Study On The Druggability Of Crotonoside On Anti-acute Myeloid Leukemia

Objective:By studying the pharmacodynamics,safety and pharmacokinetics of crotonosine,the druggability of crotonoside against acute myeloid leukemia were evaluated.Methods:The in vitro inhibitory activity of crotonoside on MV4-11 cells and three normal cell lines was detected by MTT assay,and to evaluate the cytotoxicity of crotonoside and study the effect of crotonoside on the proliferation of acute myeloid leukemia cells at different doses.The effect of crotonoside on the expression of FLT3and its downstream proteins in MV4-11 cells was detected by Western Blot,and to detect the effect of crotonoside on the expression of HDACs signaling pathway in MV4-11 cells.The maximal tolerable dose of crotonoside was determined by the maximum tolerance test method,and the safety of crotonoside was evaluated.The concentration of crotonoside in plasma of rats and plasma and tissues of mice was determined by HPLC.Rats were given a tail vein injection with 12.5,25.0,50.0 mg/kg crotonoside,serial blood samples were collected from the orbital veins of each rat in heparinized tubes at different time.The plasma concentration of crotonoside was determined by HPLC.The pharmacokinetic parameters were calculated by Phoenix WinNonlin pharmacokinetic software.The plasma partition coefficient of crotonoside was determined by incubating the whole blood at 37?in vitro.Mice were given a tail vein injection with 35.7 mg/kg crotonoside,tissues?including the brain,heart,spleen,lung and kidney?and plasma were collected at different time.The concentration of crotonoside in different matrices and plasma were determined by HPLC.Results:The inhibitory effect of crotonoside on MV4-11 cells was significantly higher than that of other cells,IC₅₀0 was 11.6±2.7?M.The inhibitory activity of crotonoside on normal human embryonic kidney cell line HEK293A,human normal mammary epithelial cell line MCF-10A and mouse B cell line was weaker,IC₅₀0 was more than 160?M.The expression of p-FLT3,p-STAT5,p-Erk_1/2,p-AKT,p-mTOR protein in MV4-11 cells were down-regulated by crotonoside,but the expression of Erk_1/2,AKT and mTOR total protein were not significantly affected.The expression of FLT3 total protein and HDAC3,HDAC6,NF-?B,c-Myc protein in MV4-11cells were inhibited by crotonoside,which after 20 hours of action with MV4-11 cells.After 100 mg/kg of crotonoside was injected into the tail vein of mice and continuous observation for 14 days.It was observed that the mice were to show slow action,hair care,shortness of breath and other toxic reactions,but all returned to normal after 1 day.After the end of the experiment,the animals were sacrificed and the main organs were visually inspected.The results showed that the main organ position,color,size were no abnormal changes.There was a significant difference in body weight change between the dosing group and the control group at the 1th day after administration?P<0.001?.There was no significant difference in body weight between the dosing group and the control group at the 7th and 14th day after administration.The linear range of crotonoside in plasma was 0.135⁵4.0?g/mL.The intra-and interday precision and accuracy of the crotonoside measurements were<15%,the average recovery of crotonoside was 105.3%.Rats were given a tail vein injection with 12.5,25.0,50.0 mg/kg crotonoside,C_maxax were 4.35±1.72,17.28±7.61,58.32±16.29?g/mL;t_1/2/2 were 30.77±7.44,49.43±18.76,53.26±13.73 min;AUC_0-t-t were 34.3±10.4,125.5±45.9,353.4±117.6?g/mL·min.One-way ANOVA indeed showed a difference in C_max/dose,AUC_0-t/dose and AUC_0-?/dose between the three study doses,the mean C_maxax and AUC_0-t-t values were not linear in the range of the doses administered.The K_RBC/PLBC/PL values of crotonside at the concentration of 6.75,13.5 and33.75?g/mL were 8.55±1.55,8.72±1.42 and 8.74±2.19 at 360 min,respectively.The results showed that the standard curves of crotonoside in the tissue homogenate samples and plasma samples of mice were linear and the correlation coefficients were more than 0.99.The lower limit of the determination of crotonoside in the tissue homogenate samples and plasma samples were 0.135?g/mL.The intra-and interday precision and accuracy of the crotonoside measurements were<15%.The average recovery of crotonoside in the tissue homogenate samples and plasma samples were>85%.Mice were given a tail vein injection with 35.7 mg/kg crotonoside,tissues?including the heart,liver,spleen,lung and kidney?were distributed,exceptthe brain.The concentration of crotonoside in the liver was the highest,followed by kidney tissue.The concentration of crotonoside in the heart,spleen and lung were similar,and the brain was not measuredat all time points.The peak time of the heart,lung and kidney were 5³0 min,and the peak time of the spleen and liver were 60¹50 min.At 330 min,the order of the concentration of crotonoside in the tissues of the mice was liver>kidney>spleen>heart?lung,and the concentration of crotonoside in the liver was much larger than that of other tissues.Conclusions:Crotonoside had good anti-AML activity in vitro,and crotonoside had the effect of inhibiting MV4-11 cells through inhibiting the expression of FLT3and HDACs in MV4-11 cells.The results showed that the toxicity of crotonoside was low and the maximum tolerance was 100 mg/kg.Pharmacokinetic and tissue distribution studies had shown that the pharmacokinetics of crotonoside in rats was a nonlinear kinetic model at a dose of 12.5 to 50.0 mg/kg.Crotonoside was widely distributed in mice.The amount of drugs in the liver was much larger than other tissues,and was always the dominant organization of distribution.