WO2022135278A1 - Azd3965药物的新用途 - Google Patents

Azd3965药物的新用途 Download PDF

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WO2022135278A1
WO2022135278A1 PCT/CN2021/138956 CN2021138956W WO2022135278A1 WO 2022135278 A1 WO2022135278 A1 WO 2022135278A1 CN 2021138956 W CN2021138956 W CN 2021138956W WO 2022135278 A1 WO2022135278 A1 WO 2022135278A1
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azd3965
pharmaceutically acceptable
acceptable salt
cardiomyocytes
promoting
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PCT/CN2021/138956
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French (fr)
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吴政源
陈阳
杜建勇
郑丽霞
朱小君
熊敬维
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忻佑康医药科技(南京)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

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  • the invention belongs to the field of medicine, in particular to a new use of AZD3965 medicine.
  • cardiovascular disease has become the number one killer that threatens human health.
  • the remaining cardiomyocytes have very limited proliferative capacity and are not enough to restore the systolic function of the heart. The patient eventually suffers from heart failure. die.
  • AZD3965 (CAS No.: 1448671-31-5) is a potent and selective inhibitor of the monocarboxylic acid transporter MCT1, with a binding Ki value of 1.6 nM, 6-fold higher selectivity than MCT2.
  • AZD3965 potently inhibits lactate transport and cell growth in lymphoma cell lines that preferentially express MCT1.
  • H526, HGC27 and DMS114 cells AZD3965 increased intracellular lactate and significantly decreased lactate uptake.
  • AZD3965 (100 mg/kg, p.o.) increased lactate concentration, decreased tumor growth, and increased radiosensitivity in H526 tumor-bearing mice.
  • AZD3965 (100 mg/kg, p.o.) reduced tumor growth and increased intratumoral lactate in non-obese diabetic scid- ⁇ mice bearing COR-L103 tumors.
  • the purpose of the present invention is to provide a new use of AZD3965 or a pharmaceutically acceptable salt thereof.
  • the new uses of AZD3965 or a pharmaceutically acceptable salt thereof provided by the present invention are its application in the preparation of a product for promoting the proliferation of cardiomyocytes and the application in promoting the proliferation of cardiomyocytes.
  • the cardiomyocytes can be human or mammalian cardiomyocytes.
  • the product can be a pharmaceutical product.
  • cardiomyocytes can be human or mammalian endogenous cardiomyocytes.
  • Another object of the present invention is to provide the use of AZD3965 or a pharmaceutically acceptable salt thereof in the preparation of a product for promoting myocardial regeneration and the application in promoting myocardial regeneration.
  • the myocardium can be human or mammalian myocardium.
  • the product can be a pharmaceutical product.
  • the present invention also provides the use of AZD3965 or a pharmaceutically acceptable salt thereof in the preparation of a product for promoting the recovery of cardiac function after myocardial infarction and the application in promoting the recovery of cardiac function after myocardial infarction.
  • the present invention also provides the use of AZD3965 or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and/or treating cardiovascular disease.
  • the product can be a pharmaceutical product.
  • the cardiovascular disease may be a heart disease caused by loss, injury or death of cardiomyocytes.
  • the above-mentioned heart disease includes but is not limited to myocardial infarction and cardiomyopathy caused by myocardial cell loss, injury or death caused by various other reasons.
  • Products for promoting myocardial cell proliferation, products for promoting myocardial regeneration, and products for preventing and/or treating cardiovascular diseases prepared with AZD3965 or a pharmaceutically acceptable salt thereof as active ingredients also belong to the protection scope of the present invention.
  • one or more pharmaceutically acceptable carriers can also be added to the above-mentioned drugs;
  • the carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents in the pharmaceutical field Agents, absorption accelerators, surfactants, adsorption carriers, lubricants, etc.
  • the above-mentioned medicines can be made into various forms such as injections, tablets, powders, granules, capsules, oral liquids, ointments, creams, etc.
  • the medicines of the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.
  • the above-mentioned drugs can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal instillation, eye instillation, penetration, absorption, physical or chemical mediated methods; or mixed or packaged by other substances and then introduced. body.
  • the invention also provides a method for culturing cardiomyocytes in vitro.
