evaluation of drugs used in ccf

Evaluation of Drugs used in CCF

Dr Ketan AsawalleJR3, Dept. of Pharmacology

SVNGMC, Yavatmal

Objectives• Introduction• In Vitro Methods• In Vivo Methods• Discussion and Conclusion• References

• CCF is a constellation of symptoms.• Hallmark of CCF is Fatigue and Dyspnoea.• Disease burden us growing = New treatments

needed.• Pathophysiology of CCF not yet completely

understood.• CCF model development depends on:

- Ethical and economical considerations.

- Accessibility and reproducibility od models.

Introduction

• The use of small animals has not been fruitful for a long time.

• Rat models have been used to asses efficacy of drugs and molecular therapies.

• Proof of principle approach - by manipulation of genome and exploring mechanisms of disease progression.

• Large animal models are more helpful - dogs, pigs, sheep.

Introduction

IN VITRO METHODS

• Purpose and rationale

Isolated Syrian Hamster for evaluation of cardiotonic drugs.• Procedure

Syrian Hamsters, age 50 weeks.

Normal as control and tests with cardiomyopathy.

5mg/kg Heparin IP

Heart prepared according to Langendorff method.

Perfused with RL

Allow to equilibrate for 60 mins at 32˚C with preload of 1.5g

Isolated Hamster Cardiomyopathic Heart

• Evaluation

Force of contraction is measured using force transducer attached to polygraph.

Heart rate is measured using chronometer.

Coronary flow is measured using electroflowmeter.

Test drugs are injected through the aortic cannula into the in flowing heart Ringer’s solution.

Contractile force and coronary flow in heart of treated and control group are compared using student’s ’t’ test.

Percentage improvement is calculated.



Prolonged electrical stimulation on cardiac tissue results in decrease in performance.

Cardiac glycosides restore the force of contraction.• Procedure

Cats of either sex, 2.5 to 3 kgs are anaesthetised.

Left thoracotomy done - Heart exposed.

Papillary muscles from right ventricle are isolated and fixed in Ringer’s at 37˚C.

Electrical stimulus of 4-6V are applied at 30/min and contractions are recorded.

Isolated Cat Papillary Muscle (Catell and Gold)

• Evaluation

On electrical stimulation for 1 hour muscle contraction start decreasing.

Cardiac glycosides added - restore contractile force.

Ouabain 300ng/ml.

Evaluation is based on increase in contractile force on adding glycoside.

Calculated as percentage of predose levels and compared between groups.



The binding kinetics of Ouabain are similar to cardiac glycosides• Procedure

Rats heart are submitted through coronary perfusion.

Myocytes are isolated by collagen digestion.

Myocyte sarcolemma is isolated

Radioactive ouabain (3H) with specific activity of 20Ci/mmol is incubated with ligands at 37˚C for 10 mins.

Association Process - 10/100nM ouabain + 200µg membrane preparation.

Ouabain Binding

• Procedure

Equilibrium binding -1. Carried out in the presence of increasing concentrations of (3H) ouabain

(10nM to 3µM).2. 40µg of membranes are added3. After 30 mins duplicate aliquots of 4.5 ml removed and filtered.

Dissociation process -4. Experimental conditions are used to study association.5. 10ml of pre warmed Mg2+ and Pi Tris-HCl added to 0.2mM unlabelled ouabain.

• Evaluation

Radioactivity bound to the filters and specific binding measures are determined.

Kinetic parameters of association and dissociation calculated.

Data analysed by Scatchard plots.

Ouabain Binding

IN VIVO METHODS

In Vivo Models• Rat Models

• Dog Models

• Rabbit Models

• Guinea Pig Models

• Syrian Hamster

• Transgenic Mice

• Newer Therapeutic Targets

In Vivo Models• Rat Models• Dog Models

• Rabbit Models


• Syrian Hamster

• Transgenic Mice



Incomplete or complete ligation of left coronary artery causes ischemia of cardiac muscle.

Failure is associated with left ventricular dilatation, reduced systolic flow and increase in filling pressure.• Procedure

Male Sprague Dawley rats are anaesthetised with 200mg/kg hexobarbital.

Trachea cannulated - artificial respiration provided.

Chest cavity exposed - LAD coronary artery isolated.

Ligature placed and cavity sutured back.

After 4 weeks chest cavity opened - carotid and jugular vein cannulated.

1. Rat Coronary Ligation Model

• Procedure

Filling pressure, systolic, diastolic and mean blood pressure are measured.

