Introduction

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Among the human liver cytochrome P450 (P450¹) isozymes, CYP3A4 is present in the highest concentration and responsible for the metabolism of various kinds of drugs. For this reason, it is considered an important isozyme for the investigation of drug interactions via P450. In a previous study, we reported that mexazolam, a benzodiazepine antianxiety agent, was metabolized by CYP3A4 (Ono et al., 1993). Using mexazolam as a probe, the inhibition of CYP3A4 activity by lactone or acid forms of HMG-CoA reductase inhibitors was investigated in a series of in vitro studies using human liver microsomes. Each of the HMG-CoA reductase inhibitors, except for pravastatin, (simvastatin, simvastatin acid, lovastatin, fluvastatin, atorvastatin, and cerivastatin) was found to inhibit mexazolam metabolism in human liver competitively (Ishigami et al., 2001a).

Although the inhibition of CYP3A4/5 by HMG-CoA reductase inhibitors can now be tested directly in man, if the question had been raised preclinically, the choice of a suitable in vivo animal model would be of primary importance, since interspecies differences in drug disposition are well known (Nelson et al., 1996). Sex differences have also been observed in liver content and activity of P450 isozymes (Kato and Kamataki, 1982; Kamataki et al., 1983), providing another important reason why results obtained in experimental animals do not always reflect drug interactions observed in humans. Therefore, we carried out an investigation to see whether the inhibition of mexazolam metabolism by HMG-CoA reductase inhibitors observed in human liver microsomes could occur in both male and female rats, the most commonly used animal species in pharmacokinetic studies. We previously found that the in vitro metabolism of simvastatin was inhibited by itraconazole in female rat liver microsomes but not in male rat liver microsomes (Ishigami et al., 2001b). Although female rat liver contains CYP3A at an extremely low level (Cooper et al., 1993), its activity is considered to be strongly inhibited by itraconazole. On the other hand, male rat liver contains much higher levels of CYP2C11 than CYP3A (Cooper et al., 1993), and CYP2C11 seems to be mainly responsible for the metabolism of simvastatin, which is not inhibited by itraconazole. Thus, there has been speculation that the sex difference in the inhibition of simvastatin metabolism by itraconazole may be attributable to both the sex difference in P450 isozymes responsible for simvastatin metabolism and the difference in the inhibition potency of itraconazole against CYP3A and CYP2C11.

In the present study, taking into account the sex difference in the drug interactions observed between simvastatin and itraconazole, the inhibition of mexazolam metabolism by HMG-CoA reductase inhibitors (Fig. 1) was investigated in an in vitro system using female and male rat liver microsomes, and the results were compared with those obtained in humans to identify an appropriate animal model for the study of drug interaction via CYP3A4 inhibition. In addition, the inhibition of mexazolam metabolism by HMG-CoA reductase inhibitors and itraconazole was also investigated under the condition in which the content of P450 isozymes was changed, using liver microsomes of rats treated with dexamethasone, which is known to induce CYP3A in rats (Ghosal et al., 1996a).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Chemical structures of HMG-CoA reductase inhibitors

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Chemicals and Reagents. Pravastatin (acid), pravastatin lactone, simvastatin (lactone), simvastatin acid (Na salt), fluvastatin (acid), atorvastatin (acid), cerivastatin (acid), itraconazole, mexazolam, and a metabolite of mexazolam (M-1; Fig. 2) used in the present study were synthesized at Sankyo Co., Ltd. (Tokyo, Japan). Anti-rat P450 antisera preparations (anti-rat CYP2C11 prepared from goat serum and anti-rat CYP3A2 prepared from rabbit serum) were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). All other chemicals and reagents used were commercially available and of reagent grade.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Metabolism of mexazolam in rats.

Preparation of Rat Liver Microsomes. Rat liver microsomes were prepared from male and female Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan) according to standard methods. To each isolated liver sample was added 10 mM phosphate buffer containing potassium chloride at a concentration of 1.15%, and the resulting mixture was homogenized and then centrifuged at 9,000g for 20 min. The supernatant was centrifuged again at 105,000g for 1 h, and phosphate buffer containing glycerol was added to the obtained pellet to prepare a microsomal suspension. Liver microsomes were also prepared in the same manner as described above from livers of female and male rats, 24 h after a single intraperitoneal administration of dexamethasone (100 mg/kg, corn oil solution) (Dex-treated rats).

