Abstract
The plasma concentration of repaglinide is reported to increase greatly when given after repeated oral administration of itraconazole and gemfibrozil. The present study analyzed this interaction based on a physiologically based pharmacokinetic (PBPK) model incorporating inhibition of the hepatic uptake transporter and metabolic enzymes involved in repaglinide disposition. Firstly, the plasma concentration profiles of inhibitors (itraconazole, gemfibrozil, and gemfibrozil glucuronide) were reproduced by a PBPK model to obtain their pharmacokinetic parameters. The plasma concentration profiles of repaglinide were then analyzed by a PBPK model, together with those of the inhibitors, assuming a competitive inhibition of CYP3A4 by itraconazole, mechanism-based inhibition of CYP2C8 by gemfibrozil glucuronide, and inhibition of organic anion transporting polypeptide (OATP) 1B1 by gemfibrozil and its glucuronide. The plasma concentration profiles of repaglinide were well reproduced by the PBPK model based on the above assumptions, and the optimized values for the inhibition constants (0.0676 nM for itraconazole against CYP3A4; 14.2 μM for gemfibrozil against OATP1B1; and 5.48 μM for gemfibrozil glucuronide against OATP1B1) and the fraction of repaglinide metabolized by CYP2C8 (0.801) were consistent with the reported values. The validity of the obtained parameters was further confirmed by sensitivity analyses and by reproducing the repaglinide concentration increase produced by concomitant gemfibrozil administration at various timings/doses. The present findings suggested that the reported concentration increase of repaglinide, suggestive of synergistic effects of the coadministered inhibitors, can be quantitatively explained by the simultaneous inhibition of the multiple clearance pathways of repaglinide.
Introduction
Repaglinide is one of the short-acting meglitinide analogs that reduce blood glucose concentrations by enhancing glucose-stimulated insulin secretion from pancreatic beta cells (Fuhlendorff et al., 1998). It has been reported that the plasma concentration of repaglinide after an oral dose was dramatically increased when it was given following repeated oral administration of itraconazole (ICZ; 100 mg) and gemfibrozil (GEM; 600 mg) twice a day for 3 days in healthy volunteers [the area under the concentration-time curve (AUC) of repaglinide was increased 1.4-, 8.1-, and 19.4-fold by ICZ, GEM, and both, respectively] (Niemi et al., 2003a). The major route of repaglinide elimination from the body is active uptake from blood into hepatocytes by an uptake transporter, organic anion transporting polypeptide (OATP) 1B1, followed by biotransformation by cytochrome P450 (P450) enzymes (CYP3A4 and CYP2C8) into several metabolites (van Heiningen et al., 1999; Bidstrup et al., 2003; Kajosaari et al., 2005a; Niemi et al., 2005; Kalliokoski et al., 2008). Therefore, the above interactions possibly resulted from inhibitions of OATP1B1, CYP3A4, and CYP2C8.
The plasma repaglinide concentration after oral administration in humans has been reported to be affected by genetic polymorphisms of OATP1B1 and CYP2C8: for OATP1B1, subjects with the 521CC genotype have a significantly higher (1.7- to 2.9-fold) repaglinide AUC than those with the wild type (521TT) (Niemi et al., 2005; Kalliokoski et al., 2008); for CYP2C8, subjects with the CYP2C8*1/*3 genotype have a significantly lower (0.5-fold) repaglinide AUC than those with wild type (*1/*1) (Niemi et al., 2003b, 2005). On the other hand, there was no difference in the AUC of repaglinide between the subjects with the CYP3A4 *1/*18 genotype and those with the wild type (*1/*1) (Ruzilawati and Gan, 2010).
The coadministered inhibitors of OATP1B1, CYP2C8, and CYP3A4 have also been reported to affect the disposition of repaglinide: the AUC of oral repaglinide was significantly increased by coadministration of clarithromycin (1.4-fold) (Niemi et al., 2001) and cyclosporine (2.4-fold) (Kajosaari et al., 2005b), which are known to inhibit both OATP1B1 and CYP3A4; coadministration of telithromycin and trimethoprim, an inhibitor of CYP3A4 and CYP2C8, respectively, increased the AUC of oral repaglinide by 1.8-fold (Kajosaari et al., 2006) and 1.6-fold (Niemi et al., 2004), respectively. It has also been reported that repeated administration of rifampicin, an inducer of P450 enzymes, including CYP3A4 and CYP2C8, significantly reduced the AUC of oral repaglinide (0.2- or 0.4-fold) (Niemi et al., 2000; Bidstrup et al., 2004).
