Short Communication Effect of Commonly Used Organic Solvents on the Kinetics of Cytochrome P450 2B6- and 2C8-Dependent Activity in Human Liver Microsomes

The effect of common organic solvents on the activities of various human cytochromes P450 has been reported. However, very little is known about their influence on CYP2B6 and CYP2C8 enzymes. The purpose of this study was to investigate the effect of solvents on the kinetics of representative CYP2B6 (bupropion hydroxylase) and CYP2C8 (paclitaxel hydroxylase) reactions in human liver microsomes. Methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied at increasing volumes (v/v). Acetonitrile, DMSO, and ethanol were shown to increase the Km and decrease the intrinsic clearance (CLint) of CYP2B6mediated bupropion hydroxylation in a concentration-dependent manner. These solvents did not noticeably alter the Vmax at concentrations of <1% (v/v). Unlike the other solvents studied, the effect of methanol (<0.5%, v/v) on CYP2B6 kinetics was negligible. Both DMSO and ethanol increased the Km and decreased the CLint of CYP2C8-mediated paclitaxel hydroxylation in a concentration-dependent manner. Acetonitrile had minimal influence on CYP2C8 enzyme kinetics at concentrations of <1% (v/v). Methanol decreased the Km of paclitaxel at low concentrations followed by an increase at concentrations of >2% (v/v). This differential influence on Km resulted in an increased CLint at low concentrations followed by a decrease at high concentrations. The studied solvents had minimal influence on Vmax of paclitaxel. Collectively, DMSO and ethanol were not suitable for characterizing CYP2B6and CYP2C8-mediated reactions because they showed concentration-dependent inhibition. Methanol and acetonitrile at concentrations of <0.5% and <1% (v/v) appeared to be suitable for the measurement of CYP2B6and CYP2C8-mediated activities, respectively. Cytochrome P450 (P450) enzymes are a major class of hemecontaining proteins critical for the metabolism of a large number of xenobiotics and endogenous compounds. Human liver microsomes (HLM) are routinely used for identifying P450 enzymes responsible for metabolism (reaction phenotyping) and/or for investigating the enzyme kinetics and inhibitory effects of discovery and development compounds (Walsky and Obach, 2004; Obach et al., 2005). The primary function of P450 enzymes is to convert lipophilic drug molecules into hydrophilic forms that can be readily excreted from the body. Because in vitro metabolism studies are typically conducted in physiological buffers, problems related to the solubilization of substrates or inhibitors may occur. Organic solvents like methanol, ethanol, acetonitrile, and dimethyl sulfoxide (DMSO) are commonly used to facilitate solubilization of highly lipophilic compounds of interest. However, owing to their lipophilic nature, organic solvents may alter the activities of the P450 enzymes directly by either modifying the native environment surrounding these enzymes or by disrupting the integrity of the enzymes, leading to erroneous interpretation of data and misleading conclusions regarding enzyme kinetics and inhibition. CYP2C8 constitutes about 7% of the total microsomal P450 content in human liver (Rendic and Di Carlo, 1997). Despite the high expression level, this polymorphic enzyme has been a relatively less explored member of the P450 family, and it is only recently that its role in metabolism has been considered. Noteworthy drug-drug interactions involving inhibition of CYP2C8 have done much to raise awareness of its importance (Backman et al., 2002; Niemi et al., 2003). Likewise, CYP2B6 is polymorphic, and large interindividual differences in enzyme expression have generated significant interest in substrates of CYP2B6 and their associated drug-drug interactions (Shimada et al., 1994; Faucette et al., 2000). Fortunately, the availability of selective probes in recent times has enabled the in vitro assessment of CYP2B6 and CYP2C8 inhibition and P450 reaction-phenotyping (isozyme-mapping) studies. However, with any in vitro study one has to consider the effects of solvents that are used to solubilize substrates and inhibitors. Toward this end, numerous efforts have been made to evaluate the impact of organic solvents on recombinant P450s or P450 activities in HLM (Chauret et al., 1998; Hickman et al., 1998; Busby et al., 1999; Tang et al., 2000). Because the effects of organic solvents on CYP2B6and CYP2C8-mediated enzyme activities in HLM have not been thoroughly assessed, we undertook the task of evaluating the effects of organic solvents on these relatively less-studied enzymes in the present report. In the current communication, an attempt was made to systemArticle, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.016816. ABBREVIATIONS: P450, cytochrome P450; HLM, human liver microsome(s); DMSO, dimethyl sulfoxide; LC/MS/MS, liquid chromatography/ tandem mass spectrometry; CLint, intrinsic clearance. 0090-9556/07/3511-1990–1995$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 11 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 16816/3267964 DMD 35:1990–1995, 2007 Printed in U.S.A. 1990 at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from