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0090-9556/01/2903-282–288$3.00 DRUG METABOLISM AND DISPOSITION Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics DMD 29:282–288, 2001 Vol. 29, No. 3 223/886405 Printed in U.S.A. A COMPARISON OF THE EFFECTS OF 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A (HMG-CoA) REDUCTASE INHIBITORS ON THE CYP3A4-DEPENDENT OXIDATION OF MEXAZOLAM IN VITRO MICHI ISHIGAMI, TOMOYO HONDA, WATARU TAKASAKI, TOSHIHIKO IKEDA, TORU KOMAI, KIYOMI ITO, AND YUICHI SUGIYAMA Drug Metabolism and Pharmacokinetics Research Laboratories and Product Strategy Department, Sankyo Co., Ltd., Shinagawa-ku, Tokyo, Japan (M.I., T.H., W.T., T.I., T.K.); School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo, Japan (K.I.); and Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan (Y.S.) (Received August 29, 2000; accepted December 5, 2000) This paper is available online at http://dmd.aspetjournals.org HMG-CoA reductase inhibitors can be divided into two groups: those administered as the prodrug, i.e., the lactone form (e.g., simvastatin and lovastatin), and those administered in the active form, i.e., the acid form (e.g., pravastatin, fluvastatin, atorvastatin, and cerivastatin). In this study, the influence of the lactone and acid forms of various HMG-CoA reductase inhibitors on metabolism by CYP3A4, a major cytochrome P450 isoform in human liver, was investigated by determining the in vitro inhibition constant (Ki value) using an antianxiety agent, mexazolam, as a probe substrate. In human liver microsomes, all the lactone forms tested inhibited the oxidative metabolism of mexazolam more strongly than did the acid forms, which have lower partition coefficient (logD7.0) values. In addition, the degree of inhibition of mexazolam metabolism tended to increase with an increasing logD7.0 value of the HMG-CoA reductase inhibitors among the lactone and acid forms. In particular, pravastatin (acid form), which has the lowest logD7.0 value, failed to inhibit CYP3A4 activity. Taking account of the lipophilicity of the inhibitors, in conjunction with the CYP3A4inhibitory activity, could be very useful in predicting drug interactions between substrates of CYP3A4 and HMG-CoA reductase inhibitors. Drug interactions can be classified roughly into two types. In one case, the pharmacological effects or side effects of particular drugs are altered by concomitant administration of other drugs. In the other case, the effects of concomitant drugs are altered by the first drugs. In either case, the drug interactions have often been evaluated by the changes in plasma levels of drugs in clinical situations. In the case of HMG-CoA1 reductase inhibitors, plasma levels of lactone-form prodrugs simvastatin and lovastatin are reported to be increased 10 times or more by the concomitant administration of an antifungal agent, itraconazole, a potent inhibitor of CYP3A4 (Varhe et al., 1994), one of the isoforms of cytochrome P450 (Neuvonen and Jalava, 1996; Neuvonen et al., 1998). The possibility has also been discussed of predicting the drug interaction with HMG-CoA reductase inhibitors. It was considered that the increase in the plasma levels of the prodrug type, i.e., the lactone forms of HMG-CoA reductase inhibitors, caused by the concomitant administration of itraconazole may be due to inhibition of the CYP3A4-mediated metabolism of simvastatin and lovastatin (Vickers et al., 1990; Wang et al., 1991; Prueksaritanont et al., 1997) by itraconazole. We have determined the in vitro Ki values of itraconazole using human liver microsomes and applied them to a pharmacokinetic model (Ito et al., 1998), together with pharmacokinetic parameters for itraconazole. As a result, we