WO2022116369A1 - Preparation and composition of new-type sting agonist complex and use thereof in antitumor drug - Google Patents
Preparation and composition of new-type sting agonist complex and use thereof in antitumor drug Download PDFInfo
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
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- 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
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
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- A—HUMAN NECESSITIES
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Definitions
- the invention belongs to the technical field of biomedicine, and in particular relates to the preparation and composition of a new type of STING agonist complex and its application in the preparation of antitumor drugs.
- Tumor is one of the major diseases that seriously endanger human life and health. According to WHO statistics, the number of cancer cases worldwide may increase to 24 million each year. At present, the methods used to treat tumors are mainly surgery, radiotherapy and chemotherapy. Chemotherapy is the main method currently used to treat malignant tumors. The biggest advantage of chemotherapy drugs is that they can quickly inhibit tumor proliferation and metastasis, and control the disease in a short period of time. Cisplatin-based antitumor drugs have become one of the most effective drugs for the clinical treatment of malignant tumors due to their unique mechanism of action and broad anticancer spectrum. However, its toxic and side effects, drug resistance or cross-resistance, and unclear targeting have become obstacles that limit its expansion of clinical applications.
- chemotherapeutic drugs on normal cells, tissues and organs of the human body are the biggest obstacles and disadvantages that limit the further use of chemotherapeutic drugs.
- chemotherapy does not reverse the tumor tissue in the body, and when chemotherapy is stopped for a period of time, the tumor may recur and worsen. Therefore, how to seek more efficient and less toxic cisplatin metal complexes in the process of clinical treatment has become an urgent problem to be solved. Therefore, based on single-functional cisplatin metal complexes, which have targeting and complex functions, and can enhance efficacy and reduce toxicity, the research on this type of innovative anticancer platinum metal complexes has become an international hotspot.
- Tumor immunotherapy refers to the most rapidly developed method of tumor treatment in the academic and medical circles in recent years. Tumor immunotherapy refers to the therapy that kills tumor cells and tissues by activating the human immune system and immune cells and relying on their own immune function. Different from traditional surgery, chemotherapy and radiotherapy, the direct target of tumor immunotherapy is not tumor tissue, but the body's own immune system. The human immune system is complex and sophisticated. Numerous immune cells perform their respective functions and protect the body's health through different mechanisms. Immunotherapy utilizes the body's own immune function to attack tumor cells without the side effects of traditional chemotherapy and radiotherapy, nor drug resistance, and controls the development and metastasis of tumors.
- Tumor immunotherapy has been proved to be an anti-tumor therapy with significant clinical therapeutic effect and great advantages.
- immunotherapy has been successfully applied to the treatment of various tumors such as melanoma, lymphoma, breast cancer, lung cancer and leukemia in clinical work. treat.
- Folic acid is an important B-group water-soluble vitamin, also known as vitamin M. It was isolated from spinach in 1941, also known as pteroylglutamic acid. As the importance of folic acid in the diet is gradually recognized, especially the research on folic acid and birth defects, cardiovascular diseases and tumors is gradually deepening, folic acid has become an extremely important micronutrient. Folic acid plays an important role in the synthesis of purine and pyrimidine, the interaction of amino acids and some methylation reactions. Folic acid is also often used as a targeting agent for targeting folate receptors on the surface of cancer cells, and is used in the preparation of various anticancer drug targeting preparation materials.
- ER endoplasmic reticulum
- STING endoplasmic reticulum
- cGAMP cyclic cGMP-AMP dinucleotide synthase
- cGAMP is a cytoplasmic DNA sensor, which acts as a second messenger to stimulate the induction of INF- ⁇ through STING, mediates the activation of TBK1 and IRF-3, and then initiates the transcription of INF- ⁇ gene.
- cGAMP As a secondary messenger molecule, cGAMP induces the production of interferon IFN- ⁇ and other cytokines through the STING protein pathway on the endoplasmic reticulum membrane, regulates the expression of downstream proteins, induces cell growth arrest and apoptosis, and produces antiviral effects.
- the STING pathway can regulate the innate immune recognition of immunogenic tumors and promote the antitumor effect of interferons.
- cGAMP is a key stimulator of the innate immune response mediated by cGAS-cGAMP-STING-IRF3 and is an endogenous agonist/activator of STING, therefore, cGAMP has a role in promoting antitumor immune response.
- the invention researches and prepares a new type of natural immune STING agonist and a stable complex composed of folic acid and/or folic acid platinum metal complex.
- This new type of complex combines active immune STING agonist and folic acid or folate platinum metal complex into one, which has targeting and complex functions, and can synergize and reduce toxicity.
- immune STING agonist/or folate platinum metal complex alone, this new type of STING agonist complex has significantly improved anti-tumor efficacy, and can significantly reduce the toxic and side effects of cisplatin metal complex, which is an ideal
- the innovative compound anti-tumor drug has potential anti-tumor clinical application prospects.
- the present invention reports a new type of STING agonist complex, which is a stable complex formed by STING agonist and folic acid or folate platinum metal complex through hydrogen bonding and intermolecular force.
- This new type of STING agonist complex can effectively inhibit the growth and tumor metastasis of solid tumors, and its anti-tumor efficacy is significantly better than that of single use of STING agonist or folate platinum metal complex.
- the anti-tumor efficacy of the STING agonist and folate platinum metal complex complex is significantly better than that of folate platinum metal complex, and also significantly better than oxaliplatin, in particular, it can significantly reduce the toxic and side effects of cisplatin metal complex anti-tumor drugs .
- the specific invention contents include:
- composition of the novel STING agonist complex is composed of an innate immune pathway STING agonist and folic acid or a cisplatin complex containing a folic acid ligand.
- the specific features are: (1) Innate immune pathway STING agonists refer to innate immune pathway (cGAS-STING-cGAMP-IRF3 pathway) STING agonists, including but not limited to cyclic dinucleotides (such as 2'3'-cGAMP ) and its various derivatives.
- Folic acid is a water-soluble B vitamin, the molecular formula is C19H19N7O6, also known as pteroylglutamic acid;
- Folic acid platinum complex is a cisplatin metal complex containing folic acid ligands, including but not limited to cyclohexane Folic acid cisplatin metal complexes such as diamine folic acid platinum (II) and diamine folic acid platinum (II);
- the composition of the new immune agonist complex is characterized by: STING agonist and folic acid or folic acid-containing platinum metal The complexes are tightly combined into stable integrated complexes through hydrogen bonds and intermolecular interactions;
- a STING agonist such as 2'3'-cGAMP
- novel STING agonist complex in the preparation of antitumor drugs.
- This type of compound drug is used in the treatment of various tumors, including but not limited to colorectal cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, testicular cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, kidney cancer, glioma , melanoma, malignant lymphoma, head and neck cancer, thyroid cancer and osteosarcoma and other solid tumors.
- Tumor metastases include but are not limited to lung metastases, liver metastases, lymphatic metastases, brain metastases, and the like.
- This new type of STING agonist compound drug includes unit preparations of different specifications and pharmaceutically acceptable pharmaceutical preparations, including but not limited to intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, nasal drip, oral administration, etc.
- pharmaceutically acceptable pharmaceutical preparations including but not limited to intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, nasal drip, oral administration, etc.
- Cyclic dinucleotide 2'3'-cGAMP is synthesized by cyclic dinucleotide synthase (cGAS) under the activation conditions after binding to DNA according to the literature method, and the purity is above 98% (Li P.W, et al. , Immunity, 2013, 39(6), 1019-1031).
- cyclohexanediamine folic acid platinum (II) metal complex All chemical reagents were of analytical grade and were purchased from Sigma Company.
