Abstract
Drugs with potential drug-drug interactions (DDIs) may have a limited scope of use and, at worst, may have to be withdrawn from the market. Therefore, during the drug discovery process it is important to select drug candidates with reduced potential for DDIs. In the present study, we evaluated the pharmacokinetics of simvastatin (SV), a typical substrate for cytochrome P450 (P450) 3A, and examined the DDI between SV and ketoconazole (KTZ), a P450 3A inhibitor, in monkeys. SV metabolism in monkey liver and intestinal microsomes was almost completely inhibited by addition of anti-P450 3A4 antiserum. A similar effect was seen in human microsomes, and the IC50 values of KTZ for inhibition of SV metabolism were similar in monkey and human samples. In vivo, there were no significant differences in the pharmacokinetic parameters of SV and SVA after i.v. administration of SV in the presence of KTZ compared with those in controls, probably because of the limited systemic exposure to KTZ. In contrast, the pharmacokinetics of SV and SVA after p.o. administration of SV were significantly influenced by the presence of KTZ, and Cmax and area under the plasma concentration-time curve were approximately 5 to 10 times higher than those after p.o. dosing with SV alone. The increases in systemic SV exposure caused by a concomitant p.o. dose of KTZ in monkeys were similar to those observed in clinical studies, which suggests that monkeys might be a suitable animal model in which to predict DDIs involving P450 3A inhibition.
Drug-drug interactions (DDIs) caused by inhibition of drug-metabolizing enzymes can increase the risk of adverse events associated with drug administration, and some drugs with marked DDIs have had to be withdrawn from the market. Therefore, the potential for drug candidates to cause DDIs is usually assessed at an early stage in the drug discovery process. Many methods have been described for quantitative prediction of in vivo DDIs from in vitro data obtained using preparations of human tissues (Iwatsubo et al., 1997; Ito et al., 1998, 2004; Yamano et al., 2001; Obach et al., 2006). These methods are widely accepted, but they tend to be used retrospectively, and they are not applicable in all cases. In particular, knowledge of an inhibitor concentration around a drug-metabolizing enzyme is required to predict DDIs in humans accurately, but this concentration is difficult to estimate at an early stage of drug development.
There is increased interest in DDIs in the intestine, and this has further complicated the prediction of DDIs. Several approaches based on cytochrome P450 (P450) 3A inhibition in the intestine have been proposed (Wang et al., 2004; Galetin et al., 2006, 2007; Obach et al., 2006), but predictions based on experimental data obtained in an in vivo animal model are limited because of substantial species differences in substrate specificities and inhibitor sensitivities of drug-metabolizing enzymes. Monkeys may be more appropriate animal models than rodents because of the similarities in functional activities and amino acid sequences of drug-metabolizing enzymes compared with those in humans (Komori et al., 1992; Shimada et al., 1997), and it has been reported that human pharmacokinetics can be predicted more accurately from results obtained in monkeys compared with those from other animals, including rats and dogs (Ward et al., 2001; Ward and Smith, 2004). In DDI studies, Tahara et al. (2006) have reported that human renal DDIs involving inhibition of drug transporters can be reproduced in monkeys, and we have evaluated the influence of ketoconazole (KTZ), a dual inhibitor of P450 3A and P-glycoprotein, on the pharmacokinetics of midazolam and fexofenadine (as model P450 3A and P-glycoprotein substrates, respectively) in monkeys. Based on this work, we suggested that in vivo studies in monkeys might be appropriate for prediction of intestinal DDIs in humans, because our results were comparable with those in humans (Ogasawara et al., 2007). We conducted the current study of DDIs of simvastatin (SV) in a monkey model to further examine the suitability of this animal species.
SV is a HMG-CoA reductase inhibitor that is widely used for treatment of hypercholesterolemia. It is administered orally as a lactone prodrug, which is converted reversibly to active simvastatin acid (SVA) by nonspecific carboxyesterases in the intestinal wall and liver and even without enzymes (Duggan and Vickers, 1990; Vickers et al., 1990). The benefits of SV are well established, but occasionally it causes adverse events in skeletal muscle, including myopathy and rhabdomyolysis, and an increased risk of these adverse events has been reported with concomitant use of drugs such as KTZ, cyclosporine, fibrates, and erythromycin (Gilad and Lampl, 1999; Gruer et al., 1999; Williams and Feely, 2002). SV undergoes extensive first-pass metabolism mediated by P450 3A, resulting in extremely poor oral bioavailability (less than 5%) (Lilja et al., 1998), and the U.S. Food and Drug Administration has suggested use of SV as a preferred in vivo probe with which to evaluate drug interactions related to P450 3A inhibition.
