WO2024140295A1 - Pharmaceutical combination for treating tumors and use thereof - Google Patents
Pharmaceutical combination for treating tumors and use thereof Download PDFInfo
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- WO2024140295A1 WO2024140295A1 PCT/CN2023/139357 CN2023139357W WO2024140295A1 WO 2024140295 A1 WO2024140295 A1 WO 2024140295A1 CN 2023139357 W CN2023139357 W CN 2023139357W WO 2024140295 A1 WO2024140295 A1 WO 2024140295A1
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- cancer
- tumor
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Classifications
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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
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- 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
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
-
- 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 disclosure provides a method for treating a tumor, comprising administering a therapeutically effective amount of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide to a subject in need thereof.
- kits or a pharmaceutically acceptable composition comprising: (a) IN0018 or a pharmaceutically acceptable salt thereof; and (b) cyclophosphamide.
- the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in preparing a combination drug for treating tumors.
- the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof in the preparation of a drug for use in combination with cyclophosphamide in treating tumors.
- the present disclosure provides a kit, comprising: cyclophosphamide; and instructions, wherein the instructions indicate that cyclophosphamide can be used for treating tumors in combination with IN0018 or a pharmaceutically acceptable salt thereof.
- the term “inhibit” refers to a decrease in the baseline activity of a biological activity or process.
- the compound of the test kit can be contained in a separate container. Alternatively, two or more compounds are contained in the same container.
- the test kit can include a first container, a second container and a package insert, wherein the first container includes at least one dose of a drug including IN10018 or a pharmaceutically acceptable salt thereof, the second container includes at least one dose of cyclophosphamide, and the package insert includes instructions for using the drug to treat the tumor of the object.
- the first container and the second container can include the same or different shapes (e.g., vials, syringes and bottles) and/or materials (e.g., plastic or glass).
- the test kit can also include other materials that can help administer the drug, such as diluents, filters, IV bags and lines, needles and syringes.
- Breast cancer cell 4T1 (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60352) was maintained and subcultured by Yingshi Biotechnology (Nanjing) Co., Ltd.
- the cells were cultured in a monolayer in vitro, and the culture conditions were RPMI-1640 medium with 10% fetal bovine serum, cultured in a 37°C, 5% CO2 incubator. Two to three times a week, routine digestion and subculture were performed with trypsin-EDTA. When the cells were in the exponential growth phase and the confluence was 80%-90%, the cells were harvested, counted, and inoculated.
- the experimental index is to examine whether the tumor growth is inhibited, delayed or cured.
- the tumor diameter is measured with a vernier caliper three times a week.
- TGI (%) [1-(average tumor volume of a certain medication group-average tumor volume at the beginning of the medication group)/(average tumor volume of the solvent control group-average tumor volume at the beginning of the solvent control group)] ⁇ 100%.
- Panel 1 Resuspend cells in fixative and nuclear membrane disrupting solution and incubate at room temperature in the dark for 45-60 min.
- Panel 2 Resuspend cells in fixative and incubate at 4°C in the dark for 30 min.
- Panel 1 nuclear antibody (1:100 dilution in True-Nuclear TM 1X Perm Buffer) was added and incubated at 4°C in the dark for 30 min;
- tumor growth was observed every day. Groups were divided according to tumor volume on the 7th day after inoculation, and the average tumor volume was about 51 mm 3 . Due to the need for FACS detection experiments, all groups were euthanized on the 20th day after inoculation, that is, on the 13th day after group administration, and the entire experiment was terminated. On the 13th day after group administration, the tumor volume of the control group was 857.9 ⁇ 204.2 mm 3 .
- the tumor volumes of the CTX (10 mg/kg) and IN10018 (25 mg/kg) single-drug groups were 759.5 ⁇ 73.6 mm 3 and 626.0 ⁇ 108.9 mm 3 , respectively; the tumor volume of the CTX+IN10018 (10+25 mg/kg) combination group was 319.3 ⁇ 86.3 mm 3 .
- the comprehensive tumor volume was compared with the control group.
- the comprehensive tumor volume was compared with the CTX+IN10018 (10+25 mg/kg) combination group, and statistical analysis was performed.
- the P values of the control group, CTX (10 mg/kg) and IN10018 (25 mg/kg) monotherapy groups were p ⁇ 0.0001, p ⁇ 0.0001 and p ⁇ 0.0001, respectively.
- the evaluation of the tumor inhibition effect of each group is shown in Table 9, and the tumor volume of each dose group at different time periods is shown in Figure 1.
- the gating strategy for the proportion of regulatory T cells is the percentage of CD25 and FoxP3 double-positive cells in CD4+T cells among CD3+T cells in live CD45+ lymphocytes.
- the percentage of Treg cells in CD4+T cells in the control group was 8.5%; the percentage of Treg cells in CD4+T cells in the CTX (10mg/kg) and IN10018 (25mg/kg) single-drug groups was 7.0% and 9.2%, respectively; the percentage of Treg cells in CD4+T cells in the CTX+IN10018 (10+25mg/kg) combination group was 3.4%.
- the percentage of comprehensive Treg cells in CD4+T cells was compared with that of the control group for statistical analysis.
- the gating strategy for the expression level of PD-L1 on tumor cells is to look at the expression of PD-L1 in the living CD45- tumor cell population.
- the expression level of PD-L1 in tumor cells of the control group was 17.7%; the expression levels of PD-L1 in tumor cells of the CTX (10 mg/kg) and IN10018 (25 mg/kg) monotherapy groups were 9.0% and 5.2%, respectively; the expression level of PD-L1 in tumor cells of the CTX+IN10018 (10+25 mg/kg) combination group was 6.5%.
- the expression level of PD-L1 in tumor cells was compared with that of the control group for statistical analysis.
- Treg and CD11b+CD11c+DC cells in each group and the expression levels of PD-L1, CD86 and MHC II are shown in Table 11 and Figures 3, 4, 5, 6 and 7.
- the PD-L1 expression levels on CD45- tumor cells in the CTX (10 mg/kg), IN10018 (25 mg/kg) monotherapy groups and the CTX+IN10018 (10+25 mg/kg) combination group were very low, which was statistically significant compared with the CTX (10 mg/kg) monotherapy group and the CTX+IN10018 (10+25 mg/kg) combination group, indicating that the combined administration of CTX+IN10018 (10+25 mg/kg) can significantly reduce the expression of PD-L1 in CD45- tumor cells in the tumor microenvironment.
- mice Female BALB/c mice aged 7-8 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The experiment began after the animals were adapted to the experimental environment upon arrival. The animals were kept in IVC (independent ventilation system) cages (4 per cage) in an SPF animal room. All cages, bedding and drinking water must be sterilized before use. All experimental personnel should wear protective clothing and latex gloves when operating in the animal room. Cages, feed and drinking water were changed twice a week.
- IVC independent ventilation system
- the experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.
