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Pyridoxic acid (PDA) was suggested as a potential endogenous biomarker to assess in vivo renal organic anion transporter (OAT) 1 and 3 activity. Here, we first investigated the population variability in the plasma baseline levels of PDA using data from five independent studies (conducted/supported by Pfizer), and subsequently developed mechanistic physiologically based pharmacokinetic (PBPK) model to assess its effectiveness in biomarker-informed drug-drug interaction (DDI) predictions. Meta-analysis suggested that the inter-individual variability in PDA plasma concentration was ~40% across all five studies (n = 71 subjects). While sex-dependent differences were not evident, the baseline plasma PDA levels were significantly higher (38%, p < 0.05) in White males compared to Japanese males. Correspondingly, the amount of PDA excreted in urine and renal clearance were significantly higher (p < 0.05) in Japanese males (1.5- and 2.2-fold, respectively), compared to White males. A PBPK model considering relative activity factor-based scaling of in vitro transport data indicated > 80% contribution by OAT3 to the renal clearance of PDA. The baseline plasma concentrations across multiple studies were recovered by the model; and using in vitro inhibition potency data, the model predicted effect of OAT inhibitors (probenecid, ritlecitinib and tafamidis) on PDA pharmacokinetics. Furthermore, DDIs with OAT3 object drug, furosemide, were well-predicted by the biomarker-informed PBPK model. PDA data and the modeling approach indicated lack of clinically-relevant OAT inhibition with ritlecitinib and tafamidis. Overall, this study presents PDA as a reliable biomarker to assess OAT3-mediated renal DDIs with moderate inter-subject and inter-study variability., (© 2025 The Author(s). CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

In vitro assessment of the potential of compounds to affect drug metabolizing enzymes and transporters and perpetrate drug-drug interactions (DDIs) is a common practice in drug research. For the development phase, regulators define an exhaustive list of enzymes and transporters to consider, but DDIs associated with many of these are minor and can be well-managed in the clinic; thus, progression of drug candidates that address unmet medical needs should not be curtailed due to this property. However, some enzymes and transporters are very important in drug disposition, so it is important to avoid/reduce inhibition or induction of these through drug design. Herein, simplified criteria and methodologies amenable to high-throughput screening are defined to enable drug design to address DDI risk. A strategy is proposed that focuses on the most important enzymes and transporters: namely, cytochrome P450 (CYP) 3A4, CYP2C9, and CYP2D6, organic anion transporting polypeptide (OATP) 1B1, and breast cancer resistant protein (BCRP).

Rifampicin is a widely employed index inhibitor to assess the impact of organic anion transporting polypeptide 1B (OATP1B) inhibition on investigational drugs. The observation of nitrosamines in certain drug products, including rifampicin, has impacted the conduct of clinical drug-drug interaction (DDI) studies with rifampicin drug products. Cyclosporine is a recommended alternative to assess in vivo OATP1B activity; however, challenges exist in its use due to pharmacokinetic (PK) variability and non-selective inhibition of other drug disposition mechanisms., (© 2025 The Author(s). Clinical Pharmacology & Therapeutics © 2025 American Society for Clinical Pharmacology and Therapeutics.)

Background and Objective: Physiologically based pharmacokinetic (PBPK) models are valuable for translating in vitro absorption, distribution, metabolism, and excretion (ADME) data to predict clinical pharmacokinetics, and can enable discovery and early clinical stages of pharmaceutical research. However, in predicting pharmacokinetics of organic anion transporting polypeptide (OATP) 1B substrates based on in vitro transport and metabolism data, PBPK models typically require additional empirical in vitro-to-in vivo scaling factors (ESFs) in order to accurately recapitulate observed clinical profiles. As model simulation is very sensitive to ESFs, a critical evaluation of ESF estimation is prudent. Previously studies have applied classic 'two-stage' and 'naïve pooled data' approaches in identifying a set of compound independent ESFs. However, the 'two-stage' approach has the parameter identification issue in separately fitting data for individual compounds, while the 'naïve pooled data' approach ignores interstudy variability, leading to potentially biased ESF estimates., Methods: In this study, we have applied a nonlinear mixed-effect approach in estimating ESF of the PBPK model and incorporated additional data from 86 runs of in vitro uptake assay and 49 clinical studies of 12 training compounds in model development to further enhance the translation of in vitro data to predict the pharmacokinetics of OATP1B substrate drugs. To test predication accuracy of the model, a 'leave-one-out' analysis has been performed., Results: The established model can reasonably describe the clinical observations, with both mean values and interstudy variabilities quantified for ESF and volume of distribution parameters. The mean estimates are largely consistent with values in the previous reports. The interstudy variabilities of these parameters are estimated to be at least 50% (as coefficient of variation). Most compounds can be reasonably predicted in the 'leave-one-out' analysis., Conclusion: This study improves the confidence in predicting the pharmacokinetics of OATP1B substrates in individual studies of small sample sizes, and quantifies the variability associated with the prediction., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Daprodustat is the first oral hypoxia-inducible factor prolyl hydroxylase inhibitor approved recently for the treatment of anemia caused by chronic kidney disease (CKD) in adults receiving dialysis. We evaluated the role of organic anion transporting polypeptide (OATP)1B-mediated hepatic uptake transport in the pharmacokinetics (PKs) of daprodustat using in vitro and in vivo studies, and physiologically-based PK (PBPK) modeling of its drug-drug interactions (DDIs) with inhibitor drugs. In vitro, daprodustat showed specific transport by OATP1B1/1B3 in the transfected cell systems and primary human and monkey hepatocytes. A single-dose oral rifampin (OATP1B inhibitor) reduced daprodustat intravenous clearance by a notable 9.9 ± 1.2-fold (P < 0.05) in cynomolgus monkeys. Correspondingly, volume of distribution at steady-state was also reduced by 5.0 ± 1.1-fold, whereas the half-life change was minimal (1.5-fold), corroborating daprodustat hepatic uptake inhibition by rifampin. A PBPK model accounting for OATP1B-CYP2C8 interplay was developed, which well described daprodustat PK and DDIs with gemfibrozil (CYP2C8 and OATP1B inhibitor) and trimethoprim (weak CYP2C8 inhibitor) within 25% error of the observed data in healthy subjects. About 18-fold increase in daprodustat area under the curve (AUC) following gemfibrozil treatment was found to be associated with strong CYP2C8 inhibition and moderate OATP1B inhibition. Moreover, PK modulation in hepatic dysfunction and subjects with CKD, in comparison to healthy control, was well-captured by the model. CYP2C8 and/or OATP1B inhibitor drugs (e.g., gemfibrozil, clopidogrel, rifampin, and cyclosporine) were predicted to perpetrate moderate-to-strong DDIs in healthy subjects, as well as, in target CKD population. Daprodustat can be used as a sensitive CYP2C8 index substrate in the absence of OATP1B modulation., (© 2024 The Authors. Clinical Pharmacology & Therapeutics © 2024 American Society for Clinical Pharmacology and Therapeutics.)

The effect of membrane transporters on drug disposition, efficacy and safety is now well recognized. Since the initial publication from the International Transporter Consortium, significant progress has been made in understanding the roles and functions of transporters, as well as in the development of tools and models to assess and predict transporter-mediated activity, toxicity and drug-drug interactions (DDIs). Notable advances include an increased understanding of the effects of intrinsic and extrinsic factors on transporter activity, the application of physiologically based pharmacokinetic modelling in predicting transporter-mediated drug disposition, the identification of endogenous biomarkers to assess transporter-mediated DDIs and the determination of the cryogenic electron microscopy structures of SLC and ABC transporters. This article provides an overview of these key developments, highlighting unanswered questions, regulatory considerations and future directions., (© 2024. Springer Nature Limited.)

Organic anion transporting polypeptide (OATP1B) plays a key role in the hepatic clearance of a majority of high molecular weight (MW) acids and zwitterions. Here, we evaluated the role of OATP1B-mediated uptake in the clearance of novel hypoxia-inducible factor prolyl hydroxylase inhibitors ("Dustats"), which are typically low MW (300-400 daltons) aliphatic carboxylic acids. Five acid dustats, namely daprodustat, desidustat, enarodustat, roxadustat and vadadustat, showed specific transport by OATP1B1/1B3 in transporter-transfected HEK293 cells. Neutral compound, molidustat, was not a substrate to OATP1B1/1B3. None of the dustats showed transport by other hepatic uptake transporters, including NTCP, OAT2 and OAT7. In the primary human hepatocytes, uptake of all acids was significantly reduced by rifampin (OATP1B inhibitor); with an estimated fraction transported by OATP1B ( f t ,OATP1B ) of up to >80% (daprodustat). Molidustat uptake was minimally inhibited by rifampin; and low permeability acids (desidustat and enarodustat) also showed biliary efflux in sandwich culture human hepatocytes. In vivo, intravenous pharmacokinetics of all 5 acids was significantly altered by a single-dose rifampin (30 mg/kg) in Cynomolgus monkey. Hepatic clearance (non-renal) was about 4-fold (vadadustat) to >11-fod (daprodustat and roxadustat) higher in control group compared to rifampin-treated subjects. In vivo f t ,OATP1B was estimated to be ~70-90%. In the case of molidustat, rifampin had a minimal effect on overall clearance. Rifampin also considerably reduced volume of distribution of daprodustat and roxadustat. Overall, OATP1B significantly contribute to the hepatic clearance and pharmacokinetics of several dustats, which are low MW carboxylic acids. OATP1B activity should therefore by evaluated in this property space. Significance Statement Our in vitro and in vivo results suggest that OATP1B-mediated hepatic uptake play a significant role in the pharmacokinetics of low MW acidic dustats, which are being developed or approved for the treatment of anemia in chronic kidney disease. Significant active uptake mechanisms are not apparent for the neutral compound, molidustat. Characterization of uptake mechanisms is therefore important in predicting human pharmacokinetics and evaluating drug-drug interactions for low MW acids., (Copyright © 2024 American Society for Pharmacology and Experimental Therapeutics.)

