Assessing OATP1B1- and OATP1B3-Mediated Drug-Drug Interaction Potential of Vemurafenib Using R-Value and Physiologically-Based Pharmacokinetic Models.

Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important determinants of transporter-mediated drug-drug interactions (DDIs). Current studies assessed the OATP1B1 and OATP1B3-mediated DDI potential of vemurafenib, a kinase inhibitor drug with high protein binding and low aqueous solubility, using R-value and physiologically-based pharmacokinetic (PBPK) models. The total half-maximal inhibitory concentration (IC50,total) values of vemurafenib against OATP1B1 and OATP1B3 were determined in 100% human plasma in transporter-overexpressing human embryonic kidney 293 stable cell lines. The unbound fraction of vemurafenib in human plasma before (fu,plasma) and after addition into the uptake assay plate (fu,plasma,inc) were determined by rapid equilibrium dialysis. There was no statistically significant difference between fu,plasma and fu,plasma,inc. Vemurafenib IC50,total values against OATP1B1 and OATP1B3 are 175 ± 82 and 231 ± 26 µM, respectively. The R-values [R = 1 + fu,plasma × Iin,max/(fu,plasma,inc × IC50,total)] were then simplified as R = 1+Iin,max/IC50,total, and were 1.76 and 1.57 for OATP1B1 and OATP1B3, respectively. The simulated pravastatin AUC ratio was 1.28 when a single dose of pravastatin (40 mg) was co-administered with vemurafenib (960 mg, twice daily) at steady-state, compared to pravastatin alone. Both R-value and PBPK models predict that vemurafenib has the potential to cause OATP1B1- and OATP1B3-mediated DDIs.

Semi-quantitative Determination of Protein Expression using Immunohistochemistry Staining and Analysis: An Integrated Protocol.

Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed procedure of how to measure or compare protein expression. Publications that use semi-quantification methods to quantify protein expression often don't provide enough details in their methods section, which makes it difficult for the reader to reproduce their data. The current protocol for the first time provides a detailed, step-by-step instruction of conducting semi-quantitative analysis of IHC images using ImageJ Fiji software so that researchers would be able to follow this single protocol to conduct their research. The protocol uses semi-quantitative IHC of organic anion transporting polypeptide (OATP1B1) as an example, and gives detailed steps on how to deconvolute IHC images stained with hematoxylin and 3, 3 - diaminobenzidine (DAB) and how to quantify their expression using ImageJ Fiji. The protocol includes clear steps for a reader so that this method can be applied to many different proteins. We anticipate this method will provide a practical guidance to the reader and make semi-quantification of proteins an easier task to include in publications.

Genotyping of the OATP1B1 c. 521 T>C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol.

Organic anion transporting polypeptide (OATP) 1B1 is a liver-specific transport protein that plays an important role in hepatic drug disposition. It transports many drugs from the blood into the liver, including lipid-lowering statins. The c. 521 T>C polymorphism of OATP1B1 has reduced transport activity and is associated with statin-induced myopathy. Formalin-fixed paraffin-embedded (FFPE) liver tissues can be an enriched source for genotyping of this clinically significant OATP1B1 polymorphism in retrospective studies. The successfulness of genotyping using Sanger-sequencing of a PCR product from FFPE tissue relies on a successful PCR amplification using genomic DNA extracted from the FFPE tissues. Such PCR amplification is often limited by the quality of DNA extracted from the FFPE tissue. An optimized method for high-quality DNA extraction and efficient PCR amplification is highly needed in order to genotype polymorphisms such as the c. 521 T>C polymorphism using FFPE tissues. The current study established an optimized and reproducible method for a Sanger-sequencing-based genotyping method using FFPE human liver tissues that is applicable to even small FFPE tissues such as needle-core biopsy specimens.

Characterization of Plasma Membrane Localization and Phosphorylation Status of Organic Anion Transporting Polypeptide (OATP) 1B1 c.521 T>C Nonsynonymous Single-Nucleotide Polymorphism.