- the tumor volume of the control group was 2795.1 ⁇ 1412.0 mm 3
- the tumor volume of the CTX (10 mg/kg) monotherapy group was 1991.3 ⁇ 1011.1 mm 3
- the tumor volumes of the CTX+IN10018 (10+10 mg/kg) and CTX+IN10018 (10+20 mg/kg) combination groups were 1625.1 ⁇ 323.3 mm 3 and 1268.4 ⁇ 190.7 mm 3 , respectively.
- the tumor volume was compared with the CTX+IN10018 (10+20 mg/kg) combination group, and statistical analysis was performed.
- the experiment was carried out according to the dosing regimen. During the experiment, the animals' activities such as eating and drinking were observed every day, and the weight of the animals was recorded 2 to 3 times a week.
- the weight of the control group changed from 21.0g on the day of group administration (Day 0) to 25.1g, with a weight change rate of 19.4%;
- the average weight of the CTX (10mg/kg) monotherapy group changed from 20.2g on Day 0 to 21.2g, with a weight change rate of 5.2%;
- the average weight of the CTX+IN10018 (10+10mg/kg) combination group changed from 21.0g on Day 0 to 20.9g, with a weight change rate of -0.2%;
- the average weight of the CTX+IN10018 (10+20mg/kg) combination group changed from 20.7g on Day 0 to 21.4g, with a weight change rate of 3.3%;
- the animals in the treatment group were in good mental state, and their eating and movement were normal.
- CTX at a dose of 10 mg/kg and the combination treatment with IN10018 between 10 mg/kg and 20 mg/kg have a dose-dependent effect, and IN10018 can better inhibit tumor growth.
- IN10018 at a high concentration (20 mg/kg) can better enhance the therapeutic level of CTX (10 mg/kg) than at a low concentration (10 mg/kg).
- Example 3 In vivo anti-tumor efficacy study of the combination of cyclophosphamide and IN10018 in the ascites model of ovarian cancer ID8-Luc cells transplanted into the peritoneal cavity of C57BL/6 mice
- Cage Made of polycarbonate, 300mm ⁇ 180mm ⁇ 150mm. Bedding is corn cobs, changed twice a week.
- Drinking water Experimental animals were free to drink sterile water.
- mice in each group 1.
- Dosing volume 10mL/kg based on the mouse body weight. If the body weight drops by more than 15%, stop dosing the animal; resume dosing when the body weight returns to the initial value;
- the health of the animal continues to deteriorate, or if there is a serious illness or pain, it must be euthanized. If the following conditions occur, notify the veterinarian and euthanize the animal: obvious weight loss, weight loss greater than 20%; cannot freely eat and drink, the experiment is terminated. If the animal shows the following clinical manifestations and continues to deteriorate: piloerection, arched back, pale ears, nose, eyes or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.
- Body weight Record the animal's body weight 1 to 2 times a week and observe changes in the animal's body weight over time and with the occurrence of disease.
- Table 21 Evaluation of abdominal circumference changes of the test substance in the C57BL/6 mouse ascites model of mouse ovarian cancer ID8-Luc cells (based on data on days 42/46/49 after group administration) Note: 1. Data are mean ⁇ standard deviation (mean ⁇ SD); 2.*: p ⁇ 0.05, vs. control group, Two-way ANOVA;
- the weight data and abdominal circumference data of the combination therapy group of CTX+IN10018 (3+12.5mg/kg) were both smaller than those of the blank control group and the CTX (3mg/kg) monotherapy group, and there were statistical differences compared with the blank control group.
- the above data indicate that the combination therapy of CTX at a dose of 3 mg/kg and IN10018 at a dose of 12.5 mg/kg has a better therapeutic effect.
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Abstract
Use of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in the preparation of a drug for treating tumors, and a composition or kit comprising IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide.
Description
本申请要求于2022年12月28日递交的中国专利申请202211699620.X的优先权,在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。This application claims priority to Chinese patent application 202211699620.X filed on December 28, 2022. The contents of the above-mentioned Chinese patent application disclosure are hereby cited in their entirety as a part of this application.
本发明属于药物化学领域。具体地,本发明涉及粘着斑激酶(Focal Adhesion Kinase,FAK)抑制剂IN10018与其它抗肿瘤药物联用治疗肿瘤。The present invention belongs to the field of pharmaceutical chemistry. Specifically, the present invention relates to the combined use of focal adhesion kinase (FAK) inhibitor IN10018 and other anti-tumor drugs to treat tumors.
环磷酰胺(Cyclophosphamide,Cytoxan,Endoxan),CAS号50-18-0,是一种化疗药。环磷酰胺英文缩写叫CTX,是常用的化疗药物,主要有抗肿瘤作用,能够与肿瘤细胞的DNA结合,打断肿瘤细胞的DNA分子的合成,能够阻断肿瘤细胞的生长和繁殖。Cyclophosphamide (Cyclophosphamide, Cytoxan, Endoxan), CAS No. 50-18-0, is a chemotherapy drug. Cyclophosphamide, abbreviated as CTX in English, is a commonly used chemotherapy drug with anti-tumor effects. It can bind to the DNA of tumor cells, interrupt the synthesis of DNA molecules of tumor cells, and block the growth and reproduction of tumor cells.
但是在化疗过程中,环磷酰胺使用还是会给患者带来比较明显的不良反应发生,比如脱发、恶心、呕吐。如果剂量过大,超过一克每平米,会出现明显的出血性膀胱炎。However, during chemotherapy, the use of cyclophosphamide will still cause obvious adverse reactions to patients, such as hair loss, nausea, and vomiting. If the dose is too large, exceeding one gram per square meter, obvious hemorrhagic cystitis will occur.
因此,降低环磷酰胺的临床剂量,让其作用能够持续是目前临床需要解决的问题。Therefore, reducing the clinical dose of cyclophosphamide so that its effect can be sustained is a problem that needs to be solved in clinical practice.
发明内容Summary of the invention
本公开一方面提供了IN0018或其药学上可接受的盐和环磷酰胺在制备用于在对象中治疗肿瘤的药物中的用途,所述IN10018结构如下:
In one aspect, the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in the preparation of a medicament for treating a tumor in a subject, wherein the structure of IN10018 is as follows:
In one aspect, the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in the preparation of a medicament for treating a tumor in a subject, wherein the structure of IN10018 is as follows:
本公开又一方面提供了IN0018或其药学上可接受的盐和环磷酰胺的药物组合产品,其用于在对象中治疗肿瘤。Yet another aspect of the present disclosure provides a pharmaceutical combination product of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide for use in treating a tumor in a subject.
本公开又一方面提供了一种治疗肿瘤的方法,该方法包括向有需要的对象施用治疗有效量的IN0018或其药学上可接受的盐和环磷酰胺。In another aspect, the present disclosure provides a method for treating a tumor, comprising administering a therapeutically effective amount of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide to a subject in need thereof.
