Drug Discovery and Clinical Research SK Gupta, Sushma Srivastava
Page numbers followed by b refer to box, f refer to figure, fc refer to flowchart, and t refer to table.
A and B reactions, characteristics of type 326t
Accidental injury, compensation for 468
Accountability and transparency, principles of 441, 461
Acellular pertussis adsorbed 44
Acne 46
Adalimumab 46, 49
Adjuvant therapy 48
Administrative documents 408
Adventitious infections 17
Adverse drug reaction 321, 349, 451, 499
classification of 325
frequency classification of 328, 328t
reporting 232, 470, 519, 363t
comparison of 363
terminology 336
Adverse event 172, 451, 499
reporting system 258, 341
serious 77, 88, 172, 382
Advertising 238
Adynovate 44
Agent bases simulation models 301
Agranulocytosis 334
Airway epithelial cells 39
small 40
Alcohol 325
Allergen extracts 42
Allergen patch tests 42
Allergenics 42
Allergic reactions 8
American Medical Writers Association 313
American Society for Testing and Materials 439
Amoxicillin 252
Amyloidosis 46
Ancillary care 384
Animal experimentation 387
Animal mimicking diabetes 6
Animal model 19
Animal pharmacology 17
and toxicology studies 21, 60
studies 14
Animal testing facility 19f
Animal toxicity studies 15
Anthrasil 44
Anthrax immune globulin intravenous 44
Antibiotic 252
Antibody 6
Antidepressant 334
Antigen skin test 42
Antihemophilic factor 42, 44
Anti-inhibitor coagulant complex 42
Antiphospholipid syndrome 46
Antithrombin 42
Antivirus software 312
Apoptosis 37
Applicable regulatory requirement(s) 500
Application for permission 84
to Import New Drug 78
Application, submission of 463
Aseptic environment 48
Asia-Pacific Economic Cooperation 134
Association of Clinical Research Professionals 198
Association of Southeast Asian Nations 134
Astemizole 334
Asthma quality-of-life 297
Astrocytes 39
Audiovisual recording 104
Audit 470, 500, 522
certificate 500, 538
of trial 451
report 500
trail 250, 500
Auditing procedures 522
Auditors, selection and qualification of 522
Australasian Medical Writers Association 313
Autoimmune diseases 27
Autoimmune hemolytic anemia 46
respect for 145
Rights and Dignity, Principle of Respect for 440
Avastin 49
Average cost-effectiveness ratio 295
Avonex 49
AYUSH, formulation under 434b
Background information 524
Balance incomplete block design 266, 267t
Banner advertising 192
B-cells 221
BCG live 42
Belmont report 73
Beneficence 145
principle of 440
Benefit risk assessment 381, 403
Benzodiazepines 4
withdrawal 325
Bevacizumab 49
Bexsero 44
Bias assessment tool, cochrane risk of 282
Bioanalytical logistics 213
Bioavailability 208, 428
study 92
Bioequivalence 208
center, workflow at 219
study 92
elements of 209
Biologic 41
development of 45
discovery of 45
license application 42, 72
Biological materials, biobanking and datasets 447
Biological products facing shortage 49t
Biological, recently approved 44t
Biomarker 32
based-strategy 45
Biosecurity 438
practices 17
Biostatistician, role of 478
Biotechnology, department of 229, 232
Biotherapeutic monographs, Indian pharmacopoeia status of 234t
Bipolar disorder 10
Birth defect 255
Bisexual 382
and transgender community 420
Black triangle scheme 349
double 269
single 269
triple 269
Blinded trials, decoding procedures for 534
Blinding 71, 269, 452, 500
techniques 270f
Blood 42
and blood products 42
bank devices 42
components 42
donor screening tests 42
factors 350
Bone 42
cements 242
demineralized 42
Bonus material 194
Brainstem death 159
Brazilian Health Surveillance Agency 223
Breast cancer 45
Calcium ions 37
Canadian Science Writers’ Association 313
Carcinogenic effects, long-term 12
studies 484
test 17f
Cardiac side effect 334
Cardiac stents 242
Cartilage 42
Case record form 70
design 248
development 71
login and inventory 249
Case safety reports, individual 233, 337
Case study report, individual 321
Catheters 242
Causal relationship 321
Causality assessment 357
Causality classification 325
co-culture technology, integrated discrete multiple 38
culture 19
mechanisms, basic 33
types 36
Cell-based treatments 42
Cell-cell talk 44
Cell-matrix talk 44
Cellular and gene therapy products 42
Cellular metabolite content 37
Cellular toxicity 37
Center for Biologics Evaluation and Research 42, 48, 133, 339
Center for Devices and Radiological Health 339
Center for Drug Evaluation and Research 22, 23, 60, 72, 133
Center for Food Safety and Applied Nutrition 339
Center for Tobacco Products 339
Center for Veterinary Medicine 339
Central Drug Authority 242
Central Drugs Standard Control Organization 117, 222, 377, 448
guidelines 31
Central hub 197
Central Licensing Approval Authority 242
Centuries ago medicines 26
Certified copy 505
Cetuximab 49
Challenges and future strategies 34
Chickenpox 428
Chinese Food and Drug Administration 222
Chi-square test 273
Chlordiazepoxide 4
Chlorpromazine 4
Clinical Data Interchange Standards Consortium 247, 260
Clinical data management 71
process 246fc, 258fc
recent advances in 258
system 249
Clinical drug 425
Clinical effectiveness and economic resources 279t
Clinical operations 70
Clinical practice
good 70, 73, 131, 136, 197, 451, 454, 499, 501
guidelines, good 451
Clinical protocols and investigator information 60, 21
Clinical research
associate 197, 198, 246
bioethics in 143
coordinator 186, 197, 198
organization 29, 71, 305, 453
Clinical trial 80
application, data requirement for 231
during 80, 99, 113
related to 104
design 67
injury related to 104
phases of 261t
processes 245fc
randomized 171
registry–India, registration with 390
related deaths, case of 101
related injury or death, compensation for 101
Cloning 42
Closing discrepancy
per clinical team 255
per internal correction 255
Clostridium difficile toxins A and B 43
Coagadex 44
Coagulation factor IX 44
Coagulation factor X (human) 44
Cochran Q test 286
Coding checks 253
Coeliac disease 46
Cognitively impaired 421
Cohort event monitoring 337
Cohort study 331, 332
Collaborative research 391
Colorless tetrazolium salt 37
Common technical document 136
Community advisory board/ group 385
Community engagement 385
Community Outreach and Health Associations 190
Community Outreach Programs 190
Community trials 432, 444
Comparator product 453
Compensation 104
quantum of 104
principles of 461
with protocol 474, 509
Computer knowledge 311
Computerized systems, validation of 505
Conducting decision tree analysis, steps in 300
Confidentiality 453, 501
statement 526
Confirmatory safety and efficacy study 231
Conflict of interest 384
issues 387
Congenital anomaly 255
process, procedures after 413
types of 445, 445b
waiver of 413, 445
Consumer health writing 306
Contamination 47
Contraceptives, clinical trial of 436, 480
Contract 453, 468, 501
Contract Research Organization 71, 74, 167, 197, 454, 501, 515
Controlled trials, randomized 277
Convenience and responsiveness 193
Coordinating Committee 501
Coordinating investigator 453, 501
Corneas 42
Cost 292
and time in drug discovery 4t
benefit analysis 296
medical 292
nonmedical 293
effectiveness analysis 294
minimization analysis 294
utility analysis 295
Cost-benefit analysis 296
Cost-effectiveness analysis 295, 296
Cost-minimization analysis 296
Cost-of-illness evaluation 294
Cost-utility analysis 296
Council for International Organizations of Medical Sciences 131, 146, 377
Counterfeit healthcare products 350
Crohn's disease 45
Crossover design 265f
two period 69fc
Crotalidae immune 44
Cryoglobulinemic vasculitis 46
Cytotoxicity screening of drugs, in vitro assays for 36
Darbepoetin alfa 49
and safety monitoring 171, 172
and scales of measurement, types of 270f
capture and collection 247
clarification forms 256
collection process 283
consistency checks 251
elements for reporting 99
entry 250
extraction 280
inconsistencies, types of 251fc
on preclinical testing 483
on purity of recombinant product 483
preclinical supporting 457
privacy 248
quality assurance 71
review and validation 250
Safety Monitoring Board 172, 174
standards 260
storage and archival 258
validation of 457
Data checks
continuity of 253
duplicate 254
external 254
Data handling
and management 459
and record keeping 525
Data management 246, 283
plan 246
practice, good 71
Data Monitoring Committee 171, 172, 174
independent 501
Database closure 257
Database development and validation 71
Database research and monitoring 332
Decision making process 463
Decision tree models 300
Declaration of Helsinki 73, 131, 136, 138, 145
Defence Research Development Organisation 480
Demonstration projects 444
Depression 10, 334
Dermal toxicity 16
Dermatomyositis 46
Developing World Bioethics 168
Diagnostic accuracy studies, quality assessment of 281
Diagnostic agents, trials of 434
Diazepam 4
Dichotomous 273
Diet/drug interactions 32
Diphtheria 44
vaccines 428
Disability/incapacity, significant 255
Discovery and development of biologics, challenges in 47
Discrepancy management 255, 256fc
Discrete event simulation models 301
Discrete value group discrepancy 252
Discussion group 135
Disease 32
phenotype of 5
with treatment 28
without treatment 28
Disposal 238
technology, recombinant 163
vaccines 428
Document history 496
Documentation 453, 476, 501
pretrial 198, 200
quality of 356
multiple ascending 30, 65
single ascending 30, 65
Dose-response relationship 15
Double dummy technique 269
Draft rules 122
Drawbacks of in vitro cytotoxicity 38
Dropsy 4
Drug 6
absorption 29
activity, early measurement of 89
and other interventions, clinical trials of 424
banned to postmarketing surveillance 333
classification of 434b
discovery of 4
eluting stents 242
event monitoring 330
hunting, science of 41
indication, limited 321
information association 198
likeness 28
molecule, small 6
pharmacodynamics 12
rapid alert system for 232
regulation 357
response 32
safety monitoring of 319
supplies 263
therapy, current 5
withdrawals, recent 334t
Drug and biological material collection 213
administration of 213
Drug and Cosmetics Amendment Bill 242
Drug candidate
rate of 36
stages of selection of 9f
Drug Controller General of India 76, 167, 229, 333, 242
Drug development 3, 56, 58
bioequivalence in 207
cost of 36
during 40
phases of 425b
preclinical 12
process 36, 57, 171
Drug discovery 57f
and development 1
of biologics 41
phases of 56f
portfolio 11
process 3, 4, 20f
steps in 4
Drug safety 36
predict 36
Drugs and Cosmetics Act 77, 228
Drugs and Cosmetics Rules 78, 84f
Drugs and Pharmaceutical Research Programme 34
Duplicate reports, identification of 357
Duties and Responsibilities, Allocation of 468
East African community 134
Economic models 299
Education 357
Educational medical writing 306
assessment of 458, 525
guidelines 136, 137
Egyptian Drug Authority 223
Electronic consent 412
Electronic data
capture 176, 179, 247
processing 477
systems, validation of 477
Electronic standards for transfer of regulatory information, development of 136
Embryo 162
cloning 163
Employ decision analysis 302
Ensuring privacy and confidentiality, principle of 379
Environmental protection, principle of 380
Enzyme 6
Equivalence trial 70
Erbitux 49
Erythema nodosum 46
concentrated solution 234
injection 234
Escape treatment 453
Essential documents 453, 501, 530
Essentially similar products 208
Etanercept 46, 49
Ethical and safety considerations 460
Ethical challenges in India 167
Ethical considerations 458
in collaborative research 391
policy statement on 377
Ethical implications 429
Ethical inquiry 162
Ethical issues
concerning organ
donors 157
recipients 155
of epidemiological and public health research study designs 441
related to reviewing protocol 403t
Ethical principles 460
Ethical review procedures 394
Ethics 525
Ethics Committee 103, 175, 453, 461, 469
accreditation of 109
communication with 474
composition of 96
functional 200
registration of 82, 97, 105, 106, 118
researchers and institutions, responsibilities of 392
responsibilities of 88
roles and responsibilities 73
trial protocol by 98
EU technical file for medical devices, components of 242t
European Federation of Pharmaceutical Industries and Associations 132, 133
European Medical Writers Association 313
European Medicines Agency 133, 134, 222, 226, 349
European Union Regulation and Guidelines 226
Expert working group 135
Extended Program of Immunization 354
Extrapyramidal side effect 334
Facility 217
audit 70
Factorial design 68, 267, 267f
Fascia 42
Fatal adverse drug reactions 319
Federal Commission for Protection against Sanitary Risks 223
Federal Food, Drug and Cosmetics Act 228
Federal Regulations, code of 72, 175
Female fertility 16
Female reproduction and developmental toxicity 16
Fibrin sealant 44
Filgrastim concentrated solution 234
Filgrastim injection 234
Final report 453
by investigator 513
Financial aspects of trial 532
Financing 517
and insurance 525
First-in-human milestone, microdosing vs conventional pathway to 65t
Fisher's exact probability test for small numbers 273
Flu vaccine 428
Fluad 44
Flupentixol 334
