Cytochrome P450s (CYP) comprise a superfamily of enzymes that catalyzes the oxidation of a wide variety of xenobiotic chemicals, including drugs and carcinogens. [3][4][5] Multiple-drug therapy is a common therapeutic practice, particularly in patients with several diseases or conditions, and many drug-drug interactions involving metabolic inhibition are reported. 6,7) Antifungal drugs, including fluconazole, itraconazole, micafungin, miconazole, and voriconazole ( Fig. 1), are widely used in the treatment of systemic candidal infections and mycoses. The mechanism of action of these antifungal drugs is the inhibition of fungal CYP (14a-sterol demethylase), an enzyme responsible for the conversion of lanosterol to 14a-demethyllanosterol in the ergosterol biosynthetic pathway, 8,9) except that micafungin inhibits 1,3-b-D-glucan synthase, leading to disruption of the growing fungal cell wall and death of the fungal cell.10,11) Because antibiotics, including antifungal drugs, are coadministered in most cases, the possibility of interactions between them and other drugs exists. Recently, we have investigated the effects of antifungal drugs excluding voriconazole on CYP3A4 activity and multidrug resistance protein 1 (MDR1), and found that itraconazole and miconazole, as well as ketoconazole, had greater inhibitory effects on both CYP3A4 metabolic and MDR1 transport activities than fluconazole and micafungin.12) In addition, it has been demonstrated that the K i values of fluconazole and micafungin against nifedipine oxidation activity, a marker enzyme activity of CYP3A4, are 10.7 mM and 17.3 mM, respectively.13) Zhang et al. 14) reported that miconazole competitively inhibits several CYPs, including CYP2C9, CYP2C19, and CYP3A4, with K i values ranging from 0.01 to 7.3 mM. Furthermore, it is likely that fluconazole and voriconazole inhibit CYP2C9, CYP2C19, and CYP3A4. Post-marketing Development Research Center, Fujisawa Pharmaceutical Co., Ltd.; 3-4-7 Doshomachi, Chuo-ku, Osaka 541-8514, Japan: and b Biopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd.; 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan. Received March 22, 2005; accepted May 31, 2005 The effects of five antifungal drugs, fluconazole, itraconazole, micafungin, miconazole, and voriconazole, on cytochrome P450 (CYP) 2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4-hydroxylation, and CYP3A4-mediated nifedipine oxidation activities in human liver microsomes were compared. In addition, the effects of preincubation were estimated to investigate the mechanism-based inhibition. The IC 50 value against tolbutamide hydroxylation was the lowest for miconazole (2.0 m mM), followed by voriconazole (8.4 m mM) and fluconazole (30.3 m mM). Similarly, the IC 50 value against S-mephenytoin 4-hydroxylation was the lowest for miconazole (0.33 m mM), followed by voriconazole (8.7 m mM) and fluconazole (12.3 m mM). On the other hand, micafungin at a concentration of 10 or 25 m mM neither inhibited nor ...

1. The effects of substrate concentration and enzyme source (human liver microsomes and recombinant cytochrome P450s, CYP) on the activation of 7-benzyloxyresorufin O-debenzylation and nifedipine oxidation were investigated. 2. 7-Benzyloxyresorufin O-debenzylase activity in human liver microsomes was inhibited by a monoclonal antibody against CYP2B6 and a polyclonal antibody against CYP3A2 by 53-69 and 19-44%, respectively, suggesting that CYP2B6 and CYP3A4 mainly catalyse 7-benzyloxyresorufin O-debenzylation in human liver microsomes. 3. 7-Benzyloxyresorufin O-debenzylase activity at 0.2-5 micro M substrate concentrations in human liver microsomes was increased by the addition of alpha-naphthoflavone, quinidine, testosterone and progesterone, and the V(max) of 7-benzyloxyresorufin O-debenzylation increased with increasing alpha-naphthoflavone concentrations, whereas the K(m) remained constant. Additionally, 7-benzyloxyresorufin O-debenzylation by recombinant CYP3A4 was increased by the addition of alpha-naphthoflavone, testosterone and progesterone but not by quinidine, whereas no chemicals tested could activate the O-debenzylation of 7-benzyloxyresorufin by CYP2B6. 4. The K(m) for nifedipine oxidation activity by CYP3A4 decreased by the addition of progesterone, whereas the V(max) remained constant. Quinidine and testosterone increased 7-benzyloxyresorufin O-debenzylase and nifedipine oxidase activities, respectively, in human liver microsomes, whereas activation was not observed in CYP3A4. 5. The results suggest that in vitro activation patterns are substrate dependent and that selection of the enzyme source can influence the activation phenomenon.

