技术领域technical field
本发明涉及化合物N-(4，5-二氢-2-噻唑)-2-(4-甲基苯氧甲基)噻唑-4-甲酰胺，应用于动脉粥样硬化心血管疾病的治疗和/或预防，属于制药领域；本发明所述化合物用于制备ABCA1上调剂、调血脂药物、以及抗动脉粥样硬化的预防和/或治疗的用途；本发明还涉及所述化合物的药物组合物。The present invention relates to compound N-(4,5-dihydro-2-thiazole)-2-(4-methylphenoxymethyl)thiazole-4-carboxamide, which is used in the treatment and treatment of atherosclerotic cardiovascular diseases / or prevention, belonging to the field of pharmacy; the compound of the present invention is used for the preparation of ABCA1 up-regulators, blood lipid drugs, and the prevention and/or treatment of anti-atherosclerosis; the present invention also relates to the pharmaceutical composition of the compound .
背景技术Background technique
心血管疾病在发达国家和大多数发展中国家是危害人类健康的主要杀手，近年来随着人们物质生活水平的提高，心脑血管疾病的发病率呈明显上升趋势，心脑血管疾病死亡占全球人口死亡的构成已达1/3。动脉粥样硬化(Atherosclerosis)是一种慢性炎性疾病，是多种严重心血管疾病(如冠心病、心绞痛、心肌梗塞、脑卒中等)的病理基础。目前我国动脉粥样硬化呈现高发病率、年轻化趋势。Cardiovascular disease is the main killer of human health in developed countries and most developing countries. In recent years, with the improvement of people's material living standards, the incidence of cardiovascular and cerebrovascular diseases has shown an obvious upward trend, and the death rate of cardiovascular and cerebrovascular diseases accounts for the The composition of population death has reached 1/3. Atherosclerosis (Atherosclerosis) is a chronic inflammatory disease, which is the pathological basis of many serious cardiovascular diseases (such as coronary heart disease, angina pectoris, myocardial infarction, stroke, etc.). At present, atherosclerosis in my country presents a high incidence rate and a younger trend.
目前动脉粥样硬化药物治疗包括扩张血管药物、调整血脂药物、抗血小板药物等。现有方法虽可减缓该病进程，但尚不能治愈。目前临床上广泛应用的他汀类药物主要是通过抑制胆固醇的生物合成和增强外周细胞对胆固醇摄取的LDL受体调节通路来实现抗动脉粥样硬化的，但仅可以降低20％～40％的心血管事件【1】，并且目前他汀类药物发现很多肝脏、心脏等方面毒性。要进一步降低心血管疾病的危害，在降低低密度脂蛋白胆固醇的同时必须从预防和/或逆转动脉粥样硬化的新的治疗靶点出发来寻找具有新作用机制的药物。Current atherosclerosis drug treatment includes vasodilation drugs, blood lipid adjustment drugs, anti-platelet drugs and so on. Although existing methods can slow down the progress of the disease, they cannot cure it yet. At present, statins widely used in clinic mainly realize anti-atherosclerosis by inhibiting the biosynthesis of cholesterol and enhancing the LDL receptor regulation pathway of peripheral cells to cholesterol uptake, but they can only reduce the heart rate by 20% to 40%. Vascular events [1], and many statins have been found to be toxic to the liver and heart. In order to further reduce the harm of cardiovascular diseases, it is necessary to find drugs with new mechanisms of action from new therapeutic targets for preventing and/or reversing atherosclerosis while reducing low-density lipoprotein cholesterol.
外周细胞内胆固醇代谢障碍是动脉粥样硬化的重要发病机制，过量胆固醇从肝外组织中的清除是预防和治疗动脉粥样硬化的关键步骤【2】。高密度脂蛋白(HDL)将外周组织中的胆固醇转运到肝脏再次代谢或以胆酸的形式排泄，这一过程被称为胆固醇逆转运(RCT)【3】。RCT及胆固醇排除途径是治疗动脉粥样硬化的有效手段。人ATP结合盒转运体A1(ABCA1，(ATP-binding cassette transporter(ABC)A1)在RCT中发挥重要作用【4】。Cholesterol metabolism disorder in peripheral cells is an important pathogenesis of atherosclerosis, and the removal of excess cholesterol from extrahepatic tissues is a key step in the prevention and treatment of atherosclerosis [2]. High-density lipoprotein (HDL) transports cholesterol in peripheral tissues to the liver for re-metabolism or excretion in the form of bile acid, a process known as reverse cholesterol transport (RCT) [3]. RCT and cholesterol elimination approach are effective means to treat atherosclerosis. Human ATP-binding cassette transporter A1 (ABCA1, (ATP-binding cassette transporter (ABC) A1) plays an important role in RCT [4].