Creatinine, a clinical marker for kidney function, is predominantly cleared by glomerular filtration, with active tubular secretion contributing to about 30% of its renal clearance. Recent studies suggested the potential involvement of organic anion transporter (OAT)2, in addition to the previously known organic cation transporter (OCT)2-mediated basolateral uptake, in creatinine active secretion. Here we characterized the transport mechanisms of creatinine using transfected human embryonic kidney (HEK)293 cells and freshly prepared human primary renal proximal tubule epithelial cells (hPTCs). Creatinine showed transport by OAT2 in transfected HEK293 cells. In addition, both creatinine and metformin showed transport by OCT2 and multidrug and toxin extrusion pump (MATE)1 and MATE2K, while penciclovir was selective for OAT2. Time-dependent cell accumulation was observed for creatinine and metformin in hPTCs. Their accumulation was increased by pyrimethamine but inhibited by decynium-22, likely due to differential inhibition of OCT2 versus MATEs. Additionally, indomethacin (an OAT2 inhibitor) reduced penciclovir uptake (∼75%) in hPTCs illustrating functional OAT2 activity. However, no modulation of creatinine and metformin cell accumulation was apparent with indomethacin. Creatinine transport characteristics in the presence of inhibitors approached those of metformin, an OCT2/MATE substrate, but were distinct from those of penciclovir, an OAT2-selective substrate. Moreover, indomethacin showed no significant effect on the basolateral-to-apical transport and net secretion of creatinine across hPTC monolayers. Collectively, the functional studies suggest OCT2 as the primary basolateral uptake mechanism and that OAT2 has a minimal role, in creatinine renal secretion. Our results highlight the utility of hPTCs to enable the functional assessment of renal transport mechanisms. SIGNIFICANCE STATEMENT: Our results obtained with primary hPTCs indicate that OCT2/MATE (vs. OAT2) play a major role in the active renal secretion of creatinine. Quantitative pharmacokinetic models should therefore focus on OCT2/MATE when describing serum creatinine and creatinine clearance modulation by inhibitor drugs and genotype- or disease-related activity changes. The present study highlights the utility of freshly isolated hPTCs to support solute carrier phenotyping to enable the functional assessment of renal transport mechanisms., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

The effect of food on oral drug absorption is determined by the complex interplay among gut physiological factors and drug properties. The currently used dissolution testing and classification systems (biopharmaceutics classification system, BCS or biopharmaceutics drug disposition classification system, BDDCS) do not account for dynamic changes in gastrointestinal physiology caused by food intake. This study aimed to identify key drug properties that influence food effect (FE) using supervised machine learning approaches. The analysis showed that drugs with high logP, dose number, and extraction ratio have a higher probability of positive FE, while drugs with low permeability and high efflux saturation index have a greater likelihood of negative FE. Weakly acidic drugs also showed a greater probability of positive FE, particularly at pKa >4.3. The importance of drug properties in predicting FE was ranked as logP, dose number, extraction ratio, pKa, and permeability. The accuracy of FE prediction using the models was compared with BCS and extended clearance classification system (ECCS). Overall, the likelihood or magnitude of FE depends on physiological changes to food intake such as altered bile acid secretion rate, intestinal metabolism, transport kinetics, and gastric emptying time, which should be considered along with drug properties (e.g., solubility, logP, and ionization) in predicting FE of orally administered drugs., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Drug-drug interactions (DDIs) involving hepatic organic anion transporting polypeptides 1B1/1B3 (OATP1B) can be substantial, however, challenges remain for predicting interaction risk. Emerging evidence suggests that endogenous biomarkers, particularly coproporphyrin-I (CP-I), can be used to assess in vivo OATP1B activity. The present work under the International Consortium for Innovation and Quality in Pharmaceutical Development was aimed primarily at assessing CP-I as a biomarker for informing OATP1B DDI risk. Literature and unpublished CP-I data along with pertinent in vitro and clinical DDI information were collected to identify DDIs primarily involving OATP1B inhibition and assess the relationship between OATP1B substrate drug and CP-I exposure changes. Static models to predict changes in exposure of CP-I, as a selective OATP1B substrate, were also evaluated. Significant correlations were observed between CP-I area under the curve ratio (AUCR) or maximum concentration ratio (C max R) and AUCR of substrate drugs. In general, the CP-I C max R was equal to or greater than the CP-I AUCR. CP-I C max R < 1.25 was associated with absence of OATP1B-mediated DDIs (AUCR < 1.25) with no false negative predictions. CP-I C max R < 2 was associated with weak OATP1B-mediated DDIs (AUCR < 2). A correlation was identified between CP-I exposure changes and OATP1B1 static DDI predictions. Recommendations for collecting and interpreting CP-I data are discussed, including a decision tree for guiding DDI risk assessment. In conclusion, measurement of CP-I is recommended to inform OATP1B inhibition potential. The current analysis identified changes in CP-I exposure that may be used to prioritize, delay, or replace clinical DDI studies., (© 2023 The Authors. Clinical Pharmacology & Therapeutics © 2023 American Society for Clinical Pharmacology and Therapeutics.)

P-glycoprotein (P-gp) may limit oral drug absorption of substrate drugs due to intestinal efflux. Therefore, regulatory agencies require investigation of new chemical entities as possible inhibitors of P-gp in vitro. Unfortunately, inter-laboratory and inter-assay variability have hindered the translatability of in vitro P-gp inhibition data to predict clinical drug interaction risk. The current study was designed to evaluate the impact of potential IC 50 discrepancies between two commonly utilized assays, i.e., bi-directional Madin-Darby Canine Kidney-MDR1 cell-based and MDR1 membrane vesicle-based assays. When comparing vesicle- to cell-based IC 50 values (n = 28 inhibitors), non-P-gp substrates presented good correlation between assay formats, whereas IC 50 s of P-gp substrates were similar or lower in the vesicle assays. The IC 50 s obtained with a cell line expressing relatively low P-gp aligned more closely to those obtained from the vesicle assay, but passive permeability of the inhibitors did not appear to influence the correlation of IC 50 s, suggesting that efflux activity reduces intracellular inhibitor concentrations. IC 50 s obtained between two independent laboratories using the same assay type showed good correlation. Using the G-value (i.e., ratio of estimated gut concentration-to-inhibition potency) >10 cutoff recommended by regulatory agencies resulted in minimal differences in predictive performance, suggesting this cutoff is appropriate for either assay format., Competing Interests: Declaration of Competing Interest SOLVO Biotechnology, a Charles River Laboratories Hungary, as the employer of some of the authors, develops and commercializes reagents and assays to study membrane transporters. The authors declare no conflict of interest., (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Hepatic impairment (HI) is known to modulate drug disposition and may lead to elevated plasma exposure. The aim of this study was to quantitate the in vivo OATP1B-mediated hepatic uptake activity in populations with varying degrees of HI. First, we measured baseline levels of plasma coproporphyrin-I, an endogenous OATP1B biomarker, in an open-label, parallel cohort study in adult subjects with normal liver function and mild, moderate, and severe HI (n = 24, 6/cohort). The geometric mean plasma concentrations of coproporphyrin-I were 1.66-fold, 2.81-fold (P < 0.05), and 7.78-fold (P < 0.0001) higher in mild, moderate, and severe impairment than those healthy controls. Second, we developed a dataset of 21 OATP1B substrate drugs with HI data extracted from literature. Median disease-to-healthy plasma area under the curve (AUC) ratios for substrate drugs were ~ 1.4, 3.0, and 6.4 for mild, moderate, and severe HI, respectively. Additionally, significant linear relationship was noted between AUC ratios of substrate drugs without and with co-administration of rifampin, a prototypic OATP1B inhibitor, and AUC ratios in moderate (P < 0.01) and severe (P < 0.001) HI. Third, a physiologically-based pharmacokinetic model analysis was conducted with 10 substrate drugs following estimation of relative OATP1B functional activity in virtual disease population models using coproporphyrin-I data and verification of substrate models with rifampin drug-drug interaction data. This approach adequately predicted plasma AUC change particularly in moderate (9 of 10 within 2-fold) and severe (5 of 5 within 2-fold) HI. Collective findings indicate progressive reduction, by as much as 90-92%, in OATP1B activity in the HI population., (© 2022 The Authors. Clinical Pharmacology & Therapeutics © 2022 American Society for Clinical Pharmacology and Therapeutics.)

