Creation of Novel Sensitive Probe Substrate and Moderate Inhibitor Models for a Comprehensive Prediction of CYP2C8 Interactions for Tucatinib.

A physiologically-based pharmacokinetic (PBPK) model was developed to simulate plasma concentrations of tucatinib (TUKYSA®) after single-dose or multiple-dose administration of 300 mg b.i.d. orally. This PBPK model was subsequently applied to support evaluation of drug-drug interaction (DDI) risk as a perpetrator resulting from tucatinib inhibition of CYP3A4, CYP2C8, CYP2C9, P-gp, or MATE1/2-K. The PBPK model was also applied to support evaluation of DDI risk as a victim resulting from co-administration with CYP3A4 or CYP2C8 inhibitors, or a CYP3A4 inducer. After refinement with clinical DDI data, the final PBPK model was able to recover the clinically observed single and multiple-dose plasma concentrations for tucatinib when tucatinib was administered as a single agent in healthy subjects. In addition, the final model was able to recover clinically observed plasma concentrations of tucatinib when administered in combination with itraconazole, rifampin, or gemfibrozil as well as clinically observed plasma concentrations of probe substrates of CYP3A4, CYP2C8, CYP2C9, P-gp, or MATE1/2-K. The PBPK model was then applied to prospectively predict the potential perpetrator or victim DDIs with other substrates, inducers, or inhibitors. To simulate a potential interaction with a moderate CYP2C8 inhibitor, two novel PBPK models representing a moderate CYP2C8 inhibitor and a sensitive CYP2C8 substrate were developed based on the existing PBPK models for gemfibrozil and rosiglitazone, respectively. The simulated population geometric mean area under the curve ratio of tucatinib with a moderate CYP2C8 inhibitor ranged from 1.98- to 3.08-fold, and based on these results, no dose modifications were proposed for moderate CYP2C8 inhibitors for the tucatinib label.

Rational design and biological evaluation of gemfibrozil modified Xenopus GLP-1 derivatives as long-acting hypoglycemic agents.

Novel methods for peptides structural modification and bioactivity optimization are highly needed in peptide-based drug discovery. Herein, we explored the use gemfibrozil (GFZ) as an albumin binder to enhance the stability and improve the bioactivity of peptides. Short-acting Xenopus glucagon-like peptide-1 (xGLP-1) analogues with anti-diabetic activity were selected as the starting point. Mono-GFZ conjugation, peptide sequence hybridization, and dimeric-GFZ derivatization were successively used to generate novel GFZ-xGLP-1 conjugates, biologically screened by various in vitro and in vivo models. Dimeric-GFZ modified conjugate 3b was finally identified as a promising anti-diabetic candidate with high albumin binding affinity, enhanced in vivo stability in SD rats, and long-acting hypoglycemic activity in db/db mice. Moreover, GFZ endowed 3b with strong lipid-regulating ability in DIO and db/db mice. In a twelve-week study, chronic administration of 3b in db/db mice resulted in sustained glycemic control, to a greater extent than liraglutide and semaglutide. In addition, 3b showed comparable therapeutic efficacies to liraglutide and semaglutide on HbA1c and pancreas islets protection. Our studies reveal 3b as a potential candidate for the treatment of metabolic diseases and indicate dimeric-GFZ modification as a novel method for peptide optimization.

Effects of Strong CYP2C8 or CYP3A Inhibition and CYP3A Induction on the Pharmacokinetics of Brigatinib, an Oral Anaplastic Lymphoma Kinase Inhibitor, in Healthy Volunteers.

