Methods in Drug Evaluation Prafulla Chandra Tiwari
Page numbers followed by f refer to figure.
Acetazolamide 67
bioassay of 31
doses of 31
hydrochloride 88
Acetylsalicylic acid 89
Acid phosphatase 20
catalyzes 20
clinical significance 20
Acid secretion in perfused rat stomach 119
evaluation 120
procedure 119
Activated partial thromboplastin time 56, 57
Acute ischemia by injection of microspheres in dogs 53
evaluation 54
procedure 53
Adenosine triphosphate 11
Adrenaline 41
Akinesia 109
Alcuronium chloride 87
Alkaline phosphatase 11, 19, 23
clinical significance 19
types of 19
Allantoxanamide inhibits enzyme uricase 80
Allantoxanamide treatment in rats, hypouricemic activity after 80
Aminophylline 88
Anesthesia, endpoint of 114
agents, local 115
dose, median 113
Anesthetized dog, method using 30
Angiotensin antagonism 45
Angiotensin converting enzyme 41, 44
inhibition in isolated guinea pig ileum 44
Anococcygeus muscle 42
contractions of 43
Antacids 119
Antagonistic activity 51, 52
Antianxiety agents 98
Antiasthmatic drugs 83
Anticoagulants 56
drugs 105
Antidiarrheal effect in cold-restrained rats, evaluation of 128
evaluation 128
procedure 128
Antidiarrheic agent 128
Antiepileptic drugs 105
Anti-goitrogenic activity 39
evaluation 40
procedure 39
Antiparkinsonian drugs 108
Antitoxin 25
Antitussive activity 92
after irritant inhalation in guinea pigs 92
evaluation 93
procedure 92
Antitussive agents 92
Antiuricosuric drugs 67
Anxiety 98
Anxiolytic drugs 98
Arachidonic acid 2
effect of 88
evaluation 89
procedure 88
Arrhythmia 9
Arterial bleeding time in mesentery 65
evaluation 66
procedure 65
Arteriovenous shunt thrombosis 64
evaluation 65
procedure 64
Aspartate transaminase and alanine transaminase 14
Astemizole 9
left 44
right 43
Atropine sulfate 88
Benzodiazepine 100
receptor binding assay 102
evaluation 102
procedure 102
tissue preparation 102
Beta blocking effects in anesthetized dogs, screening for 51
Bezold-Jarisch reflex 50
evaluation 50
procedure 50
Bioassay 25
applications of 25
demerits of matching 27
of acetylcholine principle 31
of ach using rat ileum preparation 28f
of adrenaline 30
principle 30
standard preparation 30
of digitalis 32
of D-tubocurarine 32
procedure 32
rabbit head-drop method 32
of estrogen 35
of insulin 33
of noradrenaline 31
principle 31
procedure 31
of oxytocin 34
of oxytocin using rat uterus 34
evaluation 34
procedure 34
of thyroxine 38
principles of 26
procedure of
four point 29
three point 28
types of 26
Bleeding models 65
evaluation 65
procedure 65
Blind pharmacological screening 25
Blood coagulation tests 56
evaluation 57
procedure 56
Blood pressure
in conscious hypertensive rats 48
in pithed rats 47
evaluation 48
procedure 47
measurement 49
Blood sugar lowering effect in rabbits 33
procedure 33
Blood urea nitrogen 22
Body plethysmography and respiratory parameters 89
alkaline phosphatase level 12
diseases 20
Bradykinin potentiation 45
Bradykinin triacetate 88
Breathing air, normal 92
Bronchial perfusion of isolated lung 86
evaluation 86
procedure 86
Bronchodilating drug, effect of 89
Bronchospasmolytic activity in anesthetized guinea pigs 87
β-sympatholytic activity in isolated rat spleen 41
Bupivacaine 115
Calcium chloride 34
Cannula, implantation of 49
Carbachol 85
Carbamazepine 105
Carbonic anhydrase inhibition 67
evaluation 68
procedure 68
Carbonic anhydrase inhibitors 67
Cardiac arrest in guinea pigs, digitalis induced 26
Cardiac puncture technique 59
Cardiac toxicity, reports of severe 9
Cardiovascular drugs 41
Cardiovascular system 25
Carotid body denervation 91
Castor oil-induced diarrhea 127
evaluation 128
procedure 127
Cecocolic junction 126