Food and Drug Administration 60, 61, 131, 133, 237
Amendments Act 340
Foot 254
Foreign direct investment 76
Foreign sponsor, role of 471
Forest plot 282, 285f
Foxglove 4
Full committee meeting 404
Fungi, infection by 5
Gas chromatography 214
Gastric 254
Gatifloxacin 334
Gay 382, 420
Gene 6
discussion group 135
ethics of 164
Gene-based treatments 42
General ethical issues 381
General principles 139
statement of 379
General public 189
General responsibilities 198, 199
General safety test 483
General statutory orders 77
Generic instruments 297
disorders 5, 27
engineering approval committee 479
factors affect 32
manipulation, committee on 229
material, harmful effects on 16
Genotoxicity 16
studies 484
Geriatric 90
Giant cell arteritis 46
Global cooperation 134
Global harmonization task force 236
Global increase in clinical trials 181f
Global scenario examples 194
Glomerulonephritis 46
Good medical writing, fundamentals of 304
Graeco-latin square design 266, 266t
Graft disease 46
Granting waiver of consent, conditions for 413b
Grave's disease 46
Group sequential design 68
Guidelines, nomenclature and regulatory authorities of different countries 222t
Gulf Central Committee 134
Handling of product(s) 458
Harmonization, process of 135
conditions, serious 148
products and food branch 134, 223
related quality of life 297
sciences authority 222
technology assessment 274, 279
Health and Science Communications Association 313
Health Associations 191
Health Canada 134
Health Canada's Biologics and Genetic Therapies Directorate 223
Health Ministry's Screening Committee 393
attack and stroke 334
risk of 334
valves 42, 242
record 252
test 252
B vaccine 42, 234
C 46
chronic 46
chronic 46
Hepatocytes 36, 39
Herbal products, categories of 481
Herbal remedies and medicinal plants, clinical trials of 481
Herceptin 49
Heterooligomers 7
Hierarchical system 422
HIV trials 29
HIV/AIDS, interventions in 432
Homologous enzymes 7
Hormones 4
Hospitalization, prolongation of existing 255
Host disease 46
HPV vaccine 42
aortic endothelial cells 39
cells 36
clinical trials, phases of 61
cloning 162
ethics of 162
dignity 162
disease, pathophysiology of 27
effects in 486, 528
embryonic stem cell 160
embryos, unjust killing of 160
genetics testing and research 447
genome 10
publication 10
medicines, commission on 349
organs, transplantation of 159
pain and suffering 165
participants, protection of 387
pharmacology 64, 88
research 146
specific xenobiotic toxicity 38
subject 146
protection guidelines 73t
translation to 27, 28
Human/clinical pharmacology
immunogenic potency 484
trials 452
Humanistic evaluation methods 297
Humanized antibody 49
Humira 49
Hydradenitis suppurativa 46
Hyperglycemia in elderly 334
Hyperostosis 46
Hypertension 325
Hypothesis, testing of 270
Ileum preparation 13
Imlygic 44
Immune diseases 27
Immune-mediated inflammatory diseases 46, 46t
Immunodiagnostic reagents, recombinant 484
Immunogenicity 47, 484
Immunoglobulin products 350
Immunotoxicity 484
Implantable medical devices directive, active 241
Implied causality 321
In situ hybridization 32
In vitro diagnostic medical devices 241
In vitro model 6
systems 36
In vitro screening methods, preclinical 36
In vitro specific cell 38
In vivo clinical research 33
In vivo models 6
In vivo preclinical imaging 33
In vivo testing 6
In vivo toxicity 39
Independent discrete multiple organ co-culture technology, principle of 39f
Independent Ethics Committee 502
Indian Conformity Assessment Certificate 242
Indian Council of Medical Research 103, 145, 152, 377
Indian Medical Devices Regulatory Authority 480
Indian Medical Writers Association 313
Individual's worthiness, criteria of 156
Industry trends, background and 180
Infection by bacteria 5
Inflammatory demyelinating polyneuropathy, chronic 46
Infliximab 46, 49
A vaccine 42
A virus 337
vaccine 44
Information technology 218
Informed consent 87, 95, 104, 140, 146, 148, 445, 454, 502
document 382, 407, 410b
form 198, 201, 210, 382, 532
form enrollment 202
form recruitment 202
form, screening 202
in nontherapeutic study 466
of subject 464
of trial subjects 510
process 382, 404, 410, 464
Inhalational toxicity 16
Inherent limitations 48
Injury 104
Inpatient hospitalization 255
In-silico technology 9f
Institutional Animal Ethical Committee 19, 230
Institutional arrangements, principle of 379, 461
Institutional Biosafety Committee 230
Institutional Ethics Committee 73
Institutional Review Board 61, 138, 148, 189, 198, 200
Instruments, types of 297
Insulin glargine injection 234
Insurance statement 532
Interactive voice
recognition system 200
response system 247
Interchangeable products 227
Internal prosthetic replacements 242
International collaborations, types of 393b
International Conference on Harmonization 131, 132, 173, 197, 377
International Council for Harmonization 73, 132
coordinators 134
guidelines 136
process of harmonization 135f
secretariat 134
working groups 135
International drug monitoring 319
International Ethical Guidelines 146
International Federation of Catholic Medical Associations 168
International Science Writers Association 313
Internet 192
Intracellular pathways 44
Intraocular lenses 242
Investigational pharmaceutical product 457
Investigational product 454, 474, 502, 509
accountability 537
Investigator 454, 502, 508
and institution selection 468
brochure 454, 484, 502, 526, 529, 532
meeting 198, 202
qualifications and agreements 508
responsibilities 74, 87, 103, 477
selection 517
Investigator's brochure
contents of 484, 527
table of contents of 530
Irritable bowel syndrome 334
Irritation, local 8
Ixinity 44
Japan Pharmaceutical Manufacturers Association 133
Josef Mengele's twin study 323, 323f
Justice, distributive 382
Juvenile idiopathic arthritis 46
Karch and Lasagna scale 326
Kawasaki disease 46
Kidney 39
Kruskal-Wallis test 273
Laboratory practice
good 18, 213, 431
requirements, good 19f
Lactate dehydrogenase 36, 37
assay, principle of 37, 37f
Lactic acidosis 334
Langendorff's heart preparation 13
Language 188, 477
Last patient last visit 257
Latin square design 266t
Lay person(s) 397
Lead identification 3, 6, 57
Lead optimization 8, 58
delivery 8
formulation 8
Least infringement, principle of 440
Legal affairs 9
Legal expert/s 397
Legislation in India 159
Lesbian 382, 420
Life-threatening 321
illness 255
Ligaments 42
Ligand-receptor interactions 44
Liquid chromatography, high performance 214
Listen and care 193
Live and attenuated vaccines 428
Local Ethics Committee 148, 189
Log rank test 273
Luciferase assay, principle of 37f
Lung toxicity 40
Lysosomal functions 37
MabThera 49
Magic bullet 320
Male fertility studies 16
Mammary cancer cell 39
Mann-Whitney test 273
Manufacturing information 21, 60
Manufacturing practice, good 136, 138, 431, 480
Manufacturing process 230
Manufacturing, packaging, labeling, and coding investigational product(s) 518
Market authorization application, data requirement for 232
Marketing authorization approval 131
Markov model 300, 301
structure, simple 301f
Masking 452, 500
Mass media 190
Master randomization list 534
Matched pairs 264, 265f
Material transfer agreements 391
Matthews media group 182
Mature market enrollment 183f
Maximum benefit, criteria of 156
Measles 428
Medical and science writers, organizations for 312, 313
Medical application 32
Medical care
factors affecting 176f
of study subjects 473
of trial subjects 508
Medical community outreach 189
Medical device 236, 239, 349, 480
agency 341
classification of 238, 240t, 431t
clinical trials with 480
directive 241
lifespan of 237, 237f
regulations and research 236
regulatory authorities across globe 241t
reporting 243
research, ethics in 165
safety 242
Medical expertise 515
Medical management decisions and effect 175
Medical monitor
in clinical trials
responsibilities of 172
role of 172
responsibilities of 173fc
Medical review 22, 61
Medical scientist(s), basic 396
Medical termination of pregnancy 152, 436, 467
Medical worthiness and social worthiness 156
Medical writer, career opportunities for 309, 309f
Medical writing
essentials of 304
types of 306
Medication error prevention and analysis, division of 339, 340
Medicinal chemists 9
Medicinal Products for Human, committee for 133, 226
and statistics 262fc
defective 349
evidence-based 275
report center, defective 349
side effect reporting form 360f, 361f
Medicines Control Agency 341
Medicines Control Council 223
Medicomarketing writing 307
MedWatch adverse drug event reporting forms 341
Melitracen 334
Member Secretary, alternate 396
Membrane integrity 36
Meningococcal group B vaccine 44
Mental illness 421
Mentally disabled 148
Meprobamate 4
Meta-analysis 282, 332
Metabolism-dependent toxicity 39
Metabolize 6
Metamizole combinations 334
Microdosing 61
advantages of 62
limitations of 64
Microorganisms, infections caused by 27
Microsimulation models 301
Microvascular endothelial cells 40
Ministry of Health and Family Welfare 110, 116, 118, 122, 123, 126, 127, 128, 130, 222, 350
Ministry of Health, Labour and Welfare 133, 134
Ministry of Healthcare of Russian Federation 222
Ministry of Public Health 223
Mitochondrial function 37
Mobile smartphones and digital devices 192
Molecular target drugs 32
Molecular weight 7
Molecularbiology 9
Molecules 6
small 48
Monitor, selection and qualifications of 519
Monitor's responsibilities 520
Monitoring 198, 203, 470, 503, 519
and auditing of records 474
extent and nature of 520
in clinical trials 171
plan 505, 522
procedures 521
report 503, 521
final trial close-out 538
visit reports 536
Monoclonal antibodies 48
Mounting international pressure 207
Mouth 254
Moving towards telehealth technology 177
Multicausal diseases 27
Multicenter studies 471
Multicenter trial 69, 503, 523
Multicentric research 406
Multicentric reticulohistiocytosis 46
Multicentric study 455
Multicentric trials 429
Multidisciplinary guidelines 136, 137
Multifactorial diseases 5, 27
Mumps 428
Myasthenia gravis 46
Myelodysplastic syndrome 46
Naranjo algorithm
questionnaire 327t
scoring for 326
Naranjo probability scale 326
National Accreditation Board for Hospitals and Healthcare Providers 409
National Accreditation Board for Testing and Calibration Laboratories 439
National Administration of Drugs, Foods and Medical Devices 223
National AIDS Control Organization guidelines 433
National bioethics organizations 168
National cancer institute 174
National Coordination Center 233, 350
working composition of 350
National Education Technology Writers Association 313
National Guidelines for Stem Cell Research 431
National Institute for Clinical Excellence 12
National Institute of Care and Health Excellence Critical Appraisal Guidance 282
National Institute of Care and Health Excellence Tool 282
National Institute of Health 60, 173, 175
Policy 131
National Institute of Hygiene Rafael Rangel 223
National Research Act 131
Nazi doctors’ trial 323
NCC-PVPI, responsibilities of 353
Nervous system 39
Neurology trials 175
Neurotransmitters 4
New drug 82, 357
application 31, 42, 72, 261, 306
for approval to manufacture 78
investigational 21, 59, 62f, 66
clinical development of 55
develop 58
process 59f
stakeholders in 9
steps in 5f
discovery 261
investigational 8, 10, 29, 59, 60, 72, 77, 79
regulations 21
stages of development of 58
success rate of developing 58
New technology 180, 438
Newspaper advertisements 190
Nicotine 40
Nominal scale 269
Non critical devices 480
Nonclinical study 455, 485, 503, 527
Nonexploitation, principle of 379, 460
Non-governmental organization 384
Non-human cells, transplantation of 42
Non-humanized antibody 49
Noninferiority 70
Non-maleficence, principle of 440
Nonrepresentative patient selection 320
Nontherapeutic study 455
Notification/Submission to Regulatory Authority 517
Null hypothesis 270
Nuremberg code 73
Nursing women 91, 467
tests, rabbits kept ready for 16f
tissues 42
toxicity 16
Office of surveillance and epidemiology, divisions of 339fc
Oncology 29
clinical trial in 437
trials 175
Opiate abstainer, restlessness in 325
Opioid analgesics 334
Optical character recognition 248
Optical mark recognition 248
Organ 42
from alternative sources 159
from cadaveric source 157
from living donors 158
function 33
sharing, united network of 155
specific toxicity 40
toxicity, multiple 39
transplant 159
ethics in 155
Original medical record 503
Orthopedic implants 242
Osteitis 46
Packaging and labeling 238
Pain relief 334
Palivizumab 49
Pan-American network for drug regulatory harmonization 134
PaniFlow 336
Paracetamol 254, 255
Patent and commercial issues 48
Patient awareness packets 189
Patient compliance 48
Patient recruitment
and retention, principles of 179
conferences 181
landscape 195
Patient-facing strategies 189
Pediatrics 90
Pegfilgrastim 234
Pemphigus 46
Performance management 193
Pergolide 334
Perinatal study 16
Periodic benefit-risk evaluation report 332
Periodic safety update report 104, 322, 332
Persistent disability/incapacity 255
Personal digital assistants 259
