Pharmacokinetic Interaction of Warfarin And

“PHARMACOKINETIC INTERACTION OF WARFARIN AND ALLIUM SATIVUM (GARLIC) IN RABBITS.” By

Mr. KHAN SHOEB ALI B.PHARM

M. Pharmacy Dissertation Protocol Submitted to the

Rajiv Gandhi University of Health Sciences, Bangalore– 560041, Karnataka.

In partial fulfillment of the requirements for the Degree of

MASTER OF PHARMACY in PHARMACOLOGY

Under the Guidance of

Dr. KAILASAM KOUMARAVELOU M.PHARM, PhD (Guide) PROFESSOR

Department of Pharmacology and Toxicology St. John’s Pharmacy College. St. John’s Educational Institutions Bangalore – 560040.

2009-2010

1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

Mr. KHAN SHOEB ALI POST GRADUATE STUDENT, 1. Name of the candidate & Address. DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, St. JOHN’S PHARMACY COLLEGE, BANGALORE- 560040.

St. John’s Pharmacy College #6, 9th cross, 2nd main, Vijayanagar 2. Name of the Institution. 2nd stage, (Hampi nagar), Bangalore- 560040. Tel: 91-80-23300958/ 23300668 Email: [email protected]

3. Course of the study & subject. Master of Pharmacy in Pharmacology

4. Date of admission. 15th June-2008

5. Title of the Topic :-

“Pharmacokinetic Interaction of Warfarin and Allium sativum (garlic) in Rabbits.”

2 6. Brief resume of intended work

6.1 Need of the work

The issue of herb-drug interactions looms large over the practice of herbal medicine. Till now there have been very few incidents reports on herb-drug interactions ,but a grave concern have raised from past few years. A possible interaction have been reported with patients taking warfarin and garlic.1 Garlic tends to decrease concentration of warfarin.2 However some data suggest that garlic does not interact with warfarin.3Therby giving a rise to dilemma wether garlic interacts with warfarin .This may result in adverse drug reaction , especially if herbs are used ,giving rise to possible herb-drug interaction These interactions go unnoticed until a serious life threatening event occurs. Data suggests that using garlic and warfarin together may elevate International Normalized Ratio (INR). Garlic alone has been attributed to a spontaneous bleeding episode which occurred in a 87- year old man.4 It was believed that only explanation to the hematoma was chronic ingestion of garlic.

The present study is useful review over possible herb –drug interaction in animals (rabbits), the data obtained will be useful in evaluating interaction between garlic and warfarin .

3 6.2 Review of Literature

Garlic is used as serum lipid lowering agent, antithrombotic agent ,inhibition of platelet aggregation, antioxidant.5 Although touted by the herbal industry to possess various properties including but not limited to antispasmodic, antiseptic, bacteriostatic, antiviral activities, as well as a promoter of leukocytosis, the most recent use of garlic has targeted its hypotensive and hypocholesterolemic activity.6 Garlic has long been used for flavoring and potential benefits of preventing and curing ailments in many cultures. The active chemical constituents of garlic are alliin, allicin sulfur containing amino acids like methiin, cyloalliin and organosulfur volatiles like DADS(diallyl disulfide), DAS (diallyl sulfide).7 Warfarin is an anticoagulant with a narrow therapeutic window with potentially fatal consequences if if either bleeding complications arise or if subtherapeutic levels occur, thus not preventing the patients from thromboembolic events. Warfarin interacts with herbs like St john’s Wort, Ginkgo biloba ,Ginseng Kava, Garlic , Liquorice, Dan Turmeric, Danshen, devil’s claw, Green tea, Papain and Vit E.8 Two case reports suggested that concomitant use of garlic and warfarin was followed by an increased in INR (international normalized ratio). 9 Interaction between donq quai and warfarin were reported which lead to conclusive point of elevation in prothrombin time and INR .10 W Warfarin interaction with ginseng lead to decrease in INR.11 P Potential interaction between warfarin and boldo ,fenugreek were reported .12 The hypolipedemic action of garlic homogenate(GH) was found to be augmented with captopril.13 Garlic interacts with propanolol and tends to increase the cardioprotective action of propanolol in isoprotorenol induced myocardial infarction in rats.14 As As warfarin is used as an anticoagulant and garlic as an blood thinning agent if given concomitantly may prolong bleeding and ultimately death may occur.9 As warfarin has a narrow therapeutic index the interaction with garlic will allow the prescriber to alter alter its dose in patients on warfarin therapy . Because users of herbal medicines tend to have chronic conditions for which they often take Prescribed drugs concomitantly ,interactions are likely.15

4 6.3 Objective of the study

The objective of the current study is to evaluate the possible interaction between garlic and warafrin in rabbits.

