Screening methods for antianginal & antimalarial drugs

SEMINAR ON INVITRO & INVIVO STUDIES OF ANTIANGINAL & ANTIMALARIAL DRUGS

PRESENTED BY,

CHARU PUNDIR M.PHARM 1ST YEAR

DEPT. OF PHARMACOLOGY & TOXICOLOGY

ANTIANGINAL MODELS

ANGINA A pain syndrome due to induction of an

adverse oxygen supply/demand situation in a portion of the myocardium.

TYPES

Classical angina (common form): attacks are provoked by exercise, emotion, eating or coitus.

Variant/Prinzmetal’s angina (uncommon form): attacks occur at rest or during sleep and are unpredictable.

Unstable angina: due to rapid increase in duration and severity of attacks. Artheromatous plaque formation takes place.

TREATMENT•NITRATES- GTN, Isosorbide dinitrate, mononitrate•β-BLOCKERS- Propanolol, Metopronol•CCB- Verapamil, Diltiazem•POTASSIUM CHANNEL OPENER- Nicorandil

MODELS TO SCREEN ANTIANGINAL DRUGS

IN VITRO MODELS

• Langendorff heart preparation• Isolated Rabbit Aorta preparation• Calcium Antagonism in pithed rat• Relaxation of Bovine Coronary Artery• Coronary Artery Ligation in Isolated Rat Heart• Isolated Heart-lung Preparation• Plastic Casts Technique in Dogs

LANGENDORFF HEART PREPARATION

APPLICATIONS

Testing of coronary vasodilator drugs, electrophysiological evaluations

Recording positive inotropic effects, negative inotropic effects, calcium antagonism, effect on potassium outflow induced by glycosides and determination of hypoxic damage.

Metabolic studies- arrhythmogenic, antiarryhythmic and antifibrillatory effects.

To study EDRF release from coronary vascular bed

PRINCIPLE

Heart is perfused in a retrograde direction from the aorta either at constant pressure or constant flow with oxygenated saline solutions.

Perfusate solution do not flow via normal

ventricular circulatory pathway. Thus left ventricle do not generate pressure volume which represents typical cardiac function.

PROCEDURE

Guinea pigs (wt. 300-500g) are sacrificed by stunning.

Heart is isolated by transabdominal incision. Heart is cradled btw fingers and lifted before incising the aorta,vena cava and pulmonary veins.

After excision heart is dipped in cold perfusion solution(4◦C)

Aorta is located and cannula is inserted into it and heart is perfused with oxygenated Kreb’s solution.

Heart is transferred to a double wall Plexiglas perfusion apparatus maintained at 37◦C.

Oxygenated Kreb’s solution is perfuse at a constant pressure of 40mm Hg.

Small steel hook with a string is attached to the apex of the heart.

Contractile force is measured isometric ally by a force transducer and recorded on a polygraph.

Heart rate is measured through a chronometer coupled to the polygraph.

Antianginal effect of the test drug is indicated by an increase in coronary blood flow.

Which is then further treated with drug and compared with control.

CALCIUM ANTAGONISM IN PITHED RATS Sprague-Dawley rats

(250-350g) are anesthetized ip with Methohexitone sodium.

Trachea is cannulated Rats are provided with

artificial respiration. Pithing rod is used as a

stimulating electrode and continuous electrical stimulation producing a cardio-accelerator response.

Jugular vein is cannulated for administration of drugs and blood pressure is recorded via carotid artery using a pressure transducer.

In the femoral region, an indifferent electrode is inserted sc

CCBs & beta blockers are administered which causes tachycardia

ID50 calculated and compared.

INVIVO 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 Models of coronary flow measurement

ISOPROTERENOL-INDUCED MYOCARDIAL NECROSIS Wistar rats (150-200g) are

pretreated with test drugs orally or sc for atleast a week.

Isoproterenol is injected sc on 2 consecutive days.

Mortality as well as symptoms are recorded in each group and compared to group injected with isoproterenol only.

After 48 hrs of 1st dose animals are sacrificed.

Heart is removed , weighed and preserved for various hemodynamic parameters.

Degree of histopathological changes can be graded as follows:

Grade 0: no change Grade 1: focal areas of

necrosis Grade 2: focal areas of

necrosis and muscle fiber fragmentation

Grade 3: confluent areas of necrosis, edema and inflammation and muscle fiber fragmenation

Grade 4: massive areas of necrosis, edema and inflammation and mural thrombi

MYOCARDIAL-ISCHEMIC PRECONDITIONING MODEL Rabbits (3-4 kg) are

anesthetized with ketamine xylazine.

Trachea canulated and animal is set up for artificial respiration

Right femoral artery and vein are catheterised for measuring hemodynamic parameters.

A 4-0 suture is looped loosely around the marginal branch of left coronary artery to facilitate coronary occlusion.