Introduction: Membrane transporters are now widely recognized for their role in the absorption, distribution, clearance, and elimination of drugs. The organic cation transporters (OCTs, SLC22A) are expressed in the intestine, liver, and kidneys and are of importance in determining systemic pharmacokinetics (PK) and tissue-specific exposure of drugs and metabolites., Areas Covered: An overview of the role of OCTs in drug disposition is presented. Genetic variation in OCTs and the effects on PK and drug response were discussed., Expert Opinion: Clinical studies demonstrated significance of OCT1 and OCT2 in the hepatic uptake and renal secretion of drug, respectively. These mechanisms are important in determining the systemic PK and tissue exposure and thus pharmacodynamics of several drugs (e.g. metformin, morphine, sumatriptan). Emerging pharmacogenomic data also suggests multidrug and toxin extrusion pump (MATE1, SLC47A1) contribution to PK and response of drugs like metformin and cisplatin. Considerations to genotyping of functional and common variants of OCTs should be given, particularly for cationic drugs with hepatic elimination or renal secretion being major clearance pathways, in the clinical development. While the current evidence indicate that pharmacokinetic variability associated with known genotypes of OCTs/MATEs is relatively small, they may be of relevance in the tissue-specific effects and for drugs with low therapeutic index.

Organic anion transporter 2 (OAT2 or SLC22A7 ) plays an important role in the hepatic uptake and renal secretion of several endogenous compounds and drugs. The goal of this work is to understand the structure activity of OAT2 inhibition and assess clinical drug interaction risk. A single-point inhibition assay using OAT2-transfected HEK293 cells was employed to screen about 150 compounds; and concentration-dependent inhibition potency (IC 50 ) was measured for the identified "inhibitors". Acids represented about 65% of all inhibitors, and the frequency of bases-plus-zwitterions approximately doubled for "non-inhibitors". Interestingly, 9 of 10 most potent inhibitors (low IC 50 ) are acids (p K a ∼ 3-5). Additionally, inhibitors are significantly larger and lipophilic than non-inhibitors. In silico (binary) models were developed to identify inhibitors and non-inhibitors. Finally, in vivo risk assessed via static drug-drug interaction models identified several inhibitors with potential for renal and hepatic OAT2 inhibition at clinical doses. This is the first study assessing the global pattern of OAT2-ligand interactions.

PF-06835919 is a first-in-class ketohexokinase inhibitor (KHKi), recently under development for the treatment of metabolic and fatty liver diseases, which inhibited organic anion transporting polypeptide (OATP)1B1 in vitro and presented drug-drug interaction (DDI) risk. This study aims to investigate the dose-dependent effect of KHKi on OATP1B in vivo activity. We performed an open-label study comparing pharmacokinetics of atorvastatin (OATP1B probe) dosed alone (20 mg single dose) and coadministered with two dose strengths of KHKi (50 and 280 mg once daily) in 12 healthy participants. Additionally, changes in exposure of coproporphyrin-I (CP-I), an endogenous biomarker for OATP1B, were assessed in the atorvastatin study (1.12-fold and 1.49-fold increase in area under the plasma concentration-time profile (AUC) with once-daily 50 and 280 mg, respectively), and a separate single oral dose study of KHKi alone (100-600 mg, n = 6 healthy participants; up to a 1.80-fold increase in AUC). Geometric mean ratios (90% confidence interval) of atorvastatin AUC following 50 and 280 mg KHKi were 1.14 (1.00-1.30) and 1.54 (1.37-1.74), respectively. Physiologically-based pharmacokinetic modeling of CP-I plasma exposure following a single dose of KHKi predicted in vivo OATP1B inhibition from about 13% to 70% over the 100 to 600 mg dose range, while using the in vitro inhibition potency (1.9 µM). Model-based analysis correctly predicted "no-effect" (AUC ratio < 1.25) at the low dose range and "weak" effect (AUC ratio < 2) on atorvastatin pharmacokinetics at the high dose range of KHKi. This study exemplified the utility of biomarker-informed model-based approach in discerning even small effects on OATP1B activity in vivo, and to project DDI risk at the clinically relevant doses., (© 2022 The Authors. Clinical Pharmacology & Therapeutics © 2022 American Society for Clinical Pharmacology and Therapeutics.)

During its fourth transporter workshop in 2021, the International Transporter Consortium (ITC) provided updates on emerging clinically relevant transporters for drug development. Previously highlighted and new transporters were considered based on up-to-date clinical evidence of their importance in drug-drug interactions and potential for altered drug efficacy and safety, including drug-nutrient interactions leading to nutrient deficiencies. For the first time, folate transport pathways (PCFT, RFC, and FRα) were examined in-depth as a potential mechanism of drug-induced folate deficiency and related toxicities (e.g., neural tube defects and megaloblastic anemia). However, routine toxicology studies conducted in support of drug development appear sufficient to flag such folate deficiency toxicities, whereas prospective prediction from in vitro folate metabolism and transport inhibition is not well enough established to inform drug development. Previous suggestion of a retrospective study of intestinal OATP2B1 inhibition to explain unexpected decreases in drug exposure were updated. Furthermore, when the absorption of a new molecular entity is more rapid and extensive than can be explained by passive permeability, evaluation of the OATP2B1 transport may be considered. Emerging research on hepatic and renal OAT2 is summarized, but current understanding of the importance of OAT2 was deemed insufficient to justify specific consideration for drug development. Hepatic, renal, and intestinal MRPs (MRP2, MRP3, and MRP4) were revisited. MRPs may be considered when they are suspected to be the major determinant of drug disposition (e.g., direct glucuronide conjugates); MRP2 inhibition as a mechanistic explanation for drug-induced hyperbilirubinemia remains justified. There were no major changes in recommendations from previous ITC whitepapers., (© 2022 The Authors. Clinical Pharmacology & Therapeutics © 2022 American Society for Clinical Pharmacology and Therapeutics.)

The role of membrane transporters on pharmacokinetics (PKs), drug-drug interactions (DDIs), pharmacodynamics (PDs), and toxicity of drugs has been broadly recognized. However, our knowledge of modulation of transporter expression and/or function in the diseased patient population or specific populations, such as pediatrics or pregnancy, is still emerging. This white paper highlights recent advances in studying the changes in transporter expression and activity in various diseases (i.e., renal and hepatic impairment and cancer) and some specific populations (i.e., pediatrics and pregnancy) with the focus on clinical implications. Proposed alterations in transporter abundance and/or activity in diseased and specific populations are based on (i) quantitative transporter proteomic data and relative abundance in specific populations vs. healthy adults, (ii) clinical PKs, and emerging transporter biomarker and/or pharmacogenomic data, and (iii) physiologically-based pharmacokinetic modeling and simulation. The potential for altered PK, PD, and toxicity in these populations needs to be considered for drugs and their active metabolites in which transporter-mediated uptake/efflux is a major contributor to their absorption, distribution, and elimination pathways and/or associated DDI risk. In addition to best practices, this white paper discusses current challenges and knowledge gaps to study and quantitatively predict the effects of modulation in transporter activity in these populations, together with the perspectives from the International Transporter Consortium (ITC) on future directions., (© 2022 The Authors. Clinical Pharmacology & Therapeutics © 2022 American Society for Clinical Pharmacology and Therapeutics.)

Excess dietary fructose consumption promotes metabolic dysfunction thereby increasing the risk of obesity, type 2 diabetes, non-alcoholic steatohepatitis (NASH), and related comorbidities. PF-06835919, a first-in-class ketohexokinase (KHK) inhibitor, showed reversal of such metabolic disorders in preclinical models and clinical studies, and is under clinical development for the potential treatment of NASH. In this study, we evaluated the transport and metabolic pathways of PF-06835919 disposition and assessed pharmacokinetics in preclinical models. PF-06835919 showed active uptake in cultured primary human hepatocytes, and substrate activity to organic anion transporter (OAT)2 and organic anion transporting-polypeptide (OATP)1B1 in transfected cells. "SLC-phenotyping" studies in human hepatocytes suggested contribution of passive uptake, OAT2- and OATP1B-mediated transport to the overall uptake to be about 15%, 60% and 25%, respectively. PF-06835919 showed low intrinsic metabolic clearance in vitro, and was found to be metabolized via both oxidative pathways (58%) and acyl glucuronidation (42%) by CYP3A, CYP2C8, CYP2C9 and UGT2B7. Following intravenous dosing, PF-06835919 showed low clearance (0.4-1.3 mL/min/kg) and volume of distribution (0.17-0.38 L/kg) in rat, dog and monkey. Human oral pharmacokinetics are predicted within 20% error when considering transporter-enzyme interplay in a PBPK model. Finally, unbound liver-to-plasma ratio (Kp uu ) measured in vitro using rat, NHP and human hepatocytes was found to be approximately 4, 25 and 10, respectively. Similarly, liver Kp uu in rat and monkey following intravenous dosing of PF-06835919 was found to be 2.5 and 15, respectively, and notably higher than the muscle and brain Kp uu , consistent with the active uptake mechanisms observed in vitro. Significance Statement This work characterizes the transport/metabolic pathways in the hepatic disposition of PF-06835919, a first-in-class KHK inhibitor for the treatment of metabolic disorders and NASH. Phenotyping studies using transfected systems, human hepatocytes and liver microsomes signifies the role of OAT2 and OATP1B1 in the hepatic uptake and multiple enzymes in the metabolism of PF-06835919. Data presented suggest hepatic transporter-enzyme interplay in determining its systemic concentrations and potential enrichment in liver, a target site for KHK inhibition., (Copyright © 2020 American Society for Pharmacology and Experimental Therapeutics.)

