Doxepin and Desme??Tyldoxepn

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

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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 study in a group of male Lewis strain rats (n=8) was carried out, in which each animal received E-desmethyldoxepin, E-doxepin and doxepin by both intravenous and oral routes. Examinztion of urine samples in two segments (0-24 and 25-48 hour) indicated that there was no evidence of E- to 2- interconversion after administration of E-isomers by either intravenous or oral routes. Significant distortion in the percentage of 2-desmethyldoxepin was observed after oral but not after intravenous administration of doxepin. In vitro metabolic studies were performed with rat and human liver rnicrosornes; experiments with rat GIT tissue hornogenates; and enzyme inhibition studies with rat liver microsornes utilizing UGT inhibitors. The results fiom these studies indicated that: (i) Consistent with the in vivo studies, there was no evidence of interconversion between isomers; (ii) increase in the percentage of Z-desrnethyldoxepin was a time dependent progressive process; (G)the reason for the ratio distortion Iay in more rapid metabolic elimination of the E-desmethytdoxepin; (iv) no trace of desmethyldoxepin was found in incubates of doxepin with rat GIT subceiiular hornogenates; and (v) the N-g lucuronidation of desmethyldoxepin was very likely the stereoselective metabolic pathway responsible. The final part of the thesis descnbes cross-over, iv/oral stereoselective pharmacokïnetic studies on doxepin and desmethyldoxepin in young male volunteers (n=12) and beagle dogs (n=6). The results in human studies indicated: (i) Signifïcant ratio distortion of desmethyldoxepin isomers occurred in both plasma and urine after iv and oral dosing. The process appears to have a time dependent progressive pattern; (ii) there was no evidence of E- to Z- isorner conversion after the administration of commercial doxepin; (iii) the 2-doxepin percent ratio stayed very close to the original composition after both iv and oral administrations; (iv) signincant "first-pass" effects after oral administration were dernonstrated by the absolute bioavailabilities of 2- and E- doxepin, which were both 29%- The results fiom the dog studies were very similar to those in humans in all the above-mentioned aspects. The absolute bioavaiiabilities of Z- and E- doxepin were 27% and 24% respectively indicative of extensive "first-pars" metabolism. ACKNO WLEDGMENTS

It was my privilege and honor having been under the guidance of Drs. J.W. Hubbard and K.K. Midha to complete my Ph. D. thesis work. 1 have leamed and benefited irnmensely fiom them for the art of scientific research, the way of logical and cntical thinking, and the kindness of understanding. 1 wish to express my sincere gratitude to them for their personal interest, guidance, encouragement and patience during the course of the thesis work. As one of my CO-supervisors, I also wish to thank Dr. G. McKay for all his help. My sincere gratitude extends to Dr. E.M. Hawes and my conmittee members for helphl advice.

1 wish to thank Mr. N. Pidskalny and Dr. Q. Li for the assistance in perfomiing mass spectral analysis. 1 also wish to thank Mr. R. Mazzei and Dr. E.D. Korchinski for the excellent technicai assistance in clinical trials, and Mr. T.S. Gurnsey for the help in laboratory instrument maintenance. I want to express my gratefulness to ail other members of the Drug Metabolism, Drug Disposition Research Group for Eendly and helpful interactions, which made my stay full of good mernories. My same appreciation is also given to the College of Pharmacy and Nutrition.

The fuiancial supports fiom the Medical Research Council of Canada for Program Grant 11472 and PharmaQuest Ltd. are gratefidly acknowledged. " Where there is a will, there is a way "

- an attitude

To my mother, father and sister my wife, Zhan-Qing for their love, encouragement and patience TABLE OF COmNTS

PERMISSION TO USE ABSTRACT ACKNOWLEDGMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF SCHEMES LIST OF ABBREVLATIONS

INTRODUCTION Depression Etiologic explanation of depressive illness Treatment of depression Antidepressant agents and their action in the neuronal system Doxepin Nomenclature of geomehical isomers Clinical use of doxepin Disposition profiles Cornparison of phamiacological effects between 2.-and E- doxepin Reserpine antagonism Amphetamine stereoqpy

vii Potentiation of hexobarbital induced sleep and urethane induced sedation Histamine antagonism 5-HT antagonism Anticho lïnergic effects Desmethyldoxepin and its clinical imporîance Pharmacokinetic studies of doxepin and desmethy ldoxepin Ratio distortion in desmethyldoxepin Stereoselective methods for quantification of doxepin and desmethyldoxepin High performance liquid chromatography WPLC instrumentation Solvent reservoir Solvent purnp

Column Detector UV detector Photo diode-anay detector Electro-chernical detector Fluorescence detector Mass spectrometer detector Integrator Mass spectrometry Electron impact ionization Electrospray ionization 1.8.0.0.0. Computational methods in phannacokinetic studies 24 1.8.1.0.0. Model independent phmacokinetic approaches 24 1.8.2.0.0. Mathematical modeling of pharmacokinetic data 25 1-8.2.1 .O. Cornpartmental models 26 1.8.2.1.1. One cornpartment mode1 27 1-8.2.1 -2. Two cornpartment mode1 29 1.8.2.2.0. Physiological models 30

RATIONALE AND OBJECTfVES Experimentai results and speculations for the mechanisrn of the ratio distortion 2.2.0-0. Hypothesized process of E- to 2- isomerization 33 2.3 .O.O. CIinicai significance of the possible 2-isomer enrichment 33 2.4.0.0. Rational of the research project 35 2.5.0.0- Specific objectives of the present thesis work 2.5.1 .O. Stereoselective, sensitive and simultaneous HPLC method 35 2.5.3.0. Confinnation of the ratio distortion and selection of an animai mode1 36 2.5-3.0. Investigation for possible mechanisms of the ratio distortion 2.5.3.1. Effect of route of administration on the ratio distortion 36 2.5.3.2. Test of E- to 2- isomer conversion in vivo 2.5-3.3. Test of isomer interconversion in vitro 37 2.5.3.4. In vi~ometabolic inhibition of desmethyldoxepin 37 2.5.4-0. Pharmacokinetic studies in vivo 38 2.5.4.1. Stereoselective pharmacokinetic studies in humans 38 2.5.4.2. Stereoselective pharmacokinetic studies in dogs 38 EXPERIMENTAL Chexnicals and reagents Instrumentation Synthesis of desmethyldoxepin Doxepin fiee base chloroform solution Phosphate buffer solution (0.5 M, pH 5) Carbarnate formation Secondary amine formation Stereoselective, sensitive and simultaneous HPLC method Preparation of standard stock solutions Z- or E- doxepin, or commercial doxepin 100 ppm (fiee base) solution E-desmethyldoxepin 100 ppm (fiee base) solution Nortriptyline 100 ppm (fiee base) solution Preparation of standard curves preparation of quality control (QC) samples Extraction procedures Examination of equivalence of detector response to Z- and E- isomers Recovery studies Stability studies Stability after fieeze-thaw cycles Stability at 4°C

Stîbility of extracts in mobile phase at room temperature Linearity and reproducibility Metabolic studies HPLC analytical method Estimation of in vitro metabolic elirnination half-lives Statistics Exploratory in vivo experiments Human studies Animal in vivo studies Confirmation of significant ratio distortion of doxepin isomers in one healthy male volunteer Studies on the effect of route of administration on the urinary excretion of the isomers of doxepin and desmethy ldoxepin in rat Studies on the possibility of metabolic E- to 2- isomer conversion in rat Studies on the possibility of metabolic isomer interconversion with liver microsomes or gastrointestinal tract (GIT) homogenates Experirnent design Rat1huma.n liver and rat GIT tissue samples Rat and human liver microsorne preparations Rat GIT subcellular preparations Protein content in incubates with enzyme preparations Incubation conditions Mass spectroscopy Enzyme inhibition studies in incubation of rat liver microsomes using UGT inhibitors Preparation of stock solutions Experimental design Incubation conditions Simulation of the ratio distortion in desmethyldoxepin isomers Phannacokinetic studies Analytical methods Pharmaco kinetic analy sis Statistical analysis Pharmacokinetic studies in humans Subjects Study design Pharmacokinetic studies in dogs AnimaIs Study design Dosing procedure Sample collection

RESULTS AND DISCUSSION Synthesis of desmethyldoxepin

Desmethy ldoxeph Stereoselective, simultaneous and sensitive HPLC method Equivalence of detector response to 2- and E- isomers Plasma and derecovery Sample stability Linearity and reproducibility Discussion Metabolic studies Exploratory in vivo experirnents Discussion Significant ratio distortion of doxepin isorners in one healthy male volunteer Discussion The effect of route of administration on the urinary excretion of the isomers of doxepin and desmethyldoxepin in rat Discussion Resdts fiom the test of metabolic E- to 2- isomer conversion in rat Discussion In vipo metabolic studies with liver microsomes or gastrointestinal tract (GIT) homogenates Incubation of doxepin related xenobiotics with rat liver microsomes Incubation of doxepin related xenobiotics with rat GIT hornogenates Incubation of doxepin related xenobiotics with human liver microsomes Discussion In viho enzyme inhibition studies using UGT inhibitors Discussion Pharmacokinetic studies Clinical pharmacokinetics Discussion Preclinical phannacokinetics Pharmacokinetics of doxepin &er intravenous administration Pharmacokinetics of doxepin after oral administration Z:E Ratio of the isomers Discussion 5.0.0.0. CONCLUSIONS 205

6.0,O.O. REFERENCES 209

7.0.0.0. APPENDICES 23 1 Appendix A Speciai consent to use dmgs for experimental human studies A study of doxepin and its geometric isomers 222 Appendix B Special consent to use dmgs for experimental human studies A study on the metabolism of doxepin in humans 224 LIST OF TABLES

Incubation conditions with rat Liver microsornes 60 Incubation conditions with human liver microsornes 62 Incubation condittons with rat liver microsomes in erzyme inhibition studies 66 Equivalent detector response in day 1 plasma samples 78 Equivaient detector response in day 2 plasma samples 78 Equivalent detector response in day 3 plasma samples 79 Equivaient detector response in plasma samples (inter-assay C.V.%) 79 Equivaient detector response in day 1 urine samples 80 Equivalent detector responsè in day 2 urine samples 80 Equivaient detector response in day 3 urine samples 8 1 Equivaient detector response in urine sarnples (inter-assay C.V.%) 8 1 Xecovery data 82 Freeze/thaw stability of E-doxepin 84 Freezehhaw stability of E-desmethyldoxepin Stability of E-doxepin at 4°C

Stability of E-desmethyldoxepin at 4°C 85

Stability of extracted E-doxepin in mobile phase at 23°C 86

Stability of extracted E-desmethyldoxepin in mobile phase at 23OC 86 Accuracy and precision (E-doxepin) 87 Accuracy and precisioa (E-desmethyldoxepin) 87 Urinary excretion (0-24 h) of doxepin in human and various animal species after doxepin oral administration 92 Urinary excretion (0-24 h) of desmethyldoxepin in human and various animal species after doxepin oral dosing Mean unnary excretion (% dose, 0-24 h) of doxepin and desmethyldoxepin after oral doxepin administration to human and different animal species Isomer concentrations and 2-isomer% in plasma samples after doxepin single oral dose (75 mg base) to one heaithy male volunteer Isomer concentrations and Z-isornefi in urine samples after doxepin single oral dose (75 mg base) to one healthy male volunteer Urinary excretion (0-24 h) of doxepin and desmethyldoxepin in parallel groups of Lewis strain rat after oral, intravenous, or intraperitoneal dosing with doxepin Urinary excretion (% dose, 0-24 h) of doxepin and desmethyldoxepin after doxepin oral, intravenous, or intraperitoneal administration to parallel groups of Lewis strain rats Urinary excretion of doxepin and desmethyldoxepin after oral or intravenous dosing with doxepin and E-isomers to a group of Lewis strain rats in the cross-over studies Urinary excretion (% dose) of doxepin and desmethy ldoxepin after oral, intravenous dosing with doxepin and E-isomers to a group of Lewis strain rats in the cross-over studies Doxepin isomer concentration &er incubation of rat Liver microsomes spiked with 25 ph4 commercial doxepin Desrnethyldoxepin isomer concentration after incubation of rat liver microsomes spiked with 25 pM commercial doxepin Doxepin isomer concentration after incubation of rat liver microsomes spiked with 25 pM E-doxepin contaminated with 2-doxepin (1 -8%) Desmethyldoxepin isomer concentration &er incubation of rat liver microsomes spiked with 25 pM E-doxepin contaminated with 2-doxepin (1 -8%) Doxepin isomer concentration derincubation of rat liver microsomes spiked with 25 yM Z-doxepin Desmethyldoxepin isomer concentration after incubation of rat liver microsomes spiked with 25 pM 2-doxepin

xvi Isomer concentration derincubation of rat liver microsomes spiked with 15 pM E-desmethyldoxepin contaminated with 2- desmethyldoxepin (3 %) Half-lives of doxepin and desrnethyidoxepin isomers in incubation with rat liver microsornes Isomer concentration after incubation of rat stomach homogenates spiked with 25 pM commercial doxepin Isomer concentration after incubation of rat srnall intestine homogenates spiked with 25 pM commercial doxepin Isomer concentration derincubation of human liver microsomes spiked with 2.5 pM commercial doxepin Isomer concentration after incubation of human liver microsomes spiked with 2.5 pM Z-doxepin Isomer concentration after incubation of human liver microsomes spiked with 2.5 pM E-doxepin contaminated with 2-doxepin (1-8%) Isomer concentration after incubation of human liver microsomes spiked with 3 .O yM E-desmethyldoxepin contamhated with 2-desmethyldoxepin (3%) Isomer concentration after incubation of rat liver microsornes spiked with 25 mM commercial doxepin Isomer concentration after incubation of rat liver microsomes spiked with 25 rnM commercial doxepin and 100 pM oleoyi-CoA lsomer concentration after incubation of rat liver microsomes spiked with 25 mM commercial doxepin and 3 mM probenecid Isomer concentration after incubation of rat liver microsomes spiked with 25 mM commercial doxepin and 3 mM furosernide Simulation of Z/E ratio distortion by a stereoselective metabolic pathway Isomer concentration after incubation of rat liver microsomes spiked with 15 mM E-desmethyldoxepin contaminated with 2-desmethyldoxepin (3%)

xvii Simulation of Z/E ratio distortion by a stereoselective metabolic pathway which is inhibited by 55% Isomer concentration after incubation of rat Iiver microsomes spiked with 15 mM E-desmethyldoxepin contaminated with 2-desrnethyldoxepin (3%), and with 100 pM oleoyl-CoA Simulation of Z/E ratio distortion by a stereoselective metabolic pathway which is inhibited by 29% Isomer concentration after incubation of rat liver microsomes spiked with 15 mM E-desmethyldoxepin contaminated with 2-desmethyldoxepin (3%), and with 3 mM probenecid Isomer concentration after incubation of rat liver microsomes spiked with 15 mbf E-desmethyldoxepin contaminated with 2-desmethyldoxepin (3%), and with 3 mM furosemide In vitro half-lives of desmethyldoxepin isomers in incubates of rat ber microsornes spiked with 15 pM E-desmethyldoxepin contaminated with 2-desmethyldoxepin (3%), with or without UGT inhibitor Pharmacokuietics of 2- and E-isomers of doxepin and desrnethyldoxepin in 12 men afier doxepin intravenous administration (22.12 mg base) Pharmacokinetics of 2- and E- isomers of doxepin and desmethyldoxepin in 12 men derdoxepin oral administration (75 mg base) Pharmacokinetics of 2- and E- isorners of doxepin in 12 men aer doxepin intravenous administration (22.12 mg base) using cornpartmental analysis Urinary recovery (dose%) of doxepin and desmethyldoxepin isomers (0-120 h) in 12 men after doxepin intravenous administration (22.12 mg base) Urinary recovery (dose%) of doxepin and desmethyldoxepin isomers (0-120 h) in 12 men after doxepin oral administration (75 mg base) Pharmacokinetics of 2- and E- isomers of doxepin and desmethyldoxepin in 6 beagle dogs after doxepin intravenous administration (6 m@g base)

xviii 4.59. Pharmacokinetics of 2- and E- isomers of doxepin and desmethyldoxepin in 6 beagle dogs &er doxepin oral administration (20 mg/kg base) 193 4.60. Pharmacokinetics of 2- and E- isomers of doxeph in 6 beagle dogs after doxepin intravenous administration (mean dose 57.1 mg/dog base) using cornpartmental analysis 194 LIST OF FIGURES

Structures of geometric isomers of doxepin and desmethyldoxepin Randomized two-sequence cross-over design in which eight male Lewis strain rats received six treatments in either of the two sequences, with a two-week washout penod between treatments Experimental design for rat liver microsomal enzyme inhibition studies using UGT inhibitors Electrospray mass spectrurn of desmethyldoxepin fiom the organic synthesis Electron impact mass spectrum of desmethyldoxepin fkom the organic synthesis HPLC chromatogram of the extracts fiom blank hurnan urine HPLC chromatogram of the extracts fiom blank human plasma Mean percentages of 2-doxepin in 0-24 hour urine from various species after doxepin oral administration Mean percentages of Z-desmethyldoxepin in 0-24 hour urine from various species after doxepin oral administration HPLC chromatograms of the exmcts fiom hurnan urine after doxepin oral administration HPLC chrornatograms of the extracts fiom dog urine after doxepin oral administration Plasma isomer AUC profiles derdoxepin oral administration to one healthy male volunteer HPLC chromatograms of plasma extracts fiom the volunteer after doxepin oral administration Mean % of desrnethyldoxepin isomers in 0-24 hour rat urine der oral, intravenous or intraperitoneal dosing with doxepin HPLC chromatograms of the extracts fYom rat urine after doxepin oral administration HPLC chromatograms of the extracts f?om rat urine after doxepin intravenous administration HPLC chromatograms of the extracts fkom rat uriné after doxepin intraperitoneai administration Mean isomeric composition of desmethyldoxepin in 0-1 6 hour rat urine after doxepin oral and intravenous administration 121 HPLC chromatograms of the extracts fiom rat urine after E-isomer oral administration Mean in vitro ha-lives of doxepin and desrnethyldoxepin isomers in incubates with rat iiver microsornes 134 HPLC chromatograms of extracts fiorn the incubation mixture with rat liver microsomes Electrospray mas spectra of 2-, E- desmethyldoxepin and didesmethyldoxepin from the incubation mixture with rat liver microsomes 13 9 IH vitro half-lives of doxepin and desrnethyldoxepin isomers in incubates with human liver microsornes 147

Mean Z-desmethyldoxeph % curves in incubates of rat liver microsomes with doxepin (25 PM) and UGT inhibitor or without UGT inhibitor (control) 154 Curves of mean 2-desmethyldoxepin% in incubates of rat liver microsomes with 15 pM E-desrnethyldoxepin, with or without (control) oleoyl-CoA, and curves derived fiom relevant simulations under assumptions of non-inhibited and inhibited metabolism 159 Mean desmethyldoxepin isomer elimination curves in incubates of rat liver microsomes with 15 ISyI E-desrnethyldoxepin, with or without (control) UGT inhibitor 159 Mean in vitro half-lives of desmethyldoxepin isomers in incubates of rat liver microsomes with 15 pM E-desmethyldoxepin, with or without (control) UGT inhibitor 162 Plot of mean observed versus predicted 2-doxepin plasma concentration after doxepin intravenous administration (22.1 2 mg base) in men with a two cornpartment mode1 fitting Plot of mean observed versus predicted E-doxepin plasma concentration derdoxepin intravenous administration (22.12 mg base) in men with a MO cornpartment mode1 fiding 174 Plot of mean plasma concentration versus time cwesof Z-doxepin and E-doxepin after doxepin oral administration (75 mg base) in men 175 Plot of mean plasma concentration versus time curves of Z-desmethyldoxepin and E-desmethyldoxepin derdoxepin intravenous administration (22.12 mg base) in men 176 Plot of mean plasma concentration versus time curves of Z-desmethyldoxepin after doxepin oral administration (75 mg) in men 177 Plot of plasma concentration versus the curves of E-desmethyldoxepin afler doxepin single oral administration (75 mg base) to volunteer 11 178 Plot of AUC of desmethyldoxepin isomers after doxepin intravenous administration (22.12 mg base) in men 179 Plot of AUC of desrnethyldoxepin isomers after doxepin oral administration (75 mg base) in men Plot of mean percentage of 2-desmethyldoxepin in urine after doxepin intravenous and oral administration in men 180 Mean urinary recovery (dose%) of doxepin and desmethyldoxepin isomers (0-120 h) after doxepin intravenous (22.12 mg base) and oral (75 mg base) administration in men 183 Plot of mean observed versus predicted 2-doxepio plasma concentration after doxepin intravenous administration (6 mgkg base) in beagle dogs with a two compartment model fitting 195 Plot of mean observed versus predicted E-doxepin plasma concentration after doxepin intravenous administration (6 mgkg base) in beagle dogs with a two cornpartment model fitting 196 Plot of mean plasma concentration versus time curves of 2-desmethyldoxepin and E-desmethyldoxepin after doxepin intravenous administration in beagle dogs Plot of mean plasma concentration versus time cwesof 2-doxepin and E-doxepin &ter doxepin oral administration (20 mgkg base) in beagle dogs

xxii 4.3 9. Plot of mean plasma concentration versus time curves of 2-desmethyldoxepin and E-desmethyldoxepin after doxepin oral administration (20 mgkg base) in beagle dogs 199 4.40. Plot of AUC of desmethyldoxepin isomers afier doxepin intravenous administration (6 mgkg base) in beagle dogs 200

4.41. Plot of AUC of desmethyldoxepin isomers derdoxepin oral administration (20 mgkg base) in beagle dogs 200

4.42. Plot of mean 2-desmethyldoxepin% in plasma after doxepin intravenous and oral administration in beagle dogs 20 1 4.43. HPLC chromatograms of the extracts fiom dog plasma samples 202 LIST OF SCHEMES

1.1. Phase I & II metabolk pathways of doxepin in humans andor anirnds 9 2.1- Hypothesized process of E- to 2- isomerization 34 3.1. Synthetic route of desmethyldoxepin 42

xxiv LIST OF ABBREVIATIONS

AIC Akaike' s Wormation Criterion ANOVA Analysis of variance AUC Area under the plasma concentration-time curve AUMC Area under the kstmoment curve Clearance

Apparent oral clearance Rend clearance

~rn, Peak plasma conceneation CNS Central nervous system C.V. Coefficient of variation EI Electron impact ES1 Electrospray ionization eV Electron volt B ioavailability Glucose 6-phosphate Glucose 6-phosphate dehydrogenase GIT Gastrointestinal tract HPLC High performance liquid chromatography Molar MRT Mean resident tirne Mass spectrometry N Nomid NADP Nicotinamide adenùie dinucleotide phosphate QC Quality control psi Pounds per square inch SC Schwarz Criterion S.D. Standard deviztion ttn Elimination half-We UGT Uridine glucurono-1 trançferase UV Ultraviolet v Volume of distribution vss Apparent volume of distribution at steady state hz Elimination rate constant based on the terminal elimination phase

xxvi 1.0.0.0. INTRODUCTION

1.1.0.0. Depression Depression is a very common and serious illness. At any given moment, about 5- 6% of the population is depressed and an estimated 10% of people may becorne depressed dhgtheir lives (Hollister, 1992). The term depression is used to describe a normal mood, a medical symptom, and a collection of psychiatric syndromes. As a nomal mood, depression is a common human reaction to a significant loss. As a medical term, it is also used to describe the sadness seen in patients who have other severe medical and psychiatric disorders (Berger, 1977). Usually, depression refers to a group of psychiatric syndromes or illnesses with well-defined symptoms, only one of which is sadness. However, sadness is neither a necessary nor a sufncient part of the syndrome. Other symptoms include pessimism; loss of interest in usual activities; feelings of low self-esteem, worthlessness or puilt; and the loss of the ability to feel pleasure. Sorne depressed patients may also be anxious or agitated, while others are retarded, with extremely slow thinking and activity. Somatic symptorns can also be part of the syndrome, which include sleep disturbance, decreased appetite and weight loss, gastrointestind distress, constipation, backaches, and hypochondnasis. Of course, the most imrnediately dangerous symptorn of depressive illness is suicidal ideation (Berger, 1977, Feighner et al-, 1972, Schildkraut and Klein, 1975). Since not every patient has all of these syrnptoms, the clinical presentation of depressive illness is quite variable. Although it is sometimes hard to strictly diagnose a patient for depressive subtypes due to complexity of etilogical factors. Generally speakirig, depression can be classified into three major types based on their origin. 1) Reactive or secondary depression, occurring in response to adverse Life events (2 60%)

2) Endogenous depression, genetically detemiined biochemical disorder manifested by precipitating life event not adequate for degree of depression (25%) 3) Bipolar affective disorder, characterized by episodes of mania-depression (10-15%)

1.1.1.0. Etiologic explanation of depressive illness The ancients postulated that c'melancholia'~(depression) was caused by an excess of "black bile". Despite the failure of investigators to identie the nature of this substance in the intervening 2000 years, the search for the cause of depression continues (Leonard, 1993). The importance of such a search Lies in its emphasis on the biochemical basis of affective disorders. In recent years, there have been a plethora of studies to suggest that depression arises as a consequence of the malfunctioning of one or more neuronal pathways in limbic regions of the brain. Neurons containing the neurotransmitters noradrenaline and serotonin (5- hydroxytryptamine; 5-HT) have been considered as the major candidates that are functionally abnormal in depression (Asberg et al., 1977). Although the exact biochemical mechanisms of depression have not yet been completely understood, the biogenic amine theory of depression suggests that a deficiency of biogenic amines, such as norepinephrine and 5-HT, in the brain that alters the number of postsynaptic receptors for thes e amines, so resulting in irregulation in the transmission of neural impulses (Richardson, 1984, Siever and Davis, 1985). This theory stems fiom the observation that reserpine, an antihypertensive and neuroleptic dmg, could induce depression in patients as weii as in normal subjects. Later pharmacological studies indicated the primary mechanism of action of reserpine was to inhibit the storage of amine neurotransmitters, such as 5-HT and norepinephrine in the vesicles of presynaptic nerve endings. The association of the depression and the depletion of biogenic amine in stores of presynaptic nerve endings induced by reserpine fostered the hypothesis that depression must be related to decreased functional amine- dependent neurotrafl~mission. It is generally agreed, according to this theory, that antidepressant agents act to increase the availability of norepinephrine or 5-HT at the postsynaptic receptor sites in the brain (Rehavi et al., 198 1).

1.2.0.0. Treatment of depression Mild depression of shoa duration should probably not be treated with any medical intervention, aithough any depressive syndromes that significantly interfere with a patient's ability to fùnction in interpersonal relationships or in employment should be treated with medication (Berger.. 1977). The appropriate use of antidepressants improves the prognosis for some patients with depressive illness. Other medical intervention measures may also be used for depression, such as eIectroconvulçive therapy (ECT).

1.3.0.0. Antidepressant agents and their action in the neuronal system Shce the early 1950~~when imipramine was fxst introduced, antidepressant agents have been used clinically in the treatment of depression for over four decades. At present, antidepressants are among the most widely prescribed of drug groups, with over eight million prescriptions each year, and prescription numbers are increasing (Henry, 1994). Structurally or functionally, they may be categorized into the following 4 classes: A) tricyclics (TCAs); B) monoamine oxidase inhibitors (MAOIS); C) selective 5-HT reuptake inhibitors (SSRIs); D) sympathornimetic stimulants (Goodnick, 1994). Most of antidepressant agents are chiefly applied in management of the second type of depression, Le., "endogenous depression". It has been demonstrated experimentally that the mechanism of therapeutic effect differs among various kind of antidepressants, although they di seem to remedy a

deficiency in amine neurotransmission. TCAs block the amine (norepinephrine or 5- HT) reuptake pump, the "off switch" of amine neurotransmission. Such an action presumably pennits a longer sojoum of neurotransmitter at the receptor site. MAOIs block a major degradative pathway for the amine neurotransmitters, whic h presumably permits more amines to accumulate presynapticlly and more to be released. The apparent characteristics of SSRIs in contras to TCAs is that they block serotoM uptake b y nerve endings specifically without inhibiting norepinephrine uptake. Amphetamine-Like sympathornimetics also block the amine pump but are thought to act chiefly by increasing the release of catecholamine neurotransmitters (Hollister, 1992). In recent years, the focus of studies of the mechanisms of clinical effects of antidepressant dnigs has been on the long term neuroadaptive changes occurring in the CNS. This has arisen because the temporal difference between the onset of the therapeutic effects (7 to 14 days or even longer) and the blockade of neurotransmitter reuptake (within hours) suggests that adaptive responses are important for mediating the clinical effects (Norman and E., 1994). There has appeared some experimental evidence that the buk of clinically effective antidepressants desensitize the noradrenergic information flow following their chronic administration either by down- regulating the density of p-adrenoceptors or by de-arnplifj&g the cyclic AMP-

generating system, or both (Sulser et al., 1983). The so-called "B-down-regdation" has been thought to be the main avenue of action of antidepressant drugs dthough conwoversy remains (Okada and Tokumitsu, 1994). There is also experimental data indicating the dependence of P-receptor regulation on serotonergic transmission. It suggests that 5-HT does not play a direct role in P-receptor down-regulation, but is important in preserving the downregulated state. Experimental evidence suggests that changes in cortical adrenoceptor function are dependent on an intact serotonergic input (Sulser, 1987). Therefore. strategies based on the manipulation of the serotonergic system may be useful for rapid induction of an antidepressant effect (Asakura et al, 1987). It should aiso be indicated that other neurotransmitter systems, such as dopaminergic, peptidergic and glucocorticoid receptors, are undoubtedly invclved in the mechanism of antidepressant action (Norman and E., 1994). Furthemore, dflerent types of antidepressant treatment cause consistent changes in neurotransmitter receptor function. These facts provide a basis for the view that there may be a final common pathway through which all antidepressants produce their effects (Leonard, 1994). There is evidence that antidepressants facilitate the G-protein-linked coupling mechanism between 5-HT2 receptors and the second messenger (protein hase-phosphatidyl inositol) system in platelets of depressed patients who respond to treatment (Butler and Leonard, 1988). It has been postulated recently that depression is primarily a disorder of the G-protein coupled receptor superfâmily, arising from a genetic defect (Cassano and Marazziti, 1992). The G-protein superfamily is linked to most of the neurotransmitter receptors believed to be malfunctioning in depression (e.g. adrenoceptors, 5-HTI and 5-HT2 receptors, GAE3As receptors, muscarinic and dopamine receptors). Hence it is possible that the abnormal transduction between the receptor and the second messenger system could be corrected by long term antidepressant treatment. This might provide the ha1common pathway &ou& which all antidepressants act to produce their therapeutic effect (leonard, 1994). 1.4.0.0. Doxepin Doxepin (Sinequa@) is a tricyclic antidepressant Like other TCAs it blocks

neurotransmitter reuptake. The marketed form of doxepin is a mixture of the cis(Z)- and trans(E)- geometric isomers of 11-(3-dimethylaminopropy1idene)-6H- dibenzp,e]oxepin hydrochlonde with Z:E ratio of 15 :8S (Figure 1.lm).

