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Drug-induced liver injury (DILI) is a common reason for drug withdrawal from the market. An important cause of DILI is drug-induced cholestasis. One of the major players involved in drug-induced cholestasis is the bile salt efflux pump (BSEP; ABCB11). Inhibition of BSEP by drugs potentially leads to cholestasis due to increased (toxic) intrahepatic concentrations of bile acids with subsequent cell injury. In order to investigate the possibilities for in silico prediction of cholestatic effects of drugs, we developed a mechanistic biokinetic model for human liver bile acid handling populated with human in vitro data. For this purpose we considered nine groups of bile acids in the human bile acid pool, i.e. chenodeoxycholic acid, deoxycholic acid, the remaining unconjugated bile acids and the glycine and taurine conjugates of each of the three groups. Michaelis-Menten kinetics of the human uptake transporter Na + -taurocholate cotransporting polypeptide (NTCP; SLC10A1) and BSEP were measured using NTCP-transduced HEK293 cells and membrane vesicles from BSEP-overexpressing HEK293 cells. For in vitro-in vivo scaling, transporter abundance was determined by LC-MS/MS in these HEK293 cells and vesicles as well as in human liver tissue. Other relevant human kinetic parameters were collected from literature, such as portal bile acid levels and composition, bile acid synthesis and amidation rate. Additional empirical scaling was applied by increasing the excretion rate with a factor 2.4 to reach near physiological steady-state intracellular bile acid concentrations (80μM) after exposure to portal vein bile acid levels. Simulations showed that intracellular bile acid concentrations increase 1.7 fold in the presence of the BSEP inhibitors and cholestatic drugs cyclosporin A or glibenclamide, at intrahepatic concentrations of 6.6 and 20μM, respectively. This simplified model provides a tool for a first indication whether drugs at therapeutic concentrations might cause cholestasis by inhibiting BSEP., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug-induced cholestasis (DIC) is one of the leading manifestations of drug-induced liver injury (DILI). As the underlying mechanisms for DIC are not fully known and specific and predictive biomarkers and pre-clinical models are lacking, the occurrence of DIC is often only reported when the drug has been approved for registration. Therefore, appropriate models that predict the cholestatic potential of drug candidates and/or provide insight into the mechanism of DIC are highly needed. We investigated the application of rat precision-cut liver slices (PCLS) to predict DIC, using several biomarkers of cholestasis: hepatocyte viability, intracellular accumulation of total as well as individual bile acids and changes in the expression of genes known to play a role in cholestasis. Rat PCLS exposed to the cholestatic drugs chlorpromazine, cyclosporine A and glibenclamide for 48 h in the presence of a 60 μM physiological bile acid (BA) mix reflected various changes associated with cholestasis, such as decrease in hepatocyte viability, accumulation and changes in the composition of BA and changes in the gene expression of Fxr, Bsep and Ntcp. The toxicity of the drugs was correlated with the accumulation of BA, and especially DCA and CDCA and their conjugates, but to a different extent for different drugs, indicating that BA toxicity is not the only cause for the toxicity of cholestatic drugs. Moreover, our study supports the use of several biomarkers to test drugs for DIC. In conclusion, our results indicate that PCLS may represent a physiological and valuable model to identify cholestatic drugs and provide insight into the mechanisms underlying DIC.

