In vitro identification of the human cytochrome P-450 enzymes involved in the N-demethylation of azelastine.

Azelastine hydrochloride [4-[(4-chlorophenyl)methyl]-2-(hexahydro-1-methyl-1H-azepin-4yl )-1-(2 H)-phthalazinone monohydrochloride], is a long-acting antiallergic and antiasthmatic drug. The human cytochrome P-450 (CYP) isoform responsible for azelastine N-demethylation, the major metabolic pathway for azelastine, has been examined. Eadie-Hofstee plots of azelastine N-demethylation in human liver microsomes were biphasic. In microsomes from baculovirus-infected insect cells, recombinant CYP3A4, 2D6, 1A2, and 2C19 exhibited high azelastine N-demethylase activity. The K(m) values of the recombinant CYP2D6 (3.75 microM) and CYP3A4 (43.7 microM) were relatively close to that of high-affinity (14.1 microM) and low-affinity (54.7 microM) components in human liver microsomes, respectively. Azelastine N-demethylase activity was inhibited only by the anti-CYP3A antibody, in contrast to antibodies for CYP1A, 2D6, and 2C. In addition, desmethylazelastine formation was significantly inhibited by ketoconazole and troleandomycin but only weakly by omeprazole, sulfaphenazole, and furafylline. These observations suggested that the N-demethylation of azelastine is most extensively catalyzed by the CYP2D6 and 3A4 isoforms in humans.     

CYP2A6 AND CYP2B6 are involved in nornicotine formation from nicotine in humans: interindividual differences in these contributions.

Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent K(m) = 173 +/- 70 microM, V(max) = 57 +/- 17 pmol/min/mg) and low-affinity (apparent K(m) = 619 +/- 68 microM, V(max) = 137 +/- 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent K(m) values of CYP2A6 (49 +/- 12 microM) and CYP2B6 (550 +/- 46 microM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 microM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 microM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.     

Role of human CYP2B6 in S-mephobarbital N-demethylation.

The role of cytochrome P-450s (CYPs) in S-mephobarbital N-demethylation was investigated by using human liver microsomes and cDNA-expressed CYPs. Among the 10 cDNA-expressed CYPs studied (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), only CYP2B6 could catalyze S-mephobarbital N-demethylation. The apparent K(m) values of human liver microsomes for S-mephobarbital N-demethylation were close to that of cDNA-expressed CYP2B6 (about 250 microM). The N-demethylase activity of S-mephobarbital in 10 human liver microsomes was strongly correlated with immunodetectable CYP2B6 levels (r = 0.920, p<.001). Orphenadrine (300 microM), a CYP2B6 inhibitor, inhibited the N-demethylase activity of S-mephobarbital in human liver microsomes to 29% of control activity. Therefore, it appears that CYP2B6 mainly catalyzes S-mephobarbital N-demethylation in human liver microsomes.     

Involvement of CYP2B6 in n-demethylation of ketamine in human liver microsomes.

Ketamine is metabolized by cytochrome P450 (CYP) leading to production of pharmacologically active products and contributing to drug excretion. We identified the CYP enzymes involved in the N-demethylation of ketamine enantiomers using pooled human liver microsomes and microsomes from human B-lymphoblastoid cells that expressed CYP enzymes. The kinetic data in human liver microsomes for the (R)- and (S)-ketamine N-demethylase activities could be analyzed as two-enzyme systems. The K(m) values were 31 and 496 microM for (R)-ketamine, and 24 and 444 microM for (S)-ketamine. Among the 12 cDNA-expressed CYP enzymes examined, CYP2B6, CYP2C9, and CYP3A4 showed high activities for the N-demethylation of both enantiomers at the substrate concentration of 1 mM. CYP2B6 had the lowest K(m) value for the N-demethylation of (R)- and (S)-ketamine (74 and 44 microM, respectively). Also, the intrinsic clearance (CL(int): V(max)/K(m)) of CYP2B6 for the N-demethylation of both enantiomers were 7 to 13 times higher than those of CYP2C9 and CYP3A4. Orphenadrine (CYP2B6 inhibitor, 500 microM) and sulfaphenazole (CYP2C9 inhibitor, 100 microM) inhibited the N-demethylase activities for both enantiomers (5 microM) in human liver microsomes by 60 to 70%, whereas cyclosporin A (CYP3A4 inhibitor, 100 microM) failed to inhibit these activities. In addition, the anti-CYP2B6 antibody inhibited these activities in human liver microsomes by 80%, whereas anti-CYP2C antibody and anti-CYP3A4 antibody failed to inhibit these activities. These results suggest that the high affinity/low capacity enzyme in human liver microsomes is mediated by CYP2B6, and the low affinity/high capacity enzyme is mediated by CYP2C9 and CYP3A4. CYP2B6 mainly mediates the N-demethylation of (R)- and (S)-ketamine in human liver microsomes at therapeutic concentrations (5 microM).     

