This document provides information on screening of cardiac glycosides. It begins with the anatomy and physiology of the heart and causes of cardiac failure. It then discusses drugs used to treat cardiac failure, including their modes of action. Various in vivo and in vitro screening tests for cardiac glycosides are described, such as frog heart assays, rat and dog models of heart failure, and guinea pig aortic binding models. Parameters for evaluating test compounds like ED50 values are also mentioned. The document concludes with details on decay rate and absorption rate determination methods for cardiac glycosides.

1. 11
SCREENINGSCREENING
OF CARDIAC GLYCOSIDESOF CARDIAC GLYCOSIDES
SANDIP CHAUDHARISANDIP CHAUDHARI
DEPT. OF PHARMACOLOGYDEPT. OF PHARMACOLOGY

2. 22
CONTENTS
1) Anatomy & Physiology of Heart.1) Anatomy & Physiology of Heart.
2) Cardiac failure :- Causes.2) Cardiac failure :- Causes.
Classification.Classification.
3) Drugs use in cardiac failure.3) Drugs use in cardiac failure.
4) Mode of action.4) Mode of action.
5) Screening tests:- In Vivo.5) Screening tests:- In Vivo.
In Vitro.In Vitro.
6) References.6) References.

3. 33
Mechanism of contractionMechanism of contraction
3Na+2K+ Ca++
A
T
P
Na+
Na+3Na+2K+ Ca++
Ca++
S.R.
PLASMA MEMBRAINE
Na+/K+
ATPase
Ca++
Chanal

4. 44
Congestive Heart Failure

5. 55
Heart failure is a complex of signs and symptoms that occurs when the heart
fails to pump an adequate cardiac output.
Starling’s Curve for normal and falling heart

6. 66

7. 77
Types of Heart Failure
Acute Heart Failure
Sudden and Rapid development of heart failure
i) Larger myocardial infarction
ii) Valve rupture
iii) Cardiac tamponade
Chronic Heart Failure
 Most often
 Heart failure develops slowly
Observed in following status
 Myocardial Ischemia
 Multivalvular heart disease
 Systemic arterial hypertension
 Chronic lung disease resulting in hypoxia
 Progression of acute into chronic failure

8. 88

9. 99
Treatment with drug:2,11
Angiotensin-Converting Enzyme Inhibitors
β-Adrenoceptor Blockers
Diuretics
Vasodilators
Enhancement of Myocardial Contractility
Phosphodiesterases inhibitor

10. 1010
CARDIAC GLYCOSIDECARDIAC GLYCOSIDE
 It is +ve inotropic. (Increase force of contraction.)It is +ve inotropic. (Increase force of contraction.)
 Cardiac glycosides are drugs used in the treatment of heartCardiac glycosides are drugs used in the treatment of heart
conditions, namely atrial fibrillation, atrial flutter and cardiacconditions, namely atrial fibrillation, atrial flutter and cardiac
arrhythmia.arrhythmia.
 They increses myocardial contractality & output inThey increses myocardial contractality & output in
hypodynamic heart with incerseing Ohypodynamic heart with incerseing O22 consumtion & notconsumtion & not
increses heart rate.increses heart rate.

11. 1111
Mode of actionMode of action2,3,4,6,112,3,4,6,11
 An increase of force of contraction via inhibition of the
Na+/K+ATPase pump
 A decrease of conduction of electrical impulses through the
AV node.

12. 1212
Mode of actionMode of action
3Na+2K+ Ca++
A
T
P
Na+
Na+3Na+2K+ Ca++
Ca++
S.R.
PLASMA MEMBRAINE
Na+/K+
ATPase
Ca++
Chanal
Cardiac
glycoside
inhibit
+

13. 1313
Image from Rang and Dale
Showing entry and exit of ions

14. 1414

15. 1515
Frog Method Assay. USPFrog Method Assay. USP..11
Frog(20-30g)Frog(20-30g)
In ventral lymph sac Digitalis injectIn ventral lymph sac Digitalis inject
(0.015mg/g of body wt.)(0.015mg/g of body wt.)
Heart is removeHeart is remove
Typical resultTypical result
1) systolic arrest of ventricle.1) systolic arrest of ventricle.
2) widely dilated atrium.2) widely dilated atrium.
calculate % of animal deadcalculate % of animal dead
in test Vs international std.in test Vs international std.