Materials and methods 7.0 Source of data: 7.1 All experiments are planned to generate data from laboratory studies. Blood samples will be collected at timely intervals and subjected to analysis by HPLC (High Performance Liquid Chromatography). Experiments will be performed as described with slight modifications.

 DRUGS AND CHEMICALS:- All chemicals used will be of analytical grade and will be purchased from standard companies.  PREPARATION OF GARLIC EXTRACT- Garlic bulbs will be purchased from the local market. The cloves will be peeled, sliced and ground into a paste and suspended in distilled water. Garlic homogenate will be prepared corresponding to 130mg/kg will be administered orally to animals. The garlic homogenate (GH) will be administered within 30 min of preparation.14  EXPERIMENTAL ANIMALS:- The healthy adult rabbits (2.5  0.5 Kg.) used for our study will be supplied by St. John’s Pharmacy college , animal house. The animals will be maintained under standard conditions in an animal house as per the guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).

7.2 Method Of Collection Of Data The collection of Data will be based on laboratory animal experimentation.

(a) Selection of Experimental Animals

5  Species New Zealand albino Rabbits  Age Young adult (6-9 months )  Weight 2.5  0.5 Kg.  Gender Either Sex  Number 18 nos. Animals will be maintained under controlled temperature (25°C  2°C) and humidity (50  5%), with 12 hr light/dark cycle with food and water provided ad libitum. Animals which do not comply with above criteria and also which are found to be diseased will be excluded from the study.

(b) Experimental Design There will be 3 groups of rabbits. Each group comprising of 6 animals. i) Group I will be treated with Normal Saline and will serve as Control. ii) Group II will be treated with Warfarin. (2 mg/kg, s.c.).16 iii) Group III will be treated with Warfarin and Garlic Homogenate (GH) (130 mg/kg, p.o.)

# NOTE: 250 mg/kg (GH) is the effective dose for rats as shown by Asdaq et al (2008).14 and converted to rabbit dose by surface area factor.17

(c) Sample Collection and Blood Analysis Blood sample (1ml) will be collected by butterfly needle kept intake with marginal ear vein puncture at 0hr, 0.5, 1, 1.5, 2, 2.5, 3.0 3.5, 4.0, 8.0, 12, 16, 20, 24hr in heparinized tubes. After collection of blood sample equal volume of normal saline will be administered to maintain volume of blood in animals to prevent hypovolemia. The collected blood samples will be centrifuged to obtain plasma and analyzed by HPLC

(d) Parameters to be evaluated

6 Prothrombin Time It is the time required by the blood to form clot, prothrombin time will be measured by coagulation analyzer as described by Scott et al (1984).18

The various Pharmacokinetic parameters like Cmax, Tmax, AUC, Ke, t1/2, CL, Vd and F needed to be evaluated during the experiment will be estimated as shown by Koumaravelou et al (2002).19 and Chan et al (2008).20 From the data obtained by HPLC, the drug level time curve will be plotted from where the area under time curve (AUC) will be calculated using trapezoid rule. The maximum concentration (Cmax) and maximum time (Tmax) of the Warfarin will be obtained directly from generated data.

1. Warfarin Half life (t1/2) It is the time period required for the concentration of drug to decrease by one half. The

elimination constant (Ke) and half life (t1/2) of the Warfarin will be determined from the semi logarithmic plot of data using following equation.

t1/2 = 0.693 / Ke

2. Warfarin Clearance (CL) It is hypothetical volume of body fluids containing drug from which the drug is removed or cleared completely in a specific period of time. The clearance (CL) of the Warfarin in the animals will be calculated from the equation.

CL= (0.693 × Vd) / t1/2

3. Warfarin Volume of Distribution (Vd) It is hypothetical volume of body fluids into which a drug is dissolved or distributed.

Apparent volume of distribution (Vd) of the drug in the animals will be calculated from the following equation.

Vd = Db/Cp Where, Db is the administered does of the drug. Cp is the plasma drug concentration of drug.

4. Warfarin Bioavailability (F) It is the rate and extent of absorption of unchanged drug from its dosage form. It will be

7 calculated from the equation.

F = [AUC]test x Dstd / [AUC]std x Dtest Where, test stands for the combined value of warfarin and garlic. Std stands for the standard value of warfarin. D stands for dose administered.

(e) Statistical analysis The data obtained will be analyzed statistically using one way ANOVA followed by suitable post test. 7.3

Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly. 7.4 The study requires healthy adult Rabbits for the concerned work.

