Identification and characterization of human cytochrome P450 isoforms interacting with pimozide

Using human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP450) isoforms, we identified the major route of pimozide metabolism, the CYP450 isoforms involved, and documented the inhibitory effect of pimozide on CYP450 isoforms. Pimozide was predominantly N-dealkylated to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI). The formation rate of DHPBI showed biphasic kinetics in HLMs, which suggests the participation of at least two activities. These were characterized as high-affinity (K(m1) and Vmax1) and low-affinity (K(m2) and Vmax2) components. The ratio of Vmax1 (14 pmol/min/mg protein)/K(m1) (0.73 microM) was 5.2 times higher than the ratio of Vmax2 (244 pmol/min/mg protein)/K(m2) (34 microM). K(m2) was 91 times higher than K(m1). The formation rate of DHPBI from 25 microM pimozide in nine human livers correlated significantly with the catalytic activity of CYP3A (Spearman r = 0.79, P = .028), but not with other isoforms. Potent inhibition of DHPBI formation from 10 microM pimozide was observed with ketoconazole (88%), troleandomycin (79%), furafylline (48%) and a combination of furafylline and ketoconazole (96%). Recombinant human CYP3A4 catalyzed DHPBI formation from 10 microM pimozide at the highest rate (V = 2.2 +/- 0.89 pmol/min/pmol P450) followed by CYP1A2 (V = 0.23 +/- 0.08 pmol/min/pmol P450), but other isoforms tested did not. The K(m) values derived with recombinant CYP3A4 and CYP1A2 were 5.7 microM and 36.1 microM, respectively. Pimozide itself was a potent inhibitor of CYP2D6 in HLMs when preincubated for 15 min (Ki = 0.75 +/- 0.98 microM) and a moderate inhibitor of CYP3A (Ki = 76.7 +/- 34.5 microM), with no significant effect on other isoforms tested. Our results suggest that pimozide metabolism is catalyzed mainly by CYP3A, but CYP1A2 also contributes. Pimozide metabolism is likely to be subject to interindividual variability in CYP3A and CYP1A2 expression and to drug interactions involving these isoforms. Pimozide itself may inhibit the metabolism of drugs that are substrates of CYP2D6.     

Reappraisal of human CYP isoforms involved in imipramine N-demethylation and 2-hydroxylation: a study using microsomes obtained from putative extensive and poor metabolizers of S-mephenytoin and eleven recombinant human CYPs

Cytochrome P450 (CYP) involved in the two major pathways of imipramine (IMI) was reappraised using human liver microsomes phenotyped for S-mephenytoin 4'-hydroxylation in vitro and 11 recombinant human CYP isoforms. Individual Eadie-Hoffstee plots for IMI N-demethylation and 2-hydroxylation showed a monophasic profile in microsomes obtained from three putative S-mephenytoin poor metabolizer (PM) livers, whereas the plots gave a biphasic relationship (except for one case in 2-hydroxylation) in those from the three extensive metabolizer (EM) livers. Effects of CYP-selective inhibitor/substrate probes on the two metabolic reactions were examined at the two IMI concentrations (2 and 400 microM) with microsomes obtained from the two PM and three EM livers. S-mephenytoin inhibited IMI N-demethylation by 50% at the low concentration in microsomes from the EM livers with no discernible effect on this pathway in those from the PM livers. Furafylline inhibited the N-demethylation by about 60% at the low and high substrate concentrations in microsomes from both the EM and PM livers. Quinidine abolished the 2-hydroxylation at the low and high concentrations in microsomes from both the EM and the PM livers. Among the recombinant human CYPs, CYP2C19, 2C18, 2D6, 1A2, 3A4 and 2B6 in rank order catalyzed the N-demethylation, whereas CYP2D6, 2C19, 1A2, 2C18 and 3A4 catalyzed the 2-hydroxylation. The Km values obtained from recombinant CYP2C19 and 1A2 approximated those of the high- and low-affinity components from human liver microsomes for IMI N-demethylation, respectively. For IMI 2-hydroxylation, the respective Km values obtained from recombinant CYP2D6 and 2C19 were close to those of the high- and low-affinity components from human liver microsomes. Our human liver microsomal study using the near-therapeutic IMI concentration (2 microM) suggests that 1) CYP2C19 and 1A2 are involved in the N-demethylation and the 2-hydroxylation is mediated exclusively by CYP2D6 and partially by CYP2C19 in the EM livers, and 2) CYP1A2 and 2D6 play a major role in IMI N-demethylation and 2-hydroxylation, respectively, in the PM livers. Our recombinant human CYP isoform study, in general, supports this conclusion.     

