WO2021196335A1 - Use of pyr3 - Google Patents

Use of pyr3 Download PDF

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WO2021196335A1
WO2021196335A1 PCT/CN2020/087952 CN2020087952W WO2021196335A1 WO 2021196335 A1 WO2021196335 A1 WO 2021196335A1 CN 2020087952 W CN2020087952 W CN 2020087952W WO 2021196335 A1 WO2021196335 A1 WO 2021196335A1
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pyr3
tumor
drugs
application
analogues
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PCT/CN2020/087952
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French (fr)
Chinese (zh)
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徐迅迪
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中南大学湘雅二医院
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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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of medical technology, in particular to the use of Pyr3.
  • Cancer is a general term for malignant tumors. It is characterized by uncontrolled division of abnormal cells, unlimited proliferation, invasion and destruction of surrounding normal tissues. According to its tissue source, it can be divided into cancer, sarcoma, blastoma, malignant tumor and so on.
  • cancers those from epithelial tissue are called cancers; those that originate from adipose tissue, fibrous tissue, muscle tissue (collectively referred to as mesenchymal tissue) and lymphatic reticular tissue are called sarcomas; those derived from embryonic tissue are called blastoma; those that are not suitable for the above names are called Malignant tumors, such as malignant schwannoma; there are also cancers with special names, such as malignant tumors of blood cells, which are also called leukemias. Cancer cells metastasize through blood and lymph fluid, and can also spread directly. The cause of cancer is complex, and the main inducing factors are chemical carcinogens, special viruses, radiation, hormones, free radicals, etc.; genetics, diet, and psychological status are important contributing factors.
  • Hepatocellular carcinoma is the fifth most common type of cancer in the world and the third leading cause of cancer-related deaths worldwide.
  • HCC Hepatocellular carcinoma
  • great progress has been made in the diagnosis and treatment of HCC, but its long-term prognosis is still unsatisfactory.
  • the poor long-term prognosis of HCC is often due to late diagnosis, high recurrence rate after treatment, and chemotherapy resistance. Therefore, there is an urgent need to further explore more effective and safer anti-cancer strategies to inhibit the progression of HCC.
  • the TRPC3 channel is a member of the transient receptor potential (TRP) channel family.
  • the TRPC3 channel is a non-selective cation channel, mainly distributed in the cell membrane. When it is opened, it allows cations such as calcium and sodium ions to pass through, thereby participating in intracellular calcium. Ion steady state.
  • Pyr3 is a specific inhibitor of the TRPC3 channel, which can effectively block the TRPC3 channel, thereby affecting the biological function of the TRPC3 channel.
  • Pyr3 has been reported to have an effect on pressure load-induced left ventricular hypertrophy in rats, and there are also reports that Pyr3 can improve the prognosis of mice after cerebral hemorrhage. However, there is no relevant report on the therapeutic effect of Pyr3 or its analogues on cancer.
  • the technical problem to be solved by the present invention is to provide the use of Pyr3.
  • the research of the present invention has found that Pyr3 can reduce the tumor volume of tumor-bearing mice, inhibit the proliferation and migration of tumor cells, and has positive significance for the treatment of cancer.
  • the present invention provides the application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor cell proliferation.
  • the inhibition of tumor cell proliferation includes: inhibiting the increase in the number of cells and/or inhibiting the formation of clones.
  • the invention also provides the application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor cell migration.
  • the invention also provides the application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor volume growth.
  • the invention also provides the application of Pyr3 or its analogues in the preparation of drugs for treating tumors.
  • the tumor is a malignant tumor, selected from cancer, sarcoma and/or blastoma.
  • the site where the tumor occurs includes head and neck, brain, thyroid, esophagus, pancreas, lung, liver, stomach, breast, kidney, gallbladder, colon, rectum, ovary, cervix, uterus, prostate, One or more of bladder and testis.
  • the tumor is liver cancer.
  • the liver cancer is hepatocellular carcinoma or hepatoblastoma.
  • Huh7 cells were used to verify the therapeutic effect of Pyr3 on hepatocellular carcinoma.
  • HepG2 cells were used to verify the therapeutic effect of Pyr3 on hepatoblastoma.
