Evalution of Anti-Anginal Drugs, Animal models for Angina, Both IN VIVO and INVITRO model, Langendorff heart prepration

ANGINA PECTORIS is a characteristic sudden, severe, pressing chest pain radiating to the neck, jaw, back, and arms.
Angina is the initial stage of myocardial infarction (Heart Attack).
This happens due to spasms of the coronary artery, and less blood and oxygen to the heart muscles.
In Vitro Model
Langendorff heart preparation
Isolated Rabbit aorta preparation
Calcium antagonism in the rat
Coronary artery ligation in isolated rat heart
Isolated heart-lung preparation
In Vivo Model
Occlusion of Coronary Artery in anesthetized Dog
Isoproterenol-induced Myocardial Necrosis in Rats
Microspheres-induced acute ischemia
Model of Coronary flow measurement
Myocardial-Ischemic Preconditioning Model

Langendorff method is a powerful technique in cardiac research and has led to discover advance medicines. The primary goal of the method is to provide an isolated heart with oxygen and metabolites via a cannula inserted into the aorta. The Langendorff heart is a in vitro technique used primarily in pharmacological research that allows the evaluation of multiple cardiac hemodynamic parameters, but not limited to, contractility and heart rate.

This model consists of applying increased tolerance of the myocardial Ischemic condition to the animal. In this Model, a brief episode of Ischemic condition is induced in the animal and then treated with the specific drug
Animal – Rabbit Weight – 3-4 kg Gender – Male/Female
Anesthetized agent – Ketamine Xylazine (Composition of Ketamine hydrochloride & Xylazine hydrochloride);
Dose – 35 mg/kg Ketamine & 5mg/kg Xylazine; Route - IM

Animal – Wister Rat; Weight – 150-200 gram; Gender - Generally Male
Drug used - Isoproterenol (ISO); Route – Intra Peritoneal (IP); Concentration – 5.25 & 8.5 mg/kg; for 2 consecutive days;
Cardiac necrosis can be produced by injection of natural and synthetic sympathomimetics in high doses.
Administration of the drug caused a significant increase in heart/body weight ratio, and myocardial necrosis as evidenced by the significant increase in serum markers i.e. Serum Glutamic Oxaloacetic Transaminase (SGOT) and Creatine Kinase (CK)

1. Presented by: RITAM MUKHERJEE Dept. of Pharmacology ISF College of Pharmacy, MOGA [email protected] TOPIC : Evaluation of Anti-Anginal drugs

2. Evaluation of Anti-Anginal drugs 2 Serial Number Topic Page Number 1. Introduction 3 2. Pathophysiology of Angina 4 3. Treatment of Angina & Classification of drugs 5-6 4. Screening model of antianginal drugs 7 5. INVITRO Model Langendorff heart preparation 8-10 6. INVIVO Model Myocardial-Ischemic Preconditioning Model Induced Myocardial Necrosis in Rats Occlusion of Coronary Artery in anesthetized Dogs 11-17

3. ANGINA PECTORIS is a characteristic sudden, severe, pressing chest pain radiating to the neck, jaw, back, and arms. Angina is the initial stage of myocardial infarction (Heart Attack). This happens due to spasms of the coronary artery, and less blood and oxygen to the heart muscles. Two principal forms of angina are recognized – Classical Angina - Also called Stable Angina, it is a very common type of angina this angina is predictable and provoked by exercise, emotion, and eating. ISF College of Pharmacy 3 Prinzmetal’s Angina – Also called Varient and Vasospastic Angina, attack occurs at rest or during sleep and is unpredictable. Fig. Angina happen due fat deposition in Blood Vessel Introduction

4. ISF College of Pharmacy 4 O₂ supply decrease due to Coronary Atherosclerosis Coronary Vasospasm Coronary Thrombosis O₂ Demanded increase due to Increase Heartrate Ventricular Contractility Ventricular wall tension Atherosclerosis Vasospasm Thrombosis Pathophysiology

5. Increase O2 Supply Restore Coronary blood flow Relieves the vasospasm by drugs- Nitrates/ CCBs (Diltiazem, Amlodipin ) Break the Thrombi using Thrombolytic agents- Streptokinase, Urokinase Prevent Thrombus formation by using – Antiplatelet Drugs (Dipyridamole) Decrease O2 Demand Decreasing workload on heart Decreasing Preload- Nitrates ( Nitroglycerine ) Decreasing Afterload- CCBs, K+ Channel opener(Nicorandil) Decreasing heart rate and Contractibility- Beta Blockers(Metoprolol) ISF College of Pharmacy 5 Treatment Strategy

6. ISF College of Pharmacy 6 Classes Drugs Nitrates Nitroglycerine , Isosorbide dinitrate, Isosorbide mononitrate Beta-Blockers Propranolol, Metoprolol, Atenolol Ca²⁺ Channel Blockers Verapamil, Diltiazem, Nifedipine, Amlodipine K⁺ Channel Openers Nicorandil Others Ivabradine, Dipyridamole Classification of Anti-Anginal Drugs

