form, and the term
pseudovarium to the ovary producing it.

[520]

Balbiani, Ann. Sci. Nat. Zool. (5) xi. 1869, p. 29. For concise recent
remarks on the early embryonic states, see Lemoine, Bull. Soc.
ent. France, 1893, p. lxxxix.

[521]

Acta Ac. German. xxxiii. 1869, No. 2, p. 81.

[522]

Seventeenth Rep. Insects Illinois, 1891, p. 66.

[523]

Kessler, Acta Ac. German. li. 1887, pp. 152, 153.

[524]

In connection with this the absence of a functional mouth in the

imago state of numerous Lepidoptera, and of Oestrid Diptera,
should not be forgotten.

[525]

Horae Soc. ent. Ross. xxiv. 1890. p. 386.

[526]
Ent. Zeit. Stettin, xxxvi. 1875, p. 368.

[527]

Zool. Anz. xv. 1892, p. 220.

[528]

Arb. Inst. Wien, iv. 1882, Heft iii. p. 397; see on this organ also
Mordwilko, Zool. Anz. xviii. 1895, p. 357.

[529]

Biol. Centralbl. xi. 1891, p. 193.

[530]

See, inter alia, Webster, J. New York ent. Soc. i. 1893, p. 119.

[531]

J. New York Ent. Soc. i. 1893, p. 120. See also as to knowledge

on the part of ants, Forbes, Eighteenth Rep. Insects Illinois, 1894,
pp. 66, etc.

[532]

Monograph by Buckton, Ray Society, 4 vols. 1879-1883.

[533]

Tr. New Zealand Inst. xxviii. 1895.

[534]

A catalogue of Coccidae has recently been published by Mr. T. D.

A. Cockerell in Bull. Illinois Lab. iv. 1896, pp. 318-339.

[535]

Signoret's papers are to be found in eighteen parts in Ann. Soc.

ent. France, 1868 to 1876: the most considerable subsequent
systematic papers are those by Maskell in the Transactions of the
New Zealand Institute from 1878 to the present time.

[536]

Coccidae of Ceylon, pt. 1, 1896, p. 16.

[537]

C. R. Ac. Sci. Paris, civ. 1887, p. 449.

[538]

Arch. Naturgesch. li. i. 1885, p. 169.

[539]

Zeitschr. wiss. Zool. xliii. 1886, p. 156.

[540]

For summary as to our present knowledge of this curious condition

of Insect life, see Mayet, Ann. Soc. ent. France, 1896, p. 419.

[541]

For additional information as to useful Coccidae, see Blanchard,

Bull. Soc. Zool. France, viii. 1883, p. 217.

[542]

Rubsaamen's paper on these Insects gives references to most of

the previous literature, Berlin. ent. Zeitschr. xxxix. 1894, p. 199.

[543]

Ent. Meddel. iii. 1891, p. 82.

[544]

Cf. Graber, Zeitschr. wiss. Zool. xxii. 1872, p. 165, and Landois in
the same Journal, xiv. 1864, p. 24.
[545]

Ann. Nat. History (3), xvii. 1866, p. 213.

[546]

N. York Ent. Soc. vii. March 1899, p. 45.

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