Evaluation methods cardiotonic drugs

1. Evaluation Methods-
Cardiotonic Drugs
Dr Sneha Dange, JR3
Dept. of Pharmacology,
GMC Nagpur

2. Overview
 Introduction
 In Vivo Methods
 In Vitro Methods
 Summary
 References
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3.  Cardiotonic –life saving drugs used in congestive heart
failure
 An ideal cardiotonic agent should not increase heart rate &
should be devoid of toxic effects
 Positive inotropic drug: increases force of contractions,
causes improved heart’s pumping ability
 Negative inotropic drug: decreases force of contraction
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Introduction

4.  Classical methods-
 systolic arrest of ventricles in frogs
 producing emesis in pigeon
Indirect method for assaying crude digitalis preparations
 Hatcher’s toxicity in cats & Knaffl’s-Lenz’s method (ECG) in
dogs – limited usefulness
 Cardiac Insufficiency Model (guinea pig) & Cardiomyopathic
Syrian Hamster - first method closely reflects symptoms of
cardiac insufficiency in man
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Introduction

5. Cardiac glycosides:
Digitoxin, Digoxin
β-adrenergic agonists:
dopamine, Dobutamine
Phosphodiesterase inhibitors :
Amrinone, Milrinone
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Introduction

6.  The current American College of Cardiology Foundation
(ACCF)/American Heart Association (AHA) guidelines define
HF:-
 Complex clinical syndrome that results from structural or
functional impairment of ventricular filling or ejection of blood,
which in turn leads to the cardinal clinical symptoms of dyspnea
and fatigue and signs of HF edema and rales
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Introduction

7.  The use of small animals to study complex cardiovascular
pathophysiology 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 for the investigation of CHF and
potential therapies–
 Dogs
 Pigs
 sheep
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8. IN VIVO METHODS
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9.  Rat Models
 Dog Models
 Rabbit Models
 Guinea Pig Models
 Syrian Hamster Model
 Pigeon Model
 Transgenic mice model
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IN VIVO METHODS

10.  Rat models are relatively inexpensive & short gestation period, a large
sample size can be produced in a short period
 A very short action potential, which normally lacks a plateau phase.
 Calcium removal - predominated by the activity of sarcoplasmic reticulum
calcium pump whereas Na+/Ca2+ exchanger activity is less relevant.
 α- myosin heavy chain isoform predominates
 Resting heart rate is five times that of humans & force-frequency relation
is inverse
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Rat Models

11. 1. Rat Coronary Ligation Model
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 - anaesthetized with 200mg/kg
hexobarbital
 Trachea cannulated - artificial respiration provided.
 Chest cavity exposed – left anterior descending coronary artery
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Rat Models

12. Rat Coronary Ligation Model contd..
Procedure:
 Ligature placed and cavity sutured back.
 After 4 weeks chest cavity opened - carotid and jugular vein
cannulated.
 Filling pressure, systolic, diastolic and mean blood pressure are
measured.
 Animal sacrificed after hemodynamic parameters tested.
 Isolated hearts are used to study calcium channels,
sarcoplasmic reticulum (SR) ATPase and protein levels.
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Rat Models

13. Rat Coronary Ligation Model contd..
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. (above mentioned
parameters)
 Disadvantage - High mortality.
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Rat Models

14. 2. Rat Aortic Banding Model
Purpose and rationale
 Restriction of blood flow of aorta - Hypertension & 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.
 Heart failure develops in 4-6 weeks
 Test group is administered with drugs for 6 weeks
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Rat Models

15. Rat Aortic Banding Model contd..
Evaluation
 Total cardiac mass, weight of left and right ventricle of
treated rats are compared between the two groups.
 Heart failure increased myosin heavy chain mRNA and
atrial natriuretic factor mRNA.
 Above parameters compared in both groups.
 Good to study the transition from hypertrophy to
failure.
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Rat Models

16. 3. Dahl Salt Sensitive Rats’ Model
Purpose and rationale
 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 dahl salt diet is prepared.
 Test drug rats are administered the drug orally for one month.
 Animals sacrificed after study period is over
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Rat Models

17. Dhal Salt Sensitive Rats’ Model contd..
Evaluation :
 Hearts are removed.
 Total cardiac mass, weight of right and left ventricles are
measured and compared.
 Hearts have concentric left ventricular hypertrophy (8 weeks)
then left ventricular dilatation Failing heart dies in 15 to
20 weeks.
 The ability of the test drug to reverse these changes are
studied.
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Rat Models

