Implications of BCRP in disease modifying anti-rheumatic drugs resistance

July, 2008

Implications of BCRP in disease modifying anti-rheumatic drugs (DMARD) resistance: the example of Leflunomide and its active metabolite

Written by Judit Janossy MSc, Senior Scientific Associate, Solvo Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the lining, or synovium, of the joints. For the treatment of RA the class of disease modifying anti-rheumatic drugs (DMARDs) is commonly used because of their convenience and low costs. One of the drawbacks is that DMARDs can take several weeks or months to display clinical effects. Moreover, due to the long-term application of the drugs many RA patients demonstrate loss of efficacy over time. DMARDs resistance may be a multifactorial event including enhanced drug efflux via ABC transporters, impaired drug uptake and drug activation, enhanced drug detoxification etc.

Members of the ATP binding casette (ABC) transporter superfamily are associated with a broad spectrum of physiological functions, including detoxification, defense against xenobiotics and oxidative stress, absorption, distribution and excretion processes etc (for review see Glavinas et al. 2004). Overexpression of certain ABC transporters, like P-glycoprotein [Pgp, MDR1, ABCB1], the multidrug resistance protein 1 [MRP1, ABCC1], or the breast cancer resistance protein [BCRP, MXR, ABCG2] can mediate multidrug resistance in tumors. As mentioned earlier, previous studies indicated the involvement of P-gp, MRP1 and BCRP in drug resistance to DMARDs, such as chloroquine, sulfasalazine and leflunomide. Leflunomide is a pro-drug that undergoes rapid nonenzymatic conversion to its active form, A771726. It is used either alone or in combination therapy with methotrexate. Methotrexate, another commonly used drug in RA is also a substrate of BCRP.

By using methotrexate as a reporter substrate in the vesicular transport assay, important drug-drug interactions can be revealed between drugs used in combination therapies and sharing a common target. The recently published study of Kis et al. (Kis et al., 2008) using in vitro membrane based methods (ATPase assay and Vesicular transport assay) and cell based methods (Hoechst assay and Cytotoxicity assay) have shown that leflunomide and its active metabolite, A771726 are BCRP substrates. Both displayed dose-dependent, competitive inhibition of BCRP mediated methotrexate transport into inside-out vesicles. Similarly, both drugs inhibited the BCRP mediated efflux of Hoechst 33342 dye from BCRP overexpressing PLB985 cells (Fig. 1). The measurement of vanadate sensitive ATPase activity of BCRP pointed to the substrate nature of the drug-transporter interactions. In cytotoxicity assays, BCRP transporter overexpressing cells showed 20.6 and 7.5-fold resistance compared to the mock vector transfected HEK293 in the case of leflunomide and A771726.

This increased resistance is attributable to the BCRP transporter because it could be reversed by a known specific BCRP inhibitor, Ko134 (Fig.2). This assay set up is considered as a surrogate transport assay, as it refers directly to the involvement of a specific transporter and its role in resistance to the drug tested. The findings of Kis et al. (Kis, 2008) underlie the role of BCRP in resistance to leflunomide and its active metabolite. Moreover, the BCRP-investigated drug interactions are highly specific, as neither drugs showed any interactions with neither MDR1 nor MRP1 transporters. In case resistance is developed for methotrexate, it is administered in combination therapy with Leflunomide.

Several studies confirmed that administration of leflunomide together with methotrexate can lead to improved patient condition. This may be at least in part due to the competitive inhibition between methotrexate and leflunomide or A771726 leading to increased local concentrations of one drug or another. 56.9% of patients treated with methotrexate together with leflunomide achieved ACR20 (Kremer JM), which might be an indirect proof that either drugs efficacy is elevated in the presence of the other which can be due to their interaction with BCRP.

These findings can be the basis of new therapeutic approaches, such as the local administration of BCRP reverting agents or inhibitors in combination with DMARDs. The article of Kis et al. is another example demonstrating that SOLVOs membrane and cellular assay systems are suitable to detect transporter-drug interactions; and as seen by the cytotoxicity assay these may have functional significance for drug therapy.


1. Glavinas, H., Krajcsi, P., Cserepes, J. and Sarkadi, B. The role of ABC transporters in drug resistance, metabolism and toxicity. Curr Drug Deliv 2004,1, 27-42. 2. Kis E, Nagy T, Jani M, Molnár E, Jánossy J, Ujhelly O, Német K, Herédi-Szabó K, Krajcsi P.Leflunomide and A771726, its metabolite are high affinity substrates of BCRP - implications for drug resistance.Ann Rheum Dis. 2008 Apr 8; [Epub ahead of print] 3. Kremer JM, Genovese MC, Cannon GW, Caldwell JR, Cush JJ, Furst DE, Luggen ME, Keystone E, Weisman MH, Bensen WM, Kaine JL, Ruderman EM, Coleman P, Curtis DL, Kopp EJ, Kantor SM, Waltuck J, Lindsley HB, Markenson JA, Strand V, Crawford B, Fernando I, Simpson K, Bathon JM. Concomitant Leflunomide Therapy in Patients with Active Rheumatoid Arthritis despite Stable Doses of Methotrexate - A Randomized, Double-Blind, Placebo-Controlled Trial. Ann Intern Med. 2002;137:726-33.