1.4.1.0. NomencIature of geometrical isomers Molecules that contain a carbon-carbon double bond (aikenes) can exist as stereoisomers. Because each of these sets of stereoisomers contains no optically active mernbers, these compounds are classifieci as geometric isomers (Wainer. 1993). The way to name such isomers is based on the Cahn-Ingold-Prelog system. The two groups at each carbon are ranked by the sequence rules. Then that isomer with the two higher ranking groups on the same side of the double bond is called cis or Z (for the German word zusammen meaning together); while the isomer with the two higher mnking groups on the opposite side of the double bond is tram or E (for the German word entgegen rneaning opposite) (March, 1985).

1.4.2.0. Clinical use of doxepin Since its advent to market in the mid-sixties, doxepin has been one of the rnost commonly used tricyclics in the treatment of depression and anxiety. Doxepin is given by oral route in clhical applications. Dosage should be individualized. The usual effective dosage for most depressed patients is 75 to 150 mg daily for outpatients and 150 to 3 00 mg daily for hospitalized patients. A few patients have required larger doses. Patients with anxiety have usually been treated with 75 to 150 mg daily, but hospitaiized patients and some other patients may require larger doses. A dosage regimen based on the major portion or total daily dose given at bedtime has been of benefit in depressed patients with sleep disturbances, in the elderly and when it has been necessary to avoid any excessive daytime drowsiness. Dosage increases should always be gradual, particularly in bedtime-based schedules (P inder et al, 1977).

/C\H (CH3)2NCH2CH2

cis (2)-Doxepin trans (E)-Doxepin

cis (2)-Desmethyldoxepin tram (E)-Desmethy ldoxepin

Figure 1.1. Stmctures of geometric isomers of doxepin and desmethyldoxepin 1.4.3.0. Disposition profiles Doxepin is readily absorbed fiom the gastrointestinal tract after oral administration and measurable amounts of the dmg rapidly appear in the blood stream, uith

maximum plasma conceniration (Crnax) occufnng at approximately 2 hours. Doxepin is biotransfomed to a variety of phase 1 and phase II metabolites in humans and animals. Phase 1 metabolic routes include N-desmethylation, ring hydroxylation and N-oxidation while ring hydroxyl andlor terminal amino glucuronidation may represent major phase II routes (Scheme 1.1.) (Hobbs, 1969, Luo et al., 1991, Shu et al., 199Oa, Shu et al., 1990b: Yan et al. , 1997b). Assuming complete oral absorption and a mean liver btood flow of 1.5 L/min, an estimated mean bioavailability of 0.27-0.32 is obtained (Faulkner et al., 1983, Virtanen et al., 1980, Ziegler et al., 1978). Doxepin is widely distributed throughout the body, with the apparent volume of distribution estimated at around 20 Likg. Equilibrium dialysis gave a mean protein binding of 75.5% (Viaanen et al-, 1980). The plasma half-life of the dmg has been estimated to be 8-24 hours, which may be considerably extended in overdose (Faulkner et al., 1983, Midha et al., 1992, Virtanen et al., 1980, Wecker et al., 1986, Ziegler et al. , 1978).

14.0. Cornparison of pharmacological effects between Z and E- doxepin Although Z-doxepin is present to the extent of 15% in the mixture resulting fkom acid-cataiyzed equilibration in the final step of the synthesis (Hobbs, 1969), it is therapeutically superior to its E-isomer since in most in vitro and in vivo tests, the 2- isorner is the more potent of the two geomehic forms (Ross, 1984, Schaumann and Ribbentrop, 1966). The pharmacological effects of Z- and E-isomers of doxepin have been compared in some pharmacological test systems. It has been demonstrated that 2-isomer is generally somewhat more potent than E-doxepin (Pinder et al., 1977). Didesrnethyldoxepin 2-Hydroxydesmethyldoxepin Desrnethyldoxepin-2- hydroxy-O-glucuronide 1

Desmethyldoxepin Doxepin 2-Hydroxydoxepin

Scheme 1.1. Phase 1 & II metabolic pathways of doxepin in humans and/or animals 1.4.4.1. Reserpine antagonism It is reported that 2-doxepin was three times more potent than E-doxepin in reversing the hypothermia produced by reserpine in rnice (Schaumann and Ribbentrop, 1966).

1-4.4.2. Amphetamine stereotypy Amphetamine induced stereotyped behavior in rats was enhanced by 2-doxepin, whereas E-doxepin tended to antagonize that behavior (Otsuki et al., 1972).

1.4.4.3. Potentiation of hexobarbital induced sleep and urethane induced sedation Hexobarbital induced sleep (Otsuki et al., 1972) and the urethane induced sedation (Schaumann and Ribbentrop, 1966) in mice were both potentiated slightly more by Z- doxepin than by E-doxepin.

1.4.4-4. Histamine antagonism 2-doxepin was found to be three and hdf hesmore potent than E-doxepin in antagonizing the histamine induced contractions of isolated guinea pig ileum (Otsuki a al., 1972).

1.4.4.5. 5-EIT antagonism 2-doxepin was seven times more potent than E-doxepin in antagonikg 5-HT induced contraction of isolated guinea pig ileum (Otsuki et al., 1972).

1.4.4.6. Anticholinergic effects

The anticholinergic action of 2-doxepin was found six to seven thes more potent than that of E-doxepin as measured by the mydriatic effect in mice (Otsuki et al., 1972, Schaumann and Ribbentrop, 1966) or by the antagonism of metacholine toxicity

(three to four times) in mice (Otsuki et al., 1972). Wbile contractions of isolated guinea pig ileum by acetylcholine in vitro were also antagonized four times more potently by 2- than by E-doxepin (Otsuki er al., 1972).

1.4.5.0. Desrnethyldoxepin and its clinicaI importance As one of the important metabolites of doxepin, desmethyldoxepin (Figure 1.1.) also appears in the blood strearn shordy &er oral administration of doxepin. It is well known now that desmethyldoxepin possesses antidepressant activity and probably plays an important role in attaining therapeutic response as that of parent dmg since in most clinical efficacy studies desmethyldoxepin is associated with phamacodynamic evaluations. It has been suggested that plasma concentrations of (2 plus E) doxepin and (2 plus E) desmethyldoxepin show better correlation with antidepressant activity than (2 plus E) concentrations of the parent dmg alone (Faullcner et al., 1983, Friedel and Raskind, 1975, Green, 1978, Pinder et al., 1977, Ribbentrop and Schaumann, 1965, Ward et al., 1982). The mean protein binding value of desmethyidoxepin (76.0%) is sirnilar to that of doxepin by equilibriun dialysis (Virtanen et al., 1980). It is extensively distnbuted throughout the body with a plasma half-life longer than that of doxepin (Fauikner et al., 1983, Midha et al., 1992, Virtanen et al., 1980, Wecker et al., 1986, Ziegler et al., 1978).

1.4.6.0. Pharmacokinetic studies of doxepin and desmethyldoxepin A review of the literature reveals that only a few doxepin pharmacokinetic studies have been carried out in humans due to the low dmg level in plasma or senun and lack of availability of sensitive and validated bioanalyticai methods (Faulkner et nL, 1983, Rao et al., 1996, Virtanen et al., 1980, Ziegler et al., 1978), while single dose stereoselective pharmacokinetic studies in humans pose an even more difficult task simply because of the extremely low plasma or semm concentration of 2-doxepin caused by the commercial mixture of geome~cisomers. Up to now, there is only one published single dose stereoselective doxepin pharmacokinetic study in healthy volunteers Wdha et al., 1992). Unfominateiy plasma concentrations of 2-doxepin were so low in this study that it was possible to monitor 2-doxepin levels in only 3 out of the 29 subjects. In those 3 individuals, however, the Z:E ratio for the parent hgremained similar (=15:85) to that in the commercial dosage form adrninistered.

1.4.7.0. Ratio distortion in desmethyldoxepin In 1978, Rosseel reported the first stereoselective quantitative GLC analytical method for simultaneous quantitative detemunation of the Z- and E-isomers of doxepin and desmethyldoxepin in human plasma (Rosseel et al., 1978). The most interesting outcome fiom this study is, however, the observation of a significant ratio distortion in 2-, E- isomers of desmethyldoxepin, where it was claimed that plasma

Ievels of 2-desmethyldoxepin in two fernale patients were similar to, or higher than, those of E-isomer, despite the fact that the Z:E ratio of the parent dnig in plasma remained at approximately 15: 85. Since then, other study reports, using various isomer discrimination assay methods, have confirmed this phenomenon in either patient or healthy volunteer's plasma and/or urine. Those found it in patient's plasma were reported by Bogaert and Hrdina Pogaert et al., 198 1, Hrdina et al., 1990); while those discovered in patient's urine and senun were reported by Shu and Adarnczyk, respectively (Adarnczyk et al., 1995, Shu et al., 1990a). A similar distortion in the Z:E ratio of desmethyldoxepin isomers in plasma was observed in a single dose pharmacokinetic study in male healthy volunteers (Midha et al, 1992). Since oral administration bas been traditionally the only dosing route in clinics (Adler et al., 1997), and dl stereoselective studies published to date were carried out via oral administration, it is not clear yet if there would be any effect of route of adminiseation on the ratio distortion.

1.5.0.0. Stereoselective methods for quantification of doxepin and desmethyldoxepin There have been several published methods of stereoselective analytical procedures for 2- and E- isomers of doxepin ador desrnethyldoxepin, including gas liquid chrornatographic (GLC) methods (Hrdina et al., 1990, Midha et al., 1992, Rosseel et al., 1978); gas chromatography-mass spectrometric (GC-MS) method

(Ghabnal et al., 199 1); and high performance liquid chromatographic (HPLC) methods (Uilger et al., 1988. Li-Wan-Po and Ir&, 1979, Midha et al., 1992). Recenîiy the fit HPLC procedure that pemiitted sirnultaneous, stereoselective quantification of both doxepin and desmethyldoxepin geomeûic isomers was reported

(Adamczyk et al., 1995) although the rnethod lacked the sensitivity required for single dose pharmacokinetic studies with lower limits of quantification of 10 ng/mL for doxepin and 5 ng/mL for desmethyldoxepin in a reverse phase system. The quantitative analytical procedure developed by the author (Yan et al.. 1997a) is a sensitive and specific normal phase HPLC method which separates and quantitates Z- and E- isomers of both doxepin and desmethyldoxepin in a single run with lower limit of quantification of 1 ag/mL for each of the four analytes. By cornparison with the method of Adamczyk and CO-workers,the present procedure for sample preparation saves tirne and gives higher sensitivity. Although a back extraction step was employed in both methods, the time needed for extraction (20 min) and separation (5- 10 min) in the present study was less compared with respective values of 60 and 30 min in the earlier method of Adarnczyk et al., 1995. Moreover, concentrated extracts were reconstituted with mobile phase in a single step in contrast with the two-step procedure used by Adamczyk and CO-workers. Furthemore, the column required ody 1-2 h of equilibration time as opposed to 12 h or more in the earlier method.

1.6.0.0.0. High performance iiquid chromatography High perfo-ce iiquid chromatography (HPLC) is perhaps the fastest growing mernber of an entire fafnily of separation techniques. It is essentially a chromatographie method that is analogous to gas chromatography (GC) . The chief distinguishg feature of HPLC over GC is that al1 sarnples are separated in the Liquid phase and not as vapor. Consequently, it is able to analyze substances that are unsuitable for GC due to non-volatiliv or thermal-instability. In many instances compounds that previously required chernical derivatization for GC analysis can now be chromatographed by HPLC without any prior treatment. Quantitative analyses are easily and accurately performed and errors of less than 1% are cornmon to most HPLC methods. Depending on the sample type and detector used, it is frequently possible to measure 1 ng (nanogram) of sarnple. With special detectors, analysis down to pg (picograrn) concentrations is possible (Lough and Wainer, 1996). WLC employs a column packed with some solid material which is coated with a stationary phase through which is percolated a liquid mobile phase. Unlike GC, HPLC mobile phase can play a major role in the separation of compounds. The mobile phase can be highly organic, highly aqueous, low or high pH, buffered or unbuffered. The exact composition of the mobile phase depends largely on the fünctional group chemistry of the compounds king separated and the type of stationary phase in use. The stationary phase will separate compounds primarily on the basis of one of four mechanisms: 1) adsorption to the solid surface; 2) sieving or separation by molecular size; 3) padtioning of compounds between two liquid phases; 4) sekcted affinities such as ion exchange or selective binding. For the partitioning rnechanism, the term "normal phase" is used to indicate that the stationary phase is a polar adsorbent and the mobile phase is generally a mixture of non-aqueous solvents, whereas, "reverse phase" indicates that the mobile phase is more hydrophilic and the stationary phase is more hydrophobie.

1.6.1.0.0. aPLC instrumentation The essential components of a HPLC consist of the following seven parts.

1.6.1.1.0. Solvent reservoir It is a storage container of a material resistant to chemical attack by the mobile phase. A solvent bottle or flask with a cap and flexible hose connection to the pump is adequate. In more sophisticated instruments the solvent reservoir may also be equipped for solvent degassing (Kissinger, 1989).

1.6.1.2.0- Solvent pump The pump is one of the most important components of the Liquid chrornatography, often said to be the heart of the system (Noctor, 1996). Other components such as the check valves, flow controllers, rnixing chambers, pulse dampeners and pressure transducers may be associated with the pump operation. An additional feature found on the more elaborate pump is extemai electronic control and is a very desirable feature when automation or controlled gradients are to be run. This becomes an unnecessary expense when using only isocratic methods. The general requirements for a modem pump in liquid chromatography include (Andreolini and Trisciani, 1990, Brooks et al., 1987, Trisciani and Andreolini, 1990): Constant volume delivery independent of back pressure, to facilitate qualitative and quantitative analysis Stable, reproducibly precise flow without pulsation to minimize detector noise and providing reproducible chromatograrns Amenable to high pressure and being capable of operation at the flow rates demanded by the various HPLC modes Easily adaptable to gradient operation Facility to compensate for solvent compressibility

1.6.1.3.0. Sample injector It is a port which allows application of a sample for separation on to a HPLC column without having to stop the pump and open the top end of the column. Ideally, the sample ought to be introduced as an infinitely narrow band on to the column. There are two methods generally used. The fust method makes use of a micro syringe designed to withstand high pressures (Berry and Karger, 1973). Tne sample is injected, through a septum in an injection port directly on to the column packing. This can be done while the system is under high pressure, or the pump may be turned off before injection and when the pressure has dropped to near atmospheric, the injection is made and pump switched on again (stop-flow injection). The second method is by the loop injector (Berry and Lawson, 1987, Coq et al., 198 1). This consists of a metal loop of small volume which can be filled with the sample. B y means of an appropriate valve, the eluent fiom the pump is channeled dvough the loop, the outlet of which leads directly on to the column. The sample is thus flushed on to the colurnn by the eluent, without interruption of solvent fiow to the column. Nowadays, automated instrumentation has become more and more popular. Autoinjector, incorporated into many integrated HPLC models, offers programmable injection volume for up to over one hundred vials. The autoinjector is usually cornbined with an autosampler which permits any number of replicate injections per via1 and fast random access to any via1 (Noctor, 1996). 1,Q.1.4.01 Precolumn filter It retains any particles larger than 3-5 pn in size such as salt crystds, dust, or parts of the pump or injector seais so as to prevent damage to the stationary phase in the column.

1.6.1.5.0. Column It varies depending primariiy on the type of separation desired. With the exception of columns for gel permeation and ion exchange, the most popular colurnn length ranges from 10 to 30 cm. Analytical columns have intemal diametes of 3 to 5 mm and are precision bore stainless steel. To dmize band-broadening and thereby to Mprove column efficiency, it is necessary to use smail diameter particles for the column packing. Currently, most HPLC separations are carried out with 5 pm diarneter packing materials with material of 3 pdiarneter also king readily available.

Particle sizes as smd as 1 p may be used, although theu application is somewhat more speciaiized and the normal range of particle sizes used in analytical application with conventional columns is between 3 pm and 10 jun (Harnbleton, 1996).

1,6.1.6.0. Detector The purpose of the detector in a HPLC system is to identiS. the presence of certain compounds of interest in the eluent &om the HPLC column. Depending on chernical and physical properties of the compounds being separated there could be a varïety of choices of HPLC detectors for use. Generally speaking, the followings are some of the comrnon systems in use (Lloyd, 1996, Parriott, 1993, Poole and Poole, 1991, Vicker-y, 1983, Yeung and Synovec, 1986): 1.6.1.6.1. UV detector

As most organk compounds have some absorption in the UV region, these detectors are fairly universal in application, although sensitivity depends on how strongly the sample absorbs. Most detectors provide an output in absorbance units which is hearly related to sample coficentration. Detection Iimits are in the Iow to subnanogram range in favorable circumstances. The most commonly used is the continuously variable wavelength detector. Wavelengths are either selected manually in the range of 190-700 nm or in an automated version, the detector may be progmmmed to change wavelength during the separation and scan the complete spectnim of any peak. The detector ceU must be designed carefully as it forms an integral part of the chrornatographic and optical system. The ce11 volume should be as smail as possible to reduce contribution to extra colurnn band broadening (Dark, 1986, Yeung and Synovec, 1986).

1.6.1.6.2. Photo diode-array detector The introduction of diode array based detectors for HPLC has added a new dimension to liquid chromatography. With conventional uv/vis detectors only one wavelength at a time can be monitored. Since different analytes generally have differing absorbance maxima, the chosen wave length inevitably compromises detector sensitivity. With multichannel detectors, however, the absorbance profiles of the eluting analytes are measured over a spectrurn of wavelengths, producing a three dimensional coordination of absorbance against time (spectro-chromatogram). Thus, both chrornatographic information at any available wavelength and spectral information at any retention time are available simultaneously. The heart of the detector is the linear photo diode array which consists of a light-sensitive silicon chip compnsing a number of photosensitive diodes. Light fiom the uv/vis source passes thïough the sample ceil and into the polychrornator, where the array is positioned such that the full spectrum fdls on the diode. The Light incident on the diode sensing area generates a charge that is collected and stored during integration penod. The accumulated charges are then measured by rnicroprocessor (AEedson and Sheehan, 1986, Hayashida et aL, 1990, Jones, 1985, Marr et al., IWO).

1.6.1.6.3. EIectro-chemical detector It includes amperornetric, coulometric and polarographic detectors for the determination of compounds which can be electrolytically oxidized or reduced at a working elecîrode, and aiso the conductivity detectors for the determination of ionic species (Kissinger, 1989, Takeda et ai., 1990). These detectors are selective as not ail organic compounds are electro reduced or oxidized within a useable voltage range. Electroche~cddetectors have hi& sensitivity (picogram level). Detector operation is dependent on fiow rate, solution pH, ionic strength, temperature, ceil geornetry and presence of eletroactive impurities. Gradient elution is not normally possible. Carbon-paste or glassy carbon materials are most comrnonly used for the working electrode which is maintained at fked potential with respect to a reference eleceode. This potentiai is at or near the limiting current plateau for the compound of interest. This may be determined fiom a curent potential recording of the sample. The current is measured at a fixed potential as the sample is eluted fiom the column.

1.6.1.6.4. Fluorescence detector It is inherently more sensitive and selective than uvhis absorbance detectors. Many biologically active compounds, pharmaceutical products and environmental contaminants are fluorescent and therefore can be determined in the picogram range bjr a fluorescence detector. Both pre-column and post-column derivatization procedures are used for the trace analysis of non-fluorescent compounds. Detectors for fluorescence monitoring differ in the method used to isolate the excitation and emission wavelength. Either filters or monochromators are used for this purpose. Mercury, deuterium, tungsten or xenon sources are commonly used to provide excitation wavelength (AEedson and Sheehan, 1986, Jones, 1985).

1.6.1.6.5. Mass spectrometer detector The growth of LC-MS within the past few years has been tnily ovmvhelming. With considerable technical improvements, more and more LC-MS interfaces have been introduced into practical uses. This development has made possible a number of LC-MS approaches formerly not amenable to MS coupling, especially der the rev~lutionbrought by the introduction of the new liquid-phase atmospheric pressure ionization (MI) techniques, such as electrospray (ES) and ionspray (Hopfgartner et al., 1993, Poon et al., 1993).

1.6.1.7.0. Integrator The detector gives an electronic output signal related to the composition of the HPLC eluent. It is the job of the last element in the chain of HPLC instrumentation to display and allow the quantification of the peaks in chromatogram (Lloyd, 1996). Today these functions are usually performed by an eiectronic or a computer-based integrator. The signal fiom the detector is f~stdigitized by the integrator, typically fiom 50 to 1000 times each second (the sampling frequency). The digitized data are analyzed to recognize the presence of peaks, their start and finish, and to calculate peak height and area.

1.7.0.0. Mass spectrometry Mass spectrometers (MS), in their simplest forms, are designed to perform three basic functions. These are (1) to vaporize compounds of widely varying volatility, (2) to produce ions fkom the neutral molecules in the gas phase, and (3) to separate ions according to their mas-to-charge ratios (dz), detect, and record them (Howe et al., 1981). Generally speakùig, a mass spectrometer is composed of the following components. (i) Met system; (ii) Ion source; (üi) Analyzer (separation of ions); (iv) Detection of ions; (v) Recording of ion amivals (Rose and Johnstone, 1982). In mass spectrometry, a substance is characterized by investigating the chemistry of ions resulting fÏom that substance. The region of the mas spectrorneter where ions are generated is called the ion source. A variety of techniques may be applied to ionize motecules of vaporized sample. With regard to the limited use of mass spectrometry for this thesis work, only the foilowing two ionization techniques are described because they were applied directly during the course of implementation of this research project.

1.7.1.0. EIectron impact ionization In electron impact (EI) pattern of ionizing molecules of the vaporized sample, the substance to be examined are introduced, as a vapor, into the source at the operating pressure (ca. 1om6ton). The vapor is ailowed to pass through a slit into the ionization chamber, where it is bombarded with a beam of electrons accelerated fiom a hot filament. The energy of the electron beam cm be varied fiom 0-100 eV. The ionization energies of most organic molecules fdl in the range 7-1 3 eV (1 eV = 23 kcal mol-' = 96 kJ mol-'); consequently, it is possible to supply energy in excess of the ionization energy. The ionization energy corresponds to the energy required to remove an electron fiom the highest occupied molecdar orbital; this reaction is represented as fo Ilows: The energy given to the molecules of vapor, by the ioniPng electrons, is usudly greater than the ionization energy. Therefore. excess energy remains in the molecular ion (MC) and this excess energy may be used to break one (or possibly more) bond(s). Depending on the energy content, molecular ions may remain as such or dissociate into fiagrnent ions (Howe et al., 198 1). Further fragmentation is possible depending on interna1 energies in fragment ions. By reducing the energy of the ionizing bearn of electrons, the relative abunc'ance of the moIecular ion cm be increased. When this eiiergy approaches the ionization energy of the molecule, the excess energy remaining in the molecular ion is smdand hgrnentation is decreased-

1.7.2.0. EIectrospray ionization Although electrospray ionization (ES0 mass spectrometry has been introduced relatively recently, it is gainhg rapid acceptance and popularity in the analysis of biopolymers, drugs, dmg metabolites and chernical compounds. In ES ionization, two electrodes are king used, me hollow capillary tube and the other counter electrode, which are placed 0.3-2 cm away fiom exh other and are kept at atmospheric pressure (Chapman, 1993). nie electrospray source is typically a metal capillary incorporating a method for electncdy biasing the liquid solution relative to counter electrode. The ions are generally produced with very high efficiency in ES. The sample solution, ty pically water-methanol mixtures containing the analyte and other additives, fiom infusion pump, HPLC, or autosarnpler etc. is passed through steel capillary tube and a potential dflerence of 3-6 kV is typically applied between capillary and counter electrodes. The hi& electric potential between the electrodes causes the liquid at the tip of capillary tube to be dispersed into a fine spray of charged droplets containing analyte, electrolyte and solvent molecules. The droplets gradually becomes smaller and smaller as the solvent molecules evaporate and ion (analytes) evaporation takes place into the surroundings. Gas phase ions formed at atmospheric pressure are then sampled through a two stage momentum separator into the high vacuum of mass andyzer (F~M Meng, 1991, Smith et al., 1990). With the assistance of pneumatic nebulization, stable operation in ES-MS can be obtained for flow rate of 2- 100 PL/&.

Perhaps, the rnost distinguishable feature of ES1 techniques is its ability to produce ions with multiple charges, opening up the area of high mas analysis to the currently existing instruments. This is a consequence of the fact that a mass spectrometer measures dzratio but not mass alone, thus a molecde having a molecular weight of 10,000 that carries 10 charges will be recorded at m/z 1000. In ESI, large molecules such as protein do not just carry one charge but normally show a distribution of charges so that mass spectra display typically 5-1 0 molecular species, each differilg fiom the adjacent peak in spectrum by one chargz. The ion pattern in spectrum cmthen be deciphered to provide the molecular weight of the compound (Whitehouse et al, 1985). Multiple charging in ES1 leads to a whole series of peaks for a single compound, making spectra look unusual and cornplex. However, the coherence of the series of peaks cm actually be taken advantage of. The charge of the molecules cm be determined in a straight forward manner fiom any two peak positions aven several assumptions. The fxst is that adjacent peaks of a series differ by only one charge. Second, it is often assumed that the charging is due to proton attachent (for ES+ mode) to the molecular ion. Finally, it is assumed that ionization occurs only for intact molecules Penn et al., 1989). 1.8.0.0.0. Computational methods in pharmacokinetic studies

1.8.1.0.0. Mode1 independent pharmacokinetic approaches One important strategy used to treat pharmacokinetic data is the so-called "model- independent", "noncornpartment" approach. Formerly, this was referred to as statistical moment analysis. The conceptual basis of this approach is that no zssumptions are made about the underlying structure of the model. These methods are usually based on the estimation of the area under a plot of dmg concentration versus time (AUC). Noncornpartmental methods have been used to estimate the primary pharmacokinetic parameters such as bioavailability, clearance, apparent volume of distribution (Gibaldi and Pemer, 1982). Since the analyses focus on the estirnation of clearance and volume of distribution, Iess uiformation about the mechanisms of drug disposition could be obtained with this approach. The tirne course of dmg concentration in plasma can usually be regarded as a statistical distribution cuve (Himmelblau and Bischoff, 1968). Irrespective of the route of administration, the frst two (zero to f~st)moments are defmed as follows:

AUC = J-C dt O

- AUMC AUC

Where AUC, MRT are termed the zero and first moment, respectively, of the drug concentration-time cwe. AUMC refers to the area under the first moment curve. Once the AUC and MRT are calculated by the method of statistical moments, other pharmacokinetic parameters mentioned above can therefore be obtained. For extravascuiar administrations, bioavailability refers to the fraction (F) of a dose that reaches the systemic circulation intact. If a hgis given by oral administration, the following equation is used for calcuiation of F:

Div AUCotai F = Dora~ AUC iv

This equation assumes equal clearances in the oral and intravenous studies. Clearance (CL) is one of the most important parameters to describe the pharrnacokinetics of a drug. The CL is defmed as the volume of the dnig's distribution that is "cleared" in a unit of time. The equation for calculation of CL der single intravenous injection is as follows:

D iv CL= - AUC

The apparent volume of distribution at steady state (V,,) is simply the product of clearance and mean resident time after a single intravenous bolus dose of a dnig.

V,, = CL - MRT

1.8.2.0.0. Mathematical modeling of pharmacokinetic data Phamacokinetics cm be simply defined as the use of mathematical models to quantitate dnig concentrations in an animal (Riviere, 1997). The idea of fitting mathematical models to a data set cm be thought of as putting a calculated he through the observed data points (Bourne, 1995). Pharmacokinetic models are used to describe the the-dependent distribution and disposition of a substance in a Living system and, as such, have numerous uses in clinical applications and dmg design (Gerlowski and Jain, 1983). Experimental results coveriug pages of tabular data may be represented with a mode1 description and a few parameter values (Borne, 1995). However, it is necessary to indicate that it would be inappropriate to classi& a mode1 as being correct or incorrect, instead models should be judged as to how accurately they cm predict dmg concentrations under new exposure situations. There are numerous modeling paradigms available for quantitating phannaco kinetic data. Mo s t of these rnodels can be classified on the basis of whether the mathematical analysis makes any irnplicit assumption about the mechanism of the disposition of the dmg king studied. For example, using plasma data to rnake inferences about rend clearance of the hgrequires fust that assurnptions be made as to the excretory pathway of the hgand second, to mathematically define "via a moder' that determinations be made as to how the concentration-thne data are to be "linked" to rend clearance. The approaches to quantitating these linkages include comparûnentd and physiological models. Today as a result of great advances in microcomputer technology, more and more user-fnendly and hi& gmphics-onentated versatile pharmacokinetic programs are available on the market. Compartmental analysis-based pharmacokinetic mode1 fitting and noncornpartmental pharmacokinetic approaches are now best carried out with compter programs, the use of which has become routine in many laboratones.

