Canalicular Efflux Research Articles

Quantitative Systems Toxicology Identifies Independent Mechanisms for Hepatotoxicity and Bilirubin Elevations Due to AKR1C3 Inhibitor BAY1128688.

BAY1128688 is a selective inhibitor of AKR1C3, investigated recently in a trial that was prematurely terminated due to drug-induced liver injury. These unexpected observations prompted use of the quantitative systems toxicology model, DILIsym, to determine possible mechanisms of hepatotoxicity. Using mechanistic in vitro toxicity data as well as clinical exposure data, DILIsym predicted the potential for BAY1128688 to cause liver toxicity (elevations in serum alanine aminotransferase (ALT)) and elevations in serum bilirubin. Initial simulations overpredicted hepatotoxicity and bilirubin elevations, so the BAY1128688 representation within DILIsym underwent optimization. The liver partition coefficient Kp was altered to align simulated bilirubin elevations with those observed clinically. Altering the mode of bile acid canalicular and basolateral efflux inhibition was necessary to accurately predict ALT elevations. Optimization results support that bilirubin elevations observed early during treatment are due to altered bilirubin metabolism and transporter inhibition, which is independent of liver injury. The modeling further supports that on-treatment ALT elevations result from inhibition of bile acid transporters, particularly the bile salt excretory pump, leading to accumulation of toxic bile acids. The predicted dose-dependent intrinsic hepatotoxicity may increase patient susceptibility to an adaptive immune response, accounting for ALT elevations observed after completion of treatment. These BAY1128688 simulations provide insight into the mechanisms behind hepatotoxicity and bilirubin elevations and may inform the potential risk posed by future compounds.

Molecular and Clinical Links between Drug-Induced Cholestasis and Familial Intrahepatic Cholestasis.

Idiosyncratic Drug-Induced Liver Injury (iDILI) represents an actual health challenge, accounting for more than 40% of hepatitis cases in adults over 50 years and more than 50% of acute fulminant hepatic failure cases. In addition, approximately 30% of iDILI are cholestatic (drug-induced cholestasis (DIC)). The liver's metabolism and clearance of lipophilic drugs depend on their emission into the bile. Therefore, many medications cause cholestasis through their interaction with hepatic transporters. The main canalicular efflux transport proteins include: 1. the bile salt export pump (BSEP) protein (ABCB11); 2. the multidrug resistance protein-2 (MRP2, ABCC2) regulating the bile salts' independent flow by excretion of glutathione; 3. the multidrug resistance-1 protein (MDR1, ABCB1) that transports organic cations; 4. the multidrug resistance-3 protein (MDR3, ABCB4). Two of the most known proteins involved in bile acids' (BAs) metabolism and transport are BSEP and MDR3. BSEP inhibition by drugs leads to reduced BAs' secretion and their retention within hepatocytes, exiting in cholestasis, while mutations in the ABCB4 gene expose the biliary epithelium to the injurious detergent actions of BAs, thus increasing susceptibility to DIC. Herein, we review the leading molecular pathways behind the DIC, the links with the other clinical forms of familial intrahepatic cholestasis, and, finally, the main cholestasis-inducing drugs.

Role of Hepatocyte Transporters in Drug-Induced Liver Injury (DILI)-In Vitro Testing.

