Abstract
Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. AnN-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2–3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 μg of material injected onto the column,1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.
Footnotes
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Send reprint requests to: William J. Ehlhardt, 0825 Drug Metabolism, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
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↵1 Maple SR, Woodland JM and Ehlhardt WJ. LC-NMR detection of the metabolites of the sulfonylurea oncolytic agent LY295501 in rat urine. Submitted for publication.
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↵3 Wrighton SA, unpublished results.
- Abbreviations used are::
- WET
- water suppression enhanced through T1 effect
- CYP
- cytochrome P450
- COSY
- correlation spectroscopy
- TOCSY
- total correlation spectroscopy
- Received June 25, 1997.
- Accepted September 23, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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