have found that the predicted increase in plasma levels of simvastatin by concomitant administration of itraconazole agreed fairy well with the observed increase in clinical situations (Ishigami et al., 2001). However, as far as the water soluble pravastatin was concerned, when administered in the active acid form, there was no evidence suggesting any drug interaction with itraconazole either in clinical situations (Neuvonen and Jalava, 1996; Neuvonen et al., 1998) or in vitro (Ishigami et al., submitted). These results strongly suggest that pravastatin does not exhibit a drug interaction with itraconazole because its metabolism is not mediated by CYP3A4. As described above, most of the studies that investigated the cause of drug interactions have focused on the identification of the enzymes responsible for the metabolism of the tested drugs, and only a few studies have examined the relationship between drug interactions and the physicochemical properties of drugs. One of the most important physicochemical properties of a drug is its partition coefficient (logD7.0), but few studies have examined the relationship between logD7.0 values and the inhibition of drug-metabolizing enzymes. Therefore, in the present study, we investigated the correlation between the logD7.0 values of various HMG-CoA reductase inhibitors and the potential inhibition of P450. We selected CYP3A4 as the CYP isoform to be examined, since this isoform has been reported to be involved in the drug interaction with simvastatin. In the present study, we selected mexazolam, an anxiolytic benzodiazepine, as a substrate 1 Abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; V0, initial formation rate; logD7.0, partition coefficient; HPLC, high-performance liquid chromatography. Send reprint requests to: Yuichi Sugiyama, Professor, Graduate School of Pharmaceutical Sciences, University of Tokyo, 3-1,7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: [email protected] 282 Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017 ABSTRACT: 283 CYP3A4 INHIBITION BY HMG-CoA REDUCTASE INHIBITORS FIG. 1. Metabolism of mexazolam by CYP3A4. Metabolites in brackets are the postulated intermediates that have not yet been isolated. for CYP3A4. Previous studies with various P450 isoforms expressed in HepG2 cells demonstrated that mexazolam to M-1 via postulated intermediates (Fig. 1) is metabolized mainly by the CYP3A family (Ono et al., 1993). In the present study, we investigated the inhibitory effects of the lactone and acid forms of various HMG-CoA reductase inhibitors (Fig. 2) on CYP3A4 using mexazolam as a probe to monitor CYP3A4 activity, and we evaluated the correlation between the potential inhibition of CYP3A4 and the logD7.0 values of the inhibitors. In addition, we compared and evaluated the HMG-CoA reductase inhibitors in terms of potential drug interactions. Materials and Methods TABLE 1 HPLC condition for logD7.0 measurement of HMG-CoA reductase inhibitors Columns used were 150 ⫻ 4.6 mm. For mobile phase, 1 mM KH2PO4 (pH 7.0 NaOH): Acetonitrile was used in varying ratios as shown under Mobile Phase. The flow rate in all cases was 1 ml/min. HMG-CoA Reductase Inhibitor Pravastatin (acid) Atorvastatin (acid) Cerivastatin (acid) Fluvastatin (acid) Atorvastatin lactone Cerivastatin lactone Fluvastatin lactone Column Mobile Phase a Deverosil ODS-UG-5 Deverosil ODS-UG-5 Deverosil ODS-UG-5 Deverosil ODS-UG-5 Capcel pak C18b Capcel pak C18 Inertsil ODS-2c 80:20 65:35 60:40 65:35 50:50 30:70 65:35 Wavelength 238 238 238 238 237 237 254 nm nm nm nm nm nm nm a Chemicals and Reagents. Pravastatin (acid form), pravastatin lactone, simvastatin (lactone form), simvastatin acid (Na⫹ salt), lovastatin (lactone