A Two-Tiered In Vitro Approach to De-Risk Drug Candidates for Potential Bile Salt Export Pump Inhibition Liabilities in Drug Discovery

Abstract

Hepatocellular accumulation of bile salts by inhibition of bile salt export pump (BSEP/ABCB11) may result in cholestasis and is one proposed mechanism of drug induced liver injury (DILI). To understand the relationship between BSEP inhibition and DILI, we evaluated 64 DILI positive and 57 DILI negative compounds in BSEP, multidrug resistance protein 2 (MRP2), MRP3, and MRP4 vesicular inhibition assays. An empirical cutoff (5 μM) for BSEP inhibition was established based on a relationship between BSEP IC 50 values and the calculated maximal unbound concentration at the inlet of the human liver (fu*I in,max , assay specificity = 98%). Including inhibition of MRP2-4 did not increase DILI predictivity. To further understand the potential to inhibit bile salt transport, a selected subset of 30 compounds were tested for inhibition of taurocholate (TCA) transport in a long-term human hepatocyte micropatterned coculture (MPCC) system. The resulting IC 50 for TCA in vitro biliary clearance (Cl biliary) and biliary excretion index (BEI) in MPCCs were compared to the compound's fu*I in,max to assess potential risk for bile salt transport perturbation. The data show high specificity (89%). Nine out of 15 compounds showed an IC 50 value in the BSEP vesicular assay of <5μM, but the BEI IC 50 was greater than 10-fold the fu*I in,max , suggesting that inhibition of BSEP in vivo is unlikely. The data indicate that although BSEP inhibition measured in membrane vesicles correlates with DILI risk, that measurement of this assay activity is insufficient. A two-tiered strategy incorporating MPCCs is presented to reduce BSEP inhibition potential and improve DILI risk. Statement of Significance This work describes a two-tiered in vitro approach to de-risk compounds for potential bile salt export pump inhibition liabilities in drug discovery utilizing membrane vesicles and a long-term human hepatocyte micropatterned co-culture system. Cutoffs to maximize specificity were established based on in vitro data from a set of 121 DILI positive and negative compounds and associated calculated maximal unbound concentration at the inlet of the human liver (fu*I in,max) based on the highest clinical dose.

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

“…Major publications from both the industry and the International Transporter Consortium have recommended the testing of BSEP inhibition. The relevant papers with suggested cut-off values [ 143 , 144 , 145 , 146 , 147 , 148 , 149 ] are summarized in Table 1 . Most studies employ a vesicular transport assay with taurocholate as a probe, albeit some cholestatic compounds have been shown to act via repression of BSEP expression, which can only be investigated in cellular models [ 150 ].…”

“…For compounds in the discovery phase where the C plasma values are still only predicted and may thus be unreliable, risk assessment can be based solely on IC 50 , and different groups have suggested cut-off values of 25 [ 145 ] and 50 µM [ 147 ]. Aleo et al have applied a complex scoring system, with the highest score (most severe category) assigned to compounds with IC 50 /C max,total ≤ 1 and the safe zone for BSEP inhibition starting at IC 50 /C max,total > 100 [ 148 ]. In other studies, the utility of in vitro BSEP inhibition for predicting DILI has been confirmed without recommending a specific cut-off [ 96 , 108 ].…”

“…The genetic impairment of MDR3 function leads to PFIC-3, and the inhibition of MDR3 by antifungals has been linked to cholestasis [ 155 , 156 ]. The possible cholestatic effect of MDR3 inhibition was addressed in a large study that assayed 125 drugs on hepatocytes using d9-phosphatidyl-choline as a probe [ 148 ]. “Most-DILI concern compounds” inhibited MDR3 and BSEP with IC 50 < 20 µM.…”

“…“Most-DILI concern compounds” inhibited MDR3 and BSEP with IC 50 < 20 µM. However, whether the substrate transport was specific to MDR3 remained a concern [ 145 ], and testing the inhibition of transporters other than BSEP was found to have minimal beneficial impacts on the prediction of DILI [ 148 ].…”

“…Other than bile salt transport inhibition, new testing paradigms encompass the mitochondrial toxicity, generation of ROS, cytotoxicity that may be related to the accumulation of bile salts, and formation of reactive metabolites. High doses (≥100 mg), lipophilicity (cLogP ≥ 3), and high molecular weights (>600) are further attributes that have been linked to hepatotoxic potential [ 17 , 147 , 148 ].…”