  • the treatment concentration of Pyr3 or its analogues is 1 ⁇ mol/L to 4 ⁇ mol/L.
  • the dose of Pyr3 or its analogues is 10 mg/kg ⁇ d -1 .
  • the method of administration is intraperitoneal injection, and the injection solvent is DMSO.
  • the invention also provides a medicine for treating tumors, which comprises Pyr3 or its analogues.
  • the medicine of the present invention also includes pharmaceutically acceptable excipients.
  • the drugs of the present invention may also include other drugs with anti-tumor functions.
  • pharmaceutically acceptable excipients are flavoring agents, osmotic pressure regulators, fillers, lubricants, preservatives, suspending agents, food colorings, diluents, emulsifiers, disintegrants One or a mixture of two or more of the additives or plasticizers.
  • the medicine is an oral preparation or an injection.
  • the dosage forms of oral preparations are tablets, pills, oral liquids, capsules, syrups, drop pills or granules.
  • the capsule is a hard capsule or a soft capsule.
  • the tablets are oral tablets or oral tablets.
  • the oral tablet refers to a tablet for oral administration.
  • the drugs in most of these tablets are absorbed through the gastrointestinal tract to exert their effects, and the drugs in some tablets act locally in the gastrointestinal tract.
  • oral tablets are ordinary compressed tablets, dispersible tablets, effervescent tablets, chewable tablets, coated tablets or sustained and controlled release tablets.
  • the injection preparations include injection powder injections or injection solutions.
  • the present invention also provides a method for treating tumors, which comprises administering Pyr3 or its analogues.
  • the Pyr3 analogue is a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or clathrate of Pyr3.
  • the medicament of the present invention can be used in combination therapy, that is, combined application with one or more other agents, wherein the combined application includes administration of Pyr3 together with other agents, or sequential administration.
  • the other agent may be administered before, during, or after the administration of Pyr3 or its analog.
  • the mode of administration may be the same or different.
  • Pyr3 and other agents may be administered in the form of injection, or both may be administered in the form of oral administration, or one may be administered in the form of injection and the other may be administered in the form of oral administration.
  • the present invention proves through experiments that Pyr3 can significantly reduce the tumor volume of tumor-bearing mice, inhibit the proliferation and migration of tumor cells, and has positive significance for the treatment of cancer. Therefore, the present invention provides the application of Pyr3 in the preparation of anti-tumor drugs, and provides an anti-tumor drug containing Pyr3. Studies have shown that for tumor-bearing animals, the volume of subcutaneous tumors in the Pyr3 group was significantly smaller than that in the NC group from the 24th day after the drug intervention to the 32nd day, and the difference was statistically significant (P ⁇ 0.05).
  • FIG 1 shows the results of the mouse subcutaneous tumor experiment, where A shows the control group (NC) group and the tumor body given Pyr3 intervention; B shows the curve of the tumor volume change of the two groups of mice with time as the abscissa;
  • FIG. 2 shows the results of the MTT experiment, where A shows the proliferation of HepG2 cells; B shows the proliferation of Huh7 cells;
  • Figure 3 shows the results of plate cloning experiments; among them, A shows the clone formation of HepG2 cells in each group; B shows the clone formation of Huh7 cells in each group;
  • FIG. 4 shows the results of the scratch experiment; where A shows the migration of HepG2 cells in each group; B shows the migration of Huh7 cells in each group.
  • the present invention provides the use of Pyr3, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention.
  • Invent technology is possible.
  • test materials used in the present invention are all common commercially available products, all of which can be purchased in the market.
  • the 6-week-old male NOD/SCID mice used were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK (xiang) 2016-0002. The mice were raised in an SPF laboratory. Pyr3 (code: B7494) was purchased from ApexBio.
  • mice were divided into two groups (NC group: DMSO) Solvent group, TRPC3 inhibitor group: Pyr3 group), 5 mice in each group, the two groups of mice were intraperitoneally injected with solvent DMSO and Pyr3 (10mg/kg) every day for 32 consecutive days, and tumor volume was measured every 3-4 days. Subcutaneous tumor specimens were collected after days.
