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ABSTRACT:Organic anion-transporting polypeptides (OATPs), members of the SLCO/SLC21 family, mediate the transport of various endo-and xenobiotics. In human liver, OATP1B1, 1B3, and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However, there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study, a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction, microarray and immunoblotting analyses, along with uptake assays, illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30% of the total uptake of cerivastatin into human hepatocytes, it caused a 50% reduction in cerivastatin metabolism, which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs.

Proteasome inhibitor therapeutics (PITs) have the potential to cause peripheral neuropathy. In a mouse model of PIT-induced peripheral neuropathy, the authors demonstrated that ubiquitin-positive multifocal protein aggregates with nuclear displacement appear in dorsal root ganglion cells of animals that subsequently develop nerve injuries. This peripheral-nerve effect in nonclinical models has generally been recognized as the correlate of grade 3 neuropathy in clinical testing. In differentiated PC12 cells, the authors demonstrated perturbations correlative with the development of neuropathy in vivo, including ubiquitinated protein aggregate (UPA) formation and/or nuclear displacement associated with the degree of proteasome inhibition. They compared 7 proteasome inhibitors of 3 chemical scaffolds (peptide boronate, peptide epoxyketone, and lactacystin analog) to determine if PIT-induced peripheral neuropathy is modulated by inhibition of the proteasome (ie, a mechanism-based effect) or due to effects independent of proteasome inhibition (ie, an off target or chemical-structure-based effect). The appearance of UPAs was assayed at IC(90) +/- 5% (90% inhibition concentration +/- 5%) for 20S proteasome inhibition. Results show that each of the investigated proteasome inhibitors induced identical proteasome-inhibitor-specific ubiquitin-positive immunostaining and nuclear displacement in PC12 cells. Other agents--such as paclitaxel, cisplatin, and thalidomide, which cause neuropathy by other mechanisms--did not cause UPAs or nuclear displacement, demonstrating that the effect was specific to proteasome inhibitors. In conclusion, PIT-induced neuronal cell UPA formation and nuclear displacement are mechanism based and independent of the proteasome inhibitor scaffold. These data indicate that attempts to modulate the neuropathy associated with PIT may not benefit from changing scaffolds.

Inflammatory bowel disease (IBD) results from intermittent and severe activation of the mucosal immune system in the gastrointestinal tract to promote a chronic state of inflammation. Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of IBD. Infiltration of gut tissue by lymphocytes, neutrophils, and macrophages results in prolonged exposure to chemical agents such as pro-inflammatory cytokines and reactive oxygen/nitrogen species. Chronic exposure to these inflammation products leads to mis-regulated cell signaling, altered protein expression, and chemical damage to lipids, protein, and nucleic acids. IBD is a significant risk factor for colon cancer. IBD exhibits phases of active disease (active inflammation) and remission, which complicate therapeutic intervention. Clinical biomarkers currently in use are not predictive of the transition from remission to active disease. Our laboratory has conducted an unbiased, four-pronged approach to IBD biomarker identification in human serum utilizing proteomic discovery of acute phase proteins, cytokine profiling, quantification of oxidative tyrosine modifications (chlorotyrosine (Cl-Tyr), bromotyrosine (Br-Tyr), and nitrotyrosine (NO-Tyr)–markers of neutrophil activity), and measurement of carbonylated proteins. One hundred and ten human serum samples were analyzed. This network of data was analyzed by orthogonormalized partial least squares (OPLS) analysis to identify covariance in the data set. Variable importance in projection (VIP) scores were determined to identify which variables were most predictive of clinically diagnosed disease score (VIP >1). Using data available from the cytokine and oxidative tyrosine analysis, the data set was stratified to clinically diagnosed active and inactive disease, with the intent of identifying serum markers that were predictive of this transition. Several variables for UC and CD demonstrated strong covariance with disease score. The top VIP scores identified for UC were: Interleukin-8 (IL-8), granulocyte-colony stimulating factor (G-CSF), IL-6, and Cl-Tyr. The top VIP scores identified for CD were: Cl-Tyr, macrophage inflammatory protein-1β (MIP-1β), inflammatory chemokine-10 (IP-10), IL-6, and IL-1 receptor antagonist (IL-1ra). A Total Score was generated for each UC and CD serum sample by normalizing the raw data to a base-10 spread, scaling by the VIP factor, and summing the total. Based on the Total Score the calculated specificity and sensitivity for identifying active UC was 81.8% and 84.2%, respectively, while the specificity and sensitivity for identifying active CD was 87.5% and 75%, respectively. We anticipate that the addition of acute phase and cabonylated protein analysis will improve the Total Score assessment. Results from this study strongly suggest that a cassette of in vivo serum markers may be predictive of the transition from remission to active disease in IBD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5113. doi:10.1158/1538-7445.AM2011-5113

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