Evaluation of approach to predict the contribution of multiple cytochrome P450s in drug metabolism using relative activity factor: effects of the differences in expression levels of NADPH-cytochrome P450 reductase and cytochrome b(5) in the expression system and the differences in the marker activities

The concept of relative activity factor (RAF) to extrapolate data obtained with recombinant cytochrome P450(CYP)s to human liver microsomes has been proposed. To evaluate the approach to predict the contribution of multiple CYPs using RAF, we investigated the effects of the differences in the expression levels of NADPH-cytochrome P450 reductase (OR) and cytochrome b(5) (b(5)) in recombinant CYPs from baculovirus-infected insect cells and the differences in the marker activities. Because we previously clarified that azelastine, an antiallergy and antiasthmatic drug, is N-demethylated by CYP1A2, CYP2D6, and CYP3A4 in humans, the reaction was used as a model. For calculation of RAF, three lots of recombinant CYP1A2, CYP2D6, and CYP3A4 from baculovirus-infected insect cells with different expression levels of OR and b(5) were used. The OR/CYP ratios for recombinant CYP1A2, CYP2D6, and CYP3A4 were 3.9-4.8, 5.1-8.7, and 8.0-11.3, respectively. The b(5)/CYP ratio for recombinant CYP3A4 was 2.1-18.7. As marker activities, ethoxyresorufin O-deethylation and phenacetin O-deethylation for CYP1A2, bufuralol 1'-hydroxylation and debrisoquin 4-hydroxylation for CYP2D6, testosterone 6beta-hydroxylation and midazolam 1'-hydroxylation for CYP3A4 were compared. Our results indicated that the differences in the expression levels of OR and b(5) coexpressed in baculovirus-infected insect cells would not be a critical factor for the quantitative prediction using RAF. In addition, we confirmed that differences in the marker activities did not significantly affect the calculation of RAF values, when the marker activities are specific for a certain CYP isoform. It was suggested that the RAF approach using recombinant CYPs from baculovirus-infected insect cells coexpressing OR (and b(5) if required) could be valuable for the prediction of the contribution of each CYP in drug metabolism.     

Role of cytochrome P450 2D6 (CYP2D6) in the stereospecific metabolism of E- and Z-doxepin

The tricyclic antidepressant, doxepin, is formulated as an irrational mixture of E (trans) and Z (cis) stereoisomers (85%: 15%). We examined the stereoselective metabolism of doxepin in vitro, with the use of human liver microsomes, recombinant CYP2D6 and gas chromatography-mass spectrometry. In human liver microsomes over the concentration range 5-1500 microM, the rate of Z-doxepin N-demethylation exceeded that of E-doxepin above 100 microM in two of three livers. Eadie-Hofstee plots were curvilinear indicating the involvement of several enzymes in N-demethylation. Coincubation of doxepin with 7,8-naphthoflavone and ketoconazole reduced the rates of N-demethylation of E- and Z-doxepin by 30-50% and 40-60%, respectively, suggesting the involvement of CYP1A and CYP3A4, whilst quinidine had little effect on N-demethylation. In contrast, doxepin hydroxylation was exclusively stereo-specific; E-doxepin and E-N-desmethyldoxepin were hydroxylated with high affinity in liver microsomes and by recombinant CYP2D6 (Km in the range of 5-8 microM), but there was no evidence of Z-doxepin hydroxylation. In 'metabolic consumption' experiments with liver microsomes (having measurable CYP2D6 activity) and initial substrate concentration of 1 microM, the consumption of E-doxepin was greater (P < 0.05, n = 5) than that of Z-doxepin. Quinidine inhibited the consumption of E-doxepin but did not affect the consumption of Z-doxepin. With N-desmethyldoxepin, quinidine inhibited the consumption of E-N-desmethyl-doxepin whereas Z-N-desmethyldoxepin appeared to be a terminal oxidative metabolite. In summary, CYP2D6 is a major oxidative enzyme in doxepin metabolism; predominantly catalysing hydroxylation with an exclusive preference for the E-isomers. The relatively more rapid metabolism of E-isomeric forms, and the limited metabolic pathways for the Z-isomers may explain the apparent enrichment of Z-N-desmethyldoxepin that is observed in vivo.     