  • NC group DMSO
  • TRPC3 inhibitor group Pyr3 group
  • Hepatocarcinoma cells HepG2 and Huh7 were plated on a 96-well plate at 5 ⁇ 10 3 cells/well, and Pyr3 at different concentrations was used to intervene for 24, 48, and 72 hours. Next, 20 ⁇ L of MTT (5 mg/ml, St. Louis, USA) was added to each well, and the incubation continued for 4 hours. Crystal violet is solvent in 150 ⁇ L of DMSO under shaking. The absorbance measurement was performed at a wavelength of 490nm.
  • Hepatocarcinoma cells HepG2 and Huh7 were seeded in a 6-well plate (1.5 ⁇ 10 3 /well) in 2 mL of complete medium, and then cultured in air at 37C and 5% CO2 for 2 weeks. During this period, the medium was replaced every two days with a different concentration of Pyr3 medium. Two weeks later, the cells were stained with 0.5% crystal violet and counted at a magnification of 50 times.
  • hepatoma cells HepG2 and Huh7 were seeded into a 6-well plate and cultured to a confluent monolayer. Use 10ul sterile tip to make parallel scratches on the monolayer. Replace the medium with 1ml serum-free medium containing different concentrations of Pyr3. The scratch healing process was taken at 0, 24, and 48 hours.

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Abstract

The present invention relates to the technical field of drugs, and particularly relates to a use of Pyr3. Experiments prove that the Pyr3 can significantly reduce the tumor volume of tumor-bearing mice and inhibit proliferation and migration of tumor cells, and has positive significance for treatment of cancers. Therefore, the present invention provides the use of Pyr3 in preparation of anti-tumor drugs, and provides an anti-tumor drug containing the Pyr3. Researches show that for tumor-bearing animals, the subcutaneous tumor volume of the Pyr3 group is obviously smaller than that of an NC group from the 24th day to the 32nd day after pharmacological intervention, and the difference has statistical significance (p<0.05).

Description

Pyr3的用途Uses of Pyr3
本申请要求于2020年03月30日提交中国专利局、申请号为202010240024.X、发明名称为“Pyr3的用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of a Chinese patent application filed with the Chinese Patent Office, the application number is 202010240024.X, and the invention name is "Pyr3 Use" on March 30, 2020, the entire content of which is incorporated into this application by reference.
技术领域Technical field
本发明涉及医药技术领域，尤其涉及Pyr3的用途。The present invention relates to the field of medical technology, in particular to the use of Pyr3.
背景技术Background technique
癌症是恶性肿瘤的统称。特点为不正常细胞失控***、无限增殖、侵入并破坏周围正常组织。按其组织来源可分为癌、肉瘤、母细胞瘤、恶性瘤等。来自上皮组织的称做癌；起源于脂肪组织、纤维组织、肌肉组织(统称间叶组织)及淋巴网状组织的称为肉瘤；来源于胚胎组织的称母细胞瘤；不宜用以上名称的叫恶性瘤，如恶性神经鞘瘤；还有些特殊称呼的癌症，如血液细胞的恶性肿瘤也称做白血病。癌症细胞经血液和淋巴液转移，也可直接蔓延。癌症的病因复杂，主要诱发因素有化学致癌物、特殊的病毒、射线、激素、自由基等；遗传、饮食、心理状态是重要的促进因素。Cancer is a general term for malignant tumors. It is characterized by uncontrolled division of abnormal cells, unlimited proliferation, invasion and destruction of surrounding normal tissues. According to its tissue source, it can be divided into cancer, sarcoma, blastoma, malignant tumor and so on. Those from epithelial tissue are called cancers; those that originate from adipose tissue, fibrous tissue, muscle tissue (collectively referred to as mesenchymal tissue) and lymphatic reticular tissue are called sarcomas; those derived from embryonic tissue are called blastoma; those that are not suitable for the above names are called Malignant tumors, such as malignant schwannoma; there are also cancers with special names, such as malignant tumors of blood cells, which are also called leukemias. Cancer cells metastasize through blood and lymph fluid, and can also spread directly. The cause of cancer is complex, and the main inducing factors are chemical carcinogens, special viruses, radiation, hormones, free radicals, etc.; genetics, diet, and psychological status are important contributing factors.