- the specific synthesis steps of cyclohexanediamine folic acid platinum (II) metal complex are as follows: after potassium tetrachloroplatinate (1mmol) and potassium iodide (8mmol) are stirred and dissolved in 20 milliliters of purified water, cyclohexanediamine (DACH is added dropwise, 2mmol), continue to stir and react for 1 hour to produce a yellow precipitate, filter, wash with water to obtain a yellow powder, dry, mix the yellow powder with silver nitrate and dissolve it in 50 ml of water, stir and react overnight, filter to remove the gray-green precipitate AgI, The sodium hydroxide aqueous solution (2mmol) of folic acid was added dropwise to the filtrate, heated and refluxed for 2 days, filtered, and the filtrate was concentrated, placed in a refrigerator at 4 degrees
- Platinum(II) metal complex in 70% yield The cyclohexanediamine folic acid platinum (II) metal complex was verified to be [Pt(cyclohexanediamine)(folic acid) 2 ] by mass spectrometry, elemental analysis, and platinum metal content analysis.
- the highest nucleocytoplasmic ratio peak in the positive spectrum of mass spectrometry was 1190, which corresponds to the molecular weight of the target product of 1189.
- the results of elemental analysis and platinum metal content analysis are as follows: measured value % (theoretical value %).
- novel STING agonist complexes prepared in the examples are as follows:
- Example 2 Anti-tumor effects of novel STING agonist complexes were detected using a tumor-bearing mouse model
- BALB/C common mice C57BL/6 common mice, male, weight 20-22 g, 7-8 weeks old, SPF grade, purchased from Ncapturing Model Biotechnology Co., Ltd.
- mice All mice foraged and drank freely and were housed at room temperature (23 ⁇ 2)°C.
- the feed and water were all sterilized by autoclaving, and the whole experimental feeding process was SPF grade.
- mice were injected into the tail vein, and the STING agonist complex was set to one dose group: 10 mg/kg
- Dosing volume 100 microliters / only
- Dosing frequency once a day for 21 consecutive days
- the mouse colorectal cancer cell line CT26, the mouse breast cancer cell line 4T1, and the mouse lung cancer cell line LL/2 were purchased from the Cell Bank of the Chinese Academy of Sciences.
- the cells were cultured and passaged, and the cells were collected in the logarithmic phase of the cells to make a cell suspension with a concentration of (1.0 ⁇ 10 7 ) per milliliter.
- Each animal was successfully tumorigenic in about 8 days, and were randomly divided into 6 groups.
- the 6 groups are A: negative control group (normal saline group), B: positive control cGAMP group (10 mg/kg), C: folic acid control group (10 mg/kg), D: cyclohexanediamine folic acid platinum (II) Metal complex (10 mg/kg), E: complex I (STING agonist folate complex) (10 mg/kg), F: complex II (STING agonist folate platinum complex complex) (10 mg/kg) . It was administered once a day for 21 consecutive days. After 21 days, the mice were sacrificed and the tumor weight was weighed.
- the tumor inhibition rate [1-average tumor weight of experimental group (groups B, C, D, E, and F are experimental groups)/average tumor weight of group A]] ⁇ 100%
- the mouse colorectal cancer cell line CT26 was prepared and transplanted into BalB/C ordinary mice, the mouse breast cancer cell line 4T1 was transplanted into BalB/C ordinary mice, and the mouse lung cancer Lewis tumor line LL/2 was transplanted into C57BL /6 mice to observe the anti-tumor effects of different drugs.
- the new STING agonist complex can significantly inhibit tumor growth, and the tumor weight after 21 days of administration was significantly lower than that of the negative control group.
- the new STING agonist complex The antitumor effects were better than those of the STING agonist cGAMP alone or the cyclohexanediamine folate platinum(II) metal complex alone, indicating that the new STING agonist complex drug has a significantly improved antitumor effect. The specific results are shown in the table below.
- Example 3 The effect of novel STING agonist complex against tumor metastasis of mouse breast cancer 4T1-Luc
- mice Male, weighing 20-22 g, 7-8 weeks old, SPF grade, were purchased from Ncapturing Model Biotechnology Co., Ltd.
- mice All mice foraged and drank freely and were housed at room temperature (23 ⁇ 2)°C.
- the feed and water were all sterilized by autoclaving, and the whole experimental feeding process was SPF grade.
- mice were injected into the tail vein, and the STING agonist complex was set to one dose group: 10 mg/kg
- Dosing volume 100 microliters / only
- Dosing frequency once a day for 30 consecutive days
- the mouse breast cancer cell line 4T1-luc (luciferase-labeled tumor cells) was donated by Zhejiang University School of Medicine. The growth and metastasis characteristics of 4T1-luc mouse breast cancer cells in BALB/C mice are very similar to those of human breast cancer.
- This tumor is an animal model of human stage VI breast cancer. 4T1-luc spontaneously produces highly metastatic tumors that can metastasize to the lung, liver, lymph nodes, and brain, while forming a primary foci at the injection site.
- Luciferase is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or firefly aldehyde in living organisms, and it comes from organisms that can emit light in nature.
- the cells were cultured and passaged, and the cells were collected in the logarithmic phase of the cells to make a cell suspension with a concentration of (1.0 ⁇ 10 6 ) per milliliter.
- One/only) the drug was administered on the second day after inoculation of breast cancer cells, once a day, and the patients were randomly divided into 6 groups.
- the 6 groups were A: negative control group (normal saline group), B: positive control cGAMP group (10 mg/kg), C: folic acid group (10 mg/kg), D: cyclohexanediamine folic acid platinum (II) metal Complex (10 mg/kg), E: complex I (STING agonist folate complex) (10 mg/kg), F: complex II (STING agonist folate platinum complex complex) (10 mg/kg).
- the tumor metastasis of breast cancer in mice was detected by a small animal in vivo optical imaging system (Perkin Elmer, IVIS Lumina XRMS Series III) (completed at Shanghai Medical College of Fudan University), on the 5th, 10th, and 30th days after administration, respectively. detection. Observing the effect of different drugs against breast cancer tumor metastasis is summarized as follows:
- mice Male, weighing 20-22 g, 7-8 weeks old, SPF grade, were purchased from Ncapturing Model Biotechnology Co., Ltd.
- mice All mice foraged and drank freely and were housed at room temperature (23 ⁇ 2)°C.
- the feed and water were all sterilized by autoclaving, and the whole experimental feeding process was SPF grade.
- mice were injected into the tail vein, and the STING agonist complex was set to one dose group: 10 mg/kg
- Dosing volume 100 microliters / only
- Dosing frequency once a day for 21 consecutive days
- mice were randomly divided into 5 groups.
- the 5 groups are A: normal group; B, negative control group (normal saline group), C: positive control oxaliplatin group (5mg/kg), D: cyclohexanediamine folic acid platinum (II) metal complex (10 mg/kg), E: complex II (complex of the STING agonist folate platinum complex) (10 mg/kg). It was administered once a day for 21 consecutive days.
- mice were dosed for 21 days, and the percentage of body weight loss and survival rate of mice are summarized in Table 5.
- mice weight loss(%) Survival rate (%) A, normal mouse ——— —— B, saline control —— —— C, oxaliplatin 40 (14 days) 0 (all deaths in 14 days) D, cyclohexanediamine folic acid platinum complex 20 80 E, complex II 6 100
- platinum-containing drugs such as Oxaliplatin
- Oxaliplatin can produce toxic side effects of blood system damage in patients, showing symptoms such as anemia, thrombocytopenia, and neutropenia. Therefore, in the experiment, we collected the peripheral blood of mice administered with the new compound and the positive control group (oxaliplatin and cyclohexyldiamine folic acid platinum complex group) for routine blood test and compared with the mice.
- the normal control group was used to evaluate the effect of the drug on the blood system of mice.
- the symptoms of anemia in the body are reflected in two aspects: first, the number of red blood cells (Red blood cells, RBC) decreases; second, the content of hemoglobin (Hemoglobin) decreases.
- RBC red blood cells
- Hemoglobin the content of hemoglobin
- ALT alanine aminotransferase
- Creatinine creatinine
- Oxaliplatin caused damage to the liver and kidney of mice.
- the ALT and Creatinine indexes of the cyclohexyldiamine folic acid-platinum complex group were much better than those of oxaliplatin, but there were still some toxic effects.
- the new complex has minimal liver and kidney side effects in mice, and the immune agonist complex significantly reduces the toxic side effects of cisplatin.
- ICR mice purchased from Ncapturing Model Biotechnology Co., Ltd.
- half male and half male weighing 18-22 g, were fed with pelleted feed and had free access to food and water.