16. 1616
Digitalis action on hypodynamic frog heartDigitalis action on hypodynamic frog heart
PRINCIPLE:-PRINCIPLE:-
Inability to contract to physiological normal it is said to be aInability to contract to physiological normal it is said to be a
hypodynamic heart.hypodynamic heart.
PROCEDURE:-PROCEDURE:-
Perfuse frog heartPerfuse frog heart
Record the normal effect on heart & digoxine in increasing conc.Record the normal effect on heart & digoxine in increasing conc.
(0.1,0.2,0.4,0.5ml)(0.1,0.2,0.4,0.5ml)
Rrplace the riger solution with hypodynamic riger solution containingRrplace the riger solution with hypodynamic riger solution containing
¼ calcium then ringer solution¼ calcium then ringer solution
Note the change by recording graphNote the change by recording graph

17. 1717
Then administered digoxine inpresence of modified riger solutionThen administered digoxine inpresence of modified riger solution
(0.1,0.2,0.4,0.5ml)(0.1,0.2,0.4,0.5ml)
Note the change in contractilityNote the change in contractility
EVALUATION:-EVALUATION:-
Compare the responses of these drugs in normal &Compare the responses of these drugs in normal &
hypodynamic heart.hypodynamic heart.

18. 1818

19. 1919
CARDIAC GLYCOSIDE SCREENINGCARDIAC GLYCOSIDE SCREENING
CLASSIFICATIONCLASSIFICATION11
IN VITRO TEST.IN VITRO TEST.
1)Oubain binding.1)Oubain binding.11
2)Influence of Na+/K+ ATPase2)Influence of Na+/K+ ATPase
enz.enz. 11
On isolated tissue & organ.On isolated tissue & organ.
3) Isolated Cat papillary3) Isolated Cat papillary11
muscle.muscle. 11
4) Isolated Hamster4) Isolated Hamster
cardiomyopathic Heart.cardiomyopathic Heart. 11
5) K+ Loss from isolated Guinea5) K+ Loss from isolated Guinea
pig heart.pig heart. 11
6)Guinea pig isolated atrial6)Guinea pig isolated atrial
model.model.1717
IN VIVO TEST.IN VIVO TEST.
1)Cardiac toxicity in cats.1)Cardiac toxicity in cats. 11
(Hatcher’s Method.)(Hatcher’s Method.)
2)Rat model of heart failure2)Rat model of heart failure1616
..
3)Dog models of heart failure.3)Dog models of heart failure.1616
4)Doxirubicin cardiomyopathy in4)Doxirubicin cardiomyopathy in
rabbit heart failure.rabbit heart failure.1616
5)Gunia pig model.5)Gunia pig model.1616
6)Decay rate & enteral absorption6)Decay rate & enteral absorption
rate of cardiac glycosiderate of cardiac glycoside11

20. 2020
IN VIVO MODELSIN VIVO MODELS

21. 2121
1)1) Cardiac toxicity in Cats.Cardiac toxicity in Cats.
(Hatcher’s Method.)(Hatcher’s Method.)11
 Purpose & Rational:-Purpose & Rational:- The cat unit as the amount of crystalline ouabainThe cat unit as the amount of crystalline ouabain
Which is fatal with in about 90 minutes to kilogram of a cat when theWhich is fatal with in about 90 minutes to kilogram of a cat when the
drug in injected slowly & continuously into the femoral vein.drug in injected slowly & continuously into the femoral vein.
 ProcedureProcedure11
:-:-
Cat of either sex(2-3.5kg)Cat of either sex(2-3.5kg)
Anesthetized by ether 70mg/kgAnesthetized by ether 70mg/kg
By Tracheotomy tracheal cannula is insertedBy Tracheotomy tracheal cannula is inserted
ECG is recorded by lead IIECG is recorded by lead II
Test Solution infusedTest Solution infused
Cardiac arrest within 30-60minCardiac arrest within 30-60min
 Evaluation:-Evaluation:- Time of cardiac arrest after I.V. infusion of test drug &Time of cardiac arrest after I.V. infusion of test drug &
standard is compare.standard is compare.11

