Pharmacokinetics And Interaction Of Busulfan And Cyclophosphamide In Rabbits

Objective:To establish a pre-column derivatization HPLC?high performance liquid chroma tography?method which was used for determining the Bu concentration in vivo an d then to investigate the pharmacokinetic parameters of Bu at different routes of ad ministration.To investigate the plasma concentration of allo-HSCT patients after pre treatment with Bu.To establish a HPLC method which was used for determining th e CY concentration in vivo.To investigate the pharmacokinetic parameters of CY th rough intravenous administration.To investigate the interaction of pharmacokinetics i n the combination of Bu and CY.Mthods:1.The establishment of pre-column derivatization HPLC method for determining the Bu concentration in vivoUsing sodium diethyldithiocarbamate?DDTC?as a derivatization reagent,the derivatization reaction conditions of Bu were determined,and the derivatization conditions of Bu in serum samples were further optimized.Using 1,5-pentanediol dimethanesulfonate as an internal standard in vivo,which was derivatized with DDTC,a pre-column derivatization HPLC method was established for the analysis of Bu concentration in vivo.2.Study on the pharmacokinetics of Bu in rabbitsNine healthy rabbits were randomly divided into 3 groups.A group of rabbits were given a singal dose Bu by intragastric administration;a group of rabbits were given multiple doses Bu by intragastric administration;and the third group of rabbits were given a singal dose Bu by intravenous injection.The administration doses of different administration routes were calculated according to the doses administered to humans,and the doses of rabbits were calculated according to the rabbit conversion coefficient.The plasma concentration in vivo was determined separately for each route of administration,and pharmacokinetic parameters were obtained.And comprehensive comparison of pharmacokinetic characteristics of various routes of administration was done.3.A preliminary determination of plasma concentrations of Bu in allo-HSCT patientsPatients who were undergoing allogeneic hematopoietic stem cell transplantation?allo-HSCT?,and were given Bu combined with CY as a pretreat-mentregimen,wer e considered as research targets.The serum samples before and after the 12th-dose intravenous infusion of Bu was collected for blood concentration determination.The valley concentration values for the 11th-dose and the peak concentration values for the 12th-dose were obtained.4.The establishment of HPLC method for the determining of CY concentration in vi vo and study on the pharmacokinetics of CYWith ifosfamide as internal standard,HPLC was established to determine the blood concentration of CY in vivo.Three healthy rabbits were intravenously administered.Dosages were calculated based on the dose of humans treated with chemotherapy,a nd were converted into rabbits dosages by rabbit conversion factors.Pharmacokineti c parameters were obtained by measuring blood plasma concentrations in vivo.The pharmacokinetic characteristics of CY in vivo were evaluated initially.5.A preliminary study on the interaction between pharmacokinetics of Bu and CYTwelve healthy rabbits were randomly divided into two groups.A group of rabbits were given 3 consecutive days of Bu by gavage,3 times a day.On the fourth day,after given to the 10th dose of Bu,1h later,the rabbits were given to CY by intravenous injection.The concentration of CY at each time point was determined and the pharmacokinetic parameters were obtained.In the other group,CY was continuously administered by gavage for 3 days,once daily.On