肝细胞癌(Hepatocellular carcinoma，HCC)是全球第五常见的癌症类型，也是全球癌症相关死亡的第三大原因。在过去的几十年里，HCC的诊断和治疗取得了很大的进展，但其长期预后仍差强人意。HCC的长期预后差往往是由于诊断较晚、治疗后复发率高和化疗耐药。因此，迫切需要进一步探索更有效、更安全的抑制HCC进展的抗癌策略。Hepatocellular carcinoma (HCC) is the fifth most common type of cancer in the world and the third leading cause of cancer-related deaths worldwide. In the past few decades, great progress has been made in the diagnosis and treatment of HCC, but its long-term prognosis is still unsatisfactory. The poor long-term prognosis of HCC is often due to late diagnosis, high recurrence rate after treatment, and chemotherapy resistance. Therefore, there is an urgent need to further explore more effective and safer anti-cancer strategies to inhibit the progression of HCC.
1-(4-(2，3，3-三氯丙烯酰胺基)苯基)-5-(三氟甲基)-1H-吡唑-4羧酸乙酯，简称Pyr3，CAS NO.1160514-60-2；其结构式如式I1-(4-(2,3,3-Trichloroacrylamido)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4 ethyl carboxylate, referred to as Pyr3, CAS NO.1160514- 60-2; Its structural formula is as formula I
Figure PCTCN2020087952-appb-000001
Figure PCTCN2020087952-appb-000001
TRPC3通道是瞬时感受器电位(transient receptor potential，TRP)通道家族的成员之一，TRPC3通道是非选择性阳离子通道，主要分布细胞膜，其开放时允许钙离子及钠离子等阳离子通过，从而参与细胞内钙离子稳态。Pyr3是TRPC3通道特异性抑制剂，可以有效阻断TRPC3通道，从而影响TRPC3通道的生物学功能。现有技术中，Pyr3被报道对压力负荷诱导的大鼠左心室肥厚存在影响，也有报道称Pyr3能够改善小鼠脑出血后的预后等。但Pyr3或其类似物对于癌症的治疗作用，此前并未见相关的报道。The TRPC3 channel is a member of the transient receptor potential (TRP) channel family. The TRPC3 channel is a non-selective cation channel, mainly distributed in the cell membrane. When it is opened, it allows cations such as calcium and sodium ions to pass through, thereby participating in intracellular calcium. Ion steady state. Pyr3 is a specific inhibitor of the TRPC3 channel, which can effectively block the TRPC3 channel, thereby affecting the biological function of the TRPC3 channel. In the prior art, Pyr3 has been reported to have an effect on pressure load-induced left ventricular hypertrophy in rats, and there are also reports that Pyr3 can improve the prognosis of mice after cerebral hemorrhage. However, there is no relevant report on the therapeutic effect of Pyr3 or its analogues on cancer.
发明内容Summary of the invention
有鉴于此，本发明要解决的技术问题在于提供Pyr3的用途，本发明研究发现，Pyr3能够降低荷瘤小鼠的肿瘤体积、抑制肿瘤细胞的增殖与迁移，对癌症的治疗存在积极意义。In view of this, the technical problem to be solved by the present invention is to provide the use of Pyr3. The research of the present invention has found that Pyr3 can reduce the tumor volume of tumor-bearing mice, inhibit the proliferation and migration of tumor cells, and has positive significance for the treatment of cancer.
本发明提供了Pyr3或其类似物在制备抑制肿瘤细胞增殖的药物中的应用。The present invention provides the application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor cell proliferation.
在本发明实施例中，所述抑制肿瘤细胞增殖包括：抑制细胞数量的增加和/或抑制克隆的形成。In an embodiment of the present invention, the inhibition of tumor cell proliferation includes: inhibiting the increase in the number of cells and/or inhibiting the formation of clones.
本发明还提供了Pyr3或其类似物在制备抑制肿瘤细胞迁移的药物中的应用。The invention also provides the application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor cell migration.
本发明还提供了Pyr3或其类似物在制备抑制肿瘤体积增长的药物中的应用。The invention also provides the application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor volume growth.
本发明还提供了Pyr3或其类似物在制备***的药物中的应用。The invention also provides the application of Pyr3 or its analogues in the preparation of drugs for treating tumors.