7. In Vitro Model Langendorff heart preparation Isolated Rabbit aorta preparation Calcium antagonism in the rat Coronary artery ligation in isolated rat heart Isolated heart-lung preparation ISF College of Pharmacy 7 In Vivo Model Occlusion of Coronary Artery in anesthetized Dog Isoproterenol-induced Myocardial Necrosis in Rats Microspheres-induced acute ischemia Model of Coronary flow measurement Myocardial-Ischemic Preconditioning Model Screening Models for anti Anginal Drug

8. Langendorff method is a powerful technique in cardiac research and has led to discover advance medicines. The primary goal of the method is to provide an isolated heart with oxygen and metabolites via a cannula inserted into the aorta. The Langendorff heart is a in vitro technique used primarily in pharmacological research that allows the evaluation of multiple cardiac hemodynamic parameters, but not limited to, contractility and heart rate.  . ISF College of Pharmacy 8 Established in 1897 by Oscar Langendroff IN VITRO MODEL Langendorff Heart preparation REFERENCE - Bell RM, Mocanu MM, Yellon DM. Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion. J Mol Cell Cardiol . 2011 Jun;50(6):940-50. doi: 10.1016/j.yjmcc.2011.02.018. Epub 2011 Mar 6. PMID: 21385587. Fig.: Article demonstrating the proof of the given Model Principle

9. ISF College of Pharmacy 9 A 300-500 gram of Guinea Pig taken. Heart is isolated by transabdominal incision. Heart is cardled between fingers and lifted before incising the aorta, vena cava, and pulmonary veins. After excision heart is dipped in a cold perfusion solution (4˚C) Aorta is located and cannula is inserted into it and the heart is perfused with oxygenated Kreb’s solution. (comp.- NaCl, NaHCO₃, KCl, KH₂PO₄, D-Glucose, pH-7.2-7.4) Heart is transferred to a double wall Plexiglas perfusion apparatus maintained at 37˚C. Oxygenated Kreb’s solution is perfused at a constant pressure of 40 mmHg. Small steel hook with a string is attached to the apex of the heart. Fig.: Double wall Plexiglas perfusion apparatus Procedure

10. Contractile force is measured isometrically by a force transducer and recorded on a polygraph. Heart rate is measured through a chronometer attached with the polygraph. The antianginal effect of the test drug is indicated by an increase in coronary blood flow which is then compared with control. ISF College of Pharmacy 10 Testing coronary vasodilator drugs, Electrophysiology evolution Recording positive inotropic effect, calcium antagonism, effect on potassium outflow induced by glycosides, and determination of hypoxic damage Metabolic studies- Arrhythmogenic antiarrhythmic and anti-fibrillatory effects. To study EDRF release from coronary vascular bed. Application

11. This model consists of applying increased tolerance of the myocardial Ischemic condition to the animal. In this Model, a brief episode of Ischemic condition is induced in the animal and then treated with the specific drug Animal – Rabbit Weight – 3-4 kg Gender – Male/Female Anesthetized agent – Ketamine Xylazine (Composition of Ketamine hydrochloride & Xylazine hydrochloride); Dose – 35 mg/kg Ketamine & 5mg/kg Xylazine; Route - IM ISF College of Pharmacy 11 Myocardial-Ischemic Preconditioning Model First described in animals in 1986 IN VIVO MODEL Fig.: Article demonstrating the proof of the given model REFERENCE - M. S. Sumeray , D. M. Yellon , Myocardial preconditioning: What have we learned?,  European Heart Journal , Volume 18, Issue suppl_A , January 1997, Pages 8–14, d oi - https://doi.org/10.1093/eurheartj/18.suppl_A.8 Principle

12. Rabbits (3-4 kg) are anesthetized with ketamine xylazine. Trachea canulated and the animal is set up for artificial respiration. Right femoral artery and vein are catheterized for measuring hemodynamic parameters. A 4-0 suture is looped loosely around the marginal branch of the left coronary artery to facilitate coronary occlusion. Ischemic preconditioning is induced by tightening the loop around the coronary artery for 5 min and then loosening to re-perfuse the myocardium for 10 min prior to a subsequent 30 min occlusion. After 30 min. ischemia, ligation is released for 120 min of re-perfusion. ISF College of Pharmacy 12 Fig. Ketamine Xylazine Procedure

13. ISF College of Pharmacy 13 Myocardial ischemic preconditioning provides several benefits, including reducing the size of an infarct and providing protection against acute myocardial ischemia. Ischemic preconditioning is currently considered the best form of myocardial protection against infarction by attenuating ischemic damage. It has been shown to significantly attenuate myocardial death caused by ischemic injury. Prior to 30 min of occlusion rabbits are selected to receive ischemic preconditioning, no preconditioning or preconditioning along with the administration of test compound. Animals are sacrificed after reperfusion. Compared with the controlled groups. Data is analyzed by ANOVA using statistical software. Application Fig. IM Injection on Rabbit