In vitro data support involvement of cytochrome P450 (CYP)2C8 and CYP3A4 in the metabolism of the anaplastic lymphoma kinase inhibitor brigatinib. A 3-arm, open-label, randomized, single-dose, fixed-sequence crossover study was conducted to characterize the effects of the strong inhibitors gemfibrozil (of CYP2C8) and itraconazole (of CYP3A) and the strong inducer rifampin (of CYP3A) on the single-dose pharmacokinetics of brigatinib. Healthy subjects (n = 20 per arm) were administered a single dose of brigatinib (90 mg, arms 1 and 2; 180 mg, arm 3) alone in treatment period 1 and coadministered with multiple doses of gemfibrozil 600 mg twice daily (BID; arm 1), itraconazole 200 mg BID (arm 2), or rifampin 600 mg daily (QD; arm 3) in period 2. Compared with brigatinib alone, coadministration of gemfibrozil with brigatinib did not meaningfully affect brigatinib area under the plasma concentration-time curve (AUC0-inf ; geometric least-squares mean [LSM] ratio [90%CI], 0.88 [0.83-0.94]). Coadministration of itraconazole with brigatinib increased AUC0-inf (geometric LSM ratio [90%CI], 2.01 [1.84-2.20]). Coadministration of rifampin with brigatinib substantially reduced AUC0-inf (geometric LSM ratio [90%CI], 0.20 [0.18-0.21]) compared with brigatinib alone. The treatments were generally tolerated. Based on these results, strong CYP3A inhibitors and inducers should be avoided during brigatinib treatment. If concomitant use of a strong CYP3A inhibitor is unavoidable, the results of this study support a dose reduction of brigatinib by approximately 50%. Furthermore, CYP2C8 is not a meaningful determinant of brigatinib clearance, and no dose modifications are needed during coadministration of brigatinib with CYP2C8 inhibitors.

Physiologically Based Pharmacokinetic Models for Prediction of Complex CYP2C8 and OATP1B1 (SLCO1B1) Drug-Drug-Gene Interactions: A Modeling Network of Gemfibrozil, Repaglinide, Pioglitazone, Rifampicin, Clarithromycin and Itraconazole.

BACKGROUND: Drug-drug interactions (DDIs) and drug-gene interactions (DGIs) pose a serious health risk that can be avoided by dose adaptation. These interactions are investigated in strictly controlled setups, quantifying the effect of one perpetrator drug or polymorphism at a time, but in real life patients frequently take more than two medications and are very heterogenous regarding their genetic background. OBJECTIVES: The first objective of this study was to provide whole-body physiologically based pharmacokinetic (PBPK) models of important cytochrome P450 (CYP) 2C8 perpetrator and victim drugs, built and evaluated for DDI and DGI studies. The second objective was to apply these models to describe complex interactions with more than two interacting partners. METHODS: PBPK models of the CYP2C8 and organic-anion-transporting polypeptide (OATP) 1B1 perpetrator drug gemfibrozil (parent-metabolite model) and the CYP2C8 victim drugs repaglinide (also an OATP1B1 substrate) and pioglitazone were developed using a total of 103 clinical studies. For evaluation, these models were applied to predict 34 different DDI studies, establishing a CYP2C8 and OATP1B1 PBPK DDI modeling network. RESULTS: The newly developed models show a good performance, accurately describing plasma concentration-time profiles, area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) values, DDI studies as well as DGI studies. All 34 of the modeled DDI AUC ratios (AUC during DDI/AUC control) and DDI Cmax ratios (Cmax during DDI/Cmax control) are within twofold of the observed values. CONCLUSIONS: Whole-body PBPK models of gemfibrozil, repaglinide, and pioglitazone have been built and qualified for DDI and DGI prediction. PBPK modeling is applicable to investigate complex interactions between multiple drugs and genetic polymorphisms.

Application of a physiologically based pharmacokinetic model for the prediction of mirabegron plasma concentrations in a population with severe renal impairment.

We previously verified a physiologically based pharmacokinetic (PBPK) model for mirabegron in healthy subjects using the Simcyp Simulator by incorporating data on the inhibitory effect on cytochrome P450 (CYP) 2D6 and a multi-elimination pathway mediated by CYP3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7 and butyrylcholinesterase (BChE). The aim of this study was to use this PBPK model to assess the magnitude of drug-drug interactions (DDIs) in an elderly population with severe renal impairment (sRI), which has not been evaluated in clinical trials. We first determined the system parameters, and meta-analyses of literature data suggested that the abundance of UGT2B7 and the BChE activity in an elderly population with sRI was almost equivalent to and 20% lower than that in healthy young subjects, respectively. Other parameters, such as the CYP3A4 abundance, for an sRI population were used according to those built into the Simcyp Simulator. Second, we confirmed that the PBPK model reproduced the plasma concentration-time profile for mirabegron in an sRI population (simulated area under the plasma concentration-time curve (AUC) was within 1.5-times that of the observed value). Finally, we applied the PBPK model to simulate DDIs in an sRI population. The PBPK model predicted that the AUC for mirabegron with itraconazole (a CYP3A4 inhibitor) was 4.12-times that in healthy elderly subjects administered mirabegron alone, and predicted that the proportional change in AUC for desipramine (a CYP2D6 substrate) with mirabegron was greater than that in healthy subjects. In conclusion, the PBPK model was verified for the purpose of DDI assessment in an elderly population with sRI.

