Low molecular weight acids and OATP1B mediated hepatic clearance: In vitro and in vivo evaluation using novel hypoxia-inducible factor prolyl hydroxylase inhibitors (Dustats).

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.

Mechanistic Determinants of Daprodustat Drug-Drug Interactions and Pharmacokinetics in Hepatic Dysfunction and Chronic Kidney Disease: Significance of OATP1B-CYP2C8 Interplay.

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.

Characterization of Bile Acid Sulfate Conjugates as Substrates of Human Organic Anion Transporting Polypeptides.

Drug interactions involving the inhibition of hepatic organic anion transporting polypeptides (OATPs) 1B1 and OATP1B3 are considered important. Therefore, we sought to study various sulfated bile acids (BA-S) as potential clinical OATP1B1/3 biomarkers. It was determined that BA-S [e.g., glycochenodeoxycholic acid 3-O-sulfate (GCDCA-S) and glycodeoxycholic acid 3-O-sulfate (GDCA-S)] are substrates of OATP1B1, OATP1B3, and sodium-dependent taurocholic acid cotransporting polypeptide (NTCP) transfected into human embryonic kidney 293 cells, with minimal uptake evident for other solute carriers (SLCs) like OATP2B1, organic anion transporter 2, and organic cation transporter 1. It was also shown that BA-S uptake by plated human hepatocytes (PHH) was inhibited (≥96%) by a pan-SLC inhibitor (rifamycin SV), and there was greater inhibition (≥77% versus ≤12%) with rifampicin (OATP1B1/3-selective inhibitor) than a hepatitis B virus myristoylated-preS1 peptide (NTCP-selective inhibitor). Estrone 3-sulfate was also used as an OATP1B1-selective inhibitor. In this instance, greater inhibition was observed with GDCA-S (76%) than GCDCA-S (52%). The study was expanded to encompass the measurement of GCDCA-S and GDCA-S in plasma of SLCO1B1 genotyped subjects. The geometric mean GDCA-S concentration was 2.6-fold (90% confidence interval 1.6, 4.3; P = 2.1 × 10-4) and 1.3-fold (1.1, 1.7; P = 0.001) higher in individuals homozygous and heterozygous for the SLCO1B1 c.521T > C loss-of-function allele, respectively. For GCDCA-S, no significant difference was noted [1.2-fold (0.8, 1.7; P = 0.384) and 0.9-fold (0.8, 1.1; P = 0.190), respectively]. This supported the in vitro data indicating that GDCA-S is a more OATP1B1-selective substrate (versus GCDCA-S). It is concluded that GCDCA-S and GDCA-S are viable plasma-based OATP1B1/3 biomarkers, but they are both less OATP1B1-selective when compared to their corresponding 3-O-glucuronides (GCDCA-3G and GDCA-3G). Additional studies are needed to determine their utility versus more established biomarkers, such as coproporphyrin I, for assessing inhibitors with different OATP1B1 (versus OATP1B3) inhibition signatures.

Transporter-Enzyme Interplay in the Pharmacokinetics of PF-06835919, A First-in-class Ketohexokinase Inhibitor for Metabolic Disorders and Non-alcoholic Fatty Liver Disease.

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.

Biomarker-Informed Model-Based Risk Assessment of Organic Anion Transporting Polypeptide 1B Mediated Drug-Drug Interactions.

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 (IC50 )). 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 IC50 values and unbound liver inlet concentrations (Iin,max,u ), suggested in vivo OATP1B inhibition risk for drugs with R-value (1+ Iin,max,u /IC50 ) 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 IC50 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.

Organic Anion-Transporting Polypeptide 1B1/1B3-Mediated Hepatic Uptake Determines the Pharmacokinetics of Large Lipophilic Acids: In Vitro-In Vivo Evaluation in Cynomolgus Monkey.

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 (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.

Effect of Human Plasma on Hepatic Uptake of Organic Anion-Transporting Polypeptide 1B Substrates: Studies Using Transfected Cells and Primary Human Hepatocytes.

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.

Optimizing the Benefit/Risk of Acetyl-CoA Carboxylase Inhibitors through Liver Targeting.

Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors have the potential to rebalance disordered lipid metabolism, leading to improvements in nonalcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis (DNL), albeit with concomitant reductions in platelet count, which were attributed to the inhibition of fatty acid synthesis within bone marrow. Herein, we describe the design, synthesis, and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting polypeptide (OATP) substrate properties, which facilitated selective distribution of the compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in animals, while demonstrating considerable safety margins against platelet reduction in a nonhuman primate model.

Nicotinic acid transport into human liver involves organic anion transporter 2 (SLC22A7).

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 (Km) 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.

Quantitative Contribution of Six Major Transporters to the Hepatic Uptake of Drugs: "SLC-Phenotyping" Using Primary Human Hepatocytes.

