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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 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 solidphase 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 T[sub max], 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 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.