STEREOSELECITVE METABOLIC AND PHARMACOKINETTC STUDIES ON ANTIDEPRESSANT: DOXEPIN AND DESME??TYLDOXEPN A Thesis Submitted to the College of Graduate Studies and Research in Parliai Fulfülment of the Requirements for the Degree of Doctor of Philosophy in the College of Phannacy and Nutrition University of Saskatchewan Saskatoon, Saskatchewan, Canada BY Jing-He Yan, B.Sc. Spring 1999 O Copyright Jing-He Yan, 1998. Al1 nghts reserved. National Library Bibliothèque nationale I*m of Cana&. du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington OttawaON K1AON4 OttawaON KlAON4 Canada Canada Your file Votre réference Our lGle Notre rdérence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distnbute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/^ de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thése ni. des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced &out the author's ou autrement reproduits sans son permission. autorisation. College of Graduate Studies and Research S-Y OF DISSERTATION Submitted in partial fulfillment - of the requirements for the DEGREE OF DOCTBR OF PHILOSOPW College of Pharmacy and Nutrition University of Saskatchewan Sprhg 1999 Examining Cornmittee: Dr. J.S. Richardson Xl&i.ü k&t910&HlDWi, Dean's Designate, Chair College of Graduate Studies and Research Dr. S.J. Whiting Chair of Advisory Cornmittee, College of Pharmacy and Nutrition Dr- J.W- Hubbard Supervisor, College of Pharmacy and Nutrition Dr. K.K. Midha CO-Supervisor, College of Pharmacy and Nutrition Dr- G. McKay Co-Supervisor, College of Pharmacy and Nutrition Dr. H. Semple Collep of Pharmacy and Nutrition Dr. V.S. Gupta College of Vetennary Medicine External Examiner: Dr. 1. J. McGilveray McGilvray Phamacon Inc 1, Stonehege Park Nepean, Ontario. K2H 821 Stereoselective Metabolic and Pharrnacokinetic Studies on Antidepressants Doxepin and Desmethyldoxepin The tricyclic antidepressant doxepin is rnarketed as a mixture of geometric isomers in a cis:trans ratio of 15:85. In most in vivo and in vitro tests, the cis-isomer is the more potent of the two geometric foms. Doxepin is metabolized to a variety of phase 1 and II metabolites, in which the N-desmethyl metabolite is thought to make an important contribution to therapeutic activity and it has been suggested that plasma concentrations of (cis plus trans) doxepin and (cis plus trans) desmethyldoxepin show better correlation with antidepressant activity than (cis plus trans) concentrations of the parent drug alone. In recent years, there have been a number of research papers reporting the ratio of cis(Z)- to ûans(E)- desmethyldoxepin equals or surpasses unity in plasma or urine of patients or healthy volunteers after orai administration of doxepin, while this significant ratio distortion is not evident for the parent hg. There may be clinical significance to this observation if the increased ratio of Z-desmethyldoxepin is ascnbed to the ccenrichment"of the 2-isomer mediated by a mechanism of isomer interconversion, since 2-isomer is the more potent of the two antipodes. Therefore, an investigation into the mechanism of this significant ratio distortion was warranted. A stereoselective normal phase KPLC assay utilkg W detection was developed for this purpose. It is a sensitive, simultaneous and convenient method, which was validated for reproducibility, linearity, sarnple stability and recovery. Results from in vivo and in vitro metabolic studies in rats and humans demonstrate that the reason for the ratio distortion Iay in more rapid metabolic elfination of the E-desmethyldoxepin rather than E- to 2- isomer conversion, and that the N-glucuronidation of desmethyldoxepin was very likely the stereoselective metabolic pathway responsible. Results from cross-over, ivloral pharmacokinetic- studies on doxepin and desmethyldoxepin in humans and dogs indicate: (i) significant ratio distortion in isomers of desmethyldoxepin occurred with an evident pattern of time dependent, progressive process, which was in keepiog with the mechanism of stereoselective metabolism; (ii) no effect of route of administration was observed on the ratio distortion; (iii) extensive first- pass effects of doxepin seemed simply hepatic in nature. BIOGRAPHICAL Born in Shanghai, China B Sc., College of Phamiacy, Shanghai Medical University HONQRS The Hoechst Marion Roussel Canada Inc. Graduate Award, 1997-1998 Virginia Commonwealth Scholarship, Virginia Commonwealth University 199 1- 1992 World Bank Loan Scholarship, Chinese Government, 1987- 1988 PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, 1 agree that the libraries of this University may make it fieely available for inspection. 