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.

Effect of Hepatic Organic Anion-Transporting Polypeptide 1B Inhibition and Chronic Kidney Disease on the Pharmacokinetics of a Liver-Targeted Glucokinase Activator: A Model-Based Evaluation.

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.

Simultaneous Assessment of Transporter-Mediated Drug-Drug Interactions Using a Probe Drug Cocktail in Cynomolgus Monkey.

We aim to establish an in vivo preclinical model to enable simultaneous assessment of inhibition potential of an investigational drug on clinically relevant drug transporters, organic anion-transporting polypeptide (OATP)1B, breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), and organic anion transporter (OAT)3. Pharmacokinetics of substrate cocktail consisting of pitavastatin (OATP1B substrate), rosuvastatin (OATP1B/BCRP/OAT3), sulfasalazine (BCRP), and talinolol (P-gp) were obtained in cynomolgus monkey-alone or in combination with transporter inhibitors. Single-dose rifampicin (30 mg/kg) significantly (P < 0.01) increased the plasma exposure of all four drugs, with a marked effect on pitavastatin and rosuvastatin [area under the plasma concentration-time curve (AUC) ratio ∼21-39]. Elacridar, BCRP/P-gp inhibitor, increased the AUC of sulfasalazine, talinolol, as well as rosuvastatin and pitavastatin. An OAT1/3 inhibitor (probenecid) significantly (P < 0.05) impacted the renal clearance of rosuvastatin (∼8-fold). In vitro, rifampicin (10 µM) inhibited uptake of pitavastatin, rosuvastatin, and sulfasalazine by monkey and human primary hepatocytes. Transport studies using membrane vesicles suggested that all probe substrates, except talinolol, are transported by cynoBCRP, whereas talinolol is a cynoP-gp substrate. Elacridar and rifampicin inhibited both cynoBCRP and cynoP-gp in vitro, indicating potential for in vivo intestinal efflux inhibition. In conclusion, a probe substrate cocktail was validated to simultaneously evaluate perpetrator impact on multiple clinically relevant transporters using the cynomolgus monkey. The results support the use of the cynomolgus monkey as a model that could enable drug-drug interaction risk assessment, before advancing a new molecular entity into clinical development, as well as providing mechanistic insights on transporter-mediated interactions.

Predicting Human Clearance of Organic Anion Transporting Polypeptide Substrates Using Cynomolgus Monkey: In Vitro-In Vivo Scaling of Hepatic Uptake Clearance.

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.

In Vitro-In Vivo Extrapolation of OATP1B-Mediated Drug-Drug Interactions in Cynomolgus Monkey.

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.

Role of Hepatic Organic Anion Transporter 2 in the Pharmacokinetics of R- and S-Warfarin: In Vitro Studies and Mechanistic Evaluation.

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 ∼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 ( 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.

Organic Anion Transporter 2 Mediates Hepatic Uptake of Tolbutamide, a CYP2C9 Probe Drug.

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.

Hepatobiliary Clearance Prediction: Species Scaling From Monkey, Dog, and Rat, and In Vitro-In Vivo Extrapolation of Sandwich-Cultured Human Hepatocytes Using 17 Drugs.

Hepatobiliary elimination can be a major clearance pathway dictating the pharmacokinetics of drugs. Here, we first compared the dose eliminated in bile in preclinical species (monkey, dog, and rat) with that in human and further evaluated single-species scaling (SSS) to predict human hepatobiliary clearance. Six compounds dosed in bile duct-cannulated (BDC) monkeys showed biliary excretion comparable to human; and the SSS of hepatobiliary clearance with plasma fraction unbound correction yielded reasonable predictions (within 3-fold). Although dog SSS also showed reasonable predictions, rat overpredicted hepatobiliary clearance for 13 of 24 compounds. Second, we evaluated the translatability of in vitro sandwich-cultured human hepatocytes (SCHHs) to predict human hepatobiliary clearance for 17 drugs. For drugs with no significant active uptake in SCHH studies (i.e., with or without rifamycin SV), measured intrinsic biliary clearance was directly scalable with good predictability (absolute average fold error [AAFE] = 1.6). Drugs showing significant active uptake in SCHH, however, showed improved predictability when scaled based on extended clearance term (AAFE = 2.0), which incorporated sinusoidal uptake along with a global scaling factor for active uptake and the canalicular efflux clearance. In conclusion, SCHH is a useful tool to predict human hepatobiliary clearance, whereas BDC monkey model may provide further confidence in the prospective predictions.

Transporter-Mediated Disposition, Clinical Pharmacokinetics and Cholestatic Potential of Glyburide and Its Primary Active Metabolites.

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 (∼30-fold) and metabolites (∼4-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.

Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3-O-Sulfate Conjugates as Potential Novel Biomarkers for Organic Anion-Transporting Polypeptides.

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.