Drug screening methods for antiarrhythmic agents

1. DRUG SCREENING METHODS FOR
ANTIARRHYTHMIC
AGENTS
Dr. Abhishek Vyas
II Year PG Student
Department of Pharmacology
AMC MET Medical College
Maniangar. Ahmedabad
1

2. METHODS
In Vitro Models In Vivo Models
1. Acetylcholine or Potassium
Induced Arrhythmias
1. Chemically Induced Arrhythmia
a) Aconitine antagonism in rats
b) Digoxin induced arrhythmias in guine pigs
c) STROPHANTHIN induced Arrhythmia.
d) ADRENALINE induced Arrhythmia
2. LANGENDROFF Technic 2. Electrically induced arrhythmia
a) Ventricular fibrillation electrical threshold
b) Programmed Electrical Stimulation Induced
Arrhythmia
c) Sudden Coronary Death Models in Dogs
3. EXERCISE INDUCED VENTRICULAR FIBRILATION
2

3. IN VITRO MODELS
1. Acetylcholine or Potassium Induced Arrhythmias
New Zealand white rabbits (0.5-3kg) sacrificed and heart removed
Atria dissected from other tissue in RL and attached with electrode
Fibrillation produced when atria exposed with Acetylcholine or KCL
After 5min exposure to Acetylcholine or KCL, atria stimulated with pulses 0.75 ms , usually of 10V
and recorded on Kymograph
Control arrhythmia produced for 6-10 min followed by 30 min rest period
Test compound were added to the bath
If fibrillation doesn't disappear Test drug found to be effective If
within 8-10 min, preparation is washed to fibrillation is disappears immediately
or within 5min
allowed to normal concentration
3

4. LANGENDROFF Technic
Basic principal involved is that 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.
Guinea pigs either sex 300-500 g
Heart is removed ASAP and placed in petridish containing RL @ 37.0°C
Aorta locaeted and cut below the division
Cannula inserted into the aorta and tied. Heart is perfused with oxygenated RL
Heart is transferred to double wall Plexiglass perfussion apparatus maintained 37.0°C
Ligature is placed around LAD coronary artery & occlusion is maintained for 10 min f’by reperfussion
4

5. Test compound administered through perfusion medium either before or after occlusion.
Epicardial ECG electrode is used for pulsatile stimulation and induction of arrhythmia
[Rectengular pulces 0.75 msec duration, 10 V; 400-1800 shocks/min]
Small steel hook with string attached to apex of heart and HR measured with chronometer
Incidence & duration of VF or VT is recorded in control & test group
5

6. 1. Chemically Induced Arrhythmia
• Large number of agents alone or combination are capable to
induced arrhythmia:
 Anesthetic like Chloroform, eather ,Halothane
 IV Adrenaline, Ouabain alkaloids
IN VIVO MODELS
6

7. Aconitine antagonism in rats
Aconitine, its plant alkaloid from roots, it Acts perstintant on Na+ channels resulting
ventricular arrhythmias
Drugs which have anti-arrhythmetic property can be tested on aconitine intoxicated rats
Male Ivanovas rats (300-400 gm) anesthetized intraperitonially with urethane (1.25 g/kg)
Aconitine dissolved (5 mcg/kg) in 0.1 N HNO3 & infused into rat’s saphenous vein @ 0.1 ml/min
Lead II ECG is recorded every 30 sec.
Test compound injected orally 5 min before Aconitine
infusion
Higher dose of aconitine in the test group gives an index of
antiarrhythmic activity.
The antiarrhythmic effect of test compound is measured
by the amount of aconitine /100g animal
Higher dose of aconitine is needed as compared with
control group
7

8. Digoxin induced arrhythmias in guine pigs
Digoxine overdose causes ventricular extrasystoles, fibrillation & death
Male Marioth guinea pigs (350-500 g) anesthetize with pentobarbitone
35mg/kg intraperitonially
Tracheae, Jugular vein & caratoid artry catheterized & animal is maintained on
artificial respiration (45 breath/min)
Through jugular vein digoxin is infused @ 85mcg/kg in 0.266 ml/min until
cardiac arrest
ECG recorded with steel needle during experiment
Test drug is administered either 1hr before if orally or 1min before if IV, prior
infusion of digoxine
The onset of ventricular extrasystoles, fibrillation, cardiac arrest
Compared results in test & control group
8

9. VT & mutifocal ventricular arrhythmia with acute intoxication of cardiac glycoside like Strophanthin K
Dogs (20kg) of either sex anesthetized with pentobarbitone 30-40 mg/kg intraperitonially
Two peripheral vains cannulated for administration of arrhythmia inducing substance and for test
compound
ECG recorded at different time point from lead II
Strophanthin was infused @ 3mcg/kg/min till 30-40 min. Once VT developed infusion terminated
After stabilizing of ventricular arrhythmia
(10 min), test compound administered
If extrasystole disappears, test compound has
antiarrhythmic property
If no positive effect; gradually increase doses
@ 15 min interval
STROPHANTHIN induced Arrhythmia.
9