本公开又一方面提供了一种试剂盒或药学上可接受的组合物,其包括:(a)IN0018或其药学上可接受的盐;和(b)环磷酰胺。In another aspect, the present disclosure provides a kit or a pharmaceutically acceptable composition, comprising: (a) IN0018 or a pharmaceutically acceptable salt thereof; and (b) cyclophosphamide.
本公开又一方面提供了IN0018或其药学上可接受的盐与环磷酰胺在制备用于治疗肿瘤的联用药物中的用途。In another aspect, the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in preparing a combination drug for treating tumors.
本公开又一方面提供了IN0018或其药学上可接受的盐在制备用于与环磷酰胺治疗肿瘤的联用药物中的用途。In another aspect, the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof in the preparation of a drug for use in combination with cyclophosphamide in treating tumors.
本公开又一方面提供了环磷酰胺在制备用于与IN0018或其药学上可接受的盐治疗肿瘤的联用药物中的用途。In another aspect, the present disclosure provides use of cyclophosphamide in preparing a drug for use in combination with IN0018 or a pharmaceutically acceptable salt thereof in treating tumors.
本公开又一方面提供了一种试剂盒,其包括:IN0018或其药学上可接受的盐;和说明书,该说明书指出该IN0018或其药学上可接受的盐可用于与环磷酰胺联合治疗肿瘤。In another aspect, the present disclosure provides a kit, comprising: IN0018 or a pharmaceutically acceptable salt thereof; and instructions, wherein the instructions indicate that IN0018 or a pharmaceutically acceptable salt thereof can be used for treating tumors in combination with cyclophosphamide.
本公开又一方面提供了一种试剂盒,其包括:环磷酰胺;和说明书,该说明书指出环磷酰胺可用于与IN0018或其药学上可接受的盐联合治疗肿瘤。In another aspect, the present disclosure provides a kit, comprising: cyclophosphamide; and instructions, wherein the instructions indicate that cyclophosphamide can be used for treating tumors in combination with IN0018 or a pharmaceutically acceptable salt thereof.
本公开另一方面提供了一种治疗肿瘤的方法,该方法包括向有需要的对象施用治疗有效量的IN0018或其药学上可接受的盐和环磷酰胺。Another aspect of the present disclosure provides a method for treating a tumor, comprising administering a therapeutically effective amount of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide to a subject in need thereof.
本公开又一方面提供了IN0018或其药学上可接受的盐和环磷酰胺的药物组合产品,其用于在有需要的对象中治疗肿瘤。In another aspect, the present disclosure provides a pharmaceutical combination product of IN0018 or a pharmaceutically acceptable salt thereof and cyclophosphamide for use in treating a tumor in a subject in need thereof.
可选的,所述IN10018药学上可接受的盐为IN10018酒石酸盐。
Optionally, the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.
可选的,所述IN0018或其药学上可接受的盐和所述环磷酰胺被同时或依次施用于所述对象。Optionally, the IN0018 or a pharmaceutically acceptable salt thereof and the cyclophosphamide are administered to the subject simultaneously or sequentially.
可选的,所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。Optionally, the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland cancer, spindle cell carcinoma caused by Metastatic tumors, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML); preferably, the tumor is ovarian cancer, liver cancer, lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, gastric cancer, pancreatic cancer or colon cancer; more preferably, the tumor is ovarian cancer or breast cancer.
为了更清楚地说明本公开实施例的技术方案,下面将对实施例的附图作简单地介绍,显然下面描述中的附图仅仅涉及本公开的一些实施例,而非对本发明的限制。In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. It is obvious that the drawings in the following description only relate to some embodiments of the present disclosure, but are not intended to limit the present invention.
图1显示了实施例1小鼠乳腺癌4T1细胞BABL/c小鼠皮下同种移植瘤模型给药后的肿瘤生长曲线。数据点代表组内平均肿瘤体积,误差线代表标准误(SEM)。Figure 1 shows the tumor growth curve of the subcutaneous BABL/c mouse transplanted tumor model of mouse breast cancer 4T1 cells after administration of the drug in Example 1. The data points represent the mean tumor volume within the group, and the error bars represent the standard error (SEM).
图2显示了实施例1小鼠乳腺癌4T1细胞BABL/c小鼠皮下同种移植瘤模型给药后的体重变化曲线。数据点代表组内平均体重,误差线代表标准误(SEM)。
Figure 2 shows the body weight change curve of the mouse breast cancer 4T1 cell BABL/c mouse subcutaneous transplant tumor model after administration in Example 1. The data points represent the average body weight within the group, and the error bars represent the standard error (SEM).
图3显示了实施例1中调节性T细胞(Treg)在CD4+T细胞中的占比。数据点代表组内每只动物的Treg占比数值,误差线代表标准误(SEM)。Figure 3 shows the proportion of regulatory T cells (Treg) in CD4+ T cells in Example 1. The data points represent the numerical value of the Treg proportion of each animal in the group, and the error bars represent the standard error (SEM).
图4显示了实施例1中CD45-肿瘤细胞的PD-L1的表达水平。数据点代表组内每只动物的CD45-肿瘤细胞的PD-L1的表达水平数值,误差线代表标准误(SEM)。Figure 4 shows the expression level of PD-L1 of CD45- tumor cells in Example 1. The data points represent the expression level of PD-L1 of CD45- tumor cells of each animal in the group, and the error bars represent the standard error (SEM).
图5显示了实施例1中CD11b+CD11c+双阳DC细胞在CD45+淋巴细胞中的占比。数据点代表组内每只动物的CD11b+CD11c+DC细胞在CD45+淋巴细胞中的占比数值,误差线代表标准误(SEM)。Figure 5 shows the percentage of CD11b+CD11c+ double positive DC cells in CD45+ lymphocytes in Example 1. The data points represent the percentage of CD11b+CD11c+ DC cells in CD45+ lymphocytes for each animal in the group, and the error bars represent the standard error (SEM).
图6显示了实施例1中CD11b+CD11c+DC细胞的CD86的表达水平。数据点代表组内每只动物的CD11b+CD11c+DC细胞的CD86的表达水平数值,误差线代表标准误(SEM)。Figure 6 shows the expression level of CD86 in CD11b+CD11c+ DC cells in Example 1. The data points represent the numerical value of the expression level of CD86 in CD11b+CD11c+ DC cells of each animal in the group, and the error bars represent the standard error (SEM).
图7显示了实施例1中CD11b+CD11c+DC细胞的MHC II的表达水平。数据点代表组内每只动物的CD11b+CD11c+DC细胞的MHC II的表达水平数值,误差线代表标准误(SEM)。Figure 7 shows the expression level of MHC II of CD11b+CD11c+DC cells in Example 1. The data points represent the expression level of MHC II of CD11b+CD11c+DC cells of each animal in the group, and the error bars represent the standard error (SEM).