PURPOSE: Membrane transport protein organic anion transporting polypeptide (OATP) 1B1 mediates hepatic uptake of many drugs (e.g. statins). The OATP1B1 c.521 T > C (p. V174A) polymorphism has reduced transport activity. Conflicting in vitro results exist regarding whether V174A-OATP1B1 has reduced plasma membrane localization; no such data has been reported in physiologically relevant human liver tissue. Other potential changes, such as phosphorylation, of the V174A-OATP1B1 protein have not been explored. Current studies characterized the plasma membrane localization of V174A-OATP1B1 in genotyped human liver tissue and cell culture and compared the phosphorylation status of V174A- and wild-type (WT)-OATP1B1. METHODS: Localization of V174A- and WT-OATP1B1 were determined in OATP1B1 c.521 T > C genotyped human liver tissue (n = 79) by immunohistochemistry and in transporter-overexpressing human embryonic kidney (HEK) 293 and HeLa cells by surface biotinylation and confocal microscopy. Phosphorylation and transport of OATP1B1 was determined using 32P-orthophosphate labeling and [3H]estradiol-17ß-glucuronide accumulation, respectively. RESULTS: All three methods demonstrated predominant plasma membrane localization of both V174A- and WT-OATP1B1 in human liver tissue and in cell culture. Compared to WT-OATP1B1, the V174A-OATP1B1 has significantly increased phosphorylation and reduced transport. CONCLUSIONS: We report novel findings of increased phosphorylation, but not impaired membrane localization, in association with the reduced transport function of the V174A-OATP1B1.

Preincubation With Everolimus and Sirolimus Reduces Organic Anion-Transporting Polypeptide (OATP)1B1- and 1B3-Mediated Transport Independently of mTOR Kinase Inhibition: Implication in Assessing OATP1B1- and OATP1B3-Mediated Drug-Drug Interactions.

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 mediate hepatic uptake of many drugs including lipid-lowering statins. Current studies determined the OATP1B1/1B3-mediated drug-drug interaction (DDI) potential of mammalian target of rapamycin (mTOR) inhibitors, everolimus and sirolimus, using R-value and physiologically based pharmacokinetic models. Preincubation with everolimus and sirolimus significantly decreased OATP1B1/1B3-mediated transport even after washing and decreased inhibition constant values up to 8.3- and 2.9-fold for OATP1B1 and both 2.7-fold for OATP1B3, respectively. R-values of everolimus, but not sirolimus, were greater than the FDA-recommended cutoff value of 1.1. Physiologically based pharmacokinetic models predict that everolimus and sirolimus have low OATP1B1/1B3-mediated DDI potential against pravastatin. OATP1B1/1B3-mediated transport was not affected by preincubation with INK-128 (10 µM, 1 h), which does however abolish mTOR kinase activity. The preincubation effects of everolimus and sirolimus on OATP1B1/1B3-mediated transport were similar in cells before preincubation with vehicle control or INK-128, suggesting that inhibition of mTOR activity is not a prerequisite for the preincubation effects observed for everolimus and sirolimus. Nine potential phosphorylation sites of OATP1B1 were identified by phosphoproteomics; none of these are the predicted mTOR phosphorylation sites. We report the everolimus/sirolimus-preincubation-induced inhibitory effects on OATP1B1/1B3 and relatively low OATP1B1/1B3-mediated DDI potential of everolimus and sirolimus.

Regulation of Organic Anion Transporting Polypeptides (OATP) 1B1- and OATP1B3-Mediated Transport: An Updated Review in the Context of OATP-Mediated Drug-Drug Interactions.

Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important hepatic transporters that mediate the uptake of many clinically important drugs, including statins from the blood into the liver. Reduced transport function of OATP1B1 and OATP1B3 can lead to clinically relevant drug-drug interactions (DDIs). Considering the importance of OATP1B1 and OATP1B3 in hepatic drug disposition, substantial efforts have been given on evaluating OATP1B1/1B3-mediated DDIs in order to avoid unwanted adverse effects of drugs that are OATP substrates due to their altered pharmacokinetics. Growing evidences suggest that the transport function of OATP1B1 and OATP1B3 can be regulated at various levels such as genetic variation, transcriptional and post-translational regulation. The present review summarizes the up to date information on the regulation of OATP1B1 and OATP1B3 transport function at different levels with a focus on potential impact on OATP-mediated DDIs.

Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.