  • the method for culturing cardiomyocytes in vitro comprises adding AZD3965 or a pharmaceutically acceptable salt thereof to a culture medium containing cardiomyocytes.
  • the final concentration of the AZD3965 or its pharmaceutically acceptable salt in the culture medium is 0.5-2 ⁇ mol/L.
  • the cardiomyocytes can be human or mammalian cardiomyocytes.
  • the invention also protects a method for promoting cardiomyocyte proliferation in vitro.
  • the method for promoting cardiomyocyte proliferation in vitro comprises treating cardiomyocytes with AZD3965 or a pharmaceutically acceptable salt thereof.
  • the concentration of the AZD3965 or its pharmaceutically acceptable salt in the treatment system is 0.5-2 ⁇ mol/L.
  • the cardiomyocytes can be human or mammalian cardiomyocytes.
  • the present invention also protects a method of promoting myocardial regeneration.
  • the method for promoting myocardial regeneration includes the following steps: administering an effective dose of AZD3965 or a pharmaceutically acceptable salt thereof to a human or a recipient animal to promote myocardial regeneration.
  • the invention also protects a method for promoting the recovery of cardiac function after myocardial infarction.
  • the method for promoting cardiac function recovery after myocardial infarction comprises the following steps: administering an effective dose of AZD3965 or a pharmaceutically acceptable salt thereof to a human or recipient animal after myocardial infarction to promote cardiac function recovery.
  • the present invention also protects a method for preventing and/or treating cardiovascular disease, comprising the steps of: administering an effective dose of AZD3965 or a pharmaceutically acceptable salt thereof to a human or a recipient animal to prevent and/or treat cardiovascular disease .
  • the cardiovascular disease may be a heart disease caused by loss, injury or death of cardiomyocytes.
  • the above-mentioned heart disease includes but is not limited to myocardial infarction and cardiomyopathy caused by myocardial cell loss, injury or death caused by various other reasons.
  • Figure 1 is a graph showing the promoting effect of AZD3965 on the proliferation of neonatal rat cardiomyocytes.
  • Figure 2 is a graph showing the effect of AZD3965 on promoting the cytokinesis of cardiomyocytes in neonatal MADM mice.
  • Figure 3 is a graph showing the measurement results of AZD3965 improving cardiac function in rats after myocardial infarction.
  • the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.
  • the methods are conventional methods unless otherwise specified.
  • the raw materials can be obtained from open commercial sources unless otherwise specified.
  • AZD3965 used in the following examples is a DMSO solution of AZD3965.
  • AZD3965 Manufacturer MedChemExpress (MCE), Cat. No. HY-12750.
  • FBS fetal bovine serum
  • SD rats were purchased from Beijing Weitong Lihua Laboratory Animal Co., Ltd.; the breeding, reproduction and use of rats followed the rules and regulations of the Peking University Molecular Laboratory Animal Management Committee, and the relevant facilities have passed the international standards.
  • small animal ventilator Reward R415
  • small animal ultrasound Vevo 3100 small animal imaging system
  • AZD3965 Shanghai Haoyuan Biomedical Technology Co., Ltd.
  • 1% sodium pentobarbital (rat) 1 g of sodium pentobarbital was weighed, dissolved in 100 mL of physiological saline, filtered and sterilized and stored at room temperature.
  • cTnT cardiac muscle-specific promoter shown as sequence 1 in the sequence listing
  • mAG-hGeminin (1/110) GenBank: NM_015895
  • the culture medium containing the co-precipitated particles was removed and washed with PBS buffer), and then the cells were distributed into a 6-well plate (each Add 3 ml of DMEM medium containing 5% fetal bovine serum to the wells, and let stand for 6 hours to make it adhere to the wall; after 6 hours, cover with agarose for the formation of viral plaques (the plaques should be formed within 10-21 days, every 4- Add agarose/DMEM mixture after 5 days or when the medium turns yellow); after obtaining the initial virus, after 2-3 rounds of amplification, the adenovirus is purified by cesium chloride density gradient centrifugation.