22. 2222
TracheostomyTracheostomy

23. 2323
2) RAT MODELS OF HEART FAILURE2) RAT MODELS OF HEART FAILURE1616
 Purpose & RationalPurpose & Rational:-:-Myocardial infraction following ofMyocardial infraction following of
coronary artery ligationcoronary artery ligation..
 Procedure:-Procedure:-
Male Sprague-Dawley rat (250-300g)Male Sprague-Dawley rat (250-300g)
Anasthetized with hexobarbitalAnasthetized with hexobarbital
Trachea cannulatedTrachea cannulated
Cheast cavity exposed & LAD carotid artery isCheast cavity exposed & LAD carotid artery is
ligatedligated

24. 2424
Chest cavity is sutured backChest cavity is sutured back
After 4 week cavity is opened & carotid artery &After 4 week cavity is opened & carotid artery &
jugular vein is cannulated.jugular vein is cannulated.
Measured B.P.& administered test compoundMeasured B.P.& administered test compound
Scarifies & isolated heart is used for studying CaScarifies & isolated heart is used for studying Ca++++
channelchannel
sarcoplasmic reticulum Casarcoplasmic reticulum Ca++++
ATPase level.ATPase level.
EVALUATION:-EVALUATION:-Compare test drug with control drug &calculateCompare test drug with control drug &calculate
EDED5050

25. 2525
3)DOG MODEL OF HEART FAILURE3)DOG MODEL OF HEART FAILURE1616
 PORPOSE & RATIONALE:-PORPOSE & RATIONALE:-
Coronary rapid pacing produce the syndromeCoronary rapid pacing produce the syndrome
of CHF. In this model the study of left ventricular functionof CHF. In this model the study of left ventricular function
& volumes more accurately then rodent model.& volumes more accurately then rodent model.
 PROCEDURE:-PROCEDURE:-
Adult male dog(18-25kg) are anesthetized withAdult male dog(18-25kg) are anesthetized with
Phenobarbital(30mg/kg)Phenobarbital(30mg/kg)
Artificial respiration by cannulate trachea.Artificial respiration by cannulate trachea.
chest cavity opened & heart is exposed.chest cavity opened & heart is exposed.

26. 2626
A pacemaker is a programmed to pace at 240-260A pacemaker is a programmed to pace at 240-260
beats/min. for 2-4 weeks.beats/min. for 2-4 weeks.
Heart is placed back in chest cavity.Heart is placed back in chest cavity.
Air from thorax is removed.Air from thorax is removed.
Skin wound is closed by applying antibiotic.Skin wound is closed by applying antibiotic.
Significant heart failure is developed by 4 week andSignificant heart failure is developed by 4 week and
continue up to 10 weeks.continue up to 10 weeks.

27. 2727
test drugs administered s.c. or i.m. over a period of 14 days.test drugs administered s.c. or i.m. over a period of 14 days.
EVALUATION:EVALUATION:
Comparison between test group & control group is made on theComparison between test group & control group is made on the
basis of changes in the parameters like cardiac output &basis of changes in the parameters like cardiac output &
systemic vascular resistance.systemic vascular resistance.

28. 2828
4) DOXORUBICIN CARDIOMAYOPATHY IN4) DOXORUBICIN CARDIOMAYOPATHY IN
RABBIT HEART FAILURE.RABBIT HEART FAILURE.1616

Purpose & RationalPurpose & Rational:-:-
Doxorubicin exhibits acute & chronic cardio toxicity and hasDoxorubicin exhibits acute & chronic cardio toxicity and has
been used to induced heart failure in various animal species.been used to induced heart failure in various animal species.
 PROCEDURE :-PROCEDURE :-
Rabbit (5-6 kg) of either sex.Rabbit (5-6 kg) of either sex.
Doxorubicin (1 mg/kg i.v.twice weekly) is given for 6-9Doxorubicin (1 mg/kg i.v.twice weekly) is given for 6-9
week in control group.week in control group.
In test group test drugs are administered for 4-6 weeksIn test group test drugs are administered for 4-6 weeks
(s.c. or i.p.)(s.c. or i.p.)

