This document discusses various in vitro and in vivo methods for evaluating drugs used in congestive cardiac failure (CCF). It describes isolated tissue experiments using hamster and cat hearts to assess drug effects on contractile force. It also outlines several animal models of CCF including rat coronary ligation and aortic banding, as well as dog, rabbit, guinea pig and hamster heart failure models induced by techniques like rapid pacing or volume/pressure overload. The models aim to mimic human CCF pathophysiology for testing new treatments.

1. Evaluation of Drugs
used in CCF
Dr Ketan Asawalle
JR3, Dept. of Pharmacology
SVNGMC, Yavatmal

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

3. • 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

4. • 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

5. IN VITRO METHODS

6. • 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

7. • 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.
Isolated Hamster Cardiomyopathic Heart

8. • Purpose and rationale
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)

9. • 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.
Isolated Hamster Cardiomyopathic Heart

10. • Purpose and rationale
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

11. • Procedure
Equilibrium binding -
1. Carried out in the presence of increasing concentrations of (3H) ouabain (10nM to 3µM).
2. 40µg of membranes are added
3. After 30 mins duplicate aliquots of 4.5 ml removed and filtered.
Dissociation process -
1. Experimental conditions are used to study association.
2. 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

12. IN VIVO METHODS

13. In Vivo Models
• Rat Models
• Dog Models
• Rabbit Models
• Guinea Pig Models
• Syrian Hamster
• Transgenic Mice
• Newer Therapeutic Targets

14. In Vivo Models
• Rat Models
• Dog Models
• Rabbit Models
• Guinea Pig Models
• Syrian Hamster
• Transgenic Mice
• Newer Therapeutic Targets

15. • Purpose and rationale
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

16. • 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.
1. Rat Coronary Ligation Model

17. • Purpose and rationale
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

18. • 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

19. • Purpose and rationale
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

20. • 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

21. • Purpose and rationale
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

22. • Evaluation
After completion of experimental protocol animal is
sacrificed.
Heart is submitted for processing number of
Apoptotic cells,
Sarcoplasmic reticulum calcium pump mRNA levels and
Expression of genes encoding for extracellular matrix.
Results are compared.
4. Spontaneous Hypertensive Rat Model

23. • Purpose and rationale
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

24. • 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

25. In Vivo Models
• Rat Models
• Dog Models
• Rabbit Models
• Guinea Pig Models
• Syrian Hamster
• Transgenic Mice
• Newer Therapeutic Targets

26. • Allows more accurate study.
• Excitation contraction coupling resembles human heart.
• But they are costly and high maintenance.
Dog Models

27. • Purpose and rationale
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.

28. • 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.

29. • Purpose and rationale
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

30. • 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

31. • Purpose and rationale
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

32. • 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

33. In Vivo Models
• Rat Models
• Rabbit Models
• Dog Models
• Guinea Pig Models
• Syrian Hamster
• Transgenic Mice
• Newer Therapeutic Targets

34. • 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

35. • Purpose and rationale
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

36. • 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

37. • Purpose and rationale
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

38. • 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

39. • Purpose and rationale
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

40. • 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

41. In Vivo Models
• Rat Models
• Rabbit Models
• Dog Models
• Guinea Pig Model
• Syrian Hamster
• Transgenic Mice
• Newer Therapeutic Targets

42. • Purpose and rationale
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

43. • 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

44. In Vivo Models
• Rat Models
• Rabbit Models
• Dog Models
• Guinea Pig Model
• Syrian Hamster
• Transgenic Mice
• Newer Therapeutic Targets

45. • Purpose and rationale
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

46. • 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

47. In Vivo Models
• Rat Models
• Rabbit Models
• Dog Models
• Guinea Pig Model
• Syrian Hamster
• Transgenic Mice
• Newer Therapeutic Targets

48. • 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

49. In Vivo Models
• Rat Models
• Rabbit Models
• Dog Models
• Guinea Pig Model
• Syrian Hamster
• Transgenic Mice
• Newer Therapeutic
Targets

50. • 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

51. • 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 method
3. Delivery location.
4. Cell concentration.
• More testing is needed.
Stem Cells

52. • 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

53. Summary

54. • 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.

55. Thank You!!!

56. • 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