ICZ is known to be a potent reversible inhibitor of CYP3A4. In in vitro studies using human liver microsomes, ICZ strongly inhibited the biotransformation of substrates of CYP3A4, such as midazolam, quinidine and testosterone (von Moltke et al., 1996; Galetin et al., 2005). The AUCs of oral triazolam and midazolam, both eliminated from the body mainly by CYP3A4-mediated metabolism, were significantly increased when coadministered with ICZ (200 mg p.o., once a day, for 4 days) by 27- and 6.2-fold, respectively (Varhe et al., 1994; Backman et al., 1998).
GEM inhibited the CYP2C8-mediated metabolism of paclitaxel and cerivastatin in human liver microsomes without affecting the CYP3A4-mediated hydroxylation of testosterone (Wang et al., 2002; Shitara et al., 2004). It was also found that GEM glucuronide (GEM-glu) is a mechanism-based inhibitor of CYP2C8 (Ogilvie et al., 2006), and this was supported by a persistent interaction between GEM and repaglinide in vivo (Tornio et al., 2008). Both GEM and GEM-glu are also reported to inhibit OATP1B1 in in vitro studies using human hepatocytes and OATP1B1-expressing cells (Shitara et al., 2004; Nakagomi-Hagihara et al., 2007). The AUCs of oral pravastatin and rosuvastatin, mainly taken up into the liver by OATP1B1 and excreted into bile in unchanged forms, were significantly increased when coadministered with GEM (600 mg p.o., twice a day, for 3 days) by 2- and 1.9-fold, respectively (Kyrklund et al., 2003; Schneck et al., 2004). In addition, the AUC of cerivastatin, a substrate of OATP1B1 and CYP2C8, was significantly increased when coadministered with GEM (600 mg p.o., twice a day, for 3 days) by 4.4-fold (Backman et al., 2002), and this was suggested to result from the inhibition of OATP1B1 and CYP2C8 by both GEM and GEM-glu (Shitara et al., 2004).
Taken together, these pieces of evidence support the hypothesis that the reported drastic increase in the AUC of repaglinide resulted from the inhibition of OATP1B1, CYP3A4, and CYP2C8 by ICZ or GEM. The present study aimed to analyze this interaction quantitatively using a physiologically based pharmacokinetic (PBPK) model for all the compounds (repaglinide, GEM, and GEM-glu) involved in the interaction. Although PBPK models have been used for the quantitative prediction of the degree of drug-drug interaction involving metabolic enzymes (Kanamitsu et al., 2000; Ito et al., 2003; Kato et al., 2008; Zhang et al., 2009; Rowland-Yeo et al., 2010; Zhao et al., 2012) and also for describing the disposition of a drug transporter substrate pravastatin (Watanabe et al., 2009), only “static” approaches have been used for the interactions at drug transporters (Hinton et al., 2008; Yoshida et al., 2012), with no reports on the application of PBPK modeling to the best of our knowledge. In the present study, we investigated whether the increase in repaglinide AUC by ICZ and GEM can be quantitatively explained based on the clearance concept and a PBPK model incorporating the inhibition of both the hepatic uptake transporter and metabolic enzymes.
Materials and Methods
Calculation of Pharmacokinetic Parameters for Repaglinide
The urinary excretion of repaglinide is reported to be negligible (0.1%) after its intravenous administration to healthy volunteers (van Heiningen et al., 1999). Therefore, the total body clearance (CLtot) was assumed to be equal to the hepatic clearance (CLh) and the hepatic availability (Fh) was calculated as 1 − CLh/Qh, where Qh represents the hepatic blood flow (86.9 l/h) (Watanabe et al., 2009). As the bioavailability (0.625) estimated from an oral/intravenous administration study (Hatorp, 2002) exceeds the estimated Fh of 0.40 (0.25–0.52 using the Qh of 70–110 l/h), a product of Fa and Fg, where Fa and Fg represent the fraction absorbed and the intestinal availability, respectively, was assumed to be unity. Thus, the overall intrinsic clearance (CLint,all) for hepatic elimination of repaglinide, multiplied by the blood unbound fraction (fb), was calculated by the following equation using the AUC values reported in the interaction study with ICZ/GEM (4.74, 6.66, 38.3, and 91.5 ng/h per ml for control, +ICZ, +GEM, and +ICZ+GEM, respectively) (Niemi et al., 2003a).
(1)where Dose is the oral dose of repaglinide (0.25 mg for all conditions).
