Murine Oatp1a/1b uptake transporters control rosuvastatin systemic exposure without affecting its apparent liver exposure.

Organic anion-transporting polypeptides (OATPs) mediate the liver uptake and hence plasma clearance of a broad range of drugs. For rosuvastatin, a cholesterol-lowering drug and OATP1A/1B substrate, the liver represents both its main therapeutic target and its primary clearance organ. Here we studied the impact of Oatp1a/1b uptake transporters on the pharmacokinetics of rosuvastatin using wild-type and Oatp1a/1b-null mice. After oral administration (15 mg/kg), intestinal absorption of rosuvastatin was not impaired in Oatp1a/1b-null mice, but systemic exposure (area under the curve) was 8-fold higher in these mice compared with wild-type. Although liver exposure was comparable between the two mouse strains (despite the increased blood exposure), the liver-to-blood ratios were markedly decreased (>10-fold) in the absence of Oatp1a/1b transporters. After intravenous administration (5 mg/kg), systemic exposure was 3-fold higher in Oatp1a/1b-null mice than in the wild-type mice. Liver, small intestinal, and kidney exposure were slightly, but not significantly, increased in Oatp1a/1b-null mice. The biliary excretion of rosuvastatin was very fast, with 60% of the dose eliminated within 15 minutes after intravenous administration, and also not significantly altered in Oatp1a/1b-null mice. Rosuvastatin renal clearance, although still minor, was increased ∼15-fold in Oatp1a/1b-null males, suggesting a role of Oatp1a1 in the renal reabsorption of rosuvastatin. Absence of Oatp1a/1b uptake transporters increases the systemic exposure of rosuvastatin by reducing its hepatic extraction ratio. However, liver concentrations are not significantly affected, most likely due to the compensatory activity of high-capacity, low-affinity alternative uptake transporters at higher systemic rosuvastatin levels and the absence of efficient alternative rosuvastatin clearance mechanisms.

Cyclosporine inhibition of hepatic and intestinal CYP3A4, uptake and efflux transporters: application of PBPK modeling in the assessment of drug-drug interaction potential.

PURPOSE: To apply physiologically-based pharmacokinetic (PBPK) modeling to investigate the consequences of reduction in activity of hepatic and intestinal uptake and efflux transporters by cyclosporine and its metabolite AM1. METHODS: Inhibitory potencies of cyclosporine and AM1 against OATP1B1, OATP1B3 and OATP2B1 were investigated in HEK293 cells +/- pre-incubation. Cyclosporine PBPK model implemented in Matlab was used to assess interaction potential (+/- metabolite) against different processes (uptake, efflux and metabolism) in liver and intestine and to predict quantitatively drug-drug interaction with repaglinide. RESULTS: Cyclosporine and AM1 were potent inhibitors of OATP1B1 and OATP1B3, IC(50) ranging from 0.019-0.093 µM following pre-incubation. Cyclosporine PBPK model predicted the highest interaction potential against liver uptake transporters, with a maximal reduction of >70% in OATP1B1 activity; the effect on hepatic efflux and metabolism was minimal. In contrast, 80-97% of intestinal P-gp and CYP3A4 activity was reduced due to the 50-fold higher cyclosporine enterocytic concentrations relative to unbound hepatic inlet. The inclusion of AM1 resulted in a minor increase in the predicted maximal reduction of OATP1B1/1B3 activity. Good predictability of cyclosporine-repaglinide DDI and the impact of dose staggering are illustrated. CONCLUSIONS: This study highlights the application of PBPK modeling for quantitative prediction of transporter-mediated DDIs with concomitant consideration of P450 inhibition.

Simultaneous assessment of uptake and metabolism in rat hepatocytes: a comprehensive mechanistic model.