1 further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly pqoses may be &ranted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done- It is understood that any copying or publication or use of this thesis or parts thereof for hancial gain shali not be dlowed without rny written permission. It is also undentood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any materiai in my thesis. Requests for permission to copy or to make olher use of material in this thesis in whole or part should be addressed to: Dean of the College of Pharmacy and Nutrition University of Saskatchewan Saskatoon, Saskatchewan (Sm5C9) ABSTRACT The tricyclic antidepressant doxepin is marketed as a mixture of geometric isomers in a cis:traus ratio of 15:85. In most in vivo and in vitro tests, the cis-isomer is the more potent of the two geometric forms. Doxepin is metabolized to a variety of phase 1and phase II metabolites, in which the N-desmethyl metabolite is thought to make an important contribution to therapeutic activity and it has been suggested that plasma concentrations of (cis plus trans) doxepin and (cis plus trans) desmethyldoxepin show better correlation with antidepressant activity thm (cis plus trans) concentrations of the parent drug alone. Disposition studies carried out in humans and animals have claimed doxepin is well absorbed after oral administration and measurable arnounts of doxepin and desmethyldoxepin rapidly appear in the blood Stream. In recent years, there have been a number of research papers published reporting that the ratio of cis(Z)- to trans(E)- desmethyldoxepin equals or surpasses unity in plasma or urine of patients or heaithy volunteers after oral administration of doxepio, while this significant ratio distortion is not evident for the parent dmg. There may be clinical significance to this observation if the increased ratio of 2-desmethyldoxepin is ascribed to the "enrichment" of the 2-isomer mediated by a rnechanism of isomer interconversion, since Z-isomer is the more potent of the two antipodes. Therefore, an investigation into the rnechanism of this significant ratio distortion was warranted. A stereoselective normal phase HPLC assay utilizing W detection was developed for this purpose. It is a sensitive, simultaneous and convenient method with lower Iimits of quantification of 1 ng/rnL for each of the cis- and tram- isomers of doxepin and desmethyldoxepin. The assay method was validated for reproducibility, linearity, sample stability and recovery. The first pilot studies involved oral doses of doxepin in male volunteers (n=4) and four animal species (dog, rabbit, guinea pig, and rat) in mal1 groups (n=4 or n=3). Ratio distortion of desmethyldoxepin was found with varying degrees in cumulative 24 hour urine. Analysis of variance (ANOVA) indicated no significant effect of subject @=0.2369), but the effect of species on the percentage of Z- desmethyldoxepin was highly significant @=0.000 1). The ratio distortion was found to be highest in human and rat, which were not significantly different fiom each other as indicated by the multiple comparison tests (Student-Newman-Keulç, Tukey- Kramer and SpjotvoI1-Stoline) . A parallel study with three gmps of male Lewis strain rats (n=4) was carried out to investigate the effects of route of administration on the 24 hour urinary excretion of the isomers of doxepin and desmethyldoxepin. There was no signincant effect of route on percentage 2-doxepin (p=0.3262), but the effect of route on percentage desmethyldoxepin was highly significant @=0.000 1). Al1 three multiple comparison tests showed the percentage of 2-desmethyldoxepin after oral adminis~ationto be significantiy different fkom the percentages O btained after either intravenous or intraperitoneal injection, whereas there were no ciifferences detected in percentage Z- desmethyldoxepin after intravenous or intraperitoneal administration. In both latter cases no obvious distortion in the ratio was observed. A cross-over