Quantitative Analysis of Complex Drug-Drug Interactions between Cerivastatin and Metabolism/Transport Inhibitors Using Physiologically Based Pharmacokinetic Modeling.

Cerivastatin (CER) was withdrawn from the world market because of lethal rhabdomyolysis. Coadministrations of CER and cyclosporine A (CsA) or gemfibrozil (GEM) have been reported to increase the CER blood concentration. CsA is an inhibitor of organic anion transporting polypeptide (OATP)1B1 and CYP3A4, and GEM and its glucuronide (GEM-glu) inhibit OATP1B1 and CYP2C8. The purpose of this study was to describe the transporter-/enzyme-mediated drug-drug interactions (DDIs) of CER with CsA or GEM based on unified physiologically based pharmacokinetic (PBPK) models and to investigate whether the DDIs can be quantitatively analyzed by a bottom-up approach. Initially, the PBPK models for CER and GEM/GEM-glu were constructed based on the previously reported standard protocols. Next, the drug-dependent parameters were optimized by Cluster Newton Method. Thus, described concentration-time profiles for CER and GEM/GEM-glu agreed well with the clinically observed data. The DDIs were then simulated using the established PBPK models with previously obtained in vitro inhibition constants of CsA or GEM/GEM-glu against the OATP1B1 and cytochrome P450s. DDIs with the inhibitors were underestimated compared with observed data using the geometric means of reported values. To search for better described parameters within the range of in vitro values, sensitivity analyses were performed for DDIs of CER. Using the in vitro parameter sets selected by sensitivity analyses, these DDIs were well reproduced, indicating that the present PBPK models were able to describe adequately the clinical DDIs based on a bottom-up approach. The approaches in this study would be applicable to the prediction of other DDIs involving both transporters and metabolic enzymes.

Investigating Transporter-Mediated Drug-Drug Interactions Using a Physiologically Based Pharmacokinetic Model of Rosuvastatin.

Rosuvastatin is a frequently used probe in transporter-mediated drug-drug interaction (DDI) studies. This report describes the development of a physiologically based pharmacokinetic (PBPK) model of rosuvastatin for prediction of pharmacokinetic (PK) DDIs. The rosuvastatin model predicted the observed single (i.v. and oral) and multiple dose PK profiles, as well as the impact of coadministration with transporter inhibitors. The predicted effects of rifampin and cyclosporine (6.58-fold and 5.07-fold increase in rosuvastatin area under the curve (AUC), respectively) were mediated primarily via inhibition of hepatic organic anion-transporting polypeptide (OATP)1B1 (Inhibition constant (Ki ) ∼1.1 and 0.014 µM, respectively) and OATP1B3 (Ki ∼0.3 and 0.007 µM, respectively), with cyclosporine also inhibiting intestinal breast cancer resistance protein (BCRP; Ki ∼0.07 µM). The predicted effects of gemfibrozil and its metabolite were moderate (1.88-fold increase in rosuvastatin AUC) and mediated primarily via inhibition of hepatic OATP1B1 and renal organic cation transporter 3. This model of rosuvastatin will be useful in prospectively predicting transporter-mediated DDIs with novel pharmaceutical agents in development.

Quantitative Rationalization of Gemfibrozil Drug Interactions: Consideration of Transporters-Enzyme Interplay and the Role of Circulating Metabolite Gemfibrozil 1-O-ß-Glucuronide.

