【作者】 王娟；

【导师】 李娟；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2014， 博士

【摘要】 慢性乙型肝炎属于重大传染性疾病，其传染性强，患病率高，对人体健康危害严重。我国是乙肝高发区之一，约有乙肝病毒携带者近1.3亿人。目前临床使用的抗病毒药物主要为核苷类似物，但其缺点是易产生耐药性，毒副作用大，因此开发高效低毒的抗病毒药物十分重要。本研究首次选用杂多化合物为主要研究体系，利用其分子可设计性的特点，设计、合成了系列稀土杂多化合物。在此基础上，按照临床前新药研发标准的要求，全面系统地开展了稀土杂多化合物抗HBV的药学、药效学、毒理学和药代动力学研究。同时，首次应用先进的同步辐射成像技术，探讨了杂多化合物抗病毒作用机制。药学研究中，采用红外光谱、紫外光谱、X射线单晶衍射及核磁共振光谱等检测方法，对受试化合物进行结构确证性和稳定性研究。多种检测方法共同表明，受试化合物化学结构明确，稳定性良好。药效学研究中，分别以HepG2.2.15细胞和HBV转基因鼠为研究模型，检测受试化合物对HBeAg、HBsAg和HBV DNA的抑制作用，结果表明，受试化合物对抗原分泌和病毒DNA合成有较强的抑制作用。经药效学筛选，选取受试化合物9开展急性毒性、长期毒性和遗传毒性等安全性评价研究。毒性评价结果表明，受试化合物9属于基本无毒化合物，长期毒性实验未发现毒性作用，无致畸、致突变作用。采用ICP-MS技术评价受试化合物9经口途径给药的药代动力学特征，结果表明，受试化合物9在大鼠体内的动力学过程符合二室模型，其吸收过程迅速，肝脏靶向性好，无组织蓄积，与血浆蛋白呈中度结合。X射线纳米CT细胞成像结果表明，杂多化合物的抗病毒机理是通过抑制病毒侵入宿主细胞过程发挥作用。本研究首次系统的研究稀土杂多化合物抗HBV的活性，课题研究成果已获得国家发明专利。本实验研究为开发具有自主知识产权的新型非核苷类抗HBV药物奠定了坚实的基础。更多还原

