different methods to screen anti arrythmic drugs In vitro & In vivo methods to screen anti arrythmic drugs

1. EVALUATION OF
ANTIARRHYTHMIC DRUGS
1
Presented by:
Syed Kashif
Department of Pharmacology
AACP

2. CONTENTS
 Introduction- Basic Understanding of Arrhythmia & ECG.
 Classification Of Drugs
 Conclusion.
In – Vitro methods:
1) Isolated guinea pig papillary
muscle.
2) Langendorff technique
3) Acetylcholine & potassium Induced
arrhythmia
In-Vivo Methods:
Chemically induced arrhythmia
Electrically induced arrhythmia
Mechanically induced arrhythmia
Exercise Induced Arrhythmia
2

3. Cardiac Arrhythmias
3

4. Arrhythmia means an Abnormal heart
rhythm
Results from the abnormalities of:
 Impulse generation (Rate or Site of origin)
 Conduction
 Both
4

5. If the arrhythmia
arises from the
ventricles it is
called ventricular
arrhythmia
If the arrhythmia
arises from atria,
SA node, or AV
node it is called
supraventricular
arrhythmia
Causes of
arrhythmia
arteriosclerosis
Coronary artery
spasm
Heart block
Myocardial
ischemia

6. ARRHYTHMIA
If the arrhythmia
arises from the
ventricles it is
called ventricular
arrhythmia
If the arrhythmia
arises from atria,
SA node, or AV
node it is called
supraventricular
arrhythmia
Causes of
arrhythmia
arteriosclerosis
Coronary artery
spasm
Heart block
Myocardial
ischemia

7. Non-pacemaker action potential
Phase 0: fast
upstroke
Due to Na+
influx
Phase 3:
repolarization
Due to K+ efflux
Phase 4: resting
membrane potential
Phase 2: plateu
Due to Ca++
influx
Phase 1: partial
repolarization
Due to rapid efflux of K+
N.B. The slope of phase 0 = conduction velocity
Also the peak of phase 0 = Vmax

8. CLASSIFICATION OF ARRHYTHMIAS
1. Characteristics:
a. Flutter – very rapid but regular contractions
b. Tachyarrhthmia – increased rate
c. Bradyarrhythmia – decreased rate
d. Fibrillation – disorganized contractile activity
2. Sites involved:
a. Ventricular
b. Atrial
c. SA Node
d. AV Node
Supraventricular
8

9. MECHANISMS OF CARDIAC ARRHYTHMIAS
(A) Enhanced Automaticity:
 In cells which normally display spontaneous
diastolic depolarization (SA Node, AV Node, His-
Purkinje System)
 Automatic behavior in sites that ordinarily lack
pacemaker activity
9

10. A normal cardiac action potential may be
interrupted or followed by an abnormal
depolarization
Reaches threshold & causes secondary upstrokes
2 Major forms:
1. Early Afterdepolarization
2. Late Afterdepolarization
(B) After depolarization and
Triggered Automaticity
10

11. (C) Re-entrant Arrhythmia
Defined as circulation of an
activation wave around an
inexitable object
3 requirements for Re-entrant
Arrhythmia:
1. Obstacle to conduction
2. Unidirectional block
3. CT>ERP
11

12. Unidirectional
Block
Establishment of Re-entrant circuit
12

13. NORMAL HEARTBEAT AND ATRIAL ARRHYTHMIA
Normal rhythm Atrial arrhythmia
AV septum

14. NORMAL ECG :
 P wave – Atrial depolarisation.
 QRS wave – ventricular depolarisation.
 T wave – ventricular repolarisation.
14

15. 15

16. Management Of
Arrhythmias
16

17. MANAGEMENT
 Non Pharmacological
 Acute Management
 Prophylaxis
 Pharmacological
17

18. NON PHARMACOLOGICAL
 Acute
1. Vagal Maneuvers
2. DC Cardioversion
 Prophylaxis
1. Radiofrequency Ablation
2. Implantable Defibrillator
18

