RESUMO
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 (â¼75%) 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.
Assuntos
Metformina , Transportadores de Ânions Orgânicos , Humanos , Transportador 2 de Cátion Orgânico , Creatinina , Proteínas de Transporte de Cátions Orgânicos , Células HEK293 , Rim , Metformina/farmacologia , IndometacinaRESUMO
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 (ft ,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 ft ,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.
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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 (Kpuu) measured in vitro using rat, NHP and human hepatocytes was found to be approximately 4, 25 and 10, respectively. Similarly, liver Kpuu 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 Kpuu, 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.
RESUMO
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 â¼400-730 Da, logP â¼3.5-8, 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 (ft,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 ft,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.
Assuntos
Inibidores Enzimáticos/farmacocinética , Hepatócitos/metabolismo , Hipoglicemiantes/farmacocinética , Transportadores de Ânions Orgânicos/metabolismo , Ácidos/administração & dosagem , Ácidos/farmacocinética , Administração Oral , Animais , Células Cultivadas , Vias de Eliminação de Fármacos , Inibidores Enzimáticos/administração & dosagem , Feminino , Células HEK293 , Humanos , Hipoglicemiantes/administração & dosagem , Macaca fascicularis , MasculinoRESUMO
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 (PSinf,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 PSinf,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 PSinf,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. PSinf,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 PSinf,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.
Assuntos
Eliminação Hepatobiliar/fisiologia , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Plasma/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Transporte Biológico , Células HEK293 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Taxa de Depuração Metabólica/fisiologia , Redes e Vias Metabólicas , Farmacocinética , TransfecçãoRESUMO
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.
Assuntos
Interações Medicamentosas/fisiologia , Intestinos/fisiologia , Fígado/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Hepatócitos/metabolismo , HumanosRESUMO
About 30% of approved drugs are cleared predominantly by renal clearance (CLr). Of these, many are secreted by transporters. For these drugs, in vitro-to-in vivo extrapolation of transporter-mediated renal secretory clearance (CLsec,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 CLsec,plasma of 26 organic anion transporter (OAT) substrates assuming that OAT-mediated uptake is the rate-determining step in the CLsec,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 CLsec,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 CLsec,plasma or CLr of OAT drugs. We recommend that the REF and RAF approaches can be used interchangeably to predict OAT-mediated CLsec,plasma Further research is warranted to evaluate the ability of the REF or RAF approach to predict CLsec,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 (CLr). The RAF, but not REF, approach requires transporter-selective probes and that the basolateral uptake is the rate-determining step in the CLr of drugs. Given that there is no difference in predictive capability of the REF and RAF approach for organic anion transporter-mediated CLr, the REF approach should be explored further to assess its ability to predict CLr when basolateral uptake is not the sole rate-determining step.
Assuntos
Vias de Eliminação de Fármacos/fisiologia , Interações Medicamentosas , Transportadores de Ânions Orgânicos , Eliminação Renal/efeitos dos fármacos , Transporte Biológico/fisiologia , Desenvolvimento de Medicamentos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/farmacocinética , Preparações Farmacêuticas/metabolismo , Farmacocinética , Valor Preditivo dos TestesRESUMO
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.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas/fisiologia , Taxa de Depuração Metabólica/fisiologia , Preparações Farmacêuticas/metabolismo , Animais , Linhagem Celular , Inibidores das Enzimas do Citocromo P-450/metabolismo , Cães , Cinética , Fígado/metabolismo , Células Madin Darby de Rim Canino , Oxirredução , PermeabilidadeRESUMO
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 µM) inhibits three OATPs, while rifampicin (5 µM) inhibits OATP1B1/1B3 only. Further, hepatitis B virus myristoylated-preS1 peptide (0.1 µM), quinidine (100 µM), and ketoprofen (100-300 µM) are relatively selective against NTCP, OCT1, and OAT2, respectively. Second, using these inhibitory conditions, the fraction transported (ft ) 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 ft values were corrected using quantitative proteomics data to obtain "scaled ft " Fourth, in vitro-in vivo extrapolation of the scaled OATP1B1/1B3 ft 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.
Assuntos
Hepatócitos/metabolismo , Fígado/citologia , Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Fenótipo , Proteínas Carreadoras de Solutos/metabolismo , Transporte Biológico/efeitos dos fármacos , Interações Medicamentosas , Células HEK293 , Hepatócitos/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fígado/efeitos dos fármacos , Rifampina/metabolismo , Rifampina/farmacologiaRESUMO
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 â¼5 µM). 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 µM, 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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas/fisiologia , Hepatócitos/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/metabolismo , Maraviroc/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Células HEK293 , Humanos , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Microssomos Hepáticos/metabolismoRESUMO
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.
Assuntos
Fígado/efeitos dos fármacos , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Permeabilidade/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Peso MolecularRESUMO
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 Km values of 2.0 and 3.9 µM for OATP1B probe substrates, pitavastatin and rosuvastatin, respectively. Rifampicin inhibited pitavastatin and rosuvastatin active uptake in monkey hepatocytes with IC50 values of 3.0 and 0.54 µM, respectively, following preincubation with the inhibitor. Intravenous pharmacokinetics of 2H4-pitavastatin and 2H6-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 IC50 obtained following 1 hour preincubation with rifampicin (0.54 µM) 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 IC50 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.
