screening method for anti arrhythmic drugs in preclinical pharmacology. In screening rats are used and the main method Legendroff's technique, various apparatus are used like board for a animal holding, O2 cyliner etc.

1. SCREENING OF ANTI ARRHYTHMIC
ACTIVITIY OF DRUG IN ANIMALS
APURVA KERU PAWAR
AISSMS COLLEGE OF PHARMACY, PUNE

2. CONTENTS
◼ Introduction
◼ Screening methods
◼ Evaluation
◼ Conclusion

3. INTRODUCTION
◼ What is Arrhythmia? - It is deviation from normal condition,rhythm and heart rate.It mainly occurs in patients
who has acute MI,taking digitalis, or in a anaesthetised patient.
◼ Screening of anti arrhythmic drug- preclinical testing of drugs in experimental animals or invitro for their
biological and toxic effects and the potential clinical applications.
◼ Examples – Quinidine, digoxin,lidocain

4. SCREENING METHODS
◼ Invitro methods
1. LAGENDROFF technique
2. Ach or KCL induced Arrhythmia
◼ Invivo methods
1. chemically induced Arrhythmia
2. Electrically induced Arrhythmia
3. Exercise induced ventricular fibrillation
4. Mechanically induced Arrhythmia
5. Genetically induced Arrhythmia

5. INVITRO MODELS
◼ LAGENDROFF technique – The heart is perfused in retrograde direction from aorta either at constant
pressure or flow with oxygenated saline solution.
◼ It closes the aortic valves,just inside heart during diastolic.The perfusate is displaced through the coronary
sinus and opened right atrium.

6. PROCEDURE
Guinea pig of either sex (300-500gm)
Selected and sacrificed.heart is removed and placed in ringer solution(37⁰c)
Aorta located and cut,cannulated with ringer sol
Heart is transferred to double walk plexiglass perfusion apparatus maintained 37⁰c

7. Ligature is placed around LAD coronary artery& occlusion is maintained for 10min by perfusion
Test compound administered through perfusion medium before or after occlusion
Epitaxial ECG electrode is used for pulsatile stimulation & induction of arrhythmia
Small steel hook with sting attached to apex of heart & HR measured with chronometer
Incidence and duration of VF is recorded in control & test group

8. ◼ Evaluation
1. Heart rate – Chronometer
2. Contractile force- force transducer
3. Incidence & duration of of VF or tachycardia is recorded in both groups

9. ACH OR KCL INDUCED ARRHYTHMIA
New Zealand white rabbits (500 gm) sacrificed & heart removed
Atria disected from other tissue in RL & attached with electrode
Fibrillation produced when atria exposed with Ach or KCL
After 5 min exposure to Ach/ KCL,atria stimulated with pulses 0.75ms usually of 10V& chymograph recorded
Control arrhythmia produced for 6-10min followed by 30min rest period

10. Test compound were added to Bath
If fibrillation doesn’t appear within test drug found to be effective
8-10min,preparation is washed to allow if fibrillation is disappear
Normal conc. immediately or within 5min

11. INVIVO MODELS
◼ Chemically induced
1.Aconitine antagonism in rats
◼ Purpose and rationale- The plant alkaloid aconitine persistently activate Na channel.Infusion of aconitine in
anaesthesied rat produces ventricular arrhythmia.
◼ Procedure –
Male ivanovas rats (300-400gm)taken
Anaesthized by I.p route by 1.25g/kg urethane
5ųg/kg aconitine dissolved in 0.1N HNO³ administered by infusion into sephanous vein of 0.1ml/min.ECG recorded
every 30 seconds

12. test compound is injected orally kr i.v at screening dose of 3mg/kg 5min before start of the aconitine infusion
◼ Evaluation –
Activity is measured by amount of aconitine/100 gm animal which induces
1. Ventricular extrasystole
2. Ventricular tachycardia
3. Ventricular fibrillation
4. Death
◼ Higher doses are compared with untreated control group are an indication of anti arrhythmia activity.
Difference between the groups is assessed by the students t-test

13. ◼ Scores are alloted for intensity & duration of effects related to efficacy of std comp.