Gemfibrozil has been suggested as a sensitive cytochrome P450 2C8 (CYP2C8) inhibitor for clinical investigation by the U.S. Food and Drug Administration and the European Medicines Agency. However, gemfibrozil drug-drug interactions (DDIs) are complex; its major circulating metabolite, gemfibrozil 1-O-ß-glucuronide (Gem-Glu), exhibits time-dependent inhibition of CYP2C8, and both parent and metabolite also behave as moderate inhibitors of organic anion transporting polypeptide 1B1 (OATP1B1) in vitro. Additionally, parent and metabolite also inhibit renal transport mediated by OAT3. Here, in vitro inhibition data for gemfibrozil and Gem-Glu were used to assess their impact on the pharmacokinetics of several victim drugs (including rosiglitazone, pioglitazone, cerivastatin, and repaglinide) by employing both static mechanistic and dynamic physiologically based pharmacokinetic (PBPK) models. Of the 48 cases evaluated using the static models, about 75% and 98% of the DDIs were predicted within 1.5- and 2-fold of the observed values, respectively, when incorporating the interaction potential of both gemfibrozil and its 1-O-ß-glucuronide. Moreover, the PBPK model was able to recover the plasma profiles of rosiglitazone, pioglitazone, cerivastatin, and repaglinide under control and gemfibrozil treatment conditions. Analyses suggest that Gem-Glu is the major contributor to the DDIs, and its exposure needed to bring about complete inactivation of CYP2C8 is only a fraction of that achieved in the clinic after a therapeutic gemfibrozil dose. Overall, the complex interactions of gemfibrozil can be quantitatively rationalized, and the learnings from this analysis can be applied in support of future predictions of gemfibrozil DDIs.

Effect of gemfibrozil, rifampicin, or probenecid on the pharmacokinetics of the SGLT2 inhibitor empagliflozin in healthy volunteers.

BACKGROUND: Empagliflozin is a potent, oral, selective inhibitor of sodium glucose cotransporter 2 in development for the treatment of type 2 diabetes mellitus. OBJECTIVE: The goal of these studies was to investigate potential drug-drug interactions between empagliflozin and gemfibrozil (an organic anion-transporting polypeptide 1B1 [OATP1B1]/1B3 and organic anion transporter 3 [OAT3] inhibitor), rifampicin (an OATP1B1/1B3 inhibitor), or probenecid (an OAT3 and uridine diphosphate glucuronosyltransferase inhibitor). METHODS: Two open-label, randomized, crossover studies were undertaken in healthy subjects. In the first study, 18 subjects received the following in 1 of 2 randomized treatment sequences: a single dose of empagliflozin 25 mg alone and gemfibrozil 600 mg BID for 5 days with a single dose of empagliflozin 25 mg on the third day. In the second study, 18 subjects received a single dose of empagliflozin 10 mg, a single dose of empagliflozin 10 mg coadministered with a single dose of rifampicin 600 mg, and probenecid 500 mg BID for 4 days with a single dose of empagliflozin 10 mg on the second day in 1 of 6 randomized treatment sequences. RESULTS: In the gemfibrozil study, 11 subjects were male, mean age was 35.1 years and mean body mass index (BMI) was 23.47 kg/m(2). In the rifampicin/probenecid study, 10 subjects were male, mean age was 32.7 years and mean BMI was 23.03 kg/m(2). Exposure to empagliflozin was increased by coadministration with gemfibrozil (AUC0-∞: geometric mean ratio [GMR], 158.50% [90% CI, 151.77-165.53]; Cmax: GMR, 115.00% [90% CI, 106.15-124.59]), rifampicin (AUC0-∞: GMR, 135.20% [90% CI, 129.58-141.06]; Cmax: GMR, 175.14% [90% CI, 160.14-191.56]), and probenecid (AUC0-∞: GMR, 153.47% [90% CI, 146.41-160.88]; Cmax: GMR, 125.60% [90% CI, 113.67-138.78]). All treatments were well tolerated. CONCLUSIONS: Increases in empagliflozin exposure were <2-fold, indicating that the inhibition of the OATP1B1/1B3, OAT3 transporter, and uridine diphosphate glucuronosyltransferases did not have a clinically relevant effect on empagliflozin exposure. No dose adjustments of empagliflozin were necessary when it was coadministered with gemfibrozil, rifampicin, or probenecid. ClinicalTrials.gov identifiers: NCT01301742 and NCT01634100.