Has ethical clearance been obtained from your institution in case of 7.3? The study is cleared from Ethical Committee of the institution. (Certificate enclosed).

List of references:

8 1. Subhuti D. Checking for possible herb-drug interactions. Available from:URL:http://www.itmonline.org/arts/herbdrug2.htm (Access on 24th Nov 2008). 2. Izzo AA, Ernst E. Interaction between herbal medicines and prescribed drugs. Drugs 2001;61 (15): 2163-75. 3. Mohammed A, Jiang X, Williams KM, Day KO, Roufagalis BD, Liauw WS, Xu H, Mclachlan AJ. Pharmacodynamic interaction of warfarin with cranberry but not with garlic in healthy subjects. Br J Pharmcol 2008;154 (8): 1691-70. 4. Rose KD, Crossant PD, Parliament CF, Levin MP. Spontaneous spinal epidural hematoma with associated platelet dysfunction from excessive garlic ingestion. A case report. Neurosurgery 1990;26 (5): 880-82. 5. Williams MJ, Sutherland WH, McCormick MP, Yeoman DJ de Jong SA.Phytotherapy research 2005;19 (4) :314-19. 6. Miller LG. Herbal medicines: Selected clinical consideration focusing on known or potential drug herb. Arch Intern med 1998; 158 (20): 2200-11 7. Harunobu A. Supplement: Significance of garlic and its constituents in cancer and cardiovascular disease. J Nutr 2006;136: 716-25. 8. Heck AM, DeWitt BA, Lukes AL. Potential interactions between alternative therapies and warfarin. Am J Health System Pharm 2000;57 (13): 1221-27. 9. Sunter WH . Warfarin and garlic. Pharm J 1991; 246: 772. 10. Page RL, Lawrence JD. Potentiation of warfarin by dong quai. Pharmacotherapy 1999; 19 (7): 870-76. 11. Janetzky K , Morreale AP. Probable interactions between warfarin and ginseng . . Am J Health System Pharm 1997; 54: 692-93 12. Lambert JP , Cormier J. Potential interaction between warfarin and boldo fenugreek. Pharmacotherapy2001; 21 (4): 509-12. 13. Asdaq SMB, Inamdar MN. Pharmacodynamic interaction of garlic with captopril in ischemia perfusion induced myocardial damage in rats. Pharmacologyonline 2008; 2: 875-88 14. Asdaq S M B, Inamdar M N, Asad M & Nanjundan P K. Interaction of propranolol with garlic in isoproterenol induced myocardial infarction in rat. J Pharmacol Toxicol 2008; 3 (6): 414-24 15. Eisenberg DM, Roger DB, Ettner SL, Scott A, Sonja W, Rompay MV, Ronald CK. Trends in alternative medicine use in United States, 1990-1997. JAMA 1998; 280: 1569-75. 16. Lo ACT, Chan K, Yeung JHK, Woo KS . Danggui (Angelica Sinensis) affects pharmacodynamics but not pharmacokinetics of warfarin in rabbits. Eur J Drug Metab Pharmacokinet 1995; 20 (1):55-60

9 17. Ghosh MN. Fundamentals of Experimental Pharmacology. 3rd ed. Hilton & company, Kolkata; 2005. 18. Scott AK, Park BK, Breckendrige AM. Interaction between warfarin and propanolol. Br J Clin Pharmac 1984; 17:559-64. 19. Koumaravelou K, Adithan C, Shashidran CH, Asad M, Abraham BK. Effect of honey on carbamazipine kinetics in rabbits. Indian J Exp Biol 2002; 40(5):560-63. 20. Chan K, Woo KS. Determination of warfarin in himan plasma by high performance liquid chromatography. Methods Find Clin Pharmacol 1998 ;10 (11): 699-03.

9. Signature of the candidate

10. Remarks of the guide

10 11. Name & Designation of

11.1 Guide Dr. KAILASAM KOUMARAVELOU M.PHARM, PhD PROFESSOR, DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, St. JOHN’S PHARMACY COLLEGE, VIJAYANAGAR-2ND STAGE, BANGALORE-560040. 11.2 Signature of Guide

11.3 Co – Guide

11.4 Signature of Co Guide

11.5 Head of the Department Dr. E P KUMAR M.PHARM, PhD PROFESSOR & PRINCIPAL, HEAD, DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, St. JOHN’S PHARMACY COLLEGE, BANGALORE- 560040. 11.6 Signature of HOD

12. 12.1 Remarks of the Chairman & Principal

12.2 Signature