Metabolite Generation via Microbial Biotransformations with Actinomycetes: Rapid Screening for Active Strains and Biosynthesis of Important Human Metabolites of Two Development-Stage Compounds, 5-[(5S,9R)-9-(4-Cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non7-yl-methyl]-3-thiophenecarboxylic Acid (BMS-587101) and Dasatinib

Abstract

ABSTRACT:The enzymes present in many microbial strains are capable of carrying out a variety of biotransformations when presented with drug-like molecules. Although the enzymes responsible for the biotransformations are not well characterized, microbial strains can often be found that produce metabolites identical to those found in mammalian systems. However, traditional screening for microbial strains that produce metabolites of interest is done with many labor intensive steps that include multiple shake flasks and many manual manipulations, which hinder the application of these techniques in drug metabolite preparation. A 24-well microtiter plate screening system was developed for rapid screening of actinomycetes strains for their ability to selectively produce metabolites of interest. The utility of this system was first demonstrated with the well characterized cytochrome P450 substrate diclofenac. Subsequently, the use of this system allowed the rapid identification of several actinomycetes strains that were capable of converting two drug candidates under development, 5-[(5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non7-yl-methyl]-3-thiophenecarboxylic acid and N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)- 1-piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, Sprycel, BMS-345825), to mammalian metabolites of interest. Milligram quantities of the metaboliteswere then prepared by scaling-up the microbial biotransformation reactions. These quantities were sufficient for initial characterization, such as testing for pharmacological activity and use as analytical standards, prior to the availability of authentic chemically synthesized compounds.An integral part of the drug discovery and development process involves characterization of the metabolites of a drug candidate. The major purposes of these characterizations are to: 1) help ensure that human metabolites are adequately tested in toxicology species, 2) determine whether any of the pharmacological activity of the drug is due to active metabolites, and 3) aid in the determination of the mechanism of metabolic clearance of the parent drug (Baillie et al., 2002; http://www.fda. gov/cder/guidance). Standard testing paradigms for ensuring the safety of drug metabolites have been recently proposed, and aspects of these proposals were recently reviewed (Davis-Bruno and Atrakchi, 2006;Guengerich, 2006;Humphreys and Unger, 2006;Smith and Obach, 2006; http://www.fda.gov/cder/guidance). Considerable effort is often required to prepare sufficient quantities of key mammalian metabolites of drug candidates for biological activity evaluation or for use as analytical standards. Metabolite biosynthesis methods using mammalian systems (microsomes, S9, hepatocytes, in vivo, etc.) are useful for generating limited quantities of metabolites for structure elucidation by LC/MS/MS and NMR analysis. Chemical synthesis is the preferred method for larger scale metabolite preparation, but it is often ...

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