Using the lentiviral vector system to stably express chicken P-gp and BCRP in MDCK cells for screening the substrates and studying the interplay of both transporters

Abstract

Transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are known to influence the pharmacokinetics and toxicity of substrate drugs. However, no detailed information is as yet available about functional activity and substrate spectra of chicken P-gp and BCRP. In this study, BCRP single and BCRP/P-gp double-transfected MDCK cell lines (named MDCK-chAbcg2 and MDCK-chAbcg2/Abcb1, respectively) were generated using lentiviral vector system to develop reliable systems for screening the substrates for these two transporters and study the interplay between them. The constructed cell lines significantly expressed functional exogenous proteins and expression persisted for at least 50 generations with no decrease. Enrofloxacin, ciprofloxacin, tilmicosin, sulfadiazine, ampicillin and clindamycin were classified as the substrates of chicken P-gp according to the rules suggested by FDA, as their net efflux ratios were greater than two. Similarly, enrofloxacin, ciprofloxacin, tilmicosin, florfenicol, ampicillin and clindamycin were classified as the substrates of BCRP. Among these drugs, enrofloxacin, ciprofloxacin, tilmicosin, ampicillin, and clindamycin were the cosubstrates of P-gp and BCRP, however, chicken BCRP and P-gp exhibit different affinities to the shared substrates at different concentrations by blocking either one or both transport with specific inhibitors in the coexpression system. It was also found that ceftiofur, amoxicillin and doxycycline were not substrates of either chicken BCRP or the substrates of chicken P-gp. These constructed cell models provide useful systems for high-throughput screening of the potential substrates of chicken BCRP and P-gp as well as the drug-drug interaction mediated via chicken BCRP and P-gp.

Synthesis of Tilmicosin Nanostructured Lipid Carriers for Improved Oral Delivery in Broilers: Physiochemical Characterization and Cellular Permeation

“…In order to elucidate the mechanism of TMS-NLCs enhancing the oral absorption of TMS, the effect of NLCs on the TMS permeability across Caco-2 cell monolayers was investigated. In our previous study, tilmicosin was proved to be a substrate of P-gp, a drug efflux transporter [35]. Herein, we found that the efflux rate of 10% TMS in Caco-2 cell monolayers was 2.29 (Table 2), far higher than the 1.5 regulated by the United States Food and Drug Administration [36], further demonstrating that TMS as the substrate of P-gp could be removed from intestinal epithelial cells and its oral absorption thereby decreased [25].…”

Considerations for application of biopharmaceutics classification system in chicken: Exemplified by seven drugs classification

“…The borderline for the low/high permeability must be defined when evaluating permeability ( P app ) data from cell monolayer, and metoprolol is currently an acceptable and widely used marker for this purpose (Incecayir, Tsume, & Amidon, ; Zur et al, ), so metoprolol was used to be a reference standard for class in our research, and the classification results formed a good correlation with the absorption of drugs in chickens. LogP has been used for the permeability classification of canine BCS and pediatric BCS, and a LogP value greater than 1.72 as high permeability (Guimaraes et al, ; Papich, ), but the LogP only represents the lipophilic of drug molecules, and it ignores the enzymes and transporters inherent in human or animal organisms, thus, it is not surprising that predictions based only on LogP may frequently be in error, often because most drugs may be substrates for some transporter (Papich, ; Wu & Benet, ), such as some substrates (ciprofloxacin hydrochloride for P‐gp and BCRP; sulfadiazine for P‐gp) in our study (Zhang et al, ), which with a higher LogP value but a lower absorption in chicken. Moreover, we also discussed the role of pH on permeability class, and the result showed that pH has less influence on classification, except sulfamethoxazole, which was an acid drug (pKa 5.6), exhibits high class under pH 5.5 but low under pH 7.4, and it is well interpreted that acid drugs have higher permeability values at lower pH (Tolle‐Sander, ; Yang et al, ).…”

“…The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% glutamine and 1% penicillin under 37°C, and 5% CO 2 . MDCK-chAbcb1/Abcg2 cell line stably expressing chicken P-gp and BCRP was established in our laboratory (Zhang, Huang, Liu, Guo, & Wang, 2018). The cell lines were cultured as the above and supplemented with 1.0 μg/ml puromycin and 1 200 µg/ml neomycin.…”

Identification of Functional Transcriptional Binding Sites within Chicken Abcg2 Gene Promoter and Screening Its Regulators

“…Xenobiotics, including natural compounds (daidzein and berberine), commonly used drugs (rifampicin, clotrimazole, enrofloxacin, florfenicol, tilmicosin, sulfadiazine, ciprofloxacin, doxycycline, cefuroxime sodium, and ivermectin) and LPS, were used in the transfection assay. The concentrations of the above xenobiotics were selected according to the cytotoxicity assay results, where the concentrations were based on cell viabilities of no less than 90% compared with the control group [34]. The final concentrations of all of the xenobiotics were 30 µM, except ivermectin (10 µM) and LPS (50 µg/mL).…”