19. PHARMACOLOGICAL APPROACH
Drugs may be antiarrhythmic by:
 Suppressing the initiator mechanism
 Altering the re-entrant circuit
1. Terminate an ongoing arrhythmia
2. Prevent an arrhythmia
19

20. VAUGHAN WILLIAMS CLASSIFICATION
Phase 4
Phase 0
Phase 1
Phase 2
Phase 3
0 mV
-
80m
V
II
I
III
IV
Class I: block Na+ channels
Ia (quinidine, procainamide,
disopyramide) (1-10s)
Ib (lignocaine) (<1s)
Ic (flecainide) (>10s)
Class II: ß-adrenoceptor
antagonists (atenolol, sotalol)
Class III: block K+ channels
(amiodarone, dofetilide,sotalol)
Class IV: Ca2+ channel
antagonists (verapamil, diltiazem)
20

21. CLASSIFICATION OF ANTIARRHYTHMICS
(BASED ON MECHANISMS OF ACTION)
 Class I – Blockers of fast Na+ channels
 Subclass IA
 Cause moderate Phase 0 depression
 Prolong repolarization
 Increased duration of action potential
 Includes
 Quinidine – 1st antiarrhythmic used, treat both atrial and
ventricular arrhythmias, increases refractory period
 Procainamide - increases refractory period but side effects
 Disopyramide – extended duration of action, used only for
treating ventricular arrthymias
21

22. CLASSIFICATION OF ANTIARRHYTHMICS
(BASED ON MECHANISMS OF ACTION)
 Subclass IB
 Weak phase 0 depression
 Shortened depolarization
 Decreased action potential duration
 Includes
 Lidocane (also acts as local anesthetic) – blocks Na+
channels mostly in ventricular cells, also good for digitalis-
associated arrhythmias
 Mexiletine - oral lidocaine derivative, similar activity
 Phenytoin – anticonvulsant that also works as antiarrhythmic
similar to lidocane
22

23. CLASSIFICATION OF ANTIARRHYTHMICS
(BASED ON MECHANISMS OF ACTION)
 Subclass IC
 Strong Phase 0 depression
 No effect on depolarization
 No effect on action potential duration
 Includes
 Flecainide (initially developed as a local anesthetic)
 Slows conduction in all parts of heart,
 Also inhibits abnormal automaticity
 Propafenone
 Also slows conduction
 Weak β – blocker
 Also some Ca2+ channel blockade
23

24. CLASSIFICATION OF ANTIARRHYTHMICS
(BASED ON MECHANISMS OF ACTION)
 Class II – β–adrenergic blockers
 Based on two major actions
1) blockade of myocardial β–adrenergic receptors
2) Direct membrane-stabilizing effects related to Na+ channel blockade
 Includes
 Propranolol
 causes both myocardial β–adrenergic blockade and membrane-
stabilizing effects
 Slows SA node and ectopic pacemaking
 Can block arrhythmias induced by exercise or apprehension
 Other β–adrenergic blockers have similar therapeutic effect
 Metoprolol
 Nadolol
 Atenolol
 Acebutolol
 Pindolol
 Stalol
 Timolol
 Esmolol 24

25. CLASSIFICATION OF ANTIARRHYTHMICS
(BASED ON MECHANISMS OF ACTION)
 Class III – K+ channel blockers
 Developed because some patients negatively
sensitive to Na+ channel blockers (died!)
 Cause delay in repolarization and prolonged
refractory period
 Includes
 Amiodarone – Prolongs action potential by delaying K+ efflux
but many other effects characteristic of other classes
 Ibutilide – slows inward movement of na+ in addition to
delaying K + influx.
 Bretylium – First developed to treat hypertension but found to
also suppress ventricular fibrillation associated with
myocardial infarction
 Dofetilide - Prolongs action potential by delaying K+ efflux
with no other effects
25