Mechanistic modeling to predict the transporter- and enzyme-mediated drug-drug interactions of repaglinide.

PURPOSE: Quantitative prediction of complex drug-drug interactions (DDIs) is challenging. Repaglinide is mainly metabolized by cytochrome-P-450 (CYP)2C8 and CYP3A4, and is also a substrate of organic anion transporting polypeptide (OATP)1B1. The purpose is to develop a physiologically based pharmacokinetic (PBPK) model to predict the pharmacokinetics and DDIs of repaglinide. METHODS: In vitro hepatic transport of repaglinide, gemfibrozil and gemfibrozil 1-O-ß-glucuronide was characterized using sandwich-culture human hepatocytes. A PBPK model, implemented in Simcyp (Sheffield, UK), was developed utilizing in vitro transport and metabolic clearance data. RESULTS: In vitro studies suggested significant active hepatic uptake of repaglinide. Mechanistic model adequately described repaglinide pharmacokinetics, and successfully predicted DDIs with several OATP1B1 and CYP3A4 inhibitors (<10% error). Furthermore, repaglinide-gemfibrozil interaction at therapeutic dose was closely predicted using in vitro fraction metabolism for CYP2C8 (0.71), when primarily considering reversible inhibition of OATP1B1 and mechanism-based inactivation of CYP2C8 by gemfibrozil and gemfibrozil 1-O-ß-glucuronide. CONCLUSIONS: This study demonstrated that hepatic uptake is rate-determining in the systemic clearance of repaglinide. The model quantitatively predicted several repaglinide DDIs, including the complex interactions with gemfibrozil. Both OATP1B1 and CYP2C8 inhibition contribute significantly to repaglinide-gemfibrozil interaction, and need to be considered for quantitative rationalization of DDIs with either drug.

Analysis of the repaglinide concentration increase produced by gemfibrozil and itraconazole based on the inhibition of the hepatic uptake transporter and metabolic enzymes.

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.

Physiologically based modeling of pravastatin transporter-mediated hepatobiliary disposition and drug-drug interactions.

PURPOSE: To develop physiologically based pharmacokinetic (PBPK) model to predict the pharmacokinetics and drug-drug interactions (DDI) of pravastatin, using the in vitro transport parameters. METHODS: In vitro hepatic sinusoidal active uptake, passive diffusion and canalicular efflux intrinsic clearance values were determined using sandwich-culture human hepatocytes (SCHH) model. PBPK modeling and simulations were implemented in Simcyp (Sheffield, UK). DDI with OATP1B1 inhibitors, cyclosporine, gemfibrozil and rifampin, was also simulated using inhibition constant (Ki) values. RESULTS: SCHH studies suggested active uptake, passive diffusion and efflux intrinsic clearance values of 1.9, 0.5 and 1.2 µL/min/10(6)cells, respectively, for pravastatin. PBPK model developed, using transport kinetics and scaling factors, adequately described pravastatin oral plasma concentration-time profiles at different doses (within 20% error). Model based prediction of DDIs with gemfibrozil and rifampin was similar to that observed. However, pravastatin-cyclosporine DDI was underpredicted (AUC ratio 4.4 Vs ~10). Static (R-value) model predicted higher magnitude of DDI compared to the AUC ratio predicted by the PBPK modeling. CONCLUSIONS: PBPK model of pravastatin, based on in vitro transport parameters and scaling factors, was developed. The approach described can be used to predict the pharmacokinetics and DDIs associated with hepatic uptake transporters.

Gemfibrozil is a strong inactivator of CYP2C8 in very small multiple doses.