Involvement of CYP2E1 as A low-affinity enzyme in phenacetin O-deethylation in human liver microsomes.

Phenacetin O-deethylation (POD) exhibits biphasic kinetics in human liver microsomes. Although cytochrome P-450 (CYP) 1A2 is responsible for the high-affinity component of POD, the enzyme(s) that catalyzes the low-affinity reaction is still unknown. We examined the roles of human CYPs in POD by using human liver microsomes and recombinant CYPs from baculovirus-infected insect cells. Of the recombinant CYPs studied, CYP1A2 showed the highest POD activity. CYP1A1, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 also showed POD activity at 500 microM phenacetin. K(M) values of recombinant CYP1A2 and CYP2E1 (28 +/- 2 microM and 785 +/- 125 microM, respectively) were similar to those of the high- and low-affinity components of POD in pooled human liver microsomes (15 +/- 5 and 894 +/- 189 microM, respectively). Fluvoxamine (10 microM) and anti-CYP1A2 antibodies potently inhibited POD activity at 500 microM phenacetin in pooled human liver microsomes to 22.8 and 34.2% of controls, respectively. CYP2E1 inhibitors diethyldithiocarbamate and aniline also reduced POD activity. The combination of fluvoxamine (10 microM) and aniline (1 mM) further inhibited the residual POD activity not inhibited by fluvoxamine alone. Microsomal POD activity in 12 human livers in the absence of fluvoxamine was correlated with immunoquantified CYP1A2 levels (r = 0.961, p <.001) and, in the presence of 10 microM fluvoxamine, was correlated with immunoquantified CYP2E1 levels (r = 0.589, p <.01) or chlorzoxazone 6-hydroxylase activity (r = 0.823, p <.001). These results suggest that CYP2E1 is responsible for the low-affinity component of POD in human liver microsomes.     

Contribution of CYP3A4, CYP2B6, and CYP2C9 isoforms to N-demethylation of ketamine in human liver microsomes.

Ketamine is a widely used drug for its anesthetic and analgesic properties; it is also considered as a drug of abuse, as many cases of ketamine illegal consumption were reported. Ketamine is N-demethylated by liver microsomal cytochrome P450 into norketamine. The identification of the enzymes responsible for ketamine metabolism is of great importance in clinical practice. In the present study, we investigated the metabolism of ketamine in human liver microsomes at clinically relevant concentrations. Liver to plasma concentration ratio of ketamine was taken into consideration. Pooled human liver microsomes and human lymphoblast-expressed P450 isoforms were used. N-demethylation of ketamine was correlated with nifedipine oxidase activity (CYP3A4-specific marker reaction), and it was also correlated with S-mephenytoin N-demethylase activity (CYP2B6-specific marker reaction). Orphenadrine, a specific inhibitor to CYP2B6, and ketoconazole, a specific inhibitor to CYP3A4, inhibited the N-demethylation of ketamine in human liver microsomes. In human lymphoblast-expressed P450, the activities of CYP2B6 were higher than those of CYP3A4 and CYP2C9 at three concentrations of ketamine, 0.005, 0.05, and 0.5 mM. When these results were extrapolated using the average relative content of these P450 isoforms in human liver, CYP3A4 was the major enzyme involved in ketamine N-demethylation. The present study demonstrates that CYP3A4 is the principal enzyme responsible for ketamine N-demethylation in human liver microsomes and that CYP2B6 and CYP2C9 have a minor contribution to ketamine N-demethylation at therapeutic concentrations of the drug.     

CYP3A4 is the major CYP isoform mediating the in vitro hydroxylation and demethylation of flunitrazepam.