26. CLASSIFICATION OF ANTIARRHYTHMICS
(BASED ON MECHANISMS OF ACTION)
 Class IV – Ca2+ channel blockers
 slow rate of AV-conduction in patients with atrial
fibrillation
 Includes
 Verapamil – blocks Na+ channels in addition to Ca2+;
also slows SA node in tachycardia
 Diltiazem
26

27. Bradyarrhythmias
Resting heart rate of <60/min
Classified as Atrial/AV Nodal/Ventricular
Management:
• Acute→ i/v atropine
• Permanent→ Pacemakers.
27

28. EXPERIMENTAL
EVALUATION OF
ANTIARRHYTHMIC DRUG
ACTION…………..
28

29. Evaluation of Antiarrhythmic Drug Action
 In-Vitro Models:
1) Isolated guinea pig papillary muscle.
2) Langendorff technique
3) Acetylcholine & potassium Induced arrhythmia
In-Vivo Methods:
Chemically induced arrhythmia
Electrically induced arrhythmia
Mechanically induced arrhythmia
Exercise Induced Arrhythmia
29

30. EVALUATION OF ANTIARRHYTHMIC DRUGS
30
IN VIVO METHODS

31. IN-VIVO METHODS :
I. Chemically induced arrhythmia
II. Electrically induced arrhythmia
III. Mechanically induced arrhythmia
IV. Exercise Induced arrhythmia.
31

32. I.CHEMICALLY INDUCED ARRHTHMIA
 A large number of chemical agents alone or
in combination are capable of inducing
arrhythmia.
32

33. ACONITINE ANTAGONISM IN RATS
PRINCIPLE: Aconite persistently activate sodium
channel.
METHOD:
Continous infusion
in saphenous vein
ACONITE 5mg/kg
dissolved in 0.1 N HN03
MONITOR LEAD II
ECG EVERY 30
SECONDS
Anaesthetize with
Urathene 1.25 g/kg
Test compound
3 mg/kg orally
or IV 5 minutes
before aconite
infusion
33

34. EVALUATION
 The anti-arrhythmic effect of the test compound is
measured by the amount of test drug, required to
precipitate aconitine/100 gm animal
 Ventricular extra systoles.
 Ventricular tachycardia.
 Ventricular fibrillation and death.
 E.g- procainamide 5 mg/kg IV & lidocaine 5 mg/kg IV leads
to increase in lethal dose by 65%.
34

35. STROPHANTHIN OR OUABAIN INDUCED ARRHYTHMIA
IN DOG:
Principle :Strophanthin K induces ventricular
tachycardia and multifocal ventricular arrhythmia
in dogs.
METHOD: Dogs of either sex are first anesthetized &
artificial respiration is given
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..
35

36. METHOD CTD..
Strophanthin K administered by
continuous i.v infusion at a rate of
3mg/kg/min
When the arrhythmias are stable for 10 min,
the test substance is administered i.v ( 1.0
and 5.0 mg/kg) or i.d ( 10 and 30 mg/kg)
ECG recordings are obtained
at 0.5, 1, 2, 5 and 10 min
following administration of
test drugs and further
duration increased if required
36

37. EVALUATION
Ajmalin , quinidine and lidocaine reestablish normal sinus
rhythm at 1 mg/kg ,3 mg/kg iv and 10 mg/kg id.
Test compound is said to
have an anti arrhythmic
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 min.
Test compound is said to have an
anti arrhythmic 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.D
I.V
37

38. ADRENALINE INDUCED ARRHTHMIA
 PRINCIPLE: Adrenaline at high dose may
precipitate arrhythmia.
38

39. METHOD:
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
pentobarbitone (30-40 mg/kg ) i.p
Femoral vein is cannulated and adrenaline
is infused at a rate of 2-2.5 mg/kg via
femoral cannula
Test drug is administered 3 mins. After adrenaline infusion and
Lead II ECG Recorded.
39