alternatives 207
equivalents 207
industry 10
manufacturers association 134
products, supply, storage and handling of 469
Pharmaceutical Research and Manufacturers of America 133, 134
Pharmaceuticals and Medical Devices Agency 133, 134
and safety studies 226
and toxicology studies 231
properties 8
complicated 47
studies 231
analysis 292t
types of 293, 296t
applications of 297
evaluation 301
method 298
principles of 291
problem 301
studies, conducting literature of 298
techniques 291
Pharmacokinetic 12, 18, 20, 89
and product metabolism
in animals 485
in humans 486
parameters 214
pharmacodynamic modeling and simulation 33
properties 8
properties, complicated 47
studies 226, 231
Pharmacological classification 325
Pharmacology review 22
Pharmacovigilance 319
division of 339
in different countries 339
in India 350
in United Kingdom 341
in United States of America 339
methods of 329
objectives of 325
office of 339
plan 232
Program of India 350, 353fc
softwares for 365, 365t
system 233, 362t
comparison of 362
Phenformin 334
Phosphotidylserine, translocation of 37
Photo-allergy 16
Physician Referral Programs 189
Physicochemical properties 8
Physiotherapy 255
Phytopharmaceutical drugs 429
Pilot studies 240
Pioglitazone 334
Pivotal studies 240
Placebo 141
Plasma concentration 29
time curve, non-transformed 215f
Policy 387
for handling misconduct 390
Poliovirus vaccine, inactivated 44
Polyarteritis nodosa 46
Polymyalgia rheumatic 46
Polymyositis 46
Population level, translation to 27, 31
Positron emission tomography 64
Postmarket data requirement 232
Postmarketing studies 232, 240
Postmarketing surveillance 91, 104, 332
methods of 332
studies 240
Postmarketing trials 67, 90
Postresearch access and benefit sharing 385
Power of test 271, 271f
Practice and research, ethics in 143
Precaution and risk minimization, principles of 460
Preclinical studies
data requirement for 230
types of 13
Preclinical work, purpose of 17
Pregnancy and clinical trials 436
Pregnant women 91, 152, 467
and foetuses 437b
Pre-market approval 241
Premature termination 471, 513, 523
President's council on bioethics 162
Pre-trial monitoring report 534
PRISMA guidelines 283t
Privacy and confidentiality 140, 382
principles of 460
Private information 146
Product characterization 230
Professional competence, principle of 379, 461
Professional gains 312
Progress reports 476, 513
Propidium iodide exclusion assay 37
Proposal-related documents 408
Propoxyphene 334
Prospective research participants, essential information for 410
Protein 6
complexes 7
Protocol 455, 457, 503
amendment 455, 503
develop 278
relevant components of 457
Protozoa, infection by 5
Provisions, post-trial 142
Proximal tubule epithelial cells 39
Pseudomonas aeruginosa infections 43
Public domain, principles of 461
Public health
concern 3
interventions 432
research 440, 446b
ethics, principles of 440
proposal 441b
stakeholders in 446
studies, epidemiological and 441
Publication policy 460, 525
Publishing tools 312
Pustulosis 46
Pyoderma gangrenosum 46
Quadracel 44
Qualifications and skills needed 309
Quality adjusted life years 295
Quality assessment/appraisal 281
Quality assurance 455, 477, 503
and quality control 257, 515
systems 469
Quality comparability study 230
Quality control 455, 503
and quality assurance 459, 525
Quality guidelines 136, 137
Quality index 257
Quality management 514
Quality review panel 354
Quality-based considerations 230
Query writing 256
Radio advertising 190
Radiofrequency identifications 166
Randomization 71, 212, 455, 503
and blinding 478
procedures 510
techniques 267
Raplixa 44
Ratio analysis 217
Rats, reproductive studies done on 16f
Raw data 456
Reaction, serious 357
Rebif 49
Reciprocity, principle of 441
Record access 519
Record and report 476, 512
Record keeping 464
and archiving 408
and data handling 476
Record, maintenance of 98
Recruitment, strategies for 191
Rectal tolerance test 16
Rectus abdominus muscle preparation 13
Refractory asthma 46
Regional harmonization initiatives 134
Regional training centers 350
Registration of Ethics Committee, requirements and guidelines for 119
Regulation and Guidelines in India 228
Regulations for conducting clinical trials in India 76
Regulations for development and clinical trials of biosimilars 220
Regulations in India for clinical trials 77
Regulatory affairs, office of 339
Regulatory and postmarketing experiences 486
Regulatory approvals 73
Regulatory authority 455, 503, 533, 535
Regulatory medical writing 306
Relapsing polychondritis 46
Relational database management system 336
Relevant communications 536
Religion 188
Remicade 49
Remote data
capture 248
entry 248
Renal proximal tubule cells 39
Replicated crossover design 211
Reporting forms, types of 355
Reporting, advice about 359
Reproductive and developmental toxicity 484
Research 146
administration 9
among tribal population 421
and therapy, creating embryos for 162
basic 27
during humanitarian emergencies and disasters 447
ethics 144
committees 140
involving human subjects 145
involving vulnerable subjects, ethics of 147
misconduct 390
on children, conditions for 419b
on human subjects 377
participants privacy and confidentiality, protection of 404
pharmacy and drug accountability 198, 202
protocol, preparation of 278
publications, types of 307
related to healthcare in developing countries, ethics of 168
responsible conduct of 386
values of 386
Researchers, responsibility of 411
Research-related harm, compensation for 383
Responsibility, allocation of 517
Risk communication 514
Risk reporting 514
Rituxan 49
Rituximab 46, 49
Rofecoxib 334
Roman technique, old 55
Rubella 428
Safeguarding confidentiality 466
Safeguards, additional 416
and efficacy 225, 486
and immunogenicity data 232
and medical monitoring 171
practices, good 175, 176t
and tolerability, initial 29
assessment of 459, 525
biologics 50
evaluation 9
guidelines 136, 137
implications 47
information 470, 519
limited 48
management teams, establishing formal multidisciplinary 177
pharmacology endpoints 15
reporting 513
Sapho syndrome 46
Sarcoidosis 46
Saudi Food and Drug Authority 222
Scalp vein sets 242
Schizophrenia 10
Scientific medical writing 305
Scientific writing 306
Sclera 42
Sclerosis, multiple 46
Scurvy 56
Search strategy theory 279f
Selecting suitable test, guidelines for 273t
Sentinel sites 330
Sepsis 45
Seriousness classification 325, 327
Severity classification 325, 327
Sex workers 420
Sexual minority 420
group 420
Sham surgery, conditions for 432b
Sibutramine 334
Signal detection procedures 333
Signal generation and strengthening 357
Signal Review Panel 354
Signal strengthening 333
Signature sheet 537
Simple randomization 267, 268f
Simulation models 301
Single cell
in vitro systems 38
system 38
type cultures 38
Single cell-culture system, conventional 38
Site initiation visit 70, 198, 201
Site management organization 197
Site staff, training of 198, 201
Skin 16, 42
Social and behavioral sciences research for health 447
Social justice, principle of 441
Social networks 192
Social responsibility, principle of 379
Social values 403
Social worthiness 156
Societal perspective 292
Society for clinical research associates 198
Society of biomedical technology 480
Solidarity, principle of 441
Somatic cell nuclear transfer 163
Somatropin 234
concentrated solution 234
for injection 234
Sophisticated husbandry 17
Spare embryos, fate of 161
Special populations, studies in 90
Sperm 42
Sponsor 174, 456, 468, 504, 514
and monitor, responsibilities of 477
investigator 504
name 529
notification 23
pre-study site visit 70
responsibilities of 74, 86, 103
to pay, obligation of 468
Staffing plan, create 193
duties of 416
obligations/duties of 416, 417t
Standard operating procedures 73, 200, 214, 246, 319, 384, 456, 468, 504
Statistical analysis 22, 61, 216, 263, 478
Statistical classification 325, 329
Statistical issues 281
Statistical significance 270
Statistical techniques 284
Statistics 459, 478, 525
Steering committee 132, 353
Stem cell 42
clinical trials with 431
ethics in 160
institutional committee for 401
Steven Johnson syndrome 334
Still's disease, adult onset 46
Stratified randomization 267, 268f
Streptococcal infection 252
Streptokinase bulk solution 234
recombinant 234
Streptokinase for injection, recombinant 234
Streptomycin 252
Students T-test
paired 273
unpaired 273
design 210, 458, 478
feasibility analysis 187
flyers, distribution of 190
management 457
data handling and record keeping 468
perspective 298
population 263
information on 209
procedures 263
product 456
protocol 209
reports 470
results 299
subjects, selection and recruitment of 475
Subject enrolment log 537
Subject identification code 456, 504
list 537
completed 538
Subject recruitment, advertisement for 532
Subject screening log 537
Subjects and investigators, compensation to 517
Suicidal tendency and seizures 334
Sulphanilamide tragedy 322
Surgical interventions 432
Surgical procedures, clinical trials with 480
Surveillance 442
active 330
Switchover design 265, 266t
Synagis 49
Synovitis 46
Synthetic biology 438
Systematic literature reviews 274, 275
Systematic review 275
and meta-analysis 274
demerits of 287
and narrative review 276t
methodology 286f
over narrative reviews, advantages of 275
steps of 275
Systemic lupus erythematosus 46
Systemic toxicity studies 15
repeated-dose 15
Takayasu's arteritis 46
Talimogene laherparepvec 44
T-cell 221
Technical skill 311
Tegaserod 334
Telephone contact report 205
Television advertising 190
Tendons 42
Teratogenicity study 16
Terminal deoxynucleotidyl transferase 37
Terminally ill 422
Termination and final report 476
toxoids 44
vaccines 428
Textual data checks 255
Thalidomide tragedy 324, 324f
Therapeutic agent 6
Therapeutic biologic applications 72
Therapeutic confirmatory 31
trials 66, 89
Therapeutic effects 320
Therapeutic equivalents 208
Therapeutic exploratory 30
trials 66, 89
Therapeutic goods administration 224
Tissue 33, 42
and tissue products 42
Title 307
page stating 333
Tools 311
major critical appraisal 282t
web 312
Torsades de Pointes 334
Toxic effects, categorization of 12
Toxicity 20
local 16
studies, acute 15
Toxicokinetic studies 15
Toxicological evaluation 39
Toxicology 486
and pharmacokinetics 484
predictive 33
review 61
testing 17
Toxin cancer drug 17f
Toxoid vaccines 428
Traditional medicines 433
Training 399
Transformed plasma concentration time curve 216f
Transgender 382
Transgenic animal models 13
Translating research 34
Translational biology 33
Translational medicine, development of 32
Translational research 26
phases of 26, 27fc
tools of 32, 32f
Transparency and accountability, principle of 380
Trastuzumab 49
Trial initiation monitoring report 534
Trials, types of 69, 69t
Tuberculin testing 42
Tumor necrosis factor 45
Tunel assay 37
Tuskegee syphilis study 322
Ulcers 254
Union Ministry of Health, Government of India 159
Uppsala monitoring centre 322
Urinary bladder cancer 334
US Food and Drug Administration 133, 173, 222
Useful products, shortage of 48
Vaccine 42, 155
clinical trials of 479
development 425
for children and adults 42
in trial 155
inactivated 428
research, ethics in 153
trials, phases of 479
types of 428b
Vaginal mucous membrane 16
Vaginal toxicity 16
Valdecoxib 334
Valvulopathy 334
Vascular endothelium 39
Vector vaccines, recombinant 428
VigiAccess 336
result output screen 337f
VigiBase 336
VigiLyze 336
VigiMed 336
VigiMine 336
VigiRank 336
Viruses, infection by 5
informed consent and community agreement, principles of 460
principle of 379
hospitalization and discharge 212
recruitment and selection 212
von Willebrand factor complex 42
Vulnerability 147, 415
Vulnerability, causes of 148
Vulnerable groups 422
and individuals 140
and protection 148
Vulnerable populations 415, 415b
Wegener's granulomatosis 46
Wegener's vasculitis 46
Weight loss pill 334
Wellcome trust's 168
Wilcoxon rank sum test 273
Willowbrook study 324
Witness, impartial 454, 501
Women participants, risks for 418b
Working group 354
World Association of Medical Editors 389
World Federation of Science Journalists 313
World Health Organization 133, 224, 319, 333, 335
Worst-case analysis 297
Writer's role 305
Writing career, initiating 309
Writing research articles, requirements for 307
Written information 532
Xenotransplantation 42
Yellow card 329, 349
reporting form 351f
scheme 349
Chapter Notes

Save Clear

  • Drug Discovery Process
  • Translational Research
  • Preclinical In Vitro Screening Methods to Predict Drug Safety during Drug Development Process
  • Drug Discovery and Development of Biologics2

Drug Discovery ProcessChapter 1

Pravina Koteshwar,
Shubha R,
Diana Francis,
SK Gupta
Drug development is a scientific endeavor which is highly regulated due to public health concern. A promising new molecule identified in drug discovery has to go through the complex and time-consuming process of drug development before it becomes available to patients.