Therapeutic doses of gemfibrozil cause mechanism-based inactivation of CYP2C8 via formation of gemfibrozil 1-O-ß-glucuronide. We investigated the extent of CYP2C8 inactivation caused by three different doses of gemfibrozil twice dailyfor 5 days, using repaglinide as a probe drug, in 10 healthy volunteers. At the end of this 5-day regimen, there were dose-dependent increases in the area under the plasma concentration­time curve from 0 to infinity (AUC0­∞) of repaglinide by3.4-, 5.5-, and 7.0-fold corresponding to 30, 100, and 600 mg of gemfibrozil, respectively, as compared with the control phase (P < 0.001). On the basis of a mechanism-based inactivation model involving gemfibrozil 1-O-ß-glucuronide, a gemfibrozil dose of 30 mg twice daily was estimated to inhibit CYP2C8 by >70% and 100 mg twice daily was estimated to inhibit it by >90%. Hence, gemfibrozil is a strong inactivator of CYP2C8 even in very small, subtherapeutic, multiple doses. Administration of small gemfibrozil doses may be useful in optimizing the pharmacokinetics of CYP2C8 substrate drugs and in reducing the formation of their potentially toxic metabolites via CYP2C8.

Dose-dependent interaction between gemfibrozil and repaglinide in humans: strong inhibition of CYP2C8 with subtherapeutic gemfibrozil doses.

Gemfibrozil 1-O-ß-glucuronide inactivates CYP2C8 irreversibly. We investigated the effect of gemfibrozil dose on CYP2C8 activity in humans using repaglinide as a probe drug. In a randomized, five-phase crossover study, 10 healthy volunteers ingested 0.25 mg of repaglinide 1 h after different doses of gemfibrozil or placebo. Concentrations of plasma repaglinide, gemfibrozil, their metabolites, and blood glucose were measured. A single gemfibrozil dose of 30, 100, 300, and 900 mg increased the area under the concentration-time curve of repaglinide 1.8-, 4.5-, 6.7-, and 8.3-fold (P < 0.001), and its peak concentration 1.4-, 1.7-, 2.1-, and 2.4-fold (P < 0.05), compared with placebo, respectively. Gemfibrozil pharmacokinetics was characterized by a slightly more than dose-proportional increase in the area under the curve of gemfibrozil and its glucuronide. The gemfibrozil-repaglinide interaction could be mainly explained by gemfibrozil 1-O-ß-glucuronide concentration-dependent, mechanism-based inhibition of CYP2C8, with a minor contribution by competitive inhibition of organic anion-transporting polypeptide 1B1 at the highest gemfibrozil dose. The findings are consistent with ∼50% inhibition of CYP2C8 already with a single 30-mg dose of gemfibrozil and >95% inhibition with 900 mg. In clinical drug-drug interaction studies, a single 900-mg dose of gemfibrozil can be used to achieve nearly complete inactivation of CYP2C8.

Mechanism-based inactivation of CYP2C8 by gemfibrozil occurs rapidly in humans.

To study the time to onset of mechanism-based inactivation of cytochrome P450 (CYP) 2C8 by gemfibrozil in vivo, we conducted a randomized five-phase crossover study in 10 healthy volunteers. In one phase the volunteers ingested 0.25 mg of repaglinide alone (control), and in the other phases they received 600 mg of gemfibrozil 0-6 h prior to the repaglinide dose. When gemfibrozil was taken 0, 1, 3, or 6 h before repaglinide, the geometric mean ratio relative to control (90% confidence interval (CI)) of repaglinide area under the plasma concentration-time curve (AUC(0-∞)) was 5.0-fold (4.3-5.7-fold), 6.3-fold (5.4-7.5-fold), 6.6-fold (5.6-7.7-fold), and 5.4-fold (4.8-6.1-fold), respectively (P < 0.001 vs. control). The geometric mean ratio relative to control (90% CI) of the maximum plasma concentration (C(max)) of the CYP2C8-mediated metabolite M4 was 1.0-fold (0.8-1.3-fold), 0.10-fold (0.06-0.17-fold, P < 0.001), 0.06-fold (0.04-0.10-fold, P < 0.001), and 0.09-fold (0.05-0.14-fold, P < 0.001), respectively. The strong inactivation of CYP2C8, evident as soon as 1 h after gemfibrozil dosing, has implications in clinical practice and in studies with gemfibrozil as a CYP2C8 model inhibitor.

The CYP2C8 inhibitor gemfibrozil does not affect the pharmacokinetics of zafirlukast.