ABCA1基因突变导致Tangier disease(TD)病、组织巨噬细胞内胆固醇聚积，血浆HDL胆固醇降低(HDL cholesterol，HDL-C)，增加心血管疾病的风险【5】。高表达ABCA1的转基因小鼠可以升高血浆HDL、apoA-I的水平，并且使巨噬细胞胆固醇流出明显增加，从而降低动脉粥样硬化的危险性。ABCA1的主要功能是将细胞内游离的胆固醇及磷脂转运至贫脂或无脂的载脂蛋白A-I(apoA-I)，这是胆固醇逆转运(RCT)和HDL生成的第一个限速环节，因此，ABCA1对脂质代谢和动脉粥样硬化的发生、发展具有重要影响；越来越多的临床试验表明升高HDL水平对心血管疾病有益，并且不依赖降低LDL【6】。研究表明，ABCA1在肝脏中的表达对于HDL代谢以至体内整个胆固醇的内环境稳定而言都很重要【7，8】，对于体内巨噬细胞中的RCT也起到正调控的作用【9】。ABCA1被认为是发现新型心血管药物的潜在的新靶标【10-13】。Mutations in the ABCA1 gene lead to Tangier disease (TD) disease, accumulation of cholesterol in tissue macrophages, reduction of plasma HDL cholesterol (HDL cholesterol, HDL-C), and increased risk of cardiovascular disease [5]. Transgenic mice with high expression of ABCA1 can increase the levels of plasma HDL and apoA-I, and significantly increase the outflow of macrophage cholesterol, thereby reducing the risk of atherosclerosis. The main function of ABCA1 is to transport free cholesterol and phospholipids in cells to fat-poor or fat-free apolipoprotein A-I (apoA-I), which is the first rate-limiting link of reverse cholesterol transport (RCT) and HDL production. Therefore, ABCA1 has an important impact on lipid metabolism and the occurrence and development of atherosclerosis; more and more clinical trials have shown that increasing HDL levels is beneficial to cardiovascular diseases, and does not rely on lowering LDL [6]. Studies have shown that the expression of ABCA1 in the liver is very important for the metabolism of HDL and the stability of the internal environment of the whole cholesterol in the body [7,8], and it also plays a positive regulatory role in the RCT of macrophages in the body [9]. ABCA1 is considered to be a potential new target for the discovery of novel cardiovascular drugs [10-13].
总之，临床上使用的他汀类治疗药物也存在较重的副作用，目前尚缺乏治疗动脉粥样硬 化的特效药物。为获得组织特异性更强、通过调节RCT关键受体从而促进胆固醇外排、减少脂质聚积，从而起到预防和/或治疗动脉粥样硬化新型药物，利用中国医学科学院医药生物技术研究所国家新药(微生物)筛选实验室构建的ABCA1上调剂筛选模型进行广泛筛选，发现化合物N-(4，5-二氢-2-噻唑)-2-(4-甲基苯氧甲基)噻唑-4-甲酰胺具有明显上调ABCA1的活性，并且在体外测定了确认其有抗动脉粥样硬化心血管疾病活性。N-(4，5-二氢-2-噻唑)-2-(4-甲基苯氧甲基)噻唑-4-甲酰胺在抗动脉粥样硬化作用方面未见有文献报道，系本专利申请的首次发现。这组胺甲酰基苯磺酰类化合物与临床上使用的他汀类药物相比，结构没有相似性，并且发挥抗动脉粥样硬化作用机制不同，有望成为特异性调节RCT关键受体ABCA1表达、降低脂质、降低胆固醇，从而成为抗动脉粥样硬化乃至保护心血管作用的治疗药物。因此，该化合物有可能是具有新作用机制的抗动脉粥样硬化化合物，具有广阔的开发应用前景。In conclusion, clinically used statin drugs also have serious side effects, and currently there is no specific drug for the treatment of atherosclerosis. In order to obtain new drugs with stronger tissue specificity, promote cholesterol efflux and reduce lipid accumulation by regulating RCT key receptors, thereby preventing and/or treating atherosclerosis, the National Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences The ABCA1 up-regulator screening model built by the New Drug (Microbial) Screening Laboratory conducted extensive screening and found the compound N-(4,5-dihydro-2-thiazole)-2-(4-methylphenoxymethyl)thiazole-4 -Formamide has the activity of significantly up-regulating ABCA1, and in vitro assays confirmed its anti-atherosclerotic cardiovascular disease activity. N-(4,5-dihydro-2-thiazole)-2-(4-methylphenoxymethyl)thiazole-4-carboxamide has no literature report on the anti-atherosclerosis effect, which is the patent The first discovery of the application. Compared with clinically used statins, this group of carbamoylbenzenesulfonyl compounds has no similarity in structure, and has a different mechanism of anti-atherosclerosis. Lipids, lower cholesterol, and thus become a therapeutic drug for anti-atherosclerosis and even cardiovascular protection. Therefore, the compound may be an anti-atherosclerotic compound with a new mechanism of action, and has broad development and application prospects.
发明内容Contents of the invention
本发明发现涉及化合物N-(4，5-二氢-2-噻唑)-2-(4-甲基苯氧甲基)噻唑-4-甲酰胺，在制备抗动脉粥样硬化心血管疾病的药物中的应用，该化合物具有结构新颖、作用机制独特、良好的抗动脉粥样硬化效果的特点，其结构如式(I)所示：The discovery of the present invention relates to the compound N-(4,5-dihydro-2-thiazole)-2-(4-methylphenoxymethyl)thiazole-4-carboxamide, which is used in the preparation of anti-atherosclerotic cardiovascular disease For application in medicine, the compound has the characteristics of novel structure, unique mechanism of action, and good anti-atherosclerotic effect, and its structure is shown in formula (I):
应当理解，本发明的化合物进行结构改造所采取的方法，可以通过药学领域众所周知的任何方法制备成所需的结构。It should be understood that the methods adopted for structural modification of the compounds of the present invention can be prepared into desired structures by any method well known in the field of pharmacy.
