This document provides an overview of preclinical screening methods for antiarrhythmic agents. It discusses various in vivo and in vitro models used to study cardiac arrhythmias and evaluate potential antiarrhythmic drugs. In vivo models include chemically-induced arrhythmias using drugs like aconitine or digoxin in rats or guinea pigs. Electrically-induced models use techniques like ventricular fibrillation threshold testing in dogs. Mechanically-induced models involve coronary artery ligation and reperfusion in rats. In vitro preparations include isolated tissues like guinea pig papillary muscle to study action potentials. The goal is to find safe and effective therapies for treating abnormal heart rhythms.

1. Preclinical
Screening of
Antiarrhythmatic
Agents
PRESENTER: ASHISH KUMAR JHA
(M.PHARM SEM-1)
GUIDE: DR.DEVANG SHETH
(ASSOCIATE PROFESSOR,
DEPT OF PHARMACOLOGY,
L.M.COLLEGE OF PHARMACY)

2.  Table of Contents
Sr. No Content Slide no.
1 About the disease 3
2 Types of arrythmia 4
3 Normal Electrical Signal Conduction Pathway 7
4 Pathophysiology of Cardiac arrhythmia 8
5 Classification of Anti – arrhythmic agents 17
6 Classification of Animal Models for Arrhythmia 18
7 IN-VIVO MODELS 19
8 IN-VITRO MODELS 34
9 References 37

3.  About the disease
 An arrhythmia is a problem with the rate or rhythm of the heartbeat. During an
arrhythmia, the heart can beat too fast, too slowly, or with an irregular rhythm.
 If they start in the ventricles, or lower chambers of your heart, they’re called
Ventricular.
 When they begin in the atria, or upper chambers, they’re called Supraventricular.
 Bradycardia is a heart rate of fewer than 60 beats per minute. Tachycardia is more
than 100 beats per minute.

4.  Types of arrythmia
Condition &
heart rate
range
Fibrillation (>350 b.p.m)
Flutter (251 – 350 b.p.m)
Paroxysmal tachyarrhythmia (151 – 250 b.p.m)
Simple tachyarrhythmia (100 – 150 b.p.m)
Normal (60 – 100 b.p.m)
Mild Bradyarrhythmia (40 – 60 b.p.m)
Moderate Bradyarrhythmia (20 – 40 b.p.m)
Severe Bradyarrhythmia (< 20 b.p.m)
 Based on heart rate :

5. Continue….
 Based on site of origin
A. Supraventricular arrhythmia
1. SA node (Sinus arrhythmia)
2. Atrial myocardial tissues (Atrial arrhythmias)
3. AV node / Junction (Junctional arrhythmia / nodal arrhythmia)
B. Ventricular arrhythmia
1. Ventricles (Ventricular arrhythmias)

7.  Normal Electrical Signal Conduction Pathway

8.  Pathophysiology of Cardiac arrhythmia
1
• Increased Automaticity
• (ectopic activity)
2
• Triggered Automaticity
• (after depolarization)
3
• Reentry
• (circus movement)

9. 1. Increased Automaticity
What is Automaticity?
▪ Property of any excitable tissue which can undergo depolarization spontaneously (e.g. Sinus
and AV nodes and the His-Purkinje system)
 Normal SA node action potential
How Automaticity increases?
▪ β – adrenergic stimulation, hypokalemia, and mechanical stretch of cardiac muscle cell
increase phase 4 slope and so accelerate pacemaker rate.

10. Continue…
 Acetylcholine reduces pacemaker rate both by decreasing phase 4 slope and by
hyperpolarization (making the maximum diastolic potential more negative).
 In addition, automatic behavior may occur in sites that ordinarily lack spontaneous
pacemaker activity ; e.g., depolarization of ventricular cells (e.g., by ischemia) may produce
such “abnormal” automaticity.
 When impulses enhanced normal or abnormal automaticity to excite the rest of the heart ,
arrhythmias result.

