Evaluation methods of anti-aarhythmics

Evaluation
methods
Anti-Arrhythmic
s
Presenter-
Dr Nikita Ingale
JR3
Pharmacology
Guide-
Dr Vijay Motghare
Professor and Head
Pharmacology

EM- Antiarrhythmics 25/11/19
Cardiac Arrhythmias…
-
- Cardiac arrhythmia is an abnormal heart rhythm
- A heart rate that is too fast – above 100 beats per minute in adults – is called tachycardia, and a heart rate that is
too slow – below 60 beats per minute – is called bradycardia
- Many types of arrhythmia have no symptoms. When symptoms are present, these may include palpitations or
feeling a pause between heartbeats, shortness of breath or chest pain.
- There are four main types of arrhythmia:
- Extra Beats  premature atrial & ventricular contractions, premature junctional contractions
- Supraventricular Tachycardias atrial fibrillation, atrial flutter, paroxysmal supraventricular
tachycardia
- Ventricular Arrhythmias  ventricular fibrillation, ventricular tachycardia
- Bradyarrhythmias  sinus bradycardia, sick sinus syndrome

Phase 0 rapid depolarisation
(inflow of Na+)
Phase 1 partial repolarisation
(inward Na+ current closed,outflow of K+)
Phase 2 plateau
(slow inward calcium current)
Phase 3 repolarisation
(calcium current closed, K+ outflow)
Phase 4 pacemaker potential
(Slow Na+ inflow, slowing of K+ outflow)
Phase 4
Phase 0
Phase 1
Phase 2
Phase 3
0 mV
-90mV
Phases of action potential in heart…

Current therapy for arrhythmias…
K+ channel blockers Ca+ channel blockers
Na+ channel blockers Na+ channel blockers Na+ channel blockers

Evaluation
methods
for
Anti-Arrhythmi
cs

Experimental animals used-
Rats
Dogs
Guinea pigs
Rabbits
Mice

Evaluation methods
In vivo methodsIn vitro methods Clinical evaluation
-Chemically induced
-Electrically induced
-Exercise induced
-Mechanically induced
-Genetically induced
-Isolated Guinea pig papillary m
uscle
-Langendorff technique
-Acetylcholine or potassium ind
uced arrhythmia

In vitro
methods for
Evaluation
of
Arrhythmias

In vitro methods…
Isolated Guinea pig papillary muscle
Langendorff technique
Acetylcholine or potassium induced
arrhythmia

Isolated Guinea pig papillary muscle…
Principle:
In right ventricular guinea pig papillary muscle, developed tension (DT),
excitability (EX), and effective refractory period (ERP) are measured.
Non-electrode method
Animal- Guinea pig(200-400g)

The tendinous end of the papillary muscle is ligated with a silk thread , chordae tendinae are freed
from the ventricle
The preparation is mounted in organ bath & experimental conditions are maintained
muscles are field stimulated to contract isometrically at a duration of 1 ms.
Method …

Pulses are delivered using constant voltage stimulator, the developed tension is recorded
using polygraph recorder.
The force frequency curve is obtained by measuring the developed tension over a range of stimulus
frequencies (0.3,0.5,0.8,1.0,1.2 HZ)
The percentage change in post treatment versus pretreatment developed tension at 1 HZ is used to
quantitate the agents inotropic effect.
Method …

Evaluation …
- Change in Effective Refractory Period (Post treatment minus pretreatment)
- Degree of shift in the strength-duration curve.i.e.(area between post and pre treatment curve)
- Percentage changes in post treatment developed tension, Duration of action potential.
- Contraction force
The results of above calculations are to classify the compounds as class I ,III or IV antiarrhythmic
agents on the basis of its effect on developed tension, excitability and effective refractory period.
EVALUATION MOA OF DRUG
EXCITABILITY Na+ Channel
Contraction Force Ca+2 Channel
Effective Refractory period K + Channel

Langendorff technique …
PRINCIPLE:
Heart is perfused in a retrograde direction from aorta either at constant
pressure or at constant flow with oxygenated saline solution
Animal – guinea pig heart

Langendorff technique …
Animal Preparation –
Animal is heparinized before the experiment

Method …

Method continued…

Evaluation …

Evaluation …
- Heart rate measured through chronometer coupled to polygraph
- Contractile force measured by force transducer.
- Incidence and duration of ventricular fibrillation or ventricular tachycardia is recorded in the
control as well as test group.