10. ADRENALINE induced Arrhythmia
Adrenaline at higher dose induce arrhythmia.
Dogs (10-11 kg) anesthetized with pentobarbitone (30-40mg/kg) intraperitonially
Adrenaline infused @ 2-2.5 mcg/kg through femoral vein
Test compound administered 3min after adrenaline infusion
If extrasystole disappear, test compound has antiarrhythmic property
10

11. 2. Electrically induced arrhythmia
• Serial electrical stimulation results in flutter and fibrillation and it is possible to
reproduce some of the main type of arrhythmias of clinical importance. The flutter
threshold or the ventricular multiple response threshold may be determine in
anesthetized dogs before or after administration of test drug.
1. Ventricular fibrillation electrical threshold
2. Programmed Electrical Stimulation Induced Arrhythmia
3. Sudden Coronary Death Models in Dogs
11

12. Ventricular fibrillation electrical threshold
• The maximum frequency at which atria would follow a stimulus used to compare
antifibrilati compound. Several methods:
• Single pulse method
• Train of pulses stimulation
• Continue 50Hz stimulation
• Sequential pulse method
Dogs (8-12 kg) anesthetized with pentobarbitone intraperitonially and maintained on artificial
respiration
Chest opened & heart is suspended on pericardial cradle
SA node crushed & Ag-AgCl stimulating electrode embedded with Teflon disc sutured to anterior
surface of LV
Anodal constant current for 400ms is applied through electrode and which was programmed by
digital stimulator
ECG recorded through lead II of body ECG monitoring
12

13. To determine Ventricular Fibrillation (VFT) 0.2 to 1.8 s train of 50Hz pulses delivered
The current intensity of pulse train required to induce sustained ventricular fibrillation is define as
VFT
When VF occurs, heart immediately defibrillate & allow to recover as controlled condition for 15-20
min
Test drug administered through femoral vein
VFT is determined before and after administration of test drug & compared by using student T-Test
13

14. Programmed Electrical Stimulation Induced Arrhythmia
Dogs (8-12 kg) anesthetized with 30mg/kg pentobarbitone IV & maintained on artificial respiration
Left thoracotomy performed in between 4th and 5th ribs & heart exposed
Left anterior coronary artery isolated, 20 G needle placed, ligature is tied around the artery and needle &
needle removed, result in stenosis of the vessel
LAD perfused for 5min
Ischemic injury to myocardium is achieved by 2hrs occlusion of LAD by
silicon rubber snare
Vessel than allowed to reperfuse for 2hrs in presence of stenosis
14

15. Meanwhile epicardial bipolar electrode sutured on intraventricular septum, adjument to occlusion site
Silver disc electrode implanted for ECG
After 6-9 days, chest reopened & programmed electrical stimulation is performed at non infarct zone
The pace of stimuli is set @ 200 ms and followed
by extra stimuli after each 15 pacing stimulation
Test compound administered 30 min after the stimulus
The minimum current intensity of pulse required to
induce sustain ventricular fibrillation is recorded before &
after test compound administered
15

16. Sudden Coronary Death Models in Dogs
Sudden coronary death is one of the most leading causes in developed countries
This model of dogs used to test protection offered against sudden coronary death
Male mongrel dogs (14-22 kg) anesthetized with pentobarbitone IV
Trachea is cannulated and maintained artificial breathing; Jugular vein cannulated for test drug
administration
Chest opened & heart exposed, LAD isolated & 20 G needle placed on LAD
Ligature tide across artery and needle followed by removal of needle resulting critical stenosis of
vessel
LAD is occluded for 2hrs using rubber snare & allowed to reperfuse for 2 hrs in presence of
stenosis
An epicardial bipolar electrode is sutured on left ventricular wall; one @ the distribution of LAD
distal to the occlusion & second in distribution of Left Circumflex Coronary (LCX) artery
Silver coted electrodes is passed through the wall & into lumen of LCX sutured to the adjunct
surface of heart
16

17. Surgical incision closed & animal is allowed to recover
After recovery, they are treated with test drug
Anodal 15 µA current from 9 V Ni-Ca battery is passed
through 25 Ω resister & applied to electrode in luman of
LCX
Animals are sacrificed after 24 hrs on constant anodal
current
Hearts are removed & thrombus mass in LCX is removed &
wighed
Heart is sectioned & stained with Tetrazolium Triphynyl
Chloride (TTC) to study the area of infarct
Time of onset of ventricular ectopy & lethal arrhythmia is
studied using recording of cardiac cassette recorder.
17

18. 3. EXERCISE INDUCED VENTRICULAR FIBRILATION
Mongral dogs (15-19 kg) anesthetized with pentobarbitone IV (10mg/kg)
Chest cavity opened, heart exposed & supported with pericardial cradle
Around LCX 20 MHz pulsed Doppler flow transducer & hydraulic accluder are placed
Pair of insulated silver wire sutured to epicardial surface of both left and right ventricle. ECG & HR
ministered
Pre calibrated solid state pressure transducer is inserted into left ventricle & finally, a two stage
occlusion of LAD is performed
Leads from cardiovascular instrumentation are tunneled under skin to exit on the back of
animal’s neck
3-4 weeks after the production of myocardial ischemia; animal are walked on treadmill & trained
to lie quietly on lab table during recovery period
18

