Metabolic interactions between mibefradil and HMG-CoA reductase inhibitors: an in vitro investigation with human liver preparations.
(17/1962)
AIMS: To determine the effects of mibefradil on the nletabolism in human liver microsomal preparations of the HMG-CoA reductase inhibitors simvastatin, lovastatin, atorvastatin, cerivastatin and fluvastatin. METHODS: Metabolism of the above five statins (0.5, 5 or 10 microM), as well as of specific CYP3A4/5 and CYP2C8/9 marker substrates, was examined in human liver microsomal preparations in the presence and absence of mibefradil (0.1-50 microM). RESULTS: Mibefradil inhibited, in a concentration-dependent fashion, the metabolism of the four statins (simvastatin, lovastatin, atorvastatin and cerivastatin) known to be substrates for CYP3A. The potency of inhibition was such that the IC50 values (<1 microM) for inhibition of all of the CYP3A substrates fell within the therapeutic plasma concentrations of mibefradil, and was comparable with that of ketoconazole. However, the inhibition by mibefradil, unlike that of ketoconazole, was at least in part mechanism-based. Based on the kinetics of its inhibition of hepatic testosterone 6beta-hydroxylase activity, mibefradil was judged to be a powerful mechanism-based inhibitor of CYP3A4/5, with values for Kinactivation, Ki and partition ratio (moles of mibefradil metabolized per moles of enzyme inactivated) of 0.4 min(-1), 2.3 microM and 1.7, respectively. In contrast to the results with substrates of CYP3A, metabolism of fluvastatin, a substrate of CYP2C8/9, and the hydroxylation of tolbutamide, a functional probe for CYP2C8/9, were not inhibited by mibefradil. CONCLUSION: Mibefradil, at therapeutically relevant concentrations, strongly suppressed the metabolism in human liver microsomes of simvastatin, lovastatin, atorvastatin and cerivastatin through its inhibitory effects on CYP3A4/5, while the effects of mibefradil on fluvastatin, a substrate for CYP2C8/9, were minimal in this system. Since mibefradil is a potent mechanism-based inhibitor of CYP3A4/5, it is anticipated that clinically significant drug-drug interactions will likely ensue when mibefradil is coadministered with agents which are cleared primarily by CYP3A-mediated pathways. (+info)
6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes.
(18/1962)
The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 microM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6beta-hydroxylation (r2=0.9). There was also a strong correlation between 6alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6beta-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 microM concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 microM). This might give an explanation for the limited formation of 6alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver. (+info)
Effect of selected antimalarial drugs and inhibitors of cytochrome P-450 3A4 on halofantrine metabolism by human liver microsomes.
(19/1962)
Halofantrine (HF) is used in the treatment of uncomplicated multidrug-resistant Plasmodium falciparum malaria. Severe cardiotoxicity has been reported to be correlated with high plasma concentrations of HF but not with that of its metabolite N-debutylhalofantrine. The aim of this study was to investigate the effects of other antimalarial drugs and of ketoconazole, a typical cytochrome P-450 3A4 inhibitor, on HF metabolism by human liver microsomes. Antimalarial drug inhibitory effects were ranked as follows: primaquine > proguanil > mefloquine > quinine > quinidine > artemether > amodiaquine. Artemisine, doxycycline, sulfadoxine, and pyrimethamine showed little or no inhibition of HF metabolism. Mefloquine, quinine, quinidine, and ketoconazole used at maximal plasma concentrations inhibited N-debutylhalofantrine formation noncompetitively with Ki values of 70 microM, 49 microM, 62 microM, and 0.05 microM resulting in 7%, 49%, 26%, and 99% inhibition, respectively, in HF metabolism. In conclusion, we showed that quinine and quinidine coadministered with HF might inhibit its metabolism resulting in the potentiation of HF-induced cardiotoxicity in patients. This requires a close monitoring of ECG. For the same reasons, the concomitant administration of HF and ketoconazole must be avoided. By contrast, none of the other antimalarials studied inhibited HF metabolism and, by extrapolation, cytochrome P-450 3A4 activity. (+info)
Effect of inhibitor depletion on inhibitory potency: tight binding inhibition of CYP3A by clotrimazole.
