3. ANGINA
A pain syndrome due to induction of an adverse
oxygen supply/demand situation in a portion of the
myocardium.
4. 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.
9. 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
10. 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.
11. 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.
12. 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.
13. 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.
14. 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
15. 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
16. 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.
18. 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 )
21. 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
22. 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
23. 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.
24. 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
25. 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
26. 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.
27. 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.