Received for publication October 18, 2007.
Revised November 9, 2007.
Accepted for publication November 12, 2007.

Biosynthesis of Drug Metabolites Using Microbes in Hollow Fiber Cartridge Reactors: Case Study of Diclofenac Metabolism by Actinoplanes sp

Abstract

Fungal and bacterial microbes are known to mimic mammalian cytochrome P450 metabolism. Traditionally, microbial biotransformation screening and small scale-ups (< 1L) are performed in shake-flask reactors. An alternative approach is the use of hollow fiber cartridge (HFC) reactors. The performance of HFC reactors is compared to shake-flask reactors using diclofenac as a model substrate. Actinoplanes sp. (ATCC-53771) in a shake-flask reactor hydroxylated diclofenac (50 µM) with 100% turnover in less than 5 hours. A scaled-up production resulted in the formation of 4'-hydroxy (169 mg, 54% yield), 5-hydroxy (42 mg, 13 % yield) and 4’,5-dihydroxy (25 mg, 7.7% yield) metabolites. HFC reactors with Teflon, poly-sulfone, and cellulose membranes were screened for non-specific binding of diclofenac. Concentration-time profiles for turnover of 50-2000 µM diclofenac by Actinoplanes sp. were then determined at 22ºC and 30 °C in an HFC reactor. Cellulose-based HFC reactors exhibited the lowest non-specific binding (87% of 50 µM diclofenac remaining after 5 hr) and offered the best conditions for its biotransformation (100% conversion; < 5hr at 30 °C at 50 µM; 25 hr at 500 µM). The time profile for substrate turnover was equivalent in both a cellulose membrane HFC reactor and shake-flask reactor. Two cellulose membrane HFC reactors were also tested to evaluate the reusability of the cartridges for diclofenac metabolism (50 µM, 22 °C, 15 hr; 500 µM, 30 °C, 36 hr). Up to seven reaction cycles with intermediate wash cycles were tested. At least 98 % conversion was observed in each reaction cycle at both diclofenac concentrations.

Key words: anti-inflammatory drugs, cytochrome P450 catalyzed oxidations, HPLC, mass spectrometry, metabolite identification, structure elucidation