Identification of reversible OATP1B1 and time-dependent CYP3A4 inhibition as the major risk factors for drug-induced cholestasis (DIC).

Hepatic bile acid regulation is a multifaceted process modulated by several hepatic transporters and enzymes. Drug-induced cholestasis (DIC), a main type of drug-induced liver injury (DILI), denotes any drug-mediated condition in which hepatic bile flow is impaired. Our ability in translating preclinical toxicological findings to human DIC risk is currently very limited, mainly due to important interspecies differences. Accordingly, the anticipation of clinical DIC with available in vitro or in silico models is also challenging, due to the complexity of the bile acid homeostasis. Herein, we assessed the in vitro inhibition potential of 47 marketed drugs with various degrees of reported DILI severity towards all metabolic and transport mechanisms currently known to be involved in the hepatic regulation of bile acids. The reported DILI concern and/or cholestatic annotation correlated with the number of investigated processes being inhibited. Furthermore, we employed univariate and multivariate statistical methods to determine the important processes for DILI discrimination. We identified time-dependent inhibition (TDI) of cytochrome P450 (CYP) 3A4 and reversible inhibition of the organic anion transporting polypeptide (OATP) 1B1 as the major risk factors for DIC among the tested mechanisms related to bile acid transport and metabolism. These results were consistent across multiple statistical methods and DILI classification systems applied in our dataset. We anticipate that our assessment of the two most important processes in the development of cholestasis will enable a risk assessment for DIC to be efficiently integrated into the preclinical development process.

Applicability of MDR1 Overexpressing Abcb1KO-MDCKII Cell Lines for Investigating In Vitro Species Differences and Brain Penetration Prediction.

Implementing the 3R initiative to reduce animal experiments in brain penetration prediction for CNS-targeting drugs requires more predictive in vitro and in silico models. However, animal studies are still indispensable to obtaining brain concentration and determining the prediction performance of in vitro models. To reveal species differences and provide reliable data for IVIVE, in vitro models are required. Systems overexpressing MDR1 and BCRP are widely used to predict BBB penetration, highlighting the impact of the in vitro system on predictive performance. In this study, endogenous Abcb1 knock-out MDCKII cells overexpressing MDR1 of human, mouse, rat or cynomolgus monkey origin were used. Good correlations between ERs of 83 drugs determined in each cell line suggest limited species specificities. All cell lines differentiated CNS-penetrating compounds based on ERs with high efficiency and sensitivity. The correlation between in vivo and predicted Kp,uu,brain was the highest using total ER of human MDR1 and BCRP and optimized scaling factors. MDR1 interactors were tested on all MDR1 orthologs using digoxin and quinidine as substrates. We found several examples of inhibition dependent on either substrate or transporter abundance. In summary, this assay system has the potential for early-stage brain penetration screening. IC50 comparison between orthologs is complex; correlation with transporter abundance data is not necessarily proportional and requires the understanding of modes of transporter inhibition.

Interactions of the Anti-SARS-CoV-2 Agents Molnupiravir and Nirmatrelvir/Paxlovid with Human Drug Transporters.

Orally administered small molecules may have important therapeutic potential in treating COVID-19 disease. The recently developed antiviral agents, Molnupiravir and Nirmatrelvir, have been reported to be efficient treatments, with only moderate side effects, especially when applied in the early phases of this disease. However, drug-drug and drug-transporter interactions have already been noted by the drug development companies and in the application notes. In the present work, we have studied some of the key human transporters interacting with these agents. The nucleoside analog Molnupiravir (EIDD-2801) and its main metabolite (EIDD-1931) were found to inhibit CNT1,2 in addition to the ENT1,2 nucleoside transporters; however, it did not significantly influence the relevant OATP transporters or the ABCC4 nucleoside efflux transporter. The active component of Paxlovid (PF-07321332, Nirmatrelvir) inhibited the function of several OATPs and of ABCB1 but did not affect ABCG2. However, significant inhibition was observed only at high concentrations of Nirmatrelvir and probably did not occur in vivo. Paxlovid, as used in the clinic, is a combination of Nirmatrelvir (viral protease inhibitor) and Ritonavir (a "booster" inhibitor of Nirmatrelvir metabolism). Ritonavir is known to inhibit several drug transporters; therefore, we have examined these compounds together, in relevant concentrations and ratios. No additional inhibitory effect of Nirmatrelvir was observed compared to the strong transporter inhibition caused by Ritonavir. Our current in vitro results should help to estimate the potential drug-drug interactions of these newly developed agents during COVID-19 treatment.