the fourth day,after given to the 4th dose of CY,1h later,the rabbits were given to Bu by intravenous injection.The blood concentration of Bu was measured at each time point to obtain the pharmacokinetic parameters.The SPSS 22.0 software was used to statistically analyze the pre-treatment and post-treatment of Bu and CY respectively,so as to preliminarily study whether there was interaction between Bu and CY in pharmacokinetics.Results:1.The establishment of pre-column derivatization HPLC method for determining the Bu concentration in vivoThe derivatization chromatography conditions of Bu:Diamonsil?II?C₁₈8 colum n?150 mm×4.6 mm,5?m?;column temperature:30°C;mobile phase:methanol-water?60:40?;flow rate:1.0 mL·min^-1,detection wavelength:280 nm,injection vol ume:10?L.The derivatization reaction conditions of Bu:the reaction temperature was 30°C and the reaction time was 30 minutes.The derivatization chromatography conditions of Bu in serum sample was as fo llows.A diamonsil?II?C₁₈8 column?150 mm×4.6 mm,5?m?was used for separ ation.The column temperature was 30°C.The mobile phase consisted of methanol and water?54:46,v/v?.The flow rate was 1.0 mL·min^-11 in 0-20 min,1.3 mL·min^-1in 20-27 min.The detection wavelength was 280 nm and the injection volume was25?L.The derivatization reaction conditions of Bu in serum sample:the reaction t emperature was 30°C;the reaction time was 30 minutes;and the final derivatizin g agent concentration was 0.08 g·mL^-11 DDTC solution.The plasma concentration of Bu was linear in the range of 0.1-3.4 mg·L^-1?r=0.9997?.The lowest limit of quan tification was 0.1 mg·L^-1.The recoveries of low,medium and high concentrations were 90.02%,89.03%,and 91.54%,respectively.The inter-day precision,intra-day p recision,and sample stability were in line with the provisions of the Chinese Phar macopoeia?2015 version?.2.Study on the pharmacokinetics of Bu in rabbitsThe pharmacokinetic parameters of Bu by different routes of administration wer e as follows.When a single dose was taken orally,t_1/2/2 was 2.26±0.66 h,k was 0.33±0.12 h^-1,t_maxax was 1.20±0.76 h,C_maxax was 0.59±0.05 mg·L^-1,k_a was 2.54±1.3 h^-1,t_1/2?a?was 0.36±0.25 h,AUC_0?t?t was 1.95±0.18h·?g·mL^-1,and AUC_0??was 2.72±0.60.When a single dose was taken by intravenous injection,t_1/2/2 was 1.53±0.09 h,k was 0.454±0.03 h^-1,C₀ was 1.99±0.01 mg·L^-1,V was 3.08±1.08L,Cl was 1.39±0.42 L·h^-1,AUC_0??was 4.38±0.26 h·mg·L^-1.When multi-dose was taken orally,t_maxax was 1.36±0.72 h,C_minwas 0.18±0.08mg·L^-1,C_maxax was 0.81±0.17mg·L^-1,?C_SSS was 0.484±0.032mg·L^-1,and AUC_0??was3.87±0.26h·mg·L^-1.3.A preliminary determination of plasma concentrations of Bu in allo-HSCT patientsTwo patients receiving allo-HSCT were collected from Shanxi University Hospi tal of Shanxi Medical Sciences.The valley concentration was 0.125,0.225 mg·L^-1,and the concentration after 1h of intravenous infusion was 0.733,0.779 mg·L^-1.A patient receiveing allo-HSCT was collected from the Second Hospital of Shanxi Me dical University Affiliated Hospital.The valley concentration was 0.18mg·L^-1,and t he peak concentration was 1.78 mg·L^-1.4.The establishment of HPLC method for determining CY concentration in vivo and study on the pharmacokineticsThe chromatographic conditions for the analysis of CY were as follows.An d iamonsil?II?C₁₈8 column?150 mm×4.6 mm,5?m?was used for separation.The column temperature was 30 degrees.The mobile phase consisted of acetonitrile an d water?20:80,v/v?.The flow rate was set at 1.0mL·min^-1.The detection wavelength was 197 nm and injection volume was 25?L.The plasma concentration of CY wa s linear in the range of 2.4-150 