18. 4. Spontaneous Hypertensive Rat Model
Purpose and rationale
 Model of genetic hypertension.
 At 18 - 24 months cardiac failure develops.
 Altered calcium cycling is observed.
Procedure
 Animals are divided in two groups.
 Test drug is given for 1 month.
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Rat Models

19. Spontaneous Hypertensive Rat Model contd..
Evaluation
 After completion of experimental protocol animal is
sacrificed.
 Heart is submitted for processing
• Number of Apoptotic cells
• Sarcoplasmic reticulum calcium pump mRNA levels
• Expression of genes encoding for extracellular matrix.
 Results are compared.
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Rat Models

20. 5. Spontaneous Hypertensive-Heart Failure
Rat Model
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.
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Rat Models

21. Spontaneous Hypertensive-Heart Failure Rat Model contd..
Evaluation
 Plasma renin activity, ANP, aldosterone, ryanodine
receptor density, sarcoplasmic reticulum calcium uptake
and endothelial nitric oxide synthase activity is tested.
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Rat Models

22. Dog Models
 Allows more accurate study.
 Excitation contraction coupling resembles human
heart.
 But they are costly and high maintenance.
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23. 1. Chronic Rapid Pacing Model
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 anaesthetized with pentobarbital
30mg/kg.
 Airway maintained.
 Chest cavity opened by 3-4 cm long thoracotomy - heart exposed.
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Dog Models

24. Chronic Rapid Pacing Model contd..
Procedure:
 Ventricular pacing lead is attached to apex of heart
 Cavity closed
 Significant heart failure develops in 4 weeks.
 There is bilateral ventricular dilatation over 3-4 weeks.
 Test drug is administered by SC and IM injections over
period of 14 days
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Dog Models

25. Chronic Rapid Pacing Model contd..
Evaluation :
 Ejection fraction decreases followed by decreased cardiac
output and increased resistance.
 There is time dependent neurohormonal and hemodynamic
abnormalities.
 Heart failure is reversible if pacing is stopped.
 Two groups are compared for parameters like ejection
fraction, cardiac output and systemic vascular
resistance.
 Plasma renin and ANP levels are also compared.
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Dog Models

26. 2. Volume Overload Model
Purpose and rationale:
 Prolonged volume overload can lead to CCF.
 In dog it is created by formation of A-V fistula or Mitral valve
is destroyed.
Procedure:
 Adult male dog, 10-12 kg, are anaesthetized with pentobarbital
30mg/kg.
 Airway maintained.
 Chest cavity opened by thoracotomy - heart exposed.
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Dog Models

27. Volume Overload Model contd..
 Chronic experimental mitral regurgitation is developed by
disruption of mitral chordae or leaflets
 Significant heart failure develops in 4 weeks - continued
upto 10th week.
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Dog Models
Evaluation :
 Neurohormonal activation of RAS is observed in CHF dogs
 Test and control treated groups are compared.
 Used to study influence of chronic ß-adrenoceptor blockade
on myocytes and left ventricular function

28. Rabbit Models
 Less expensive than dog models.
 Rabbit myocardium has many similarities to
human heart
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29. 1. Volume and Pressure Overload
Purpose and rationale
 Volume overload, pressure overload or combination of both are
used to induce heart failure.
 Chronic severe aortic regurgitation- systolic dysfunction- heart
failure.
Procedure
 Rabbits are anaesthetized with pentobarbitone sodium 35mg/kg IP.
 Trachea cannulated to maintain artificial respiration.
 Chest cavity opened and carotid artery cannulated
 Aortic insufficiency created by destroying valve.
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Rabbit Models

30. Volume and Pressure Overload contd..
 After 14 days - aortic constriction using PVC clamp.
 Test drug given – 2 weeks
 Heart failure occurs within 4 weeks
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Rabbit Models
Evaluation :
 Animal sacrificed after experimental protocol.
 Heart failure is associated with alterations in ß-adrenoceptor
levels.
 Protein, mRNA levels of Na+/Ca2+ and Sarcoplasmic Ca2+
ATPase is measured
 The ability of test drug to reverse these changes is observed

31. 2. Tachycardia Pacing Model
Purpose and rationale
 Chronic rapid pacing - 350-400 beats/min.
 Myocardial depression, hemodynamic and neurohormonal signs of
heart failure.
Procedure
 Rabbits are anaesthetized 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.
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Rabbit Models

32. Tachycardia Pacing Model contd..
 Heart failure occurs in 4-6 weeks
 Two groups are formed- Test (sc or ip for 2 weeks) and
control
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Rabbit Models
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.

