Kinetic Characterization of Sulfasalazine Transport by Human ATP-Binding Cassette G2

Abstract

Sulfasalazine is an anti-inflammatory agent used for the treatment of chronic inflammatory bowel diseases and rheumatoid arthritis. It displays low passive permeability and low oral bioavailability. ATP-binding cassette G2 (ABCG2) (breast cancer resistance protein (BCRP), mitoxantrone resistance protein (MXR)), an ATP driven efflux pump present at many important pharmacological barriers, most notably in the apical membrane of enterocytes, brain microcapillary endothelial cells, placenta, as well as in the canalicular membrane of hepatocytes 1) is known to influence the pharmacokinetic properties of many low and high permeability drugs. 2)Cells overexpressing the transporter show drug resistance to multiple anti-proliferative drugs used in cancer treatment. 3)Earlier studies indicated that sulfasalazine is a substrate of ABCG2. Its high efflux ratio on Caco-2 monolayer was unresponsive to verapamil, an ATP-binding cassette B1 (ABCB1) (P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)) inhibitor, 4) but was decreased by fumitremorgin C, an ABCG2 inhibitor. 5) Resistance to sulfasalazine was linked to ABCG2 overexpression in T-lymphocytes. 6) In abcg2 knock out mice compared to control animals the area under curve (area under blood concentration vs. time curve) (AUC ) and the C max level of sulfasalazine was over 100 fold and over 70 fold elevated respectively. 7) Clinical studies revealed that in vitro sulfasalazine transport 8) and in vivo measured plasma concentrations 9) correlate to single nucleotide polymorphisms in the ABCG2 gene. The studies suggest that sulfasalazine is an ideal probe substrate for ABCG2.Yet, hitherto the kinetic parameters of the interaction were still undetermined. Due to its low passive permeability, sulfasalazine requires a presently unknown uptake transporter. Therefore, its intracellular concentration is also largely unknown. Thus, kinetic characterization of the ABCG2-sulfasalazine interaction in cell based experiments is difficult. In contrast, membrane preparations contain inside out vesicles with the ABCG2 binding sites facing the external buffer. This enables precise control of the sulfasalazine concentration the transporter is exposed to.It was reported that sulfasalazine activates ABCG2 ATPase 10) in membrane based assays. In this work the detailed kinetic characterization of sulfasalazine-ABCG2 interaction is described using direct vesicular transport and ATPase assays at pH 7.0 and pH 5.5. MATERIALS AND METHODSAll chemicals were purchased from Sigma (Sigma-Aldrich Kft, Budapest, Hungary). Membrane preparations from mammalian cells selectively overexpressing ABCG2 were obtained from Solvo (Solvo Biotechnology, Budaors, Hungary).ATPase activity was measured as described earlier. 11)Membrane vesicles (SB-BCRP-M-ATPase; 20 mg/well) were incubated in 10 mM MgCl 2 , 40 mM MOPS-Tris (pH 7.0), 50 mM KCl, 5 mM dithiothreitol, 0.1 mM EGTA, 4 mM sodium azide, 1 mM ouabain, 5 mM ATP, and in the presence of various concentrations of test drugs with or without 1.2 mM sodium ort...

Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

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The role of drug transporters in the kidney: lessons from tenofovir

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Deletion ofAbcg2Has Differential Effects on Excretion and Pharmacokinetics of Probe Substrates in Rats

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