PURPOSE: Gemfibrozil, a strong inhibitor of cytochrome P450 (CYP) 2C8 in vivo, was recently found to markedly increase the plasma concentrations of montelukast in humans. Like montelukast, zafirlukast is a substrate of CYP2C9 and CYP3A4 and a potent inhibitor of CYP2C8 in vitro. To investigate the contribution of CYP2C8 to the metabolism of zafirlukast in vivo, we studied the effect of gemfibrozil on the pharmacokinetics of zafirlukast. METHODS: Ten healthy subjects in a randomized cross-over study took gemfibrozil 600 mg or placebo twice daily for 5 days, and on day 3, a single oral dose of 20 mg zafirlukast. The plasma concentrations of zafirlukast were measured for 72 h postdose. RESULTS: The mean total area under the plasma concentration-time curve of zafirlukast during the gemfibrozil phase was 102% (geometric mean ratio; 95% confidence interval 89-116%) of that during the placebo phase. Furthermore, there were no statistically significant differences in the peak plasma concentration, time of peak concentration, or elimination half-life of zafirlukast between the phases. CONCLUSIONS: Gemfibrozil has no effect on the pharmacokinetics of zafirlukast, indicating that CYP2C8 does not play a significant role in the elimination of zafirlukast.

Validation of cell-based OATP1B1 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions.

Transporters are carrier proteins that may influence pharmacokinetic, pharmacodynamic, and toxicological characteristics of drugs. The development of validated in vitro transporter models is imperative to support regulatory submissions of drug candidates. This study is focused on utilizing human embryonic kidney (HEK) 293 cell cultures genetically transfected with the human organic anion transporting polypeptides (OATP) 1B1 transporter to identify substrates and inhibitors in drug development. The kinetics of OATP1B1-mediated uptake of [(3)H]-oestradiol 17beta-glucuronide and inhibition of uptake by rifamycin SV were used to determine K(m), V(max), and IC(50) values over a range of passage numbers to investigate accuracy and precision. The mean K(m) and V(max) values were found to be 6.3 +/- 1.2 microM and 460 +/- 96 pmol min(-1) mg(-1), respectively. The mean IC(50) value for rifamycin SV was 0.23 +/- 0.07 microM on uptake of 1 microM [(3)H]-oestradiol 17beta-glucuronide. These data were similar to previously reported values (accuracy greater than 82%), reproducible (precision less than 29%) and exhibited low standard deviations (SDs) obviating the need to study test compounds on more than one occasion. [(3)H]-oestrone 3-sulfate and [(3)H]-pravastatin exhibited concentration-dependent OATP1B1 uptake, and statistically significant differences were observed at each concentration between uptake rates of HEK293-OATP1B1 and HEK293-MOCK cells (uptake ratios greater than or equal to 3). Propranolol showed no positive uptake ratio. Bezafibrate and gemfibrozil exhibited concentration-dependent inhibition of OATP1B1-mediated uptake of [(3)H]-oestradiol 17beta-glucuronide with mean IC(50) values of 16 and 27 microM, respectively. Based on the validation results, acceptance criteria to identify a test compound as a substrate and/or inhibitor using these specific cell lines were determined. These validated OATP1B1 assays were robust, reproducible, and suitable for routine in vitro evaluation of candidate drugs.

CYP2C8 activity recovers within 96 hours after gemfibrozil dosing: estimation of CYP2C8 half-life using repaglinide as an in vivo probe.