Bile acids and bile salts (BA/BS) are substrates of both influx and efflux transporters on hepatocytes. Canalicular efflux transporters, such as BSEP and MRP2, are crucial for the removal of BA/BS to the bile. Basolateral influx transporters, such as NTCP, OATP1B1/1B3, and OSTα/β, cooperate with canalicular transporters in the transcellular vectorial flux of BA/BS from the sinusoids to the bile. The blockage of canalicular transporters not only impairs the bile flow but also causes the intracellular accumulation of BA/BS in hepatocytes that contributes to, or even triggers, liver injury. In the case of BA/BS overload, the efflux of these toxic substances back to the blood via MRP3, MRP4, and OST α/β is considered a relief function. FXR, a key regulator of defense against BA/BS toxicity suppresses de novo bile acid synthesis and bile acid uptake, and promotes bile acid removal via increased efflux. In drug development, the early testing of the inhibition of these transporters, BSEP in particular, is important to flag compounds that could potentially inflict drug-induced liver injury (DILI). In vitro test systems for efflux transporters employ membrane vesicles, whereas those for influx transporters employ whole cells. Additional in vitro pharmaceutical testing panels usually include cellular toxicity tests using hepatocytes, as well as assessments of the mitochondrial toxicity and accumulation of reactive oxygen species (ROS). Primary hepatocytes are the cells of choice for toxicity testing, with HepaRG cells emerging as an alternative. Inhibition of the FXR function is also included in some testing panels. The molecular weight and hydrophobicity of the drug, as well as the steady-state total plasma levels, may positively correlate with the DILI potential. Depending on the phase of drug development, the physicochemical properties, dosing, and cut-off values of BSEP IC50 ≤ 25-50 µM or total Css,plasma/BSEP IC50 ≥ 0.1 may be an indication for further testing to minimize the risk of DILI liability.

Interleukin-6 regulates the expression of hepatic canalicular efflux drug transporters after cecal ligation and puncture-induced sepsis: A comparison with lipopolysaccharide treatment

Hepatic multidrug transporters expressed on the canalicular membrane play a role in the hepatobiliary excretion of xenobiotics and endogenous substrates. The aim of this study was to elucidate the role of pro-inflammatory cytokines in the regulation of hepatic drug transporter expression after cecal ligation and puncture (CLP), a valuable tool for studying polymicrobial sepsis, and to compare CLP with lipopolysaccharide (LPS) treatment. CLP reduced the expression of Mdr2/Abcb4, Mrp2/Abcc2, Bsep/Abcb11, Bcrp/Abcg2, and Mate1/Slc47a1 mRNAs in wild-type (WT) mouse livers in a time-dependent manner up to 48 h postoperation. LPS also reduced the expression of all transporters in WT mouse livers 24 h posttreatment; thereafter, expression levels tended to return to normal by 48 h posttreatment. IL-6−/− mice exhibited inhibited downregulation of drug transporters following CLP, although IL-1−/− and TNFα−/− mice exhibited the reduced expression of all transporters in a manner similar to that found in WT mice. Compared with CLP, LPS treatment reduced the expression of all transporters in all cytokine-deficient mouse livers, except for the expression of Mrp2/Abcc2 in IL-6−/− mice. Overall, these findings suggest that IL-6 is major factor in the downregulation of hepatic multidrug transporters following the onset of polymicrobial sepsis but not after LPS treatment.

Inhibition of canalicular and sinusoidal taurocholate efflux by cholestatic drugs in human hepatoma HepaRG cells.

HepaRG cells are highly-differentiated human hepatoma cells, which are increasingly recognized as a convenient cellular model for in vitro evaluation of hepatic metabolism, transport, and/or toxicity of drugs. The present study was designed to evaluate whether HepaRG cells can also be useful for studying drug-mediated inhibition of canalicular and/or sinusoidal hepatic efflux of bile acids, which constitutes a major mechanism of drug-induced liver toxicity. For this purpose, HepaRG cells, initially loaded with the bile acid taurocholate (TC), were reincubated in TC-free transport assay medium, in the presence or absence of calcium or drugs, before analysis of TC retention. This method allowed us to objectivize and quantitatively measure biliary and sinusoidal efflux of TC from HepaRG cells, through distinguishing cellular and canalicular compartments. In particular, time-course analysis of the TC-free reincubation period of HepaRG cells, that is, the efflux period, indicated that a 20min-efflux period allowed reaching biliary and sinusoidal excretion indexes for TC around 80% and 60%, respectively. Addition of the prototypical cholestatic drugs bosentan, cyclosporin A, glibenclamide, or troglitazone during the TC-free efflux phase period was demonstrated to markedly inhibit canalicular and sinusoidal secretion of TC, whereas, by contrast, incubation with the noncholestatic compounds salicylic acid or flumazenil was without effect. Such data therefore support the use of human HepaRG cells for in vitro predicting drug-induced liver toxicity (DILI) due to the inhibition of hepatic bile acid secretion, using a biphasic TC loading/efflux assay.

