Biotransformation of Flurbiprofen by Cunninghamella Species

The biotransformation of the fluorinated anti-inflammatory drug flurbiprofen was investigated in Cunninghamella spp. Mono- and dihydroxylated metabolites were detected using gas chromatography-mass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy, and the major metabolite 4′-hydroxyflurbiprofen was isolated by preparative high-pressure liquid chromatography (HPLC). Cunninghamella elegans DSM 1908 and C. blakesleeana DSM 1906 also produced a phase II (conjugated) metabolite, which was identified as the sulfated drug via deconjugation experiments.One of the objectives of the recent European Union legislation governing the testing and evaluation of chemicals, REACH (Regulation, Evaluation, Authorisation and Restriction of Chemicals), is to further reduce the need for animals in the testing process. Some microorganisms, such as the zygomycete fungus Cunninghamella and actinomycetes bacteria, have been shown to metabolize xenobiotic compounds in a fashion analogous to that of mammals (3, 5, 11, 17). It was suggested over 3 decades ago that microorganisms had potential as models of mammalian metabolism (16), although there are concerns about their predictive value (8). Nevertheless, certain microorganisms can be applied to the generation of useful quantities of drug metabolic intermediates (13), which is more desirable than isolation of these compounds from dosed animals, and avoids the concerns often associated with chemical synthesis, such as the use of toxic reagents and harsh reaction conditions.Owing to the desirable physicochemical properties of the fluorine atom (small Van der Waals radius, electronegativity, and strength of the carbon-fluorine bond), approximately 25% of drugs either currently on the market or in the pipeline are fluorinated (12). One such example is flurbiprofen (RS)-2-(2-fluoro-4-biphenyl)propionic acid], which is a nonsteroidal anti-inflammatory drug (NSAID) used in the treatment of inflammation caused by arthritis. In humans it is transformed to the phase I (oxidative) metabolites 4′-hydroxyflurbiprofen, 3′,4′-dihydroxyflurbiprofen, and 3′-hydroxy,4′-methoxyflurbiprofen; glucuronide and sulfate conjugates (phase II metabolites) have also been detected (9, 15). In equine urine additional hydroxylated and methoxylated metabolites were detected (20). Tracy et al. (18) demonstrated that only one cytochrome P450 isoform (2C9) is involved in the oxidation of flurbiprofen, which makes the drug a potentially useful in vivo probe for this particular isoform. Despite the prevalence of fluorinated drugs, only a few investigations have been undertaken to determine the microbial biotransformation of these compounds (7, 21). Here we describe the biotransformation of flurbiprofen by Cunninghamella species and the determination of the metabolites by nuclear magnetic resonance (NMR) spectroscopy (¹H and ¹⁹F), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC).Three species of Cunninghamella were selected for the biotransformation experiments: C. elegans (strains DSM 1908, DSM 8217, and DSM 63299), C. echinulata DSM 1905, and C. blakesleeana DSM 1906. The fungi were grown on Sabouraud dextrose agar plates (Sigma) for 5 days at 26°C before being homogenized in 100 ml of sterile saline solution. The homogenate (10%, vol/vol) was used to inoculate 50 ml of fresh Sabouraud dextrose broth in 250-ml Erlenmeyer flasks, which were incubated at 28°C with shaking at 150 rpm. Following previously established procedures (2), 5 mg of flurbiprofen (Sigma) dissolved in dimethyl formamide (20 μl) was added to the cultures after 72 h, and the incubation was continued up to a further 120 h. Control experiments were conducted in the absence of either flurbiprofen or fungus. The cultures (supernatant and cells) were sonicated on ice (Sonicator U200S control; IKA Labortechnik) for 5 min at 50% amplitude, with intervals of 30 s after each minute to prevent overheating. The sonicates were centrifuged, the supernatant was extracted with 50 ml of ethyl acetate, and the extracts were evaporated to dryness.