(20/1962)
The purpose of this work was to evaluate the effect of mutual unbound inhibitor and unbound enzyme depletion on the potency of three antifungal cytochrome P-450 (CYP)3A inhibitors with over 1000-fold range in enzyme affinity. Incubations were performed with human liver microsomal protein concentrations that varied from 25 to 1000 microg/ml. The effect of each inhibitor was evaluated using midazolam as a CYP3A probe. Clotrimazole was found to be a tight binding inhibitor of CYP3A with a Ki of 250 pM. Analysis of percent inhibition data by stepwise linear regression for the matrix of inhibitor and enzyme concentrations used showed that protein concentrations predicted the percent inhibition by clotrimazole (r2 = 0.60, p <.001). When clotrimazole concentrations were added to the model, the r2 improved to 0.81, p =.003. Clotrimazole concentrations alone were not a significant predictor of percent inhibition (r2 = 0. 21, p =.08). For ketoconazole, protein concentrations provided a weak prediction of the percent inhibition (r2 = 0.39, p =.006). Conversely, ketoconazole concentrations alone were a good predictor of percent inhibition (r2 = 0.55, p <.001). In contrast to results with clotrimazole and ketoconazole, percent inhibition by fluconazole was not dependent on protein concentrations (r2 = 0.06, p =.39). We conclude that microsomal inhibitory potency can be affected by incubation conditions that deplete the unbound concentration of inhibitor available to the enzyme. This may introduce serious error into a quantitative prediction of an in vivo drug-drug interaction based on an in vitro derived Ki value. (+info)
Methadone N-demethylation in human liver microsomes: lack of stereoselectivity and involvement of CYP3A4.
(21/1962)
AIMS: To investigate the kinetics of CYP-mediated N-demethylation of methadone in human liver microsomes, and examine the role of stereoselectivity and CYP isoforms involved. METHODS: The kinetics of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) formation via N-demethylation of rac-, (R)- and (S)-methadone in human liver microsomes prepared from six liver samples were determined by h.p.l.c., and inhibition of metabolic function was studied using isoform-specific chemical inhibitors and monoclonal antibodies. Microsomes containing expressed CYP3A4, CYP2D6 and CYP2C19 were also used to examine the formation of EDDP. RESULTS: The V max, Km, and CLint values for the formation of EDDP from rac-, (R)- and (S)-methadone were in the ranges of 20-77 nmol mg-1 protein h-1, 125-252 microm, and 91-494 ml h-1 g-1 protein. Km and CLint values for (R)- and (S)-methadone were not statistically significantly different (P >0.05), while V max values for (S)-methadone were 15% (P=0.045) lower than for (R)-methadone. Expressed CYP3A4 and CYP2C19 showed similar reaction rates for both (R)- and (S)-methadone, while CYP2D6 did not catalyse this reaction. Selective chemical inhibitors of CYP3A (troleandomycin, ketoconazole) and monoclonal human CYP3A4 antibodies significantly inhibited (P<0.05) the formation of EDDP in a concentration dependent manner by up to 80%. Sulphaphenazole (CYP2C9) also significantly inhibited (P<0.05) EDDP formation (range 14-25%). There were no statistically significant differences in the inhibition observed between the three substrates. Selective inhibitors of CYP1A2 (furafylline), CYP2A6 (coumarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (quinidine) and CYP2E1 (diethyldithiocarbamic acid sodium salt and monoclonal human CYP2E1 antibodies) had no significant (P >0.05) effect. CONCLUSIONS: The N-demethylation of methadone in human liver microsomes is not markedly stereoselective, and is mediated mainly by CYP3A4 with the possible involvement of CYP2C9 and CYP2C19. Thus, the large interindividual variation reported for methadone pharmacokinetics may be due to variability in the expression of these CYP isoforms, and the reported stereoselectivity in the systemic clearance of methadone in vivo is not due to stereoselectivity in N-demethylation. (+info)
Ticlopidine decreases the in vivo activity of CYP2C19 as measured by omeprazole metabolism.
(22/1962)
AIMS: To examine the effect of ticlopidine administration on the activities CYP2C19 and CYP3 A in vivo using omeprazole as a model substrate. METHODS: A single dose of 40 mg omeprazole was administered orally with or without ticlopidine (300 mg daily for 6 days) to six Japanese extensive metabolisers with respect to CYP2C19. Blood samples were taken for the measurement of plasma concentrations of omeprazole, 5-hydroxyomeprazole and omeprazole sulphone. RESULTS: Ticlopidine administration increased omeprazole Cmax (1978+/-859/ 3442+/-569 (control phase/ticlopidine phase, nm )) and decreased the oral clearance of omeprazole (CL/F; 25.70+/-16. 17/10.76+/-1.16 (control phase/ticlopidine phase, l h-1 )) significantly. The 5-hydroxyomeprazole to omeprazole AUC ratio (0. 817+/-0.448/0.236+/-0.053 (control phase/ticlopidine phase)) and the 5-hydroxyomeprazole to omeprazole sulphone AUC ratio (1.114+/-0. 782/0.256+/-0.051 (control phase/ticlopidine phase)) were decreased significantly after ticlopidine administration. The decrease in omeprazole CL/F and the 5-hydroxyomeprazole to omeprazole AUC ratio correlated significantly with their respective absolute values when the drug was given alone. The decrease in CL/F following ticlopidine administration correlated with that in the 5-hydroxyomeprazole to omeprazole AUC ratio. CONCLUSIONS: These findings suggest that ticlopidine inhibited the in vivo activity of CYP2C19, but not, or to a lesser extent CYP3 A4, and that the magnitude of inhibition by ticlopidine is related to the in vivo activity of CYP2C19 before inhibition. (+info)
Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil.