本发明中，所述肿瘤为恶性肿瘤，选自癌、肉瘤和/或母细胞瘤。In the present invention, the tumor is a malignant tumor, selected from cancer, sarcoma and/or blastoma.
一些实施例中，所述肿瘤发生的部位包括头颈部、脑部、甲状腺、食管、胰腺、肺脏、肝脏、胃、乳腺、肾脏、胆囊、结肠、直肠、卵巢、子宫颈、子宫、***、膀胱、睾丸中的一种或多种。In some embodiments, the site where the tumor occurs includes head and neck, brain, thyroid, esophagus, pancreas, lung, liver, stomach, breast, kidney, gallbladder, colon, rectum, ovary, cervix, uterus, prostate, One or more of bladder and testis.
一些具体实施例中，所述肿瘤为肝癌。In some specific embodiments, the tumor is liver cancer.
一些具体实施例中，所述肝癌为肝细胞肝癌或肝母细胞瘤。In some specific embodiments, the liver cancer is hepatocellular carcinoma or hepatoblastoma.
在具体实施例中，以Huh7细胞验证Pyr3对肝细胞肝癌的治疗作用。以HepG2细胞验证Pyr3对肝母细胞瘤的治疗作用。In a specific example, Huh7 cells were used to verify the therapeutic effect of Pyr3 on hepatocellular carcinoma. HepG2 cells were used to verify the therapeutic effect of Pyr3 on hepatoblastoma.
在本发明中，对于细胞实验，Pyr3或其类似物的处理浓度为1μmol/L～4μmol/L。对于动物实验，Pyr3或其类似物的剂量为10mg/kg·d -1。给予方式为腹腔注射，注射的溶剂为DMSO。 In the present invention, for cell experiments, the treatment concentration of Pyr3 or its analogues is 1 μmol/L to 4 μmol/L. For animal experiments, the dose of Pyr3 or its analogues is 10 mg/kg·d -1 . The method of administration is intraperitoneal injection, and the injection solvent is DMSO.
本发明还提供了一种***的药物，其包括Pyr3或其类似物。The invention also provides a medicine for treating tumors, which comprises Pyr3 or its analogues.
本发明所述的药物中，还包括药学上可接受的辅料。The medicine of the present invention also includes pharmaceutically acceptable excipients.
本发明所述的药物中，还可以包括其他具有抗肿瘤功能的药物。The drugs of the present invention may also include other drugs with anti-tumor functions.
在本发明提供的一些实施例中，药学上可接受的辅料为矫味剂、渗透压调节剂、填充剂、润滑剂、防腐剂、助悬剂、食用色素、稀释剂、乳化剂、崩解剂或增塑剂中的一种或两者以上的混合物。In some embodiments provided by the present invention, pharmaceutically acceptable excipients are flavoring agents, osmotic pressure regulators, fillers, lubricants, preservatives, suspending agents, food colorings, diluents, emulsifiers, disintegrants One or a mixture of two or more of the additives or plasticizers.
作为优选，所述药物为口服制剂或注射用剂。其中口服制剂的剂型为片剂、丸剂、口服液剂、胶囊剂、糖浆剂、滴丸剂或颗粒剂。优选的，胶囊剂为硬胶囊剂或软胶囊剂。片剂为口服片剂或口腔片剂。所述口服片剂指供口服的片剂，多数此类片剂中的药物是经胃肠道吸收而发挥作用，也有的片剂中的药物是在胃肠道局部发挥作用。在本发明提供的一些实施例中，口服片剂为普通压制片、分散片、泡腾片、咀嚼片、包衣片或缓控释片。所述注射用剂包括注射粉针剂或注射液。Preferably, the medicine is an oral preparation or an injection. The dosage forms of oral preparations are tablets, pills, oral liquids, capsules, syrups, drop pills or granules. Preferably, the capsule is a hard capsule or a soft capsule. The tablets are oral tablets or oral tablets. The oral tablet refers to a tablet for oral administration. The drugs in most of these tablets are absorbed through the gastrointestinal tract to exert their effects, and the drugs in some tablets act locally in the gastrointestinal tract. In some embodiments provided by the present invention, oral tablets are ordinary compressed tablets, dispersible tablets, effervescent tablets, chewable tablets, coated tablets or sustained and controlled release tablets. The injection preparations include injection powder injections or injection solutions.
