Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

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 K_i 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 (logD_7.0), but few studies have examined the relationship between logD_7.0 values and the inhibition of drug-metabolizing enzymes. Therefore, in the present study, we investigated the correlation between the logD_7.0 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 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).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Metabolism of mexazolam by CYP3A4. Metabolites in brackets are the postulated intermediates that have not yet been isolated.

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 logD_7.0 values of the inhibitors. In addition, we compared and evaluated the HMG-CoA reductase inhibitors in terms of potential drug interactions.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Structures of HMG-CoA reductase inhibitors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

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 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 µmol of potassium phosphate buffer (pH 7.4), 2 µmol of MgCl₂, 0.5 µmol of NADP, 5 µmol of glucose 6-phosphate, and 0.4 units of glucose-6-phosphate dehydrogenase. To the enzyme source preincubated at 37°C for 3 min, mexazolam 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 µM 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 K_m, V_max, and n:

(1)

where V₀ is the initial formation rate, V_max is the maximum metabolic rate, S is the substrate concentration, K_m is the substrate concentration showing a half-maximal velocity, and n is the Hill coefficient. The intrinsic metabolic clearance (CL_int) was evaluated using the following equation:

(2)

For immunoinhibition studies, human liver microsomes (pooled from 10 male or 10 female subjects, 10 mg of protein/ml, 10 µl) were first incubated with antiserum or corresponding control serum (total, 25 µl) at room temperature for 30 min. The mixture was added with the cofactor solution and mexazolam solution (final concentration, 20 µM), 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 µM) was coincubated with various HMG-CoA reductase inhibitors [50 to 400 µM pravastatin (acid form); 20 to 200 µM simvastatin acid, fluvastatin (acid form), atorvastatin (acid form), and cerivastatin (acid form); and 2 to 20 µM simvastatin (lactone form)] in the enzyme source mixture.

(3)

(4)

Measurement of the logD_7.0 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 centrifugation, and the drug concentrations in each layer were determined by HPLC as described in Table 1.

(5)

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

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 anti-CYP3A4 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.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effects of anti-CYP sera on the formation of M-1 from mexazolam in male (a) and female (b) human liver microsomes. Pooled human liver microsomes (10 male subjects or 10 female subjects, 0.1 mg of protein/10 µl) 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 µM) at 37°C for 2 min. Results (mean ± S.E.) were based on triplicate determinations. , anti-CYP1A; , anti-CYP2C; , anti-CYP2D6; , anti-CYP3A4.

View larger version (14K): [in this window] [in a new window]   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. , female; , male. Solid lines, theoretical curves calculated using the obtained values of Km, Vmax, and n.

Influence of HMG-CoA Reductase Inhibitors on Mexazolam Metabolism in Human Liver Microsomes. The K_i values for the 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 HMG-CoA reductase inhibitors. Also, there were no significant differences in the K_i 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).

View larger version (34K): [in this window] [in a new window]   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 µM) 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 µM; simvastatin, 5-20 µM; simvastatin Na+, fluvastatin, atorvastatin, and cerivastatin, 20-200 µM). , mexazolam 5 µM; , mexazolam 10 µM; , mexazolam 20 µM; , mexazolam 50 µM; V0, M-1 formation rate (nmol/min/mg). Solid lines, theoretical curves calculated using the obtained values of Km, Vmax, n, and Ki.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effects of HMG-CoA reductase inhibitors on the formation of M-1 from mexazolam in female human liver microsomes. Mexazolam (20 µM) 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 µM). Control activities were obtained in the absence of HMG-CoA reductase inhibitors. Results (mean ± S.E.) were based on triplicate determinations. , acid form; , lactone form. Solid lines, theoretical curves calculated using the obtained values of Km, n, and Ki.

Correlation between logD_7.0 and K_i for CYP3A4 Inhibition of HMG-CoA Reductase Inhibitors. Table 4 summarizes the logD_7.0 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 logD_7.0 values for other HMG-CoA reductase inhibitors (Watanabe, 1990). The logD_7.0 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 logD_7.0 values compared with their corresponding acid forms. The difference in logD_7.0 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.

View larger version (9K): [in this window] [in a new window]   Fig. 7.   logD7.0 of HMG-CoA reductase inhibitor and Ki for mexazolam metabolism in human liver microsomes. Ki values were calculated from data shown in Fig. 6 according to eq. 4. , acid form; , lactone form. Solid line, linear regression line.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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 Eadie-Hofstee 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 (logD_7.0 value of the lactone forms was 2.54 units higher on average). Among the acid forms, only pravastatin (acid) showed a negative logD_7.0 value, while all the other HMG-CoA reductase inhibitors, such as fluvastatin (acid), atorvastatin (acid), and cerivastatin (acid), had positive logD_7.0 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 K_i values for the inhibition of CYP3A4 activity were plotted against the logD_7.0 values of HMG-CoA reductase inhibitors for every lactone and acid form to determine the correlation between logD_7.0 and K_i 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.