Disposition Of Disodium Norcantharidate In Vivo And Preparation Of Disodium Norcantharidate Liposome

Objectives:1.To synthesize disodium norcantharidate(DSNC) as the test drug; 2.To establish and validate a sensitive and high specificity HPLC-MS/MS method for determination of DSNC in mice plsma and tissues;3.To study the pharmacokinetic and tissue distriburion of DSNC solution(DSNC-sol) in mice;4. To study the metabolite and metabolic pathway of DSNC in vivo and in vitro;5.To prepare DSNC liposomes by reverse phase evaporation method and evaluate its pharmaceutical characterater;6.To study the pharmacokinetics and tissu distribution of DSNC liposomes(DSNC-lip) to evaluate its delayed releasing and targeting character.Methods:1.Disodium norcantharidate was synthesized by acid-base neutralization reaction and purified by recrystallization in ethanol.Its structure was identified by infrared absorption(IR) spectrum and mass spectrum.2.Biological samples were extracted by protein precipitation with acetone from 0.3mL plasma or tissue homogenates under acid condition.The samples were analysized on a C₁₈ column with a mobile phase of acetonitrile-ammonium acetate (2mM,0.1%formic acid)(20:80,v/v) and ribavirin was used as internal standard (IS).DSNC was quantified using a triple quadrupole mass spectrometer operating in positive electrospray ionization(ESI) in multiple reaction monitoring(MRM) mode, ion transitions of m/z 169.0→123.0 for DSNC and m/z 267.1→135.1 for I.S.were selected.The pecificity,matrix effect,absolute recovery,calibration curve,LLOQ, precision,accuracy and stability were studied to validate the method.3.102 Kunming mice were divided into 12 groups randomly,one was blank group and others were experiment groups.The DSNC solution(1mg·mL^-1) was administered to experiment groups via intragastric administration at a dose of 20mg·kg^-1.Blood samples were obtained from the fossa orbitalis vein at 0.083,0.25, 0.5,1,1.5,2,4,6,8,10,24h after administration.After centrifuged,the plasma was separated and stored at -20℃for analysis.Heart,liver,spleen,lung,kidney,brain, stomach,small intestine,testis,uterus were rapidly collected at 0.25,1,2,4,8h following blood collection,respectively.The tissue samples were washed with 0.9% NaCl and blotted on filter paper,then weighted accurately and homogenized in 0.9% NaCl(2mL·g^-1 tissue),and the homogenates were stored at -20℃for analysis.The samples were analysized by etablished HPLC-MS/MS method,and pharmacokinetic parameters were calculated by DAS 2.0 software.4.The metabolite and metabolic pathway of DSNC in vivo and in vitro were studied.In vivo,rat urine and feces were collected after administration of DSNC-sol and were analysized by HPLC-MSⁿ method after disposition.In vitro,rat liver microsome was prepared with Ca²⁺ precipitation method,assisted with oxidoreduction coenzyme to simulate the conditiong of phaseâ… and phaseâ…¡metabolism in vivo.The reaction was terminated with acetonitrile and products were analysized by HPLC-MSⁿ method after disposition.5.The reverse phase evaporation method was used to prepare disodium norcantharidate liposome(DSNC-Lip).The orthogonal experimental design was applied to optimize the prescription and the technology of preparation.The diameter and size distribution properties of disodium norcantharidate liposome were observed by transmission electric microscop and laser dispersive analyzing apparatus for granularity,and Zeta potential was measured by micro-electrophoresis apparatus. Liposome containing drug was separated from free drug by sephadex gel column method.The encapsulation efficiency and drug loading capacity were assayed by HPLC method.In vitro releases of liposome were performed by dialysis method.6.The pharmacokinetics and tissue distribution of DSNC in mice after administration of DSNC-lip were investigated.The main pharmacokinetic parameters and targeting parameters were calculated to evaluate the delayed releasing and targeting characters.Results:1.The average yield of synthesis was about 94%.The compoud was validated to be DSNC by IR spectrum and Mass spectrum.2.Good linearity of DSNC in plasma,liver,lung,kidney,stamoch,small intestine,uterus and testis were observed in the range of 0.01-5.0μg·mL^-1,the LLOQ was 0.01μg·mL^-1,and good linearity was observed in the range of 0.005-0.5μg·mL^-1 in other tissus(heart,spleen and brain) too,the LLOQ was 0.005μg·mL^-1,the correlation coefficients(r²) were between 0.9918 and 0.9976.The intra-day and inter-day RSD were less than 15%.All absolute recoveries exceeded 50%and relative recoveries were between 91%and 107%.DSNC in plasma and all tissues placed at room temprature for 4h and frozen at -20℃for 24 hours and seven days and after two freeze-thawing cycles was stable,the RSD were less than 15%. This method was suitable for DSNC pharmacokinetic and tissue distribution studies.3.After fitting,the pharmacokinetic process of DSNC in mice after administration of DSNC-sol was coincidence with double compartment model with weight of 1.The main pharmacokinetic parameters of DSNC after administration of DSNC-sol were as follows:t_1/2α 0.196±0.117h;t_1/2β 6.345±3.321h;Tmax 0.375±0.137h;Cmax 3.973±0.656mg·L^-1;AUC_(0-24) 11.22±2.614mg·L^-1·h;AUC_(0-∞) 11.894±2.805mg·L^-1·h;MRT_(0-24) 5.392±1.12h;MRT_(0-∞) 8.285±4.941h;Vl/F 0.089±0.039L·kg^-1;CL/F 0.088±0.021L·h^-1·kg^-1.The AUC of DSNC in tissues was in the order as follows:small intestine＞stomach＞uterus＞kidney＞testis＞liver＞lung＞spleen＞heart＞brain.The MRT of DSNC in tissues were lung＞liver＞testis＞brain＞spleen＞kidney＞uterus＞heart＞stomach＞small intestine. 