Experimentalevaluationofanti Arrhythmicdrugs 150209013417 Conversion Gate02

Experimental evaluation of anti-arrhythmic drugs

Experimental evaluation of anti-arrhythmic drugsDr Kirtan BhattPost-graduate,KIMS, BangaloreProtocol IntroductionRecording of ECG in experimental animalsExperimental modelsIn vitro modelsIn vivo modelsChemically induced arrhythmiasElectrically induced arrhythmiasMechanically induced arrhythmiasExercise induced arrhythmiasGenetic modelsCell culture modelsConclusion

Introduction and historical landmarks 1897 Oskar/Oscar Langeddorrf1975 Laszlo Szekeres, Papp 1979 Laszlo Szekeres1984 Winslow1984 Wilson1988 Walker1993 Cheung

Laszlo SzekeresRecording of ECG in experimental animalsRecording of the ECG is an essential tool in the evaluation of anti-arrhythmic drugs. The pattern of ECG varies between species.Some changes: Lead II between right foreleg and left hindleg, which is in line with neutrally placed heartLead I between right and left foreleg stated to lie in the axis of horizontal heartLead III between left foreleg and left hindleg in line with the vertical heart.Along with these, unipolar leads (V1 to V6) and aVL, aVR and aVF.The procedure for recording of ECG in rats is as follows:

Male Sprague Dawley rats 250-300 g anaethetised by IP pentobarbitone 50 mg/kgRight jugular vein injections and left coronary for BPLead IIElectrode constructed with 26 gauge needles placed subcutaneouslyPaper speed 100 mm/s on a student physiographSensitivity of student physiograph adjusted to provide deflection of 30 mm for 1mV standard square wave

Precautions handle the animal with care, keep the room calmStorage oscilloscope5ECG recording (cont)Variables measured: TP-R intervalQRSQ-TRShSome differences between ECG of humans and smaller animalsHeart rate is very highMore prominent QRSST segment is generally absent

In vitro methodsIsolated Guinea Pig papillary muscleCharacterization of anti-arrhythmic activityAction potential and refractory periodLagendorff techniqueAch or potassium induced arrhythmiasIsolated guinea pig papillary muscleA simple and accurate method to classify anti-arrhythmic drugs into class I, II, III and IV.Based on the changes seen in Tension developed in papillary muscle (DT)Excitability (EX)Effective refractory periodAction potential and refractory period in Guinea pig muscleGuinea pigs of Marioth strain of 250-300 gTwo strongest papillary muscles from LV takenStandard microelectrode technique is used to measure APStimulation is given by rectangular pulses of 1 V and 1ms duration at 500ms intervalSecond stimuli given in decremental intervals till contraction ceases Compounds affecting ERP have either pro or anti-arrhythmic effects.Inotropic effects can also be determined.11Lagendorff techniquePrincipleThe heart is perfused in a retrograde direction from the aorta either at a constant pressure or a constant flow with oxygenated saline solutionsAnimalsAlbino rats (300g and at least 1 year of age)New Zealand rabbits (1.5-3 kg and 3 years of age)Guinea pig (300-450 g and 2-3 years of age)

In 1897.was considered a breakthrough discovery in cardiovascular research.12Animal housing conditionsHoused at ambient temperature (23C 2C)12:12 hour light and dark cycleFree access to tap waterFood ad libitum

PrecautionsPretreatment with heparinMaintenance of PSS flow rate to prevent edema of cardiac tissuePrevent air bubble entryMaintain sufficient hydrostatic pressure by maintaining distance between heart and the PSS reservoirCannula shouldnt penetrate the aortic valve

Heparin to prevent thrombus formation in heartFlow rate: 7-9 ml/min for rat,20 ml/min for rabbit13

Rat, guinea pig Krebs solutionRabbit McEwens solution15Ach or potassium induced arrhythmiasNew Zealand white rabbits weighing 0.5-3 kg are used

Chemically induced arrhythmias

Chemical agents capable of producing arrhythmias are:Anaesthetic agents like chloroform, ether, halothane (sensitizing agents) followed by a precipitating stimulus such as IV adrenaline, aconitine, cardiac glycosides (ouabain), veratrum alkaloids.The sensitivity of these arrhythmogenic substances differs from species to species.

