Drug Metab. Pharmacokinet. 26 (6): 559­568 (2011). Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) Regular Article Inhibition of Cytochrome P450 2C8-mediated Drug Metabolism by the Flavonoid Diosmetin Luigi QUINTIERI1,PietroPALATINI1,StefanoMORO2 and Maura FLOREANI1,* 1Department of Pharmacology and Anaesthesiology, University of Padova, Italy 2Molecular Modeling Section (MMS), Department of Pharmaceutical Sciences, University of Padova, Italy Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: The aim of this study was to assess the effects of diosmetin and hesperetin, two flavonoids present in various medicinal products, on CYP2C8 activity of human liver microsomes using paclitaxel oxidation to 6¡-hydroxy-paclitaxel as a probe reaction. Diosmetin and hesperetin inhibited 6¡-hydroxy- paclitaxel production in a concentration-dependent manner, diosmetin being about 16-fold more potent than hesperetin (mean IC50 values 4.25 « 0.02 and 68.5 « 3.3 µM for diosmetin and hesperetin, respectively). Due to the low inhibitory potency of hesperetin, we characterized the mechanism of diosmetin-induced inhibition only. This flavonoid proved to be a reversible, dead-end, full inhibitor of CYP2C8, its mean inhibition constant (Ki)being3.13« 0.11 µM. Kinetic analysis showed that diosmetin caused mixed-type inhibition, since it significantly decreased the Vmax (maximum velocity) and increased the Km value (substrate concentration yielding 50% of Vmax) of the reaction. The results of kinetic analyses were consistent with those of molecular docking simulation, which showed that the putative binding site of diosmetin coincided with the CYP2C8 substrate binding site. The demonstration that diosmetin inhibits CYP2C8 at concentrations similar to those observed after in vivo administration (in the low micromolar range) is of potential clinical relevance, since it may cause pharmacokinetic interactions with co- administered drugs metabolized by this CYP. Keywords: CYP2C8 inhibition; diosmetin; paclitaxel; drug­drug interactions, molecular docking simulations present in pharmaceutical preparations. For example, Introduction diosmin, the 7-rutinoside ¤rhamnosyl-D-glucoside¥ of the Flavonoids are polyphenolic compounds ¤produced as flavone diosmetin ¤3$,5,7-trihydroxy-4-methoxyflavone¥, secondary plant metabolites¥ widely ingested with the diet alone or in association ¤9:1, w/w¥ with hesperidin, the by humans. Their beneficial effects against oxidative stress 7-rutinoside of the flavanone hesperetin ¤3$,5,7-trihydroxy- and related pathologies1®3¥ have been mainly ascribed to their 4-methoxyflavanone¥¤Fig. 1¥, is widely used for the antioxidant activity,4¥ although flavonoids appear to exert management of chronic venous insufficiency, venous ulcers more complex biological effects, including inhibition of and hemorrhoids.9¥ However, the safe use of herbs or tumor proliferation, modulation of several enzymes and medications containing flavonoids should take adequate interaction with different signal transduction pathways.5¥ account of their potential for interactions with co- Moreover, they seem to exhibit cancer-preventive proper- administered drugs, since various clinical studies have ties, possibly due to inhibition of cytochromes P450 ¤CYP¥ demonstrated that some flavonoids modify the oral 1A1, CYP1A2 and CYP1B1, the CYP forms principally disposition kinetics of numerous drugs.10®15¥ For example, involved in the metabolism of pro-carcinogens.6®8¥ For these Rajnarayana et al. showed that oral treatment of healthy reasons, the use of dietary supplements and/or herbal volunteers with diosmin increases significantly the bioavail- medications containing flavonoids has consistently increased ability of metronidazole,12¥ diclofenac14¥ and chlorzoxa- in recent years. In many countries, some flavonoids are also zone,15¥ and hypothesized that diosmin-induced inhibition Received; May 30, 2011, Accepted; July 14, 2011 J-STAGE Advance Published Date: July 26, 2011, doi:10.2133/dmpk.DMPK-11-RG-048 *To whom correspondence should be addressed: Prof. Maura FLOREANI, Department of Pharmacology and Anaesthesiology, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy. Tel. +39 049 827 5088, Fax. +39 049 827 5093, E-mail: m.fl[email protected] This work was supported by grants from the University of Padova, Italy. The molecular modeling work coordinated by S. M. was carried out also with financial support from the Italian Ministry of Education, University and Research (MIUR), Rome, Italy. 