本发明所述化合物的应用，其特征在于所述药物为可上调ATP结合盒转运子(ABCA1)表达活性的药物。The application of the compound of the present invention is characterized in that the drug is a drug capable of up-regulating the expression activity of the ATP-binding cassette transporter (ABCA1).
本发明所述化合物的应用，其特征在于所述药物为调节血脂的药物。The application of the compound of the present invention is characterized in that the drug is a drug for regulating blood lipid.
一种药物组合物在制备抗动脉粥样硬化心血管疾病的药物的应用，其特征在于所述药物组合物含有治疗有效量的所述化合物作为活性成分，以及一种或多种药学上可接受的载体。An application of a pharmaceutical composition in the preparation of a drug against atherosclerotic cardiovascular disease, characterized in that the pharmaceutical composition contains a therapeutically effective amount of the compound as an active ingredient, and one or more pharmaceutically acceptable Carrier.
本发明所述药物组合物的应用，其特征在于所述药物组合物含有重量比为0.1％-99.5％的活性成分，优选含有重量比为0.5％-99.5％的活性成分。The application of the pharmaceutical composition of the present invention is characterized in that the pharmaceutical composition contains 0.1%-99.5% active ingredient by weight, preferably 0.5%-99.5% active ingredient by weight.
本发明所述药物组合物的应用，其特征在于所述药物为可上调ATP结合盒转运子(ABCA1)表达活性、调节血脂的药物。The application of the pharmaceutical composition of the present invention is characterized in that the drug is a drug that can up-regulate the expression activity of the ATP-binding cassette transporter (ABCA1) and regulate blood lipids.
本发明所述药物组合物的应用，其特征在于所述药物为抗动脉粥样硬化的药物。The application of the pharmaceutical composition of the present invention is characterized in that the drug is an anti-atherosclerosis drug.
本发明所述药物组合物，其各种剂型可以按照药学领域的常规生产方法制备，如使活性成分与一种或多种载体混合，然后将其制成所需的剂型，适于通过任何合适的途径治疗施用的形式(例如口服、皮下、肌内、静脉内和皮内等途径)。The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the field of pharmacy, such as mixing the active ingredient with one or more carriers, and then making it into the desired dosage form. Routes of therapeutic administration and forms of administration (for example, oral, subcutaneous, intramuscular, intravenous and intradermal routes).
本发明所述化合物，可以按照药学领域的合成方法制备。The compounds of the present invention can be prepared according to the synthetic methods in the field of pharmacy.
应当理解，本发明的化合物及其组合物的适当剂量可能取决于疾病的类型、严重程度和阶段，并且随患者各不相同。确定最佳剂量一般包括使治疗优点水平与本发明的治疗的任何危险或有害副作用相平衡。It is to be understood that appropriate dosages of the compounds of the present invention and compositions thereof may depend on the type, severity and stage of the disease and will vary from patient to patient. Determining the optimal dosage will generally involve balancing the level of therapeutic advantage against any risk or deleterious side effects of the treatments of the invention.
本发明人利用本实验室建立的人ABCA1筛选模型对专利化合物的活性进行评价，选用0.1％DMSO为阴性对照，9CRA为阳性对照，测定化合物在10μg/ml浓度下在ABCA1模型中的上调率，多次活性测定实验结果证明，本发明化合物(编号为T61)对ABCA1有很好的上调作用，并提供了它在治疗动脉粥样硬化方面的应用，为该化合物发展成为新型抗动脉粥样硬化药物奠定了基础。化合物作用于人ABCA1上调剂筛选模型，结果显示其能剂量依赖性上调ABCA1的表达，EC50为0.04μg/ml(图1)。将化合物T61在(0.05、0.24、1.2、6.0μg/ml)作用于HepG2细胞18-24h，western blot结果显示，化合物能在蛋白(图2)水平明显上调HepG2细胞中ABCA1的表达。由此证明，本发明所述化合物或组合物可以作为上调ATP结合盒转运子ABCA1表达活性的药物。The inventors used the human ABCA1 screening model established in this laboratory to evaluate the activity of the patented compound, selected 0.1% DMSO as a negative control, and 9CRA as a positive control, and determined the up-regulation rate of the compound in the ABCA1 model at a concentration of 10 μg/ml. The results of multiple activity measurement experiments prove that the compound of the present invention (coded as T61) has a good up-regulation effect on ABCA1, and provides its application in the treatment of atherosclerosis, which provides a basis for the development of this compound into a new type of anti-atherosclerosis. Drugs lay the groundwork. The compound acts on the human ABCA1 up-regulator screening model, and the results show that it can dose-dependently up-regulate the expression of ABCA1, with an EC50 of 0.04 μg/ml (Figure 1). Compound T61 was applied to HepG2 cells at (0.05, 0.24, 1.2, 6.0 μg/ml) for 18-24 hours. The results of western blot showed that the compound could significantly up-regulate the expression of ABCA1 in HepG2 cells at the protein level (Figure 2). This proves that the compound or composition of the present invention can be used as a drug for up-regulating the expression activity of the ATP-binding cassette transporter ABCA1.