14. Animal – Wister Rat; Weight – 150-200 gram; Gender - Generally Male Drug used - Isoproterenol (ISO); Route – Intra Peritoneal (IP); Concentration – 5.25 & 8.5 mg/kg; for 2 consecutive days ; Cardiac necrosis can be produced by injection of natural and synthetic sympathomimetics in high doses. A dministration of the drug caused a significant increase in heart/body weight ratio, and myocardial necrosis  as evidenced by the significant increase in serum markers i.e. Serum Glutamic Oxaloacetic Transaminase (SGOT) and Creatine Kinase (CK) ISF College of Pharmacy 14 Induced Myocardial Necrosis in Rats Myocardial Necrosis in the rat by isoproterenol have been described by Rona et al. (1959) Fig. Article demonstrating the proof of the given model REFERENCE - Sumitra M, Manikandan P, Kumar DA, Arutselvan N, Balakrishna K, Manohar BM, Puvanakrishnan R. Experimental myocardial necrosis in rats: role of arjunolic acid on platelet aggregation, coagulation and antioxidant status. Mol Cell Biochem . 2001 Aug;224(1-2):135-42. doi: 10.1023/a:1011927812753 Principle

15. Groups of 10 male Wistar rats weighing 150-200 g are pretreated with the test drug or the standard either S.C. or orally. Then, they receive 5.25 and 8.5 mg/kg Isoproterenol on two consecutive days. Symptoms and mortality in each group are recorded and compared with those of rats given isoproterenol alone. After 48 hours of the first administration of isoproterenol the rats are sacrificed. The hearts are removed and weighed, and frontal sections are embedded for histological examination. If it is found that heart weight is decreased then it signifies that necrosis occurs in the heart muscle. ISF College of Pharmacy 15 The model has several advantages over other animal models, including lower mortality and higher reproducibility . Fig.: Isoproterenol HCL Injection Procedure Application

16. ISF College of Pharmacy 16 Occlusion of Coronary Artery in anesthetized Dogs Dogs of either gender weighing approximately 30 kg are used. The animals are anesthetized by intravenous injection of Pentobarbital sodium (bolus of 35 mg/kg followed by continuous infusion of 4 mg/kg/h). The animals are placed in the right lateral position. Animal Used – Dog; Weight – 25-30 kg; Gender – Male Preferable Anaesthetic agent – Pentobarbital Sodium; Route – IV and the continuous Infusion of drug; Concentration of drug – Bolus 35 mg/kg then followed by continuous infusion of 4 mg/kg/h. Principle Procedure Fig. Article demonstrating the proof of the given model REFERENCE – Krishnan S., Levy M.N.; Effects of coronary artery occlusion and reperfusion on the idioventricular rate in anesthetized dogs; Journal of the American College of Cardiology; 1994 May, doi- 10.1016/0735-1097(94)90396-4

17. Respiration is maintained through a tracheal tube. The animal is on artificial respiration. Arterial blood gases are checked, and the ventilation rate and/or oxygen flow rate are adjusted to achieve physiological blood gas values (pO2: 100-140 mmHg, pCO2: 32-40 mmHg, and pH 7.47). A peripheral vein (Saphenous vein) is cannulated for the administration of the test compound (Labetalol). The ECG is recorded continuously from the beginning of the experiment. ISF College of Pharmacy 17 Mortality and the different hemodynamic parameters are determined. Changes in parameters in drug-treated animals are compared to vehicle controls. The different characteristics are evaluated separately. Statistical analyses consist of regression and correlation analyses and of the Student's t-test. Application

18. Tripathi K.D., Essentials of Medical Pharmacology, Jaypee Brothers Medical Publishers; 8 th edition 2019, page no. 584-588 Schlaifer J.D., Kerensky R.A.; Ischemic Preconditioning: ClinicalRelevance and Investigative Studies; Clinical Cardiology 2009, doi - 10.1002/clc.4960200705 Liao J., Huang W., Liu G.; Animal models of coronary heart disease; The Journal of Biomedical Research, 2017, doi-10.7555/JBR.30.20150051 Zaragoza C., Guerrero C.G., Ventura J.S.M., Colio L.B., Lavin B., Mallavia B., Tarin C., Mas S., Ortiz A., Egido J.; Animal Models of Cardiovascular Diseases; Journal of Biomedicine and BiotechnologyVolume 2011; doi:10.1155/2011/497841 Vogels Gerhard, Drug discovery and evaluation Pharmacological assays. Springer publications, 3th edition, 2008, 253-257 . ISF College of Pharmacy 18