The changes of hepatic bile acid synthesis and transport and bile acids profiles in isopsoralen-induced liver injury C57BL/6J mice

Contest Isopsoralen, one of the main active and quality-control compounds in Psoralea corylifolia L. (Fabaceae), has antitumor and oestrogen-like effects. Previous studies demonstrated that isopsoralen induced hepatotoxicity and its long-term exposure led to cholestatic liver injury. Objective This study investigates the effect of three- or seven-day exposure of low dose isopsoralen (80 mg/kg) on bile acid homeostasis in C57BL/6J mice. Materials and methods Forty-two C57BL/6J mice were randomly divided into control, three- and seven-day groups (n = 14 per group, half female and half male). Isopsoralen suspension was administrated intragastrically at 80 mg/kg once a day. Blood and liver samples were collected to measure biochemical indices and transport of BAs. The histopathology of the liver was also observed. HPLC–MS/MS was also used to measure the BAs profiles and transport activity. Results In the study, isopsoralen increased the levels of serum AST, ALT in three- and seven-day groups, and caused vacuolar degeneration and swelling in the liver. Canalicular efflux transporters BSEP, OSTα, MRP2, MRP3, and basolateral uptake transporters NTCP, OATP4 were inhibited after seven-day-administration. Moreover, amino acid binding enzymes (BAAT and BACS) were also inhibited after seven-day-administration. The composition of BAs changed greatly and the concentration of some unconjugated-BAs which have stronger hydrophobicity, such as CA, CDCA, was significantly increased. Conclusions Isopsoralen (80 mg/kg) caused hepatotoxicity after short-term exposure by inhibiting the expression of efflux transporters, amino acid binding enzymes, and disrupting BAs spectrum.

Pharmacokinetic Imaging Using 99mTc-Mebrofenin to Untangle the Pattern of Hepatocyte Transporter Disruptions Induced by Endotoxemia in Rats

Endotoxemia-induced inflammation may impact the activity of hepatocyte transporters, which control the hepatobiliary elimination of drugs and bile acids. 99mTc-mebrofenin is a non-metabolized substrate of transporters expressed at the different poles of hepatocytes. 99mTc-mebrofenin imaging was performed in rats after the injection of lipopolysaccharide (LPS). Changes in transporter expression were assessed using quantitative polymerase chain reaction of resected liver samples. Moreover, the particular impact of pharmacokinetic drug–drug interactions in the context of endotoxemia was investigated using rifampicin (40 mg/kg), a potent inhibitor of hepatocyte transporters. LPS increased 99mTc-mebrofenin exposure in the liver (1.7 ± 0.4-fold). Kinetic modeling revealed that endotoxemia did not impact the blood-to-liver uptake of 99mTc-mebrofenin, which is mediated by organic anion-transporting polypeptide (Oatp) transporters. However, liver-to-bile and liver-to-blood efflux rates were dramatically decreased, leading to liver accumulation. The transcriptomic profile of hepatocyte transporters consistently showed a downregulation of multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), which mediate the canalicular and sinusoidal efflux of 99mTc-mebrofenin in hepatocytes, respectively. Rifampicin effectively blocked both the Oatp-mediated influx and the Mrp2/3-related efflux of 99mTc-mebrofenin. The additive impact of endotoxemia and rifampicin led to a 3.0 ± 1.3-fold increase in blood exposure compared with healthy non-treated animals. 99mTc-mebrofenin imaging is useful to investigate disease-associated change in hepatocyte transporter function.

