Enzyme Kinetics Of Koumine In Liver Microsomes In Vitro And Its Toxicokinetics In Rhesus Monkeys,SD Rats In Vivo

Koumine is one of highest active components of Gelsemium alkaloids with high efficiency and low toxicity,and a great prospect in new drugs creation.The metabolic research of drugs is an important part of the research and development of new drug.The research group had made a preliminary comparative study about the metabolism of koumine in liver microsomes of human and various animals in vitro,and the analysis of metabolized enzyme subtype.This research would further carry out the analysis of enzyme kinetic parameters of koumine in liver microsomes of different species in vitro,to predict its metabolic kinetics in vivo.The koumine in plasma samples was quantified by LC-MS/MS method,to carry out toxicokinetic studies of koumine in rhesus monkeys and SD rats,and therefore to evaluate their systemic exposure levels and to confirm the relationship between the exposure and dose or the time of administration.And providing a reference for clinical safety evaluation.Part one: The study of enzyme kinetics of koumine in liver microsomes in vitroThe degradation curves of the series of concentration of koumine（0.15,0.29,0.73,1.83,3.65 mmol·L-1）and incubated various liver microsomes were determined by the optimized reaction system and UPLC detection method.The kinetic parameters of koumine in various species enzymes were calculated by substrate elimination method.Results: In liver microsomes of human,the K_m,V_max,T_1/2 and CL_int of koumine were（0.135 ± 0.067）mmol·L-1,（1.89 ± 0.19）μmol·min-1·g^-1pro,（712 ± 23）min and（15.7 ± 5.2）mL·min-1·g^-1pro.Compared with human,dog and pig had larger K_m,and their K_m were（0.673 ± 0.042）and（0.556 ± 0.188）mmol·L-1;Rat and monkey had higher CL_int,and their CL_int were（144.1±18.1）and（230.1±95.6）mL·min-1·g^-1pro;The V_max of koumine in the four laboratory animals were large,and their V_max ranged from（5.69 ± 1.13）to（19.16 ± 1.14）μmol·min-1·g^-1pro;The T_1/2 of koumine in the four laboratory animals were short,and their T_1/2 ranged from（120 ± 11）to（362 ± 38）min.Part two: The establishment of LC-MS/MS method for determining the content of koumine in rhesus monkey and SD rat plasmaThe plasma samples were treated with liquid-liquid extraction method,and the internal standard was Gelsemine.HPLC conditions: The analysis was achieved by Gemini 5μ C18 chromatogram column（110A,50×2.0 mm）.The mobile phase was composed of methanol-water-ammonia（49∶51∶0.00625）at a flow rate of 0.2 mL·min-1.The temperature of column was 30 ℃.The injection volume was 5 μL.MS conditions: Detection was performed by positive ion electrospray ionization（ESI）in multiple-reaction monitoring（MRM）mode;Monitoring the transitions m/z 307.2→m/z 180.2（koumine）,m/z 323.2→m/z 236.1（Gelsemine）;The flow rate of dry gas was 10 L·min-1;The temperature of dry gas was 350 ℃;Spray gas,25 psi;Vcap,3500 V;Dissociation voltage of collision induced,140 V（koumine）,135 V（Gelsemine）;Collision energy,56 V（koumine）,25 V（Gelsemine）;The dwell time was 200 ms.Results: The method of LC-MS/MS was established for determining the content of koumine in plasma,had a good specificity,and the linearity was good in the range of 0.1²400 ng·mL-1 and 0.1¹60 ng·mL-1.The lower limit of quantification was 0.1 ng·mL-1.The precision of intra-day and inter-day were good,and their RSD were both less than 15%.The recoveries were more than 70%.The recovery of the method and matrix effect ranged from 85% to 115%.Plasma samples were placed at room temperature for six hours and three freeze-thaw cycles,the samples were placed in the injector for 24 hours and then analyed were stable.Part three: The study of the acute toxicity test accompanied toxicokinetics in rhesus monkeys by nasal feeding koumineThe acute toxicity test in rhesus monkeys by nasal