Drug Metabolism-mediated Tripterygium Wilfordii Chronohepatotoxicity

ObjectivesTripterygium wilfordii Hook F.（TW）is a traditional Chinese medicine with a property of“bitterness and coldness”.Numerous clinical trials have confirmed the efficacy of TW extracts in treatment of rheumatoid arthritis and chronic kidney disease.However,therapeutic applications of TW are limited by the narrow therapeutic window and severe toxic effects（e.g.hepatotoxicity and nephrotoxicity）.Many drug-metabolizing enzymes and transporters are rhythmically expressed in the liver,intestine and kidney,leading to chronopharmacokinetics and thus dosing time-dependent drug effect.However,whether and how the TW-induced hepatotoxicity is regulated by the circadian clock system remain to be explored.Thus,we aimed to determine the diurnal rhythm of Tripterygium wilfordii（TW）hepatotoxicity and to investigate a potential role of clock–controlled drug metabolism and pharmacokinetics in generating chronotoxicity.Methods1.To evaluate the dose-and time-dependency of TW hepatotoxicity,wild-type mice were treated with different doses of TW suspension（equivalent to triptolide doses of 0.225,0.45,0.9,1.35 and 1.8 mg/kg,oral gavage）at ZT2 or ZT14.24 h later,mice were sacrificed and plasma samples were collected.Plasma ALT and AST levels were determined using commercial assay kits according to the manufacturer’s instructions.2.WT mice received a single dose of TW suspension（1.35 mg/kg,oral gavage）or vehicle at each of six circadian time points（ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22）.24 h later,mice were sacrificed and plasma and liver samples were collected.Plasma levels of ALT and AST were determined as described above.Liver tissues were fixed in 4%paraformaldehyde and embedded in paraffin,followed by H&E staining.3.Clock^+/+and Clock^-/-mice received a single dose of TW suspension（1.35 mg/kg,oral gavage）at ZT2 or ZT14.24 h later,mice were sacrificed and plasma and liver samples were collected.Plasma ALT and AST and hepatic GSH levels were determined.Additionally,liver tissues were subjected to histopathological examinations as described above.4.Clock^+/+and Clock^-/-mice were treated with TW（1.35 mg/kg,oral gavage）at ZT2 or ZT14.At each time point（5,15,30,60,120,240 and 480 min）,mice were sacrificed to collect plasma samples.Then UPLC-QTOF/MS was used to detect the main components of TW in plasma（triptolide,tripdiolide,celastrol,wilfordine and wilfortrine）.Pharmacokinetic parameters were determined by using Win Nonlin software.5.Liver samples from Clock^-/-mice and Clock^+/+mice were collected at ZT2 and ZT14,and liver microsomes were prepared by sequential ultracentrifugation.Metabolism of TP was performed using liver microsomes,and TP metabolites were quantified using UPLC-MS.6.Total m RNA was extracted from the livers of Clock^-/-mice and Clock^+/+mice collected at ZT2 and ZT14.The expression levels of CYP genes involved in triptolide metabolism（Cyp3a11,Cyp2c29,Cyp2c37,Cyp2c38,Cyp2c39,Cyp2c50 and Cyp2c54）were determined using RT-q PCR method.Results1.Circadian variations in TW hepatotoxicity in mice.We determined the hepatotoxicity of TW at a dose range of 0.225–1.8 mg/kg.Interestingly,hepatotoxicity of TW was dosing time-dependent at doses of 0.45-1.35 mg/kg.Mice dosed at ZT2 showed more severe toxicity than animals dosed at ZT14.We further determined the hepatotoxicity of TW（1.35 mg/kg）dosed at each of six circadian time points（ZT2,6,10,14,18and 22）.TW hepatotoxicity displayed significant diurnal variations with a peak level at ZT2 and a trough level at ZT14,Histopathological examinations（H&E staining）showed a higher incidence of balloon-like degeneration,vacuolization,cell swelling and lymphocyte infiltration in mouse livers at dosing time of ZT2 than at ZT14.Taken together,these data indicated circadian time-dependent toxicity（chronotoxicity）of TW in mice.2.Circadian time-dependent pharmacokinetics of TW in mice.It was postulated that the circadian variations in TW hepatotoxicity might be associated with dosing time-dependent exposure of active and toxic constituents in TW.We determined the pharmacokinetic profiles of these compounds after TW treatment at ZT2 or ZT14.TW dosing at ZT2 generated higher plasma concentrations of triptolide compared with ZT14 dosing.The systemic exposure（reflected by AUC,area under the curve）for triptolide at ZT2 was higher than that at ZT14.By contrast,pharmacokinetic behaviours of the other four constituents（tripdiolide,celastrol,wilfordine and wilfortrine）were not different between two dosing times.The circadian variation in triptolide pharmacokinetics（AUC:ZT2>ZT14）may account for time-dependent triptolide hepatotoxicity（ZT2>ZT14）.3.Clock ablation abrogates time dependency of TW toxicity and of pharmacokinetics.Compared with Clock^+/+mice,Clock^-/-mice were more sensitive to TW-induced toxicity at ZT14 dosing（with an increased plasma ALT level,decreased liver GSH and higher histopathological score）.By contrast,the pharmacokinetic profiles of triptolide and its metabolites did not show significant differences between two dosing times in Clock^-/-mice.As a result,the time dependency of TW toxicity was abolished in Clock^-/-mice.These data indicated a regulatory role for circadian clock in generation of TW chronotoxicity.4.Clock regulates hepatic metabolism of triptolide.In line with the in vivo pharmacokinetic study,the generation of TP metabolites in liver microsomes from wild-type Clock^+/+mice also showed a significant time-dependent variation,with a higher level at ZT14 than at ZT2.In the absence of clock gene,the metabolic rate of TP was largely reduced and the rhythmicity in TP metabolism was abolished.5.Clock ablation down-regulates Cyp3a11 expression.A circadian variation in the expression level of Cyp3a11 m RNA was observed in wild-type Clock^+/+mice,with a higher level at ZT14 than at ZT2.In contrast,ablation of Clock gene not only decreased Cyp3a11 m RNA level at ZT14 but also blunted the circadian rhythm.In addition,hepatic Cyp2c genes were not rhythmic and their expression levels were not altered after Clock ablation.ConclusionWe have observed a diurnal rhythmicity in TW hepatotoxicity.TW chronotoxicity was associated with diurnal variations in triptolide pharmacokinetics and circadian expression of hepatic CYP3A11 regulated by circadian clock.Our findings may have implications for improving TW treatment outcome with a chronotherapeutic approach.