Pharmacokinetics-based Studies On Chronotoxicity And Chronoefficacy Of Semen Strychni

Objectives Semen Strychni(also known as Nux vomica)is the dried seed of Strychnos nux-vomica L.(Loganiaceae),widely used to treat rheumatic diseases in Asia.Previous studies showed that BMAL1 could regulate the toxicity of the compounds derived from Semen Strychni,but the relationship between toxicity/efficacy of Semen Strychni and the biological clock is still unclear.This study aimed to determine the time dependent-toxicity/efficacy of Semen Strychni in mice and to investigate the role of chronopharmacokinetics in generating chronotoxicity and chronoefficacy of Semen Strychni,and to provide a theoretical basis for optimization of the administration time of Semen Strychni.Methods 1.Wild-type mice were used as the experimental objects,and administered with the Semen Strychni extract(SSE,0.4 g/kg,oral gavage)at different time points(ZT2,6,10,14,18 or 22).The nephrotoxicity and neurotoxicity of Semen Strychni were evaluated by measuring plasma creatinine and CK-BB(creatine kinase-BB).In addition,mice were administered with a high dose of SSE(0.8 g/kg,oral gavage)at different time points.The survival curve and the average survival time of mice were recorded to verify the time-dependent toxicity of SSE.In order to investigate the chronopharmacokinetics of SSE,mice were administered with SSE(0.4 g/kg,oral gavage)at ZT6 or ZT18(two time points with the largest difference in SSE toxicity),and the pharmacokinetic parameters were analyzed.2.Bmal1-/-(a diurnal rhythm disrupted model)mice were used as the experimental objects.Bmal1-/-mice were administered with the SSE(0.4 g/kg,oral gavage)at ZT6 or ZT18.Plasma creatinine and CK-BB were determined and pharmacokinetic parameters of SSE were analyzed.Furthermore,metabolism and transport of SSE in wild type and Bmal1-/-mice were determined by microsomal metabolism assays and everted gut sac experiments..3.Wild-type and Bmal1-/-mice were used as the experimental objects.q PCR assay was performed to detect the Semen Strychni-related metabolic enzymes and transporters,and to analyze the temporal variation in expression.4.The wild-type mice were used as the experimental objects,and collagen was used to establish a mouse rheumatoid arthritis model.Different concentrations of Semen Strychni were used to determine the therapeutic effect on CIA(collagen-induced arthritis)mice.To assess the chronoefficacy of Semen Strychni,Enzyme linked immunosorbent assay(ELISA),q PCR,and H&E staining were performed.ELISA was used to detect the levels of inflammatory factors.q PCR was used to analyze the m RNA expression of disease-related factors.H&E staining was used to characterize the tissue damage.In addition,SSE was administrated to CIA mice at different time points,the plasma levels of the main components of SSE were analyzed by mass spectrometry and the pharmacokinetic parameters were estimated.5.Primary fibroblast-like synoviocytes were used to evaluate the anti-inflammatory activities of strychnine and brucine.Results 1.The toxicity and pharmacokinetics of Semen Strychni were time-dependent.Semen Strychni nephrotoxicity and neurotoxicity as well as average survival time of mice,displayed significant diurnal rhythms.The toxicity was the most severe at ZT18 and less severe at ZT2 to ZT6.According to pharmacokinetic experiments,Semen Strychni dosing at ZT18 generated higher plasma concentrations(and systemic exposure)of strychnine and brucine(two toxic constituents)compared with ZT6 dosing.This was accompanied by the lower formation of both dihydroxystrychnine and strychinine glucuronide(two strychnine metabolites)at ZT18.2.The time-dependent pharmacokinetics and toxicity of Semen Strychni are related to the regulation of metabolism and transport by BMAL1.Bmal1 ablation sensitized mice to Semen Strychni-induced nephrotoxicity and neurotoxicity(with increased levels of plasma creatinine and CK-BB),and abolished the time-dependency of toxicity.Metabolism of Semen Strychni(represented by strychnine and brucine)in liver and intestine microsomes of wild-type mice was more extensive at ZT6 than at ZT18.The time-dependency in hepatic and intestinal metabolism were lost in Bmal1-/-mice.Additionally,efflux transport of Semen Strychni(strychnine and brucine)was more extensive at ZT6 than ZT18 in wild-type mice.However,the time-varying transport difference was abolished in Bmal1-/-mice.The expression of metabolic enzymes such as CYP3A11,CYP3A13,UGT2B5 and transporters MRP2,BCRP and P-gp in wild-type mice were higher at ZT6 than ZT18.However,in Bmal1-/-mice,the expression levels of metabolic enzymes and transporters were decreased and the rhythms were lost.These results suggested that BMAL1 can affect the exposure of Semen Strychni in plasma by affecting the rhythmic expression of related metabolic enzymes and transporters,so that the metabolism and toxicity of Semen Strychni were time-dependent.3.The anti-inflammatory effect of Semen Strychni was time-varying and related to chronopharmacokinetics.Semen Strychni dosed at ZT18 was more effective in protecting mice from rheumatoid arthritis than drug dosed at ZT6.Specifically,the levels of inflammatory factors in CIA mice dosed at ZT18 were significantly lower than those dosed at ZT6.In the meantime,the histopathological examinations also indicated a better drug effect at ZT18.The systemic exposure of strychnine and brucine in CIA mice treated at ZT18 were higher than at ZT6.In addition,Semen Strychni didn’t show a significant toxic effect when it was given at the effective dose.4.The main active components of Semen Strychni showed anti-inflammatory effects.Strychnine and brucine significantly inhibited the abnormal proliferation of primary fibroblast-like synoviocytes,reduced the production of TNF-α and IL-6,and reduced the m RNA expression of Tnf-α,Il-6,Cox-2 and i NOS,suggesting that they are active components of Semen Strychni against rheumatoid arthritis.Conclusion 1.The toxicity and efficacy of Semen Strychni are time-dependent and related to chronopharmacokinetics;2.BMAL1 regulates the chronopharmacokinetics of Semen Strychni by regulating metabolic enzymes/transporters;3.Strychnine and brucine show an anti-inflammatory effect.