Study On In Vivo Pharmacokinetics And In Vitro Metabolism Of Betulin

Betulin is a lupinane type pentacyclic triterpene compound,which has mainly been extracted from the bark of Betula series in amounts up to 30%dry weight.Recent pharmacology studies have shown that betulin has anti-tumor,anti-virus,anti-inflammatory,antioxidant and liver protection activity,it is a very promising plant drug candidate.This thesis presented the pharmacokinetic study of betulin in rat plasma,and the excretion of betulin in rat feces.Metabolic pathways of betulin in rat and human were studied,and metabolic isozymes involved in rat glucuronidation and sulfonation were determined.All the results provide the data basisfor the development and future clinical application of betulin.To solve the problem that betulin cannot be ionization in electrospray ionization(ESI)source,an LC/MS/MS method was developed for the quantification of betulin in rat plasma based on precolumn derivatization.In the present study,p-toluenesulfonyl isocyanate(PTSI)was pro-ven to be the optimal,which generated an ionizable and fragmentable derivative in a negative mode by liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI/MS/MS).After optimizing the content of derivatization reagent,reaction time and temperature,an LC-MS/MS method was developed for betulin quantification,the detection sensitivity of 1 ng/mL.The specificity linear,the minimum quantitative limit,accuracy,precision,recovery,stability and biological matrix effect of this method were validated,all these validations meet Analytical Procedures and Methods Validation Guidance requirement.The method was applied to a pharmacokinetic study in rats after oral administration of betulin at a single dose of 500 mg/kg.Betulin showed slow oral absorption in rat,with a Cmax of 59.6±23.0 ng/mL at 8 h after administration.The mean residence time(MRT)and T1/2 were 26.3±7.6 h and 16.9±2.7 h,respectively.An ultra-performance liquid chromatography(UPLC)with ultraviolet(UV)detection method was established to detect betulin concentration in rat feces.The specificity,accuracy,precision,recovery and stability of this method were validated,suggesting this method meets the requirement for biological sample analysis.After oral administration of betulin at a single dose of 500 mg/kg,betulin cumulative excretion reached 47.21%in rat feces in 96 h,and betulin was not detected in the rat urine.Using 14C labeled uridine diphosphate glucuronic acid(UDPGA)as a cosubstrate,betulin glucuronidation metabolism in rat and human liver microsomes were studied.One glucoronate conjugate of betulin was found in both adult rat and human liver microsomes.In adult human microsome,maximum reaction rate(Vmax)was 19.18±1.97 pmol/min/mg protein,Michaelis-Menten constant(Km)was 21.10±5.93 μM.In 52 weeks adult rat microsome,the level of betulin glucoronate conjugate was only 15%of that in human microsome.Because of the low enzyme activity,enzyme kinetics study could not be performed in adult rat microsome.No betulin glucoronate metabolite was observed in 4-week rat microsome.Eleven cDNA-transfected insect cells expressing individual human UGTs were screened for catalyzing betulin glucuronidation.The results showed that UGT1A3 and UGT1A4 were involved in the betulin glucuronidation metabolism.There is no difference between the efficiency of betulin glucuronidation in UGT1A3 and UGT1A4.Using 35S labeled 3’-phosphoadenosine-5’-phosphosulfate(PAPS)as cosubstrate,betulin sulfation metabolism in rat and human liver cytosols were studied.Two betulin sulfate conjugates were found in both adult rat and human liver cytosol,namely betulin sulfates Ⅰ andⅡ.In rat cytosol.Vmax of betulin sulfate Ⅰ was 9.8 times higher than that of betulin sulfate Ⅱ;as for Km value,there was no obvious difference between two metabolites.In rat cytosol,Vmax of betulin sulfate Ⅰ was 3.7 times higher than that of betulin sulfate Ⅱ;as for Km value,there was also no obvious difference between two metabolites.Rat cytosol showed higher efficiency in catalyzing betulin sulfate I than human cytosol did;No interspecific difference was observed on betulin sulfate Ⅱ formation.Five E.coli cell-free extracts expressing recombinant Human SULTs were screened on the catalytic activity of betulin sulfate.The results showed that SULT2A1 is only involved in the betulin sulfation metabolism.The enzyme kinetics parameters for hSULT2Al were obtained after optimizing incubation time and protein concentration.There was no difference between Km values in human cytosol and hSULT2A1,further proving that hSULT2Al is the major isoenzyme participated in betulin sulfation in human liver.