Research On The Construction Of Biological Analysis Methods In Animals,Pharmacokinetics And Anti-breast Cancer Effect Of CU-PTX

Objective:Paclitaxel?PTX?is a first-line drug for the treatment of breast cancer,but it can lead to serious adverse reactions such as bone marrow suppression and neurotoxicity,etc.Therefore,it is necessary and urgent to find a safe and effective drug for PTX.The traditional Chinese medicine monomer CU has multi-target anti-tumor effect and is a safe candidate for tumor.In this study,we considered to combine CU and PTX?CU-PTX?and investigate its anti-tumor effect.Methods:1.High performance liquid chromatography?HPLC?method was established for the determination of CU-PTX in rat plasma and tissue samples.2.High performance liquid chromatography?HPLC?method was established for the determination of CU-PTX in rabbit plasma samples.3.Rabbits were selected as the animal model,CU 12mg/kg,PTX 12mg/kg and CU-PTX?1:1,0.5:1,w/w?were given to each group by the ear vein injection.Then blood was taken at 0.083 h,0.167 h,0.25 h,0.5 h,1 h,2 h,4 h,6 h,8 h,12 h and 24 h,and after the biological sample treatment,the plasma concentration was determined by HPLC,and the pharmacokinetic software DAS 2.0 was used to calculate the pharmacokinetic parameters.4.MTT method was used to detect the inhibitory effect of CU?2.3,4.6,9.2,18.4 and 36.8?g/mL?and PTX?8:1 and 80:1,w/w?on breast cancer T47D cell line for 48 h and 72 h,respectively.5.The subcutaneous transplanted tumor model of human breast cancer cell line T47D was constructed in nude mice.The tumor growth and the inhibition rate were taken as the index,intraperitoneal injection of drugs to investigate the anti-tumor effects of CU 80mg/kg,PTX 10 mg/kg and CU-PTX?80:10,80:1,80:0.5,80:0.25,w/w?.Results:1.Chromatographic condition:the column was Luna 5?m C18 column?4.6 mm×250 mm?.The guard column was Phenomenex C18?4.0 mm×3.0mm?.Acetonitrile and phosphate buffer?pH 3.5??55:45,v/v?was chosen as mobile phase and the detection wavelength was 425 nm for 0-9.5 min.As for9.51-11.5 min,ratio of acetonitrile and phosphate buffer was turned to 40:60?v/v?and the detection wavelength was 227 nm.Mobile phase and wavelength were returned to 425 nm and 55:45?v/v?for 11.51-19.0 min,respectively.The flow rate was 1.0 mL/min at 30°C,and the injection volume was 20?L.2.Establishment and validation of analytical methods for rat biological samples:100?L of rat plasma and 25?L of citrate buffer?pH 3.6?was added into a 5 mL centrifuge tube and whirled for 30 s.Then,0.5 mL of extractant with ethyl acetate and methanol?90:10,v/v?was added and whirled for 3 min.The supernatant was obtained by centrifugating for 3 min?8000 rpm/min?.Added 0.5 mL of extractant again and extracted again.The two supernatant were merged and was dried with nitrogen.Added 200?L extractant?pH 3.5?,mixed for 4 min,ultrasonic for 4 min,centrifugated for 10 min?10000 rpm/min?at high speed to obtain supernatant.And 20?L of the supernatant were used to detected by HPLC.Results of the standard curve showed that CU,PTX and CU-PTX were good linearity in the range of 0.01-4?g/mL?R²>0.999?,the minimum detection limit was 0.01?g/mL.The recoveries of CU and PTX were 76.89%-97.49%and 72.96%-92.87%,respectively.The inter-and intra-day precision were2.14%-14.91%,and the stability coefficient was less than 14.70%at room temperature for 24 h,7 days and 14 days.CU,PTX and CU-PTX in the seven tissues?heart,liver,spleen,lung,kidney,stomach and brain?of the rats were good linearly in the range of 0.01-4?g/mL?R²>0.999?,the minimum detection limit was 0.01?g/mL.The inter-and intra-day precision of the seven tissues were 1.87%-14.81%,and the stability coefficient was less than 14.99%at room temperature for 24 h and frozen for 3 days and 7 days.Sample recovery rate in gastric tissue of CU and PTX were 65.13%-74.83%and74.98%-103.13%,respectively.In the heart tissue,sample recovery rate of CU and PTX were 69.25%-78.64%and 92.97%-102.72%,respectively.In spleen tissue,sample recovery rate of CU and PTX were 74.95%-98.14%and88.51%-100.20%,respectively.In liver tissue,sample recovery rate of CU and PTX were 84.42%-97.92%and 68.36%-92.59%,respectively.In lung tissue,sample recovery rate of CU and PTX were 82.80%-99.76%and75.68%-101.50%,respectively.In kidney tissues,sample recovery rate of CU and PTX were 79.46%-94.63%and 84.5%-98.04%,respectively.In brain tissue,sample recovery rate of CU and PTX were 69.81%-93.87%and81.64%-97.66%,respectively.3.Establishment and validation of analytical methods for biological samples of plasma in rabbits:500?L of rabbit plasma and 25?L of citrate buffer?pH 3.6?was added into a 5 mL centrifuge