Regioselectivity and substrate concentration-dependency of involvement of the CYP2D subfamily in oxidative metabolism of amitriptyline and nortriptyline in rat liver microsomes

Kinetic analysis of the metabolism of amitriptyline and nortriptyline using liver microsomes from Wister rats showed that more than one enzyme was involved in each reaction except for monophasic amitriptyline N-demethylation. The Vmax values particularly in the high-affinity sites for E-10-hydroxylation of both drugs were larger than those for Z-10-hydroxylations. Their E- and E-10-hydroxylase activities in Dark-Agouti rats, which are deficient for CYP2D1, were significantly lower than those in Wistar rats at a lower substrate concentration (5 microM). The strain difference was reduced at a higher substrate concentration (500 microM). A similar but a smaller strain difference was also observed in nortriptyline N-demethylase activity, and a pronounced sex difference (male > female) was observed in N-demethylation of both drugs in Wistar and Dark-Agouti rats. The reactions with the strain difference were inhibited concentration-dependently by sparteine, a substrate of the CYP2D subfamily, and an antibody against a CYP2D isoenzyme. The profiles of these decreased metabolic activities corresponded to that of the lower metabolic activities in Dark-Agouti rats. These results indicated that a cytochrome P450 isozyme in the CYP2D subfamily was involved in E- and Z-10-hydroxylations of amitriptyline and nortriptyline in rat liver microsomes as a major isozyme in a low substrate concentration range. It seems likely that the CYP2D enzyme contributes to nortriptyline N-demethylation.     

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

Metabolism of clozapine by cDNA-expressed human cytochrome P450 enzymes

The metabolism of clozapine was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6, and 2E1. CYP1A2, 3A4, 2C9, 2C19, and 2D6 were able to N-demethylate clozapine. N-Oxide formation was exclusively catalyzed by CYP3A4. CYP2E1 did not metabolize clozapine. With regard to quantitative relationships, CYP1A2, 2C9, 2C19, and 2D6 displayed KM values ranging from 13 to 25 microM, whereas CYP3A4 had a 5-10 times higher KM value. CYP2C19 and 2D6 had the highest Vmax values (149-366 mol/hr/mol CYP). Taking into account the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that at therapeutic concentrations CYP2C19 and CYP3A4 each accounted for about 35% of the metabolism. At toxic concentrations, the relative importance of CYP3A4 increased.     

Identification of amino acid substitutions that confer a high affinity for sulfaphenazole binding and a high catalytic efficiency for warfarin metabolism to P450 2C19

Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, KS, of <1 microM. P450 2C19 is not affected by SFZ at concentrations up to 100 microM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227-338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (KS approximately 4 microM) that was not seen when a shorter fragment of 2C9 was substituted (227-282). However, replacement of amino acids 283-338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a KS < 10 microM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (KS < 5 microM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower KM for (S)-warfarin metabolism (<10 microM) typical of 2C9.     