【Abstract】 Hepatitis B is an infectious disease caused by the hepatitis B virus (hepatitis Bvirus, HBV) So far there are about3.5to400million chronic hepatitis B virusinfection in the world. There are more than one million patients die from diseasescaused by HBV in each year. Our country is a high incidence of HBV infection. It iscalculated that there are existing120million people carriers chronic HBV, and about30million cases of hepatitis B patients. Moreover the number of new cases is around10to30thousand per year. Chronic HBV infection has become a serious public healthproblem. The direct economic losses caused by chronic hepatitis B disease are up toabout900billion Yuan each year in China.The target of anti-HBV therapy is to suppress or remove the body of HBV in thebody, reducing and preventing complications, thereby, improving life quality ofpatients and prolong survival. Drug therapy is the most important means of clinicalanti-HBV treatment. Currently, drug therapy is the most important means of clinicalanti-HBV treatment. The anti-HBV drugs are mainly divided into three categories,interferon, and nucleoside and non-nucleoside drugs. However, these drugs in usehave different levels of drug resistance and side effects. Such as interferon withserious side effects is low response rate; THF can lead to emergence oflamivudine-resistant mutants.Although the effect of these drugs in inhibiting orremoving serum HBV DNA is rapid and significant, however, the drug concentrationin the liver is low. It is difficult to remove the virus in the liver cells, so the viruspersists in the patient body. Therefore, the development of efficiency, low toxicity,low-cost anti-HBV drugs has still great importance.Polyoxometalates are a class of metal oxide cluster compounds. POMs have beenstudied on antiviral activity for decade years. It is found that POMs have a stronginhibitory effect for HIV, influenza virus and other RNA viruses and some DNA viruses. It has been reported POMs may achieve its antiviral activity by inhibiting thereverse transcriptase activity or blocked viruses’ entry. So far its antiviralunderpinning mechanisms remain elusive.Polyoxometalates application in the field of biology has broad prospects forsynthesize of POMs entering into the molecular Design phase. Our previous studyfound that, the new compound (PTW-6) has excellent anti-HBV activity in vitro.With the inspiration our research group designed and synthesized a series of new rareearth polyoxometalates according to the structure and replication characteristics ofHBV. In accordance with the standard of pre-clinical drug development andrequirements, we carried out a comprehensive and systematic studies on POMs inpharmacy, pharmacodynamics, toxicology and pharmacokinetic. Last but not the mostimportant, the antiviral mechanism of POMs had been revealed directly by CellImaging using advanced X-ray nano-CT technology.1. Pharmaceutical StudyIn this study,11new rare earth POMs have been self-designed and synthesizedby general inorganic synthesis method. Confirmatory studies on compound structureshave been detected using IR, UV, X-ray diffraction and NMR spectroscopy and othermethods. PH stability of compounds was determined by conductance method. Theresults showed that the conductivity remained unchanged in the range of PH3~9.Compounds long-term placement stability and thermal stability were detected usingIR, UV, NMR and TG analysis. The results showed that the properties and structure ofthe compound is not changed. All the results in this study indicate a series of POMsshowing good drug ability with definite structure and excellent stability.2. Pharmacodynamic StudyCytotoxicity of compounds was detected by MTT assay. The results showcytotoxic of all compounds was low, in which the compound3,4,9and11hadoptimal results by comparing the CC50value.HBeAg, HBsAg secretion inhibition ofcompounds was detected by HBV e (s) antigen diagnostic test kit. The results showHBeAg, HBsAg secretion inhibitory effect of compound1,3,4and6is strong.Using quantitative PCR assay to test extracellular HBV DNA levels, the results showedHBV DNA replication inhibitory effect of compounds9and11were stronger than thepositive control drugs by comparing the IC50values. Then, the compounds9and11with the strongest anti-HBV activity and less toxic were selected to be conductedefficacy experiments in vivo. Quantitative PCR and ELISA were used detection oftransgenic mouse serum HBV DNA and HBsAg levels. The results showed that bothcompounds existed stronger effect than the positive control drugs on the replication ofHBV DNA and HBsAg secretion inhibitory effect.3. Safety Evaluation StudyIn this study, Kunming mice and Wistar rats were used as experimental animalmodels.The compounds were tested in acute toxicity, chronic toxicity andgenotoxicity. Acute toxicity test results showed that compounds3,4and11belong tolow toxicity compounds and compound9is non-toxic compound. Then, thecompound9was selected for long-term toxicity studies. The results show that thegeneral normal conditions, blood biochemical parameters and histopathologicalexamination were found no abnormalities in rats. The genetic toxicity results showedthe compound9has no teratogenic and mutagenic effects.4. Pharmacokinetics StudyThe quantitative analysis method was established to test the drug concentrationin Wistar rats by inductively coupled plasma (ICP-MS) technology. Then thepharmacokinetic studies of compound9were conducted based on this method. Thedynamic process of the test compound9complied with the two-compartment modelby calculate the compound concentration in rats through gavage (or oraladministration). The main pharmacokinetic parameters,AUC0~t, AUC0-∞andCmax, were dose related, and t1/2, CL, MRT and Vd and other pharmacokineticparameters Displays linear kinetics characteristics, which did not increase with thedose varies. The compound9can be quickly absorbed into the blood circulationsystem after oral administration with Tmax between0.5~2h, t1/2between20~26h. The absolute bioavailability in rats on different doses of compound9was13.76%, 6.39%and5.79%respectively. The result indicates that most of the test compoundmay be biotransformation after absorbing into the body. Tissue distribution result ofthe test compound9showed the concentration of the small intestine and the stomachthan other organizations, and liver tissue concentration was up to1579.10ng/g,indicating that the test compound has a good liver targeting property. Usingequilibrium dialysis method to test plasma protein binding, the results showed that thetest compound9showed moderate intensity to plasma proteins. The excretion resultsshow that about24.30%of the drug dose was excreted with urine, feces and bilewithin72h after administration. Prototype content in feces, urine and bile were low.This further indicates the test compound9conducted extensive metabolism in rats.In summary, we have synthesized a series of rare earth polyoxometalates withexcellent anti-HBV activity. The achievement of polyoxometalates in pharmaceutical,pharmacodynamics, toxicology and pharmacokinetic created a new era in theanti-virus field. The Comprehensive study also laid a solid foundation for thedevelopment of new non-nucleoside with independent intellectual property rights ofanti-HBV drugs.更多还原