The sodium taurocholate cotransporting polypeptide (NTCP; gene name
) is the primary hepatic basolateral uptake transporter for conjugated bile acids and the entry receptor for the hepatitis B and D ...virus (HBV/HDV). Regulation of human NTCP remains a knowledge gap due to significant species differences in substrate and inhibitor selectivity and plasma membrane expression. In the present study, various kinase inhibitors were screened for inhibition of NTCP function and taurocholate (TCA) uptake using NTCP-transfected HuH-7 cells. This study identified everolimus, an mTOR inhibitor and macrocyclic immunosuppressive drug, as an NTCP inhibitor with modest potency (IC
= 6.7-8.0 µM). Further investigation in differentiated HuH-7 cells expressing NTCP and NTCP-overexpressing Flp-In T-REx 293 cells revealed that the mechanism of action of everolimus on NTCP is direct inhibition and mTOR-independent. Structural analogs of everolimus inhibited NTCP-mediated TCA uptake, however, functional analogs did not affect NTCP-mediated TCA transport, providing further evidence for direct inhibition. This work contributes to the growing body of literature suggesting that NTCP-mediated bile acid uptake may be inhibited by macrocyclic peptides, which may be further exploited to develop novel medications against HBV/HDV.
Hepatobiliary imaging is increasingly used by pharmacologists to quantify liver concentrations of transporter-dependent drugs. However, liver imaging does not quantify concentrations in extracellular ...space, hepatocytes, and bile canaliculi. Our study compared the compartmental distribution of two hepatobiliary substrates gadobenate dimeglumine BOPTA; 0.08 liver extraction ratio (ER) and mebrofenin (MEB; 0.93 ER) in a model of perfused rat liver. A gamma counter placed over livers measured liver concentrations. Livers were preperfused with gadopentetate dimeglumine to measure extracellular concentrations. Concentrations coming from bile canaliculi and hepatocytes were calculated. Transporter activities were assessed by concentration ratios between compartments and pharmacokinetic parameters that describe the accumulation and decay profiles of hepatocyte concentrations. The high liver concentrations of MEB relied mainly on hepatocyte and bile canaliculi concentrations. In contrast, the three compartments contributed to the low liver concentrations obtained during BOPTA perfusion. Nonlinear regression analysis of substrate accumulation in hepatocytes revealed that cellular efflux is measurable ∼4 minutes after the start of perfusion. The hepatocyte-to-extracellular concentration ratio measured at this time point was much higher during MEB perfusion. BOPTA transport by multidrug resistance associated protein 2 induced an aquaporin-mediated water transport, whereas MEB transport did not. BOPTA clearance from hepatocytes to bile canaliculi was higher than MEB clearance. MEB did not efflux back to sinusoids, whereas BOPTA basolateral efflux contributed to the decrease in hepatocyte concentrations. In conclusion, our ex vivo model quantifies substrate compartmental distribution and transport across hepatocyte membranes and provides an additional understanding of substrate distribution in the liver. SIGNIFICANCE STATEMENT: When transporter-dependent drugs target hepatocytes, cellular concentrations are important to investigate. Low concentrations on cellular targets impair drug therapeutic effects, whereas excessive hepatocyte concentrations may induce cellular toxicity. With a gamma counter placed over rat perfused livers, we measured substrate concentrations in the extracellular space, hepatocytes, and bile canaliculi. Transport across hepatocyte membranes was calculated. The study provides an additional understanding of substrate distribution in the liver.
Membrane transport proteins are involved in the absorption, disposition, efficacy, and/or toxicity of many drugs. Numerous mechanisms (e.g., nuclear receptors, epigenetic gene regulation, microRNAs, ...alternative splicing, post‐translational modifications, and trafficking) regulate transport protein levels, localization, and function. Various factors associated with disease, medications, and dietary constituents, for example, may alter the regulation and activity of transport proteins in the intestine, liver, kidneys, brain, lungs, placenta, and other important sites, such as tumor tissue. This white paper reviews key mechanisms and regulatory factors that alter the function of clinically relevant transport proteins involved in drug disposition. Current considerations with in vitro and in vivo models that are used to investigate transporter regulation are discussed, including strengths, limitations, and the inherent challenges in predicting the impact of changes due to regulation of one transporter on compensatory pathways and overall drug disposition. In addition, translation and scaling of in vitro observations to in vivo outcomes are considered. The importance of incorporating altered transporter regulation in modeling and simulation approaches to predict the clinical impact on drug disposition is also discussed. Regulation of transporters is highly complex and, therefore, identification of knowledge gaps will aid in directing future research to expand our understanding of clinically relevant molecular mechanisms of transporter regulation. This information is critical to the development of tools and approaches to improve therapeutic outcomes by predicting more accurately the impact of regulation‐mediated changes in transporter function on drug disposition and response.