图8显示了实施例2小鼠乳腺癌4T1细胞BABL/c小鼠皮下同种移植瘤模型给药后的肿瘤生长曲线。数据点代表组内平均肿瘤体积,误差线代表标准误(SEM)。Figure 8 shows the tumor growth curve of the subcutaneous BABL/c mouse homograft tumor model of mouse breast cancer 4T1 cells after administration of the drug in Example 2. The data points represent the mean tumor volume within the group, and the error bars represent the standard error (SEM).
图9显示实施例2小鼠乳腺癌4T1细胞BABL/c小鼠皮下同种移植瘤模型给药后的体重变化曲线。数据点代表组内平均体重,误差线代表标准误(SEM)。Figure 9 shows the body weight change curve of the mouse breast cancer 4T1 cell BABL/c mouse subcutaneous transplant tumor model after administration of the drug in Example 2. The data points represent the average body weight within the group, and the error bars represent the standard error (SEM).
图10显示实施例3小鼠卵巢癌ID8-Luc细胞C57BL/6小鼠腹水模型给药后的发病率曲线。FIG. 10 shows the incidence curve of the mouse ovarian cancer ID8-Luc cell C57BL/6 mouse ascites model after administration of Example 3.
图11显示实施例3中小鼠卵巢癌ID8-Luc细胞C57BL/6小鼠腹水模型给药后的体重变化曲线。数据点代表组内平均体重,误差线代表标准误(SEM)。Figure 11 shows the body weight change curve after administration of mouse ovarian cancer ID8-Luc cells to the C57BL/6 mouse ascites model in Example 3. The data points represent the mean body weight within the group, and the error bars represent the standard error (SEM).
图12显示实施例3中小鼠卵巢癌ID8-Luc细胞C57BL/6小鼠腹水模型给药后的腹围变化曲线。数据点代表组内平均腹围,误差线代表标准误(SEM)。Figure 12 shows the abdominal circumference change curve after administration of mouse ovarian cancer ID8-Luc cells to the C57BL/6 mouse ascites model in Example 3. The data points represent the average abdominal circumference within the group, and the error bars represent the standard error (SEM).
为使本公开实施例的目的、技术方案和优点更加清楚,下面将结合本公开实施例的附图,对本公开实施例的技术方案进行清楚、完整地描述。显然,所描述的实施例是本公开的一部分实施例,而不是全部的实施例。基于所描述的本公开的实施例,本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
本发明可在不偏离本发明基本属性的情况下以其它具体形式来实施。应该理解的是,在不冲突的前提下,本发明的任一和所有实施方案都可与任一其它实施方案或多个其它实施方案中的技术特征进行组合以得到另外的实施方案。本发明包括这样的组合得到的另外的实施方案。The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.
本公开中提及的所有出版物和专利在此通过引用以它们的全部内容纳入本公开。如果通过引用纳入的任何出版物和专利中使用的用途或术语与本公开中使用的用途或术语冲突,那么以本公开的用途和术语为准。All publications and patents mentioned in this disclosure are hereby incorporated by reference into this disclosure in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.
本文所用的章节标题仅用于组织文章的目的,而不应被解释为对所述主题的限制。The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
除非另有规定,本文使用的所有技术术语和科学术语具有要求保护主题所属领域的通常含义。倘若对于某术语存在多个定义,则以本文定义为准。Unless otherwise specified, all technical and scientific terms used herein have the common meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.
本申请中所用的下列术语和符号具有如下所述的含义,其所处的上下文中另有说明的除外。The following terms and symbols used in this application have the meanings described below, unless otherwise indicated in the context in which they are used.
除了在工作实施例中或另外指出之外,在说明书和权利要求中陈述的定量性质例如剂量的所有数字应理解为在所有情况中被术语“约”修饰。还应理解的是,本申请列举的任何数字范围意在包括该范围内的所有的子范围和该范围或子范围的各个端点的任何组合。
Except in the working examples or otherwise indicated, all numbers of quantitative properties such as dosage stated in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited in the application is intended to include all subranges within the range and any combination of the respective endpoints of the range or subrange.
本公开中使用的“包括”、“含有”或者“包含”等类似的词语意指出现该词前面的要素涵盖出现在该词后面列举的要素及其等同,而不排除未记载的要素。本文所用的术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之,所述术语也包括“基本上由…组成”、或“由…组成”。The words "include", "comprise", "comprising" and the like used in the present disclosure mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "comprise" or "comprising" used herein may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of", or "consisting of".
本文所用的“药物组合”或者“药物组合产品”既可以指采用一个剂量单位形式的固定组合(例如所有药物活性成分以一种剂型存在)或者成套药盒以组合施用的产品的情形,也可以指一种药物与指示该药物可与另外的一种或多种药物联合使用的说明书的组合情形。As used herein, "drug combination" or "drug combination product" may refer to a fixed combination in the form of one dosage unit (for example, all active pharmaceutical ingredients are present in one dosage form) or a kit for combined administration, or it may refer to a combination of one drug and instructions indicating that the drug can be used in combination with one or more other drugs.
本文所用的“联合治疗”或者“联用药物”是指一种药物与另外的一种或多种药物联合使用来治疗疾病,既包括一种药物与另外的一种或多种药物的组合的情形,也包括一种药物与指示该药物可与另外的一种或多种药物联合使用的说明书的组合情形。As used herein, "combination therapy" or "combination drug" refers to the use of a drug in combination with one or more other drugs to treat a disease, including both the combination of a drug with one or more other drugs and the combination of a drug with instructions indicating that the drug can be used in combination with one or more other drugs.
“同时或依次施用”在本申请中是指一个给药周期内(例如4周内、3周内、2周内、1周内或24小时以内)两种以上的药物同时或以一定时间间隔先后施用,药物施用的方式(例如口服、静脉、肌肉或皮下施用等)可以相同或不同,两种以上的药物的给药频率/周期可以相同或不同。当本公开的治疗方法、产品或用途涉及两种药物时,两种药物可同时或以一定时间间隔分别单独施用。"Administered simultaneously or sequentially" in this application means that two or more drugs are administered simultaneously or sequentially at certain time intervals within one administration cycle (e.g., within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours), the modes of drug administration (e.g., oral, intravenous, intramuscular or subcutaneous administration, etc.) may be the same or different, and the administration frequency/cycle of the two or more drugs may be the same or different. When the method of treatment, product or use disclosed herein involves two drugs, the two drugs may be administered separately at the same time or at certain time intervals.
本文所用的术语“治疗”是指给患有疾病或者具有所述疾病的症状的对象施用一种或多种药物物质,用以治愈、缓解、减轻、改变、医治、改善、改进或影响所述疾病或者所述疾病的症状。在一些实施方案中,所述疾病是肿瘤或者癌症。The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a subject suffering from a disease or symptoms of the disease to cure, alleviate, mitigate, alter, cure, improve, ameliorate or affect the disease or symptoms of the disease. In some embodiments, the disease is a tumor or cancer.