- mice were injected with 2 g/kg of STING agonist complex (I, II) into the tail vein according to body weight, and the toxicity and death of mice were observed within 14 days after administration. It was found that the mice moved normally after a single tail vein injection. Within 14 days after administration, the mice did not die. On the 15th day, all mice were sacrificed, dissected, and each organ was examined with the naked eye, and no obvious lesions were found.
- STING agonist complex I, II
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Abstract
Description
本发明属于生物医药技术领域，具体涉及一类新型STING激动剂复合物的制备、组成及其在制备抗肿瘤药物中的应用。The invention belongs to the technical field of biomedicine, and in particular relates to the preparation and composition of a new type of STING agonist complex and its application in the preparation of antitumor drugs.
肿瘤是一类严重危害人类生命健康的重大疾病之一。据WHO统计，全球范围内每年癌症病例可能增加到2400万。目前用于治疗肿瘤的手段主要是手术、放疗和化疗。化疗是目前用于治疗恶性肿瘤的主要手段。化疗药物的最大优点是能够快速抑制肿瘤增殖与转移，在短时间内可将病情控制。顺铂类抗肿瘤药物因其独特的作用机制、广泛的抗癌谱成为目前临床治疗恶性肿瘤最为有效的药物之一。但其毒副作用大，耐药性或交叉耐药性、靶向性不明确等问题,成为了限制其扩大临床应用的障碍。化疗药物对人体正常细胞、组织和器官的毒副作用是限制化疗药物进一步使用的最大障碍和弊端。此外，化疗并不能逆转体内肿瘤组织，当化疗停止一段时间后，肿瘤可能会重新复发导致病情恶化。因此，如何寻求临床治疗过程中，具有更高效、更低毒性的顺铂金属配合物类药物成为人们迫切需要解决的问题。因此，基于单功能顺铂金属配合物，使其具有靶向、复合功能，又能增效减毒，该类创新抗癌铂金属配合物物的研究成为一个国际热点。Tumor is one of the major diseases that seriously endanger human life and health. According to WHO statistics, the number of cancer cases worldwide may increase to 24 million each year. At present, the methods used to treat tumors are mainly surgery, radiotherapy and chemotherapy. Chemotherapy is the main method currently used to treat malignant tumors. The biggest advantage of chemotherapy drugs is that they can quickly inhibit tumor proliferation and metastasis, and control the disease in a short period of time. Cisplatin-based antitumor drugs have become one of the most effective drugs for the clinical treatment of malignant tumors due to their unique mechanism of action and broad anticancer spectrum. However, its toxic and side effects, drug resistance or cross-resistance, and unclear targeting have become obstacles that limit its expansion of clinical applications. The toxic and side effects of chemotherapeutic drugs on normal cells, tissues and organs of the human body are the biggest obstacles and disadvantages that limit the further use of chemotherapeutic drugs. In addition, chemotherapy does not reverse the tumor tissue in the body, and when chemotherapy is stopped for a period of time, the tumor may recur and worsen. Therefore, how to seek more efficient and less toxic cisplatin metal complexes in the process of clinical treatment has become an urgent problem to be solved. Therefore, based on single-functional cisplatin metal complexes, which have targeting and complex functions, and can enhance efficacy and reduce toxicity, the research on this type of innovative anticancer platinum metal complexes has become an international hotspot.
肿瘤免疫治疗是指近几年学术界和医学界发展最为迅速的治疗肿瘤的手段。 肿瘤免疫治疗是指通过激活人体免疫系统和免疫细胞，依靠自身的免疫机能，对肿瘤细胞和组织进行杀灭的疗法。与传统的手术、化疗、放疗不同，肿瘤免疫治疗的直接作用靶标不是肿瘤组织，而是人体自身的免疫系统。人体免疫系统复杂而精密，众多免疫细胞各司其职，通过不同的机制保护机体健康。免疫治疗利用人体自身的免疫功能，对肿瘤细胞进行攻击，不会产生传统化疗及放疗的副作用，也不会产生耐药性，并控制肿瘤的发展及转移。但是，由于肿瘤在癌变之前难于被免疫监视系统发现；另一个方面，肿瘤发生发展中，免疫细胞功能通过免疫检查站受到抑制，因此，利用自身免疫抗肿瘤是几十年来难于实现的梦想。能让自身免疫抗肿瘤梦想成真的两个关键点是：(1)通过免疫通路激活剂/激动剂，增强天然免疫能力；(2)破坏免疫检查站对免疫细胞的抑制。人为提高机体对抗肿瘤的免疫反应成为癌症治疗中最令人振奋的科学进展。随着这类药物在临床上的推广应用，其抗肿瘤疗效逐渐得到了临床的认可。利用免疫系统治疗肿瘤，有望成为继手术，化疗，放疗，靶向治疗后肿瘤治疗领域的一场革新。肿瘤免疫治疗已被证明是具有显著临床治疗效果、极具优势的抗肿瘤疗法，目前在临床工作中免疫疗法已被成功应用于黑色素瘤、淋巴瘤、乳腺癌、肺癌、白血病等多种肿瘤的治疗。Tumor immunotherapy refers to the most rapidly developed method of tumor treatment in the academic and medical circles in recent years. Tumor immunotherapy refers to the therapy that kills tumor cells and tissues by activating the human immune system and immune cells and relying on their own immune function. Different from traditional surgery, chemotherapy and radiotherapy, the direct target of tumor immunotherapy is not tumor tissue, but the body's own immune system. The human immune system is complex and sophisticated. Numerous immune cells perform their respective functions and protect the body's health through different mechanisms. Immunotherapy utilizes the body's own immune function to attack tumor cells without the side effects of traditional chemotherapy and radiotherapy, nor drug resistance, and controls the development and metastasis of tumors. However, because tumors are difficult to be detected by the immune surveillance system before they become cancerous; on the other hand, during the development of tumors, the function of immune cells is inhibited through immune checkpoints, so using autoimmunity to fight tumors has been a dream that has been difficult to achieve for decades. The two key points that can make the autoimmune anti-tumor dream come true are: (1) enhancing innate immunity through immune pathway activators/agonists; (2) destroying the inhibition of immune cells by immune checkpoints. Artificially enhancing the body's immune response against tumors has become the most exciting scientific advance in cancer treatment. With the clinical application of these drugs, their anti-tumor efficacy has gradually been recognized by the clinic. Using the immune system to treat tumors is expected to become an innovation in the field of tumor treatment following surgery, chemotherapy, radiotherapy, and targeted therapy. Tumor immunotherapy has been proved to be an anti-tumor therapy with significant clinical therapeutic effect and great advantages. At present, immunotherapy has been successfully applied to the treatment of various tumors such as melanoma, lymphoma, breast cancer, lung cancer and leukemia in clinical work. treat.
叶酸是一种重要的B族水溶性维生素，又称维生素M，1941年从菠菜中分离而出，又名喋酰谷氨酸。由于叶酸在膳食中的重要性逐渐被认识，特别是叶酸与出生缺陷、心血管病及肿瘤的研究逐步深入，叶酸已成为极其重要的微量营养素。叶酸在嘌呤与嘧啶的合成、氨基酸的相互转化作用以及某些甲基化的反应中都发挥着重要作用。叶酸也常被用作靶向癌细胞表面叶酸受体的靶向试剂，而被应用于制备各种各样的抗癌药物靶向制剂材料。Folic acid is an important B-group water-soluble vitamin, also known as vitamin M. It was isolated from spinach in 1941, also known as pteroylglutamic acid. As the importance of folic acid in the diet is gradually recognized, especially the research on folic acid and birth defects, cardiovascular diseases and tumors is gradually deepening, folic acid has become an extremely important micronutrient. Folic acid plays an important role in the synthesis of purine and pyrimidine, the interaction of amino acids and some methylation reactions. Folic acid is also often used as a targeting agent for targeting folate receptors on the surface of cancer cells, and is used in the preparation of various anticancer drug targeting preparation materials.