Central nervous system 25, 94, 113, 125
diseases 17
stimulants and depressants 94
Chandler loop 58
evaluation 59
procedure 58
Charcoal Norit A 35
Cheng-Prusoff equation 104
Chicken blood pressure 34
evaluation 35
procedure 34
Chinchilla rabbits 45
Chloride secretion in rabbit colon, inhibition of 127
evaluation 127
procedure 127
Chronic gastric fistula 119
in rats 121
evaluation 121
procedure 121
Chronic renal failure
after subtotal nephrectomy in rats 76
in rats 75
evaluation 76
procedure 75
test 67
Clonazepam 102
Conduction anesthesia in sciatic nerve of frog 115
evaluation 116
procedure 115
Conduction anesthesia in sciatic nerve of rat 116
evaluation 116
procedure 116
Conduction anesthesia on mouse tail 116
evaluation 117
procedure 116
Congestive heart failure 41
Corneal reflex present 118
Coronary artery, left circumflex 53
Coronary thrombosis induced
by electrical stimulation 62
by stenosis 61
Cortical collecting duct 69
Cough induced by mechanical stimulation 93
evaluation 93
procedure 93
Creatine kinase 15, 16
Cromakalim 45
Cyclic flow reductions 61
Cyclooxygenase 2
enzyme 2
inhibitor of 89
Cytosol preparation 35
Cytosolic fraction binding 35
Dazoxiben HCl 89
Deep vein thrombosis 56
Dextran 35, 53
coated charcoal adsorption technique 35
Diarrhea-free period 128
Diarrheal excretion, early 128
Disodium hydrogen orthophosphate 34
Diuretic activity in rats 72
evaluation 73
procedure 72
Diuretic and saluretic activity in dogs 74
evaluation 74
procedure 74
Diuretics, clearance method of evaluation of 74
evaluation 75
procedure 74
Dopamine, prodrug of 111
Dose response curve 27
Dose studies
multiple ascending 8
single ascending 8
Drug 25
acting on lower urinary tract, evaluation of 80
acting on urinary tract 67
acting, screening of 25
affecting motility, evaluation of 94
classification of 24
development, stages 1
in process of 1
discovery 1
high throughput screening 2
latency, duration of 118
test 99
using isolated rat stomach, evaluation of 120
D-tubocurarine, test solution of 32
Duloxetine 67
Electric shock 107
Electrical-induced thrombosis 64
evaluation 64
procedure 64
Electroencephalogram threshold test in rats 113
evaluation 113
procedure 113
Electroshock in mice 107
evaluation 107
procedure 107
Elevated body swing test 111
evaluation 111
procedure 111
Endralazine 49
Enflurane 112
Enteric nervous system 125
Enteropooling test 126
evaluation 126
procedure 126
Enzymes 10, 11
in clinical practice 13
in diagnosis of diseases, application of 10
increased in serum in
cholestasis 23
liver damage 23
leak from cell 11
levels in serum and plasma, regulation of 11
production, alteration in 12
tests, selection of 12
Enzymology, clinical 10
Epilepsy 105
treatment of 105
Equivalent short circuit current 71
Estrogen receptor binding 35
procedure 35
Estrogen responsive element gene 37
Estrogens, transactivation assay for 37
evaluation 38
procedure 37
Ethosuximide 105
Etomidate 112
Ex vivo-assays 61
Eyelid closure 109
Fanconi's syndrome 20
Fecal pellet output 128
Fixed dose test 6
Flexed posture 109
Fluoroquinolone 9
Food effect 8
Functional plasma enzymes 10
GABAA receptor binding 100
evaluation 101
materials 100
procedure 100
GABA-mimetic agents 107
Gabapentin 105
Gama-amino butyric acid 100
Gamma-glutamyl transferase 23
Gastric acidity, measurement of 21
Gastric fluid, functional tests of 20
Gastric function tests 20
Gastric oxyntic glands 11
Gastrin, determination of activity of 122
evaluation 122
procedure 122
Gastrointestinal system, complex 125
Gastrointestinal tract 125
Gatifloxacin 9
General anesthetic 112
agents 112
Glomerular filtration rate 72
Glomerular function tests 22
Gould pressure transducer 91