  • the cTnT cardiac muscle-specific promoter (shown as SEQ ID NO: 1 in the sequence listing), Cre (GenBank: NC_005856.1), pAdeno vector (Obio Technology, H202) were assembled by NEBuilder (NEB, E2621). The assembled products were transformed into DH5 ⁇ competent cells, the recombinant plasmids were screened and amplified, and then 293A cells were transfected with the recombinant plasmids (7.5 ⁇ 10 5 293A cells were plated in a 60 mm petri dish one day before transfection, and the cells were plated with 5% fetal bovine serum.
  • the cells were cultured in DMEM medium until the number of cells reached 1.0-1.5 ⁇ 10 6 , and then the cells were transfected with the recombinant plasmid by calcium phosphate co-precipitation method. On the second day after transfection, the medium containing the co-precipitated particles was removed, and PBS buffer was used.
  • the cells were divided into a 6-well plate (add 3 ml of DMEM medium containing 5% fetal bovine serum to each well), and let stand for 6 hours to adhere to the wall; after 6 hours, cover with agarose for virus formation Plaques (the plaques should be formed within 10-21 days, and the agarose/DMEM mixture should be added every 4-5 days or when the medium turns yellow); 2-3 rounds of amplification after obtaining the initial virus, cesium chloride density gradient centrifugation Purified adenovirus.
  • Example 1 In vitro test of AZD3965 promoting the proliferation of rat cardiomyocytes
  • Cardiomyocytes of SD rats on day 3 were isolated and cultured in DMEM high glucose medium (Hyclone) + 5% horse serum (GIBCO) in a 37 degree, 5% carbon dioxide incubator.
  • Hyclone high glucose medium
  • GIBCO horse serum
  • Experimental group treated with AZD3965 (the final concentration in the medium was 2 ⁇ mol/L) for 24 hours.
  • Blank control group treated with the same amount of DMSO as in experimental group a.
  • the nuclei were stained with Hoechst fluorescent dye, and then photographed with a high-content viable cell analyzer (Molecular Device) to analyze the mAG-hGeminin (1/110) positive cardiomyocytes and the total number of cells.
  • a high-content viable cell analyzer Molecular Device
  • Example 2 In vitro experiment of AZD3965 promoting the cytokinesis of cardiomyocytes in neonatal mice with MADM
  • P3MADM-ML-11 TG/GT mouse cardiomyocytes were isolated by a two-step digestion method. Left ventricular tissue from P3MADM-ML-11 TG/GT mice was taken and digested in 0.25% trypsin (including EDTA) overnight at 4°C. The heart tissue was transferred to digestion solution containing 0.5 mg/ml collagenase (Gibco, 216320) and 5 mg/ml BSA (MP, Y180306) and digested at 37°C for a total of about 30 minutes, 6 minutes per digestion.
  • Each cell suspension was collected, centrifuged at 1000 g for 5 min, the supernatant was discarded, the cells were resuspended in DMEM high-glucose medium containing 10% FBS, and adhered at differential speed for 2 h. After 2 h, the cells were resuspended, centrifuged at 1000 g for 5 min, the supernatant was discarded, and the cardiomyocytes were resuspended in high glucose DMEM medium containing 20 ⁇ M cytarabine, 10% FBS and 1% penicillin/streptomycin. Incubate at 37 degrees, 5% carbon dioxide incubator for 48h.
  • the groups are as follows:
  • Experimental group treated with AZD3965 (the final concentration in the medium was 2 ⁇ mol/L) for 48 hours.
  • Blank control group treated with the same amount of DMSO as in experimental group a.
  • the nuclei were stained with Hoechst fluorescent dye, and then photographed with a high-content viable cell analyzer (Molecular Device), and the numbers of red-green monochromatic cardiomyocytes and yellow cardiomyocytes were analyzed.
  • Example 3 AZD3965 improves cardiac function after myocardial infarction in rats
  • the rat model of myocardial infarction was established by ligation of the anterior descending coronary artery. Specific steps are as follows:
  • Rats weight 200-250g were anesthetized with 1% sodium pentobarbital intraperitoneal injection (6mL/1000g), and the limbs and head were fixed supine on the operating table.
  • Cardiac function was detected by echocardiography after 1 day, 7 days, 14 days and 28 days after coronary artery ligation. The results are shown in Figure 3.