Estimation of the Inhibition Ratio of Intrinsic Clearances for Each Process Based on the AUC Increase produced by ICZ and GEM
The CLint,all calculated above is expressed as follows (Shitara et al., 2006):
(2)where PSinf and PSeff represent the intrinsic clearance for hepatic uptake from blood and the efflux from hepatocytes to blood, respectively; γ is defined as CLint/PSeff; β, defined as CLint/(CLint+PSeff), can be used for evaluating the rate-limiting process of hepatic intrinsic clearance. CLint represents the intrinsic clearance for hepatic metabolism mediated by CYP2C8 and CYP3A4, which can be expressed as follows using the fraction metabolized by CYP2C8 (fm2C8):
(3)For the control (administration of repaglinide alone), the following equations can be derived from the above equations:
(4)assuming that ICZ inhibits CYP3A4, the following equations can be derived for +ICZ (coadministration of ICZ) condition:
(5)
(6)where R3A4 represents the inhibition ratio of CYP3A4 by ICZ [CLint,3A4(+ICZ)/CLint,3A4(control)]. Assuming that GEM (and its glucuronide) inhibits both OATP1B1 and CYP2C8, the following equations can be derived for the +GEM (coadministration of GEM) condition:
(7)
(8)where Rinf and R2C8 represents the inhibition ratio of hepatic uptake and CYP2C8, respectively, by GEM [PSinf(+GEM)/PSinf(control) and CLint,2C8(+GEM)/CLint,2C8(control), respectively]. Finally, the AUC and R values can be expressed as follows for the +ICZ+GEM (coadministration of both ICZ and GEM) condition:
(9)
(10)The observed AUC values for the four conditions (control, +ICZ, +GEM, and +ICZ+GEM) were analyzed by the above equations using a multipurpose nonlinear least-squares fitting computer program Napp (version 2.26) (Hisaka and Sugiyama, 1998) to obtain the optimized values for Rinf, R2C8, R3A4, and fm2C8, fixing the fb·PSinf value at 100 l/h.
Fitting Analyses of the Blood Concentration-Time Profiles of Inhibitors (ICZ, GEM, and GEM-glu) Based on a Simple PBPK Model
ICZ.
A simple PBPK model for ICZ disposition, consisting of a blood compartment, a gastrointestinal compartment, and a liver compartment, was constructed as shown in Fig. 1A. According to this model, the concentration profile of ICZ can be expressed by the following differential equations:
(11)
(12)where Cb and Ch represent the concentrations in the blood and liver compartments, respectively; Vb represents the distribution volume in the blood compartment; Vh represents the volume of liver; ka represents the absorption rate constant; Rb represents the blood-to-plasma concentration ratio; Kp represents the liver-to-plasma concentration ratio; and fh represents the unbound fraction in the liver. The subscript icz after each parameter indicates the parameters for ICZ.
Simple PBPK models for ICZ (A), GEM/GEM-glu (B), and repaglinide (C).
Because the concentration profile of ICZ was not shown in the interaction study with repaglinide (Niemi et al., 2003a), the values of Vb,icz, ka,icz, and CLint,icz were optimized by fitting analyses based on the concentration profile reported in another paper (Jaakkola et al., 2005). The dose was set at 100,000 μg (Niemi et al., 2003a; Jaakkola et al., 2005), and values of Qh = 86.9 l/h and Vh=1.22 l were used for physiologic parameters (Watanabe et al., 2009). The FaFg was estimated by a similar method as that for repaglinide described above using the values of CLtot and bioavailability (Table 1), The fh value was calculated based on the following equation (Poulin and Theil, 2002):
(13)where fp represents the unbound fraction in the plasma. The Kp value was calculated as follows (Poulin and Theil, 2002):
(14)
(15)where cLogP represents the logarithm of the computer-calculated partition coefficient between n-octanol and water. The initial ICZ concentration in the blood compartment (Cb,0) after 3 days of repeated oral administration was obtained from the reported concentration profile (Jaakkola et al., 2005). The initial ICZ concentration in the liver compartment (Ch,0) was assumed to be equal to Cb,0 × Kp / Rb.
Pharmacokinetic parameters for inhibitors
GEM and GEM-glu.
