ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2.

Introduction: ABCG2 has a broad substrate specificity and is one of the most important efflux proteins modulating pharmacokinetics of drugs, nutrients, and toxicokinetics of toxicants. ABCG2 is an important player in transporter-mediated drug-drug interactions (tDDI). Areas covered: The aims of the review are i) to cover transporter interaction profile of substrates and inhibitors that can be utilized to test interaction of drug candidates with ABCG2, ii) to highlight main characteristics of in vitro testing and iii) to describe the structural basis of the broad substrate specificity of the protein. Preclinical data utilizing Abcg2/Bcrp1 knockouts and clinical studies showing effect of ABCG2 c.421C>A polymorphism on pharmacokinetics of drugs have provided evidence for a broad array of drug substrates and support drug – ABCG2 interaction testing. A consensus on using rosuvastatin and sulfasalazine as intestinal substrates for clinical studies is in the formation. Other substrates relevant to the therapeutic area can be considered. Monolayer efflux assays and vesicular transport assays have been extensively utilized in vitro. Expert opinion: Clinical substrates display complex pharmacokinetics due to broad interaction profiles with multiple transporters and metabolic enzymes. Substrate-dependent inhibition has been observed for several inhibitors. Harmonization of in vitro and in vivo testing makes sense. However, rosuvastatin and sulfasalazine are not efficiently transported in either MDCKII or LLC-PK1 based monolayers. Caco-2 monolayer assays and vesicular transport assays are potential alternatives.

The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

Ivermectin has long been considered a model substrate of ABCB1 (P-glycoprotein, MDR1). Ivermectin brain exposure increased 70-fold in CF-1 mice deficient in Abcb1a,¹ x 1. Kwei, G.Y., Alvaro, R.F., Chen, Q., Jenkins, H.J., Hop, C.E., Keohane, C.A., Ly, V.T., Strauss, J.R., Wang, R.W., Wang, Z., Pippert, T.R., and Umbenhauer, D.R. Disposition of ivermectin and cyclosporin A in CF-1 mice deficient in mdr1a P-glycoprotein. Drug Metab Dispos. 1999; 27: 581–587 PubMed | Google Scholar See all References 87-fold in Abcb1a knockout mice² x 2. Schinkel, A.H., Smit, J.J., van Tellingen, O., Beijnen, J.H., Wagenaar, E., van Deemter, L., Mol, C.A., van der Valk, M.A., Robanus-Maandag, E.C., te Riele, H.P., Riele, H.P., Berns, A.J., and Borst, P. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to increased sensitivity to drugs. Cell. 1994; 77: 491–502 Abstract | Full Text PDF | PubMed | Scopus (1832) | Google Scholar See all References leading to a 100-fold increase in neurotoxicity.² x 2. Schinkel, A.H., Smit, J.J., van Tellingen, O., Beijnen, J.H., Wagenaar, E., van Deemter, L., Mol, C.A., van der Valk, M.A., Robanus-Maandag, E.C., te Riele, H.P., Riele, H.P., Berns, A.J., and Borst, P. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to increased sensitivity to drugs. Cell. 1994; 77: 491–502 Abstract | Full Text PDF | PubMed | Scopus (1832) | Google Scholar See all References Abcb1 is a crucial determinant of ivermectin pharmacokinetics inasmuch as several coadministered Abcb1-interacting drugs have been shown to significantly alter pharmacokinetics of ivermectin across a board of different species (reviewed in 3. x 3. Lespine, A., Dupuy, J., Alvinerie, M., Comera, C., Nagy, T., Krajcsi, P., and Orlowski, S. Interaction of macrocyclic lactones with the multidrug transporters: The bases of the pharmacokinetics of lipid-like drugs. Curr Drug Metab. 2009; 10: 272–288 Crossref | PubMed | Scopus (35) | Google Scholar See all References , 4. x 4. Gonzalez-Canga, A., Fernandez-Martinez, N., Sahagun-Prieto, A., Diez-Liebana, M.J., Sierra-Vega, M., and Garcia-Vieitez, J.J. A review of the pharmacological interactions of ivermectin in several animal species. Curr Drug Metab. 2009; 10: 359–368 Crossref | PubMed | Scopus (7) | Google Scholar See all References ).