The discovery process begins with an understanding of the disease mechanism(s) or cause of the disease and discovery (or identification) of genes and/or proteins involved in causing certain diseases. The identification of genes/proteins responsible for the disease condition is referred to as target identification. These identified targets (gene/proteins) are the potential targets for drugs to interact and to bring about a beneficial effect in a patient. Next step is target validation, where certain studies are performed to confirm that targets (genes/proteins) are actually involved in the disease. In this stage, along with validation of the target, ability of the target to bind to a drug is identified.
After target identification and validation, a lead compound needs to be identified. A lead compound is a substance which has the greatest potential for successful interaction with the identified target. A lead compound is generally selected from libraries containing thousands of compounds. This step of the drug discovery process is known as lead identification. After the lead compound is identified it goes through an optimization process wherein the structure of the compound may be altered to make it safe and efficacious. Once this process is completed the compound is tested first in animal models (such as rats and mice), then in humans to further ascertain its properties.
It takes about ten to twelve years to develop a new drug and the cost is over €800 million, about 60% of which is spent on necessary rigorous clinical trials. For a variety of reasons, fewer than one or two compounds per ten thousand tested actually make it to the market and are authorized for use in patients. In view of the high cost of the drug development process, the industry has to be careful and has to look into the factors that have significant impact on the process and should form basis for allocation of resources.
The decision to develop a new drug by a pharmaceutical company depends on the various factors and one of the key factors is to review and find out the unmet medical needs in the specific therapeutic area in which the company is interested due to strategic reasons. In some cases there may be industry—university or industry—government scientific institutes collaboration that may help to develop a new molecule. New and interesting findings may also come from university, institutes and the lead may be taken over by the pharmaceutical companies for further research.4
Table 1.1   Cost and time involved in drug discovery
Target discovery
2.5 years ↓ 4%
Lead generation and lead optimization
3.0 years ↓ 15%
Preclinical development
1 year ↓ 10%
Phase I, II and III clinical trials
6 years ↓ 68%
FDA review and approval
1.5 years ↓ 3%
Drug to the market
14 years € 880 million
The drug discovery and development process is designed to ensure that only those pharmaceutical products that are both safe and effective are brought to market for the benefit of the patients (Table 1.1).
Overview of Drug Discovery Process
During the last 50 years the philosophy of valuable drugs discovery has evolved from one that was mostly based around chemistry to one that has more biological approach to treat a disease. These changes were not only driven by strategic imperative, but are enabled also by the significant changes in technology that has occurred during the past half century.
Historical Background
Before the existence of pharmaceutical industry, medicines were discovered by accident, and their use was passed down by written and verbal tradition. For example, digitalis is an active principal of a natural product, namely foxglove leaf used to treat dropsy or edema, in which liquids accumulate in the body and causes swelling of tissues and body cavities.
This remedy was described and used some hundred(s) of years before the isolation of the active components. In 1776, William Withering, a physician in England treated a lady who was dying from a disease called dropsy. He left her, expecting her to die shortly, but he later learned that she had recovered after taking an old cure of a garden plant called foxglove. For ten years, Withering conducted experiments to demonstrate the uses of foxglove and discovered that dropsy is actually a symptom of heart disease in which the heart does not pump hard enough to get rid of urine. He showed that foxglove stimulated urination by pumping more liquids to the kidneys. He published his results in 1785, but it was not until the 20th century that the cardiac glycosides, the component of the foxglove plant, were structurally and pharmacologically described
In 1950s and 1960s, pharmaceutical industries’ success in drug discovery had its origins in serendipity, i.e. discovery by accident/chance. Lead molecules were found by chance or from screening the chemical diversity available. These were then optimized by medicinal chemists to produce candidates, which were passed to development and eventually into the market. This method led to discovery of drugs such as Chlorpromazine, Meprobamate, and Benzodiazepines (Chlordiazepoxide, Diazepam) all of which have gone on to become successful medicines.
However, this approach at that time suffered from lack of sufficient molecules with high enough structural diversity, and the common use of animal models meant that other factors such as absorption, metabolism, brain penetration, and pharmacokinetics had profound effects on the number of active molecules found. In addition, many molecules that showed activity in the models were of unknown mechanism. This greatly impeded the development of back-ups when the lead failed due to toxicity or poor pharmacokinetics.
To combat these problems, a more rational approach was developed based around the structure of the agonist (i.e., hormones and neurotransmitters) and its receptor. This was set against a background of studying biological/physiological systems in animal tissues. Thus, knowledge around molecular determinants that contribute to affinity and efficacy enabled a generation of specific and potent agonists and antagonists to be developed.
Steps in Drug Discovery
The advent of molecular biology, coupled with advances in screening and synthetic chemistry technologies, has allowed a combination of both knowledge around the receptor and random screening to be used for drug discovery.5
zoom view
Fig. 1.1: Steps in new drug development
The process of drug development is divided into two stages: New lead discovery and new product development (clinical development) (Fig. 1.1).
Target Identification
Before any potential new medicine can be discovered the disease to be treated needs to be understood, to unravel the underlying cause of the condition. Even with new tools and insights, research on disease mechanism takes many years of work and, too often, leads to frustrating dead ends. And even if the research is successful, it will take many more years of work to turn this basic understanding of what causes a disease into a new treatment.
The disease mechanism defines the possible cause or causes of a particular disorder, as well as the path or phenotype of the disease. Understanding the disease mechanism directs research and formulates a possible treatment to slow or reverse the disease process. It also predicts a change of the disease pattern and its implications.
Disease mechanisms can be broadly classified into the following groups:
  • Defects in distinct genes—genetic disorders
  • Infection by bacteria, fungi, viruses, protozoa or worms.
  • Immune/autoimmune disease
  • Trauma and acute disease based on injury or organ failure
  • Multicausal disease.
The identification of new and clinically relevant molecular targets for drug intervention is of outstanding importance to the discovery of innovative drugs.
It is estimated that up to 10 genes contribute to multifactorial diseases, which are linked to another 5–10 gene products in physiological and pathophysiological circuits which are also suitable for intervention with drugs. Environmental factors such as diet, exposure to toxins, trauma, stress, and other life experiences are assumed to interact with genetic susceptible factors to result in disease. Thus, drug targets may include molecular pathways related to environmental factors.
Current drug therapy is based on less than 500 molecular targets with potential to exploit at least 10 times the number of targets in the future. Targets for therapeutic intervention can be broadly classified into these categories:
  • Receptors
  • Proteins and enzymes
  • DNA
  • RNA and ribosomal targets
Methods used for target identification include classical methods such as cellular and molecular biology and newer technique such as genomics and proteomics.
In the classical method, animal and human cell lines are used to identify the potential target of drug action. Two key research avenues involve the enzymes that metabolize the molecules (drugs) and proteins that act as receptors.6
The newer methods like genomics and proteomics along with bioinformatics are aimed at discovering new genes and proteins and quantifying and analyzing gene and protein expression between diseased and normal cells.
Target Validation
Target validation requires a demonstration that a molecular target (such as an enzyme, gene or protein) is actually involved in a disease process, and that binding of a drug to the target is likely to have a curative effect.
The validation of a molecular target in vitro (in an artificial environment) usually precedes the validation of the therapeutic concept in vivo (in a living organism); together this defines its clinical potential. Validation involves studies in intact animals or disease-related cell-based models that can provide information about the integrative response of an organism to a pharmacological intervention and thereby help to predict the possible profile of new drugs in patients.
Targets are validated with:
  • In vitro models: RNA and protein expression analysis and cell based assays for inhibitors, agonists (substances which activate the target) and antagonists (counteracts the effect of a target). In vitro assays are more robust and cost-effective, and have fewer ethical implications than whole-animal experiments. For these reasons they are usually chosen for high-throughput screening, a process through which active compounds, antibodies or genes which modify a particular biomolecular pathway can be identified rapidly.
  • In vivo models: In vivo testing involves testing in whole animals. It assesses both pharmacology and biological efficacy in parallel. Animal models that are capable of mimicking the disease state (e.g. animals mimicking diabetes), by adding/modifying or deleting certain genes are used. These animal models are referred to as knock-in and knock-out animal models.
Along with validation of the target it is essential to predict the “druggability” of the target. The “druggability” of a given target is defined either by how well a therapeutic agent, such as small drug molecule or antibody, can access the target (i.e. ability of a target to bind to drug).
Knowledge of three–dimensional structure will help to unravel the physiological roles of target proteins and contribute to “chemical” target validation and also enable the prediction of “druggability” of the protein. One of the successfully targeted targets is G-protein coupled receptors (GPCRs), and a sizable number of drugs prescribed today hit this particular class. Therefore, the GPCR target type is considered druggable.
In summary, target validation is one of the bottlenecks in drug discovery, as this phase is less adaptable to automation. Careful validation of target not only with respect to relevance to disease but also druggability will reduce the failure rate and increase the efficiency of drug discovery.
Lead Identification
In this phase, compounds which interact with the target protein and modulate its activity are identified.
The lead identification process starts with the development of an assay which will be followed by screening of compound libraries. The quality of an assay determines the quality of data. The assay used should fulfill these criteria: relevance, reliability, practicability, feasibility, automation and cost effectiveness.
Primary screens will identify hits. Subsequently, confirmation screens and counter screens will identify leads out of the pool of hits. This winnowing process is commonly referred to as “hits-to-leads.”
The success of screening depends on the availability of compounds, as well as their quality and diversity. Efforts to synthesize, collect, and characterize compounds are an essential and costly part of drug discovery.
There are several sources for compounds:
  • Natural products (NPs) from microbes, plants, or animals. NPs are usually tested as crude extracts first, followed by isolation and identification of active compounds.
  • (Random) collections of discreetly synthesized compounds.
  • Focused libraries around certain pharmacophores.7
  • Random libraries exploring “chemical space.”
  • Combinatorial libraries.
A primary screen is designed to rapidly identify hits from compound libraries. The goals are to minimize the number of false positives and to maximize the number of confirmed hits. Depending on the assay, hit rates typically range between 0.1% and 5%. This number also depends on the cutoff parameters set by the researchers, as well as the dynamic range of a given assay.
Typically, primary screens are initially run in multiplets of single compound concentrations. Readouts are expressed as percent activity in comparison to a positive (100%) and a negative (0%) control. Hits are then retested a second time (or more often, depending on the assays’ robustness). The retest is usually run independently of the first assay, on a different day. If a compound exhibits the same activity within a statistically significant range, it is termed a confirmed hit, which can proceed to dose-response screening.
