1.
Screening for anxiolytic activity
IN VITRO METHODS
1. In vitro assay for GABA-ergic compounds
- GABA receptor binding
2. Benzodiazepine receptor flunitrazepam
binding assay
3. Serotonin receptor binding
-binding of 8 hydroxy-2-tetralin
-5 HT3 receptor in rat entorhinal cortex
membrane
4. Histamine H3 receptor binding in brain
IN VIVO METHODS
1 Anti- anxiety test (light-dark model)
2 Anticipatory anxiety in mice
3 Social interation in rats
4 Elevated plus maze test
5 Open field test
6 Hole-board test
7 Staircase test
8 Cork gnawing test
9 Four plate test in mice
10 Foot shock induced freezing behaviour in rats
2.Anti-aggressive activity
Foot-shock induced aggression
Isolation-induced aggression
Resident-intruder aggression test
Water competition test
Maternal aggression in rats
3.SCREENING METHODS OF ANTIEPILEPTIC
AGENTS
PRECLINICAL EVALUATION models
IN-VITRO MODELS
[3H] GABA receptor binding
GABA(A) receptor binding
GABA(B) receptor binding
3H-GABA uptake in rat cerebral cortex synaptosome
TBPS binding in rat cortical homogenate and section
Electrical recordings from hippocampal slices in vitro
Electrical recordings isolated brain cells
Glutamate receptors: [3H]CPP binding
NMDA receptor complex: [3H]TCP binding
[3H]glycine binding in rat cerebral cortex
INVIVO Methods
1. Electroshock seizures
1. Threshold model
2. Maximal electroshock seizures (MES)test
3 . Focal electrical stimulation such as kindling
-Corneal electroshock kindling
-Kindling by stimulation of the other brain areas
-Chemically induced kindling
2. Chemical induced seizures :
1. Pentylenetetrazol ( Leptazol/PTZ) induced seizures.
2. Strychnine-induced convulsions.
3. Picrotoxin-induced convulsions.
4. Isoniazid-induced convulsions.
5. Bicuculine tests in rats.
6. 4-Aminopyridine induced seizures in mice.
3. Epilepsy induced by Focal Seizures.
4. Model of status epilepticus.
Pilocarpine induced status epilepticus.
Lithium pilocarpine induced status epilepticus.
Lithium methomyl induced seizure in rats.
Electrical stimulation of hippocampal perforant pathways.
D,L-homocysteine induced status epilepticus.
Generalized myoclonic seizures in baboons.
GENETIC ANIMAL MODELS OF EPILEPSY
Seizure-prone mice strains
-Totterer Mice
-Lethargic mice
-DBA/2J mice
-E1 Mice
-Quaking Mice
Genetically epilepsy prone Rats
Photosensitive baboons
Mongolian gerbils
Recent advance in animal model
Zebrafish as a model for studying genetic aspects of epilepsy
4.Screening of antiulcer agents
Part A
Methods to produce experimental ulcers and evaluation of antiulcer
activity
Pylorus ligation in rats
Stress ulcer models
Histamine induced gastric ulcers
Indomethacin induced ulcers in rats
Ethanol induced mucosal damage
Acetic acid induced gastric ulcers
Reserpine induced chronic ulcers
Cysteamine induced duodenal ulcers
Dimaprit induced duodenal ulcers
Mepirizole induced duodenal ulcers
Gastric mucosal injury by local ischemia-reperfusion in rats
Part B
Anti-secretory activity
H+/K+-ATPase (proton pump) inhibition
H2 antagonism
Anti-cholinergic activity
Evaluation of antacids
Mucus secretion
Gastric mucosal blood flow
Models of Helicobacter pylori infection
Other methods-
Measurement of gastric emptying rate (GER)
Determination of the prostacyclin levels of the gastric mucosa in rats
Measurement of gastric potential difference in rats
Estimation of dissolved mucosubstances
5.SCREENING OF ANTIPSYCHOTIC DRUGS
IN-VITRO METHODS
D-2 receptor assay [H3 ] SPIROPERIDOL
Dopamine D-2 receptor autoradiography
Binding to D-3 receptor
Determination of Dopamine autoreceptor activity
Serotonoin 5HT-2receptor autoradiography
Measurement of neurotransmitters by intracranial microdialysis
Push-pull cannulae to determine release of endogenous
neurotransmitters
IN-VIVO METHODS
Behavioural test:
1. Golden Hamster test
2. Influence on behaviour of cotton rat
3. Catalepsy in rodents
4. Pole climb avoidance in rats
5. Foot shock induced aggression
