This article is available online at http://dmd.aspetjournals.org ABSTRACT:Azamulin [14-O-(5-(2-amino-1,3,4-triazolyl)thioacetyl)-dihydromutilin] is an azole derivative of the pleuromutilin class of antiinfectives. We tested the inhibition potency of azamulin toward 18 cytochromes P450 using human liver microsomes or microsomes from insect cells expressing single isoforms. In a competitive inhibition model, IC 50 values for CYP3A (0.03-0.24 M) were at least 100-fold lower than all other non-CYP3A enzymes except CYP2J2 (ϳ50-fold lower). The IC 50 value with heterologously expressed CYP3A4 was 15-fold and 13-fold less than those of CYP3A5 and CYP3A7, respectively. The reference inhibitor ketoconazole was less selective and exhibited potent inhibition (IC 50 values <10 M) for CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP4F2, and CYP4F12. Inhibition of CYP3A by azamulin appeared sigmoidal and well behaved with the substrates 7-benzyloxy-4-trifluoromethylcoumarin, testosterone, and midazolam. Preincubation of 4.8 M azamulin in the presence of NADPH for 10 min inhibited ϳ95% of testosterone 6␤-hydroxylase activity compared with preincubation in the absence of NADPH. Catalytic activities of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 were unaffected by similar experiments. Incubation of azamulin with heterologously expressed CYP3A4 yielded a type I binding spectrum with a spectral dissociation constant of 3.5 M, whereas no interaction was found with CYP2D6. Azamulin exhibited good chemical stability when stored in acetonitrile for up to 12 days. Aqueous solubility was found to be >300 M. Azamulin represents an important new chemical tool for use in characterizing the contribution of CYP3A to the metabolism of xenobiotics.Along with other experimental approaches, enzyme-selective chemical inhibitors are commonly used in reaction phenotyping studies to determine cytochrome P450 isoform contribution to a metabolic reaction (Clarke, 1998). Chemical inhibitors provide a simpler and more cost-effective alternative to immunoinhibitory antibodies and can be used in cells. However, proper use of chemicals may require foreknowledge of the reaction kinetics under investigation. For example, competitive inhibitors should be used with a substrate concentration near or below the apparent K M . In addition, specificity is often lost when the inhibitor concentration is too high. The effect of microsomal protein concentration and incubation time may also need to be considered, if the inhibitor is also a substrate.Chemicals used as selective inhibitors of CYP3A include triacetyloleandomycin, gestodene, and ketoconazole. Ketoconazole is most widely used, probably because of advantages in potency, selectivity, commercial availability, and ease of use (e.g., preincubation steps are not required) (Maurice et al., 1992;Baldwin et al., 1995;Newton et al., 1995;Bourrie et al., 1996;Sai et al., 2000;Zhang et al., 2002). However, selectivity of ketoconazole for CYP3A is often less than ideal. For example, CYP1B1, CYP2B6, and CYP2C8...

ABSTRACT:We have tested a panel of 29 cDNA-expressed rat and human enzymes with 9 fluorometric substrates to determine the P450 isoform selectivity in the catalysis of the substrates to fluorescent products. The substrates examined were dibenzyl fluorescein, 7-benzyloxyquinoline (BQ), 3-cyano-7-ethoxycoumarin, 3-cyano-7-methoxycoumarin, 7-methoxy-4-trifluoromethylcoumarin, 3- Cytochrome P450 (P450 1 ) enzymes are the principal enzymes that catalyze the metabolism of drugs and other xenobiotics. Analysis of drug metabolism by the cytochrome P450 system has become an important part of the drug discovery/development process, and numerous assay methodologies have been developed. Cytochrome P450 activity assays which have fluorometric endpoints are advantageous in that they offer high sensitivity and are often direct and homogeneous assays. These properties enable testing larger numbers of experimental conditions with cost effective, higher-throughput methodologies. Indeed, fluorometric P450 substrates have been used for many applications in toxicology and drug discovery and development (Ullrich and