Acetylcholine and potassium induced arrhythmia…
Animals - New Zealand White rabbits 0.5 to 3 kg
Method -
The animals are sacrified and heart removed immediately
Atria dissected and kept in Ringer’s solution
Fibrillation is produced when atria are exposed to Acetylcholine 3x10-4 g/ml
or 0.1 gm potassium chloride

Acetylcholine and potassium induced arrhythmia…
A mechanical record is taken on kymograph
Controlled arrhythmia are produced and allowed to continue for 6 to 10 mins
After rest period of 30 mins test compound is added to bath.
Evaluation:- Test compound is found to be effective if fibrillation disappears
immediately or within 5 mins following test drug supplementation to organ bath.

In vivo
methods for
Evaluation
of
Arrhythmias

In vivo methods…
Chemically induced
Electrically induced
Exercise induced
Mechanically induced
Genetically induced

Chemical methods…
Aconitine
Digoxin
Adrenaline
Strophanthin

Aconitine induced chemical method…
PRINCIPLE: Aconite persistently activates sodium channel present on the cardiac muscle
Continues infusion
in saphenous vein
ACONITE 5mg/kg
dissolved in 0.1% HN03
MONITOR
ECG EVERY 30
SECONDS
Anaesthetize with
Urathene 1.25 g/kg
Test compound
orally or IV 5 minutes
before aconite infusion
- Alkaloid
- Toxin
- Aconitum
plant
- Devil’s
helmet
- cardio toxin

Evaluation …
- The anti-arrhythmic effect of the test compound is measured by the amount of
aconitine/100 gm animal (infusion duration ), required to precipitate
Ventricular extrasystoles Ventricular tachycardia Ventricular fibrillation

Digoxin induced chemical method …
PRINICPLE: Over dose of cardiac glycosides induces ventricular extrasystoles, ventricular fibrillation,
and finally death.
Anesthetised guinea pigs.( 350-500 gms)
Trachea, jugular vein or carotid artery are catheterized.
Test grp receives Test drug either orally 1 hr or iv 30 min prior to the infusion while control group
receives digoxin infusion only at rate of 85 mg / kg in 0.266 ml/min until cardiac arrest occurs.
ECG recorded

Evaluation …
 The period until the onset of ventricular extra systoles, ventricular fibrillation and cardiac
arrest is recorded.
 Total amount of infused Digoxin to induce ventricular extra systoles, or ventricular fibrillation
and cardiac arrest is calculated.

Strophanthin / ouabin induced chemical method…
Principle :Strophanthin induces ventricular tachycardia and multifocal ventricular arrhythmia in dogs.
Strophanthin administered to dogs by continuous i.v infusion at a rate of 3mg/kg/min
30-40 min later ventricular tachycardia / multifocal ventricular arrhythmias occur,
strophanthin infusion is terminated.
When the arrhythmias are stable for 10 min, the test substance is administered IV or ID
ECG recordings are obtained at 0.5, 1, 2, 5 and 10 min following administration
- Cardiac glycoside
- Arrow Toxin
- Strophanthus gratus
- Inhibits Na+k+
ATPase
- Overdose  cardiac
arrhythmias