Graphical bioassay 28
of acetylcholine employing rat ileum 28f
Guinea pig 43, 44
ileum 45
method 32
Halothane 112
Heart diseases 15, 16, 18
Helicobacter pylori 20
Hemodynamic measurements, preparation for 53
Histamine 85
dihydrochloride 84, 88
H3 receptor binding in brain 104
evaluation 104
procedure 104
receptor binding assay 83
evaluation 84
procedure 83
Histamine-induced bronchoconstriction 89
in anesthetized guinea pigs 89
Hole-board test 94, 95
procedure 95
Homogenates 35
Hormones 25
Human pharmacology 8
Hydroxy dopamine 110
Hypercholesterolemia 56
Hypertension 41
Hypoglycemic seizures in mice 33
evaluation 34
procedure 33
Imipramine-HCl 88
In vitro assay for GABAergic compounds 98
evaluation 100
procedure 98
In vitro methods 79
for evaluation of
antianxiety agents 98
diuretic and saluretic activity 67
In vitro tests 56, 83
In vivo methods 72
In vivo models 61
In vivo tests 87
Indomethacin-induced ulcers in rats 124
evaluation 124
procedure 124
Inhalation anesthetics, efficacy and safety of 114
evaluation 114
procedure 114
Inhalation toxicity, acute 7
Inhalational anesthetics 112
Inhibition of acid secretion 120
International Conference on Harmonization 4
Intracellular electrical measurements 71
Intracellular enzymes 12
Intravenous anesthetics, screening of 112
evaluation 113
procedure 112
Intravenous bolus injection 53
Intravenous injections of histamine 90
Intrinsic agonistic activity 51
Iodine release, inhibition of 39
evaluation 39
procedure 39
Isocitrate dehydrogenase of heart muscles 13
Isoenzymes 11, 18
and isoforms, measurement of 13
subunits 18
clinical significance 18
Isoflurane 112
Isoguvacine 99
Isolated blood vessels, contractile and relaxing activity on 45
Isolated kidney tubules, perfusion of 69
evaluation 70
procedure 69
Isolated perfused kidney method 72
evaluation 72
procedure 72
Isoniazid-induced convulsions 106
evaluation 107
procedure 106
Isonicotinic acid hydrazide 106
Isozymes 11
Kidney 25
diseases 19
Konzett-Rössler method 87
Krebs-Henseleit solution 85, 120
Lactate dehydrogenase 10, 17
enzyme 11
Late diarrheal excretion 128
Laxative activity in rats 125
evaluation 126
procedure 125
Laxative and antidiarrheal agents 125
Leukotriene 84, 88
LTC4 85
Levothyroxine 40
Lewy bodies 108
Lichtfield-Wilcoxon procedure 107
Lidocaine 116
Lipophilic character of drugs 5
Lipschitz test 72
cirrhosis 15
diseases 15, 18, 19
function tests 20, 22
L-triiodothyronine 40
Lysosomes 20
Magnesium chloride 34
Medullary collecting duct 69
Meissner's plexus 125
Membrane preparation 100
Methacholine 88
Microperfusion experiments 76
Micturition studies 80
evaluation 81
procedure 80
Milk ejection in lactating rabbit 35
evaluation 35
procedure 35
Mouse uterus cytosol, determination of specific binding in 36
Muscarinic agonists tremorine 108
Muscimol 99
Muscle coordination, tests for 96
grip strength 97
inclined plane 96
rotarod method 97
Muscle diseases 18
Myocardial infarction 13, 15, 56
after coronary ligation 52
evaluation 53
procedure 52
biomarker for 10
Nephrectomy, two-thirds 76
Nephritis, experimental 77
Nephrotoxic serum nephritis 77
evaluation 79
experimental protocol 78
procedure 78
Nephrotoxic serum, preparation of 78
Nerves, extrinsic 42
New drug application 8
New drug development process 1
Nicorandil 45
Nifedipine 49
Nuclear extract preparation 36
Nuclear uptake 36
Omperazole 119
Open field test 95
procedure 95
Ovalbumin 88
Oxaloacetate 14
Oxotremorine 108
antagonism 108
Oxygen consumption 38
evaluation 39
procedure 38
Oxygen-enriched air 47
Oxyhemoglobin 47
Paget's disease 20
Pancreas 11
Papillary collecting duct 69
Paracetamol 4
Parkinson's disease 108, 110
Patch clamp technique in kidney cells 68
