title: Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance.
abstract: BACKGROUND
  Earlier publications have suggested a possible role for the efflux transporter breast cancer resistance protein (BCRP) in acquired resistance to disease-modifying antirheumatic drugs (DMARDs) such as leflunomide and its metabolite A771726 (teriflunomide). However, there is no direct evidence that BCRP interacts with these drugs.
  
  
  OBJECTIVE
  To characterise the interaction between BCRP transporter and leflunomide and its active metabolite A771726, with emphasis on the nature of the interaction (substrate or inhibitor) and the kinetic characterisation 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) were performed on PLB985-BCRP and HEK293-BCRP cell lines overexpressing BCRP.
  
  
  RESULTS
  In all assays used, an interaction between the investigated drugs and BCRP was detected. In the vesicular transport assay, both leflunomide and its metabolite inhibited BCRP-mediated methotrexate transport. Both compounds are likely substrates of BCRP as shown 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 conferred 20.6-fold 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.
  
  
  CONCLUSIONS
  Based on these results, BCRP could play an important role in the resistance to leflunomide and A771726 via interactions with these drugs. BCRP may also mediate drug-drug interactions when leflunomide is administered with other BCRP substrate drugs such as methotrexate.
authors.affiliation: Solvo Biotechnology, Szeged, Hungary.
authors.forename: E
authors.initials: E
authors.lastname: Kis
authors.forename: T
authors.initials: T
authors.lastname: Nagy
authors.forename: M
authors.initials: M
authors.lastname: Jani
authors.forename: E
authors.initials: E
authors.lastname: Molnár
authors.forename: J
authors.initials: J
authors.lastname: Jánossy
authors.forename: O
authors.initials: O
authors.lastname: Ujhellyi
authors.forename: K
authors.initials: K
authors.lastname: Német
authors.forename: K
authors.initials: K
authors.lastname: Herédi-Szabó
authors.forename: P
authors.initials: P
authors.lastname: Krajcsi
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000187:drug effects*; D000251:Adenosine Triphosphatases / Q000187:drug effects; D000814:Aniline Compounds / Q000494:pharmacology*; D018501:Antirheumatic Agents / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003437:Crotonates; D004351:Drug Resistance; D006801:Humans; D006885:Hydroxybutyrates / Q000494:pharmacology*; D007555:Isoxazoles / Q000494:pharmacology*; D000077339:Leflunomide; D009363:Neoplasm Proteins / Q000187:drug effects*; D009570:Nitriles; D014052:Toluidines
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000814:Aniline Compounds; D018501:Antirheumatic Agents; D003437:Crotonates; D006885:Hydroxybutyrates; D007555:Isoxazoles; D009363:Neoplasm Proteins; D009570:Nitriles; D014052:Toluidines; C527525:teriflunomide; D000251:Adenosine Triphosphatases; D000077339:Leflunomide