  • Echocardiography and related measurement results showed that compared with preoperative, functional indicators such as cardiac EF (cardiac ejection fraction) and FS (left ventricular short-axis shortening rate) in the control group were significantly reduced, indicating that the group after myocardial infarction Myocardial infarction and reduced cardiac function.
  • cardiac EF cardiac ejection fraction
  • FS left ventricular short-axis shortening rate
  • the present invention proves through experiments that AZD3965 can promote the proliferation of rat cardiomyocytes and the cytokinesis of cardiomyocytes in MADM neonatal mice, and significantly improve the cardiac function of rats with myocardial infarction. Therefore, AZD3965 can be used in the preparation of drugs for promoting myocardial cell proliferation and drugs for the treatment or prevention of heart disease and other related fields, providing a new drug and treatment idea for the treatment or prevention of heart disease such as myocardial infarction.

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Abstract

本发明公开了AZD3965的一种药物新用途。所述新用途为AZD3965或其药学上可接受的盐在制备促进心肌细胞增殖药物中的应用。本发明通过实验证明，AZD3965可以促进大鼠心肌细胞增殖以及促进小鼠心肌细胞胞质分裂。因此，AZD3965可以用于制备促进心肌细胞增殖药物以及治疗或预防心肌梗死或其它各种原因导致的心肌细胞缺失、损伤或死亡而造成的心脏病的药物等相关领域，为治疗或预防心脏病例如心肌梗死提供一种新的药物和治疗思路。

Description

AZD3965药物的新用途 技术领域
本发明属于医药领域，具体涉及一种AZD3965药物的新用途。
背景技术
当前心血管疾病已成为威胁人类健康的头号杀手，仅心力衰竭患者全球就有4000万，已经成为人类死亡的主要原因。研究发现哺乳动物成年后心肌细胞逐渐失去增殖能力，一旦发生心梗，心肌细胞的损失将不可逆转。成年人约有20-40亿个心肌细胞，心梗发生后数小时内会造成约25％的心肌细胞损失，剩余的心肌细胞增殖能力非常有限，不足以恢复心脏的收缩功能，患者最终心衰死亡。为解决这一难题，除外科手术外，基础转化研究主要包括寻找心脏干细胞或前体细胞，通过诱导分化移植到心梗区域，然而缺乏足够确凿的分子标记物，移植效率低限制了这一技术的使用；利用重编程技术将成纤维细胞转分化为心肌细胞，或通过促进内源心肌细胞增殖的方式以补充丢失的心肌。已有的研究表明低等的脊椎动物如斑马鱼、蝾螈等心脏具有很强的再生能力，新生乳鼠也具有一定的再生能力，主要通过损伤诱导的心肌细胞增殖来实现，而这种能力在成年之后就消失了。