Gemfibrozil 1-O-beta-glucuronide is a mechanism-based inhibitor of cytochrome P450 2C8. We studied the recovery of CYP2C8 activity after discontinuation of gemfibrozil treatment using repaglinide as a probe drug, to estimate the in vivo turnover half-life of CYP2C8. In a randomized five-phase crossover study, nine healthy volunteers ingested 0.25 mg of repaglinide alone or after different time intervals after a 3-day treatment with 600 mg of gemfibrozil twice daily. The area under the plasma concentration-time curve (AUC) from time 0 to infinity of repaglinide was 7.6-, 2.9-, 1.4- and 1.0-fold compared with the control phase when it was administered 1, 24, 48, or 96 h after the last gemfibrozil dose, respectively (P < 0.001 versus control for 1, 24, and 48 h after gemfibrozil). Thus, a strong CYP2C8 inhibitory effect persisted even after gemfibrozil and gemfibrozil 1-O-beta-glucuronide concentrations had decreased to less than 1% of their maximum (24-h dosing interval). In addition, the metabolite to repaglinide AUC ratios indicated that significant (P < 0.05) inhibition of repaglinide metabolism continued up to 48 h after gemfibrozil administration. Based on the recovery of repaglinide oral clearance, the in vivo turnover half-life of CYP2C8 was estimated to average 22 +/- 6 h (mean +/- S.D.). In summary, CYP2C8 activity is recovered gradually during days 1 to 4 after gemfibrozil discontinuation, which should be considered when CYP2C8 substrate dosing is planned. The estimated CYP2C8 half-life will be useful for in vitro-in vivo extrapolations of drug-drug interactions involving induction or mechanism-based inhibition of CYP2C8.

The effect of gemfibrozil on repaglinide pharmacokinetics persists for at least 12 h after the dose: evidence for mechanism-based inhibition of CYP2C8 in vivo.

Repaglinide is metabolized by cytochrome P450 (CYP) 2C8 and 3A4. Gemfibrozil has the effect of increasing the area under the concentration-time curve (AUC) of repaglinide eightfold. We studied the effect of dosing interval on the extent of the gemfibrozil-repaglinide interaction. In a randomized five-phase crossover study, 10 healthy volunteers ingested 0.25 mg repaglinide, with or without gemfibrozil pretreatment. Plasma repaglinide, gemfibrozil, their metabolites, and blood glucose were measured. When the last dose of 600 mg gemfibrozil was ingested simultaneously with repaglinide, or 3, 6, or 12 h before, it increased the AUC(0-infinity) of repaglinide 7.0-, 6.5-, 6.2- and 5.0-fold, respectively (P < 0.001). The peak repaglinide concentration increased approximately twofold (P < 0.001), and the half-life was prolonged from 1.2 h to 2-3 h (P < 0.001) during all the gemfibrozil phases. The drug interaction effects persisted at least 12 h after gemfibrozil was administered, although plasma gemfibrozil and gemfibrozil 1-O-beta-glucuronide concentrations were only 5 and 10% of their peak values, respectively. The long-lasting interaction is likely caused by mechanism-based inhibition of CYP2C8 by gemfibrozil glucuronide.

Multiple inhibition mechanisms and prediction of drug-drug interactions: status of metabolism and transporter models as exemplified by gemfibrozil-drug interactions.

PURPOSE: To assess the consequences of multiple inhibitors and differential inhibition mechanisms on the prediction of 12 gemfibrozil drug-drug interactions (DDIs). In addition, qualitative zoning of transporter-related gemfibrozil and cyclosporine DDIs was investigated. METHODS: The effect of gemfibrozil and its acyl-glucuronide on different enzymes was incorporated into a metabolic prediction model. The impact of CYP2C8 time-dependent inhibition by gemfibrozil acyl-glucuronide was assessed using repaglinide, cerivastatin, loperamide, rosiglitazone and pioglitazone DDIs. Gemfibrozil and cyclosporine inhibition data obtained in human embryonic kidney cells expressing OATP1B1 and hepatic input concentration ([I]in) were used for qualitative zoning of 14 transporter-mediated DDIs. RESULTS: Incorporation of time-dependent inhibition by gemfibrozil glucuronide showed no significant improvement in the prediction, as CYP2C8 contributed <65% to the overall elimination of the victim drugs investigated. Qualitative zoning of OATP1B1 DDIs resulted in no false negative predictions; yet the magnitude of observed interactions was significantly over-predicted. CONCLUSIONS: Time-dependent inhibition by gemfibrozil glucuronide is only important for victim drugs eliminated predominantly (>80%) via CYP2C8. Qualitative zoning of OATP1B1 inhibitors based on [I]in/K (i) is valid in drug screening to avoid false negatives. Refinement of the transporter model by incorporating the fraction of drug transported by a particular transporter is recommended.