4.The rat urine and feces were analysized by HPLC-MS/MS method after disposition.The molecular ion peak of molecule weight 169 and 205 were detected in rat urine,which were norcantharidin(M0) and the product of epoxide hydration reaction of phaseâ… metabolism(M1).The molecular ion peak of molecule weight 169 and 539 were detected in rat feces,which were norcantharidin(M0) and the glucuronate conjugation metabolite of phaseâ…¡metabolism(M2).The metablic reaction products in vitro were analysized by HPLC-MS/MS after filteration with 0.45μm millipore filter.Norcantharidin and glucuronate conjugation metabolite were detected in phaseâ…¡metabolism product.Only norcantharidin was detected in phaseâ… metabolism product,no metabolite of phase metabolism was found.5.HPLC method was established for the determination of DSNC in liposome, there was a good linearity of the drug concentration within the range of 50.0～350.0μg·ml^-1,the correlation coefficients was 0.9998.The intra-day and inter-day RSD were less than 2%.The recovery rate was between 90%and 110%. The liposome was all spherically shaped with uniform size.The mean diameter of DSNC liposome and blank liposome was 243.1nm and 209.6nm;Zeta potential was -22.94mv and -14.05mv,respectively.PH was 7.54±0.13.The incorporation efficiency was(34.34±1.21)%and the incorporation efficiency didn't change much during the storage period at 4℃for three month.6.After fitting,the pharmacokinetic process of DSNC in mice after administration of DSNC-lip was coincidence with double compartment model with weight of 1.The main pharmacokinetic parameters of DSNC after administration of DSNC-sol were as follows:t_1/2α 0.763±0.101h;t_1/2β 12.235±5.279h;Tmax 0.708±0.459h;Cmax 3.286±1.114mg·L^-1;AUC_(0-48)17.555±2.941mg·L^-1·h;AUC_(0-∞) 18.231±3.209mg·L^-1·h;MRT_(0-24) 11.631±1.282h;MRT_(0-∞) 12.395±1.4331h; Vl/F 0.169±0.138L·kg^-1;CL/F 0.057±0.012L·h^-1·kg^-1.The AUC of DSNC in tissues was in the order as follows:uterus＞stomach＞small intestine＞kidney＞liver＞lung＞heart＞spleen＞testis＞brain.The MRT of DSNC in tissues were spleen＞liver＞small intestine＞testis＞heart＞brain＞lung＞uterus＞kidney＞stomach. The elimination half life(t_1/2β) and the mean residence time(MRT) in plasma of DSNC-lip increased from 6.354h to 12.235h and 8.285h to 12.395h,respectly, which indicated that the time of drug in plasma was prolonged distinguishly.The apparent volume of distribution(Vl/F) increased from 0.089L·kg^-1 to 0.169L·kg^-1, which implied the liposolubility was enhanced than DSNC-sol.The plasma clearance rate(CL/F) decreased from 0.088L·h^-1·kg^-1 to 0.057L·h^-1·kg^-1,which indicated that the elimination of DSNC-lip was more slow than DSNC-sol.The AUC_0-∞ of DSNC-sol and DSNC-lip were 11.894mg·L^-1·h and 18.231mg·L^-1·h, respectly,the bioavailability(F) was 153.3%.DSNC-lip changed the distribution of DSNC in mice obviously.Except testis, the relative uptake efficency(r_e) of DSNC-lip in other tissues were＞1,which indicated that the liposolubility of DSNC-lip was enhanced than DSNC-sol.The targeting efficency(T_e) of DSNC-lip in brain,heart,lung,uterus and stomach were increased from 0.42%,2.58%,2.88%,18.38%and 18.40%to 1.13%,5.58%,6.00%, 22.44%and 20.73%,respectly.The highest distribution was in uterus.The T_e in liver and kidney were no change.The T_e in small intestin,testis decreased from 21.40%and 8.61%to 12.12%and 3.81%oConclusion:1.The HPLC-MS/MS method was simple,rapid and sensitive enough to meet the requirements for the quantitation of DSNC in biology samples.The comparision between HPLC-MS/MS method and isotope labelling method indicated that the HPLC-MS/MS method can describe the disposition of DSNC in vivo more exactly.2.In this study,the results of tissue distribution indicated that uterus was a targeting tissue of DSNC.It hinted us that the further researches were worth to carry out and supplied evidences for clinical applying in future.3.The results of metabolism study in vivo and in vitro demonstrated that DSNC was excrected in the form of norcantharidin,the product of epoxide hydration reaction of phaseâ… metabolism and the glucuronate conjugation metabolite of phaseâ…¡metabolism. 4.DSNC-lip was prepared successfully by reverse phase evaporation method with higher entrapment efficiency.The initial stability and the delayed releasing character were observed to be good in vitro.5.DSNC-lip can increase distributions in most tissues and can prolong the action time,which can decrease the administration dose and reduce adverse effect.