Relative Activity Factor Research Articles

Utility of recombinant enzyme kinetics in prediction of human clearance: impact of variability, CYP3A5, and CYP2C19 on CYP3A4 probe substrates.

A systematic kinetic analysis of the metabolism of five benzodiazepines (low to high clearance compounds) was performed in CYP3A4, CYP3A5, and CYP2C19 baculovirus-expressed recombinant systems. The data obtained in the expression systems were scaled and compared with human liver microsomal predicted clearance and observed in vivo values, using either cytochrome P450 relative activity factors (RAFs) or the relative abundance approach. Interindividual variability, both in content (CYP3A4, CYP3A5) and activity (CYP3A4, CYP3A5, and CYP2C19), were incorporated in the clearance prediction by bootstrap analysis. These resampling Monte Carlo-based simulations were performed to justify any distribution assumptions in the generated range of the predicted clearance due to a limited sample size. This approach allowed extrapolation of the recombinant clearance data to specific population groups and investigation of the role of "minor" forms like CYP3A5 and CYP2C19 in comparison to the most prolific CYP3A4. The use of quinidine 3-hydroxylation and alprazolam 1'-hydroxylation as RAF markers for CYP3A4 and CYP3A5 activity, respectively, and the incorporation of variability improved the clearance prediction of the selected benzodiazepines (apart from flunitrazepam) to within 2-fold of the in vivo value. Clearance estimates from the immunoquantified protein levels were approximately 8-fold lower in comparison to the RAF approach. The differences observed in the benzodiazepine metabolite pathway ratios between CYP3A4 and CYP3A5, particularly for 1'- to 4-hydroxymidazolam and alprazolam, provided a useful measure of interindividual differences within the CYP3A family.

Prediction of differences in in vivo oral clearance of N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100) between extensive and poor metabolizers from in vitro metabolic data in human liver microsomes lacking CYP2D6 activity and recombinant CYPs

It has previously been reported that N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) was predominantly metabolized by cytochrome P450 (CYP) 2D6 in human liver microsomes (HLM). In the present study, the contribution of CYP forms involved in the formation of the major metabolites of NE-100 in human liver lacking CYP2D6 activity (PM-HLM) has been predicted by use of in vitro kinetic data on recombinant CYPs microsomes (rCYPs).In PM-HLM, NE-100 is predicted to be metabolized to N-despropyl-NE-100 (NE-098), p-hydroxy-NE-100 (NE-152) and m-hydroxyl-NE-100 (NE-163), but not to O-demethy-NE-100 (NE-125), which is a major metabolite in pooled human liver microsomes (EM-HLM). The relative activity factor approach assumed that NE-098 formation is predominantly catalysed by CYP3A4 and CYP2C9 and the NE-152 + 163mix (a mixture of two hydroxylated metabolites, NE-152 and NE-163) formation is only catalysed by CYP3A4.The predicted contribution rates of CYP3A4 and CYP2C9 for NE-098 formation were 58.1 and 34.6%, respectively, in PM-HLM. These predicted results were strongly supported by kinetic and inhibition studies using PM-HLM. The intrinsic clearance of NE-100 predicted from rCYPs (the predicted CLint-HLM-total) corresponded to those observed from EM- and PM-HLM (the observed CLint-HLM).The in vivo oral clearance (CLoral) of NE-100 in extensive metabolizers and poor metabolizers of CYP2D6 was predicted to be 50 times higher in extensive metabolizers than poor metabolizers using in vitro–in vivo scaling method based on the dispersion model. These data suggest that polymorphism of CYP2D6 might greatly affect NE-100 metabolism in vivo.

Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the hepatic uptake of pitavastatin in humans.