以往人们认为成年心脏不能再生，但是大量证据表明哺乳动物心肌细胞存在缓慢的更新，并且研究发现新生的心肌细胞来源于已有的心肌。因此，越来越多的科学家们对诱导内源性心肌细胞的增殖这一策略感兴趣。因此寻找能够诱导内源性心肌细胞增殖的药物对于人类心血管疾病的治疗具有重大意义。AZD3965(CAS No.：1448671-31-5)是一种高效的单羧酸转运体MCT1选择性抑制剂，结合Ki值为1.6nM，比MCT2高6倍的选择性。AZD3965在优先表达MCT1的淋巴瘤细胞系中，有效的抑制了乳酸的转运以及细胞的生长。在H526、HGC27和DMS114细胞中，AZD3965增加了细胞内的乳酸，并显著地减少了乳酸的摄入。AZD3965(100mg/kg,p.o.)在含有H526肿瘤的小鼠中，增加了乳酸浓度，减少了肿瘤生长，增加了放射的敏感性。AZD3965(100mg/kg,p.o.)在含有COR-L103肿瘤的非肥胖糖尿病scid-γ小鼠中，减少了肿瘤生长并增加了瘤内的乳酸。但并没有AZD3965在诱导内源性心肌细胞增殖方面的相关报道。
发明公开
本发明的目的是提供一种AZD3965或其药学上可接受的盐的新用途。
所述AZD3965，CAS No.：1448671-31-5，其结构式如式I所示：
Figure PCTCN2021138956-appb-000001
本发明所提供的AZD3965或其药学上可接受的盐的新用途是其在制备促进心肌细胞增殖产品中的应用以及在促进心肌细胞增殖中的应用。
所述心肌细胞可为人或哺乳动物的心肌细胞。所述产品可为药品。
进一步的，所述心肌细胞可为人或哺乳动物的内源性心肌细胞。
本发明另一个目的是提供AZD3965或其药学上可接受的盐在制备促进心肌再生的产品中的应用以及在促进心肌再生中的应用。
所述心肌可为人或哺乳动物的心肌。所述产品可为药品。
本发明还提供AZD3965或其药学上可接受的盐在制备促进心肌梗死后心脏功能恢复的产品中的应用以及在促进心肌梗死后心脏功能恢复中的应用。
本发明还提供AZD3965或其药学上可接受的盐在制备预防和/或治疗心血管疾病产品中的应用。所述产品可为药品。
进一步地，在本发明的一些实施方案中，所述心血管疾病可为由心肌细胞缺失、损伤或死亡而导致的心脏病。
进一步地，在本发明的一些实施方案中，上述心脏病包括但不限于心肌梗死以及其它各种原因导致的心肌细胞缺失、损伤或死亡造成的心肌病等。
以AZD3965或其药学上可接受的盐为活性成分制备的促进心肌细胞增殖产品、促进心肌再生的产品、以及预防和/或治疗心血管疾病的产品也均属于本发明的保护范围。
需要的时候，在上述药物中还可以加入一种或多种药学上可接受的载体；所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
上述药物可以制成注射液、片剂、粉剂、颗粒剂、胶囊、口服液、膏剂、霜剂等多种形式；上述各种剂型的药物均可以按照药学领域的常规方法制备。
上述药物可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹后导入机体。
本发明还提供一种体外培养心肌细胞的方法。
本发明所提供的体外培养心肌细胞的方法，包括在含心肌细胞的培养基中加入AZD3965或其药学上可接受的盐。
所述AZD3965或其药学上可接受的盐在所述培养基中的终浓度为0.5-2μmol/L。
所述心肌细胞可为人或哺乳动物的心肌细胞。
本发明还保护一种体外促进心肌细胞增殖的方法。
本发明所提供的体外促进心肌细胞增殖的方法，包括用AZD3965或其药学上可接受的盐处理心肌细胞。
所述AZD3965或其药学上可接受的盐在处理体系中的浓度为0.5-2μmol/L。
所述心肌细胞可为人或哺乳动物的心肌细胞。
本发明还保护一种促进心肌再生的方法。
所述促进心肌再生的方法，包括下述步骤：给人或受体动物施用有效剂量的AZD3965或其药学上可接受的盐以促进心肌再生。
本发明还保护一种促进心肌梗死后心脏功能恢复的方法。
所述促进心肌梗死后心脏功能恢复的方法，包括下述步骤：给心肌梗死后的人或受体动物施用有效剂量的AZD3965或其药学上可接受的盐以促进心脏功能恢复。