(23/1962)
AIMS: The present study was conducted to evaluate metabolism of the enantiomers of verapamil and norverapamil using a broad range of cytochrome P450 isoforms and measure the kinetic parameters of these processes. METHODS: Cytochrome P450 cDNA-expressed cells and microsomes from a P450-expressed lymphoblastoid cell line were incubated with 40 microm concentrations of R- or S-verapamil and R- or S-norverapamil and metabolite formation measured by h.p.l.c. as an initial screening. Those isoforms exhibiting substantial activity were then studied over a range of substrate concentrations (2.5-450 microm ) to estimate the kinetic parameters for metabolite formation. RESULTS: P450s 3A4, 3A5, 2C8 and to a minor extent 2E1 were involved in the metabolism of the enantiomers of verapamil. Estimated Km values for the production of D-617 and norverapamil by P450 s 3A4 and 3A5 were similar (range=60-127 microm ) regardless of the enantiomer of verapamil studied while the Vmax estimates were also similar (range=4-8 pmol min-1 pmol-1 P450). Only nominal production of D-620 by these isoforms was noted. Interestingly, P450 2C8 readily metabolized both S- and R-verapamil to D-617, norverapamil and PR-22 with only slightly higher Km values than noted for P450s 3A4 and 3A5. However, the Vmax estimates for P450 2C8 metabolism of S- and R-verapamil were in general greater (range=8-15 pmol min-1 pmol-1 P450) than those noted for P450 s 3A4 and 3A5 with preference noted for metabolism of the S-enantiomer. Similarly, P450 s 3A4, 3A5 and 2C8 also mediated the metabolism of the enantiomers of norverapamil with minor contributions by P450 s 2D6 and 2E1. P450s 3A4 and 3A5 readily formed the D-620 metabolite with generally a lower Km and higher Vmax for S-norverapamil than for the R-enantiomer. In contrast, P450 2C8 produced both the D-620 and PR-22 metabolites from the enantiomers of norverapamil, again with stereoselective preference seen for the S-enantiomer. CONCLUSIONS: These results confirm that P450s 3A4, 3A5 and 2C8 play a major role in verapamil metabolism and demonstrate that norverapamil can also be further metabolized by the P450s. (+info)
Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles.
(24/1962)
The contributions of specific human liver cytochrome P-450 (CYP) enzymes to the activation, via 4-hydroxylation, of the oxazaphosphorine anticancer prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) were investigated. Analysis of a panel of 15 human P-450 cDNAs expressed in human lymphoblasts and/or baculovirus-infected insect cells (Supersomes) demonstrated that CYPs 2A6, 2B6, 3A4, 3A5, and three CYP2C enzymes (2C9, 2C18, 2C19) exhibited significant oxazaphosphorine 4-hydroxylase activity, with 2B6 and 3A4 displaying the highest activity toward CPA and IFA, respectively. CYP2B6 metabolized CPA at a approximately 16-fold higher in vitro intrinsic clearance (apparent Vmax/Km) than IFA, whereas 3A4 demonstrated approximately 2-fold higher Vmax/Km toward IFA. A relative substrate-activity factor (RSF)-based method was developed to calculate the contributions of individual P-450s to total human liver microsomal metabolism based on cDNA-expressed P-450 activity data and measurements of the liver microsomal activity of each P-450 form. Using this method, excellent correlations were obtained when comparing measured versus predicted (calculated) microsomal 4-hydroxylase activities for both CPA (r = 0. 96, p <.001) and IFA (r = 0.90, p <.001) in a panel of 17 livers. The RSF method identified CYP2B6 as a major CPA 4-hydroxylase and CYP3A4 as the dominant IFA 4-hydroxylase in the majority of livers, with CYPs 2C9 and 2A6 making more minor contributions. These predicted P-450 enzyme contributions were verified using an inhibitory monoclonal antibody for 2B6 and the P-450 form-specific chemical inhibitors troleandomycin for 3A4 and sulfaphenazole for 2C9, thus validating the RSF approach. Finally, Western blot analysis using anti-2B6 monoclonal antibody demonstrated the presence of 2B6 protein at a readily detectable level in all but one of 17 livers. These data further establish the significance of human liver CYP2B6 for the activation of the clinically important cancer chemotherapeutic prodrug CPA. (+info)