本文所用的术语“肿瘤”是指机体在各种致瘤因素的作用下,局部组织的细胞在基因水平上失去对其生长的正常调控,从而导致其克隆型异常增生而形成的异常病变。实施例包括,但不限于:膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤,神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。The term "tumor" as used herein refers to an abnormal lesion formed when the cells of local tissues lose normal regulation of their growth at the genetic level under the influence of various tumorigenic factors, thereby causing abnormal proliferation of their clonal types. Examples include, but are not limited to: bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland cancer, spindle cell carcinoma Metastatic tumors, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML); preferably, the tumor is ovarian cancer, liver cancer, lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, gastric cancer, pancreatic cancer or colon cancer; more preferably, the tumor is ovarian cancer or breast cancer.
本文所用的术语“对象”或“受试者”是指哺乳动物和非哺乳动物。哺乳动物是指哺乳类的任何成员,其包括但不限于:人;非人灵长类动物,如黑猩猩及其它猿类和猴类物种;农场动物,如牛、马、绵羊、山羊和猪;家畜,如兔、狗和猫;实验室动物,包括啮齿类动物,如大鼠、小鼠和豚鼠;等等。非哺乳动物的例子包括但不限于鸟等。术语“对象”并不限定特定的年龄或性别。在一些实施方案中,对象是人。As used herein, the term "subject" or "subject" refers to mammals and non-mammals. Mammals refer to any member of the class mammals, including but not limited to: humans; non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, horses, sheep, goats, and pigs; livestock, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds, and the like. The term "subject" does not limit a particular age or sex. In some embodiments, the subject is a human.
本文所用的术语“药学上可接受的”指的是无毒的、生物学上可耐受的,适合给对象施用的。As used herein, the term "pharmaceutically acceptable" means non-toxic, biologically tolerable, and suitable for administration to a subject.
本文所用的术语“药学上可接受的盐”指的是无毒的、生物学上可耐受的适合给对象施用的酸加成盐,包括但不限于:与无机酸形成的酸加成盐,例如盐酸盐、氢溴酸盐、碳酸盐、碳酸氢盐、磷酸盐、硫酸盐、亚硫酸盐、硝酸盐等;以及与有机酸形成的酸加成盐,例如甲酸盐、乙酸盐、苹果酸盐、马来酸盐、富马酸盐、酒石酸盐、琥珀酸盐、柠檬酸盐、乳酸盐、甲磺酸盐、对甲苯磺酸盐、2-羟基乙磺酸盐、苯甲酸盐、水杨酸盐、硬脂酸盐和与式HOOC-(CH2)n-COOH(其中n是0-4)的链烷二羧酸形成的盐等。
The term "pharmaceutically acceptable salt" as used herein refers to non-toxic, biologically tolerable acid addition salts suitable for administration to a subject, including, but not limited to: acid addition salts formed with inorganic acids, such as hydrochlorides, hydrobromides, carbonates, bicarbonates, phosphates, sulfates, sulfites, nitrates, etc.; and acid addition salts formed with organic acids, such as formate, acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, and salts formed with alkanedicarboxylic acids of the formula HOOC-( CH2 ) n -COOH (wherein n is 0-4), etc.
此外,药学上可接受的酸加成盐可以按照由碱性化合物制备酸加成盐的常规操作通过将游离碱溶于合适的溶剂并用酸处理该溶液来得到。本领域技术人员无需过多实验即可确定各种可用来制备无毒的药学上可接受的酸加成盐的合成方法。在一些实施例中,IN10018药学上可接受的盐为酒石酸盐。In addition, pharmaceutically acceptable acid addition salts can be prepared by dissolving the free base in a suitable solvent and treating the solution with an acid according to the conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art can determine various synthetic methods that can be used to prepare non-toxic pharmaceutically acceptable acid addition salts without excessive experimentation. In some embodiments, the pharmaceutically acceptable salt of IN10018 is a tartrate.
本文所用的术语“药学上可接受的组合物”是指必须在化学和/或毒理学上与包括制剂的其他成分相容,和/或与接受其治疗的对象相容。本文所用的术语“治疗有效量”是指通常足以对对象产生有益治疗效果的量。可以通过常规方法(例如建模、剂量递增研究或临床试验)结合常规影响因素(例如给药方式、化合物的药代动力学、疾病的严重程度和病程、对象的病史、对象的健康状况、对象对药物的响应程度等)来确定本发明的治疗有效量。The term "pharmaceutically acceptable composition" as used herein means that it must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation, and/or compatible with the subject receiving the treatment thereof. The term "therapeutically effective amount" as used herein means an amount that is generally sufficient to produce a beneficial therapeutic effect on the subject. The therapeutically effective amount of the present invention can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., mode of administration, pharmacokinetics of the compound, severity and course of the disease, medical history of the subject, health status of the subject, degree of response of the subject to the drug, etc.).
本文所用的术语“抑制”是指生物活动或过程的基线活性的降低。As used herein, the term "inhibit" refers to a decrease in the baseline activity of a biological activity or process.
本文所用的术语“试剂盒”是指用于盛放检测化学成分、药物残留、病毒种类等化学试剂的盒子。本发明所述试剂盒可以是包括(i)IN10018或其药学上可接受的盐和环磷酰胺中的一种或二种;以及(ii)说明书,所述说明书指出可使用IN10018或其药学上可接受的盐和环磷酰胺来在对象中治疗肿瘤。在一种实施方案中,试剂盒包括(i)IN10018或其药学上可接受的盐;以及(ii)说明书,所述说明书指出可使用IN10018或其药学上可接受的盐和环磷酰胺来在对象中治疗肿瘤。在一种实施方案中,试剂盒包括(i)环磷酰胺;以及(ii)说明书,所述说明书指出可使用IN10018或其药学上可接受的盐和环磷酰胺来在对象中治疗肿瘤。在一种实施方案中,试剂盒包括(i)IN10018或其药学上可接受的盐和环磷酰胺;以及(ii)说明书,所述说明书指出可使用IN10018或其药学上可接受的盐和环磷酰胺来在对象中治疗肿瘤。The term "kit" as used herein refers to a box for containing chemical reagents for detecting chemical components, drug residues, virus species, etc. The kit of the present invention may include (i) one or two of IN10018 or its pharmaceutically acceptable salt and cyclophosphamide; and (ii) instructions, which indicate that IN10018 or its pharmaceutically acceptable salt and cyclophosphamide can be used to treat tumors in subjects. In one embodiment, the kit includes (i) IN10018 or its pharmaceutically acceptable salt; and (ii) instructions, which indicate that IN10018 or its pharmaceutically acceptable salt and cyclophosphamide can be used to treat tumors in subjects. In one embodiment, the kit includes (i) cyclophosphamide; and (ii) instructions, which indicate that IN10018 or its pharmaceutically acceptable salt and cyclophosphamide can be used to treat tumors in subjects. In one embodiment, the kit includes (i) IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide can be used to treat a tumor in a subject.
