This document describes various in vitro and in vivo models used to screen for anti-anginal and anti-allergic drugs. It discusses models such as isolated heart perfusion using the Langendorff technique to study coronary blood flow and drugs for angina. For allergy screening, it covers techniques like inhibiting histamine release from mast cells and testing anti-anaphylactic activity using Schultz-Dale reaction in guinea pigs. In vivo models described are coronary artery ligation in anesthetized dogs to study infarct size and isoproterenol-induced myocardial necrosis in rats. The document provides detailed procedures for these screening methods.

1. By
Gurubarath M
M.Pharm 1st year
Dept of Pharmacology
PSG College of Pharmacy
Screening of Anti-Anginal and
Anti-Allergic Drugs
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2. Angina
Angina Pectoris is a clinical symptom of IHD resulting from
transient myocardial Ischemia
It is Characterized by Paroxysmal and
usually recurrent attacks of substernal or
precordial chest discomfort
Caused by transient myocardial Ischemia that is insufficient to
induce myocyte necrosis
Often, the pain radiates to the left arm, neck, jaw or right arm
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3. Stable
Angina
Unstable
Angina
Prinzmeta
l Variant
Angina
Types of Angina
Characterized by regular episodes of
pain triggered by Physical activity,
emotional excitement or
psychological stress
Sudden pain - doesn’t go away on its
own or respond to rest or medication
Caused by Blood clot – blocks blood
vessel
Caused by Spasm in Coronary artery –
temporary
Specific form of Unstable Angina
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4. ↓ Coronary Blood Flow
ANGINA
PATHOPHYSIOL
OGY
Supply Demand
Vasospasm
Fixed Stenosis
Thrombosis
↑ O2 consumption
↑ Heart rate
↑ Contractility
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5. TREATMENT
2. Increasing blood supply – by dilating coronaries
1. Reduce Oxygen demand – by decreasing work
Immediate relief of Angina
Chronic Stable Angina
1. Rest
2. Sublingual Nitroglycerin
1. Long Acting nitrates
2. Beta blockers
3. CCBs
4. K+ channel openers
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6. In Vitro Models
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7. With perfusion buffer flowing retrogradely down the aorta,
opposite to normal physiologic flow, the aorta valve is closed
pressure just as during Diastole
normal physiologic flow,
Langendorff technique of
Isolated Heart perfusion
Principle The heart is perfused by cannulating the aorta.
The Perfusate then passes through vascular bed – drawn off via
the coronary veins to the coronary sinus in the right atria
With free drainage of right atrium, the preparation is maintained
without filling the ventricular chambers
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8. Procedure Sprague-Dawley Rats
Anaesthetize – corneal reflexes no longer
present
Dissection
Transversely cut the abdomen and cut midaxillary line to open
ribcage and lift up the front
Could not touch the heart tissue
Had to be done quickly
Oxygenated Mammalian Ringer’s solution
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9. Procedure
Identify the Aorta – fit the cannula – clip and tip it off because
it builds pressure in the aorta – left ventricle pushes and
coronary arteries can be perfused
Perfusate – similar to blood has same ionic conc., pH and
osmolarity – provides Oxygen and Nutrients
Ringer solution via Aorta – coronary arteries to coronary vein in
right side of heart – spilling what left after the pulmonary
artery
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10. Procedure
Force Transducer setup – small incision is made on the left atria -
insert the balloon into the left ventricle through the left atria
through the AV valve
Keep left ventricle under pressure
Collecting Data – electrical leads on the bath measuring the
electrical activity of the Heart
Results
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11. Procedure
Future labs - Ischemia
Anterior Intraventricular coronary artery – suture to block
Blue Dye into cannula
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12. Coronary Artery Ligation in
Isolated Rat Heart
Principle Langendorff Technique – regional Ischemia by clamping the left coronary
artery close to its origin
Prevention of these symptoms – indicator of coronary drugs
After removal – changes in reperfusion period can be measured
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13. Procedure Wistar rats (280-300g) – sacrificed by decapitation
Hearts are removed dissected free from Epicardium and surrounding
Connective tissue
Cannula is introduced into Aorta, in left ventricle Balloon closely fitting the
ventricular cavity is placed – artificial circulation