The kinetics of flunitrazepam (FNTZ) N-demethylation to desmethylflunitrazepam (DM FNTZ), and 3-hydroxylation to 3-hydroxyflunitrazepam (3-OH FNTZ), were studied in human liver microsomes and in microsomes containing heterologously expressed individual human CYPs. FNTZ was N-demethylated by cDNA-expressed CYP2A6 (K(m) = 1921 microM), CYP2B6 (K(m) = 101 microM), CYP2C9 (K(m) = 50 microM), CYP2C19 (K(m) = 60 microM), and CYP3A4 (K(m) = 155 microM), and 3-hydroxylated by cDNA-expressed CYP2A6 (K(m) = 298 microM) and CYP3A4 (K(m) = 286 microM). The 3-hydroxylation pathway was predominant in liver microsomes, accounting for more than 80% of intrinsic clearance compared with the N-demethylation pathway. After adjusting for estimated relative abundance, CYP3A accounted for the majority of intrinsic clearance via both pathways. This finding was supported by chemical inhibition studies in human liver microsomes. Formation of 3-OH FNTZ was reduced to 10% or less of control values by ketoconazole (IC(50) = 0.11 microM) and ritonavir (IC(50) = 0.041 microM). Formation of DM FNTZ was inhibited to 40% of control velocity by 2.5 microM ketoconazole and to 30% of control by 2.5 microM ritonavir. Neither 3-OH FNTZ nor DM FNTZ formation was inhibited to less than 85% of control activity by alpha-naphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), omeprazole (CYP2C19), or quinidine (CYP2D6). Thus, CYP-dependent FNTZ biotransformation, like that of many benzodiazepine derivatives, is mediated mainly by CYP3A. Clinical interactions of FNTZ with CYP3A inhibitors can be anticipated.     

The role of CYP2C19 in the metabolism of (+/-) bufuralol, the prototypic substrate of CYP2D6.

Upon characterization of baculovirus-expressed cytochrome P-450 (CYP) 2C19, it was observed that this enzyme metabolized (+/-) bufuralol to 1'hydroxybufuralol, a reaction previously understood to be selectively catalyzed by CYP2D6. The apparent K(m) for this reaction was 36 microM with recombinant CYP2C19, approximately 7-fold higher than for recombinant CYP2D6. The intrinsic clearance for this reaction was 37-fold higher with CYP2D6 than for CYP2C19. The involvement of human CYP1A2 in bufuralol 1'-hydroxylation was also confirmed using the recombinant enzyme. Using S-mephenytoin as an inhibitor, the K(i) for inhibition of recombinant CYP2C19-mediated bufuralol hydroxylation was 42 microM, which is the approximate K(m) for recombinant CYP2C19-mediated S-mephenytoin metabolism. The classic CYP2D6 inhibitors quinidine and quinine showed no inhibition of CYP2C19-catalyzed bufuralol metabolism at concentrations that abolished CYP2D6-mediated bufuralol metabolism. Ticlopidine, a potent inhibitor of CYP2C19 and CYP2D6, inhibited bufuralol 1'-hydroxylation by each of these enzymes equipotently. In human liver microsomes that are known to be deficient in CYP2D6 activity, it was shown that in the presence of quinidine, the K(m) shifted from 14 to 38 microM. This is consistent with the K(m) determination for recombinant CYP2C19 of 36 microM. In human liver microsomes that have high CYP2D6 and CYP2C19 activity, the K(m) shifted to 145 microM in the presence of S-mephenytoin and quinidine, consistent with the K(m) determined for CYP1A2. This data suggests that bufuralol, and possibly other CYP2D6 substrates, have the potential to be metabolized by CYP2C19.     

Quantitative contribution of CYP2D6 and CYP3A to oxycodone metabolism in human liver and intestinal microsomes.

Oxycodone undergoes N-demethylation to noroxycodone and O-demethylation to oxymorphone. The cytochrome P450 (P450) isoforms capable of mediating the oxidation of oxycodone to oxymorphone and noroxycodone were identified using a panel of recombinant human P450s. CYP3A4 and CYP3A5 displayed the highest activity for oxycodone N-demethylation; intrinsic clearance for CYP3A5 was slightly higher than that for CYP3A4. CYP2D6 had the highest activity for O-demethylation. Multienzyme, Michaelis-Menten kinetics were observed for both oxidative reactions in microsomes prepared from five human livers. Inhibition with ketoconazole showed that CYP3A is the high affinity enzyme for oxycodone N-demethylation; ketoconazole inhibited >90% of noroxycodone formation at low substrate concentrations. CYP3A-mediated noroxycodone formation exhibited a mean K(m) of 600 +/- 119 microM and a V(max) that ranged from 716 to 14523 pmol/mg/min. Contribution from the low affinity enzyme(s) did not exceed 8% of total intrinsic clearance for N-demethylation. Quinidine inhibition showed that CYP2D6 is the high affinity enzyme for O-demethylation with a mean K(m) of 130 +/- 33 microM and a V(max) that ranged from 89 to 356 pmol/mg/min. Activity of the low affinity enzyme(s) accounted for 10 to 26% of total intrinsic clearance for O-demethylation. On average, the total intrinsic clearance for noroxycodone formation was 8 times greater than that for oxymorphone formation across the five liver microsomal preparations (10.5 microl/min/mg versus 1.5 microl/min/mg). Experiments with human intestinal mucosal microsomes indicated lower N-demethylation activity (20-50%) compared with liver microsomes and negligible O-demethylation activity, which predict a minimal contribution of intestinal mucosa in the first-pass oxidative metabolism of oxycodone.     