试剂盒的化合物可以包含在分开的容器中。可选地,两种或更多种化合物包含在同一容器中。例如,试剂盒可以包括第一容器、第二容器和包装插页,其中第一容器包括至少一个剂量的包括IN10018或其药学上可接受的盐的药物,第二容器包括至少一个剂量的环磷酰胺,且所述包装插页包括使用药物治疗对象的肿瘤的说明。第一容器和第二容器可以包含相同或不同形状(例如,小瓶、注射器和瓶)和/或材料(例如,塑料或玻璃)。试剂盒还可以包括可以有助于施用药物的其他材料,如稀释剂、过滤器、IV袋和管线、针和注射器。The compound of the test kit can be contained in a separate container. Alternatively, two or more compounds are contained in the same container. For example, the test kit can include a first container, a second container and a package insert, wherein the first container includes at least one dose of a drug including IN10018 or a pharmaceutically acceptable salt thereof, the second container includes at least one dose of cyclophosphamide, and the package insert includes instructions for using the drug to treat the tumor of the object. The first container and the second container can include the same or different shapes (e.g., vials, syringes and bottles) and/or materials (e.g., plastic or glass). The test kit can also include other materials that can help administer the drug, such as diluents, filters, IV bags and lines, needles and syringes.
给予受试者的IN10018或其药学上可接受的盐和环磷酰胺的精确量将取决于各种因素,例如给定的药物或化合物,药物制剂,给药途径,疾病类型,病症,所治疗的受试者或宿主的身份等,但是仍然可以由本领域技术人员常规确定。例如,确定有效量还取决于细胞增殖的程度,严重性和类型。技术人员将能够根据这些和其他因素确定合适的剂量。The exact amount of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide administered to a subject will depend on various factors, such as a given drug or compound, pharmaceutical formulation, route of administration, type of disease, condition, identity of the subject or host being treated, etc., but can still be routinely determined by one skilled in the art. For example, determining an effective amount also depends on the extent, severity, and type of cell proliferation. A skilled person will be able to determine an appropriate dosage based on these and other factors.
IN10018或其药学上可接受的盐和环磷酰胺可选择合适的方式例如口服、静脉、肌肉或皮下施用给药。IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide can be administered by a suitable route such as oral, intravenous, intramuscular or subcutaneous administration.
例如,口服给药时,可以将药物与药学上可接受的载体例如惰性稀释剂或可吸收的食用载体一起口服给药。它们可以封装在硬壳或软壳明胶胶囊中,可以压制成片剂,或者可以直接与患者的食物混合。例如,药物可以与一种或多种赋形剂组合,并以可摄取的片剂,口腔片剂,锭剂,胶囊,酏剂,悬浮液,糖浆或糯米纸囊剂等形式使用。片剂,锭剂,丸剂,胶囊剂等可进一步包括:粘合剂,例如黄芪胶,阿拉伯胶,玉米淀粉或明胶;赋形剂,如磷酸二钙;崩解剂,例如玉米淀粉,马铃薯淀粉,海藻酸等;润滑剂,例如硬脂酸镁;或甜味剂,例如蔗糖,果糖,乳糖或阿斯巴甜;或调味剂。For example, when administered orally, the drug can be administered orally with a pharmaceutically acceptable carrier such as an inert diluent or an absorbable edible carrier. They can be encapsulated in hard or soft shell gelatin capsules, can be compressed into tablets, or can be mixed directly with the patient's food. For example, the drug can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, or wafers. Tablets, lozenges, pills, capsules, etc. may further include: binders, such as tragacanth, gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; or sweeteners, such as sucrose, fructose, lactose, or aspartame; or flavoring agents.
例如,输注或注射静脉内或腹膜内给药时,药物的溶液可以在水中制备,任选地与无毒的表面活性剂混合。For example, when administered intravenously or intraperitoneally by infusion or injection, solutions of the drug can be prepared in water, optionally mixed with a nontoxic surfactant.
用于注射或输注的示例性药物剂型包括:无菌水溶液,分散液,或包含活性成分的无菌粉末,该无菌粉末适合于临时制备无菌注射或输注溶液或分散液。无论如何,最终剂型在生产和储存条件下均应无菌,流动且稳定。Exemplary pharmaceutical dosage forms for injection or infusion include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In any event, the final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
无菌注射溶液可以通过将所需量的药物与所需的上述各种其他成分掺入适当的溶剂中,然后过滤灭菌来制备。对于用于制备无菌注射溶液的无菌粉末,优选的制备方法可以是真空干燥和冷冻干燥技术,其可以产生活性成分加上先前无菌过滤后存在的任何其他所需成分的粉末。Sterile injectable solutions can be prepared by mixing the required amount of the drug with the various other ingredients listed above in an appropriate solvent, followed by filtered sterilization. For sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods may be vacuum drying and freeze drying techniques, which can produce a powder of the active ingredient plus any other desired ingredients present after previous sterile filtration.
用于治疗所需的IN10018或其药学上可接受的盐和环磷酰胺的量不仅可以随所选择的特定试剂而变化,还可以随给药途径,所治疗疾病的性质以及患者的年龄和状况而变化,并且最终可以由主治医师或临床医生自行决定。然而,一般而言,剂量可以在每天约0.1至约50mg/kg体重的范围内。The amount of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide required for treatment may vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician. However, in general, the dosage may be in the range of about 0.1 to about 50 mg/kg body weight per day.
在一些实施方案中,IN10018或其药学上可接受盐以成年人中5mg/天-100mg/天,例如20mg/天的剂量施用,所述剂量以游离碱计。In some embodiments, IN10018 or a pharmaceutically acceptable salt thereof is administered at a dose of 5 mg/day to 100 mg/day, for example 20 mg/day, in adults, calculated as the free base.
所述环磷酰胺以成年人中每天0.1-5mg/kg的剂量范围施用。The cyclophosphamide is administered in a dosage range of 0.1-5 mg/kg per day in adults.
本文所用的未具体定义的技术和科学术语具有本发明所属领域的技术人员通常理解的含义。Technical and scientific terms used herein without specific definition have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
在一些实施方案中,本公开还公开了以下:In some embodiments, the present disclosure also discloses the following:
1.IN10018或其药学上可接受的盐和环磷酰胺在制备用于在对象中治疗肿瘤的药物中的用途,所述IN10018结构如下:
1. Use of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in the preparation of a medicament for treating a tumor in a subject, wherein the structure of IN10018 is as follows:
1. Use of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in the preparation of a medicament for treating a tumor in a subject, wherein the structure of IN10018 is as follows:
2.IN10018或其药学上可接受的盐和环磷酰胺的药物组合产品,其用于在对象中治疗肿瘤,所述IN10018结构如下:
2. A pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide for use in treating a tumor in a subject, wherein the structure of IN10018 is as follows:
2. A pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide for use in treating a tumor in a subject, wherein the structure of IN10018 is as follows:
3.一种治疗肿瘤的方法,该方法包括向有需要的对象施用治疗有效量的IN10018或其药学上可接受的盐和环磷酰胺,所述IN10018结构如下:
3. A method for treating a tumor, comprising administering a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide to a subject in need thereof, wherein the structure of IN10018 is as follows:
3. A method for treating a tumor, comprising administering a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide to a subject in need thereof, wherein the structure of IN10018 is as follows:
4.如实施方案1-3任一项所述的用途、药物组合产品或者方法,其中所述IN10018药学上可接受的盐为酒石酸盐。4. The use, pharmaceutical combination or method according to any one of embodiments 1 to 3, wherein the pharmaceutically acceptable salt of IN10018 is tartrate.