In the present study, we first determined the P450 isozyme involved in SV metabolism in monkey liver and intestinal microsomes. We then investigated the pharmacokinetics of SV after i.v. and p.o. administration to monkeys to clarify the hepatic and intestinal contributions to the overall first-pass metabolism. Finally, we evaluated the effects of KTZ on the pharmacokinetics of i.v. and p.o. administered SV to discriminate between hepatic and intestinal DDIs in monkeys.
Materials and Methods
Chemicals. SV, SVA, SV d6-form, and SVA d6-form were purchased from Toronto Research Chemicals (North York, ON, Canada). KTZ and polyethylene glycol 400 were purchased from Wako Pure Chemical Industries (Osaka, Japan). Reserpine was obtained from Nacalai Tesque (Kyoto, Japan). Ibuprofen sodium salt and methyl cellulose (400 cps) were obtained from Sigma-Aldrich (St. Louis, MO). Pooled liver and intestinal microsomes of human (liver microsomes pooled from 50 donors; intestinal microsomes pooled from 11 donors) and monkey (liver microsomes pooled from 5 animals; intestinal microsomes pooled from 7 animals) and anti-human P450 antisera preparations (anti-P450 2C8, anti-P450 2C9, and anti-P450 3A4) were provided by XenoTech, LLS (Lenexa, KS). All other reagents and solvents were of analytical grade and were commercially available.
Animals. Male cynomolgus monkeys, 3.0 to 4.5 kg, were supplied by the Yulin Hongfeng experimental animal's domesticating and breeding center (Guangxi, China) and Guangdong Zhaoquing Laboratory Animals Research Center Xiangang (Guangdong, China). Animals were housed in a temperature- and humidity-controlled room with a 12-h light/dark cycle. Animals were fed a standard animal diet (Teklad Global Certified 25% Protein Primate Diet; Harlan Sprague-Dawley, Indianapolis, IN); food was provided ad libitum except for during the overnight periods before dosing. Whenever overnight fasting was used, food was provided after an 8-h blood sample was obtained. All procedures for the animal experiments were approved by the Animal Ethics Committee of Tanabe Seiyaku.
In Vitro Inhibition Studies. Preliminary experiments were performed to determine incubation conditions showing a linearity of the disappearance of SV with respect to the protein concentration and incubation time. Furthermore, it was confirmed that the elimination half-life of SV was almost constant at SV concentrations less than 0.5 μM. All incubations were performed in duplicate. A typical incubation contained 0.08 M potassium phosphate (pH 7.4), 1 mM EDTA, 5 mM MgCl2, 1 mM NADPH, 0.5 μM SV, and microsomal protein in a final volume of 200 μl. The concentrations of microsomal protein in the reaction mixtures were as follows: human liver microsomes, 0.06 mg/ml; human intestinal microsomes, 0.2 mg/ml; monkey liver microsomes, 0.02 mg/ml; and monkey intestinal microsomes, 0.06 mg/ml. After preincubation at 37°C for 5 min, the reactions were initiated by the addition of the NADPH solution. After incubation at 37°C for 3 min, aliquots of 50 μl were removed and added to 50 μl of an acetonitrile and methanol mixture (1:1, v/v). The samples were spiked with 20 μl of internal standard solution containing 50 ng/ml ibuprofen. After vortexing and centrifugation, the supernatants were transferred to an HPLC vial to determine the SV concentration by HPLC analysis.
The effect of P450 3A-selective inhibitor on SV metabolism was investigated using KTZ. Each microsomal protein was incubated with SV (0.5 μM) in the presence of KTZ at the concentrations of 0.01, 0.05, 0.25, and 1.25 μM. The final concentration of organic solvent in the incubation mixture was 1% (v/v).
In immunoinhibition studies, human and monkey microsomes were preincubated at room temperature for 20 min with 50 or 200 μl/mg microsomal protein rabbit serum for human P450 2C8, 2C9, or 3A4 or control serum. The SV metabolic reaction and subsequent treatment were carried out as described above.
