Microbial oxidative metabolism of diclofenac: production of 4′-hydroxydiclofenac using Epiccocum nigrum IMI354292

Abstract

The 4'-hydroxylated metabolite of diclofenac was produced by biocatalysis for probing specific human drug-metabolising enzymes (CYP2C9). An initial screen of 11 microorganisms was carried out (50 ml scale) to identify the organism best suited to the regioselective conversion of diclofenac to its 4'-hydroxylated metabolite. From this screen, the fungus Epicoccum nigrum IMI354292 was selected as the most suitable microorganism. Scale-up was carried out in a 30-l fermenter to which 2 g diclofenac was added. After 48 h, 50% of the diclofenac had been converted to it 4'-hydroxylated metabolite. The broth was then extracted with ethyl acetate and purified by chromatography and crystallisation. This yielded 0.3 g 4'-hydroxydiclofenac with a purity of at least 99%. The 4'-hydroxydiclofenac produced by E. nigrum was characterised by HPLC, mass spectrometry and NMR.

Efficient biotransformation of non-steroid anti-inflammatory drugs by endophytic and epiphytic fungi from dried leaves of a medicinal plant, Plantago lanceolata L.

“…Several other peaks likely to be metabolites of the NSAIDs could be detected, but unfortunately, their abundance was insufficient to subject them to MS Table 1.). These were identified as hydroxy-DIC derivatives as compared with the MS/MS data (Webster et al, 1998;Kallio et al, 2010). Although the actual position of the hydroxyl group could not be determined from the MS/MS alone, the fragmentation fully supported the proposed structure.…”

“…The ibuprofen metabolism also consisted of oxidation (hydroxylation) steps in the case of Trametes versicolor, but the final product (1,2-dihydroxyibuprofen) was found to be new, and not previously described in biological systems (Marco-Urrea et al, 2009b). Production of 4'-hydroxy-diclofenac by a non-white-rot fungal strain, Epicoccum nigrum was described by (Webster et al, 1998).…”

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

“…One hundred sixty-nine milligrams of 4Ј-hydroxy diclofenac was obtained, and a 54% yield was significantly higher than previously reported bioreactor scale-ups. Previously obtained yields were 15% from Epicoccum nigrum (Webster et al, 1998), 26% from human P450 2C9 expressed in Sf21 insect cells (Rushmore et al, 2000), and 35% from human P450 2C9 expressed in an Escherichia coli system (Vail et al, 2005). 5-Hydroxy diclofenac has been previously reported to be formed in fungal incubations (Webster et al, 1998) but was never isolated.…”

“…Previously obtained yields were 15% from Epicoccum nigrum (Webster et al, 1998), 26% from human P450 2C9 expressed in Sf21 insect cells (Rushmore et al, 2000), and 35% from human P450 2C9 expressed in an Escherichia coli system (Vail et al, 2005). 5-Hydroxy diclofenac has been previously reported to be formed in fungal incubations (Webster et al, 1998) but was never isolated. It has been isolated from human urine (Stierlin et al, 1979) as well as synthesized (Bort et al, 1996;Tang et al, 1999;Kenney et al, 2004).…”

Highly Selective and Asymmetric Reductive Biotransformation of ^|^alpha;-Ionone by Epicoccum purpurascens

“…This conversion route reduced α, β-unsaturated ketones via enzymatic reactions catalyzed by E. purpurascens. Because the Eipcoccum species is suitable for screening specific human drug-metabolizing enzymes CYP2C9 25 , this biocatalysis may be useful as a means for screening tests. …”