11. 2. Triggered automaticity ( after depolarizations )
▪ Under some pathophysiological conditions, a normal cardiac action potential may be
interrupted or followed by an abnormal depolarization if this abnormal depolarization
reaches threshold, it may, in turn, give rise to secondary upstrokes that can propagate and
create abnormal rhythms.
▪ This abnormal secondary upstrokes occur only after an initial normal, or “triggering’’,
upstroke and so are termed triggered rhythms.
▪ Two major forms of triggered rhythms are recognized. (DAD and EAD)
A) Delay after depolarization(DAD)
▪ In the first case, under conditions of intracellular Ca2+ overload (e.g., myocardial ischemia,
adrenergic stress, digitalis intoxication, or heart failure), a normal action potential may be
followed by delayed after depolarization(DAD)
▪ If this after depolarization reaches threshold, a secondary triggered beat or beats may occur.

12. Continue….
B) Early after depolarization:
▪ In the second type of triggered activity, the key abnormality is marked prolongation of the
cardiac action potential. When this occurs, phase 3 repolarization may be interrupted by an
early after depolarization(EAD).
▪ EAD-related triggered upstrokes probably reflect inward current through Na+ or Ca2+
channels
▪ EADs are induced much more readily in Purkinje cells and in mid myocardial (or M) cells
than in epicardial or endocardial cells.
▪ When cardiac repolarization is markedly prolonged, polymorphic ventricular tachycardia
with a long QT interval, known as the torsades de pointes syndrome, may occur.
▪ This arrhythmia is thought to be caused by EADs, which trigger functional re-entry owing to
heterogeneity of action potential durations across the ventricular wall.
▪ Congenital long QT syndrome, a disease in which torsades de pointes is common, can be
caused by mutations in the genes encoding the Na+ channels or the channels underlying the
repolarizing currents Ikr and Iks.

13. 3. Re-entry (circus movement)
▪ Normally, a cardiac action potential dies out after it has activated the ventricles because it is
surrounded by refractory tissue, which it has just traversed.
▪ Re-entry describes a situation in which the impulse re-excites regions of the myocardium
after the refractory period has subsided, causing continuous circulation of action potentials.
 2 types of re-entry a) Anatomically defined re-entry
b) Functionally defined re-entry

14. a) Anatomically Defined Re-entry
Re-entry determined by :
 Presence of anatomically defined circuit,
 Heterogeneity in refractoriness among regions in circuit, and
 Slow conduction in one part of the circuit.
~ e.g., Patient with Wolff-Parkinson-White(WPW) syndrome, and it shows AV re-entrant
tachyarrhythmia.
▪ Similar “anatomically defined” re-entry commonly occurs in the region of the-
o AV node ( AV nodal re-entrant tachycardia)
o Atrium (atrial flutter)
o Both AV and AV nodal re-entry (Paroxysmal Supraventricular tachycardia-PSVT)

15. b) Functionally Defined Re-entry
▪ Here no fixed ‘obstacle’ or ‘pathway’, rather functional obstacle (core of circuit)and
unidirectional conduction pathway is created by a premature impulse which travels through
electro physiologically inhomogeneous myocardium.
▪ On encountering refractory tissue in one direction, the wavefront travels through partially
recovered fibers – gets markedly slowed and can set up small reentry circuits which may
constantly shift location.
▪ Example of “functionally defined” (or “leading circle”) re-entry is Atrial or Ventricular
fibrillation: cells are re-excited as soon as they are repolarized sufficiently to allow enough
Na+ channels to recover from inactivation.
▪ Ventricular extrasystoles, polymorphic ventricular tachycardia.

16. Difference of anatomically and functionally defined reentry

17. Classification of Anti – arrhythmic agents

18.  Classification of Animal Models for Arrhythmia
Animal Model for
Arrhythmia
IN-VIVO
Chemically Induced Electrically Induced
Mechanically
Induced
Exercise Induced
IN-VITRO
Acetylcholine and
Potassium Induced
Arrhythmia
Langendroff
Technique
Isolated Right
Ventricular of
Guinea Pig’s
Papillary Muscle
Action Potential and
Refractory Period in
Isolated left
Ventricular Guinea
Pig Papillary Muscle