本发明还提供了一种***的方法，其包括给予Pyr3或其类似物。The present invention also provides a method for treating tumors, which comprises administering Pyr3 or its analogues.
本发明中，所述Pyr3的类似物为Pyr3的可药用的盐、溶剂化物、水合物、立体异构体或包合物。In the present invention, the Pyr3 analogue is a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or clathrate of Pyr3.
本发明所述的药物可在联合治疗中使用，即与一种或多种其它药剂联合应用，其中所述联合应用包括Pyr3与其它药剂一起施用，或者依次施用。例如，所述其它药剂可在施用Pyr3或其类似物之前、期间或之后施用。施用的方式可相同也可不同，例如Pyr3与其它药剂可皆以注射形式给予，也可皆通过口服形式给予，也可一者通过注射形式给予另一者通过口服形式给予。The medicament of the present invention can be used in combination therapy, that is, combined application with one or more other agents, wherein the combined application includes administration of Pyr3 together with other agents, or sequential administration. For example, the other agent may be administered before, during, or after the administration of Pyr3 or its analog. The mode of administration may be the same or different. For example, Pyr3 and other agents may be administered in the form of injection, or both may be administered in the form of oral administration, or one may be administered in the form of injection and the other may be administered in the form of oral administration.
本发明通过实验证明，Pyr3能够显著降低荷瘤小鼠的肿瘤体积、抑制肿瘤细胞的增殖与迁移，对癌症的治疗存在积极意义。因此，本发明提供了Pyr3在制备治疗抗肿瘤的药物中的应用，并提供了一种含有Pyr3的抗肿瘤药物。研究表明，对于荷瘤动物而言，从药物干预后的第24天开 始直至32天，Pyr3组的皮下瘤体积明显小于NC组，差异有统计学意义(P＜0.05)。The present invention proves through experiments that Pyr3 can significantly reduce the tumor volume of tumor-bearing mice, inhibit the proliferation and migration of tumor cells, and has positive significance for the treatment of cancer. Therefore, the present invention provides the application of Pyr3 in the preparation of anti-tumor drugs, and provides an anti-tumor drug containing Pyr3. Studies have shown that for tumor-bearing animals, the volume of subcutaneous tumors in the Pyr3 group was significantly smaller than that in the NC group from the 24th day after the drug intervention to the 32nd day, and the difference was statistically significant (P<0.05).
附图说明Description of the drawings
图1示小鼠皮下瘤实验结果，其中，A示对照组(NC)组和给予Pyr3干预的瘤体；B示以时间为横坐标两组小鼠瘤体积变化的曲线；Figure 1 shows the results of the mouse subcutaneous tumor experiment, where A shows the control group (NC) group and the tumor body given Pyr3 intervention; B shows the curve of the tumor volume change of the two groups of mice with time as the abscissa;
图2示MTT实验结果，其中，A示HepG2细胞的增殖；B示Huh7细胞的增殖；Figure 2 shows the results of the MTT experiment, where A shows the proliferation of HepG2 cells; B shows the proliferation of Huh7 cells;
图3示平板克隆实验结果；其中，A示各组HepG2细胞的克隆形成情况；B示各组Huh7细胞的克隆形成情况；Figure 3 shows the results of plate cloning experiments; among them, A shows the clone formation of HepG2 cells in each group; B shows the clone formation of Huh7 cells in each group;
图4示划痕实验结果；其中，A示各组HepG2细胞的迁移情况；B示各组Huh7细胞的迁移情况。Figure 4 shows the results of the scratch experiment; where A shows the migration of HepG2 cells in each group; B shows the migration of Huh7 cells in each group.
具体实施方式Detailed ways
本发明提供了Pyr3的用途，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The present invention provides the use of Pyr3, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invent technology.
本发明采用的试材皆为普通市售品，皆可于市场购得。The test materials used in the present invention are all common commercially available products, all of which can be purchased in the market.