Pitavastatin, a novel potent 3-hydroxymethylglutaryl-CoA reductase inhibitor, is selectively distributed to the liver in rats. However, the hepatic uptake mechanism of pitavastatin has not been clarified yet. In the present study, we investigated the contribution of organic anion transporting polypeptide 2 (OATP2/OATP1B1) and OATP8 (OATP1B3) to pitavastatin uptake using transporter-expressing HEK293 cells and human cryopreserved hepatocytes. Uptake studies using OATP2- and OATP8-expressing cells revealed a saturable and Na(+)-independent uptake, with K(m) values of 3.0 and 3.3 microM for OATP2 and OATP8, respectively. To determine which transporter is more important for its hepatic uptake, we proposed a methodology for estimating their quantitative contribution to the overall hepatic uptake by comparing the uptake clearance of pitavastatin with that of reference compounds (a selective substrate for OATP2 (estrone-3-sulfate) and OATP8 (cholecystokinin octapeptide) in expression systems and human hepatocytes. The concept of this method is similar to the so-called relative activity factor method often used in estimating the contribution of each cytochrome P450 isoform to the overall metabolism. Applying this method to pitavastatin, the observed uptake clearance in human hepatocytes could be almost completely accounted for by OATP2 and OATP8, and about 90% of the total hepatic clearance could be accounted for by OATP2. This result was also supported by estimating the relative expression level of each transporter in expression systems and hepatocytes by Western blot analysis. These results suggest that OATP2 is the most important transporter for the hepatic uptake of pitavastatin in humans.

Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate

1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug.2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CLint) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively.3. CLint of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CLint of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CLint of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4.4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.

Uridine diphosphate sugar-selective conjugation of an aldose reductase inhibitor (AS-3201) by UDP-glucuronosyltransferase 2B subfamily in human liver microsomes

N-Glucosidation is known as a major metabolic reaction for barbiturates in humans. However, the enzyme(s) involved in this N-glucosidation has not been clarified yet. Thus, to clarify the enzyme(s) involved in the N-glucosidation in human liver microsomes, we investigated the N-glucosyltransferase activity in recombinant UDP-glucuronosyltransferases (UGTs) using AS-3201, an aldose reductase inhibitor, as a substrate. AS-3201 was found to be biotransformed to both N-glucoside and N-glucuronide in human liver microsomes. The N-glucosyltransferase activities were detectable with multiple UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT2B4, UGT2B7, and UGT2B15). In contrast, the N-glucuronyltransferase activities for the same substrate were seen with UGT1A (UGT1A1, UGT1A3, UGT1A4, and UGT1A9) but not UGT2B isoforms. We then determined the relative activity factor of each recombinant UGT and estimated the contribution of each UGT isoform to the N-glucosidation in human liver microsomes. The results showed that UGT2B isoforms mainly contribute to AS-3201 N-glucosidation in human liver microsomes. In addition, the activity of AS-3201 N-glucosyltransferase significantly correlated with that of amobarbital N-glucosyltransferase in microsomes from sixteen human livers ( r=0.964, P<0.01), indicating that UGT2B isoforms were also involved in the barbiturate N-glucosidation in humans. The findings of this study clearly show that UGT2B specifically utilizes UDP-glucose but not UDP-glucuronic acid as a sugar donor for the conjugation of AS-3201 in human liver microsomes.

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.

Identification of cytochrome P450 enzymes involved in the metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a designer drug, in human liver microsomes

1. The metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a novel designer drug, to its demethylated major metabolite 4′-hydroxy-pyrrolidinopropio-phenone (HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes.2. CYP2C19 catalysed the demethylation with apparent Km and Vmax values of 373.4 ± 45.1 μM and 6.0 ± 0.3 pmol min−1 pmol−1 CYP, respectively (mean ± SD). Both CYP2D6 and HLM exhibited clear biphasic profiles with apparent Km,1 values of 1.3 ± 0.4 and 22.0 ± 6.5 μM, respectively, and Vmax,1 values of 1.1 ± 0.1 pmol min−1 pmol−1 CYP and 169.1 ± 20.5 pmol min−1 mg−1 protein, respectively.3. Percentages of intrinsic clearances of MOPPP by particular CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively.4. MOPPP, HO-PPP and the standard 3′,4′-methylenedioxy-pyrrolidinopropio-phenone (MDPPP) were separated and analysed by liquid chromatography–mass spectrometry in the selected-ion monitoring (SIM) mode.5. The CYP2D6 specific chemical inhibitor quinidine (3 μM) significantly ( p<0.0001) inhibited HO-PPP formation by 91.8 ± 0.5% (mean ± SEM) in incubation mixtures with HLM and 2 μM MOPPP.6. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MOPPP demethylation.