Clinical evaluation of [18F]pitavastatin for quantitative analysis of hepatobiliary transporter activity

It is widely accepted that uptake and efflux transporters on clearance organs play crucial roles in drug disposition. Although in vitro transporter assay system can identify the intrinsic properties of the target transporters, it is not so easy to precisely predict in vivo pharmacokinetic parameters from in vitro data. Positron emission tomography (PET) imaging is a useful tool to directly assess the activity of drug transporters in humans. We recently developed a practical synthetic method for fluorine-18-labeled pitavastatin ([18F]PTV) as a PET probe for quantitative evaluation of hepatobiliary transport. In the present study, we conducted clinical PET imaging with [18F]PTV and compared the pharmacokinetic properties of the probe for healthy subjects with or without rifampicin pretreatment. Rifampicin pretreatment significantly suppressed the hepatic maximum concentration and biliary excretion of the probe to 52% and 34% of the control values, respectively. Rifampicin treatment markedly decreased hepatic uptake clearance (21% of the control), and moderately canalicular efflux clearance with regard to hepatic concentration (52% of the control). These results demonstrate that [18F]PTV is a useful probe for clinical investigation of the activities of hepatobiliary uptake/efflux transporters in humans.

Mechanism of hepatobiliary toxicity of the LPA1 antagonist BMS-986020 developed to treat idiopathic pulmonary fibrosis: Contrasts with BMS-986234 and BMS-986278

In a Phase 2 clinical trial, BMS-986020, a lysophosphatidic acid receptor-1 (LPA1) antagonist, produced hepatobiliary toxicity (increased ALT, AST, and ALP; cholecystitis) and increases in plasma bile acids (BA). Nonclinical investigations conducted to identify a potential mechanism(s) for this toxicity examined BMS-986020 and two LPA1 antagonists structurally distinct from BMS-986020 (BMS-986234 and BMS-986278). BMS-986020 inhibited hepatic BA efflux transporters BSEP (IC50 1.8 μM), MRP3 (IC50 22 μM), and MRP4 (IC50 6.2 μM) and inhibited BA canalicular efflux in human hepatocytes (68% at 10 μM). BMS-986020 inhibited mitochondrial function (basal and maximal respiration, ATP production, and spare capacity) in human hepatocytes and cholangiocytes at ≥10 μM and inhibited phospholipid efflux in human hepatocytes (MDR3 IC50 7.5 μM). A quantitative systems toxicology analysis (DILIsym®), considering pharmacokinetics, BA homeostasis, mitochondrial function, oxidative phosphorylation, and reactive intermediates performed for BMS-986020 recapitulated clinical findings ascribing the effects to BA transporter and mitochondrial electron transport chain inhibition. BMS-986234 and BMS-986278 minimally inhibited hepatic BA transporters (IC50 ≥20 μM) and did not inhibit MDR3 activity (IC50 >100 μM), nor did BMS-986234 inhibit BA efflux (≤50 μM) or mitochondrial function (≤30 μM) (BMS-986278 not evaluated). Multiple mechanisms may be involved in the clinical toxicity observed with BMS-986020. The data indicate that this toxicity was unrelated to LPA1 antagonism since the mechanisms that likely influenced the adverse clinical outcome of BMS-986020 were not observed with equipotent LPA1 antagonists BMS-986234 and BMS-986278. This conclusion is consistent with the lack of hepatobiliary toxicity in nonclinical and clinical safety studies with BMS-986278.

Tanshinone IIA alleviate rifampicin-induced cholestasis by regulating the expression and function of bile salt export pump.

Objective:Rifampicin (RFP) induces cholestasis due to long-term tubercular therapy. Impairment of the canalicular bile acids efflux via the bile salt export pump (BSEP) is a well-recognized cause of cholestasis. Tanshinone IIA (TAN IIA) has a protective effect on the liver. However, there are limited studies on the effects of RFP and TAN IIA on BSEP. In present study, we aimed to elucidate the effects of RFP and TAN IIA on BSEP and provide evidence to support the treatment of RFP-induced cholestasis with TAN IIA.Methods:Firstly, liver histopathological examination and serum biochemical tests were evaluated in rats. Secondly, we evaluated BSEP expression by qRT-PCR and western blotting to explore whether RFP and TAN IIA influence liver function through BSEP. Thirdly, the accumulation of BSEP substrate taurocholic acid (TCA) in bile ducts was determined to investigate the effects of RFP and TAN IIA on BSEP function.Results:Apparent histopathological alterations and significantly increased serum biomarkers were observed in the RFP group (200 mg/kg), while these changes were attenuated in the combination groups. The mRNA and protein levels of BSEP were decreased by RFP. Whereas TAN IIA reversed the downward regulation of BSEP caused by RFP. And RFP primarily inhibited TCA excretion but co-administration of TAN IIA markedly induced TCA excretion mediated by BSEP.Conclusion:Our findings collectively demonstrated that RFP-induced cholestasis could be related to the inhibition of BSEP, and TAN IIA had the potential to prevent RFP-induced cholestasis by regulating BSEP.

