1.双环醇对免疫性肝纤维化大鼠的保护作用及机制研究 2.Beagle犬口服富马酸泰诺福韦二吡呋酯和BP0018后血浆药代动力学及生物利用度研究

【作者】 谷雨；

【导师】 郭顺星； 李燕； 程桂芳； 陈晓光； 申竹芳；

【作者基本信息】 中国协和医科大学 ， 生药学， 2010， 博士

【摘要】 第一部分双环醇对免疫性肝纤维化大鼠的保护作用及机制研究肝纤维化(liver fibrosis,)是指肝细胞发生坏死及炎症刺激时,肝脏内纤维结缔组织异常增生的病理过程,不仅是慢性肝病最重要的病理特征之一,也是慢性肝炎、肝硬化进一步发展、恶化的重要原因。虽然目前认为肝纤维化是可逆的病理过程,但若不进行及时有效的治疗,最终可发展为不可逆转的肝硬化、甚至肝癌。因此,研究肝纤维化的形成机理及预防和治疗肝纤维化已成为世界医药学攻关中的热点课题之一。目前用于研究肝纤维化机制的常用动物模型包括CCl4、二亚基亚硝胺、胆管结扎、免疫性肝纤维化模型等,其中免疫性肝纤维化模型具有与临床慢性肝炎所致的肝纤维化在发病机制上更加接近,适合于慢性肝炎的免疫机制研究的特点。双环醇是中国医学科学院药物研究所研制的抗肝炎药,已用于治疗慢性病毒性肝炎。临床资料显示,双环醇可明显改善慢性乙型、丙型病毒性肝炎患者临床症状和肝功能损伤,兼有一定抑制乙型肝炎病毒复制的作用,同时具有反跳率低、不良反应小、服用方便的特点。以往研究表明,双环醇对多种实验性肝损伤均有明显的保护作用,其保护机制与清除活性氧自由基、调控细胞因子分泌、抑制免疫损伤诱发的凋亡等相关。本文应用牛血清白蛋白诱导的大鼠免疫性肝纤维化模型,进一步了解双环醇对肝纤维化的保护作用特点,并从细胞核转录因子、细胞因子调控等方面探讨双环醇保护作用机制,为临床应用提供科学可靠的实验依据。双环醇对免疫性肝纤维化大鼠肝损伤的保护作用及机制研究牛血清白蛋白(Bovine Serum Albumin, BSA)诱导大鼠肝纤维化模型的发生机制是基于异种血清白蛋白进入体内后可刺激机体产生抗体,继而形成免疫复合物激活补体,引发Ⅲ型变态反应造成血管炎、血管周围炎等局部免疫炎症反应,进一步刺激胶原的增生而形成肝纤维化。本研究应用的BSA诱导大鼠肝纤维化模型可见肝脏羟脯氨酸(Hydroxyproline,Hyp)水平升高,血清透明质酸(Hyaluronic Acid, HA)和三型前胶原肽(Procollagen TypeⅢ, PⅢP)含量显著增加,肝间质胶原纤维和胶原蛋白增多,汇管区出现网状纤维和胶原纤维的超常增生等纤维化病理变化,与以往研究相符。给药组于攻击阶段开始四周后开始给予双环醇(100,200,300 mg／ml)每日一次,连续给药5周可显著降低BSA诱导肝纤维化大鼠肝脏Hyp、血清HA及PIIIP水平的升高,上述炎症细胞浸润、纤维组织增生等肝脏病理形态学改变也明显减轻。已知肝纤维化形成过程与肝细胞外基质沉积的增加和改变密切相关,主要特征为肝星状细胞激活,转化生长因子(Transforming growth factorβ-1, TGF-β1)及其下游细胞调控子活性发生改变。TGF-β1可调节细胞外基质(Extracellularmatrix, ECM)网状系统成分如纤维胶原和纤维链接蛋白的表达,同时也对蛋白酶抑制因子包括基质金属蛋白酶抑制剂(Tissue inhibitor of metalloproteinase,TIMPs)进行调节。以往研究表明,TGF-β1信号传导通路已成为预防、治疗肝纤维化的一个重要靶点。本研究结果显示,双环醇可在mRNA和蛋白水平显著抑制肝TGF-β1的过表达,提示双环醇可通过下调肝TGF-β1从而防止肝纤维化的形成。肝脏受损后TGF-β可通过细胞内TGF-β/Smad信号通路激活并加速肝星状细胞分化为成肌纤维细胞。此外,TGF-β／Smad通路还可与其他信号通路相互作用,增加或抑制Smad蛋白的磷酸化,其中最重要的是活化蛋白激酶通路(MAPK),如p38 MAPK。已有研究证实,Smad3的磷酸化作用依赖p38 MAPK,促进肌成纤维细胞体外和体内ECM的产生,而P38 MAPK在多重刺激下可被各种MAPKKKs激酶激活,进而在不同水平促进成肌细胞分化。本研究可见,双环醇给药组中Smad2／3磷酸化程度明显降低,且p38 MAPK的磷酸化也相应受到抑制,提示双环醇对TGF-β1表达的抑制作用至少与Smad2／3和p38 MAPK信号通路调控相关。IL-1是重要的炎症因子之一,其促进肝纤维化的机制主要包括以下两方面：1)IL-1在肝组织中所产生的炎症反应可激活p38、JNK,从而导致肝纤维化生成；2)IL-1能够上调TIMP-1mRNA表达,促进ECM的蓄积从而发生肝纤维化。本研究中可见BSA诱导大鼠血浆白介素-1β(Interleukin-1β, IL-1β) mRNA水平明显升高,双环醇可抑制IL-1β的升高,可能与双环醇通过抑制p38MAPK从而减轻肝脏炎症反应有关。此外,在肝纤维化过程中,TNF-α对HSC的增殖、活化以及ECM的合成、基质金属蛋白酶及其组织抑制剂的释放起重要作用。BSA诱导肝纤维化大鼠TNF-αmRNA水平明显升高,双环醇可显著降低其表达。白介素-10(Interleukin-10, IL-10)是体内重要的抗炎因子,与其受体结合后可抑制多种炎症因子和促纤维化因子包括IL-1β等。肝脏免疫炎症过程中可见Kupffer细胞、HSCs和肝浸润T细胞中IL-10 mRNA的高表达。以往研究显示,外源性IL-10能拮抗CCl4诱导的大鼠肝纤维化,并能抑制体外培养活化HSCTGF-β1和碱性成纤维生长因子mRNA表达及促进HSC凋亡。本研究发现,BSA攻击大鼠后肝IL-10反应性升高,而双环醇可减少IL-10的过表达。推测由于双环醇显著下调肝脏前炎症因子的表达,抑制肝脏炎症反应,从而反馈式降低了IL-10的增加。以往研究表明,肝纤维化中胶原的沉积与降解和基质金属蛋白酶(Matrix metalloproteinase, MMPs)及TIMPs密切相关。在健康肝脏中,ECM稳态环境通过MMPs指导精确调节。而在慢性肝损伤中,肝星状细胞被激活、分化为成纤维细胞样表型。与此同时,在活化HSCs中TIMP-1表达上调导致MMP活性的抑制以及随后细胞外间隙基质蛋白蓄积。本研究可见,双环醇使BSA诱导的MMP-2和TIMP-1 mRNA过表达显著降低。