OATP1B1 and OATP1B3 mediate hepatic uptake of many drugs (e.g., statins) and can mediate transporter-mediated drug-drug-interactions (DDIs). Bortezomib is the first-in-class proteasome inhibitor drug approved by the U. S. Food and Drug Administration for the treatment of multiple myeloma. The potential of bortezomib to cause OATP-mediated DDIs has not been assessed. The current study investigated the involvement of the ubiquitin-proteasome system (UPS) in OATP1B1 and OATP1B3 degradation and determined the effects of proteasome inhibitors on OATP1B1- and OATP1B3-mediated transport. Co-immunoprecipitation of FLAG-OATP1B1/1B3 and HA-ubiquitin was observed in human embryonic kidney (HEK) 293 cells co-transfected with FLAG-tagged OATP1B1/OATP1B3 and hemagglutinin (HA)-tagged ubiquitin, suggesting that OATP1B1 and OATP1B3 can be ubiquitin-modified. Although blocking proteasome activity by bortezomib treatment (50 nM, 7 h) increased the endogenous ubiquitin-conjugated FLAG-OATP1B1 and FLAG-OATP1B3 in HEK293-FLAG-OATP1B1 and-OATP1B3 cells, such treatment did not affect the total protein levels of OATP1B1 and OATP1B3, suggesting that the UPS plays a minor role in degradation of OATP1B1 and OATP1B3 under current constitutive conditions. Pretreatment with bortezomib (50-250 nM, 2-7 h) significantly decreased transport of [3H]CCK-8, a specific OATP1B3 substrate, in HEK293-OATP1B3 and human sandwich-cultured hepatocytes (SCH). However, bortezomib pretreatment had negligible effects on the transport of [3H]E217ßG and [3H]pitavastatin, dual substrates of OATP1B1 and OATP1B3, in HEK293-OATP1B1/1B3 cells and/or human SCH. Compared with vehicle control treatment, bortezomib pretreatment significantly decreased the maximal transport velocity (Vmax) of OATP1B3-mediated transport of CCK-8 (92.25 ± 14.2 vs. 133.95 ± 15.5 pmol/mg protein/min) without affecting the affinity constant (Km) values. Treatment with other proteasome inhibitors MG132, epoxomicin, and carfilzomib also significantly decreased OATP1B3-mediated [3H]CCK-8 transport. In summary, the current studies for the first time report ubiquitination of OATP1B1 and OATP1B3 and the apparent substrate-dependent inhibitory effect of bortezomib on OATP1B3-mediated transport. The data suggest that bortezomib has a low risk of causing OATP-mediated DDIs.

Pretreatment With Rifampicin and Tyrosine Kinase Inhibitor Dasatinib Potentiates the Inhibitory Effects Toward OATP1B1- and OATP1B3-Mediated Transport.

Present studies determined the effects of pretreatment with rifampicin, an organic anion-transporting polypeptide (OATP) inhibitor, and the tyrosine kinase inhibitor dasatinib on OATP1B1- and OATP1B3-mediated transport, and evaluated the OATP-mediated drug-drug interaction potential of dasatinib using the static R-value and dynamic physiologically based pharmacokinetic models. Rifampicin and dasatinib pretreatment significantly decreased OATP1B1- and OATP1B3-mediated transport. Rifampicin pretreatment also significantly decreased [3H]-pitavastatin and [3H]-CCK-8 accumulation in human sandwich-cultured hepatocytes. Present studies revealed that estrone-3-sulfate is a less-sensitive OATP1B1 substrate than estradiol-17ß-glucuronide in assessing rifampicin pretreatment effects. Pretreatment with rifampicin and dasatinib reduced the inhibition constant (Ki) values against OATP1B1 by 3 and 2.1 fold and against OATP1B3 by 2.4 and 2.1 fold, respectively. The in vitro rifampicin Ki values after preincubation are comparable to the estimated in vivo Ki reported previously. Models predict that dasatinib has a low potential to cause OATP1B1- and OATP1B3-mediated drug-drug interactions. Time-lapse confocal microscopy demonstrated that rifampicin and dasatinib pretreatment did not affect plasma membrane localization of green-fluorescent protein-tagged OATP1B1 (GFP-OATP1B1) and GFP-OATP1B3 in human embryonic kidney 293 stable cell lines. In summary, we report novel findings that pretreatment with rifampicin and dasatinib potentiates the inhibitory effects toward OATP1B1 and OATP1B3 without affecting plasma membrane levels of the transporters.