CYP2B6, CYP3A4, and CYP2C19 are responsible for the in vitro N-demethylation of meperidine in human liver microsomes.

Meperidine is an opioid analgesic metabolized in the liver by N-demethylation to normeperidine, a potent stimulant of the central nervous system. The purpose of this study was to identify the human cytochrome P450 (P450) enzymes involved in normeperidine formation. Our in vitro studies included 1) screening 16 expressed P450s for normeperidine formation, 2) kinetic experiments on human liver microsomes and candidate P450s, and 3) correlation and inhibition experiments using human hepatic microsomes. After normalization by its relative abundance in human liver microsomes, CYP2B6, CYP3A4, and CYP2C19 accounted for 57, 28, and 15% of the total intrinsic clearance of meperidine. CYP3A5 and CYP2D6 contributed to < 1%. Formation of normeperidine significantly correlated with CYP2B6-selective S-mephenytoin N-demethylation (r = 0.88, p < 0.0001 at 75 > microM meperidine, and r = 0.89, p < 0.0001 at 350 microM meperidine, n = 21) and CYP3A4-selective midazolam 1'-hydroxylation (r = 0.59, p < 0.01 at 75 microM meperidine, and r = 0.55, p < 0.01 at 350 microM meperidine, n = 23). No significant correlation was observed with CYP2C19-selective S-mephenytoin 4'-hydroxylation (r = 0.36, p = 0.2 at 75 microM meperidine, and r = 0.02, p = 0.9 at 350 microM meperidine, n = 13). An anti-CYP2B6 antibody inhibited normeperidine formation by 46%. In contrast, antibodies inhibitory to CYP3A4 and CYP2C8/9/18/19 had little effect (<14% inhibition). Experiments with thiotepa and ketoconazole suggested inhibition of microsomal CYP2B6 and CYP3A4 activity, whereas studies with fluvoxamine (a substrate of CYP2C19) were inconclusive due to lack of specificity. We conclude that normeperidine formation in human liver microsomes is mainly catalyzed by CYP2B6 and CYP3A4, with a minor contribution from CYP2C19.     

Expression, purification, and characterization of mouse CYP2d22.

Metabolism of the prototype human CYP2D6 substrates debrisoquine and bufuralol proceeds at a much slower rate in mice; therefore, the mouse has been proposed as an animal model for the human CYP2D6 genetic deficiency. To interpret the molecular mechanism of this deficiency, a cDNA belonging to the CYP2D gene subfamily (Cyp2d22) has been cloned and sequenced from a mouse mammary tumor-derived cell line. In the current study, Cyp2d22 enzyme was overexpressed and purified from insect cells using a baculovirus-mediated system. The activity of this purified enzyme was directly compared with purified human CYP2D6 toward codeine, dextromethorphan, and methadone as substrates. Purified Cyp2d22 was found to catalyze the O-demethylation of dextromethorphan with significantly higher K(m) values (250 microM) than that (4.2 microM) exhibited by purified human CYP2D6. The K(m) for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 microM, much lower than that observed with human CYP2D6 and near the K(m) for dextromethorphan N-demethylation catalyzed by CYP3A4. CYP2D6 catalyzed codeine O-demethylation, whereas Cyp2d22 and CYP3A4 mediated codeine N-demethylation. Furthermore, methadone, a known CYP3A4 substrate and CYP2D6 inhibitor, was N-demethylated by Cyp2d22 with a K(m) of 517 microM and V(max) of 4.9 pmol/pmol/min. Quinidine and ketoconazole, potent inhibitors to CYP2D6 and CYP3A4, respectively, did not show strong inhibition toward Cyp2d22-mediated dextromethorphan O- or N-demethylation. These results suggest that mouse Cyp2d22 has its own substrate specificity beyond CYP2D6-like-deficient activity.     

Metabolism of the antidepressant mirtazapine in vitro: contribution of cytochromes P-450 1A2, 2D6, and 3A4.