Quantitative prediction of drug-drug interactions (DDIs) involving organic anion transporting polypeptide (OATP)1B1/1B3 inhibition is limited by uncertainty in the translatability of experimentally determined in vitro inhibition potency (half-maximal inhibitory concentration (IC 50 )). This study used an OATP1B endogenous biomarker-informed physiologically-based pharmacokinetic (PBPK) modeling approach to predict the effect of inhibitor drugs on the pharmacokinetics (PKs) of OATP1B substrates. Initial static analysis with about 42 inhibitor drugs, using in vitro IC 50 values and unbound liver inlet concentrations (I in,max,u ), suggested in vivo OATP1B inhibition risk for drugs with R-value (1+ I in,max,u /IC 50 ) above 1.5. A full-PBPK model accounting for transporter-mediated hepatic disposition was developed for coproporphyrin I (CP-I), an endogenous OATP1B biomarker. For several inhibitors (cyclosporine, diltiazem, fenebrutinib, GDC-0810, itraconazole, probenecid, and rifampicin at 3 different doses), PBPK models were developed and verified against available CP-I plasma exposure data to obtain in vivo OATP1B inhibition potency-which tend to be lower than the experimentally measured in vitro IC 50 by about 2-fold (probenecid and rifampicin) to 37-fold (GDC-0810). Models verified with CP-I data are subsequently used to predict DDIs with OATP1B probe drugs, rosuvastatin and pitavastatin. The predicted and observed area under the plasma concentration-time curve ratios are within 20% error in 55% cases, and within 30% error in 89% cases. Collectively, this comprehensive study illustrates the adequacy and utility of endogenous biomarker-informed PBPK modeling in mechanistic understanding and quantitative predictions of OATP1B-mediated DDIs in drug development., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics.)

Quantitative assessment of drug-drug interactions (DDIs) involving breast cancer resistance protein (BCRP) inhibition is challenged by overlapping substrate/inhibitor specificity. This study used physiologically-based pharmacokinetic (PBPK) modeling to delineate the effects of inhibitor drugs on BCRP- and organic anion transporting polypeptide (OATP)1B-mediated disposition of rosuvastatin, which is a recommended BCRP clinical probe. Initial static model analysis using in vitro inhibition data suggested BCRP/OATP1B DDI risk while considering regulatory cutoff criteria for a majority of inhibitors assessed (25 of 27), which increased rosuvastatin plasma exposure to varying degree (~ 0-600%). However, rosuvastatin area under plasma concentration-time curve (AUC) was minimally impacted by BCRP inhibitors with calculated G-value (= gut concentration/inhibition potency) below 100. A comprehensive PBPK model accounting for intestinal (OATP2B1 and BCRP), hepatic (OATP1B, BCRP, and MRP4), and renal (OAT3) transport mechanisms was developed for rosuvastatin. Adopting in vitro inhibition data, rosuvastatin plasma AUC changes were predicted within 25% error for 9 of 12 inhibitors evaluated via PBPK modeling. This study illustrates the adequacy and utility of a mechanistic model-informed approach in quantitatively assessing BCRP-mediated DDIs., (© 2021 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Background and Objective: The use of herbal medicines is common in Africa, and patients often use a combination of herbs and drugs. Concurrent herbal and pharmaceuticals treatments can cause adverse effects through herb-drug interactions (HDI). This study evaluated the potential risk of HDI for five medicinal plants, Vernonia amygdalina, Ocimum gratissimum, Moringa oleifera, Azadirachta indica, and Picralima nitida, using in vitro assays. Patients with diabetes and some other disease conditions commonly use these medicinal plants in Nigeria, and little is known regarding their potential for drug interaction, despite their enormous use., Methods: Crude extracts of the medicinal plants were evaluated for reversible and time-dependent inhibition (TDI) activity of six cytochrome P450 (CYP) enzymes using pooled human liver microsomes and cocktail probe-based assays. Enzyme activity was determined by quantifying marker metabolites' formation using liquid chromatography-mass spectrometry/mass spectrometry. The drug interaction potential was predicted for each herbal extract using the in vitro half-maximal inhibitory concentration (IC 50 ) values and the percentage yield., Results: O. gratissimum methanol extracts reversibly inhibited CYP 1A2, 2C8, 2C9 and 2C19 enzymes (IC 50 : 6.21 µg/ml, 2.96 µg/ml, 3.33 µg/ml and 1.37 µg/ml, respectively). Additionally, V. amygdalina methanol extract inhibited CYP2C8 activity (IC 50 : 5.71 µg/ml); P. nitida methanol and aqueous extracts inhibited CYP2D6 activity (IC 50 : 1.99 µg/ml and 2.36 µg/ml, respectively) while A. indica methanol extract inhibited CYP 3A4/5, 2C8 and 2C9 activity (IC 50 : 7.31 µg/ml, 9.97 µg/ml and 9.20 µg/ml, respectively). The extracts showed a potential for TDI of the enzymes when incubated at 200 µg/ml; V. amygdalina and A. indica methanol extracts exhibited TDI potential for all the major CYPs., Conclusions: The medicinal plants inhibited CYP activity in vitro, with the potential to cause in vivo HDI. Clinical risk assessment and proactive monitoring are recommended for patients who use these medicinal plants concurrently with drugs that are cleared through CYP metabolism.