apoE-/-小鼠主动脉en face进行油红染色，结果如图3。从图3A中可以看出，高脂饮食模型组(图3b)的整个主动脉出现明显的动脉硬化斑块，说明动脉硬化模型构建成功。与高脂饮食模型组相比，治疗组(图3c)小鼠主动脉斑块明显较少、减小，说明T61能对动脉硬化的治疗起到积极作用。阴性对照组(图3a)也可以看到在主动脉头皮干出现较小的动脉斑块，可能由于apoE-/-小鼠自发产生。The oil red staining of apoE-/- mouse aorta en face is shown in Figure 3. It can be seen from Fig. 3A that obvious arteriosclerotic plaques appeared in the whole aorta of the high-fat diet model group (Fig. 3b), indicating that the arteriosclerosis model was successfully constructed. Compared with the high-fat diet model group, the mice in the treatment group (Figure 3c) had significantly fewer and smaller aortic plaques, indicating that T61 can play a positive role in the treatment of arteriosclerosis. In the negative control group (Fig. 3a), smaller arterial plaques could also be seen in the aortic scalp, probably due to spontaneous generation in apoE-/- mice.
将apoE-/-小鼠肝脏冰冻切片油红染色结果如图4。高脂饮食组(图4b)肝脏利用油红O染色的病理切片，油红染色很深，脂肪沉积面积很大。给药组(图4c)肝细胞内有很少量的脂滴存在，与阴性对照组的效果相似。The oil red staining results of frozen sections of apoE-/- mouse livers are shown in Figure 4. The oil red O-stained pathological sections of the liver in the high-fat diet group (Figure 4b) showed deep oil red staining and a large area of fat deposition. There were very few lipid droplets in the hepatocytes of the administration group (Fig. 4c), which was similar to the effect of the negative control group.
T61对血脂的调节水平如表1。表1显示，与高脂饮食组相比，给药组中总胆固醇TC、低密度脂蛋白胆固醇LDL-C、甘油三酯TG显著降低，说明化合物具有调节血脂的作用。The regulation level of T61 on blood lipids is shown in Table 1. Table 1 shows that compared with the high-fat diet group, the total cholesterol TC, low-density lipoprotein cholesterol LDL-C, and triglyceride TG in the administration group were significantly reduced, indicating that the compound has the effect of regulating blood lipids.
表1IMB-T61对高脂饮食的apoE-/-小鼠血脂的影响Table 1 Effect of IMB-T61 on blood lipids of apoE-/- mice fed a high-fat diet
综合以上结果，说明T61有良好的抗动脉粥样硬化作用。Based on the above results, it shows that T61 has a good anti-atherosclerosis effect.
发明的有益效果Beneficial Effects of the Invention
1)作用靶点的新颖性：目前研究表明ABCA1是抗动脉粥样硬化的新靶点，在动脉粥样硬化的发生、发展具有重要影响，目前国内外致力于寻找ABCA1的上调剂或者激动剂，但尚未有作用于此靶点的药物出现；1) Novelty of the target: current studies have shown that ABCA1 is a new target for anti-atherosclerosis, which has an important impact on the occurrence and development of atherosclerosis. At present, domestic and foreign efforts are being made to find up-regulators or agonists of ABCA1 , but there are no drugs acting on this target;
2)化合物的新颖性：本发明第一次阐述了化合物N-(4，5-二氢-2-噻唑)-2-(4-甲基苯氧甲基)噻唑-4-甲酰胺在动脉硬化心血管方面的确切作用，并且从首次从分子机制阐述其抗动脉硬化作用的原因，这在国内外属首次，这对于我国开发具有自主知识产权的抗动脉粥样硬化药物具有重要的意义。2) Novelty of the compound: the present invention firstly illustrates the effect of the compound N-(4,5-dihydro-2-thiazole)-2-(4-methylphenoxymethyl)thiazole-4-carboxamide on arterial It is the first time at home and abroad to explain the exact role of cardiovascular hardening and explain its anti-atherosclerotic effect from the molecular mechanism. This is of great significance for the development of anti-atherosclerotic drugs with independent intellectual property rights in my country.
3)动脉硬化的预防和治疗：心脑血管疾病尤其是动脉粥样硬化已严重威胁到人类的生命健康，已有的药物早已不能满足临床的需求，不能从根本上治疗。本发明发现的化合物在动物体内具有良好的抗动脉粥样硬化效果，为开发抗动脉硬化药物提供了很好的先导化合物，具有广阔的应用前景。3) Prevention and treatment of arteriosclerosis: Cardiovascular and cerebrovascular diseases, especially atherosclerosis, have seriously threatened human life and health. Existing drugs have long been unable to meet clinical needs and cannot be fundamentally treated. The compound found in the invention has good anti-atherosclerosis effect in animals, provides a good lead compound for the development of anti-arteriosclerosis drugs, and has broad application prospects.
附图说明Description of drawings
图1为T61在ABCA1表达上调剂筛选模型中的量效关系曲线；Figure 1 is the dose-effect relationship curve of T61 in the ABCA1 expression up-regulation screening model;
图2为T61(0.05、0.24、1.20、6.0μg/ml)增加HepG2细胞中ABCA1的蛋白表达；Figure 2 shows that T61 (0.05, 0.24, 1.20, 6.0 μg/ml) increases the protein expression of ABCA1 in HepG2 cells;
图3为T61减少高脂饮食的apoE-/-小鼠主动脉斑块；Fig. 3 is that T61 reduces the apoE-/- mouse aortic plaque of high-fat diet;
图4为T61减少高脂饮食的apoE-/-小鼠肝脏中脂质含量；Fig. 4 is that T61 reduces the lipid content in the liver of apoE-/- mice of high-fat diet;
具体实施方式Detailed ways
以下实施例可以使专业技术人员更全面的理解本发明，但不以任何方式限制本发明。The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.
