HPLC ANALYSIS AND PHARMACOKINETICS OF CYCLIZINE ~~ : - by Roderick Bryan Walker A Thesis Submitted to Rhodes tJ niversity in Fulfilment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY January 1995 School of Pharmaceutical Sciences Rhodes University Grahamstown II STUDY OBJECTIVES Introduction Cyc1izine, is a piperazine derivative that exhibits antihistaminic activity. It is particularly useful for the prevention and treatment of nausea and vomiting associated with motion sickness. Cyclizine is available as an intramuscular injection, tablet, syrup and suppository for both adult and paediatric use. In spite of widespread use of cyclizine as an anti-emetic, there is a paucity of information regarding the pharmacokinetics of the compound, which can probably be accounted for by the lack of a suitable, sensitive analytical method that is necessary to measure the low concentrations of cyclizine likely to be found after administration. Ohjectives The objectives of this study were therefore:- 1. To develop and validate a suitable High-Performance Liquid Chromatographic (HPLC) method with the necessary sensitivity to accurately and precisely quantitate cyc1izine and its major metabolite, norcyclizine in serum and urine. 2. To characterize the pharmacokinetics of cyclizine by studying the absorption and disposition of the drug in healthy human volunteers. 3. To evaluate, using both compartmental and non-compartmental pharmacokinetic analyses the data obtained following administration of cyclizine as single doses (intravenous and oral) and following fixed multiple dosing (oral) in man. iii ABSTRACT The investigations detailed in this dissertation < h,ave been conducted to address the paucity of pharrnacokinetic information, in published literature, pertaining to cyclizine. The areas of investigation have included the selective quantitation of both cyclizine and its demethylated metabolite, norcyclizine in serum and urine, assessment of stability of both_ cOI?pounds in stored biological samples, dosage form analysis, dissolution rate testing of tablets, and bioavailability and pharmacokinetics following administration of an intravenous solution, and tablets to humans. High-performance liquid chromatography (HPLC) was used as the main analytical technique throughout these studies. An original HPLC method employing ultraviolet detection with a limit of quantitation of 5jLg/f was developed for the determination of cyclizine in serum and both cyclizine and norcyclizine in urine, Solid-phase extraction using extraction columns packed with reversed-phase C 18 material, and followed by a simple phase-separation step proved successful for the accurate and precise isolation of the compounds. The validated method was applied to the analysis of serum and urine samples from a pilot study in which a single volunteer was administered 50mg of cyclizine hydrochloride. Several samples collected during the pilot study revealed the presence of both drug and metabolite in concentrations below the limit of detection. In order to improve the selectivity and sensitivity of the analytical method an HPLC method with electrochemical detection operating in the "oxidative-screen" mode was developed. The solid-phase extraction procedure was modified slightly and the method found to be precise, accurate, selective and highly sensitive with a limit of quantitation of IjLg/f for both cyclizine and norcyclizine in both serum and urine. This method was applied to the determination of both compounds after intravenous and oral administration of cyclizine to humans. HPLC with electrochemical detection was used for the analysis of samples collected during dissolution studies on the batch of tablets used for pharmacokinetic studies. In addition, this method was used to assess content uniformity of the tablets and of samples from the batch of intravenous ampoules of cyclizine lactate. Dissolution studies showed that all tablets tested passed the compendial specifications for cyclizine. Content uniformity assessment revealed that within-batch uniformity existed for both the tablets and ampoules and, therefore, variations in pharmacokinetic parameters for the drug would more than likely be as a result of inter- and intra-individual variability within the subject population. IV Pharmacokinetic information for cyclizine was obtained following administration of an intravenous bolus dose of cyclizine lactate as a solution, oral administration of cyclizine hydrochloride as a single dose of 50mg and as fixed multiple doses of 50mg every 8 hours for five days. Further information was acquired following administration of single doses of 100mg and 150mg cyclizine hydrochloride. Data collected from these studies were evaluated using both compartmental and non-compartmental