Digitalis-like compounds (DLCs) such as digoxin, digitoxin, and ouabain, also known as cardiac glycosides, are among the oldest pharmacological treatments for heart failure. The compounds have a narrow therapeutic window, while at the same time, DLC pharmacokinetics is prone to drug-drug interactions at the transport level. Hepatic transporters organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and Na(+)-dependent taurocholate co-transporting polypeptide (NTCP) influence the disposition of a variety of drugs by mediating their uptake from blood into hepatocytes. The interaction of digoxin, digitoxin, and ouabain with hepatic uptake transporters has been studied before. However, here, we systematically investigated a much wider range of structurally related DLCs for their capability to inhibit or to be transported by these transporters in order to better understand the relation between the activity and chemical structure of this compound type. We studied the uptake and inhibitory potency of a series of 14 structurally related DLCs in Chinese hamster ovary cells expressing NTCP (CHO-NTCP) and human embryonic kidney cells expressing OATP1B1 and OATP1B3 (HEK-OATP1B1 and HEK-OATP1B3). The inhibitory effect of the DLCs was measured against taurocholic acid (TCA) uptake in CHO-NTCP cells and against uptake of β-estradiol 17-β-d-glucuronide (E217βG) in HEK-OATP1B1 and HEK-OATP1B3 cells. Proscillaridin A was the most effective inhibitor of NTCP-mediated TCA transport (IC50 = 22 μM), whereas digitoxin and digitoxigenin were the most potent inhibitors of OATP1B1 and OAPTP1B3, with IC50 values of 14.2 and 36 μM, respectively. Additionally, we found that the sugar moiety and hydroxyl groups of the DLCs play different roles in their interaction with NTCP, OATP1B1, and OATP1B3. The sugar moiety decreases the inhibition of NTCP and OATP1B3 transport activity, whereas it enhances the inhibitory potency against OATP1B1. Moreover, the hydroxyl group at position 12 reinforces the inhibition of NTCP but decreases the inhibition of OATP1B1 and OATP1B3. To investigate whether DLCs can be translocated, we quantified their uptake in transporter-expressing cells by LC-MS. We demonstrated that convallatoxin, ouabain, dihydroouabain, and ouabagenin are substrates of OATP1B3. No transport was observed for the other compounds in any of the studied transporters. In summary, this work provides a step toward an improved understanding of the interaction of DLCs with three major hepatic uptake transporters. Ultimately, this can be of use in the development of DLCs that are less prone to transporter-mediated drug-drug interactions.

Purpose: We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate., Methods: AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with (99m)Tc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of (99m)Tc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration., Results: HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with (99m)Tc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected (99m)Tc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater (99m)Tc-HYNIC-cys-AnxA5 accumulation., Conclusion: (99m)Tc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.

Purpose: Delivery of apoptosis-inducing compounds to hepatic stellate cells (HSC) may be an effective strategy to reverse liver fibrosis. The aim of this study was therefore to examine the selective targeting of the apoptosis-inducing drug 15-deoxy-delta12,14-prostaglandin J2 (15dPGJ2) with two different HSC-carriers: human serum albumin modified with the sugar mannose-6-phosphate (M6PHSA) or albumin modified with PDGF-receptor recognizing peptides (pPBHSA)., Methods and Results: After chemical conjugation of 15dPGJ2 to the carriers, the constructs displayed pharmacological activity and specific receptor-mediated binding to HSC in vitro. Unlike 15dPGJ2-pPBHSA, the cellular binding of 15dPGJ2-M6PHSA was reduced by a scavenger receptor antagonist. In vivo, both conjugates rapidly accumulated in fibrotic livers. Intrahepatic analysis revealed that 15dPGJ2-M6PHSA mainly accumulated in HSC, and to a lesser extent in Kupffer cells. 15dPGJ2-pPBHSA also predominantly accumulated in HSC with additional uptake in hepatocytes. Assessment of target receptors in human cirrhotic livers revealed that M6P/IGFII-receptor expression was present in fibrotic areas. PDGF-P receptor expression was abundantly expressed on human fibroblasts., Conclusions: These studies show that 15dPGJ2 coupled to either M6PHSA or pPBHSA is specifically taken up by HSC and is highly effective within these cells. Both carriers differ with respect to receptor specificity, leading to differences in intrahepatic distribution. Nevertheless, both carriers can be used to deliver the apoptosis-inducing drug 15dPGJ2 to HSC in vivo.