The Effects of Polyphenol-Rich Black Elderberry on Oxidative Stress and Hepatic Cholesterol Metabolism

Black elderberry (Sambucus Nigar) with high polyphenol content has been reported to have a hypolipidemic effect, but its underlying mechanisms have not been well characterized. In the present study, we investigated the effects of polyphenol-rich black elderberry (BEE) on oxidative stress and hepatic lipid metabolism. The total antioxidant activity of BEE was evaluated. The expression of genes for lipid metabolism was measured in 50 or 100 μg/mL of BEE-treated HepG2 cells. The mRNA and protein levels of genes for cholesterol metabolisms, i.e., sterol regulatory element-binding protein 2, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and low-density lipoprotein receptor, were decreased by BEE. There was marked induction of genes for high-density lipoprotein metabolism, i.e., scavenger receptor class B type 1and ATP-binding cassette (ABC) transporter A1 in BEE-treated cells. The expression of canalicular efflux transporter for hepatic cholesterol and bile acids, such as ABCG5/G8 and ABCB11, was significantly increased by BEE treatment. There was no alteration of the lipogenic genes, whereas BEE significantly decreased the expression of genes for fatty acid oxidation. BEE significantly altered the expression of histone deacetylase and sirtuins. These data suggest that the hypocholesterolemic effects of BEE may be attributed to the alteration of genes for hepatic cholesterol synthesis and flux.

Bosentan Alters Endo- and Exogenous Bile Salt Disposition in Sandwich-Cultured Human Hepatocytes.

Bosentan, a well-known cholestatic agent, was not identified as cholestatic at concentrations up to 200 µM based on the drug-induced cholestasis (DIC) index value, determined in a sandwich-cultured human hepatocyte (SCHH)-based DIC assay. To obtain further quantitative insights into the effects of bosentan on cellular bile salt handling by human hepatocytes, the present study determined the effect of 2.5-25 µM bosentan on endogenous bile salt levels and on the disposition of 10 µM chenodeoxycholic acid (CDCA) added to the medium in SCHHs. Bosentan reduced intracellular as well as extracellular concentrations of both endogenous glycochenodeoxycholic acid (GCDCA) and glycocholic acid in a concentration-dependent manner. When exposed to 10 µM CDCA, bosentan caused a shift from canalicular efflux to sinusoidal efflux of GCDCA. CDCA levels were not affected. Our mechanistic model confirmed the inhibitory effect of bosentan on canalicular GCDCA clearance. Moreover, our results in SCHHs also indicated reduced GCDCA formation. We confirmed the direct inhibitory effect of bosentan on CDCA conjugation with glycine in incubations with liver S9 fraction. SIGNIFICANCE STATEMENT: Bosentan was evaluated at therapeutically relevant concentrations (2.5-25 µM) in sandwich-cultured human hepatocytes. It altered bile salt disposition and inhibited canalicular secretion of glycochenodeoxycholic acid (GCDCA). Within 24 hours, bosentan caused a shift from canalicular to sinusoidal efflux of GCDCA. These results also indicated reduced GCDCA formation. This study confirmed a direct effect of bosentan on chenodeoxycholic acid conjugation with glycine in liver S9 fraction.