1.8.2.1.0. Compartmental models The most widely used modeling paradigm in preclinical and clinical pharmacokinetic (PK) studies is the cornpartmental approach. In this analysis, the body is viewed as being composed of a number of so-called equilibrium compartments, each defined as representing specific body regions where the rates of dmg decay are of a similar order of magnitude. The elimuiation of a dmg fkom such a compartment occurs at a constant rate. These compartments are thus defined and grouped on the basis of sunilar rates of drug movement witbin a heterogeneous groups of tissues and not on anatomical or physiological principles (Riviere, 1997).

1.8.2.1.1. One compartment model One compartment model is the simplest in this approach, in which the body is considered a single homogeneous compartment, Le., drug is assurned to move into and within the body at a single rate. The equation for intravenous administration using this model is as follows:

Concentration = Co e-"

In Concentration = ln Co - kt

Where Co is the concentration at the O (units of mass/volurne), k is the dope (units of Utime), and t is the the after injection. A number of usefül pharmacokinetic parameters may be defined fkom diis simple analysis. The first is the elimination half- life (tin), which is defined as the time it takes for one haif (50%) of the administered dmg to be eliminated fkom the body. Ha-life may be calculated as:

The second parameter that can be easily calculated korn this simple analysis is the volume of distribution (V) (unïts of volume): The V is the effective volume hto which the dose distributes at time O to result in the observed concentration in an accessible body fluid. This value could be viewed as a proportionately constant relating the amount of hgin the body to the observed concen~ation;it descnbes the apparent volume into which a drug is dissolved. The body clearance (CL) (uni& of volume/time) of the hgcan be easily calcdated once the k and V are obtained:

If a hg were eliminated by both the kidney and liver (biliary and/or biotransformation) only, the observed CL would be the sum of the contribution of both organs. One important point should be made clear that the V and CL are both independent parameters, while k and tlB are physiologically dependent on both V and CL of the drug. From this biological perspective, the true relationship is:

It is important to stress the reason why intravenous pharmacokinetic studies should dways be used to define a species' dmg disposition. If an extravascdar route of administration is used, the investigator can never be guaranteed that the plasma profile is not dependent upon a slow and thus rate-limiting absorptive process due to a formulation factor or the nature of the dmg. TIn may therefore be overestimated. Complete absorption can not be assured either, thus one never truly knows the size of the administered dose. Without knowing dose, neither V nor CL can be accurately estimated.

1.8.2.1.2. Two compartment model Aithough a one compartment model is simple and maight forward, unfominately, most dmgs are not descnbed by it because the log transformed plasma concentration- thne profde is not a straight line. In reality, the body is not a single homogeneous compartment; instead, it is composed of regions that are defmed by having different rates of hgdisposition. Such a situation is reflected in the two compartment model. In the two compartment model, the body is hypothetically divided into two compartments, the compartment 1 (central), which norrnaliy represents the well perfused body regions, and the compartment 2 (penpheral), which usually represents poorly perfused body regions. The dmg initially is distnbuted in the central compartrnent and by definition is elirninated fiom this compartment. Now the hg also moves into other body regions, i.e., compartment 2 at a rate that is different from that of the central compartrnent. Two rate constants govem this movement the rate out of compartment 1 and into compartment 2, and the rate back into compartment 1 fiorn compartment 2. In analyzing a two compartment model after intravenous administration, the fundamental principle involved is that the observed serum or plasma concentration-thne profile is actually the result of two separate phannacokinetic processes, which can be described by two separate exponential terms :

Where C is the plasma concentration at tirne t, A is the zero time intercept associated with the alpha phase, a is the rnacro rate constant associated with the distribution phase, B is the zero the intercept associated with the beta phase, and P is the macro

rate constant associated with the elimination phase. By definition, CC »P.

The finai level of cornpartmental model complexity encountered is the three cornpartment model. The dnig now distributes into two more different compments (e.g., rates of distribution are different). The third cornpartment is usually a so called deep compartment characterized by very slow rate constants. Again, dmg is always eliminated fkom the central comparûnent and by convention, a dose is always administered into the central compartment. The equation required to hanclle three multicompartmental model gets more complex.

1.8.2.2.0. Physiological models There are numerous other approaches to pharmacokinetic modeling that would have applications to different animal species. The most prornising strategy to this area would be the use of physiologically based models. Physiological modehg is different from classical cornpartmental modeling, which is ernpirical in nature. In this approach, the body is descnbed by anatomically distinct organs (e.g., liver, kidney) and tissue groups (e-g., muscle, fat) connected by the circulatory system. Each physiologie compartment is then linked to form a whole body system. The dnig is then described as partitionhg between the blood and the tissue, quantitated as the partition coefficient (FC = concentrationti,e/concen~ationbi,,d). A senes of differential equations are written using mas balance techniques to describe the rate of change of dmg concentration in the blood and in each organ. The complexity of each organ's equation will be a hction of how much is known about the disposition in that organ. Experimental data are then used to solve the unknown parameters and to partition the dmg between rnodel components. In these experirnents, multiple doses are often used. Animais must be euthanized at different time points, and dmg concentrations must be assayed in the various tissues cornprising the model. Specific organs not included are lumped into undefined residual organs. These studies are experimentally more cornplex, and the mathematics are easily hded in various simulation programs. The major advantage of this approach is that interspecies scaling is easy because species-specinc anatomy and physiology may be iocorporated hto the mode1 (Colbum, 1988, Gerlowski and Jain, 1983, Riviere, 1997). CHAPTIER 2

2.0.0.0. RATIONALE AND OBTEC'PIVES

2.1.0.0. Experimental results and speculations for the mechanism of the ratio distortion There are several mechanisms that could explain the obsewed distortion in the ratio of desmethy 1 isomers includuig: (i) stereoselective metabolic clearance of E- desmethyldoxepin; (ii) stereoselective renal clearance of desmethyldoxepin; (iii) higher rate of formation of 2-desmethyldoxepin than E-desmethyldoxepin; (iv) metabolic interconversion of E- to 2- isomer. Experbnental evidence for the latter possibility was reported in a study in which a mixture of 25 mg of 2-doxepin and 25 mg of deuterium labeled E-doxepin was administered to 8 healthy volunteers (Ghabrial et al., 1991). Whereas there was some evidence of stereoselective renal clearance of doxepin, there was no apparent interconversion between the isomers of the parent dmg. There were, however, traces cf deutenum labeled 2-desmethyldoxepin detected in senun and urine der administration of deuterium labeled E-doxepin. It has been suggested (Ghabnal et al., 199 1) that E and Z interconversion might take place by way of a desmethylated intermediate metabolite in which the exocyclic double bond is hydrated and subsequently dehydrated to provide an opportunity for the formation of both 2- and E- isomers of desmethyldoxepin. In a separate study, on the urinary metabolites of doxepin in patients under medication with the dmg, mass specaometric evidence was obtained for the presence of a hydroxy desmethyldoxepin in which the exocyclic double bond was hydrated (Shu et al., 1990a). 2.2.0.0. Hypothesized process of E- to 2- isomerization Based on these reported observations, we postulated a possible process for the conversion of E- to Z-isomer of desmethyldoxepin (Seheme 2.1.). As shown in Scheme Z.I., loss of the hydroxyl ion fiom the hydrated metabolite would give a carbonium ion in which H-bonding transannuiar interaction between the protonated amino group of the side chain and the oqgen function of the oxepin ring is possible. Subsequent loss of a proton to regenerate the exocyclic double bond would then Iead to the formation of the 2-isomer in which the side chain remains on the same side as the oxygen atom of the oxepin ring. In a similar carbonium ion intermediate fkom the parent drug doxepin, molecular models suggest that the presence of the second N- methyl group may provide sufncient steric hindrance to hamper such interaction between the protonated amino group and the ring oxygen. Therefore, there would be no preference for the reformation of either isomer of the parent dmg, and no net change in Z/E ratio.

2.3.0.0. Clinical significance of the possible 2-isomer enrichment Obviously, it would have therapeutic signincance if the rise in 2- desrnethyIdoxepin ratio is attributed to the "enrichment" of the Z-isomer at the expense of its geometric antipode, since Z-isorner of the dnig is pharmacologicaily more potent than the E-isorner and desmethyldoxepin is considered to contribute in the treatment of depression as mentioned before (sections 1.4.4.0. - 1A.5.O.). If so, the E- to Z- isomerization must be taken into account, as partial contribution to the overall therapeutic effect, when clinicai phmacodynamic evaluations are to be carried out. On the other hand, this factor of complexity in kùietics needs to be kept in mind if the clinical stereoselective pharrnacokinetic profiles of desmethyldoxepin are to be appropriately explained. 2-Desrnethy ldoxepin

Scheme 2.1. Hypothesized process of E- to 2- isomerization 2.4.0.0. Rationale of the research project The above hypothesis of E- to Z-isomer conversion sounds plausible with the dues from the experimental outcomes cited previously (section 2.1.0.0.). The ciinical signincance accompanyhg this possible mechanism has aiready been stated. Thus, Merexploration of this concept by carrying out an investigation that would cl&@ issues such as the mechanism of the ratio distortion and the effect of route of administration on the ratio distortion is warranted. Other questions such as oral absolute bioavailability and stereoselective pharmacokinetics of the doxepin isomers wiil aiso be explored.

2.5.0.0. Specific objectives of the present thesis work The implementation of this research project comprises several stages of development in a progressive manner. It covers metabolic and pharmacokinetic studies in both prec1Ïnica.l and clinical settings, and both i~ vivo and in vih-O conditions. For an explicit description of objectives correlated to various research plans at diKerent stages, the following sections are written out.

2.5.1.0. Stereoselective, sensitive and simultaneous EWLC method Pnor to the present studies, there was no published HPLC method that permitted simultaneous, stereoselective quantification of both doxepin and desmethyldoxepin isomers, although there hacl been two publications of discriminative HPLC methods for assay of doxepin isomers (Li-Wan-Po and M,1979, Midha et al., 1992). Dilger et al. reported a HPLC method for sensitive and selective determination of E-doxepin and desmethyldoxepin in 1988 @figer et al., 1988), but this method is not stereoselective for desmethyldoxepin isomers. Hence, the first task in the present studies was to develop a convenient, sensitive, simultaneous, and stereoselective HPLC method for the investigations of mechanistic aspects of the ratio distortion and of pharmacokinetic profiles of the four isomers.

2.5.2.0. Confirmation of the ratio distortion and selection of an animal model After a sensitive E-PLC method for simultaneous quantification of the isomers of doxepin and desmethyldoxepin was developed, the second and third steps were to confum the reported ratio distortion cf desmethyldoxepin in humans and to identfi a suitable animal model. The purpose of the preliminary animal tests was to hdan animal species in which the percentages of 2-doxepin and 2-desrnethyldoxepin were similar to those in humans so that Merexperiments of mechanistic study would be carried out in this particular species to avoid restrictions often encountered in carrying out human studies.

2.5.3.0. Investigation for possible mechanisms of the ratio distortion

2.5.3.1. Effect of route of administration on the ratio distortion It was planned to investigate in the animal model and in hurnans the effect of route of administration on the distortion of the isomers of desmethyldoxepin.

2.5.3.2. Test of E- to Z- isomer conversion in vivo Observation of 2-desmethyldoxepin in the plasma and/or urine after administration of E-isorner wouid cobthe suggested mechankm of isomer interconversion. On the other hand, failure to observe Z-desmethyldoxepin afier E- isomer administration would suggest other mechanism are responsible.

2.5.3.3, Test of isomer interconversion in vitro Ln the same sense as that in vivo, separate incubation of pure isomers with Liver microsornes would help elucidate the mechanism of isomeric distortion since appreciable amount of 2-desmethyldoxepin would be detected in incubates of E- isorners if liver is involved in the interconversion.

2.5.3-4. In vitra metabolic inhibition of desmethyldoxepin Inhibition of biotransformation pathway(s) of desmethyldoxepin would further support the responsible mechanism developed fiom the studies described above (sections 2.5.3.2. - 2.5.3.3.). If the mechanism was simply the E- to Z-isomer conversion, the ratio distortion would not be affected by enzyme inhibition because the proposed process (section 2.2.0.0.) of isomerization would probably not be enzyme mediated. On the other hand, if the rnechanism was not isomerization but another mechanism such as preferential metabolic clearance of E-desmethyldoxepin, the reduced elimination rate in the biotransformation pathway(s) responsible would curb the development of the ratio distortion so that it would occur to a lesser extent. 2.5.4.0. Pharmacokinetic studies in vivo

2.5.4.1. Stereoselective pharmacokinetic studies in humans As mentioned previously stereoselecnve pharmacokinetics of doxepin and desmethyldoxepin has been far iess studied due to unavailability of suitable analytical approaches. At the time of commencement of this project there was only one stereoselective pharmacokinetic study of doxepin and desmethyldoxepin published, due to extremely low plasma concentrations of 2-doxepin encountered it was possible to monitor Z-doxepin levels in only 3 out of the 29 subjects (Midha et al, 1992). Moreover, there were no data on the uitravenous pharmacokinetics of doxepui. Hence, it was planned to cany out a two-treatment, randomized cross-over intravenous versus oral single dose pharmacokinetic study in healthy volunteers.

2.5.4.2. Stereoselective pharmacokinetic studies in dogs Although dogs had been used in animal disposition and pharmacodynarnic studies of doxepin (Brogden et al., 1971, Hobbs, 1969, Pinder et al., 1977), there had not been any report published on stereoselective pharmacokinetic profiles of doxepin and desmethyldoxepin in dogs. It was plaaned to conduct a randomized cross-over stereoselective p harmacokinetic studies in dogs with both oral and intravenous administration of doxepin to elucidate some interesting issues such as the extent of the ratio distortion; the effect of dosing route on the ratio distortion; stereoselective pharmacokinetics of doxepin and desmethyldoxepin; and oral absolute bioavailability. 3.1.0.0. Chernicals and reagents

Doxepin hydrochloride (Z:E = 16:84) and nortriptyline hydrochloride were purchased fiom Sigma (St. Louis, MO, USA). E-Desmethyldoxepin hydrochloride, and 2- and E- doxepin hydrochloride were kindly supplied by Pfizer (Groton, CT, USA). The isomeric purities of the isomers were determined by the HPLC to be: E- desmethyldoxepin 97%; Z-doxepin, 100%; and E-doxepin, 98 2%. 2,2,2,- trichloroethylchlorofonnate, N,N-diis~propylethylarnine~triethylarnine, nonylamine, MCdust, and silica gel (60-100 mesh, type 150A) were purchased fiom Aldrich Chernical Company (Milwaukee, WI, USA). Chloroform (CHC13), methylene chioride (CH2C12), tetrahydro furan, ethy lacetate, pentane, hexane, methanol, and isopropanol were purchased fiom Fisher Scientinc (Nepean, Ontario, Canada). Sodium hydroxide (NaOH), sodium sulfate (Na2S03, potassium dihydrogen orthophcsphate (KH2P04), and dipotassium hydrogen orthophospliate 0(IHP04), ammonia (NH40H), and hydrochloric acid (KC1) were purchased fiom BDH Inc. (Toronto, Ontario, Canada). All solvents and chernicals used in the HPLC system and those used in extraction were of analfical grade or higher. Chernicals for in vitro studies with her microsornai and gastrointestinal tract liornogenates were as follows. Glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (GoPD, type XII), f3-nicotuiarnide adenine dinucleotide phosphate

(NADPC), potassium chloride (KCl), oleoyl coenzyme A, probenecid, furosernide, and bovine semalbumin were obtained fiom Sigma Chemical Co. (St. Louis, MO, USA). Magnesium chloride (MgC12), disodium hydrogen orthophosphate (NaJ3POd and sodium dihydrogen orthophosphate (N-POJ were purchased fiom BDH Inc. (Toronto, Ontario, Canada).

Commercially available capsules containing 25 mg doxepin hydrochloride (Z:E =

16:84, ~ovo-~oxe~in~)were purchased fiom the Royal University Hospital (RUH) in Saskatoon for the in vivo human rnetabolic study. In the human pharmacokinetic study, commercially available capsules containhg 85.5 mg doxepin hydrochlonde (75 mg fiee base) (APO-DOXEPIN, APOTEX, Toronto, Ontario, Canada) were purchased fkom the RUH. USP grade doxepin (Geneva Pharmaceutical Inc.) was used for the preparation of iv dosage form which was forrnulated in 5 mg/& hydrochloride aqueous solution by the RUH dispensary. This formulation was shown to be sterile and pyrogen fiee (Nucro Technics, Scarborough, Ont. Canada). The quality of both iv and oral formulations was examined by the HPLC to establish the content of active ingredient. The isomenc ratio in both formulations was deterrnined to be 1694 (Z:E). No detectable impurities were seen on the HPLC chromatograms.

7 Sterilization of solutions intended for parenteral administration to animais was carried out as follows. Glass vials, rubber caps and metal rings were stenlized in an autoclave for 25 minutes at 121°C and 20 psi. Solutions of the various doxepin related hydrochlorides were prepared in sterile normal saline by aseptic transfer under a laminar flow hood. Finally the solutions were passed through a 0.2 micron sterile filter (Coming Glass Works, Corning, New York NY, USA) into a sterile via1 which was seaIed immediately. 3.2.0.0. Instrumentation High resolution mass spectra were detemiined on z VG analytical 70 SQ hybrid mass spectrometer equipped with a PDP 11-230J data system under electron impact (EI+) conditions. The mass spectrometer was operated at 70 eV with an accelerating voltage of 6 kV. The emission curent and the source temperature were held constant at 100 pA and 230°C, respectively. Electrospray (ES? mass spectra were collected on a Fisons Bio-Q mass spectrometer operated at cone voltage of 45 eV and a source temperature of 60°C.

The HPLC system consisted of a Waters WSP Model 712 intelligent sample processor, a Waters Lambda-Max Model 480 W detector, a Waters Model 501 HPLC pump; and either a Shimadni (Kyoto, Japan) Chromatopac C-IUA integrator or a Waters Maxima 820 data system. A 150 x 4.5 mm I.D. column packed with 3 pm Spherisorb silica was used for the separation. Chromatographie conditions were as follows. The mobile phase was an organic solution (filtered and degassed in situ) consisting of hexane, rnethanol and nonylarnine in the ratio of 9550.3 (v/v/v) . The flow-rate was 1.0 rnL/min (back pressure: 70 kg cm-2), the W detector was operated at 254 nm, and the injection volume was 50-100 pL. The chromatography was performed under temperature controlled conditions (23OC).

3.3.0.0. Synthesis of desrnethyldoxepin For the purpose of the reference smple used in the HPLC assay, a mixture of desrnethyldoxepin cis(Z)-, tram@)- isomers was synthesized according to a literature

method (Adamczyk et al., 1992). The synthesis was a two step procedure as shown in Scheme 3.1 .. Scheme 3.1. Synthetic route of desmethyldoxepin

33.1.0. Doxepin free base chloroform solution Doxepin hydrochloride 1.13 g was dissolved in 50 rnL water. To this aqueous solution, was added 2 mL NaOH (2 N) with constant stirring (theoretical neutralkation equivalent = 1.7 mL). The resulting oily opaque suspension was transferred to a separation funoel where it was extracted using chloroform (10 mL x 5). The pooled chloroform solution was fkst dned with anhydrous sodium sulfate, then evaporated in vacuo at 60°C to yield 1 g yellow oil. To this oil, 17.24 mL chloroform was added to form doxepin f?ee base solution, of which 3 mL was equivalent to 174 mg doxepin fiee base.

3.3.2.0. Phosphate buffer solution (0.5 M, pH 5) 50 mL 0.5 M KJ&P04 (3.40 g =O, + 50 mL water) mixed with 50 mL 0.5 M K2HP04(4.36 g K2HP04 + 50 mL water). This solution (pH 6.88) was adjusted to pH 5.0 with 10% HCI.

3.3.3.0. Carbamate formation To a 25 mL flask were added 3 mL doxepin f?ee base chloroform solution (0.623 mol)and 0.25 mL N,N-diisopropylethylamine (1.4 mol). The solution was cooled in an ice-bath at O°C, into which 0.19 rnL 2,2,2-trichloroethylchIorofonnate (1 -38 rnrnol) was added with constant agitation. The reaction mixhire was dowed to corne to room temperature and stood for 3 hours under a continuous flow of nitrogen gas. The reaction mixture was then poured into 25 rnL water, and the pH adjusted fiom 9 to 13 with 2 N NaOH. Extraction was performed using chloroform (20 mL x 2; 10 mL x 1). After the solution was ciried with aahydrous sodium sulfate, solvent was removed by evaporation in vacuo to give crude product which was purifed by column chromatography (2 cm diameter colm, silica gel 40 g, 25% ethylacetate / 75% hexane / 0.1% ûiethylamine; v/v/v). The separated chromatographie eluent was collected in test tubes (10 mT, each). The segments collected were examined with silica gel thin-layer-chromatography (TLC) with the same mobile phase sy stem as the column chramatography. Al1 eluents that showed a single spot of the same retention tirne under UV light (254 Mi> were therefore combined. Solvents were removed by evaporation in vacuo to lave the carbarnate, I 1-[3 -N-methyl-N-(2,2,2- tnchloroethoxyciubonyl)-aminopropylidene]-6H-~be~~,e]oxepin. 3.3.4.0. Secondary amine formation Zinc powder (456 mg, 6.98 mmol) was added to a solution of the carbamate (205 mg, 0.465 mmol) in 8 mL tetrahydrofûran and 1.5 mL 0.5 M phosphate buffer @H = 5 .O) and stirred for 4 hours at room temperature. The mixture was filtered through a sintered glass funnel, washed with 0.3 N HCI (10 rnL x 2) and chioroform (20 mL x 2). The filbate was transferred to a separatory funne1 and the aqueous layer was adjusted to pH 12 with 2 N NaOH. The lower chloroforrn layer was separated and the remaining aqueous layer was extracted with chloroform (20 mL x 2). The combined organic extracts were fust dried with nahydrous Na2S04 and then evaporated in vacuo. The residual was pUnfied by column chromatography as described in the previous section except for a different mobile phase system (20% methano1 / 80% methylene chlonde / 0.5% triethylamine; v/v/v) to give desmethyldoxepin. The identity of the product was confrmed by rnass spectrometry (ESf and high resolution EIf). 3.4.0.0. Steseoselective, sensitive and sirnultaneous HPLC method

3.4.1.0. Preparation of standard stock solutions

3.4.1.1. 2- or E- doxepin, or commercial doxepin 100 ppm (free base) solution 1.131 mg doxepin or 2- or E- doxepin hydrochloride was dissolved in 10 mL of a mixtue of double distilled water and methanol (5050, v/v). The solution was stored in -20°C.

3.4.1.2. E-desmethyldoxepin 100 ppm (free base) solution 1.138 mg E-desmethyldoxepin hydrochloride was dissolved in 10 mL of a mixture of double distilled water and methanol(50:50, v/v). The solution was stored in -20°C.

3.4.1.3. Nortriptyline 100 ppm (free base) solution 1.139 mg nortriptyline hydrochloride was dissolved in 10 rnL mumire of double distiiled water and methanol (50:50, v/v). The solution was stored in -20°C.

3.4.2.8. Preparation of standard curves Standard stock solutions were diiuted to 0.1-10 @rnL with a mixture of double distilled water and rnethanol (85:15, v/v) for spiking control biofluids (2 rnL) to achieve fuial concentrations (in duplicate) of 1, 10,50, 100,200, and 400 ng/mL of E- doxepin and E-desmethyldoxepin (urine) or l, 3, 10, 50, 100, 200 ng/mL (plasma). The concentration of intemal standard nortriptyline was 400 ng/mL (urine) or 200 ng/mL (plasma). Standard calibration curves were made fiesh for each day of assay. 3.4.3.0. Preparation of quality control (QC) samples Three concentrations across the range of standard curve (lower, miclde and upper regions) were used for preparation of quality control (QC) samples (duplicates, operator blind). 15% Deviation fiom the quotient of the determined value and the nominal value was the cntenon of acceptance except at the lowest concentration in which 20% deviation was allowed. No more than two out of six QCs were dowed to fail to meet the acceptance critena (no more than one at any given concentration). Quality control samples were made fkesh for each day assay.

3.4.4.0. Extraction procedures To samples of spiked or unlmown plasma or urine (2.0 mL), 0.5 mL 3 N arnmonia solution (solution A) and 7 rnL mixture of n-pentane-isopropanol (955 v/v) (solution B) were added. The tube was capped and shaken in an overhead shaker for 20 minutes. The organic layer was separated by centrifugation (1500 g 5 min) and then transferred to a clean test tube containing 0.1 N hydrochlonc acid (1.0 mL). The mixture was shaken for 20 minutes and then alIowed to stand for separation for 5 minutes. The organic phase was aspirated to waste. Pentane (3-4 mL) was used to wash the remaining aqueous layer (shaken for 10 min) and then discarded after separation. To the washed aqueous residue, solution A (0.5 rnL) and solution B (6 rnL) were added and the mixture was shaken for 20 minutes. After separation of layes, the organic phase was transferred to a cleau test tube and evaporated under vacuum in a SpeedVac Concentrator svc200H (Savant Instruments, New York, NY, USA). The resulting dry residue was reconstituted with 120-200 pL of the mobile phase and aliquots (20-1 00 pL) were injected ont0 the column (autosampler). 3.4.5.0. Examination of equivalenee of detector respoose to 2- and E- isomers In order to compare detector response to the Z- and E- isomers, 2.5 ,yg/m.L standard aqueous solutions (50 pL 100 cl8/mL stock solution + 1950 ~.LLD.D. water) of Z- and E- doxepîn were added to control plasma or urine (2.0 mL) to give different 2-isomer percentages [Z/(Z + E) x 100%]. The total concentration of the two isomers in each sample was 125 ng/mL while the lowest concentration of individuai isomer was 12.5 ng/mL. The percentage 2-doxepui was composed at 10, 20, 40, 60, and 80, respectively. The solutions were analyzed in replicates of three or five on each of three consecutive days. QC sarnples (operator blind) were used to assure the validity of each day's results. The original peak area data of 2- and E- doxepin were used directly in the calculation of Z-doxepin percentage. Averages of observed ratios were tested against nominal ratios by Linear regression (no weighting factor). The corresponding calibration curves were used in back calculation. Intra- and inter-assay CVs (%) and percent accuracy were based on back caiculated values.

3.4.6.0. Recovery studies Recovery was uivestigated with cornmercially available doxepin because supplies of the pure isomers were limited. Doxepin fiee base chloroform solution was prepared as previously described (Section 3.3.1.0.). 20 pL of this solution was transferred to a 10 rnL test tube, dried ai 42OC with constant flow of nitrogen (Nz) gas to leave the oily fiee base (1 mg). Hexane (10 rnL) was added to make 100 p@L doxepin fiee base stock solution. A solution (!O yg/mL) was made by diluting the 100 pg/mL stock with hexane. Three concentrations (20, 80 and 400 ng/mL) were selected in the study. Samples were duplicated for direct injection, while the extracted samples were analyzed as replicates of six at each concentration. The extracted sarnples were made first by spiking blank plasma or urine (2 rnL) with standard aqueous solution and finally reconstituting with 200 pL mobile phase. The direct injection samples were prepared by mixing 20 a,10 @mL f?ee base solution with 980 pL mobile phase (20 ng/rnL), or 80 pL 10 pg/rnL f?ee base solution with 920 pL mobile phase (80 ng/mL), or 40 pL

100 pghL fiee base solution with 960 pL mobile phase (400 ng/mL). From these solutions of fiee base in mobile phase, 200 pL daliquots were transferred to injection vials. For both direct injection and extraction samples, 80 pL was injected for the lower and the middle levels, while 20 ILL was injected for the upper level.

3.4.7.0. Stability studies Stability in plasma and urine was studied under three different conditions: (i) stability after two freeze-thaw cycles (-20°C); (ii) stability &er two days of storage at 4°C (day 1 and day 2); and (iii) stability of extracts reconstituted in mobile phase and stored at room temperatue (up to 16.5 hours).

3.4.7.1. Stability after freeze-thaw cycles Two fieeze-thaw cycle stability tests were carried out at two concentrations (1 0 and 100 ngmL for plasma, 20 and 100 ng/mL for urine) in triplicate. Sarnples fkom two cycles were ûssayed together with fiesh QC samples (triplicate).

3.4.7.2. Stability at 4°C

Stability after two days of storage at 4°C test was also carried out at two concentrations (10 and 100 ngMfor plasma, 20 and 90 ng/mL for urine) in triplicate. Samples fiom day 1 and day 2 were quantified on the same day with fiesh QC samples (ûiplicate). 3.4.7.3. Stabiüty of extracts in mobile phase at room temperature Three concentrations in duplicate were selected for this test (10, 50, and 100 ng/mL for plasma, 20,50, and 90 ng/mL for urine). Cornparisons were made between samples injected at O hour and those injected at 16.5 hours.

