Pharmacokinetics-based Studies On Chronotoxicity And Chronoefficacy Of Fuzi

ObjectivesThe lateral root of Aconitum carmichaeli Debx（Fuzi）is a well-known traditional Chinese medicine.Fuzi has been widely used as a cardiotonic,analgesic,anti-inflammatory,and diuretic agent to treat colds,polyarthralgia,diarrhea,heart failure,beriberi,and edema.It is noteworthy that main components of Fuzi（such as aconitine,hypaconitine and mesaconitine）show both pharma-cological and toxicological effects.It has been reported that BMAL1 can regulate the metabolism of aconitine and hypaconitine（the main active/toxic components of Fuzi）,and the chronotoxicity of single compound is caused by chronopharmacokinetics.However,it is still unclear whether Fuzi shows chronotoxicity and chronoefficacy.Therefore,this thesis aims to explore whether Fuzi has chronotoxicity and chronoefficacy and its regulatary mechanism,which has implications for optimization of drug efficacy and toxicity.Methods1.Wild-type mice were used as the experimental objects.The toxicity of Fuzi at six circadian points was detected by measuring plasma CK-MB and LDH.Chronopharmacokinetic studies were performed with wild-type mice to estimate the time-varying Fuzi pharmacokinetics.2.Bmal1^-/-mice were used as the experimental objects.Bmal1^-/-mice were administered with Fuzi at ZT10 or ZT22.The toxicity of Fuzi in Bmal1^-/-mice was detected by measuring plasma CK-MB and LDH.Chronopharmacokinetic studies were performed with Bmal1^-/-mice to estimate the time-varying Fuzi pharmacokinetics.Everted gut sac experiments and microsomal enzyme incubation experiments were used to elucidate the regulatory mechanism of chronotoxicity.3.Wild-type and Bmal1^-/-mice with CKD（chronic kidney disease）were used as the experi-mental objects.Renal function test,q PCR assay and histopathological analysis were used to assess the chronoefficacy of Fuzi.Chronopharmacokinetic studies were performed with CKD mice to assess the time-varying Fuzi pharmacokinetics.4.Renal function test and q PCR assay were used to evaluate the therapeutic effects of com-pounds（aconitine,hypaconitine and mesaconitine,three major active compounds of Fuzi）on CKD mice.Chronopharmacokinetic experiments were performed with CKD mice to assess the time-varying pharmacokinetics of three compounds.Results1.Fuzi shows dosing time-dependent toxicity and pharmacokinetics in wild-type mice.Fuzi exhibited a diurnal rhythmicity in cardiotoxicity.The highest level of toxicity was ob-served at ZT10,while the lowest level of toxicity occurred at ZT22.In pharmacokinetic experi-ments,ZT10 dosing of Fuzi generated higher both C_max and AUC（area under the curve,represent-ing systemic exposure of drug）values of aconitine,hypaconitine and mesaconitine compared to ZT22 dosing.2.BMAL1 regulates chronotoxicity and chronopharmacokinetics of Fuzi through modu-lating hepatic metabolism.Bmal1 ablation resulted in an increased level of Fuzi toxicity（i.e.,elevations in CK-MB and LDH levels）at ZT22 dosing,while having no influences on Fuzi toxicity when drug was dosed at ZT10.Circadian time-dependent toxicity of Fuzi was lost in Bmal1^-/-mice.In pharmacokinetic experiments,Bmal1 ablation increased the plasma concentrations of aconitine,hypaconitine and mesaconitine,and reduced the formation of their metabolites.Bmal1 ablation abrogated circadian time-dependency of hepatic Fuzi metabolism.By contrast,intestinal metabolism of Fuzi was cir-cadian time-independent,and was not regulated by BMAL1.Additionally,there were no signifi-cant differences in intestinal efflux transport of Fuzi（aconitine,hypaconitine and mesaconitine）in wild-type mice between ZT10 and ZT22 according to everted gut sac experiments.Taken together,these data suggested that circadian clock（BMAL1）regulated chronotoxicity of Fuzi through mod-ulating hepatic metabolism.3.Fuzi efficacy and pharmacokinetics depend on the dosing time in CKD mice.The Fuzi efficacy against CKD was higher when the drug was dosed at ZT10,and was lower when the drug was dosed at other times.The AUC values of aconitine,hypaconitine and mesaco-nitine for Fuzi dosing at ZT10 were significantly larger than those for herb dosing at ZT22.Alto-gether,Fuzi pharmacokinetics displayed a dosing time-dependency,contributing to the chronoef-ficacy of this herb medicine.4.Aconitine,hypaconitine and mesaconitine are the active ingredients of Fuzi,exerting ef-ficacy against CKD in mice.CKD mice were orally treated with single compound（aconitine,hypaconitine or mesaco-nitine）,and renal functions were tested.All three compounds decreased the levels of plasma CRE and BUN as well as urinary NAG in CKD mice.In addition,all three compounds reduced the levels of inflammatory and fibrotic factors（IL-1β,IL-6,Tnf-α,Ccl2,Tgfb1 and Col1a1）in the kidney.5.Bmal1 ablation abrogates dosing time-dependency in Fuzi pharmacokinetics and effi-cacy.Bmal1 ablation abrogated the dosing time-dependency in Fuzi pharmacokinetics as evidenced by similar plasma concentration-time profiles of aconitine,hypaconitine and mesaconitine be-tween two dosing times（ZT10 and ZT22）.Likewise,dosing time-dependency in renal drug distri-bution was lost in Bmal1^-/-mice.Bmal1 ablation also abrogated the dosing time-dependency in Fuzi efficacy according to biomarker measurements（i.e.,plasma CRE and BUN and urinary NAG）,as well as inflammation,fibrosis and histological analyses.Conclusion1.Fuzi shows dosing time-dependent toxicity and pharmacokinetics in wild-type mice.2.BMAL1 regulates chronotoxicity and chronopharmacokinetics of Fuzi through modulating he-patic metabolism.3.Fuzi efficacy and pharmacokinetics depend on the dosing time in CKD mice.4.Aconitine,hypaconitine and mesaconitine are the active ingredients of Fuzi,exerting efficacy against CKD in mice.5.Bmal1 ablation abrogates dosing time-dependency in Fuzi pharmacokinetics and efficacy.