| ObjectiveFuzi is a Traditional Chinese Medicinine(TCM)which has been used in China for thousands years.Fuzi appear to possess excellent pharmacoligical activities,such as cardiotonic,dilated vessel,antiarrhythmia,strengthen the immune system,analgesia,antiinflammation and some other effects.Nevertheless,Fuzi is a toxic herb that can cause fatal poisoning,since therapeutic dose of Fuzi is very close to its toxic dose,frequent cases of poisoning after the intake of Fuzi are encountered in clinics.In recent decades a lot of researches illustrated that aconitine not only with pharmacological activity,but also is one of the highly toxic alkaloid of Fuzi,it was often studied as a marker ofFuzi.The efflux transporters P-glycoprotein(P-gp)and breast cancer resistance protein(BCRP)transport their substrate drugs out of the cell from intracellular,this process regulate the drug transportation and disposition in vivo,such as absorption,distribution and excretion of drugs.Our previous study indicated that P-gp and BCRP involved in the transportation of aconitine in vitro,but the related in vivo study was not published at present So,we further investigate the effect of P-gp and BCRP on aconitine disposition in mice.The results of the present study could boost the scientific recognition of relationship between toxicity and effectiveness of Fuzi and provide significant information for the safe clinical practice ofFuzi.Methods1.Pharmacokinetics study of aconitine following oral administration in mice To investigate the effect of efflux transporters on aconitine disposition in vivo,the gene knockout mice Mdrl a-/-,Bcrpl-/-mice with FVB background and wild type FVB mice were used.Blood samples were collected after a single oral dose of aconitine(0.1 mg/kg or 0.2 mg/kg),concentration of aconitine was detected by UHPLC-MS/MS,the pharmacokinetic parameters(Tmax,Cmax,t1/2,CL,AUC0-12,MRT0-12h)were calculated by WinNonLin3.3.2.The tissue distribution of aconitine in miceTo investigate the effect of efflux transporters on tissue distribution of aconitine,the gene knockout mice Mdr1a-/-,Bcrp1-/-mice with FVB background and wild type FVB mice were used.After a single oral dose of aconitine(0.1 mg/kg or 0.2 mg/kg),mice were anesthetized and sacrificed after blood collection at scheduled time point,tissues were taken out of mice and homogenized,the concentration of aconitine in homogenate wre detected by UHPLC-MS/MS.3.Statistical analysisData were expressed as means ± standard errors of the means.Statistical evaluation was carried out by Students't-test or One-way ANOVA.A value of p<0.05 was regarded as being statistically significant.Results1.The effect of efflux transporter on aconitine pharmacokinetics in miceThe pharmacokinetic parameter t1/2 was significantly increased in Mdr1a-/-and Bcrp1-/-mice compared with FVB mice(p<0.05).After 0.1 mg/kg aconitine was administrated in male FVB,Mdr1a-/-,Bcrp1-/-mice the t1/2 of aconitine were 58.8 ± 13.4,105.4 ± 19.1 min,respectively,and 110.0 ± 33.6 min,and the t1/2 of aconitine were 51.5 ± 12.8?107.9 ± 32.6?95.5 ± 15.9 min in female FVB,Mdrla-/-,Bcrp1-/-mice.The t1/2 of aconitine of male FVB,Mdrla-/1,Bcrpr1-/-mice were 69.1 ± 23.3,277.0 ± 52.3,and 110.0 ± 32.9 min,respectively,4-fold increased in Mdrl a-/-mice and 2-fold increased in Bcrp1-/-mice compared with FVB mice after oral administration of 0.2 mg/kg aconitine.In FVB mice the pharmacokinetic parameters Cmax and AUC0-12h was significantly increased in female compared with male(p<0.05).The pharmacokinetic parameters Cmax and AUC0-12h of aconitine were 1.7-fold increased in female mice compared with male mice(p<0.05)after oral administration of 0.1 mg/kg aconitine.The Cmax of aconitine were 6.5± 1.2 and 11.3 ± 2.3 ng/mL,AUC0-12h of aconitine were 671.4 ± 154.4 and 1108.4 ± 285.9 min·ng/mL,respectively,after 0.1 mg/kg aconitine oral administrated in male and female FVB mice.2.Physiological symptoms after aconitine was administered in miceAfter 0.1 mg/kg aconitine was administrated,Mdrla-/-mice suffered severe aconitine poisoning,while aconitine poisoning symptoms did not arise in female FVB mice.The male Mdrla-/-mice suffered vomiting,salivation,diarrhea,convulsion,abnormal breathing,and even death after oral administration of 0.2 mg/kg aconitine.All the female Mdrla-/-mice were dead within 8 hours after oral administration of 0.2 mg/kg aconitine.Vomiting,inactivity,unsteady walking,and abnormal breathing occurred on male FVB mice,only slight vomiting was observed on female FVB mice after 0.2 mg/kg aconitine oral administrated.3.The effect of efflux transporter on aconitine tissue distribution in miceThe concentration of aconitine in Mdrla-/-mice brain,brain-to-blood ratio and brain-to-heart ratio were significantly increased compared with male FVB mice(p<0.05).After oral administration of 0.1 mg/kg aconitine in male FVB and Mdrla-/-mice at time point 4 h,the concentration of aconitine in brain were 0.2 ± 0.2 and 7.7 ± 2.6 ng/g,the brain-to-blood ratio were 1.3 ± 1.4 and 5.5 ± 1.8 and the brain-to-heart ratio were 0.5 ± 0.5 and 2.4 ± 1.4,respectively.After 0.2 mg/kg aconitine was administrated in male FVB and Mdrla-?-mice at time point 4 h,the concentration of aconitine in brain were 0.9 ± 0.5 and 8.7 ± 0.6ng/g,the brain-to-blood ratio were 1.3 ± 0.9 and 4.7 ± 1.0,the brain-to-heart ratio were 0.5 ± 0.3 and 2.7 ± 0.9 respectively.In FVB mice the concentration of aconitine in brain,brain-to-blood ratio and brain-to-heart ratio were significantly increased in male mice compared with female mice(p<0.05).At time point 30 min,2 h after 0.2 mg/kg aconitine oral administrated in FVB mice the concentrations of aconitine in brain were 6.5 ± 2.8 and 4.6 ± 2.2 ng/g for malemice,werel.4 ± 0.7and 0.8 ± 0.3 ng/g for female FVB mice,respectively.At time point 10 min and 30 min after administration of aconitine 0.2 mg/kg,the brain-to-blood ratio were 0.11 ± 0.03 and 0.68 ± 0.30 for male mice,were 0.06 ± 0.01 and 0.08 ± 0.05 for female mice,at time point 10 min,30 min,2 h the brain-to-heart ratio were 0.05 ± 0.01,0.68 ±0.30 and 0.4 ± 0.1 for male mice,were 0.06 ± 0.01,0.09 ± 0.04 and 0.1 ± 0.07 for female mice respectively.Conclusions1.The biological half-life of aconitine in mice was extended when P-gp and BCRP were knockout2.Aconitine can pass through blood-brain barrier easier and distribute in brain when P-gp was knockout,and toxicity of aconitine was increased in P-gp knockout mice.3.In FVB mice the brian distribution of aconitine in male mice is singnificantly higher than female mice,so that the toxicity of aconitine in male mice was more serious than male mice.however,the aconitine exposure level in female mice blood is singnificantly higher than male mice. |
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