Study On The Liver Targeting Of Norcantharidin Enhanced By Oleanolic Acid Based On Traditional Chinese Medicine Guide Theory

Liver cancer remains the sixth most frequently diagnosed cancer andthe third leading cause of cancer-related mortality globally.It is estimatedthat695,900cancer deaths and748,300new liver cancer cases occurred in2008.More seriously,liver cancer incidence rates are still increasing inmany parts of the world including the Central Europe and United States,andthe incidence range has doubled in Europe over the last20years. In Asiacountries,the situation could be more worse.Half of the diagnosed livercancer deaths and cases were estimated to occur inChina.Norcantharidin,the demethylated analogue of cantharidin,inhibits theproliferation of a variety of human cancer cells,especially liver cancer,andappears to cause the least inflammatory and nephrotoxic side effects.It isnoteworthy that NCTD can increase the peripheral white blood cell andstimulate the bone marrow significantly,which makes it more valuable incombination chemotherapy since many of the chemotherapeutic agents inuse nowadays have the side effect of bone marrow suppression. Oleanolicacid is pentacyclic triterpene acid naturally occurring in many plants suchas ginseng root and olive plant.The inhibitory effects of this compounds onthe growth of human bladder,prostate,pancreatic and liver cancer cell lineshave been reported.The study on the liver targeting of norcantharidinenhanced by oleanolic acid based on traditional Chinese medicine guidetheory is to explore the relationship between traditional Chinese medicine guide theory and medicine targeting,which may provide references forstudying the modern targeting delivery system based on traditional Chinesemedicine guide theory.The first part:Objective: A high performance liquid chromatography(HPLC) methodhas been established for the determination of encapsulation efficiency ofnorcantharidin and oleanolic acid liposome.Methods:Separation wasachieved using an Thermo Sycronis C18column(150mm×4.6mm,5μm)and a gradient mobile phase.The samples were monitored at210nm andthe column temperature was30°C.The free components in the liposomewere separated by ultracentrifugation.Results:The linear range ofnorcantharidin was in the range of20.0～300.0μg/mL(r=0.9998) and theaverage recoveries were99.15%～100.11%with the RSD below3.20%.The linear range of oleanolic acid was in the range of10.0～150.0μg/mL(r=0.9997) and the average recoveries were99.22%～100.30%withthe RSD below3.35%.Conclusion:The established HPLC method wassimple,fast and thus could be applied to the determination of eencapsulation efficiency of norcantharidin and oleanolic acid liposome.The second part:Objective:To prepare norcantharidin and oleanolic acid liposome(NCTD-OA-LP).Method:Prepared liposome by film hydration method.Central composite design-response surface method and overall desirabilitywere used to optimize the formula，the encapsulation efficiency of NCTDand OA as indicators.Result:The optimum conditions of formulation weremedicine/phosphatidylcholine11.8%,cholesterol/phosphatidylcholine17.2%and ultrasonic time20.0min.The encapsulation efficiency of NCTDand OA were45.60%and80.69%， respectively.Conclusion:The NCTD-OA-LP showed good encapsulation efficiency after being optimizedwith central composite design-response surface method.The third part:Objective: A reliable and sensitive liquid chromatography/massspectrometry(LC-MS) method has been developed and validated fordetermination of norcantharidin in mouse plasma，heart，liver，spleen，lungand kidney after intravenous administration.To study the liver targeting ofnorcantharidin enhanced by oleanolic acid based on traditional Chinesemedicine guide theory,to explore the relationship between traditionalChinese medicine guide theory and medicine targeting.Methods:LCseparation was achieved on C18column(50mm×2.1mm,1.7μm) using amobile phase consisting of methanol-0.25%formic acid in water at a flowrate of0.3mL/min.The detection was carried out on a quantum triple stagequadrupole mass spectrometer with electrospray ionization(ESI) inlet in thepositive ion SIM mode. Tail intravenous injection,NCTD dosage is5mg/kg.Detect the concentration of NCTD in plasma and tissues.Calculatethe pharmacokinetic parameters.Study the liver targeting of norcantharidinenhanced by oleanolic acid with tce,re,TE.Results:The linear range ofnorcantharidin in plasma was in the range of0.15～12.00μg/mL(r=0.9997)and the average recoveries were84.32%～91.06%with the RSD below8.28%.The linear range of norcantharidin in heart was in the range of0.10～1.00μg/g(r=0.9988) and the average recoveries were80.08%～86.61%with the RSD below12.17%.The linear range of norcantharidin inliver was in the range of0.40～12.80μg/g(r=0.9990) and the averagerecoveries were83.62%～90.30%with the RSD below8.90%.The linearrange of norcantharidin in spleen was in the range of0.20～6.40μg/g(r=0.9993) and the average recoveries were82.32%～89.92%with the RSD below9.15%.The linear range of norcantharidin in lung was in therange of0.20～2.40μg/g(r=0.9994) and the average recoveries were81.89%～88.10%with the RSD below10.95%.The linear range ofnorcantharidin in kidney was in the range of0.20～2.40μg/g(r=0.9987)and the average recoveries were99.15%～100.11%with the RSD below3.20%.t1/2,MRT,clearance,Vssof norcantharidin liposome group is3.46h,4.80h,223.99mL/h kg,1075.99mL/kg. t1/2,MRT,clearance,Vssofnorcantharidin and oleanolic acid liposome group is3.46h,4.78h,248.59mL/h kg,1189.56mL/kg.te-NCTD-OAand TEcNCTD-OAof norcantharidin andoleanolic acid liposome group was higher than te-NCTDand TEcNCTDofnorcantharidin liposome group.Conclusion: The established LC-MSmethod was simple,fast, accurate,sensitive and thus could be applied to thedetermination of norcantharidin in mouse plasma,heart,liver,spleen,lungand kidney.Norcantharidin and oleanolic acid liposome had the ability ofliver targeting,the liver targeting of norcantharidin enhanced by oleanolicacid.