在感染的哺乳动物细胞中微生物和病毒DNA能通过刺激干扰素分泌诱导內源强有力的免疫应答。内质网(ER)受体蛋白(STING)对胞质DNA的免疫应答是必需的因素。最近的研究表明，环化cGMP-AMP二核苷酸合成酶(cGAS)在结合DNA后的活化条件下，内源性地催化cGAMP的合成。cGAMP是一种胞质DNA传感器，它作为第二信使通过STING刺激INF-β的感应，介导TBK1和IRF-3的活化，进而启动INF-β基因的转录。cGAMP作为二级信使分子通过内质网膜上的STING蛋白通路诱导干扰素IFN-β和其他细胞因子的产生，调节 下游蛋白质表达，诱导细胞生长停滞和凋亡，产生抗病毒效应。STING通路可以调节免疫原性肿瘤的先天免疫识别，促进干扰素的抗肿瘤作用。cGAMP是cGAS-cGAMP-STING-IRF3介导的先天免疫反应的关键刺激物，是STING的内源性激动剂/激活剂，因此，cGAMP具有促进抗肿瘤免疫应答的作用。Microbial and viral DNA can induce potent endogenous immune responses by stimulating interferon secretion in infected mammalian cells. The endoplasmic reticulum (ER) receptor protein (STING) is an essential factor in the immune response to cytoplasmic DNA. Recent studies have shown that cyclic cGMP-AMP dinucleotide synthase (cGAS) endogenously catalyzes the synthesis of cGAMP under activated conditions after DNA binding. cGAMP is a cytoplasmic DNA sensor, which acts as a second messenger to stimulate the induction of INF-β through STING, mediates the activation of TBK1 and IRF-3, and then initiates the transcription of INF-β gene. As a secondary messenger molecule, cGAMP induces the production of interferon IFN-β and other cytokines through the STING protein pathway on the endoplasmic reticulum membrane, regulates the expression of downstream proteins, induces cell growth arrest and apoptosis, and produces antiviral effects. The STING pathway can regulate the innate immune recognition of immunogenic tumors and promote the antitumor effect of interferons. cGAMP is a key stimulator of the innate immune response mediated by cGAS-cGAMP-STING-IRF3 and is an endogenous agonist/activator of STING, therefore, cGAMP has a role in promoting antitumor immune response.
本发明研究制备了一类新型天然免疫STING激动剂与叶酸/或叶酸铂金属配合物组成的稳定复合物。该类新型复合物将主动免疫STING激动剂和叶酸或叶酸铂金属配合物组合形成一体，具有靶向、复合功能，又能增效减毒。相比于单独使用免疫STING激动剂/或叶酸铂金属配合物，该类新型STING激动剂复合物抗肿瘤药效显著提高，又能显著降低顺铂金属配合物的毒副作用，是一种理想的创新复合物抗肿瘤药物，具有潜在的抗肿瘤临床应用前景。The invention researches and prepares a new type of natural immune STING agonist and a stable complex composed of folic acid and/or folic acid platinum metal complex. This new type of complex combines active immune STING agonist and folic acid or folate platinum metal complex into one, which has targeting and complex functions, and can synergize and reduce toxicity. Compared with the use of immune STING agonist/or folate platinum metal complex alone, this new type of STING agonist complex has significantly improved anti-tumor efficacy, and can significantly reduce the toxic and side effects of cisplatin metal complex, which is an ideal The innovative compound anti-tumor drug has potential anti-tumor clinical application prospects.
发明内容SUMMARY OF THE INVENTION
本发明报道了一类新型STING激动剂复合物，该新型复合物由STING激动剂与叶酸或叶酸铂金属配合物通过氢键和分子间作用力形成的稳定复合体。该类新型STING激动剂复合物可以有效抑制实体肿瘤的生长、肿瘤转移，抗肿瘤药效显著优于单一使用STING激动剂或叶酸铂金属配合物。该STING激动剂与叶酸铂金属配合物复合物抗肿瘤药效明显好于叶酸铂金属配合物，也明显优于奥沙利铂，特别是能显著降低顺铂金属配合物抗肿瘤药物的毒副作用。具体发明内容包括：The present invention reports a new type of STING agonist complex, which is a stable complex formed by STING agonist and folic acid or folate platinum metal complex through hydrogen bonding and intermolecular force. This new type of STING agonist complex can effectively inhibit the growth and tumor metastasis of solid tumors, and its anti-tumor efficacy is significantly better than that of single use of STING agonist or folate platinum metal complex. The anti-tumor efficacy of the STING agonist and folate platinum metal complex complex is significantly better than that of folate platinum metal complex, and also significantly better than oxaliplatin, in particular, it can significantly reduce the toxic and side effects of cisplatin metal complex anti-tumor drugs . The specific invention contents include:
1、新型STING激动剂复合物的组成。其特征为：新型STING激动剂复合物由先天免疫通路STING激动剂和叶酸或含叶酸配体的顺铂配合物组成。具体特征为：(1)先天免疫通路STING激动剂是指天然免疫通路(cGAS-STING-cGAMP-IRF3通路)STING的激动剂，包括但不限于环二核苷酸(如2’3’-cGAMP)及其各种衍生物。(2)叶酸是一种水溶性B类维生素，分子式是C19H19N7O6，又名喋酰谷氨酸；(3)叶酸铂配合物是含有叶酸配体的顺铂金属配合物，包括但不限于环己二胺叶酸合铂(II)、二氨叶酸合铂(II)等叶酸顺铂金属配合物；(4)新型免疫激动剂复合物的组成特征为：STING激动剂与叶酸或含叶酸的铂金属配合物通过氢键和分子间相互作用紧密结合成稳定 一体复合物；1. Composition of the novel STING agonist complex. It is characterized in that the novel STING agonist complex is composed of an innate immune pathway STING agonist and folic acid or a cisplatin complex containing a folic acid ligand. The specific features are: (1) Innate immune pathway STING agonists refer to innate immune pathway (cGAS-STING-cGAMP-IRF3 pathway) STING agonists, including but not limited to cyclic dinucleotides (such as 2'3'-cGAMP ) and its various derivatives. (2) Folic acid is a water-soluble B vitamin, the molecular formula is C19H19N7O6, also known as pteroylglutamic acid; (3) Folic acid platinum complex is a cisplatin metal complex containing folic acid ligands, including but not limited to cyclohexane Folic acid cisplatin metal complexes such as diamine folic acid platinum (II) and diamine folic acid platinum (II); (4) the composition of the new immune agonist complex is characterized by: STING agonist and folic acid or folic acid-containing platinum metal The complexes are tightly combined into stable integrated complexes through hydrogen bonds and intermolecular interactions;
2、该类新型STING激动剂复合物的制备方法，其特征在于，将STING激动剂(如2’3’-cGAMP)与叶酸或含有叶酸的铂配合物按1:1摩尔比混合溶于水中，调pH＝8，加热(60度)搅拌4小时，过滤加乙醇后在冷藏冰箱放置过夜，析出固体，过滤，干燥，得到新型STING激动剂复合物。2. A method for preparing the new type of STING agonist complex, characterized in that, a STING agonist (such as 2'3'-cGAMP) and folic acid or a platinum complex containing folic acid are mixed in a 1:1 molar ratio and dissolved in water , adjust pH=8, heat (60 degrees) and stir for 4 hours, filter and add ethanol, place in refrigerator overnight, separate out solid, filter and dry to obtain novel STING agonist complex.
3、该类新型STING激动剂复合物在制备抗肿瘤药物中的应用。该类复合物药物应用于治疗各种肿瘤，包括但不限于结直肠癌、乳腺癌、卵巢癌、前列腺癌、胰腺癌、睾丸癌、肺癌、鼻咽癌、食道癌、肾癌、胶质瘤、黑色素瘤、恶性淋巴瘤、头颈部癌、甲状腺癌及成骨肉瘤等多种实体肿瘤。3. The application of the novel STING agonist complex in the preparation of antitumor drugs. This type of compound drug is used in the treatment of various tumors, including but not limited to colorectal cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, testicular cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, kidney cancer, glioma , melanoma, malignant lymphoma, head and neck cancer, thyroid cancer and osteosarcoma and other solid tumors.