Test based on mechanism of action ;
1. Amphetamine group toxicity
2. inhibition of amphetamine stereotypy in rats
3. Inhibition of apomorphine stereotypy in rats
4. Inhibition of apomorphine climbing in mice
5. Yawning and penile erection syndrome in rats
6. inhibition of mouse jumping
6.SCREENING OF ANTI-INFLAMMATORY DRUGS
IN VIVO METHODS
For acute and subacute inflammation
1.UV-erythema in guinea pigs
2.Vascular permeability
3.Oxazolone-induced ear edema in mice
4.Croton-oil ear edema in rats and mice
5.Paw edema in rats
6.Pleurisy tests
7.Granuloma pouch technique
8.Urate induced synovitis
9. PIFIR(Pain induced functional index in rats)
For testing chronic inflammation
1.Cotton wool granuloma
2.PVC sponge granuloma
3.Glass rod granuloma
IN VITRO METHODS
1. 3H- Bradykinin Receptor Assay
2. 3H-Substance P Receptor Binding
3. Assay of PMNL Chemotaxis in Vitro
4. Neurokinin Receptor Binding
5. PMNL Aggregation Induced By FMLP(formyl-L-methionyl-L-leucyl-L-
phenylalanine)
6. Constitutive and Inducible Cellular Arachidonic Acid Metabolism In
Vitro
7. Flow Cytometric Analysis of Intracellular Cytokines
8. TNF- Antagonism
9. Bindind to Interferon Receptors
10.Screening for Interleukin-1 Antagonists
11.Inhibition of Interleukin-1 Converting Enzyme
12.Induced Release of Cytokines from Human White Blood Cells In Vitro
NOTE:-Methods for testing immunological factors
1.Adjuvant arthritis in rats
2.Experimental allergic encephalomyelitis
3.Schultz-Dale reaction
4.Passive cutaneous anaphylaxis
5.Arthus type immediate hypersensitivity
6.Delayed type hypersensitivity
7.Screening of antihypertensive drugs
Animal models I
Renovascular hypertension :
Acute renal hypertension in rats: Goldblatt method
Chronic renal hypertension in rats
Chronic renal hypertension in dogs
Endocrine hypertension:
DOCA-salt induced hypertension in rats.
Fructose induced hypertension in rats.
Adrenal regeneration hypertension in rats.
Neurogenic hypertension:
Neurogenic hypertension in dogs(denervation of sinoaortic
baroreceptors)
Hypertension in rats by electrical stimulation of different
areas of brain.
Animal models II
Psychogenic hypertension:
Borderline hypertensive rats.
Air-jet stimulation.
Genetic hypertension:
Hypertension in spontaneous hypertensive rats(SHR)
Other models:
Obesity related hypertension
Hypertension induced by cholinomimetic agents.
Angiotensin-II induced hypertension.
Hypertension induced by cadmium.
Chronic nitric oxide inhibition induced hypertension in
SHR.
Transgenic rat models
Uterine ischemia induced hypertension in rats.
In-vitro models:-
-Sympatholytic activity in isolated vascular smooth muscle
-Sympatholytic activity in the isolated guinea pig seminal vesicle
1-Sympatholytic activity in isolated guinea pig atria
Angiotensin converting enzyme inhibition in the isolated guinea pig ileum
8.SCREENING OF ANTIFERTILITY DRUGS:-
SCREENING OF ANTIFERTILITY AGENTS IN FEMALES
1.Antiovulatory Activity:-
HCG-induced ovulation in Rat
Cupric acetate induced ovulation in Rabbits
2.Estrogenic Activity:-
INVIVO METHODS
Vaginal Opening
Assay for water upatake
Four day Uterine weight Assay
Vaginal cornification
Chick oviduct method
INVITRO METHODS
Estrogenic receptor-binding Assay
Dextran-coated charcoal (DCC) adsorption technique
Potency Assay
3.Antiestrogenic Activity
INVIVO METHOD
Antagonism of physiological effects of estrogen
INVIVO METHODS
Aromatase inhibition
4.Progestational activity
INVIVO METHODS
Pregnancy maintain test
Proliferation of uterine endometrium in estrogen primed Rabbits( Claudberg
Mcphail Test
Carbonic anhydrase activity in Rabbits endometrium
Deciduoma reaction in Rats
Prevention of abortion in oxytocin treated pregnant rabbit
INVITRO TEST
Progesterone receptor binding assay
5.anti-Progestational activity
Anti-progestational activity in immature rabbits(Mc ginty test)
6.Anti-implantation activity
7.Abortifacient activity
SCREENING OF ANTIFERTILITY AGENTS IN MALES
9.Screening of anti diabetic agents:-