Metabolism of Mexazolam in Rat Liver Microsomes. An NADPH-generating system containing 2.5 mM NADP, 25 mM glucose 6-phosphate, 2 units glucose-6-phosphate dehydrogenase, and 10 mM MgCl₂ was added to rat liver microsomes (0.2 mg of protein/ml) in a total volume of 0.2 ml, and the resulting mixtures were preincubated at 37°C for 3 min. Then 2 µl of an ethanol solution of mexazolam was added to each reaction mixture to make concentrations of 5 to 100 µM. After incubation at 37°C for 2 min, 0.4 ml of methanol was added followed by vortex mixing to stop the reaction and centrifugation at 19,000g for 3 min. The amount of mexazolam metabolite (M-1; Fig. 2) in the supernatants was analyzed by high-performance liquid chromatography (HPLC). For the HPLC operating conditions, a Deverosil ODS-UG-5 column (250 × 4.6 mm; Nomura Chemical Co., Ltd., Aichi, Japan), a mobile phase of acetonitrile/50 mM phosphate buffer (pH 8.0) (55:45, v/v), a flow rate of 1 ml/min, and a wavelength at 235 nm for UV measurement were used.

Kinetic Analysis of Mexazolam Metabolism. The formation of a metabolite from mexazolam by rat liver microsomes (pooled liver microsomes from five male or five female rats) was fitted to eq. 1 using a nonlinear least-squares regression program (WinNonlin; Scientific Consulting, Inc., Apex, NC), and the K_m and V_max values were calculated:
where V₀, V_max, S, and K_m represent the initial rate of metabolism, maximum rate of metabolism, substrate concentration, and Michaelis constant, respectively. Intrinsic metabolic clearance (CL_int) was calculated by the following equation:

Inhibition of Mexazolam Metabolism by Anti-P450 Antisera. To identify the enzymes responsible for the metabolism of mexazolam, inhibition experiments using anti-rat P450 antisera were performed. Anti-rat P450 antisera or corresponding control sera (0-0.25 mg of IgG, 25 µl) were added to rat liver microsomes (pooled from five male rats or five female rats, 10 mg of protein/ml, 10 µl) separately, and the resulting mixtures were preincubated at room temperature for 30 min. Then, as described above for the metabolism of mexazolam in rat liver microsomes, an NADPH-generating system was added to each mixture, and the resulting solutions were preincubated at 37°C for 3 min. Subsequently, an ethanolic solution of mexazolam was added (final concentration, 20 µM mexazolam) and incubated at 37°C for 2 min (0.2 mg of protein/ml; volume of reaction mixture, 0.5 ml). The reaction was stopped by the addition of 1 ml of methanol and the amount of metabolite (M-1) formed in the supernatants obtained after centrifugation was determined by HPLC.

Inhibition of Mexazolam Metabolism by HMG-CoA Reductase Inhibitors and Itraconazole. To 0.2 ml of each of male and female rat liver microsomal preparations (0.2 mg of protein/ml), 2 µl of an ethanol solution of each of the HMG-CoA reductase inhibitors [pravastatin (acid), pravastatin lactone, simvastatin acid, fluvastatin (acid), atorvastatin (acid), and cerivastatin (acid), 20 to 200 µM; and simvastatin (lactone), 2 to 20 µM] was added separately. The resulting mixtures were preincubated at 37°C for 3 min, and an ethanol solution of mexazolam (2 µl) was added to each microsomal preparation to give a final concentration of 10 to 50 µM. The amount of metabolite, M-1, formed in each reaction mixture was determined in the same manner as described above for the metabolism of mexazolam in rat liver microsomes. Inhibition by itraconazole was also examined in the same manner as mentioned above by adding 2 µl of a dimethylacetamide solution of itraconazole (0.1-1 µM) to each of the male and female rat liver microsomal preparations, separately.

