Transporter-transporter interplay determines the renal-predominant elimination of the O-glucuronide metabolite (BI 689875) of vicadrostat in humans.

Nuclear Receptor Ligands: Rational and Effective Therapy for Chronic Cholestatic Liver Disease?

Renal glucuronidation and multidrug resistance protein 2-/ multidrug resistance protein 4-mediated efflux of mycophenolic acid: interaction with cyclosporine and tacrolimus

A major concern in drug development is the characterization of new molecular entities (NMEs) with respect to their safety and efficacy. Both factors are determined by the drug’s exposure within the body which itself is affected by drug clearance processes. The major clearance organs are the liver and the kidney, where an interplay of metabolic enzymes and drug transporters mediates the elimination of drugs by metabolism and/or secretion. By that, inhibition of active clearance pathways, as observed from drug-drug interactions (DDIs), can result in alterations in a drug’s exposure. Therefore, the early characterization of the pharmacokinetic profile (PK) of NMEs is a major goal in preclinical drug development. However, due to lacking human in vivo PK data in this early phase of drug development, in vitro-based methods are commonly used to make a first assessment of the PK profile of NMEs. Consequently, the development, validation, and characterization of these methods is of major importance. Therefore, it was the aim of this work to investigate the prediction of human renal and hepatic drug clearances by in vitro-in vivo extrapolation (IVIVE) models and assess their feasibility to predict the DDI potential of drugs in human. To date, only few IVIVE approaches have been described to predict the human renal organ clearance based on filtration, secretion, and reabsorption. In a first study, we measured in LLC-PK1 cells the transport of 20 compounds with various physiochemical and PK properties. These data were incorporated into a novel kidney model to predict all renal clearance processes in human. Compared to reported renal clearances from clinical studies, the prediction accuracy in terms of percentage within three-fold error was 95%. Moreover, our model allowed the assessment of the contribution of filtration, secretion, and reabsorption to the net renal organ clearance in human. In a second study, we investigated the contribution of the organic anion transporting polypeptides (OATP) 1 and OATP1B3 to the net hepatic uptake clearance of statins. For this purpose, the absolute transporter protein abundances were determined by liquid chromatography-tandem mass spectrometry in cryopreserved human hepatocytes and single-transporter expressing HEK293 cells. Subsequently, uptake kinetics of eight statins and OATP1B1 and OATP1B3-specific reference substrates were determined in all expression systems. Transporter activity data generated in recombinant cell lines were extrapolated to hepatocyte values using relative transporter expression factors (REF) or relative activity factors (RAF). We showed that REF and RAF-based predictions were highly similar indicating a direct transporter expression-activity relationship. Moreover, we demonstrated that the REF-scaling method provided a powerful tool to quantitatively assess the transporter-specific contributions to the net uptake clearance of statins in hepatocytes. In a third study, we applied a recently developed IVIVE method to predict the human hepatic clearance and the DDI potential of eight statins. Application of the recently established Extended Clearance Concept Classification System (ECCCS), demonstrated a good predictability of the human hepatic clearance with six out of eight statins projected within a two-fold deviation to reported values. Furthermore, the DDI potential of the statins was assessed with respect to the impact of possible perpetrator drugs on hepatic uptake, metabolism, and biliary secretion and subsequently compared with reported clinical DDI effects. The predicted DDIs for statins showed excellent quantitative correlations with clinical observations. The ECCCS thus represents a powerful tool to anticipate the DDI potential of victim drugs based on in vitro drug metabolism and transport data. In a last study, we assessed the inhibitory potential of telaprevir, a new, direct-acting antiviral drug, on major human renal and hepatic drug transporters. By that, co-incubations of drug-transporter reference substrates and telaprevir in stable, single-transporter transfected HEK293 cells was investigated. Our data showed that telaprevir exhibited significant potential to inhibit major renal and hepatic drug transporters in human. Therefore, clinical co-administration of telaprevir together with drugs that are substrates of renal and hepatic transporters should be carefully monitored. Taken together, with the help of this work the safety profiles of NMEs can now be assessed in preclinical drug development based on in vitro methods. It is therefore expected, that the establishment, validation, and application of novel in vitro based methods, described in this work, will add significant value in the early assessment of the PK profile of NMEs.