3.4.8.0. Linearity and reproducibillity

Tests on intra- and inter- assay variability as weIl as finearity were carrïed out on three consecutive days with five or three samples at each concentration. nie tests were performed on plasma and urine samples at various concentration levels as specified previously (section 3.4.2.0). 3.5.0.0. Metabolic studies

3.5.1.0. KPLC analytical method The same HPLC method as described previously was used for in vivo studies, while the ranges of the standard curves for the in vitro studies were adjusted as appropnate for the concentration ranges in the quenched incubation mixtures. Standard curves for the in viho studies were prepared by spiking diluted E-doxepin and E-desmethyldoxepin standard solutions into dmg fiee incubation blanks (1 mL) after quenching to make concentrations at 10,50, 100,200, 400, 800,2000, and 3000 nghd (in duplicate) for experirnents with rat liver microsornes and GIT homogenates. while those prepared for incubation with the human liver microsornes were at 5, 25, 50, 100, 200, 400 and 800 ng,.Nortnptyline as interna1 standard was spiked at the concentration of 800 ng/mL. Acceptance critena were based on quality control sarnples (duplicate, analyst "blind") representing the low, medium and high regions of the standard curves. Liquid-Liquid extraction was done with the same procedure as described previously (section 3.4.4.0.). Eventually the dried residue was reconstituted with 170 mobile phase and 80 pL of ththe solution was injected.

3.5.2.0. Estimation of in vitro metabolic elimination half-lives Metabolic elimination half-lives of the 2- and E- isomers of doxepin and desmethyldoxepin were computed fiom isorner concentrations over sequential time points (10-60 minutes for rat, and 40-240 minutes for human) after incubations with 2-doxepin, E-doxepin, or E-desmethyldoxepin (concentrations of Z- desmethyldoxepin arising fiom geometric impurity was used for calculation of half- lives of 2-desmethyldoxepin), using the PC pharmacokinetic program TopFit. In the enzyme inhibition studies, metabolic elimination half-lives of 2- and E- isomers of desmethyldoxepin in incubates of E-desmethyldoxepin with inhibitor were calculated with same approach.

3.5.3.0. Statistics Many of the calculations for means and standard deviations were performed with computer program Microsof? Excel (version 5 .O). Analysis of variance (ANOVA) was carried out on a Power Macintosh computer by means of SuperANOVA or StatView 4.5 (Abacus Concepts, Inc., Berkeley, CA, USA). Multiple cornparison tests applied were Student-Newman-Keuls, Tukey-Krarner and SpjotvolLStoline. The level of significance was set at a=0.05.

3.5.4.0. Exploratory in vivo experiments

3.5.4.1. Human studies Four young male volunteers (1 Caucasian and 3 Oriental, age of 3 1-35 years, weight 67.500 + 6.455 kg) were recpired to be fiee of cardiovascular, hepatic rend, or gastrointestinal diseases, dmg abuse or alcohol dependence, as assessed by physical examination and a review of medical history. Blood pressure and clinical laboratory tests (blood chemistry, hematology and urinalysis) were required to be within normal ranges. AU subjects were required to abstain fiom the use of al1 other dmgs, including non-prescription hgs, for at least one week pnor to and until after completion of the study. The subjects were also required to refiain £kom the use of alcohol or caf5eine containing beverages or foodstu-flfs for 24 hours prior to dosing until 24 hours post dose. The study protocol and informed consent forms were approved by the local ethics review board. A consent form was signed by each subject before the administration of the dmg. Afier an ovemight fast, each subject was first asked to give a sarnple of control urine, and then to take orally 3 capsules of 25 mg (free base) doxepin (Novo-doxepP, NOP) with 100 mL water. A standard lunch meal was provided 4 hours later. Cumulative urine was collected over a 24 hou period post dose. At the end of the collection penod, urine samples were fkozen (-20°C) until analy sis. Stereoselective quantification of doxepin and desmethy ldoxepin isomers were performed on urine samples to confïrm the ratio distortion in Z-, E- isomers of desmethyldoxepin.

3.5.4.2. Animal in vivo studies Experiments with small groups of various animal species were carried out in which a.illnals were dosed orally with doxepin and then housed in metabolism cages for total collection of 0-24 hou urine as follows: (a) n=4 female beagle dogs (weight 6.457 + 0.1 7 1 kg) were dosed with commercial doxepin (1 0 m@g) in capsule form; (b) n=2 female and n=l male New Zealand White rabbits (weight 3.582 + 0.664 kg) were dosed (30 mgkg) by oral intubation; (c) n=3 male Dunkin-Hartley Guinea-pigs (weight 1.1 17 t 0.106 kg) were dosed (30 mgkg) by orai intubation; and (d) n=4 male Lewis strain rats (weight 0.403 f0.03 1 kg) were dosed (30 mgkg) by oral intubation. Control urine (0-24 hour) was collected before the dmg administration. Throughout the experiment, the animals were maintained on their regular diet with water accessible ad Libitum. Post-dose urine collection penod, storage conditions, and the method of quantitative assay were same as those described in the previous section. Dilutions were made and assays were repeated for those urine samples in which concentration(s) of the individual isomer(s) were beyond the upper Limit of the standard cunre(s). The objective of this experiment was to investigate the urinary stereoselective metaboiic profiles of doxepin and the metabolite in different animal species and to select an animal mode1 for Merinvestigation. 3.5.5.0. Confirmation of significant ratio distortion of doxepin isomers in one healthy male volunteer Since a special case of significant ratio distortion of parent drug in 0-24 hour urine was found in the previous human study (section 3.5.4.1.), it was of interest to confïrm this observation by doing a separate study with the same subject. This experirnent was conducted under the same ethical standards as descnbed previously, and the same physical examination and clinical laboratory test were done before the study. Other requirements for this single dose studÿ were just same as before. The subject was asked to give control blood and urine samples first der an overnight fast, and then oraily received 3 capsules of 25 mg (fiee base) doxepin (Novo-doxepinTM,

NOP) with 100 mL water. A standard lunch meal was provided 4 hours later. Cumulative blood and urine samples were collected up to 120 hour period post dose. The blood samples were taken at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 3 6, 48, 72, 96, 120 hour, while urine was collected in 12 hour segments for 120 hours post dose. Heparinized blood samples were centrifuged (1500 g) imrnediately to separate plasma. The pH and volume of each urine segment was recorded before a s~~allsarnple (15 mL) was taken for storage. Both plasma and urine samples were stored at -20°C until analysis. The HPLC method for quantification of these plasma and urine samples was same as descnbed before. Area (extrapolated) under the curve of plasma isomer concentration versus time (AUC) was calculated using the pharmacokinetic cornputer program (TopFit). 3.5.6.0. Studies on the effect of route of administration on the urinary excretion of the isomers of doxepin and desmethyldoxepin in rat A cohort of 12 male Lewis strain rats (weight 460 + 22g) were randomly divided into 3 sub-groups of n=4 animals. Each sub-group received commercial doxepin hydrochloride by one of three routes of administration: (a) by oral @O) intubation (30 mg/kg); or @) by inbapentoneal (ip) injection (15 mgkg); or (c) by intravenous (iv) injection (7.5 mgkg) into tail vein. The rats were housed in metabolism cages for 24 hours post dose for urine collection, with access to the nomial diet and water ad libitum. Control urines were harvested over 24 hour penod before dosing. The urine samples were processed and quantified as described in the previous section.

3.5.7.0. Studies on the possibility of metabolic E- to 2- isomer conversion in rat Rats were dosed with E-doxepin and E-desrnethyldoxepin to test the hypothesized mechanism of isomerization. In order to investigate the influence of route of administration on the ratio distortion, rats were dosed via both iv and po routes on separate occasions. These experirnents were carried out according to a cross-over design in eight male Lewis strain rats (initial weight 273 f 12 g), each of which was marked with an ear punch label. The rats were randody assiped to two groups (Group 1 and Group 2). Each group of rats received six treatments according to the sequences shown in Figure 3.1., with two week washout periods between treatments. The animais gained weight during the course of the study which took three months to complete. They were therefore reweighed before each phase for the calculation of doses (expressed as the appropriate base) as follows: doxepin iv 9 mgkg, po 30 mgkg; E-doxepin iv 7 mgkg, po 15 mgkg; E-desmethytdoxepin (E- NorDox) iv 4 mgkg, po 8 mgkg. The rats were placed in metabolism cages for 24 hours before dosing for the collection of control urine samples. After administration of doxepin, urine samples were collected over the periods 0-1 6 h and 17-41 hours post dose. Afier administration of either E-doxepin or E-desmethy ldoxepin, urine samples were collected over the periods of 0-24 and 25-48 hours post dose. The rats were given access to their regular diet and water ad libitum duriog the experimental penod. The urine samples were stored at -20°C until analysis. Quantification of urine samples was same as described previously. Urine samples fiom the treatments of iv and po dosing with same dmg were assayed on same day. iv E - NorDox 6 a PO iv

iv PO E - Doxepin PO iv

iv PO Doxepin PO iv

Figure 3.1. Randomized two-sequence cross-over design in which eight male Lewis strain rats received six treatments in either of the two sequences, with a two-week washout period between treatments 3.5.8.0. Studies on the possibility of metabolic isomer interconversion with ber microsomes or gastrointestinal tract (GIT) homogenates

3.5.8.1. Experinrent design Each experiment used rat liver microsomes or GIT homogenates fiom an individual rat. For incubations with rat or human liver microsomes, the four substrates (commercial doxepin, 2-doxepin, E-doxepin, or E-desmethyldoxepin) were added separately to the labeled incubatioii tubes. A single substrate, commercial doxepin, was incubated with rat GIT homogenates, experiments in which Liver microsomes fiom the same anunal were used as controls. Incubations were quenched at four different time points for incubations with rat liver microsomes (10, 20, 40, and 60 min) and humanLiver microsorne (40, 120, 180, and 240 min). Only two time points (30 and 60 min) were chosen for termination of incubations with rat GIT homogenates and controls. Al1 incubation samples were carried out in duplicate.

3.5.8.2. Rathuman iiver and rat GIT tissue samples Eight male Lewis rats (ca. 250 g, 56-65 days old) were dowed to acclirnatize for one week after which they were killed by decapitation and livers and GIT tissues quickly excized. The human liver sample was obtained through The Royal University Hospital (Saskatoon, SK) fiom a fernale Caucasian who died fiom congestive heart failure at age 80 years. Her medicai records indicated no history of liver disease. AU liver and GIT tissue samples were immediately frozen in liquid nitrogen and stored at -70°C until used.

3.5.8.3. Rat and human liver microsome preparations AU liver microsomes were prepared at 4OC by homogenization of hepatic tissue with 150 mM potassium chloride in a quartz tube (Polytron motor-driven homogenator, Brinkmann Instruments, Westbury, NY, USA). The homogenate was £irst centrifùged at 9000 g for 20 minutes. The supematant was decanted and centrifuged for 60 minutes at 105000 g. The microsomal pellet was resuspended in 150 mM potassium chloride solution and stored at -70°C until use. Total protein concentrations for each liver microsomal preparation was deterrnined according to a published method (Lowry, 1951 #793). The enzyme activity of the human liver microsomal preparation was comparable to that of homogenates prepared fiom other human Livers, as judged by the tum-over rate of the N-desmethylation and oxidation of clozapine after 30 minutes of incubation (data not shown).

3.5.8.4. Rat GIT subcellular preparations Immediately after removal of rat liver, the stomach and small intestine (duodenum, jejunum, and ileum) were removed fkom the two of the eight rats. These tissues were dit open and washed with ice water three tirnes before plunging into liquid nitrogen and subçequent storage at -70°C until required for preparation of homogenates. Three fiactions of subcelIuIar homogenates (mitochondnd, microsomal and cytosolic) were prepared fiom stomach and srnall intestine respectively. The procedure for the preparation of GIT microsornai preparations and estirnates of total protein concentration for these fractions were exactly the same as descnbed for preparation of liver microsomes. The mitochondrial fkaction was collected as the separated solid afler the first cenûïiùgation (9000 g x 20 min), while the cytosolic fraction was simply the supematant after the second centrifugation (105000 g x 60 min).

3.5.8.5. Protein content in incubates with enzyme preparations Hepatic microsomal protein content in the incubation mixtures ranged fiom 0.87 to 1.28 mg/mL (rat), and 1.O7 mg/mL (human). In incubations with rat stomach homogenates, the protein content ranged fkom 0.20 to 0.89 mg/mL. The protein content in rat small intestine homogenates was 0.60 - 0.88 mg/mL.

3.5.8.6. Incubation conditions For experiments with rat Liver microsomes (Table 3.1.) or gastrointestind tract subcellular preparations, the incubation mixtures containeci a NADPH cofactor regeneration system (2 rnM MgCl,-Y 2 mM G6P, 1 unit G6PD, 0.4 rmM NADP+) in a final volume of 1 mL of 100 mM phosphate baer at pH of 7.4, and one of the four substrates at concentration of 25 pM (doxeph, Z-doxepin or E-doxepïn,) or 15 pM (E-desmethy ldoxepin). For experiments with human Ever microsomes (Table 3.2.), higher content of the NADPH cofactor regeneration system (2 mM MgCG, 3 mM

G6P, 1.5 unit G6PD, 0.75 rnM NADPC) and lower substrate concentration (2.5 instead of 25 pM and 3 instead of 15 pM) were employed, and the other incubation conditions remained the same as those for rat preparations. Substrate double distilled (D.D.) water solutions were made fiesh for spiking into the incubates. The incubation mixtures were prepared by adding (i) the NADPH CO-factor regeneration system, (ii) the substrate in double distilled water, (iii) phosphate buffer and (iv) liver rnicrosome homogenates or gastrointestinal tract subcellular preparations. The tissue homogenates were diluted with 150 mM potassium chionde as appropnate, depending on the protein concentration of the homogenate in question. Incubation mixtures were then transferred immediately to a GCAPrecision Scientific Dubnoff metabolic shaking incubator ScientSc Products, West Cheater, Pennsylvania, USA) rnaintained at 37OC. Termination of the incubations was performed by inserling incubation tubes into ice and then adding and mixing immediately with 0.5 rnL 3 N ammonia solution (NH,OH). The quenched incubation mixtures were kept at 4OC untîl extraction. Extraction of incubation samples were done on the same day and HPLC quantification was nin ovemight-

Table 3.1. Incubation conditions with rat liver microsornes

Chemicals or Stock solution Volume hcubate tissue homogenate concentration (fi) concentration

Substrate (use only one) Dox, 2-Dox, E-DOX E-NorDox

Phosphate buffer (NaH2POd Na2HP04) 0.2 M (pH 7.4) 500

Liver microsornes in Protein content @iIute appropnate) 0.87-1.28 mg/mL 1.15% KCl ca. 10-20 mg/rnL 200

D.D. H20

.-. .. . - . 1000 (total) 3.5.8.4. Mass spectrometry HPLC eluents corresponding to individuai peaks were collected and the mobile phase evaporated under vacuo in a SpeedVac Concentrator svc2OOH (Savant Instniments, New York, NY, USA). The residue was redissolved in methanol and the solution injected înto a mass spectrometer (VG Quatro II. Micrornass, Manchester, England), which was operated at cone voltage of 25 eV and a source temperature of 50°C, for characterization using positive ion electrospray . The mobile phase used for this mass spectroscopy was 0.2% formic acid in methanol. Corresponding background mass spectra were obtained fiom evaporated HPLC eluates of control incubation mixtures containing no doxepin related material. Table 3.2. Incubation conditions with hurnan her microsornes

Chemicals or Stock solution Volume Incubate tissue homogenate concentration (PL) concentration

Substrate (use only one) Dox, Z-Dox,E-Dox E-NorDox

0.2 M (pH 7.4)

Liver microsorne in Protein content @ ilute appropriate) 1.15% KC1 6.71 mg/mL 300 1.O7 m8/mL

D.D. H20

1000 (total) 3.5.9.0. Enzyme inhibition studies in incubation of rat liver microsomes using UGT inhibitors

3.5.9.1. Preparation of stock solutions Standard solution of E-doxepin, E-desrnethyldoxepin and nortriptyline (intemal standard) hydrochlorides were prepared in a solvent mixture of D.D. water and methanol (50:50, VN) at a concentration of 100 j@mL (eee base) and stored at -

30°C. This 100 WrnL stock solution was mixed in situ with a solvent of 15% methanol in D.D. water to designated concentrations by a seriai dilution procedure. While standard curves were prepared by spiking these dilutions or the original IO0 @rnL stock solutions into incubation blanks (no substrate added) after quenching. 250 pM D.D. water solutions of doxepui hydrochloride, 150 pM D.D. water solution of E-desrnethyldoxepin hydrochloride were prepared fiesh for addition of the substrate in incubates. Phosphate buffer (0.2 M, pH 7.4) was used as solvent for preparation of UGT inhibitor solutions at the foIlowulg conceneations: 1 mM (oleoyl-CoA), and 30 mM (both probenecid and furosernide). These inhibitor buffer solutions were temporarily stored at 4OC before use.

3.5.9.2. Experirnental design In each experiment, liver microsomes f?om one rat was used. One of the two substrates, either doxepin or E-desmethyldoxepin, was spiked into each labeled incubation tube with one of the three UGT inhibitors (Figure 3.2.), while control sarnples received same volume of extra blank phosphate bufTer, instead of one of the inhibitor buffer solutions to match the rest of the incubation conditions. Incubations were terminated at four different thepoints (20,40, 60, and 80 min). AU incubation samples were carried out in duplicate. A regeneration CO-enzymesyçtem 1 for incubation at W°C & pH 7.4

a Zhong et al., 1992 11

\ f Quenching points: / ' 1 20 rnins; 40 rnins; ( 60 rnins; 80 rninç;

Figure 3.2. Experimental design for rat liver microsomal enzyme inhibition studies using UGT inhibitors

3.5.9.3. Incubation conditions The rat hepatic microsomal protein concentrations in final incubation mixtures ranged fiom 0.49 to 0.95 mg/mL (mean protein concentration 0.68 f 0.19 mglmL). The incubation mixtures contained a NADPH cofactor regeneration system (2 mM

MgCl, 2 rnM G6P, 1 unit GBPD, 0.4 mM NADPr, one of the two substrates at a concentration of 25 pM (doxepin) or 15 pM (E-desmethyldoxepin) and one of the three UGT inhibitors at concentration of 100 pM(oleoyl-CoA) or 3 rnM (probenecid and furosemide) in a final volume of 1 mL of 120 mM phosphate buffer at pH 7.4 (Table 3.3). Following the sequential additions of the NADPH cofactor regeneration system, substrate, phosphate buffer, and inhibitor, liver microsomal preparation (diluted with 150 mM KC1 and maintained at 4°C in situ) was added to incubate at last. Incubations were then started ünmediately at 37°C in a GCAPrecision Scienfific

Dubnoff metabolic shaking Uicubator. Termination of the incubations was done with the same approach as described previously (section 3 -5.8.6.). Controls were carried out in an identical manner in the absence of inhibitor.

3.5.9.4. Simulation of the ratio distortion in desmethyldoxepin isomers To more explicitly demonstrate the effect of enzyme inhibition on the ratio distortion as stated previously, the simulation of the stereoselective metabolic pathway with or without inhibition and the resulting ratio distortion in desmethyldoxepin isomers was performed by iteration with the assistance of computer program Microsoft Excel (version 5.0). Different metabolic elimuiation rates were assigned to individual isomers, while arbitrary starting concentration values such as 3 (percent 2-isomer impurity) and 97 (Z:E = 3:97) were used for 2- and E- desmethyldoxepin isomers, respectively. Table 3.3. Incubation conditions with rat tiver microsornes in enzyme inhibition studies

Chemicds or Stock solution Volume Incubate tissue homogenate concentration (PL) concentration

Substrate (use one) Dox 250 pM E-NorDox 150 pM

Phosphate buf5er (Nal&POd Na2HP04) 0.2 M (pH 7.4)

UGT inhibitors (use one) oleoyl-CoA ImM pro benecid & furosemide 30 IIM

Liver microsornes in Protein content (Dilute appropriate) 1.15% KC1 ca. 10-20 mg/mL 200 0.49-0.95 mm

D.D. H20

1000 (total) 3.6.0.0. Pharrnacokinetic studies

3.6.1.0. AnaIytical methods The plasma andior urine samples were quantitatively analyzed by means of the HPLC method descnbed previously (section 3 A.O.0.) with minor modification in the procedure of preparation of standard solutions and the volume spiked. Standard stock solutions (200 @mL) were prepared by diçsolving E-doxepin, or E- desmethyldoxepin hydrochloride in a solution of nortriptyline (4 pg/mL) in double distilled water and methanol (50:50 v/v). MWng equal volumes of these two stock solutions (20 0 pg/mL) produced a solution containing E-doxepin, E-desmethy ldoxepin at concentration of 100 @mL, and nortriptyline at concentration of 4 pg/mL. The standard solution was stored at -20°C until required for use. The standard solution was then diluted as appropriate with nortriptyline solution (4 pg/mL) in a mixture of double distilled water and methanol (85:15 v/v). The dihted solutions (200 PL) were spiked into dnig kee plasma (2 mL) andlor urine (2 mL). Standard cuves were run daily. Acceptance criteria were based on quality control samples (analyst "blind") representing the low, medium and high regions of the standard curve. Plasma andor urine samples of the same individual (volunteer or animal) from both iv and oral treatments were analyzed on same day. Extraction was done by adding 0.5 rnL 3 N NH40H solution and 8 mC 5% isopropanol in pentane into plasma or urine sample, vortex 25 minutes. Mer centnfiiging at 1000 g for 5 minutes, the organic layer was transferred to a clean test tube containing 1 mL 0.1 N HCl solution. The mkhxe was vortexed for 20 minuies after which the upper organic Iayer was discarded afkr separation. The remaining aqueous solution was washed with 3 mL pentane. The acidic aqueous solution was adjusted to an alkaline pH by adding 0.5 mL 3 N NH40H. Pentane (7 mL) was used for the final extraction (vortex 20 min). The pentane was dned under vacuo in a SpeedVac Concentrator svc2OOH (Savant Instruments, New York, NY, USA). The dried residue was reconstituted with 160 pL mobile phase, of which 100 pL was injected into the HPLC column. Long term stability control samples (5 ng/mL) for E-doxepin and E-desmethyldoxepin in both plasma and urine were analyzed during the 3-month (plasma) and 5-month (urine) period of storage. Total of eight samples were analyzed for each of the two isomers spiked in each of the two matrices.

3.6.2.0. Pharmacokinetic analysis Bo th noncompartmental and cornpartmental pharmaco kinetic methods were applied to calculate pharmacokinetic parameters, using pharmacokinetic cornputer program WinNonlin (ScientSc Consulting Inc., Apex, North Carolina, USA). In the noncompartmental anaiysis, the elirnination rate constant, &,, w-as determined by log- linear regression of the terminal phase (last four plasma concenû-ations) of the concentration-tirne curve. Linear trapezoidal dewas used to compute the area under the plasma concentration versus time curve (AUC) up to t,,, then log trapezoidal dewas used for the declinùig portions of the cwetill tl, the last time showing a measurable concentration (C ,,J. For intravenous (iv) administration log trapezo idal dealone was used for the calculation of AUC. AUC(0-=) values were determined by adding to the appropriate AUC(0-t), the quotient of CI, and the appropnate value of &. Terminai phase half-lives (tin) were calculated fkom the quotient of ln2 and A,.

Clearance was detemiined by the following ways: (i) After iv administration clearance (CL) was calculated as the quotient of dose and extrapolated area; (ii) Apparent oral clearance (CL,) was calculated as the quotient of dose and extrapolated area after oral administrati O n. Doseiv CL= - (Equation 3.1 .) AUC

(Equation 3 -2.)

Where the absolute bioavailabilities F were determined as the quotient of appropriate values of CL and CLo. The apparent volume of distribution at steady state (V,,) was simply estimated as the product of clearance and mean residence time after iv bolus dose of the hg.

Doseiv - AUMC V,, = CL - MRT = (Equation 3 -3.) AUC?

The mean residence times (MRT) were estimated as the quotient of area under the first moment curve (AUMC) and AUC. The amount of drug eluninated in the urine, Ae,, was estirnated fkom the total urine collection period (up to 120 hows after the hgadministration in human studies). The percent of the dose excreted unchanged was calcdated based on Ae, and the total administrated dose. Rend clearance (CLR) was calcdated as follows:

CLR- -Aem (Equation 3.4.) AUC

Comparûnental pharmacokinetic analysis using a two-cornpartment mode1 (no lag time, and first order elimination) was performed on the mean 2- and E- doxepin concentratiodtime data afier iv administration using WioNonlin. The mode1 equation is show as folows:

C = A~-~~+ Be-Pt (Equation 3 S.)

Where C is the plasma concentration at tirne t, A is the zero time intercept associated with the alpha phase, a is the macro rate constant associated with the distribution phase, B is the zero time intercept associated with the beta phase, and P is the macro rate constant associated with the elhination phase. Various weighting schemes were evaluated for their ability to accommodate the heteroscedasticity of the data, and a weighting factor of K2(predicted values) was selected to minimize optimaliy the weighted sum of squared residuals. The method of Gauss-Newton (Levenberg and Hartley) was used for minimization. Model selection was based on evaluation of the value of Akaike's Information Criterion (Akaike, 1974, Yamaoka, 1978) and Schwarz Critenon (Schwarz, 1978). The limited information of plasma concentration versus thedata afier oral administration was not suficient for a valid pharmacokinetic modeling. Hence, only noncompartmental method was applied for oral data.

3.6.3.0. Statistical analysis Statistics of the pharmacokinetic parameters in noncornpartmental and cornpartmental analy sis were performed in WinNoniin. Various other programs were also applied (Microsofi Excel 5.0; SuperANOVA and StatView 4.5). The level of sigrrificance was set at a=0.05. 3.6.4.0. Pharmacolainetic studies in humans

3.6.4.1. Subjects Twelve healthy male volunteers (non-smokers, 21 to 36 years of age inclusive, and deviakg by no more than I 10% fkom the ideal weight for height according to the

Metropolitan LXe Insurance Company Bulletin, 1983) were enrolled in the study. 'ïhe study protocol was approved by the local institutional review board. The subjects were required to refkain fiom consuming alcoholic or cafZeine containing beverages from 48 hours prior to dosing until after the collection of the last blood sarnple. All the other requirements concerning the physical and medicd conditions for the participation were just same as described before (section 3.5.4.1.). Written informed consent was obtained fiom al1 volunteers prior to their participation in the study .

3.6.4.2. Study design The study consisted of two dosing periods with a balanced randomized two treament (iv and oral), two sequence, cross-over design. The wash-out penod between the two dosing periods was two weeks. The volunteers remained under close medical supervision for 8 hours post dose in each phase. After an overnight fast, the iv group received 25 mg doxepin hydrochlonde (22.12 mg fiee base) by a bohs injection (5 mL in 2 min). Blood samples (14 mL) were drawn in heparinised evacuated tubes, immediately before dosing, and at 5, 10,20,30,43 minutes, and then at 1,2,3, 5, 7, 10, 12,24,36,48, 72,96, and 120 hours. The oral group received 85.5 mg doxepin hydrochlonde (75 mg fkee base) in capsule with 100 mL water. Blood samples (14 mL) were drawn in heparinised evacuated tubes, immediately before dosing, and at 15,30 minutes, and then at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 36, 48, 72, 96, and 120 hours. For the first 12 hours, each volunteer was required to drink 150 mC water per hour to guarantee the urine collection. Total urine was collected and saved at the segments of 0-2,2-4,4-6,6-8,8-10, 10-12, 12-24: 24-36, 36- 48, 60- 72, 72-84, 84-96, 96-108, and 108-120 hours der both iv and oral administration, while a sample of blank urine was collected immediately before dosing. Each blood sample was ïmrnediately centrifuged at 1000 g to separate plasma which was then divided into two aliquots and fiozen at -20°C until analysis. Volume and pH of urine in each segment were recorded and a sample (15 mL) was taken to store at -20°C until analysis.

3.6.5.0. Pharmacokinetic studies in dogs

3.6.5.1. Anirnals Six beagle dogs (4 female # 1, 3, 4, 6; 2 male #2, 5), weighing 9- 1 1 kg, were used. The animais were fasted overnight pnor to administration and were fed 4 hours after dosing to match the protocol used for human subjects. The standard feeding schedule was resumed as usual on day 2. The dogs had access to water ad libitum throughout the course of the experiment.

3.6.5.2. Study design Each dog was randomly assigned a number. These numbers were randomly matched with one of the two dosing sequences, i.e. idoral or ordiv, so that there were three dogs with each dosing sequence in each of the two dosing penods. The washout interval between two penods was 40 days. 3.6.5.3. Dosing procedure lmmediately before the administration of doxepin in each period, jugular vein catheterïzation was first established in three dogs receiving iv administration. The catheter was kept for blood sampling untii 24 hours post dose. Blood samples after 24 hours were obtained by venipuncture at the cubital fossa. Doses of 6 mgkg and 20 mg/kg of doxepin base were applied to iv and oral administrations, respectively. The sterile saline solution of the dnig was injected directly into the cubital fossa of comcious dogs. In oral administration, dogs were forced to swallow the capsule.