4、该类新型STING激动剂复合物在制备抗肿瘤转移药物中的应用，肿瘤转移包括但不限于肺转移、肝转移、淋巴转移、脑转移等。4. The application of the novel STING agonist complex in the preparation of anti-tumor metastases. Tumor metastases include but are not limited to lung metastases, liver metastases, lymphatic metastases, brain metastases, and the like.
5、该类新型STING激动剂复合物药物，包含不同规格的单位制剂及药学上可接受的药物制剂，包括但不限于静脉注射、肌肉注射、皮下注射、静脉滴注、鼻腔滴注、口服等中的一种或多种，进行上述相关疾病的预防或治疗。5. This new type of STING agonist compound drug includes unit preparations of different specifications and pharmaceutically acceptable pharmaceutical preparations, including but not limited to intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, nasal drip, oral administration, etc. One or more of the above-mentioned related diseases are prevented or treated.
下面通过实施例具体说明本发明的内容。在本发明中，以下所述的实施例是为了更好地阐述本发明，并不是用来限制本发明的范围。The content of the present invention is specifically described below by means of examples. In the present invention, the embodiments described below are for better illustrating the present invention, and are not intended to limit the scope of the present invention.
实施例1：新型STING激动剂复合物的制备与表征Example 1: Preparation and characterization of novel STING agonist complexes
(1)STING激动剂的制备。环二核苷酸2’3’-cGAMP按文献方法在结合DNA后的活化条件下，由环化二核苷酸合成酶(cGAS)催化合成，纯度在98％以上(Li P.W，et al.,Immunity,2013,39(6),1019-1031)。(1) Preparation of STING agonists. Cyclic dinucleotide 2'3'-cGAMP is synthesized by cyclic dinucleotide synthase (cGAS) under the activation conditions after binding to DNA according to the literature method, and the purity is above 98% (Li P.W, et al. , Immunity, 2013, 39(6), 1019-1031).
(2)环己二胺叶酸合铂(II)金属配合物的制备与表征(2) Preparation and characterization of cyclohexanediamine folic acid platinum (II) metal complexes
化学试剂均为分析纯，购于Sigma公司。环己二胺叶酸合铂(II)金属配合物的具体合成步骤如下：四氯铂酸钾(1mmol)和碘化钾(8mmol)在20毫升纯净水中搅拌溶解后，滴加环己二胺(DACH，2mmol)，继续搅拌反应1小时，产生黄色沉淀，过滤，用水洗涤，得到黄色粉末，干燥，将该黄色粉末与硝酸银混 合溶解在50毫升水中，搅拌反应过夜，过滤除掉灰绿色沉淀AgI，向滤液中滴加叶酸的氢氧化钠水溶液(2mmol),加热回流反应2天，过滤，滤液浓缩，放置4度冷藏过夜，过滤，得到棕黄色微晶粉末，干燥，得到环己二胺叶酸合铂(II)金属配合物，产率为70％。环己二胺叶酸合铂(II)金属配合物经质谱、元素分析、铂金属含量分析验证为[Pt(环己二胺)(叶酸) 2]。质谱分析正谱最高核质比峰值为1190，与目标产物分子量1189相对应。元素分析及铂金属含量分析结果如下：实测值％(理论值％)。 All chemical reagents were of analytical grade and were purchased from Sigma Company. The specific synthesis steps of cyclohexanediamine folic acid platinum (II) metal complex are as follows: after potassium tetrachloroplatinate (1mmol) and potassium iodide (8mmol) are stirred and dissolved in 20 milliliters of purified water, cyclohexanediamine (DACH is added dropwise, 2mmol), continue to stir and react for 1 hour to produce a yellow precipitate, filter, wash with water to obtain a yellow powder, dry, mix the yellow powder with silver nitrate and dissolve it in 50 ml of water, stir and react overnight, filter to remove the gray-green precipitate AgI, The sodium hydroxide aqueous solution (2mmol) of folic acid was added dropwise to the filtrate, heated and refluxed for 2 days, filtered, and the filtrate was concentrated, placed in a refrigerator at 4 degrees overnight, filtered to obtain brown-yellow microcrystalline powder, and dried to obtain cyclohexanediamine folic acid complex. Platinum(II) metal complex in 70% yield. The cyclohexanediamine folic acid platinum (II) metal complex was verified to be [Pt(cyclohexanediamine)(folic acid) 2 ] by mass spectrometry, elemental analysis, and platinum metal content analysis. The highest nucleocytoplasmic ratio peak in the positive spectrum of mass spectrometry was 1190, which corresponds to the molecular weight of the target product of 1189. The results of elemental analysis and platinum metal content analysis are as follows: measured value % (theoretical value %).
C：16.78(16.40)；H：43.89(44.41)；N：4.36(4.20)；Pt:19.26(18.84)。C: 16.78 (16.40); H: 43.89 (44.41); N: 4.36 (4.20); Pt: 19.26 (18.84).
(3)新型STING激动剂复合物制备与表征(3) Preparation and characterization of novel STING agonist complexes
将STING激动剂2’3’-cGAMP分别与叶酸或环己二胺叶酸合铂(II)金属配合物按1:1摩尔比混合溶于水中，调pH＝8，加热(60度)搅拌4小时，过滤后的滤液加等体积乙醇后在冷藏冰箱放置过夜，析出固体粉末，过滤，干燥，得到新型STING激动剂复合物。The STING agonist 2'3'-cGAMP was mixed with folic acid or cyclohexanediamine folic acid platinum (II) metal complex in a 1:1 molar ratio and dissolved in water, adjusted to pH=8, heated (60 degrees) and stirred for 4 hours, the filtered filtrate was added with an equal volume of ethanol and then placed in a refrigerator overnight, solid powder was precipitated, filtered and dried to obtain a novel STING agonist complex.
实施例中制备的新型STING激动剂复合物如下：The novel STING agonist complexes prepared in the examples are as follows:
复合物I：STING激动剂叶酸复合物：「2’3’-cGAMP：FA」Complex I: STING agonist Folic acid complex: "2'3'-cGAMP:FA"
复合物II：STING激动剂叶酸铂配合物的复合物：「(2’3’-cGAMP) 2： Complex II: Complex of the STING agonist folate platinum complex: "(2'3'-cGAMP) 2 :
[Pt(DACH)(FA) 2]」 [Pt(DACH)(FA) 2 ]”
(4)STING激动剂复合物亲合常数测定(4) Determination of STING agonist complex affinity constant
(A)仪器：等温滴定微量热仪MicroCal ITC200(GE)(A) Instrument: isothermal titration microcalorimeter MicroCal ITC200 (GE)
(B)实验条件参数：Syringe转速1000rpm；仪器温度25℃；参比热量10μcal/s；第一滴样品0.4μL，此后每滴样品2μL，滴数位15滴；进样时间2s；平衡时间90s。(B) Experimental conditions parameters: Syringe speed 1000rpm; instrument temperature 25°C; reference heat 10μcal/s; first drop of sample 0.4μL, after that each drop of sample 2μL, 15 drops; injection time 2s; equilibration time 90s.
(C)样品准备：(1)STING激动剂cGAMP(2mM)；(2)叶酸(20mM)；(3)环己二胺叶酸合铂(II)配合物(20mM)。溶剂均为Tris-HCl缓冲液pH 8.0。(C) Sample preparation: (1) STING agonist cGAMP (2 mM); (2) folic acid (20 mM); (3) cyclohexanediamine folate platinum(II) complex (20 mM). All solvents were Tris-HCl buffer pH 8.0.
(D)实验步骤：用手动注射器分别吸取20mM叶酸溶液(300μl)(或环己二胺叶酸合铂(II)配合物)，缓慢注入到ITC样品池(Cell)中(禁止产生气泡)，随后操作仪器，使进样(Syringe)吸取2mM cGAMP溶液，操作完成后将Syringe与Cell连接，实验参数设置完成后仪器开始滴定。(D) Experimental steps: use a manual syringe to draw 20 mM folic acid solution (300 μl) (or cyclohexanediamine folic acid platinum (II) complex), and slowly inject it into the ITC sample cell (cell) (no air bubbles), then Operate the instrument so that the injection (Syringe) sucks 2mM cGAMP solution. After the operation is completed, connect the Syringe to the Cell. After the experimental parameters are set, the instrument starts to titrate.