Various models for chemically induced arrhythmias are:Aconitine induced arrhythmia in ratsDigoxin-induced arrhythmia in Guinea pigsStrophanthin/Ouabain induced arrhythmia in dogAdrenaline induced arrhythmia in dogsCalcium-induced arrhythmia in Wistar albino ratsAconitine induced arrhythmia in rats

Aconitine is a plant alkaloid - Aconitum napellus root Can persistently activate sodium channels --> ventricular arrhythmia in anaesthetised rats. Drugs with anti-arrhythmic property can be tested in aconitine-intoxicated rats.

Procedure:Animal Male Ivanovas rats weighing 300-400 g Anaesthesia Intraperitoneal injection 1.25 g/kg urethane5 g/kg aconitine dissolved in 0.1 N HNO3 is infused into saphenous vein at 0.1 ml/minLead II is recorded every 30 secondsTest compound given oral or IV (3 mg/kg 5 minutes before aconitine)8-10 animals are used per compoundUrethane 21EvaluationThe anti-arrhythmic effects are measured by amount of aconitine/100g (duration of infusion) which includesVentricular extra-systolesVTVFDeath

Statistical significance is assessed by students t-test Digoxin induced ventricular arrhythmiasOverdose of cardiac glycosides can cause ventricular extra-systoles, VF and deathThe occurrence of these symptoms can be delayed by anti-arrhythmic drugs

ProcedureAnimal Male guinea pig (Marioth strain) weighing 350-500 gAnaesthesia 35 mg/kg pentobarbital sodium intraperitoneal

23Evaluation

Doses of digoxin required to induce ventricular extra-systoles or VF or cardiac arrest after treatment with anti-arrhythmic drugs are compared statistically with students t-test Strophanthin/Ouabain induced arrhythmia in dogsAnimal Male or female dogs weighing 20 kg approximatelyAnaesthesia IV pentobarbital sodium 30-40 mg/kg

Two peripheral veins are cannulated for administration of arrhythmia inducing substance (V. brachialis) and for test substance (V. cephalica antebrachii)Duodenum is cannulated for intraduodenal administrationECG is recorded with needle electrodes from lead II. Heart frequency is derived from R-peaks of ECG. Strophanthin K is given continuous IV infusion at 3g/kg/min.Signs of intoxication in the form of VT or multifocal ventricular arrhythmias are seen in 30-40 minutes. Infusion is stopped. When the arrhythmia is stable for 10 minutes, the test substance is given IV 1-5 mg/kg or intraduodenally 10-30 mg/kg.ECG is recorded at -0.5, 1, 2, 5 and 10 minutes following the test drug administrationEvaluation:For IV test drug administrationConsidered anti-arrhythmic if extra-systoles disappear immediatelyIncrease the dose every 15 minutes if they dont

For ID test drug administrationConsidered anti-arrhythmic if extra-systoles disappear in 15 minutesno effect if it doesnt improve intoxication in 60 minutesCalcium induced arrhythmiasAl-Obaid et al in 1998 used calcium chloride induced arrhythmias for anti-arrhythmic activity evaluation in anaesthetized male rats

Wistar albino rats weighing 60-130 g are usedAnaesthesia pentobarbital 60 mg/kg intraperitonealAdrenaline induced arrhythmiasAdrenaline can precipitate arrhythmia at high dosesDogs of 10-11 kg are anaesthetized by pentobarbitone sodium 30-40 mg/kg intraperitoneally.Adrenaline is given through femoral vein at 2-2.5 mg/kgLead II ECG and atrial ECG are recordedTest drug given 3 minutes after adrenaline infusionSome other models for chemically induced arrhythmiasMouse chloroform model (Lawson, 1968 and Vargafting, 1969)BaCl2 model (Papp et al, 1967)Benzene vapours induced arrhythmia (Tripathi and Thomas, 1986)Wenzel and Kloeppel demonstrated that arrhythmias could be induced by changing the medium of cultured heart cellsVF production by isoprenaline and COMT inhibitor at high temperature (Sono et al, 1985)Grayanotoxin I induced arrhythmia in guinea pigs (Takei et al, 1994)Electrically induced arrhythmiasVentricular fibrillation electrical thresholdProgrammed electrical stimulation induced arrhythmiaSudden coronary death model in dogs (Harris dog model)Ventricular fibrillation electrical thresholdSeveral electrical stimulation techniques are used to measure VF threshold:Single pulse stimulationTrain of pulses stimulationContinuous 50 Hz stimulationSequential pulse stimulation

Animals adult dogs weighing 8-12 kgAnaesthesia sodium pentobarbital 35 mg/kg intraperitoneal

The minimal current intensity of the pulse train required to induce sustained VF is defined as VFT.