Drug-induced liver injury (DILI) is a leading cause of acute liver failure and transplantation. DILI can be the result of impaired hepatobiliary transporters, with altered bile formation, flow, and ...subsequent cholestasis. We used gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), combined with pharmacokinetic modelling, to measure hepatobiliary transporter function in vivo in rats. The sensitivity and robustness of the method was tested by evaluating the effect of a clinical dose of the antibiotic rifampicin in four different preclinical imaging centers. The mean gadoxetate uptake rate constant for the vehicle groups at all centers was 39.3 +/- 3.4 s-1 (n = 23) and 11.7 +/- 1.3 s-1 (n = 20) for the rifampicin groups. The mean gadoxetate efflux rate constant for the vehicle groups was 1.53 +/- 0.08 s-1 (n = 23) and for the rifampicin treated groups was 0.94 +/- 0.08 s-1 (n = 20). Both the uptake and excretion transporters of gadoxetate were statistically significantly inhibited by the clinical dose of rifampicin at all centers and the size of this treatment group effect was consistent across the centers. Gadoxetate is a clinically approved MRI contrast agent, so this method is readily transferable to the clinic.
Rate constants of gadoxetate uptake and excretion are sensitive and robust biomarkers to detect early changes in hepatobiliary transporter function in vivo in rats prior to established biomarkers of liver toxicity.
Nonalcoholic fatty liver disease (NAFLD), representing a clinical spectrum ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), is rapidly evolving into a global ...pandemic. Patients with NAFLD are burdened with high rates of metabolic syndrome‐related comorbidities resulting in polypharmacy. Therefore, it is crucial to gain a better understanding of NAFLD‐mediated changes in drug disposition and efficacy/toxicity. Despite extensive clinical pharmacokinetic data in cirrhosis, current knowledge concerning pharmacokinetic alterations in NAFLD, particularly at different stages of disease progression, is relatively limited. In vitro‐to‐in vivo extrapolation coupled with physiologically based pharmacokinetic and pharmacodynamic (IVIVE‐PBPK/PD) modeling offers a promising approach for optimizing pharmacologic predictions while refining and reducing clinical studies in this population. Use of IVIVE‐PBPK to predict intra‐organ drug concentrations at pharmacologically relevant sites of action is particularly advantageous when it can be linked to pharmacodynamic effects. Quantitative systems pharmacology/toxicology (QSP/QST) modeling can be used to translate pharmacokinetic and pharmacodynamic data from PBPK/PD models into clinically relevant predictions of drug response and toxicity. In this review, a detailed summary of NAFLD‐mediated alterations in human physiology relevant to drug absorption, distribution, metabolism, and excretion (ADME) is provided. The application of literature‐derived physiologic parameters and ADME‐associated protein abundance data to inform virtual NAFLD population development and facilitate PBPK/PD, QSP, and QST predictions is discussed along with current limitations of these methodologies and knowledge gaps. The proposed methodologic framework offers great potential for meaningful prediction of pharmacological outcomes in patients with NAFLD and can inform both drug development and clinical practice for this population.
Purpose
Transport of the hepatobiliary scintigraphy agent Tc-99m mebrofenin (MEB) was characterized and simulation studies were conducted to examine the effects of altered hepatic transport on MEB ...pharmacokinetics in humans.
Methods
MEB transport was investigated in
Xenopus laevis
oocytes expressing OATP1B1 or OATP1B3, and in membrane vesicles prepared from HEK293 cells transfected with MRP2 or MRP3. A pharmacokinetic model was developed based on blood, urine and bile concentration-time profiles obtained in healthy humans, and the effect of changes in hepatic uptake and/or excretion associated with disease states (hyperbilirubinemia and cholestasis) on MEB disposition was simulated.
Results
MEB (80 pM) transport by OATP1B1 and OATP1B3 was inhibited by rifampicin (50 μM) to 10% and 4% of control, respectively. MEB (0.4 nM) transport by MRP2 was inhibited to 12% of control by MK571 (50 μM); MRP3-mediated transport was inhibited to 5% of control by estradiol-17-beta-glucuronide (100 μM). A two-compartment model described MEB (2.5 mCi) systemic disposition in humans (systemic clearance = 16.2 ± 2.7 ml min
−1
kg
−1
); biliary excretion was the predominant route of hepatic elimination (efflux rate constants ratio canalicular/sinusoidal = 3.4 ± 0.8). Based on simulations, altered hepatic transport markedly influenced MEB systemic and hepatic exposure.