In Vivo Studies. The p.o. and i.v. dosing solutions of SV were prepared in 0.5% (w/v) methylcellulose aqueous vehicle and saline containing 60% (v/v) polyethylene glycol 400 vehicle, respectively. The dosing solution of KTZ was prepared in ultrapure water containing 0.01 M hydrochloric acid. The same four male cynomolgus moneys were used again, after a washout period of at least 2 weeks, to investigate the effects of KTZ on the SV pharmacokinetics. Immediately after p.o. administration of KTZ (20 mg/kg), SV was administered p.o. or i.v. to the monkeys. The dosages of SV were 20 mg/kg and 1 mg/kg for p.o. and i.v. administration, respectively. To obtain the control values of the pharmacokinetic parameters for SV and SVA, the vehicle for KTZ was administered p.o. immediately before p.o. and i.v. administration of SV. For all studies, blood was collected from the femoral vein at the desired time points and centrifuged at 1800g for 15 min at 4°C to obtain plasma.
Analytical Procedure.Determination of SV concentrations in the microsomal reaction mixtures. The SV concentrations in the microsomal reaction mixtures were measured using an Agilent 1100 system (Agilent Technologies, Palo Alto, CA). SV was eluted from a Capcell Pak C18 column (5-μm particle size, 4.6 × 150 mm; Shiseido, Tokyo, Japan) with a mobile phase of 0.025% (v/v) trifluoroacetic acid aqueous solution (pH 2.5) and acetonitrile. The flow rate was 1 ml/min, and the initial mobile phase was 50% of trifluoroacetate aqueous solution (0.025%) and 50% of acetonitrile. The acetonitrile concentration was increased linearly to 90% over 8 min and held at 90% for 2 min. The eluates were monitored at a wavelength of 240 nm. SV and the corresponding acid were eluted at 8.3 and 6.9 min, respectively. The concentrations of SV were determined by its peak area ratio relative to an internal standard with reference to a standard curve using the ChemiStation software (version 10.02; Agilent Technologies).
Determination of SV and SVA concentrations in plasma samples. Twenty microliters of 60% (v/v) acetonitrile aqueous solution, 20 μl of internal standard solution containing 50 ng/ml SV-d6 and SVA-d6, and 500 μl of 1 mM ammonium acetate buffer solution were added to 100-μl aliquots of the plasma samples. The mixtures were loaded on Oasis HLB extraction cartridges (Waters, Milford, MA), which were preactivated with 500 μl of methanol, water, and 1 mM ammonium acetate buffer solution. The cartridges were subsequently washed twice with 500 μl of 5% (v/v) methanol aqueous solution and then eluted twice with 100 μl of acetonitrile. One hundred microliters of the acetonitrile extracts were mixed with 100 μl of ammonium acetate buffer solution (1 mM), and aliquots of the mixtures were analyzed by liquid chromatography-tandem mass spectrometry. Chromatography was performed using an Inertsil ODS-3 column (5-μm particle size, 2.1 × 50 mm; GL Science, Tokyo, Japan) and a HPLC system consisting of a Shimazu 10A series (Shimazu, Kyoto, Japan) using a mobile phase consisting of 20% 1 mM ammonium acetate buffer solution and 80% acetonitrile. The mobile phase was delivered at a flow rate of 0.3 ml/min. The analyte and internal standard were detected using a Sciex API 4000 mass spectrometer (Applied Biosystems, Foster City, CA) in the positive ion mode for SV and SV-d6 and negative ion mode for SVA and SVA-d6 using a TurboIonspray source at 300°C. The collision gas was set at level 7. Mass transitions (m/z) monitored were 419 → 199 for SV, 435 → 319 for SVA, 425 → 199 for SV-d6, and 441 → 319 for SVA-d6 at collision energies of 17, –24, 17, and –24 eV, respectively. The lower limit of quantification was 0.1 ng/ml for both SV and SVA. The standard curves were linear from 0.1 to 200 ng/ml for both analytes. The between-day coefficients of variation (CV) with CV and relative error (RE) values for SV were 7.4 and –4.8% at 0.2 ng/ml (n = 8), 2.9 and 1.8% at 10 ng/ml (n = 8), and 3.5 and 0.9% at 160 ng/ml (n = 8). The between-day coefficients of variation with CV and RE values for SVA were 11.7 and 3.1% at 0.2 ng/ml (n = 8), 2.6 and 1.0% at 10 ng/ml (n = 8), and 2.4 and 0.4% at 160 ng/ml (n = 8).
