Leflunomide and A771726, its metabolite are high affinity substrates of BCRP – implications for drug resistance

E Kis ¹, T Nagy ¹, M Jani ¹, É Molnár ¹, J Jánossy ¹, O Ujhelly ², K Német ², K Herédi-Szabó ¹ and P Krajcsi ^1*

¹ Solvo Biotechnology, Hungary
² National Blood Transfusion Service, Hungary

^* To whom correspondence should be addressed. E-mail: krajcsi{at}solvo.com.

Accepted 29 March 2008

Abstract

Background: Earlier publications have suggested a possible role of the efflux transporter, BCRP (ABCG2, MXR) in acquired resistance to DMARDs, like leflunomide and it’s metabolite, A771726 (Teriflunomide). However, direct evidence that BCRP interacts with these drugs was missing.

Objectives: To characterize the interaction between BCRP transporter and leflunomide and its active metabolite, A771726, with emphasis on the nature of interaction (substrate or inhibitor) as well as the kinetic characterization of the interactions.

Methods: Different in vitro membrane based methods (ATPase and Vesicular transport assay) using BCRP-HAM-Sf9 membrane preparations and cellular assays (Hoechst assay and Cytotoxicity assay) on PLB985-BCRP and HEK293-BCRP cell lines overexpressing BCRP were performed.

Results: In all assays applied interaction between the investigated drugs and BCRP was detected. In the vesicular transport assay both leflunomide and its metabolite inhibited the BCRP mediated methotrexate transport. Both compounds are likely substrates of BCRP as revealed by the vanadate sensitive ATPase assay. In line with the membrane assays leflunomide and A771726 inhibited BCRP mediated Hoechst efflux from PLB985-BCRP cells. In the cytotoxicity assay overexpression of BCRP confered 20.6 and 7.5-fold resistance to HEK293 cells against leflunomide and A771726, respectively. The resistance could be reversed by Ko134, a specific inhibitor of BCRP.

Conclusion: Based on the results, we suggest that BCRP via interactions with leflunomide and A771726 could play an important role in the resistance to these drugs. In addition, BCRP may mediate drug-drug interactions when leflunomide is coadministered with other BCRP substrate drugs such as methotrexate.