evaluation 69
procedure 68
Pentobarbital 118
sodium 53, 88
Pentylenetetrazole test 105
Pentylenetetrazole-induced convulsions 105
evaluation 106
procedure 105
Perineal nerve 42
Peripheral venous catheter 125
Pharmacodynamics 4
Pharmacokinetics 3, 4
Pharmacological screening
and bioassay 24
types of 24
Photinus pyralis 37
Photocells 95
Picrotoxin 106
Picrotoxin-induced convulsions 106
evaluation 106
procedure 106
Pinacidil 45
Pirbright guinea-pigs 87
Pirbright white guinea pigs 90
creatinine ratio 22
enzymes, non-functional 11
specific enzymes 10
Platelet aggregation 59, 60
substances 59
Platelet rich plasma, deaggregation in 59
Pneumotachography in anesthetized guinea pigs 90
Post-marketing surveillance 9
Postural tremor 109
Potassium channel openers, effects of 45
Potassium chloride 34
Preclinical studies 3
Procaine 116
Product characterization 3
Propofol 112
Prostaglandin synthesis inhibition 85
Prostaglandin treated animals 126
Prostate specific antigen 12
Prothrombin time 56, 57
Proton pump inhibitors 119
Psoas muscle 49
Pulmonary artery pressure 53
Pulmonary capillary pressure, mean 53
Pulmonary mechanics 91
Pulmonary resistance, calculation of 90
Pylorus ligation method 122
evaluation 123
procedure 122
Rabbit tooth pulp assay 117
evaluation 118
procedure 117
Rabbit's duodenum, method using 31
Rabeprazole 119
Rat anococcygeus muscle 42
isolated 42
Rat brain by telemetry, EEG analysis from 96
Renal function
evaluation of 74
impaired 75
tests 21
Renal pelvis, studies on 81
evaluation 82
procedure 81
Reserpine antagonism 110
evaluation 110
procedure 110
Respiratory depressant drugs
effects of 91
in conscious rats, effects of 91
Respiratory function in vivo 88
Retrobulbar block in dogs 118
evaluation 118
procedure 118
Rigidity 109
Salivary glands 11
Saluretic activity in rats 73
evaluation 73
procedure 73
Secreted enzymes 11
Serotonin receptor binding assay 103
evaluation 104
procedure 103
tissue preparation 103
albumin 22
bilirubin 22
creatine kinase 16
enzymes 22
Sevoflurane 112
Simple pharmacological screening 25
Skeletal muscle disease 15, 17
Sodium chloride 34
Sodium cyanide 91
Sodium dihydrogen orthophosphate 34
Sodium hydrogen carbonates 34
Spasmolytic activity in isolated
guinea pig lung strips 84
trachea 85
evaluation 85
procedure 85
Sprague-Dawley or Wistar rats, female 34
Sprague-Dawley rats 41, 120
adult 113
male 39, 47, 50, 91
Stress ulcer through immobilization stress 123
evaluation 123
procedure 123
Strychnine-induced convulsions 106
evaluation 106
procedure 106
Subaqueous tail bleeding time in rodents 65
Subcritical stenosis 61
Succinylcholine 89
Sucrose 99
Synthetic function, tests for 23
Systolic blood pressure 48
Tail cuff method 48
Tensile strength of connective tissue in rats 40
Test tube to new drug application, steps from 2f
Thiazides 67
Thrombin time 56
Thromboelastography 57
alpha angle 58
formation time 58
strength 58
evaluation 58
procedure 57
reaction time 58
Thromboxane synthetase, inhibitor of 89
diseases 17
enlargement 39
hormones, modified for 40
Tiagabine 105
Tissue preparation 99
Tolpropamine-HCl 88
Toxic class method, acute 6
Toxicological studies 6
Toxins 25
Transepithelial electrical measurements 71
Transpulmonary pressure 90
Tremorine 108
Tris-sucrose buffer 35
Tubular function tests 22
Tumor lysis syndrome, acute 67
Tumors 19
Tyrode's solution 44
Urapidil 49
Uricosuric and hypouricemic activity 79
Urinary bladder and internal urethral sphincter 82
Urinary incontinence, management of 67
Vaginal cornification 38
evaluation 38
procedure 38
Valproic acid 105
Vigabatrin 105
Vitamins 25
Volatile anesthetics, screening of 113
evaluation 114
procedure 113