Effects of ablation and activation of Nrf2 on bile acid homeostasis in male mice

The role of nuclear factor erythroid 2-related factor 2 (Nrf2) in bile acid (BA) homeostasis remains controversial. In this study, activation of Nrf2 was achieved either pharmacologically by CDDO-imidazolide (CDDO-Im) or genetically through a “gene dose-response” model consisting of Nrf2-null, wild-type (WT), Keap1-knockdown (Keap1-KD), and Keap1-hepatocyte knockout (Keap1-HKO) mice. In WT mice, CDDO-Im increased bile flow and decreased hepatic BAs, which was associated with a down-regulation of the canalicular BA efflux transporter Bsep and an increase in biliary BA excretion. In contrast, hepatic Bsep and biliary BA excretion were not altered in Keap1-KD or Keap1-HKO mice, suggesting that Nrf2 is not important for regulating Bsep or BA-dependent bile flow. In contrast, hepatic Mrp2 and Mrp3 were up-regulated by both pharmacological and genetic activations of Nrf2. Furthermore, ileal BA transporters (Asbt and Ostβ) and cholesterol transporters (Abcg5 and Abcg8) were down-regulated by both pharmacological and genetic activations of Nrf2, suggesting a role of Nrf2 in intestinal absorption of BAs and cholesterol. In Nrf2-null mice, CDDO-Im down-regulated hepatic BA uptake transporters (Ntcp, Oatp1a1, and Oatp1b2), leading to a 39-fold increase of serum BAs. To conclude, the present study demonstrates that activation of Nrf2 in mice up-regulates Mrp2 and Mrp3 in the liver and down-regulates BA and cholesterol transporters in the intestine.

Measurement of Hepatic ABCB1 and ABCG2 Transport Activity with [11C]Tariquidar and PET in Humans and Mice.

P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in the canalicular membrane of hepatocytes mediate the biliary excretion of drugs and drug metabolites. To measure hepatic ABCB1 and ABCG2 activity, we performed positron emission tomography (PET) scans with the ABCB1/ABCG2 substrate [11C]tariquidar in healthy volunteers and wild-type, Abcb1a/b(-/-), Abcg2(-/-), and Abcb1a/b(-/-)Abcg2(-/-) mice without and with coadministration of unlabeled tariquidar. PET data were analyzed with a three-compartment pharmacokinetic model. [11C]Tariquidar underwent hepatobiliary excretion in both humans and mice, and tariquidar coadministration caused a significant reduction in the rate constant for the transfer of radioactivity from the liver into bile (by -74% in humans and by -62% in wild-type mice), suggesting inhibition of canalicular efflux transporter activity. Radio-thin-layer chromatography analysis revealed that the majority of radioactivity (>87%) in the mouse liver and bile was composed of unmetabolized [11C]tariquidar. PET data in transporter knockout mice revealed that both ABCB1 and ABCG2 mediated biliary excretion of [11C]tariquidar. In vitro experiments indicated that tariquidar is not a substrate of major hepatic basolateral uptake transporters (SLCO1B1, SLCO1B3, SLCO2B1, SLC22A1, and SLC22A3). Our data suggest that [11C]tariquidar can be used to measure hepatic canalicular ABCB1/ABCG2 transport activity without a confounding effect of uptake transporters.

Identification and Characterization of Efflux Transporters That Modulate the Subtoxic Disposition of Diclofenac and Its Metabolites.

In the present work, in vivo transporter knockout (KO) mouse models were used to characterize the disposition of diclofenac (DCF) and its primary metabolites following a single subtoxic dose in mice lacking breast cancer resistance protein (Bcrp) or multidrug resistance-associated protein (Mrp)3. The results indicate that Bcrp acts as a canalicular efflux mediator for DCF, as wild-type (WT) mice had biliary excretion values that were 2.2- to 2.6-fold greater than Bcrp KO mice, although DCF plasma levels were not affected. The loss of Bcrp resulted in a 1.8- to 3.2-fold increase of diclofenac acyl glucuronide (DCF-AG) plasma concentrations in KO animals compared with WT mice, while the biliary excretion of DCF-AG increased 1.4-fold in WT versus KO mice. Furthermore, Mrp3 was found to mediate the basolateral transport of DCF-AG, but not DCF or 4'-hydroxy diclofenac. WT mice had DCF-AG plasma concentrations 7.0- to 8.6-fold higher than Mrp3 KO animals; however, there were no changes in biliary excretion of DCF-AG. Vesicular transport experiments with human MRP3 demonstrated that MRP3 is able to transport DCF-AG via low- and high-affinity binding sites. The low-affinity MRP3 transport had a V max and K m of 170 pmol/min/mg and 98.2 µM, respectively, while the high-affinity V max and K m parameters were estimated to be 71.9 pmol/min/mg and 1.78 µM, respectively. In summary, we offer evidence that the disposition of DCF-AG can be affected by both Bcrp and Mrp3, and these findings may be applicable to humans.