Female exposure to high G: chronic adaptations of cardiovascular functions

The substrate structure-activity relationships described for the major human drug-metabolizing cytochrome P450 (P450 or CYP) enzymes suggest that the H1 receptor antagonist terfenadine could interact with CYP2D6 either as a substrate or as an inhibitor, in addition to its known ability to act as a substrate for CYP3A4. Based on this substrate structure-activity relationship, computer modeling studies were undertaken to explore the likely interactions of terfenadine with CYP2D6. An overlay of terfenadine and dextromethorphan, a known substrate of CYP2D6, showed that it was possible to superimpose the site of hydroxylation (t-butyl group) and the nitrogen atom of terfenadine with similar regions in dextromethorphan. These observations were substantiated by the ease of docking of terfenadine into a protein model of CYP2D6. Experimentally, terfenadine inhibited CYP2D6 activity in human liver microsomes with an IC50 of 14-27 microM, depending on the CYP2D6 substrate used. The inhibition of CYP2D6 was further defined by determining the Ki for terfenadine against bufuralol 1'-hydroxylase activity in four human livers. Terfenadine inhibited bufuralol 1'-hydroxylase activity with a Ki of approximately 3.6 microM. The formation of the hydroxylated metabolite (hydroxyterfenadine) in microsomes prepared from human liver and specific P450 cDNA-transfected B lymphoblastoid cells indicated that only CYP2D6 and CYP3A4 were involved in this transformation. As expected, the rate of formation was greatest with CYP3A4 (Vmax = 1257 pmol/min/nmol of P450), with CYP2D6 forming the metabolite at a 6-fold lower rate (Vmax = 206 pmol/min/nmol of P450). The two enzymes had similar KM values (9 and 13 microM, respectively). These data indicate that, as predicted from modeling studies, terfenadine has the structural features necessary for interaction with CYP2D6.     

Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricyclic structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver microsomes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoconazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies directed against CYP1A1/2 and CYP3A4 inhibited the demethylation reaction whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did not show any inhibitory effects. When a panel of microsomes prepared from human B-lymphoblastoid cells that expressed specific human cytochrome P450 isoforms were used, only microsomes containing cytochromes P4501A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylation catalyzed by these recombinant cytochromes P450 can be completely inhibited with selective inhibitors at concentrations as low as 1 to 20 microM. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 beta-hydroxylation (3A4) but not with dextromethorphan O-demethylation (2D6) in human liver microsomes. To further determine the involvement of cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes from a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occurred in the incubation with this microsome. These results suggested that cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-demethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenzaprine metabolism in human liver microsomes. Due to the minimum involvement of CYP2D6 in the vitro metabolism of cyclobenzaprine, the polymorphism of cytochrome P4502D6 in man should not be of muci concern in the clinical use of cyclobenzaprine.     

MR imaging of rabbit hip cartilage with a clinical imager and specifically designed surface coils

To study whether an in-vitro model for three different genotypes of human CYP2C9*3 polymorphism would be useful for predicting the in-vivo kinetics of (S)-warfarin in patients with the corresponding genotypes, the intrinsic clearance (Cl(int) or Vmax/Km) for (S)-warfarin 7-hydroxylation obtained from recombinant human CYP2C9*1 [wild-type (wt)] and CYP2C9*3 (Leu359/Leu) expressed in yeast and the mixture of equal amounts of these were compared with the in-vivo unbound oral CI (CI(po,u)) of (S)-warfarin obtained from 47 Japanese cardiac patients with the corresponding CYP2C9 genotypes. The in-vitro study revealed that the recombinant CYP2C9*1 (wt/wt), 2C9*3 (Leu359/Leu) and their mixture (Ile359/Leu) possessed a mean Km of 2.6, 10.4 and 6.6 microM and Vmax of 280, 67 and 246 pmol/min/nmol P450, respectively. Thus, the mean in-vitro Cl(int) obtained from recombinant CYP2C9*3 (Leu359/Leu) and the mixture (Ile359/Leu) of 2C9*3 and 2C9*1 were 94% and 65% lower than that obtained from CYP2C9*1 (wt/wt) (6.7 versus 38 versus 108 ml/min/micromol P450, respectively). The in-vivo study showed that the median Cl(po,u) for (S)-warfarin obtained from patients with homozygous (Leu359/Leu, n = 1) and heterozygous (Ile359/Leu, n = 4) CYP2C9*3 mutations were reduced by 90% (62 ml/min) and 66% (212 ml/min, P < 0.05) compared with that obtained from those with homozygous 2C9*1 (625 ml/min, n = 42). Consequently, there was a significant correlation (r = 0.99, P < 0.05) between the in-vitro Cl(int) for (S)-warfarin 7-hydroxylation and the in-vivo Cl(po,u) for (S)-warfarin in relation to the CYP2C9*3 polymorphism. In conclusion, the in-vitro model for human CYP2C9*3 polymorphism using recombinant cytochrome P450 proteins would serve as a useful means for predicting changes in in-vivo kinetics for (S)-warfarin and possibly other CYP2C9 substrates in relation to CYP2C9*3 polymorphism.     

