Optimization of chromatography to overcome matrix effect for reliable estimation of four small molecular drugs from biological fluids using LC-MS/MS.

The article describes a systematic study to overcome the matrix effect during chromatographic analysis of gemfibrozil, rivastigmine, telmisartan and tacrolimus from biological fluids using LC-ESI-MS/MS. All four methods were thoroughly developed by the appropriate choice of analytical column, elution mode and pH of mobile phase for improved chromatography and overall method performance. Matrix effect was assessed by post-column analyte infusion, slope of calibration line approach and post-extraction spiking. The best chromatographic conditions established were: Acquity BEH C18 (50 × 2.1 mm, 1.7 µm) column with 5.0 mm ammonium acetate, pH 6.0-methanol as the mobile phase under gradient program for gemfibrozil; Luna CN (50 × 2.0 mm, 3 µm) column with a mobile phase consisting of acetonitrile-10 mm ammonium acetate, pH 7.0 (90:10, v/v) for rivastigmine; Inertsustain C18 (100 × 2.0 mm, 5 µm) column using methanol-2.0 mm ammonium formate, pH 5.5 (80: 20, v/v) as the mobile phase for isocratic elution of telmisartan; and Acquity BEH C18 (50 × 2.1 mm, 1.7 µm) with methanol-10 mm ammonium acetate, pH 6.0 (95:5, v/v) as mobile phase for tacrolimus. The methods were thoroughly validated as per European Medicines Agency and US Food and Drug Administration guidance and were successfully applied for pharmacokinetic studies in healthy subjects.

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.

Species-related exposure of phase II metabolite gemfibrozil 1-O-ß-glucuronide between human and mice: A net induction of mouse P450 activity was revealed.

Gemfibrozil is a fibrate drug used widely for dyslipidemia associated with atherosclerosis. Clinically, both gemfibrozil and its phase II metabolite gemfibrozil 1-O-ß-glucuronide (gem-glu) are involved in drug-drug interaction (DDI). But the DDI risk caused by gem-glu between human and mice has not been compared. In this study, six volunteers were recruited and took a therapeutic dose of gemfibrozil for 3 days for examination of the gemfibrozil and gem-glu level in human. Male mice were fed a gemfibrozil diet (0.75%) for 7 days, following which a cocktail-based inhibitory DDI experiment was performed. Plasma samples and liver tissues from mice were collected for determination of gemfibrozil, gem-glu concentration and cytochrome p450 enzyme (P450) induction analysis. In human, the molar ratio of gem-glu/gemfibrozil was 15% and 10% at the trough concentration and the concentration at 1.5 h after the 6th dose. In contrast, this molar ratio at steady state in mice was 91%, demonstrating a 6- to 9-fold difference compared with that in human. Interestingly, a net induction of P450 activity and in vivo inductive DDI potential in mice was revealed. The P450 activity was not inhibited although the gem-glu concentration was high. These data suggested species difference of relative gem-glu exposure between human and mice, as well as a net inductive DDI potential of gemfibrozil in mouse model.

Alteration of the intravenous and oral pharmacokinetics of valsartan via the concurrent use of gemfibrozil in rats.

The present study aimed to examine the potential pharmacokinetic drug interaction between valsartan and gemfibrozil. Compared with the control given valsartan (10 mg/kg) alone, the concurrent use of gemfibrozil (10 mg/kg) significantly (p < 0.05) increased the oral exposure of valsartan in rats. In the presence of gemfibrozil, the Cmax and AUC of oral valsartan increased by 1.7- and 2.5-fold, respectively. Consequently, the oral bioavailability of valsartan was significantly higher (p < 0.05) in the presence of gemfibrozil compared with that of the control group. Furthermore, the intravenous pharmacokinetics of valsartan (1 mg/kg) was also altered by pretreatment with oral gemfibrozil (10 mg/kg). The plasma clearance of valsartan was decreased by two-fold in the presence of gemfibrozil, while the plasma half-life was not altered. In contrast, both the oral and intravenous pharmacokinetics of gemfibrozil were not affected by the concurrent use of valsartan. The cellular uptake of valsartan and gemfibrozil was also investigated by using cells overexpressing OATP1B1 or OATP1B3. Gemfibrozil and gemfibrozil 1-O-ß glucuronide inhibited the cellular uptake of valsartan with IC50 values (µm) of 39.3 and 20.4, respectively, in MDCK/OATP1B1, while they were less interactive with OATP1B3. The cellular uptake of gemfibrozil was not affected by co-incubation with valsartan in both cells. Taken together, the present study suggests the potential drug interaction between valsartan and gemfibrozil, at least in part, via the OATP1B1-mediated transport pathways during hepatic uptake. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

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.

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.

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.

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.

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.

Pharmacokinetic drug interaction between gemfibrozil and sitagliptin in healthy Indian male volunteers.