实施例一本发明化合物对ABCA1上调剂筛选模型的EC50测定Example 1 The EC50 determination of the compound of the present invention on the ABCA1 up-regulation screening model
将化合物溶于DMSO配成10mg/ml的母液。将ABCA1-LUC HepG2细胞以5×104个/孔接种于96孔细胞培养板中，约6h待细胞贴壁后，移去含血清的培养基，用PBS轻轻漂洗细胞一次。用无血清RPMI1640或MEM稀释的化合物成一系列浓度，0.001-100μmol/L，200μl/孔。终浓度0.1％DMSO培养基的孔作为空白对照。继续于37℃，5％CO2条件下培养18-24h后，PBS(200μl/孔)洗板2次，弃PBS。加入细胞裂解液(20μl/孔)(Promega)，15-30min后，显微镜下观察细胞裂解完全后，加入荧光素酶(60μl/孔)，立即测定荧光素酶活性(酶标仪读数)，计算待测样品对荧光素酶活性的改变率。利用Origin8.5计算EC50。The compound was dissolved in DMSO to prepare a 10 mg/ml stock solution. ABCA1-LUC HepG2 cells were seeded in a 96-well cell culture plate at 5×104 cells/well, and after about 6 hours for the cells to adhere to the wall, the serum-containing medium was removed, and the cells were gently rinsed once with PBS. Compounds diluted with serum-free RPMI1640 or MEM were made into a series of concentrations, 0.001-100 μmol/L, 200 μl/well. Wells with a final concentration of 0.1% DMSO medium were used as blank controls. After continuing to culture at 37° C. and 5% CO 2 for 18-24 h, the plate was washed twice with PBS (200 μl/well), and the PBS was discarded. Add cell lysate (20 μl/well) (Promega), after 15-30min, observe under the microscope that the cell lysis is complete, add luciferase (60 μl/well), immediately measure luciferase activity (microplate reader reading), calculate The rate of change of the luciferase activity of the test sample.EC50 was calculated using Origin8.5.
实施例二本发明化合物对ABCA1蛋白表达水平影响的测定Example 2 Determination of the impact of the compound of the present invention on the expression level of ABCA1 protein
利用western blot法检测化合物对ABCA1蛋白表达的影响方法同前。HepG2细胞铺于六孔板(Costar)，设立加药组T61(0.05、0.24、1.20、6.0μg/ml)，以及阴性对照组(不加入待测样品)。RIPA细胞裂解液提取细胞总蛋白，BCA试剂盒(Pierce)进行蛋白定量，然后进行10％SDS-PAGE电泳，30μg蛋白每孔。各蛋白抗体使用浓度：ABCA1(NB400-105；1：500dilution；Novus Biologicals Inc.，San Jose，CA)；β-actin(1∶5000；Invitrogen)；peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody(1∶3000dilution；Santa Cruz)；peroxidase(HRP)-conjugated rabbit anti-mouse IgG antibody(1∶3000dilution；Santa Cruz)。用化学发光法(ECL，Millipore)试剂盒检测，照相。The effect of the compound on the expression of ABCA1 protein was detected by western blot method as before. HepG2 cells were plated in a six-well plate (Costar), and a drug-dosed group T61 (0.05, 0.24, 1.20, 6.0 μg/ml) and a negative control group (no sample to be tested) were set up. RIPA cell lysate was used to extract total cell protein, and BCA kit (Pierce) was used for protein quantification, followed by 10% SDS-PAGE electrophoresis, 30 μg protein per well. Concentration of each protein antibody used: ABCA1 (NB400-105; 1:500dilution; Novus Biologicals Inc., San Jose, CA); β-actin (1:5000; Invitrogen); peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:3000 dilution; Santa Cruz); peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibody (1:3000 dilution; Santa Cruz). It was detected by chemiluminescence (ECL, Millipore) kit and photographed.
实施例三在apoE-/-小鼠体内的药效学评价Example 3 Pharmacodynamic evaluation in apoE-/- mice
apoE-/-小鼠动脉硬化模型的构建Construction of an arteriosclerosis model in apoE-/- mice
apoE-/-小鼠，7周龄，普通饲料喂养一周；apoE-/- mice, 7 weeks old, fed with normal diet for one week;
将apoE-/-小鼠称体重，随机分组，共分为4组(模型组、给药组、阴性对照组)，每组6-8只；The apoE-/- mice were weighed, randomly grouped, and divided into 4 groups (model group, drug administration group, negative control group), 6-8 in each group;
从8周龄开始，模型组和给药组喂养高脂饲料，阴性对照组继续喂养普通饲料；From the age of 8 weeks, the model group and the treatment group were fed with high-fat feed, and the negative control group continued to be fed with common feed;
T61(20mg/Kg)灌胃给药；阴性对照组和模型组给羧甲基纤维素钠溶液，灌胃给药；给药8周；T61 (20mg/Kg) was administered by intragastric administration; the negative control group and the model group were given sodium carboxymethyl cellulose solution, administered by intragastric administration; administration for 8 weeks;
将给药8周的apoE-/-小鼠禁食6h；摘眼球取血，用肝素润洗过的EP管收集血，上下颠倒，置于冰上，4000rpm/min，4℃离心3min，将上清液转移至新的EP管中(分两份)，置于-20℃保存，采用中生北控公司血脂测定试剂盒测定血脂水平。The apoE-/- mice administered for 8 weeks were fasted for 6 hours; the eyeballs were removed to collect blood, and the blood was collected in an EP tube washed with heparin, turned upside down, placed on ice, 4000rpm/min, and centrifuged at 4°C for 3min. The supernatant was transferred to a new EP tube (divided into two), stored at -20°C, and the blood lipid level was measured using the Zhongsheng Beikong Company Blood Lipid Measurement Kit.