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known. We synthesized [(125)I]11-azidophenyl agosterol A ([(125)I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.

Cabotegravir, a novel integrase inhibitor under development for treatment and prevention of HIV, is primarily metabolized by UDP-glucuronosyltransferase (UGT)1A1 and UGT1A9 to a direct ether glucuronide metabolite. The aim of these studies was to elucidate the mechanistic basis of cabotegravir-glucuronide disposition in humans. Cabotegravir glucuronidation was predominantly hepatic (>95%) with minimal intestinal and renal contribution. Rat liver perfusions demonstrated that cabotegravir-glucuronide formed in the liver undergoes comparable biliary and sinusoidal excretion, consistent with high concentrations of the glucuronide in human bile and urine. Cabotegravir-glucuronide biliary excretion was mediated by multidrug resistance-associated protein (MRP)2 (not transported by breast cancer resistance protein or P-glycoprotein), whereas hepatic basolateral excretion into sinusoidal blood was via both MRP3 [fraction transport (Ft) = 0.81] and MRP4 (Ft = 0.19). Surprisingly, despite high urinary recovery of hepatically-formed cabotegravir-glucuronide, metabolite levels in circulation were negligible, a phenomenon consistent with rapid metabolite clearance. Cabotegravir-glucuronide was transported by hepatic uptake transporters organic anion-transporting (OAT) polypeptide (OATP)1B1 and OATP1B3; however, metabolite clearance by hepatic uptake from circulation was low (2.7% of hepatic blood flow) and unable to explain the minimal systemic exposure. Instead, circulating cabotegravir-glucuronide undergoes efficient renal clearance, where uptake into the proximal tubule would be mediated by OAT3 (not transported by OAT1), and subsequent secretion into urine by MRP2 (Ft = 0.66) and MRP4 (Ft = 0.34). These studies provide mechanistic insight into the disposition of cabotegravir-glucuronide, a hepatically-formed metabolite with appreciable urinary recovery and minimal systemic exposure, including fractional contribution of redundant transporters to any given process based on quantitative proteomics. SIGNIFICANCE STATEMENT: The role of membrane transporters in metabolite disposition, especially glucuronides, and as sites of unexpected drug-drug interactions, which alter drug efficacy and safety, has been established. Cabotegravir-glucuronide, formed predominantly by direct glucuronidation of parent drug in liver, was the major metabolite recovered in human urine (27% of oral dose) but was surprisingly not detected in systemic circulation. To our knowledge, this is the first mechanistic description of this phenomenon for a major hepatically-formed metabolite to be excreted in the urine to a large extent, but not circulate at detectable levels. The present study elucidates the mechanistic basis of cabotegravir-glucuronide disposition in humans. Specific hepatic and renal transporters involved in the disposition of cabotegravir-glucuronide, with their fractional contribution, have been provided.

This study characterized 99mTc-Mebrofenin (MEB) and 99mTc-Sestamibi (MIBI) hepatic transport and preferential efflux routes (canalicular vs. basolateral) in rat and human sandwich-cultured hepatocytes (SCH). 99mTc-MEB and 99mTc-MIBI disposition was determined in suspended hepatocytes and in SCH in the presence and absence of inhibitors and genetic knockdown of breast cancer resistance protein (Bcrp). The general organic anion transporting polypeptide (Oatp/OATP) inhibitor rifamycin SV reduced initial 99mTc-MEB uptake in rat and human suspended hepatocytes. Initial 99mTc-MIBI uptake in suspended rat hepatocytes was not Na+-dependent or influenced by inhibitors. Multidrug resistance-associated protein (Mrp2/MRP2) inhibitors decreased 99mTc-MEB canalicular efflux in rat and human SCH. 99mTc-MEB efflux in human SCH was predominantly canalicular (45.8 +/- 8.6%) and approximately 3-fold greater than in rat SCH. 99mTc-MIBI canalicular efflux was similar in human and rat SCH; basolateral efflux was 37% greater in human than rat SCH. 99mTc-MIBI cellular accumulation, biliary excretion index and in vitro biliary clearance in rat SCH were unaffected by Bcrp knockdown. 99mTc-MEB hepatic uptake is predominantly Oatp-mediated with biliary excretion by Mrp2. 99mTc-MIBI appears to passively diffuse into hepatocytes; biliary excretion is mediated by P-gp. The SCH model is useful to investigate factors that may alter the route and/or extent of hepatic basolateral and canalicular efflux of substrates.