A simple PBPK model constructed for GEM and GEM-glu disposition, consisting of a blood compartment, a gastrointestinal compartment, a liver extracellular space compartment, and a liver compartment for GEM and a blood compartment, a liver extracellular space compartment, and a liver compartment for GEM-glu, is shown in Fig. 1B. According to this model, the concentration profiles of GEM and GEM-glu can be expressed by the following differential equations:
(16)
(17)
(18)
(19)
(20)
(21)where Ve represents the volume of the liver extracellular space (0.469 l) (Watanabe et al., 2009); Lag represents the lag time for absorption of GEM; CLint1 and CLint2 were defined as the hepatic intrinsic clearances for glucuronidation and other metabolic pathway(s), respectively, for GEM; CLNH represents the nonhepatic clearance for elimination of GEM-glu from the blood compartment; CLint,h represents the hepatic intrinsic clearance for elimination of GEM-glu from the liver compartment. The subscripts gem and glu after each parameter indicate the parameters for GEM and GEM-glu, respectively.
Because the concentration profiles of GEM and GEM-glu were not shown in the interaction study with repaglinide (Niemi et al., 2003a), the values of the pharmacokinetic parameters for GEM and GEM-glu were estimated based on their concentration profiles after repeated oral administration of GEM according to the same dosage schedule, as reported in another paper (Tornio et al., 2008). The dose was set at 600,000 μg (Niemi et al., 2003a; Tornio et al., 2008), and the FaFg for GEM was estimated by a similar method as that for repaglinide described above using the values of CLtot and bioavailability (Table 1). The CLint1/CLint2 ratio was fixed at 6.1 (= 0.86/0.14) because the fraction of GEM metabolized by the UDP-glucuronosyltransferase is reported to be 0.86 (Kilford et al., 2009). The PSinf,gem was assumed to be equal to 10-fold the value of CLint,all for GEM that was calculated by Eq. 1 using the reported AUC value (Tornio et al., 2008); the PSinf,glu was calculated based on an assumption that fb·PSinf,glu was equal to fb·PSinf,gem; the CLNH,glu was assumed to be equal to the value of the glomerular filtration rate (Davies and Morris, 1993) multiplied by fb,glu. Initially, the GEM blood concentration profile was fitted to Eq. 16 based on the simultaneous analysis of Eqs. 16–18 to obtain the optimized values of ka,gem, Vb,gem, PSeff,gem, and CLint1. Then, fixing the above parameters for GEM at the optimized values, the GEM-glu blood concentration profile was fitted to Eq. 19 based on the simultaneous analysis of Eqs. 19–21 to obtain the optimized values of PSeff,glu and CLint,h,glu.
Fitting Analyses of Repaglinide Concentration Profiles Based on a Simple PBPK Model
A simple PBPK model constructed for repaglinide disposition, consisting of a blood compartment, a gastrointestinal compartment, a peripheral compartment, a liver extracellular space compartment, and a liver compartment, is shown in Fig. 1C. According to this model, the concentration profile of repaglinide can be expressed by the following differential equations considering the inhibition of hepatic uptake by GEM and GEM-glu, a mechanism-based inhibition of CYP2C8 by GEM-glu and a competitive inhibition of CYP3A4 by ICZ:
(22)
(23)
(24)
(25)where the subscript s after each parameter indicates the parameters for repaglinide; Xsub represents the amount of repaglinide in the peripheral compartment; k12 and k21 represent the transfer rate constant from the blood compartment to the peripheral compartment and that from the peripheral compartment to the blood compartment, respectively; Ki,1B1 and Ki,3A4 represent the inhibition constant for hepatic uptake and that for CYP3A4-mediated hepatic metabolism, respectively. It was assumed that the inhibition of hepatic uptake and metabolism depends on the inhibitor concentration in the liver extracellular space compartment (Ce) and that in the liver compartment (Ch), respectively. Lag for repaglinide was assumed in the present analysis to include the 1-hour lag time between the doses of ICZ/GEM and repaglinide (Niemi et al., 2003a).
The differential equation for REact2C8, the fraction of active CYP2C8, can be expressed as follows considering the mechanism-based inhibition by GEM-glu:
(26)where kinact,2C8 and Ki,app,2C8 represent the maximum inactivation rate constant and the apparent inhibition constant, respectively, and kdeg,2C8 represents the first-order rate constant for degradation of CYP2C8.