Identification of cytochrome p450 enzymes involved in the metabolism of 4'-methyl-alpha-pyrrolidinopropiophenone, a novel scheduled designer drug, in human liver microsomes.

4'-Methyl-alpha-pyrrolidinopropiophenone (MPPP) is a new drug of abuse. It is believed to have an abuse potential similar to that of amphetamines. Previous studies with Wistar rats had shown that MPPP was metabolized mainly by hydroxylation in position 4' followed by dehydrogenation to the corresponding carboxylic acid. The aim of the study presented here was to identify the human hepatic cytochrome p450 (p450) enzymes involved in the biotransformation of MPPP to 4'-hydroxymethyl-pyrrolidinopropiophenone. Baculovirus-infected insect cell microsomes and human liver microsomes were used for this purpose. Only CYP2C19 and CYP2D6 catalyzed this hydroxylation. The apparent Km and Vmax values for the latter were 9.8 +/- 2.5 microM and 13.6 +/- 0.7 pmol/min/pmol p450, respectively. CYP2C19 was not saturable over the tested substrate range (2-1000 microM) and interestingly showed a biphasic kinetic profile with apparent Km,1 and Vmax,1 values of 47.2 +/- 12.5 microM and 8.1 +/- 1.4 pmol/min/pmol p450, respectively. Experiments with pooled human liver microsomes also revealed biphasic nonsaturable kinetics with apparent Km,1 and Vmax,1 values of 57.0 +/- 20.9 microM and 199.7 +/- 59.7 pmol/min/mg of protein for the high affinity enzyme, respectively. Incubation of 2 microM MPPP with 3 microM of the CYP2D6-specific inhibitor quinidine resulted in significant (p < 0.01) turnover inhibition (11.8 +/- 1.6% of control). Based on kinetic data corrected for the relative activity factors, CYP2D6 is the enzyme mainly responsible for MPPP hydroxylation, confirmed by CYP2D6 inhibition studies.

Metabolism of N,N-Dipropyl-2-[4-Methoxy-3-(2-Phenyl-Ethoxy)-Phenyl]-Ethyl-Amine-Monohydrochloride (NE-100), A Novel Sigma Ligand: Contribution of Cytochrome P450 Forms Involved in the Formation of Individual Metabolites in Human Liver and Small Intestine

In the present study, human cytochrome P450 (CYP) forms involved in producing the primary metabolites of NE-100 were identified. Major metabolites of NE-100 in human liver microsomes (HLM) were N-depropylation of NE-100 (NE-098), p-hydroxylation of phenyl group of NE-100 (NE-152), m-hydroxylation of phenyl group of NE-100 (NE-163) and O-demethylation of NE-100 (NE-125). Judging from the correlation and inhibition studies, NE-125 and NE-152+163mix formations were predominantly mediated by CYP2D6 and NE-098 formation was mediated by multiple CYP forms at a low NE-100 concentration (0.1 microM) in the HLM. According to relative activity factor (RAF) approaches, all these reactions were predominantly catalyzed by CYP2D6 at a substrate concentration assuming a plasma level of NE-100 (K(m)>>S) in case of the human liver. Depending on the increase in NE-100 concentrations, the rate of contribution for NE-098 and NE-152+163mix formations increased in CYP3A4, although the predominant contribution of CYP2D6 for NE-125 formation did not change. In human intestinal microsomes (HIM), NE-100 was mainly metabolized to NE-098 and NE-152+163mix by CYP3A4. The intrinsic clearance for their formations in HIM was 3.2 and 14.9 times less than those in HLM, respectively, and no formation of NE-125 was observed in HIM. These results strongly suggest that CYP2D6 is the predominant form for NE-100 metabolism in the human liver in in vivo conditions (K(m)>>S) and the liver plays a more important role than does the small intestine in the first pass metabolism.

Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate.