29. 2929
Anesthetized with phenobarbiton sodium ( 35 mg/kg i.p.)Anesthetized with phenobarbiton sodium ( 35 mg/kg i.p.)
Carotid artery is cannuleted for measuring the blood pressure.Carotid artery is cannuleted for measuring the blood pressure.
Heart is exposed and cannula is inserted into left ventricle toHeart is exposed and cannula is inserted into left ventricle to
measure LVEDP & dp/dt.measure LVEDP & dp/dt.
Animals are sacrified & heart is processed forAnimals are sacrified & heart is processed for
immunohistochemical studies.immunohistochemical studies.

30. 3030
 EVALUATION :-EVALUATION :-
Left ventricular fractional shortening & ratio ofLeft ventricular fractional shortening & ratio of
RYR/Calcium magnesium ATPase mRNA level in the leftRYR/Calcium magnesium ATPase mRNA level in the left
ventricle.Studied in the test drug group & comparison are madeventricle.Studied in the test drug group & comparison are made
with the control group.with the control group.

31. 3131
5) GUINEA PIG AORTIC BINDING MODEL.5) GUINEA PIG AORTIC BINDING MODEL.1616

Purpose & RationalPurpose & Rational:-:-
Alteration of myocardial function in guinea pig model hasAlteration of myocardial function in guinea pig model has
some similarity to end stage failing human myocardium.some similarity to end stage failing human myocardium.
 PROCEDURE :-PROCEDURE :-
Male guinea pig (250-400 g)Male guinea pig (250-400 g)
Anesthetized with etherAnesthetized with ether
chest cavity is open and heart is exposed without closing ofchest cavity is open and heart is exposed without closing of
blood circulation.blood circulation.

32. 3232
Third of the both ventricle tied off with disinfectant threadThird of the both ventricle tied off with disinfectant thread
and clamp it.and clamp it.
After removal of the clamp heart is placed back.After removal of the clamp heart is placed back.
The test drugs are administered s.c. or i.m. for 14 days.The test drugs are administered s.c. or i.m. for 14 days.
Animals developed symptoms of C.H.F. with death rate ofAnimals developed symptoms of C.H.F. with death rate of
80% within 1 day.80% within 1 day.
lung edema and liver congestion are observed histological.lung edema and liver congestion are observed histological.

33. 3333
 EVALUATION :-EVALUATION :-
TheThe ability of the test drug to reverse this studies. ED50 of testability of the test drug to reverse this studies. ED50 of test
drug is calculated to assess the survival rate.drug is calculated to assess the survival rate.

34. 3434
6)Decay rate & enteral absorption rate of cardiac6)Decay rate & enteral absorption rate of cardiac
glycosideglycoside11
 Purpose & Rational:-Purpose & Rational:- Determine decay rate of cardiac glycosideDetermine decay rate of cardiac glycoside
by excretion & degradation.by excretion & degradation.11
 ProcedureProcedure11
:-:- Beagal dog(8-20kg)Beagal dog(8-20kg)
Tracheostomy- Tracheal cannula insertedTracheostomy- Tracheal cannula inserted
Vena fumoralis cannulated for infusionVena fumoralis cannulated for infusion
ECG is recorded from lead IIECG is recorded from lead II
First toxic dose is infused- Blok AV node & extra systole.First toxic dose is infused- Blok AV node & extra systole.
After 4,8,12,24hrs infusion with cardiac glycoside continueAfter 4,8,12,24hrs infusion with cardiac glycoside continue

35. 3535
first dose is partially metabolize so second dose administer less than firstfirst dose is partially metabolize so second dose administer less than first
dose.dose.
Evaluation:-Evaluation:- Second dose expected to change ECG = amount of FirstSecond dose expected to change ECG = amount of First
dose to be metabolize or excreted.dose to be metabolize or excreted.11

36. 3636
IN VITRO MODELSIN VITRO MODELS

37. 3737
 Purpose & Rational:-Purpose & Rational:-To determine K+ loss from heart dueTo determine K+ loss from heart due
to inhibition of Na+/K+ ATPase pump.to inhibition of Na+/K+ ATPase pump.11
 ProcedureProcedure11
:-:- Heart ofHeart of Guinea pig is isolateGuinea pig is isolate
Coronary outflow is measured by counting drop ofCoronary outflow is measured by counting drop of
effluent by photo celleffluent by photo cell
Effluent is analyze by Flame photometerEffluent is analyze by Flame photometer
Determine K+ con. from the affluent & effluentDetermine K+ con. from the affluent & effluent
& compare with Std.& compare with Std.
 Evaluation:-Evaluation:- Following parameter , K+ concentration ,Following parameter , K+ concentration ,
1)K+ loss from Isolated guinea pig heart1)K+ loss from Isolated guinea pig heart11

