cardiac glycoside

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SCREENING SCREENING OF CARDIAC GLYCOSIDESOF CARDIAC GLYCOSIDES

SANDIP CHAUDHARISANDIP CHAUDHARIDEPT. OF PHARMACOLOGYDEPT. OF PHARMACOLOGY

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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.

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Mechanism of contractionMechanism of contraction

3Na+2K+ Ca++A TP

Na+

Na+ 3Na+2K+ Ca++

Ca++

S.R.

PLASMA MEMBRAINE

Na+/K+ATPase Ca++

Chanal

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Congestive Heart Failure

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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

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Types of Heart Failure

Acute Heart Failure

Sudden and Rapid development of heart failurei) Larger myocardial infarctionii) Valve ruptureiii) 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

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Treatment with drug:2,11

Angiotensin-Converting Enzyme Inhibitors

β-Adrenoceptor Blockers

Diuretics

Vasodilators

Enhancement of Myocardial Contractility

Phosphodiesterases inhibitor

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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 heart Cardiac glycosides are drugs used in the treatment of heart conditions, namely atrial fibrillation, atrial flutter and cardiac conditions, namely atrial fibrillation, atrial flutter and cardiac arrhythmia.arrhythmia.

They increses myocardial contractality & output in They increses myocardial contractality & output in hypodynamic heart with incerseing Ohypodynamic heart with incerseing O2 2 consumtion & not consumtion & not increses heart rate.increses heart rate.

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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.

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Mode of actionMode of action

3Na+2K+ Ca++A TP

Na+

Na+ 3Na+2K+ Ca++

Ca++

S.R.

PLASMA MEMBRAINE

Na+/K+

ATPase

Ca++Chanal

Cardiac glycoside inhibit

+

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Image from Rang and Dale

Showing entry and exit of ions

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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 dead calculate % of animal dead in test Vs international std.in test Vs international std.

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Digitalis action on hypodynamic frog heartDigitalis action on hypodynamic frog heart

PRINCIPLE:-PRINCIPLE:- Inability to contract to physiological normal it is said to be a Inability 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 containing Rrplace the riger solution with hypodynamic riger solution containing ¼ calcium then ringer solution ¼ calcium then ringer solution

Note the change by recording graphNote the change by recording graph

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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.

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CARDIAC GLYCOSIDE SCREENING CARDIAC GLYCOSIDE SCREENING CLASSIFICATIONCLASSIFICATION11

IN VITRO TEST.IN VITRO TEST.1)Oubain binding.1)Oubain binding.11

2)Influence of Na+/K+ ATPase 2)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 Hamster 4) Isolated Hamster cardiomyopathic Heart. cardiomyopathic Heart. 11

5) K+ Loss from isolated Guinea 5) K+ Loss from isolated Guinea pig heart. pig heart. 11

6)Guinea pig isolated atrial 6)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 in 4)Doxirubicin cardiomyopathy in rabbit heart failure.rabbit heart failure.1616

5)Gunia pig model.5)Gunia pig model.1616

6)Decay rate & enteral absorption 6)Decay rate & enteral absorption rate of cardiac glycosiderate of cardiac glycoside11

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IN VIVO MODELSIN VIVO MODELS

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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 the Which 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/kg Anesthetized by ether 70mg/kg By Tracheotomy tracheal cannula is inserted By Tracheotomy tracheal cannula is inserted

ECG is recorded by lead II ECG is recorded by lead II

Test Solution infused Test 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

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TracheostomyTracheostomy

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2) RAT MODELS OF HEART FAILURE2) RAT MODELS OF HEART FAILURE1616

Purpose & RationalPurpose & Rational:-:-Myocardial infraction following of Myocardial 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

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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 compound Measured B.P.& administered test compound

Scarifies & isolated heart is used for studying CaScarifies & isolated heart is used for studying Ca++++ channel channel sarcoplasmic reticulum Casarcoplasmic reticulum Ca++ ++ ATPase level.ATPase level.