33. 3. Doxorubicin Cardiomyopathy Model
Purpose and rationale
 Doxorubicin exhibits acute and chronic cardiotoxicity.
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 experimental duration, animal anaesthetized
 Carotid artery – cannulated
 Canula inserted into left ventricle- left ventricular end
diastolic pressure (LVEDP)
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Rabbit Models

34. Doxorubicin Cardiomyopathy Model contd..
Evaluation:
 Animals are sacrificed - Heart is processed for immunohistochemical
tests.
 Chronic doxorubicin causes impairment of cardiac contractility.
 Decreased gene expression of sarcoplasmic reticulum and Ryanodine
receptor.
 The ability of test drug to reverse these conditions is observed in
both groups
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Rabbit Models

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Guinea Pig Models

36. 1. Cardiac Insufficiency Model
Purpose and rationale
 8 weeks of cardiac banding of the descending thoracic aorta
in guinea pigs – CHF develops
 Very similar to human heart failure
Procedure
 Male guinea pigs, 250-400g are anaesthetized with ether.
 Chest cavity opened, heart exposed, Aorta located and ligated.
 Symptoms of CCF are developed with death rate of 80% in one
day.
 Lung weight, relative heart weight are increased.
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Guinea Pig Model

37. Cardiac Insufficiency Model
Evaluation :
 Lung Weight and heart weight increases due to failure
 Decrease in SR Ca2+ ATPase and phospho-lamban is seen in
failing heart of guinea pig
 Ability of test drug to reverse these signs are observed
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Guinea Pig Model

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Syrian Hamster Models

39. 1. Cardiomyopathic Hamster
Purpose and rationale
 Cardiomyopathic strains of Syrian hamsters are used - Autosomal
recessive.
 Degenerative changes in striated muscles - cardiomyopathy
Procedure
 These animals develop failure after 7-10 months.
 Cardiomyopathic disease-(Stages)
 Pre necrotic stage.
 Fibrosis and calcium deposition.
 Overlapping period of reactive hypertrophy.
 Depressed myocardial function
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Syrian Hamster Model

40. Cardiomyopathic Hamster
Evaluation
 Test drugs are administered by SC and IM route for 14 days.
 Ability of drug to reverse the condition is observed.
 Advantages of this model are absence of surgical manipulations,
low cost and the ease with which large number of animals can be
studied
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Syrian Hamster Model

41.  Depends on the observation that intravenously injected cardiac
glycosides have an emetic action in pigeons
 Adult pigeons weighing 300–400 g are injected with a solution of the
cardiac glycoside into a suitable wing vein in the axillary region
 Vomiting occurring within 15 min is regarded as positive result
 Two doses of test solution and standard are injected and percentage
of vomiting pigeons registered
 This 4 point assay allows calculation of ED50 values and of the potency
ratio compared with the standard
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Pigeon Models

42.  Techniques – to alter expression of genes
 Help in understanding pathophysiology of disease
 Gene targeted disruption of muscle LIM protein (MLP)- new model
of heart failure
 Homozygous deletion of MLP gene - dilated cardiomyopathy with
hypertrophy
 Transgenic mice overexpressing either β-adrenergic receptor
kinase or G-protein coupled receptor kinase 5, resulting in
uncoupling of the β-adrenergic receptor, also exhibit reduced
contractility
 Heart failure resembles as seen in humans
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Transgenic Mice