19. Protocol starts with 3 min warm up followed by run @ 6.4 km/hr (0 % grade)
Grade is increased every 3min run 0% 4% 8% 12% and 16%
During last min of exercise LCX is occluded, treadmill stopped & occlusion maintained for
additional 1min
Occlusion is released if VF occurs
Exercise and ischemia test is repeated after pretreated with test drug & compared with
control reading
19

20. MECHANICALLY INDUCED ARRHYTHMIA
• Arrhythmia can be induced directly by ischemia or by reperfusion. By
ischemia induced infarction or by coronary ligation several phases or
arrhythmia can be studied. The two stage coronary ligation technique
focuses on late arrhythmia.
1. Reperfusion Arrhythmia in Rats
2. Reperfusion Arrhythmia in Dogs
20

21. Reperfusion Arrhythmia in Rats
Ligature of left main coronary artery results in ventricular arrhythmia and MI
ECG is recorded during ligation & subsequent reperfusion
The amount of infracted tissue is measured by means of P-nitro blue tetrazolium chloride
staining in mayocardium section
Spargue-Dawley rats (350-400 g) are anesthetized with pentobarbitone (60 mg/kg)
intraperitonially and maintained on artificial respiration
Jugular vain is cannulated for administration of test drug
BP is recorded from the caratoid artery using pressure transducer connected to polygraph
Chest is opened and heart is exposed. The left coronary arery is located & ligated for 15-90
min & subsequently reperfusion for 30 min.
21

22. Test drug is administered 5min before the ligation
BP & ECG lead II recorded continuously during experiment
The number of ventricular premature beats, ventricular tachycardia & fibrillation are counted
in occlusion and reperfusion period
At the end of reperfusion period, the animal is sacrificed and TTC staining is done to quantify
the infarct size
Heart is dissected & cut into transverse section & stained with TTC in order to visualize the
infarct (Blue/violate stained healthy; Unstained necrotic tissue)
Slices are photographed & infarct area is measured by planimetry from projection of all slice
Changes in hemodynamic parameters & infarct size in drug treated animal are compared to
control values
22

23. Reperfusion Arrhythmia in Dogs
CA ligation in dogs may results in increase HR, heart contractility, left ventricular end
diastolic pressure, BP & ventricular arrhythmia, especially in the reperfusion duration
Dogs (20-25 kg) anesthetized with thiobutobarbital (30 mg/kg) intraperitonially &
maintained on IV chloralose (20 mg/kg) & 250 mg/kg urethane IV followed by
morphine 2mg/kg Sq
Animal maintained on artificial respiration
A sephenous vein cannulated for test drug administration. ECG recorded with lead II
Femoral artery cannulated to measure BP
23

24. • Left ventricular end diastolic pressure & HR are determined from the left
ventricular pressure curves
• Myocardial contractility is measured as arise of left ventricular pressure
• Coronary artery is ligated for 90 min
• 20 min prior to ligation, test drug is administrated
• Animal is reperfused for 30 min, All parameters are recorded
• Changes in parameter like mortality, hemodynamic & arrhythmia in drug treated
animal are compared to control group
24

25. Two Stage Coronary Ligation in Dogs
Mortality in dogs after coronary occlusion with two stage ligation procedure is
lower than single stage ligation
Dogs (8-11 kg) are anesthetized with IV methohexitone (10 mg/kg) & maintained
on artificial respiration
Chest is opened & heart is exposed
Left coronary artery located & coronary ligature is performed in two stage.
Two ligatures are placed around the artery & 21G needle
First ligature tied around the needle & artery followed by removal of needle.
25

26. 30 min after 2nd ligature is tide tightly around the artery
Chest is closed in layers, 30 min after 2nd ligature & dogs are allowed to recover
After 24-48 hrs of ligature arrhythmia developed & abate within 3-5 days
Lead II ECG, atrial ECG & mean BP are measured
Test drugs are given as infusion for 10 min after coronary artery ligature
Changes in parameter like mortality, hemodynamic & arrhythmia in drug treated
animal are compared to control group
26

27. CONCLUSION
• Antiarrhythmic drugs exert their effect by modulating conduction velocity
or refrective period duration or both
• Conduction velocity depends on passive electrical properties of cardiac
tissue or characteristics of Na+ and Ca2+ channel
• In contrast there are marked difference among species in the K+ currents
that largely determine repolarization, so that AP duration & duration of
refractory period differ widely various species
• It is clear that species difference do exist with respect to factors that
determine arrhythmogenesis & it ia also clear that no animal models will
mimic the human suffering from or threatened by arrhythmia
• Electrophysiological studies should be encouraged in animals with
naturally occurring cardiovascular disease.
27

28. 28