Amodiaquine (AQ) metabolism to N-desethylamodiaquine (DEAQ) is the principal route of disposition in humans. Using human liver microsomes and two sets of recombinant human cytochrome P450 isoforms (from lymphoblastoids and yeast) we performed studies to identify the CYP isoform(s) involved in the metabolism of AQ. CYP2C8 was the main hepatic isoform that cleared AQ and catalyzed the formation of DEAQ. The extrahepatic P450s, 1A1 and 1B1, also cleared AQ and catalyzed the formation of an unknown metabolite M2. The K(m) and V(max) values for AQ N-desethylation were 1.2 microM and 2.6 pmol/min/pmol of CYP2C8 for recombinant CYP2C8, and 2.4 microM and 1462 pmol/min/mg of protein for human liver microsomes (HLMs), respectively. Relative contribution of CYP2C8 in the formation of DEAQ was estimated at 100% using the relative activity factor method. Correlation analyses between AQ metabolism and the activities of eight hepatic P450s were made on 10 different HLM samples. Both the formation of DEAQ and the clearance of AQ showed excellent correlations (r(2) = 0.98 and 0.95) with 6alpha-hydroxylation of paclitaxel, a marker substrate for CYP2C8. The inhibition of DEAQ formation by quercetin was competitive with K(i) values of 1.96 for CYP2C8 and 1.56 microM for HLMs. Docking of AQ into the active site homology models of the CYP2C isoforms showed favorable interactions with CYP2C8, which supported the likelihood of an N-desethylation reaction. These data show that CYP2C8 is the main hepatic isoform responsible for the metabolism of AQ. The specificity, high affinity, and high turnover make AQ desethylation an excellent marker reaction for CYP2C8 activity.

Relative Contribution of CYP3A to Amitriptyline Clearance in Humans: In Vitro and In Vivo Studies

The relative contribution of cytochrome P450 3A (CYP3A) to the oral clearance of amitriptyline in humans has been assessed using a combination of in vitro approaches together with a clinical pharmacokinetic interaction study using the CYP3A-selective inhibitor ketoconazole. Lymphoblast-expressed CYPs were used to study amitriptyline N-demethylation and E-10 hydroxylation in vitro. The relative activity factor (RAF) approach was used to predict the relative contribution of each CYP isoform to the net hepatic intrinsic clearance (sum of N-demethylation and E-10 hydroxylation). Assuming no extrahepatic metabolism, the model-predicted contribution of CYP3A to net intrinsic clearance should equal the fractional decrement in apparent oral clearance of amitriptyline upon complete inhibition of the enzyme. This hypothesis was tested in a clinical study of amitriptyline (50 mg, p.o.) with ketoconazole (three 200 mg doses spaced 12 hours apart) in 8 healthy volunteers. The RAF approach predicted CYP2C19 to be the dominant contributor (34%), with a mean 21% contribution of CYP3A (range: 8%-42% in a panel of 12 human livers). The mean apparent oral clearance of amitriptyline in 8 human volunteers was decreased from 2791 ml/min in the control condition to 2069 ml/min with ketoconazole. The average 21% decrement (range: 2%-40%) was identical to the mean value predicted in vitro using the RAF approach. The central nervous system (CNS) sedative effects of amitriptyline were slightly greater when ketoconazole was coadministered, but the differences were not statistically significant. In conclusion, CYP3A plays a relatively minor role in amitriptyline clearance in vivo, which is consistent with in vitro predictions using the RAF approach.

Differential N-Demethylation of l-α-Acetylmethadol (LAAM) and norLAAM by Cytochrome P450s 2B6, 2C18, and 3A4

Incubation of l-α-acetylmethadol (LAAM) or norLAAM with cDNA-expressed P450s 3A4, 2B6, and 2C18 produced significant N-demethylation products. P450s 2C19, 2C8, 3A5, 2C9, 3A7, 1A1, and 2D6 (norLAAM only), also produced detectable product. Coexpression of cytochrome b5 enhanced LAAM N-demethylation, most dramatically for 3A4, but had marginal effects on norLAAM N-demethylation. Modeling total liver metabolism using immunoquantification and relative activity factors of P450s suggests contributions of P450 3A4 > 2B6 > 2C18, with the importance of 2B6 to 2C isozymes enhanced by relative activity factors. The ratio of dinorLAAM to norLAAM plus dinorLAAM formed from LAAM did not exceed 20%, and was isozyme and cytochrome b5 coexpression dependent. This ratio decreased with concentration with 3A4, but was relatively constant for 2B6 and 2C18. The human liver microsomes substrate-concentration response was similar to cDNA-expressed 3A4, but the ratio was higher. Changes in the environment of cDNA-expressed 3A4 also effected the magnitude of the ratio, but not the concentration-dependent decrease. These studies show that the N-demethylation of LAAM and norLAAM is not restricted to P450 3A4, particularly P450s 2B6 and 2C18, and suggest that the mechanism of sequential metabolism for 3A4 differs from that of 2B6 and 2C18.

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.

The substrate-independence assumption of the relative activity factor (RAF) approach

The substrate-independence assumption of the relative activity factor (RAF) approach

Relative Quantities of Catalytically Active CYP 2C9 and 2C19 in Human Liver Microsomes: Application of the Relative Activity Factor Approach

The relative catalytic activities of CYP2C9 and CYP2C19 in human liver microsomes has been determined using the approach of relative activity factors (RAFs). Tolbutamide methylhydroxylation and S-mephenytoin 4′-hydroxylation were used as measures of CYP2C9 and CYP2C19 activity, respectively. The kinetics of these reactions were studied in human liver microsomes, in microsomes from human lymphoblastoid cells, and in insect cells expressing CYP2C9 and CYP2C19. RAFs were calculated as the ratio of Vmax (reaction velocity at saturating substrate concentrations) in human liver microsomes of the isoform-specific index reaction divided by the Vmax of the reaction catalyzed by the cDNA expressed isoform. RAFs were also determined for SUPERMIX, a commercially available mixture of cDNA expressed human drug metabolizing CYPs formulated to achieve a balance of enzyme activities similar to that found in human liver microsomes. Lymphoblast RAF2C9 in human liver microsomes ranged from 54 to 145 pmol CYP/mg protein (mean value: 87), while a value of 251 pmol CYP/mg protein was obtained for SUPERMIX. Insect cell RAF2C9 in human liver microsomes ranged from 1.6 to 143 pmol CYP/mg protein (mean value: 49), while a value of 201 pmol CyP/ mg protein was obtained for SUPERMIX. Both lymphoblast and insect cell RAF2C19 in human liver microsomes ranged from 4 to 45 pmol CYP/mg protein (mean values: 29 and 28, respectively), while a value of 29 pmol CYP/mg protein was obtained for SUPERMIX. The nature of the cDNA expression system used had no effect on the kinetic parameters of CYP2C9 as a tolbutamide methylhydroxylase, or of CYP2C19 as a S-mephenytoin hydroxylase. However insect cell expressed CYP2C19 (which includes oxidoreductase) had substantially greater activity as a tolbutamide methylhydroxylase when compared to lymphoblast expressed CYP2C19. The ratio of mean lymphoblast-determined RAF2C9 to RAF2C19 in human livers was 3.0 (range 1.6–17.9; n=10), while this ratio for SUPERMIX was 8.6. The ratio of mean insect cell-determined RAF2C9 to RAF2C19 in human livers was 1.7 (range 0.04–16.2; n=10), while this ratio for SUPERMIX was 7.0. Neither ratio is in agreement with the 20:1 ratio of immuno-quantified levels of CYP2C9 and 2C19 in human liver microsomes reported in previous studies. SUPERMIX may contain catalytically active CYP2C9 in levels higher than those in human liver microsomes.

Involvement of CYP1A2 in mexiletine metabolism.

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. 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). 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 CYPIA2 to both mexiletine p- and 2-hydroxylase activities was 7-30% in human liver microsomes. However, the Km values of the expressed CYP1A2 (approximately 15 microM) were almost identical with those of the expressed CYP2D6 (approximately 22 microM) and human liver microsomes. 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.