mg·L^-1?r=0.9996?.The lowest limit of quantific ation was 2.4 mg·L^-1.The recoveries of low,medium and high concentrations were90.02%,89.03%and 91.54%respectively,The recoveries were stable.The inter-day precision,intra-day precision,and sample stability were in line with the provisions of the Chinese Pharmacopoeia?2015 version?.The pharmacokinetic parameters of CY were as follows.t_1/2/2 was 0.439±0.091 h,k was 1.641±0.298 h^-1,AUC_0??was 87.736±17.160 h·mg·L^-1,C₀ was 187.130±3.518mg·L^-1,V was 2.688±0.125 L,Cl was 4.392±0.745 L·h^-1?5.A preliminary study on the interaction between pharmacokinetics of Bu and CYThe pharmacokinetic parameters of Bu by pretreatment with CY were as follo ws.t_1/2/2 was 0.867±0.167 h,k was 0.831±0.168 h^-1,AUC_0??was 4.203±0.286h·mg·L^-1,C₀ was 3.271±0.269 mg·L^-1,V was 1.694±0.145 L,Cl was 1.399±0.261 L·h-¹.The statistical analysis result of each parameter was as follows.The pharmacok inetic parameters k?0.526±0.103?h^-1?C₀?2.175±0.272?mg·L^-11 when intraveno usly injected Bu alone were lower than the k?0.831±0.168?h^-1??P<0.05?.C₀?3.271±0.269?mg·L^-1?P<0.05?when combined with CY.The pharmacokineti c parameters V?2.883±0.681?L?t_1/2?1.363±0.240?h?Cl?1.486±0.281?L·h^-1?AUC_0???4.203±0.286?h·mg·L^-11 when intravenously injected Bu alone were hi gher than the V?1.694±0.145?L?P>0.05??t_1/2?0.867±0.167?h?P<0.05??Cl?1.399±0.261?L·h^-1?P>0.05??AUC_0???4.038±0.548?h·mg·L^-1?P>0.05?when combined with CY.Through statistical analysis,it was initially determi ned that the pharmacokinetic parameters k,C₀,t_1/2/2 of Bu were affected by CY,and V,Cl,AUC_0??were not affected.The pharmacokinetic parameters of CY by pretreatment with Bu were as follo ws.t_1/2/2 was 0.373±0.021h,k was 1.865±0.101 h^-1,AUC_0??was 118.099±22.367 h·mg·L^-1,C₀ was 164.636±3.813 mg·L^-1,V was 2.544±1.212 L,Cl was 4.801±2.241 L·h^-1.The statistical analysis result of each parameter was as follows.The pharmacok inetic parameters k?1.641±0.298?h^-1?Cl?4.392±0.745?L·h^-1when intravenousl y injected CY alone were lower than the k?1.865±0.101?h^-1?P>0.05??Cl?4.801±2.241?L·h^-1?P>0.05?when combined with Bu.The pharmacokinetic par ameters t_1/2?0.439±0.091?h?C₀?187.130±3.518??V?2.688±0.125?L?AU C_0???118.099±22.367?h·mg·L^-1when intravenously injected CY alone were highe r than the t_1/2?0.373±0.021?h?P>0.05?,C₀?164.636±38.139?mg·L^-1?P>0.05??V?2.544±1.212?L?P>0.05??AUC_0???87.736±17.160?h·m g·L^-1?P>0.05?when combined with Bu.By comparing the pharmacokinetic para meters before and after the pre-treatment,there was no significant difference.The p reliminary determination was that the pharmacokinetic parameters of CY will not be affected by Bu.Conclusion:1.The rabbits were treated with busulfan in parallel and the DAS 2.0 software“Intelligent Analysis”module was used to determine the pharmacokinetic data,which basically belonged to a one-compartment model.From the value of blood drug concentration,it could be seen that the individual differences of Bu were great.The steady-state plasma concentration of multi-dose oral was between?0.18±0.08?mg·L^-11 to?0.81±0.17?mg·L^-1,so a preliminary investigation of the steady-state plasma concentration range in vivo was done.The metabolism of intravenous administration was faster.2.The results of the combination of Bu and CY showed that after being pretreated with CY,the metabolic rate of Bu was significantly increased.There was no significant difference in the metabolic rate and distribution of CY after pretreatment with Bu.Being consistent with the relevant reports,it was necessary to monitor the blood concentration of Bu to adjust the dosage when it was administrated with CY.