Received for publication January 30, 2006.
Revised February 22, 2006.
Accepted for publication February 22, 2006.

APPLICATION OF BIOCATALYSIS TO DRUG METABOLISM: PREPARATION OF MAMMALIAN METABOLITES OF A BIARYL-BIS-SULFONAMIDE AMPA RECEPTOR POTENTIATOR USING ACTINOPLANES MISSOURIENSIS

Abstract

LY451395 is a potent and highly selective potentiator of the -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. It is a biaryl-bis-sulfonamide and is known to be highly metabolized in preclinical species. In those metabolism studies, the metabolite structures were proposed exclusively by the analysis of mass spectrometric data. Although mass spectrometry is clearly a technique of choice for rapid identification of drug metabolites, occasionally nuclear magnetic resonance spectroscopy is required to unambiguously assign and characterize, particularly, the regio- and stereochemistry of metabolic changes. Nuclear magnetic resonance spectroscopy, in general, is less sensitive than other detection methods and demands several micrograms of material for the analysis. To support full structure characterization of metabolites by NMR, in this study we demonstrated the application of a microbial-based surrogate biocatalytic system to produce sufficient amounts of the mammalian metabolites of LY451395. The results revealed that incubation of LY451395 with Actinoplanes missouriensis NRRL B3342 generated several metabolites that were previously detected in the in vivo metabolism studies of the preclinical species. Subsequent large-scale bioconversion resulted in the isolation of seven mammalian metabolites in milligram quantities for structural characterization by nuclear magnetic resonance spectroscopy. Furthermore, a selected group of metabolites generated from the microbial conversion served as analytical standards to monitor and quantify drug metabolites during clinical investigations.

Key words: cytochrome P450 catalyzed oxidations, HPLC, mass spectrometry, metabolite identification, structure elucidation

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