Volatile anesthetics, testing of 113
Washed platelets (born method) 59
Wilson's disease 23
Wistar rats, male 72
Xanthine oxidase catalyzes 79
Yohimbine-induced convulsions 107
evaluation 107
procedure 107
Zollinger-Ellison syndrome 21
Chapter Notes

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Introduction to Various Stages in the Process of Drug DevelopmentChapter 1

A drug development process has to pass through various stages to produce a drug product which is safe, efficacious, and has passed all drug regulatory requirements. Drug discovery program initiates because there is a medical problem for which there is no efficient treatment is available. The process of New Drug Development is explained in Figure 1.
Various stages of drug development include:
  1. Drug discovery
  2. Product characterization
  3. Formulation of drug product
  4. Pharmacokinetics
  5. Preclinical studies
  6. Clinical trials
Drug Discovery
Phase of drug discovery start from target identification. Choosing a right target is essential for drug discovery, such as choosing a correct biochemical mechanism of a disease. The initial research on drug discovery usually occurs in academia or pharmaceutical industries. Drug candidates synthesized in research laboratory are tested for their interaction with drug targets. Usually, 5000–10,000 molecules are screened rigorously. This high number screening of molecules has been made possible with the advent of high throughput screening. Without high throughput screening it will not be possible to screen such high number of molecules in a day.2
zoom view
Figure 1: Steps from test tube to new drug applicationCourtesy: USFDA
High throughput screening (HTS) can be defined as a process in which batches of potential leads are tested for their biological activity on the target. The leads obtained after screening of compounds through HTS are called as hits. These hits are obtained through automatized miniature robotic assay.
High Throughput Screening
A typical high throughput screening will contain seven components, namely:
  1. Target: A target can be defined as a substance which has key role in any biological process, for example, cyclooxygenase enzyme responsible for breakdown of cell arachidonic acid ‘a key factor in inflammation’ is drug target of NSAIDs like aspirin which acts on both COX 1 and COX 2 variants of cyclooxygenase to show its anti-inflammatory effect.
  2. Assay: An assay is usually performed to check the biological activity of the test compound with reference to its activity on the target. Fluorescence and radioactivity are generally used as indicators in the assay.
  3. A library of compounds should contain a wide range of structural diversity. Library size does not matter as compared to its diversity, quality and management.
  4. Automation: Automation of screening process is required to perform large number of assays in shorter duration.3
  5. Information systems: Information systems are necessary for any type of data handling. These include resource scheduling, assay tracking, compound management, and data collection.
  6. Facilities: Facilities required are adequate laboratory space and environmental control.
  7. Scientist.
Product Characterization
Characterization of a potential drug candidate is done if it shows some promising therapeutic effects. Product characterization includes determination of shape and size of molecule, preferred storage conditions. Early pharmacological studies helps in determination of mechanism of action of the molecule. Moreover with the help of pharmacological studies we also determine toxicity and bioavailability of the compound.