19.  IN-VIVO MODELS
1.1)Chemically
induced
arrhythmia:
• Aconitine
antagonism in
rats.
• Digoxin
induced
arrhythmias
in guinea pig.
• Stropanthin
induced
arrhythmia
• Adrenaline
induced
arrhythmia
1.2)Electrically
induced
arrhythmia:
• Ventricular
fibrillation
electrical
threshold
• Programmed
Electrical
stimulation
induced
arrhythmia
• Sudden
coronary
death models
in dogs
1.3)Mechanically
induced:
• Coronary artery
ligation,
reperfusion
arrhythmia &
infarct size
• Ventricular
arrhythmia after
coronary
occlusion.
• -VF after
coronary
occlusion and
perfusion.
• -Harris dog
model for
ventricular
tachycardia
1.4)Exercise
induced
ventricular
fibrillation :
• VF induced
by cardiac
ischemia
during
exercise

20. 1) Chemically Induced Arrhythmia:
Aconitine Antagonism in rats:
 Purpose and rationale:
▪ The plant alkaloid aconitine activates sodium channels.
▪ Infusion of aconitine in the anesthetized rat causes ventricular arrhythmias.
 Procedure:
▪ Animal : Male Ivanovas rat (300-400g)
▪ No. of animals : 8 to 10
▪ Standard drug : Procainamide 5 mg/kg i.v.,
Lidocaine 5 mg/kg i.v.
▪ Rat anesthetized with Urethane 1.25g/kg i.p
▪ Give test compound 3mg/kg orally or i.v 5 min before aconite infusion
▪ Aconitine 5μg/kg dissolved in 0.1N HNO3 continuous infusion into saphenous vein of
0.1ml/min & ECG Lead II is recorded every 30 seconds.

21. Continue….
 Evaluation
▪ The anti arrhythmic effect of a test compound is measured by the amount of Aconitine/100 g
animal (duration of infusion) which induces
-ventricular extrasystoles
-ventricular tachycardia
-ventricular fibrillation
-Death.
▪ Higher doses of Aconitine in the treated group as compared to an untreated control group are
an indication of antiarrhythmic activity.
▪ Statistical significance between the groups is assessed by the Student’s t-test.

22. 2) :
 Strophanthin Induced Arrhythmia:
 Purpose and Rationale:
Acute intoxication with the cardiac glycoside strophanthin K induces
▪ ventricular tachycardia
▪ multifocal ventricular arrhythmias in dogs
This can be used as a test model to evaluate the effect of potential anti-arrhythmic drugs on
ventricular arrhythmias.
 Procedure :
 Animal : Male or Female Dog (approx. 20 kg)
 No.of animal: 2-3 in one group
 Anesthesia : pentobarbital sodium (30-40 mg/kg) i.v
 Standard drugs : Quinidine & Lidocaine (1 and 3 mg/kg) i.v
 Male or female dogs of either sex are used and are anesthetized.
 Two peripheral veins are cannulated for the administration of the arrhythmia-inducing
substance and the test compound. For intraduodenal administration of the test drug, the
duodenum is cannulated.
 Electrocardiogram is registered

23.  Strophanthin K is administered by continuous i.v.- infusion at a rate of 3 μg/kg/min
 30-40 min later, signs of cardiac glycoside intoxication appear leading to ventricular
tachycardia or to multifocal ventricular arrhythmias. When this state is achieved, the
strophanthin infusion is terminated.
 When the arrhythmias are stable for 10 min, the test substance is administered intravenously
or intraduodenally.
 For i.v. administration: A test compound is considered to have an anti-arrhythmic effect if
the extrasystoles immediately disappear
 For i.d. administration: A test compound is considered to have a definite anti-arrhythmic
effect if the extrasystoles disappear within 15 min.
 The test drug is considered to have “no effect” if it does not improve strophanthin
intoxication within 60 min following drug administration.
 Evaluation:
 Test compound considered to have an anti-arrhythmic effect if the extrasystole immediately
disappear,
 If not then the increasing doses are administered at the 15 min. intervals.

24. 2) Electrically Induced Arrhythmia.
 Ventricular fibrillation electrical threshold:
 Purpose and rationale :
▪ The use of anti arrhythmic drugs in treatment of ventricular arrhythmias aims to prevent
the development of Ventricular fibrillation.
▪ Several electrical stimulation techniques have been used to measure ventricular fibrillation
threshold such as –
- Single pulse stimulation
- Train of the pulses stimulation
- Continuous 50Hz stimulation
- Sequential pulse stimulation
 Procedure:
 Dogs (8-12kg) anesthetized with phenobarbitone intraperitoniallly and maintained on
artificial respiration.

