ISOLATION, IDENTIFICATION AND SYNTHESIS OF HYDROMORPHONE i METABOLITES: ANALYSIS AND ANTINOCICEPTIVE ACTIVITIES IN COMPARISON TO MORPHINE by MING ZHENG B.Sc. (Pharm.), Shanghai Medical University, 1987 M.Sc, The University of Ghent, 1990 M.Sc, The University of British Columbia, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1997 © Ming Zheng, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT As active drug metabolites may have profound pharmacological and toxicological effects on patients, a thorough understanding of the metabolic fate of the parent drug and the potential pharmacological and toxicological activities of its metabolite(s) is essential to the effective and safe usage of the drug. Although hydromorphone is commonly used for the management of cancer and postoperative pain, information about the metabolic fate of this opioid analgesic is scant. Only hydromorphone-3-glucuronide and dihydromorphine-3-glucuronide have been reported in urine samples of rabbits following hydromorphone administration, while conjugates of unknown identities were reported for hydromorphone, dihydromorphine and dihydroisomorphine in urine samples from healthy male subjects. Thus the objectives of this study were to investigate the metabolic fate of hydromorphone in patient(s) receiving chronic hydromorphone therapy and to test for antinociceptive activities of the metabolites thus identified and synthesized. Hydromorphone-3-glucuronide, hydromorphone-3-sulfate, dihydromorphine, dihydroisomorphine, dihydromorphine-3-glucuronide, dihydroisomorphine-3- glucuronide and norhydromorphone were isolated from pooled urine samples of a cancer patient receiving chronic hydromorphone therapy. The metabolites were analyzed using a high-performance liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method. Synthetic standards of these metabolites were prepared by adaptation of published methods for the synthesis of the same or structurally similar compounds. The identities of these metabolites in urine were thus confirmed by Ill comparison of the retention times and ion transitions with those of the synthetic standards using the LC-MS-MS method. The presence of hydromorphone-3-glucuronide, dihydromorphine-3-glucuronide, and unconjugated dihydromorphine and dihydroisomorphine was confirmed for the first time in pooled urine samples of a human subject. Hydromorphone-3-sulfate, dihydroisomorphine-3-glucuronide and norhydromorphone were novel metabolites of hydromorphone identified for the first time. Another novel metabolite of hydromorphone, nordihydroisomorphine, was tentatively identified. The urinary recovery of dihydroisomorphine-3-glucuronide was estimated to be 17-fold higher than the previously published result of conjugated dihydroisomorphine by other investigators. LC-MS-MS assays were also developed for the determination of morphine and its metabolites, and hydromorphone and its metabolites in plasma samples of male Sprague Dawley rats. The analytes were extracted from plasma samples by solid- phase extraction using C2 cartridges. The assays were linear over the concentration ranges determined. Both intra- and inter-assay variabilities were less than or equal to 12%. The LC-MS-MS assays developed were applied to pharmacokinetic studies of morphine, hydromorphone, dihydromorphine, dihydroisomorphine and norhydromorphone in male Sprague Dawley rats following intraperitoneal administration. Following morphine administration, morphine-3-glucuronide was detected as the predominant metabolite of morphine, whereas normorphine was detected as a minor metabolite. The slopes of the apparent elimination phases for morphine-3-glucuronide IV and morphine were not significantly different, suggesting the elimination rate constant of morphine-3-glucuronide is much greater than that of morphine and the formation of morphine-3-glucuronide is the rate-limiting step for the elimination of this metabolite following morphine administration. Normorphine glucuronide was tentatively detected as a minor metabolite of morphine. Morphine-6-glucuronide was not detected, indicating that this metabolite is not formed in rats. The 3-O-glucuronide metabolites were detected as the common major metabolites following administration of hydromorphone, dihydromorphine and dihydroisomorphine. Norhydromorphone glucuronide was tentatively detected as a major metabolite following