38. 3838

39. 3939
 Purpose & RationalPurpose & Rational :-:- Decrease performance of papillaryDecrease performance of papillary
muscle, after prolong electrical stimulation & duringmuscle, after prolong electrical stimulation & during
restoration of force under influence of cardiac glycoside.restoration of force under influence of cardiac glycoside.11
 ProcedureProcedure11
:-:- either sex of cats(2.5-3kg)either sex of cats(2.5-3kg)
Anesthetized with ether, opening of thorax & isolate papillaryAnesthetized with ether, opening of thorax & isolate papillary
muscle from right ventricle of heart & placed in organ bathmuscle from right ventricle of heart & placed in organ bath
containing oxygenated ringer sol. at(36containing oxygenated ringer sol. at(3600
c)c)
Stimulated electrically 4-5V at 30/min rate & contraction isStimulated electrically 4-5V at 30/min rate & contraction is
record.record.
slowly contractility of muscle decreasesslowly contractility of muscle decreases
Test cardiac glycoside is added in bath to restore theTest cardiac glycoside is added in bath to restore the
contraction forcecontraction force
2)ISOLATED2)ISOLATED CAT PAPILLARY MUSCLE.CAT PAPILLARY MUSCLE.11

40. 4040
Evaluation :-Evaluation :-
Contractility of muscle calculated as % of predose level.Contractility of muscle calculated as % of predose level.
Dose response curve can be established using various doses.Dose response curve can be established using various doses.

41. 4141

42. 4242
3)GUINEA PIG’S ISOLATED ATRIAL MODEL3)GUINEA PIG’S ISOLATED ATRIAL MODEL..1717
 PURPOSE & RATIONAL:-PURPOSE & RATIONAL:-
Cardiotonic activity by evaluating their ability to increase the forceCardiotonic activity by evaluating their ability to increase the force
of myocardial contraction in isolated atrial tissue from guinea pig.of myocardial contraction in isolated atrial tissue from guinea pig.
Thus test compound may be evaluated for their ability to augment theThus test compound may be evaluated for their ability to augment the
contractile response.contractile response.
 PROCEDURE:-PROCEDURE:-
Adult male guinea pig (300-400gm)Adult male guinea pig (300-400gm)
Anasthetized with etherAnasthetized with ether
Chest cavity is openedChest cavity is opened

43. 4343
Remove the heart& place in already prepared ice cold locke ringerRemove the heart& place in already prepared ice cold locke ringer
solution & aerated with a mixture of 95% of oxygen & 5% co2solution & aerated with a mixture of 95% of oxygen & 5% co2
Separate the atria from ventricle.Separate the atria from ventricle.
Submerge in locke ringer solution & connect with stimulator.Submerge in locke ringer solution & connect with stimulator.
Three consecutive reading are taken of the spontaneous atrialThree consecutive reading are taken of the spontaneous atrial
rate at 5 minute interval. Record atrial rate on physiograph.rate at 5 minute interval. Record atrial rate on physiograph.

44. 4444
Add oubain in increasing concentration( 0.1,0.3,1.0,3.0 mcg/ml)Add oubain in increasing concentration( 0.1,0.3,1.0,3.0 mcg/ml)
Use the same approach for unknown compound & record theUse the same approach for unknown compound & record the
amplitude on contraction of physiograph.amplitude on contraction of physiograph.
Repeat this experiment for all concentrationRepeat this experiment for all concentration

EVALUATIONEVALUATION:-:-
Determine the ECDetermine the EC5050 for atrial rate& ECfor atrial rate& EC5050 for atrial contractionfor atrial contraction
& check the potency of unknown drug by following formula& check the potency of unknown drug by following formula
ECEC5050 for unknown compoundfor unknown compound
POTENCY=POTENCY= --------------------------------------------------------------------------
ECEC5050 of oubainof oubain

45. 4545

46. 4646

47. 4747
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