Identification of the human liver cytochrome P450 isoenzymes responsible for the 5-methylhydroxylation of the novel anti-fibrotic drug AKF-PD

Identification of cytochrome P450 isoforms (CYPs) involved in flourofenidone (5-methyl-1-(3-fluorophenyl)-2-[1H]-pyridone, AKF-PD) 5-methylhydroxylation was carried out using human liver microsomes and cDNA-expressed human CYPs (supersomes). The experiments were performed in the following in vitro models: (A) a study of AKF-PD metabolism in liver microsomes: (a) correlations study between the rate of AKF-PD 5-methylhydroxylation and activity of CYPs; (b) the effect of specific CYPs inhibitors on the rate of AKF-PD 5-methylhydroxylation; (B) AKF-PD biotransformation by cDNA-expressed human CYPs (1A2, 2D6, 2C9, 2C19, 2E1, 3A4). In human liver microsomes, the formation of AKF-PD 5-methylhydroxylation metabolite significantly correlated with the caffeine N3-demethylase (CYP1A2), chlorzoxazone 6-hydroxylase (CYP2E1), midazolam 1'- hydroxylase (CYP3A4), tolbutamide 4-hydroxylase (CYP2C9), and debrisoquin 4-hydroxylase (CYP2D6) activities. The production of AKF-PD 5-methylhydroxylation metabolite was completely inhibited by a-naphthoflavone (a CYP1A2 inhibitor) with the IC50 value of 0.12 μM in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of AKF-PD 5-methylhydroxylation metabolites, but the preference of CYP isoforms to catalyze AKF-PD metabolism was as follows: 2D6?>?2C19?>?1A2?>?2E1?>?2C9?>?3A4. The results demonstrated that CYP1A2 is the main isoform catalyzing AKF-PD 5-methylhydroxylation while CYP3A4, CYP2C9, CYP2E1, CYP2C19, and CYP2D6 are engaged to a lesser degree. Potential drug-drug interactions involving CYP1A2 may be noticed when AKF-PD is used combined with CYP1A2 inducers or inhibitors.     

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.     

Evaluation of Supermix as an in vitro model of human liver microsomal drug metabolism

SUPERMIX is a commercially available formulation of insect cell-expressed human drug-metabolizing cytochrome P450 (CYP) isoforms, mixed in proportions that are optimized to parallel their relative activities in human liver microsomes. We have evaluated the apparent functional affinity and capacity of individual CYP isoforms in SUPERMIX in comparison with microsomes from a panel of 12 human livers, using enzyme kinetic studies of isoform-selective index reactions. In addition, we have measured the concentration of NADPH cytochrome P450 oxidoreductase (OR) in SUPERMIX and compared it with the concentrations of this accessory electron transfer protein in human liver microsomes. No important differences were evident in the catalytic activities of CYPs 1A2, 2C8, 2C9, 2C19, 2D6 and 3A4 between SUPERMIX and human liver microsomes. However, SUPERMIX lacks CYP2B6 activity and did not hydroxylate the antidepressant bupropion, a clinically relevant substrate of this enzyme. In addition, the concentration of OR in SUPERMIX (1198 pmol mg protein(-1)) is 17-fold higher than the mean value in human liver microsomes (70 pmol mg protein(-1)). In conclusion, SUPERMIX lacks CYP2B6 activity and contains supraphysiological concentrations of the accessory electron transfer protein OR. These factors should be considered when this formulation is used as an in vitro model in human liver microsomal drug metabolism studies.     

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.     

Identification of the human liver cytochrome P450 isoenzymes responsible for the 5-methylhydroxylation of the novel anti-fibrotic drug AKF-PD

1. Identification of cytochrome P450 isoforms (CYPs) involved in flourofenidone (5-methyl-1-(3-fluorophenyl)-2-[1H]-pyridone, AKF-PD) 5-methylhydroxylation was carried out using human liver microsomes and cDNA-expressed human CYPs (supersomes). The experiments were performed in the following in vitro models: (A) a study of AKF-PD metabolism in liver microsomes: (a) correlations study between the rate of AKF-PD 5-methylhydroxylation and activity of CYPs; (b) the effect of specific CYPs inhibitors on the rate of AKF-PD 5-methylhydroxylation; (B) AKF-PD biotransformation by cDNA-expressed human CYPs (1A2, 2D6, 2C9, 2C19, 2E1, 3A4).

