title: Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity.
abstract: OBJECTIVE
  Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo.
  
  
  METHODS
  The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg · kg(-1) · d(-1), ig) for 4 weeks, their blood samples were analyzed.
  
  
  RESULTS
  A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 μmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 μmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg(-1) · d(-1)) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg(-1) · d(-1), all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats.
  
  
  CONCLUSIONS
  Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major metabolite teriflunomide suppresses the expression and function of NTCP, leading to potential cholestasis.
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|Hongqiao International Institute of Medicine, Shanghai Tongren Hospital and Faculty of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Lei-lei
authors.initials: LL
authors.lastname: Ma
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Zhi-tao
authors.initials: ZT
authors.lastname: Wu
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Le
authors.initials: L
authors.lastname: Wang
authors.affiliation: Jiangsu Tripod Preclinical Research Laboratories Inc, Nanjing 211800, China.
authors.forename: Xue-feng
authors.initials: XF
authors.lastname: Zhang
authors.affiliation: Jiangsu Tripod Preclinical Research Laboratories Inc, Nanjing 211800, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Ni
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Yun-fei
authors.initials: YF
authors.lastname: Lin
authors.affiliation: Hongqiao International Institute of Medicine, Shanghai Tongren Hospital and Faculty of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Yi-yi
authors.initials: YY
authors.lastname: Cao
authors.affiliation: Hongqiao International Institute of Medicine, Shanghai Tongren Hospital and Faculty of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Luan
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Guo-yu
authors.initials: GY
authors.lastname: Pan
mesh_terms: D000818:Animals; D018501:Antirheumatic Agents / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity*; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism* / Q000473:pathology; D003437:Crotonates / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D006885:Hydroxybutyrates; D007555:Isoxazoles / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity*; D000077339:Leflunomide; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008810:Mice, Inbred C57BL; D009570:Nitriles; D029363:Organic Anion Transporters, Sodium-Dependent / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014052:Toluidines / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity
chemical_list: D018501:Antirheumatic Agents; D003437:Crotonates; D065607:Cytochrome P-450 Enzyme Inhibitors; D006885:Hydroxybutyrates; D007555:Isoxazoles; D009570:Nitriles; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; D014052:Toluidines; C071908:sodium-bile acid cotransporter; C527525:teriflunomide; D003577:Cytochrome P-450 Enzyme System; D000077339:Leflunomide