pH Dependence of Organic Anion-Transporting Polypeptide 2B1 in Caco-2 Cells: Potential Role in Antiretroviral Drug Oral Bioavailability and Drug–Drug Interactions

Abstract

Human intestinal epithelium expresses a number of drug efflux and influx transporters that can restrict and/or facilitate intestinal drug uptake during absorption. Organic anion-transporting polypeptide 2B1 (OATP2B1), a multispecific organic anion uptake transporter localized at the brush-border membrane of intestinal epithelial cells, is known to transport many endogenous substrates (e.g., steroid conjugates) and xenobiotics (e.g., statins). At present, limited information is available on the mechanism of HIV protease inhibitor (PIs) intestinal uptake. In this study, we examined the interaction of PIs with the OATP2B1 transport system in Caco-2 cells, an in vitro model of human intestinal epithelium, and Madin-Darby canine kidney II cells stably transfected with OATP2B1. The expression of OATP2B1 transcript and protein was confirmed by reverse transcription-polymerase chain reaction and immunoblot analysis, respectively. Estrone-3-sulfate (E3S) uptake demonstrated biphasic saturation kinetics in Caco-2 cells, with dissociation constants (K M ) of 6 Ϯ 2 M and 1.5 Ϯ 0.2 mM. Several PIs potently inhibited OATP2B1-mediated transport in Caco-2 cells at clinically relevant IC 50 concentrations for ritonavir (0.93 M), atazanavir (2.2 M), lopinavir (1.7 M), tipranavir (0.77 M), and nelfinavir (2.2 M). An inwardly directed proton gradient was identified as the driving force of E3S uptake through NH 4 Cl intracellular acidification studies with a H ϩ :E3S stoichiometry for OATP2B1 of 1:1. In contrast, although atazanavir and ritonavir uptake by Caco-2 cells was stimulated by low extracellular pH, this process was not mediated by OATP2B1 and was not affected by an outwardly directed H ϩ gradient. Because OATP2B1 exhibits an increasing number of drug substrates, including several statins, alterations of its function by PIs could result in clinically significant drug-drug interactions in the intestine.

Differential Role of Organic Anion-Transporting Polypeptides in Estrone-3-Sulphate Uptake by Breast Epithelial Cells and Breast Cancer Cells

“…The MDCKII/OATP2B1 medium was supplemented with 1% L-glutamine and 400 g/ml hygromycin B (Sigma-Aldrich, St. Louis, MO) to provide selection pressure for stably transfected cells (Kis et al, 2010b). HEK/OATP3A1 and HEK/ OATP4A1 medium was supplemented with 800 g/ml G-418 as described previously .…”

“…Immunoblotting was performed as described previously (Kis et al, 2010b;Ronaldson et al, 2010) with minor modifications. Cells from three different passages were harvested, and lysates were prepared as described previously.…”

Intestinal transporters for endogenic and pharmaceutical organic anions: the challenges of deriving in-vitro kinetic parameters for the prediction of clinically relevant drug–drug interactions

“…Atorvastatin [164] 0.2 Atazanavir [165] 3.6 a Estriol [158] Benzylpenicillin [35] Atorvastatin [164] 0.7 Pregnenolone sulfate [158] Estrone [158] Bosentan [166] 202 Benzylpenicillin [36] 16 000 a Prostaglandin E2 [35] Pregnenolone sulfate [157,158] 3.5 a Fexofenadine [41] Cerivastatin [164] 66 a Taurocholate [41] 72 Testosterone [158] 15 a /21 a Fluvastatin [162,167] 0.7 Cimetidine [36] Glibenclamide [163] 6.3 Ciclosporin [154] 0.07 a Pitavastatin [161] 1.2 Darunavir [165] 26 a Pravastatin [37,41] 2250 Digoxin [164] Rosuvastatin [87,154] 2.4/6.4 Efavirenz [165] 9.6 a Tebipenem pivoxil [156] >1000 Gemfibrozil [154,162,164] 8 a Glibenclamide [164] Indinavir [168] 3.0 Indometacin [36,164] Lopinavir [165] 0.7 a Lovastatin [164] Memantine [164] Mifepristone [158] 2.2 a /4.7 a Nelfinavir [165] 0.9 a Pravastatin [36,37] 5 500 a /1 020 a Probenecid …”

“…[178,179] Thus, the inhibition of the enterocytic influx via OSTa/b is probably not of concern with regards to DDIs. However, OSTa/b is also an intestinal basolateral absorptive transporter (along [165,168] 4.0/4.6 a Simvastatin [164] 85 a Tipranavir [165] 0.9 a OATP3A1_v1 (OATP-D)…”

Identification of natural products as modulators of OATP2B1 using LC-MS/MS to quantify OATP-mediated uptake

“…The absolute amount of E3S transported by OATP2B1 into cells in the absence of inhibitor in a 24-well plate was $70 pmol/well. BSP, a known OATP2B1 inhibitor (Kis et al, 2010;Lan et al, 2009), was used as a positive control and it strongly inhibited OATP2B1-mediated E3S uptake by approximately 90%. Chrysanthemum stem-leaf flavonoids, glycyrrhetinic acid, mulberrin, quercetin, and scutellarin were strong inhibitors of OATP2B1 with more than 80% inhibition.…”