Inhibition of transport across the hepatocyte canalicular membrane by the antibiotic fusidate

In vitro and in silico characterization of the transport of selected perfluoroalkyl carboxylic acids and perfluoroalkyl sulfonic acids by human organic anion transporter 1 (OAT1), OAT2 and OAT3

The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition.

Transporters responsible for hepatic uptake and biliary clearance (CLBile) of rosuvastatin (RSV) have been well characterized. However, the contribution of basolateral efflux clearance (CLBL) to hepatic and systemic exposure of RSV is unknown. Additionally, the appropriate design of in vitro hepatocyte efflux experiments to estimate CLBile versus CLBL remains to be established. A novel uptake and efflux protocol was developed in sandwich-cultured hepatocytes (SCH) to achieve desired tight junction modulation while maintaining cell viability. Subsequently, studies were conducted to determine the role of CLBL in the hepatic disposition of RSV using SCH from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(-)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor elacridar (GF120918). RSV CLBile was nearly ablated by GF120918 in TR(-) SCH, confirming that Mrp2 and Bcrp are responsible for the majority of RSV CLBile. Pharmacokinetic modeling revealed that CLBL and CLBile represent alternative elimination routes with quantitatively similar contributions to the overall hepatocellular excretion of RSV in rat SCH under baseline conditions (WT SCH in the absence of GF120918) and also in human SCH. Membrane vesicle experiments revealed that RSV is a substrate of MRP4 (Km = 21 ± 7 µM, Vmax = 1140 ± 210 pmol/min per milligram of protein). Alterations in MRP4-mediated RSV CLBL due to drug-drug interactions, genetic polymorphisms, or disease states may lead to changes in hepatic and systemic exposure of RSV, with implications for the safety and efficacy of this commonly used medication.

Saikosaponin b2 enhances the hepatotargeting effect of anticancer drugs through inhibition of multidrug resistance-associated drug transporters

Background: Intestinal drug metabolism and transport are now well recognized determinants of drug disposition in humans. During the last decade, various animal models lacking drug transporters have been generated in order to investigate the role of transporters for drug absorption, distribution and elimination. Objective: In this review the use of the animal models for the investigation of intestinal drug transport will be discussed. Methods: Publications describing the use of knockout animals (e.g., P-glycoprotein, Bcrp, and Oct1) regarding intestinal drug transport and animals characterized by mutations in transporters genes (e.g., Mrp2) were mainly considered for this review. Results/conclusion: Knockout mouse models for ABC transporters are highly valuable tools to investigate the role of intestinal efflux transporters for the bioavailability of various compounds.

