Pharmacokinetic Study Of Tongguangsan C₂₁Steroidal Compound MT2 And Its Synergistic Effect On Antitumor Activity Of Paclitaxel

Background and objectiveOur research team have found previously that the C21 steroids rich total aglycones(ETA)prepared from Marsdenia tenacissima(Tongguansan)effectively enhanced the antitumor activity of paclitaxel both in vjtro and jn vivo,while 11?-O-2 methybutyryl-12 ?-O-tigloyl-tenacigenin B(MT2),which is a main C21 steroidal component of ETA,showed the capability to directly inhibit metabolic activity of CYP450 enzumes(3A4,2C8,2B6 and 2C19)and the transport function of P-glycoprotein(P-gp),and enhanced paclitaxel-induced growth inhibition to the growth of' tumor cells.This study intended to investigate whether MT2 enhance in vivo antitumor activity of paclitaxel and if yes,the pharmacokinetics of MT2 and its influence on pharmacokinetics of paclitaxel,in order to provide pharmacodynamic and pharmacokinetic basis for developing MT2 as a candidate adjuvant for tumor chemotherapy.Methods1.Effect of MT2 on in vivo antitumor activity of paclitaxelHela tumor xenograft model was estabolisned by subsconeously implantation with HeLa cells.For drug treat.ment,mice were int raperitoneally(i.p.)gi ven paclitaxel or MT2 alone,or the combination of paclitaxel and MT2 every ot:her day for 5 doses(six mice for each treatment).Mice given equivalent amount of vehicle were set as control.Body weight and cleath case were monit.ored routinely.Six days after the last treatment,mice were sacrificed,tumors were stripped and weighed for assessing the inhibitory cffects of different treatments on tumor growth.2.Effect of MT2 on pharmacokinetics of paclitaxel in ratsEighteen male SD rats were randomly assigned tc)3 groups and preteated with 20 mg/kg(MT2-L+paclitaxel group),40 mg/kg(MT2-H+paclitaxel group)of MT2 or equivalent volume of the vehicle(paclitaxel group)respectively(i.p.).Twenty-four h later,rats were given 10 mg/kg of paclitaxel(i.p.),with MT2 given as the day before.Blood samples were collected at designated time-point after paclitaxel injection for quantitative analysis of paelitaxel.Data obtained were analyzed using Drug and Statistic 3.0(DAS 3.0)pharmacokinetic software.A non-compartmental model was selected to fit plasma concentration versus time data to obtain pharmacokinetic parameters.3.Pharmacokinetic study of MT2 in ratsSD rats were orally given 100 mg/kg or 40mg/kg of MT2,or intravenously(i.v.)given 20 mg/kg,10 mg/kg or 5 mg/kg of MT2(N=6 for each group).Blood samples were collected at designated time-point after drug treatment.Concentration of MT2 in rat plasma was measured by UPLC-MS/MS method.Data obtained from the animal study were analyzed using Drug and Statistic 3.0(DAS 3.0)pharmacokinetic software.A non-compartmental model was selected to fit plasma concentration versus time data to obtain pharmacokinetic parameters.4.Plasma protein binding ratio of MT2 in ratsUPLC-MS/MS method was used for quantitative determination of MT2 in dialysate.Equilibrium dialysis method was used to determine the plasma protein binding ratio(PPB)of MT2 in three different concentration.5.Excretion study of MT2 in ratsUPLC-MS/MS method was used for quantitative determination of MT2 in rat bile,urine and fece collected at different time period after i.v.given of MT2.Excretion rates and the cumulative excretion obtained were used to analyze the primary excretion pathway.6.Distribution study of MT2 in ratsUPLC-MS/MS method was used for quantitative analysis of MT2 in the heart,liver,spleen,lung,kidney,brain,pancreas,fat,muscle,testicle,uterus and ovary at designated time-point after intravenous injection of MT2(20 mg/kg).Data obtained were used to evaluate the tissue distribution of MT2.7.Metabolism study of MT2 in rat liver microsomesMT2 was incubated with rat liver microsomes at 37? for 60 min.UPLC-Q-TOF-MS/MS method was used for identification of the metabolites of MT2 using the Metabolite Pilot 1.5 and Peakview 2.0 software.Results1.MT2 significantly enhanced in vivo antitumor activity of paclitaxelTreatment with paclitaxel(8 mg/kg)alone achieved 18%of reduction in tumor weight(P>0.05 vs Vehicle),while treatment with MT2(30 mg/kg)alone achieved no reduction in tumor weight at all.Treatment with paclitaxel in combination with MT2 achieved 96%of reduction in tumor weight(P=0.006 vs Vehicle).In addition,coadministration of MT2 with paclitaxel caused tumor disappearance in 3 mice.No death case or significant body weight loss was observed.2.Effect of MT2 on pharmacokineties of paclitaxel in ratsCompared to administration of paclitaxel alone(Paclitaxel group),coadministration of paclitaxel with MT2(40 mg/kg)(MT2-H+paclitaxel group)achieved approximately an 8-fold increase in peak plasma concentration(Cmax)(P=0.002<0.01).and a 5-fold increase in area under the concentration versus time curve(AUC0-t)of paclitaxel(p=0.002<0.01).Despite of the unchanged the terminal half-life(t1/2),there were significant differences between the two groups in the time to reach peak concentration(tmax),apparent volume of distribution(Vz/F),and total body clearance(Clz/F).Changes in these pharmacokinetic parameters were also observed in group co-administered with 20 mg/kg MT2(MT2-L+paclitaxel group),though without statistical implication(P>0.05)due to the great differences