(E)实验数据分析：利用MicroCal ITC200分析软件中的One set of binding sites模式拟合等温线并删除第一滴的滴定误差，数据处理完成后得到反应复合物亲和力的解离常数(k d＝1/K)。解离常数如下：复合物I(k d＝5.1μM)；复合物II(k d＝4.3μM)。 (E) Experimental data analysis: Use the One set of binding sites mode in the MicroCal ITC200 analysis software to fit the isotherm and delete the titration error of the first drop. After the data processing is completed, the dissociation constant of the affinity of the reaction complex (k d = 1/K). The dissociation constants were as follows: complex I (k d =5.1 μM); complex II (k d =4.3 μM).
实施例2：采用荷瘤鼠模型进行检测新型STING激动剂复合物的抗肿瘤作用Example 2: Anti-tumor effects of novel STING agonist complexes were detected using a tumor-bearing mouse model
动物种属、品系、性别、体重、来源、合格证Animal species, strain, sex, weight, source, certificate
BALB/C普通小鼠、C57BL/6普通小鼠，雄性，体重20－22g，7-8周龄，SPF级，购于南方模式生物科技有限公司。BALB/C common mice, C57BL/6 common mice, male, weight 20-22 g, 7-8 weeks old, SPF grade, purchased from Nanfang Model Biotechnology Co., Ltd.
饲养条件rearing conditions
所有小鼠均自由觅食和饮水，在室温(23±2)℃下饲养。饲料及水均经高压灭菌处理，全部实验饲养过程为SPF级。All mice foraged and drank freely and were housed at room temperature (23±2)°C. The feed and water were all sterilized by autoclaving, and the whole experimental feeding process was SPF grade.
剂量设置dose setting
尾静脉注射小鼠，STING激动剂复合物均设置1个剂量组：10mg/kgMice were injected into the tail vein, and the STING agonist complex was set to one dose group: 10 mg/kg
试验对照test control
阴性对照：生理盐水溶液Negative control: saline solution
阳性对照：(1)cGAMP，剂量10mg/kg；(2)叶酸，剂量10mg/kgPositive control: (1) cGAMP, dose 10mg/kg; (2) folic acid, dose 10mg/kg
(2)环己二胺叶酸合铂(II)金属配合物，剂量10mg/kg；(2) cyclohexanediamine folic acid platinum (II) metal complex, the dose is 10mg/kg;
给药方法method of administration
给药途径：尾静脉注射给药Route of administration: Tail vein injection
给药体积：100微升/只Dosing volume: 100 microliters / only
给药次数：每天1次，连续21天Dosing frequency: once a day for 21 consecutive days
每组动物数：10只Number of animals per group: 10
肿瘤细胞株tumor cell line
小鼠结直肠癌细胞株CT26，小鼠乳腺癌细胞株4T1，小鼠肺癌细胞株LL/2，均购自中国科学院细胞库。The mouse colorectal cancer cell line CT26, the mouse breast cancer cell line 4T1, and the mouse lung cancer cell line LL/2 were purchased from the Cell Bank of the Chinese Academy of Sciences.
试验主要步骤The main steps of the test
肿瘤模型鼠的建立与干预Establishment and intervention of tumor model mice
细胞培养，传代，在细胞对数期收集细胞，做成浓度为(1.0×10 7)每毫升的细 胞悬液，小鼠右前肢腋下注射0.2ml细胞悬液(细胞数目为2.0×10 6个/只)，8天左右致瘤成功，随机均分为6组。6组分别为A：阴性对照组(生理盐水组)、B：阳性对照cGAMP组(10mg/kg)、C：叶酸对照组(10mg/kg)、D：环己二胺叶酸合铂(II)金属配合物(10mg/kg)、E：复合物I(STING激动剂叶酸复合物)(10mg/kg)、F:复合物II(STING激动剂叶酸铂配合物的复合物)(10mg/kg)。每天给药1次，连续给药21天。21天后，处死小鼠并称瘤体重量，抑瘤率＝[1-实验组平均瘤重(B、C、D、E、F组为实验组)/A组平均瘤重]]×100％。 The cells were cultured and passaged, and the cells were collected in the logarithmic phase of the cells to make a cell suspension with a concentration of (1.0× 10 7 ) per milliliter. Each animal was successfully tumorigenic in about 8 days, and were randomly divided into 6 groups. The 6 groups are A: negative control group (normal saline group), B: positive control cGAMP group (10 mg/kg), C: folic acid control group (10 mg/kg), D: cyclohexanediamine folic acid platinum (II) Metal complex (10 mg/kg), E: complex I (STING agonist folate complex) (10 mg/kg), F: complex II (STING agonist folate platinum complex complex) (10 mg/kg) . It was administered once a day for 21 consecutive days. After 21 days, the mice were sacrificed and the tumor weight was weighed. The tumor inhibition rate=[1-average tumor weight of experimental group (groups B, C, D, E, and F are experimental groups)/average tumor weight of group A]]×100% .
分别制备小鼠结直肠癌细胞株CT26，移植到BalB/C普通小鼠，小鼠乳腺癌细胞株4T1，移植到BalB/C普通小鼠,小鼠肺癌Lewis瘤株LL/2，移植到C57BL/6小鼠，观察不同药物抗肿瘤效果。The mouse colorectal cancer cell line CT26 was prepared and transplanted into BalB/C ordinary mice, the mouse breast cancer cell line 4T1 was transplanted into BalB/C ordinary mice, and the mouse lung cancer Lewis tumor line LL/2 was transplanted into C57BL /6 mice to observe the anti-tumor effects of different drugs.
统计分析Statistical Analysis
数据用x±s表示，利用SPSS10.0软件进行处理，采用单因素方差分析(one-way ANOVA)检验比较各组瘤重差异的显著性，显著性水平a＝0.05。The data were expressed as x±s, processed by SPSS 10.0 software, and one-way ANOVA was used to test the significance of the difference in tumor weight among the groups, and the significance level was a=0.05.
实验结果Experimental results
小鼠皮下接种肿瘤细胞后制备成功皮下移植瘤模型，新型STING激动剂复合物复合物均可明显抑制肿瘤生长，给药21天后的瘤重均显著低于阴性对照组，新型STING激动剂复合物抑瘤效果均优于单独使用STING激动剂cGAMP或环己二胺叶酸合铂(II)金属配合物单独用药，表明新型STING激动剂复合物药物具有显著提高的抗肿瘤作用。具体结果见下表。After subcutaneous inoculation of tumor cells in mice, a successful subcutaneous transplanted tumor model was prepared. The new STING agonist complex can significantly inhibit tumor growth, and the tumor weight after 21 days of administration was significantly lower than that of the negative control group. The new STING agonist complex The antitumor effects were better than those of the STING agonist cGAMP alone or the cyclohexanediamine folate platinum(II) metal complex alone, indicating that the new STING agonist complex drug has a significantly improved antitumor effect. The specific results are shown in the table below.
表1.新型STING激动剂复合物对BalB/C鼠结直肠癌细胞CT26皮下移植瘤的作用Table 1. Effects of novel STING agonist complexes on subcutaneous xenografts of BalB/C mouse colorectal cancer cell CT26
(n＝10，mean±SD)(n=10, mean±SD)
表2新型免疫激动剂复合物对小鼠肺癌Lewis瘤株LL-2皮下移植瘤的抑制作用Table 2 Inhibitory effect of novel immune agonist complex on mouse lung cancer Lewis tumor line LL-2 subcutaneously transplanted tumor
(n＝10，mean±SD)(n=10, mean±SD)
表3.新型免疫激动剂复合物对BalB/C鼠乳腺癌4T1皮下移植瘤的抑制作用Table 3. Inhibitory effect of novel immune agonist complexes on subcutaneous xenografts of BalB/C mouse breast cancer 4T1
(n＝10，mean±SD)(n=10, mean±SD)
实施例3新型STING激动剂复合物抗小鼠乳腺癌4T1－Luc肿瘤转移的作用Example 3 The effect of novel STING agonist complex against tumor metastasis of mouse breast cancer 4T1-Luc
(n＝10，mean±SD)(n=10, mean±SD)
动物种属、品系、性别、体重、来源、合格证Animal species, strain, sex, weight, source, certificate
BALB/C普通小鼠，雄性，体重20－22g，7-8周龄，SPF级，购于南方模式生物科技有限公司。BALB/C ordinary mice, male, weighing 20-22 g, 7-8 weeks old, SPF grade, were purchased from Nanfang Model Biotechnology Co., Ltd.