Formulation, Delivery, and Packaging Development
A proper formulation of the drug molecule is required to ensure proper drug delivery. The drug product thus developed must be stable and easy to administer. If administration of drug product will not be simple then it will increase patient noncompliance. Therefore, it is a very critical stage in drug development as drug developer has to design a formulation which is stable, easy and safe to administer and look attractive.
With the help of pharmacokinetic study we determine parameters such as auc (area under the curve) which gives us the bioavailability of the drug, Cmax, i.e. maximum concentration of drug in the blood, Tmax, i.e. time required to reach the maximum concentration, T1/2, i.e. time in which half of drug is removed from the body, i.e. volume of distribution (Vd) of drug. Vd is the total volume in which the drug is distributed. Vd is a hypothetical volume and gives us information regarding the tissue binding of the drug. Sometimes Vd can be even larger than the total body volume.
Preclinical Studies
Preclinical study is a risk based process which involves safety and efficacy evaluation of new drugs in animals. The results obtained from preclinical studies are extrapolated to obtain human outcomes. A new compound has to be tested for its safety and efficacy in animals before its use as a drug product in humans. The preclinical pharmacological and toxicological drug responses with respect to dosage form used, dosage regimen of the drug product and route of administration helps in initiating clinical studies on humans. Preclinical studies should be approved by the governing body (CPCSEA in 4India). Care must be taken while preclinical studies to avoid cruelty on animals and if animal has to be sacrificed it should be done duly under anesthesia.
International Conference on Harmonization (ICH) has issued guidelines outlining the technical requirements of acceptable preclinical studies of drug.
Preclinical studies are generally performed in two disciplines namely (1) general pharmacology and (2) toxicology.
General Pharmacology
Pharmacology is the study of effect of drug on the function of various body systems. There are various other factors along with the pharmacological action of a drug in a specific tissue at a particular receptor, which contribute in the success of drug therapy. When a drug enters the body processes of absorption, distribution, metabolism and excretion or elimination starts simultaneously. All these processes occur parallel. These are the processes of pharmacokinetics. Pharmacokinetics is what body does to the drug. The drug also acts on the body and shows its pharmacological and therapeutic action with the help of drugs binding to the receptor at tissue specific site. This is what drug does to the body and is called as ‘pharmacodynamics’.
Pharmacokinetics involves absorption, distribution, metabolism and excretion (ADME) of the drug, e.g. paracetamol is rapidly absorbed orally, attains peak plasma level in 30 to 60 minutes, 25% of drug is bound from plasma protein and remaining occur in plasma as free drug, Vd is 1 L/kg, extensively metabolized in liver and excreted in urine. Process of drug metabolism is explained in Figure 2.
Movement of drug from its site of administration to the central compartment or systemic circulation is called absorption. Rate and extent of absorption is termed as bioavailability. It is bioavailability about which clinicians are generally considered.
zoom view
Figure 2: Absorption, distribution, metabolism, and excretion of a drugAbbreviation: GI, Gastrointestinal
When, a drug is administered orally, it is first absorbed from stomach and intestine, comes to liver through hepatic portal system, where it is metabolized extensively. This metabolism of drug into liver is termed as first pass metabolism or first pass effect. Along with the metabolism, biliary excretion of drug also occurs in liver before entering the drug into systemic circulation. If the metabolizing and excretory capacity of liver for a particular drug is comparatively higher, then it will greatly reduce the bioavailability of the drug, which may lead to therapeutic failure.
Following absorption, drug reaches in systemic circulation and is distributed to interstitial and intracellular fluid. This process of distribution, displays various physiological factors and physicochemical properties of the individual drug. Cardiac output, blood flow to the underlying area, blood vessel permeability and tissue volume govern the delivery rate and amount of drug distributed into tissues. Initially, drug is mostly distributed into well perfused organs such as liver, kidney and brain, whereas delivery to least perfused areas such as muscles, viscera, skin and fat is slower. Redistribution or second phase of drug distribution may take several minutes to few hours before the concentration of drug in tissues reaches equilibrium with that of in blood. Redistribution of drug involves larger fraction of body area in comparison to initial phase of drug distribution and generally accounts for most of the extravascular drug distribution. With, the exception of brain, drug diffuses readily into interstitial fluid because of the highly permeable nature of capillary endothelial membrane. Thus, tissue distribution is determined by the partitioning of drug between blood and particular tissue. Relative binding of drug to plasma proteins and tissue macromolecules is crucial in determination of drug distribution.