固定小鼠，用手术剪沿着颈部的正中线剪开皮肤直至腹部，剪开腹腔，先留取新鲜的肝脏组织，放入EP管中(分三份)，立即放入液氮，留做RNA和蛋白实验；Fix the mouse, use surgical scissors to cut the skin along the midline of the neck to the abdomen, cut open the abdominal cavity, first take fresh liver tissue, put it into EP tubes (divided into three parts), put it into liquid nitrogen immediately, and leave Do RNA and protein experiments;
取好肝脏后，剪开胸腔，剪去胸骨，暴露心脏，立即进行心脏灌流，先用4％的多聚甲醛灌入大约1ml，然后换成PBS灌入大约4ml，待肝脏变白后停止；After taking the liver, cut open the thorax, cut off the sternum, expose the heart, and immediately perfuse the heart with about 1ml of 4% paraformaldehyde, then replace it with PBS and pour about 4ml, and stop after the liver turns white;
依次取出肝脏和小肠；分离自主动脉至髂骨总分支的动脉全长，取出心脏和动脉。解剖后，将肝脏、心脏和主动脉放入4％的多聚甲醛中，37℃固定2h，然后放入20％的蔗糖溶液中，4℃过夜。The liver and small intestine were removed in sequence; the arteries from the aorta to the common iliac branch were separated for the full length, and the heart and arteries were removed. After dissection, the liver, heart and aorta were fixed in 4% paraformaldehyde at 37°C for 2h, and then placed in 20% sucrose solution at 4°C overnight.
B)冰冻切片的制作方法B) Method for making frozen sections
将1.5ml EP管从中部切断，留下带盖的部分，将盖子盖上，做好标记；Cut off the 1.5ml EP tube from the middle, leave the part with the cover, cover the cover, and make a mark;
将OCT包埋剂加到EP管中，注意不要有气泡；Add the OCT embedding agent to the EP tube, be careful not to have air bubbles;
将20％蔗糖中浸泡的组织放入到OCT中，心脏包埋时，留大约1/3心脏，切平，将心尖部分朝上，慢慢放入OCT中；肝脏包埋时，保持切面放入OCT中时在一个平面上；Put the tissue soaked in 20% sucrose into the OCT. When embedding the heart, leave about 1/3 of the heart, cut it flat, put the apex up, and slowly put it into the OCT; when embedding the liver, keep the cut surface On one plane when entering OCT;
将包有组织的EP管慢慢放入到液氮中；Slowly put the organized EP tube into the liquid nitrogen;
用锡纸立即包好冷冻好的EP管，-20℃避光保存，长期保存放在-80℃。Immediately wrap the frozen EP tubes with tin foil, store them in the dark at -20°C, and store them at -80°C for long-term storage.
C)主动脉油红O染色C) Oil red O staining of aorta
将主动脉从20％蔗糖取出，PBS洗一次；Take out the aorta from 20% sucrose, and wash once with PBS;
在解剖显微镜下，将主动脉纵向剖开；Under a dissecting microscope, the aorta was dissected longitudinally;
将剪开的主动脉用蒸馏水洗3次；The cut aorta was washed 3 times with distilled water;
60％异丙醇浸泡10min，进行同步化；Soak in 60% isopropanol for 10 minutes for synchronization;
将同步化好的放入新配好的油红工作液中(油红储存液配置，过滤1-2h内用)，30min；Put the synchronized product into the newly prepared oil red working solution (oil red storage solution configuration, use within 1-2 hours of filtration), 30min;
放入60％异丙醇分色1min；Put in 60% isopropanol for color separation for 1min;
双蒸水洗3次；Wash with double distilled water 3 times;
将解剖开的主动脉平铺在黑蜡上，照相机立即照相。The dissected aorta was tiled on black wax, and the camera took pictures immediately.
结果显示：见附图The result shows: see attached picture
D)冰冻切片油红O染色(心脏流出道和肝脏组织)D) Oil red O staining of frozen sections (cardiac outflow tract and liver tissue)
将切片在室温下放置30min，吹干冰冻切片；Place the slices at room temperature for 30 min, and dry the frozen slices;
将切片放入4％多聚甲醛中，固定10min；Put the slices into 4% paraformaldehyde and fix for 10min;
弃多聚甲醛溶液，加入双蒸水，洗3次，每次3min；Discard the paraformaldehyde solution, add double distilled water, wash 3 times, 3min each time;
60％异丙醇浸泡切片3min，进行同步化；Soak slices in 60% isopropanol for 3 minutes for synchronization;
然后将同步化好的切片放入新配好的油红工作液中(油红储存液配置，过滤1-2h内用)，30min；Then put the synchronized slices into the newly prepared oil red working solution (oil red storage solution configuration, use within 1-2 hours of filtration), 30min;
将切片放入60％异丙醇分色，随时在显微镜下观察；Put the slices into 60% isopropanol for color separation, and observe under the microscope at any time;
双蒸水洗3次；Wash with double distilled water 3 times;
苏木精染2-3min，复染细胞核；Hematoxylin stained for 2-3min, counterstained cell nuclei;
双蒸水洗3次后，将切片放在蒸馏水中；After washing with double distilled water for 3 times, the slices were placed in distilled water;
水性封片剂封片(9份医用甘油+1份双蒸水)；Water-based mounting agent for sealing (9 parts of medical glycerin + 1 part of double distilled water);
将封好的片子四周涂指甲油，阴凉处阴干，立即照相。Apply nail polish around the sealed film, dry it in the shade, and take a picture immediately.