form), lovastatin acid (Na⫹ salt), fluvastatin (acid form), fluvastatin lactone, atorvastatin (acid form), atorvastatin lactone, cerivastatin (acid form), cerivastatin lactone, mexazolam, and a metabolite of mexazolam (M-1) were synthesized at Sankyo Co., Ltd. (Tokyo, Japan). Human liver microsomes were obtained from the International Institute for the Advancement of Medicine (Exton, PA). Polyclonal antibodies for human P450 (anti-CYP2C antiserum from goat, and anti-CYP1A, -CYP2D6, and -CYP3A4 antisera from rabbit) were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). All Deverosil: Nomura Chemical Co., Ltd. (Aichi, Japan). Capcel pak: Shiseido Co., Ltd. (Tokyo, Japan). Inertsil: GL Science Inc. (Tokyo, Japan). b c other chemicals and reagents used were of analytical grade and were obtained commercially. In Vitro Metabolism of Mexazolam. A final volume (0.2 ml) of a typical enzyme source mixture consisted of 0.04 mg of liver microsomal protein, 2 mol of potassium phosphate buffer (pH 7.4), 2 mol of MgCl2, 0.5 mol of NADP, 5 mol of glucose 6-phosphate, and 0.4 units of glucose-6-phosphate dehydrogenase. To the enzyme source preincubated at 37°C for 3 min, mex- Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017 FIG. 2. Structures of HMG-CoA reductase inhibitors. 284 ISHIGAMI ET AL. FIG. 3. Effects of anti-CYP sera on the formation of M-1 from mexazolam in male (a) and female (b) human liver microsomes. half-maximal velocity, and n is the Hill coefficient. The intrinsic metabolic clearance (CLint) was evaluated using the following equation: CL int ⫽ Vmax/Km FIG. 4. Eadie-Hofstee plots for the formation of M-1 from mexazolam in male and female human liver microsomes. Mexazolam was incubated for 2 min at 37°C with pooled human liver microsomes (10 male subjects or 10 female subjects, 0.2 mg of protein/ml). Each value represents the mean ⫾ S.E. of triplicate determinations. ⽧, female; 䡺, male. Solid lines, theoretical curves calculated using the obtained values of Km, Vmax, and n. azolam dissolved in ethanol (final concentration, 1%) was added, and the reaction was continued for 2 min at 37°C and then terminated by the addition of methanol (0.4 ml). Next, the reaction mixture was centrifuged at 19,000g for 3 min, and the resulting supernatant was analyzed for the mexazolam metabolite (M-1, Fig. 2) by an HPLC method. The HPLC conditions were as follows: column, Deverosil ODS-UG-5 (250 ⫻ 4.6 mm, Nomura Chemical Co., Ltd., Aichi, Japan); mobile phase, acetonitrile/50 mM phosphate buffer (pH 8.0) ⫽ 55/45; flow rate, 1 ml/min; and UV-detection wavelength, 235 nm. Kinetic Analysis of Mexazolam Metabolism. Mexazolam was tested at a final concentration range of 2 to 100 M in human liver microsomes to determine the kinetic parameters. The production of the mexazolam metabolite determined was fitted to the Hill equation (eq. 1) using a nonlinear regression program (WinNonlin, Scientific Consulting, Inc., Apex, NC) to estimate Km, Vmax, and n: V 0 ⫽ V max ⫻ Sn /共Kmn ⫹ Sn 兲 (1) where V0 is the initial formation rate, Vmax is the maximum metabolic rate, S is the substrate concentration, Km is the substrate concentration showing a (2) For immunoinhibition studies, human liver microsomes (pooled from 10 male or 10 female subjects, 10 mg of protein/ml, 10 l) were first incubated with antiserum or corresponding control serum (total, 25 l) at room temperature for 30 min. The mixture was added with the cofactor solution and mexazolam solution (final concentration, 20 M), and then a final volume of 0.5 ml was incubated in a similar manner as described under In Vitro Metabolism of Mexazolam. Finally, 1 ml of methanol was added to terminate the reaction, and after centrifugation the