Lipophilic character of drugs promotes there passage through various biological membranes and there access to site of action. This lipophilic nature of drug serves as hindrance in excretion or removal of drug from the body. Only, a small fraction of drug is excreted unchanged through the kidneys, because lipophilic compounds filtered through glomerulus are largely reabsorbed into the systemic circulation. Thus, metabolism of drug is required to convert it into water soluble hydrophilic form, which is essential for elimination of drug from body as well as termination of their biological and pharmacological activity. Biotransformation or metabolism reactions are generally classified into:
  • Nonsynthetic/phase 1/functionalization reaction
  • Synthetic/phase 2/conjugation reaction.
Excretion of drug is its passage out from the systemic circulation. Drugs and drug metabolites are generally excreted in urine, feces, saliva and exhaled air, etc.6
In pharmacokinetics, we have studied the action of body on the drug, while in pharmacodynamics we study the effect of drug on body. Pharmacodynamics involves study of biochemical and physiological effects of drugs and their mechanism of action.
Toxicological Studies
Toxicological studies are performed to ensure safety of drug before its administration into human beings. Toxicological activity of a drug product is determined by in vitro and in vivo assay. In vitro studies are performed to examine the direct effect of drug on cell phenotype and proliferation. In vivo studies are performed for qualitative and quantitative determination of toxicological effects. LD50 values are also determined in toxicological studies.
Concept of LD50 (lethal dose 50) was first introduced in 1927 for establishing the toxic potency of biologically active compounds. LD50 value can be defined as ‘the statistically derived single dose of a substance which can cause death in 50% of the animals in an experimental group’. LD50 value of a drug can be determined using following methods:
  • Fixed dose test: This test is useful in minimizing the number of animals required for LD50 value determination.
    Test procedure: Rats of either sex (preferably female) are dosed in a stepwise manner using the fixed doses of 5, 50, 300, and 2000 mg/kg (exceptionally an additional high dose of 5000 mg/kg may be also used). The initial dose level is selected as the dose expected to produce some toxicity other than mortality (if there is no information regarding initial dose and toxicity, initial dose will be 300 mg/kg). Animals may be further dosed of fixed doses at higher or lower level depending upon the presence or absence of signs and symptoms of toxicity in the animals.
    Observation: Animals are observed daily for 14 days. The animals are observed for changes in skin, fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behavior pattern. Attention should be given to tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
  • Acute toxic class method: The test consists on a stepwise procedure with the use of three animals of either sex (normally females) per step. Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step (i.e. no further testing is needed; dosing of three additional animals with the same dose; dosing of three additional animals at the next higher or lower dose level). The starting dose level is selected from one of four fixed levels (5, 50, 300, and 2000) and it should be that which is most likely to produce mortality in some of the dosed animals. When there is no information on a substance to be tested, it is recommended to use the starting dose of 300 mg/kg.7
Observations are similar to first dose test.
  • Up-and-down method: The test consists of a single ordered dose progression in which animals of a single sex (normally females) are dosed, one at a time. The first animal receives a dose step below the level of the best estimate of the LD50 (when no information is available to make a preliminary estimate of the LD50, the suggested starting dose is 175 mg/kg). If the animal survives, the dose for the next animal is increased by a factor of 3.2. Times the original dose; if it dies, the dose for the next animal is decreased by a similar dose progression.
Observations are also similar to fix dose test.
  • Acute inhalation toxicity: This test provides information on health hazards likely to arise from short-term exposure by the inhalation route. Several groups of animals are exposed for a defined period to the test substance in graduated concentrations (at least three), one concentration being used per group. The preferred species is the rat and at least 10 animals (5 female and 5 male) are used at each concentration level. Animals should be tested with inhalation equipment designed to sustain a dynamic airflow of 12 to 15 air changes per hour, ensure adequate oxygen content of 19% and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance.