Kinetic parameters describing hepatic uptake in hepatocytes are frequently estimated without appropriate incorporation of bidirectional passive diffusion, intracellular binding, and metabolism. A mechanistic two-compartment model was developed to describe all of the processes occurring during the in vitro uptake experiments performed in freshly isolated rat hepatocytes plated for 2 h. Uptake of rosuvastatin, pravastatin, pitavastatin, valsartan, bosentan, telmisartan, and repaglinide was investigated over a 0.1 to 300 µM concentration range at 37°C for 2 or 45-90 min; nonspecific binding was taken into account. All concentration-time points were analyzed simultaneously by using a mechanistic two-compartment model describing uptake kinetics [unbound affinity constant (K(m,u)), maximum uptake rate (V(max)), unbound active uptake clearance (CL(active,u))], passive diffusion [unbound passive diffusion clearance (P(diff,u))], and intracellular binding [intracellular unbound fraction (fu(cell))]. When required (telmisartan and repaglinide), the model was extended to account for the metabolism [unbound metabolic clearance (CL(met,u))]. The CL(active,u) ranged 8-fold, reflecting a 11-fold range in uptake K(m,u), with telmisartan and valsartan showing the highest affinity for uptake transporters (K(m,u) <10 µM). Both P(diff,u) and fu(cell) span over two orders of magnitude and reflected the lipophilicity of the drugs in the dataset. An extended incubation time allowed steady state to be reached between media and intracellular compartment concentrations and reduced the error in certain parameter estimates observed with shorter incubation times. Active transport accounted for >70% of total uptake for all drugs investigated and was 4- and 112-fold greater than CL(met,u) for telmisartan and repaglinide, respectively. Modeling of uptake kinetics in conjunction with metabolism improved the precision of the uptake parameter estimates for repaglinide and telmisartan. Recommendations are made for uptake experimental design and modeling strategies.

Use of mechanistic modeling to assess interindividual variability and interspecies differences in active uptake in human and rat hepatocytes.

Interindividual variability in activity of uptake transporters is evident in vivo, yet limited data exist in vitro, confounding in vitro-in vivo extrapolation. The uptake kinetics of seven organic anion-transporting polypeptide substrates was investigated over a concentration range in plated cryopreserved human hepatocytes. Active uptake clearance (CL(active, u)), bidirectional passive diffusion (P(diff)), intracellular binding, and metabolism were estimated for bosentan, pitavastatin, pravastatin, repaglinide, rosuvastatin, telmisartan, and valsartan in HU4122 donor using a mechanistic two-compartment model in Matlab. Full uptake kinetics of rosuvastatin and repaglinide were also characterized in two additional donors, whereas for the remaining drugs CL(active, u) was estimated at a single concentration. The unbound affinity constant (K(m, u)) and P(diff) values were consistent across donors, whereas V(max) was on average up to 2.8-fold greater in donor HU4122. Consistency in K(m, u) values allowed extrapolation of single concentration uptake activity data and assessment of interindividual variability in CL(active) across donors. The maximal contribution of active transport to total uptake differed among donors, for example, 85 to 96% and 68 to 87% for rosuvastatin and repaglinide, respectively; however, in all cases the active process was the major contributor. In vitro-in vivo extrapolation indicated a general underprediction of hepatic intrinsic clearance, an average empirical scaling factor of 17.1 was estimated on the basis of seven drugs investigated in three hepatocyte donors, and donor-specific differences in empirical factors are discussed. Uptake K(m, u) and CL(active, u) were on average 4.3- and 7.1-fold lower in human hepatocytes compared with our previously published rat data. A strategy for the use of rat uptake data to facilitate the experimental design in human hepatocytes is discussed.

Organic anion-transporting polypeptides 1a/1b control the hepatic uptake of pravastatin in mice.

Organic anion-transporting polypeptides (OATPs) mediate the hepatic uptake of many drugs. Hepatic uptake is crucial for the therapeutic effect of pravastatin, a cholesterol-lowering drug and OATP1A/1B substrate. We aimed to gain empirical insight into the relationship between OATPs and pravastatin pharmacokinetics and toxicity. We therefore compared the distribution and toxicity of pravastatin in wild-type and Oatp1a/1b-null mice. Intestinal absorption of pravastatin was not affected by Oatp1a/1b absence, but systemic plasma exposure (AUC) increased up to 30-fold after oral bolus administration. This increased plasma exposure resulted from reduced hepatic uptake, as evident from 10 to 100-fold lower liver-to-plasma concentration ratios. However, the reductions in liver exposure were far smaller (<2-fold) than the increases in plasma exposure. Reduced pravastatin liver uptake in Oatp1a/1b-null mice was more obvious shortly after intravenous administration, with 8-fold lower biliary pravastatin excretion. Although mice chronically exposed to pravastatin for 60 days evinced little muscular toxicity, Oatp1a/1b-null mice displayed 10-fold higher plasma concentrations and 8-fold lower liver concentrations than wild-type mice. Thus, Oatp1a/1b transporters importantly control the hepatic uptake of pravastatin. Activity-reducing human OATP1B polymorphisms may therefore both reduce pravastatin therapeutic efficacy in the liver and increase systemic toxicity risks, thus compromising its therapeutic index in a two-edged way.