In vivo models
Pancreatectomy in dogs
Alloxan induced diabetes
Streptozotocin induced diabetes
Virus induced diabetes
Insulin deficiency due to insulin antibodies
Genetically diabetic animals
Spontaneously diabetic rats
Spontaneously diabetic mice
Transgenic animals
Euglycemic clamp technique
Glucose absorption model
Animal models for type1DM
Animal models for type2 DM
Transgenic animals/Knock out animals
In vitro models
Glucose transporters in adipocytes
Perfusion of isolated rat pancreas
Perfusion of isolated rat liver
Inhibition of glucosidase in vitro
Inhibition of aldose reductase
Inhibition of -amylase in vitro
10. Screening methods for anti-anginal drugs
In- vitro models
Langendorff heart preparation
Isolated rabbit aorta preparation
Relaxation of bovine coronary artery
Coronary artery ligation in isolated rat heart
Isolated heart lung preparation
Plastic cast technique in dogs
In-vivo models
Occlusion of coronary artery
Microspheres induced acute ischemia
Isoproterenol induced myocardial necrosis
Stenosis-induced coronary thrombosis model
Electrical-stimulation induced coronary thrombosis
Myocardial ischemic preconditioning model
11.SCREENING OF ANALGESICS
Invivo Methods
Thermal methods
-ELECTRIC HOT PLATE METHOD
-Radiant heat method (analgesiometer)
-Tail warm water immersion method
-Neuropathic pain model
Mechanical methods
-Haffners Tail clip method
-Randall selitto test
-Measured Caudal Compression
Electrical methods
-Tooth pulp electrical stimulation method
-Monkey shock test
-Pododolimeter
-Rectodolorimeter
Ischemic methods
Chemical methods
-Formalin test
-Writhing test
-Frog foot immersion method
Human method:
Hollanders method
Achilles tendon method
Other methods:
Pethidine pottentiation
Nalorphine antagonism
Oxytocin cramping
Lenticular opacity
In vitro methods-
H-naloxone binding assay
- opiate receptor binding assay
Assay to study canabinoid activity
12.SCREENING OF Antiarrhythmic Drug:-
In-Vitro Models:
1) Isolated guinea pig papillary muscle.
2) Action potential and refractory period in isolated left ventricular guinea pig
papillary muscle
3) Langendorff technique
4) Acetylcholine & potassium Induced arrhythmia
In-Vivo Methods:
Chemically induced arrhythmia
Electrically induced arrhythmia
Mechanically induced arrhythmia
Exercise Induced Arrhythmia
Canine model of two stage ligation
Animal models to induce cardiac arrhythmias
Type of arrhythmia Model
Supraventricular tachycardia
Wolf- Parkinson-White syndrome Pre-excitation in dogs
Transgenic mice (PRKAG2 over expression)
Re-entrant arrhythmia of AV node Isolated rabbit heart preparation
Isolated rabbit heart atrium preparation, including the AV node and Bundle of His
Blocked of atrial impulses from the anterior input site to the AV node in dogs
Isolated canine AV nodal preparation
Atrial Flutter Canine right atrial crush injury model
Atrial flutter induced by acetylcholine (ACh) and rapid pacing in the dog
Atrial flutter by aconitine
Right atrial enlargement model of atrial flutter
Atrial Fibrillation Atrial fibrillation by atrial ischaemia in dogs
PACAP-27 induced biphasic chronotropic effect and atrial fibrillation.
Atrial fibrillation in dogs by atrial burst pacing
Canine model of chronic atrial fibrillation
Vagal atrial fibrillation
Atrial Fibrillation in the isolated Langendorff-perfused rabbit heart
Atrial Fibrillation by fibrillation pacemaker
Atrial fibrillation by aconitine
Ventricular fibrillation Porcine model of VF
Ventricular fibrillation induction by 60-hz alternating current in isolated swine right ventricle
Ventricular arrhythmia by various chemicals
Ischaemia induced ventricular arrhythmia
Ventricular arrhythmia during exercise by ischaemia
Stretch induced arrhythmias in isolated canine ventricle
Model for sudden cardiac death
13.Screening of Antidepressant Drugs
METHODS
Behavioral tests
-Catalepsy antagonism in chicken.
-Despair swim test.
-Tail suspension test in mice.