It is generally presumed that uptake transport mechanisms are of limited significance in hepatic clearance for lipophilic or high passive-permeability drugs. In this study, we evaluated the mechanistic role of the hepato-selective organic anion-transporting polypeptides (OATPs) 1B1/1B3 in the pharmacokinetics of compounds representing large lipophilic acid space. Intravenous pharmacokinetics of 16 compounds with molecular mass ∼400-730 Da, logP ∼3.5-8, and acid pKa <6 were obtained in cynomolgus monkey after dosing without and with a single-dose rifampicin-OATP1B1/1B3 probe inhibitor. Rifampicin (30 mg/kg oral) significantly ( P < 0.05) reduced monkey clearance and/or steady-state volume of distribution (VDss) for 15 of 16 acids evaluated. Additionally, clearance of danoprevir was reduced by about 35%, although statistical significance was not reached. A significant linear relationship was noted between the clearance ratio (i.e., ratio of control to treatment groups) and VDss ratio, suggesting hepatic uptake contributes to the systemic clearance and distribution simultaneously. In vitro transport studies using primary monkey and human hepatocytes showed uptake inhibition by rifampicin (100 µM) for compounds with logP ≤6.5 but not for the very lipophilic acids (logP > 6.5), which generally showed high nonspecific binding in hepatocyte incubations. In vitro uptake clearance and fraction transported by OATP1B1/1B3 (f t,OATP1B ) were found to be similar in monkey and human hepatocytes. Finally, for compounds with logP ≤6.5, good agreement was noted between in vitro f t,OATP1B and clearance ratio (as well as VDss ratio) in cynomolgus monkey. In conclusion, this study provides mechanistic evidence for the pivotal role of OATP1B-mediated hepatic uptake in the pharmacokinetics across a wide, large lipophilic acid space. SIGNIFICANCE STATEMENT: This study provides mechanistic insight into the pharmacokinetics of a broad range of large lipophilic acids. Organic anion-transporting polypeptides 1B1/1B3-mediated hepatic uptake is of key importance in the pharmacokinetics and drug-drug interactions of almost all drugs and new molecular entities in this space. Diligent in vitro and in vivo transport characterization is needed to avoid the false negatives often noted because of general limitations in the in vitro assays while handling compounds with such physicochemical attributes., Competing Interests: All authors are full-time employees of Pfizer Inc. The authors have no conflicts of interest that are directly relevant to this study. No funding was received for the work reported here., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Current challenges with the in vitro-in vivo extrapolation (IVIVE) of hepatic uptake clearance involving organic anion-transporting polypeptide (OATP) 1B1/1B3 hinder drug design strategies. Here we evaluated the effect of 100% human plasma on the uptake clearance using transfected human embryonic kidney (HEK) 293 cells and primary human hepatocytes and assessed IVIVE. Apparent unbound uptake clearance (PS inf,u ) increased significantly ( P < 0.05) in the presence of plasma (vs. buffer incubations) for about 50% of compounds in both OATP1B1-transfected and wild-type HEK cells. Thus, plasma showed a minimal effect on the uptake ratios. With cultured human hepatocytes, plasma significantly ( P < 0.05) increased PS inf,u for 11 of 19 OATP1B substrates (median change of 2.1-fold). Cell accumulation in HEK cells and hepatocytes was also increased for tolbutamide, which is not an OATP substrate. Plasma-to-buffer ratio of PS inf,u obtained in hepatocytes showed a good correlation with unbound fraction in plasma, and the relationship was best described by a "facilitated-dissociation" model. IVIVE was evaluated for the 19 OATP1B substrates using hepatocyte data in the presence of buffer and plasma. PS inf,u from buffer incubations markedly underpredicted hepatic intrinsic clearance (calculated via well stirred and parallel tube models) with an estimated bias of 0.10-0.13. Predictions improved when using PS inf,u from plasma incubations; however, considerable systemic underprediction was still apparent (0.19-0.26 bias). Plasma data with a global scaling factor of 3.8-5.3 showed good prediction accuracy (95% predictions within 3-fold; average fold error = 1.7, bias = 1). In summary, this study offers insight into the effect of plasma on the uptake clearance and its scope in improving IVIVE. SIGNIFICANCE STATEMENT: Our study using diverse anionic compounds shows that human plasma facilitates organic anion-transporting polypeptide 1B-mediated as well as passive uptake clearance, particularly for the highly bound compounds. Leveraging data from transfected human embryonic kidney 293 cells and primary human hepatocytes, we further evaluated mechanisms involved in the observed plasma-facilitated uptake transport. Enhanced hepatic uptake rate in the presence of plasma could be of relevance, as such mechanisms likely prevail in vivo and emphasize the need to maintain physiologically relevant assay conditions to achieve improved translation of transport data., Competing Interests: All authors are full-time employees of Pfizer Inc. The authors have no conflicts of interest that are directly relevant to this study., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Hepatic uptake clearance has been measured in suspended human hepatocytes (SHH). Plated human hepatocytes (PHH) after short-term culturing are increasingly employed to study hepatic transport driven mainly by its higher throughput. To know pros/cons of both systems, the hepatic uptake clearances of several organic anion transporting polypeptide 1B substrates were compared between PHH and SHH by determining the initial uptake velocities or through dynamic model-based analyses. For cerivastatin, pitavastatin and rosuvastatin, initial uptake clearances (PS inf ) obtained using PHH were comparable to those using SHH, while cell-to-medium concentration (C/M) ratios were 2.7- to 5.4-fold higher. For pravastatin and dehydropravastatin, hydrophilic compounds with low uptake/cellular binding, their PS inf and C/M ratio in PHH were 1.8- to 3.2-fold lower than those in SHH. These hydrophilic substrates are more prone to wash-off during the uptake study using PHH, which may explain the apparently lower uptake than SHH. The C/M ratios obtained using PHH were stable over an extended time, making PHH suitable to estimate the C/M ratios and hepatocyte-to-medium unbound concentration ratios (K p,uu ). In conclusion, PHH is useful in evaluating hepatic uptake/efflux clearances and K p,uu of OATP1B substrates in a high-throughput manner, however, a caution is warranted for hydrophilic drugs with low uptake/cellular binding., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Herbal medicine represents a significant component of disease prevention and therapy in most African countries. Herb-drug interactions (HDI) can arise from the co-administration of herbal and orthodox medicines., Objective: This study assessed the potential for HDI of V. amygdalina, O. gratissimum, M. oleifera, A. indica, and P. nitida extracts using in vitro assays. Little is known about these medicinal plants' potential for drug interaction despite their extensive use in Nigeria for several disease conditions., Method: The medicinal plant crude extracts were evaluated for Cytochrome P450 (CYP) enzyme induction using cryopreserved human hepatocytes. Enzyme activity was determined by quantifying probe substrate metabolism and metabolite formation using liquid chromatography-mass spectrometry/mass spectrometry. The extracts were evaluated for the potential to inhibit P-glycoprotein (P-gp) activity using human embryonic kidney membrane vesicles over-expressing human P-gp. The herbal extracts in vivo drug interaction potential was predicted based on the USFDA drug interaction guidance., Result: O. gratissimum and P. nitida methanol extracts induced CYP1A2 enzyme activity by greater than 3-fold. P. nitida methanol extracts showed over 2-fold induction of CYP1A2 mRNA expression. O. gratissimum methanol extract induced CYP2B6 mRNA expression over 2-fold. P. nitida and A. indica methanol extracts showed potent inhibition of P-gp activity (IC 50 : 3.8 and 5.4 μg/mL), respectively, while V. amygdalina and M. oleifera methanol extracts showed moderate P-gp inhibition (IC50: 12.1 and 37.2 μg/mL, respectively)., Conclusion: Our studies suggested that the medicinal plants' extracts can modulate CYP enzymes and P-gp activity with the potential to cause herb-drug interaction in vivo., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Oral drug absorption is a complex process depending on many factors, including the physicochemical properties of the drug, formulation characteristics and their interplay with gastrointestinal physiology and biology. Physiological-based pharmacokinetic (PBPK) models integrate all available information on gastro-intestinal system with drug and formulation data to predict oral drug absorption. The latter together with in vitro-in vivo extrapolation and other preclinical data on drug disposition can be used to predict plasma concentration-time profiles in silico. Despite recent successes of PBPK in many areas of drug development, an improvement in their utility for evaluating oral absorption is much needed. Current status of predictive performance, within the confinement of commonly available in vitro data on drugs and formulations alongside systems information, were tested using 3 PBPK software packages (GI-Sim (ver.4.1), Simcyp® Simulator (ver.15.0.86.0), and GastroPlus™ (ver.9.0.00xx)). This was part of the Innovative Medicines Initiative (IMI) Oral Biopharmaceutics Tools (OrBiTo) project. Fifty eight active pharmaceutical ingredients (APIs) were qualified from the OrBiTo database to be part of the investigation based on a priori set criteria on availability of minimum necessary information to allow modelling exercise. The set entailed over 200 human clinical studies with over 700 study arms. These were simulated using input parameters which had been harmonised by a panel of experts across different software packages prior to conduct of any simulation. Overall prediction performance and software packages comparison were evaluated based on performance indicators (Fold error (FE), Average fold error (AFE) and absolute average fold error (AAFE)) of pharmacokinetic (PK) parameters. On average, PK parameters (Area Under the Concentration-time curve (AUC 0-tlast ), Maximal concentration (C max ), half-life (t 1/2 )) were predicted with AFE values between 1.11 and 1.97. Variability in FEs of these PK parameters was relatively high with AAFE values ranging from 2.08 to 2.74. Around half of the simulations were within the 2-fold error for AUC 0-tlast and around 90% of the simulations were within 10-fold error for AUC 0-tlast . Oral bioavailability (F oral ) predictions, which were limited to 19 APIs having intravenous (i.v.) human data, showed AFE and AAFE of values 1.37 and 1.75 respectively. Across different APIs, AFE of AUC 0-tlast predictions were between 0.22 and 22.76 with 70% of the APIs showing an AFE > 1. When compared across different formulations and routes of administration, AUC 0-tlast for oral controlled release and i.v. administration were better predicted than that for oral immediate release formulations. Average predictive performance did not clearly differ between software packages but some APIs showed a high level of variability in predictive performance across different software packages. This variability could be related to several factors such as compound specific properties, the quality and availability of information, and errors in scaling from in vitro and preclinical in vivo data to human in vivo behaviour which will be explored further. Results were compared with previous similar exercise when the input data selection was carried by the modeller rather than a panel of experts on each in vitro test. Overall, average predictive performance was increased as reflected in smaller AAFE value of 2.8 as compared to AAFE value of 3.8 in case of previous exercise., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prospective predictions of human hepatic clearance for anionic/zwitterionic compounds, which are oftentimes subjected to transporter-mediated uptake, are challenging in drug discovery. We evaluated the utility of preclinical species, rats and cynomolgus monkeys [nonhuman primates (NHPs)], to predict the human hepatic clearance using a diverse set of acidic/zwitterionic drugs. Preclinical clearance data were generated following intravenous dosing in rats/NHPs and compared to the human clearance data ( n = 18/27). Single-species scaling of NHP clearance with an allometric exponent of 0.50 allowed for good prediction of human clearance (fold error ∼2.1, bias ∼1.0), with ∼86% predictions within 3-fold. In comparison, rats underpredicted the clearance of lipophilic acids, while overprediction was noted for hydrophilic acids. Finally, an in vitro clearance assay based on human hepatocytes, which is routinely used in discovery setting, markedly underpredicted human clearance (bias ∼0.12). Collectively, this study provides insights into the usefulness of the preclinical models in enabling pharmacokinetic optimization for acid/zwitterionic drug candidates.

Cytochrome-P450 (P450) isoforms are major drug-metabolizing enzymes implicated in the clearance and drug-drug interactions (DDIs) of diverse small-molecule drugs. Here, we evaluated the association between primary physicochemical descriptors of substrate drugs and their clinical DDI risk with P450 index (probe) inhibitors using an exhaustive clinical data set ( n = 397, substrate-inhibitor pairs). Additionally, the ability of extended clearance classification system (ECCS), a categorical clearance mechanism model, to predict P450 DDI risk was assessed. The clinical data set indicated that basic and neutral compounds are probable candidates to show a higher magnitude of DDIs on P450 inhibition (i.e., plasma exposure change > twofold). Additionally, trends with lipophilicity were apparent for P450-based DDIs. ECCS class 2 drugs (high-permeability bases/neutrals) have higher probability to show moderate-to-strong DDIs with probe inhibitors of CYP1A2/2C19/2C9/2D6/3A, while ECCS class 1A/1B drugs are prone to interactions with inhibitors of CYP2C8 and CYP2C9. On the other hand, P450-based DDIs are notably small for classes 3A/3B/4. In conclusion, this study emphasizes the relevance of the ECCS framework in clearance characterization to evaluate victim DDI liabilities and aid chemists in mitigating risk during drug design.