40. II.ELECTRICALLY INDUCED ARHYTHMIAS
40

41. VENTRICULAR FIBRILLATION ELECTRICAL
THRESHOLD
 Principle :Ventricular fibrillations can be induced
by various techniques of electrical stimulation
like single pulse stimulation , train of pulse
stimulation , continous 50 HZ stimulation.
 Ventricular fibrillation threshold :The minimal
current intensity of the pulse train required to
induce sustained ventricular fibrillation.
 Requirement:
 Animals – Adult dogs (8-12kg)
 Anesthetic – Sodium pentobarbital (35mg/kg)
41

42. PROCEDURE
↓
Sinus node is crushed and electrical
stimulation is provided with Ag-AgCl
stimulating electrode embedded in a Teflon
disc sutured to anterior surface of left venticle.
Chest cavity is opened
Heart suspended in pericardial cradle
Artificial respiration – Harvard
respiratory pump
B.P – monitored
Body temperature – maintained by
thermal blanket
Adult dogs are anaesthetized and
heart is exposed.
42

43. PROCEDURE CTD…
Test drug/ std/control Drugs are
administered through the femoral
vein.
A nodal const. current for 400ms is
applied through the driving
electrode
ECG of lead II is recorded
43

44. EVALUATION
 To determine VFT :- A 0.2 to 1.8 second train of
50 Hz pulses is delivered 100 ms after every
18thbasic driving stimulus. The current intensity
is increased from diastolic threshold. When
ventricular fibrillation occurs, the heart is
immedietely defibrilleted and allowed to recover
for 15-20 mins.
 VFT is determined before and after
administration of test drugs at given time
intervals.
 Compared using student - t Test.
44

45. III.MECHANICALLY INDUCED ARRHYTHMIA
 Coronary artery occlusion/reperfusion
arrhythmia
45

46. CORONARY ARTERY
OCCLUSION/REPERFUSION ARRHYTHMIA
Arrhythmias –induced directly by ischemia and
reperfusion
• Coronary artery ligation in anesthetized dog
results in:
 ↑ in HR
 ↑in heart contractility
 ↑ in BP
 Ventricular arrhythmias
46

47. REQUIREMENTS
Animals – Dogs (20-25Kg)
Anesthetic – Thiobutobarbital sodium (30mg/kg)
PROCEDURE:
Animals – selected
↓
Anesthetized
↓
Artificial respiration – positive pressure respirator
↓
Femoral artery – cannulated and connected to pressure
transducer
↓
Chest cavity –opened
LAD is exposed
↓
47

48. Silk suture is placed around LAD
↓
After 45 min(equilibration) – test/std/control-
administered through saphenous vein
↓
After 20 min- ligature of coronary artery is closed for
90 min
↓
Occlusion released – reperfusion period maintained
for 30 min.
• All the parameters – recorded
• At the end – surviving animals are sacrificed by an
overdose of Pentobarbital sodium.
48

49. EVALUATION
 Mortality
 Hemodynamics
 Arrhythmia
 Ventricular fibrillation
 % animals with VF.
49

50. IV .EXERCISE
INDUCED
ARRHYTHMIA
50

51. EXERCISE INDUCED VENTRICULAR
FIBRILLATION
 PURPOSE AND RATIONALE:
Tests combining coronary constriction with physical
exercise may resemble most closely the situation in
coronary patients
51

52. • REQUIREMENTS:
Animals – Mongrel dogs (15 -19 kg)
Anesthetic – Sodium pentobarbitone (10mg/kg) i.v.
SURGICAL PREPARATION:
Animals – selected
↓
Anesthetized
↓
Chest cavity – opened
↓
Heart suspended in pericardial cradle
↓
Around left circumflex artery – Doppler flow inducer-
to measure blood pressure
- Hydraulic occluder - to occlude coronary artery
are placed
52