Animal sacrificed after hemodynamic parameters tested.

Isolated hearts are used study calcium channels, SR ATPase and protein levels.• Evaluation

In control group the progression of left ventricular dysfunction and myocardial failure is associated with neurohormonal activation as seen in CCF patients.

Depressed myocardial function - altered calcium transients.

Density of L-type calcium channels, SR Ca+2-ATPase and protein levels decreased.

Test group and control groups are compared.

Disadvantage - High mortality and induction of Mild CCF.

1. Rat Coronary Ligation Model


Restriction of blood flow to aorta - Hypertension and CCF.• Procedure

Sprague Dawley rats are anaesthetised with 200mg/kg hexobarbital.

Trachea cannulated - artificial respiration provided.

Abdominal cavity exposed - Aorta is isolated.

Ligature placed and cavity sutured back.

In sham operated groups no banding done.

Test group is administered with drugs.

2. Rat Aortic Banding Model

• Evaluation

Total cardiac mass, weight of left and right ventricle of treated rats are compared between the two groups.

Heart failure = ++ myosin heavy chain mRNA atrial natriuretic factor mRNA.

During compensated hypertrophy - local RAS is active - CCF

Above parameters compared in both groups.

good to study the transition to failure at level of myocardium.

2. Rat Aortic Banding Model


Used to study the transition from compensated hypertrophy to failure.

This strain of rats develop systemic hypertension after receiving high salt diet.• Procedure

Sprague Dawley rats are selected for the study.

Drinking water is replaced with 1% NaCl saline water.

High dhal salt diet is prepared.

Test drug rats are administered the drug for one month.

Animals sacrificed after study period is over - heart observed.

3. Dhal Salt Sensitive Rats’ Model

• Evaluation

Hearts are removed.

Total cardiac mass, weight go right and left ventricles are measured and compared.

Sham control hearts have concentric left ventricular hypertrophy - left ventricular dilatation.

failing heart dies in 15 to 20 weeks.

The ability of the test drug to reverse these changes are studied.

3. Dhal Salt Sensitive Rats’ Model


Model of genetic hypertension.

At 18 - 24 months cardiac failure develops.

Alter calcium cycling is observed.

Transition to failure is associated with alterations in gene expression encoding for extracellular matrix.

Increased number of apoptotic myocytes are observed.• Procedure

Animals are divided in two groups.

Test drug is given for 1 month.

4. Spontaneous Hypertensive Rat Model

• Evaluation

After completion of experimental protocol animal is sacrificed.

Heart is submitted for processing number ofApoptotic cells,Sarcoplasmic reticulum calcium pump mRNA levels andExpression of genes encoding for extracellular matrix.

Results are compared.

4. Spontaneous Hypertensive Rat Model


Spontaneous hypertensive rats that develop heart failure before 18 months of age are selectively bred.

Heart failure develops - gene facp.

These animals have increased plasma renin activity, ANP and aldosterone levels.• Procedure

Animals are divided in two groups.

Test drug is given for 1 month.

4. Spontaneous Hypertensive-Heart Failure Rat Model

• Evaluation

Plasma renin activity, ANP, aldosterone, rynodine receptor density, sarcoplasmic reticulum calcium uptake and endothelial nitric oxide synthase activity is tested.

4. Spontaneous Hypertensive-Heart Failure Rat Model


• Dog Models• Rabbit Models


• Syrian Hamster

• Transgenic Mice


• Allows more accurate study.• Excitation contraction coupling resembles human

heart.• But they are costly and high maintenance.

Dog Models


Chronic rapid pacing of previously normal heart causes syndrome of CCF.

Beats are > 200 per minute.• Procedure

Adult male dog, 18 to 25 kg, are anaesthetised with pentobarbital 30mg/kg.

Airway maintained.

Chest cavity opened by 3-4 cm long thoracotomy - heart exposed.

Ventricular pacing lead is attached to apex of heart.

Cavity closed after placing heart back.

Significant heart failure develops in 4 weeks.

1. Chronic Rapid Pacing Model.

• Procedure

Heart failure is developed for 6 more weeks.

There is bilateral ventricular dilatation over 3-4 weeks.

Test drug is administered by SC and IM injections.• Evaluation

Ejection fraction decreases - decreased CO and increased resistance.

There is time dependent neurohormonal and hemodynamic abnormalities.

Heart failure is reversible if pacing os stopped.

Two groups are compared for parameters like ejection fraction, CO and systemic vascular resistance.

