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DRUG SCREENING METHODS FOR ANTIARRHYTHMIC

AGENTS

Dr. Abhishek VyasII Year PG Student

Department of PharmacologyAMC MET Medical College

Maniangar. Ahmedabad

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METHODSIn Vitro Models In Vivo Models

1. Acetylcholine or Potassium Induced Arrhythmias

1. Chemically Induced Arrhythmiaa) Aconitine antagonism in ratsb) Digoxin induced arrhythmias in guine pigsc) STROPHANTHIN induced Arrhythmia.d) ADRENALINE induced Arrhythmia

2. LANGENDROFF Technic 2. Electrically induced arrhythmiaa) Ventricular fibrillation electrical thresholdb) Programmed Electrical Stimulation Induced

Arrhythmiac) Sudden Coronary Death Models in Dogs

3. EXERCISE INDUCED VENTRICULAR FIBRILATION

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IN VITRO MODELS1. 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 Ifwithin 8-10 min, preparation is washed to fibrillation is disappears immediately

or within 5min allowed to normal concentration

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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°CAorta 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

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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

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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

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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/minLead 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

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Digoxin induced arrhythmias in guine pigsDigoxine 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

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VT & mutifocal ventricular arrhythmia with acute intoxication of cardiac glycoside like Strophanthin KDogs (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.

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ADRENALINE induced ArrhythmiaAdrenaline 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

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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 threshold2. Programmed Electrical Stimulation Induced Arrhythmia3. Sudden Coronary Death Models in Dogs

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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

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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

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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

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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

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Sudden Coronary Death Models in DogsSudden 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

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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.

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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

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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

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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 Rats2. Reperfusion Arrhythmia in Dogs

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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.

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

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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

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• 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

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.

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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

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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.

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THANK YOU…