Determination of KTZ concentrations in plasma samples. Ten microliters of acetonitrile and 10 μl of internal standard solution containing 1 μg/ml reserpine were added to 100-μl aliquots of the plasma samples. Aliquots of acetonitrile (200 μl) were added to the mixtures, and then they were stored at 4°C for at least 1 h. After centrifugation of 15,300g at 4°C for 5 min, 100 μl of the supernatants were added to the equal volume of 10 mM formate ammonium solution, and aliquots of the mixtures were analyzed by liquid chromatography-tandem mass spectrometry. Chromatography was performed using an Unison UK-C8 column (3-μm particle size, 2.0 × 75 mm; Silvertone Science, Philadelphia, PA) and a Shimazu 10A HPLC series system (Shimazu). The mobile phase consisted of 10 mM formate ammonium solution and acetonitrile. The flow rate was 0.3 ml/min, and the initial mobile phase was 80% formate ammonium solution (10 mM) and 20% acetonitrile. The acetonitrile concentration was increased linearly to 90% over 10 min. Detection of the analyte and internal standard was performed using a Sciex API 4000 mass spectrometer in the positive ion mode using a TurboIonspray source at 300°C. The collision gas was set at level 7. Mass transitions (m/z) monitored were 531 → 489 for KTZ and 609 → 195 for the internal standard (reserpine), respectively. The standard curves were linear from 0.1 to 100 ng/ml for KTZ. The between-day coefficients of variation with CV and RE values for KTZ were 9.3 and –3.8% at 0.3 ng/ml (n = 6), 3.4% and –1.1% at 10 ng/ml (n = 6), and 4.7% and –2.7% at 80 ng/ml (n = 6).
Data Analysis. The percentage activity of SV metabolism remaining was plotted against the range of KTZ concentrations on a semilog scale. The IC50 values were determined by nonlinear regression analysis with the Prism software package (version 3.02; GraphPad Software Inc., San Diego, CA).
Pharmacokinetic parameters were calculated in individual animals by noncompartmental analysis using WinNonlin Professional (version 4.0.1; Pharsight, Mountain View, CA). The maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) were directly recorded from experimental observations. The area under the plasma concentration-time curve (AUC) was calculated using the trapezoidal rule up to the last measurable concentrations. The bioavailability (F) was determined from the dose-corrected AUC after p.o. and i.v. administration. The components of SV availability were further predicted on the basis of the following equation: F = FABS · FG · FH, in which FABS refers to the fraction of the dose absorbed from the gut lumen, FG refers to the fraction of the dose not metabolized by intestinal metabolic enzymes, and FH refers to the fraction of the dose absorbed into the hepatic vein that escapes first-pass effect in the liver. FH was defined as follows: FH = 1 – ERH, in which ERH refers to the hepatic extraction ratio defined as the clearance of i.v. administered SV divided by the hepatic blood flow rate in monkeys (45 ml/min/kg) (Davies and Morris, 1993), presuming that the nonhepatic contribution to the clearance of SV was negligible and that SV exhibited linear pharmacokinetics in the range of plasma concentrations observed in this study. The products FABS · FG and FH were determined and compared with and without the presence of KTZ.
Statistical Analysis. All statistical tests were performed using SAS software (version 9.1.3; SAS Institute, Cary, NC). Statistical differences in the pharmacokinetic parameters were calculated by a two-tailed paired Student's t test. In all cases, a probability level of p < 0.05 was considered significant.