    Observations: Animals are observed daily for a total of 14 days. Observations include changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, and autonomic and central nervous systems, and somatomotor activity and behavior pattern. Attention should be given to tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Clinical Trials
After conducting preclinical studies pharma companies submit data collected in preclinical studies to the Food and Drug Administration (FDA) and file NDA (New Drug Approval). If after 30 days of filing the NDA, there is no response from FDA then it is considered that the trial is not put on hold and companies can go ahead with the trials.
Clinical trials can be defined broadly as ‘the medical research involving the people’. Clinical trials are performed:
  • To establish the safety of new drug or treatment in the human beings
  • To determine its side effects
  • To prove the efficacy of newer drug over the currently available one.
Phases of Clinical Trials
Clinical trials are generally classified into three phases according to their objectives. However, there is also a phase IV of clinical trials, which involves monitoring of drug, when it comes into market.8
Phase 1 Trials
Phase 1 trials are the earliest use of a new medicine in the human beings. Phase 1 trials are also called as ‘clinical development or human pharmacology’ phase. Phase 1 trials are small trials recruiting less than thirty healthy human volunteers. Phase 1 trials help us to determine:
  • Safe dose range
  • Side effects of drug
  • Adverse effects of drug if any
Phase 1 trials are classified into:
  • Single ascending dose studies (SAD)
  • Multiple ascending dose studies (MAD)
  • Food effect
Single ascending dose (SAD) studies: In SAD studies a single dose of drug is given to the volunteers. The volunteers are kept under observation for a specific time and their blood samples are collected and assayed at regular intervals. If, volunteers do not exhibit any prominent adverse effect, then dose of drug is increased and is given to newer group of volunteers. This, process is continued until and unless we reach maximum safe level of the drug or intolerable side effects occur in the volunteers.
Multiple ascending dose (MAD) studies: In (MAD) studies small group of volunteers are given small multiple doses of a drug and are kept under observation. Their bloods samples are collected at regular intervals are analyzed. Dose of drug is increased for the next group.
Food effect: Food effect is a short trial which is designed to check the effect of food on the bioavailability of the drug.
Phase 2/Phase II Clinical Trials
After confirming the initial safety of drug in phase 1 trials, phase 21 trial is performed on large number peoples (200–300). In phase 2 of clinical trials effect of drug on the body is explored and phase 1 studies are continued in the phase 2.
Phase 2 studies are sometimes divided into phase 2A and phase 2B.
  • Phase 2A or IIA is designed to prepare dosage regimen
  • Phase 2B or IIB is designed to determine the efficacy of the drug.
Phase 3/Phase III Trials
Phase 3 trials compare the newer drug/treatment with the currently available standard treatment for a particular disease. Phase 3 trials involve large number of patients.
During the phase III studies, preparations are made for submitting the Biologics License Application (BLA) or the New Drug Application (NDA).
Phase 3 trials are generally multicentric so that the researchers can collect more information regarding drug and its effects in different environmental condition in a shorter duration. Phase 3 trials are generally randomized control 9trials. Randomization of trial means that the patients are grouped into two groups randomly. One group will receive standard drug and other will receive test drug.
Phase 4/Phase IV Trial
It is also called as post-marketing surveillance. It involves monitoring of safety data of drug, even after the drug obtains license for its sale. Several drugs have been withdrawn from market due to monitoring of their safety data in phase IV of clinical trials, e.g. Astemizole an H1 antagonist was withdrawn from the market after reports of severe cardiac toxicity, several coxibs (selective COX 2 inhibitors) were withdrawn from market on allegedly causing arrhythmia, gatifloxacin, a fluoroquinolone was withdrawn from market on causing photo toxicity.
Recently, a new phase, zero (Phase 0) has been added. Phase 0 trials are performed, while testing for new anticancer/anti HIV medicine in the cancer/HIV patients as these medicines cannot be used in normal individuals.