About 30% of approved drugs are cleared predominantly by renal clearance (CL r ). Of these, many are secreted by transporters. For these drugs, in vitro-to-in vivo extrapolation of transporter-mediated renal secretory clearance (CL sec,plasma ) is important to prospectively predict their renal clearance and to assess the impact of drug-drug interactions and pharmacogenetics on their pharmacokinetics. Here we compared the ability of the relative expression factor (REF) and the relative activity factor (RAF) approaches to quantitatively predict the in vivo CL sec,plasma of 26 organic anion transporter (OAT) substrates assuming that OAT-mediated uptake is the rate-determining step in the CL sec,plasma of the drugs. The REF approach requires protein quantification of each transporter in the tissue (e.g., kidney) and transporter-expressing cells, whereas the RAF approach requires the use of a transporter-selective probe substrate (both in vitro and in vivo) for each transporter of interest. For the REF approach, 50% and 69% of the CL sec,plasma predictions were within 2- and 3-fold of the observed values, respectively; the corresponding values for the RAF approach were 65% and 81%. We found no significant difference between the two approaches in their predictive capability (as measured by accuracy and bias) of the CL sec,plasma or CL r of OAT drugs. We recommend that the REF and RAF approaches can be used interchangeably to predict OAT-mediated CL sec,plasma Further research is warranted to evaluate the ability of the REF or RAF approach to predict CL sec,plasma of drugs when uptake is not the rate-determining step. SIGNIFICANCE STATEMENT: This is the first direct comparison of the relative expression factor (REF) and relative activity factor (RAF) approaches to predict transporter-mediated renal clearance (CL r ). The RAF, but not REF, approach requires transporter-selective probes and that the basolateral uptake is the rate-determining step in the CL r of drugs. Given that there is no difference in predictive capability of the REF and RAF approach for organic anion transporter-mediated CL r , the REF approach should be explored further to assess its ability to predict CL r when basolateral uptake is not the sole rate-determining step., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction : Transporters and enzymes play an important role in absorption, distribution, clearance and elimination of drugs. Areas covered : This review provides an overview of the extended clearance concept and usefulness of extended clearance classification system (ECCS) in early identification of predominant clearance mechanisms. Clinical studies demonstrating transporter-enzyme interplay, challenges in scaling clearance from in vitro systems, utility of animal models and modeling approaches for evaluating hepatic clearance and drug-drug interactions are reviewed. Expert opinion : Clinical evidence exists supporting organic anion transporting peptide (OATP)1B and drug metabolizing enzymes involvement in clearance of ECCS class 1B drugs. Emerging evidence point toward contribution of organic cation transporter (OCT)1 to hepatic uptake of cationic drugs. Although, limited clinical evidence is presented, preclinical studies and modeling suggests organic anion transporter (OAT)2-enzyme interplay in clearance of class 1A drugs. Data from in vitro assays and preclinical models coupled with physiologically based modeling approaches are key for understanding transporter-enzyme interplay, enabling prediction of pharmacokinetics, tissue exposure and drug interactions. Current methodologies incur limitations and emphasis should be placed on the development of physiologically relevant in vitro models and characterize in vivo animal models to inform mechanistic modeling and improve confidence in prospective predictions.

Understanding transporter-mediated drug disposition and pharmacokinetics (PK) in patients with nonalcoholic fatty liver disease (NAFLD) is critical in developing treatment options. Here, we quantified the expression levels of major drug transporters in healthy, steatosis, and nonalcoholic steatohepatitis (NASH) liver samples, via liquid-chromatography tandem mass spectrometry-based proteomics, and used the data to predict the PK of substrate drugs in the disease state. Expression of organic anion transporting polypeptides (OATPs) and multidrug resistance-associated protein (MRP)2 is significantly lower in NASH livers; whereas MRP3 is induced while no change was observed for organic cation transporter (OCT)1. Physiologically-based pharmacokinetic models verified with PK data from healthy subjects well recovered the PK in NASH subjects for morphine (involving OCT1) and its glucuronide metabolites (MRP2/MRP3/OATP1B), 99m TC-mebrofenen (OATP1B/MRP2/MRP3), and rosuvastatin (OATP1B/breast cancer resistance protein). Overall, considerations to altered protein expression can enable quantitative prediction of PK changes in subjects with NAFLD., (© 2019 The Authors Clinical Pharmacology & Therapeutics © 2019 American Society for Clinical Pharmacology and Therapeutics.)

In the development of new drugs, the prediction of metabolite-to-parent plasma exposure ratio in humans prior to administration in a clinical study has emerged as an important need. In this work, we derived a mechanistic static model based on first principles to estimate metabolite-to-parent plasma exposure ratio, considering the contribution of liver and gut metabolism and drug transport. Knowledge (or assumptions) of mechanisms of clearance and organs involved is required. Input parameters needed included intrinsic clearance, fraction of clearance to the metabolite of interest, various binding values, and, in some cases, active transport clearance. The principles are illustrated with four drugs that yield six metabolites, with one in which clearance is dependent on a pathway subject to genetic polymorphism. Overall, the approach yielded metabolite-to-parent ratios within about twofold of the actual values and, thus, can be valuable in decision making in the drug development process., (© 2019 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Nicotinic acid (NA) and nicotinamide (NAM) are biosynthetic precursors of nicotinamide adenine dinucleotide (NAD + ) - a physiologically important coenzyme that maintains the redox state of cells. Mechanisms driving their entry into cells are not well understood. Here we evaluated the hepatic uptake mechanism(s) of NA and NAM using transporter-transfected cell systems and primary human hepatocytes. NA showed robust organic anion transporter (OAT)2-mediated transport with an uptake ratio (i.e., ratio of accumulation in transfect cells to wild-type cells) of 9.7 ± 0.3, and a Michaelis-Menten constant (K m ) of 13.5 ± 3.3 µM. However, no transport was apparent via other major hepatic uptake and renal secretory transporters, including OAT1/3/4, organic anion transporting polypeptide (OATP)1B1/1B3/2B1, sodium-taurocholate co-transporting polypeptide, organ cation transporter 1/2/3. OAT2-specific transport of NA was inhibited by ketoprofen and indomethacin (known OAT2 inhibitors) in a concentration-dependent manner. Similarly, NA uptake into primary human hepatocytes showed pH- and concentration-dependence and was subject to inhibition by specific OAT2 inhibitors. Unlike NA, NAM was not transported by the hepatic and renal solute carriers upon assessment in transfected cells, although its uptake into human hepatocytes was significantly inhibited by excess unlabelled NAM and a pan-SLC inhibitor (rifamycin SV 1 mM). In conclusion, these studies demonstrate, for the first time, a specific transport mechanism for NA uptake in the human liver and suggest that OAT2 (SLC22A7) has a critical role in its physiological and pharmacological functions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Organic anion transporting polypeptides (OATPs), expressed in human liver (OATP1B1, OATP1B3, and OATP2B1) and intestine (OATP2B1), govern the pharmacokinetics (PK) of drugs (e.g., statins) and endogenous substrates (e.g., coproporphyrin I [CPI]). Their expression is known to be modulated (e.g., disease, age, and environmental factors), and they also present as the loci of clinically relevant polymorphisms and drug interactions involving inhibition. In comparison, relatively few clinical reports describe the induction of OATPs, although the effect of inducers (e.g., rifampicin [RIF], carbamazepine [CBZ]) on OATP biomarker plasma levels and statin PK has been reported. Of note, available human tissue (e.g., biopsy) protein and messenger RNA expression profiling data indicate that OATPs in gut and liver are not induced by prototypical inducers such as RIF when compared with cytochrome P450 3A4 (CYP3A4), P-glycoprotein (Pgp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). Such results are consistent with in vitro human hepatocyte data. Therefore, the observed impact of RIF, and possibly CBZ, on statin PK (>20% decrease in the area under the plasma concentration vs. time curve) cannot be ascribed to OATP induction with certainty. In fact, most statins and CPI have been shown to present variously as substrates of RIF-inducible proteins such as CYP3A4, Pgp, MRP2, and BCRP. Interpretation of multidose RIF data is further complicated by its autoinduction, which likely leads to decreased inhibition of OATP. In the absence of more conclusive OATP induction data, caution is needed when modeling drug-drug interactions involving multidose inducers such as RIF. SIGNIFICANCE STATEMENT: Presently, there is limited direct clinical evidence supporting the notion that human liver and gut organic anion transporting polypeptides (OATPs) are inducible by agents like rifampicin (RIF). Such data need to be reconciled and will pose challenges for attempting to incorporate OATP induction into physiologically based pharmacokinetics models. Although disparate sets of tissue biopsy (atorvastatin and carbamazepine) and in vitro hepatocyte (phenobarbital, chenodeoxycholate, and amprenavir) data present OATP messenger RNA induction (≥2-fold) by agents beyond RIF, the clinical relevance of such data needs to be determined., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Drug-drug interactions (DDIs) involving drug-metabolizing enzymes and membrane transporters can lead to alteration in substrate drug (victim) exposure, and can influence the pharmacological and toxicological effects. In order to predict DDI potential, it is important to quantitatively characterize the major enzyme(s) and/or transporter(s) involved in the clearance of drugs, in terms of fraction metabolized (f m ) and fraction transported (f t ). In this review, we discuss a strategy using Extended Clearance Classification System (ECCS) to identify the clearance mechanism(s) early in drug discovery, and subsequently rational staging of in vitro characterization to determine f m and f t . In addition, the examples of complex DDIs due to involvement of transporter-enzyme interplay in the hepatic clearance are discussed., Competing Interests: Declaration of competing interest There are no conflict of interest pertaining to the content of this manuscript., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

PF-04991532 ((S)-6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-yl) propanamido) nicotinic acid) is a glucokinase activator designed to achieve hepato-selectivity via organic anion-transporting polypeptides (OATP)s, so as to minimize systemic hypoglycemic effects. This study investigated the effect of OATP1B1/1B3 inhibition and renal impairment on PF-04991532 oral pharmacokinetics. Cyclosporine (600 mg single dose) increased mean area under the plasma curve (AUC) of PF-04991532 by approximately threefold in healthy subjects. In a renal impairment study, PF-04991532 AUC values were ~ 2.3-fold greater in subjects with mild, moderate, and severe kidney dysfunction, compared with healthy subjects. Physiologically-based pharmacokinetic (PBPK) model parameterizing hepatic and renal transporter-mediated disposition based on in vitro inputs, and verified using first-in-human data, indicated the key role of OATP-mediated hepatic uptake in the systematic and target-tissue exposure of PF-04991532. Mechanistic evaluation of the clinical data suggest reduced hepatic OATPs (~ 35%) and renal organic anion transporter (OAT)3 (80-90%) function with renal impairment. This study illustrates the adequacy and utility of the PBPK approach in assessing the impact of drug interactions and kidney dysfunction on transporter-mediated disposition., (© 2019 Pfizer Inc. Clinical Pharmacology & Therapeutics © 2019 American Society for Clinical Pharmacology and Therapeutics.)