本发明还保护一种预防和/或治疗心血管疾病的方法，包括下述步骤：给人或受体动物施用有效剂量的AZD3965或其药学上可接受的盐进行预防和/或治疗心血管疾病。
进一步地，在本发明的一些实施方案中，所述心血管疾病可为由心肌细胞缺失、损伤或死亡而导致的心脏病。
进一步地，在本发明的一些实施方案中，上述心脏病包括但不限于心肌梗死以及其它各种原因导致的心肌细胞缺失、损伤或死亡造成的心肌病等。
附图说明
图1为AZD3965对新生大鼠心肌细胞增殖的促进作用图。
图2为AZD3965促进新生MADM小鼠心肌细胞胞质分裂的作用图。
图3为AZD3965对于大鼠心梗后心脏功能改善的测量结果图。
实施发明的最佳方式
下面结合具体实施例对本发明作进一步阐述，但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所使用的AZD3965，为AZD3965的DMSO溶液。AZD3965生产商：MedChemExpress(MCE)，货号HY-12750。
下述实施例中“FBS”为胎牛血清。
下述实施例中所用的实验动物：SD大鼠购自北京维通利华实验动物有限公司；大鼠的饲养、繁殖和使用遵循北京大学分子实验动物管理委员会的规章制度，相关设施已通过国际实验动物AAALAC认证。
下述实施例中所用的实验仪器和耗材：小动物呼吸机：瑞沃德R415；小动物超声：Vevo 3100小动物影像系统；AZD3965：上海皓元生物医药科技有限公司。
下述实施例中所用的主要溶液配方：
1％戊巴比妥钠(大鼠)：称取1g戊巴比妥钠，溶于100mL生理盐水，过滤除菌后室温保存。
下述实施例中使用的cTnT-mAG-hGeminin(1/110)病毒的构建方法如下：
cTnT心肌特异性启动子(如序列表中序列1所示)和mAG-hGeminin(1/110)(GenBank:NM_015895)通过pEASY-Uni Seamless Cloning and Assembly Kit(全式金CU101-01)组装克隆到pShuttle载体(Addgene 16402)上，然后采用限制性内切酶PmeI进行酶切，收集线性化质粒；将线性化质粒和pAdEasy1DNA(Addgene 16400)共转化BJ5183感受态，筛选重组质粒并扩增，然后用重组质粒转染293A细胞(转染前一天将7.5×10 5个293A细胞在60mm培养皿中铺板，用含5％胎牛血清的DMEM培养液培养，至细胞数量达到1.0-1.5×10 6，然后利用磷酸钙共沉淀法将重组质粒转染细胞，转染后第二天，去除含共沉淀颗粒的培养液，用PBS缓冲液清洗)，然后将细胞分装入一块6孔板中(每孔加入3ml含5％胎牛血清的DMEM培养液)，静置6小时使其贴壁；6小时后覆盖琼脂糖以供形成病毒空斑(空斑应在10-21天内形成，每4-5天或培养基变黄时追加琼脂糖/DMEM混合物)；得到初始病毒后经过2-3轮扩增，氯化铯密度梯度离心纯化腺病毒。
下述实施例中使用的cTnT-cre病毒的构建方法如下：
cTnT心肌特异性启动子(如序列表中序列1所示)、Cre(GenBank:NC_005856.1)、pAdeno载体(Obio Technology，H202)通过NEBuilder(NEB，E2621)进行组装。组装产物转化DH5α感受态，筛选重组质粒并扩增，然后用重组质粒转染293A细胞(转染前一天将7.5×10 5个293A细胞在60mm培养皿中铺板，用含5％胎牛血清的DMEM培养液培养，至细胞数量达到1.0-1.5×10 6，然后利用磷酸钙共沉淀法将重组质粒转染细胞，转染后第二天，去除含共沉淀颗粒的培养液，用PBS缓冲液清洗)，然后将细胞分装入一块6孔板中(每孔加入3ml含5％胎牛血清的DMEM培养液)，静置6小时使其贴壁；6小时后覆盖琼脂糖以供形成病毒空斑(空斑应在10-21天内形成，每4-5天或培养基变黄时追加琼脂糖/DMEM混合物)；得到初始病毒后经过2-3轮扩增，氯化铯密度梯度离心纯化腺病毒。
实施例1、AZD3965促进大鼠心肌细胞增殖的体外试验
(1)SD大鼠的心肌细胞的培养
分离出生3天SD大鼠的心肌细胞进行培养，DMEM高糖培养基(Hyclone)+5％马血清(GIBCO)，在37度，5％二氧化碳培养箱培养。