During each Heart beat, flowmeter probe measures the fluid volume pressed
from the balloon corresponding to the stroke volume
Preload and afterload are adjusted and perfusate flow is recorded
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14. Isolated working hearts are perfused with kreb’s buffer for 20min at 60mmHg
Left coronary artery clamped for 15min – Ischemia produced
Clip opened – Changes during reperfusion period are monitored for 30min
Hemodynamic Parameters are measured
Experimental
Procedure
Test drug is given – either before occlusion or 5min before reperfusion
Incidence and duration of Ventricular fibrillation after treatment with test
drugs are compared with controls
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15. In Vivo Models
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16. Occlusion of Coronary Artery in
Anesthetized Dogs
Principle
Compounds that reduce infarct size are studied using this model
Infarct size is studied after paroxysmal occlusion of left descending
coronary artery in open chest dogs
Nitro-blue tetrazolium chloride stain – visualize infarct size in
coronary arteriograms made after injection of BaSO4 gelatin mass
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17. Dogs of either sex are used in this model
Procedure
Femoral vein – connected to pressure transducer – measure
peripheral systolic and diastolic pressure
Trachea – artificial respiration
Anaesthetized with Phenobarbitone sodium (35mg/kg) followed
by continuous infusion (4mg/kg)
Peripheral vein – Administration of test compound
Millar microtip catheter – via coronary artery measures left ventricular end diastolic, left
ventricular pressure and heart rate
Cannulation
Heart is exposed through a left thoracotomy between 4th and 5th intercoastal space
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18. Experimental
Procedure
Heart is exposed through a left thoracotomy between 4th and 5th intercoastal
space
Pericardium is opened – left coronary descending artery is exposed
– ligated for 6hrs
Test substance is administered and Haemodynamic parameters are
monitored – animals sacrificed with overdose
Left ventricle is cut into transverse sections
Evaluation - Mortality, Haemodynamic parameters and infarct size
are determined comparing the control
Slices are incubated with stain to visualize infarct tissue
From each slice angiograms are made – assess area at risk of
infarction
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19. Isoproterenol-induced Myocardial Necrosis
Principle
Isoproterenol at high doses produce cardiac necrosis
Rona et al. have studied the infarct like lesions in rat myocardium
Sympatholytic or Calcium antagonists can totally or partially prevent these
lesions
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20. Wistar rats – pretreated with test drug or standard drug orally or
subcutaneously for at least a weekProcedure
Isoproterenol (85mg/kg) S.C. – for two consecutive days
Mortality as well as symptoms – compared with group injected with
isoproterenol only
After 48hr animals are sacrificed – before sacrificing, hemodynamic
parameters are recorded – cannulating carotid artery and connecting to
pressure transducer
Degree of Histopathological changes are Graded as follows
Grade 0 : No change
Grade 1 : Focal areas of Necrosis
Grade 2 : Focal areas of Necrosis and muscle fiber fragmentation
Grade 3 : Confluent areas of necrosis, edema and inflammation and Muscle fiber fragmentation
Grade 4 : Massive areas of Necrosis, edema and inflammation and mural thrombi
Changes of Parameters of drug treated animals are compared with
Isoproterenol controls
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21. Allergy
May range from mild irritation to life-threatening anaphylactic
shock
Hypersensitivity refers to undesirable immune reactions produced
by the normal immune system
Distinguished into four types
Type I
Type II
Type III
Type IV
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22. Types of Hypersensitivity
TYPE I
TYPE II
TYPE III
Immediate Hypersensitivity Reactions
Antibody-Mediated Hypersensitivity Reactions
Immune Complex-Mediated Reactions
TYPE IVDelayed Hypersensitivity Reactions
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23. In Vitro Models
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24. Inhibition of Histamine release from Rat
Peritoneal Mast cells
Inhibition of Calcium Ionophore (A23187) – Stimulated
Histamine release
Ability of the test drug – Inhibit the Calcium ionophore induced
Histamine release was investigated and compared with selected
Anti-Allergic drugs
Principle
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25. Mixed Peritoneal cells (3-5% Mast cells) – harvested in Tris-Gel Buffer
from Peritoneal cavity of S-D rats (300-700g)