3.6.5.4. Sample collection Blood samples (5 mL) were drawn in heparinised evacuated tubes dediately before dosing, andat 10,20,30 minutes, and then at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, 20, 24, 36, 48, 72 hours after both iv and oral administration. The plasma was separated by centrifugation at 1000 g for 10 minutes immediately after collection and stored at -30°C until analyzed. CHAPTER 4

4.0.0.0. RESULTS AND DISCUSSION

4.1.0.0. Synthesis of desmethyldoxepin

4.1.1.0. 11-[3-N-methyl-N-(2,2,2-tti~hloroethonycarbony~)-amho- propylidenej-6H-dibenz[b,e]oxepin The yellow oily carbarnate, 11-[3-N-methyl-N-(2,2,2-trichloroethoxycarbony~)- aminopropy lidene]-6H-dibe@~,e]oxepin prepared fiom doxepin, was used directly in the next step. The results £iom TLC indicated the reaction completed with no detectable starting rnatenal left. The yield matched the literature level(90 %, 248 mg)

4.1.2.0. Desmethyldoxepin Desmethyldoxepin kee base (MW: 265.1537) was a yellow oil. The yield for this step was 82 % (101 mg). The identity of desmethyldoxepin was confirmeci by both the ESC/MS (Figure 4.1.) and the EI+/MS (Figure 4.2.). Under the electron impact conditions employed and the use of desmethyldoxepin fiee base a pseudomolecular ion was observed at m/z 266 indicating the addition of a proton. This observation is not unusual for primary and secondary amuies which ofien demonstrate protonated molecular ions even under conditions of electron impact. The Er/MS: m/z (M+H)+ values were: calc 266.1545, obs 266.1548 (literature, obs 266.1544). The purity of this synthesized metabolite was later assured by its HPLC chromatogram in which (figure not shown here). The isomeric ratio of diis product tumed out to be 1j:85 (Z:E).

Fi le:C368A LAB-BASE - The HS Data System Sam~le:SAHPLE JY-1-36 COHE 45V ESI+

Figure 4.1. Electrospray mass spectrum of desmethyldoxepin from the organic synthesis Figure 4.2. Electron impact mass spectrum of desmethyldoxepin from the organic synthesis 4.2.0.0. Stereoselective, simukaneous and sensitive HPLC method Standard curves were based on the E-isomers of parent dmg and metabolite since only trace quantities of the 2-doxepin and no 2-desmethyldoxepin were available. The method was therefore based on the assumption that the 2- and E-isomers should have the same peak area if their W detector responses were equivalent, notwithstanding any merence in retention times and peak sharpness. Accordingly, peak area was used throughout as the basis of quantincation.

4.2.1.0. Equivalence of detector response to 2- and E- isomers The original peak area data of 2- and E- doxepin were used directly in the calculation of 2-doxepin percentage [Z/(Z + E) x 100%] .. Averages of observed ratios were tested against nominal ratios by linear regression (no weighting factor). The corresponding calibration curves were used in back calculation. htra- and inter- assay C.V.s (%) and percent accuracy were based on back calculated values. Coefficients of determination (2)ranged fÏom 0.999 to unity. Tables 4.1.43. list back calculated Z-doxepin%, standard deviation (S.D.), and intra-assay coefficient of variation (C.V.%) in spiked plasma sample for day 1, day 2, and day 3, respectively. Table 4.4. lists the results of inter-assay C.V.% for these plasma samples. While Tables 45-4.7. list back calculated Z-doxepin%, standard deviation (S.D.), and intra- assay coefficient of variation (C.V.%) in spiked urine sample for day 1, day 2, and day 3, respectively. Table 4.8. lists the results of inter-assay C.V.% for these urine samples.

4.2.2.0. Plasma and urine recovery The recovery was calcdated by comparing the peak height (su of 2- and E- doxepin) of extracted samples (n=6) with those of directly injected samples (n=2). The results of the mean recovery in plasma and urine are Listed in Table 4.9.. Table 4.1. Equivalent detector response in day 1 plasma samples

Back caiculated 2-doxepin %

- X 10.59 19.16 40.48 59.54 80.28 + S.D. 0.94 0.13 0.73 2.78 1.83 C.V. % 8.83 0.69 1.79 4.67 2.28

Table 4.2. Equivalent detector response in day 2 plasma samples

Nominal 10 % 20 % 40 % 60 % 80 %

- -- Back calculated Z-doxepin %

- X 10.28 19.80 39.84 59.98 80.15 t S.D. 1.37 0.63 1.55 2.46 1.44 C.V. % 13.30 3.19 3.89 4.1 1 1.80 Table 4.3. Equivalent detector response in day 3 plasma samples

Nominal

Sample # Back calculated 2-doxepin %

X k S.D. C.V.%

Table 4.4. Equivalent detector response in plasma samples (inter-assay C.V.%)

- - Sample # Back calcdated 2-doxepin %

Day 1 10.17 19.04 40.48 56.78 79.9 1 10.08 L9.30 40 .O4 62.3 3 8 1.98 12.13 19.15 3 9.5 2 59.52 81.1 1 9.79 4 1.O8 81.1 1 10.79 41.28 77.28 Day 2 8.13 19.35 42 -42 60-99 77.78 10.90 20.52 38.53 57-17 8 1.27 10.56 19.53 40.07 6 1.77 81.35 11.81 39.23 80.28

Day 3 10.8 1 20.46 9.98 19.77 10.92 19.95 10.19 10.81

C.V. % 8.84 2.77 Accuracy % 95.30 98.40 Table 4.5. Equivalent detector response in day 1 urine samples

------Nominal IO % 20 % 40 % 60 % 80 %

Sample # Back calcdated 2-doxepin %

- X k S.D. C.V. %

Table 4.6. Equivalent detector response in day 2 urine sarnples

------Nominal 10 % 20 % 40 % 60 96 80 %

Sample # Back calcdated 2-doxepin %

- X 10.63 S.D. 1.14 C.V. % 10.70 Table 4.7. Equivalent detector response in day 3 urine samples

------Nominal 10 % 20 % 40 % 60 % 80 %

Sample # Back caIculated Z-doxepin %

- X 9.77 19.82 40.49 60.44 79.53 + S.D. 0.49 0.59 1.22 O -40 2.86 C.V. % 5 -05 2.96 3 .O2 0.66 3 -60

Table 4.8. Equivalent detector response in urine sampies (inter-assay

Sample # Back calculated Z-doxepin %

Day 1 10.43 19.8 1 40.2 1 58.49 77.42 10.50 19.27 39.58 62.3 8 83.17 10.71 19.98 39.9 1 59.23 80.32 10.12 38.96 78.02 10.48 39.52 8 1.66 Day 2 9.90 18.3 1 40.34 58.70 80.54 12.63 19.89 41.14 62.1 1 80.22 9.98 19.96 39.07 58.61 80.47 10.43 37.93 80.01 10.22 41 -34 80.25 Day 3 9.63 20.48 4 1.62 60.52 74.88 9.42 19.36 41.05 60.79 79.50 9.38 19.61 4 1.27 60.0 1 80.02 10.59 39.79 82.66 9.82 3 8.68 80.59

C.V. % 7.53 3.12 2.76 2 -46 2.54 Accuracy % 97.20 98.15 99.93 99.85 99.98 Table 4.9. Recovery data

------Plasma recovery (%) Urine recovery (%)

Sample # 20 ng/mL 80 ng/d 400 ng/rnL 20ng/mL 8Ong/mL 400ogh.L

1 -3 3 4 5 5

- X i S.D. C.V. %

4.2.3.0. Sample stability The stability data of E-doxepin and E-desmethyldoxepin for freeze-thaw cycles are listed in Table 4.10. and Table 4.11. respectiveiy. Table 4.12. and Table 4.13. show the results of stability test of samples at 4OC, while those of e-acts in mobile phase at room temperature are depicted in Table 4.14. and Table 4.15..

4.2.4.0. Linearity and reproducibiiity

Mer iinear regression @eak response weighting: Vconcentration), the back calcdated concentrations were cornpared to nominal concentrations ushg the same criterion described previously for QCs (page 46). A typical linear regression equation 2 for E-doxepin was y = 0.003~- 0.002 (r = 0.9997), whereas it was y = 0.003~- 0.001 2 (r = 0.9997) for E-desmethyldoxepin in validation studies with plasma. For the 2 studies with urine, they were y = 0.008~- 0.028 (r = 0.9994) for E-doxepin and y = 2 0.006~- 0.001 (r = 0.9996) for E-desmethyldoxepin. htra- and inter- assay C.V.s were both 45% across the range of the standard curves. Table 4.16. and 4.17. list the data on rnean back-calculated concentrations, accuracy and precision (inter-assay C.V.%) for E-doxepin and E-desmethyldoxepin, respectively. The rnean values of accuracy and precision (mean + S.D.) for E-doxepin were 97.53 + 1.67 and 3.89 it 1.65 (plasma), 97.10 f 2.40 and 3.82 + 1.14 (urine), while those for E- desmethyldoxepin were 97.57 + 2.06 and 4.3 8 k 3.24 (plasma), 97.64 + 3.32 and 5.26 lr 1.83 (urine), respectively.

4.2.5.0- Discussion The stability data showed no evidence of degradation of E-doxepin and E- desmethyldoxepin under the three storage conditions described previously (section 3.2.8.1. - 3.2.8.3.). AU results fiom the other tests indicate this HPLC procedure is a reliable approach for simultaneous quantification of 2-, E- isomers of doxepin and its desrnethyl metabolite. Figure 4.3. shows spectra (C-IUA integrator) of standard reference samples (A) and extracts of control human urine (B), while Figure 4.4. shows similar spectra (data system) of standard reference samples (A) and extracts ficm control human plasma (B). Table 4.10. Freezefthaw stability of E-doxepin

Urine 20 ng/mL 100 ng/mL - One cycle 10.16 20.14 100.38 10.29 20.14 94.86 9.76 19.90 100.42 - X 10.07 20.06 98.55 C.V. % 2.74 O -69 3 -25

Two cycles 10.62 10.21 10.62 - X 10.48 C.V. % 2.26

Table 4.11. Freezefthaw stability of E-desmethyldoxepin

Plasma Urine Cycle 10 ng/mL 100 ng/mL 20 nghL 100 ng/mL One cycle 10.44 99.34 18.2 1 97.69 10.54 98.69 19.17 93.1 1 19.50 - 10.12 98.87 93.29 X 10.37 98.97 18.96 94-70 C.V. % 2.12 0.34 3 -53 2.74

Two cycles 9.87 98.62 10.08 98.25 10.53 98.38 - X 10.16 98.42 C.V. % 3.32 0.19 Table 4.12. Stability of E-doxepin at 4°C

Plasma Urine Cycle 10 ng/mL 100 ng/mL 20 ng/mL 90 ndmL Day one 10.24 107.43 10.90 110.85 10.47 110.61 X 10.54 109.63 C.V. % 3.18 1.74

Day two 9.99 104.23 10.73 105.10 10.41 104.3 5 - X 10.38 104.56 C.V. % 3 -58 0.45

Table 4.13. Stability of E-desmethyldoxepin at 4OC

P Iasma Urine Cycle 10 nghL 100 ng/mL 20 ng/mL 90 nghL Day one 10.36 97.13 19.46 84.98 10.38 99.48 18.15 86.60 10.52 100.42 19.70 87.7 1 - X 10.42 99.01 19.10 86.43 C.V. % O. 84 1.71 4.37 1.59

Day two 16-63 97.32 10.24 98.16 10.34 97.47 - X 10.40 97.65 C.V. % 1.95 0 -46 Table 4.14. Stabiiity of extracted E-dompin in mobile phase at 23OC

Plasma Urine Time IO ng/rnL 50 ng/mL 1 O0 20 ng/mL 50 ng/mL 90 ng/rnL O hou 10.42 52.85 102.5 1 20.07 5 1.32 96.14 - 10.43 50.52 104.13 20.52 47 -40 97.46 X 10.43 51.69 103.32 20.30 49.3 6 96.80 Difference % 4.25 3.3 7 3.32 1.48 1.28 7.56

16.5 hour 9.47 48.98 96.17 8.79 49.23 98-72 - X 9.13 49.11 97.45 Difference % 8.70 1.79 2.56

Table 4.15. Stability of extracted E-desmethyldoxepin in mobile phase at

Plasma Urine Tirne 10 ng/mL 50 ng/mL 100 20 ng/mL 50 ng/mL 90 ng/mL O hour 8.56 46.46 103.87 9 -34 53.95 103.45 - X 8.95 50.21 103.66 Difference % 10.50 0.41 3.66

16.5 hour 9 -60 48.38 100.75 9 -65 50.24 101.33 - X 9.63 49.31 10 1.04 Difference % 3 -75 1.38 1.O4 Table 4.16. Accuracy and precision @-doxepin)

-- Matrix n Added (ng/rnL) Found (ng/mt) Accuracy (%) Precision (%) PIasma 15 1 1.O5 95.00 5.75 9 3 2-90 96.67 6.04 15 1O 9.7 1 97-10 3 -79 9 50 48.90 97.80 2.97 15 100 101.I1 98.89 2.66 9 200 199.41 99.7 1 2.1 1

Urine t3 1 1.07 93.00 S. 15 9 1 O 10.44 95.60 3.41 L 5 50 48.68 97.36 2.84 9 1O0 98.84 98.84 5.27 9 200 197.32 98.66 3 -63 15 400 403.41 99.15 2.62

Table 4.17. Accuracy and precision (E-desmethyldoxepin)

------..- - -- - Matrix n Added (ng/mL) Found (ng/mL) Accuracy (%) Precision (%) Plasma 13 1 1.O6 94.00 10.01 8 3 2.97 99.00 5.62 15 10 9-74 97.40 4.26 9 50 48.3 3 96.66 1.O7 15 100 99.55 99.55 3.73 9 200 202.37 98.82 1.59

Urine 14 1 1 .O9 9 1 .O0 6.79 9 1O IO. 14 98.60 2.9 1 15 50 48.96 97.92 7.44 9 100 99.55 99.55 3.3 3 9 200 198.00 99.00 5.86 15 400 400.81 99.80 5.23 Figure 4.3. HPLC chrornatogram of the extracts from blank human urine A, mixture of standard reference samples in mobile phase B, bIank human urine Peak identifications: 1, Zdoxepin; 2, E-doxepin; 3, nortriptyIine; 4, Zdesmethyldoxepin 5, E-desmcthyldoxepin

r 1 1 I O Figure 4.3. A 5 10 u üme (min)

Figure 43. B 1 L i 1 O s 10 U îimo (min) Figure 4.4. HPLC chromatogram of the extracts from biank human plasma A, mixture of standard reference samples in mobile phase B, blank human plasma Peak identifications: 1, Zdoxepin; 2, E-doxepin; 3, nortriptyline; 4, Z-desmethyidoxepin 5, E-desmethyldoxepin 2

Figure 4.4. A

Figure 4.4. B 4.3.0.0. Meiabolic studies

4.3.1.0. Exploratory in vivo experiments The doxepin treatments were generally well tolerated by all species, the only observable side effect king sedation which was particularly marked for 2-4 hours after parenterai administration. Total recoveries of the isomers of doxepin plus desmethyldoxepin in 0-24 hour urine after oral dosing were genedy very low when expressed as percentages of the dose administered. Of the species examine& recovery was highest in the dog (1.1%) and lowest in the rat (0.03%) and the mean recovery in humans (0.47%) was some 15 fold greater than that in rats (Table 4.20.). The results of the studies on human volunteers and various animal species are summarized in Tables 4.18. to 4.19. in which the urinary recoveries of 2-doxepin or 2-desmethyldoxepin were expressed as percentages of total doxepin or desmethyldoxepin respectively. ANOVA was carried out in which the effects in the mode1 were species and subject with % 2-desmethyldoxepin or % 2-doxepin as the dependent vanable. The results showed no significant effect of subject @=0.2369), but the effect of species was highly significant @=0.0001). The multiple cornparison tests (Student-Newman-Keuls, Tukey-Kramer and Spjotvoll-Stoline) all showed rat and human desmethyldoxepin data to be significantly different from those of dog, guinea-pig and rabbit, but not significantly different from each other. No corresponding interspecies differences were detected with % 2-doxepin as the dependent variable. Figures 4.5. and 4.6. depict mean percentages of Z-doxepin and 2- desmethyldoxepin in 0-24 hour post-dose urine fiom various species. Figures 4.7. and 4.8. show HPLC chrornatograms of standard reference samples in mobile phase (A), blank urine (B) and 0-24 hour post-dose urine (C) fkom human and dog afier doxepin oral dose. Interestingly, one case of significant ratio distortion in % Z-doxepin was observed in human studies. The % Z-doxepin in 0-24 hour urine in this subject tunied out to be 42% compared to the other three subjects ranged kom 17-2 1%. Table 4.18. Urinary excretion (0-24 h) of doxepin in human and various animal species after doxepin oral administration

Subject or Urine volume 2-doxepin E-doxepin Z-doxepin Species Animal # (mu (ng/mL) (ng/mL) (%)

4 2475 23.28 31.91 42 - X + S.D. 1318 f 801 30t 7 114 f 58 24 f 12

Rat 1 20 8.12 36.33 18 2 20 20.37 92.23 18 3 23 5.88 24.97 19 4 25 5.43 17.46 24 - X + S.D. 22 k 2 IO* 7 43 -C- 34 20t 3

Rabbit 1 100 7.7 1 27.54 22 2 150 79.88 297.58 2 1 3 1O0 6.25 2 1.O0 23 - X I S.D. 117 + 29 31 f 42 115 f 158 22t 1

Dog 1 190 358.80 1873 -43 16 2 200 775.79 2810.37 22 3 250 542.5 O 1 168.53 32 4 170 438.58 987.48 3 1 - X f S.D. 202 f 34 529 + 181 1710 f 827 25 t 8 Table 4.19. Urinary excretion (0-24 h) of desmethyldoxepin (NorDox) in human and various animal species afier doxeph oral dosing

Subject or Urine volume 2-NorDox E-NorDox 2-NorDox S pecies Animal # (mL) (ndmL) (ng/mL) (%) Human 1 1030 83.20 57.52 59 2 63 5 185.14 202.98 48 3 1130 132.80 69.65 66 4 2475 32.17 16.25 66 - X IS.D. 1318 C 801 108 I 66 87f 81 60f 9

Rat 1 20 41.33 38.85 52 2 20 63.37 70.67 47 3 23 39.64 3 1 .O9 56 4 25 1 1.39 5 -90 66 - X + S.D. 22- 2 39 k 21 37 k 27 55+ 8

X IS.D. 117 t- 86 31 + 36 52 + 43 32k 8

Rab bit 1 I O0 987.35 19 12.5 1 34 2 150 1748.36 3435.52 34 3 1 O0 86.65 2 10.83 29

X + S.D. 117 f 29 941 I 832 1853 +- 32f 3

Dog 1 190 200.7 1 636.83 24 2 200 409.83 1278.96 24 3 250 400.86 978.64 29 4 170 350.15 883.7 1 25 - X + S.D. 202 f 34 340 I 97 945 + 265 26f 3 Man (n = 4) - . .rr@ y Dog (n = 4)

Rabbit

Rat (n = 4)

f I 1 I . 1 O 20 40 6 0 80 100

Figure 4.5. Mean percentages of Zdoxepin in 0-24 hour urine from various species after doxeph oral administration

Man (n = 4)

Dog (n = 4)

Rabbit (n = 3)

Guinea-pig (n = 3)

Rat (n = 4)

O 20 40 60 80 100

Figure 4.6. Mean percentages of Zdesmethyldoxepin in 0-24 hour urine from various species after doxepin oral administration Table 4.20. Mean urinary excretion (Oh dose, 0-24 h) of doxepin @ox) and desmethyldoxepin (NorDox) after doxepin oral administration to human and different animal species

Dox NorDox Dox + NorDox

S pecies Z E Z+E Z E ZiE

- ~urnan~

~at~

Guinea-

P igc

ab bit^

Iloge

*, negligible Figure 4.7. HPLC chromatograms of the extracts from hurnan urine A, mixture of standard reference samples in mobile phase B, blank human urine C, 0-24 hour buman urine after doxepin oral administration Peak identifications: 1, Zdoxepin; 2, E-doxepin; 3, nortriptyline; 4, 2-desmethyldoxepin 5, E-desmethyldoxepin

t L k i O s 10 l3 thi8 (min) Figure 4.7. A t I L 1 O 5 10 Ls tirne (min) Figure 4.7. B

t L A 1 O 5 IO U the (min) Figure 4.7. C Figure 4.8. HPLC chromatograms of the extracts from dog urine A, mixture of standard reference samples in mobile phase B, blank dog urine C, 0-24 hour dog urine after doxepin oral administration Peak identifications: 1, Zdoxepin; 2, E-doxepin; 3, nortriptyline; 4, 2-desmethyldoxepin 5, E-desmethyldoxepin

i A i 1 O s 10 U the (min) Figure 4.8. A Figure 4.8. B

i 1 1 1 O s 10 U the (min) Figure 4.8. C 431.1 Discussion The pharmacokinetic and pharmacodynamic consequences of stereoisomensm have received considerable attention in recent years. Stereoselective studies on doxepin have been difficult to pursue because the low percentage of the Z-doxepin in commercial dosage forms leads to extremely low plasma concentrations of this active isomer. Another problem is that tncyclic antidepressants have been known to induce sinus tachycardia, changes in conduction time and arrhythmia (Braden et al., 1986, F lsoli and Glauser, 198 1, Pellinen et al.,1987, Pente1 and Senowitz, 1986) so that assay sensitivity problems cannot be overcome by the use of high doses in healthy human volunteers. The problem of assay sensitivity has been resolved somewhat by the development of the present HPLC procedure with lower limits of quantification of 1.O ng/mL for each isomer of the parent drug and the desmethyl metabolite. A third diniiculty is that the impact of "enrichment" of the active 2-isorners (Ghabrial et al., 1991) cannot be resolved easily without administration of the pure E-isomer which was not available in quantities suficient to administer at a reasonable dosage level in humans. Consequently the decision was made to carry out the investigation in animais. Use of the phrase "animal model" is often viewed with justifable skepticism because of wide interspecies variation in metabolism and pharmacokinetics. In the present context, the term "animal modei'' refers to an animal species in which the percentages of 2-doxepin and 2-desmethyldoxepin in 0-24 hour cumulative urine were similar to those in human urine. Cumulative urinary excretion data reflect an overall end-result of a variety of pharmacokinetic events (absorption, distribution, phase 1 and phase II metabolism, various excretion pathways and, in the present case, the possibility of interconversion between isomers). Thus an experiment was carried out to find an animal species in which the "overall end result" was similar to that in humans. The percentages of Z- doxepin in the 0-24 hour urine was reasonably consistent amongst the species examined (TabIe 4.18.) with mean values ranging 5om 20 to 25%. There was wider interspecies variability in terms of the percentages of 2-desmethyldoxepin excreted which in dog, rabbit and guinea-pig urine were only about half that in human urine (Table 4.19.). ANOVA and the multiple cornparison tests showed rat and hurnan data to be significantly different @=0.000 1) fiom those of the other species, but not significantly difYerent fiorn each other. Therefore, the remaining in vivo experirnents were carried out in Lewis strain rats. The low urinary recoveries of doxepin and desmethyldoxepin may be explained by significant "first-pass" effects involving a variety of other phase 1 and phase II metabolic pathways. Nevertheless, urùiary excretion data would give valuable information on the issue of interconversion between the isomers.

4.3.2.0. Significant ratio distortion of doxepin isomers in one healthy male volunteer The resdts codbned the previous observation (section 4.3.1 .O.) and indicated significant percent ratio increase for 2-isomer of parent clmg in addition to its desmethyl metabolite in both urine (mean + SD: 53 f 11 for doxepin, and 82 + 14 for the metabolite) and plasma (mean f SD: 33 + 5 for doxepin, and 55 + 10 for the metabolite) samples at various tirne periods although a the dependent, progressive process was observed for the ratio distortions (Tables 4.21. and 4.22.). Z-isomer percent ratios based on AUCs of the individual isomers tunied out to be 31% and 84% for doxepin and desmethyldoxepin, respectively (Figure 4.9.). The total 0-120 hour urinary recovery (doxepin + desmethyldoxepin) was 0.92% (0.36% + 0.56%) of the dose. Figure 4.10. shows the HPLC chromatograms of plasma samples, in which signincant ratio distortion of doxepin can readily be seen. Table 4.21. Isomer concentrations and 2-isomer% in plasma samples after doxepin single oral dose (75 mg base) to one healthy male volunteer

Time (hour)

Mean + S.D. a ND, non-detected b *, c 1 ng/d

4.3.2.1. Discussion There have been no such cases of signincant ratio distortion in doxepin isorners reported in literature. The mechanisrn for this distortion is unknown although speculations could be made similar to those to account for the signincant ratio distortion in desmethyldoxepin isomers. The fact that there is evidence of a tirne dependent, progressive process for the ratio distortion in both doxepin and desmethyldoxepin isomers may indicate the ratio distortion is related to stereoselective biotransformation.

Table 4.22. Isomer concentrations and Z-isomer% in urine samples after doxepin single oral dose (75 mg base) to one healthy male volunteer

Time (hour)

a ND, non-detected b *, < i ng/rnL AUC (ng -h/mL)

Dox NorDox

Figure 4.9. Plasma isorner AUC profdes after doxepin @ox) oral administration to one healthy male volunteer (2-Dox % = 31%; ZNorDoxOh= 84%) Figure 4.10. IIPLC chromatograms of pIasma extracts from the volunteer A, mixture of standard reference samples in mobile phase B, blank plasma C, 8 hour plasma after doxepin oral administration Peak identifications: 1, Zdoxepin; 2, E-doxepin; 3, nortriptyline; 4, Zdesmethyldoxepin 5, E-desmethyldoxepin

Figure 4.10. A Figure 4.10. B

Figure 4.10. C 4.3.3.0. The effect of route of administration on the urinary excretion of the isomers of doxepin and desmethyldoxepin in rat Table 4.23. shows data on the effect of route of administration on urinmy recovery in three paralle1 groups of rats (n=4). ANOVA was carried out in which the

effect in the mode1 was route of administration and the dependent variable was either

% Z-doxepin or % 2-desmethyldoxepin. The results showed no sipiiicant effect of route on % Z-doxepin @=0.3262, +0.220), but the effect of route on % Z- desmethyldoxepin was highly signiscant @=0.000 1, ?=0.926). Ail three multiple cornparison tests (Student-Newman-Keuls, Tukey-Kramer and Spjo~oll-Stoline) showed the % Z-desrnethyldoxepin afier oral (po) administration to be signifïcantly dEerent fiom the percentages Obtained after either intravenous (iv) or intraperitoneal (ip) injection, whereas there were no ciifferences detected in % 2-desrnethyldoxepin after iv or ip administration. Figure 4.11. depicts the mean % composition of desmethyldoxepin isomers in 0-24 hou urine after dosing with doxepin via these three routes. The urinary recovery (0-24 hour) relative to the doxepin dose (% dose) is shown in Table 4.24.. Figures 4.12.-4.14- are HPLC chromatograms fkom the studies after po, iv, and ip administration of the hg.

4.3.3.1. Discussion This experiment was carried out in three pardel groups of four rats to investigate the effects of route of administration on the urinary output. As anticipated, urinary concentrations of both isomers of the parent dnig were substantially lower after oral administration than after parenteral administration (Table 4.23.). Nevertheless, ANOVA revealed no significant ciifferences in the Z:E ratios expressed in terms of percentages of 2-doxepin which were consistent across all three routes of administration at approximately 20%. In the case of the desmethyl metabolite, however, ANOVA and the multiple comparison tests showed that the percentage of Z-desmethyldoxepin deroral administration of commercial doxepin was signincantly different @=0.0001) fiom the percentages of 2-desmethyldoxepin excreted afier either parenterd route. These data suggest that the change in Z:E ratio of the metabolite after oral administration of commercial doxepin may be due to phase 1 and/or phase U metaboiism of the drug andor the desmethyl metabolites during the first pass through the GIT, and ïiver before reaching the systemic circulation. A cornparison of 0-24 hour urinary recovery ('%O dose) of doxepin between oral and intrapentoneaf administrations (0.0 1 versus 0 -26) seems to suggest the extensive fist- pass effect der oral dosing is not solely attnbuted to hepatic biotransformation, but loss in GIT before reaching systemic circulation due to various elimination events (e.g. excretion through feces, andor metabolism occurred in gut lumen and wall) plays a significant role (Table 4-23.). Table 4.23. Urinary excretion (0-24 h) of doxepin @ox) and desmethyldoxepin (NorDox) in parauel groups of Lewis strain rat after oral (po), intravenous (iv), or intraperitoneal (ip) doshg with doxepin (Z:E=16:84)

------Urine 2-Dox E-Dox Z-Dox 2- E- Z- Route volume (ng/mL) (ng/rnL) (%) NorDox NorDox NorDo (mu (ng/mL) (ng/mL) x (Oh)

AI1 data are expressed as mean + SB. from four Lewis strain rats a 30 mgkg oral dose 7.5 mgkg intravenous dose C 15 mgkg intraperitoneal dose

Mean isomer percentage

Figure 4.11. Mean % of desmethyldoxepin isomers in 0-24 hour rat urine after oral, intravenous or intraperitoneal dosing with doxepin Table 4.24. Urinary excretion (% dose, 0-24 h) of doxepin @ox) and

desmethyldoxepin (NorDox) after doxepin oral @O), inéravenous (iv), or intraperitoneal (ip) administration to paralle1 groups of Lewis strain rats

Dox NorDox Dox + NorDox Rat # z E Z+E Z E Z+E total (~0~) 1 2 3 4 - X Rat # (ivb) 1 2 3 4 - X Rat # OpC) 1 2 3 4 - X a 30 mg/kg oral dose 7.5 mgkg intravenouç dose C 15 mgkg intraperitoneal dose Figure 4.12. WLCchromatograms of the extracts from rat urine A, mixture of standard reference sampies in mobile phase B, blank rat urine C, 0-24 hour rat urine after doxepin oral administration Peak identifications: 1, Zdoxepin; 2, E-doxepin; 3, nortriptyline; 4, 2-desmethyldoxepin 5, E-desmethyldoxepin

I L I 1 O 5 10 L5 the (min) Figure 4.12. A Figure 4.12. B tSme (min)

Figure 4.12. C Figure 4.13. HPLC chromatograms of the extracts from rat urine A, mixture of standard reference samples in mobile phase B, bIank rat urine C, 0-24 hour rat urine after doxepin intravenous administration Peak identifications: 1, Zdoxepin; 2, E-doxepin; 3, nortriptyline; 4, 2-desmethyldoxepin 5, E-desmethyldoxepin

4 ; 10 the (min) Figure 4.13. A Figure 4.13- B

Figure 4.13. C Figure 4.14. WLC chromatograms of the extracts from rat urine A, mixture of standard reference samples in mobile phase B, blank rat urine C, 0-24 hour rat urine after doxepin inbaperitoneal administration Peak identifications: 1, Z-doxepin; 2, E-doxepin; 3, nortriptyline; 4, Z-desmethyldoxepin 5, E-desmethyldoxepin

tirne (min) Figure 4.14. A L 3 L 1 O 5 10 U lime (min) Figure 4.14. B

1 1 i 10 U ha (min) Figure 4.14. C 4.3.4.0. Results from the test of metabolic E- to 2- isomer conversion in rat No 2-, but only E- desmethyldoxepin was detected after E-desmethyldoxepin or E-doxepin administration via either iv or po routes. In many experiments, no dmg related peaks were detected in chromatograms of urines cokcted 24-48 hours post dose and these scant data were neglected in the present treatment. Table 4.25. summarizes data kom the cross-over study in the group of rats (n=8), each of which received six treabnents according to the design shown in Figure 3.1. (page 56). Thus each animal received E-desmethyldoxepin, E-doxepin and commercial doxepin through iv and po routes with a washout period of two weeks between treatrnents. Urine samples were coUected in two segments (0-24 and 24-48 h) except for the administrations of doxepin after which the £ïrst segment was collected over 0-16 hours due to circumstances beyond our control. Nevertheless, the % 2-desmethyldoxepin was still ~i~cantiydifferent (ANOVA, p=0.000 1) &om that of Z-doxepin after oral administration (Figure 4-15.), consistent with previcus observations in the rat (Tables 4.18.-4.19., Table 4.23. and Figure 4.11.). Table 4.26. lists 0-24 hour mean urinary recovery (% dose) in this group of rats with cross-over administration of these three dmgs via both iv and po routes. The most important observation eom this experiment, however, is that there was no evidence of E to Z isomer conversion der administration of E-doxepin or E-desmethyldoxepin by either iv or po route. Figure 4.16. shows HPLC chrornatograms of standard references (A), blauk urine (B), and 0-24 hour cumulative urine after dosing with E-isomers (C & D).

