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

Abstract

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.

Quercetin Is a Flavonoid Breast Cancer Resistance Protein Inhibitor with an Impact on the Oral Pharmacokinetics of Sulfasalazine in Rats

“…Co-incubation with quercetin increased the P app , A-to-B of prazosin ( Figure 4A) while simultaneously decreasing the P app , B-to-A ( Figure 4B) with an increasing concentration of quercetin, leading to a concentration-dependent decrease in the overall ER ( Figure 4C). Additionally, the functional expression of the efflux transporter in MDCKII/BCRP cells was also confirmed in this study, with an ER of 5.4 for prazosin (the stereotypical substrate of BCRP [27,35,36]), which decreased to 0.9 in the presence of the known inhibitor Ko143 (Figure 5C). Notably, the inhibitory effect of 10 µM quercetin on the B-to-A transport and efflux ratio was comparable to 1 µM Ko143 ( Figure 5; p > 0.05).…”

“…In addition, pharmacokinetically relevant parameters were obtained in a bi-directional transport study using MDCKII/BCRP cells, where the IC 50 values of quercetin for the inhibition of BCRP-mediated efflux were estimated to be 4.22 µM. Assuming the mechanism of inhibition to be competitive, the IC 50 value was further transformed to a Ki value of 3.91 µM, using the K m value of 128 µM [27] for prazosin. The values obtained in the bi-directional transport studies were comparable to those previously observed in MCF-7/MX and MDCKII/BCRP cells using Hoechst 33342 accumulation (IC 50 values of 7.6 and 6.9 µM, respectively) [21].…”

“…For the cellular accumulation and cytotoxicity studies, HeLa (human cervical cancer) cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Welgene Inc., Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Welgene Inc., Daegu, Korea) and 100 U/mL penicillin-100 µg/mL streptomycin at 37 • C in a humidified incubator with 5% CO 2 . For the bi-directional transport study, previously established human BCRP-overexpressing MDCKII cells [27] were used. Briefly, a plasmid construct containing cDNA for human BCRP was transfected into wildtype MDCKII cells to functionally express the transporter.…”

“…Kinetic analysis of the transport process yielded an estimated IC 50 value of 4.22 µM for quercetin. Assuming the mechanism of inhibition to be competitive, the inhibitory constant (Ki) value was then estimated to be 3.91 µM using the K m value of 128 µM [27] for prazosin. 2.…”

Renal Excretion of Dabigatran: The Potential Role of Multidrug and Toxin Extrusion (MATE) Proteins

“…In the presence of both P-gp-MDCK (Figure B) and BCRP-MDCK (Figure C) monolayers, dabigatran exhibited comparable or slightly greater vectorial B-A transport versus A-B transport, yielding B-A/A-B ER values of 1.8 and 2.3 for P-gp- and BCRP-MDCK cells, respectively. The efflux ratios were reduced by cyclosporine A, Ko143, and elacridar, which are dual inhibitors of P-gp and BCRP. − These data indicate that dabigatran behaves as a poor P-gp and BCRP substrate in vitro. It is worth noting that the inhibitor concentrations used in this study (i.e., 10 μM for Ko143, 50 μM for elacridar, and 20 μM for cyclosporine A) are relatively high, and thus, the inhibition of endogenous canine transporters expressed in the MDCK cells might occur.…”