Hepatic uptake transporters [solute carriers (SLCs)], including organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, sodium-dependent taurocholate cotransporting polypeptide (NTCP), and organic anion (OAT2) and organic cation (OCT1) transporters, play a key role in determining the systemic and liver exposure of chemically diverse drugs. Here, we established a phenotyping approach to quantify the contribution of the six SLCs, and passive diffusion, to the overall uptake using plated human hepatocytes (PHHs). First, selective inhibitor conditions were identified by screening about 20 inhibitors across the six SLCs using single-transfected human embryonic kidney 293 cells. Data implied rifamycin SV (20 µ M) inhibits three OATPs, while rifampicin (5 µ M) inhibits OATP1B1/1B3 only. Further, hepatitis B virus myristoylated-preS1 peptide (0.1 µ M), quinidine (100 µ M), and ketoprofen (100-300 µ M) are relatively selective against NTCP, OCT1, and OAT2, respectively. Second, using these inhibitory conditions, the fraction transported ( f t ) by the individual SLCs was characterized for 20 substrates with PHH. Generally, extended clearance classification system class 1A/3A (e.g., warfarin) and 1B/3B compounds (e.g., statins) showed predominant OAT2 and OATP1B1/1B3 contribution, respectively. OCT1-mediated uptake was prominent for class 2/4 compounds (e.g., metformin). Third, in vitro f t values were corrected using quantitative proteomics data to obtain "scaled f t " Fourth, in vitro-in vivo extrapolation of the scaled OATP1B1/1B3 f t was assessed, leveraging statin clinical drug-drug interaction data with rifampicin as the perpetrator. Finally, we outlined a novel stepwise strategy to implement phenotypic characterization of SLC-mediated hepatic uptake for new molecular entities and drugs in a drug discovery and development setting., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The aim of the present study was to quantitatively evaluate the drug-drug interactions (DDIs) of maraviroc (MVC) with various perpetrator drugs, including telaprevir (TVR), using an in vitro data-informed physiologically based pharmacokinetic (PBPK) model. MVC showed significant active uptake and biliary excretion in sandwich-cultured human hepatocytes, and biphasic organic anion transporting polypeptide (OATP)1B1-mediated uptake kinetics in transfected cells (high-affinity K m ∼5 µ M). No measureable active uptake was noted in OATP1B3- and OATP2B1-transfceted cells. TVR inhibited OATP1B1-mediated MVC transport in vitro, and also exhibited CYP3A time-dependent inhibition in human hepatocytes (inactivation constant, K I = 2.24 µ M, and maximum inactivation rate constant, k inact = 0.0112 minute -1 ). The inactivation efficiency ( k inact / K I ) was approximately 34-fold lower in human hepatocytes compared with liver microsomes. A PBPK model accounting for interactions involving CYP3A, P-glycoprotein (P-gp), and OATP1B1 was developed based on in vitro mechanistic data. MVC DDIs with ketoconazole (inhibition of CYP3A and P-gp), ritonavir (inhibition of CYP3A and P-gp), efavirenz (induction of CYP3A), rifampicin (induction of CYP3A and P-gp; inhibition of OATP1B1), and TVR (inhibition of CYP3A, P-gp, and OATP1B1) were well described by the PBPK model with optimized transporter K i values implying that OATP1B1-mediated uptake along with CYP3A metabolism determines the hepatic clearance of MVC, and P-gp-mediated efflux limits its intestinal absorption. In summary, MVC disposition involves intestinal P-gp/CYP3A and hepatic OATP1B1/CYP3A interplay, and TVR simultaneously inhibits these multiple mechanisms leading to a strong DDI-about 9.5-fold increase in MVC oral exposure., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Chronic kidney disease (CKD) differentially affects the pharmacokinetics (PK) of nonrenally cleared drugs via certain pathways (e.g., cytochrome P450 (CYP)2D6); however, the effect on CYP2C8-mediated clearance is not well understood because of overlapping substrate specificity with hepatic organic anion-transporting polypeptides (OATPs). This study used physiologically based pharmacokinetic (PBPK) modeling to delineate potential changes in CYP2C8 or OATP1B activity in patients with CKD. Drugs analyzed are predominantly substrates of CYP2C8 (rosiglitazone and pioglitazone), OATP1B (pitavastatin), or both (repaglinide). Following initial model verification, pharmacokinetics (PK) of these drugs were simulated in patients with severe CKD considering changes in glomerular filtration rate (GFR), plasma protein binding, and activity of either CYP2C8 and/or OATP1B in a stepwise manner. The PBPK analysis suggests that OATP1B activity could be decreased up to 60% in severe CKD, whereas changes to CYP2C8 are negligible. This improved understanding of CKD effect on clearance pathways could be important to inform the optimal use of nonrenally eliminated drugs in patients with CKD., (2018 The Authors. Clinical Pharmacology and Therapeutics published by Wiley Periodicals, Inc on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

High-permeability-low-molecular-weight acids/zwitterions [i.e., extended clearance classification system class 1A (ECCS 1A) drugs] are considered to be cleared by metabolism with a minimal role of membrane transporters in their hepatic clearance. However, a marked disconnect in the in vitro-in vivo (IVIV) translation of hepatic clearance is often noted for these drugs. Metabolic rates measured using human liver microsomes and primary hepatocytes tend to underpredict. Here, we evaluated the role of organic anion transporter 2 (OAT2)-mediated hepatic uptake in the clearance of ECCS 1A drugs. For a set of 25 ECCS 1A drugs, in vitro transport activity was assessed using transporter-transfected cells and primary human hepatocytes. All but two drugs showed substrate affinity to OAT2, whereas four (bromfenac, entacapone, fluorescein, and nateglinide) also showed OATP1B1 activity in transfected cells. Most of these drugs (21 of 25) showed active uptake by plated human hepatocytes, with rifamycin SV (pan-transporter inhibitor) reducing the uptake by about 25%-95%. Metabolic turnover was estimated for 19 drugs after a few showed no measurable substrate depletion in liver microsomal incubations. IVIV extrapolation using in vitro data was evaluated to project human hepatic clearance of OAT2-alone substrates considering 1) uptake transport only, 2) metabolism only, and 3) transporter-enzyme interplay (extended clearance model). The transporter-enzyme interplay approach achieved improved prediction accuracy (average fold error = 1.9 and bias = 0.93) compared with the other two approaches. In conclusion, this study provides functional evidence for the role of OAT2-mediated hepatic uptake in determining the pharmacokinetics of several clinically important ECCS 1A drugs., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Organic anion transporter (OAT) 2 and OAT7 were recently shown to be involved in the hepatic uptake of drugs; however, there is limited understanding of the population variability in the expression of these transporters in liver. There is also a need to derive relative expression-based scaling factors (REFs) that can be used to bridge in vitro functional data to the in vivo drug disposition. To this end, we quantified OAT2 and OAT7 surrogate peptide abundance in a large number of human liver tissue samples ( n = 52), as well as several single-donor cryopreserved human hepatocyte lots ( n = 30) by a novel, validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The average surrogate peptide expression of OAT2 and OAT7 in the liver samples was 1.52 ± 0.57 and 4.63 ± 1.58 fmol/μg membrane protein, respectively. While we noted statistically significant differences ( p < 0.05) in hepatocyte and liver tissue abundances for both OAT2 and OAT7, the differences were relatively small (1.8- and 1.5-fold difference in median values, respectively). Large interindividual variability was noted in the hepatic expression of OAT2 (16-fold in liver tissue and 23-fold in hepatocytes). OAT7, on the other hand, showed less interindividual variability (4-fold) in the livers, but high variability for the hepatocyte lots (27-fold). A significant positive correlation in OAT2 and OAT7 expression was observed, but expression levels were neither associated with age nor sex. In conclusion, our data suggest marked interindividual variability in the hepatic expression of OAT2/7, which may contribute to the pharmacokinetic variability of their substrates. Because both transporters were less abundant in hepatocytes than livers, a REF-based approach is recommended when scaling in vitro hepatocyte transport data to predict hepatic drug clearance and liver exposure of OAT2/7 substrates.