norhydromorphone administration. The slopes of the apparent elimination phases for the glucuronide metabolites were not significantly different from the slopes of the respective parent compounds, also suggesting that the elimination rate constants of these glucuronide metabolites are much greater than those of their respective parent compounds and the formation of these glucuronide metabolites is the rate-limiting step for the elimination of these glucuronides following administration of the parent compounds. The antinociceptive activities of hydromorphone, dihydromorphine, dihydroisomorphine and norhydromorphone were determined using the formalin test. Morphine was also tested as a reference standard for comparison of antinociceptive activities between these compounds. The values of Tmax, the time when maximum plasma concentrations are reached, for the above compounds determined from the pharmacokinetic studies were used for the design of the formalin test. The results showed that morphine, hydromorphone, dihydromorphine and dihydroisomorphine were equally effective in the formalin test. However, hydromorphone was five times as V potent as morphine, while dihydromorphine was equipotent to morphine and dihydroisomorphine was less potent than morphine. The antinociceptive activities of norhydromorphone were low and not significantly different at the three different doses tested. Preliminary tests were also conducted for hydromorphone-3-glucuronide, dihydromorphine-3-glucuronide and dihydroisomorphine-3-glucuronide. No antinociception was observed for these glucuronide metabolites at the doses tested. Research Supervisor Keith McErlane, Ph.D. vi TABLE OF CONTENTS Abstract ii Table of Contents vi List of Figures xi List of Tables xv List of Abbreviations xvii Acknowledgment xx Dedication xxi 1. INTRODUCTION 1 1.1 Morphine 4 1.1.1 The Chemical Structure of Morphine 4 1.1.2 Mechanisms of Action 6 1.1.2.1 Opioid Receptors 6 1.1.2.2 Nociceptive Pain 7 1.1.2.3 Mechanisms of Action 8 1.1.2.4 Peripheral Analgesia 10 .1.1.3 Clinical Pharmacokinetics of Morphine 11 1.1.4 Metabolism of Morphine 13 1.1.4.1 Glucuronidation of Morphine 14 1.1.4.2 N-Demethylation of Morphine 15 1.1.5 Pharmacology of the Metabolites of Morphine 16 1.1.5.1 Morphine-6-Glucuronide 16 1.1.5.2 Morphine-3-Glucuronide 18 1.1.5.3 Other Metabolites of Morphine 19 1.1.6 Accumulation of Morphine-3-Glucuronide and Morphine-6-Glucuronide in Patients with Renal Impairment 20 1.2 Hydromorphone 23 1.2.1 The Chemical Structure of Hydromorphone 23 1.2.2 Clinical Uses of Hydromorphone 24 1.2.3 Clinical Pharmacokinetics of Hydromorphone 25 1.2.4 Metabolism of Hydromorphone 27 1.2.5 Antinociceptive Activities of Dihydromorphine and Dihydroisomorphine 28 vii 1.3 Analytical Methods for the Measurement of Morphine, Hydromorphone and Their Metabolites in Biological Samples 29 1.3.1 Radioimmunoassays 29 1.3.2 Chromatographic Methods 30 1.3.2.1 Chromatographic Analysis of Morphine and Its Metabolites 30 1.3.2.2 Chromatographic Analysis of Hydromorphone and Its Metabolites 32 1.4 Antinociceptive Tests 33 1.4.1 Thermal Methods 34 1.4.1.1 Tail-Flick Test 34 1.4.1.2 Hot-Plate Test 35 1.4.2 Mechanical Method: the Tail-Clip Test 35 1.4.3 Chemical Methods 37 1.4.3.1 Writhing Test 37 1.4.3.2 Formalin Test 37 1.5 Thesis Hypothesis and Objectives 39 2. EXPERIMENTAL 41 2.1 Materials and Supplies 41 2.1.1 Drugs 41 2.1.2 Chemicals and Solvents 41 2.1.3 Animals 42 2.2 LC-MS-MS Instrumentation 42 2.3 Isolation and Identification of Hydromorphone and Its Metabolites from Pooled Urine Samples of a Cancer Patient 43 2.3.1 Isolation of Hydromorphone and Its Metabolites from Pooled Urine Samples of a Cancer Patient 43 2.3.2 LC-MS-MS Conditions 44 2.3.2.1 LC-MS-MS Conditions for Identification of Hydromorphone, Dihydromorphine, Dihydroisomorphine, Hydromorphone- 3-Glucuronide, Dihydromorphine-3-Glucuronide and Dihydroisomorphine-3-Glucuronide 44 2.3.2.2 LC-MS-MS Conditions for Identification of Norhydromorphone and Hydromorphone-3-Sulfate 45 2.3.3 Chemical Synthesis 46 2.3.3.1 Synthesis of Dihydromorphine 46 2.3.3.2 Synthesis of Dihydroisomorphine 47 2.3.3.3 Synthesis of Hydromorphone-3-Glucuronide 48 2.3.3.4 Synthesis of Dihydromorphine-3-Glucuronide 49 2.3.3.5 Synthesis of Dihydroisomorphine-3-Glucuronide 50 VIII 2.3.3.6 Synthesis of Norhydromorphone 50 2.3.3.7 Synthesis of Hydromorphone-3-Sulfate 51 2.4