Preclinical assessment of drug-drug interaction of fb-PMT, a novel anti-cancer thyrointegrin αvß3 antagonist.

The objective of the current study was to identify potential drug-drug interactions (DDIs) with the drug candidate fb-PMT, a novel anticancer thyrointegrin αvß3 antagonist. This was accomplished by using several in vitro assays to study interactions of fb-PMT with both cytochrome P450 (CYP) enzymes and drug transporters, two common mechanisms leading to adverse drug effects. In vitro experiments showed that fb-PMT exhibited weak reversible inhibition of CYP2C19 and CYP3A4. In addition, fb-PMT did not show time-dependent inhibition with any of the seven CYP isoforms tested, including 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. Human liver microsomal incubations demonstrated that fb-PMT is stable. Potential transporter-mediated DDIs with fb-PMT were assessed with two ATP binding cassette (ABC) family transporters (P-glycoprotein and breast cancer resistance protein) using Caco2 cells and seven solute carrier family (SLC) transporters (organic cation transporter OCT2, organic anion transporters OAT1 and OAT3, organic anion transporter peptides OATP1B1 and OATP1B3, and the multidrug and toxic extrusion proteins MATE1 and MATE2-K using transfected HEK293 cells). Fb-PMT was not a substrate for any of the nine transporters tested in this study, nor did it inhibit the activity of seven of the transporters tested. However, fb-PMT inhibited the uptake of rosuvastatin by both OATP1B1 and OATP1B3 with half-maximal inhibitory concentrations greater than 3 and less than 10 µM. In summary, data suggest that the systemic administration of fb-PMT is unlikely to lead to DDIs through CYP enzymes or ABC and SLC transporters in humans.

Development and Application of Human Renal Proximal Tubule Epithelial Cells for Assessment of Compound Toxicity.

Kidney toxicity is a major problem both in drug development and clinical settings. It is difficult to predict nephrotoxicity in part because of the lack of appropriate in vitro cell models, limited endpoints, and the observation that the activity of membrane transporters which plays important roles in nephrotoxicity by affecting the pharmacokinetic profile of drugs is often not taken into account. We developed a new cell model using pseudo-immortalized human primary renal proximal tubule epithelial cells. This cell line (SA7K) was characterized by the presence of proximal tubule cell markers as well as several functional properties, including transporter activity and response to a few well-characterized nephrotoxicants. We subsequently evaluated a group of potential nephrotoxic compounds in SA7K cells and compared them to a commonly used human immortalized kidney cell line (HK-2). Cells were treated with test compounds and three endpoints were analyzed, including cell viability, apoptosis and mitochondrial membrane potential. The results showed that most of the known nephrotoxic compounds could be detected in one or more of these endpoints. There were sensitivity differences in response to several of the chemicals between HK-2 and SA7K cells, which may relate to differences in expressions of key transporters or other components of nephrotoxicity pathways. Our data suggest that SA7K cells appear as promising for the early detection of renal toxicants.

Zinc finger nuclease-mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 cells.

Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 µM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-2',7'-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches.

A nanofiber membrane maintains the quiescent phenotype of hepatic stellate cells.

BACKGROUND: Hepatic stellate cells (HSC) play a major role in the progression of liver fibrosis. AIM: The aim of our study was to investigate whether rat HSC cultured on a nanofiber membrane (NM) retain their quiescent phenotype during both short- and long-term culture and whether activated HSC revert to a quiescent form when re-cultured on NM. METHODS: Rat HSC cultured for 1 day on plastic plates (PP) were used as quiescent HSC, while cells cultured for 1 week on PP were considered to be activated HSC. Quiescent or activated HSC were subsequently plated on PP or NM and cultured for an additional 4 days at which time their gene expression, stress fiber development, and growth factor production were determined. For long-term culture, HSC were grown on NM for 20 days and the cells then replated on PP and cultured for another 10 days. RESULTS: Expression of marker genes for HSC activation, stress fiber development, and growth factor production were significantly lower in both quiescent and activated HSC cultured on NM than in those cultured on PP. After long-term culture on NM, activation marker gene expression and stress fiber development were still significantly lower in HSC than in PP, and HSC still retained the ability to activate when replated onto PP. CONCLUSIONS: HSC cultured on NM retained quiescent characteristics after both short- and long-term culture while activated HSC reverted toward a quiescent state when cultured on NM. Cultures of HSC grown on NM are a useful in vitro model to investigate the mechanisms of activation and deactivation.

Biochemical, cellular, and anti-inflammatory properties of a potent, selective, orally bioavailable benzamide inhibitor of Rho kinase activity.

Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.