Results
Identification of P450 Isoenzymes Responsible for SV Metabolism. To identify the P450 isoform involved in SV metabolism, we evaluated the effects of KTZ, a P450 3A-specific inhibitor, on the metabolism of SV in human and monkey microsomes. SV metabolism in human liver and intestinal microsomes was inhibited by KTZ in a concentration-dependent manner, and the values of IC50 were estimated to be 0.023 and 0.051 μM, respectively (Table 1). As in human liver and intestinal microsomes, KTZ inhibited SV metabolism in monkey liver and intestinal microsomes with IC50 values of 0.012 and 0.007 μM, respectively, which were similar to those in the respective human sample (Table 1). Furthermore, we evaluated the effects of anti-P450 antisera on SV metabolism to confirm the involvement of the P450 3A isoform in SV metabolism. Anti-human P450 antisera was used in the immunoinhibition studies because it has been reported that these antibodies exhibit cross-reactivity with the corresponding monkey hepatic P450 isoforms (Shimada et al., 1997). In human and monkey microsomal samples, almost no effects on SV metabolism were observed in the presence of antibodies against human P450 2C8 and 2C9 even at the concentration of 200 μl/mg microsomal protein (Fig. 1). In contrast, SV metabolism in human and monkey microsomal samples was strongly inhibited by antibody against human P450 3A4. These results suggest that SV is metabolized primarily by P450 3A isozymes in humans and monkeys.
Pharmacokinetics of SV in Cynomolgus Monkeys. The mean plasma concentration-time profiles and pharmacokinetic parameters of SV and SVA after a single i.v. administration of SV to the monkeys are presented in Fig. 2A and Table 2, respectively. The SV plasma concentrations declined rapidly in a monophasic manner with t1/2 of 2.1 ± 0.8 h. The total body clearance (CLtot) and distribution volume at steady state (Vdss) were 1288 ± 110 ml/min/kg and 2567 ± 245 ml/kg, respectively. The maximum concentration of SVA (144.9 ± 10.2 ng/ml) was achieved at 0.083 h, the first collection time, after i.v. dosing of SV (Fig. 3A; Table 3), and, subsequently, the SVA concentration declined in parallel with that of the parent drug.
The plasma concentrations of SV reached a Cmax of 7.9 ± 6.1 ng/ml at 5.0 ± 2.6 h after p.o. dosing of SV (Fig. 2B; Table 2). The oral bioavailability of SV was remarkably low (0.8 ± 0.6%). After p.o. dosing of SV, measurable amounts of SVA appeared in plasma at the first sampling time (0.25 h) (Fig. 3B; Table 3). Thereafter, the SVA concentration reached a Cmax of 2.9 ± 0.5 ng/ml at 1.5 ± 0.6 h.
Effect of KTZ on the Pharmacokinetics of SV and SVA in Monkeys. To evaluate the effects of KTZ on the pharmacokinetics of SV and SVA, we determined SV and SVA concentrations in plasma after i.v. and p.o. administration of SV to the monkeys with concomitant p.o. dosing of KTZ. Figs. 2 and 3 depict the plasma concentration-time profiles of SV and SVA, respectively, in the presence and absence of KTZ. The effects of KTZ on the pharmacokinetic parameters of SV are summarized in Table 2. As shown in Fig. 2A and Table 2, the pharmacokinetic parameters of SV after i.v. dosing were not significantly affected with a concomitant p.o. dose of 20 mg/kg KTZ. Furthermore, there were also no differences in the pharmacokinetic profiles of SVA with and without KTZ treatment (Fig. 3A; Table 3). In contrast, when SV was administered p.o. with a concomitant p.o. dose of KTZ, the plasma concentrations of SV increased markedly (Fig. 2B; Table 2). The values of Cmax and AUC for SV were increased 12.9- and 6.3-fold, respectively. Statistically significant increases were observed in the AUC and F for SV after p.o. coadministration with 20 mg/kg KTZ, compared with the control values. Cmax for SV showed a tendency to increase, but this change was not statistically significant (p = 0.0968), probably because of the individual variability observed in monkeys with KTZ coadministration. Moreover, the plasma concentrations of SVA increased extremely as observed with the parent drug (Fig. 3B; Table 3). The values of Cmax and AUC for SVA were increased 7.2- and 6.0-fold, respectively, and statistically significant increases were observed in these parameters.
Figure 4 and Table 4 represent the concentration-time profiles and pharmacokinetic parameters of KTZ, respectively, during both p.o. and i.v. administration of SV. There were no obvious differences between the pharmacokinetic parameters for KTZ with concomitant p.o. and i.v. dosing of SV.