techniques. Cyclizine was rapidly absorbed following oral administration with ~ ~ean k. = 1.54 hr- I and was found to have an absolute bioavailability (F) of 0.47. Tne presence of norcyclizine in serum following oral and not intravenous dosing suggests cyclizine is susceptible to "first-pass" metabolism in either the gut wall or the I iver. Mean CIToT determined following the intravenous dose was 0.865 f1hr/kg. The mean CI ToT of 0.823 flhr/kg calculated following oral dosing, using a unique value of F for each subject compared favourahly with that obtained following intravenous dosing. Renal clearance of cyclizine is negligihle indicating that non-renal routes of elimination account for the majority of removal of cyclizine form the body. Cyclizine is extensively distributed and the mean Vz following an intravenous dose was 16.70 f1kg. This value is lower than that calculated from all oral studies from which the mean Vz was determined to be 25.74 f1kg. Cyclizine is eliminated l slowly with a mean elimination t /2 = 20.11 hours. Cyclizine dose not appear to follow dose­ dependent kinetics and therefore, inability to predict steady state levels are more than likely due to accumulation as a result of frequent dosing rather than saturation of elimination mechanisms. Modelling of intravenous data to one-compartment (lBCM), two-compartment (2BCM) and three­ compartment models indicated that the pharmacokinetics of cyclizine can be adequately described by I a 3BCM. The drug is rapidly distributed into a "shallow" peripheral compartment (O! = 9.44 hr- , I and k21 = 2.09 hr- ), and slowly distributed to the "deep" peripheral compartment (/3 = 0.451 hr\ 1 and k31 = 0.120 hr- ). Modelling of all oral data indicated that a 2BCM best described the pharmacokinetics of the drug, however, distribution to the peripheral compartment is not as rapid as to the "shallow" peripheral compartment following the intravenous dose. Mean distribution 1 parameters were O! = 0.64 hr- and, k21 = 0.39 hr-l. Mean CI TOT following intravenous dosing of 0.70 f1hr/kg was similar to the mean CI ToT of 0.73 f1hr/kg determined after oral dosing. The mean distribution volume at steady state determined following intravenous dosing (17.78 f1kg) was lower than that obtained from the oral studies (25.52 f1kg). The mean terminal elimination half-lives calculated for cyclizine following fitting of intravenous and oral data was 25.09 hours. v In general, mean pharmacokinetic parameters calculated following titting of data to a 2BCM after oral administration correlate closely with those calculated using non-compartmental techniques. However, the pharmacokinetics following intrav~l1ous dosing are better described by- a 3BCM and a close correlation between parameters estimated using noncompartmental techniques and compartmental techniques is evident when a 3BCM model is used. vi ACKNOWLEDGEMENTS I would like to express my sincere thanks to the.-. following. people:- My supervisor, Professor 1. Kanfer for his patience, guidance, understanding and assistance throughout the course of my studies. Also, for providing superb laboratory facilities, financial ~ - ~ support and the opportunity to attend the Eleventh International Symposium on Column Liquid Chromatography in Amsterdam and the Pharmacy World Congress '91 in Washington, DC. All my colleagues in the laboratory for their congenial company, support and numerous forms of assistance. Specific thanks to Mike Skinner, Chris Stubbs and Neil Sparrow for their assistance in the beginning. Also my colleagues, Roslind Dowse and Eric Smith in the Division of Pharmaceutics for their encouragement and support. Beverley Glass for her help in preparation of molecular structures and Susan Abrahams for her help in preparing chromatograms for this dissertation. Carol Hubbard for her patience and help with the printing of this thesis. Andy Soper and Owen Campbell for maintaining and repairing equipment. To Sagaran Abboo for his help in the laboratory and during bioavailability trials and also Leon Purdon for his various forms of assistance. Special thanks also go to Dr Ann Kench, and Sisters Penny Bernard and Penny Jaffray for their invaluable help during bioavailability trials. The Head, Professor H. Parolis, the Dean, Dr BJ. Wilson and the staff of the School of Pharmaceutical Sciences for their assistance, advice and use of departmental facilities. Also for affording me the opportunity to gain invaluable teaching experience during this time. The Council for Scientific and Industrial Research, Foundation for Pharmaceutical Education and Millipore (SA) for their generous financial assistance. My parents and family for their continued support, encouragement and assistance throughout my studies. My wife, Rosy, for her