5.如实施方案1-4任一项所述的用途、药物组合产品或者方法,其中所述IN10018或其药学上可接受的盐和所述环磷酰胺被同时或依次施用于所述对象。5. The use, pharmaceutical combination or method according to any one of embodiments 1 to 4, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the cyclophosphamide are administered to the subject simultaneously or sequentially.
6.如实施方案1-5任一项所述的用途、药物组合产品或者方法,其中所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。6. The use, pharmaceutical combination product or method according to any one of embodiments 1 to 5, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer , salivary gland cancer, metastases caused by spindle cell cancer, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML); preferably, the tumor is ovarian cancer, liver cancer, lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, gastric cancer, pancreatic cancer or colon cancer; more preferably, the tumor is ovarian cancer or breast cancer.
7.一种试剂盒或药学上可接受的组合物,其包括:7. A kit or a pharmaceutically acceptable composition comprising:
(a)IN10018或其药学上可接受的盐;和
(a) IN10018 or a pharmaceutically acceptable salt thereof; and
(b)环磷酰胺,(b) cyclophosphamide,
所述IN10018结构如下:
The structure of IN10018 is as follows:
The structure of IN10018 is as follows:
8.如实施方案7所述的试剂盒或组合物,其用作药物。8. A kit or composition according to embodiment 7 for use as a medicament.
9.如实施方案7-8任一项所述的试剂盒或组合物,所述IN10018药学上可接受的盐为酒石酸盐。9. The kit or composition according to any one of embodiments 7-8, wherein the pharmaceutically acceptable salt of IN10018 is tartrate.
10.一种在对象中治疗肿瘤的方法,其中所述方法包括将根据实施方案7-9任一项所述的试剂盒或组合物中的化合物同时或依次施用至所述对象。10. A method for treating a tumor in a subject, wherein the method comprises administering to the subject simultaneously or sequentially a compound in the kit or composition according to any one of embodiments 7-9.
11.如实施方案10所述的方法,所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。11. As described in embodiment 10, the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland cancer, spindle cell carcinoma The invention relates to a method for treating a tumour of a certain type, wherein the tumour is a metastatic tumour caused by a cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML); preferably, the tumour is ovarian cancer, liver cancer, lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, gastric cancer, pancreatic cancer or colon cancer; more preferably, the tumour is ovarian cancer or breast cancer.
12.一种在对象中治疗肿瘤的方法,其中所述方法包括向所述对象施用治疗有效量的IN10018或其药学上可接受的盐和环磷酰胺;所述IN10018结构如下:
12. A method for treating a tumor in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide; the structure of IN10018 is as follows:
12. A method for treating a tumor in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide; the structure of IN10018 is as follows:
13.如实施方案12所述的方法,其中所述IN10018其药学上可接受的盐是IN10018酒石酸盐。13. The method according to embodiment 12, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.
14.如实施方案12-13任一项所述的方法,其中所述IN10018或其药学上可接受的盐和所述环磷酰胺被同时或依次施用于所述对象。14. The method of any one of embodiments 12-13, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the cyclophosphamide are administered to the subject simultaneously or sequentially.
15.如实施方案12-14任一项所述的方法,其中所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。15. A method as described in any one of embodiments 12-14, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland cancer The invention relates to a method for treating a thyroid cancer of the present invention and comprising the following steps: the method comprises: treating a thyroid cancer of the present invention and/or treating a thyroid cancer of the present invention; ...
16.IN10018或其药学上可接受的盐在制备用于在对象中治疗肿瘤的药物中的用途,其中将所述IN10018或其药学上可接受的盐和环磷酰胺施用于所述对象,所述IN10018结构如下:
16. Use of IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a tumor in a subject, wherein the IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide are administered to the subject, and the structure of IN10018 is as follows:
16. Use of IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a tumor in a subject, wherein the IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide are administered to the subject, and the structure of IN10018 is as follows:
17.如实施方案16所述的用途,其中所述IN10018药学上可接受的盐是IN10018酒石酸盐。17. The use according to embodiment 16, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.
18.如实施方案16-17任一项所述的用途,其中所述IN10018或其药学上可接受的盐和所述环磷酰胺被同时或依次施用于所述对象。18. The use according to any one of embodiments 16-17, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the cyclophosphamide are administered to the subject simultaneously or sequentially.
19.如实施方案16-18任一项所述的用途,其中所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤,神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。19. The use as described in any one of embodiments 16-18, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland The invention relates to a method for treating a thyroid cancer of the present invention and comprising the following steps: the method comprises: treating a thyroid cancer of the present invention and/or treating a thyroid cancer of the present invention; ...
20.环磷酰胺在制备用于在对象中治疗肿瘤的药物中的用途,其中将所述环磷酰胺和IN10018或其药学上可接受的盐施用于所述对象,所述IN10018结构如下:
20. Use of cyclophosphamide in the preparation of a medicament for treating a tumor in a subject, wherein the cyclophosphamide and IN10018 or a pharmaceutically acceptable salt thereof are administered to the subject, wherein the structure of IN10018 is as follows:
20. Use of cyclophosphamide in the preparation of a medicament for treating a tumor in a subject, wherein the cyclophosphamide and IN10018 or a pharmaceutically acceptable salt thereof are administered to the subject, wherein the structure of IN10018 is as follows:
21.如实施方案20所述的用途,其中所述IN10018其药学上可接受的盐是IN10018酒石酸盐。21. The use according to embodiment 20, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.
22.如实施方案20-21任一项所述的用途,其中所述IN10018或其药学上可接受的盐和所述环磷酰胺被同时或依次施用于所述对象。22. The use according to any one of embodiments 20-21, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the cyclophosphamide are administered to the subject simultaneously or sequentially.
23.如实施方案20-22任一项所述的用途,其中所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤,神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。23. The use as described in any one of embodiments 20-22, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland The invention relates to a method for treating a thyroid cancer of the present invention and comprising the following steps: the method comprises: treating a thyroid cancer of the present invention and/or treating a thyroid cancer of the present invention; ...