4.3.4.1. Discussion This in vivo experiment was based on a cross-over design (Figure 3.1., page 42) in which eight rats each received oral and intravenous doses of E-doxepin, E- desmethyldoxepin and commercial doxepin (Table 4.25.). The two sequences (Figure 3.1.), together with the two week washout periods between treatrnents, were selected to space the intravenous administrations one month apart to give the tail vein injection sites time to heal between injections. The percentage of 2-desrnethyldoxepin measured as Z/Z+E after oral administration of commercial doxepin was signifkantly different from the percentage of 2-doxepin (ANOVA) but there was no such difference after intravenous administration (Table 4.25.). No 2-isomers were detected after either oral or intravenous administration of E-doxepin or E- desmethyldoxepin. Thus there was no evidence of isomerization fiom E- to Z- desmethyldoxepin in these in vivo experirnents. The present data suggests that stereoselective metabolism is most probably the mechanism responsible to the ratio distortion. The O ther two possible mechanisms. Le. stereoselective rend clearance of desmethyldoxepin, or higher rate of formation of Z-desmethyldoxepin than E- desmethyldoxepin, appear to be unlikely since the significantly dif5erent 2- desmethyldoxepin percentages between oral and intravenous, and the % 2-doxepin data do not lend support to these hypotheses. Stereoselective metabolic pathway(s) could be either phase 1 or phase II. For example, E-desmethyldoxepin could undergo faster oxidation, andlor more extensive phase II metabolism than its Z couterpart. The Merence between the oral and intravenous administration indicates the effect of extensive first-pass metabolism. Further in viîro studies with rat liver microsornes and gastrointestinal tract homogenates may bring us closer to the clarification of these remaining questions. Table 4.25. Urinary excretion of doxepin (Dox) and desmethyldoxepin (NorDox) after oral (po) or intravenous (iv) dosing with doxepin and E-isomers to Lewis strain rats in the cross-over studies

Dmg Urine Z-Dox E-Dox Z-Dox Adm inistered segment (h) wmL) (ng/mL) (%)

po Dox a 0-16 25 I 15 113 + 63 183

po Dox a iv Dox b O- 16 32 + 34 183 -t 208 18k4

Al1 data are mean f S.D. in groups of 8 Lewis strain rats a 30 mglkg oral dose of commercial doxepin 9 mgkg intravenous dose 15 mgtkg oral dose 7 mgkg intravenous dose 8 mgkg oraI dose 4 mgkg intravenous dose ND, Not detected NA, Not applicable Table 4.26. Urinary excretion (% dose) of doxepin (Dox) and desrnethyldoxepin (NorDox) after oral (po), intravenous (iv) dosing with doxepin, E-doxepin, and E-desmethyldoxepin to Lewis strain rats

-- -- -

Drug Urine Dox adminis- segment Z E Z+E trated (hl

po DOX~

iv Dox b

PO E-DOX' iv E-Dox d

po E- NO~DOX~

iv E- ~or~ox' Al1 data are mean in a group of 8 Lewis strain rats a 30 mgkg oral dose of commercial doxepin b 9 mgkg intravenous dose 15 mgkg oral dose d 7 mgkg intravenous dose 8 mgkg oral dose f 4 mgkg intravenous dose

*, negligible ND, Not detected Figure 4.15. Mean isorneric composition of desmethyldoxepin in 0-16 hour rat urine after doxepin oral (po) and intravenous (iv) administration Figure 4.16. HPLC chromatograms of the exh-acts from rat urine A, mixture of standard reference samptes in mobile phase B, blank rat urine C, 0-24 hour rat urine after E-desmethyldoxepin oral administration D, 0-24 hour rat urine after E-doxepin oral administration Peak identifications: 1, Zdoxepin; 2, E-doxepin; 3, nortriptyline; 4, Z-desmethyldoxepin 5, E-desmethyldoxepin

Figure 4.16. A Figure 4.16. B

Figure 4.16. C Figure 4.16. D

4.3.5.0. In vitro metabolic studies with liver microsomes or gastrointestinal tract (GIT) homogenates

4.3.5.1. Incubation of doxepin related xenobiotics with rat liver microsomes

Incubation of a commercial preparation of doxepin (Z:E = 16:84) with rat Lver microsomes showed time dependent progressive increases in the percentage of the Z- isomer, which was particularly marked in the case of the desmethyl metabolite (Tables 4.27.-4.28.). Exmination of the concentration data, ho wever, suggests that the reason for the ratio distortion lies in more rapid metabolic eIiminiition of the E- isomers rather than "enrichment" of the 2-isomers. These observations were connmied when E-doxepin was incubated with rat iïver microsomes (Tables 4.29.- 4.30.). Small amounts of 2-desmethyldoxepin detected in the homogenates were attributable to Z-isomer contamination (1.8%) rather than interconversion between the isomers. Moreover, no E-isorners were detected upon incubation of pure Z- doxepin with the rat liver microsamal preparation (Tables 4.31.-4.32). Incubation of E-desmethyldoxepin contaminated with 3% Z-desmethyldoxepin with the rat liver microsomes showed evidence (Table 4.33.) of more rapid metabolic elimination of the E-isomer of which concentrations fell 7-8 fold over one hour while those of the Z- isomer feU by only 2 fold. This phenornenon is iIlustrated graphically in Figure 4.17. which shows the metabolic elimination half-life of 2-desmethyldoxepin in the rat to be 5-7 fold greater than those of the other three analytes. The half-Me data (Table 4.34.) were examined by ANOVA in which the effects in the mode1 were subject (i-e. rat) and analyte, with half-He as the dependent variable. While there was no significant effect of subject @=0.2283), there was a highiy significant effect of anaiyte (p=0.0001). Al1 three multiple cornparison tests (Student-Newman-Keuls, Tukey- Kramer and Spjotvoll-Stoline) showed that the half-life for Z-desmethyldoxepin was significantly différent Çom those of the other three analytes. HPLC chromatograms of doxepin related isomers and the intemal standard in the extracts of incubations of commercial doxepin or individual isomers with rat Liver microsomes are show in Figure 4.18. A-F. Traces of the didesmethyl isomers (peaks 6 and 7) appeared in the chromatograms at long retention times and did not interfere with the quantification of either isomer of doxepin or desmethyldoxepin. The structural identities of the desmethyl (moleculzr weight of 265) and the didesmethyl (molecular weight of 25 1) metabolites were codhned by electrospray mass spectrometry (Figure 4.19. A-E). Isomers of desmethyl ador didesmethyl metabolites were not detected in incubation mixtures containing doxepin in the absence of iiver microsomes. Based on these HPLC chromatograms, deduction can be made to assign the isomenc identity to each of the peak pair of didesmethyldoxepin. Since only peak 6 but no peak 7 appeared fiom the incubation of 2-doxepin, while peak 7 (but no peak 6 beyond a trace isorneric impurity) after incubations of either E-doxepin or E-

desmethyldoxepin, it is rational to idenw peaks 6 and 7 as 2- and E- didesmethyldoxepin, respectively. Table 4.27. Doxepin isorner concentration after incubation of rat tiver microsornes spiked with 25 pM commercial doxepin (Z-Dox, 1116

10 1 0.96 13 1 1134 10 2 1.28 19 188 9 3 O. 8 8 104 1111 9 4 0.87 270 2170 11 5 O. 8 8 144 1439 9 6 0.98 69 744 8 7 0.87 83 1108 7 8 0.87 111 1335 8 - X t S.D. 0.95 + 0.14 116 2 73 1154 + 567 911

- X + S.D. 0.95 5 0.14 47 & 30 395 t 297 13 + 5

40 1 0.96 20 28 42 2 1.28 12 28 30 3 0.88 20 26 43 4 0.87 63 393 14 5 0.88 33 122 2 1 6 0.98 26 53 33 7 0.87 14 8 1 15 8 0.87 16 97 14 - X k S.D. 0.95 k 0.14 26 + 17 104 t 122 27 I 12

60 1 0.96 14 22 39 2 1.28 14 3 5 29 3 0.88 19 27 4 L 4 0.87 55 308 15 5 0.88 23 4 1 36 6 0.98 22 46 32 7 0.87 8 25 24 - 8 0.87 1 O 26 28 X + S.D. 0.95 & 0.14 21 $r 15 66 +, 98 31k9 Table 4.28. Desrnethyldoxepin (NorDox) isomer concentration after incubation of rat liver microsornes spiked with 25 pM

commercial doxepin (ZDox, 1116 ng/mL; E-Dox, 5789 nglmL)

Time Protein Z-NorDox E-NorDox Z-NorDox Rat # (min) (mg/ mi;) (ngfmL) (ng/mL) % 10 1 0.96 610 1198 34 2 1.28 625 994 39 3 0.88 642 1258 34 4 0.87 509 1040 3 3 5 0.88 578 1228 32 6 0.98 625 1283 3 3 7 0.87 682 1577 30 8 O. 8 7 670 1573 30 - X f S.D. 0.95 k 0.14 618 k 55 1269 & 215 33 +3

20 1 O -96 593 86 1 4 1 2 1.28 519 358 59 3 0.88 607 803 43 4 0.87 599 1115 3 5 5 0.88 594 1063 36 O 0.98 5 72 85 1 40 7 0.87 672 1342 33 8 0.87 676 1397 3 3 - X + S.D. 0.95 f: 0.14 604 + 51 974 + 334 40 t 9

- X k S.D. 0.95 + 0.14 SOS + 117 476 f 316 58 + 18

60 1 0.96 415 8 1 84 2 1.28 150 12 93 3 0.88 405 62 87 4 0.87 53 1 629 46 5 0.88 505 303 63 6 0.98 381 228 63 7 0.87 576 505 53 8 0.87 575 505 53 - X & S.D. 0.95 f: 0.14 442 k 141 291 st 234 68 I 18 Table 4.29. Doxepin isomer concentration after incubation of rat ber microsornes spiked with 25 pME-doxepin (E-Dox, 6849 nglmL) contaminated with Zdoxepin (Z-Dox, 126 ag/mL)

- Time Protein Z-Dox E-Dox Z-Dox Rat # (min) (mgfmL) (ng/mL) (WmL) Oh 10 1 0.96 ND 1260 NA 2 1.28 II 259 II 3 0.88 II 1222 II 4 0.87 II 2507 11 5 0.88 II 1703 II 6 0.98 II 828 11 7 0.87 Il 1234 II II II - 8 0.87 1453 X + S.D. 0.95 t 0.14 1308 k 650

20 1 0.96 ND 327 NA 2 1.28 Il 58 11 II 3 0.88 369 II 4 0.87 11 1279 II 5 0.88 II 571 Il 11 6 0.98 11 23 O 7 0.87 II 485 11 8 0.87 II 576 11 - X -t S.D. 0.95 f 0.14 487 + 365

ND, not detected NA, not applicable Table 4.30. Desmethyldoxepin (NorDox) isomer concentration after incubation of rat ber microsornes spiked with 25 pM E-doxepin (6849 ng/mL) contaminated with Zdoxepin (126 ng/mL)

-- - 10 1 0.96 27 1532 2 2 1.28 27 1294 2 3 0.88 26 1612 2 4 0.87 2 1 1304 2 5 0.88 24 1546 2 6 O -98 24 1607 1 7 0.87 27 1923 f 8 0.87 28 1916 1 - X * S.D. 0.95 + 0.14 26 I 2 1592 I1: 237 2f0 20 1 0.96 25 1141 2 2 1.28 24 551 4 3 0.88 22 1128 2 4 0.87 24 1440 2 5 0.88 25 1413 2 6 0.98 22 1088 2 7 0.87 26 1614 2 8 0.87 27 1691 2 - X f S.D. 0.95 -t 0.14 24 F 2 1258 + 365 2+1

40 1 0.96 22 419 5 2 1.28 13 25 34 3 O. 8 8 19 518 4 4 0.87 23 1076 2 5 0.88 23 838 3 6 0.98 18 464 4 7 0.87 24 1041 2 8 0.87 26 1102 2 - X + S.D. 0.95 + 0.14 21 +4 685 k 389 7f11

60 I 0.96 i 9 135 12 2 1.28 8 13 38 3 0.88 17 112 13 4 O. 87 2 1 842 2 5 0.88 2 1 596 3 6 0.98 14 103 12 7 0.87 23 655 3 8 0.87 24 736 3 - X -t S.D. 0.95 + 0.14 18 f 5 399 f 339 11 Il2 Table 4.31. Doxepin isomer concentration after incubation of rat Iiver

microsornes spiked with 25 j.M Zdoxepin (Z-Dox, 6975 ng/mL)

Time Protein Z-Dox E-Dox Rat # Z-Dox (min) (W/mL) (ng/mL) WmL) %

- X k S.D. 0.95 + 0.14 1207 + 756 20 t O .96 242 ND NA 2 1.28 78 11 II 3 0.88 178 11 11 4 0.87 Il32 11 II 5 0.88 522 Il II 6 0.98 152 Il II 7 0.87 240 II II 8 0.87 333 Il Il - X + S.D. 0.95 + 0.14 360 & 340

40 1 0 -96 54 ND NA 2 1.28 118 t1 11 3 0.88 60 11 II 4 0.87 732 11 Il 5 0.88 113 Il II 6 0.98 65 II II 7 0.87 102 11 II 8 0.87 83 Il 11 - X t- S.D. 0.95 & 0.14 166 t 230

1 0.96 4 1 ND NA 2 1.28 54 II II 3 0.88 26 II II 4 0.87 371 II Il 5 0.88 57 11 11 6 O -98 90 11 Il 7 0.87 29 II 11 8 0.87 46 11 Il - X + S.D. 0.95 I 0.14 89 k 116 ND, not detected NA, not applicable Table 4.32. Desmethyldoxepin (NorDox) isomer concentration after incubation of rat ber microsomes spiked with 25 pM 2-doxepin (6975 ng/mL)

Time Protein Z-NorDox E-NorDox Z-NorDox Rat # (min) (mgfd) (ng/mL) (ng/mL) Oh 10 1 0.96 2552 ND NA 2 1.28 2615 11 11 3 0.88 2669 11 11 4 0.87 1913 19 11 5 0.88 2352 11 11 6 0.98 271 1 11 Ir 7 0.87 2960 Il lr 8 0.87 2886 11 11 - X + S.D. 0.95 4: 0.14 2582 4 330 20 1 0.96 2292 ND NA

2 1.28 1851 11 11

3 0.88 2319 n 11

4 0.87 2445 lq 11 5 0.8 8 2616 II 11

6 0.98 2264 11 11

7 0.87 2903 11 11 8 0.87 3002 Il 11 - X + S.D. 0.95 + 0.14 2462 + 373 40 1 0.96 1612 ND NA 2 1.28 1052 II Il 3 0.88 1649 Ir 11 4 0.87 2120 It rl 5 O. 8 8 2165 11 rl 6 0.98 l504 11 11 7 0.87 23 18 11 11 8 0.87 2474 11 11 - X + S.D. 0.95 + 0.14 1862 I 483 60 1 0.96 1284 ND NA 2 1.28 796 11 11 3 0.88 1261 11 11 4 0.87 1923 rl 11 5 0.88 1759 11 11 6 0.98 1501 11 11 7 0.87 2056 11 11 Il 8 0.87 2118 11 - X & S.D. 0.95 + 0.14 1587 .t 459

ND, not detected NA, not applicable Table 4.33. Isomer concentration after incubation of rat ber rnicrosomes spiked with 15 pM E-desmethyldoxepin (E-NorDox, 3856 ng/mL) contaminated with 2-desmethyldoxepin (2-NorDox, 119 ng/mL)

-- - - 10 1 O .96 9 1 1623 5 2 1.28 79 871 8 3 0.88 84 1549 5 4 0.87 92 2083 4 5 0.8 8 9 1 1952 4 6 0.98 83 1482 5 7 0.87 93 1953 5 8 0.87 95 2049 4 - X t S.D. 0.95 + 0-14 89 + 6 1695 + 407 5+1

20 1 0.96 8 1 900 8 2 1.28 6 1 170 26 3 0.88 75 890 8 4 0.87 89 1615 5 5 0.88 85 1329 6 6 0.9 8 73 807 8 7 0.87 87 1385 6 8 O. 8 7 90 1510 6 - X t S.D. 0.95 + 0-14 80 & IO 1076 + 478 9f7

40 1 0.96 65 176 27 2 1.28 6 14 30 3 0.88 60 149 29 4 0.87 78 912 8 5 0.88 75 655 1O 6 O. 9 8 5 5 145 28 7 0.87 78 732 IO 8 0.87 8 1 827 9 k S.D. 0.95 h 41-14 62 + 25 451 li: 364 19 t 10 Table 4.34. ~alf-livesaof doxepin Oox) and desmethyldoxepin (NorDox) isomeas in incubation with rat Iliver microsornes

Rat Z-Dox E-Dox 2-NorDox E-NorDox # (min) (min) (min) (min)

1 11.1 9 .O 45.8 8.8 2 14.0 14.9 26.8 5.1 3 10.1 8.8 28.8 9.0 4 19.2 19.5 123 -0 23.1 5 10.2 9.0 125.0 19.2 6 9.8 10-4 25.2 8 -4 7 10.4 11.3 136.0 22.4 8 10.7 9.8 146.0 22.9 - x + S.D. 11.9 $1 3.2 11.6 + 3.8 82.1 + 54.7 14.9 k 7.7 a, denved fkom parallel observations, sample in duplicate at each time point

Rat liver (n=8)

Dox NorDox

Figure 4.17. Mean NI vitro metabolic elimination half-lives of doxepin @ox) and desmethyldoxepin (NorDox) isomers Figure 4.18. HPLC chromatograms of extracts from the incubation mixture with rat liver microsornes A, mixture of standard reference samples in mobile phase B, incubation blank C, incubation of doxepin at 20 minutes D, incubation of Zdoxepin at 40 minutes & incubation of E-doxepin ai 40 minutes F, incubation of E-desmethyldoxepin at 40 minutes Peak identifications: 1, 2-doxepin; 2, E-doxepin; 3, nortriptyline; 4, 2-desmethyldoxepin 5, E-desmethyldoxepin 6, 2-didesmethyldoxepin 7, E-didesmethyidoxepin

time (min)

Figure 4.18. A 1 I I I 8 12 16 20

time (min)

Figure 4.18. B

I 1 I L 1 1 r 1 1 1 1 O 4 8 12 16 20 time (min)

Figure 4.18. C time (min)

Figure 4.18. D

time (min)

Figure 4.18. E time (min)

Figure 4.18. F Figure 4.19. Mass spectra (ES+) of Z,E- desmethyl- and didesmethyldoxepin A, controI incubation mixture B, Z-desrnethyldoxepin (HPLC peak 4) C, E-desmethyldoxepin (WLC peak 5) D, Z-didesmethyldoxepin (HPLC peak 6) E, E-didesmethyldoxepin (HPLC peak 7)

San ES* 3.1387

Figure 4.19. A: control incubation mixture Scrr ES- 4.lkl

2-Desmethy Idoxepin

Figure 4.19. B: Zdesmethyldoxepin (HPLC peak 4)

Sca ES- 4.1 187

Figure 4.19. C: E-desmethyldoxepin @PLCpeak 5) SM ES* 2 94.7

Figure 4.19. D: Z-didesmethyldoxepin (HPLC peak 5)

JY-RATl IIVUU Dotu~S1 i (O.ô21] Sm (SG. 2~0.70):Cm (5341 San ES- ZSh7

Figure 4.19. E: E-didesmethyldoxepin (HPLC peak 7) 4.3.5.2. Incubation of doxepin related xenobiotics with rat GIT homogenates No trace of desmethyldoxepin was found in incubates of doxepin with any of the six hctions of GIT homogenates, whereas controls with liver microsomes from the same rats demonstrated sdarresults as those in the previous experiments using rat liver microsomes (Tables 4.27.-4.28.). On the other hand, reduction in concentrations of the parent dmg (Tables 4.35.-4.36.) compared to controls with no GIT homogenates may be an indication that ebation through other metabolic pathways might have occurred.

4.3.5.3. Incubation of doxepin related xenobiotics with human liver microsomes Metabolism was generaüy slower in the human Liver microsomal preparations compared to corresponding rat preparations. Mer incubation with commercial doxepin, the Z:E ratio of the parent cornpounds remained stable at 15235 (Table 4.37.), and there was no time dependent progressive change in the Z:E ratio of the desmethyl metabolite. The results of incubating 2-doxepin (Table 4.38.), E-doxepin (Table 4.39.) or E-desmethyldoxepin (Table 4.40.) with human liver microsomes were in keeping with rat data (Tables 4.29.-4.34.) in that there was no evidence of interconversion between the isorners. HaK-Lives in human liver homogenate were registered in hours rather than minutes as was the case with rat liver preparations, although the half-Me of Z-desmethyldoxepin was still considerably longer than those of the other analytes in human liver homogenates (Figure 4.20.). TabIe 4.35. Isomer concentration after incubation of rat stomach homogenates spiked with 25 yM commercial doxepin (ZDox, 1116 nghL; E-Dox, 5789 ng/mL)

subcellular Protein Z-Dox E-Dox 2-Dox Tirne Ra* # (min) fraction (mgfmL) (ng/mL) (ng/mL) YO 30 1 mitochondri 0.87 762 4543 14 2 II 0.89 815 4809 14 - X 0.88 789 4676 14

68 1 mitochondri 0.87 752 454 1 14

30 1 microsomal 0.60 752 4550 14

60 1 microsomal 0.60 747 4526 14

2 II 0.80 803 4803 14 - X 0.70 775 4665 14

30 1 cytosolic 0.20 760 4573 14

60 1 cytosolic 0.20 742 4469 14 2 11 0.20 795 483 1 14 - X 0.20 769 4650 14 Table 4.36. Isomer concentration after incubation of rat small intestine homogenates spiked with 25 pM commerciaI doxepin (ZDox, 1116

subcellular Protein Z-Dox E-Dox Z-Dox Tirne Rat # (min) portion (mg/mL) (ng/mL) (ng/mL) Oh

30 1 mitochondri 0.88 837 4575 15

60 1 mitochondri 0.88 766 4470 15 2 Ir 0.88 827 4795 15 - X 0.88 797 4633 15

30 1 microsomal 0.87 785 4610 15 2 II O.ES 885 4762 16 - X 0.87 835 4686 16

60 1 microsomal 0.87 749 4604 14

30 1 cytoso lic 0.60 808 4619 15 2 I1 0.80 83 4 4924 14 - X 0.70 82 1 4772 15

60 1 cytoso lic 0.60 770 4548 14 II 0.80 819 4858 14 -2 X 0.70 795 4703 14 Table 4.37, Isomer concentration after incubation of human liver microsornesa spiked with 2.5 pM commercial doxepin (2-Dox, 112

40 85 498 15 7 19 27 120 75 423 15 13 41 24 180 70 401 15 16 52 24 240 65 364 15 20 58 26 Data are presented as the average of two replicates a Protein concentration 1-07 mg/mL

Table 4.38. Isomer concentration after incubation of human liver microsornesa spiked with 2.5 pM Z-doxepin (Z-Dox, 698 ng/mL)

------Time ZDox E-Dox ZDox Z-NorDox E-NorDox ZNsrDox

(min) (n91mU (WW Oh (ng/mL) (WmL) Oh 40 578 ND NA 35 ND NA 120 507 ND NA 80 ND NA 180 486 ND NA 1 07 ND NA 240 478 ND NA 123 ND NA Data are presented as the average of two replicates a Protein concentration 1.07 mg/rnL ND, not detected NA, not applicable Table 4.39. Isomer concentration after incubation of hurnan liver microsornesa spiked with 2.5 pM E-doxepin (E-Dox, 685 ng/mL) contaminated with Z-doxepin (Z-Dos, 13 ng/mL)

Data are presented as the average of two replicates a Protein concentration 1.07 mghL WD, not detected NA, not applicable

Table 4.40. Isomer concentration after incubation of human Iiver microsornesa spiked with 3.0 pM E-desmethyldoxepin (E-NorDox, 771 ng/mL) contaminated with 2-desmethyldoxepin (2-NorDox, 24 ng/mL)

Data are presented as the average of two replicates a Protein concentration 1.07 mg/mL ND, not detected NA, not appticable Human liver (n= 1)

Dox NorDox

Figure 4.20. In vitro metabolic elimination half-iives of doxepin vox) and desmethyldoxepin (NorDox) isomers