Methotrexate pharmacokinetics in transgenic mice with liver-specific expression of human organic anion-transporting polypeptide 1B1 (SLCO1B1).

Human organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter that can transport a wide variety of drugs. In the present study, we have generated and characterized a transgenic mouse model with specific and functional expression of human OATP1B1 (SLCO1B1) in the liver. Immunohistochemical staining revealed basolateral localization of transgenic OATP1B1 in the liver, whereas no expression of OATP1B1 was found in the kidney and small intestine. Using this transgenic model, the in vivo role of human OATP1B1 in the disposition of the anticancer drug methotrexate (MTX) was studied. In mice on a semisynthetic diet, the area under the plasma concentration-time curve for intravenous methotrexate in SLCO1B1 transgenic mice was 1.5-fold decreased compared with wild-type mice. Furthermore, the amount of MTX in the liver was markedly higher ( approximately 2-fold) in the SLCO1B1 transgenic mice compared with wild-type mice, resulting in 2- to 4-fold higher liver-plasma ratios of MTX. Some murine liver Slco genes were markedly down-regulated on the semisynthetic diet compared with a standard diet, which probably reduced murine Oatp-mediated MTX uptake in the liver and therefore facilitated detection of the function of the transgenic OATP1B1. Taken together, these data demonstrate a marked and possibly rate-limiting role for human OATP1B1 in MTX elimination in vivo. Variation in OATP1B1 activity due to genetic polymorphisms, drug-drug interactions, and possibly dietary conditions may therefore play a role in the severity of MTX-related toxicity. SLCO1B1 transgenic mice could be a useful tool in studying the in vivo role of human OATP1B1 in drug pharmacokinetics.

Organic anion-transporting polypeptide 1B1 mediates transport of Gimatecan and BNP1350 and can be inhibited by several classic ATP-binding cassette (ABC) B1 and/or ABCG2 inhibitors.

Organic anion-transporting polypeptides (OATPs) are important uptake transporters that can have a profound impact on the systemic pharmacokinetics, tissue distribution, and elimination of several drugs. Previous in vivo studies of the pharmacokinetics of the lipophilic camptothecin (CPT) analog gimatecan suggested that the ATP-binding cassette (ABC) B1 (P-glycoprotein) and/or ABCG2 (breast cancer resistance protein) inhibitors elacridar and pantoprazole could inhibit transporters other than ABCB1 and ABCG2. In this study, we tested the possible role of OATP1B1 in this interaction by screening a number of CPT analogs for their transport affinity by human OATP1B1 in vitro. In addition, the impact of several widely used ABCB1 and/or ABCG2 modulators on this OATP1B1-mediated transport was assessed. We identified two novel CPT anticancer drugs, gimatecan and BNP1350, as OATP1B1 substrates, whereas irinotecan, topotecan, and lurtotecan were not transported by OATP1B1. It is interesting to note that transport of 17beta-estradiol 17beta-d-glucuronide (control), gimatecan, and BNP1350 by OATP1B1 could be completely inhibited by the classic ABCB1 and/or ABCG2 inhibitors elacridar, valspodar, pantoprazole, and, to a lesser extent, zosuquidar and verapamil. Therefore, the effect of these ABCB1 and ABCG2 modulators on the plasma pharmacokinetics of gimatecan and BNP1350 (and possibly also other OATP1B1 substrates) may be partly because of inhibition of OATP1B1 besides inhibition of ABCB1 and/or ABCG2. The findings of this study suggest that OATP1B1 polymorphisms or coadministration with one of the ABCB1/ABCG2 inhibitors could affect drug uptake, tissue distribution, and elimination of some CPT anticancer drugs, thereby modifying their efficacy and/or safety profile.