Plasma renin and ANP levels are also compared.

1. Chronic Rapid Pacing Model.


Prolonged volume overload can lead to CCF.

In dog it is created by formation of AV fistula.

Mitral valve is also destroyed.• Procedure

Adult male dog, 10-12 kg, are anaesthetised with pentobarbital 30mg/kg.

Airway maintained.

Chest cavity opened by 3-4 cm long thoracotomy - heart exposed.

Chronic experimental mitral regurgitation is developed.

Significant heart failure develops in 4 weeks - continued upto 10th week.

2. Volume Overload

• Evaluation

Neurohormonal activation of RAS is observed in CHF dogs.

Test and sham treated groups are compared.

Used to study influence of chronic ß-adrenoceptor blockade on myocytes and left ventricular function

2. Volume Overload


Has been used to induce infarction and CCF in dogs.• Procedure

Adult male dog, 10-12 kg, are anaesthetised with pentobarbital 30mg/kg.

Airway maintained.

Transducer introduced from femoral artery for peripheral pressure data.

A microtip catheter inserted in carotid for measuring ventricular pressures.

Heart is exposed - Polystearyl microspores are injected through angiogram catheter - stepwise elevation of LVEDP - target 16-18mmHg.

3. Coronary Artery Ligation and Microembolization

• Evaluation

Recordings are obtained before and after treatment with test drug.• Disadvantages

Time consuming and costly.

Co-lateral circulation - comparison between man and dog is difficult.

High mortality and morbidity (arrhythmia)

3. Coronary Artery Ligation and Microembolization


• Rabbit Models• Dog Models


• Syrian Hamster

• Transgenic Mice


• Less expensive that dog models.• ß myosin heavy chain isoforms predominate in

adult models.• SR contributes to 70% and Na+/Ca2+ 30% of

calcium estimation.

Rabbit Models


Volume overload, pressure overload or combination of both are used to induce heart failure.

Chronic sever aortic regurgitation - systolic dysfunction - heart failure.• Procedure

Rabbits are anaesthetised with pentobarbitone sodium 35mg/kg IP.

Trachea cannulated to maintain artificial respiration.

Chest cavity opened and aortic insufficiency created by destroying valve.

After 14 days - aortic constriction using PVC clamp.

Heart failure occurs within 4 weeks.

1. Volume and Pressure Overload

• Evaluation

Animal sacrificed after experimental protocol.

Heart failure is associated with alterations in ß-adrenoceptor levels.

Protein and mRNA levels of Na+/Ca2+ are increased.

Sarcoplasmic Ca2+ ATPase is not altered.

The ability of test drug to reverse these changes is observed.

Mimics alteration of myocardial function observed in end stage failing heart.

Used to study the changes in excitation contraction coupling in hypertrophy an failing heart.

1. Volume and Pressure Overload


Chronic rapid pacing - 350-400 beats/min.

Myocardial depression, hemodynamic and neurohormonal signs of heart failure.• Procedure

Rabbits are anaesthetised with pentobarbitone sodium 35mg/kg IP.

Trachea cannulated to maintain artificial respiration.

Chest cavity opened and ventricular pacing lead is attached at apex.

A pace of 350-400 beats/min is set.

Heart failure occurs in 4-6 weeks.

Two groups are formed- Test and Sham.

2. Tachycardia Pacing Model

• Evaluation

Animal sacrificed after experimental protocol.

Heart is weighed.

Parameters that are used to compare the two groups are;hemodynamic parameters, plasma renin activity and weight of hearts.

Ability of test drug to reverse these parameters are assessed.

2. Tachycardia Pacing Model


Doxorubicin exhibits acute and chronic cardiotoxicity.

Free radical generation, lipid per oxidation,reactive sulphydryl groups, binding to channel regulatory sites, inhibition of protein synthesis and mRNA.• Procedure

Rabbits of both sexes and various strains (5-6kg).

Doxorubicin 1mg/kg IV twice weekly for 6-9 weeks in both groups.

In test group drug is administered for 4-6 weeks SC or IP.

After protocol animal anaesthetised and LVEDP is measured in situ followed by sacrifice.

3. Doxorubicin Cardiomyopathy Model

• Evaluation

Heart is processed for immunohistochemical tests.

Chronic doxorubicin causes impairment of cardiac contractility.

Decreased gene expression of Ca induced Ca release channels in SR - Rynodine receptor.

RYR2/Ca-Mg ATPase ration significant reduction.