559 560 Luigi QUINTIERI, et al. Fig. 1. Basic structures and numbering system of flavones, flavanones and flavonols The figure also shows the substitutions for diosmetin (3′,5,7-trihydroxy-4-methoxyflavone) and hesperetin (3′,5,7-trihydroxy-4-methoxy- flavanone), and for some flavonols which also inhibit CYP2C8.24) Moreover, the structure of diosmin and hesperidin, natural glycosides of diosmetin and hesperetin, respectively, are reported. of the CYP enzymes catalyzing the biotransformation of terize the mechanism of CYP2C8 inhibition caused by these drugs was responsible for the observed pharmacoki- diosmetin, we performed both enzyme kinetic studies and netic interactions. We have recently demonstrated that molecular docking simulations, since these two techniques diosmetin, the absorbable aglycone of diosmin,16¥ to which provide useful complementary information on the mecha- orally administered diosmin is converted by rhamnosidases nisms of drug-enzyme interactions. of Enterobacteriaceae,17¥ is a potent in vitro inhibitor of human ¥ ¥ Materials and Methods CYP3A4/518 and CYP2C9,19 the CYP forms responsible for the metabolism of metronidazole and diclofenac, Human liver microsomes ¤HLMs¥ and reagents: respectively,12,14¥ thereby confirming the hypothesis of Pooled mixed-gender HLMs ¤obtained from 25 female and Rajnarayana et al.12,14¥ 27 male donors¥ were provided by Xenotech LLC ¤Lenexa, Since CYP2C9 shares more than 80% amino acid KS, USA¥. The microsomal fractions were stored in aliquots sequence identity with CYP2C8,20¥ a CYP form expressed at %80ôC until use. at relatively high levels ¤6®7% of total CYP content¥ in Diosmetin and hesperetin, purchased from Chromadex human liver21¥ and which plays a major role in the ¤Irvine, CA, USA¥, were dissolved in N,N-dimethylform- 22¥ ¤ ¥ ¤ ¥ fl metabolism of several therapeutically important drugs, amide DMF /H2O 50:50, v/v . Solutions of the avonoid the main aim of the present study was to assess whether were prepared daily and kept in ice until use. The final diosmetin is also an effective inhibitor of human CYP2C8. solvent concentration in the incubation medium was 0.5%. Because in some pharmaceutical preparations the flavone Paclitaxel was a kind gift from Indena ¤Milan, Italy¥, diosmin is associated with the flavanone hesperidin, which is whereas its 6Æ-hydroxy metabolite ¤6Æ-OH paclitaxel¥ was also likely to be hydrolyzed by intestinal microflora to its purchased from SPI-Bio ¤Montigny le Bretonneux, France¥. aglycone hesperetin,17¥ a further aim of our study was to test Paclitaxel and 6Æ-OH paclitaxel were dissolved in aceto- ¤ ¥ ¤ ¥ the effect of hesperetin on CYP2C8 activity. Although this nitrile CH3CN /H2O 50:50, v/v .CH3CN was chosen as enzyme has been shown to be inhibited by some flavonols, the solvent for paclitaxel since, at the final concentration such as quercetin, morin and fisetin ¤see Fig. 1¥,23,24¥ to the used in our incubation mixtures ¤1%¥, it had been shown best of our knowledge, no data are available in the literature to produce a negligible effect on CYP2C8-mediated concerning the effects of flavones and/or flavanones on paclitaxel hydroxylation.27¥ NADPH and DMF were CYP2C8-catalyzed reactions. In this study, we used the purchased from Sigma-Aldrich Italy ¤Milan, Italy¥. Potassium ¤ ¥ antitumor agent paclitaxel as a probe substrate for CYP2C8 dihydrogen phosphate KH2PO4 ,CH3CN and methanol activity of human liver microsomes ¤HLMs¥ for two main ¤all of HPLC grade¥ were from Carlo Erba Reagenti reasons: ¤1¥ 6Æ-hydroxylation of paclitaxel is a validated ¤Milan, Italy¥. Ultrapure water was obtained by means of a marker reaction of CYP2C8 activity,22,23¥ since CYP2C8 is Pure-Lab Option Q ¤Elga Lab-Water, High Wycombe, UK¥ the only CYP form catalyzing this reaction in humans;25¥ ¤2¥ apparatus. a possible inhibitory effect of diosmetin and/or hesperetin on Determination of 6Æ-OH-paclitaxel formation this metabolic reaction would be of great clinical relevance, rate by HLMs: Incubation of paclitaxel with HLMs was since formation of the inactive metabolite 6Æ-hydroxy- carried out in conditions yielding linear reaction rates with paclitaxel ¤6Æ-OH-paclitaxel¥ accounts for more than 60% of respect to microsomal protein concentration and incubation paclitaxel metabolic disposition in humans.25,26¥ To charac- time at all paclitaxel concentrations used for our assays. Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) Inhibition of CYP2C8 by Diosmetin 561 HLMs ¤final protein concentration: 0.5 mg/ml¥ were n © 6¥ in the presence of heat-inactivated microsomes ¤final incubated in a mixture ¤total volume: 200 µl¥ containing concentration: 0.5 mg/ml¥ processed exactly as the samples ¤ ¥ 100 mM KH2PO4 pH 7.4 , 0.5 mM NADPH, and increas- obtained from kinetic experiments. The calibration curves ing concentrations ¤n © 9¥ of paclitaxel ¤from 3 to 40 µM¥. were linear in the above-reported concentration range Moreover, in some experiments the incubation medium ¤r2 ; 0.99¥,