supernatant was subjected to HPLC. Inhibition of Mexazolam Metabolism by HMG-CoA Reductase Inhibitors. To define the type of inhibition by HMG-CoA reductase inhibitors on mexazolam metabolism, mexazolam (5–50 M) was coincubated with various HMG-CoA reductase inhibitors [50 to 400 M pravastatin (acid form); 20 to 200 M simvastatin acid, fluvastatin (acid form), atorvastatin (acid form), and cerivastatin (acid form); and 2 to 20 M simvastatin (lactone form)] in the enzyme source mixture. The 1/V0(⫹I) values obtained for each concentration of HMG-CoA reductase inhibitor were applied to Dixon plots, and the Ki values were obtained by simultaneous fitting using WinNonlin (eq. 3): 1/V 0共⫹I兲 ⫽ 1 ⫹ K mn /Sn I 䡠 K mn ⫹ Vmax Ki 䡠 Sn 䡠 Vmax (3) where “(⫹I)” represents the value after alteration by the drug-drug interaction, and S and I represent the concentration of substrate (mexazolam) and inhibitor (HMG-CoA reductase inhibitor), respectively. In addition, to compare the Ki values for lactone and acid forms of HMGCoA reductase inhibitors, HMG-CoA reductase inhibitors (0.1–200 M) and mexazolam (20 M) were incubated with human liver microsomes (pooled from 10 female subjects) for 2 min at 37°C, and the amount of the mexazolam metabolite M-1 formed was determined. The Ki value was calculated by fitting the data to the following equation using WinNonlin: V 0共⫹I兲/V 0 ⫽ K mn ⫹ Sn Kmn 䡠 共1 ⫹ I/Ki兲 ⫹ Sn (4) Measurement of the logD7.0 Value of HMG-CoA Reductase Inhibitors. Partition coefficients between phosphate buffer (pH 7.0, Britton Robinson Buffer) and 1-octanol for fluvastatin lactone, cerivastatin lactone, atorvastatin lactone, fluvastatin (acid form), cerivastatin (acid form), atorvastatin (acid form), and pravastatin (acid form) were determined by the flask-shaking method (OECD, 1981). Each drug was dissolved in 1-octanol-saturated buffer, and then the solution was mixed with buffer-saturated 1-octanol solution. After shaking for 30 min at 25°C, the 1-octanol and buffer layers were separated by Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017 Pooled human liver microsomes (10 male subjects or 10 female subjects, 0.1 mg of protein/10 l) were preincubated for 30 min at room temperature with 0.05 to 0.25 mg IgG of anti-human CYP sera or control sera, preincubated for 3 min at 37°C with NADPH-generating system, and then incubated with mexazolam (20 M) at 37°C for 2 min. Results (mean ⫾ S.E.) were based on triplicate determinations. 䡺, anti-CYP1A; Œ, anti-CYP2C; 〫, anti-CYP2D6; F, anti-CYP3A4. 285 CYP3A4 INHIBITION BY HMG-CoA REDUCTASE INHIBITORS TABLE 2 Kinetics of mexazolam metabolite formation in human liver microsomes The kinetic parameters Km, Vmax, and n (mean ⫾ calculated S.D.) were calculated from data shown in Fig. 4 according to eq. 1. The CLint was estimated by dividing Vmax by Km. Male Female Km Vmax M nmol/min/mg n 29.2 ⫾ 10.6 26.5 ⫾ 3.8 0.68 ⫾ 0.12 1.64 ⫾ 0.11 CLint ml/min/mg 1.34 ⫾ 0.26 1.30 ⫾ 0.10 0.023 0.062 CLint, intrinsic clearance. centrifugation, and the drug concentrations in each layer were determined by HPLC as described in Table 1. The distribution coefficients and logD7.0 values were calculated from the following equation: Distribution coefficients 共Dow兲 ⫽ Co/Cw, logD7.0 ⫽ log10Dow (5) in which CO and CW represent the concentration (mol/liter) of test drug in octanol and buffer layer, respectively. Results The Characteristics of Mexazolam Metabolism by Human Liver Microsomes. The formation of the mexazolam metabolite (M-1) in male and female human liver microsomes was inhibited by addition of antiCYP3A4 serum to the reaction mixture in a concentration-dependent manner, but was not affected by the addition of anti-CYP1A, -CYP2C or -CYP2D6 sera (Fig. 3, a and