Evaluation of transporter-mediated hepatobiliary transport of newly developed 18F-labeled pitavastatin derivative, PTV-F1, in rats by PET imaging

Quantitative evaluations of the functions of uptake and efflux transporters directly in vivo is desired to understand an efficient hepatobiliary transport of substrate drugs. Pitavastatin is a substrate of organic anion transporting polypeptides (OATPs) and canalicular efflux transporters; thus, it can be a suitable probe for positron-emission tomography (PET) imaging of hepatic transporter functions. To characterize the performance of [18F]PTV-F1, an analogue of pitavastatin, we investigated the impact of rifampicin (a typical OATP inhibitor) coadministration or Bcrp (breast cancer resistance protein) knockout on [18F]PTV-F1 hepatic uptake and efflux in rats by PET imaging. After intravenous administration, [18F]PTV-F1 selectively accumulated in the liver, and the radioactivity detected in plasma, liver, and bile mainly derived from the parent PTV-F1 during the PET study (∼40 min). Coadministration of rifampicin largely decreased the hepatic uptake of [18F]PTV-F1 by 73%. Because of its lower clearance in rats, [18F]PTV-F1 is more sensitive for monitoring changes in hepatic OATP1B function that other previously reported OATP1B PET probes. Rifampicin coadministration also significantly decreased the biliary excretion of radioactivity by 65%. Bcrp knockout did not show a significant impact on its biliary excretion.[18F]PTV-F1 enables quantitative analysis of the hepatobiliary transport system for organic anions.

Towards Improved Pharmacokinetic Models for the Analysis of Transporter-Mediated Hepatic Disposition of Drug Molecules with Positron Emission Tomography

Positron emission tomography (PET) imaging with radiolabeled drugs holds great promise to assess the influence of membrane transporters on hepatobiliary clearance of drugs. To exploit the full potential of PET, quantitative pharmacokinetic models are required. In this study, we evaluated the suitability of different compartment models to describe the hepatic disposition of [11C]erlotinib as a small-molecule model drug which undergoes transporter-mediated hepatobiliary excretion. We analyzed two different, previously published data sets in healthy volunteers, in which a baseline [11C]erlotinib PET scan was followed by a second PET scan either after oral intake of unlabeled erlotinib (300 mg) or after intravenous infusion of the prototypical organic anion-transporting polypeptide inhibitor rifampicin (600 mg). We assessed a three-compartment (3C) and a four-compartment (4C) model, in which either a sampled arterial blood input function or a mathematically derived dual input function (DIF), which takes the contribution of the portal vein to the liver blood supply into account, was used. Both models provided acceptable fits of the observed PET data in the liver and extrahepatic bile duct and gall bladder. Changes in model outcome parameters between scans were consistent with the involvement of basolateral hepatocyte uptake and canalicular efflux transporters in the hepatobiliary clearance of [11C]erlotinib. Our results demonstrated that inclusion of a DIF did not lead to substantial improvements in model fits. The models developed in this work represent a step forward in applying PET as a tool to assess the impact of hepatic transporters on drug disposition and their involvement in drug-drug interactions.