	Results

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Effects of Anti-P450 Antisera on Mexazolam Metabolism in Rat Liver Microsomes. The mexazolam metabolism in male rat liver microsomes was inhibited by about 80% after the addition of anti-CYP2C11 antiserum (0.25 mg as IgG), whereas it was scarcely affected by the addition of anti-CYP3A2 antiserum (Fig. 3a). On the other hand, the mexazolam metabolism in female rat liver microsomes was inhibited by about 80% after the addition of anti-CYP3A2 antiseram (0.25 mg as IgG) but was scarcely affected by the addition of anti-CYP2C11 antiserum (Fig. 3b). In the Dex-treated rats, however, the mexazolam metabolism in liver microsomes was inhibited by about 80% in male rats and by about 90% in female rats, after the addition of anti-CYP3A2 antiseram (0.25 mg as IgG). In addition, following the addition of anti-CYP2C11 antiserum (0.25 mg as IgG), the metabolism in male and female rats was also inhibited by about 40% and by about 50%, respectively (Fig. 4, a and b).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effects of anti-P450 antisera on the formation of M-1 from mexazolam in male (a) and female (b) rat liver microsomes. Pooled rat liver microsomes (5 male rats or 5 female rats, 0.1 mg of protein/10 µl) were preincubated for 30 min at room temperature with anti-rat P450 antisera (0.05 to 0.25 mg as IgG) or control sera, preincubated for 3 min at 37°C with an NADPH-generating system, and incubated with mexazolam (20 µM) at 37°C for 2 min. Results (mean ± S.E.) were based on triplicate determinations. , anti-CYP2C11; , anti-CYP3A2.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effects of anti-P450 antisera on the formation of M-1 from mexazolam in Dex-treated male (a) and female (b) rat liver microsomes. Pooled rat liver microsomes (5 Dex-treated male rats or 5 Dex-treated female rats, 0.1 mg of protein/10 µl) were preincubated for 30 min at room temperature with anti-rat P450 antisera (0.05 to 0.25 mg as IgG) of anti-rat P450 antisera or control sera, preincubated for 3 min at 37°C with an NADPH-generating system, and incubated with mexazolam (20 µM) at 37°C for 2 min. Results (mean ± S.E.) were based on triplicate determinations. , anti-CYP2C11; , anti-CYP3A2.

Evaluation of Kinetic Parameters for Mexazolam Metabolism in Rat Liver Microsomes. The concentration dependence of mexazolam metabolism in control and Dex-treated rat liver microsomes was examined by Eadie-Hofstee plots in Fig. 5. As can be seen, saturation was reached in the formation of mexazolam metabolite, M-1. The calculated kinetic parameters of K_m and V_max are summarized in Table 1. In the control rats, the calculated K_m values in female and male rats were almost identical, but the V_max value and the CL_int in male rats were about 11 times higher than those in female rats. In the Dex-treated rats as well, the calculated K_m values in the female and male rats were almost identical, and no difference was observed between the control and Dex-treated rats. The V_max value in the Dex-treated female rats was about 1.3 times higher than that in the Dex-treated male rats, and the V_max values in the Dex-treated female and male rats were higher than those in the control female and male rats by about 39- and 2.7-fold, respectively. The intrinsic clearance values in Dex-treated female and male rats were also higher than those obtained in the control female and male rats by about 37- and 4.0-fold, respectively.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Eadie-Hofstee plots for the formation of M-1 from mexazolam in control (a) and Dex-treated (b) rat liver microsomes. Mexazolam was incubated for 2 min at 37°C with pooled rat liver microsomes (5 rats, 0.2 mg of protein/ml). Each value represents the mean ± S.E. of triplicate determinations. , female; , male. Solid lines represent the theoretical lines calculated using the obtained values of Km and Vmax.