2. In human liver microsomes, the formation of AKF-PD 5-methylhydroxylation metabolite significantly correlated with the caffeine N3-demethylase (CYP1A2), chlorzoxazone 6-hydroxylase (CYP2E1), midazolam 1’- hydroxylase (CYP3A4), tolbutamide 4-hydroxylase (CYP2C9), and debrisoquin 4-hydroxylase (CYP2D6) activities. The production of AKF-PD 5-methylhydroxylation metabolite was completely inhibited by a-naphthoflavone (a CYP1A2 inhibitor) with the IC50 value of 0.12 μM in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of AKF-PD 5-methylhydroxylation metabolites, but the preference of CYP isoforms to catalyze AKF-PD metabolism was as follows: 2D6?>?2C19?>?1A2?>?2E1?>?2C9?>?3A4.

3. The results demonstrated that CYP1A2 is the main isoform catalyzing AKF-PD 5-methylhydroxylation while CYP3A4, CYP2C9, CYP2E1, CYP2C19, and CYP2D6 are engaged to a lesser degree. Potential drug–drug interactions involving CYP1A2 may be noticed when AKF-PD is used combined with CYP1A2 inducers or inhibitors.

     

Contribution of human cytochrome p-450 isoforms to the metabolism of the simplest phenothiazine neuroleptic promazine

1. The aim of the present study was to identify human cytochrome p-450 isoforms (CYPs) involved in 5-sulphoxidation and N-demethylation of the simplest phenothiazine neuroleptic promazine in human liver. 2. The experiments were performed in the following in vitro models: (A). a study of promazine metabolism in liver microsomes-(a). correlations between the rate of promazine metabolism and the level and activity of CYPs; (b). the effect of specific inhibitors on the rate of promazine metabolism (inhibitors: CYP1A2-furafylline, CYP2D6-quinidine, CYP2A6+CYP2E1-diethyldithiocarbamic acid, CYP2C9-sulfaphenazole, CYP2C19-ticlopidine, CYP3A4-ketoconazole); (B). promazine biotransformation by cDNA-expressed human CYPs (Supersomes 1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2E1, 3A4); (C). promazine metabolism in a primary culture of human hepatocytes treated with specific inducers (rifampicin-CYP3A4, CYP2B6 and CYP2C inducer, 2,3,7,8-tetrachlordibenzeno-p-dioxin (TCDD)-CYP1A1/1A2 inducer). 3. In human liver microsomes, the formation of promazine 5-sulphoxide and N-desmethylpromazine was significantly correlated with the level of CYP1A2 and ethoxyresorufin O-deethylase and acetanilide 4-hydroxylase activities, as well as with the level of CYP3A4 and cyclosporin A oxidase activity. Moreover, the formation of N-desmethylpromazine was correlated well with S-mephenytoin 4'-hydroxylation. 4. Furafylline (a CYP1A2 inhibitor) and ketoconazole (a CYP3A4 inhibitor) significantly decreased the rate of promazine 5-sulphoxidation, while furafylline and ticlopidine (a CYP2C19 inhibitor) significantly decreased the rate of promazine N-demethylation in human liver microsomes. 5. The cDNA-expressed human CYPs generated different amounts of promazine metabolites, but the rates of CYP isoforms to catalyse promazine metabolism at therapeutic concentration (10 microM) was as follows: 1A1>2B6>1A2>2C9>3A4>2E1>2A6>2D6>2C19 for 5-sulphoxidation and 2C19>2B6>1A1>1A2>2D6>3A4>2C9>2E1>2A6 for N-demethylation. The highest intrinsic clearance (V(max)/K(m)) was found for CYP1A subfamily, CYP3A4 and CYP2B6 in the case of 5- sulphoxidation, and for CYP2C19, CYP1A subfamily and CYP2B6 in the case of N-demethylation. 6. In a primary culture of human hepatocytes, TCDD (a CYP1A subfamily inducer), as well as rifampicin (mainly a CYP3A4 inducer) induced the formation of promazine 5-sulphoxide and N-desmethylpromazine. 7. Regarding the relative expression of various CYPs in human liver, the obtained results indicate that CYP1A2 and CYP3A4 are the main isoforms responsible for 5-sulphoxidation, while CYP1A2 and CYP2C19 are the basic isoforms that catalyse N-demethylation of promazine in human liver. Of the other isoforms studied, CYP2C9 and CYP3A4 contribute to a lesser degree to promazine 5-sulphoxidation and N-demethylation, respectively. The role of CYP2A6, CYP2B6, CYP2D6 and CYP2E1 in the investigated metabolic pathways of promazine seems negligible.     