Ischemic preconditioning is induced by tightening the loop around the coronary artery for 5 min and then loosening to reperfuse the myocardium for 10 min prior to a subsequent 30 min occlusion.

After 30 min. ischemia, ligation is released for 120 min of reperfusion.

Prior to 30 min of occlusion rabbits are selected to receive ischemic preconditioning, no preconditioning or preconditioning along with the administration of test compound.

Animals are sacrificed after reperfusion duration.

Compared with the controlled groups.

Data is analysed by ANOVA using statistical software.

ANTIMALARIAL MODELS

MALARIA A Protozoal disease caused by parasites of the genus

Plasmodium and transmitted to man by certain species of infected female anopheles mosquito.

Five species of the genus Plasmodium cause nearly all malarial infections in humans.

Falciparum – life threatening Vivax Ovale Malariae Knowlesi

(in Southeast Asia—the monkey malaria parasite )

LIFE CYCLE OF PLASMODIUM

TREATMENT Quinolones Cinchona Biguanides Diaminopyrimidines Sulfonamides and sulfone Tetracyclines Sesquiterpine Amino alcohols Mannich base naphthoquinone

IN VITRO METHODS FOR SCREENING ANTIMALARIAL COMPOUNDS

3H Hypoxanthine uptake Giemsa stained slide method Micro test Flow cytometry Measurement of LDH activity of P. falciparum Isobologram analysis

3H HYPOXANTHINE UPTAKE Parasites are cultured in the presence of different

concentration of test compounds in media containing reduced concentration of hypoxanthine.

3H Hypoxanthine (for Purine salvage and DNA synthesis) is added for incubation.

Cells harvested and radioactivity is measured by

a 1205 Betaplate reader (20,000-60,000) % Reduction in 3H Hypoxantine uptake = 100* (Geometric mean cpm of no drug sample) – (mean cpm of test samples)

Geometric mean cpm of no sample

GIEMSA STAINED SLIDE METHOD (MIC) MIN. INHIBITORY CONC.

METHOD)

Parasites are incubated in a 5% suspension of erythrocytes with an initial parasite density (1-2%) at 37◦C.

A sealed incubation chamber continuously gassed with a mixture of 2% O2, 8%CO2, 90%N2 is used.

Increase in the proportion of infected RBCS is assessed at the end of 72 hour incubation period in control samples and at various concentrations of each drug.

IN VIVO METHODS

Plasmodium berghei 4 day suppression test Hill’s test for causal prophylaxix and residual

activity Sporonoicidal activity testing Plasmodium cynomolgi rhesus model

PLASMODIUM BERGHEI 4 DAY SUPPRESSION DAY A group of 5 mice is injected with 0.2ml of aliquot (2*107

parasitized erythrocytes. Plasmodium berghei ANKA strain) iv/ip on day 0.

Vehicle treated mice (control group) is compared with test drug treated group using chloroquine as reference drug.

Experiment is again repeated day 1-3. Day 4- 24 hour after the last dose blood smears from all

animals are prepared with Giemsa stain. Parasitemia is determined microscopically. Difference

between mean value of the control group and those of the experimental groups is calculated and expressed as % reduction or activity using:

activity= 100 - mean parasitemia treated *100 Mean parasitemia control

HILL ’S TEST FOR CAUSAL PROPHYLAXIS AND RESIDUAL

ACTIVITY Mice inoculated with P. yoelii (N67 strain) sporozoites

from A. Stephensi. Test compound have to pass through all the 4 phase.

Phase 1: test compound is given 3 hr after sporozoites inoculation and checked for Parasitemia.

Phase 2: compound is tested for residual activity directed against blood stage parasites by administrating a single dose of the test compound 48hr before 104 trophozoites are injected iv. Time should be same as that of control group.

Phase 3: compound is checked for prolonged residual activity by administrating sporozoites followed by the drug 3h later.

Phase 4: additional procedure is done to clarify whether or not a compound has residual effect on erythrocytic stages during the 48 hr period of drug exposure in vivo.

REFERENCES Vogels Gerhard, Drug discovery and evaluation

Pharmacological assays, Springer publications, 3rd edition, 2008, 253-257.

Gupta S.K, Drug screening methods (preclinical evaluation of new drugs), Jaypee Publishers, 2nd edition, 2009, 314-327.

B.S. Kalra, S. Chawla, P. Gupta, N. Valecha*, Screening of antimalarial drug – An overview, Indian J Pharmacol , February 2006, Vol. 38, Issue 1, 5-12.

Tripathi KD, Essential of medical pharmacology, Jaypee publishers, 6th edition, 2010, 521-780.

Ross and Wilson, Anatomy and Physiology, Churchill Livingstone, 10th edition, 2006, 75-89.

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