参考文献references
[1]Shah PK.Emerging non-statin LDL-lowering therapies fordyslipidemia and atherosclerosis.Rev Cardiovase Med，2003，4(3)：136-141.[1] Shah PK. Emerging non-statin LDL-lowering therapies for dyslipidemia and atherosclerosis. Rev Cardiovase Med, 2003, 4(3): 136-141.
[2]Tall，A.R.2008.Cholesterol efflux pathways and other potentialmechanisms involved in the athero-protective effect of high densitylipoproteins.Journal of internal medicine263：256-273.[2] Tall, A.R. 2008. Cholesterol efflux pathways and other potential mechanisms involved in the athero-protective effect of high density lipoproteins. Journal of internal medicine 263: 256-273.
[3]Ohashi，R.，H.Mu，X.Wang，Q.Yao，and C.Chen.2005.Reverse cholesteroltransport and cholesterol efflux in atherosclerosis.QJM：monthly journal ofthe Association of Physicians98：845-856.[3] Ohashi, R., H.Mu, X.Wang, Q.Yao, and C.Chen. 2005. Reverse cholesterol transport and cholesterol efflux in atherosclerosis. QJM: monthly journal of the Association of Physicians 98: 845-856.
[4]Yancey，P.G.，A.E.Bortnick，G.Kellner-Weibel，M.de la Llera-Moya，M.C.Phillips，and G.H.Rothblat.2003.Importance of different pathways ofcellular cholesterol efflux.Arteriosclerosis，thrombosis，and vascularbiology23：712-719.[4] Yancey, P.G., A.E. Bortnick, G. Kellner-Weibel, M.de la Llera-Moya, M.C.Phillips, and G.H.Rothblat. 2003. Importance of different pathways of cellular cholesterol efflux. 719.
[5]Joyce C，Freeman L，Jr Brewer HB，Santamarina-Fojo S.Study of ABCAlFunction in Transgenic Mice，Arteroscler Thromb Vasc Biol.2003，23(6)：965-971.[5] Joyce C, Freeman L, Jr Brewer HB, Santamarina-Fojo S. Study of ABC Al Function in Transgenic Mice, Arteroscler Thromb Vasc Biol. 2003, 23(6): 965-971.
[6]Price MJ，Shah PK.New Strategies in Managing and PreventingAtherosclerosis：Focus on HDL.Rev Cardiovasc Med，2002，3(3)：129-137.[6] Price MJ, Shah PK. New Strategies in Managing and Preventing Atherosclerosis: Focus on HDL. Rev Cardiovasc Med, 2002, 3(3): 129-137.
[7]Timmins JM，Lee JY，Boudyguina E，et al.Targeted activation ofhepatic Abcal causes profound hypoalphalipoproteinemia and kidneyhypercatabolism of apoA-I.J Clin Invest，2005， 115(15)：1333-1342.[7] Timmins JM, Lee JY, Boudyguina E, et al. Targeted activation of hepatic Abcal causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I. J Clin Invest, 2005, 115(15): 1333-1342.
[8]Brunham LR，Kruit JK，Iqbal J，et al.Intestinal ABCA1directlycontributes to HDL biogenesis in vivo.J Clin Invest，2006，116(4)：1052-1062.[8] Brunham LR, Kruit JK, Iqbal J, et al. Intestinal ABCA1 directly contributes to HDL biogenesis in vivo. J Clin Invest, 2006, 116(4): 1052-1062.
[9]F.Basso，L.Freeman，C.L.Knapper，et al.Role of the hepaticABCA1transporter in modulating intrahepatic cholesterol and plasma HDLcholesterol concentrations.J Lipid Res，2003，44(2)：296-302.[9] F.Basso, L.Freeman, C.L.Knapper, et al.Role of the hepaticABCA1transporter in modulating intrahepatic cholesterol and plasma HDLcholesterol concentrations. J Lipid Res, 2003, 44(2): 296-302.
[10]Attie AD，Kastelein JP，Hayden MR.Pivotal role of ABCA1in reversecholesterol transport influencing HDL levels and susceptibitoatherosclerosis.J Lipid Res，2001，42(11)：1717-1726.[10]Attie AD, Kastelein JP, Hayden MR. Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibitoatherosclerosis. J Lipid Res, 2001, 42(11): 1717-1726.
[11]Soumian S，Albrecht C，Davies AH，Gibbs RG.ABCA1andatherosclerosis.Vascular Medicine，2005，10(2)：109-119.[11] Soumian S, Albrecht C, Davies AH, Gibbs RG. ABCA1andatherosclerosis. Vascular Medicine, 2005, 10(2): 109-119.