Establishing a dose-response relationship is an important step in hit verification. It typically involves a so-called secondary screen. In the secondary screen, a range of compound concentrations usually prepared by serial dilution is tested in an assay to assess the concentration or dose dependence of the assay's readout. Typically, this dose-response is expressed as an IC50 in enzyme-, protein-, antibody-, or cell-based assays or as an EC50 in in vivo experiments. IC50 is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug is needed to inhibit a given biological process by half. EC50 (half maximal effective concentration) refers to the concentration of a drug or antibody which induces a response halfway between the baseline and maximum. The EC50 of a graded dose response curve therefore represents the concentration of a compound where 50% of its maximal effect is observed.
Confirmed hits proceed to a series of counterscreens. These assays usually include drug targets of the same protein or receptor family, for example, panels of GPCRs (G-protein coupled receptors) or kinases. In cases where selectivity between subtypes is important, counterscreens might include a panel of homologous enzymes, different protein complexes, or heterooligomers. Counterscreens profile the action of a confirmed hit on a defined spectrum of biological target classes. The number and stringency of counterscreens can vary widely and depend on the drug target.
One of the goals throughout the discovery of novel drugs is to establish and confirm the mechanism of action (MOA). In an ideal scenario, the MOA remains consistent from the level of molecular interaction of a drug molecule at the target site through the physiological response in a disease model, and ultimately in the patient.
The tools used for lead identification are: High throughput screening, in silico/virtual screening, NMR-based screening and X-ray crystallography.
  • High-throughput screening (HTS) aims to rapidly assess the activity of a large number of compounds or extracts on a given target. Entire in-house compound libraries with millions of compounds can be screened with a throughput of 10,000 (HTS) up to 100,000 compounds per day (ultra HTS) using robust test assays.
  • Virtual (in silico) screening sifts through large numbers of compounds based on a user-defined set of selection criteria. Selection criteria can be as simple as a physical molecular property such as molecular weight or charge, a chemical property such as number of heteroatoms, number of hydrogen-bond acceptors or donors. Selection criteria can be as complex as a three dimensional description of a binding pocket of the target protein, including chemical functionality and solvation parameters. In silico screening can involve simple filtering based on static selection criteria (i.e., molecular weight) or it can involve actual docking of ligands to a target site, which requires computer-intensive algorithms for conformational analysis, as well as binding energies.
  • NMR-based screening fills the gap between HTS and virtual screening. This method combines the random screening approach with the rational structure-based approach to lead discovery.
  • X-ray crystallography: X-ray crystallography uses X-rays to determine the structure and functioning of biological molecules. The point at which X-ray crystallography comes into the drug discovery and development process depends on the purpose for which it is used. X-ray crystallography is being increasingly used to determine the three-dimensional structure of a lead compound. The information accumulated during the process of lead identification by means of X-ray crystallography is essential for the next stage of drug development which is lead optimization.8
Following are the criteria for hits to be regarded as leads:
  • Pharmacodynamic properties: Efficacy, potency and selectivity in vitro and in vivo;
  • Physicochemical properties: For example, Lipinski's “rule of five”
  • Pharmacokinetic properties: For example, permeability in the Caco-2 assays
  • Chemical optimization potential
  • Patentability.
Lead Optimization
Lead optimization is the complex, nonlinear process of refining the chemical structure of a confirmed hit to improve its drug characteristics with the goal of producing a preclinical drug candidate. This stage constitutes the tightest bottleneck in drug discovery.
Lead optimization employs a combination of empirical, combinatorial, and rational approaches that optimize leads through a continuous, multi-step process based on knowledge gained at each stage. Typically, one or more confirmed hits are evaluated in secondary assays, and a set of related compounds, called analogs, are synthesized and screened.
The testing of analog series results in quantitative information that correlates changes in chemical structure to biological and pharmacological data generated to establish structure activity relationships (SAR).
The lead optimization process is highly iterative. Leads are assessed in pharmacological assays for their “drug-likeness.” Medicinal chemists change the lead molecules based on these results in order to optimize pharmacological properties such as bioavailability or stability. At that point the new analogs are fed back into the screening hierarchy for the determination of potency, selectivity, and mechanism of action.
Pharmacokinetics (PK)/Pharmacodynamics (PD)/Absorption, Distribution, Metabolism, Excretion (ADME) studies are an integral part of lead optimization. They feed back into the medicinal chemistry effort aiming to optimize the physicochemical properties of new leads in terms of minimal toxicity and side effects, as well as of maximum efficacy toward disease. PK/PD/ADME studies rely heavily on analytical methods and instrumentation. The recent innovation and progress in mass spectroscopy, (whole-body) imaging, and chromatography technology (HPLC, LC-MS, MS) have tremendously increased the quantity and quality of data generated in PK/PD experiments.
This data is then fed into the next optimization cycle. The lead optimization process continues for as long as it takes to achieve a defined drug profile that warrants testing of the new drug in humans (Fig. 1.2).
Lead Optimization: Formulation and Delivery
Formulation development: It is the process of turning an active compound into a form and strength suitable for human use.
Formulation and delivery of drugs is an integral part of the drug discovery and development process. Indeed, formulation problems and solutions influence the design of the lead molecules; they feed back into the iterative lead optimization cycle, as well as the preclinical and clinical evaluations.
If formulation substances are not generally recognized as safe (GRAS), they become part of the safety assessment and their PK/PD/ADME behavior, as well as toxicity profile, needs to be documented in the IND (investigational new drug) application. In fact, side effects such as local irritation or allergic reactions are often attributable to drug formulation, not the active pharmaceutical ingredient (API).
Formulation substances might exhibit different biological activity than the actual drug.
Indeed, a sizable number of drug discovery and development programs in the pharmaceutical and biotech industry are centered on new ways of formulating already known and even marketed drugs to increase their efficacy or safety profiles.9
zoom view
Fig. 1.2: Depicts use of In-Silico technology in various stages of selection of a drug candidateSource: www.scfbio-iitd.res.in/image/insilicodrug.JPG
Stakeholders in New Drug Development
Expertise involved to achieve goal of new drug development are numerous, once the management team sets therapeutic targets, budgets and resources, departments involved in drug discovery include:
  • Research and development: It is responsible for finding new compounds and assuring that they are safe enough to test in humans.
  • Medicinal chemists: Whose responsibilities are to prepare new chemical entities which can be screened for biological activity and to prepare compounds which have been found to be active (new leads) in quantities sufficient for advanced testing.
  • Pharmacology/molecularbiology/screening: Examines each New Chemical Entity (NCE) in a set of high throughput screens.
  • Safety evaluation: Demonstrates that the NCE and its metabolites do not accumulate and do not cause harm during short-term administration.
  • Formulations research: Develop a dosage form that is absorbed into the bloodstream when administered and is stable when stored for long periods of time. The concentration in the blood is an important factor in early development. The potential new drug must reach and maintain a level sufficient to sustain its biological effect; these studies are initially conducted in animals, to establish the doses for human studies.
  • Process research: Manufacture the NCE in sufficient quantity for advanced testing, dosage form development.
  • Legal affairs: Writes and files the patents necessary to protect a company's inventions.
  • Research administration: Collects the material generated by all of the departments and formats it into a request for exemption so that the NCE can be tested in humans. This submission is the Investigational New Drug (IND).10
Need for Systematic Approach in New Drug Discovery
The pharmaceutical industry is operating in a world where medicines have to add real value in an environment in which costs are under constant pressure. This high cost is causing the evolution of the drug discovery process so that high percentages of efficient pipeline molecules are delivered to market quickly. The following needs to be considered to have a systematic approach in drug discovery:
Unmet Medical Needs
A constant driver for developing new medicines has always been the unmet medical need. However, there are now strong pressures to treat the underlying cause of the disease rather than provide symptomatic relief alone. This is reinventing the biological systems approach, but using humans rather than animals. In order to accomplish this, the investment that has already been initiated in technologies such as noninvasive imaging, clinical genetics and genomics will increase. This is now assured with the publication of the human genome.
The lack of disease models in animals in some therapeutic areas is a major driver to understand the human pathology. This is particularly relevant in the central nervous system (such as depression, bipolar disorder, schizophrenia) area. In these diseases, with no simple ways to validate the targets in the complex intact system, option left is targeting components such as receptor or biochemical systems. In these cases, the scientist is constrained to collecting a logical series of evidence that associates the target with the disease. Along with the existing imaging methods such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), application of technologies like clinical genetics and genomics will strengthen the understanding of the correlation between disease and specific receptors.
Clinical genetics networks are being put into place to allow sufficient information on probands (proband denotes a particular subject (person or animal) first affected with genetics disorder) to be collected, such that associations between particular gene(s) and disease (target validation) can be made and eventually resulting in identification of a lead compound.
The advent of the human genome's publication now offers a great opportunity for the understanding of the genetic make-up of disease and will furnish specific gene products and/or pathways as new targets that would not have been previously identified. Importantly, they will be born out of human data, so again adding to the level of confidence in the validity of the target.
Attrition is another driver for systematic approach in drug discovery for overall success rate. Attrition has remained static despite the investment in the new technologies. This reflects the fact that good molecules need more than potency and selectivity to be successful, and it is in these areas where technology has been concentrating in the last few years. The challenges ahead lie in reducing the risk of not obtaining efficacy in humans, and in increasing the developability of the molecules.
Efficacy: Many new mechanisms fail when they get into humans through lack of efficacy. This is one of the risks that the industry takes when developing such molecules. One way to diminish risk is to get better validation in humans (proof-of-concept i.e. proof of efficacy) as soon as possible. The use of imaging, genetics, and genomics has already been discussed earlier as a way to help build early confidence in the target.
It is now recognized that fast decision making saves money and allows resources to be more effectively used. Proof-of-concept is generally obtained in phase III. Killing compounds in Phase III is extremely costly; therefore it is a disadvantage to obtain proof of concept at such a late stage. Thus, simple proof-of-concept (POC) studies are being sought in phase I or phase II. If POC were to be obtained during phase I and phase II instead of phase III it will provide sponsors with sufficient evidence which can be used to assess the clinical and commercial potential of the drug and in turn eliminate potential failures from the drug discovery pipeline.11
In addition, diagnostics will play a greater role in helping to choose patient populations, at least initially to show that the mechanism works. This will see greater use of imaging, proteomics and genetics in helping to identify the right patient group.
In the meantime, a better balance of novel molecules and those that are precedented will be seen in the drug discovery portfolio. This will mean that a higher proportion of molecules will not fail for efficacy. However, this strategy creates its own problems in that to be successful in the marketplace the molecule will need to be differentiated from those already present. To do this in the clinic will add to the cost and to the overall cycle time, thus these problems will need to be addressed much earlier in the process.
Developability: A large proportion of molecules fail due to of lack of developability. Prentis et al. suggest that this proportion is as high as 69%, broken down as toxicity (22%), poor biopharmaceutical properties (41%) and market reasons (6%). This is not a new revelation, and efforts have been actively followed to automate and miniaturize methods to measure solubility, stability, pKa (value which describes the acid and basic properties of a substance), bioavailability, brain penetration, and various toxicity. These methods (combinatorial lead optimization) are being applied to leads during optimization, but need to be developed further and applied even earlier to maximize their impact. This is particularly true for toxicity screens, where it can be predicted that a great deal of effort will be done in the next few years.
Great extent of work is being done in the field of predictive algorithms, and Pfizer has developed tool known as the “rule of 5”. This is an awareness tool for medicinal chemists that suggest that there will be poor absorption if a molecule has two or more of the following: more than 5 H-bond donors; a molecular weight >500; c log P >5; the sum of Ns and Os (a rough measure of H-bond acceptors) >10.
While it is inherently costly to try to fix poor developability by formulation, pharmaceutical development will become more actively engaged in alternative formulations and delivery systems during the lead optimization phase. The trend toward higher potency compounds, that reduces cost of goods, also allows, due to the smaller dose, alternative delivery systems such as inhalation, nasal, buccal and sublingual absorption.
Cycle Times
The need to speedup the delivery of molecules to the market is another driver to have systematic approach in drug discovery. The regulatory environment and the growing complexity of drug development affect the time taken for a drug to reach the market.
Screening automation and combinatorial chemistry have greatly reduced the time to candidate selection. This will almost certainly decrease again by further application of techniques like chemoinformatics to aid library design, both for those to be used for random screening and those within the process of lead optimization. As mentioned above, continual automation of developability criteria will also speed up the process by selecting compounds with a high probability of succeeding. This raises the concept that speed in each phase should not always be the major driver. A candidate for development goes forward with all of its associated baggage. Fixing problems becomes costly and may lead to a suboptimal product that cannot fulfill its medical and commercial potential. Thus, spending time choosing the right candidate will have major benefits downstream, both in terms of speed and value. The same concept applies to development candidates in phase III. Differentiation may not be obvious if the mechanism is precedented with another marketed product. Thus, differentiation will become a challenge, which potentially will increase the time in phase III. To aid in this process and help in choosing which differentiators to pursue, this problem will need to be addressed much earlier. This might stimulate automated assays for common side effects of drugs as part of the candidate selection criteria during the lead optimization stage.