(2)实验分组和处理
分离SD大鼠的心肌细胞，加入5％马血清(GIBCO)+DMEM高糖培养基培养液(Hyclone)，加阿糖胞苷(终浓度20umol/L)处理以抑制非心肌细胞的生长，细胞贴壁48h后感染cTnT-mAG-hGeminin(1/110)病毒(病毒感染的MOI值＝100)，再过24小时后换成含0.5％FBS的DMEM培养液，分组加药处理，分组如下：
a、实验组：加AZD3965处理(培养基中的终浓度为2μmol/L)24小时。
b、空白对照组：加与a实验组等量DMSO处理。
(3)试验方法
上述2个分组分别处理24h后，用hoechst荧光染料染细胞核，然后用高内涵活细胞分析仪(Molecular Device)进行拍照，并分析mAG-hGeminin(1/110)阳性心肌细胞及总细胞数。
(4)结果
试验结果如图1所示。由图1可知，AZD3965处理后mAG-hGeminin(1/110)阳性心肌细胞相比于空白对照组(0.5％FBS+DMSO)增加了2倍。Mean±SEM；**P＜0.01。
实施例2、AZD3965促进MADM新生小鼠心肌细胞胞质分裂的体外实验
(1)P3MADM小鼠的心肌细胞的分离及培养
采用两步消化法分离P3MADM-ML-11 TG/GT小鼠心肌细胞。取P3MADM-ML-11 TG/GT小鼠的左心室组织，在0.25％胰蛋白酶(包括EDTA)中4℃过夜消化。将心脏组织转移到含有0.5mg/ml胶原酶(Gibco，216320)和5mg/ml BSA(MP，Y180306)的消化液中，在37℃下一共消化约30分钟，每次消化6分钟。收集每次的细胞悬液，1000g离心5min，弃去上清，用含10％FBS的DMEM高糖培养基重悬细胞，差速贴壁2h。2h后重悬细胞，1000g离心5min，弃去上清，用含20μM阿糖胞苷、10％FBS和1％青霉素/链霉素的高糖DMEM培养基重悬心肌细胞。在37度，5％二氧化碳培养箱中贴壁培养48h。
(2)实验分组和处理
P3MADM小鼠心肌细胞贴壁48h后感染cTnT-cre病毒(病毒感染的MOI值＝200)，再过24小时后换成含0.5％FBS的DMEM培养液，分组加药处理，分组如下：
a、实验组：加AZD3965处理(培养基中的终浓度为2μmol/L)48小时。
b、空白对照组：加与a实验组等量DMSO处理。
(3)试验方法
上述2个分组分别处理48h后，用hoechst荧光染料染细胞核，然后用高内涵活细胞分析仪(Molecular Device)进行拍照，并分析红绿单色心肌细胞及黄色心肌细胞数。
(4)结果
试验结果如图2所示。由图2可知，AZD3965处理后红绿单色心肌细胞占所有有色细胞数的比例明显增加。Mean±SEM；**P＜0.01。
实施例3、AZD3965对于大鼠心梗后心脏功能改善
一、心梗模型的制备
采用冠状动脉前降支结扎法制作大鼠心梗模型。具体步骤如下：
1、大鼠(体重200-250g)用1％戊巴比妥钠腹腔注射麻醉(6mL/1000g)，仰卧固定四肢及头部于手术台上。
2、行气管插管，连接呼吸机，按体重调节参数。观察大鼠胸廓起伏与呼吸机频率完全一致后，用酒精进行术野消毒，在左胸心脏搏动最明显处剪开皮肤。钝性分离胸大肌和前锯肌，在第四肋间刺破肋间肌开胸。用开胸夹撑开肋骨，除去心包，找到冠状动脉前降支位置(自左心耳与肺动脉圆锥交界处发出，向心尖方向走行)。
3、用6-0带线缝合针结扎，观察到结扎部位下方心肌大面积变为苍白色即为成功。
4、结扎后，准备0.3mg AZD3965溶于0.1ml生理盐水中，使用31G针头注射在白色心肌区域的中间及周围；对照组注射生理盐水。
5、清理胸腔积血，逐层关胸缝合。至大鼠恢复自主呼吸后拔去呼吸机插管，用酒精再次消毒，腹腔注射氨苄青霉素以防止术后感染(注射量为50mg/200g)，置于电热毯上维持体温，至其苏醒后放回笼中饲养。
二、心脏结构和功能检测
冠状动脉结扎1天、7天14天及28天后超声心动图检测心脏功能。结果见图3。