Effects of HMG-CoA Reductase Inhibitors on Mexazolam Metabolism in Rat Liver Microsomes. The K_i values of various HMG-CoA reductase inhibitors for mexazolam metabolism in female and male rat liver microsomes were obtained from Dixon plots (Fig. 6, a and b) and summarized in Table 2. Mexazolam metabolism in female rat liver microsomes was not inhibited by pravastatin (acid) but was inhibited competitively by other HMG-CoA reductase inhibitors (Fig. 6b). The order of inhibition potency of these HMG-CoA reductase inhibitors observed, in decreasing order, was simvastatin (lactone), pravastatin (lactone), atorvastatin (acid), cerivastatin (acid), fluvastatin (acid), and simvastatin acid. On the other hand, mexazolam metabolism in male rat liver microsomes was inhibited by all the HMG-CoA reductase inhibitors except simvastatin acid and pravastatin (acid) (Fig. 6a). The order of inhibition potency in decreasing order was simvastatin (lactone), atorvastatin (acid), cerivastatin (acid), fluvastatin (acid), and pravastatin (lactone), demonstrating that the order of inhibition potency differed between female and male rats. Furthermore, the HMG-CoA reductase inhibitors were less potent as inhibitors of mexazolam metabolism in male rat liver microsomes than in female rat liver microsomes (Table 2). In the Dex-treated female and male rat liver microsomes, mexazolam metabolism was inhibited competitively by simvastatin (lactone), pravastatin (lactone), and simvastatin acid but was scarcely affected by pravastatin (acid) (Fig. 7, a and b).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Dixon plots for the inhibition of M-1 formation from mexazolam in male (a) and female (b) rat liver microsomes by HMG-CoA reductase inhibitors. Mexazolam (5-50 µM) was incubated for 2 min at 37°C with rat liver microsomes (one male rat or one female rat, 0.2 mg of protein/ml) in the presence or absence of HMG-CoA reductase inhibitors [pravastatin (acid), 50-400 µM; simvastatin (lactone), 5-20 µM; pravastatin lactone, simvastatin acid, fluvastatin (acid), atorvastatin (acid), and cerivastatin (acid), 20-200 µM]. , 5 µM mexazolam; , 10 µM mexazolam; , 20 µM mexazolam; , 50 µM mexazolam. V0, M-1 formation rate (nanomoles per minute per milligram of protein)

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Dixon plots for the inhibition of M-1 formation from mexazolam in Dex-treated male (a) and Dex-treated female (b) rat liver microsomes by simvastatin (lactone), simvastatin acid, pravastatin lactone, and pravastatin (acid). Mexazolam (5-50 µM) was incubated for 2 min at 37°C with pooled rat liver microsomes (5 Dex-treated male rats or 5 Dex-treated female rats; 0.2 mg of protein/ml) in the presence or absence of simvastatin acid, pravastatin lactone, and pravastatin (acid) (20-200 µM); and simvastatin (lactone) (5-20 µM). , 5 µM mexazolam; , 10 µM mexazolam; , 20 µM mexazolam; , 50 µM mexazolam. V0, M-1 formation rate (nanomoles per minute per milligram of protein)

Effects of Itraconazole on Mexazolam Metabolism in Rat Liver Microsomes. The formation of mexazolam metabolite in control female rat liver microsomes was considered to be inhibited by the dimethylacetamide used to dissolve the itraconazole, and the amount of metabolite formed was less than the detection limit. The mexazolam metabolism in male rat liver microsomes was not inhibited by itraconazole (Fig. 8a). On the other hand, in the Dex-treated female and male rat liver microsomes, mexazolam metabolism was noncompetitively inhibited by itraconazole, with K_i values of 0.693 µM in female rats and 0.877 µM in male rats (Fig. 8, b and c).

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Dixon plots for the inhibition of M-1 formation from mexazolam in male (a), Dex-treated male (b), and Dex-treated female (c) rat liver microsomes by itraconazole. Mexazolam (5-50 µM) was incubated for 2 min at 37°C with pooled rat liver microsomes (5 male rats, 5 Dex-treated male rats, or 5 Dex-treated female rats; 0.2 mg of protein/ml) in the presence or absence of itraconazole (0.1-1 µM). , 5 µM mexazolam; , 10 µM mexazolam; , 20 µM mexazolam; , 50 µM mexazolam. V0, M-1 formation rate (nanomoles per minute per milligram of protein)

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have previously reported that mexazolam is metabolized mainly by CYP3A4 in humans (Ono et al., 1993) and that this metabolism by human liver microsomes is inhibited by a variety of HMG-CoA reductase inhibitors (Ishigami et al., 2001a). In the present study, we investigated the inhibition of mexazolam metabolism in male and female rat liver microsomes in vitro by HMG-CoA reductase inhibitors and compared the results with those previously reported in humans to identify an appropriate animal model for the study of drug interaction via CYP3A4 inhibition. Experiments using anti-rat P450 antisera demonstrated that the P450 isozymes responsible for mexazolam metabolism in rat liver microsomes differ between female and male rats. In the male rat liver microsomes, CYP2C11 was the major contributor to mexazolam metabolism, whereas in the female rat liver microsomes, the CYP3A isoforms were the main contributors (Fig. 3).

