The Study On The Identification And Pharmacokinetics Of Heparan Sulfate In Rats

In recent years,glycosaminoglycans(GAGs)have gradually become the one of the hot spots in the research area of biochemical drugs,and among which heparan sulfate(HS)is one of the most important ones.A variety of activities of HS have been found,such as anticoagulation,antitumor,antiviral,prevention and treatment of Alzheimer’s disease.HS and heparin are both composed of the same structural units repeatedly,the only difference between them is the degree of sulfuration,and having no specific quantitative indexes or effective methods.Therefore,the problems in quality evaluation are needed to be solved urgently with the development of HS.In addition,pharmacokinetic study of heparin-related medicines was still characterized by the anticoagulant activity of chromogenic substrate assay currently,which could not really reflect the medicine content in the circulatory system since only the interaction between heparin and clotting-related serine protease inhibitors was considered,and the accuracy was also susceptible to macromolecules with similar structures and endogenous substances in the process of analysis.Therefore,this paper carried out the following researches under the above backgrounds.1.Identification of heparin and HS by HILIC-MS/MS method.In this paper,a hydrophilic interaction chromatography-mass spectrometry(HILIC-MS/MS)method was developed for the determination of eight disaccharide structural units of heparin and HS and was applied to identify them.The method was simple,rapid,accurate and reliable,and had the advantages of high sensitivity andspecificity.The linear relationships of the measured components were good in the determination range of molar concentrations,and the precision and repeatability of the method were well.The stability and recovery rate of the samples were ideal during the test.According to the total molar content of 8 disaccharides,the △UA2S-GlcNS6S(TriS)had the largest proportion in heparin,with an average rate of 69.8%(67.2%～72.4%).The ΔUA-GlcNAc(OS)is the largest disaccharide in HS,with an average rate of 51%(45.0%～66.7%).Also,the contents of other disaccharides are different.The above results provide a method and data references for the identification and the development of identification indexs for heparin and HS,as well as provide the references for the construction of a more complete quality evaluation standard system of GAGs2.Establishment of a method for the analysis of HS in rat plasmaIn order to solve the problems of lacking quantitative method for HS,a HILIC-MS/MS method was established to quantify representive disaccharide of HS with high specificity and sensitivity.The disaccharide produced by heparinase treatment of HS was quantified to indirectly represent HS in biological samples UA-GlcNAc(OS)was selected as the representative disaccharide of HS after overall consideration of the results of disaccharides analysis of HS both in water and in plasma.3%ammonium hydroxide in acetonitrile was determined as plasma protein deposition solvent and the linear,precision,accuracy,recovery rate,matrix effect,stability and other aspects of methodology were validated respectively according to the Guidelines for Validation of Quantitative Analysis Methods of Biological Samples The results showed that the proposed method had high sensitivity,good precision and accuracy,and samples were stable during tests.The linear relationship of OS in plasma samples was well with the limit of quantification of 10 ng/mL.The average recovery was 67.7%and the matrix effect was 88.3%～92.2%.This study provides a methodological basis for the pharmacokinetic study of HS,and also provides a new idea for the pharmacokinetic study of other GAGs3.Pharmacokinetic study of HS in ratsThe above verified HILIC-MS/MS method of representive disaccharide of HS in biological samples was applied to study preclinical pharmacokinetics in SD rats.HS was given to rats at the dose of 2 mg/kg intravenously,50,200 and 400 mg/kg by gavage,50 mg/kg by subcutaneous injection to obtain the plasma concentration-time curves.The tissue distribution experiment and excretion experiment were carried out by gavage at the dose of 400 mg/kg and 10 mg/kg,respectively.The OS in plasma,tissue,urine and fece samples of rats were quatified and the pharmacokinetic process and characteristics were analyzed and summarized as follows:HS presented nonlinear pharmacokinetic characteristics by gavage and the absolute bioavailabilities of low,intermediate and high doses were 2.2%,0.88%and 0.56%,respectively,while absolute bioavailability of subcutaneous injection was 26.3%.There was no obvious tissue distribution and accumulation and the fecal excretion rate was 74.7%by gavage.In conclusion,an accurate,sensitive and simple indirect quantitative method for HS was established and successfully applied to preclinical pharmacokinetic study in rats,the results showed that oral HS was absorbed lowly in the blood circulation and tissues,and eliminated mainly through fecal excretion.This study was significant for the further development,rational medicine use and medicine monitoring of HS and GAGs.