Identification of the human liver cytochrome P450 enzymes involved in the in vitro metabolism of a novel 5-lipoxygenase inhibitor

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) forms involved in the oxidative metabolism of [14C]ABT-761 and its N-dehydroxylated metabolite, [14C]ABT-438, by human liver microsomes. The two compounds were metabolized by parallel pathways, to form the corresponding methylene bridge hydroxy metabolites. There was no evidence of sulfoxidation and/or ring hydroxylation. Over the ABT-761 and ABT-438 concentration ranges studied (1-300 microM), the rate of NADPH-dependent hydroxylation was linear with respect to substrate concentration ([S]) and did not conform to saturable Michaelis-Menten kinetics. Under these conditions ([S] < KM), the intrinsic clearance (Vmax/KM) of ABT-438 was 10-fold higher than that of ABT-761 (1.7 +/- 0.8 vs. 0.17 +/- 0.06 microl/min/mg, mean +/- SD, N = 3 livers). The hydroxylation of both compounds was shown to be highly correlated (r = 0.83, p < 0.01, N = 11 different human livers) with CYP3A-selective erythromycin N-demethylase activity, and the correlation between ABT-761 hydroxylation and tolbutamide hydroxylase (CYP2C9-selective) activity (r = 0.63, p < 0.05, N = 10) was also statistically significant. Ketoconazole (2.0 microM), a CYP3A-selective inhibitor, inhibited the hydroxylation of both compounds by 53-67%, and sulfaphenazole (CYP2C9-selective) decreased activity by 10-20%. By comparison, alpha-naphthoflavone, a known activator of CYP3A, stimulated the hydroxylation of ABT-761 (8-fold) and ABT-438 (4-fold). In addition, the abundance-normalized rates of cDNA-expressed CYP-dependent metabolism indicated that hydroxylation was largely mediated (66-86%) by CYP3A(4). Therefore, it is concluded that the hydroxylation of ABT-761 and ABT-438 (相似文献   

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human cytochrome P4501A1, P4501A2 and P4501B1

While the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by N-hydroxylation has been well documented, the relative roles of the human cytochrome P450 (CYP) enzymes that catalyze this reaction have not been established. Previous studies indicated that the mutagenic activation product, 2-hydroxyamino-PhIP (N2-OH-PhIP), is produced primarily by CYP1A2, and to a lesser extent by CYP1A1. We recently reported that human CYP1B1 also produces N2-OH-PhIP (Carcinogenesis, 18, 1793-1798, 1997). In the present study, we examined PhIP metabolism by microsomes containing recombinant human CYP1A1, 1A2 or 1B1 expressed in Sf9 insect cells and compared the kinetic values for PhIP metabolite formation. PhIP metabolites were analyzed by high pressure liquid chromatography with fluorescence and absorbance detection. Vmax values for N2-OH-PhIP formation were 90, 16 and 0.2 nmol/min/nmol P450, and the apparent Km values were 79, 5.1 and 4.5 microM for human CYP1A2, 1A1 and 1B1, respectively. The non-mutagenic metabolite, 4'-hydroxy-PhIP, was also formed by all three CYP enzymes with Vmax values of 1.5, 7.8 and 0.3 nmol/ min/nmol P450 and apparent Km values of 43, 8.2 and 2.2 microM for human CYP1A2, 1A1 and 1B1, respectively. Although the Vmax for N2-OH-PhIP production was highest for CYP1A2, the catalytic efficiency (Vmax/Km) of CYP1A1 was greater than that of CYP1A2. These results suggest that, for humans, extrahepatic CYP1A1 may be more important than previously thought for the metabolic activation of the dietary carcinogen PhIP.     