尽管尚不能确定胶原活性的增加是否是双环醇抑制TIMP-1表达的结果,但TIMP-1的减少以及胶原酶活性的增加常伴随肝纤维化的逆转。有报道显示,IL-1激活p38 MAPK可导致TIMP-1的上调。综合上述结果,双环醇可能通过调节TGF-β1,抑制炎症因子表达(抑制IL-1β),抑制p38MAPK的磷酸化,干扰TIMP-1从而抑制胶原表达,防止肝纤维化的发生。由此可见,其机制作用与防止炎症反应的发生,从而使IL-10水平反馈性降低,与此同时,双环醇的治疗可使MMP-2和TIMP-1表达显著降低,分析是通过抑制p38 MAPK的磷酸化,干扰TIMP-1抑制胶原表达,最终防止肝纤维化的发生。综上所述,双环醇对BSA诱导的肝纤维化具有明显保护作用,其作用机制可归纳为：1)通过Smad2／3和p38MAPK信号通路调控TGF-β1和IL-1β的表达,抑制肝细胞外基质沉积；2)抑制炎症因子TNF-α和IL-1的表达,从而改善炎症因子／抗炎因子平衡；3)通过调控MMP-2及TIMP-1,抑制胶原表达从而治疗肝纤维化。上述研究为进一步了解双环醇的肝保护作用特点以及临床上应用于肝纤维化的治疗提供了有参考价值的实验依据。第二部分Beagle犬口服富马酸泰诺福韦二吡呋酯和BP0018后血浆药代动力学及生物利用度我国是肝病大国,以慢性乙肝为主,携带HBsAg的人数已经达到总人口的10%,每年新增乙肝患者约为50万,而每年死于乙肝的人数约为28万。与此同时,据最新统计数据显示,全球已有2500万人死于HIV感染,目前HIV感染者尚有3300万人,在我国,现存HIV病毒感染者人和病人已超过70万人。抗HIV和抗HBV药物研发对我国病毒感染性疾病的治疗有着巨大社会和经济意义。Viread一种具有高度抗HBV和HIV活性的新型核苷类逆转录酶抑制剂,已在美国和欧洲获批治疗人类免疫缺陷病毒(HIV)感染,同时也是抗慢性乙肝药物的有效药物之一,可通过干扰乙肝病毒DNA聚合酶的功能,抑制乙肝病毒复制,降低血清及肝组织内的病毒载量。BP0018是江苏正大天晴药业研制的抗病毒新药,与Viread一样,均为泰诺福韦(PMPA)的前药。本文旨在研究Beagle犬单次口服viread和BP0018后血浆药代动力学特点,并计算生物利用度,为BP0018临床合理用药的安全性和有效性提供依据。本研究根据化学药物制剂生物利用度和生物等效性研究技术指导原则,对Beagle犬单次口服BP0018和参比制剂viread的体内血浆药代动力学和生物利用度进行研究。研究结果表明：(1)本研究所建立的用于测定生物样品中泰诺福韦、BP0018和泰诺福韦二吡呋酯的HPLC-MS／MS方法不受杂质干扰,日内精密度RSD小于2.8%,日间精密度RSD小于10.1%,回收率在80.9%至93.1%之间,RSD均小于8.2%,且稳定性良好,可满足体内药代动力学研究要求。(2)Beagle犬单次口服BP0018和参比制剂后各时间点原型药浓度均低于最低检测限(5ng／mL),但血中均可检测到泰诺福韦(PMPA),其浓度随给药后不同时间而变化；(3)BP0018和参比制剂PMPA的达峰时间分别为0.25-1h和0.25-2h,达峰浓度分别为328.06-65.74ng／mL和331.21±22.74ng／mL,体内平均滞留时间(MRT)分别为6.87±1.52h和4.35±1.61h；(4)与参比制剂相比BP0018平均滞留时间(6.876.87±1.52h)和消除半衰期(14.95±9.49h)均有所延长,而AUC(0-t)、AUC(0-∞)、Cmax和Tmax无显著性差别。(5) Beagle犬单次静脉注射PMPA后,血浆药物浓度于给药后2min迅速达峰,24h仍能检测到一定浓度PMPA,36h血浆中PMPA浓度低于检测限。(6) Beagle犬单次口服给药BP0018的体内生物利用度为20.7±4.3%,而参比制剂富马酸泰诺福韦二吡呋酯的生物利用度为16.8±6.2%。综上所述,本文对Beagle犬单次口服富马酸泰诺福韦二吡呋酯和BP0018后血浆药代动力学特点进行了研究,并计算其生物利用度,结果显示,两药主要药代动力学参数无明显差异,生物利用度结果提示基本等效,为BP0018临床合理用药的安全性和有效性提供实验依据。更多还原

【Abstract】 Liver fibrosis is the pathological process of fibrous connective tissue development secondary to inflammatory necrosis of liver cells. It is not only the pathological characteristic of chronic liver disease, but also the reason which leads to the development of chronic hepatitis and cirrhosis.Studies on the mechanism of liver fibrosis and prevention against its formation have become the hot spot on all of world. CCl4, dimethyl nitrosamine, bile duct ligation and immunological liver fibrosis are the common models to study the mechanism of liver fibrosis.Since immunological liver fibrosis is more closed to the pathogenesy of liver fibrosis which is caused by chronic hepatitis in clinic, so it’s used to study the immunological mechanism of action of chronic hepatitis.Liver fibrosis is a scarring process associated with an increased and altered deposition of liver extracellular matrix. It is mainly characterized