Drug-mediated modulation of hepatic bile salt transporters

Transporters are key players in the disposition of both endogenous and exogenous compounds, thereby impacting safety and efficacy of numerous medicines. This becomes clear when drugs interfere with transporter function leading to drug-drug or drug-endogenous compound interactions. Evaluation of a novel pharmaceutical agent for inhibition of certain transport proteins is included in several regulatory agency guidelines before this molecule is allowed on the market. Beyond the direct pharmacokinetic and toxicological relevance, several transporters have been demonstrated to be true pharmacological targets. Sodium taurocholate co-transporting polypeptide (NTCP) is the primary transporter responsible for the uptake of bile salts from the blood. The discovery of NTCP as the receptor of the hepatitis B virus (HBV) has fostered the search for HBV entry blockers. It became clear that functional inhibition of NTCP could also prevent viral entry. As currently existing therapy is not able to eradicate the virus, HBV entry inhibitors could complement the treatment options. At the canalicular side of the hepatocyte, bile salt export pump (BSEP) almost exclusively translocates bile salts to the bile. Inhibition of BSEP has been shown to result in drug-induced cholestasis (DIC) and should therefore be avoided. Consequently, the substrate and inhibitor overlap between NTCP and BSEP poses a critical challenge during the development of NTCP inhibitors as novel antiviral therapeutics. BSEP inhibition is a well-established mechanism underlying DIC. Nevertheless, bile salt homeostasis is a complex process not only involving transporters, but also enzymes and nuclear receptors. In addition, a potential BSEP inhibitor can only exert its activity if sufficiently high unbound concentrations can be reached at the binding site of BSEP. Sandwich-cultured human hepatocytes (SCHH) are considered the gold standard tool for detecting DIC in vitro. They preserve transporter and enzyme expression and require the compound of interest to reach the intracellular compartment. Bosentan, a known cholestatic agent in humans, did not cause cholestasis in preclinical animal studies. It is currently still marketed but received an FDA black box warning and requires monitoring of liver function during treatment. Although in vitro BSEP inhibition was previously demonstrated, bosentan did not provoke cholestasis in our SCHH based DIC assay. Other mechanisms than BSEP inhibition must therefore be involved in bosentan-mediated bile salt alterations as well as in cholestatic patients. The overarching goal of this research was to generate novel pharmacological and toxicological insights regarding the hepatic bile salt transporters NTCP and BSEP, respectively. The specific objectives were (1) to identify novel NTCP inhibitors that may be used for the treatment of chronic HBV infection. (2) To develop a sensitive BSEP inhibition assay using glycocholic acid (GCA) as a substrate that could be used to evaluate the BSEP inhibitory potential of the NTCP inhibitors. (3) To quantify the effect of therapeutically relevant bosentan concentrations on bile salt disposition in SCHH. In the first part of this work (Chapter 3), we evaluated over 2500 compounds for their inhibitory potential towards NTCP in vitro. We used an easy and reproducible assay based on NTCP-transfected Chinese hamster ovary cells and tauro-nor-THCA-24-DBD as fluorescent substrate. Thirty compounds showed concentration-dependent inhibition of NTCP. The inhibition data was used to develop overlay and virtual screening models. The overlay model consists of a hydrophobic core surrounded by hydrophilic groups and additional hydrophobic groups that interact with NTCP. This is consistent with previously published models for NTCP substrates and inhibitors. Importantly, our overlay model lacked an anion group that is present in NTCP substrates, indicating that absence of this group might be a desirable feature of NTCP inhibitors. We subsequently applied our models to the ChEMBL database. Additional NTCP inhibitors were identified in silico of which the activity was confirmed in vitro. Our models were thereby validated and could be of use in industry. To determine the BSEP inhibitory potential of our NTCP inhibitors, we developed a sensitive and reproducible in vitro assay (Chapter 4). We used inside-out membrane vesicles obtained from BSEP-transfected HEK293 cells and non-radioactive GCA as a substrate. This unique combination enables quantification of GCA at very low concentrations using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis without the disadvantages of using a radioactive substrate. While some of the NTCP inhibitors showed to be potent BSEP inhibitors, others lacked BSEP inhibition. This indicates that distinguishing between NTCP and BSEP inhibition is feasible. We also used the assay to assess bosentan and its major metabolites. We discovered that desmethyl bosentan was a more potent BSEP inhibitor than bosentan, thereby likely contributing to the cholestatic effect observed for bosentan. The IC50 values were 56.5 microM and 162.0 microM, respectively. Hydroxy bosentan and hydroxy desmethyl bosentan inhibited BSEP with IC50 values of 461.5 microM and 512.4 microM, respectively. Hence, their contribution to bosentan-induced toxicity is expected to be limited. Despite BSEP inhibition being the key event in DIC, other factors may be involved in the DIC potential of a certain agent. This is exemplified by bosentan. Previously performed research by our group demonstrated that bosentan did not cause in vitro cholestasis at supratherapeutic concentrations up to 200 microM. In Chapter 5, we further investigated the effect of bosentan at therapeutically relevant concentrations on bile salt handling in SCHH. Bosentan reduced both the endogenous intracellular and extracellular glycochenodeoxycholic acid (GCDCA) and GCA amounts in SCHH. We therefore hypothesized that bosentan inhibits both de novo synthesis of bile salts and the conjugation of unconjugated bile salts. In a second phase of this work, SCHH were exposed to exogenously added chenodeoxycholic acid (CDCA) in the presence and absence of bosentan. We observed a reduced intracellular and canalicular GCDCA accumulation with a shift towards sinusoidal efflux of GCDCA while the amounts of CDCA remained unaltered. We developed a mechanistic model to quantify the effects of bosentan on CDCA and GCDCA disposition. Our model confirmed the inhibitory effect of bosentan on canalicular GCDCA clearance. This research provided understanding of the bile salt alterations induced by bosentan at clinically relevant concentrations. Reduced GCDCA formation and a shift towards sinusoidal efflux of GCDCA might explain these observations. In conclusion, this doctoral research advanced knowledge concerning the effect of small molecules on the hepatic transporters NTCP and BSEP. Based on a combination of in vitro and in silico approaches novel NTCP inhibitors were identified that could lead to a new treatment option for chronic HBV infection. Our chemometric model could be of interest for industry to identify and verify additional NTCP inhibitors. A sensitive and reproducible BSEP inhibition assay was established. Several NTCP inhibitors concomitantly inhibited BSEP, increasing the risk for inducing DIC. Based on the substrate overlap between NTCP and BSEP, we propose to verify absence of BSEP inhibition among NTCP inhibitors. These data could be used to develop a QSAR model which allows early and low-cost identification of BSEP inhibitors. We evaluated bosentan and its three major metabolites in humans in our BSEP inhibitions assay. Desmethyl bosentan was discovered to be a more potent BSEP inhibitor than bosentan. Exposing SCHH to therapeutically relevant bosentan concentrations provoked alterations of both endo- and exogenous bile salts. Mechanistic modeling revealed inhibition of the biliary clearance of GCDCA as predominant mechanism of the reduced GCDCA amounts in the canaliculi. Moreover, we also observed reduced intracellular GCDCA which might be the result of direct inhibition of CDCA conjugation with glycine and might contribute to this effect. Further investigation unravelling potential other mechanisms involved in bosentan-induced cholestasis are pursued to lay the foundation for a novel biomarker for DIC.

Jaar van publicatie:2021

Toegankelijkheid:Open