The repaglinide blood concentration profiles for the four conditions (control, +ICZ, +GEM, and +ICZ+GEM) were fitted to Eq. 22 based on the simultaneous analysis of Eqs. 11, 12 and Eqs. 16–26 to obtain the optimized values of ka,s, Lags, Vb,s, fb,s·PSinf,s, fh,s·CLint,s, k12,s, k21,s, Ki,3A4,icz, and Ki,1B1,gem. The dose of repaglinide was set at 250 μg, which is the oral dose used in the reported interaction study (Niemi et al., 2003a); the values of Ki,app,2C8, kinact,2C8, and kdeg,2C8 were fixed at the reported values (20 μM, 12.6 h−1 and 0.030 h−1, respectively) (Ogilvie et al., 2006; Yang et al., 2008); the ratio of Ki,1B1,glu/Ki,1B1,gem was fixed at the mean of the reported values obtained in in vitro uptake studies using human hepatocytes and OATP1B1-expressing cells (0.385) (Shitara et al., 2004; Nakagomi-Hagihara et al., 2007); the values of PSeff,s was calculated as the geometric mean of the reported intrinsic clearances for passive diffusion of repaglinide into human and rat hepatocytes (Yabe et al., 2011; Ménochet et al., 2012; Jones et al., 2012) (Table 2). The PK parameters for ICZ, GEM, and GEM-glu were fixed at the values obtained by the above-mentioned analyses of respective concentration profiles (Table 1).
Pharmacokinetic parameters for repaglinide and inhibition parameters
Simulation of the Repaglinide Concentration Profiles in Other Interaction Studies with GEM
Simulation studies were performed to investigate whether the repaglinide concentration increase produced by concomitant GEM administration at various timing/doses, reported in other papers, can be reproduced using the same PBPK model (Figs. 1, B and C, Eqs. 16–26) together with the parameters estimated in the present study (Table 2). The following three studies were investigated:
Report 1: repaglinide was taken 1, 24, 48, or 96 hours after the last administration of gemfibrozil (600 mg p.o., twice a day, for 3 days) (Backman et al., 2009).
Report 2: repaglinide was taken 0, 1, 3, or 6 hours after a single administration of gemfibrozil (600 mg p.o.) (Honkalammi et al., 2011).
Report 3: repaglinide was taken 1 hour after the last administration of gemfibrozil (30, 100, or 600 mg p.o., twice a day, for 3 days) (Honkalammi et al., 2012).
Analysis
A multipurpose nonlinear least-squares fitting computer program Napp (version 2.26) (Hisaka and Sugiyama, 1998) was used for all the fitting and simulation analyses. The differential equations were numerically solved by using the Runge-Kutta-Fehlberg method. The fitting analysis was carried out based on a nonlinear least-squares procedure with a weight value fixed at 1.
Results
Analysis of the Increased Repaglinide AUC Produced by ICZ and GEM.
The inhibition ratios of repaglinide intrinsic clearances as well as the fm2C8 were estimated by regression analysis of the AUC increase by ICZ and GEM using Eqs. 4–10. The R3A4, R2C8, Rinf, and fm2C8 were optimized at 0.22, 0.045, 0.50, and 0.85, respectively, showing that the reported AUC increase for each condition (control, +ICZ, +GEM, and +ICZ+GEM) can be explained by the CYP3A4, CYP2C8, and OATP1B1 all being inhibited by coadministration of ICZ and GEM. The estimated fm2C8 value was used as its initial value in the final fitting analysis of the repaglinide concentration profiles.
Analysis of the ICZ Concentration Profile.
The blood concentration profile of ICZ was fitted to Eq. 11 based on the simple PBPK model (Fig. 1A) and the values of ka,icz, Vb,icz, and CLint,icz were optimized to best fit the observed concentration profile. All the parameters for ICZ used in the analyses are summarized in Table 1 together with the estimated values. As shown in Fig. 2A, the obtained concentration profile of ICZ after 3 days of repeated administration was close to the observed values (Jaakkola et al., 2005).
Concentration profiles of ICZ (A), GEM and GEM-glu (B), and repaglinide (C) in blood. (A) On the basis of the PBPK model (Fig. 1A) and differential equations (Eqs. 11, 12), the time course of the ICZ concentration in blood (Cb,icz) after 3 days of repeated administration (100 mg p.o., twice a day) was reproduced using the pharmacokinetic (PK) parameters shown in Table 1. The solid line and the open circles indicate the simulated time course and the observed values (Jaakkola et al., 2005), respectively. (B) On the basis of the PBPK model (Fig. 1B) and differential equations (Eqs. 16–21), the time courses of the GEM and GEM-glu concentration in blood (Cb,gem and Cb,glu, respectively) after 3 days of repeated administration of GEM (600 mg p.o., twice a day) were reproduced using the PK parameters shown in Table 1. The solid line and the open circles indicate the simulated time course and the observed values (Tornio et al., 2008), respectively, for GEM; the dotted line and the closed circles indicate the simulated time course and the observed values (Tornio et al., 2008), respectively, for GEM-glu. (C) On the basis of the PBPK model (Fig. 1C) and differential equations (Eqs. 22–26), the time courses of the repaglinide concentration in blood (Cb,s) for the four conditions (control, +ICZ, +GEM and +ICZ+GEM) were reproduced using the parameters shown in Table 2. The concentration profiles for ICZ and GEM/GEM-glu were simultaneously analyzed using Eqs. 11, 12, and 16–21, respectively, to provide the inhibitor concentrations. The solid line and open circles indicate the simulated time course and the observed values, respectively, for the control condition; the dotted line and open squares indicate the simulated time course and the observed values, respectively, for the +ICZ condition; the broken line and open triangles indicate the simulated time course and the observed values, respectively, for the +GEM condition; and the chain line and closed circles indicate the simulated time course and the observed values, respectively, for the +ICZ+GEM condition.