36Evaluation:VF threshold is determined before and after test drug administration and compared using students t-test. Programmed electrical stimulation induced arrhythmias1. 20 gauge needle.2. by a silicon rubber snare (produces anterior wall infarction) 3. adjacent to the occlusion site38Test drug is given 30 minutes after the stimulus.The minimum intensity of current needed for sustained VF is recorded before and after test drugs and mean values of 10 experiments are compared by students t-test.

39Sudden coronary death model in dogsThe group of Lucchesi described the experimental dog model for protection against sudden coronary death.

Animal - Male Mongrel dogs of 14-22 kg weightAnaesthesia - pentobarbital sodium 30 mg/kg IV.

Sudden coronary death being the leading cause of death, it warrants for use of more complicated models in higher animals for discovery of active drugs.

41Mechanically induced arrhythmiasArrhythmias can be induced by directly by ischemia or and also by re-perfusion. Studies involving both the mechanisms to produce arrhythmias have been demonstrated.Reperfusion arrhythmias in ratsVF after coronary occlusion and reperfusion in dogsTwo stage ligation in dogs (The Harris dog model)Reperfusion arrhythmias in ratsLigation of left main coronary artery results in ventricular arrhythmias and MI. ECG is recorded during ligation and also during reperfusion. The infarcted tissue is measured by tetrazolium triphenyl chloride staining

The heart is dissected and cut into transverse sections (1 mm thick) and stained with TTC prepared in Sorensen phosphate buffer containing 100 mM, L-maleate to visualise the infarct tissue (blue/ violet stained healthy tissue and unstained necrotic tissue).Slices are photographed and infarct area is measured by planimetry from projections of all slices

45Reperfusion arrhythmias in dogsLigation of coronary artery in dogs may lead to increase in HR, LV end diastolic pressure, BP and ventricular arrhythmias especially in reperfusion.

Animal - Dogs of 20-25 kg weightAnaesthesia:thio-butobarbital sodium 30 mg/kg IPmaintained on IV chloralose 20 mg/kg and 250 mg/kg urethane IV followed by SC morphine 2 mg/kg.The experimental procedure is similar to rats.

47Two stage ligation in dogs (Harris dog model)In 1950, Harris found that mortality in dogs after coronary occlusion with a 2 stage ligation procedure was lower than with 1 stage ligationLead II ECG, atrial electrogram and mean BP are measured.Test drugs are infused 10 minutes after coronary ligation.Changes in mortality, hemodynamics and arrhythmias in treated animals are compared with controls.

Exercise induced ventricular fibrillationBillman and his group developed methods to evaluate anti-arrhythmic drugs for their activity in exercise-plus-ischemia induced arrhythmia test.

3. from which HR is determined by Gould Biotachometer.4. first partially occluded for 20 minutes and then tied off.

522. Analgesics and antibiotics are given to minimize the discomfort.Defibrillation is given if animal becomes unconscious and the occlusion is released if VF occurs during experiment.

53Repeated after the test drug and the findings are compared with the control (saline) group.All hemodynamic data (rate of change of LV pressure) are recorded. The refractory period data, reactive hyperemia response to each occlusion is averaged and data analysed using ANOVA.The effect of intervention on arrhythmia formation are determined by chi-square test with Yates correction.

Cell culture techniquesVentricular arrhythmias, especially torsades de pointes can be evaluated using isolated ventricular myocytes.Analysis of action potential and patch clamp techniques in isolated ventricular myocytes helps us to clarify mechanisms underlying development of torsades de pointes.

4. The heart is digested and cut into small pieces, placed in a 20 ml HEPES buffered saline containing calcium chloride and shaken until single cells are dissociated.

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A list EPC-7 clamp amplifier is used to voltage clamp the isolated cells.AP are assessed with three way ANOVA to determine significance within treatment variations. Dunetts t test s used for significance of individual treatment means compared with control mean values.

58Genetically induced arrhythmiasA certain breed of dogs German shepherds exhibit propensity to sudden death that occurs due to inherited ventricular arrhythmias.Death usually occurs in them during sleep or at rest after exercise or excitement.These dogs can be used for screening of potential anti-arrhythmic drugs.

Conclusion Species differences do exist in mechanisms producing arrhythmias and no animal can exactly resemble humansHowever, knowledge acquired from animal studies can help a great deal in devising therapeutic strategies for both ventricular and supraventricular arrhythmiasVarious animal models can be combined to build novel strategies in management of arrhythmias