Economic Value
There is growing internal pressure to increase productivity while controlling costs. This has led to the drive for high-value molecules in diseases with high unmet need. An extension of this concept is the “blockbuster” approach where projects that deliver medicines with potential peak sales greater than 1 billion pounds are given the highest priority. This means that portfolio management will become even more important with an associated greater interaction between R&D and the commercial functions.12
Thus, new portfolio tools will also be major contributors to the future process of drug development. The real value of medicines to the health of society is only now beginning to be recognized. It has taken many years of persuasion that medicines can have profound economic benefit.
The push to raise health, economic, and quality-of-life issues has produced a counter response from some regulators that the industry demonstrates added value in its novel medicines. Thus, committees like National Institute for Clinical Excellence (NICE) in the UK will put pressure on the process to produce medicines that have significant value for society. This will mean that in the future more outcome studies will be needed to demonstrate quality-of-life and economic benefit.
Preclinical drug development is a stage that begins before clinical trials (testing in humans) during which important safety and pharmacology data is collected. Clinicians and regulators need to be reassured that information concerning all of these different aspects is available to enable clinical trials to progress and ultimately to support regulatory decisions on whether a new drug can be approved for marketing. Most regulatory toxicity studies are conducted in animals to identify possible hazards from which an assessment of risk to humans is made by extrapolation. Regulatory agencies request studies in a rodent (e.g. rat) and a non rodent (e.g. dog). The choice of animal species is made based on the similarities of its metabolism to humans or the applicability of desired pharmacological properties to humans. It is not possible or ethical to use animals in large numbers, to compensate for the same it is assumed that increasing the dose and prolonging the duration of exposure will improve both sensitivity and predictivity of the tests.
Preclinical research includes synthesis, purification and animal testing which is done to measure the biological activity and safety of an investigational drug or device. Preclinical research is conducted by pharmaceutical companies early in the process of new drug development. This research takes place in either the part or whole animal to determine important information, including: therapeutic effects the drug may have, potential side effects and toxicities and metabolism and clearance of the drug in the body. Good results in the preclinical or animal stage do not necessarily mean that similar results will be found when the drug is given to healthy volunteers or patients.
The main goals of preclinical studies are to determine a drug's pharmacodynamics (PD), pharmacokinetics (PK) and toxicity through animal testing. This data allows researchers to estimate a safe starting dose of the drug for clinical trials in humans.
The goals of the nonclinical safety evaluation include:
  • Categorization of toxic effects with respect to target organs, dose dependence, relationship to exposure, and potential reversibility. This information is important for the estimation of an initial safe starting dose for the human trials
  • The identification of specific parameters for clinical monitoring for potential adverse effects.
  • The nonclinical safety studies, although limited at the beginning of clinical development, should be adequate to characterize potential toxic effects.
The need for nonclinical information including toxicology, pharmacology and pharmacokinetics to support clinical trials is addressed in the ICH (International Conference on Harmonization) Safety guidelines. Typically, both in vitro and in vivo tests will be performed. Studies of a drug's toxicity on organs targeted by that drug, as well as any long-term carcinogenic effects or toxic effects on mammalian reproduction will be estimated in preclinical studies.13
Types of Preclinical Studies
  • In vitro studies
  • In vivo studies
  • Ex vivo studies.
In Vitro Studies
In vitro studies are done for testing of a drug or chemical's effect on a specific isolated tissue or organ maintaining its body functions. Basic instruments used for isolated tissue experiments are organ baths, recording devices.
Few examples of in vitro studies include:
Langendorff's heart preparation: The objective is to study the effect of drugs—noradrenaline, acetylcholine and isoprenaline on the coronary blood flow and heart rate and force of contraction using rat isolated heart.
  • Ileum preparation: The objective is to record the effect of drugs—histamine and antihistamine by using segment of ileal portion of Guinea pig.
  • Rectus abdominus muscle preparation: The objective is to record the effect of drugs d-tubocurarine by using rectus abdominis muscle of frog.
In Vivo Studies
It is a Latin term meaning (with) “in the living”. It indicates the use of a whole organism/animals (for an experiment). Researchers use laboratory animals as models of humans or some other target species to achieve long-term objective, such as developing a new drug for a particular disease, screening a particular compound for human toxicity, studying a gene or mutation found in both animals and human; to achieve short-term objective to find out how the animal responds to the treatment. If it is a faithful model of humans, then humans should respond in the same way. Animals, and other models, are used because the research cannot be done on humans for practical or ethical reasons.
Purpose of models: A specific model is chosen because it is believed to be appropriate to the condition being investigated and is thought likely to respond in the same way as humans to the proposed treatment(s) for the character being investigated.
Having chosen the model it is essential that any experiments in which it is used are well designed, i.e. are capable of demonstrating a response to a treatment. If the model happens to be insensitive or the experiments are badly designed so that they are incapable of distinguishing between treated and control groups, say as a result of using too few animals, then the model is not appropriate to its purpose
Animal models are used to define a new molecule's:
  • Therapeutic potential
  • Toxicity potential
  • Pharmaceutical properties and metabolic pathways
  • Mechanism and specificity of action (lead molecules).
In vivo studies are preferred over in vitro studies for the following reasons:
  • Greater similarity to human studies when compared to in vitro screening
  • Drug effects modified by physiological mechanisms can be studied
  • ADME of drugs that modifies drug effects are also factored
  • Most animal systems are similar to human systems
  • Effect of drug is studied on complete systems rather than tissues and organs
  • Drugs acting on central nervous system, cardiovascular system, gastrointestinal system, and other systems can be studied
  • Results easier to interpret and extrapolate
Some of the examples of in vivo studies are:
  • Non-invasive method—rat tail cuff method
  • Invasive methods—BP recording in anesthetized dog or cat
Transgenic animal models: Partly due to the low speed and high cost of conventional animal models (typically rodents) and the relatively high number of preliminary hits from HTS (High Throughput Screening), alternative small-animal models have emerged. The small size, high utility, and experimental tractability (i.e. easy to manage) of these animals enable cost-effective and rapid screening of numerous compounds. 14Technologies for engineering the mouse genome have made it possible to create various disease models for use in screening corresponding therapeutic compounds. Knockout mouse models have been shown to be highly predictive of the effects of drugs that act on target specific gene-sequence alterations or manipulate the levels and patterns of target-gene expression. Using these techniques, researchers can generate specific disease models to validate targets as therapeutic intervention points and screen drug candidates. Transgenic technology represents an attractive approach to reduce the attrition rate of compounds entering clinical trials by increasing the quality of the target and compound combinations making the transition from discovery into development. Some of the transgenic animal models are Obese Zucker rats for testing obesity related hypertension, genetically epilepsy prone rats for testing antiepileptic drugs.
Ex Vivo Studies
In ex vivo, experiment is performed in vivo and then analyzed in vitro. The organs of the animals are detached from the body and replaced once an experiment is performed. Then the animals are kept under observation and findings recorded for a set duration.
General requirements for conducting preclinical studies are:
  • Toxicity studies should comply with Good Laboratory Practice (GLP).
  • These studies should be performed by suitably trained and qualified staff employing properly calibrated and standardized equipment, done as per written protocols.
  • Standard operating procedures (SOPs) should be followed.
  • Test substances and test systems (in vitro or in vivo) should be properly characterized and standardized.
  • All documents belonging to each study, including its approved protocol, raw data, draft report, final report, and histology slides and paraffin tissue blocks should be preserved for a minimum of 5 years after marketing of the drug.
Animal Pharmacology Studies
Safety pharmacology studies are studies that investigate potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure within the therapeutic range or above. Specific pharmacological actions are those which demonstrate the therapeutic potential for humans.
Based on the individual properties and intended uses of an investigational drug, specific studies that need to be conducted and their design will vary. Only scientifically validated methods should be used.
The essential safety pharmacology is to study the effects of the test drug on vital functions. Vital organ systems such as cardiovascular, respiratory and central nervous systems should be studied.
In addition to the essential safety pharmacological studies, additional supplemental and follow-up safety pharmacology studies may need to be conducted as appropriate. These depend on the pharmacological properties or chemical class of the test substance, and the data generated from safety pharmacology studies
Specific and essential pharmacological studies should be conducted to support use of therapeutics in humans. Essential safety pharmacology studies may be excluded or supplemented based on scientific rationale. Also, the exclusion of certain test(s) or exploration(s) of certain organs, systems or functions should be scientifically justified. Supplemental Safety Pharmacology Studies are required to investigate the possible adverse pharmacological effects that are not assessed in the essential safety pharmacological studies and are a cause for concern.
The following factors are to be considered when specific tests are to be conducted:
  • Mechanism of action
  • Class specific effects
  • Ligand binding or enzyme assay suggesting a potential for adverse events
Safety pharmacology studies are usually not required when:
  • Product is to be used for local application, e.g. dermal or ocular,
  • The pharmacology of the investigational drug is well known, and/or15
  • Systemic absorption from the site of application is low.
Safety pharmacology testing is also not necessary, in case of a new derivative having similar pharmacokinetics and pharmacodynamics. For biotechnology-derived products that achieve highly specific receptor targeting, it is often sufficient to evaluate safety pharmacology endpoints as a part of toxicology and/or pharmacodynamic studies; therefore, safety pharmacology studies can be reduced or eliminated for these products. For biotechnology-derived products that represent a novel therapeutic class and/or those products that do not achieve highly specific receptor targeting, a more extensive evaluation by safety pharmacology studies should be considered.
In vivo safety pharmacology studies should be designed to define the dose-response relationship of the adverse effect observed. When feasible the time course (e.g. onset and duration of response) of the adverse effect should be investigated.
In vitro studies should be designed to establish a concentration-effect relationship. The range of concentrations used should be selected to increase the likelihood of detecting an effect on the test system. The upper limit of this range may be influenced by physicochemical properties of the test substance and other assay specific factors (Figs 1.3A and B).
Animal Toxicity Studies
Toxicokinetic studies should be conducted to assess the systemic exposure achieved in animals and its relationship to dose level and the time course of the toxicity study. Other objectives of toxicokinetic studies include:
  • To relate the toxicological findings to clinical safety.
  • To support in selecting species, treatment regimen and designing subsequent non-clinical toxicity studies.
Several toxicity studies need to done before a drug goes into the clinical phase. They are:
Systemic Toxicity Studies
Single dose study (acute toxicity studies): Single dose studies in animals are essential for any pharmaceutical product intended for human use. The information obtained from these studies is useful in choosing doses for repeat-dose studies, providing preliminary identification of target organs of toxicity, and occasionally, revealing delayed toxicity. Acute toxicity studies may also aid in the selection of starting doses for phase I human studies, and provide information relevant to acute overdosing in humans.
Repeated-dose systemic toxicity studies: The primary goal of repeated dose toxicity studies is to characterize the toxicological profile of the test compound following repeated administration. This includes identification of potential target organs of toxicity and exposure/response relationships, and may include the potential reversibility of toxic effects. This information should be part of the safety assessment to support the conduct of human clinical trials and the approval of a marketing authorization.
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Figs 1.3A and B: A researcher studies a rat being used in medical experiment
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Figs 1.4A and B: Reproductive studies done on rats
The decision whether a developmental toxicity study needs to be performed should be made on a case-by-case basis taking into consideration historical use, product features, intended target population and intended clinical use.
Male Fertility Studies
Male fertility studies are designed to provide general information concerning the effects of a test substance on male reproductive system such as gonadal function.
Female Reproduction and Developmental Toxicity Studies
Female fertility studies are designed to provide general information concerning the effects of a test substance on female reproductive system such as ovary function and lactation. These studies need to be carried out for all drugs proposed to be studied or used in women of child-bearing age (Figs 1.4A and B).
Teratogenicity Study
The drug should be administered throughout the period of organogenesis in animals if the test drug is intended for women of child-bearing age and if women of child-bearing age are to be included as subjects in the clinical trial stage.
Perinatal Study
This study is specially recommended if the drug is to be given to pregnant or nursing mothers for long periods or where there are indications of possible adverse effects on fetal development.