by the cellular activation of HSC, aberrant activity of TGF-β1 and its downstream cellular mediators. TGF-P was known to regulate the expression of both components of ECM network, such as fibrillar collagens, fibronectin and protease inhibitors including TIMPs. Based on the previous studies, TGF-β1 signal transduction pathway has become an effective target for the prevention or treatment of liver fibrosis. As shown in the present study, bicyclol was found to reduce the expression of liver TGF-β1 with significant difference both on mRNA and protein levels. It suggested that bicyclol may protect liver from fibrogenesis through the down regulation of liver TGF-β1.Previous reports showed that TGF-P can activate and promote the transdifferentiation of HSCs to myofibroblasts via intracellular TGF-β/Smad signaling pathway during the process of liver injury. It is admitted that TGF-β/Smad pathway interacts with other signaling pathways to increase or inhibit Smad phosphorylation. Of most important is the mitogen activated protein kinase (MAPK) pathways, such as p38 MAPK. It has been proved that the phosphrylation of Smad3 is p38 MAPK-dependent. p38 MAPK is activated by various MAPK kinase kinases (MAPKKKs) in response to many stimuli. Then it promotes the progression of myoblasts differentiation at multiple levels. Our results showed that bicyclol inhibited the phosphorylation of Smad2/3, correspondently, the phosphorylation of p38 was also reduced as well. Thus, it suggested that the inhibitory effect of bicyclol on TGF-β1 expression was at least in part via the Smad2/3 and p38 signaling pathways.IL-1, as an inflammatory cytokine, was involved in the liver fibrosis mainly through the activation of p38 and up-regulation of TIMP-1 mRNA expression in HSCs. In the present study, we found that the mRNA level of IL-1βwas enhanced and bicyclol can inhibit the elevation of IL-1βin BSA-induced rats. The above results suggested that the effect of bicyclol on preventing the inflammation in liver via inhibiting p38.Interleukin-10 (IL-10) is considered as an anti-inflammatory cytokine which inhibit the production of several inflammatory cytokines, including IL-1β. Several reports demonstrated that the exogenous IL-10 can inhibited the liver fibrosis in CCl4 induced rats, the level of TGF-β1 expressed in activated HSCs cultured in vitro and mRNA level of basic fibroblast growth factor, enhanced the apoptosis of HSCs. The high mRNA expression of IL-10 in Kupffer cell, HSCs and liver-infiltrating T cells was found in the course of liver inflammation previously. It was found that the hepatic IL-10 was increased in BSA treated rats, and the overexpression of IL-10 can be reduced by bicyclol