53. ↓
Pair of insulated silver coated wires – sutured on left
and right ventricles- measure HR (By Gould
biotachometer)
↓
Occlusion of LAD(Two stage)
↓
Myocardial infarction
↓
After 24 hrs- test drug/std/control - administered
During this experiment:
• Transdermal fentanyl patch (↓post operative
discomfort)
• Bupivacaine HCl
• Antibiotic therapy(amoxicillin)
53

54. IN VITRO METHOD:
 1) Isolated guinea pig papillary muscle.
 2) Langendorff technique
 3) Acetylcholine & potassium Induced
arrhythmia
54

55. 1)ANTI-ARRHYTHMIC ACTIVITY IN THE ISOLATED
RIGHT VENTRICULAR GUINEA-PIG PAPILLARY
MUSCLE:
 Principle: In right ventricular guinea pig papillary muscle,
developed tension (DT), excitability (EX) and effective
refractory period (ERP) are measured.
55

56.  METHOD:
The heart is removed & placed into a pre-warmed, pre-
oxygenated PSS & right ventricle is opened
The tendinous end of the papillary muscle is ligated with a
silk thread and the chordae tendinae are freed from the
ventricle
The preparation is mounted in organ bath &
experimental conditions are maintained
The silk thread is used to connect the muscle to
forced transducer and muscles are field stimulated to
contract isometrically at a duration of 1 ms.
56

57. Pulses are delivered using constant voltage
stimulator and
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.
57

58. 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
58

59. LANGENDORFF TECHNIQUE
59

60.  PRINCIPLE: Heart is perfused in a retrograde direction from
aorta either at constant pressure or at constant flow with
oxygenated saline soln.
 Retrograde perfusion closes the aortic valve , just as in situ
heart during diastole .
 The perfusate is displaced through the coronary artery using a
canula inserted in the ascending aorta following of the
coronary sinus and opened right atrium and flows out via the
right ventricle and pulmonary artery.
60

61. PROCEDURE
Animal – Guinea pig (300-500g)
Animal – selected

Sacrificed (stunning)

heart – removed – placed in Ringer’s solution
(37⁰C)

Aorta – located and cut – cannulated with Ringer’s
solution (perfused at 40 mm Hg)

Ligature – placed around LAD
61

62. Ligature – placed around LAD

Test /std/control - administered.

Occluded for 10 minutes

reperfusion

ECG electrode – pulsatile stimulation, induction of
arrhythmia

Heart rate and contractile force measured
62

63.  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.
63

64. 64

65. 3)ACETYLCHOLINE & POTASSIUM INDUCED
ARRHYTHMIA
 New Zealand White rabbits 0.5 to 3 kg .
 The animals are sacrified and heart removed
immediately.Atria dissected from other tissue in
Ringer’s solution.
 Fibrillation is produced when atria are exposed
to Acetylcholine 3x10-4 g/ml or 0.1 gm potassium
chloride.
 After 5 mins exposure to Ach. Or KCL the atria
are stimulated with rectangular pulses of 0.75
ms duration of 10 V.
65

66.  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.
66

67. CURRENT STATUS OF ANTIARRHYTHMIC DRUGS
 Based on results of large Randomized controlled trials
 Cardioversion /defibrillator devices as compared to
Antiarrhythmic drugs have proved to be superior in
prolonging survival
67

68. CONCLUSION
 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.
68

69. REFERENCES
 Vogel WH, Schölkens BA. Drug Discovery and Evaluation [Internet]. Vogel HG, Vogel WH,
Schölkens BA, Sandow J, Müller G, Vogel WF, editors. Berlin, Heidelberg: Springer Berlin
Heidelberg; 2002. P.208-29.
 V.Gerald ,Drug Discovery and Evaluation Pharmacological Assay; Second Edition;2002; Springer;
pg no. 236- 242
 Tripathy .K D ,Essitial of Medical Pharmacology; sixth Edition;2008; Jaypee Brothers Medical
Publishers LTD; New Delhi; pg no 508- 520
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