tube and whirled for 30 s.Then,3 mL of extractant with ethyl acetate and methanol?90:10,v/v?was added and whirled for 3 min.The supernatant was obtained by centrifugating for 3 min?8000 rpm/min?.Added 3 mL of extractant again and extracted again.The two supernatant were merged and was dried with nitrogen.Added 200?L extractant?pH 3.5?,mixed for 4 min,ultrasonic for 4 min,centrifugated for 10min?10000 rpm/min?at high speed to obtain supernatant.And 20?L of the supernatant were used to detected by HPLC.Results of the standard curve showed that CU,PTX and CU-PTX were good linearity in the range of 0.008-30?g/mL?R²>0.999?,the minimum detection limit was 0.008?g/mL.The recoveries of CU and PTX were77%-93%and 75%-89%,respectively.Inter-and intra-day precision were1.13%-14.9%and stability coefficient at room temperature for 24 h and frozen for 7 days,14 days was all less than 14.38%.4.Study on pharmacokinetics in rabbits.A group was administrated 12mg/kg CU to rabbits by intraperitoneal injection,results showed that t_1/2,C_max,AUC and CL were 1.442 h,4685.732?g/L,1111.898?g/L*h and 10.7921L/h/kg,respectively.B group was given 12mg/kg PTX,results showed that t_1/2,C_max,AUC and CL were 1.041 h,17825.902?g/L,6853.791?g/L*h and 1.751L/h/kg,respectively.C group was given 12 mg/kg CU and 12 mg/kg PTX,t_1/2,C_max,AUC and CL of CU were 1.316 h,6227.152?g/L,1655.028?g/L*h and7.251 L/h/kg,respectively.While t_1/2,C_max,AUC and CL of PTX were 1.041 h,26931.173?g/L,11413.512?g/L*h and 1.051 L/h/kg,respectively.D group was given 6 mg/kg CU and 12 mg/kg PTX,t_1/2,C_max,AUC and CL of CU were1.448 h,2806.965?g/L,696.521?g/L*h and 8.614 L/h/kg,respectively while t_1/2,C_max,AUC and CL of PTX were 1.043 h,27847.556?g/L,15139.43?g/L*h and 0.793 L/h/kg,respectively.Compared with PTX alone,the CL of PTX was decreased in the CU-PTX group,AUC and C_maxax were increased by 0.67 and0.51 times,respectively.CL of CU in the CU-PTX group was lower than that of the same dose CU,AUC and C_maxax were increased from 1111.898 g/L*h to1655.028 g/L*h,increased from 4685.732 g/L to 6227.152 g/L,respectively.5.When the drug was used to affect T47D cells for 48 h,the IC₅₀0 0f CU and PTX was 20.17 and 30.46?g/mL,respectively.When CU combined with PTX at the mass ratio of 8:1,the IC50 of CU and PTX were 12.32?g/mL?reduced by 38.92%?and 1.54?g/mL?reduced by 94.94%?,respectively.When CU combined with PTX at the mass ratio of 16:1,the IC50 of CU and PTX were 15.91?g/mL?reduced by 21.12%?and 0.20?g/mL?reduced by99.43%?,respectively.As for 72 h,the IC₅₀0 0f CU and PTX were 19.10 and28.63?g/mL,respectively.When CU combined with PTX at the mass ratio of8:1,the IC50 of CU and PTX were 10.41?g/mL?reduced by 46.91%?and 1.30?g/mL?reduced by 95.46%?,respectively.When CU combined with PTX at the mass ratio of 16:1,the IC50 of CU and PTX were 14.12?g/mL?reduced by25.97%?and 0.18?g/mL?reduced by 99.37%?,respectively.6.According to the experimental results of nude mice,compared with saline group,tumor inhibition rates of A group?CU 80 mg/kg?,B group?PTX10 mg/kg?,C group?CU 80 mg/kg and PTX 10 mg/kg?,D group?CU 80 mg/kg and PTX 1 mg/kg?,E group?CU 80 mg/kg and PTX 0.5 mg/kg?and F group?CU 80 mg/kg and PTX 0.25 mg/kg?were 29.55%,46.25%,63.64%,47.73%,44.32%and 42.05%,respectively.Conclusion:1.This paper successfully established the analysis method of CU-PTX rat and rabbit biological samples,the method had high specificity,good linearity,high sensitivity,good precision,high recovery rate and could simultaneously detect the concentration of CU and PTX in rats and rabbits in biological samples.It was successfully applied to the pharmacokinetics of CU-PTX in rabbits in vivo.This method has not been reported in literature with definite innovation.2.Compared with the same dose of CU and PTX alone,CL of CU-PTX is relatively reduced,the AUC and Cmax of two drugs significantly increased,and the elimination of the role was inhibited in the body.3.The inhibitory effects of CU and PTX on the growth of human breast cancer cell line T47D showed a significant dose-and time-effect relationship.The IC50 values of the two drugs were significantly reduced after combined treatment with 8:1 and 80:1,which indicated that different doses of the two drugs were better than than CU or PTX alone.It suggested that CU-PTX inhibits tumor synergistic effect.4.According to the inhibition test in nude mice,it was indicated that CU-PTX has a good synergistic inhibitory effect on subcutaneous solid tumors of human breast cancer T47D,suggesting that CU could be used to reduce the amount of PTX to achieve therapeutic effect,and provide a certain experimental basis for clinical application of PTX.