25.  Chest opened and heart is suspended on pericardial cradle.
 SA node crushed and Ag-AgCl+-- simulating electrode embedded with Teflon disc sutured to
anterior surface of left ventricle.
 Constant current of 400ms is applied through electrode and which was programmed by
digital stimulator
 ECG recorded and to determine ventricular fibrillation 0.2 to 1.8s train of 50hz pulses is
delivered. (VFT)
 Heart is immediately defibrillate and allow to recover as controlled condition for 15mins.
 Test drug administered through femoral vein.
 VFT is determined before and after administration of test drug.
Evaluation :
 Ventricular fibrillation threshold is
determined before and after
administration of test drugs at
given time intervals.
 The mean values of 10
experiments are compared using
Student’s T-test.

26. 3) Mechanically induced arrhythmias
Coronary artery ligation, Reperfusion arrhythmia and infarct size in rats
Purpose and rationale :
▪ Coronary artery ligation in anesthetized rats results in arrhythmias and myocardial infraction,
Following occlusion of the left main coronary artery, very marked ventricular dysrhythmias
occur. Electrocardiogram is recorded.
▪ The amount of infracted tissue in measured by means of p-nitro blue tetrazolium chloride-
staining in myocardial sections.
▪ The model is used to test drugs with potential antiarrhythmic activities.
Procedure :
▪ Animal : male Sprague-Dawley rats(350-400 g )
▪ No. of animals : 8-10
▪ Anesthesia : pentobarbital sodium(60 mg/kg) i.p

27. • The trachea is intubated to allow artificial ventilation.
• A catheter is placed in an external jugular vein for administration of test compounds.
• Peripheral blood pressure is recorded from the common carotid artery using a pressure
transducer and a polygraph.
• The chest is opened by thoracotomy at the 4th intercostal space.
• A thin silk thread ( Ethicon 1.5 metric, 4-0) attached to a needle is placed around the left
coronary artery about 2-3 mm distal of the origin of the left coronary artery for later ligation.
• The trachea is intubated to allow artificial ventilation.
• A catheter is placed in an external jugular vein for administration of test compounds.
• Peripheral blood pressure is recorded from the common carotid artery using a pressure
transducer and a polygraph.
• The chest is opened by thoracotomy at the 4th intercostal space.
• A thin silk thread ( Ethicon 1.5 metric, 4-0) attached to a needle is placed around the left
coronary artery about 2-3 mm distal of the origin of the left coronary artery for later ligation.

28. Preparation to determine infract size:
 At the end of the reperfusion period, the animal is sacrificed with an overdose of
pentobarbital sodium, the heart is dissected and cut into transversal sections (approx. 1mm
thick) from the apex to the base.
 The slices are stained with p-nitro-blue tetrazolium chloride solution in order to visualize the
infract tissue(blue/violet-stained healthy tissue, unstained necrotic tissue).
 The slices are photographed on color transparency film for the determination of infract area.
 Evaluation:
The following parameters are evaluated;
1. Mortality
2. Hemodynamics
-Peripheral blood pressure[mmHg]
-Heart rate [beats/min]
-Pressure rate index[ BP * HR ]

29. 3. Arrhythmias-
- ventricular extrasystoles ( premature ventricular contractions-PVC)
- ventricular tachycardia(VT)
- ventricular fibrillation (VF)
% animals with PVC, VT, VF
No. of PVC, VT, VF/ 5 or 30min
- infarct size (area)
These parameters are less in treated animals as compared to control are an indication of anti
arrhythmic activity.
The no. of ventricular premature contractions, ventricular tachycardia and ventricular
fibrillation are counted in the occlusion and reperfusion periods.