treatment. It was presumed that bicyclol significantly down regulated the gene expression of liver pro-inflammatory cytokines, and thereby attenuated feedback increase of IL-10.During the process of liver fibrosis, TNF-a may active HSCs, and play an important role in the synthesis of ECM, the release of MMPs and TIMPs. The mRNA level of TNF-a is induced in the BSA-induced liver fibrosis rats, and bicyclol can inhibite the expression of it.Previous studies indicated that the deposition and degradation of collagen in liver fibrosis were related to MMPs and TIMPs closely. The homeostasis of ECM in healthy liver is sustained by a precisely regulated permanent turn-over directed by MMPs. Upon chronic liver damage, HSCs was activated and differentiate into fibroblast-like phenotype. At the same time, the expression of TIMP-1 especially in activated HSCs is upregulated leading to the inhibition of MMP activity and subsequent accumulation of matrix proteins in extracellular space. In the present study, the treatment of bicyclol resulted in a significant reduction of MMP-2 and TIMP-1 mRNA expression. Although it is still uncertain whether the increase of collagenase activity was a result of bicyclol’s inhibitory effect on TIMP-1 expression, the decrease of TIMP-1 and the increase of collagenase activity often coincide when liver fibrosis begin to reverse. In addition, it has been reported that IL-1 activated P38 MAPK and resulted in the up regulation of TIMP-1. Considered the results above all, bicyclol may protect liver from fibrogenesis through regulating TGF-β1, preventing the inflammation (as evidenced by the inhibition of IL-1βand IL-10) by suppressing the phosphorylation of p38 MAPK, thus interfering with TIMP-1 and inhibiting the collagen expression.In conclusion, bicyclol showed significant protective effect on BSA-induced liver fibrosis. The hepatoprotection of bicyclol is mostly due to the following aspects: bicyclol can modulate TGF-β1 through Smad2/3 and p38 MAPK signaling pathways; it inhibit IL-1βvia regulating TGF-β1 to protect liver from fibrogenesis, then attenuated feedback increase of IL-10; at the same time, the treatment of bicyclol lower the levels of MMP-2 and TIMP-1, it’s hypothesized that bicyclol can regulate TIMP-1 and inhibit the expression of collagen by reducing the phosphrylation of p38 MAPK, the balance of fibrogenesis and fibrolysis was maintained. BP0018 is a novel anti-hepatitis drug which is developed by Jiang Su Chia-Tai Tianqing Pharmacy Co. Ltd. As a new type of nucleosidereverse transcriptase inhibitor (NRTI), BP0018 and Viread both are the prodrugs of tenofovir (PMPA) Viread was known to be efficient in inhibition of HBV and HIV, and was