饲养条件rearing conditions
所有小鼠均自由觅食和饮水，在室温(23±2)℃下饲养。饲料及水均经高压灭菌处理，全部实验饲养过程为SPF级。All mice foraged and drank freely and were housed at room temperature (23±2)°C. The feed and water were all sterilized by autoclaving, and the whole experimental feeding process was SPF grade.
剂量设置dose setting
尾静脉注射小鼠，STING激动剂复合物均设置1个剂量组：10mg/kgMice were injected into the tail vein, and the STING agonist complex was set to one dose group: 10 mg/kg
试验对照test control
阴性对照：生理盐水溶液Negative control: saline solution
阳性对照：(1)cGAMP，剂量10mg/kg；(2)叶酸，剂量10mg/kgPositive control: (1) cGAMP, dose 10mg/kg; (2) folic acid, dose 10mg/kg
(2)环己二胺叶酸合铂(II)金属配合物，剂量10mg/kg；(2) cyclohexanediamine folic acid platinum (II) metal complex, the dose is 10mg/kg;
给药方法method of administration
给药途径：尾静脉注射给药Route of administration: Tail vein injection
给药体积：100微升/只Dosing volume: 100 microliters / only
给药次数：每天1次，连续30天Dosing frequency: once a day for 30 consecutive days
每组动物数：10只Number of animals per group: 10
肿瘤细胞株tumor cell line
小鼠乳腺癌细胞株4T1－luc(荧光素酶标记肿瘤细胞)，由浙江大学医学院赠送。4T1－luc小鼠乳腺癌细胞在BALB/C小鼠中的生长与转移特性与人体中的乳腺癌十分相近。这种肿瘤是人VI期乳腺癌的动物模型。4T1－luc自发产生高转移 肿瘤，可转移到肺，肝，淋巴结和大脑，同时在注射部位形成始发灶。荧光素酶(Luciferase)是生物体内催化荧光素(luciferin)或脂肪醛(firefly aldehyde)氧化发光的一类酶的总称，来自于自然界能够发光的生物。The mouse breast cancer cell line 4T1-luc (luciferase-labeled tumor cells) was donated by Zhejiang University School of Medicine. The growth and metastasis characteristics of 4T1-luc mouse breast cancer cells in BALB/C mice are very similar to those of human breast cancer. This tumor is an animal model of human stage VI breast cancer. 4T1-luc spontaneously produces highly metastatic tumors that can metastasize to the lung, liver, lymph nodes, and brain, while forming a primary foci at the injection site. Luciferase is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or firefly aldehyde in living organisms, and it comes from organisms that can emit light in nature.
试验主要步骤The main steps of the test
1.肿瘤模型鼠的建立与干预1. Establishment and intervention of tumor model mice
细胞培养，传代，在细胞对数期收集细胞，做成浓度为(1.0×10 6)每毫升的细胞悬液，小鼠右前肢腋下注射0.2ml细胞悬液(细胞数目为2.0×10 5个/只)，接种乳腺癌细胞后第二天开始给药，每天一次，随机均分为6组。6组分别为A：阴性对照组(生理盐水组)、B：阳性对照cGAMP组(10mg/kg)、C：叶酸组(10mg/kg)、D：环己二胺叶酸合铂(II)金属配合物(10mg/kg)、E：复合物I(STING激动剂叶酸复合物)(10mg/kg)、F:复合物II(STING激动剂叶酸铂配合物的复合物)(10mg/kg)。每天给药1次，连续给药30天。 The cells were cultured and passaged, and the cells were collected in the logarithmic phase of the cells to make a cell suspension with a concentration of (1.0× 10 6 ) per milliliter. One/only), the drug was administered on the second day after inoculation of breast cancer cells, once a day, and the patients were randomly divided into 6 groups. The 6 groups were A: negative control group (normal saline group), B: positive control cGAMP group (10 mg/kg), C: folic acid group (10 mg/kg), D: cyclohexanediamine folic acid platinum (II) metal Complex (10 mg/kg), E: complex I (STING agonist folate complex) (10 mg/kg), F: complex II (STING agonist folate platinum complex complex) (10 mg/kg). Administer once a day for 30 consecutive days.
2、不同药物抗乳腺癌肿瘤转移检测方法2. Different drugs for the detection of breast cancer metastasis
小鼠乳腺癌肿瘤转移情况使用小动物活体光学成像系统(Perkin Elmer,IVIS Lumina XRMS Series III)检测(在复旦大学上海医学院完成)，分别在给药后的第5天、10天、30天检测。观察不同药物抗乳腺癌肿瘤转移的效果总结如下：The tumor metastasis of breast cancer in mice was detected by a small animal in vivo optical imaging system (Perkin Elmer, IVIS Lumina XRMS Series III) (completed at Shanghai Medical College of Fudan University), on the 5th, 10th, and 30th days after administration, respectively. detection. Observing the effect of different drugs against breast cancer tumor metastasis is summarized as follows:
表4、不同药物抗乳腺癌肿瘤转移随时间变化情况表Table 4. Changes of different drugs against breast cancer tumor metastasis over time
实验结果表明，新型STING激动剂复合物抗BalB/C鼠乳腺癌4T1－Luc肿瘤转移的作用显著优于单独使用STING激动剂及叶酸铂金属配合物。该类新型复合 物药物具有抗肿瘤转移的潜在临床应用价值。The experimental results showed that the anti-metastasis effect of the new STING agonist complex against BalB/C mouse breast cancer 4T1-Luc tumor was significantly better than that of STING agonist and folate platinum metal complex alone. This new type of compound drug has potential clinical application value in anti-tumor metastasis.
实施例4新型STING激动剂复合物降低铂金属配合物的毒副作用Example 4 Novel STING agonist complexes reduce the toxic side effects of platinum metal complexes
(n＝10，mean±SD)(n=10, mean±SD)
动物种属、品系、性别、体重、来源、合格证Animal species, strain, sex, weight, source, certificate
BALB/C普通小鼠，雄性，体重20－22g，7-8周龄，SPF级，购于南方模式生物科技有限公司。BALB/C ordinary mice, male, weighing 20-22 g, 7-8 weeks old, SPF grade, were purchased from Nanfang Model Biotechnology Co., Ltd.
饲养条件rearing conditions
所有小鼠均自由觅食和饮水，在室温(23±2)℃下饲养。饲料及水均经高压灭菌处理，全部实验饲养过程为SPF级。All mice foraged and drank freely and were housed at room temperature (23±2)°C. The feed and water were all sterilized by autoclaving, and the whole experimental feeding process was SPF grade.
剂量设置dose setting
尾静脉注射小鼠，STING激动剂复合物均设置1个剂量组：10mg/kgMice were injected into the tail vein, and the STING agonist complex was set to one dose group: 10 mg/kg
试验对照test control
阴性对照：生理盐水溶液Negative control: saline solution
阳性对照：(1)奥沙利铂5mg/kg；(2)环己二胺叶酸合铂(II)金属配合物，剂量10mg/kg；Positive control: (1) oxaliplatin 5mg/kg; (2) cyclohexanediamine folic acid platinum (II) metal complex, dose 10mg/kg;
给药方法method of administration
给药途径：尾静脉注射给药Route of administration: Tail vein injection
给药体积：100微升/只Dosing volume: 100 microliters / only
给药次数：每天1次，连续21天Dosing frequency: once a day for 21 consecutive days
每组动物数：10只Number of animals per group: 10
小鼠随机均分为5组。5组分别为A：正常组；B，阴性对照组(生理盐水组)、C：阳性对照奥沙利铂组(5mg/kg)、D：环己二胺叶酸合铂(II)金属配合物(10mg/kg)、E：复合物II(STING激动剂叶酸铂配合物的复合物)(10mg/kg)。每天给药1次，连续给药21天。Mice were randomly divided into 5 groups. The 5 groups are A: normal group; B, negative control group (normal saline group), C: positive control oxaliplatin group (5mg/kg), D: cyclohexanediamine folic acid platinum (II) metal complex (10 mg/kg), E: complex II (complex of the STING agonist folate platinum complex) (10 mg/kg). It was administered once a day for 21 consecutive days.
