Biotransformation of Flurbiprofen by Cunninghamella Species

Abstract

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 antiinflammatory drug (NSAID) used in the treatment of inflammation caused by arthritis. In humans it is transformed to the phase I (oxidative) metabolites 4Ј-hydroxyflurbiprofen, 3Ј,4Ј-dihydroxyflurbiprofen, and 3Ј-hydroxy,4Ј-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 ( 1 H and 19 F), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC).Three species of Cunninghamella were selected for the biotransformation experiments: C. ...

Filamentous fungal biofilm for production of human drug metabolites

“…elegans DSM 1908 is very well known to be a model for mammalian drug metabolism (Asha and Vidyavathi 2009), and we have shown previously that the suspended mycelia transform the drug flurbiprofen to mono-and di-hydroxylated metabolites (Amadio et al 2010). This drug was used to study the capability of the biofilm for biotransformation.…”

“…In contrast, the fungus Cunninghamella elegans has been shown to transform a broad range of drugs and xenobiotics to the same metabolites as humans (Asha and Vidyavathi 2009;Amadio et al 2010;Amadio and Murphy 2011).…”

Biodegradation and biotransformation of polycyclic non-steroidal anti-inflammatory drugs

“…However, they and their metabolites are detected in the aquatic environment (Chowdhury et al 2003;Amadio et al 2010;Hata et al 2010).…”

“…In the environment, fungi which able to transform this drug are nevertheless found. Amadio et al (2010) revealed flurbiprofen transformation to 4 0 -hydroxyflurbiprofen and 3 0 -hydroxy-4 0 -methoxyflurbiprofen, as well as to 3 0 ,4 0 -dihydroxyflurbiprofen by Cunninghamella species: C. elegans DSM 1908, C. echinulata DSM 1905 and C. blakesleeana DSM 1906. C. elegans strains DSM 8217 and DSM 63299 were able to transform flurbiprofen only to 4 0 -hydroxyflurbiprofen and 3 0 -hydroxy-4 0 -methoxyflurbiprofen.…”

“…Moreover, C. elegans DSM 1908 and C. blakesleeana DSM 1906 also produced a sulfated drug-an analog of the II phase metabolite of the mammalian detoxification system (Fig. 5) (Amadio et al 2010).…”