The metabolism of the antidepressant mirtazapine (MIR) was investigated in vitro using human liver microsomes (HLM) and recombinant enzymes. Mean K(m) values (+/-S.D., n = 4) were 136 (+/-44) microM for MIR-hydroxylation, 242 (+/-34) microM for N-demethylation, and 570 (+/-281) microM for N-oxidation in HLM. Based on the K(m) and V(max) values, MIR-8-hydroxylation, N-demethylation, and N-oxidation contributed 55, 35, and 10%, respectively, to MIR biotransformation in HLM at an anticipated in vivo liver MIR concentration of 2 microM. Recombinant CYP predicted a 65% contribution of CYP2D6 to MIR-hydroxylation at 2 microM MIR, decreasing to 20% at 250 microM. CYP1A2 contribution increased correspondingly from 30 to 50%. In HLM, quinidine and alpha-naphthoflavone reduced MIR-hydroxylation to 75 and 45% of control, respectively, at 250 microM MIR. A >50% contribution of CYP3A4 to MIR-N-demethylation at <1 microM MIR was indicated by recombinant enzymes. In HLM, ketoconazole (1 microM) reduced N-desmethylmirtazapine formation rates to 60% of control at 250 microM. Twenty percent of MIR-N-oxidation was accounted for by CYP3A4 at 2 microM MIR, increasing to 85% at 250 microM, while CYP1A2 contribution decreased from 80 to 15%. Ketoconazole reduced MIR-N-oxidation to 50% of control at 250 microM. MIR did not substantially inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP1E2, and CYP3A4 activity in vitro. Induction/inhibition or genetic polymorphisms of CYP2D6, CYP1A2, and CYP3A4 may affect MIR metabolism, but involvement of several enzymes in different metabolic pathways may prevent large alterations in in vivo drug clearance.     

Involvement of CYP1A2 in mexiletine metabolism

Aims Mexiletine has been reported to be hydroxylated by cytochrome P450 2D6 (CYP2D6) in humans. However, the involvement of CYP1A2 in the metabolism of mexiletine has been proposed based on the interaction with theophylline which is mainly metabolized by CYP1A2. The aim of this study was to clarify the role of human CYP1A2 in mexiletine metabolism.
Methods Human CYP isoforms involved in mexiletine metabolism were investigated using microsomes from human liver and B-lymphoblastoid cells expressing human CYPs. The contributions of CYP1A2 and CYP2D6 to mexiletine metabolism were estimated by the relative activity factor (RAF).
Results Mexiletine p - and 2-hydroxylase activities in human liver microsomes were inhibited by ethoxyresorufin and furafylline as well as quinidine. Mexiletine p - and 2-hydroxylase activities in microsomes from nine human livers correlated significantly with bufuralol 1'-hydroxylase activity ( r =0.907, P <0.001 and r =0.886, P <0.01, respectively). Microsomes of B-lymphoblastoid cells expressing human CYP1A2 exhibited lower mexiletine p - and 2-hydroxylase activities than those expressing human CYP2D6. It was estimated by RAF that the major isoform involved in mexiletine metabolism was CYP2D6, and the contribution of CYP1A2 to both mexiletine p - and 2-hydroxylase activities was 7–30% in human liver microsomes. However, the K _m values of the expressed CYP1A2 (∼15  μm ) were almost identical with those of the expressed CYP2D6 (∼22  μm ) and human liver microsomes.
Conclusions Mexiletine is a substrate of CYP1A2. The data obtained in this study suggest that the interaction of mexiletine with theophylline might be due to competitive inhibition of CYP1A2.     

Trimethoprim and sulfamethoxazole are selective inhibitors of CYP2C8 and CYP2C9, respectively.

To evaluate the inhibitory effects of trimethoprim and sulfamethoxazole on cytochrome P450 (P450) isoforms, selective marker reactions for CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were examined in human liver microsomes and recombinant CYP2C8 and CYP2C9. The in vivo drug interactions of trimethoprim and sulfamethoxazole were predicted in vitro using [I]/([I] + K(i)) values. With concentrations ranging from 5 to 100 microM, trimethoprim exhibited a selective inhibitory effect on CYP2C8-mediated paclitaxel 6alpha-hydroxylation in human liver microsomes and recombinant CYP2C8, with apparent IC(50) (K(i)) values of 54 microM (32 microM) and 75 microM, respectively. With concentrations ranging from 50 to 500 microM, sulfamethoxazole was a selective inhibitor of CYP2C9-mediated tolbutamide hydroxylation in human liver microsomes and recombinant CYP2C9, with apparent IC(50) (K(i)) values of 544 microM (271 microM) and 456 microM, respectively. With concentrations higher than 100 microM trimethoprim and 500 microM sulfamethoxazole, both drugs lost their selectivity for the P450 isoforms. Based on estimated total hepatic concentrations (or free plasma concentrations) of the drugs and the scaling model, one would expect in vivo in humans 80% (26%) and 13% (24%) inhibition of the metabolic clearance of CYP2C8 and CYP2C9 substrates by trimethoprim and sulfamethoxazole, respectively. In conclusion, trimethoprim and sulfamethoxazole can be used as selective inhibitors of CYP2C8 and CYP2C9 in in vitro studies. In humans, trimethoprim and sulfamethoxazole may inhibit the activities of CYP2C8 and CYP2C9, respectively.     