结果分析：Result analysis:
(1)新型复合物对小鼠体重和生存率的影响(1) Effects of novel complexes on body weight and survival rate of mice
小鼠给药21天，小鼠体重下降百分数和生存率总结于表5.The mice were dosed for 21 days, and the percentage of body weight loss and survival rate of mice are summarized in Table 5.
Table 5.小鼠体重下降百分数和生存率Table 5. Percentage weight loss and survival rate of mice
表5结果显示，新型免疫激动剂复合物(II)显著降低了铂金属配合物的毒副作用。奥沙利铂给药组14天全部死亡，体重下降了40％；环己二胺叶酸合铂(II)配合物比奥沙利铂毒性小很多，但是仍然具有明显毒副作用，小鼠体重下降了20％；新型复合物给药组生存率100％，体重基本没有明显变化。The results in Table 5 show that the novel immune agonist complex (II) significantly reduces the toxic and side effects of platinum metal complexes. The oxaliplatin-administered group all died within 14 days, and the body weight decreased by 40%; the cyclohexyldiamine folic acid platinum (II) complex was much less toxic than oxaliplatin, but still had obvious toxic and side effects, and the weight of the mice decreased The survival rate of the new compound administration group was 100%, and there was basically no significant change in body weight.
(2)新型复合物对小鼠血液系统的影响(2) Effects of the new complex on the blood system of mice
在临床上，含有铂的药物，如Oxaliplatin会产生患者血液系统损伤的毒副作用，呈现出贫血、血小板和中性粒细胞减少等症状。因此，在实验中，我们对新型复合物给药与阳性对照组(奥沙利铂和环己二胺叶酸合铂配合物组)的小鼠进行外周血的采集，用于血常规检测并与正常组对照，以此来评估药物对小鼠血液系统的影响。机体出现贫血症状体现在两方面：第一，红细胞数目(Red blood cells，RBC)减少；第二，血红蛋白(Hemoglobin)含量降低。我们对实验各组的 这两项指标进行了统计总结，见表6(Table 6)。Clinically, platinum-containing drugs, such as Oxaliplatin, can produce toxic side effects of blood system damage in patients, showing symptoms such as anemia, thrombocytopenia, and neutropenia. Therefore, in the experiment, we collected the peripheral blood of mice administered with the new compound and the positive control group (oxaliplatin and cyclohexyldiamine folic acid platinum complex group) for routine blood test and compared with the mice. The normal control group was used to evaluate the effect of the drug on the blood system of mice. The symptoms of anemia in the body are reflected in two aspects: first, the number of red blood cells (Red blood cells, RBC) decreases; second, the content of hemoglobin (Hemoglobin) decreases. We made a statistical summary of these two indicators of each group in the experiment, as shown in Table 6 (Table 6).
Table 6.The routine blood testsTable 6. The routine blood tests
表6结果显示，阳性药Oxaliplatin组的红细胞数目和血红蛋白含量分别与正常组比较明显低于正常值。环己二胺叶酸合铂配合物各组的红细胞数目和血红蛋白含量均比奥沙利铂组好，但是仍然在正常值边沿。新型复合物给药组的血液检测指标明显优于阳性对照组，血常规指标均在正常范围，与正常组数据一致。The results in Table 6 show that the number of red blood cells and the content of hemoglobin in the positive drug Oxaliplatin group were significantly lower than those in the normal group, respectively. The number of erythrocytes and hemoglobin content in each group of cyclohexanediamine folate-platinum complex were better than those in oxaliplatin group, but they were still at the border of normal value. The blood test indexes of the new compound administration group were significantly better than those of the positive control group, and the blood routine indexes were all within the normal range, which were consistent with the data of the normal group.
(3)新型复合物对小鼠肝肾的影响(3) Effects of the new complex on the liver and kidney of mice
我们对实验各组小鼠血清中丙氨酸氨基转移酶(ALT)和肌酐(Creatinine)含量进行了统计(Table 7)。数据显示，在肝功能指标上，与正常组相比，阳性药Oxaliplatin组ALT的含量明显超出了正常值，环己二胺叶酸合铂配合物给药组处在最高正常参考值的边缘。Oxaliplatin组Creatinine的含量明显偏高；环己二胺叶酸合铂配合物组与正常组含量略有偏高。新型复合物给药组与正常组含量基本一致，没有呈现明显的变化。根据实验结果，Oxaliplatin对小鼠的肝脏和肾脏产生了损伤，环己二胺叶酸合铂配合物组的ALT和Creatinine指标均比奥沙利铂好很多，但仍然有些毒性影响。新型复合物对小鼠 产生的肝肾副作用极小，免疫激动剂复合物显著降低了顺铂药物的毒副作用。We counted the levels of alanine aminotransferase (ALT) and creatinine (Creatinine) in the serum of each group of mice (Table 7). The data showed that in terms of liver function indexes, compared with the normal group, the ALT content of the positive drug Oxaliplatin group was significantly higher than the normal value, and the cyclohexanediamine folic acid-platinum complex administration group was on the edge of the highest normal reference value. The content of Creatinine in the Oxaliplatin group was significantly higher; the content of the cyclohexanediamine folic acid-platinum complex group and the normal group was slightly higher. The content of the new compound administration group was basically the same as that of the normal group, and there was no obvious change. According to the experimental results, Oxaliplatin caused damage to the liver and kidney of mice. The ALT and Creatinine indexes of the cyclohexyldiamine folic acid-platinum complex group were much better than those of oxaliplatin, but there were still some toxic effects. The new complex has minimal liver and kidney side effects in mice, and the immune agonist complex significantly reduces the toxic side effects of cisplatin.
Table 7.Summary of biochemical markers in serumTable 7.Summary of biochemical markers in serum
实施例5新型STING激动剂复合物的急性毒性研究Example 5 Acute toxicity studies of novel STING agonist complexes
实验材料Experimental Materials
ICR小鼠20只(购于南方模式生物科技有限公司)，雌雄各半，体重18～22g，动物以颗粒饲料喂养，自由摄食和饮水。20 ICR mice (purchased from Nanfang Model Biotechnology Co., Ltd.), half male and half male, weighing 18-22 g, were fed with pelleted feed and had free access to food and water.
实验方法experimental method
ICR小鼠按体重分别单次尾静脉注射2g/kg的STING激动剂复合物(I，II)药物，观察给药后小鼠14天内的毒性反应及死亡情况。结果发现，小鼠单次尾静脉注射给药后，小鼠活动正常。给药后14天内，小鼠未出现死亡，第15天，全部小鼠处死，解剖，肉眼检查各脏器，均未见明显病变。ICR mice were injected with 2 g/kg of STING agonist complex (I, II) into the tail vein according to body weight, and the toxicity and death of mice were observed within 14 days after administration. It was found that the mice moved normally after a single tail vein injection. Within 14 days after administration, the mice did not die. On the 15th day, all mice were sacrificed, dissected, and each organ was examined with the naked eye, and no obvious lesions were found.
实验结果Experimental results
上述急性毒性实验结果表明，静脉注射给药最大耐受量MTD不低于2g/Kg，说明新型STING激动剂复合物(I，II)药物的急性毒性低。The above acute toxicity test results show that the maximum tolerated dose MTD of intravenous injection is not less than 2g/Kg, indicating that the acute toxicity of the new STING agonist complex (I, II) is low.
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| CN108892700A (en) * | 2018-05-27 | 2018-11-27 | 杭州星鳌生物科技有限公司 | A kind of new antitumoral compounds and its application in preparation of anti-tumor drugs |
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