approved by FDA in America and Europe to cure HIV. At the same time, viread is an efficient inhibitior of anti-HBV, it can interfere with the HBV DNA polymerase to stop the virus from reproducing. As a result, the copies of hepatitis virus and serologic markers are reduced.However, for treatment of HIV or HBV, the paients need take medicine for long term, so the economic of the treatment is one of the most important elements. Viread as an import durg increases the cost of therapy and the economic burden of the patients. In China, most anti-hepatitis drugs and anti-HIV durgs are imported drugs or Generic drugs, theres no efficient new drugs with independent intellectual property in clinic. BP0018 as a new drug researched by Jiang Su Chia-Tai Tianqing Pharmacy Co. Ltd, has clinical significance. The aim of this paper is to study the plasma pharmacokinetics of viread and BP0018 after being given orally in beagle dogs. The bioavailabilities of this two drugs were analyzed to provide evidence for safety and effectivity of BP0018 in clinic.According to the instruction of chemical pharmaceutical preparations bioavailability and bioequiavailability, the plasma pharmacokinetic and bioavailabilities of BP0018 and its reference preparation viread were studied after single dose in beagle dogs.The results showed:(1) The HPLC/MS/MS analysis method for the simultaneous quantification of tenofovir, viread and BP0018 in biological samples showed good sensitivity, linearity of response, high precision and appropriate for pharmacokinetic study.(2) After single dose of BP0018 and reference preparation in beagle dogs, the concentration of drug prototype at every time point is lower than the detectable limit, but PMPA can be detected in plasma, the concentrations of PMPA vary with time.(3) Tmax of BP0018 and reference preparation PMPA are 0.25-1h and 0.25-2h, Cmax are 328.06±65.74ng/mL and 331.21±22.74ng/mL, and mean residence time (MRT) are 6.87±1.52h and 4.35±1.61h, respectively.(4) Compared with the reference preparation, the MRT and elimination half life (tl/2z) of are extended, but there’s no siginificant difference of AUC(0-t)、AUC(0-∞)、Cmax and Tmax.(5) After single intravenous injection of PMPA, the concentration of it reached to the peak value immediately, PMPA can still be detected at 24h, the concentration of PMPA is lower the detectable limit at 36h.(6) After single dose of BP0018, the availability of it is 20.7±4.3%, and the availability of viread is 16.8±6.2%.In conclusion, study of relative system pharmacokinetic research was performed on viread and BP0018, bioavailabilities of this two drugs were analyzed to provide basis for safety and effectivity of BP0018 in clinic.更多还原