The ability of test drug to reverse these conditions is observed in both groups

3. Doxorubicin Cardiomyopathy Model


• Rabbit Models

• Dog Models

• Guinea Pig Model

• Syrian Hamster

• Transgenic Mice



8 weeks of cardiac binding of the descending thoracic aorta in guinea pigs -overt CHF.

Very much similar to human heart failure.• Procedure

Male guinea pigs, 250-400g are anaesthetised with ether.

Chest cavity opened, heart exposed, Aorta located and ligated.

Symptoms of CCF are developed 80% in one day.

Lung weight, relative heart weight are increased.

1. Cardiac Insufficiency Model

• Evaluation

Lung Weight and heart weight increases due to failure.

Ascites is seen and fluid in thoracic cavity - 3.5-7.5ml.

Decrease in SR Ca2+ ATPase and phospholamban is seen in failing heart.

Signs snd symptoms of heart failure seen

Ability of test drug to reverse these signs are observed.

1. Cardiac Insufficiency Model


• Rabbit Models

• Dog Models


• Syrian Hamster• Transgenic Mice



Cardiomyopathic strains of Syrian hamsters are used - Autosomal recessive.

Degenerative cagnges in started muscles - cardiomyopathy - CCF• Procedure

These animals develop failure after 7-10 months.Time dependent change in myosin isoform expression -

Cardiomyopathy:Pre necrotic stage.Fibrosis and calcium deposition.overlapping period of reactive hypertrophy.Depressed myocardial function.

Cardiomyopathic Hamster

• Evaluation

Test drugs are administered by SC and IM route for 14 days.

Ability of drug to reverse the condition is observed.

This model uses animals with natural disease.

So the time of assessment is very important.

Cardiomyopathic Hamster


• Rabbit Models

• Dog Models


• Syrian Hamster

• Transgenic Mice• Newer Therapeutic Targets

• Specific alteration is expression of genes.• Help in understanding pathophysiology of disease.• Gene targeted disruption of muscle LIM protein (MLP)

Homozygous deletion of MLP gene - dilated cardiomyopathy with hypertrophy.

Heart failure resembles as seen in humans.• Knockout of myogenic factor 5 - cardiomyopathy.• Over expression of - adrenergic receptor kinase or G-protein coupled

receptor kinase 5 - reduced contractility but no HF.• Over expression of tropomodulin model - CCF in 2-4weeks of birth.• In all models test drugs is used to observe reversal of changes in

failure.

Transgenic Mice


• Rabbit Models

• Dog Models


• Syrian Hamster

• Transgenic Mice


• Morbidity and cost to treat CCF is more - important to understand the cellular and molecular derangements.

• Models have been developed targeting molecular causes of HF - regulation of cardiomyocyte calcium model.

• Kaye et al. - Sheep model of rapid pacing induced DCM.

• Tawase et al.- Therapeutic potential of test drug using pig model with volume overload HF - SERCA2a gene.

• First human gene therapy trial has been initiated - patients with CCF receiving SERC2a via myocardial gene therapy.

Gene Therapy

• Stem cells derived from various tissues have been introduced in post-MI myocardium to view attenuation of cardiac re-modeling.

• Initial studies showed promising results.• Recent trials with mesenchymal stem cells have

failed.• Reason for failure

1. Lack of consensus regarding type of stem cell.2. Delivery method3. Delivery location.4. Cell concentration.

• More testing is needed.

Stem Cells

• LV assist devices with totally implantable bi-ventricular assist systems.

• Haithcock et al. used canine micro-embolization model of HF to demonstrate benefits pf LV unloading, established the basis of percutaneous continuous aortic augmentation device (in clinical trial).

• Chakir et al. used canine rapid pacing model to demonstrate reduced myocyte apoptosis and improved stress response molecular signalling with cardiac resynchronising therapy.

Device and Mechanical Support

Summary

• Large animal models are more similar to human HF.

• Pathophysiology of CCF is not clear.• Help to understand the pathophysiology of

disease.• Animal studies are loosing their hold as newer

invasive techniques are being developed to be used in patients.

• Animal models will still be useful for testing new pharmacological agents.

Thank You!!!

• Drug screening methods; S K Gupta; 3rd Edition.• Drug Discovery and Evaluation: Pharmacological

Assays; Hans Gerhard Vogel; 3rd edition• Essentials of Medical Pharmacology; K D Tripathi;

7th Edition.

References

evaluation of drugs used in ccf

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