Influence of human OATP1B1, OATP1B3, and OATP1A2 on the pharmacokinetics of methotrexate and paclitaxel in humanized transgenic mice.

PURPOSE: Organic anion-transporting polypeptide (OATP) drug uptake transporters are thought to play an important role in drug pharmacokinetics and toxicokinetics. We aimed to determine the influence of the individual human OATP1B1, OATP1B3, and OATP1A2 transporters on the in vivo disposition of the anticancer drugs methotrexate and paclitaxel by using liver-specific humanized OATP1A/1B transgenic mice. EXPERIMENTAL DESIGN: Wild-type, Slco1a/1b(-/-) (Oatp1a/1b knockout), Slco1a/1b(-/-);1B1(tg), Slco1a/1b(-/-);1B3(tg), and newly generated Slco1a/1b(-/-);1A2(tg) (humanized OATP1B1, OATP1B3, and OATP1A2 transgenic) mice were characterized biochemically and physiologically, and subsequently intravenously dosed with methotrexate or paclitaxel (2 or 10 mg/kg each) for pharmacokinetic analyses. RESULTS: Humanized OATP1B1, OATP1B3, and OATP1A2 transgenic mice all showed partial or complete rescue of increased plasma bilirubin levels, but also of the increased plasma levels and decreased liver and small intestinal accumulation of methotrexate observed in Slco1a/1b(-/-) mice. Furthermore, hepatic expression of OATP1B3 and OATP1A2, but not OATP1B1, resulted in increased liver uptake of paclitaxel (2 mg/kg). At 10 mg/kg, a modest effect of only OATP1A2 on paclitaxel liver uptake was observed. CONCLUSION: Human OATP1A/1B transporters play an important role in plasma and tissue distribution of the structurally diverse chemotherapeutics methotrexate (organic anion) and paclitaxel (hydrophobic, bulky). Variation in OATP1A/1B activity due to genetic variation and pharmacologic inhibition, or differences in tumor-specific expression levels might therefore affect plasma, tissue, and tumor levels of these drugs in patients, and hence their therapeutic efficacy. Humanized transgenic OATP1A/1B mice will provide excellent tools to further study these aspects in vivo for many (anticancer) drugs.

Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.

High impact of Oatp1a/1b transporters on in vivo disposition of the hydrophobic anticancer drug paclitaxel.

PURPOSE: Organic anion-transporting polypeptides (OATP) mediate the cellular uptake of a broad range of drugs. The hydrophobic anticancer drug, paclitaxel (PTX), was recently identified as a substrate for OATP1B3 in vitro. We investigated the role of Oatp1a/1b transporters in the pharmacokinetics of PTX in vivo, as well as their impact at different dose levels of PTX and methotrexate (MTX). EXPERIMENTAL DESIGN: Recently generated Slco1a/1b(-/-) (lacking all Oatp1a/1b transporters) and wild-type mice were intravenously dosed with 2, 10, or 50 mg/kg of PTX, or with 10, 50, or 500 mg/kg of MTX, and plasma and tissue drug concentrations were measured. RESULTS: In spite of its hydrophobicity, PTX systemic exposure (at 10 mg/kg) was increased by greater than 2-fold in Slco1a/1b(-/-) mice compared with wild-type, whereas PTX liver uptake was reduced by about 2-fold. Oatp1a/1b transporters displayed a high impact on PTX and MTX pharmacokinetics over a broad dose range. For MTX, even at 500 mg/kg, saturation of Oatp1a/1b was not observed, with a 3.4-fold increase in plasma and 30-fold decrease in liver levels in Slco1a/1b(-/-) mice compared with wild-type. Although beginning saturation of Oatp1a/1b was observed at the highest dose of PTX, plasma levels in Slco1a/1b(-/-) mice were still 1.7-fold increased and liver levels 1.5-fold decreased compared with wild-type. CONCLUSION: Oatp1a/1b transporters play a pronounced role in determining plasma levels and tissue distribution of MTX and PTX, thus affecting even highly hydrophobic drugs. Variation in OATP1A/1B transporter activity, due to genetic variation, inhibition, and/or tumor expression might affect toxicity and therapeutic efficacy of these anticancer drugs.

Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs.

Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b-/- mice, as SLCO genes encode OATPs). Slco1a/1b-/- mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b-/- mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b-/- mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b-/- mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.