在本发明的实施例中，使用的6周龄雄性NOD/SCID小鼠由湖南斯莱克景达实验动物有限公司提供，许可证号：SCXK(湘)2016-0002。小鼠饲养于SPF级实验室。Pyr3(编号：B7494)购自ApexBio公司。In the embodiment of the present invention, the 6-week-old male NOD/SCID mice used were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK (xiang) 2016-0002. The mice were raised in an SPF laboratory. Pyr3 (code: B7494) was purchased from ApexBio.
下面结合实施例，进一步阐述本发明：The following examples further illustrate the present invention:
实施例1：皮下瘤模型试验Example 1: Subcutaneous tumor model test
清洁级饲养，1周适应期后以HepG2肝癌细胞2*10^6/只接种于小鼠颈背部皮下，待皮下瘤体积达到约100mm 3后将小鼠平均分为两组(NC组： DMSO溶剂组，TRPC3抑制剂组：Pyr3组)，每组5只，两组小鼠每天分别腹腔注射溶剂DMSO和Pyr3(10mg/kg)，连续32天，每隔3-4天测量肿瘤体积，32天后收集皮下瘤标本。 Raised at a clean level. After 1 week of adaptation period, HepG2 liver cancer cells 2*10^6/mouse were inoculated subcutaneously on the back of the neck of the mouse. After the subcutaneous tumor volume reached about 100mm 3, the mice were divided into two groups (NC group: DMSO) Solvent group, TRPC3 inhibitor group: Pyr3 group), 5 mice in each group, the two groups of mice were intraperitoneally injected with solvent DMSO and Pyr3 (10mg/kg) every day for 32 consecutive days, and tumor volume was measured every 3-4 days. Subcutaneous tumor specimens were collected after days.
如图1A、1B所示，从药物干预后的第24天开始直至32天，Pyr3组的皮下瘤体积明显小于NC组，差异有统计学意义(P＜0.05)。As shown in Figures 1A and 1B, from day 24 to day 32 after drug intervention, the volume of subcutaneous tumors in the Pyr3 group was significantly smaller than that in the NC group, and the difference was statistically significant (P<0.05).
实施例2：MTT实验Example 2: MTT experiment
肝癌细胞HepG2和Huh7以5×10 3细胞/孔铺板于96孔板，用不同浓度的Pyr3干预24、48、72h。接下来，20μL MTT(5mg/ml，圣路易斯，美国)添加到每个孔，继续孵育4h。结晶紫在150μL DMSO的振荡下溶剂。以490nm波长进行吸光度测量。 Hepatocarcinoma cells HepG2 and Huh7 were plated on a 96-well plate at 5×10 3 cells/well, and Pyr3 at different concentrations was used to intervene for 24, 48, and 72 hours. Next, 20 μL of MTT (5 mg/ml, St. Louis, USA) was added to each well, and the incubation continued for 4 hours. Crystal violet is solvent in 150μL of DMSO under shaking. The absorbance measurement was performed at a wavelength of 490nm.
如图2A、2B所示，分别以4uM和1uM的Pyr3干预肝癌细胞HepG2和Huh7，肝癌细胞的增殖能力与NC组比较明显降低，差异有统计学意义(P＜0.0001)。As shown in Figures 2A and 2B, when 4uM and 1uM Pyr3 were used to intervene hepatocarcinoma cells HepG2 and Huh7, the proliferation ability of hepatocarcinoma cells was significantly lower than that of the NC group, and the difference was statistically significant (P<0.0001).
实施例3：平板克隆实验Example 3: Plate cloning experiment
将肝癌细胞HepG2和Huh7接种于2mL完全培养基中的6孔板(1.5×10 3/孔)，然后在37C、含5％CO2的空气中培养2周。期间以不同浓度Pyr3的培养基每两天更换一次培养基。2周后用0.5％结晶紫对细胞进行染色，放大50倍计数。 Hepatocarcinoma cells HepG2 and Huh7 were seeded in a 6-well plate (1.5×10 3 /well) in 2 mL of complete medium, and then cultured in air at 37C and 5% CO2 for 2 weeks. During this period, the medium was replaced every two days with a different concentration of Pyr3 medium. Two weeks later, the cells were stained with 0.5% crystal violet and counted at a magnification of 50 times.