The Inhibitor Preincubation Effect Is Universal to SLC Transporter Assays and Is Only Partially Eliminated in the Presence of Extracellular Protein.

Variation in the methodology of in vitro transporter inhibition assays causes wide divergence in reported IC50/Ki data. Notably, although potentiation of transporter inhibition by preincubation (PTIP) has been described, current guidelines do not specifically recommend inhibitor preincubation; they only encourage sponsors to follow emerging literature. To clarify how generally preincubation should be considered in transporter inhibition studies and whether PTIP can be solely explained by protein binding of the respective inhibitors, we performed in vitro inhibition assays on solute carrier (SLC) and ATP-binding cassette transporters scarcely or not covered in prior research and examined the effect of extracellular protein in preincubation and washout experiments. In SLC assays without extracellular protein, a 30-minute preincubation caused significant > twofold change of IC50 in 21/33 transporter-inhibitor combinations involving 19 evolutionarily disparate transporters. The preincubation effect correlated with inhibitor properties like protein binding and aqueous solubility. In vesicular transport assays of multidrug resistance protein 1, breast cancer resistance protein, multidrug resistance-associated protein 2, and bile salt export pump, sizable PTIP was observed for only 2/23 combinations, and preincubation was practically inconsequential in breast cancer resistance protein or multidrug resistance protein 1 monolayer assays. In SLC assays, PTIP partly persisted in the presence of 5% albumin, indicating that the absence of extracellular protein does not fully explain PTIP. The presence of protein, however, complicated the interpretation of results. Overall, while preincubating without protein may overpredict inhibitory potency, adding protein compromises clarity, and omitting preincubation altogether may miss clinically relevant inhibitors. Therefore, we propose that protein-free preincubation should be considered in all SLC inhibition assays. ATP-binding cassette transporter inhibition seems less commonly affected by preincubation, but conclusions require further investigation. SIGNIFICANCE STATEMENT: Drugs may inhibit transporter proteins in the body, which may precipitate drug interactions. In vitro transporter inhibition assays help predict such drug interactions. Some inhibitors act more potently when preincubated with the transporter prior to the assay. Here we argue that this effect is not a mere in vitro artifact due to the lack of plasma proteins and should be considered in all uptake inhibition assays to model the worst-case scenario. Preincubation in efflux transporter inhibition assays is likely dispensable.

A Unique In Vitro Assay to Investigate ABCB4 Transport Function.

ABCB4 is almost exclusively expressed in the liver, where it plays an essential role in bile formation by transporting phospholipids into the bile. ABCB4 polymorphisms and deficiencies in humans are associated with a wide spectrum of hepatobiliary disorders, attesting to its crucial physiological function. Inhibition of ABCB4 by drugs may lead to cholestasis and drug-induced liver injury (DILI), although compared with other drug transporters, there are only a few identified substrates and inhibitors of ABCB4. Since ABCB4 shares up to 76% identity and 86% similarity in the amino acid sequence with ABCB1, also known to have common drug substrates and inhibitors, we aimed to develop an ABCB4 expressing Abcb1-knockout MDCKII cell line for transcellular transport assays. This in vitro system allows the screening of ABCB4-specific drug substrates and inhibitors independently of ABCB1 activity. Abcb1KO-MDCKII-ABCB4 cells constitute a reproducible, conclusive, and easy to use assay to study drug interactions with digoxin as a substrate. Screening a set of drugs with different DILI outcomes proved that this assay is applicable to test ABCB4 inhibitory potency. Our results are consistent with prior findings concerning hepatotoxicity causality and provide new insights for identifying drugs as potential ABCB4 inhibitors and substrates.

Translatability of in vitro Inhibition Potency to in vivo P-Glycoprotein Mediated Drug Interaction Risk.