[12]Oram JF，Heinecke JW.ATP-binding cassette transporter A1：a cellcholesterol exporter that protects against cardiovascular disease.PhysiolRev，2005，85(4)：1343-1372.[12] Oram JF, Heinecke JW. ATP-binding cassette transporter A1: a cellcholesterol exporter that protects against cardiovascular disease. PhysiolRev, 2005, 85(4): 1343-1372.
[13]Srivastava N.ATP binding cassette transporter A1-key roles incellular lipid transport and atherosclerosis.Molecular and CellularBiochemistr，2002，237(1-2)：155-164.[13] Srivastava N. ATP binding cassette transporter A1-key roles cellular lipid transport and atherosclerosis. Molecular and Cellular Biochemistr, 2002, 237(1-2): 155-164.
[14]Robins SJ，Collins D，Wittes JT，et al.Relation of gemfibroziltreatment and lipid levels with major coronary events：VA-HIT：a randomizedcontrolled trial.Journal of theAmerican Medical Association，2001，285(12)：1585-1591。[14] Robins SJ, Collins D, Wittes JT, et al. Relation of gemfibrozil treatment and lipid levels with major coronary events: VA-HIT: a randomized controlled trial. Journal of the American Medical Association, 2001, 285(12): 1585-1591 .
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310329640.2ACN104337809B (en) | 2013-08-01 | 2013-08-01 | The purposes of N- (4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310329640.2ACN104337809B (en) | 2013-08-01 | 2013-08-01 | The purposes of N- (4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide |
| Publication Number | Publication Date |
|---|---|
| CN104337809A CN104337809A (en) | 2015-02-11 |
| CN104337809Btrue CN104337809B (en) | 2018-08-14 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310329640.2AActiveCN104337809B (en) | 2013-08-01 | 2013-08-01 | The purposes of N- (4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide |
| Country | Link |
|---|---|
| CN (1) | CN104337809B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0013378D0 (en)* | 2000-06-01 | 2000-07-26 | Glaxo Group Ltd | Use of therapeutic benzamide derivatives |
| Title |
|---|
| 人膜转运蛋白ABCA1及清道夫受体CLA-1表达上调剂的筛选和活性研究;许艳妮等;《中国医药生物技术》;20130630;第8卷(第3期);161-165* |
| 人高密度脂蛋白受体CLA-1上调剂9179D的分离、结构鉴定和活性研究;徐扬等;《中国抗生素杂志》;20120531;第37卷(第5期);377-382* |
| Publication number | Publication date |
|---|---|
| CN104337809A (en) | 2015-02-11 |
| Publication | Publication Date | Title |
|---|---|---|
| US9610251B2 (en) | Pharmaceutical compositions for substituted quinazolinones | |
| WO2018215795A2 (en) | Senolytic compounds | |
| ES2753639T3 (en) | Tetrahydrocannabinol-11-oic acids for use in the treatment of fibrotic diseases | |
| EA030651B1 (en) | Substituted aromatic compounds and related method for the treatment of fibrosis | |
| AU2016349634A1 (en) | Treatment of mixed dyslipidemia | |
| JPH09505809A (en) | Inhibition of smooth muscle migration and proliferation by hydroxycarbazole compounds | |
| JP2021091713A (en) | Use of kaurane compounds in manufacture of medicament for treatment of cardiac hypertrophy and pulmonary hypertension | |
| CN113840598A (en) | Cannabinoid ester compositions and uses thereof | |
| KR20120018761A (en) | Methods and compositions of pi-3 kinase inhibitors for treating fibrosis | |
| CN102688242B (en) | Novel application of rutaecarpine compound | |
| Theurl et al. | Secretoneurin gene therapy improves hind limb and cardiac ischaemia in Apo E−/− mice without influencing systemic atherosclerosis | |
| JP2010509247A (en) | ACAT inhibitors and their use in the prevention or treatment of fibrosis | |
| CN113181160A (en) | Application of isothiocyanate compounds | |
| AU2017214157A1 (en) | Application of phosphodiesterase 4 inhibitor ZL-n-91 in preparation of medications for lung cancer proliferation and metastasis | |
| KR20110042108A (en) | New methylenedioxy phenolic compounds and their use in the treatment of diseases | |
| CN104337809B (en) | The purposes of N- (4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide | |
| CN117257803B (en) | Application of lurasidone in preparing drugs for treating or preventing ischemia/reperfusion injury and cell protection drugs | |
| US20230041138A1 (en) | Treatment of his hyporesponders | |
| US20210290580A1 (en) | New use of carbamate ß phenylethanolamine analogues for enhancing intracellular clearance of LDL cholesterol and for combining therapy with statins to enhance the efficacy and reduce adverse effects | |
| CN104211695B (en) | Use of group of carbamyl phenylsulfonyl compounds | |
| WO2013103148A1 (en) | Collateral blood circulation development promoter | |
| WO2007094193A1 (en) | Anti-angiogenic agent, prophylactic or therapeutic agent for disease accompanied by angiogenesis, and food | |
| US20160193179A1 (en) | Treatment of Cardiovascular Risk in Diabetic Patients | |
| US9216163B2 (en) | Method of inhibiting angiogenesis | |
| US20220008435A1 (en) | Therapeutic, preventive, or improvement agent for inflammatory disease and allergic disease |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |