Cytochrome P4502B6 and 2C9 do not metabolize midazolam: kinetic analysis and inhibition study with monoclonal antibodies
N. Hamaoka,
Y. Oda,
I. Hase and
A. Asada
Department of Anesthesiology and Intensive Care Medicine, Osaka City University Medical School, 1-5-7 Asahimachi, Abeno-ku, Osaka 545-8586, Japan*Corresponding author
Accepted for publication: October 13, 2000
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Abstract
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We determined the contribution of cytochrome P450 (CYP) isoforms to the metabolism of midazolam by kinetic analysis of human liver microsomes and CYP isoforms and by examining the effect of chemical inhibitors and monoclonal antibodies against CYP isoforms in vitro. Midazolam was metabolized to 1'-hydroxymidazolam (1'-OH MDZ) by human liver microsomes with a MichaelisMenten constant (Km) of 4.1 (1.0) (mean (SD)) µmol litre1 and a maximum rate of metabolism (Vmax) of 5.5 (1.1) nmol min1 mg protein1 (n=6). Of the nine representative human liver CYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, three (CYP2B6, 3A4 and 3A5) showed midazolam 1'-hydroxylation activity, with Kms of 40.7, 1.7 and 3.0 µmol litre1, respectively, and Vmax values of 12.0, 3.3 and 13.2 nmol min1 nmol P4501, respectively (n=4). Midazolam 1'-hydroxylation activity of human liver microsomes correlated significantly with testosterone 6ß-hydroxylation activity, a marker of CYP3A activity (r2=0.77, P=0.0001), but not with S-mephenytoin N-demethylation activity, a marker of CYP2B6 activity (r2<0.01, P=0.84) (n=11). Troleandomycin and orphenadrine, chemical inhibitors of CYP isoforms, inhibited the formation of 1'-OH MDZ by human liver microsomes. Monoclonal antibody against CYP3A4 inhibited the formation of 1'-OH MDZ by 79%, whereas monoclonal antibody against CYP2B6 had no effect on midazolam 1'-hydroxylation by human liver microsomes (n=5). These results indicate that only CYP3A4, but not CYP2B6 or CYP2C, is involved in the metabolism of midazolam in vitro.
Br J Anaesth 2001; 86: 5404
Keywords: hypnotics benzodiazepine, midazolam; enzymes, cytochrome P4502B6; enzymes, cytochrome P4503A4
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Introduction
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Midazolam is an intravenous anaesthetic used widely in clinical practice. Numerous studies of the metabolism of midazolam have been performed. Microsomal cytochrome P450 (CYP) 3A in liver is the predominant CYP isoform involved in the metabolism of this agent.13 Metabolism of midazolam is inhibited by concomitantly administered agents such as antimycotics, calcium channel blocking agents and opioids, resulting in significant prolongation of amnesia and disturbance of psychomotor performance.46 Inhibition of CYP3A4 activity by these agents has been considered responsible for inhibiting midazolam metabolism.4 7 According to a recent in vivo and in vitro study, propofol decreases the clearance of midazolam by competitively inhibiting CYP3A activity.8 Since competitive inhibition of one drug by another drug is commonly observed when the two drugs are metabolized by the same CYP isoform,9 the findings of inhibition of metabolism of midazolam by propofol suggest that the same CYP isoforms may be involved in the metabolism of these anaesthetics. Several lines of evidence, however, have shown that propofol is metabolized predominantly by CYP2B6 or CYP2C9, and that CYP3A4 does not metabolize propofol (Y.Oda, personal communication and ref. 10), suggesting that CYP2B6 and/or 2C9 as well as CYP3A4 may be involved in the metabolism of midazolam. According to a recent study,11 both midazolam 1'-hydroxylation activity and immunoquantified CYP3A4 levels significantly correlated with CYP2B6 level in human liver microsomes, suggesting that genetic expression of CYP2B6 and CYP3A4 is related, and that CYP2B6 contributes to the metabolism of midazolam.
Although there have been several studies of the metabolism of midazolam by CYP isoforms,2 7 11 few studies have compared the metabolism of midazolam by CYP2B6 and 2C9 with that by other CYP isoforms, possibly because the human liver contains less of these CYP isoforms than other isoforms.11 12 CYP isoforms belonging to the CYP2B and 2C families are involved in the metabolism of various agents and are inducible by commonly used agents such as barbiturates and steroids.13 In addition, the pharmacokinetics of agents metabolized by CYP2B and 2C are significantly influenced by the levels of these isoforms in liver microsomes,14 15 suggesting that examination of the roles of CYP2B6 and 2C9 in the metabolism of midazolam may shed light on the pharmacokinetic interactions of midazolam with other agents, as well as for testing the possibility of induction and inhibition of metabolism of midazolam by various agents. The objective of this study was to determine the contribution of CYP isoforms including CYP2B6 and 2C9 to the metabolism of midazolam in vitro by kinetic analysis and by examination of the effects of chemical inhibitors and monoclonal antibodies on the metabolism of midazolam by liver microsomes.
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Materials and methods
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Materials
The experimental protocol was approved by the Institutional Human Investigational Committee of Osaka City University Medical School. Midazolam and its metabolites, 1'-hydroxymidazolam (1'-OH MDZ), 4'-hydroxymidazolam (4'-OH MDZ) and 1',4'-hydroxymidazolam (1',4'-OH MDZ), were kind gifts from Hoffmann-La Roche Ltd (Nutley, NJ, USA). Testosterone, 6ß-hydroxytestosterone, S-mephenytoin and its N-demethylated metabolite, nirvanol, were obtained from Ultrafine Chemicals (Manchester, UK). Troleando mycin and orphenadrine were obtained from Sigma Chemical Co. (St Louis, MO, USA). Human liver microsomes were obtained from the International Institute for the Advancement of Medicine (Scranton, PA, USA). These microsomes were prepared from liver samples of kidney donors by differential centrifugation; the protein content was measured using Lowrys method,16 with bovine serum albumin as standard. P450 content was determined from the differential spectrum of carbon monoxide-reduced versus oxidized microsomes as described by Omura and Sato.17 Specific contents of P450 in human liver microsomes were 0.330.66 nmol mg protein1. Recombinant human P450s expressed in human lymphoblast cells with cytochrome P450 reductase were obtained from Gentest (Woburn, MA, USA). These P450s were supplied as microsomes. Monoclonal antibodies against CYP3A4 and 2B6 were obtained from Gentest. The selectivity and inhibitory activity of these antibodies were confirmed as described in the manufacturers instructions. In our preliminary experiments, monoclonal antibody against CYP2B6 inhibited S-mephenytoin N-demethylation by human liver microsomes by 75% at 1.0 mg IgG (mg of microsomal protein)1 and midazolam 1'-hydroxylation by recombinant CYP2B6 by 85% at 0.5 mg IgG (mg of microsomal protein)1. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was obtained from the Oriental Yeast Co. (Tokyo, Japan). C18 columns (TSK gel ODS-120T, 4.6x150 mm) were obtained from Tosoh Corp. (Tokyo, Japan). Other reagents and organic solvents were from Wako Pure Chemical Industries (Osaka, Japan).
Assay of midazolam metabolism
The metabolism of midazolam by liver microsomes from six individuals was measured as reported previously.7 There was 100 µg of microsomal protein in each 500 µl incubation mixture. When metabolism of midazolam was measured with each of the CYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, 30 pmol of P450 was used instead of human liver microsomes (n=4). The midazolam concentration was between 0.2 and 20 µmol litre1. Incubation times were 2 and 10 min for experiments with microsomes and CYP isoforms, respectively. The P450 chemical inhibitors, orphenadrine and troleandomycin, were used at a concentration of 100 µmol litre1, which is sufficient to suppress >80% of CYP2B and CYP3A activities in human liver microsomes.7 18 19 The Michaelis Menten constant (Km) and maximum rate of metabolism (Vmax) for the formation of 1'-OH MDZ from midazolam were determined by linear regression from Lineweaver Burk double-reciprocal plots. When antibodies against CYP isoforms were used, microsomes and antibodies were preincubated in test tubes at room temperature for 20 min, followed by addition of midazolam and NADPH. The metabolites were extracted and measured by high-performance liquid chromatography (HPLC) as previously reported.7 The lower limit of quantification of 1'-OH MDZ, 4'-OH MDZ and 1',4'-OH MDZ was 20 nmol litre1. Intra-assay and inter-assay coefficients of variations were <4.8% and 7.0%, respectively.
Assay of S-mephenytoin N-demethylation and testosterone 6ß-hydroxylation
S-Mephenytoin N-demethylation and testosterone 6ß-hydroxylation activities of human liver microsomes from 11 individuals were determined as measures of CYP2B6 and 3A4 activities, respectively, following the method described previously.11 20 S-mephenytoin and testosterone were used at 1 mmol litre1 and 500 µmol litre1, respectively; these concentrations were chosen based on previously reported Km values.20 21 The content of microsomal protein was 400 mg litre1 and incubation time was 20 min for both substrates. The lower limit of detection of nirvanol and 6ß-hydroxytestosterone was 50 nmol litre1 and 1 µmol litre1, respectively. Correlations between midazolam 1'-hydroxylation and S-mephenytoin N-demethylation and testosterone 6ß-hydroxylation activity were determined by linear regression analysis using Statview J version 4.5 (Abacus Concepts, Inc., Berkley, CA, USA). All values are presented as mean (SD); P<0.05 was considered statistically significant.
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Results
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Metabolism of midazolam by human liver microsomes and CYP isoforms
When midazolam was incubated with human liver microsomes, 1'-OH MDZ was the only metabolite detected by HPLC. The total amount of residual midazolam and 1'-OH MDZ formed in the incubation mixture was approximately equal to the total amount of midazolam added to the reaction, suggesting that 1'-OH MDZ was the predominant midazolam metabolite. Rates of formation of 1'-OH MDZ were linear up to 15 and 30 min for the substrate concentrations used with microsomes and P450s, respectively. Single-phase straight curves were obtained from the LineweaverBurk plots for 1'-hydroxylation of midazolam by microsomes, with a mean Km and Vmax of 4.1 (SD 1.0) µmol litre1 and 5.5 (1.1) nmol min1 mg protein1, respectively (n=6). These values are comparable to those reported previously.1 3 7 Of the nine representative CYP isoforms examined, only CYP2B6, 3A4 and 3A5 catalysed midazolam 1'-hydroxylation at a substrate concentration of 10 µmol litre1. The mean rates of formation of 1'-OH MDZ by CYP2B6, 3A4 and 3A5 were 2.6 (0.7), 2.6 (0.3) and 10.2 (0.6) nmol min1 nmol P4501, respectively (n=4). CYP2B6 had midazolam metabolic activity comparable to that of CYP3A4, suggesting that CYP2B6 may contribute to the metabolism of midazolam by human liver microsomes.
The Kms of CYP2B6, 3A4 and 3A5 for midazolam 1'-hydroxylation were 40.7, 1.7, 3.0 µmol litre1, respectively, and the corresponding Vmax values were 12.0, 3.3, 13.2 nmol min1 nmol P4501, respectively (n=4) (Figure 1). These values were obtained from LineweaverBurk plots showing the correlation between the reciprocal concentration of midazolam and the mean formation rates of 1'-OH MDZ obtained in four different experiments. Km values for CYP3A4 and 3A5 were comparable to those obtained with human liver microsomes. The Vmax for CYP3A5 was higher than that for CYP3A4; however, CYP3A5 is found in only 2030% of adult human livers, indicating that the contribution of CYP3A5 to midazolam 1'-hydroxylation in liver microsomes is small.13 Both Km and Vmax for CYP2B6 were higher than those for CYP3A4.

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Fig 1 LineweaverBurk plots of midazolam 1'-hydroxylation by recombinant CYP2B6, 3A4 and 3A5. Midazolam (0.520 µmol litre1) was incubated at 37°C for 10 mins with recombinant P450 (30 pmol) and 0.4 mmol litre1 reduced NADP in 0.1 mol litre1 potassium phosphate buffer (pH 7.4) in a final volume of 500 µl. Each plot depicts the mean of four experiments.
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Correlation of midazolam 1'-hydroxylation activity with S-mephenytoin N-demethylation and testosterone 6ß-hydroxylation activities
There was 13-fold variation between individuals in the midazolam 1'-hydroxylation activity of human liver microsomes obtained from 11 individuals at a substrate concentration of 10 µmol litre1. Preliminary studies indicated that the rates of formation of nirvanol from S-mephenytoin (1 mmol litre1) and of 6ß-hydroxytestosterone from testosterone (500 µmol litre1) were linear for
1.0 h incubation when microsomal protein content was 400 mg litre1 and for
2.0 g litre1 microsomal protein when the incubation time was 20 min. The mean S-mephenytoin N-demethylation and testosterone 6ß-hydroxylation activity of human liver microsomes was 339 (185) pmol min1 mg protein1 and 5.4 (2.5) nmol min1 mg protein1, respectively. These values were comparable to those reported previously.20 21 There was a significant correlation between midazolam 1'-hydroxylation and testosterone 6ß-hydroxylation, a marker of CYP3A4 activity (r2=0.77, P=0.0001), whereas no correlation was found between midazolam 1'-hydroxylation and S-mephenytoin N-demethylation, a marker of CYP2B6 activity (r2<0.01, P=0.84) (Figure 2).

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Fig 2 Correlations between midazolam 1'-hydroxylation and S-mephenytoin N-demethylation, a marker of CYP2B6 activity (A) and testosterone 6ß-hydroxylation, a marker of CYP3A4 activity (B) with human liver microsomes obtained from 11 individuals. Concentrations of midazolam, S-mephenytoin and testosterone were 10 µmol litre1, 1 mmol litre1 and 500 µmol litre1, respectively. Each plot is the mean of three experiments.
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Inhibition of midazolam metabolism by orphenadrine, troleandomycin and monoclonal antibodies
Both orphenadrine, an inhibitor of CYP2B6, and troleandomycin, an inhibitor of CYP3A4, inhibited the formation of 1'-OH MDZ from midazolam (10 µmol litre1) by human liver microsomes by 54 (3.6)% and 95 (0.8)%, respectively (n=6). Monoclonal antibody against CYP3A4 inhibited the formation of 1'-OH MDZ from midazolam (10 µmol litre1) by human liver microsomes by 79 (5.1)%, whereas antibody against CYP2B6 had no effect (n=5) (Figure 3). Since the results obtained with antibodies are not consistent with those obtained with chemical inhibitors of CYP isoforms, chemical inhibitors of CYP isoforms were added to the reaction mixture containing CYP2B6 or 3A4 to elucidate the specificities of those chemical inhibitors to each CYP isoform at a midazolam concentration of 10 µmol litre1. Troleandomycin completely inhibited the formation of 1'-OH MDZ by CYP3A4 and decreased the formation of 1'-OH MDZ by CYP2B6 by 26 (range 2230)%. Orphenadrine decreased the formation of 1'-OH MDZ by CYP2B6 by 67 (range 6568)% and that by CYP3A4 by 62 (range 6066)% (mean of three experiments). These results demonstrated that orphenadrine inhibits CYP3A4 as well as CYP2B6 activities and is not a selective inhibitor of CYP2B6.

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Fig 3 Effects of monoclonal antibodies against CYP2B6 and 3A4 on midazolam 1'-hydroxylation. Human liver microsomes (100 µg of protein) obtained from five individuals were preincubated with antibodies at room temperature for 20 min followed by incubation with midazolam (10 µmol litre1) and reduced NADP (0.4 mmol litre1) in 0.1 mol litre1 potassium phosphate buffer (pH 7.4) at 37°C for 2 min in a final volume of 500 µl. Midazolam 1'-hydroxylation activity without antibodies was 6.8 (2.0) nmol min1 mg protein1. Each plot depicts the mean±SD of five experiments.
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Discussion
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In this study, we have shown that neither CYP2C9 nor 2C19 metabolizes midazolam, consistent with the findings of our previous study.7 Of the representative CYP isoforms in liver, CYP2B6 as well as 3A4 and 3A5 had midazolam 1'-hydroxylation activity. However, the Km value for midazolam 1'-hydroxylation by CYP2B6 was approximately 10-fold that with microsomes and CYP3A4. Midazolam 1'-hydroxylation activity of microsomes correlated significantly with testosterone 6ß-hydroxylation, a specific marker of CYP3A4, but not with S-mephenytoin N-demethylation, a specific marker of CYP2B6. Antibody against CYP2B6 had no effect on midazolam metabolism with human liver microsomes. These results suggest that CYP2B6 is not involved in the metabolism of midazolam. CYP isoforms which metabolize a substrate do not always contribute to the metabolism of the substrate by microsomes or in vivo. These findings have been reported previously.22 According to one study,11 CYP2B6 has midazolam 1'-hydroxylation activity and a correlation exists between midazolam 1'-hydroxylation and S-mephenytoin N-demethylation activity, suggesting coregulation of CYP2B6 and 3A4 activities in microsomes. In the present study, however, no relationships were found between CYP2B6 and 3A4 activities, although the reason for this is not clear. Our study also suggests that competitive inhibition of the metabolism of two substrates, such as midazolam and propofol, does not always imply that these two substrates are metabolized by the same CYP isoforms.
Antibody against CYP3A4 inhibited midazolam 1'-hydroxylation by approximately 80%. Orphenadrine has been used as a chemical inhibitor of CYP2B6.19 In this study, however, orphenadrine inhibited midazolam 1'-hydroxylation by CYP3A4 as well as that by CYP2B6. Orphenadrine could have inhibited the metabolism of midazolam by inhibiting CYP3A4 activity, and not by inhibiting CYP2B6 activity. These results suggest that orphenadrine lacks specificity as a selective CYP2B6 inhibitor at 100 µmol litre1. To determine the involvement of CYP2B6 in metabolism of a substrate, inhibition studies with characterized antibodies or examination of the correlation of metabolic ratios with typical substrates of CYP2B6 such as S-mephenytoin N-demethylation and 7-ethoxytrifluoromethylcoumarin would be required.21
In conclusion, midazolam is selectively metabolized by CYP3A4, but neither CYP2B6, 2C9 nor 2C19 is involved in the metabolism of midazolam in vitro.
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Acknowledgements
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This study was supported in part by the Fund for Medical Research from Osaka City University Medical Research Foundation and a Grant-in-Aid for Research from the Ministry of Education, Science and Culture of Japan, nos 09771183 and 11671517.
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