Hepatic stellate cell (HSC) proliferation is a key event in liver fibrosis; therefore, pharmacological intervention with antiproliferative drugs may result in antifibrotic effects. In this article, the antiproliferative effect of three cytostatic drugs was tested in cultured rat HSC. Subsequently, the antifibrotic potential of the most potent drug was evaluated in vivo. As a strategy to overcome drug-related toxicity, we additionally studied how to deliver this drug specifically to HSC by conjugating it to the HSC-selective drug carrier mannose-6-phosphate-modified human serum albumin (M6PHSA). We investigated the effect of cisplatin, chlorambucil, and doxorubicin (DOX) on 5-bromo-2'-deoxyuridine incorporation in cultured HSC and found DOX to be the most potent drug. Treatment of bile duct-ligated (BDL) rats with daily i.v. injections of 0.35 mg/kg DOX from day 3 to 10 after BDL reduced alpha-smooth muscle actin-stained area in liver sections from 8.5 +/- 0.8 to 5.1 +/- 0.9% (P < 0.01) and collagen-stained area from 13.1 +/- 1.3 to 8.9 +/- 1.5% (P < 0.05). DOX was coupled to M6PHSA, and the organ distribution of this construct (M6PHSA-DOX) was investigated. Twenty minutes after i.v. administration, 50 +/- 6% of the dose was present in the livers, and colocalization of M6PHSA-DOX with HSC markers was observed. In addition, in vitro studies showed selective binding of M6PHSA-DOX to activated HSC. Moreover, M6PHSA-DOX strongly attenuated HSC proliferation in vitro, indicating that active drug is released after uptake of the conjugate. DOX inhibits liver fibrosis in BDL rats, and HSC-selective targeting of this drug is possible. This may offer perspectives for the application of antiproliferative drugs for antifibrotic purposes.

Targeting of antifibrotic drugs to hepatic stellate cells (HSC) is a promising strategy to block fibrotic processes leading to liver cirrhosis. For this purpose, we utilized the neo-glycoprotein mannose-6-phosphate-albumin (M6PHSA) that accumulates efficiently in HSC during liver fibrosis. Pentoxifylline (PTX), an antifibrotic compound that inhibits HSC proliferation and activation in vitro, was conjugated to M6PHSA. We employed a new type of platinum-based linker, which conjugates PTX via coordination chemistry rather than via covalent linkage. When incubated in plasma or in the presence of thiol compounds, free PTX was released from PTX-M6PHSA at a sustained slow rate. PTX-M6PHSA displayed pharmacological activity in cultured HSC as evidenced by changes in cell morphology and reduction of collagen I production. PTX-M6PHSA and platinum coupled PTX did not induce platinum-related toxicity (Alamar Blue viability assay) or apoptosis (caspase activation and TUNEL staining). In vivo distribution studies in fibrotic rats demonstrated specific accumulation of the conjugate in nonparenchymal cells in the fibrotic liver. In conclusion, we have developed PTX-M6PHSA employing a novel type of platinum linker, which allows sustained delivery of the drug to HSC in the fibrotic liver.

Background/aims: Hepatic stellate cell (HSC) activation and proliferation are key events in the pathology of liver fibrosis. Inhibiting these parameters, therefore, is a relevant option to treat liver fibrosis pharmacologically. The immunosuppressive drug mycophenolic acid (MPA) has been shown to inhibit proliferation and activation of various types of fibroblasts. In an effort to circumvent the immunosuppression and at the same time enhance this antifibrotic effect, we coupled MPA to the HSC-selective drug carrier mannose-6-phosphate modified human serum albumin and evaluated this conjugate for its specificity and antifibrotic activity., Methods/results: We found that MPA inhibited proliferation of HSC in vitro. The drug coupled to the drug carrier bound specifically to HSC and reduced HSC proliferation in vitro. In vivo studies demonstrated that our conjugate accumulated selectively in the liver with significant uptake in HSC apart from Kupffer and endothelial cells, whereas primary and secondary lymphoid tissues were avoided. Treatment of bile duct-ligated rats with this conjugate reduced hepatic inflammation and hepatic alpha-beta-Crystallin mRNA expression, a marker for HSC activation., Conclusions: This study shows that targeted delivery of MPA to HSC results in a decrease in HSC activation, making it the first drug that is successfully delivered to this cell type.