In Vitro Drug-Induced Liver Injury Prediction: Criteria Optimization of Efflux Transporter IC50 and Physicochemical Properties
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Drug-induced liver injury (DILI) is a severe drug adverse response, which cannot always be reliably predicted in preclinical or clinical studies. Lack of observation of DILI during preclinical and clinical drug development has led to DILI being a leading cause of drug withdrawal from the market. As DILI is potentially fatal, pharmaceutical companies have been developing in vitro tools to screen for potential liver injury. Screens for physicochemical properties, mitochondrial function, and transport protein inhibition have all been employed to varying degrees of success. In vitro inhibition of the bile salt export pump (BSEP) has become a major risk factor for in vivo DILI predictions, yet discrepancies exist in which methods to use and the extent to which BSEP inhibition predicts clinical DILI. The presented work focuses on optimizing DILI predictions by comparing BSEP inhibition via the membrane vesicle assay and the hepatocyte-based BSEPcyte assay, as well as dual and triple liabilities. BSEP transport inhibition of taurcholic acids and glycocholic acids were similar for up to 29 drugs tested, in both the vesicle and hepatocyte-based assays. Positive and negative DILI predictions were optimized at a 50-µM cutoff value for 50 drugs using both NIH Livertox and PharmaPendium databases. Additionally, dual inhibition of BSEP and other efflux transporters (multidrug resistance-associated protein [MRP]2, MRP3, or MRP4) provided no observable predictive benefit compared with BSEP inhibition alone. Eighty-five percent of drugs with high molecular weight (>600 Da), high cLogP (>3), or a daily dose >100 mg and BSEP inhibition were associated with DILI. Triple liability of BSEP inhibition, high molecular weight, and high cLogP attained a 100% positive prediction rate.Keywords:
Efflux
Efflux
P-glycoprotein
Multidrug Resistance-Associated Proteins
Bone canaliculus
Organic anion-transporting polypeptide
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Abstract Purpose: To compare side-by-side the uptake of sorafenib and sunitinib in vitro by human uptake solute carriers of the SLC22A and SLCO families, the transport by and inhibition of efflux ATP-binding cassette (ABC) transporters, and the role of ABCB1 in the plasma pharmacokinetics and brain penetration of these agents. Experimental Design: Uptake of [3H]sorafenib or [3H]sunitinib was assessed in Xenopus laevis oocytes or mammalian cells transfected with cDNAs coding for human OATP1A2, OATP1B1, OATP1B3, OCT1, OAT2, OAT3, OCTN1, or OCTN2. Efflux and inhibition experiments were conducted in cells transfected with human ABCB1, ABCG2, ABCC2, or ABCC4. In vivo pharmacokinetic studies were done in knockout mice lacking Abcb1-type transporters. Results: Intracellular uptake was not appreciably affected by any of the studied solute carriers and was minute relative to the respective prototypical substrates. Sorafenib and sunitinib showed concentration-dependent (1 and 10 μmol/L), low to moderate affinity for ABCB1 but were not affected by the other ABC transporters. Both agents inhibited all tested ABC transporters. The absence of Abcb1 had no affect on plasma pharmacokinetics, but brain penetration was moderately increased by 1.9- and 2.9-fold for sorafenib and sunitinib, respectively, in knockout animals versus controls. Conclusions: Unlike other tyrosine kinase inhibitors, sorafenib and sunitinib do not appear to rely on active transport to enter the cell nor are they high-affinity substrates for ABC efflux transporters. Based on these characteristics, these two drugs may be less susceptible to transporter-mediated alterations in systemic exposure and transporter-related resistance mechanisms. (Clin Cancer Res 2009;15(19):6062–9)
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Transporter proteins mediate the cellular uptake and efflux of a broad variety of endogenous compounds, drugs, and their metabolites. Their systemic plasma concentrations are determined, in particular, by drug transporters expressed in the small intestine, liver, and kidney. In addition, drug transporters expressed in peripheral tissues (e.g., skeletal muscle) are likely to influence organ-specific drug concentrations and side effects. This review summarizes current findings regarding the association between adverse drug reactions in humans and modification of the functions of certain transporters caused by genetic factors or simultaneously administered inhibitors. We focus on adverse drug reactions occurring in humans due to transport in the small intestine, liver, kidneys, and blood–brain barrier. Clinical Pharmacology & Therapeutics (2011) 89 6, 798–805. doi:10.1038/clpt.2010.354
Efflux
Clinical Pharmacology
Drug reaction
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The important role of drug transporters in drug absorption and disposition has been well documented. Statins are subjected to active transport of membrane proteins of the superfamilies ATP-binding cassette and solute carrier, and there is limited understanding of the mechanisms by which differences in transporter expression and activity contributes to variability of pharmacokinetics (PKs)/pharmacodynamics (PDs) of statins.This review aims to discuss the roles of drug transporters in the PKs and PDs of statins, and in drug interactions with statins.A comprehensive summary of the literature on this subject including in vitro and in vivo observations.In vivo and in vitro studies have shown that efflux and uptake transporters modulate the PKs/PDs of statins. Until now organic anion transporting polypeptides (OATP)1B1 variants have been considered major factors in limiting the uptake of statins and increasing statin exposure, and, consequently, increasing risk of myopathy. Further studies in pharmacogenetics and in vitro models to assess statin disposition and toxicity are required to understand the contribution of others transporters, such as multidrug resistance-associated protein (MRP)1, MRP2, breast cancer resistance protein, OATP2B1, OAT1B3 and OATP1A2, in interindividual variability to statins efficacy and safety.