Evaluation…
- Test compound is said to have an
antiarrhythmic effect if extra systoles
disappear within 15 min .
- The test drug is considered to have No
effect if it does not improve
strophanthin intoxication within 60
mins
- Test compound is said to have antiarrhythmic
effect if the extra systoles immediately
disappear .
- If not then the increasing doses are
administered at 15 min- intervals.
- If the test substance does reverse the
arrhythmias, the next dose is administered
after the reappearance of stable arrhythmias.
I.DI.V

Adrenaline induced chemical method…
PRINCIPLE: Sympathomimetic drugs are known to increase pacemaker activity
Adrenaline at high dosemay precipitate arrhythmia.
Evaluation: A test compound is said to have antiarrhythmic effect if the extrasystole
disappears immediately after drug administration.
Dogs (10-11 kg) are anaesthetized with Halothane 1% vapourised by 100%O2
Femoral vein is cannulated and adrenaline is infused at a rate of 2-2.5 mg/kg
Test drug is administered 3 mins after adrenaline infusion
ECG Recorded.
Why halothane??
Highly sensitize the myocardium to
catecholamines

Electrical method…
Principle :Ventricular fibrillations can be induced by various techniques-
1] Single pulse stimulation
Requirement:
Animals – Adult dogs (8-12kg)
Anesthetic – Sodium pentobarbital (35mg/kg)
3] Continous 50 HZ stimulation
2] Train of pulse stimulation

Methods …
Adult dogs are anaesthetized and heart is exposed
Artificial respiration – respiratory pump
B.P – monitored
Body temperature – maintained by thermal blanket
Chest cavity is opened
Heart suspended in pericardial cradle
Sinus node is crushed and electrical stimulation is provided with Ag-AgCl stimulating
electrode sutured to anterior surface of left venticle.

Method continued…
Test drug/ std/control Drugs are administered through the femoral vein
Anodal current for 400ms is applied through the driving electrode
ECG is recorded
Evaluation:-
- 0.2 to 1.8 second of 50 Hz pulses is delivered for every 100 ms
- The current intensity is increased . When ventricular fibrillation occurs, the heart is
immedietely defibrillated and allowed to recover for 15-20 mins.
- VFT is determined before and after administration of test drugs at given time intervals.

Exercise induced ventricular fibrillation…
Purpose: Tests combining coronary constriction with physical exercise resembles most closely the situation in
coronary patients
Animals – Mongrel dogs (15 -19 kg)
Anesthetic – Sodium pentobarbitone (10mg/kg) IV
Animals – selected, Anesthetized
↓
Chest cavity – opened
↓
Heart suspended in pericardial cradle
↓
Around left circumflex artery – Doppler flow inducer- to measure blood pressure
Method …

Method continued…
Pair of insulated silver coated wires – sutured on left and right ventricles- measure HR
Occlusion of LAD(Two stage)
Myocardial infarction
After 24 hrs- test drug/std/control – administered
• Transdermal fentanyl patch (↓pain and discomfort)
• Antibiotic therapy(amoxicillin)

Method continued…
3-4 weeks after the production of MI
↓
Animals – run on a motor –driven treadmill at speed
6.4 km/hr
↓
Work load - ↑every 3 min for total of 18 mins
↓
During last minute-treadmill is stopped
↓
After 10-20 sec of VF – defibrillation is achieved without any delay
EVALUATION:
The exercise plus ischemia test is repeated after administration of the test drug and
compared to control (saline) group.

Two stage coronary ligation…
Two stages
First stage
- Partial occlusion
Of the artery
Second stage
- Ligature tightly tied
around artery
- Total occlusion

Method…
In the first stage, LAD surgically isolated
The first ligature drawn around artery and needle but not tightly around the artery
State of partial occlusion for 30 min
Then another suture is used to completely occlude the LAD
Arrhythmia develops 24-48 hrs after ligation and abate in 3-5 days
Test drug infusion 10 min after coronary ligation
Mortality, Hemodynamics in animals with VF are seen
Drug treated animals are compared with controls.
Evaluation.…

Genetic models…
- Genetic arrhythmia – in German Shepherd dogs
- Inherited ventricular arrhythmia- sudden cardiac death
- These dog can used to screen potential anti arrhythmic drug.