Conclusions
MEB may be a useful probe to assess how altered hepatic function at the transport level modulates hepatobiliary drug disposition.
Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of ...endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA), as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR® technology, BAs were measured in cells+bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells+bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3±5.9μM in CTL rat and 183±56μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16±0.21μM in CTL rat SCH and 9.61±6.36μM in CTL human SCH. Treatment of cells for 24h with 10μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na+-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account.
► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA concentrations were estimated using B-CLEAR® technology. ► Endogenous BA profiles in SCH were similar to those reported in vivo for each species. ► Species differences were evident in endogenous BA profiles of rat vs human SCH. ► 10µM troglitazone had no effect on endogenous BA profiles in rat or human SCH at 24h.
Abstract
Organic solute transporter (OST) α/β is a key bile acid transporter expressed in various organs, including the liver under cholestatic conditions. However, little is known about the ...involvement of OSTα/β in bile acid-mediated drug-induced liver injury (DILI), a major safety concern in drug development. This study investigated whether OSTα/β preferentially transports more hepatotoxic, conjugated, primary bile acids and to what extent xenobiotics inhibit this transport. Kinetic studies with OSTα/β-overexpressing cells revealed that OSTα/β preferentially transported bile acids in the following order: taurochenodeoxycholate > glycochenodeoxycholate > taurocholate > glycocholate. The apparent half-maximal inhibitory concentrations for OSTα/β-mediated bile acid (5 µM) transport inhibition by fidaxomicin, troglitazone sulfate, and ethinyl estradiol were: 210, 334, and 1050 µM, respectively, for taurochenodeoxycholate; 97.6, 333, and 337 µM, respectively, for glycochenodeoxycholate; 140, 265, and 527 µM, respectively, for taurocholate; 59.8, 102, and 117 µM, respectively, for glycocholate. The potential role of OSTα/β in hepatocellular glycine-conjugated bile acid accumulation and cholestatic DILI was evaluated using sandwich-cultured human hepatocytes (SCHH). Treatment of SCHH with the farnesoid X receptor agonist chenodeoxycholate (100 µM) resulted in substantial OSTα/β induction, among other proteomic alterations, reducing glycochenodeoxycholate and glycocholate accumulation in cells+bile 4.0- and 4.5-fold, respectively. Treatment of SCHH with troglitazone and fidaxomicin together under cholestatic conditions resulted in increased hepatocellular toxicity compared with either compound alone, suggesting that OSTα/β inhibition may accentuate DILI. In conclusion, this study provides insights into the role of OSTα/β in preferential disposition of bile acids associated with hepatotoxicity, the impact of xenobiotics on OSTα/β-mediated bile acid transport, and the role of this transporter in SCHH and cholestatic DILI.
Tolvaptan is a selective vasopressin V2 receptor antagonist, approved in several countries for the treatment of hyponatremia and autosomal dominant polycystic kidney disease (ADPKD). No liver injury ...has been observed with tolvaptan treatment in healthy subjects and in non-ADPKD indications, but ADPKD clinical trials showed evidence of drug-induced liver injury (DILI). Although all DILI events resolved, additional monitoring in tolvaptan-treated ADPKD patients is required. In vitro assays identified alterations in bile acid disposition and inhibition of mitochondrial respiration as potential mechanisms underlying tolvaptan hepatotoxicity. This report details the application of DILIsym software to determine whether these mechanisms could account for the liver safety profile of tolvaptan observed in ADPKD clinical trials. DILIsym simulations included physiologically based pharmacokinetic estimates of hepatic exposure for tolvaptan and2 metabolites, and their effects on hepatocyte bile acid transporters and mitochondrial respiration. The frequency of predicted alanine aminotransferase (ALT) elevations, following simulated 90/30 mg split daily dosing, was 7.9% compared with clinical observations of 4.4% in ADPKD trials. Toxicity was multifactorial as inhibition of bile acid transporters and mitochondrial respiration contributed to the simulated DILI. Furthermore, simulation analysis identified both pre-treatment risk factors and on-treatment biomarkers predictive of simulated DILI. The simulations demonstrated that in vivo hepatic exposure to tolvaptan and the DM-4103 metabolite, combined with these 2 mechanisms of toxicity, were sufficient to account for the initiation of tolvaptan-mediated DILI. Identification of putative risk-factors and potential novel biomarkers provided insight for the development of mechanism-based tolvaptan risk-mitigation strategies.
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