Cell suspensions centrifuged and resuspended in Tris-
Gel Buffer
Peritoneal cell suspensions in T-G Buffer (37oC, 10min) with gentle shaking
Procedure
Isolation of Peritoneal Mast cells
Assay of Histamine Release
Incubated with Ca2+ ionophore (37oC) shaking at 60-80 oscillations/min and
Centrifuged at 2000rpm (5min, 4oC)
Histamine extracted and measured by fluorometric method
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26. Procedure
Effect of test solution or suspension
A fine suspension of test in T-G buffer was prepared
Reaction mixtures containing cell suspension and drug solution – incubated
(37oC, 10min)
A23187 induced Histamine release – assayed in presence and absence of drugs – Total cell
Histamine content
On A23187 – stimulated Histamine release was studied
Inhibition of Histamine release by test and selected antiallergic
drugs
% Inhibition = ( (A-P) / A ) x 100
where, A = % Histamine release in the absence of drug
P = % Histamine release in the presence of drug
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27. Anti-Anaphylactic activity
(Schultz-Dale reaction)
Guinea pigs are sensitized against egg albumin
Principle
Challenge after 3 weeks causes in isolated organ release of
mediators
e.g. histamine, which induce contraction in isolated ileum
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28. Procedure Guinea pigs (300-350g) – sensitized with alum precipitated egg albumin
Each animal receives (0.125ml) injection in each foot pad and 0.5ml
subcutaneously
Results are expressed in presence and absence of blocking activity
If Anaphylactic activity is observed, ED50 values are calculated
After 4 weeks animals are killed – ileum is dissected out
Mounted in organ bath – Tyrode solution at 37oC
Contractility is tested (BaCL2) – standard (Tribenosid) in one bath and to
others, test compounds are added
Evaluation
Ovalbumin (2x10-6g/ml) is added and recorded
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29. In Vivo Models
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30. Acute Systemic Anaphylaxis in Rats
Rats are immunized with Ovalbumin and Bordetella pertussis
suspension as adjuant
Principle
After 11 days – challenged by inj. Of Ovalbumin
Shock symptoms are inhibited by Corticosteroids and i.v.
Disodium cromoglycate
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31. Procedure Sprague-Dawley rats (120g) – immunized by i.m. Inj. of Highly purified
Ovalbumin – 1ml of Bordetella pertussis suspension i.p.
IgE Abs are induced
The shock symptoms are scored and mortality counted
Results after treatment are compared with untreated controls
11 days later – animals are challenged by i.v. Inj. Of purified Ovalbumin
(25mg/kg)
Results in release of various mediators of AnaphylaxisResults in release of various mediators of Anaphylaxis
Corticosteroids are given 18hr prior to challenge, or disodium
cromoglycate i.v. before injection of Ovalbumin
Evaluation
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32. Passive cutaneous anaphylaxis
Passive immunization of rats in skin with anti-ovalbumin serum
Principle
After 2 days – challenged with ovalbumin at same skin area
Results in Vasodilatation, ↑ permeability of vessel walls and
plasma leakage
Evan’s blue dye – attached to albumin fraction in plasma – blue
spot
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33. Procedure Male rats (200-250g) are adrenalectomized – allowed to recover for 3
days
Sensitized with alum precipitated egg albumin (0.125ml) in each foot pad
and 0.5ml s.c.
After 8 days, animals are bled and antiserum is collected
For test, antiserum is diluted to give a wheal of 15-20mm diameter
Antiserum (100mc.l Aliquots) injected i.d. into shaved dorsal skin of male
rats
Preparation of antiserum
After 24hr, challenged with i.v. 2.5% Evan’s blue dye containing egg
albumin (25mg/ml)
Test compound is administered simultaneously with the dye and antigen
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34. Procedure
Amount of Dye leaked at site of Passive cutaneous Anaphylactic reaction
is extracted and calorimetrically determined (620nm)
Animals are sacrificed after 30min after challenge
Amount of Dye extracted from anaphylactic reaction is taken 100%
Evaluation
Percent inhibition in test treated rats in calculated using ED50.
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35. Reference
Drug discovery and evaluation, Pharmacological Assays; H.Gerhard Vogel,
2nd edition; Page no.797 and 798
Inhibition of calcium ionophore-stimulated histamine release from RPMC;
Naresh Chand et. al 1983
Retrograde heart perfusion: The Langendorff technique of isolated heart
perfusion; M.Bell et. al 2011
Drug screening methods; SK Gupta; 3rd edition; Page no.
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