4.3.5.4. Discussion Incubation of commercial doxepin with rat liver homogenates (Tables 4.27.-4.28.) revealed relatively high concentrations of both Z- and E- desmethyldoxepin were produced very rapidly (within 10 min) after which concentrations of ail anaiytes feu progressively over incubation tirne period (60 min). ANOVA and the multiple cornparison tests revealed that the half-life for the deche in 2-desmethyldoxepin concentrations (Table 4.34.; Figure 4.17.) was significantly different @=O. 000 1) fiom those of the other three analytes. Meanwhile there was no evidence of statistical difference between the half-lives of 2- and E- doxepin (p=0.442). The relatively rapid rate of metabolism of E-desmethyldoxepin accounts for the substantial time dependent increase in percentage of 2-desmethyldoxepin. Moreover, incubation of rat liver rnicrosomes with E-doxepin (Tables 4.29.-4.30.), Z-doxepin (Tables 4.31.- 4.32) or E-desmethyldoxepin (Table 4.33.) revealed no evidence of interconversion between the isomers since the presence of small amounts of the 2-isorner in incubates of the E-isomer could be accounted for by the presence of trace isomeric contaminants in the starting material. Furthemore there was no evidence of preferential N- desmethylation of 2-doxepin. Based on the original geometric isomer ratio (Z:E=16:84) of the commercial doxepin, the concentration of 2-desmethyldoxepin to match the E-isomer for the same ratio may be calculated. The differences between the calculated and the observed values could be translated into fairly large amounts of Z-desmethyldoxepin to produce such ratio distortion (316, 352, 348, 325 nghL at time points of 10, 20, 40, 60 min respectively, derived fkom mean values, Table 4.28.). If these 2-desmethyldoxepin molecules were formed via an E- to Z- isomerization process as hypothesized previously (section 2.2.0.0., page 33-34), this process should also be effective in incubations of E-isomers alone such that considerable amounts of Z- desmethyldoxepin would be generated thereupon. Only trace amounts of 2- desmethyldoxepin were detected, however, in incubates of E-doxepin or E- desmethyldoxepin, and these concentrations were equivalent to the levels of the geometric impurities in the starting E-isomers (Tables 4.30. and 4.33.). When 2-desmethyldoxepin concentrations (Table 4.32.) in incubates of 2- doxepin and E-desmethyldoxepin concentrations (Table 4.30.) in incubates of E- doxepin are compared, it is apparent that the concentrations or their mean values of 2-desmethyldoxepin at every time point are notably higher than those of E- desmethyldoxepin (1.6, 2.0, 2.7, and 4.0 rimes of the E-desmethyldoxepin concentrations as 2-desmethyldoxepin concentrations for mean values at points of 10, 20,40, and 60 min respectively), whiie the mean concentrations of 2-doxepin (Table 4.31.) and E-doxepin (Table 4.29.) are relatively close in these incubates. These data aiso indicated thar desmethyldoxepin undenvent stereoselective metabolkm in which the E-isomer was eliminated the faster of the two. On the other hand, it also suggested the process was not related to cornpetitive mechanisms. This tendency was also mdested by the incubations of E-desmethyldoxepin (Table 4.33.), in which the rate of decline of 2-desmethyldoxepin concentrations (geornetric impurity) was much slower than that of E-desmethyldoxepin such that the percent ratio of 2- desmethyldoxepin progressively increased. The discrepancy might be caused by stereochemistry of the individual isomers. Z- desmethyldoxepin could have transannular interaction between the ring oxygen and the protonated terminal side-chah nitrogen (Figure 2. l., page 34). The hydrogen bonding interaction characterizes its steric image and modifies its electronic orientation. This steric configuration could harnper the Z-isomer's approach to the active binding site of certain types of metabolic enzyme(s) which possess signiticant active-site restraints and require ngid orientation of the substrate. Whereas E- desmethyldoxepin does not have such transannuiar interaction between the bridge oxygen and the side chah nitrogen, so it may have a greater &mity for enzyme(s) and/or more effective binding. This could have resulted in faster biotransformation rate of E-desmethyldoxepin and, of course, progressively increase the percent ratio of Z-desmethyldoxepin. Stereoselective metabolic pathway(s) could be either phase 1 or phase II or boa. For example, E-desmethyldoxepin codd undergo faster oxidation, and/or more extensive phase II conjugation than the 2-isomer. The enzymatic activity and capacity in the human liver microsomes was relatively much lower than those of rat. Incubation of the same doxepin related compounds with microsomes of a human liver (Tables 437.-4.40.) gave essentidy simila. results to those obtained fkom rat liver homogenates, although the ratio distortion occuned to a lesser extent. No evidence of isomer interconversion was found in these experiments. Since the most striking difference between the two species was one of metabolic rate, the half-lives with human liver homogenates were measured in hours rather than minutes (F'igure 4.20.). No statistical analysis is possible with data fiom a single liver, but Figure 4.20. shows that the half-He of 2-desrnethyldoxepin is by far the longest of al1 analytes, in keeping with data fiom the rat livers. The present N? vitro study also suggests GIT enzyme system(s) may not play any relevant role in the ratio distortion of desmethyldoxepin isomers, which may be entirely attributed to the metabolic functions of hepatic microsomal proteins. The cytochrome P-450 isozymes responsiblz for the N-desmethylation of doxepin are probably those only expressed in liver, e.g. CYP 1A2 and CYP2C 19 (Gonzalez, 1992, Romkes et al., 1991). 4.3.6.0. Irr vitro enzyme inhibition studies using UGT inhibitors Mean isorner concentrations (n=8) and the percentages of 2-isomer in incubates of doxepin are lïsted in Table 4-41.? while those in incubates of commercial doxepin with one of the three UGT inhibitors are shown in Tables 4.42.-4.44. individually. As was expected, the effect of enzyme inhibition on the consequent percent ratio of 2- desmethyldoxepin is clearly demonstrated. Cornpared to controls, test samples with any one of the three inhibitors aü have an attenuated development of the ratio distortion. Among them oleoyl-CoA shows most evident inhibition effect (Figure 4.21.). Since E-desmethyldoxepin samples were contaminated with 3% of 2- desmethyldoxepin, the latter was detectable in the incubates of E-desmethyldoxepin. Taking advantage of the presence of Z-isomer, however, pennitted simulation of Z:E ratio distortion of desmethyldoxepin isomers for incubates of E-desrnethyldoxepin with or without enzyme inhibitor. The simulated data reasonably match the experirnental outcornes. Tables 4.45. and 4.46. summarize simulations and experimental data individualiy in the absence of inhibition. Table 4-48., 4.50. and 4.51. show data for incubation of contaminated Edesmethyldoxepin in the presence of oleoyl-CoA (4.48.), probenecid (4.50.), or furosemide (4.51.) whereas tables 4.47. and 1.49. represent simulated inhibition. Figure 4.22. shows the curves of 2- desmethyldoxepin % in incubates of E-desmethyldoxepin with or without oleoyl- CoA, as well as curves derived fiom relevant simulations. Inhibited biotransformation in incubates of E-desmethyldoxepui with UGT inhibitors are clearly demonstrated by comparing mean isorner concentrations (n=8) at each time point between control and test samples (Tables 4.46., 4.48.: 4.50., and 4.51., and Figures 4.23. A - 4.23. B). The presence of 2-isomer in E-desmethyldoxepin due to the geometric impurity also makes it possible to calculate the in vitro rnetabolic elimination half-lives for both isorners in each rat, which are derived fYom individual isomer concentrations at sequential tùne points. These half-Eves and their means (n=8) are listed in Table

4.52.. ANOVA (ha-life = treatment, isomer, Liver, treatment * liver, isomer * ber, isomer * treatment) was performed. The effects of treatment (error tem liver * treatment), isomer (error term liver * isomer) and liver (error term residual) were all significant at a=O.OS. The SNK post hoc test showed that the effect of oleoyl-CoA on half-life was significantly different fiom conrrols. Ln order to detemiine whether the inhibitors affected both isomers in the same way a simpler ANOVA was also performed (ha-life = treatment, liver). The effect of treatment was signifïcant with E-desmethyldoxepin as the dependent variable. Dunnett's one tailed test (meamcontrol) showed that half-life was significantly higher dian control in the presence of oleoyl-CoA or furosemide. With 2-desmethyldoxepin as the dependent variable, however, cnly oleoy 1-CoA caused a significant increase in half-life compared with control. The ANOVA aiso indicated liver 5 was significantly dEerent fiom the other livers. Figure 4.24. de picts msan metabolic elimination half-lives of desmethyldoxepin isomers in control and test groups.

Table 4.41. Isomer concentration after incubation of rat Iiver microsornesa spiked with 25 mM commercial doxepin (1116 ng/mL 2-Dox plus 5789 ng/rnL E-Dox)

Time Z-Dox E-Dox 2-Dox 2-NorDox E-NorDox Z-NorDox (min) (ng/mL) (ng/mL) (%) (WmL) (WmL) (%)

80 11 +5 22 t 13 33k8 350t117 92 I 74 83 59 Data are given as rnean t S.D. from 8 rats a Mean pmtein concentration 0.68 k 0.19 mglmL Table 4.42. Psomer concentration after incubation of rat iiver microsomesa spiked with 25 mM commercial doxepin (1116 nghL Z-Dox plus 5789 nghL E-Dox) and 100 pMoleoyl-CoA

Time - 2-Dox E-Dox 2-Dox Z-NorDox E-NorDox 2-NorDox (min) (ng/mL) (nghL) (%) (ng/mL) (ng/mL) (%)

80 37 + 29 245 + 225 14 + 3 616 t 49 918 t 234 41 +7 Data are given as mean + S.D. from 8 rats a Mean protein concentration 0.68 I 0.19 mg/mL

Table 4.43. fsomer concentration after incubation of rat liver microsornesa spiked with 25 mM commercial doxepin (1116 ng/mL Z-Dox plus 5789 ng/mL E-Dox) and 3 mM probenecid

Time 2-Dox E-Dox 2-Dox Z-NorDox E-NorDox 2-NorDox (min) (ng/mL) (ng/mL) (%) WmL) (ng/mL) (%)

Data are given as mean + S.D.fiom 8 rats a Mean protein concentration 0.68 + 0.19 mg/mL Table 4.44. Isomer concentration after incubation of rat iiver microsornesa spiked with 25 mM commercial doxepin (1116 nghL 2-Dor plus 5789 ng/mL E-Dox)and 3 mM furosemide

Time Z-Dox E-Dox Z-Dox 2-NorDox E-NorDox 2-NorDox (min) (ng/mL) (ng/mL) (%) (ng/mL) (%/mu (%)

80 66k25 314+ 146 18+2 410 t72 187 t- 136 73 k 15 Data are given as mean f S.D. fiorn 8 rats a Mean protein concentration 0.68 f 0.19 rng/mL

* control

4oleoyl-CoA - probenecid U furosemide

O 20 40 60 80 100 time (min)

Figure 4.21. Mean (n=8) Z-desmethyldoxepin (ZNorDox) % curves in incubates of rat hermicrosomes with doxepin (25 pM) and

UGT inhibitor or without UGT inhibitor (control) Table 4.45. Simulation of ZIE ratio distortion by a stereoselective metabolic pathwaya

Time 2-NorDox E-NorDox 2-NorDox point concentration concentration (%)

- - a It is supposed to be the incubation of E-NorDox, assume 16 time difference in stereoselectivity and 52% of E-NorDox is biotransfomed by the pathway after each incubation period, then there would be 3.25% of 2-NorDox is biotransformed after the same period. Arbitrary data are used.

Table 4.46. Isomer concentration after incubation of rat liver microsornesa spiked with 15 mM E-desmethyldoxepin (E-NorDox, 3856 ng/mL) contaminated with 3% Zdesmethyldoxepin (ZNorDox, 119 ng/mL)

Data are given as mean + S.D. fkom 8 rats a Mean protein concentration 0.68 f 0.19 mg/rnL Table 4.47. Simulation of uE ratio distortion by a stereoselective metaboiic pathwaya which is inhibited by 55%

Time 2-NorDox E-NorDox Z-NorDox point concentration concentration (%>

a It is supposed to be the incubation of E-NorDox, 16 time difference in stereoselectivity remains sarne. 55% inhibition results in 23.40% (0.45 x 52%) of E-NorDox is biotransformed by the pathway after each incubation period and 1.46% of Z-NorDox is biotransformed afier the same period. Arbitrary data are used.

Table 4.48. Isomer concentration after incubation of rat liver microsomesa spiked with 15 mM E-desmethyldoxepin (E-NorDox, 3856 ng/mL) contarninated with 3% Z-desrnethyidoxepin (ZNorDox, 119 ng/mL), and with 100 pM oleoyl-CoA

Data are given as mean + S.D. fiom 8 rats a Mean protein concentration 0.68 f 0.19 mghL Table 4.49. Simulation of Z/E ratio distortion by a stereoselective metabolie pathwaya which is inhibited by 29%

Tirne Z-NorDox E-NorDox 2-NorDox point concentration concentration (%)

a It is supposed to be the incubation of E-NorDox, 16 tirne difference in stereoselectivity rernains same. 29% inhibition results in 36.92% (0.71 x 52%) of E-NorDox is biotransforrned by the pathway afier each incubation period and 2.3 1% of 2-NorDox is biotransformed after the same period. Arbitrary data are used.

Table 4.50. Isomer concentration after incubation of rat liver microsornesa spiked with 15 mM E-desmethyldoxepin (E-NorDox, 3856 ng/mL) contaminated with 3% 2-desmethyldoxepin (2-NorDox, 119 ng/mL), and with 3 mM probenecid

- - Data are given as mean t S.D. fiom 8 rats a Mean protein concentration 0.68 + 0.19 mg/mL Table 4.51. Isomer concentration after incubation of rat ber microsornesa spiked with 15 mM E-desmethyldoxepin (E-NorDox, 3856 ng/mL) contaminated with 3% Zdesmethyldoxepin (ZNorDou, 119 ng/mL), and with 3 mM furosemide

Time 2-NorDox E-NorDox Z-NorDox (min) (ndmL) WmL) (%>

Data are given as mean t S.D. frorn 8 rats a Mean pratein concentration 0.68 + 0.19 mg/mL obsewed (control)

sirnulated (no inhibition)

observed (oleoyl-CoA)

simulated (inhibition)

tirne (min)

Figure 4.22. Curves of mean Z-desmethyldoxepin% in incubates of rat liver microsomes with 15 pM E-desmethyldoxepin

(contaminated with 3% Zdesmethyldoxepin) and oleoyl-CoA, or without oleoyECoA (control), and cuwes derived from relevant simulations under assumptions of non-inhibited and inhibited metabolism

Figure 4.23. Mean (n=8) desmethyldoxepin isomer elimination cuwes in incubates of rat liver microsomes with 15 pM E-desmethyl-

doxepin (3856 ng/mL) contaminated with 3% Z-desmethyldoxepin (119 ng;/mL), with or without (control) UGT inhibitor A: Z-desmethy ldoxepin B: E-desmethyldoxepin - Z-NorDox (control) - Z-NorDox (oleoyl-CoA) Z-NorDox (probenecid)

Z-NorDox (furosemide)

time (min)

Figure 4.23. A

A E-NorDox (control)

+ E-NorDox (oleoyl-CoA) * E-NorDox (probenecid) E-NorDox (furosernide)

O 25 50 75 100 time (min)

Figure 4.23. B Table 4.52. In vitro half-üves of desmethyldoxepin isomers in incubates of rat liver microsornes spiked with 15 pM E-desmethyldoxepin

contaminated with Zdesmethyldoxepio (3%), with or without UGT inhibitor

Rat 2-NorDox E-NorDox # (min) (min) (min) (min)

- -

mean _+ S.D.

3 mM Probenecid Rat 2-NorDox E-NorDox # (rn in) (min) (min) (min)

mean $: S.D.

Data are denved hm parallel observations, al1 samples in duplicate at each tirne point between 20-80 min Figure 4.24. Mean (n=8) in vitro half-lives of desmethyldoxepin isomers in incubates of rat river microsornes with 15 pM E-desmethyl-

doxepin (contaminated with 3% Z-desmethyi-doxepin), with or without (control) UGT inhibitor

4.3.6.1. Discussion The conclusion that the distortion in desmethyldoxepin ratio is not due to isomer interconversion but stereoselective biotransformation fiom the previous in vivo and in vitro rat studies (sections 4.3.4.0. - 4.3.5.0.) is reinforced by the evidence of effect of enzyme inhibition on extent of the ratio distortion £i-omthe curent experiment. Stereoselective metabolic pathway(s) could be either phase 1 or phase II, or both. For exarnple, E-desmethyldoxepin could undergo faster oxidation, and/or more extensive phase II conjugation metabolism than 2-desrnethyldoxepin. Although it is not possible, at this tune, to codkm the exact pathway responsible, speculations rnay still be made. As a major type of phase II metabolism, glucuronidation plays an important role in metabolism of xenobiotics as weil as endobiotics. The pathway of the glucuronidation has a relatively high capacity of biotransformation. UDP- glucuronosyl~msferase(UGT) is a known microsomal eqme (Boutin et al., 1985, Tephly and Burchell, 1990). Biochemical separation and stmctural analysis of UGT isoforms fiom rat and human liver microsomes have provided definitive co~ation of multiplicity of UGTs (Bock el al, 1979, Burchell, 1981, Falany et al., 1983, Falany and Tephley, 1983, Kroemer and Klotz, 1992, Roy Chowdhury et al., 1993, Roy Chowdhury et al., 1986% Roy Chowdhury et al., 1986b, Weatherill and Burchell, 1980). Studies at various laboratmies have suggested the presence of at least 11 UGT isoforms in rat (Mackenzie et al., 1989). The pathway of glucuronidation should be viable under the incubation condition specined in the current study. The availability of the cofactor UDPGA could be either fiom the endogenous UDPGA in liver tissue (Arias, 1961) or fiom the in-situ biosynthesis utilizing cofactors in the regeneration system provided and enzymatic proteins native to liver microsomes. N- glucuronidation is one type of metabolic reactions, which may include primary amines (Arias,196 1, Green and Tephly, 1W6), secondary amuies (Green and Tephly, 1996, Lemberger et al., l985), or tertiary amines (Green and Tephly, 1996, Luo et al, 1991, Pemer et al., 1994) as substrates. It is hthat the ratio distortion was caused by stereoselective biotransforrnation of desmethyldoxepin isomers. If glucuronidation was the pathway solely or partially responsible, it must be the N-glucuronidation of desmethyldoxepin. It is well known that the UGT mediated glucuronidation reaction can be stereoselective. In vitro studies on glucuronidation of a variety of drugs have indicated considerable enantioselectivity (Armstrong et aL, 1988, Fujirnaki and Hakusui, 1990, Koster et al., 1986, Okazaki et al., 1991, Sisenwine et aL, 1982, Sweeny and Nellans, 1995, Vermeulen, 1986, Wilson and Thompson, 1984). Similar to the arguments in section 4.3.5.4., the occurrence of such stereoselectivity for geometnc isomers of desmethyldoxepin is possible in light of their steric molecdar structures. Z-desmethyldoxepin may have hydrogen bonding between ring oxygen and the terminal protonated side-chain nitrogen (Scheme 2.1.). This interaction may characterize the steric image of the 2-isomer and modi@ its electronic orientation. Such a steric configuration could obstnict its approaching the active substrate binding site of the UGT isozyme(s), which may possess signincant active-site restraints, or result in a lower product formation rate if the enzyme requires rigid orientation of the substrate to initialize a highly efficient process of the enzyme catalyzed conjugation reaction. Lack of such interaction between the bridge oxygen and the side chain nitrogen rnay provide E-desmethyldoxepin a greater fityto the enzyme ador more effective binding for the subsequent reaction. This of course would give rise to a raised 2-desrnethyldoxepin percentage appearing as an apparent distortion in Z:E isomer ratio. Although three UGT inhibitors used al1 form O-glucuronides (KamaIi, 1993, Sorgel et al., 1979, Zhong et al., 1991), UGT isoforms could have partly overlapping substrate specificity (Roy Chowdhiiry et al., l986a). On the other hand, non-specific inhibition of the reaction may be exerted by binding the enzyme with inhibitors that form different types of glucuronides, e.g. O-glucuronidation of the substrate can be inhibited by an inhibitor that cm only form N-glucuronide (Dumont et al., 1987). As a matter of fact, the current study shows apparent inhibition of metabolism by these inhibitors as demonstrated by the in vitro metabolic elimination half-lives of the desmethyldoxepin isomers (Table 4.52., Figure 4.24.). The inhibition effect on consequent percent ratio of 2-desmethyldoxepin was, as expected, to curb the development of the ratio distortion and hally to render the distortion to a Iesser extent (Tables 4.41.-4.44., Tables 4.46., 4.48., 4.50. and 4.51.). This effect is also clearly demonstrated by the simulation of stereoselective biotransformation (Tables 4-45,, 4.47. and 4.49,). Based on current experimental observations, it is reasonable to conclude that stereoselective glucuronidation is very possibly the pathway involved in the ratio distortion of desmethyldoxepin isomers. 4.4.0.0. Pharmacokinetic s tudies

4.4.1.0. Clinical pharmacokinetics AU twelve subjects who met the entry requirements completed the study. No blood or urine samples were missed during the two phases of the study. Generally, the doses were well tolerated in ail subjects der both intravenous (iv) and oral doxepin administration. The most common side effects included dry mouth, drowsiness, and sleep, the onset of which started earlier after iv than oral adminiskation. In the latter case, it usudy started one hour afker the administration. Al1 symptorns of these side effects generally disappeared after 24 hours. Results from the stability test indicated quality of plasma and urine samples were well maintained under the storage condition for the penods specifïed (3-5 months). The quotient of the deviations of the mean values against the nominal concentration ranged from 1.0 - 2.6%. The assay procedure was sensitive enough to permit pharmacokinetic anaiysis of both isomers of doxepin after iv administration of 22.12 mg (3 -54 mg 2-doxepin + 18.58 mg E-doxepin) or oral administration of 75 mg (12 mg 2-doxepin + 63 mg E-Doxepin). Pharmacokuietic parameters denved fkom non-cornpartmental analysis are listed in Tables 4.53.-4.54. for iv and oral administration respectively. The mean absolute bioavailability is 29% for both doxepin isorners (Table 4.54.). Table 4.55. shows the results of cornpartmental pharmacokinetic analysis (two-cornpartment model), while the cuves of the observed data (iv) versus predicted ones (Figures 4.25.-4.26.) indicate reasonable model fitting (2-doxepin: AIC = -7.62; SC = -10.49. E-doxepin:

AIC = 5.98; SC = 3.40). The mean plasma concentrations of the doxepin isomers versus tirne curves after oral administration are show in Figure 4.27., and curves of desmethyldoxepin isomer concentration versus time afier iv administration are presented in Figure 4.28.. Plasma concentrations of desmethyldoxepin isomers were so low after iv administration of doxepin that their pharmacokinetics could be determined only in seven volunteers for Z-isomer and in two volunteers for E-iscmer. In contrat, the pharmacokinetics of 2-desmethyldoxepin after oral administration could be determined in all twelve subjects whereas E-desmethyldoxepin could be determined in eleven out of twelve volunteers. Figure 4.29. shows the mean plasma concentration versus the profile of 2-desmethyldoxepin after orai administration, but there was large between-subject variation in plasma concentrations of E- desmethyldoxepin after oral administration, such that an example of a plot korn a randomly selected volunteer is shown in figure 4.30. as an illustration. The ratio of 2-doxepin appeared very close to that in the administered dosage form (16%) in that the mean percentage of 2-doxepin in both plasma and urine ranged fiom 18-20 in all volunteers after both iv and oral administrations. However, there was significant ratio distortion in percentage of 2-desmethyldoxepin, of which the mean ratio in urine ranged fiom 43-87% (iv) and 3 5-82% (oral). An apparent pattern of the ratio distortion was observed in most subjects along the time course in that it was a time dependent, progressive process except for subjects 4 and 6, in whom no such pattern was obvious. Figure 4.33. shows the curves of mean urinary 2- desmethyldoxepin% along the time course in the first 48 hours. The extent and the pattern of the ratio distortion along the time course in plasma were similar to those in urine. Repeated measures ANOVA was used to examine within-subject variability across tirne. The dependent variable was urinary recovery of doxepin or desmethyldoxepin isomers (in 12 hour segments) expressed as percentages of dose which pennitted simultaneous cornparison of the effect of time, isomer and route of administration. As expected, the mode1 showed significant effects of time for both doxepin and desrnethyldoxepin because percentages of dose recovered for both dependent variables diminished progressively across tune segments. Similarly the eEect of isomer was significant for both dependent variables because of a general tendency for greater percentage recoveries of 2-isomer (iv + oral) than E-isomer (iv + oml) across the segments. The effect of route of administration was signincant with doxepin as the dependent variable, Iargely because extensive first pass metabolism of both isomers after oral administration meant that the percentages of dose recovered as total (Z+E) doxepin was very low compared with corresponding iv data in the fist two time segments (0-12 and 12-24 hours), whereas the percentages recovered der the two routes were sunilar in later time segments. The effect of route w-as not signincant with desmethyldoxepin, however, because the percentages of dose recovered as (Z+E) desmethyldoxepin tended to be similar in any given time segment after iv and oral administrations. Factorial AVOVA, carried out io examine between-subject effects within time segments, showed that the effect of route of administration with doxepin as the dependent variable was significant only in the earliest tune segments (0-12 and 12-24 hours), presurnably as a consequence of extensive first pass metabolism after oral administration. The effect of route was not significant, however, in later he segments. Similady, the effect of route was signifïcant only in the earliest time segment (0-12 hours) with desmethyldoxepin as the dependent variable. The effect of doxepin isomer was signScant in the earlier time segments because there was a tendency for the percentage of the dose of the Z-isomer excreted in the urine to be higher than that of the E-isomer in aU subjects. The same trend was also apparent with desmethyldoxepin as the dependent variable such that the effect of isomer was significant in al1 time segments. The plasma AUCs of 2-desmethyldoxepin after both iv and oral administration were rnuch greater than those of its antipode in al1 subjects except for subjects 4 and 6 (Figures 4.31.-4-32.), in whom AUC of E-isomer was greater than that of Z-isomer and to a lesser extent the ratio distortion occurred. Noticeably, this study did not demonstrate any effect of route of administration on the ratio distortion of desrnethyldoxepin isomers as confïtmed by the ANOVA that the effect of route was not significant for the urinary recovery of desmethyldoxepin but the effect of isomer was. The urinary recoveries of 2-, E-isomers of doxepin and desrnethyldoxepin up to 120 hours, expressed as the percentage of the dose, are listed in Tables 4.56.-4.57.. while the Figure 4.34. shows the mean (n=12) urinary recoveries of these isomers up to 120 hours. TabIe 4.53. Phamacokinetics of 2- and E- isomers of doxepin and desmethyldoxepin in 12 men after doxepin intravenous administration (22.12 mg base, Z:E=16:84)

- - -- - The figures in parentheses are percentage coefficients of variation a n=7

n = 2 (E-desmethyldoxepin detectable in subjects 4 and 6 who appeared to be outliers) C Plasma concentrations at 5 min Table 4.54. Pharmacokinetics of 2- and E- isomers of doxepin and desmethyldoxepin in 12 men after doxepin oral administration (75 mg base,

The figures in parentheses are percentage coefficients of variation a n = 12 (no outliers detected)

n = 9 (volunteers 4 and 6 are not included in calculations for the outlier çtatus, vohnteer 3 could not be calculated due to low plasma concentrations) C The half-lives of subject 4 was 83.01 hours and that of subject 6 was 56.55 hours Table 4.55. Pharmacokinetics of 2- and E- isorners of doaepui in 12 men after doxepin intravenous (iv) administration (22.12 mg base, Z/E=16/84) using cornpartmental analysisa

The figures in parentheses are percentage coeffkients of variation a Two-cornpartment iv-bolus, no lag time, first order elirnination C - 2-dox (observed) -O-- 2-dox (predicted) l

t I 1 t 1 I

Time (hr)

Figure 4.25. Plot of mean (n = 12 except where otherwise indicated) observed (solid circles) versus predicted (open circles) Z- doxepin (Zdox) plasma concentration after doxepin intravenous administration (22.12 mg base) in men with a two cornpartment mode1 fitting using WinNonlin E-dox (observed) - - -

b------'1 --Cf-- E-dox (predicted)

O 12 24 36 48 60 Time (hr)

Figure 4.26. Plot of mean (n = 12 except where otherwise indicated) observed (sof d squares) versus predicted (open squares) E-doxepin (E-dox) plasma concentration after doxepin intravenous administration (22.12 mg base) in men with a two cornpartment mode1 fifting using WinNonlin time (h)

Figure 4.27. Plot of mean (n = 12 except where otherwise indicated) plasma

concentration versus thecurves of 2-doxepin (Zdox, solid circles) and E-doxepin (E-dox, solid squares) after doxepin oral (po) administration (75 mg base) in men 2-5 ---+--- 2-Norl-dox (iv) 2 - E-Nor 1-dox (iv) 1-5

I I I I I O 24 48 72 96 120 144

t ime (h)

Figure 4.28. Plot of mean (number of observations indicated) plasma concentration versus time curves of 2-desmethyldoxepin (Z- Norl-dox, diarnonds) and E-desmethyldoxepin (E-Nori-dox, crosses) after doxepin intraveoous (iv) administration (22.12 mg base) in men "1 - 2-Nor 1-dox (po)

O 24 48 72 96 120 144

time (h)

Figure 4.29. Plot of mean (n = 12 except where othenvise indicated) piasma concentration versus tirne curves of 2-desmethyldoxepin (Z- Norl-dox, diamonds) after doxepin oral (po) administration (75 mg base) in men time (h)

Figure 4.30. Plot of plasma concentration versus time curves of E- desmethyldoxepin @-Nor,-do~,crosses) after doxepin oral (PO) administration (75 mg base) in volunteer 11

2-Nor 1-dox% (iv)

h v 2-NorF-dox% (po)

12 24 36

time (h)

Figure 4.33. Plot of mean (n = 12 except where otherwise indicated) percentage of Zdesmethyldoxepin (Z-Norl-dox%) in urine after doxepin intravenous (iv, diamonds) and oral (po, eircles) administration in men Table 4.50. Urinary recovery (dose%) of doxepin @ox) and desmethyldoxepin (NorDox) isomers (0-120 h) in 12 men after doxepin intravenous administration (22.12 mg base, WE=16/84)

Subject Z-Dox E-Dox Z-NorDox E-NorDox Table 4.57. Unnary recovery (dose%) of doxepin @ox) and desmethyldoxepin (NorDox) isomers (0-120 h) in 12 men after doxepin oral administration (75 mg base, Z/E=16/84)

Subject ZDox E-Dox Z-NorDox E-NorDox Figure 4.34. Mean (n=12) urinary recovery (dose%) of doxepin (Dox) and desmethyldoxepin (NorDox) isomers (0-120 h) after doxepin intravenous (iv, 22.12 mg base) and oral (po, 75 mg base) administration in men

4.4.1.1. Discussion The challenge in carrying out stereoselective single dose pharmacokinetic studies of doxepîn and desmethyldoxepin has been facilitated to some degree with the newly developed stereoselective HPLC method (Yan et al., I997a), although a more sensitive analytical method is required if plasma concentrations of the hgand the metabolite are to be foilowed for longer penods of time. The studies have indisputably demonstrated that significant ratio distortion of desmethyldoxepin isomers occurred regardless of the route of aGsûation. The ratio of doxepin isomers, on the other hand, stayed very close to that in the original formulation after both iv and oral administration. Intravenous administration of doxepin in healthy volunteers reveded both isomers to have huge volumes of distribution and relatively short ha-lives (Table 5.43.) which suggests that both isomers werc subject to extensive tissue distribution andor binding. As a consequence there may have been a late phase of elimination that was not measurable in the plasma because of low concentrations of the isomen. The observed mean plasma concentrations, however, fitted a two-cornpartment mode1 which gave reasonable estimates of clearances and volurnes of distribution of the doxepin isomers (Table 4.55.). The absolute bioavailability of both 2-doxepin and E-doxepin was 29% indicative of extensive metabolisrn of both isomers. The foregoing factors resulted in very low rend clearances (Table 4.53.) for both isomers and the fact that low but quantifiable concentrations of both isomers appeared in the segmented urines over prolonged penods after iv or oral dosing (Table 4.56.-4.57.). The present studies detected no evidence of interconversion between the isomers as proposed by Ghabrial and CO-workers(199 1). The distortion in the ratio between the desmethyl isomers appears to be the result of stereoselective metabolism such that the half-life of 2-desmethyldoxepin is much longer than that of the E-isomer. After intravenous administration, the ha-Ue of E-desrnethyldoxepin was estimated in only 211 2 subjects whereas the 2-isomer was measured in 7/12 individuals (Table 4.53.). In vivo and in vitro studies in rats (sections 4.3 -4.0. - 4.3.5.0.) proved that the signifîcant ratio distortion in rat was mediated by stereoselective metabolism with no evidence of any isomer interconversion. This mechanism was Mercohed in enzyme inhibition studies (section 4.3.6.0.) in which the UGT inhibitor oleoyl-CoA signiscantly increased the half-lives of both desmethy ldoxepin isomers, and diminished the progressive process of the ratio distortion. After oral administration, both doxepin isomers exhibited relatively short half lives and veiy low rend clearances (Table 4.54.) consistent with iv data. The fact that the ratio of the doxepin isomers in plasma remained close to that in the dosage form administered (16234) suggested stereoselective metabolism was not a factor after either route of administration. Furthemore, rend clearances were viirtually identical for the two isomers after iv (Table 4.53.) or oral (Table 4.54.) administration which suggests that stereoselective rend excretion was not a factor either. Nevertheless, the effect of

isomer was significant in factorial MOVA of the early urinq besegments because

the recovery of 2-doxepin (iv t oral) was slightly but consistenw higher than that of the E-isomer in al1 subjects. The plasma AUC values of 2-desmethyldoxepin were higher than the corresponding values for the E-isomer after both iv and oral adminimation, wîth the exception of subjects 4 and 6 (17% of the sample of the population), who had the largest total AUC (Figures 4.31.-4.32.) and highest total urinary recovery of desmethyldoxepin (Tables 4.56.-4.57.). This phenornenon has been observed in a previous investigation (Midha et al., 1992) of the stereoselective oral pharmacokinetics of doxepin in which two out of twenty nine subjects (7% of the sample of the population) also appeared to be outliers in terms of hi& plasma concentrations of the E-desmethyl metabolite, whereas in both studies, the outliers had concentrations of the Z-isomer in the sarne range as the other volunteers. In view of this situation, it was decided not to include data fiom subjects 4 and 6 in pharmacokinetic calculations involving the E-desmethyl isorner after oral dosing.