1. Recent guidance from the US Food and Drug Administration (USFDA) has advocated testing of time-dependent inhibition of cytochrome P450 (CYP), which can be addressed by performing IC(50) shift as well as K(I)/k(inact) determinations. 2. Direct (IC(50), K(i)) and time-dependent inhibition (IC(50) shift, K(I)/k(inact)) assays were validated in human liver microsomes with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis for the following enzyme/substrate/inhibitor combinations: CYP1A2/phenacetin/alpha-naphthoflavone/furafylline, CYP2C8/amodiaquine/montelukast/gemfibrozil-1-O-beta-glucuronide, CYP2C9/diclofenac/sulfaphenazole/tienilic acid, CYP2C19/S-mephenytoin/S-benzylnirvanol/S-fluoxetine, CYP2D6/dextromethorphan/quinidine/paroxetine, and CYP3A4/midazolam/testosterone/ketoconazole/azamulin/verapamil/diltiazem. IC(50) shift assays were performed with two pre-incubation time points (10 and 30 min) to facilitate k(inact) assay design. 3. Data obtained show good agreement with literature values. For rapid acting inhibitors, such as azamulin/CYP3A4 and tienilic acid/CYP2C9, the IC(50) shifts were similar at both time points suggesting a short maximum pre-incubation time with closely spaced time points for the K(I)/k(inact) assay. Slow acting inhibitors (such as verapamil/CYP3A4 or S-fluoxetine/CYP2C19) showed an increase in IC(50) shift between 10 and 30 min suggesting a longer maximum pre-incubation time with wider spacing of time points for K(I)/k(inact). 4. The two-time point IC(50) shift experiment proved to be an excellent method for the selection of appropriate K(I)/k(inact) assay parameters and is suitable for the routine analysis of P450 inhibition by drug candidates.

Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug‐drug interactions and associated toxicities. We have developed microtiter plate‐based, direct, fluorometric assays for the activities of the principal human drug‐metabolizing enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as well as for CYP2A6, which is an important enzyme in environmental toxicology. These assays are rapid and compatible with existing high‐throughput assay instrumentation. For CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, the potency of enzyme inhibition (IC50) is consistent regardless of the probe substrate or assay method employed. In contrast, CYP3A4 inhibition for an individual inhibitor shows significant differences in potency (>300‐fold) depending on the probe substrate being used. We have investigated these differences through the use of several structurally distinct fluorescent substrates for CYP3A4 and several classical substrate probes (e.g., testosterone, nifedipine, and midazolam), with a panel of known, clinically significant, CYP3A4 inhibitors. The use of multiple probe substrates appears to be needed to characterize the inhibition potential of xenobiotics for CYP3A4.

ABSTRACT:Clevidipine is a short-acting dihydropyridine calcium channel antagonist under development for treatment of perioperative hypertension. Patients treated with clevidipine are likely to be comedicated. Therefore, the potential for clevidipine and its major metabolite H152/81 to elicit drug interactions by induction or inhibition of cytochrome P450 was investigated. Induction of CYP1A2, CYP2C9, and CYP3A4 was examined in primary human hepatocytes treated with clevidipine at 1, 10, and 100 M. Clevidipine was found to be an inducer of CYP3A4, but not of CYP1A2 or CYP2C9, at the 10 M and 100 M concentrations of clevidipine tested. Induction response for CYP3A4 to 100 M clevidipine was approximately 20% of that of the positive control inducer rifampicin. The response of H152/81 was similar. Using cDNA-expressed enzymes, clevidipine inhibited CYP2C9, CYP2C19, and CYP3A4 activities with IC 50 values below 10 M, whereas CYP1A2, CYP2D6, and CYP2E1 activities were not substantially inhibited (IC 50 values >70 M). The K i values for CYP2C9 and CYP2C19 were 1.7 and 3.3 M, respectively, and those for CYP3A4 were 8.3 and 2.9 M, using two substrates, testosterone and midazolam, respectively. These values are at least 10 times higher than the highest clevidipine concentration typically seen in the clinic. Little or no inhibition by H152/81 was found for the enzyme activities mentioned above (IC 50 values > 69 M). The present study demonstrates that it is highly unlikely for clevidipine or its major metabolite to cause cytochrome P450-related drug interactions when used in the dose range required to manage hypertension in humans.Clevidipine (CLE), butyroxymethyl methyl 4-(2Ј, 3Ј-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate ( Fig. 1), is in phase III clinical trials as a rapid acting calcium channel antagonist for intravenous control of blood pressure (Nordlander et al., 2004). Clevidipine is rapidly hydrolyzed and inactivated by esterases in blood and extravascular tissues to its primary metabolite H152/81 (Ericsson et al., 1999(Ericsson et al., , 2000. CLE may have advantages over other antihypertensives. For example, CLE effectively controlled blood pressure in hypertensive patients after undergoing elective coronary bypass grafting, and hemodynamic changes were less pronounced with CLE, compared with sodium nitroprusside (Powroznyk et al., 2003). CLE successfully decreased systemic vascular resistance and mean arterial pressure without changing heart rate, cardiac index, or cardiac filling pressures (Bailey et al., 2002).Clevidipine is a member of the dihydropyridine class of compounds, which have been shown to be inducers and/or inhibitors of cytochromes P450. For example, nifedipine, nicardipine, and isradipine are potent inducers for CYP3A4, CYP2B, and CYP2C in human hepatocyte cultures (Drocourt et al., 2001), and nifedipine induces CYP2C in coronary artery segment endothelial cells (Fisslthaler et al., 2000). In addition, nicardipine, benidipine, manifipine, and barnidipine are potent inhibit...