P-glycoprotein (P-gp) may limit oral drug absorption of substrate drugs due to intestinal efflux. Therefore, regulatory agencies require investigation of new chemical entities as possible inhibitors of P-gp in vitro. Unfortunately, inter-laboratory and inter-assay variability have hindered the translatability of in vitro P-gp inhibition data to predict clinical drug interaction risk. The current study was designed to evaluate the impact of potential IC50 discrepancies between two commonly utilized assays, i.e., bi-directional Madin-Darby Canine Kidney-MDR1 cell-based and MDR1 membrane vesicle-based assays. When comparing vesicle- to cell-based IC50 values (n = 28 inhibitors), non-P-gp substrates presented good correlation between assay formats, whereas IC50s of P-gp substrates were similar or lower in the vesicle assays. The IC50s obtained with a cell line expressing relatively low P-gp aligned more closely to those obtained from the vesicle assay, but passive permeability of the inhibitors did not appear to influence the correlation of IC50s, suggesting that efflux activity reduces intracellular inhibitor concentrations. IC50s obtained between two independent laboratories using the same assay type showed good correlation. Using the G-value (i.e., ratio of estimated gut concentration-to-inhibition potency) >10 cutoff recommended by regulatory agencies resulted in minimal differences in predictive performance, suggesting this cutoff is appropriate for either assay format.

Intracellular Metabolomics Identifies Efflux Transporter Inhibitors in a Routine Caco-2 Cell Permeability Assay-Biological Implications.

Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.

Characterization of new, efficient Mycobacterium tuberculosis topoisomerase-I inhibitors and their interaction with human ABC multidrug transporters.

Drug resistant tuberculosis (TB) is a major worldwide health problem. In addition to the bacterial mechanisms, human drug transporters limiting the cellular accumulation and the pharmacological disposition of drugs also influence the efficacy of treatment. Mycobacterium tuberculosis topoisomerase-I (MtTopo-I) is a promising target for antimicrobial treatment. In our previous work we have identified several hit compounds targeting the MtTopo-I by in silico docking. Here we expand the scope of the compounds around three scaffolds associated with potent MtTopo-I inhibition. In addition to measuring the effect of newly generated compounds on MtTopo-I activity, we characterized the compounds' antimicrobial activity, toxicity in human cells, and interactions with human multidrug transporters. Some of the newly developed MtTopo-I inhibitors have strong antimicrobial activity and do not harm mammalian cells. Moreover, our studies revealed significant human ABC drug transporter interactions for several MtTopo-I compounds that may modify their ADME-Tox parameters and cellular effects. Promising new drug candidates may be selected based on these studies for further anti-TB drug development.

Baicalin is a substrate of OATP2B1 and OATP1B3.

The use and significance of baicalin, the main bioactive component found in Radix Scutellaria, have been on the rise due to its interesting pharmacological properties. Baicalin, a low passive permeability compound, is directly absorbed from the upper intestine and its hepatic elimination is dominant. However, interaction but no transport studies have implicated organic anion­transporting polypeptides in its cellular uptake. By using mammalian cells stably expressing the uptake transporters of interest, we are showing that baicalin is a potent substrate of Organic anion­transporting polypeptide 2B1 (OATP2B1) and less potent substrate of OATP1B3. OATP2B1 and OATP1B3 transport baicalin and may play a role in the hepatic uptake of baicalin formed in the intestine.

[Role of drug transporters in the central nervous system]. / Gyógyszertranszporterek szerepe a központi idegrendszerben.

Although the presence of blood-brain barrier in the mammalian organisms was discovered in the early 1900s, its precise structure and the drug transporter proteins localized in the blood-brain barrier were identified only in the last decades. Beside the ATP-binding cassette transporter proteins responsible for the protection of the brain, the Solute Carrier transporters play also an important role in the function of the central nervous system by its feeding, energy supply and cleaning function during the metabolism. This review provides an overview on the main types of transporters located in the brain, on their localization in different cell types and the main techniques for their investigation. In the second part of this article various neurodegenerative disorders and the pathology-related transporter proteins are presented. In the light of recent experimental results new therapeutic strategies may come into the focus of research for the treatment of disorders currently without effective therapy.