In mature female rat liver, CYP3A2 and CYP3A1 are reported to be expressed at extremely low levels (Cooper et al., 1993). Therefore, the significantly lower V_max value for mexazolam metabolism in female rats than that in male rats may be due to the low expression level of the CYP3A isoforms responsible for mexazolam metabolism in female rats. On the other hand, CYP2C11, the male rat-specific P450 isozyme is expressed in the liver to a greater extent than the CYP3A isoforms, leading to a much higher level of mexazolam metabolism in male than in female rats. However, it is not possible to draw a definitive conclusion based on our limited findings as to whether the substrates of CYP3A4 in humans serve as the substrates for CYP2C11 in the male rat and for CYP3A in the female rat without exception. Antisera against rat CYP2C11 and CYP3A have recently become commercially available, and we believe that with their growing use, an increasing number of examples similar to mexazolam will be reported in the future, as we have previously found with simvastatin (Ishigami et al., 2001b).

As shown in the results (Fig. 6, a and b), mexazolam metabolism in rat liver microsomes was inhibited competitively by HMG-CoA reductase inhibitors. Taking simvastatin as a representative HMG-CoA reductase inhibitor and plotting its inhibition data for both male and female rats according to the Eadie-Hofstee method, the resulting plot demonstrated that the mode of inhibition was of the competitive type rather than of the noncompetitive or mixed type, since the lines intersected the y-axis. A Dixon plot also indicated that the mode of inhibition was competitive.

The finding that mexazolam metabolism was not inhibited by simvastatin acid in male rats but was inhibited in female rats with a K_i value of 81 µM further suggests that simvastatin acid exhibits an inhibitory effect on the CYP3A isoforms but not on CYP2C11. Mexazolam metabolism in human liver microsomes was inhibited competitively by simvastatin acid with K_i values of 23 (female) to 31 µM (male) (Ishigami et al., 2001a). Based on the present findings, the results obtained in female rats rather than male rats more accurately reflect the situation in humans, as far as the in vitro inhibition of mexazolam metabolism by simvastatin acid is concerned. In addition, a difference was also observed in the inhibition potency of other inhibitors against mexazolam metabolism between female and male rats, as seen by the lower K_i values obtained in female rats.

Another interesting finding was that more potent inhibition was observed for the lactone forms of simvastatin and pravastatin, compared with the corresponding acid forms. This result suggests that compounds with a higher lipophilicity exhibit a higher inhibition potency against CYP3A4 in humans, although the correlation between lipophilicity and inhibition potency among the acid forms was found to be relatively low compared with that in humans (Table 2).

Mexazolam metabolism in male rat liver microsomes was not inhibited by simvastatin acid, unless the rats were treated with Dex (K_i value, 60 µM). Thus, Dex-treatment made the male rat microsomes respond similarly to human microsomes with respect to simvastatin acid inhibition. The inhibition potency of other inhibitors in the Dex-treated male rats tended to be greater than that in the control male rats. Mexazolam metabolism in Dex-treated rats involved mainly the CYP3A isoforms in both female and male rats, although the CYP2C11 isozyme also appeared to participate in mexazolam metabolism in Dex-treated rats (Fig. 4). It has been reported that CYP3A1, CYP3A2, and CYP2C11 are induced by dexamethasone in female rats, whereas in male rats, CYP3A1 and CYP3A2 are induced, but CYP2C11 is reduced by dexamethasone (Ghosal et al., 1996a). These findings are consistent with the results obtained in the present study.

Mexazolam metabolism in male rats was not inhibited by itraconazole, whereas in the Dex-treated rats, mexazolam metabolism was inhibited by itraconazole in both female and male rats. Furthermore, we previously found that itraconazole did not inhibit simvastatin metabolism in male rats but inhibited it in female rats (Ishigami et al., 2001b), which was consistent with the results obtained in the present study. Taking into account that the midazolam metabolism mediated by CYP3A2 in male rats (Ghosal et al., 1996b) was inhibited by itraconazole (Yamano et al., 1999), the sex difference in the inhibition by itraconazole, like simvastatin acid, may be due to the considerably higher inhibition potency of itraconazole against CYP3A than CYP2C11.

In conclusion, since species and sex differences are observed in P450 isozymes, appropriate experimental conditions that take into account P450 isozymes responsible for the drug metabolism in question will be indispensable for the investigation of drug interactions using rats as a model animal for humans.