Inhibitory effects of azelastine and its metabolites on drug oxidation catalyzed by human cytochrome P-450 enzymes.

Azelastine, an antiallergy and antiasthmatic drug, has been reported to be metabolized mainly to desmethylazelastine and 6-hydroxyazelastine in mammals. In the present study, the inhibitory effects of azelastine and its two metabolites on human cytochrome P-450 (CYP) isoform-dependent reactions were investigated to predict the drug interactions of azelastine using microsomes from human B-lymphoblast cells expressing CYP. The specific activities for human CYP isoforms included: 7-ethoxyresorufin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), 7-benzyloxyresorufin O-dealkylation (CYP2B6), S-warfarin 7-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and testosterone 6beta-hydroxylation (CYP3A4). In almost all the activities, desmethylazelastine exhibited stronger inhibition than azelastine and 6-hydroxyazelastine. Desmethylazelastine, but not azelastine and 6-hydroxyazelastine, uncompetitively inhibited CYP2B6 activity (Ki = 32.6 +/- 4.8 microM). Azelastine, desmethylazelastine, and 6-hydroxyazelastine competitively inhibited CYP2C9 activity (Ki = 13. 9 +/- 1.8, 15.0 +/- 3.1, and 17.0 +/- 4.1 microM, respectively), CYP2C19 activity (Ki = 21.9 +/- 2.2, 7.3 +/- 1.6, and 9.3 +/- 1.6 microM, respectively), and CYP2D6 activity (Ki = 1.2 +/- 0.1, 1.5 +/- 0.2, and 3.0 +/- 0.5 microM, respectively). Azelastine and desmethylazelastine competitively inhibited CYP3A4 activity (Ki = 23. 7 +/- 4.6 and 13.2 +/- 2.3 microM). 6-Hydroxyazelastine interfered with the determination of testosterone 6beta-hydroxylation by HPLC. CYP1A2, CYP2A6, and CYP2E1 activities were not significantly inhibited by azelastine and the two metabolites. Among the human CYPs tested, the inhibitory effects of azelastine and its two metabolites were the most potent on human CYP2D6. In consideration of the Ki values and the concentration of azelastine and desmethylazelastine in human livers after chronic oral administration of azelastine, the possibility of in vivo drug interaction of azelastine and other drugs that are mainly metabolized by CYP2D6 was suggested although it might not cause critical side effects. The inhibition of CYP2C9, CYP2C19, and CYP3A4 activity by azelastine and its two metabolites might be clinically insignificant.     

2,5-Dimethoxyamphetamine-derived designer drugs: studies on the identification of cytochrome P450 (CYP) isoenzymes involved in formation of their main metabolites and on their capability to inhibit CYP2D6

The designer drugs 4-methyl-2,5-dimethoxy-amphetamine (DOM), 4-iodo-2,5-dimethoxy-amphetamine (DOI), 4-chloro-2,5-dimethoxy-amphetamine (DOC), 4-bromo-2,5-dimethoxy-amphetamine (DOB), 4-bromo-2,5-dimethoxy-methamphetamine (MDOB), and 2,4,5-trimethoxy-amphetamine (TMA-2) are potent serotonin 5HT(2) receptor agonists and have appeared on the illicit drug market. These drugs are mainly metabolized by O-demethylation or in case of DOM by hydroxylation of the methyl moiety. In an initial activity screening using microsomes of insect cells heterologously expressing human CYPs, CYP2D6 was found to be the only CYP isoenzyme involved in the above-mentioned main metabolic steps whereas the amounts of metabolites formed were very small. As inhibition of CYP2D6 by other amphetamines had been described, the inhibitory effects of the 2,5-dimethoxyamphetamine derivatives were studied using insect cell microsomes with heterologously expressed human CYP2D6 and pooled human liver microsomes (HLM) as enzyme sources and dextromethorphan O-demethylation as probe reaction. All studied drugs were observed to be non-mechanism-based competitive inhibitors of CYP2D6 with inhibition constants (K(i)) from 7.1 to 296microM using recombinant CYP2D6 and 2.7-19.9microM using HLM. For comparison, the K(i) values for quinidine and fluoxetine were 0.0092 and 8.2microM using recombinant CYP2D6 and 0.019 and 0.93microM using HLM. As the K(i) values of the drugs were much higher than that of quinidine and, with the exception of DOI, higher than that of fluoxetine, interactions with other CYP2D6 substrates are possible but rather unlikely.     

Involvement of CYP2A6 in the formation of a novel metabolite, 3-hydroxypilocarpine, from pilocarpine in human liver microsomes.

Pilocarpine is a cholinergic agonist that is metabolized to pilocarpic acid by serum esterase. In this study, we discovered a novel metabolite in human urine after the oral administration of pilocarpine hydrochloride, and we investigated the metabolic enzyme responsible for the metabolite formation. The structure of the metabolite was identified as 3-hydroxypilocarpine by liquid chromatography-tandem mass spectrometry and NMR analyses and by comparing to the authentic metabolite. To clarify the human cytochrome P450 (P450) responsible for the metabolite formation, in vitro experiments using P450 isoform-selective inhibitors, cDNA-expressed human P450s (Supersomes; CYP1A2, -2A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4), and liver microsomes from different donors were conducted. The formation of 3-hydroxypilocarpine in human liver microsomes was strongly inhibited (>90%) by 200 microM coumarin. Other selective inhibitors of CYP1A2 (furafylline and alpha-naphthoflavone), CYP2C9 (sulfaphenazole), CYP2C19 [(S)-mephenytoin], CYP2E1 (4-methylpyrazole), CYP2D6 (quinidine), and CYP3A4 (troleandomycin) had a weak inhibitory effect (<20%) on the formation. The highest formation activity was expressed by recombinant CYP2A6. The K(m) value for recombinant CYP2A6 was 3.1 microM, and this value is comparable with that of human liver microsomes (1.5 microM). The pilocarpine 3-hydroxylation activity was correlated with coumarin 7-hydroxylation activity in 16 human liver microsomes (r = 0.98). These data indicated that CYP2A6 is the main enzyme responsible for the 3-hydroxylation of pilocarpine. In conclusion, we identified a novel metabolite of pilocarpine, 3-hydroxypilocarpine, and we clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes.     

Sertraline N-demethylation is catalyzed by multiple isoforms of human cytochrome P-450 in vitro.

Sertraline, a new antidepressant of the selective serotonin reuptake inhibitor class, is extensively metabolized to desmethylsertraline in humans. We identified the cytochrome P-450 (CYP) isoforms involved in sertraline N-demethylation using pooled human liver microsomes and cDNA-expressed CYP isoforms. Eadie-Hofstee plots for the sertraline N-demethylation in human liver microsomes were monophasic. The estimated Michaelis-Menten kinetic parameters were: KM = 18.1 +/- 2.0 microM, Vmax = 0.45 +/- 0.03 nmol/min/mg of protein, and Vmax/KM = 25.2 +/- 4.3 microl/min/mg of protein. At the substrate concentration of 20 microM, which approximated the apparent KM value, sulfaphenazole (CYP2C9 inhibitor) and triazolam (CYP3A substrate) reduced the N-demethylation activities by 20 to 35% in human liver microsomes, whereas the inhibition induced by mephenytoin (CYP2C19 substrate) or quinidine (CYP2D6 inhibitor) was marginal. The anti-CYP2B6 antibody inhibited the sertraline N-demethylation activities by 35%. Sertraline N-demethylation activities were detected in all cDNA-expressed CYP isoforms studied. In particular, CYP2C19, CYP2B6, CYP2C9-Arg, CYP2D6-Val, and CYP3A4 all showed relatively high activity. When the contributions of CYP2D6, CYP2C9, CYP2B6, CYP2C19, and CYP3A4 were estimated from the Vmax/KM of cDNA-expressed CYP isoforms and from their contents in pooled human liver microsomes, the values were found to be 35, 29, 14, 13, and 9%, respectively. The results suggest that at least five isoforms of CYP (CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4) are involved in the sertraline N-demethylation in human liver microsomes and that the contribution of any individual isoform does not exceed 40% of overall metabolism. Therefore, concurrent administration of a drug that inhibits a specific CYP isoform is unlikely to cause a marked increase in the plasma concentration of sertraline.