如图3A、3B所示，分别以4uM和1uM的Pyr3干预肝癌细胞HepG2和Huh7，肝癌细胞的增殖能力与NC组比较明显降低，差异有统计学意义(P＜0.01)。As shown in Figures 3A and 3B, when 4uM and 1uM Pyr3 were used to intervene hepatocarcinoma cells HepG2 and Huh7, the proliferation ability of liver cancer cells was significantly lower than that of the NC group, and the difference was statistically significant (P<0.01).
实施例4：划痕实验Example 4: Scratch test
在划痕实验中，将肝癌细胞HepG2和Huh7接种到6孔板中，培养至融合单层。用10ul无菌tip头对单层细胞进行平行划痕。用1ml含不同浓度Pyr3的无血清培养基代替培养基。划痕愈合过程分别在0、24和48小时拍摄。In the scratch experiment, hepatoma cells HepG2 and Huh7 were seeded into a 6-well plate and cultured to a confluent monolayer. Use 10ul sterile tip to make parallel scratches on the monolayer. Replace the medium with 1ml serum-free medium containing different concentrations of Pyr3. The scratch healing process was taken at 0, 24, and 48 hours.
如图4A、4B所示，分别以4uM和1uM的Pyr3干预肝癌细胞HepG2和Huh7，肝癌细胞的迁移能力与NC组比较明显降低，差异有统计学意义(P＜0.01)。As shown in Figures 4A and 4B, when 4uM and 1uM Pyr3 were used to intervene hepatocarcinoma cells HepG2 and Huh7, the migration ability of liver cancer cells was significantly lower than that of the NC group, and the difference was statistically significant (P<0.01).
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be considered This is the protection scope of the present invention.

Claims (10)

  1. Pyr3或其类似物在制备抑制肿瘤细胞增殖的药物中的应用。Application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor cell proliferation.
  2. 根据权利要求1所述的应用，其特征在于，所述抑制肿瘤细胞增殖包括：抑制细胞数量的增加和/或抑制克隆的形成。The application according to claim 1, wherein the inhibition of tumor cell proliferation comprises: inhibiting the increase in the number of cells and/or inhibiting the formation of clones.
  3. Pyr3或其类似物在制备抑制肿瘤细胞迁移的药物中的应用。Application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor cell migration.
  4. Pyr3或其类似物在制备抑制肿瘤体积增长的药物中的应用。Application of Pyr3 or its analogues in the preparation of drugs for inhibiting tumor volume growth.
  5. Pyr3或其类似物在制备***的药物中的应用。Application of Pyr3 or its analogues in the preparation of drugs for treating tumors.
  6. 根据权利要求1～5任一项所述的应用，其特征在于，所述肿瘤为恶性肿瘤，选自癌、肉瘤和/或母细胞瘤。The use according to any one of claims 1 to 5, wherein the tumor is a malignant tumor, selected from cancer, sarcoma and/or blastoma.
  7. 根据权利要求6所述的应用，其特征在于，所述肿瘤发生的部位包括头颈部、脑部、甲状腺、食管、胰腺、肺脏、肝脏、胃、乳腺、肾脏、胆囊、结肠、直肠、卵巢、子宫颈、子宫、***、膀胱、睾丸中的一种或多种。The application according to claim 6, wherein the site where the tumor occurs includes head and neck, brain, thyroid, esophagus, pancreas, lung, liver, stomach, breast, kidney, gallbladder, colon, rectum, ovary , Cervix, uterus, prostate, bladder, testis, one or more.
  8. 根据权利要求1～7任一项所述的应用，其特征在于，所述肿瘤为肝癌；所述肝癌为肝细胞肝癌或肝母细胞瘤。The use according to any one of claims 1 to 7, wherein the tumor is liver cancer; and the liver cancer is hepatocellular carcinoma or hepatoblastoma.
  9. 根据权利要求1～8任一项所述的应用，其特征在于，Pyr3或其类似物的剂量为10mg/kg·d -1。 The use according to any one of claims 1 to 8, wherein the dose of Pyr3 or its analogue is 10 mg/kg·d -1 .
  10. 一种***的药物，其特征在于，包括Pyr3或其类似物。A medicine for treating tumors, which is characterized by comprising Pyr3 or its analogues.
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