30.  Harris dog model of ventricular tachycardia (canine model of two stage ligation)
 Purpose and rationale :
▪ In 1950, Harris found that the mortality in the dogs after coronary occlusion with a 2- stage
ligation procedure was lower than with 1- stage ligation.
▪ The left descending coronary artery is partially occluded for 30 min after which time total
ligation is performed
▪ Under these conditions arrhythmias develop within 4-7 hr, reach a peak between 24 and 48
hr. and abate within 3-5 days.
 Procedure :
▪ Animal : male or female dog
▪ Anesthesia : methohexitone sodium (10 mg/kg) i,.v
maintained with halothane.
 The heart is exposed through an incision in the 4th or 5th intercostal space. The anterior
descending branch of the left coronary artery is dissected ligated in two stages.
 Two ligatures are placed around the artery and 21 gauge needle. The first ligature is tied
around the artery and the needle, which is then removed. 30min later, the second ligature is
tied tightly round the artery.

31.  The chest is closed and the dog is allowed to recover.
 Further observations are made when the dogs are conscious after ligation of the coronary
artery.
 The dogs are positioned to lie on their side and remain in this position throughout the
experiment.
 Mean blood pressure is recorded from a catheter placed in the femoral artery.
 Lead II of the ECG and BP are continuously recorded for a control period of 30 min
before and during drug administration.
 Drugs are administered either by injection or by continuous infusion.
 Evaluation :
 The number of sinus and ectopic beats are counted for each successive 5-min. period.

32. 4) Exercise Induced Arrhythmia:
 Ventricular fibrillation induced by cardial ischemia during exercise
Purpose and rationale:
▪ Billman and his group developed method to evaluate antiarrhythmic drugs for their activity in
cardiovascular parameters in an exercise-plus-ischemia test.
Procedure:
▪ Animal : Mongrel dogs, (15.4 to 19.1 kg)
▪ Anesthesia : anesthesia is induced with pentobarbital (10 mg/kg) i.v
In this model a major surgery is done in dogs, in which transducers are fixed in body followed
by 2-stage left anterior descending coronary artery ligation.
After 28 days animals were prepared for test, in which animals are allowed to walk on a motor
driven treadmill.

33. The animals run on the treadmill simultaneously the workload was increase in every 3 min for 18
min.
During the last minute of exercise, the left circumflex coronary artery is occluded, the treadmill is
stopped.
Large metal plates are placed across the animal’s chest so that electrical defibrillation can be
achieved with minimal delay.
The occlusion is immediately released if VF occurs.
The animals receive one or more of the following treatments:
1) Standard drug glibenclamide (1 mg/kg). The drug is injected in a cephalic
vein; 3 min before exercise begins.
2) The exercise-plus-ischemia test is repeated after pretreatment with the
test drug.
3) A second control(saline) exercise plus ischemia test is performed 1 week after the last drug test.
 Evaluation :
Coronary blood flow
The data are analyzed using analysis of variance (ANOVA) for repeated measures.

34.  IN-VITRO MODELS
 Langendroff Technique :
Principle :
▪ Heart is perfused in RETROGRADE direction from aorta either at constant pressure or
constant flow with oxygenated saline solution.
▪ It closes aortic valves , just in situ heart during diastole.
▪ The perfusate is displaced through the coronary sinus and the opened right atrium.
Procedure:
Guinea pig of either sex (300-500 g)
Heart is removed as soon as possible and place in Petri dish containing RL at 37◦C aorta located
and cut below the division
Cannula inserted into the aorta and tied. Heart is perfused with oxygenated RL

35. Heart is transferred to double wall Plexi glass perfusion apparatus maintained 37ºC.
Ligature is placed around LAD coronary artery & occlusion is maintained for 10 min by
reperfusion
Test compound administered through perfusion medium either before or after occlusion.
Epicardial ECG electrode is used for pulsatile stimulation and induction of arrhythmia
(rectangular pulses 0.75msec duration, 10V; 400-1800 shocks/min)
Small steel hook with string attached to apex of heart and HR measured with chronometer.
 Evaluation:
Incidence & duration of Ventricular fibrillation or ventricular tachycardia recorded in control
& test groups.

37.  References
 Essentials of Medical Pharmacology seventh edition by K.D.Tripathi.
 Drug Discovery and evaluation second edition by H. Gerhard vogel
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164530/
 https://en.wikipedia.org/wiki/Antiarrhythmic_agent
 https://www.webmd.com/heart-disease/atrial-fibrillation/heart-disease-abnormal-heart-
rhythm

38. THANK YOU….