Suppression of Canine ATP Binding Cassette ABCB1 in Madin-Darby Canine Kidney Type II Cells Unmasks Human ABCG2-Mediated Efflux of Olaparib.

Endogenous canine ATP binding cassette B1 (cABCB1) is expressed abundantly in Madin-Darby canine kidney type II (MDCKII) cells, and its presence often complicates phenotyping of the transport process. Errors in estimating the corrected efflux ratio (cER), as a result of the variable expression of cABCB1, were examined for the dual substrates of ABCB1 and ABCG2 in MDCKII cells expressing human ABCG2 (hABCG2). cABCB1 mRNA and protein expression was 60% and 55% lower, respectively, in MDCKII cells expressing hABCG2 compared with the wild type, suggesting that the expression of endogenous cABCB1 became variable after the expression of hABCG2. To minimize the contribution of endogenous efflux, cABCB1 was suppressed kinetically (using verapamil as a selective inhibitor) or biochemically (transfecting short-hairpin RNA against cABCB1). Under these suppression conditions, cER values for irinotecan and topotecan (dual substrates of ABCB1 and ABCG2) were elevated by more than 4-fold and 2-fold, respectively, compared with cER values without the suppression. The cER of olaparib was similarly increased to 3- and 5-fold in MDCKII cells under the kinetic and biochemical suppression of cABCB1, respectively, suggesting that hABCG2-mediated efflux cannot be ruled out for olaparib. Since the substrate selectivity for ABCB1 and ABCG2 overlapped considerably, the possibility of an inaccurate estimation of cER must be considered for dual substrates in the case of the variable expression of cABCB1 in MDCKII cells.

Identification of the primary determining factor(s) governing the oral absorption of edaravone in rats.

This study was performed to determine the primary factor(s) governing the oral absorption of edaravone, a novel anti-oxidant for the treatment of amyotrophic lateral sclerosis, in rats. While the aqueous solubility of edaravone widely varied depending on the vehicle used, the oral bioavailability of the drug was not low when it was adequately solubilized, as evidenced by the fact that the oral exposure was high (in terms of the absolute bioavailability of 50-90%) at all dose ranges (i.e., 0.5-27 mg/kg) under solubilized conditions in rats. The sum of the in vitro clearance values for edaravone, 12.7 mL/(min × kg), obtained from metabolic stability studies with tissue-homogenates from the rat liver, kidney, intestine, and with the rat plasma, was found to be virtually identical to the systemic clearance of the drug in rats. It was noted that the liver represented over 83.9% of the total elimination with a hepatic extraction ratio of approximately 0.137, indicative of the minor role of hepatic first pass metabolism in the systemic absorption of edaravone after its oral administration. In studies with Ussing chamber with rat intestinal segments and Madin-Darby canine kidney (MDCKII) cells, edaravone was found to be highly permeable (i.e., Papp over 10 × 10-6 cm/s), and appeared to be a substrate for rat P-glycoprotein (P-gp; estimated Km of 421 µM). In contrast, however, the drug did not appear to be a substrate for human P-gp in transport studies with MDCKII-hMDR1 cells. Collectively, these observations suggest that the primary determining factor for the intestinal absorption of edaravone is its solubilization in vehicle/intestinal fluids, rather than permeability, pre-systemic first-pass metabolism, or efflux transport. Considering the fact that the newly approved indication of the drug would require prolonged administration, probably via oral administration, the findings reported herein provide relevant information regarding its use.

The conditional stimulation of rat organic cation transporter 2, but not its human ortholog, by mesoridazine: the possibility of the involvement of the high-affinity binding site of the transporter in the stimulation.

OBJECTIVES: To study the functional consequences of the human and rat forms of OCT2 in the presence of phenothiazines. METHODS: MDCK cells expressing human or rat OCT2 were established, and MPP+ transport was determined by uptake assays. Concentration dependency was studied for the stimulatory/inhibitory effects of phenothiazines on MPP+ transport. KEY FINDINGS: Among the 11 phenothiazines examined, the majority were found to have comparable effects on transporter function between the orthologous forms, while three phenothiazines, particularly mesoridazine, had complex impacts on transporter function. For rOCT2, mesoridazine stimulated transport at 0.1 and 1 µmMPP+ with the mesoridazine concentration-uptake curve becoming bell-shaped. This conditional effect became less pronounced at 30 µmMPP+, resulting in an inhibition curve with a typical profile. For hOCT2, mesoridazine behaved as a typical inhibitor of transporter function at all MPP+ concentrations, although the kinetics of inhibition were still affected by the substrate concentration. CONCLUSIONS: The conditional stimulation by mesoridazine in rOCT2, and the lack thereof in hOCT2, may be a manifestation of the interaction of phenothiazine with substrate binding at the high-affinity site of the OCT2. As OCT2 was previously indicated in some drug-drug interactions, the conditional stimulation of OCT2 and its potential species-differences may be of practical relevance.