Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity

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, ig) for 4 weeks, their blood samples were analyzed. Results: A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC 50 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 (AUC 0-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, 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.

Effects of paeoniflorin‐6′‐O‐benzene sulfonate on the pharmacokinetics, excretion and tissue distribution of leflunomide in rats

“…However, in primary rat hepatocytes, CYP enzymes are only directly involved in LEF-related detoxification and not in TER-related detoxification. 36 Because LEF in vivo levels following its oral administration are usually very low, and the downstream metabolites of TER are not known, it is difficult to determine whether CP-25 can promote TER metabolism. Transporters, rather than CYP enzymes, play a unique role in TER-induced liver toxicity.…”

“…TER can significantly reduce the expression and function of the sodium/bile acid cotransporter (NTCP), thereby interfering with bile acid circulation and causing potential bile acid-related liver problems. 36 Pae exerts significant therapeutic effects against alphanaphthylisothiocyanate (ANIT)-induced cholestasis, and the potential mechanism underlying the effects of Pae in alleviating ANIT-induced cholestasis appears to be related to increased expression of NTCP, the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2). 37 Therefore, CP-25 may promote TER excretion and reduce its exposure in the liver by increasing the expression of efflux transporters such as NTCP, BSEP and MRP2.…”

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2

“…In the present study, the inhibitory effect of LEF and its metabolite TER on OAT1/3 was evaluated. Because LEF will be converted to its active metabolite TER in a very short time in vivo [ 2 , 27 ], TER was employed to assess the impact of LEF on the related transporters in subsequent in vitro studies. The calculated IC 50 values of TER on OAT1 and OAT3 were 3.39 μmol/L and 0.87 μmol/L, respectively (Fig.…”

“…The plasma concentration and the urinary concentration of acyclovir were determined by liquid chromatography–mass spectrometry tandem mass spectrometry (LC–MS/MS) (LCMS-8030; Shimadzu, Kyoto, Japan). The plasma concentration of TER was determined by an LC-MS/MS method as previously established in our lab [ 27 ].…”

Hepatic CYP3A4 Enzyme Compensatively Maintains Endogenous Geranylgeranoic Acid Levels in MAOB-Knockout Human Hepatoma Cells

“…First, we examined the sensitivity of endogenous GGA levels in Hep3B/MAOB-KO cells to cytochrome P450 enzyme inhibitors. A well-known nonspecific and irreversible inhibitor of cytochrome P450 enzymes [18], 1-aminobenzotriazole (ABT), dose-dependently decreased the endogenous GGA content in Hep3B/MAOB-KO cells with an IC 50 (the half maximal inhibitory concentration) of 10.9 µM, whereas it did not affect the endogenous GGA levels in MAOB wild-type Hep3B cells (Hep3B/MAOB-WT cells) (Figure 1A). Bergamottin (BG), another broad-specific inhibitor of cytochrome P450 enzymes, also dosedependently reduced the amount of endogenous GGA only in Hep3B/MAOB-KO cells with an IC 50 of 7.5 µM (Figure 1B), but not in Hep3B/MAOB-WT cells.…”