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Fig. 1.5: Rabbits kept ready for ocular tests
Local Toxicity
These studies are required when the new drug is proposed to be used by some special route (other than oral) in humans. The drug should be applied to an appropriate site (e.g. skin or vaginal mucous membrane) to determine local effects in a suitable species. If the drug is absorbed from the site of application, appropriate systemic toxicity studies will also be required. Examples of Local Toxicity are Dermal Toxicity Study, Vaginal Toxicity Study, Photo-Allergy, Rectal Tolerance Test, Ocular Toxicity Studies, Inhalational Toxicity Studies, Hypersensitivity (Fig. 1.5).
Genotoxicity refers to potentially harmful effects on genetic material (DNA) which may occur 17directly through the induction of permanent transmissible changes (mutations) in the amount or structure of the DNA within cells. In vitro (artificial environment) and in vivo (in living organisms) genotoxicity tests are conducted to detect compounds which induce genetic damage directly or indirectly. These tests should enable hazard identification with respect to damage to DNA and its fixation. Damage to DNA can occur at three levels:
  • Point mutations
  • Chromosomal mutations
  • Genomic mutations
The following standard test battery is generally expected to be conducted:
  • A test for gene mutation in bacteria (Ames Test).
  • An in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma assay.
  • An in vivo test for chromosomal damage using rodent hematopoietic cells.
Studies should be performed for all drugs that are expected to be clinically used for six months or more than six month as well as for drugs used frequently in an intermittent manner in the treatment of chronic or recurrent condition (Figs 1.6 and 1.7).
Limitations of Preclinical Studies
The purpose of preclinical work (animal pharmacology/toxicology testing) is to develop adequate data to undergird a decision that it is reasonably safe to proceed with human trials of the drug. Mice and rats are the most widely used host species for preclinical drug development for a variety of important reasons. First, rodents have a comparatively short life cycle. Rodent research studies can be time-compressed to evaluate disease progression with or without therapeutic intervention. The short life cycle has also lent itself to the development of many unique inbred strains. In addition, rodents, especially mice, have been thoroughly characterized genetically and were the first animal species to be genetically modified by transgenic and gene knock-out methods. The microbiology of rats and mice has been extensively studied. Sophisticated husbandry, biosecurity practices, and diagnostic testing effectively control environmental conditions and adventitious infections with pathogenic microorganisms that might cloud the interpretation of experimental findings.
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Fig. 1.6: Carcinogenicity test done on mice
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Fig. 1.7: Lab mice showing one with a tumor, the other treated with toxin cancer drug
Because genetic, environmental, and microbiological variables can be comprehensively defined and carefully controlled, data from studies using rodents are invaluable for characterizing disease conditions and therapies. Also, research reagents are more widely available for biochemical testing of rodents then for testing other laboratory animal species.
However, animal studies have certain limitations:
  • Not reliably predictive of human responses due to species variation and extrapolation, Poor disease models, confounding effects of laboratory confinement, stress, environment and food.18
  • Repeatability/reproducibility is difficult
  • Expensive, time-consuming, and not amenable to high throughput. Toxicity studies are costly in terms of animals and resources. For a product developed for chronic oral therapy approximately 4,000 rats, 1300 mice, 100 rabbits, 50 guinea pigs and 160 dogs, a total of nearly 5,000 animals are used. If the fetus and offspring from the reproductive toxicity studies are included, the number doubles.
  • Attempting to translate research from animals to humans not as efficient as studying humans directly—92% of drugs that pass preclinical testing, almost all in vivo animal-based, fail in clinical trials.
  • Ethical issues in using animal for studies.
Predictive software and advanced in vitro technologies, have improved both the efficiency of laboratory animal experiments and the quality of data to make decisions about dosing with New Chemical Entity. It is very clear that animals are not the way to explore libraries of 1 million or even 25,000 compounds. On the other hand, much can be done when the number that survives the in silico and in vitro process reaches 1000 or fewer. There are several very compelling new technologies now available that include: whole-body imaging, protein biomarkers monitoring by multichannel immunoassays, flow cytometry of blood components, metabonomic component monitoring using in vivo microdialysis and in vivo ultrafiltration, automated blood sampling for awake, freely-moving animals [pharmacokinetics (PK) and biomarkers] and parallel monitoring of physiological and electrocardiogram and psychological parameters. While not all of these data sources can be enabled simultaneously, many of them can be accomplished automatically, raising the quality of information available from animal models dramatically.
FDA Requirements for Preclinical Studies
It is essential to ensure the quality and reliability of safety studies and this can be achieved by adhering to Good Laboratory Practices (GLP). The purpose of GLP is to obtain data on properties and safety of these substances with respect to human health and environment, to promote development of quality test data, such comparable data forms the basis of mutual acceptance across organizations/countries, confidence in and reliability of data from different countries will prevent duplicating tests, saves time, energy and resources.
For every 5000 drug compounds that enter preclinical testing in the United States, only about 5 will eventually be considered acceptable to test in humans. Of those final 5, only 1 drug may actually receive approval for use in patient care.
Under FDA requirements, a sponsor must first submit data showing that the drug is reasonably safe for use in initial, small-scale clinical studies.
Depending on whether the compound has been studied or marketed previously, the sponsor may have several options for fulfilling this requirement:
  • Compiling existing nonclinical data from past in vitro laboratory or animal studies on the compound
  • Compiling data from previous clinical testing or marketing of the drug in the United States or another country whose population is relevant to the US population or
  • Undertaking new preclinical studies designed to provide the evidence necessary to support the safety of administering the compound to humans.
At the preclinical stage, the FDA will generally ask, at a minimum that sponsors:
  • Develop a pharmacological profile of the drug;
  • Determine the acute toxicity of the drug in at least two species of animals
  • Conduct short-term toxicity studies ranging from 2 weeks to 3 months, depending on the proposed duration of use of the substance in the proposed clinical studies.
Organization of Economic Cooperation and Development (OECD) framed guidelines known as Good Laboratory Practice (GLP). GLP gives guidelines for animal testing facility (Fig. 1.8), housing the animals, responsibilities and duties of personnel conducting the animal studies, equipment, quality control, etc.
In India, the Committee for the Purpose of Control and Supervision for Experiments on Animals (CPCSEA) ensures that the animal facilities are well maintained and experiments are conducted as per internationally accepted norms. Organizations or individuals that use animals for research, testing and teaching are required to have a code of ethical conduct which sets out the policies and procedures which must be followed when using animals for research, testing or teaching19
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Fig. 1.8: Animal testing facility according to good laboratory practice (GLP) requirements
It needs to specify provisions for compliance monitoring, the collection and maintenance of information on projects involving animals and animal management practices and facilities, and allow the fair and prompt handlings of complaints from any member of the animal ethics committee An Institutional Animal Ethical Committee (IAEC) must be established by an institution (or group of organizations) which has an approved code of ethical conduct.
A final report shall be prepared for each non clinical laboratory study and shall include:
  • Name and address of the facility performing the study and the dates on which the study was initiated and completed.
  • Objectives and procedures stated in the approved protocol, including any changes in the original protocol.
  • Statistical methods employed for analyzing the data.
  • The test and control articles identified by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics.
  • Stability of the test and control articles under the conditions of administration.
  • A description of the methods used.
  • A description of the test system used. Where applicable, the final report shall include the number of animals used, sex, body weight range, source of supply, species, strain and sub strain, age, and procedure used for identification.
  • A description of the dosage, dosage regimen, route of administration, and duration.
  • A description of all circumstances that may have affected the quality or integrity of the data.
  • The name of the study director, the names of other scientists or professionals, and the names of all supervisory personnel, involved in the study.
  • A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the data, and a statement of the conclusions drawn from the analysis.
  • The signed and dated reports of each of the individual scientists or other professionals involved in the study.
  • The locations where all specimens, raw data, and the final report are to be stored.
  • A statement prepared and signed by the quality assurance unit and the final report signed and dated by the study director.
Drug discovery and drug development is being revolutionized due to changes in technology. Technologies like genomics, proteomics, high throughput screening and structure-based design have enabled the process of drug discovery to evolve into a system where new lead molecules can be rapidly found against novel, and difficult targets. Preclinical testing of pharmaceuticals in animals has been instrumental in the development of modern therapeutic regimens. Unquestionably, human quality of life (and life expectancy) has flourished as a result of preclinical testing of drugs in animals. However, drug development remains extremely challenging, with numerous obstacles to overcome. The transition from activity in vitro (cell culture) to activity in vivo (animal model) can be especially challenging. Obtaining pharmacokinetic behavior consistent with the desired reactivity can be very difficult and the use of animals in toxicity testing has not progressed without controversy. Objections to animal testing have emphasized that the results obtained from animal tests do not always correlate well with human experience.20
Attrition rates remain high, and generally only one out of ten thousand drugs tested will enter clinical development and make it all the way to regulatory approval and find a place in the market. Drugs most frequently fail in the clinic because of poor pharmacokinetics or toxicity.
Despite the fact that drug development remains a long and arduous journey, the prospect of genome-targeted individualization of therapy remains an extremely exciting one. The possibility of personalized treatments (right drug for the right patient) based on the genomic or proteomic readout of the particular patient is now becoming a reality.
It is envisaged that more and more strategic alliances will be formed between biotechnology and small pharmaceutical companies to make the most of all of the opportunities like human genome data (Fig. 1.9).
During a new drug's early preclinical development, the sponsor's primary goal is to determine that the product is reasonably safe for initial use in humans and that compound exhibits pharmacological activity that justifies commercial development. When a product is identified as a viable candidate for further development, the sponsor then focuses on collecting the data and information necessary to establish that the product will not expose humans to unreasonable risks when used in limited, early-stage clinical studies.
FDA's role in the development of a new drug begins when the drug's sponsor (usually the manufacturer or potential marketer) having screened the new molecule for pharmacological activity and acute toxicity potential in animals, wants to test its diagnostic or therapeutic potential in humans. At that point, the molecule changes in legal status under the Federal Food, Drug, and Cosmetic Act and becomes a new drug subject to specific requirements of the drug regulatory system.
Before the sponsor proceeds to study a new drug in human, approval has to be obtained by through Investigational New Drug Application.
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Fig. 1.9: The drug discovery process
An Investigational New Drug (IND) application is to provide the data showing that it is reasonable to begin tests of a new drug on humans. In many ways, the IND application is the result of a successful preclinical development program. The IND application is also the vehicle through which a sponsor advances to the next stage of drug development known as clinical trials (human trials). Current Federal law requires that a drug be the subject of an approved marketing application before it is transported or distributed across state lines. Because a sponsor will probably want to ship the investigational drug to clinical investigators in many states, it must seek an exemption from that legal requirement. The IND application is the means through which the sponsor technically obtains this exemption from the FDA. The IND application shows results of previous experiments, how, where and by whom the new studies will be conducted, the chemical structure of the compound; how the compound is manufactured and any toxic effects in the animal studies.
There are two IND application categories:
  1. Commercial
  2. Research (noncommercial)
    There are three IND application types:
    1. An investigator IND application: It is submitted by a physician who both initiates and conducts an investigation, and under whose immediate direction the investigational drug is administered or dispensed. A physician might submit a research IND application to propose studying an unapproved drug, or an approved product for a new indication or in a new patient population.
    2. Emergency use IND application: It allows the FDA to authorize use of an experimental drug in an emergency situation that does not allow time for submission of an IND application. It is also used for patients who do not meet the criteria of an existing study protocol, or if an approved study protocol does not exist. In such a case, FDA may authorize shipment of the drug for a specified use in advance of submission of an IND application.
    3. Treatment IND application: It is submitted for experimental drugs showing promise in clinical testing for serious or immediately life-threatening conditions while the final clinical work is conducted and the FDA review takes place. A drug that is not approved for marketing may be under clinical investigation for a serious or immediately life-threatening disease condition in patients for whom no comparable or satisfactory alternative drug or other therapy is available. The purpose is to facilitate the availability of promising new drugs to desperately ill patients as early in the drug development process as possible, before general marketing begins, and to obtain additional data on the drug's safety and effectiveness. In the case of a serious disease, a drug ordinarily may be made available for treatment use during phase III investigations or after all clinical trials have been completed. In the case of an immediately life-threatening disease, a drug may be made available for treatment use earlier than phase III, but ordinarily not earlier than phase II.
  • The IND application must contain information in three broad areas:
  • Animal pharmacology and toxicology studies: Preclinical data to permit an assessment as to whether the product is reasonably safe for initial testing in humans.