Discussion
The risk of drug candidates causing pharmacokinetic DDIs is generally evaluated in in vitro studies of inhibition of drug-metabolizing enzymes at an early stage of drug development. However, the success of this approach varies because of the difficulty in estimating drug concentrations around drug-metabolizing enzymes, which is the most influential factor in predictive accuracy. It has been proposed that prediction of DDIs in in vivo animal models is necessary for more accurate results (Ohba et al., 1996; Tsuruta et al., 1997; Ward et al., 2001), but a number of studies have demonstrated species-related differences in the catalytic roles of P450 isoforms for drugs in animals and humans. In the present study, we determined the P450 isozymes involving in SV metabolism in monkeys using anti-P450 antisera and KTZ, a potent P450 3A inhibitor. SV metabolism in monkey liver and intestinal microsomes was almost completely inhibited by addition of anti-P450 3A4 antiserum, and a similar effect was observed in human microsomes. In contrast, anti-P450 2C8 or 2C9 antiserum had no inhibitory effect in either human or monkey microsomes. The IC50 values of KTZ in monkey liver and intestinal microsomes were extremely low as was also observed in human microsomes.
On the basis of the in vitro results, we conclude that the P450 3A isozyme expressed in monkey liver and intestine is predominantly involved in SV metabolism. In a study of inhibition of midazolam metabolism, Kanazu et al. (2004) showed that the apparent Ki values and inhibitory mechanisms of KTZ, erythromycin, and diltiazem, all of which are typical P450 3A inhibitors, were similar in human and monkey liver microsomes, and we have demonstrated the similarity of inhibitor sensitivities for midazolam hydroxylation in human and monkey intestinal and liver microsomes (Ogasawara et al., 2007). Taken together, these results suggest that a monkey model may be useful for assessment of human DDIs associated with P450 3A inhibition in the liver and intestine. P450 3A8, which exhibits more than 90% amino acid sequence identity with P450 3A4, is the only reported P450 3A isoform in cynomolgus monkeys (Komori et al., 1992) and represents approximately 20% of the P450 content in monkey liver, comparable with the proportion of P450 3A4 in human liver. In monkey intestine, P450 MI-1, MI-2, and MI-3 have been identified as P450 isoforms associated with ebastine metabolism (Hashizume et al., 2001). It has been suggested that P450 MI-3 belongs to the P450 3A subfamily on the basis of the N-terminal amino acid sequence, immunoblot analysis, and inhibition studies using antibodies and KTZ.
It is widely believed that the liver is the major site of first-pass metabolism of orally administered drugs, but recent studies have indicated that the intestine also makes a significant contribution to the overall first-pass metabolism. In fact, a number of drugs that are solely metabolized by P450 3A undergo significant first-pass metabolism in the intestine (Gomez et al., 1995; Thummel et al., 1996; Floren et al., 1997; Masica et al., 2004). In the present study, we evaluated SV pharmacokinetics in monkeys to estimate the relative significance of the liver and intestine in first-pass metabolism of SV. Our results indicate that monkey intestine is a predominant site for first-pass metabolism of SV because the intestinal availability (FG) was much lower than the hepatic availability (FH) (1.6 versus 52%) (Table 2), assuming that the p.o. dose of SV was almost completely absorbed into the gut wall regardless of species (Vickers et al., 1990) and that the nonhepatic contribution to the clearance of SV was negligible. Regarding the intestinal contribution to first-pass metabolism of SV in humans, Obach et al. (2006) estimated FH and FG to be 0.67 and 0.66, respectively. Therefore, our results suggest that FH values in monkeys are comparable with those in humans but that there is a significant difference in the intestinal contribution to the overall first-pass metabolism in monkeys and humans. However, Lilja et al. (1998) reported that concomitant administration of SV with grapefruit juice, which influences intestinal P450 3A expression with no effect on hepatic P450 3A expression and activity, increased the AUC of SV by 16-fold in humans. The increase in the AUC of SV with grapefruit juice suggests that FG for SV may be extremely low compared with FH in humans, as in monkeys. Further investigations are required to clarify the relative contribution of the liver and intestine to the overall first-pass metabolism of SV in humans.