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Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Quinolinas/farmacologia , Rosuvastatina Cálcica/farmacologia , Administração Oral , Animais , Transporte Biológico , Relação Dose-Resposta a Droga , Interações Medicamentosas , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Macaca fascicularis , Masculino , Quinolinas/administração & dosagem , Quinolinas/metabolismo , Quinolinas/farmacocinética , Rosuvastatina Cálcica/administração & dosagem , Rosuvastatina Cálcica/metabolismo , Rosuvastatina Cálcica/farmacocinética , Distribuição TecidualRESUMO
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 (Km)] of 19.5 ± 4.3 µM. 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 Km and Vmax values of 39.3 ± 6.6 µM 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.
Assuntos
Citocromo P-450 CYP2C9/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Tolbutamida/metabolismo , Transporte Biológico , Células HEK293 , Hepatócitos/metabolismo , Humanos , Distribuição Tecidual , Tolbutamida/farmacocinética , Tolbutamida/farmacologiaRESUMO
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.
Assuntos
Transporte Biológico/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Interações Medicamentosas/fisiologia , Humanos , CinéticaRESUMO
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.
Assuntos
Transporte Biológico/fisiologia , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Cromatografia Líquida/métodos , Hepatócitos/metabolismo , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
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.
Assuntos
Hepatócitos/metabolismo , Fígado/metabolismo , Taxa de Depuração Metabólica/fisiologia , Transportadores de Ânions Orgânicos/metabolismo , Preparações Farmacêuticas/metabolismo , Adulto , Animais , Transporte Biológico/fisiologia , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Cinética , Macaca fascicularis , Peptídeos/metabolismoRESUMO
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.
Assuntos
Hepatócitos/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteômica/métodos , Adulto , Fatores Etários , Idoso , Variação Biológica da População , Criança , Cromatografia Líquida , Feminino , Células HEK293 , Humanos , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Cultura Primária de Células , Fatores Sexuais , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
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 ( Km) of â¼7-12 µM but did not show substrate affinity for other major hepatic uptake transporters. Uptake of both enantiomers by primary human hepatocytes was saturable ( Km ≈ 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.
Assuntos
Anticoagulantes/farmacocinética , Hepatócitos/metabolismo , Modelos Biológicos , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Varfarina/farmacocinética , Adulto , Ciclosporina/farmacologia , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Interações Medicamentosas , Feminino , Células HEK293 , Hepatócitos/efeitos dos fármacos , Humanos , Cetoprofeno/farmacologia , Fígado/citologia , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/antagonistas & inibidores , EstereoisomerismoRESUMO
Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion-transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (â¼30-fold) and metabolites (â¼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability.
Assuntos
Transporte Biológico/fisiologia , Colestase/metabolismo , Glibureto/metabolismo , Glibureto/farmacocinética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Teorema de Bayes , Linhagem Celular , Células HEK293 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/metabolismoRESUMO
In the search for novel bile acid (BA) biomarkers of liver organic anion-transporting polypeptides (OATPs), cynomolgus monkeys received oral rifampicin (RIF) at four dose levels (1, 3, 10, and 30 mg/kg) that generated plasma-free Cmax values (0.06, 0.66, 2.57, and 7.79 µM, respectively) spanning the reported in vitro IC50 values for OATP1B1 and OATP1B3 (≤1.7 µM). As expected, the area under the plasma concentration-time curve (AUC) of an OATP probe drug (i.v. 2H4-pitavastatin, 0.2 mg/kg) was increased 1.2-, 2.4-, 3.8-, and 4.5-fold, respectively. Plasma of RIF-dosed cynomolgus monkeys was subjected to a liquid chromatography-tandem mass spectrometry method that supported the analysis of 30 different BAs. Monkey urine was profiled, and we also determined that the impact of RIF on BA renal clearance was minimal. Although sulfated BAs comprised only 1% of the plasma BA pool, a robust RIF dose response (maximal ≥50-fold increase in plasma AUC) was observed for the sulfates of five BAs [glycodeoxycholate (GDCA-S), glycochenodeoxycholate (GCDCA-S), taurochenodeoxycholate, deoxycholate (DCA-S), and taurodeoxycholate (TDCA-S)]. In vitro, RIF (≤100 µM) did not inhibit cynomolgus monkey liver cytosol-catalyzed BA sulfation and cynomolgus monkey hepatocyte-mediated uptake of representative sulfated BAs (GDCA-S, GCDCA-S, DCA-S, and TDCA-S) was sodium-independent and inhibited (≥70%) by RIF (5 µM); uptake of taurocholic acid was sensitive to sodium removal (74% decrease) and relatively refractory to RIF (≤21% inhibition). We concluded that sulfated BAs may serve as sensitive biomarkers of cynomolgus monkey OATPs and that exploration of their utility as circulating human OATP biomarkers is warranted.