Analysis of the GEM and GEM-glu Concentration Profiles.
The blood concentration profiles of GEM and GEM-glu were fitted to Eqs. 16 and 19, respectively, based on the simple PBPK model (Fig. 1B) and the values of ka,gem, Vb,gem, PSeff,gem, PSeff,glu, CLint1, and CLint,h,glu were optimized to best fit the observed concentration profiles. All parameters used in the analysis are summarized in Table 1 together with the estimated values. As shown in Fig. 2B, the obtained concentration profiles of GEM and GEM-glu after 3 days of repeated administration of GEM were close to the observed values (Tornio et al., 2008).
Analysis of the Repaglinide Concentration Profiles Considering the Interaction with ICZ/GEM.
The blood concentration profiles of repaglinide for the four conditions (control, +ICZ, +GEM, and +ICZ+GEM) were analyzed based on the simple PBPK model (Fig. 1C) and differential equations (Eqs. 22–26) together with the concentration profiles of ICZ, GEM, and GEM-glu (Eqs. 11, 12, 16–21). The values of ka,s, Vb,s, k12,s, k21,s, Lags, fm2C8, Ki,3A4,icz, and Ki,1B1,gem were optimized by simultaneous fitting analysis fixing all the parameters for the inhibitors (ICZ, GEM, and GEM-glu) at values shown in Table 1. All the parameters for repaglinide used in the analyses are summarized in Table 2 together with the estimated values. The obtained concentration profiles of repaglinide were close to the observed values for each of the four conditions as shown in Fig. 2C. The repaglinide AUC ratios for +ICZ, +GEM, and +ICZ+GEM conditions were calculated to be 1.2-, 6.5-, 24.3-fold, respectively.
Simulation of the Repaglinide Concentration Profiles in Other Interaction Studies with GEM.
The repaglinide concentration increase produced by GEM reported in other interaction studies (Backman et al., 2009; Honkalammi et al., 2011, 2012) were simulated based on the same PBPK model (Figs. 1, B and C; Eqs. 16–26) together with the parameters estimated in the present study (Table 2). Figure 3 shows the observed (Backman et al., 2009) and simulated concentration profiles of repaglinide administered at various intervals after repeated GEM dosing. The persistent interaction, which recovers after 96 hours, was well reproduced in our simulation analysis. Furthermore, the estimated AUC increase agreed well with the observed values for all the conditions investigated (Table 3).
Observed (A) and simulated (B) concentration profiles of repaglinide in an interaction study with GEM administered at different dosing intervals. (A) Reported repaglinide concentration profiles in healthy volunteers who received a single oral dose of repaglinide (0.25 mg) at different times after GEM (600 mg p.o., twice a day, for 3 days) (Backman et al., 2009). The solid line and open circles represent the profiles for the control condition (without GEM); the broken line and closed squares for the 1-hour interval condition; the chain line and closed triangles for the 24-hour interval condition; the double chain line and closed circles for the 48-hour interval condition; and the dotted line and asterisks for the 96-hour interval condition. (B) Blood concentration profiles of GEM and repaglinide, assuming the same dosing regimen as in (A), were simulated based on the PBPK model shown in Figs. 1, B and C together with the parameters estimated in the present study (Tables 2). The same line as in Fig. 3A was used for each condition.
Repaglinide AUC increase produced by concomitant GEM administration at various timings/doses
Discussion
The AUC of repaglinide has been reported to be increased 1.4-, 8.1-, or 19.4-fold when healthy volunteers received repaglinide after 3 days of treatment with ICZ, GEM, or both GEM and ICZ, respectively (Niemi et al., 2003a). The present study investigated whether these interactions can be quantitatively explained by the inhibition of the transporter and enzymes involved in repaglinide disposition on the basis of the clearance concept and simple PBPK modeling.