feeding koumine.Using pyramiding dosage design in the doses of 5,10,20,40 and 80 mg·kg^-1.There were 4 animals（half males and females）in each group（negative control and administration）.Serial blood samples were collected at pre-dose and 5,20,60,120,240,480,1440 min post-dose,when first dose(5 mg·kg^-1)and possible toxic dose(40 mg·kg^-1).Animals within the dose of 5 to 40 mg·kg^-1 were not observed obvious toxic symptoms.A female animal appeared a transient foot and head tremor at 80 mg·kg^-1 dose.Hematology,blood biochemical and general anatomy were not obtained any obvious abnormalities.The results of accompanied toxicokinetics:（1）Compared 40 mg·kg^-1 with 5 mg·kg^-1: The Cmax and AUC0-t of koumine were 8.73 and 14.99 times in female animals,and the Cmax and AUC0-t of koumine were 10.64 and 24.24 times in male animals.Prompted that the increase of exposure is higher than the dose;The Tmax was delayed(Their were 60 min at 5 mg·kg^-1.Their were 120 min in 3/4 animals at 40 mg·kg^-1);The T_1/2 of koumine has a certain extension in male animals.The T_1/2 of koumine were 64.44 and 69.62 min at 40 mg·kg^-1.The T_1/2 of koumine were 39.65 and 44.62 min at 5 mg·kg^-1.（2）Comprared in gender,the exposure of koumine in female animals was higher.The Cmax and AUC0-t of koumine in the female animals were 2.25 and 2.57 times than that in males at the dose of 5 mg·kg^-1,and the Cmax and AUC0-t of koumine in the female animals were 1.84 and 1.59 times than that in males at the dose of 40 mg·kg^-1.Part four: The study of the toxicokinetics in SD rats by gavage koumine repeatedlyThe toxicokinetics test in SD rats by gavage koumine repeatedly for two weeks.There were 6 animals（half males and females）in negative control group and administration groups(10,30,90 mg·kg^-1),respectively.Serial blood samples were collected at pre-dose and 5,10,20,60,240,480 min post-dose.The animals of every group were not observed obvious abnormal reaction.Hematology,blood biochemistry and histopathological examination were not observed significant toxicological changes.Supplementary 150 mg·kg^-1 trial（4 animals,7 days of administration）showed symptoms such as low activity and tremor for 5 days,of which one female died after the first administration.The results of toxicokinetics:（1）The exposure of koumine in SD rats increased with the dose.（2）The Cmax and AUC0-t of koumine in the last administration increased than the first at 30 and 90 mg·kg^-1.Suggested that there may be some accumulation.（3）The exposure of koumine in rats had a significant difference in gender.Comparied in same dose,the Cmax of koumine in the female rats was 1.66 to 7.40 times than that in males in the first administration.The Cmax of koumine in the female rats was 2.10 to 11.58 times than that in males in the last administration;The AUC0-t of koumine in the female rats was 2.68 to 11.42 times than that in males in the first administration.The AUC0-t of koumine in the female rats was 4.39 to 13.74 times than that in males in the last administration.In summary:1.There is a significant difference in the enzyme kinetics of koumine between human and different species liver microsomes.The K_m of koumine in dog and pig,CL_int in rat and monkey,V_max in all four laboratory animals were larger,and T_1/2 in the four laboratory animals were shorter than the values from human.2.The exposure of koumine in rhesus monkeys increased with the dose,and the multiple is higher than the doses.Suggested that there may be some accumulation at high doses.The exposure of koumine in female rhesus monkeys is significantly higher than that in males.3.Gavaged koumine repeatedly for 14 days,the exposure of koumine in SD rats increased with the dose and time,and there may be some accumulation in higher doses.The exposure of koumine in the female rats is significantly higher than that in males.