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ABSTRACT:Some inhibitory agents against CYP2B6 have been reported, but none of these has been extensively characterized or compared with others, as to the potency and selectivity of inhibition toward CYP2B6. The goal of this work was to find a selective and potent chemical in vitro inhibitor toward CYP2B6 using bupropion hydroxylation as a model reaction. At the initial screening of more than 30 substances, ticlopidine, triethylenethiophosphoramide (thioTEPA), metyrapone, xanthate C8, and benzylisothiocyanate displayed IC 50 values of <10 M and were selected for a more detailed analysis. Metyrapone, xanthate C8, and benzylisothiocyanate inhibited several other cytochrome P450 activities rather effectively, some of them even more potently than CYP2B6, and consequently are unsuitable as CYP2B6-selective probes. Ticlopidine and thioTEPA were the most potent inhibitors of bupropion hydroxylation with K i values of 0.2 and 2.8 M, respectively. The inhibition type of ticlopidine was found to be mixed type, with a component of mechanism-based inhibition, whereas thioTEPA inhibited CYP2B6 in a competitive manner. In addition to CYP2B6, ticlopidine also inhibited both mephenytoin 4-hydroxylation (CYP2C19) (IC 50 , 2.7 M) and dextromethorphan O-demethylation (CYP2D6) (IC 50 , 4.4 M). For thioTEPA the next sensitive P450 activity after CYP2B6 was coumarin 7-hydroxylation (IC 50 , 256 M). Thus, although both compounds proved to be relatively potent inhibitors of CYP2B6, thioTEPA was about 2 orders of magnitude more selective than ticlopidine. Thus, thioTEPA is a drug of choice when high CYP2B6 selectivity among major P450 enzymes is required. Ticlopidine is a useful alternative under a controlled experimental setup and when higher potency is needed.

Abstract:The inhibitory effects of five calcium channel blockers (diltiazem, isradipine, mibefradil, nifedipine and verapamil) and three azole antifungal agents (itraconazole, hydroxyitraconazole and ketoconazole) on the a-hydroxylation of midazolam, a probe drug for CYP3A4-mediated interactions in humans, were studied in vifro using human liver microsomes. ICso and K, values were determined for each inhibitor. The kinetics of the formation of a-hydroxymidazolam were best described by simple Michaelis-Menten kinetics. The estimated values of V, , , and K, were 696 pmol min.-' mg-' and 7.46 pmol I-', respectively. All the compounds studied inhibited midazolam a-hydroxylation activity in a concentration-dependent manner, but there were marked differences in their relative inhibitory potency. Ketoconazole was the most potent inhibitor of midazolam a-hydroxylation (ICso 0.12 pmol I-'), being 10 times more potent than itraconazole (1Cs0 1.2 pmol I-'). The inhibitory effect of hydroxyitraconazole (ICso 2.3 pmol I-') was almost as large as that of itraconazole. Among the calcium channel blockers, mibefradil was the most potent inhibitor of the a-hydroxylation of midazolam, with an ICs0 value (1.6 pmol 1-l) similar to that of itraconazole. The other calcium channel blockers were much weaker inhibitors than mibefradil: verapamil exhibited a modest inhibitory effect with an ICso of 23 p o l I-', while isradipine, nifedipine and diltiazem, with IC50 values ranging from 57 to >lo0 pmol I-', were weak inhibitors. This rank order of potency against the a-hydroxylation of midazolam was verified by the K, values. With the exception of diltiazem, these in vitro results conform with the observed interaction potential of these agents with midazolam and many other CYP3A4 substrates in vivo in man.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Selegiline was used as a model compound in a project aimed at comparing, evaluating, and integrating different in vitro approaches for the prediction of cytochrome P450 (P450)-catalyzed hepatic drug metabolism in humans (EUROCYP). Metabolic predictions were generated using homology modeling, cDNA-expressed P450 enzymes, human liver microsomes, primary cultured human hepatocytes, and precision-cut human liver slices. All of the in vitro systems correctly indicated the formation of two dealkylated metabolites, desmethylselegiline and methamphetamine. The metabolic instability of selegiline was demonstrated by all of the in vitro systems studied. Estimates of clearance varied from 16 l/h to 223 l/h. With the exception of one approach, all systems underpredicted the in vivo clearance in humans (236 l/h). Despite this, all approaches successfully classified selegiline as a high clearance compound. Homology modeling suggested the participation of CYP2B6 in the demethylation of selegiline and of CYP2D6 in the depropargylation of the drug. Studies with recombinant expressed enzymes and with human hepatic microsomal fraction supported the involvement of CYP2B6 but not of CYP2D6. These techniques also suggested the involvement of CYP1A2, CYP2C8, and CYP2C19 in the biotransformation of selegiline. In vitro, CYP2B6 was the most active form of P450 involved in selegiline metabolism. Metabolism by several enzymes operating in parallel implies a low interaction potential for the drug. None of the techniques alone was able to predict all aspects of the metabolic and kinetic behavior of selegiline in vivo. However, when used as an integrated package, all significant characteristics were predictable.For more than a decade, in vitro techniques have had a well established role in the early phases of drug development in predicting the pharmacokinetic and metabolic behavior of new chemical entities. When properly used, predictions based on results from in vitro studies will save both development costs and time. In addition, they greatly reduce the number of experimental animals used by the industry in absorption, distribution, metabolism, and excretion studies. Support for the increased utilization of in vitro techniques in drug metabolism studies has come from regulatory authorities (CDER/FDA, 1997). Most of these techniques are easily applicable to high throughput screening, which has further promoted their use recently (Rodrigues, 1997).Several in vitro methods for metabolic predictions are in common use, and even commercially available, but little attention has been paid to the validation of the systems in terms of quality of the predictions obtained and their usefulness in the process of drug development. In particular, comparisons of the data produced by different systems have been scarce. The present study was part of the EUROCYP project within the Biomed2 (Framework IV) program of the European Union. The goals of the project were to evaluate, compare, a...

All sartans except eprosartan have at least some affinity for CYP2C9, but only losartan has an affinity for CYP2C19. Losartan and irbesartan have modest affinity for CYP1A2 and CYP3A4. This would suggest that the theoretical potential for drug interactions is likely to be quite low, with the possible exceptions of losartan and irbesartan for CYP2C9. Based on these findings, further studies on the interaction potential of losartan and irbesartan are warranted.