The eŠects of cyclosporine and tacrolimus on cytochrome P450 (CYP) 1A2-mediated 7-ethoxyresoruˆn O-deethylation, CYP2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4′ -hydroxylation, CYP2D6-mediated debrisoquine 4-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, and CYP3A4-mediated testosterone 6b-hydroxylation activities in human liver microsomes were compared. Cyclosporine and tacrolimus, at concentrations of 0.2 or 2 mM, neither inhibited nor stimulated any of the metabolic activities except for those of CYP3A4. On the other hand, cyclosporine and tacrolimus competitively inhibited CYP3A4-mediated nifedipine oxidation activity, with inhibition constants (K i ) of 1.42 and 0.36 mM, respectively. In addition, 20 mM cyclosporine inhibited CYP2C19 and CYP2D6 activities by 29％ and 30％, respectively. These results suggest that tacrolimus would not cause clinically signiˆcant interactions with other drugs, which are metabolized by CYPs, via the inhibition of hepatic metabolism and that the reason why cyclosporine, but not tacrolimus, has a pharmacokinetic inhibitory eŠect might be that the dosage and/or the unbound concentrations around its metabolic enzymes are higher than those of tacrolimus, rather than the diŠerences in the inhibition potential. Obvious substrate-dependent eŠects on CYP3A4-inhibition potential were not observed.

Antifungal drugs, including fluconazole, itraconazole, micafungin, miconazole, and voriconazole, are widely used in the treatment of systemic candidal infections and mycoses. The mechanism of action of these antifungal drugs except for micafungin is the inhibition of fungal cytochrome P450 (CYP, 14a-sterol demethylase), an enzyme responsible for the conversion of lanosterol to 14a-demethyllanosterol in the ergosterol biosynthetic pathway, 1,2) whereas micafungin inhibits 1,3-b-D-glucan synthase, leading to disruption of growing fungal cell wall and death of the fungal cell. 3,4)Multiple drug therapy is a common therapeutic practice, particularly in patients with several diseases or conditions, and many drug-drug interactions involving metabolic inhibition are being reported. 5,6) Recently, we have demonstrated that itraconazole and miconazole, as well as ketoconazole, had higher inhibitory effects on CYP3A4 metabolic activities than fluconazole and micafungin.7) In addition, the K i values of fluconazole and micafungin against nifedipine oxidation activity, a marker enzyme activity of CYP3A4, have been reported to be 10.7 mM and 17.3 mM, respectively. 8) Zhang et al. 9) reported that miconazole inhibits several CYPs, including CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, with K i values ranging from 0.01 to 7.3 mM. Furthermore, it is likely that fluconazole and voriconazole inhibit CYP2C9, CYP2C19, and CYP3A4.2,10-13) On the other hand, fluconazole and itraconazole are reported to exhibit no inhibition of CYP2E1, whereas the K i value of miconazole against CYP2E1 activity is 4 mM. 14) However, there are few studies comparing the effect of antifungal drugs on the drugmetabolizing activity by human hepatic CYPs, such as CYP1A2, CYP2D6, and CYP2E1, under the same experimental conditions.Many inhibitors are known to be activated metabolically to a reactive intermediate(s) that, in turn, is irreversibly or quasi-irreversibly bound to the enzyme(s).15) For example, some acetylenes, including those synthetic steroids such as gestodene and ethinyl estradiol, cause mechanism-based inactivation of CYP3A4.16) Sorivudine is converted by gut flora to (E)-5-(2-bromovinyl)uracil (BVU), which is metabolized to dihydro-BVU by dihydropyrimidine dehydrogenase (DPD), and the dihydro-BVU binds to DPD itself.17) Numerous laboratories have indicated that these mechanism-based inactivators exhibit preincubation time-dependence of inhibition. [16][17][18][19][20][21][22][23][24][25][26][27] In the present study, we compared the effects of five antifungal drugs on specific activities by CYP1A2, CYP2D6, and CYP2E1 in human liver microsomes under the same experimental conditions. In addition, the effect of preincubation was estimated in order to investigate whether these antifungal drugs are the mechanism-based inhibitors. ) and itraconazole from Janssen-Kyowa (Tokyo, Japan). 7-Ethoxyresorufin, resorufin, debrisoquine sulfate, and chlorzoxazone were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). 4-Hydroxydebrisoquine ...

Triple-stage quadrupole (TSQ) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and ion trap ESI-MS/MScan be used to cleave protonated molecules to produce carbocations and neutral molecules in the positive ion mode. Dissociation products which correspond to protonated forms of neutral fragment molecules can also be trapped and detected. These protonated molecules in turn can cleave via carbocation cleavage, ipso cleavage, onium cleavage or McLafferty or related rearrangements. One can elucidate the structures of metabolites from the differences in m/z ratios of the fragments arising from the original drug compound and its metabolite. This strategy for structural elucidation is further facilitated by estimates of the reactivity of drugs with oxygen diradicals involved in cytochrome P-450 cycles.