PURPOSE: To study the impact of gemfibrozil co-administration on the pharmacokinetics of sitagliptin in healthy Indian male volunteers. METHODS: A randomized open label two-period crossover study involving 12 healthy Indian male volunteers was conducted at a single center. In each phase, the volunteers were administered sitagliptin as 100 mg tablets, either alone or co-administered with gemfibrozil as 600 mg tablets twice daily for 3 days. There was a 2-week washout period between phases. The venous blood samples were serially collected at 0-12 h post-dose, and plasma concentrations of the study drugs were estimated by a validated high-performance liquid chromatography-ultraviolet method. RESULTS: Relative to the administration of sitagliptin alone, co-administration with gemfibrozil increased the AUC0₋12 (2,167 ± 82.9 vs. 2,970 ± 76.4 ng h/ml; p < 0.0001), AUC(0-∞) (3,621 ± 222.5 vs. 5,574 ± 249.6 ng h/ml; p < 0.0002), C(max) (282.9 ± 7.7 vs. 344.1 ± 5.9 ng/ml; p < 0.0001), and t(½) (7.4 ± 0.6 vs. 10 ± 0.6 h; p = 0.0076) to statistically significant levels. The interindividual differences in the pharmacokinetic parameters of sitagliptin were found to be within acceptable limits (coefficient of variation <20%). No adverse drug events associated with sitagliptin occurred in the subjects during the study period. CONCLUSION: Although the bioavailability of sitagliptin was increased by 54% when co-administered with gemfibrozil, this interaction may not have any clinical significance as sitagliptin has a wide therapeutic index. Hence, in clinical practice, sitagliptin as 100 mg tablets and gemfibrozil as 600 mg tablets may be co-prescribed without much threat of sitagliptin toxicity. However, these results may not hold if the dose of sitagliptin is increased or if is co-prescribed with other antidiabetic drugs and/or cytochrome P450 2C8/human organic anion transporter-3 inhibitors. Further studies are needed to confirm these results in patients.

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.

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.

Validation of an LC/MS method for the determination of gemfibrozil in human plasma and its application to a pharmacokinetic study.

Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug-drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA-anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra-assay precision between 1.6 and 10.7%, and inter-assay precision ranging from 4.4 to 7.8%. The assay also showed good accuracy, with intra-assay concentrations within 85.6-108.7% of the expected value, and inter-assay concentrations within 89.4-104.0% of the expected value. The linear concentration range was between 0.5 and 50 µg/mL with a lower limit of quantitation of 0.5 µg/mL when 125 µL of plasma were extracted. This LC/MS method yielded a quick, simple and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies.

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.

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.

Myotoxicity of gemfibrozil in cynomolgus monkey model and its relationship to pharmacokinetic properties.

Fibrate drugs are PPARalpha agonists prescribed for the treatment of dyslipidemia. Severe myotoxicity has been reportedly associated with their use albeit at a low frequency, especially for gemfibrozil. Few studies have investigated the mechanism of fibrate-induced myotoxicity in vivo. Considering the apparent species-related differences in PPARalpha agonist-induced hepatotoxicity, we studied the myotoxicity of gemfibrozil in a Cynomolgus monkey model and explored the relationship between myotoxicity and pharmacokinetics. Six Cynomolgus monkeys were dosed with gemfibrozil twice daily at 600 mg/kg/day for the first two periods (P1 and P2, 8 days and 9 days respectively) and 300 mg/kg/day for the third period (P3, 14 days). Creatine kinase and myoglobin were measured, together with hepatotoxicity and nephrotoxicity markers. Behavioral responses were recorded for indication of toxicity. Pharmacokinetics was carried out following the 16th dosage of P1 and 17th dosage of P2 when myotoxicity was identified. Multivariable data analysis was employed to explore the relationship between pharmacokinetic parameters and myotoxicity markers. Consequently, myotoxicity occurred in monkey #2 (M2) and M6 in P1, M3 and M4 in P2, M3 and M6 in P3. Data analysis showed T80-150 (sustained time above the given concentration) contributed for myotoxicity discriminance and correlated with myotoxicity risk. This study revealed Cynomolgus monkey may be a good animal model for myotoxicity evaluation with sensitivity, reproducibility and similarities to humans. More interestingly, they exhibited a much higher incidence of myotoxicity than that of humans. Sustained high drug concentration plays an important role for the occurrence of myotoxicity. This may suggest an influence of drug transport and metabolism on myotoxicity.

UPLC-MS/MS method for gemfibrozil determination in plasma with application to a pharmacokinetic study in healthy Brazilian volunteers.

Gemfibrozil (GFZ) is a derivative of fibric acid and is used in the treatment of dyslipidemia. GFZ may affect the metabolism of various drugs, including statins, by inhibiting the sinusoidal influx transporter OATP1B1 and also CYP2C9 and CYP2C8 enzymes. This study presents the development and validation of a rapid, simple, sensitive and reproducible method of GFZ analysis in human plasma using UPLC-MS/MS. The method was applied in a pharmacokinetic study following administration of multiple doses of 600 mg GFZ every 12 h in healthy volunteers (n = 15). GFZ was separated on a C18 column using a mixture of 0.01% formic acid and acetonitrile (40:60, v/v) as the mobile phase at a flow rate of 0.4 mL/min. The method showed linearity in the range from 0.01 µg/mL to 100 µg/mL plasma. The coefficients of variation and the relative standard errors of the accuracy and precision analyses were <15%. The method allowed quantification of plasma concentrations of GFZ in the dose interval of the sixth day of administration of multiple oral doses of GFZ every 12 h. The pharmacokinetic parameters are presented as mean (95% CI): area under the plasma concentration versus time curve 88.84 (72.72-104.96) µg·h/mL, steady state mean plasma concentration 7.40 (6.06-8.75) µg/mL, minimum plasma concentration 1.24 (0.87-1.61) µg/mL, maximum plasma concentration 26.73 (21.31-32.15) µg/mL, time to reach maximum plasma concentration 2.28 (1.42-3.13) h, elimination half-life 2.81 (2.22-3.40) h, apparent total clearance 7.72 (5.85-9.58) L/h, apparent distribution volume 33.97 (18.41-49.53) L. In conclusion, the method for analysis of GFZ in human plasma showed sensitivity, linearity, precision and accuracy compatible with application in pharmacokinetic studies of multiple oral dose of 600 mg GFZ every 12 h.