Evidence that decreased expression of sinusoidal bile acid transporters accounts for the inhibition by rapamycin of bile flow recovery following liver ischemia

Rapamycin is employed as an immunosuppressant following organ transplant and, in patients with hepatocellular carcinoma, to inhibit cancer cell regrowth following liver surgery. Preconditioning the liver with rapamycin to induce the expression of antioxidant enzymes is a potential strategy to reduce ischemia reperfusion (IR) injury. However, pre-treatment with rapamycin inhibits bile flow, especially following ischemia. The aim was to investigate the mechanisms involved in this inhibition. In a rat model of segmental hepatic ischemia and reperfusion, acute administration of rapamycin by intravenous injection did not inhibit the basal rate of bile flow. Pre-treatment of rats with rapamycin for 24 h by intraperitoneal injection inhibited the expression of mRNA encoding the sinusoidal influx transporters Ntcp, Oatp1 and 2 and the canalicular efflux transporter Bsep, and increased expression of canalicular Mrp2. Dose-response curves for the actions of rapamycin on the expression of Bsep and Ntcp in cultured rat hepatocytes were biphasic, and monophasic for effects on Oatp1. In cultured tumorigenic H4IIE liver cells, several bile acid transporters were not expressed, or were expressed at very low levels compared to hepatocytes. In H4IIE cells, rapamycin increased expression of Ntcp, Oatp1 and Mrp2, but decreased expression of Oatp2. It is concluded that the inhibition of bile flow recovery following ischemia observed in rapamycin-treated livers is principally due to inhibition of the expression of sinusoidal bile acid transporters. Moreover, in tumorigenic liver tissue the contribution of tumorigenic hepatocytes to total liver bile flow is likely to be small and is unlikely to be greatly affected by rapamycin.

Evaluation of Drug Biliary Excretion Using Sandwich-Cultured Human Hepatocytes.

Evaluation of hepatobiliary transport of drugs is an important challenge, notably during the development of new molecular identities. In this context, sandwich-cultured human hepatocytes (SCHH) have been proposed as an interesting and integrated tool for predicting in vitro biliary excretion of drugs. The present review was therefore designed to summarize key findings about SCHH, including their establishment, their main functional features and their use for the determination of canalicular transport and the prediction of in vivo biliary clearance and hepatobiliary excretion-related drug-drug interactions. Reviewed data highlight the fact that SCHH represent an original and probably unique holistic in vitro approach to predict biliary clearance in humans, through taking into account sinusoidal drug uptake, passive drug diffusion, drug metabolism and sinusoidal and canalicular drug efflux. Limits and proposed refinements for SCHH-based analysis of drug biliary excretion, as well as putative human alternative in vitro models to SCHH are also discussed.

Hepatobiliary Disposition of Atovaquone: A Case of Mechanistically Unusual Biliary Clearance.

Atovaquone, an antiprotozoal and antipneumocystic agent, is predominantly cleared by biliary excretion of unchanged parent drug. Atovaquone is ≥10,000-fold concentrated in human bile relative to unbound plasma. Even after correcting for apparent nonspecific binding and incomplete solubility in bile, atovaquone is still concentrated ≥100-fold in bile, consistent with active biliary excretion. Mechanisms of atovaquone hepatobiliary disposition were studied using a multiexperimental in vitro and in vivo approach. Atovaquone uptake was not elevated in HEK293 cells singly overexpressing OATP1B1, OATP1B3, OATP2B1, OCT1, NTCP, or OAT2. Hepatocyte uptake of atovaquone was not impaired by OATP and OCT inhibitor cocktail (rifamycin and imipramine). Atovaquone liver-to-blood ratio at distributional equilibrium was not reduced in Oatp1a/1b and Oct1/2 knockout mice. Atovaquone exhibited efflux ratios of approximately unity in P-gp and BCRP overexpressing MDCK cell monolayers and did not display enhanced uptake in MRP2 vesicles. Biliary and canalicular clearance were not decreased in P-gp, Bcrp, Mrp2, and Bsep knockout rats. In the present study, we rule out the involvement of major known basolateral uptake and bile canalicular efflux transporters in the hepatic uptake and biliary excretion of atovaquone. This is the first known example of a drug cleared by biliary excretion in humans, with extensive biliary concentration, which is not transported by the mechanisms investigated herein.