The major route of repaglinide elimination from the body is hepatic metabolism, with a negligible contribution from renal clearance (fe = 0.001) (van Heiningen et al., 1999), and the plasma concentration profile of repaglinide has been shown to be affected by the genetic polymorphism of OATP1B1 (Niemi et al., 2005; Kalliokoski et al., 2008) or CYP2C8 (Niemi et al., 2003b, 2005). In the present analysis, therefore, it was assumed that repaglinide is eliminated only from the liver and that both hepatic uptake and metabolism contribute to its hepatic elimination. As a result, the β value in Eq. 2 was calculated to be 0.27 using the optimized values of PSeff and CLint (Table 2), which indicates that its hepatic elimination is not completely limited by the hepatic uptake.
It has been reported that about 66% of orally administered [14C]repaglinide is excreted as a metabolite M2 in the feces and urine in healthy volunteers (van Heiningen et al., 1999). In in vitro studies, M0-OH, M1, M2, M4, and M5 have been reported as metabolites of repaglinide (Bidstrup et al., 2003). At a relatively high concentration of repaglinide (22 μM), formation of M1 by CYP3A4 and M4 by CYP2C8 were the major metabolic pathways in human liver microsomes (Bidstrup et al., 2003). Kajosaari et al. (2005a) investigated the contributions of CYP3A4 and CYP2C8 to the metabolism of repaglinide at a therapeutic repaglinide concentration (< 0.4 μM) using recombinant enzymes, but the results were highly dependent on the scaling factor used. It has also been reported recently that the fm2C8 value was estimated to be 0.49, 0.41, and 0.63 in an in vitro study using human hepatocytes, S9 fractions, and liver microsomes, respectively (Säll et al., 2012). In the present study, the estimated fm2C8 value of 0.801 (Table 2) indicates a larger contribution of CYP2C8 than CYP3A4, which is consistent with the previous findings that the AUC of repaglinide was affected by the genetic polymorphism of CYP2C8 (Niemi et al., 2003b, 2005) but not CYP3A4 (Ruzilawati and Gan, 2010).
In vitro studies have demonstrated that both GEM and GEM-glu inhibit OATP1B1 (Shitara et al., 2004; Nakagomi-Hagihara et al., 2007; Hinton et al., 2008). The Ki or IC50 values for GEM and of GEM-glu were reported to be 72.4 and 24.3 μM, respectively, in OATP1B1-expressing Madin-Darby canine kidney cells (Shitara et al., 2004); 35.8 μM and 9.3 μM, respectively, in human hepatocytes (Nakagomi-Hagihara et al., 2007); and 15.5 and 7.9 μM, respectively, in OATP1B1-expressing Xenopus oocytes (Nakagomi-Hagihara et al., 2007). In the present fitting analysis, the ratio of Ki,1B1glu to Ki,1B1gem was fixed at 0.385, the mean ratio calculated from these reported values (0.26–0.51), to reduce the number of unknown parameters. As a result, the estimated Ki values (Ki,1B1gem = 14.2±10.1 μM, Ki,1B1glu = 5.48 μM) (Table 2) were close to the reported values described above.
Although both GEM and GEM-glu have been also reported to inhibit CYP2C8, the IC50 value for GEM was sevenfold higher than that for GEM-glu (Shitara et al., 2004), which was shown to be a mechanism-based inhibitor of CYP2C8 (Ogilvie et al., 2006). In the present study, therefore, only the mechanism-based inhibition of CYP2C8 by GEM-glu was incorporated in the model fixing the kinact,2C8 and Ki,app,2C8 at the reported values (12.6 h−1 and 52 μM, respectively) (Ogilvie et al., 2006).
On the basis of a PBPK analysis of reported in vivo interactions between ICZ and CYP3A4 substrates, Kato et al. (2008) reported an in vivo Ki value of 0.282 μg/l (0.4 nM) for ICZ against CYP3A4, which is nearly 500-fold lower than the average Ki value obtained in in vitro studies using human liver microsomes. The Ki,3A4,icz value of 0.0477 ± 0.0365 μg/l (0.0676 ± 0.0518 nM) estimated by the present fitting analysis (Table 2) was even smaller than the reported in vivo Ki value, although the contribution of CYP3A4 inhibition to the overall change in repaglinide pharmacokinetics should not be large in the present analysis based on the fm2C8 value of 0.801 and the hepatic disposition limited in part by the uptake (β = 0.27).