43. IN VITRO
METHOD
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44. 1. Isolated Hamster Cardiomyopathic
Heart
Purpose and rationale
 Isolated Syrian Hamster hearts for evaluation of cardiotonic
drugs
Procedure
 Syrian Hamsters, age 50 weeks.
 Heart prepared according to Langendorff method.
 Perfused with RL
 Allow to equilibrate for 60 mins at 32˚C with preload of 1.5g
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45. Isolated Hamster Cardiomyopathic Heart
 Force of contractions is measured using polygraph
 Heart rate is measured using a chronometer
 Coronary flow is measured using an electro-flowmeter.
 Test compounds are injected via the aortic cannula into the inflowing
heart–Ringer's solution.
 Contractile force and coronary flow in heart of treated and control
group are compared, percentage improvement is calculated
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46. 2.Isolated Cat Papillary Muscle (Catell
and Gold)
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 4-6 V are applied at 30/min and contractions are
recorded
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47. Isolated Cat Papillary Muscle
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
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48. Isolated Cat Papillary Muscle
Evaluation :
 Calculated as percentage of pre-dose levels and compared
between groups
 The potency of natural and semisynthetic glycosides determined
with this method
 Catecholamines, like adrenaline (10 ng/ml) or isoprenaline (10
ng/ml), are active as well
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49. 3. Isolated frog heart Method
 Frog method was used for standardization
 Healthy frogs (Rana pipiens) weighing 20–30 g were
selected
 The doses of digitalis were calculated so that they
approximated 0.015 ml/g body weight
 Injections were made into the ventral lymph sac
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50. Isolated frog heart Method
 One hour later, the animals were pithed and the heart
removed and examined
 Systolic arrest of the ventricle and widely dilated
atrium indicated the typical result
 Calculations were made from the percentage of dead
animals in the test group versus those in the group
receiving the international standard
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51. Clinical evaluation
 The distinction between patients with HFrEF & HFpEF is important
because they shows different underlying pathophysiologic,
haemodynamic and neurohormonal abnormalities
 Main therapeutic goals –
To reduce cardiovascular mortality
To prevent deterioration of the clinical status and hospitalization
 These goals should represent the primary aim of new agents
developed for the treatment of CHF
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52. Assessment of efficacy
 Mortality -primary endpoint
 Time to first Heart Failure Hospitalisation - primary endpoint or
secondary endpoint
 Effect of the treatment on exercise capacity
 Improvement of symptoms & quality of life (QoL)
 Events from implantable devices
 Measurements of changes in haemodynamic parameters- mode of
action & required dose of a therapeutic agent in early phase studies
 Biomarkers cannot be included as primary endpoints in phase III
clinical trials in CHF
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53. Selection of Patients
 Include >75 years of age
 CHF must be differentiated – HFrEF ( LVEF <40%), HFmrEF (LVEF 40-49%),
HFpEF (LVEF >50%)
 Patients hospitalised because of an acute episode who are stabilized on standard
therapy and are not receiving parenteral treatments but remain hospitalized
 Distinct subsets of chronic HF patients other than LVF may be conducted like
(e.g. amyloid, drug-induced, diabetic, hypertrophic cardiomyopathies)
 Patients entering phase IIb and III clinical trials with agents for the treatment
of CHF (NYHA class II-IV) should be treated at study entry as per GCP
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54. Study Design
 Patients with CHF, a period of stability of CHF medications is
required before inclusion
 In patients with recent decompensation (e.g. patients included in
the study during a hospital stay) - first line therapies according to
current clinical practice guidelines
 Pharmacodynamic
 Pharmacokinetic
 Interactions
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55. Study Design
 Exploratory therapeutic studies- A parallel, fixed dose, double blind
placebo controlled design – for therapeutic dose-range
 Confirmatory therapeutic studies- A controlled, double blind,
randomised studies- to confirm the efficacy of a new drug
 Studies in special populations-
 Subgroup analyses for gender, race, age
 Elderly and very-old patients
 Diabetes mellitus, COPD, renal failure, cachexia and/or sarcopenia, anaemia
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56. Assessment of Safety
 long-term data on adverse effects should be provided & should be
fully characterized and documented
 Particular attention should be paid to the following specific side
effects-
 Cardiovascular safety
 Hypotension/bradycardia
 End-organ consequences
 Effect on cardiac rhythm
 Pro-ischaemic effects
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57. Summary :
 In order to study transition processes occurring in heart failure,
animal models are very important
 Animal models of heart failure used to study the effects of new
pharmacological strategies on hemodynamics, neurohumoral activation
and survival
 At present, transgenic animal models of heart failure are important
to understand the molecular alterations underlying the development
of the disease
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58. Summary :
In vivo models
In vitro models
 Isolated hamster
cardiomyopathic heart
 Isolated cat papillary
muscle
 Isolated frog heart
Method
 Rat models
• Rat coronary ligation model
• Rat aortic banding
• Dahl salt sensitive rats
• Spontaneous hypertensive rat
• Spontaneous hypertensive-heart failure
rats (SH-HF)
 Dog models
• Chronic rapid pacing
• Volume overload
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 Rabbit models of heart failure
• Volume and pressure overload
• Tachycardia pacing
• Doxorubicin cardiomyopathy
 Guinea pig model
• Cardiac insufficiency
 Syrian hamster
• Cardiomyopathic hamster
 Genetic model
• Transgenic mice

59. References :
 Drug Discovery and Evaluation: Pharmacological Assays; Hans Gerhard
Vogel; 4th edition
 Screening methods in pharmacology; N. Parmar.
 Drug screening methods; S K Gupta; 3rd Edition
 Ema. Guideline on Clinical Investigation of Medicinal Products for the
treatment of Chronic Heart Failure. Gl [Internet]. 2006;44(1):12.
Available from:
http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/
general_content_000425.jsp&mid=WC0b01ac0580034cf5
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