14. DIOXIN INDUCED VENTRICULAR ARRHYTHMIA IN
GUINEA PIG
◼ Purpose & rationale – Overdose of cardiac glucoside induces Ventricular extrasystole,VF and finally
death.The occurrence of these can be delayed by anti arrhythmic drugs.
◼ Procedure –
Male guinea pig(350-500gm) Anaesthized with 35mg/kg phenobarbital sodium I.p
Trachea,on juglar vein nd one cartridge artery are catherized.positive pressure is applied with respiratory pump
at 45braths/min.
Caretoid artery is used for monitoring system BP via pressure transducer

15. Digoxin is infused into jugular vein with perfusion pump at rate of 85ųg/kg in0.266ml/min until cardiac arrest.
ECG is recorded with steel needle electrodes
Treated grps(5-10) receive the test drug orally or I.v 1 min prior to infusion .control grp receive digoxin only
until effects are recorded.
Total amount of Infused digoxin to induce VF is calculated.
Eg- std lidocaine 3mg/kg I.v
ramipril 1mg/kg I.o

16. ◼ Evaluation
◼ Using student’s t- test the doses of digoxin needed to induce ventricular extrasystole, Vf, or cardiac arrest.
◼ After treatment with anti arrhythmic drugs are compared statistically with control receiving digoxin only.

17. STROPHANTIN INDUCED ARRHYTHMIA
◼ Purpose and rationale- Acute intoxication with the cardiac glucoside strophantin K induce V.tachycardia and
multifocal Ventricular arrhythmia in dogs.used as test model to evaluate effect of potential and arrhythmic
drugs on Ventricular arrhythmia.
◼ Procedure –
Male or female dog (20kg) anaesthised by phenobarbital sodium 30-40mg/kg
Two peripheral veins are cannulated for administration of arrhythmia inducing substance and for the test
compound
ECG recorded at different time point from lead II
Strophantine was infused at 3ùg/kg/min till 30-40min.Once VT developed infusion is terminated

18. After stabilising ventricular arrhythmia (10min),test compound is administered
If extrasystole disappar,test compound has anti arrhythmic activity
If no positive effect, gradually increase dose at 15min interval.
◼ For I.d a test compound issue considered effective if extrasystole disappear within 15min. Test drug is considered
to have NO EFFECT if it does not improve straphonine intoxication within 60 min following the drug
administration.
◼ Evaluation- therapeutic Evaluation of drug is difficult because there is no clear correlation between effectiveness
of test drug and duration of its effects.The std drugs ajamalinr,quinidine, and lidocaine re establish normal sinus
rhythm at dose of 1 and 3mg/kg I.v and 10mg/kg I.d.

19. ADRENALINE INDUCED ARRHYTHMIA
◼ Pocedure- Adrenalin at higher doses induces arrhythmia
Dogs( 10-11kg) anaesthised with phenobarbitone(30-40mg/kg) intrperitonially
Adrenalin infused at 2-2.5 ųg/kg through femoral vein
Test compound administred 3 min after Adrenalin infusion
If extrasystole disappar, test compound has anti arrhythmic activity

20. ELECTRICALLY INDUCED ARRHYTHMIA
◼ Purpose and Rationale- electrical stimulation- flutter and fibrillation
◼ Ventricular fibrillation method
◼ Programmed electrical stimulation induced Arrhythmia
◼ Sudden coronary death model in dogs

21. 1.VENTRICULAR FIBRILLATION ELECTRIC THRESHOLD
◼ Purpose and rationale – several electrical stimulation techniques have been used to measure ventricular
fibrillation threshold such as single pulse stimulation, train of pulse stimulation, continues 50-Hz stimulation and
sequential pulse stimulation.
◼ Procedure - adult dog (8-12kg) anaesthised with sodium phenobarbital (35mg/kg)& ventilated with air using
Harvard respiratory pump.
arterial pressure is monitored and temp is maintained.the heart is removed and suspended in pericardial cradle
SA node crushed and Ag-Agcl stimulating electrode embedded with Teflon disc saturated to anterior surface of LV
Anodal constant current for 400ms is applied through electrode and which was programmed by digital stimulator

22. ECG recorded through lead II of body ECG monitoring
To determine ventricular fibrillation 0.2 to 1.8 strain of 50Hz pulse delivered
The current intensity of pulse strain required to induce sustained ventricular fibrillation
When VFT occurs heart immediately deliberate and allow to recover as controlled condition for 20min
Test drug is administered through femoral vein.