The validity of the California Q-set Alexithymia Prototype

Classic antihistamines, namely diphenhydramine, chlorpheniramine, clemastine, perphenazine, hydroxyzine, and tripelennamine, share structural features with substrates and inhibitors of the polymorphic cytochrome P450 (CYP) isozyme CYP2D6. Therefore, the current study was undertaken to characterize the in vitro inhibition of CYP2D6 by these commonly used, histamine H1 receptor antagonists. Microsomal incubations were performed using bufuralol as a specific CYP2D6 substrate and microsomes derived from human cells transfected with CYP2D6 cDNA. Reaction velocities were assessed in the absence and presence of antihistamines (20 microM) at 11 substrate concentrations (1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, and 100 microM), as well as at three nonsaturating substrate concentrations (2.5, 5, and 20 microM) and three inhibitor concentrations (5, 20, and 50 microM). In the presence of all antihistamines, the Vmax and KM of bufuralol 1'-hydroxylation were significantly altered, compared with the uninhibited reaction (p < 0.05). Lineweaver-Burke plots suggested competitive inhibition of the reaction by diphenhydramine and mixed inhibition by all other antihistamines tested. Diphenhydramine and chlorpheniramine, with estimated Ki values of approximately 11 microM, were the weakest inhibitors of CYP2D6 in vitro. Whereas tripelennamine, promethazine, and hydroxyzine were similar in their inhibitory capacities (Ki approximately 4-6 microM), clemastine appeared to be significantly more potent, with a Ki of approximately 2 microM. These data demonstrate that classic histamine H1 receptor antagonists, available in over-the-counter preparations, inhibit CYP2D6 in vitro. Furthermore, the CYP2D6-inhibitory concentrations of these antihistamines are in the range of their expected hepatic blood concentrations, suggesting that, under specific circumstances, clinically relevant interactions between classic antihistamines and CYP2D6 substrates might occur.     

Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to the CYP52 multigene family occurring in the alkane-assimilating yeast Candida maltosa, have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the p450 proteins and the corresponding C. maltosa NADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs in Saccharomyces cerevisiae, purified, and reconstituted to active monooxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity. P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding of n-hexadecane (Ks= 26 micron) and n-octadecane (Ks = 27 microM) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid (Ks = 127 microM) and myristic acid (Ks = 134 microM) as substrates. These substrate selectivities were further substantiated by kinetic parameters, determined for n-alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured for n-hexadecane hydroxylation to 1-hexadecanol by P450Cm1 (Vmax = 27 microM x min-1, Km = 54 microM) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 (Vmax = 30 microM x min-1, Km = 61 microM). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for either n-alkanes or fatty acids.     

Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.     

Metabolism of epinastine, a histamine H1 receptor antagonist, in human liver microsomes in comparison with that of terfenadine

Epinastine is a non-sedative second-generation antiallergic drug, like terfenadine. In the present study, the metabolism of epinastine in human liver microsomes was investigated and compared with that of terfenadine. Terfenadine was extensively metabolized to terfenadine acid with a Km value of 1.78 microM, a Vmax value of 173.8 pmol/min/mg and a metabolic clearance (Vmax/Km) of 103.9. Epinastine, in contrast, was poorly metabolized by microsomes from the same source with a high Km value of 232 microM. Metabolic clearance of epinastine was only 0.832, which was lower by three orders of magnitude than that of terfenadine. Studies with microsomes expressing recombinant cytochrome P450 (CYP) species revealed that the CYP isoforms responsible for epinastine metabolism are CYP3A4, 2D6 and (to a minor extent) 2B6. Epinastine and terfenadine had no effect on CYP1A2 (theophylline 1-demethylation), 2C8/9 (tolbutamide hydroxylation) or 2E1 (chlorzoxazone 6-hydroxylation) activity, but weakly inhibited CYP2D6 (debrisoquine 4-hydroxylation) activity. CYP3A4 (testosterone 6 beta-hydroxylation) activity was strongly inhibited by terfenadine with a Ki value of 25 microM, whereas epinastine had no effect at up to 100 microM. Thus, epinastine is very poorly metabolized compared to terfenadine in human liver microsomes and does not inhibit CYP3A4 activity in vitro, unlike terfenadine.     