b). When 0.25 mg of IgG of anti-CYP3A4 serum was added, the M-1 formation in human liver microsomes was inhibited by about 80% in both males and females. The formation of M-1 at various concentrations of mexazolam in human liver microsomes was investigated to calculate the kinetic parameters for mexazolam metabolism. As shown in Fig. 4, an EadieHofstee plot showed a convex curve, and kinetic parameters such as Km, Vmax, and the Hill coefficient (n) are shown in Table 2. The Km and n values were similar for males and females, but the Vmax value in females was about 2.4 times higher than that in males. In addition, the mean intrinsic clearance (Vmax/Km) in females was about 2.6 times higher than that in males. Influence of HMG-CoA Reductase Inhibitors on Mexazolam Metabolism in Human Liver Microsomes. The Ki values for the Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017 FIG. 5. Dixon plots for the inhibition of M-1 formation from mexazolam in male (a) and female (b) human liver microsomes by HMG-CoA reductase inhibitors. Mexazolam (5–50 M) was incubated for 2 min at 37°C with human liver microsomes (one female subject or one male subject, 0.2 mg of protein/ml) in the absence or presence of HMG-CoA reductase inhibitors (pravastatin, 50 – 400 M; simvastatin, 5–20 M; simvastatin Na⫹, fluvastatin, atorvastatin, and cerivastatin, 20 –200 M). E, mexazolam 5 M; ⽧, mexazolam 10 M; 䡺, mexazolam 20 M; Œ, mexazolam 50 M; V0, M-1 formation rate (nmol/min/mg). Solid lines, theoretical curves calculated using the obtained values of Km, Vmax, n, and Ki. 286 ISHIGAMI ET AL. TABLE 3 Ki of various HMG-CoA reductase inhibitors for the inhibition of mexazolam metabolism by human liver microsomes Ki values were calculated from data shown in Fig. 5 according to eq. 3. HMG-CoA Reductase Inhibitor Km Ki Male Female No inhibition 91.9 ⫾ 8.8 3.57 ⫾ 0.48 31.9 ⫾ 4.8 112 ⫾ 14 56.8 ⫾ 5.6 18.7 ⫾ 3.6 No inhibition 90.8 ⫾ 26.4 3.25 ⫾ 0.70 23.0 ⫾ 4.4 90.9 ⫾ 15.0 63.3 ⫾ 11.8 25.1 ⫾ 3.3 Male Female No inhibition 10.7 ⫾ 0.7 10.0 ⫾ 2.1 8.57 ⫾ 1.27 10.3 ⫾ 1.0 9.78 ⫾ 1.05 6.53 ⫾ 1.04 No inhibition 10.8 ⫾ 1.3 12.1 ⫾ 0.6 14.2 ⫾ 0.7 15.0 ⫾ 0.8 10.7 ⫾ 0.5 13.8 ⫾ 0.4 M Pravastatin (acid) Pravastatin lactone Simvastatin (lactone) Simvastatin acid Fluvastatin (acid) Atorvastatin (acid) Cerivastatin (acid) inhibition of mexazolam metabolism in male and female human liver microsomes by HMG-CoA reductase inhibitors were determined by a Dixon plot (Fig. 5, a and b; Table 3). The formation of M-1 from mexazolam in human liver microsomes was not inhibited by pravastatin (acid form), but it was inhibited competitively by other HMGCoA reductase inhibitors. Also, there were no significant differences in the Ki values of various inhibitors between males and females. The extent of inhibition was in the following order: simvastatin (lactone form), cerivastatin (acid form), simvastatin acid, atorvastatin (acid form) and fluvastatin (acid form). The effects of HMG-CoA reductase inhibitors (lactone and acid forms) on the formation of M-1 in human liver microsomes (female) are shown in Fig. 6. In this figure, the extent of inhibition of the formation of M-1 was plotted against the concentrations of inhibitors, and the results were analyzed by regression analysis. The calculated Ki values are summarized in Table 4. When the effect on mexazolam metabolism in human liver microsomes was compared for various HMG-CoA reductase inhibitors, it was found that the inhibitory activity of the lactone forms was higher than that of the acid forms for all HMG-CoA reductase inhibitors tested. Correlation between logD7.0 and Ki for CYP3A4 Inhibition of HMG-CoA Reductase Inhibitors. Table 4 summarizes the logD7.0 values of HMG-CoA reductase