The risk of myopathy with SV alone is dose-related, and this risk is increased by concomitant use of the P450 3A inhibitors such as KTZ, cyclosporine, fibrates, and erythromycin (Gilad and Lampl, 1999; Gruer et al., 1999; Williams and Feely, 2002). In the present study, KTZ had a significant influence on the pharmacokinetic profiles of SV and SVA after p.o. administration of SV to monkeys, with the Cmax and AUC of SV increasing by 12.9- and 6.3-fold, respectively, in the presence of KTZ (Fig. 2; Table 2). Moreover, coadministration of KTZ led to a significant increase in Cmax and AUC of SVA, the main active metabolite of SV (Fig. 3; Table 3). In contrast, there were no significant changes in the pharmacokinetic parameters of SV and SVA after i.v. administration of SV in the presence of KTZ. These results indicate that KTZ does not inhibit hepatic SV metabolism in monkeys, suggesting that the KTZ-mediated increase in SV bioavailability may be attributable predominantly to decreased intestinal metabolism, resulting in the increased FG. A clinical study showed that Cmax and AUC of SV after p.o. administration to humans increased by 7.4- and 12.6-fold, respectively, after pretreatment with 400 mg of KTZ once a day for 10 days (Chung et al., 2006). Furthermore, unlike in monkeys, KTZ also influenced hepatic SV metabolism, resulting in prolongation of the terminal half-life of p.o. SV by 1.9-fold in the presence of KTZ. The KTZ plasma concentration in humans reaches 10.2 μg/ml after p.o. administration of 400 mg of KTZ once a day for 10 days (Khaliq et al., 2000), whereas the plasma concentration of KTZ in monkeys after 20 mg/kg p.o. dosing was approximately one-sixteenth of that in humans. Therefore, the apparent species differences in the effect of KTZ on hepatic SV metabolism may be due to a difference in the systemic exposure level to KTZ in monkeys and humans. A further investigation in which KTZ is dosed i.v. before SV administration may be necessary to clarify the effect of KTZ on hepatic SV metabolism in monkeys. Regarding DDIs associated with SV in humans, there are no data that permit discrimination of the influence of KTZ on FG and FH of p.o. SV. However, considering the significant SV metabolism in the intestine and the susceptibility of SV metabolism to KTZ, it is possible that the increased Cmax and AUC of SV can be attributed predominantly to decreased intestinal metabolism, as observed for other drugs metabolized by P450 3A4 (Gomez et al., 1995; Floren et al., 1997).
Several studies have evaluated the applicability of animal models for prediction of human DDIs. Ishigami et al. (2001) examined whether the interaction between SV and itraconazole observed in humans also occurs in rats. The AUC of SV was slightly increased (approximately 1.6-fold) in female rats with concomitant use of itraconazole, even at a dose of 50 mg/kg, but this increase was significantly less than that observed in a clinical situation (more than 10-fold) (Neuvonen et al., 1998). It was concluded that this result might be due to species differences in the metabolic pathways and P450 isoforms involved in SV metabolism. Likewise, Kotegawa et al. (2002) evaluated the DDI between midazolam and KTZ in rats and concluded that the clinical applicability of the rat model is limited by species differences in patterns of metabolic biotransformation and systemic pharmacokinetics, as well as susceptibility to inhibition by chemical inhibitors. In contrast, the results presented herein indicate that the DDI between SV and KTZ in humans is reproduced in monkeys, as observed in our previous work using midazolam and KTZ (Ogasawara et al., 2007).
In conclusion, we have demonstrated that the oral bioavailability of SV is extremely low in monkeys and that intestinal metabolism contributes extensively to the overall availability. When SV was administered intravenously, the plasma concentration-time profiles of SV and SVA were not influenced by oral coadministration of KTZ because of the limited systemic exposure of KTZ. In contrast, SV oral bioavailability was increased significantly by a concomitant oral dose of KTZ, probably because of inhibition of SV intestinal metabolism. Because these results are comparable with those in humans, we suggest that monkeys might be a suitable animal model in which to predict DDIs involving P450 3A inhibition. Further studies using other P450 3A substrates and inhibitors are needed to confirm the suitability of this model.
Acknowledgments
We express our deep gratitude to Kazuyuki Hirakoso, general manager of DMPK Research Laboratories at Mitsubishi Tanabe Pharma Corporation, for his interest and encouragement.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.022574.
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ABBREVIATIONS: DDI, drug-drug interaction; P450, cytochrome P450; KTZ, ketoconazole; SV, simvastatin; SVA, simvastatin acid; HPLC, high-performance liquid chromatography; AUC, area under the plasma concentration-time curve; CV, coefficient of variation; RE, relative error.
- Received May 28, 2008.
- Accepted October 9, 2008.
- The American Society for Pharmacology and Experimental Therapeutics