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Multidrug Resistance-Associated Proteins
Abcg2
Solute carrier family
Organic anion-transporting polypeptide
P-glycoprotein
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Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies.
Efflux
P-glycoprotein
Paracellular transport
Bone canaliculus
Organic anion-transporting polypeptide
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Bile acids and bile salts (BA/BS) are substrates of both influx and efflux transporters on hepatocytes. Canalicular efflux transporters, such as BSEP and MRP2, are crucial for the removal of BA/BS to the bile. Basolateral influx transporters, such as NTCP, OATP1B1/1B3, and OSTα/β, cooperate with canalicular transporters in the transcellular vectorial flux of BA/BS from the sinusoids to the bile. The blockage of canalicular transporters not only impairs the bile flow but also causes the intracellular accumulation of BA/BS in hepatocytes that contributes to, or even triggers, liver injury. In the case of BA/BS overload, the efflux of these toxic substances back to the blood via MRP3, MRP4, and OST α/β is considered a relief function. FXR, a key regulator of defense against BA/BS toxicity suppresses de novo bile acid synthesis and bile acid uptake, and promotes bile acid removal via increased efflux. In drug development, the early testing of the inhibition of these transporters, BSEP in particular, is important to flag compounds that could potentially inflict drug-induced liver injury (DILI). In vitro test systems for efflux transporters employ membrane vesicles, whereas those for influx transporters employ whole cells. Additional in vitro pharmaceutical testing panels usually include cellular toxicity tests using hepatocytes, as well as assessments of the mitochondrial toxicity and accumulation of reactive oxygen species (ROS). Primary hepatocytes are the cells of choice for toxicity testing, with HepaRG cells emerging as an alternative. Inhibition of the FXR function is also included in some testing panels. The molecular weight and hydrophobicity of the drug, as well as the steady-state total plasma levels, may positively correlate with the DILI potential. Depending on the phase of drug development, the physicochemical properties, dosing, and cut-off values of BSEP IC50 ≤ 25–50 µM or total Css,plasma/BSEP IC50 ≥ 0.1 may be an indication for further testing to minimize the risk of DILI liability.
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Transporter gene knockout models are a practical and widely used tool for pharmacokinetic studies in drug discovery. P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are major efflux transporters that control absorption and bioavailability, and are important when determining oral drug disposition. To the best of our knowledge, beyond the rule of five (bRo5) molecules launched on the market to date tend to be substrates for efflux transporters. The purpose of this study is to evaluate in vivo the impact of efflux transporters on the oral absorption process and systemic clearance using rats which lack P-gp and/or Bcrp expression. We administered five bRo5 substrates (asunaprevir, cyclosporine, danoprevir, ledipasvir, and simeprevir) intravenously or orally to wild-type and Mdr1a, Bcrp, and Mdr1a/Bcrp knockout rats, calculated the clearance, oral bioavailability, and absorption rate profile of each substrate, and compared the results. Systemic clearance of the substrates in knockout rats changed within approximately ±40% compared to wild-types, suggesting the efflux transporters do not have a significant influence on clearance in rats. On the other hand, the oral absorption of substrates in the knockout rats, especially those lacking Mdr1a, increased greatly—between 2- and 5-fold more than in wild-types. This suggests that rat efflux transporters, especially P-gp, greatly reduce the oral exposure of these substrates. Moreover, results on the absorption rate–time profile suggest that efflux transporters are constantly active during the absorption period in rats. Transporter knockout rats are a useful in vivo tool for estimating the transporter-mediated disposition of bRo5 molecules in drug discovery.
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Abcg2
P-glycoprotein
Knockout mouse
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Oral bioavailability (F) is determined as fraction of the drug dose absorbed through the gastrointestinal membranes (Fa), the unmetabolized fraction of the absorbed dose that passes through the gut into the portal blood (Fg), and the hepatic first pass availability (Fh), namely F is expressed as the product of Fa, Fg and Fh (F = Fa.Fg.Fh). Current evidence suggests that transporter proteins play a role in intestinal absorption and hepatobiliary clearance of drugs. Among those transporters, this review will focus on PEPT1 and OATP2B1 as influx transporter and p-glycoprotein (P-gp) and BCRP as efflux transporter in intestinal epithelial cells, and on OATP1B1 and 1B3 as influx transporter and MRP2 as efflux transporter in hepatocytes, respectively, because drug-drug (DDI) and -food (DFI) interactions on these transporter are considered to affect bioavailability of their substrate drugs. DDI and DFI may reduce systemic exposure to drug by blocking influx transporters in intestine, but increase it by modulating influx and efflux transporters in liver and efflux transporters in intestines. Namely, drug disposition and efficacy are likely affected by DDI and DFI, resulting in treatment failures or increase in adverse effect. Therefore, it is of significantly importance to understand precise mechanism of DDI and DFI. This review will present information about transporter-based DDI and DFI in the processes of intestinal absorption and hepatic clearance of drugs, and discuss about their clinical implication.
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