inhibitors, including the values measured in this study [pravastatin (acid form), atorvastatin (acid form), atorvastatin lactone, fluvastatin (acid form), fluvastatin lactone, and cerivastatin lactone] and previously reported logD7.0 values for other HMG-CoA reductase inhibitors (Watanabe, 1990). The logD7.0 values of all HMG-CoA reductase inhibitors examined were positive, except for the negative value of pravastatin (acid form). All the lactone forms examined showed higher logD7.0 values compared with their corresponding acid forms. The difference in logD7.0 values between the lactone form and the corresponding acid form ranged from 2.9 to 1.9 units, and the mean difference was 2.54 units. The logD7.0 values of HMG-CoA reductase inhibitors and Ki values for CYP3A4 inhibition by HMG-CoA reductase inhibitors are shown in Table 4, and the correlation between logD7.0 and Ki values are plotted in Fig. 7. The extent of inhibition of mexazolam metabolism increased with the inhibitor’s lipophilicity among the acid forms, except for fluvastatin, and water-soluble pravastatin produced no inhibition at all. The same tendency of stronger inhibition with higher lipophilicity was observed among the lactone forms, and the Ki value of 110 M for pravastatin lactone was much higher than that of 10 M or less for atorvastatin lactone, cerivastatin lactone, lovastatin (lactone), simvastatin (lactone), and fluvastatin lactone. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017 FIG. 6. Effects of HMG-CoA reductase inhibitors on the formation of M-1 from mexazolam in female human liver microsomes. Mexazolam (20 M) was incubated for 2 min at 37°C with pooled human liver microsomes (10 female subjects, 0.2 mg of protein/ml) in the absence or presence of HMG-CoA reductase inhibitors (0.1–200 M). Control activities were obtained in the absence of HMG-CoA reductase inhibitors. Results (mean ⫾ S.E.) were based on triplicate determinations. 䡺, acid form; ⽧, lactone form. Solid lines, theoretical curves calculated using the obtained values of Km, n, and Ki. 287 CYP3A4 INHIBITION BY HMG-CoA REDUCTASE INHIBITORS TABLE 4 logD7.0 and Ki values of various HMG-CoA reductase inhibitors for the inhibition of mexazolam metabolism by female human liver microsomes Ki values (mean ⫾ calculated S.D.) were calculated from data shown in Fig. 6 according to eq. 4. Ki value logD7.0 Inhibitor Lactone Acid Lactone 2.42 4.40 3.91 3.65 4.2 5.2 ⫺0.47 1.88 1.51 1.75 1.53 2.32 115 ⫾ 9 2.13 ⫾ 0.14 2.98 ⫾ 0.18 7.10 ⫾ 0.39 2.54 ⫾ 0.12 2.18 ⫾ 0.10 Acid M Pravastatin Simvastatin Lovastatin Fluvastatin Atorvastatin Cerivastatin Ki values were calculated from data shown in Fig. 6 according to eq. 4. 䡺, acid form; ⽧, lactone form. Solid line, linear regression line. Discussion It has been reported from a study with cDNA-expressed human P450 isoforms (CYP1A2, -2A6, -2B6, -2C8, -2C9, -2D6, -2E1, -3A3, -3A4, and -3A5) in HepG2 cells that mexazolam is mainly metabolized by the CYP3A family (Ono et al., 1993). In the present study, the inhibitory activity by anti-CYP1A, -2C, -2D6, and -3A4 antisera on mexazolam metabolism in human liver microsomes was investigated, and it was shown that CYP3A4 was the main CYP isoform responsible for mexazolam metabolism (Fig. 3). Furthermore, an EadieHofstee plot for the formation of mexazolam metabolite, M-1, showed a convex curve (Fig. 4), which is characteristic of metabolism by CYP3A4 (Ueng et al., 1997). The metabolic activities (testosterone 6-hydroxylation) of pooled human liver microsomes used in the present study were 3.7 nmol/min/mg of protein and 2.1 nmol/min/mg of protein for female and male human liver microsomes, respectively, i.e., about 1.8 times higher in females than in males (data