-Learned helplessness in rat.
-Muricide behavior in rat
Tests for antidepressant activity based on mechanism of
action
-Potentiation of norepinephrine toxicity.
-Compulsive gnawing in mice.
-Apomorphine induced hypothermia in mice.
-Tetrabenazine antagonism in mice.
-Reserpine induced hypothermia.
-5-Hydroxytryptophan potentiation in rats.
-Yohimbine toxicity enhancement.
In vitro methods
1. Inhibition of [3H]-norepinephrine uptake in rat brain
synaptosomes.
2. Inhibition of [3H]-dopamine uptake in rat striatal
synaptosomes.
3. Inhibition of [3H]-serotonin uptake in rat brain
synaptosomes.
4. Binding to monoamine transporters.
5. Antagonism of p-chloramphetamine toxicity by
inhibitors of serotonin uptake.
6. Receptor sub sensitivity after treatment with
antidepressants.
7. Measurement of beta adrenoreceptor stimulated
adenylate cyclase.
8. [3H]-yohimbine binding to alfa-2 adrenoceptors in rat
cerebral cortex.
6. Test for anti cholinergic properties by [3H]-QNB
binding to muscarinic receptors in rat brain.
7. Monoamine oxidase inhibition.
14.SCREENING OF ANTICANCER DRUGS
Animal Models of Cancer
ECTOPIC- with tumours implanted or induced in superficial tissues ear pinna / dermal /
subcutaneous, mammary fatpad and footpad.
INTERNAL/ORTHOTOPIC- where tumours are implanted or induced internally or
orthotopically in the tissues of origin
GENETICALLY ALTERED MODELS- derived from spontaneous mutations or generated
by genetic manipulation
In Vitro Cell Line Screening Project (IVCLSP): The operation of this screen utilizes 60
different human tumor cell lines, representing leukemia, melanoma and cancers of the
lung, colon, brain, ovary, breast, prostate, and kidney.
IN-VITRO METHODS
Tetrazolium salt assay
Sulphorhodamine B assay
H3 thymidimine uptake assay
Flouroscence
Dye exclusion tests
Clonogenic assay
Cell counting assay
IN-VIVO METHODS
A) CHEMICALLY INDUCED TUMOUR MODEL
DMBA-induced mouse skin papillomas
N-methyl N-nitrosoureas (MNU) induced rat mammary gland carcinogenesis
DMBA-induced rat mammary gland carcinogenesis
MNU induced tracheal squamous cell carcinoma in hamster
N-N Diethyl (DEN) induced lung adenocarcinoma in hamster
1,2 Dimethylhydralazine (DMH) induced colorectal adenocarcinoma in rat and
mouse
Azoxymethane induced Aberrant crypt foci in rats
B) Models involving cell lines/ tumour piece implantation :
Hollow fibre technique
Use of xenograft
Nude mouse model
Newborn rat model
Transgenic mouse model
15.Screening of drugs used in migraine
In- vitro tests
Isolated human coronary artery contraction
( vascular)
Isolated human middle meningeal artery
constriction (vascular)
Neurotransmitter release by sliced trigeminal
nucleus of rats ( neurogenic )
5-HT binding assay in rats or bovine brain tissue
In-vivo tests
Based on vascular involvement:
1. Constriction of carotid AV anastomoses
in anaesthetized pigs
2. Constriction of external carotid artery
bed in anaesthetized dogs.