Organic anion transporting polypeptide (OATP)1B1 and OATP1B3 (OATP1B) are clinically important transporters that mediate active hepatic uptake for a broad range of drugs and endogenous compounds and play a pivotal role in hepatic disposition and drug-drug interactions (DDIs) of substrate drugs. Efforts have been made on developing humanized transgenic OATP1B rodent models to mechanistically understand the role of OATP1B in drug disposition and DDIs. However, the lack of robust OATP1B functional activities limits their utility. We therefore developed humanized OATP1B1 and OATP1B3 and double-humanized OATP1B1/1B3 rat models in an Oatp1a1/1a4/1b2 knockout rat background. The knockout of rat Oatp1a1/1a4/1b2 and humanized transgenic overexpression of OATP1B1 and OATP1B3 in rat liver did not cause profound compensatory changes in gene expression of other transporters and metabolizing enzymes. The protein expression of OATP1B1 and OATP1B3 in humanized OATP1B rat liver was 4.7-fold and 22.3-fold higher than those reported in human liver tissues, respectively, and higher than those reported in humanized OATP1B mice. This has subsequently led to a robust in vitro and in vivo functional activity for a range of OATP1B substrate drugs. A translational analysis indicated a good correlation between in vitro hepatic uptake clearance measured in these models and human hepatocytes after correcting for OATP1B protein expression. Furthermore, these humanized OATP1B rats predicted the relative contribution of OATP1B in hepatic uptake of several OATP1B substrate drugs. Overall, these data suggest that our humanized OATP1B rat model is a promising tool to study OATP1B-mediated drug disposition in humans, advancing preclinical to clinical translation. SIGNIFICANCE STATEMENT: Humanized OATP1B1 and OATP1B3 and double-humanized OATP1B1/1B3 rat models were successfully generated and characterized in an Oatp1a1/1a4/1b2 knockout rat background. These novel humanized OATP1B models demonstrate greater functional activity than the existing humanized OATP1B rodent models, enabling evaluation of human OATP1B activity in vitro and in vivo in a preclinical setting. This study showed that these models are promising tools to improve the prediction of OATP1B-mediated hepatic uptake and DDIs in humans.

Hepatic uptake and efflux transporters govern the systemic and hepatic exposure of many drugs and metabolites. Enalapril is a pharmacologically inactive prodrug of enalaprilat. Following oral administration, enalapril is converted to enalaprilat in hepatocytes and undergoes translocation into the systemic circulation to exert its pharmacologic effect by inhibiting angiotensin-converting enzyme. Although the transport proteins governing hepatic uptake of enalapril and the biliary excretion of enalapril and enalaprilat are well established, it remains unknown how hepatically derived enalaprilat translocates across the basolateral membrane into the systemic circulation. In this study, the role of ATP-binding cassette transporters in the hepatic basolateral efflux of enalaprilat was investigated using membrane vesicles. ATP-dependent uptake of enalaprilat into vesicles expressing multidrug resistance-associated protein (MRP) 4 was significantly greater (∼3.8-fold) than in control vesicles. In contrast, enalaprilat was not transported to a significant extent by MRP3, and enalapril was not transported by either MRP3 or MRP4. The functional importance of MRP4 in the basolateral excretion of derived enalaprilat was evaluated using a novel basolateral efflux protocol developed in human sandwich-cultured hepatocytes. Under normal culture conditions, the mean intrinsic basolateral efflux clearance (CLint ,basolateral) of enalaprilat was 0.026 ± 0.012 µl/min; enalaprilat CLint,basolateral was significantly reduced to 0.009 ± 0.009 µl/min by pretreatment with the pan-MRP inhibitor MK-571. Results suggest that hepatically derived enalaprilat is excreted across the hepatic basolateral membrane by MRP4. Changes in MRP4-mediated basolateral efflux may alter the systemic concentrations of this active metabolite, and potentially the efficacy of enalapril.

Despite the existence of established, effective therapies for hypertension, new methods of blood pressure and cardiovascular risk reduction are still needed. Novel approaches are targeted towards treating resistant hypertension, improving blood-pressure control, and achieving further risk reduction beyond blood-pressure lowering. Modulation of the renin-angiotensin-aldosterone system (RAAS) provides the rationale for current antihypertensive therapies, including the relatively new agents eplerenone and aliskiren. Novel targets for antihypertensive therapy are also likely to be RAAS-related. The stimulation of angiotensin II type 2 receptors, or supplementation with renalase, could counteract the effects of angiotensin II type 1 receptor stimulation or catecholamine release. Combined angiotensin-converting-enzyme and neutral endopeptidase blockade decreases blood pressure, but is associated with a high incidence of angioedema. Aldosterone synthase inhibitors might improve tolerability in aldosterone antagonism. A (pro)renin-receptor blocker could prevent the deleterious angiotensin-independent actions of renin that are not inhibited by aliskiren. Finally, new minimally invasive surgical procedures have revived the concept of renal denervation, and could be a therapeutic option for patients with resistant hypertension. All of these strategies are exciting prospects, but which of them will prove valuable in clinical setting remains to be discovered.