超声心动图及相关测量结果显示，与术前相比，对照组大鼠心脏EF(心脏射血分数)、FS(左室短轴缩短率)等功能性指标明显降低，表明心梗后该组心脏出现心肌梗死、心脏功能降低。与对照组相比，AZD3965给药组大鼠心脏功能下降与心室腔扩张程度均明显减轻，且术后4周内EF(心脏射血分数)、FS(左室短轴缩短率)等功能性指标明显上升。以上结果表明AZD3965药物治疗能促进大鼠心梗后心功能的恢复，改善心脏功能。
工业应用
本发明通过实验证明，AZD3965可以促进大鼠心肌细胞增殖以及促进MADM新生小鼠心肌细胞胞质分裂，显著改善心梗大鼠的心脏功能。因此，AZD3965可以用于制备促进心肌细胞增殖药物以及治疗或预防心脏病的药物等相关领域，为治疗或预防心脏病例如心肌梗死提供一种新的药物和治疗思路。

Claims (21)

  1. AZD3965或其药学上可接受的盐在制备促进心肌细胞增殖产品中的应用；
    所述AZD3965，CAS No.：1448671-31-5。
  2. AZD3965或其药学上可接受的盐在促进心肌细胞增殖中的应用；
    所述AZD3965，CAS No.：1448671-31-5。
  3. 根据权利要求1或2所述的应用，其特征在于：所述心肌细胞为人或哺乳动物的心肌细胞。
  4. AZD3965或其药学上可接受的盐在制备促进心肌再生产品中的应用；
    所述AZD3965，CAS No.：1448671-31-5。
  5. AZD3965或其药学上可接受的盐在促进心肌再生中的应用；
    所述AZD3965，CAS No.：1448671-31-5。
  6. AZD3965或其药学上可接受的盐在制备促进心肌梗死后心脏功能恢复的产品中的应用；
    所述AZD3965，CAS No.：1448671-31-5。
  7. AZD3965或其药学上可接受的盐在促进心肌梗死后心脏功能恢复中的应用；
    所述AZD3965，CAS No.：1448671-31-5。
  8. AZD3965或其药学上可接受的盐在制备预防和/或治疗心血管疾病产品中的应用；所述AZD3965，CAS No.：1448671-31-5。
  9. 根据权利要求8所述的应用，其特征在于：所述心血管疾病为各种原因导致的心肌细胞缺失、损伤或死亡而造成的心脏病。
  10. 根据权利要求9所述的应用，其特征在于：所述心血管疾病包括心肌梗死、心力衰竭以及其它各种原因导致的心肌细胞缺失、损伤或死亡造成的心肌病。
  11. 根据权利要求1、3、4、6、7或8所述的应用，其特征在于：所述产品为药品。
  12. 一种体外培养心肌细胞的方法，包括在含心肌细胞的培养基中加入AZD3965或其药学上可接受的盐。
  13. 根据权利要求12所述的方法，其特征在于：所述AZD3965或其药学上可接受的盐在所述培养基中的终浓度为0.5-2μmol/L。
  14. 一种体外促进心肌细胞增殖的方法，包括用AZD3965或其药学上可接受的盐处理心肌细胞。
  15. 根据权利要求14所述的方法，其特征在于：所述AZD3965或其药学上可接受的盐在处理体系中的浓度为0.5-2μmol/L。
  16. 根据权利要求12-15中任一项所述的方法，其特征在于：所述心肌细胞为人或哺乳动物的心肌细胞。
  17. 一种促进心肌再生的方法，包括下述步骤：给人或受体动物施用有效剂量的AZD3965或其药学上可接受的盐以促进心肌再生。
  18. 一种促进心肌梗死后心脏功能恢复的方法，包括下述步骤：给心肌梗死后的人或受体动物施用有效剂量的AZD3965或其药学上可接受的盐以促进心脏功能恢复。
  19. 一种预防和/或治疗心血管疾病的方法，包括下述步骤：给人或受体动物施用有效剂量的AZD3965或其药学上可接受的盐进行预防和/或治疗心血管疾病。
  20. 根据权利要求19所述的方法，其特征在于：所述心血管疾病为各种原因导致的心肌细胞缺失、损伤或死亡而造成的心脏病。
  21. 根据权利要求19所述的方法，其特征在于：所述心血管疾病包括心肌梗死、心力衰竭以及其它各种原因导致的心肌细胞缺失、损伤或死亡造成的心肌病。
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