The metabolism of the piperazine-type phenothiazine neuroleptic perazine by the human cytochrome P-450 isoenzymes.

Identification of cytochrome P-450 isoenzymes (CYPs) involved in perazine 5-sulphoxidation and N-demethylation was carried out using human liver microsomes and cDNA-expressed human CYPs (Supersomes). In human liver microsomes, the formation of perazine metabolites correlated significantly with the level of CYP1A2 and ethoxyrezorufin O-deethylase activity, as well as with the level of CYP3A4 and cyclosporin A oxidase activity. Moreover, the formation of N-desmethylperazine also correlated well with S-mephenytoin 4'-hydroxylase activity (CYP2C19). alpha-Naphthoflavone (a CYP1A2 inhibitor) and ketoconazole (a CYP3A4 inhibitor) significantly decreased the rate of perazine 5-sulphoxidation, while ticlopidine (a CYP2C19 inhibitor) strongly reduced the rate of perazine N-demethylation in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of perazine metabolites, but the preference of CYP isoforms to catalyze perazine metabolism was as follows (pmol of product/pmol of CYP isoform/min): 1A1>2D6>2C19>1A2>2B6>2E1>2A6 approximately 3A4>2C9 for 5-sulphoxidation and 2C19>2D6>1A1>1A2>2B6>3A4>2C9>2A6 for N-demethylation. In the light of the obtained results and regarding the contribution of each isoform to the total amount of CYP in human liver, it is concluded that CYP1A2 and CYP3A4 are the main isoenzymes catalyzing 5-sulphoxidation (32% and 30%, respectively), while CYP2C19 is the main isoform catalyzing perazine N-demethylation (68%). CYP2C9, CYP2E1 CYP2C19 and CYP2D6 are engaged to a lesser degree in 5-sulphoxidation, while CYP1A2, CYP3A4 and CYP2D6 in perazine N-demethylation (6-10%, depending on the isoform).     

In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450

Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4'-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4'-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4'-OH-NBL. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.     

Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4

1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (+/- TSEM) Km and Vmax = 4.6 +/- 0.3 microM and 20.0 +/- 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of lO microM (i.e. around twice Km). Good correlations (r2 = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10O microM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 microM BFBFC in human liver microsomes was markedly inhibited by 5-50 microM troleandomycin and 0.2-5 microM ketoconazole, but stimulated by 0.2-10 microM alpha-naphthoflavone. The metabolism of 10 microM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed Km and Vmax = 70 microM and 3.39 nmol/min/mg protein, respectively. 8. While 80 microM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYPIA2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.     

Metabolism of Zaleplon by human hepatic microsomal cytochrome P450 isoforms

1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Human liver microsomes catalysed the NADPH-dependent N -deethylation of ZAL to DZAL (CL-284,859), but not to two other known in vivo metabolites, namely M1 (CL345,644) and M2 (CL-345,905). Sigmoidal kinetic plots were observed for ZAL deethylation indicating positive cooperativity. 3. The metabolism of ZAL to DZAL was determined in a characterized bank of 24 human liver microsomalpreparations.Good correlations (r² = 0.734-0.937) were observed with caffeine 8-hydroxylase, diazepam 3-hydroxylase, dextromethorphan N-demethylase and testosterone 2β-, 6β- and 15β-hydroxylase activities, which are allcatalysed by CYP3A isoforms. In contrast, poor correlations (r² 0.152-0.428) were observed for enzymatic markers for CYP1A2, CYP2A6, CYP2C9 10, CYP2D6, CYP2E1 and CYP4A9 11. 4. The metabolism of ZAL to DZAL in human liver microsomes was inhibited to 6-15% of control by 5-50 μM of the mechanism-based CYP3A inhibitor troleandomycin. Whereas some inhibition of DZAL formation was observed in the presence of 200 μM diethyldithiocarbamate, 5-50 μM furafylline, 2-20 μM sulphaphenazole, 50-500 μM S-mephenytoin and 1-10 μM quinidine had little effect. 5. Using human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, ZAL was metabolized to DZAL by CYP3A4, but not to any great extent by CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. In contrast with ZAL, the NADPH-dependent N-deethylation of M2 to M1 proceeded at only a very low rate with both human liver microsomes and cDNA-expressed CYP3A4. 7. In summary, by correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, ZAL N -deethylation to DZAL in human liver appears to be catalysed by CYP3A isoforms.     

Nortriptyline E-10-hydroxylation in vitro is mediated by human CYP2D6 (high affinity) and CYP3A4 (low affinity): implications for interactions with enzyme-inducing drugs.

The human cytochrome P450 (CYP) isoforms mediating nortriptyline 10-hydroxylation have been identified using kinetic studies on heterologously expressed human CYPs and chemical inhibition studies on human liver microsomes. Nortriptyline was metabolized to E-10-hydroxynortriptyline by human lymphoblast-expressed CYPs 2D6 (Km 2.1 microM) and 3A4 (Km 37.4 microM) with high and low affinity, respectively, whereas CYPs 1A2, 2A6, 2B6, 2C9, 2C19, and 2E1 had no detectable activity. Human liver microsomal nortriptyline E-10-hydroxylation displayed biphasic kinetics. The high-affinity component (Km 1.3 +/- 0.4 microM, n = 11 livers) was selectively inhibited by the CYP 2D6 inhibitor quinidine, whereas the CYP3A4 inhibitor ketoconazole selectively inhibited the low-affinity component (K(m) 24.4 +/- 7 microM, n = 11 livers). Inhibition by ketoconazole increased with increasing substrate concentration, whereas the reverse was true for quinidine. The Vmax of the low-affinity component in human liver microsomes was significantly correlated (r2 = 0.84) with the relative activity factor for CYP3A4, a measure of the amount of catalytically active enzyme. A simulation of the relative contribution of CYPs 2D6 and 3A4 to net nortriptyline hydroxylation rate suggested that the relative contribution of CYP3A4 is only 20% even at the higher end of the therapeutic range. Induction of CYP3A4 will increase its importance and increase the net metabolic rate, whereas inhibition of CYP3A4 will be of little importance due to its minimal relative contribution under uninduced conditions. The identification of CYP3A4 as a low-affinity nortriptyline E-10-hydroxylase explains the ability of poor metabolizers of debrisoquin to hydroxylate nortriptyline, as well as the increased in vivo clearance via this pathway caused by CYP3A4-inducing drugs such as pentobarbital, carbamazepine, and rifampin.     

In vitro metabolism of tributyltin and triphenyltin by human cytochrome P-450 isoforms

The metabolic fate of tributyltin and triphenyltin may contribute to the toxicity of these chemicals. We used human hepatic cytochrome P-450 (CYP) systems to confirm the specific CYP(s) involved in the in vitro metabolism of tributyltin and triphenyltin. There were no significant sex differences in the metabolic pattern of tributyltin or triphenyltin, indicating that the CYP(s) responsible for the metabolism of these chemicals in humans is/are not sex-specific form(s). Six major drug-metabolizing isoforms of cDNA-expressed human CYPs and the CYP2C subfamily were tested to determine their metabolic capacities for tributyltin and triphenyltin. CYP2C9, 2C18, 2C19, and 3A4 significantly mediated both dealkylation and dearylation of these triorganotins. Furthermore, the metabolism of tributyltin and triphenyltin was significantly inhibited in vitro by pretreatment with selective inhibitors, azamulin for CYP3A4 and N-3-benzylnirvanol for CYP2C19. Since the CYP2C18 content of hepatic microsomes in humans is relatively low, CYP2C9, 2C19, and 3A4 might be the main isoforms of CYP that are responsible for tributyltin and triphenyltin metabolism in the human liver.     

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.     

In vitro biotransformation of sildenafil (Viagra) in the male rat: the role of CYP2C11.

To assess the suitability of the male rat model for human studies on sildenafil metabolism, we examined the biotransformation of sildenafil in male rat liver microsomes and identified the role of specific cytochrome P450s (P450) using inhibitory antibodies and cDNA-expressed P450s. Rates of formation of the major circulating metabolite of sildenafil, UK-103,320, were 11-fold greater in the male rat than in human liver microsomes at 36 microM sildenafil, whereas substrate concentration corresponding to 50% V(max) (K(m) values) were 2.9-fold lower in the male rat. Although sildenafil is largely metabolized by CYP3A isoforms in humans, coincubation of rat liver microsomes with immunoinhibitory antibodies (CYP1A1/2, 2B1/2, 2C11, 2E1, and 3A1/2) revealed that metabolite formation was inhibited only by an antirat CYP2C11 antibody. Incubation of sildenafil with a cDNA-expressed CYP2C11 produced 10-fold higher levels of UK-103,320 than other P450s (CYP1A1, 1A2, 2B1, 2C6, 2C12, 2C13, 2E1, 3A1, and 3A2). Thus CYP2C11 contributes in a major way to the metabolism of sildenafil in the male rat. P450 isoforms mediating sildenafil biotransformation differ substantially between humans and the male rat, thereby limiting the applicability of this species as a model for sildenafil metabolism and drug interactions in humans.     

Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes

1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.     

Cytochrome P450 fluorometric substrates: identification of isoform-selective probes for rat CYP2D2 and human CYP3A4.

We have tested a panel of 29 cDNA-expressed rat and human enzymes with 9 fluorometric substrates to determine the P450 isoform selectivity in the catalysis of the substrates to fluorescent products. The substrates examined were dibenzyl fluorescein, 7-benzyloxyquinoline (BQ), 3-cyano-7-ethoxycoumarin, 3-cyano-7-methoxycoumarin, 7-methoxy-4-trifluoromethylcoumarin, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and 7-benzyloxy-4-trifluoromethylcoumarin (BFC). For most substrates, multiple cDNA-expressed cytochrome P450 isoforms were found to catalyze the formation of the fluorescent product. However, among the combinations tested, rat CYP2D2 displayed high selectivity for AMMC demethylation (a substrate selective for CYP2D6 in human liver microsomes). AMMC demethylation activity was 15-fold lower in microsomes isolated from female Dark Agouti rats, a model known to have a low abundance of CYP2D2, and apparent K(M) values were similar for cDNA-expressed CYP2D2 and male Sprague-Dawley liver microsomes. BFC dealkylation and BQ dealkylation were selective but not exclusive for human CYP3A4. A small role for CYP1A2 could be demonstrated. The CYP3A4 selectivity in hepatic microsomes was supported by studies using chemical and antibody inhibitors and a correlation analysis within a panel of liver microsomes from individual donors. BQ demonstrated a higher degree of selectivity for and higher rates of metabolism by CYP3A than BFC. However, per unit enzyme the fluorescent signal is lower for BQ than BFC. AMMC, BQ, and BFC should find uses as enzyme-selective probe substrates.     

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.     

Human cytochromes P450 mediating phenacetin O-deethylation in vitro: validation of the high affinity component as an index of CYP1A2 activity

Phenacetin O-deethylation, widely used as an index reaction for cytochrome P450 1A2 (CYP1A2) activity, displays biphasic kinetics in human liver microsomes. CYP1A2 has been identified as contributing to the high affinity component, but is not verified as the sole contributor to the high affinity phase. In addition, the human CYP isoforms accounting for the low affinity phase have not been identified. We have used heterologously expressed human CYP isoforms to identify, kinetically characterize, and predict the relative contribution of the major human liver CYP isoforms mediating phenacetin O-deethylation. CYP1A2 (Km 31 microM) is the only high affinity phenacetin O-deethylase in human liver microsomes, while CYPs 2A6 (Km 4098 microM), 2C9 (Km 566 microM), 2C19 (Km 656 microM), 2D6 (Km 1021 microM), and 2E1 (Km 1257 microM) all contribute to the low affinity phase of the reaction. Considering the relative abundance of the various CYPs in human liver, CYP1A2 accounts for 86% of net reaction velocity at a substrate concentration of 100 microM, while CYP2C9 becomes the primary phenacetin O-deethylase at substrate concentrations of 865 microM and higher and accounts for 31% of the net Vmax of the reaction. Predictions from kinetic studies on heterologously expressed CYPs are consistent with chemical inhibition studies on human liver microsomes with sulfaphenazole and alpha-naphthoflavone that suggest a greater role for CYP2C9, and a smaller role for CYP1A2, at higher substrate concentrations. Thus CYP1A2 is the only high affinity human liver phenacetin O-deethylase, thereby validating the use of the high affinity component as an index of CYP1A2 activity in human liver microsomes.     

