Abstract
Aim:
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.
Conclusion:
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.
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Acknowledgements
We are grateful to Cinkate Pharmaceutical Intermediates Co, Ltd (Shanghai, China) for providing leflunomide and teriflunomide as gifts. We thank Dr Lei GUO (NCTR, USA) for helpful discussions and comments. We thank Dr Jun GU (New York State Department of Health, USA) for kindly providing the HRN mice.
This project is supported by the Key Projects of National Science and Technology Program, China (Grant No 2012ZX09301001-006, 012ZX09302003 and 2012ZX09301001), and National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program”, China (Grant No 2012ZX09303-001).
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Supplemental Figure S1
Cytotoxicity of LEF and TER in primary rat hepatocytes. (JPG 149 kb)
Supplemental Figure S2
Effect of LEF and TER on the biliary excretion of d8-TCA. (JPG 224 kb)
Supplemental Methods
Measurement of the mitochondrial membrane potential (MMP) (DOC 126 kb)
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Ma, Ll., Wu, Zt., Wang, L. et al. Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity. Acta Pharmacol Sin 37, 415–424 (2016). https://doi.org/10.1038/aps.2015.157
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DOI: https://doi.org/10.1038/aps.2015.157
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