Removai of data fiom the outliers led to substantial reductions in Crnax, tm~~,and tin in the case of the E-isomer, but had little effect on the same parameters in the case of the Z-isomer. The mean half-He of Z-desmethyldoxepin was very long after iv (Table 4.53.) or oral (Table 4.54.) administration. The half-life of the E-desmethyl isomer was much shorter after oral administration (Table 4.54.) in al1 but the two outliers (Subjects 4 and 6) in whom the half-lives were 83.01 and 56.55 hours respectively. One might speculate that these two outliers with the Ieast distorted ratio rnight be poor metabokers of desmethy ldoxepin due to inherited status of deficiency of enzyrnatic protein or 1ow activity of relevant isozymes. This speculation is in keeping with the results fiom rat in vitro enzyme inhibition studies f section 4.3 -6.0.). which demonstrated a lesser extent of ratio distortion when metabolism of desmethyldoxepin by glucuronosyl tramferases was inhibited. The absolute bioavailability (29%) of both doxepin isomers matched with the estimated bioavailability values of doxepin in previous single or multiple oral dose studies conducted by Ziegler et al in 1978 (F=0.27), Virtanen et al in 1980 (F=0.29) and Faulkner et al in 1983 (P0.31) (Faulkner et al., 1983, Virtanen et al., 1980,

Ziegler et al., 1978). Ln these studies, Gibaldi's method (Gibaldi, 1971) for the caiculation of hepatic availability was applied to estimate the oral bioavailability, wbere 100% availability of the dose fiom gastrointestinal tract hto the hepatoportal system and a liver blood flow of 1.5 Llmin were assumed. Hence, in retrospect, these assumptions appear very likely to be the real circumstances encountered by doxepin after oral administration. Therefore, the first-pass effect for doxepin is simply limited to the metabolic function of hepatic isozymes. These hepatic isozymes, rnediating a variety of active pathways, contribute to the elimination of doxepin, in which N- desmethylation comprises only a smd part of it. This doubtless explains the low urinary recovery of doxepin and desmethyldoxepin isomers (Tables 4.56.-1.57. and Figure 4.34.). The effect of route of administration on the ratio distortion in rat in vivo studies

(sections 4.3-3 -0. - 4.3.4.0.)appears to be caused by extensive first-pass elhination. Specuiations for this effect may be made to explain the Merence observed. Due to a hi& hepatic extraction plus possible elimination through feces excretion in the GIT, relatively very small fiactioc of the dosed amount of parent drug was able to reach the systemic circulation, while a relatively large amount of desmethyldoxepin was produced due to the first-pass metabolism after an oral administration. After an intravenous bolus injection, ali hgor a very high fiaction of the dosed amount (small loss might be the case due to possible metabolic clearance occurring in lung ador heart) was put into the systemic circulation imnediately. The net result of the ratio distortion was a reflection of the balance in a dynamic process between the formation rate and the stereoselective elimination rate of the desmethyldoxepin. New1y formed desmethyldoxepin with the original ratio of 16% 2-isomer flowed into the desmethyldoxepin pool. This event was a perturbation to the course of the ratio distortion development. The degree of this perturbation depended on the tuni-over rate and the stereoselective elirnination rate of desmethyldoxepin. This tum-over rate was low due to a small size of parent dnig pool after oral dosing. Hence, the distortion in the ratio of the geometric isomers became evident as the time went on. The story of stereoselective elimination was same with iv administration, but the absence of the first-pass metabolism let relatively very large amount of parent drug enter the systemic circulation, continuous N-desmethylation of doxepin provided

sufficient desmethyldoxepin isomers with original ratio (Z:E = L6:84) to curb the development of the ratio distortion. Therefore, the significant ratio distortion was not observed in early penod of time although the tendency of the ratio distortion might still be seen when a cornparison was made between early (0-16 hou) and later (17-41 hou) segments of urine collections (data not shown). The sharp contrast in size of parent drug pool between intravenous and oral dosing was reflected clearly by urine concentrations of doxepin isomers (Tables 4.23. and 4.25.) even tkough the size of the oral dose was generally about 3-4 limes bigger than that of the intravenous dose. The tum-over rate of desmethyldoxepin after iv administration of doxepin was relatively slow in humans due to a much larger volume of distribution and a smder unit dose. Hence such discrepancy of dosing route on the ratio distortion was not observed in hriman studies. In conclusion, the present cross-over ivloral single dose study has demonstrated no effect of route of administration on the ratio distortion of 2-desmethyldoxepin. The mechanism for the ratio distortion in human appears to be the stereoselective metabolism, in keeping with the conclusion fiom the rat studies. The values of absolute bioavailability of 2- and E- doxepin tends to support the notion that the dmg is completely absorbed fiom the gastrointestind tract and the extensive first-pass metaboiism is essentially hepatic in nature. The clinically relevant outcome of this investigation is the discovery of extensive tissue distribution and/'inding of the more active 2-doxepin isomer and low but sustained plasma levels of the more active 2- desmethyl isomer in all subjects, ùicluding the two subjects who were outliers in terms of E-desmethyldcxepin. 4.4.2.0. Preclinical p harmacokinetics Generally, the doses were well tolerated in all dogs der both intravenous (iv) and oral doxepin administration. The most common side effect was apparent sedation, the omet of which started earlier deriv (5-10 min) than oral administration (20-30 min). But the sedation seemed much more severe &er oral than after iv administration. One male dog (dog $2) had seinire about 25 minutes afler the oral dosing. The symptorns included uncontrolled urination, pupil dilation, and reduced rate of heart beat. It was resuscitated using oxygen and had recovered in the for the collection of the one hour blood sample, althoilgh the halfhour sample could not be obtained. AU symptoms of these side effects graddy disappeared dlning the period of first eight hours, after which dldogs resurned normal activity.

4.4.2.1. Pharmacokinetics of doxepin after intravenous administration Phmacokinetic parameters derived fÏom non-cornpartmental analysis are listed in Table 4.58.. Table 4.60. shows the results of cornpartmental pharmacolcinetic analysis. Figures 4.35-4.36. present the cwes of the mean observed plasma concentrations dong the time course versus predicted ones for 2-doxepin and E- doxepin, respectively. The curves of mean plasma concentration of desmethyldoxepin isorners versus tirne are shown in Figure 4.37.. The plot of AUC of desmethyldoxepin isomers is shown in Figure 4.40..

4.4.2.2. Pharmacokinetics of doxepin after oral administration The curves of mean plasma concentration of doxepin and desmethyldoxepin isomers versus tune are shown in Figures 4.38.-4.39.. While the Figure 4.11. presents the AUC of desmethyldoxepin isomers. Pharmacokinetic parameters, denved fiom non-cornpartmental analysis are listed in Table 4.59., where absolute bioavailabilities of 2- (27%), and E-doxepin (24%) are included. Like the situation in clinid pharmacokinetic stuclies, the limited information of plasma concentration

versus time data after oral administration was not sufficient to nui a valid pharmacokinetic modeling. Hence, only noncornpartmental cdculations of pharmacokinetic parameters for oral data were canied out. Figures 4.43. A - 4.43. C shows HPLC chromatograms of the extracts fiom dog plasma samples.

4.4.2.3. Z:E Ratio of the isomers The percent ratio of 2-doxepin was very close to the value in the adrninistered form (16%), the percentage of 2-doxepin in plasma dong the time course ranged fiom 15-21 in all dogs after iv administration (rnean=18%), while that fier oral dosing ranged fkom 16-22 (mean=l9%). However, the ratio distortion of 2- desmethyldoxepin in plasma appeared to be sigdicant, the percentage of which over the time course ranged Çom 13-6 1% in all dogs with a mean of 3 1% (iv), and 20-73% in al1 dogs with a mean of 34% (oral). An apparent pattern of the ratio distortion was observed in that it was a time dependent, progessive process except for dog #2 after iv dosing, in which no such pattern was observed although the pattern was still obvious in this dog after oral administration. It was also noted that this dog showed the Ieast distortion in the ratio after both iv (13-27%) and oral (27-37%) administrations. Meanwhile the largest total AUC of desmethyldoxepin after both iv and oral dosing was observed in the sarne dog (F'igures 4.40.-4.41.). The resuIt fiom an ANOVA test (repeated measures) indicated it was statistically significant (P=0.0001) for the ef5ect of time on plasma 2-desmethyldoxepin%. Figure 4.42. shows the cuves of the mean plasma 2-desmethyldoxepin% dong the time course in the f~st16 hours (iv) and 24 hours (oral). When route of administration was selected as the factor in the ANOVA model (repeated measures), and plasma percent ratio of Z-desmethyldoxepin as the dependent variable, the result indicated there was no si&cant effect of this factor on the ratio (P=0.6428). The inter-individual variation was evident as the ANOVA test indicated signincant effect of dog on the ratio of 2-desmethyldoxepio (P=0.0014) in the mode1 (factorid). Table 4.58. Pharmacokinetics of 2- and E- isomers of doxepin and desmethyidoxepin in 6 beagle dogs after doxepin intravenous administration (6 mg/& base, Z:E=16:84)

------The figures in parentheses are percentage coefficients of variation a Plasma concentrations at 10 min Table 4.59. Pharmacokinetics of 2 and E- isomers of doxeph and desrnethyldoxepin in 6 beagle dogs after doxepin oral administration (20 mgkg base, Z:E=16:84)

------The figures in parentheses are percentage coefficients of variation Table 4.60. Phamacokinetics of 2- and E- isomers of doxeph in 6 beagle dogs after doxepin intravenous administration (mean dose: 57.1 rng/dog base, Z:E=l6: 84) using cornpartmental analysisa

The figures in parentheses are percentage coefficients of variation a Two-cornpartment iv-bolus, no lag thne, first order elimination 1000 - Z-dox (observed)

O 4 8 12 16 20 24 Time (hr)

Figure 4.35. Plot of mean (n = 6 except where otherwise indicated) observed (solid circles) versus predicted (open circles) Zdoxepin (Z-dox) plasma concentration after doxepin intravenous administration (6 mgkg base) in beagle dogs with a two cornpartment mode1 fitting using WinNonlin - E-dox (observed) 4- E-dox (predicted) ------

Time (hr)

Figure 4.36. Plot of mean (n = 6 except where otherwise indicated) obsewed (soüd squares) versus predicted (open squares) E-doxepin (E-dox) plasma concentration after doxepin intravenous administration (6 rnglkg base) in beagle dogs with a two cornpartment mode1 fitting using WinNonlin 1 - Z-Nor 1 -dox (iv) &hl - E-Nor 1 -dox (ivj

O 4 8 12 16 20 24 28 time (h)

Figure 4.37. Plot of mean (n = 6 except where otherwise indicated) plasma concentration versus time curves of Z-desmethyldoxepin (Z-Norl-dox, diarnonds) and E-desmethyldoxepin (E-Norl-dox, crosses) after doxepin intravenous (iv) administration (6 mgkg) in beagle dogs O 6 12 18 24 30 36 42

tirne (h)

Figure 4.38. Plot of mean (n = 6 except where otherwise indicated) plasma concentration versus tirne curves of 2-doxepin (Zdox, solid circles) and E-doxepin (E-dox, solid squares) after doxepin oral (po) administration (20 mgkg base) in beagle dogs - 2-Norl-dox (po) - E-Norl-dox (po)

O 12 24 36 48 60 72 84 t ime (h)

Figure 4.39. PLot of mean (n = 6 except where otherwise indicated) plasma concentration versus time curves of 2-desmethyldoxepin (%Norl-dox, diamonds) and E-desmethyldoxepin (E-Norl-dox, crosses) after doxepin oral (po) administration (20 rnglkg base) in beagle dogs I AUC (ng .

Figure 4.40. Plot of AUC of desmethyldoxepin (Norl-dox) isomers after doxepin intravenous administration (6 mgkg base) in beagle dogs

Figure 4.41. Plot of AUC of desmethyldoxepin (Norl-dox) isomers after doxepin oral administration (20 mgkg base) in beagle dogs 2-Norl-dox% (po)

o.? 1 1 1 1 O 6 12 18 24 30 time (h)

Figure 4.42. Plot of mean (n = 6 except where otherwise indicated) Z-desmethyldoxepin% (Z-Nori-dox%) in plasma after doxepin

intravenous (iv, diamonds) and oral (PO, circles) administration in beagle dogs Figure 4.43. HPLC chromatograms of the extracts from dog plasma samples A, mixture of standard reference samples spiked in blank plasma B, blank plasma C, plasma sarnple 6 hours after an oral dose of doxepin (20 mgkg) to a beagle dog Peak identifications: 1, Z-doxepin; 2, E-doxepin; 3, nortriptyhe; 4, Zdesmethyldoxepin 5, E-desrnethyldsxepin 6, Zdidesmethyldoxepin 7, E-didesmethyldoxepin

- - 3

œ -

œ 5 -

I 1 I L I l I 1 I 1 O 4 8 12 16 20 time (min)

Figure 4.43. A time (min)

Figure 4.43. B

O ! I 1 1 I l I O 4 8 12 16 20

time (min) Figure 4.43. C 4.4.2.4. Discussion A distortion in the ratio of desmethyldoxepin isomes in plasma similar to that observed in humans (section 4.4.1.0.) was also observed in dogs although to a lesser extent. Moreover, as in the human studies, there was no significant effect of route of administration on ratio distortion of the desmethyldoxepin isorners and there was no

evidence to show such distortion took place in the ratio of parent hgisomers. The percentage of the more active Z-isomer appeared to remain close to that in the administered dosage form after either iv or orai administration. The absolute bioavailabilities of 2-doxepin and E-doxepin were 27% and 24Y0, respectively, indicating extensive fist-pass metabolism. Results in dogs Mer fIom those in humans in that the plasma AUCs of E-desmethyldoxepin are larger than the corresponding values of the Z-isomer after either iv or oral administration (Figures 4.40.-4.41.). This result is in agreement with the fact that the ratio distortions in dogs are to a lesser extent than those in humans. Dog #2 had the largest total plasma AUC of desmethyldoxepin after either iv or oral administration, and the ratio distortion occurred to a lest extent in this dog. It is interesthg to note that the ratio distortion was related to the metabolic elimination of desmethyldoxepin in rat in vitro studies (section 4.3.6.0.), where it was demonstrated that the development of ratio distortion was curbed by incubation with lïver microsornes containing UGT inhibitors, notably oleoyl-CoA, which reduced the metabolic elimination of desmethyldoxepin. Simikir to the human studies, no evidence of "e~chment"of the more active 2- desmethyldoxepin was observed in the present dog studies. The half-life of 2- desmethyldoxepin was much longer than its geometric antipode (Tables 4.58.-4.59.). These data hdicate that stereoselective metabolism is responsible for the percent ratio increase of 2-desmethyldoxepin observed in dog plasma after either iv or oral administration of doxepin. 5.0.0.0. CONCLUSIONS The pharmacokinetic and pharmacodynamie consequences of stereoisomerism have received considerable attention in recent years. Some clinical investigators have suggested that stereoselective data on doxepin and its active desmethyl metabolite are essential for studies on correlation between plasma levels and therapeutic/toxic e ffects of doxepin. One of the problems in conducting stereoselective studies on doxepin has been the low percentage of the Z-doxepin in commercial dosage forms leading to extremely low plasma concentrations of this active isomer. The fm goal of this thesis work was to develop a convenient HPLC method which wouid do simultaneous quantification for both doxepin and desmethyldoxepin isomers. The method presented proved to be a very useful tool for a direct, sensitive, stereoselective, simultaneous, and also reIiable quantitative assay of doxepin and its pharmacologicaily active metabolite desmethyldoxepin. The method gave satisfactory separation of the 2- and E- isomers of the parent dmg and the desmethyl metabolite and good accuracy and precision. The preliminary pilot investigations carried out in humans and four animal species revealed ratio distortions of desmethyldoxepin with varying degrees in cumulative 24 hour urine post doxepin oral administration. In all mamrndian species tested, the ratio distortion was found highest in human and rat which were not signincantly different fbm each other. Thus using rat as an animal species should give valuable information on the issue of rnechanism of the ratio distortion. The results from rat in vivo studies, and in vitro experiments using rat and human liver microsomes, rat GIT tissue homogenates, and enzyme inhibition studies with rat liver microsornes utilking UGT inhibitors, have indicated that systemic stereoselective metabolism of desmethyldoxepin w-as responsible for the enhanced percent ratio of Z-desmethyldoxepin. There was no evidence of E- to Z- isomer conversion. Preferential clearance of E-desmethyldoxepin occurred in stereoselective rnetabolic pathway (s), which was very likely the phase II glucuronidation mediated by microsomal UDP-glucuronosyltransferase. The clinicai pharmacokinetic studies demonstrated that sigmficant ratio distortion of desmethyldoxepin isomers occurred in both plasma and urine with no effect of the route of administration. Similar to the results from rat in vitro studies, an evident pattern of time dependent, progressive process for the ratio distortion was observed, in keeping with a mechanism of preferential rnetaboiism rather than isomer interconversion. The Z-doxepin ratio stayed very close to the original composition after both iv and oral administrations. Based on the absolute bioavailability of both 2- and E- doxepin, and the published data fkom previous nonstereoselective doxepin single or multiple dose pharmacokinetic studies, signficant frst-pass effects of doxepin in humans appeared to be solely attributabla to the metabolic capacities of hepatic cells. Si@cant ratio distortion in desmethyldoxepin isomers was observed in dog plasma, which was also a time dependent, progressive process similar to that which occurred iil human subjects in the clinical pharmacokinetic studies. There was no evidence of substantial E- to 2- isomer conversion after the dose of commercial doxepin, and there was no effect of route of administration on the ratio distortion either. It appears more likely that the change in Z:E ratio of the desmethyl isomers from 16:84 to approximately 1: 1 is the result of stereoselective phase I andor phase II metabolism of E-desmethyldoxepin. There was no evidence to show such significant ratio distortion took place in parent dnrg isomers. Rather, the percentage of 2-doxepin appeared to remain close to that in the administered dosage form after either iv or oral administrations. Extensive first-pas metabolism was indicated with the absolute bioavailabilities of 2-doxepin (27%) and E-doxepin (24%). Another noticeable phenornenon was observed in both the clinical and the preclinical phamiacokinetic studies that the inaividuals with least extent in the distortion of percent ratio of Z-desmethyldoxepin had largest AUC and highest urinary recovery of total desmethyldoxepin. These individuals appeared to be poor metabolizers of desmethyldoxepin who comprised 17% of the sample of the population in the present study. This was in keeping with the resdts fiom the enzyme inhibition study which demomtrated occurrence of a lesser extent of the ratio distortion when the metabolic elimination of desmethyldoxepin was inhibited using UGT inhibitors. The conclusion for the mechankm of ratio distortion in desmethyldoxepin isorners made from the present research is different fkom a previously published paper (Ghabrïal et al., 1991) which stated the ratio distortion was caused by isomer interconversion. Their conclusion was based on the observation that trace amounts of deuterium labeled ~-[~l&]-desmeth~ldoxe~inwere detected in healthy male volunteers' urine (n=8) after orally dosing with 25 mg each of deuterium labeled E- [2HJ-doxepin and unlabelled ~-[~~~]-doxe~inhydrochlonde. This conclusion rnay be wrong since trace arnounts of labeled ~-[~&]-desmeth~ldoxe~inobserved could be produced through an isotope exchange process. 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SPECIAL CONSENT TO USE DRUGS FOR EXPERIMENTAL HUMAN STUDIES A STUDY OF DOXEPIN AND ITS GEOMETRIC ISOMERS

VoIunteer: Date: a.m. Time: p.m.

1 have been tcld the nature of the dmg to be administered to me for the purpose of experimental human studies, the expected duration of its effect, the methods and means by which it is to be administered, and the methods, techniques and procedures to be used to assess its effects. A brief surnmary of these particulars is as follows:

Doxepin is a psychotropic dmg used rnainly in the treatment of depression. The dose adrninistered in this experiment will not exceed 75 mg of commercial doxepin. The study will consist of a single oral dose of 75 mg followed by collection of urine over the next 24 hours.

1 am fully aware of the experimental nature of the tests being performed, and the risks and potential risks involved with this type of dmg in an experimental study of this nature. The known risks were outlined to me and are as follows:

The majority of adults tolerate the dosages used in this study without adverse drug effects apart from a transient sedation. If any other adverse dmg reactions occur, they would usually be expected to be transient because of the limited dosage used, the interval between administrations and the supervision provided. Reactions after doxepin administration include postural hypotension ( a rapid fall in blood pressure on changing from lying down to standing up causing one to fell dizzy, especially when done rapidly), tachycardia (a rapid heartbeat), involuntary movements characteristic of an extrapyramidal syndrome, anticholinergic effects (which include a dry mouth, constipation, urinary retention, blurring of vision, and alteration in the extent of sweating), blood disorders, photosensitivity, changes in liver function and gastric irritation. Hypertension and cardiac arrhythmias have also been reported.

1 have been cautioned that for the 24 hours after ingestion of the drug, I must not drive any motor powered vehicle, such as a car or snowmobile, or operate any machinery, whether small such as a power drill or power saw, or heavy such as a farm tractor and farm machinery. 1 will not take any dmg (including such things as aspifin and medications for colds) for the &O weeks prior to the snidy and during the trial. If in an emergency it becomes necessary for me to take any dmg, 1 will inform the physician.

I have been told of the serious nature of a diug interaction with alcohol. As a consequence, 1 have been cautioned that 1 must abstain from alcohol for the 24 hours before as well as derdmg administration.

1 have been infomed of the interaction of doxepin with monoamine oxidase inhibitors (the marketed monoamine oxidase inhibitors are phenelzine-Nardil@, tranylcypromine-Parnate@, and isocarboxazed-Marplano. Other dmgs which may have monoamine oxidase effects are debrisoquine-DeclinaxO, isoniazid-IsotarnineO, Rimifona, metoclopramide-Mazerana, ReglanB and procarbazine-NatuIana).

1 have also been inforrned of interactions of doxepin which depresses the central nervous system with other drugs which affect the central nervous system. These include such drugs as aIcoho1, marijuana, tranquilizers such as Vahm and Librium, amphetamines, antidepressants, barbiturates and narcotics.

Also, doxepin habeen reported to give photosensitivity reactions and, therefore, as a result, 1 agree to avoid excessive exposure to direct sunlight or the use of sun Iamps or UV tanning Iights both during the study day and for the 48 hours after it.

Additionaliy, 1 will refrain from exercise on the first thouree days of dmg administration and if, at any time 1 feel dizzy or weak, 1 will lie down immediatdy.

1 have agreed to abstain from caffieine for 12 hours after dmg administration. This includes coffee, tea, chocolate and sorne cola beverages.

1 have been informed that there are possible unknown reactions from an experimental stridy of this kind, and 1 acknowledge that no guarantees have been made to me concerning the effects of the drugs to be administered. In Iight of this information which 1 have considered and fülly understand, 1 have agreed to participate in the study with the understanding that 1 am fiee to drop out at any time.

1 have been informed that if 1 have any problems 1 rnay contact Dr. Korchinski at 966-7859.

Witness Volunteer Appendix B

SPECIAL CONSENT TO USE DRUGS FOR EXPERIMENTAL HUMAN STUDIES A STUDY ON THE METABOLISM OF DOXEPIN IN FIühMNS

Volunteer: Date:

1 have been told the nature of the drug to be administered to me for the purpose of Experimenbl Human Studies, the expected duration of its effect, the methods and means by which it is to be adrninistered, and the methods, techniques and procedures to be used to assess its effects. A brief summary of these particuIars is as follows:

Doxepin is a psychotropic drug used mainiy in the treatment of depression. 1 understand that the purpose of the study is to advance knowledge on the human rnetabolism of doxepin. 1 wiI1 be required go to the Family Medicine Unit at the Royal University Hospital on two days separate6 by an interval of two weeks. On one occasion, 1 will be given an oral dose of doxepin and on the othe- 1 wiII be given an intravenous dose of doxepin. The doses will be administered in random order. The oral dose will consist of 75 mg of a commercial doxepin formulation and the intravenous dose will consist of 25 mg of doxepin hydrochloride (USP standard product) in a steriIe saline solution prepared by the Phamacy Department at the Royal University Hospital. On each occasion, 1 will be required to remain in the FamiIy Medicine Unit under the observation of Dr. Korchinski or his staff for at least eight hours following administration of either dose. Not more than 19 (14 mL each) blood sarnples will be taken by venipuncture over the 120 hours following each administration of the hg. AI1 urine will also be collected over this time period. Sarnples for clinical test (blood and urine) will be required before the trial and on completion of the study.

1 am hlly aware of the experimental nature of the tests being perforrned, and the possible risks involved with this type of dnig in an experimental study of this nature. The known risks were outlined to me and are as follows:

The majority of adula tolerate the dosages used in this study without adverse dmg effects apart fiom a temporary sedation. If any other adverse dmg reactions occur, they would usualIy be expected to be temporary because of the limited dosage used, the interval between administrations and the supervision provided. Reactions after doxepin administration include postural hypotension (a rapid fa11 in blood pressure on changing fiom iying down to standing up, which causes diuiness, especially after standing up rapidly), tachycardia (a rapid heartbeat), involuntary movements characteristic of an "extrapyramidal syndrome" (for example, trembling of the hands), anticholinergic effects (which include a dry rnouth, constipation, urinary retention, blurring of vision, and alteration in the extent of sweating), blood disorders, photosensitivity, changes in liver function and imtation of the stomach. Hypertension (hi& blood pressure) and cardiac arrhythmias (irregular heartbeat) have also been reported. 1 have been cautioned that for the 24 hours after administration of the dmg, I must -not drive any rnotor powered vehicle, such as a car or snowmobile, or operate any machinery, whether small such as a power drill or power saw, or heavy such as a farm tractor and fmrnachinery. 1 wifl not take any dmg (including such things as aspirin and medications for colds) for the two weeks prior to the study and during the trial. If in an ernergency it becomes necessary for me to take any drug, I will inform the physician.

I have been toId of the senous nature of a drug interaction with alcohol. As a consequence, 1 have been cautioned that for each dose, I must abstain from aicohol for the 24 hours before dmg administration during each study penod and until at least 24 hours after thz Iast blood sample has been taken.

1 have been informed that I must not take any of the following list of dmgs: phenelzine-(Nardil@), tranylcypromine-(Parnate@), isocarboxazid-(Marplm@), debrisoquine-(DecIinax@), isoniazid-(Isotamine8, RimifonGO), rnetoclopramide- (MazeranB, RegIanB) and procarbazine-NatulanB) or amphetamine derivatives. Mixing any of these dmgs with the study medication can lead to potentially serious cardiovascular side effects such as elevated blood pressure. 1 rnust also avoid taking benzodiazepines such as diazepam (Valium@), antidepressant, barbiturates, narcotics or marijuana which can lead to increased drowsiness when taken witn the study medication.

Afso, doxepin has been reported to give photosensitivity reactions and, therefore, as a result, I agree to avoid excessive exposure to direct sunlight or the use of Sun larnps or UV tanning !ights both during the study and for the 48 hours after the Iast bIood sample for each dose.

Additionally, I will refrain from exercise on the first three days of dmg administration and if, at any time I feeI dizzy or weak, 1 will lie down immediately. 1 have also agreed to an overnight fast and that 1 will not eat until the standardized meal, supplied at 4 hours after dosing (or a Iight breakfast 1 hour after dosing).

1 have agreed to abstain frorn caffeine for 12 hours after dmg administration. This includes coKee, tea, chocolate and some cola beverages.

I have agreed to remain under medical supervision for 8 hours after drug administration. Supervision may be lengthened if necessary.

1 have been informed that there are possible unknown reactions fiom an experimental study of this kind, and I acknowledge that no guarantees have been made to me concerning the effects of the dmgs to be administered. In Iight of this information which I have considered and fully undentand, I have agreed to participate in the study with the understanding that 1 am fiee to drop out at any time.

1 have been informed that if 1 have any problems or questions 1 may contact Dr. Korchinski at 655-6835 (hospitaI) or 477-2973 (home).

Witness Volunteer