A significant role of human cytochrome P450 2C8 in amiodarone N-deethylation: an approach to predict the contribution with relative activity factor.

Human cytochrome P450 (CYP) isoforms involved in amiodarone N-deethylation were identified, and the relative contributions of these CYP isoforms were evaluated in different human liver microsomes. The mean K(M) and V(max) values of amiodarone N-deethylation in microsomes from six human livers were 31.6 +/- 7.5 microM and 1.2 +/- 0.7 pmol/min/pmol of CYP, respectively. Ketoconazole and anti-CYP3A antibodies strongly inhibited amiodarone N-deethylase activity in human liver microsomes at a substrate concentration of 50 microM. Of 15 recombinant human CYP enzymes (19 preparations), CYP1A1, CYP3A4, CYP1A2, CYP2D6, CYP2C8, and CYP2C19 catalyzed amiodarone N-deethylation. The amiodarone N-deethylase activity at a substrate concentration of 5 microM was significantly correlated with the paclitaxel 6alpha-hydroxylase activity (r = 0.84, P <.05) in the human liver microsomes, whereas the amiodarone N-deethylase activity at 100 microM was significantly correlated with the testosterone 6beta-hydroxylase activity (r = 0.94, P <.005). According to the concept of relative activity factor, it was clarified that CYP2C8 as well as CYP3A4 were significantly involved in amiodarone N-deethylation in human livers at clinically significant concentrations and that the contributions of CYP1A2, CYP2C19, and CYP2D6 were relatively minor. However, there was a large interindividual variability in the contribution of each CYP isoform to amiodarone N-deethylase activity in human liver; the relevance of these enzymes would be dependent on the content of the respective isoforms and on the amiodarone concentration in the liver.     

Azelastine N-demethylation by cytochrome P-450 (CYP)3A4, CYP2D6, and CYP1A2 in human liver microsomes: evaluation of approach to predict the contribution of multiple CYPs.

Azelastine, an antiallergy and antiasthmatic drug, has been reported to be mainly N-demethylated to desmethylazelastine in humans. In the present study, Eadie-Hofstee plots of azelastine N-demethylation in human liver microsomes were biphasic. In microsomes from human B-lymphoblast cells, recombinant cytochrome P-450 (CYP)2D6 and CYP1A1 exhibited higher azelastine N-demethylase activity than did other CYP enzymes. On the other hand, recombinant CYP3A4 and CYP1A2 as well as CYP1A1 and CYP2D6 in microsomes from baculovirus-infected insect cells were active in azelastine N-demethylation. The K(M) value of the recombinant CYP2D6 (2.1 microM) from baculovirus-infected insect cells was similar to the K(M) value of the high-affinity (2.4+/-1.3 microM) component in human liver microsomes. On the other hand, the K(M) values of the recombinant CYP3A4 (51.1 microM) and CYP1A2 (125.4 microM) from baculovirus-infected insect cells were similar to the K(M) value of the low-affinity (79.7+/-12.8 microM) component in human liver microsomes. Bufuralol inhibited the high-affinity component, making the Eadie-Hofstee plot in human liver microsomes monophasic. Azelastine N-demethylase activity in human liver microsomes (5 microM azelastine) was inhibited by ketoconazole, erythromycin, and fluvoxamine (IC(50) = 0.08, 18.2, and 17.2 microM, respectively). Azelastine N-demethylase activity in microsomes from twelve human livers was significantly correlated with testosterone 6beta-hydroxylase activity (r = 0.849, p<.0005). The percent contributions of CYP1A2, CYP2D6, and CYP3A4 in human livers were predicted using several approaches based on the concept of correction with CYP contents or relative activity factors (RAFs). Our data suggested that the approach using RAF(CL) (RAF as the ratio of clearance) is most predictive of the N-demethylation clearance of azelastine because it best reflects the observed N-demethylation clearance in human liver microsomes. Summarizing the results, azelastine N-demethylation in humans liver microsomes is catalyzed mainly by CYP3A4 and CYP2D6, and CYP1A2 to a small extent (in average, 76.6, 21.8, and 3.9%, respectively), although the percent contribution of each isoform varied among individuals.