from International Institute for the Advancement of Medicine). These data are consistent with our finding that the intrinsic metabolic clearance of mexazolam was about 2.3 times higher in females than in males (Table 2). These results indicate that mexazolam is a suitable probe for monitoring CYP3A4 activity. HMG-CoA reductase inhibitors (Fig. 1) are classified into the following two groups: those administered as a prodrug, i.e., the lactone form (simvastatin, lovastatin, etc.) and those administered in the active form, i.e., the acid form (pravastatin, fluvastatin, atorvastatin, cerivastatin, etc.). The inhibition of CYP3A4 activity by the lactone forms is much higher than that by the acid forms. This correlated with the higher lipophilicity of the lactone form (logD7.0 value of the lactone forms was 2.54 units higher on average). Among the acid forms, only pravastatin (acid) showed a negative logD7.0 value, while all the other HMG-CoA reductase inhibitors, such as fluvastatin (acid), atorvastatin (acid), and cerivastatin (acid), had positive logD7.0 values, indicating the high lipophilicity of these compounds. This may explain the finding that only pravastatin (acid) did not inhibit CYP3A4 activity (Table 4). The Ki values for the inhibition of CYP3A4 activity were plotted against the logD7.0 values of HMG-CoA reductase inhibitors for every lactone and acid form to determine the correlation between logD7.0 and Ki values (Fig. 7). Consequently, with the exception of fluvastatin (acid) and pravastatin lactone, the extent of inhibition of CYP3A4 activity increased with the inhibitor’s lipophilicity (Fig. 7). Although pravastatin lactone and cerivastatin (acid) have a similar lipophilicity, the inhibition by pravastatin was lower than that by cerivastatin (acid), possibly because the pyridine in cerivastatin (acid) may play a key role in increasing the ability to inhibit CYP3A4 activity, or because the decalin ring structure and 6⬘-hydroxy group in pravastatin lactone may be responsible for reducing the inhibition activity. Simvastatin (lactone), lovastatin (lactone), atorvastatin (acid), and cerivastatin (acid), which were reported to be the substrates for CYP3A4 (Vickers et al., 1990; Wang et al., 1991; Boberg et al., 1997; Prueksaritanont et al., 1997; Boyd et al., 2000), inhibit mexazolam metabolism. Simvastatin (lactone), lovastatin (lactone), and atorvastatin (acid) have been reported to exhibit a significant drug interaction with itraconazole, a well known inhibitor of CYP3A4, when both drugs are coadministered (Neuvonen and Jalava, 1996; Kantola et al., 1998; Neuvonen et al., 1998). Therefore, the potential for drug interactions between HMG-CoA reductase inhibitors and other drugs may be related to the metabolism of HMG-CoA reductase inhibitors by CYP3A4. Fluvastatin (acid) has been reported to be an inhibitor of CYP2C9 (Transon et al., 1995, 1996), and in the present study it was shown that fluvastatin (acid) also inhibited microsomal CYP3A4 activity. Among the HMG-CoA reductase inhibitors examined in the present study, inhibitors with a higher lipophilicity generally tended to be stronger inhibitors of CYP3A4. P450s are involved in the conversion of a lipophilic drug to a more hydrophilic metabolite and excrete it from the body. Taking this into consideration, the present finding may reasonably be explained by the fact that lipophilic drugs have a high affinity for P450s and, as a result, produce inhibition of P450. Therefore, pravastatin is free from a drug interaction due to its high water solubility and its absence of metabolism by P450. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017 FIG. 7. logD7.0 of HMG-CoA reductase inhibitor and Ki for mexazolam metabolism in human liver microsomes. No inhibition 69.6 ⫾ 5.2 179 ⫾ 18 289 ⫾ 44 189 ⫾ 30 46.3 ⫾ 2.1 288 ISHIGAMI ET AL. References Boberg M, Angerbauer R, Fey P, Kanhai WK, Karl W, Kern A, Ploschke J and Radtke M (1997) Metabolism of cerivastatin by human liver microsomes in vitro. Characterization of primary metabolic pathways and of cytochrome P450 isozymes involved. Drug Metab Dispos 25:321– 331. Boyd RA, Stern RH, Stewart BH, Wu X, Reyner EL, Zegarac EA, Randinitis EJ and Whitfield L (2000) Atorvastatin coadministration may increase digoxin concentrations by inhibition of intestinal P-glycoprotein-mediated secretion. J Clin Pharmacol 40:91–98. Ishigami M, Uchiyama M, Kondo T, Iwabuchi H, Inoue S, Takasaki W, Ikeda T, Komai T, Ito K and Sugiyama Y (2001) Inhibition of in vitro metabolism of simvastatin by itraconazole in humans and prediction of in vivo drug-drug interactions. Pharm Res, in press. Ito K, Iwatsubo T, Kanamitsu S, Ueda K, Suzuki H and Sugiyama Y (1998) Prediction of pharmacokinetic alterations caused by drug-drug interactions: Metabolic interaction in the liver. Pharmacol Rev 50:387– 411. Kantola T, Kivisto KT and Neuvonen PJ (1998) Effect of itraconazole on pharmacokinetics of atorvastatin. Clin Pharmacol Ther 64:58 – 65. Neuvonen PJ and Jalava KM (1996) Itraconazole drastically increases plasma concentrations of lovastatin and lovastatin acid. Clin Pharmacol Ther 60:54 – 61. Neuvonen PJ, Kantola T and Kivisto KT (1998) Simvastatin but not pravastatin is very susceptible to interaction with the CYP3A4 inhibitor itraconazole. Clin Pharmacol Ther 63:332–341. OECD (1981) Section 1. Physical chemical properties, TG No. 107, Partition coefficient (n-Octanol/Water), in OECD Guidelines for the Testing of Chemicals, Organization for Economic Co-operation and Development. Ono S, Hatanaka T, Iwabuchi H, Ikeda T and Aoyama T (1993) Mexazolam metabolism by cDNA expressed human cytochrome P450 forms using a recombinant vaccinia virus (Abstract). Xenobio Metab Dispos 8:866. Prueksaritanont T, Gorham LM, Ma B, Liu L, Yu X, Zhao JJ, Slaughter DE, Arison BH and Vyas KP (1997) In vitro metabolism of simvastatin in humans [SBT] identification of metabolizing enzymes and effect of the drug on hepatic P450s. Drug Metab Dispos 25:1191–1199. Transon C, Leemann T and Dayer P (1996) In vitro comparative inhibition profiles of major human drug metabolising cytochrome P450 isozymes (CYP2C9, CYP2D6 and CYP3A4) by HMG-CoA reductase inhibitors. Eur J Clin Pharmacol 50:209 –215. Transon C, Leemann T, Vogt N and Dayer P (1995) In vivo inhibition profile of cytochrome P450TB (CYP2C9) by (⫹/⫺)-fluvastatin. Clin Pharmacol Ther 58:412– 417. Ueng YF, Kuwabara T, Chun YJ and Guengerich FP (1997) Cooperativity in oxidations catalyzed by cytochrome P450 3A4. Biochemistry 36:370 –381. Varhe A, Olkkola KT and Neuvonen PJ (1994) Oral triazolam is potentially hazardous to patients receiving systemic antimycotics ketoconazole or itraconazole. Clin Pharmacol Ther 56:601– 607. Vickers S, Duncan CA, Vyas KP, Kari PH, Arison B, Prakash SR, Ramjit HG, Pitzenberger SM, Stokker G and Duggan DE (1990) In vitro and in vivo biotransformation of simvastatin, an inhibitor of HMG-CoA reductase. Drug Metab Dispos 18:476 – 483. Wang RW, Kari PH, Lu AY, Thomas PE, Guengerich FP and Vyas KP (1991) Biotransformation of lovastatin. IV. Identification of cytochrome P450 3A proteins as the major enzymes responsible for the oxidative metabolism of lovastatin in rat and human liver microsomes. Arch Biochem Biophys 290:355–361. Watanabe H (1990) Physicochemical properties of pravastatin, lovastatin, mevastatin and simvastatin. Annu Rep Sankyo Res Lab 42:117–120. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017