23. EVALUATION
◼ Ventricular fibrillation threshold is determined before and after administration of test drugs at given intervals of
time.
◼ The mean value of 10 experiments test drugs are compared using student t-test

24. 2.PROTECTION AGAINST SUDDEN CORONARY DEATH
◼ Purpose and rationale- The group of lucchesi described an experimental dog model to test protection against
sudden coronary death.
◼ Procedure - bred make mongrel dogs (14-22kg)anaesthised with 30mg/kg pentobarbital I.v. The dogs are
ventilated with room air through a cuffed endotracheal tube & harvard respiratory.
Cannula inserted in left jugular vein.left thoractomay is done and heart is exposed and suspended in pericardial
cardiac cradle.
Ligature tide across the artery and needle followed by removal of needle resulting critical stension of vessels
LAD is occluded for 2 hrs using rubber snare and allow to reperfuse to perfuse for 2 hrs in presence of stenosis

25. Epicardial bipolar electrode is sutured on left ventricle wall @one of the distribution of LAD distal to the occlusion
& second distribution of LCX artery.
Silver cited electrode is passed through the wall and into the lumen of LCX suture to adjunct surface of heart
Surgical incision is closed and allow the animal to recover. And they treated with test drug.
Anodal 15ųA current from 9V ni ca battery is passed through 25ohm and applied to electrode in lumen of LCX
Animals are sacrificed After 24 hrs.Heart is sectioned and stained with TTC to study the area of infacrat.
Time of onset ventricular ectopy and arrhythmia is recorded.

26. 3.PROGRAMMED ELECTRICAL STIMULATION INDUCED ARRHYTHMIA
◼ Pocedure – dogs(8-11kg)anaesthised with phenobarbital I.v and maintained on artificial respiration
Eft throactomy performed between 4 and 5 rib and heart exposed
Left anterior coronary artery isolated 20 G needle placed and ligature is tied around artery and needle removed
result in stenosis
LAD perfused for 5 min and ishchemic injury to myocardium.is achieved by 2 hr occlusion of LAD by silicon rubber
snare and then vessels are allowed to perfusion for 2hrs in presence of stenosis
Meanwhile epicardial bipolar electrode sutured on Ventricular septum adjunct to occlusion site

27. Silver disc electrode implanted for ECG
After 6-9 days chest reopened and programmed electrical stimulation is performed at non infacrat zone
The piece on stimuli is set at 200ms and followed by extra stimuli After each 15 pacing stimuli
Test compound is administered after 30 min of stimulus.
◼ Evaluation- the minimum current intensity of pulse is recorded to induce a sustain ventricular fibrillation
before and after administration of test drug.

28. EXERCISE INDUCED VENTRICULAR FIBRILLATION
◼ Purpose and rationale – test combining coronary construction with physical exercise May resembles most
closely the situation in coronary patients.
◼ Procedure –
Animal mongorel dog(15-20kg)anaesthised by phenobarbital sodium 10mg/kg I.v
Chest cavity opened and heart is suspended in pericardial cradle, around left circumflex artery doppler flow inducer
to measure BP
Hydraulic occlider coronary artery are placed

29. Pair of insulated silver wire sutured on left and right ventricle – measure HR
Occlusion of LAD
Myocardial infaraction,after 24hrs test drug is administered
1. During this test trandermal fentynl patch is used to reduce post operative discomfort
2. Antibiotic therapy given (amoxicillin)

30. MECHANICALLY INDUCED ARRHYTHMIA
◼ Purpose and rationale – arrhythmia iduced directly by ischaemia and reperfusion.Coronary artery ligation in
dog results in,
◼ Increased HR, contractility ,BP, ventricular fibrillation

31. ◼ Procedure- dog (20-25kg) anaesthised with phenobarbital sod(30mg/kg)-Artificial respiration on positive
pressure respirator
Femoral artery cannulated and connected to pressure transducer . Chest cavity opened and LAD is exposed
Silk suture is placed around LAD .After 45 min test drug is administered through sephanous vein
after 20 min ligature of coronary artery closed for 90 min.occlusion released and reperfusion period maintained for
90min
◼ Evaluation – All parameters are recorded and at the end animal is sacrificed by overdose of phenobarbital
sodium.

32. CONCLUSION
◼ Antiarrhytmic drug exert their effect by modulating conduction velocity or refrative period duration or both.
◼ Conduction velocity depends on passive electrical properties of cardiac tissue or Characteristic of Na± Ca+
channels.
◼ In contrast there are marked difference among species in K current that largely determine the repolarisation,so
that AP duration and Refrative duration differ form species.
◼ Electrophysiological studies should encourage in animals with naturally occurring cardiovascular disease.

33. REFERENCE
◼ Drug discovery and evaluation by Gerhard Vogel ,Pharmacological Assay 2Edition
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