  • Manufacturing information: Information pertaining to the composition, manufacturer, stability, and controls used for manufacturing the drug substance and the drug product. This information is assessed to ensure that the company can adequately produce and supply consistent batches of the drug.
  • Clinical protocols and investigator information: Detailed protocols for proposed clinical studies to assess whether the initial-phase trials will expose subjects to unnecessary risks. Also, information on the qualifications of clinical investigators who oversee the administration of the experimental compound—to assess whether they are qualified to fulfill their clinical trial duties. Finally, commitments to obtain informed consent from the research subjects, to obtain review of the study by an institutional review board (IRB), and to adhere to the investigational new drug regulations.22
Sponsor files the IND application in Form 1571 to the FDA for review once successful series of preclinical studies are completed.
Along with the IND application, the sponsor submits the statement of the Investigator (Investigator's undertaking) in Form 1572.
Once the IND application is submitted, the sponsor must wait 30 calendar days before initiating any clinical trials. If the sponsor hears nothing from CDER (Center for Drug Evaluation and Research), then on day 31 after submission of IND application, the study may proceed as submitted. The CDER is a division of the FDA that reviews New Drug Applications to ensure that the drugs are safe and effective.
During this time, FDA has an opportunity to review the IND application for safety to assure that research subjects will not be subjected to unreasonable risk.
  • Medical review: During the IND application review process, the medical reviewer evaluates the clinical trial protocol to determine: (1) if the participants will be protected from unnecessary risks; and (2) if the study design will provide data relevant to the safety and effectiveness of the drug. Under Federal regulations, proposed phase I studies are evaluated almost exclusively for safety reasons. Since the late 1980's, FDA reviewers have been instructed to provide drug sponsors with greater freedom during phase I, as long as the investigations do not expose participants to undue risks. In evaluating phase II and III investigations, however, FDA reviewers also must ensure that these studies are of sufficient scientific quality to be capable of yielding data that can support marketing approval.
  • Chemistry reviewers: They address issues related to drug identity, manufacturing control, and analysis. The reviewing chemist evaluates the manufacturing and processing procedures for a drug to ensure that the compound is adequately reproducible and stable. At the beginning of the chemistry and manufacturing section, the drug sponsor should state whether it believes the chemistry of either the drug substance or the drug product, or the manufacturing of either the drug substance or the drug product, present any signals of potential human risk. If so, these signals should be discussed, with steps proposed to monitor for such risks. In addition, sponsors should describe any chemistry and manufacturing differences between the drug product proposed for clinical use and the drug product used in the animal toxicology trials that formed the basis for the sponsor's conclusion that it was safe to proceed with the proposed clinical study.
  • Pharmacology/toxicology review: This team is staffed by pharmacologists and toxicologists who evaluate the results of animal testing and attempt to relate animal drug effects to potential effects in humans. This section of the application should contain, if known:
    • A description of the pharmacologic effects and mechanism(s) of action of the drug in animals
    • Information on the absorption, distribution, metabolism, and excretion of the drug. The regulations do not further describe the presentation of these data, in contrast to the more detailed description of how to submit toxicology data. A summary report, without individual animal records or individual study results, usually suffices. An integrated summary of the toxicology effects of the drug in animals and in vitro the particular studies needed depend on the nature of the drug and the phase of human investigation. When species specificity, immunogenicity, or other considerations appear to make many or all toxicological models irrelevant, sponsors are encouraged to contact the agency to discuss toxicological testing.
  • Statistical analysis: The purpose of these evaluations is to give the medical officers a better idea of the power of the findings to be extrapolated to the larger patient population in the country.
  • Safety review: Following review of an initial IND application submission, CDER (Center for Drug Evaluation and Research) has 30 calendar days in which to decide if a clinical hold is necessary (i.e., if patients would be at an unacceptable risk or if CDER does not have the data to make such a determination) (Flow chart 1.1).
Generally, drug review divisions do not contact the sponsor if no concerns arise with drug safety and the proposed clinical trials. If the sponsor hears nothing from CDER (Center for Drug Evaluation and Research), then on day 31 after submission of the IND application, the study may proceed as submitted. The sponsor is notified about the deficiencies through a clinical hold. A clinical hold is issued by the FDA to the sponsor to delay a proposed clinical Investigation or to suspend a clinical Investigation.23
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Flow chart 1.1: IND Application Review Process*While sponsor answers any deficiencies
Sponsor Notification
Once a clinical hold is placed on a commercial IND application, the sponsor will be notified immediately by telephone by the division director. For both individual and commercial IND applications, the division is required to send a letter within five working days following the telephone call. The letter should describe the reasons for the clinical hold, and must bear the signature of the division director (or acting division director).
The grounds for imposition of Clinical Hold are as follows:
  • Human subjects are or would be exposed to an unreasonable and significant risk of illness or injury.
  • Clinical investigators named in IND application are not qualified.
  • Investigator brochure is misleading, erroneous or materially incomplete.
  • IND does not contain sufficient information to assess risks.
  • Protocol is deficient to meet objective of trial.
  • Mechanism that CDER uses when it does not believe, or cannot confirm that the study can be conducted.
  • CDER will contact sponsor within 30 days initial review period.
The sponsor may then respond to CDER by sending an “IND CLINICAL HOLD RESPONSE” letter to the division. To expedite processing, the letter must be clearly identified as an “IND CLINICAL HOLD RESPONSE” letter.24
The division then reviews the sponsor's response and decides within 30 days as to whether the hold should be lifted. If the division does not reply to the clinical hold response within 30 calendar days, the division director will telephone the sponsor and discuss what is being done to facilitate completion of the review.
If it is decided that the hold will not be lifted, the hold decision is automatically sent to the office director for review. The office director must decide within 14 calendar days whether or not to sustain the division's decision to maintain the clinical hold. If the decision is made to lift the hold, the division telephones the sponsor, informs them of the decision, and sends a letter confirming that the hold has been lifted. The letter will be sent within 5 working days of the telephone call. However, the trial may begin once the decision has been relayed to the sponsor by telephone.
Sponsor will be Notified
If other deficiencies are found in an IND application that the review division determines are not serious enough to justify delaying clinical studies, the division may either telephone or forward a deficiency letter to the sponsor. In either case, the division informs the sponsor that it may proceed with the planned clinical trials, but that additional information is necessary to complete or correct the IND application file.
Study Ongoing
Once the CDER's 30-day initial review period expires, clinical studies can be initiated, unless a clinical hold has been placed. Beyond the 30-day review period for an IND application, subsequent clinical trials may begin immediately upon submission of the clinical protocol to the IND application (i.e., there is no 30-day waiting period for subsequent clinical trials after the submission of the first clinical trial protocol). If the sponsor was notified of deficiencies that were not serious enough to warrant a clinical hold, the sponsor addresses these deficiencies while the study proceeds.
Exploratory IND studies are intended to provide clinical information for a new drug candidate at a much earlier phase of drug development. These studies help to identify the best candidates for continued development and eliminate those lacking promise. These clinical trials occur very early in phase I, involve very limited human exposure, and have no therapeutic intent. Exploratory IND studies are conducted prior to the traditional dose escalation, safety, and tolerance studies and provide important information on pharmacokinetics (PK) and bioavailability of a candidate drug.
In April 2005, the FDA released a draft guidance for Exploratory IND studies that clarifies preclinical and clinical approaches that should be considered when planning exploratory IND studies in humans. As part of FDA's “Critical Path Initiative”, this process is a new tool available to the industry that enables a faster, more cost-effective path to early clinical development.
  1. Abbreviated New Drug Application (ANDA) Process for Generic Drugs http://www.fda.gov/cder/regulatory/applications/ANDA.htm. (Accessed on 20/07/2008).
  1. Bianca Piachand. 2002. Challenges facing the pharmaceutical industry. http://findarticles.com/
  1. CDER Manual of Policies and Procedures (MaPP). http://www.fda.gov/cder/mapp.htm. (Accessed on 23/07/2008).
  1. Code of Federal Regulations (CFR). http://www.gpoaccess.gov/cfr/index.html. (Accessed on 22/07/2008).
  1. Dimasi JA. Risks in new drug development: Approval success rates for investigational drugs. Clin Pharmacol Ther. 2001;69:297–307.
  1. EMEA. Eudravigilance data analysis system. 2006. www.eudravigilance.emea.europa.eu
  1. EMEA. Transitional measures for submission of PSURs for centrally authorized medicinal products for human and veterinary use. 2005. www.emea.europa.
  1. FDA. Reporting adverse experience to FDA. 2007. www.fda.gov/medwatch/how/htm.
  1. Genevieve D, Buyse M. Clinical research after drug approval. What is needed and what is not. Drug Information. 1999;33:627–34.25
  1. Giersiefen H, Hilgenfeld R, Hillisch A. Modern methods of drug discovery: An introduction. In: Modern methods of drug discovery. Ed. Hilgenfeld R and Hillisch A; 2004. pp. 1–18.
  1. ICH Guidance for Industry, S7A Safety Pharmacology Studies for Human Pharmaceuticals. Available on http://www.fda.gov/cber/gdlns/ichs7a071201.htm (Accessed on 30/07/2008).
  1. IND Forms and Instructions http://www.fda.gov/cder/regulatory/applications/ind_page_1.htm#IND%20Forms. (Accessed on 20/07/2008).
  1. Investigational New Drug (IND) Application Process, http://www.fda.gov/cder/regulatory/applications/ind_page_1.htm. (Accessed on 23/07/2008).
  1. Jens Eckstein. ISOA/ARF Drug Development Tutorial. Available on http://www.alzforum.org/drg/tut/ISOATutorial.pdf (Accessed on 30/07/2008).
  1. Lindquist M. Data quality management in pharmacovigilance In: Rosie Stather (Ed). Drug Safety, 2005.
  1. Mant T. Early phase studies, pharmacokinetics and adverse drug reactions. In: Giovanna ID, Hayes G (Eds). Principles of Clinical Research; 2001. pp. 117–60.
  1. Michelle DG, Mike IW, McDonald E, Ian Judson, Paul Workman. The contemporary drug development process: advances and challenges in preclinical and clinical development. Progress in Cell Cycle Research. 2003;5:145–58.
  1. Ministry of Health and Family Welfare (n.d.) National pharmacovigilance program. www.jipmer.edu
  1. Mishra SK. Drug development and discovery. In: Gupta SK (Ed). Basic Principles of Clinical Research and Methodology, 2007.
  1. Naidu MUR, Usharani P. Drug development and discovery. In: Gupta SK (Ed). Basic Principles of Clinical Research and Methodology, 2007.
  1. New Drug Application (NDA) Process. http://www.fda.gov/cder/regulatory/applications/nda.htm. (Accessed on 23/07/2008).
  1. Posner J. Exploratory development. In: Griffin JP, O'Grady J (Eds). The Textbook of Pharmaceutical Medicine; 2005. pp. 170–213.
  1. Ratti E, Trist D. Continuing evolution of the drug discovery process in the pharmaceutical industry. Pure Appl Chem. 2001;73(1):67–75.
  1. Rolan P. Clinical pharmacokinetics. In: Griffin JP, O'Grady J (Eds). The Textbook of Pharmaceutical Medicine; 2005. pp. 214–46.
  1. Ross Tonkens. An overview of the drug development process. 2005. The Physician executive.
  1. Schedule Y (Amended Version)-CDSCO, Appendix III of Schedule Y, Animal Toxicity. Available on http://cdsco.nic.in/html/Schedule-Y%20(Amended%20Version-2005)%20original.htm. (Accessed on 23/07/2008).
  1. Schedule Y (Amended Version)-CDSCO, Appendix IV of Schedule Y, Animal Pharmacology. Available on http://cdsco.nic.in/html/Schedule-Y%20(Amended%20Version-2005)%20original.htm. Accessed on 27/07/2008).
  1. Spilker B. Classification and description of phases I, II, III Designs. In: Williams L, Wilkins (Eds). Guide to Clinical Trials; 1991. pp. 27–43.
  1. Spilker B. Classification and description of phases IV post marketing study designs. In: Williams L and Wilkins (Eds). Guide to Clinical Trials; 1991. pp. 44–58.
  1. The process of Drug Development. Available on http://www.netsci.org/Courseware/Drugs/Intro.html (Accessed on 25/07/2008).
  1. Tweats DJ, Scales MDC. Toxicity testing. In: Griffin JP and O'Grady J (Eds). The Textbook of Pharmaceutical Medicine; 2005. pp. 128–69.
  1. Workflow driven Pharmacovigilance software. http://www.assured.co.uk/pv-workdetail.htm. Downloaded on 20th August 2008.