Inclusion criteria:
 A healthy male or female 18 to 59 years of age.
BMI range - 18-35 kg/m2,
No significant disease or abnormal laboratory values
Normal 12-lead ECG
Adequately informed the nature and risks of the study
written informed consent
EXCLUSION CRITERIA
- Women who are pregnant or breast feeding.
- Known hypersensitivity or allergy to drug.
- A history or presence of asthma or other pulmonary disease, thyroid
disease (hypo- or hyperthyroidism), hepatitis or other liver disease.
- The presence of abnormal lab values which are considered clinically
significant.
- History of smoking or alcohol within past 1 year.

Outcome parameters…
Symptoms and quality of life
Recommended as secondary outcome parameter
Atrial fibrillation symptoms scale (AFSS)
EHRA atrial fibrillation symptoms classification
Death:
Most Important primary outcome.
All-cause death should be classified in the following groups.
Non-cardiovascular
Cardiovascular death
Cardiac
Sudden (including arrhythmic, myocardial infarction, others)
Treatment- or procedure-related (is also a serious adverse event)
Left ventricular function and heart failure
Transthoracic echocardiographic data at entry & follow-up.
– LA size
– LV size
– LV function
Number of hospitalizations

Summary…
- Although no animal model can accurately resemble with human disease condition and
species differences also exist, close similarities with humans suffering from or
threatened by arrhythmias can be developed by selecting appropriate model and
species.
- Rather than a single model or experimental technique, combinations of investigations,
like isolated heart (langendorff arrangement or working heart), whole hearts in
anesthetized or conscious animals, excised cardiac preparations, testing the function can
be used.

Arrhythmia type Experimental model
Atrial Flutter Atrial flutter induced by Ach
Atrial flutter by aconite.
Atrial Fibrillation Atrial fibrillation in the isolated langendorff
perfused heart.
Ventricular Fibrillation Ventricular fibrillation electrical threshold.
Canine model of two stage ligation.
Ventricular arrhythmia during exercise by ischemia
.

References…
1]Vogel WH, Schölkens BA. Drug Discovery and Evaluation [Internet]. Vogel HG, Vogel WH, Schölkens BA, Sa
ndow J, Müller G, Vogel WF, editors. Berlin, Heidelberg: Springer Berlin Heidelberg; 2002. P.208-29.
2]Pratt C, Camm A. Evaluation of antiarrhythmic drug efficacy in patients with an ICD. Unlimited potential or re
plete with complexity and problems? Eur. Heart J. [Internet]. 1999 [cited 2014 Feb 21];1538–52.
3]Mor M, Shalev A, Dror S, Pikovsky O, Beharier O, Moran A, et al. INO-8875, a highly selective A1 adenosin
e receptor agonist: evaluation of chronotropic, dromotropic, and hemodynamic effects in rats. J. Pharmacol. Exp.
Ther. [Internet]. 2013 Jan;344(1):59–67.
4]Nilles KM, London B. Knockin animal models of inherited arrhythmogenic diseases: what have we learned fr
om them? J. Cardiovasc. Electrophysiol. [Internet]. 2007 Sep [cited 2014 Feb 21];18(10):1117–25.

References…
5]Bodhankar S, Bhatt L, Nandakumar K. Experimental animal models to induce cardiac arrhythmias. I
ndian J. Pharmacol. [Internet]. 2005;37(6):348.
6] Kirchhof P, Auricchio A, Bax J, Crijns H, Camm J, Diener H-C, et al. Outcome parameters for trials
in atrial fibrillation: executive summary. Eur. Heart J. [Internet]. 2007 Nov ;28(22):2803–17.
7] Drug screening methods by S. K. Gupta
8] Evaluation of drug activities: Pharmacometrics; Dr Lawrence and Bacharach, volume 1

Next PG activity-
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