among individuals in this group.The results indicated significant drug-drug interactions(DDIs)when paclitaxel was co-administered with MT2.3.Pharmacokinetics of MT2 in ratsA UPLC-MS/MS method was established for determination of MT2 in rat plasma.Pharmacokinetie parameters of MT2 obtained from intravenous injection(data were presented in dose order of 5 mg/kg,10 mg/kg,20 mg/kg):t1/2:2.64±0.39,2.53±0.30 and 3.68±1.11 h;MRT:1.41±0.14,1.15±0.11 and 1.55±0.14 h;Vz:13.67±2.16,13.56±2.30 and 9.03±3.08 L/kg;CLz:3.59±0.26,3.71±0.38 and 1.69±0.16 L/h/kg,respectively.The resul ts indicated that MT2 was distributed and eliminated rapidly in rats without concentration dependence in Cmax and AUC values.The high Vz suggested that MT2 given by i.v.injection not only presented in blood,but also widely distributed in tissues.Results for oral administraton of MT2:MT2 could be detected in rat plasma 5 minutes after administration.The bioavailability of MT2 at 40mg/kg and 100mg/kg were 1.09%and 1.66%,respectively.Corresponding t1/2 and MRT0-t were 11.01±7.03 and 8.84±1.62 h(for 40 mg/kg),14.14±5.87 and 12.29±1.89 h(for 100 mg/kg),respectively.The results indicated that MT2 could be absorbed quickly with low bioavailability and obvious first-pass effect.The phenomenon that two or more peaks appeared in the absorption phase rather than in the eliminate phase or in intravenous administration suggested that there were no enterohepatic circulation in rats by administration of MT2?We attribute the phenomenon to the poor water solubility of MT2,which may lead to the precipitatation of MT2 in the gastrointestinal tract after intragastric administration and thus the reabsorbtion of MT2.Multilocus absorption in gastrointestinal may also contributes to the double peak or multi-peak.4.Rat plasma protein binding ratio of MT2A UPLC-MS/MS method was established for the determination of MT2 in dialysate.The protein binding ratio(PPB)of different concentrations of MT2 in rat plasma were 93.84%(200 ng/mL),94.35%(2000 ng/mL)and 94.96%(10000 ng/mL),respectively,indicating high plasma protein binding affinity and a concentration-independent binding mode of MT2.5.The excretion of MT2 in ratsA UPLC-MS/MS method was established for the quantitative determination of MT2 in rat bile,urine and feces.The concentrations of MT2 in bile,urine and feces at different time period after intravenous administration were measured and analyzed.The results showed the 24 h-cumulative excretion percentage of 0.0193%from bile,48 h-cumulative excretion percentages of 0.0030%from urine and 0.0693%from feces,respectively.The summaried cumulative excretion percentage of MT2 from above three pathways was as low as 0.0916%,indicating a very low excretion rate of MT2 as the prototype and suggesting that MT2 was mainly eliminated in rats through biotransformation and metabolism.6.Distribution of MT2 in ratsA UPLC-MS/MS method was established for quantitative determination of MT2.The concentration of MT2 in heart,liver,spleen,lung,kidney,brain,pancreas,fat,muscle,testicle,uterus and ovary at 15 min,1 h and 4 h after intravenous administration of MT2 were measured and analyzed.Results at the time-points 1 h(except fat)and 4 h showed gender difference in distribution of MT2 in above investigated tissues.Concentrations of MT2 decreased rapidly in tissues of male rats.However,in tissues of female rats,concentrations of MT2 were greater 2000 ng/g even 4 h after the drug administration,and in ovary,liver,fat and pancreas,the values were above 10000 ng/g.The results demonstrated that MT2 has long-term accumulation in all tissues of female rats,and preferably target tissues of ovary,liver,fat and pancreas.7.Metabolism of MT2 in rat liver microsomesA UPLC-Q-TOF-MS/MS qualitative analysis method was established for detecting and identifying the metabolites of MT2 in rat liver microsomes.In this study,five I phase metabolites of MT2 together with MT2 were detected.The five metabolites were identified as tri-oxidated MT2(M1 and M2),demethylated and di-oxidated MT2(M3),demethylated and monoxidated MT2(M4)and di-oxidated MT2(M5),respectively.The results suggested that the oxidation reaction was the main metabolic pathway of MT2 in rat liver.Conclusion1.This study demonstrated firstly the ability of C21 steroidal compound MT2 from Tongguangsan to effectively enhance in vivo antitumor activity of paclitaxel.Pharmacokinetic study for paclitaxel showed that,co-administration of MT2 with paclitaxel by intraperitoneal injection did not change the absorption speed and t1/2 of paclitaxel,but obviously increased the AUC and Cmax of paclitaxel and affected the apparent volume and elimination rate of paclitaxel in rats,indicating drug-drug interaction.2.UPLC-MS/MS methods were estabolished for quantitative analysis of MT2 in rat plasma,bile,urine,feces and liver.For MT2,pharmacokineties in rats,plasma protein binding rat io,excretion,tissue distribution and liver microsomal metabolism were studied systematically.MT2 showed to be highly affinitive to plasma protein binding and was absorpted fastly with low bioavailability by oral administration.In rats,gender differences in tissue distribution of MT2 were observed,while the elimination of MT2 was mainly through biological transformation,and oxidation was the main metabolic pathway in rat liver microsomes.