This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic uptake clearance mediated by organic anion transporting polypeptide (OATP) transporters. Nine OATP substrates (rosuvastatin, pravastatin, repaglinide, fexofenadine, cerivastatin, telmisartan, pitavastatin, bosentan, and valsartan) were investigated in plated cynomolgus monkey and human hepatocytes. Total uptake clearance and passive diffusion were measured in vitro from initial rates in the absence and presence of the OATP inhibitor rifamycin SV , respectively. Total uptake clearance values in plated hepatocytes ranged over three orders of magnitude in both species, with a similar rank order and good agreement in the relative contribution of active transport to total uptake between cynomolgus monkey and human. In vivo hepatic clearance for these nine drugs was determined in cynomolgus monkey after intravenous dosing. Hepatic clearances showed a range similar to human parameters and good predictions from respective hepatocyte parameters (with 2.7- and 3.8-fold bias on average, respectively). The use of cross-species empirical scaling factors (determined from cynomolgus monkey data either as the data set average or individual drug values) improved prediction (less bias, better concordance) of human hepatic clearance from human hepatocyte data alone. In vitro intracellular binding in hepatocytes also correlated well between species. It is concluded that the minimal species differences observed for the current data set between cynomolgus monkey and human hepatocyte uptake, both in vitro and in vivo, support future use of this preclinical model to delineate drug hepatic uptake and enable prediction of human in vivo intrinsic hepatic clearance., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Hepatic organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are clinically relevant transporters associated with significant drug-drug interactions (DDIs) and safety concerns. Given that OATP1Bs in cynomolgus monkey share >90% degree of gene and amino acid sequence homology with human orthologs, we evaluated the in vitro-in vivo translation of OATP1B-mediated DDI risk using this preclinical model. In vitro studies using plated cynomolgus monkey hepatocytes showed active uptake K m values of 2.0 and 3.9 µ M for OATP1B probe substrates, pitavastatin and rosuvastatin, respectively. Rifampicin inhibited pitavastatin and rosuvastatin active uptake in monkey hepatocytes with IC 50 values of 3.0 and 0.54 µ M, respectively, following preincubation with the inhibitor. Intravenous pharmacokinetics of 2 H 4 -pitavastatin and 2 H 6 -rosuvastatin (0.2 mg/kg) and the oral pharmacokinetics of cold probes (2 mg/kg) were studied in cynomolgus monkeys ( n = 4) without or with coadministration of single oral ascending doses of rifampicin (1, 3, 10, and 30 mg/kg). A rifampicin dose-dependent reduction in i.v. clearance of statins was observed. Additionally, oral pitavastatin and rosuvastatin plasma exposure increased up to 19- and 15-fold at the highest dose of rifampicin, respectively. Use of in vitro IC 50 obtained following 1 hour preincubation with rifampicin (0.54 µ M) predicted correctly the change in mean i.v. clearance and oral exposure of statins as a function of mean unbound maximum plasma concentration of rifampicin. This study demonstrates quantitative translation of in vitro OATP1B IC 50 to predict DDIs using cynomolgus monkey as a preclinical model and provides further confidence in application of in vitro hepatocyte data for the prediction of clinical OATP1B-mediated DDIs., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Targeted protein quantification using liquid chromatography-tandem mass spectrometry with stable isotope-labeled standards is recognized as the gold standard of practice for protein quantification. Such assays, however, can only cover a limited number of proteins, and developing targeted methods for larger numbers of proteins requires substantial investment. Alternatively, large-scale global proteomic experiments along with computational methods such as the "total protein approach" (TPA) have the potential to provide extensive protein quantification. In this study, we compared the TPA-based quantitation of seven major hepatic uptake transporters in four human liver tissue samples using global proteomic data obtained from two multiplexed tandem mass tag experiments (performed in two independent laboratories) to the quantitative data from targeted proteomic assays. The TPA-based quantitation of these hepatic transporters [sodium-taurocholate cotransporting polypeptide (NTCP/SLC10A1), organic anion transporter 2 (OAT2/SLC22A7), OAT7/SLC22A9, organic anion-transporting polypeptide 1B1 (OATP1B1/SLCO1B1), OATP1B3/SLCO1B3, OATP2B1/SLCO2B1, and organic cation transporter (OCT1/SLC22A1)] showed good-to-excellent correlations (Pearson r = 0.74-1.00) to the targeted data. In addition, the values were similar to those measured by targeted proteomics with 71% and 86% of the data sets falling within 3-fold of the targeted data. A comparison of the TPA-based quantifications of enzyme abundances to available literature data showed that the majority of the enzyme quantifications fell within the reference data intervals. In conclusion, these results demonstrate the capability of multiplexed global proteomic experiments to detect differences in protein expression between samples and provide reasonable estimations of protein expression levels., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Membrane transporters play an important role in the absorption, distribution, clearance, and elimination of drugs. Supported by the pharmacokinetics data in human, several transporters including organic anion transporting polypeptide (OATP)1B1, OATP1B3, organic anion transporter (OAT)1, OAT3, organic cation transporter (OCT)2, multidrug and toxin extrusion (MATE) proteins, P-glycoprotein and breast cancer resistance protein are suggested to be of clinical relevance. An early understanding of the transporter role in drug disposition and clearance allows reliable prediction/evaluation of pharmacokinetics and changes due to drug-drug interactions (DDIs) or genetic polymorphisms. We recently proposed an extended clearance classification system (ECCS) based on simple drug properties (i.e., ionization, permeability, and molecular weight) to predict the predominant clearance mechanism. According to this framework, systemic clearance of class 1B and 3B drugs is likely determined by the OATP-mediated hepatic uptake. Class 3A and 4 drugs, and certain class 3B drugs, are predominantly cleared by renal, wherein, OAT1, OAT3, OCT2, and MATE proteins could contribute to their active renal secretion. Intestinal efflux and uptake transporters largely influence the oral pharmacokinetics of class 3A, 3B, and 4 drugs. We discuss the paradigm of applying the ECCS framework in mapping the role of clinically relevant drug transporters in early discovery and development; thereby implementing the right strategy to allow optimization of drug exposure and evaluation of clinical risk due to DDIs and pharmacogenomics., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Interindividual variability in warfarin dose requirement demands personalized medicine approaches to balance its therapeutic benefits (anticoagulation) and bleeding risk. Cytochrome P450 2C9 ( CYP2C9) genotype-guided warfarin dosing is recommended in the clinic, given the more potent S-warfarin is primarily metabolized by CYP2C9. However, only about 20-30% of interpatient variability in S-warfarin clearance is associated with CYP2C9 genotype. We evaluated the role of hepatic uptake in the clearance of R- and S-warfarin. Using stably transfected HEK293 cells, both enantiomers were found to be substrates of organic anion transporter (OAT)2 with a Michaelis-Menten constant ( K m ) of ∼7-12 μM but did not show substrate affinity for other major hepatic uptake transporters. Uptake of both enantiomers by primary human hepatocytes was saturable ( K m ≈ 7-10 μM) and inhibitable by OAT2 inhibitors (e.g., ketoprofen) but not by OATP1B1/1B3 inhibitors (e.g., cyclosporine). To further evaluate the potential role of hepatic uptake in R- and S-warfarin pharmacokinetics, mechanistic modeling and simulations were conducted. A "bottom-up" PBPK model, developed assuming that OAT2-CYPs interplay, well recovered clinical pharmacokinetics, drug-drug interactions, and CYP2C9 pharmacogenomics of R- and S-warfarin. Clinical data were not available to directly verify the impact of OAT2 modulation on warfarin pharmacokinetics; however, the bottom-up PBPK model simulations suggested a proportional change in clearance of both warfarin enantiomers with inhibition of OAT2 activity. These results suggest that variable hepatic OAT2 function, in conjunction with CYP2C, may contribute to the high population variability in warfarin pharmacokinetics and possibly anticoagulation end points and thus warrant further clinical investigation.

Tolbutamide is primarily metabolized by CYP2C9, and, thus, is frequently applied as a clinical probe substrate for CYP2C9 activity. However, there is a marked discrepancy in the in vitro-in vivo extrapolation of its metabolic clearance, implying a potential for additional clearance mechanisms. The goal of this study was to evaluate the role of hepatic uptake transport in the pharmacokinetics of tolbutamide and to identify the molecular mechanism thereof. Transport studies using singly transfected cells expressing six major hepatic uptake transporters showed that tolbutamide is a substrate to organic anion transporter 2 (OAT2) alone with transporter affinity [Michaelis-Menten constant ( K m )] of 19.5 ± 4.3 µ M. Additionally, OAT2-specific transport was inhibited by ketoprofen (an OAT2 inhibitor) and 1 mM rifamycin SV (pan inhibitor), but not by cyclosporine and rifampicin (OAT polypeptides/Na + -taurocholate cotransporting polypeptide inhibitors). Uptake studies in primary human hepatocytes confirmed the predominant role of OAT2 in the active uptake with significant inhibition by rifamycin SV and ketoprofen, but not by the other inhibitors. Concentration-dependent uptake was noted in human hepatocytes with active transport characterized by K m and V max values of 39.3 ± 6.6 µ M and 426 ± 30 pmol/min per milligram protein, respectively. Bottom-up physiologically based pharmacokinetic modeling was employed to verify the proposed role of OAT2-mediated hepatic uptake. In contrast to the rapid equilibrium (CYP2C9-only) model, the permeability-limited (OAT2-CYP2C9 interplay) model better described the plasma concentration-time profiles of tolbutamide. Additionally, the latter well described tolbutamide pharmacokinetics in carriers of CYP2C9 genetic variants and quantitatively rationalized its known drug-drug interactions. Our results provide first-line evidence for the role of OAT2-mediated hepatic uptake in the pharmacokinetics of tolbutamide, and imply the need for additional clinical studies in this direction., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)