EVALUATION:-EVALUATION:-Compare test drug with control drug &calculate Compare test drug with control drug &calculate EDED5050

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3)DOG MODEL OF HEART FAILURE3)DOG MODEL OF HEART FAILURE1616

PORPOSE & RATIONALE:-PORPOSE & RATIONALE:-

Coronary rapid pacing produce the syndrome Coronary rapid pacing produce the syndrome of CHF. In this model the study of left ventricular function of 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 with Adult 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.

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A pacemaker is a programmed to pace at 240-260 A 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 and Significant heart failure is developed by 4 week and continue up to 10 weeks.continue up to 10 weeks.

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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 the Comparison 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.

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4) DOXORUBICIN CARDIOMAYOPATHY IN 4) DOXORUBICIN CARDIOMAYOPATHY IN RABBIT HEART FAILURE.RABBIT HEART FAILURE.1616

Purpose & RationalPurpose & Rational:-:- Doxorubicin exhibits acute & chronic cardio toxicity and has Doxorubicin 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.)

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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.

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EVALUATION :-EVALUATION :- Left ventricular fractional shortening & ratio of Left ventricular fractional shortening & ratio of

RYR/Calcium magnesium ATPase mRNA level in the left RYR/Calcium magnesium ATPase mRNA level in the left ventricle.Studied in the test drug group & comparison are made ventricle.Studied in the test drug group & comparison are made with the control group.with the control group.

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5) GUINEA PIG AORTIC BINDING MODEL.5) GUINEA PIG AORTIC BINDING MODEL.1616

Purpose & RationalPurpose & Rational:-:- Alteration of myocardial function in guinea pig model has Alteration 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 of chest cavity is open and heart is exposed without closing of blood circulation.blood circulation.

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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.

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EVALUATION :-EVALUATION :- TheThe ability of the test drug to reverse this studies. ED50 of test ability 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.

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6)Decay rate & enteral absorption rate of cardiac 6)Decay rate & enteral absorption rate of cardiac glycosideglycoside11

Purpose & Rational:-Purpose & Rational:- Determine decay rate of cardiac glycoside Determine 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 infusion Vena 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 continue After 4,8,12,24hrs infusion with cardiac glycoside continue

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first dose is partially metabolize so second dose administer less than first first dose is partially metabolize so second dose administer less than first dose.dose.

Evaluation:-Evaluation:- Second dose expected to change ECG = amount of First Second dose expected to change ECG = amount of First dose to be metabolize or excreted.dose to be metabolize or excreted.11

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IN VITRO MODELSIN VITRO MODELS

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Purpose & Rational:-Purpose & Rational:-To determine K+ loss from heart due to To determine K+ loss from heart due to inhibition of Na+/K+ ATPase pump.inhibition of Na+/K+ ATPase pump.11

ProcedureProcedure11:-:- Heart ofHeart of Guinea pig is isolate Guinea pig is isolate

Coronary outflow is measured by counting drop of Coronary outflow is measured by counting drop of

effluent by photo celleffluent by photo cell

Effluent is analyze by Flame photometer Effluent 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 , contractile Following parameter , K+ concentration , contractile force, coronary flow is determine.force, coronary flow is determine.11

1)K+ loss from Isolated guinea pig heart1)K+ loss from Isolated guinea pig heart11

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Purpose & RationalPurpose & Rational :- :- Decrease performance of papillary Decrease performance of papillary muscle, after prolong electrical stimulation & during muscle, 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 papillary Anesthetized with ether, opening of thorax & isolate papillary muscle from right ventricle of heart & placed in organ bath muscle 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 is Stimulated 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 the Test cardiac glycoside is added in bath to restore the contraction force contraction force

2)ISOLATED2)ISOLATED CAT PAPILLARY MUSCLE. CAT PAPILLARY MUSCLE.11

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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.

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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 force Cardiotonic 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 the Thus 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

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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 atrial Three 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.

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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 the Use 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& EC for atrial rate& EC50 50 for atrial contraction for atrial contraction

& check the potency of unknown drug by following formula& check the potency of unknown drug by following formula

ECEC5050 for unknown compound for unknown compound POTENCY=POTENCY= -------------------------------------------------------------------------- ECEC50 50 of oubain of oubain

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