Based on neurogenic involvement :
1. Plasma protein extravasation model
2. Central trigeminal neuronal inhibition
3. Inhibition of neurogenic dural
vasodilation in anaesthetized animals
4. Limitation of c-fos gene expression models
Integrated migraine models :
1. Electrical stimulation of the trigeminal
ganglia/nerves and superior saggital
sinus
2. Chemical stimulation of sensory nerve
fibres with capsaicin
3. Intravital microscopy : studying of dural
vasculature in vivo
4. Meningeal blood flow study with laser
Doppler flowmetry
16. SCREENING OF DIURETICS
In Vivo Methods
Diuretic activity in rats (LIPSCHITZ test)
Saluretic activity in rats
Diuretic and saluretic activity in dogs
Clearance methods
Micropuncture techniques in rats
Stop flow technique
In Vitro Methods
Carbonic anhydrase inhibition in vitro
Patch clamp technique in kidney cells
Perfusion of isolated kidney tubules
Isolated perfused kidney technique
17. SCREENING METHODS FOR ANTIMALARIAL
AGENTS
In vitro methods
3
H Hypoxanthine uptake
Giemsa stained slide method
Flow cytometry
LDH activity of P. falciparum
Micro-test( Mark III)
In vivo methods
Rodent models
Plasmodium berghei 4 day suppression test
Dose ranging full four day test
Onset of activity and recrudescence test
Prophylactic test
Hills test for causal prophylaxis and residual activity
Xenogenic transplantation models
Resistance
Avian models
447 species infected with malarial parasite
avian parasite infects nucleated erythrocytes
Vector- Aedes, Culex
Pre erythrocytic stage- mesodermal tissue, 2 generations in host
Wasielewski(1904)-parasite killed in vitro by 1:10,000 quinine
Primate models
Plasmodium cynomolgi rhesus model
18. Screening of Hypolipidemics
Induction of experimental atherosclerosis
Cholesterol-diet induced atherosclerosis in rabbits and other species
Hereditary hypercholesterolemia in rats
Hereditary hyperlipemia in rabbits
Transgenic animals
Triton-induced hyperlipidemia
Fructose induced hypertriglyceridemia in rats
Intravenous lipid tolerance test in rats
Influence on lipoprotein-lipase activity
Inhibition of cholesterol biosynthesis
General considerations on HMG-CoA reductase
Inhibition of the isolated enzyme HMG-CoA-reductase in vitro
Inhibition of the incorporation of 14C sodium acetate into cholesterol in isolated
liver cells
Ex vivo inhibition of cholesterol biosynthesis in isolated rat liver slices
Effect of HMG-CoA-reductase inhibitors in vivo
Inhibition of cholesterol absorption
Inhibition of ACAT (Acyl coenzyme A:cholesterol acyltransferase)
In vitro ACAT inhibitory activity
In vivo tests for ACAT inhibitory activity
Lymph fistula model for cholesterol absorption
Interruption of bile acid recirculation
Cholestyramine binding
Inhibition of lipid oxidation
Inhibition of lipid peroxidation of isolated plasma low density lipoproteins
Internalization of labeled LDL into HepG2 cells
19. Screening of Anti-Parkinson Drugs
In vivo methods
Cholinomimetic induced parkinsonism
Reserpine induced parkinsonism
Neuroleptic induced parkinsonism
MPTP induced parkinsonism
Bilateral nigrostriatal 6-OHDA administration test
Circling behaviour in rats
Elevated body swing test
FeCl3 induced parkinsonism
Skilled paw reaching in rats
Stepping test in rats
IN VITRO TESTS
Dopamine stimulated adenylyl cyclase activity
Radioligand binding studies for D1 and D2 receptors
Dopamine release from synaptosomes
In vitro neuroprotective efficacy
20. Anti-HIV Drug Screening Models
In vivo models
Nonhuman primate models: such as Chimpanzees, Pigtail macaques, Baboons
Severe combined immunodeficiency mice
Transgenic mice models
In vitro models
Mono Mac 1-cell line
Visna virus model
Blood brain barrier model for CNS infection
a) Macaque BBB Model
b) Cultured Brain Microvascular Endothelial Cells
c) DIV-BBB Model
21. Screening of sedatives and hypnotics Drugs
Observation tests:
Observational assessment
Motor activity and behaviour:
Method of intermittent observations
Open field test
Hole-board test
Combined open field test
Coordination:
Inclined plane
Chimney test
Grip strength
22. Assays of anti obesity activity
Experimental manipulation
-Diet induced obesity
-Hypothalamic obesity
Surgically induced
Chemically induced
Virus induced
Genetically obese animals
-Spontaneously obese rats
-Spontaneously obese mice
-Transgenic animals
In vivo assay
Anorectic activity
-Food consumption in rats
In vitro assays
Metabolic activity
-GDP binding in brown adipose tissue
-Uncoupling protein and GLUT4 in brown adipose tissue
Activity of 3 agonists
-cAMP response element luciferase receptor gene assay for
adrenoceptors
23. Screening of antitubercular Drugs
IN-VITRO MODELS
1.ABSOLUTE CONC METHOD
2.RADIOMETRIC METHOD
3.CALORIMETRIC NITRATE REDUCTASE ASSAY
4.MYCOBACTERIA GROWTH INDICATOR TUBE
5.PHAGE BASED ASSAY
5.RT-PCR
In-vivo models
Animals used-
Guinea pig
Mice:- Two stage chemotherapy in mice , Low-Dose Aerosol Infection
Model , Gamma Interferon Gene-Disrupted Mice
Monkey
Golden hamster
Rabbit