Identification of human cytochrome P450 isoforms involved in the 3-hydroxylation of quinine by human live microsomes and nine recombinant human cytochromes P450

Studies using human liver microsomes and nine recombinant human cytochrome P450 (CYP) isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) were performed to identify the CYP isoform(s) involved in the major metabolic pathway (3-hydroxylation) of quinine in humans. Eadie-Hofstee plots for the formation of 3-hydroxyquinine exhibited apparently monophasic behavior for all of the 10 different microsomal samples studies. There was interindividual variability in the kinetic parameters, as follows: 1.8-, 3.2- and 3.5-fold for K(m) Vmax and Vmax/K(m), respectively. The mean +/- S.D. values for K(m), Vmax and Vmax/K(m) were 106.1 +/- 19.3 microM, 1.33 +/- 0.48 nmol/mg protein/min and 12.8 +/- 5.1 microliters/mg protein/min, respectively. With 10 different human liver microsomes, the relationships between the 3-hydroxylation of quinine and the metabolic activities for substrates of the respective CYP isoforms were evaluated. The 3-hydroxylation of quinine showed an excellent correlation (r = 0.986, P < .001) with 6 beta-hydroxylation of testosterone, a marker substrate for CYP3A4. A significant correlation (r = 0.768, P < .01) between the quinine 3-hydroxylase and S-mephenytoin 4'-hydroxylase activities was also observed. However, no significant correlation existed between the 3-hydroxylation of quinine and the oxidative activities for substrates for CYP1A2 (phenacetin), 2C9 (diclofenac), 2D6 (desipramine) and 2E1 (chlorzoxazone). Ketoconazole and troleandomycin (inhibitors of CYP3A4) inhibited the 3-hydroxylation of quinine by human liver microsomes with respective mean IC50 values of 0.026 microM and 28.9 microM. Anti-CYP3A antibodies strongly inhibited quinine 3-hydroxylation, whereas weak inhibition was observed in the presence of S-mephenytoin or anti-CYP2C antibodies. Among the nine recombinant human CYP isoforms, CYP3A4 exhibited the highest catalytic activity with respect to the 3-hydroxylation of quinine, compared with the minor activity of CYP2C19 and little discernible or no effect of other CYP isoforms. Collectively, these data suggest that the 3-hydroxylation of quinine is mediated mainly by CYP3A4 and to a minor extent by CYP2C19. Other CYP isoforms used herein appear to be of negligible importance in this major pathway of quinine in humans.     

Human buprenorphine N-dealkylation is catalyzed by cytochrome P450 3A4

Buprenorphine (BN) is a thebaine derivative with analgesic properties. To identify and characterize the cytochrome P450 (CYP) enzyme(s) involved in BN N-dealkylation, in vitro studies using human liver microsomes and recombinant human CYP enzymes were performed. Norbuprenorphine formation from BN was measured by a simple HPLC-UV assay method, without extraction. The BN N-dealkylation activities in 10 human liver microsomal preparations were strongly correlated with microsomal CYP3A-specific metabolic reactions, i.e. triazolam 1'-hydroxylation (r = 0.954), midazolam 1'-hydroxylation (r = 0.928), and testosterone 6beta-hydroxylation (r = 0.897). Among the eight recombinant CYP enzymes studied (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), only CYP3A4 could catalyze BN N-dealkylation. The apparent KM value for recombinant CYP3A4 was similar to that for human liver microsomes (23.7 vs. 39.3 +/- 9.2 microM). The demonstration of BN N-dealkylation by recombinant CYP3A4 and the agreement in the affinities (apparent KM values) of human liver microsomes and recombinant CYP3A4 provide the most supportive evidence for BN N-dealkylation being catalyzed by CYP3A4.     