To confirm the validity of the parameter values obtained in the present study, sensitivity analyses were carried out for fm2C8, Ki,1B1,gem, and Ki,3A4,icz. As shown in Fig. 4, the simulated concentration profiles of repaglinide deviated greatly from the observed concentrations when altered values of fm2C8 and Ki were used, suggesting the reliability of these parameters estimated in the present analysis. Furthermore, the repaglinide concentration increase by concomitant GEM administration at various timings/doses, reported in other papers, were well reproduced using the same PBPK model together with the parameters estimated in the present study (Fig. 3; Table 3). These results indicate the validity of the PBPK model used in the present analyses as well as the estimated parameters.
Sensitivity analysis of the parameters obtained in this study. The concentration profiles of repaglinide were simulated using different values for fm2C8 (A and B), Ki,1B1,gem (C and D), or Ki,3A4,icz (E and F), with other parameters fixed at the same values as in Fig. 2C (the original values for fm2C8, Ki,1B1,gem, and Ki,3A4,icz were 0.801, 3,560 μg/l, and 0.0477 μg/l, respectively). The symbols and lines are same as in Fig. 2C.
In conclusion, the plasma concentration profiles of repaglinide in the interaction study were reproduced by the PBPK model, suggesting that the reported concentration increase by ICZ and GEM results from the inhibition of OATP1B1-mediated hepatic uptake by GEM and GEM-glu and that of CYP2C8- and CYP3A4-mediated metabolism by GEM-glu and ICZ, respectively. The present PBPK model is expected to be applicable to the analyses of other interactions involving both hepatic uptake transporters and metabolic enzymes.
Authorship Contributions
Participated in research design: Kudo, Sugiyama, Ito.
Conducted experiments: Kudo, Ito.
Contributed new reagents or analytic tools: Hisaka.
Performed data analysis: Kudo, Hisaka, Sugiyama, Ito.
Wrote or contributed to the writing of the manuscript: Kudo, Hisaka, Sugiyama, Ito.
Footnotes
This work was supported by JSPS KAKENHI Grant [23590204].
Abbreviations
- AUC
- area under the concentration-time curve
- β
- CLint divided by (CLint + PSeff)
- Cb
- concentrations in the blood compartment
- Ce
- concentration in the liver extracellular space compartment
- Ch
- concentrations in the liver compartment
- CLh
- hepatic clearance
- CLint
- intrinsic metabolic clearance
- CLint,all
- overall intrinsic clearance
- CLint,h
- hepatic intrinsic clearance
- CLNH
- non-hepatic clearance
- cLogP
- logarithm of the computer-calculated partition coefficient between n-octanol and water
- CLtot
- total body clearance
- P450
- cytochrome P450
- Fa
- fraction absorbed
- fb
- blood unbound fraction
- fe
- urinary excretion ratio of parent drug
- Fg
- intestinal availability
- Fh
- hepatic availability
- fh
- unbound fraction in the liver
- fm2C8
- fraction metabolized by CYP2C8
- GEM
- gemfibrozil
- GEM-glu
- gemfibrozil glucuronide
- IC50
- half maximal inhibitory concentration
- ICZ
- itraconazole
- k12
- transfer rate constant from the blood compartment to the peripheral compartment
- k21
- transfer rate constant from the peripheral compartment to the blood compartment
- ka
- absorption rate constant
- kdeg,2C8
- the first-order rate constant for degradation of CYP2C8
- Ki,1B1
- inhibition constant for hepatic uptake
- Ki,3A4
- the inhibition constant for CYP3A4-mediated hepatic metabolism
- Ki,app,2C8
- the apparent inhibition constant for CYP2C8-mediated hepatic metabolism
- kinact,2C8
- the maximum inactivation rate constant for CYP2C8-mediated hepatic metabolism
- Kp
- liver-to-plasma concentration ratio
- Lag
- lag time for absorption
- OATP
- organic anion transporting polypeptide
- PBPK
- physiologically based pharmacokinetic
- PSeff
- intrinsic clearance for the efflux from hepatocytes to blood
- PSinf
- intrinsic clearance for hepatic uptake from blood
- Qh
- hepatic blood flow
- R2C8
- inhibition ratio of CYP2C8
- R3A4
- inhibition ratio of CYP3A4
- Rb
- blood-to-plasma concentration ratio
- Rinf
- inhibition ratio of hepatic uptake
- Vb
- distribution volume in the blood compartment
- Ve
- volume of the liver extracellular space
- Vh
- volume of liver
- Xsub
- amount in the peripheral compartment
- Received October 11, 2012.
- Accepted November 8, 2012.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