Novobiocin inhibits both UDP-glucuronosyltransferase and cytochrome P450-mediated enzyme activities in pig liver microsomes

The effects of novobiocin (range 0.0125-2 mmol/L) on the hydroxylation of testosterone, the N-demethylation of erythromycin, and the glucuronidation of alpha-naphthol and paracetamol were studied using pig hepatic microsomes, pooled from five animals. The final concentrations of these substrates in the incubation mixtures were selected to meet Vmax conditions. Novobiocin caused a concentration-dependent inhibition of the glucuronidation of paracetamol; the formation of alpha-naphthol-glucuronide was reduced to a lesser degree. These results confirm and extend earlier findings in laboratory animal species that novobiocin inhibits UDP-glucuronosyltransferases (UDPGTs). Moreover, novobiocin strongly inhibited 6 beta-hydroxylation of testosterone. The microsomal N-demethylation of erythromycin and hydroxylation of testosterone at the 15 alpha position were less affected by novobiocin. These results suggest that novobiocin inhibits not only UDPGTs, but also cytochrome P450 (CYP) enzyme activities, probably those belonging to the CYP3A subfamily. More research is needed to reveal which CYPs and UDPGTs are affected by novobiocin in vivo, in order to improve the understanding the probably the predictability of potential drug interactions with this antibiotic.     

Kinetic analysis of propranolol-induced impairment of its own metabolism in rats

The effect of repetitive oral administration of propranolol (100 mg kg-1 day-1, 5 days) on the kinetics of liver microsomal propranolol metabolism was investigated in the rat. Vmax values of the high-affinity phase for biphasic kinetics of propranolol 4- and 5-hydroxylase activities were decreased by propranolol pretreatment, while those of the low-affinity phase were unchanged. The Vmax value of monophasic 7-hydroxylase activity was also decreased. On the other hand, the Vmax value of N-desisopropylase activity in the propranolol-treated rats was increased more than 2-fold compared with non-treated (control) rats, resulting in a change from monophasic in control rats to biphasic kinetics in propranolol-treated rats. These findings indicate that repetitive administration of propranolol selectively impairs a CYP2D isozyme that is involved in the high-affinity phases for propranolol ring-hydroxylations.     

Characterization of the cytochrome P450 isoenzymes involved in the in vitro N-dealkylation of haloperidol

AIMS: The present study was carried out to identify the cytochrome P450 isoenzyme(s) involved in the N-dealkylation of haloperidol (HAL). METHODS: In vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms. N-dealkylation of HAL was assessed by measuring the formation of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). RESULTS: There was a tenfold variation in the extent of CPHP formation amongst the nine human liver microsomal preparations. The CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. Km and Vmax values ranged respectively from 50 to 78 microM and from 180 to 412 pmol mg-1 min-1 CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the activity of dextromethorphan O-demethylase (CYP2D6), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Ketoconazole, an inhibitor of CYP3A4, inhibited competitively CPHP formation (Ki=0.1 microM), whereas sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine (CYP2D6) gave only little inhibition (IC50 > 100 microM). CPHP formation was, moreover, enhanced by apha-naphtoflavone, an effect common to CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited CPHP formation, whereas no inhibition was observed in the presence of CYP2D6 antibodies. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to CPHP formation rate, with no detectable effect of other CYP isoforms (CYP1A2, CYP2D6 and CYP2C9). HAL inhibited dextromethorphan O-demethylase (CYP2D6) with IC50 values between 2.7 and 8.5 microM, but not (IC50 > 100 microM) dextromethorphan N-demethylase (CYP3A4), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). CONCLUSIONS: These results strongly suggest that the N-dealkylation of HAL in human liver microsomal preparations is mediated by CYP3A4.