24.IN10018或其药学上可接受的盐和环磷酰胺在制备用于联合治疗肿瘤的药物中的用途;所述IN10018结构如下:
24. Use of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in the preparation of a medicament for combined treatment of tumors; the structure of IN10018 is as follows:
24. Use of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in the preparation of a medicament for combined treatment of tumors; the structure of IN10018 is as follows:
25.IN10018或其药学上可接受的盐在制备用于与环磷酰胺联合治疗肿瘤的药物中的用途;所述IN10018结构如下:
25. Use of IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors in combination with cyclophosphamide; the structure of IN10018 is as follows:
25. Use of IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors in combination with cyclophosphamide; the structure of IN10018 is as follows:
26.环磷酰胺在制备用于与IN10018或其药学上可接受的盐联合治疗肿瘤的药物中的用途;所述IN10018结构如下:
26. Use of cyclophosphamide in the preparation of a medicament for treating tumors in combination with IN10018 or a pharmaceutically acceptable salt thereof; the structure of IN10018 is as follows:
26. Use of cyclophosphamide in the preparation of a medicament for treating tumors in combination with IN10018 or a pharmaceutically acceptable salt thereof; the structure of IN10018 is as follows:
27.如实施方案24-26任一项所述的用途,其中所述IN10018或其药学上可接受的盐是IN10018酒石酸盐。27. The use according to any one of embodiments 24-26, wherein the IN10018 or a pharmaceutically acceptable salt thereof is IN10018 tartrate.
28.如实施方案24-27任一项所述的用途,其中所述IN10018或其药学上可接受的盐和所述环磷酰胺被同时或依次施用于所述对象。28. The use according to any one of embodiments 24-27, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the cyclophosphamide are administered to the subject simultaneously or sequentially.
29.如实施方案24-28任一项所述的用途,其中所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤,神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。29. The use as described in any one of embodiments 24-28, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland The invention relates to a method for treating a thyroid cancer of the present invention and comprising the following steps: the method comprises: treating a thyroid cancer of the present invention and/or treating a thyroid cancer of the present invention; ...
实施例Example
提供下面的实施例以进一步阐述本发明。应理解,这些实施例仅用于举例说明本发明,而不用于限制本发明的范围。The following examples are provided to further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
下列实施例中未注明具体条件的实验方法均可以按照这类反应的常规条件进行或者按照制造厂商所建议的条件进行。The experimental methods in the following examples without specifying specific conditions can be carried out according to conventional conditions of such reactions or according to the conditions recommended by the manufacturers.
以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.
实施例中所用的缩写含义如下:
The abbreviations used in the examples have the following meanings:
The abbreviations used in the examples have the following meanings:
实施例1:IN10018和环磷酰胺在乳腺癌4T1细胞中BALB/c小鼠皮下同种移植瘤模型中的体内抗肿瘤药效研究其抗肿瘤作用后肿瘤浸润淋巴细胞(TILs)的FACS检测分析Example 1: In vivo antitumor efficacy study of IN10018 and cyclophosphamide in a BALB/c mouse subcutaneous homograft tumor model in breast cancer 4T1 cells FACS analysis of tumor infiltrating lymphocytes (TILs) after antitumor effects
实验材料:Experimental Materials:
小鼠:6-7周龄的雌性BALB/c小鼠购自上海灵畅生物科技有限公司。动物到达后在实验环境适应性饲养后开始实验。动物在SPF级动物房以IVC(独立送风系统)笼具饲养(每笼5只)。所有笼具、垫料及饮水在使用前均需灭菌。所有实验人员在动物房操作时应穿着防护服和乳胶手套。笼具、饲料及饮水每周更换两次。饲养环境及光照情况如下:Mice: Female BALB/c mice aged 6-7 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The experiment began after the animals were adapted to the experimental environment upon arrival. The animals were kept in IVC (independent ventilation system) cages (5 per cage) in an SPF animal room. All cages, bedding and drinking water must be sterilized before use. All experimental personnel should wear protective clothing and latex gloves when operating in the animal room. Cages, feed and drinking water were changed twice a week. The breeding environment and lighting conditions are as follows:
温度:20-26℃
Temperature: 20-26℃
湿度:40-70%Humidity: 40-70%
光照周期:12小时光照,12小时无光照Photoperiod: 12 hours light, 12 hours no light
笼具:以聚碳酸酯制成,体积300mm×180mm×150mm。垫料为玉米芯,每周更换两次。Cage: Made of polycarbonate, 300mm×180mm×150mm. Bedding is corn cobs, changed twice a week.
食物:实验动物在整个实验阶段中可自由进食(辐照灭菌,干颗粒状食物)。Food: The experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.
饮水:实验动物可自由饮用灭菌水。Drinking water: Experimental animals were free to drink sterile water.
笼具标识:每笼动物信息卡应注明笼内动物数目,性别,品系,接收日期,给药方案,实验编号,组别以及实验开始日期。Cage identification: The animal information card for each cage should indicate the number of animals in the cage, gender, strain, receipt date, dosing regimen, experiment number, group and start date of the experiment.
动物标识:实验动物以耳标进行标识。Animal identification: Experimental animals were identified with ear tags.
供试品信息见表1。
The information of the test products is shown in Table 1.
The information of the test products is shown in Table 1.
原辅料信息见表2。The information of raw materials and auxiliary materials is shown in Table 2.
表2:
Table 2:
Table 2:
FACS使用抗体信息见表3。The information of antibodies used for FACS is shown in Table 3.
表3:
table 3:
table 3:
关键仪器设备信息见表4。The information of key instruments and equipment is shown in Table 4.
表4:
Table 4:
Table 4:
细胞培养Cell culture
乳腺癌细胞4T1(来源南京科佰生物科技有限公司,货号:CBP60352)由应世生物科技(南京)有限公司维持传代。细胞体外单层培养,培养条件为RPMI-1640培养基中加10%胎牛血清,37℃,5%CO2培养箱中培养。一周两到三次用胰酶-EDTA进行常规消化处理传代。当细胞处于指数生长期,汇合度为80%-90%时,收取细胞,计数后接种。Breast cancer cell 4T1 (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60352) was maintained and subcultured by Yingshi Biotechnology (Nanjing) Co., Ltd. The cells were cultured in a monolayer in vitro, and the culture conditions were RPMI-1640 medium with 10% fetal bovine serum, cultured in a 37°C, 5% CO2 incubator. Two to three times a week, routine digestion and subculture were performed with trypsin-EDTA. When the cells were in the exponential growth phase and the confluence was 80%-90%, the cells were harvested, counted, and inoculated.
细胞接种,分组及FACS检测
Cell seeding, grouping and FACS detection
将0.1mL含有2×105个细胞的细胞悬液皮下接种于每只小鼠的右后背。当肿瘤体积达到~51mm3左右时(细胞接种后的第7天),根据肿瘤体积进行随机分组并给药,分组信息见表5。0.1 mL of cell suspension containing 2×10 5 cells was subcutaneously inoculated on the right back of each mouse. When the tumor volume reached about 51 mm 3 (7 days after cell inoculation), the mice were randomly divided into groups according to the tumor volume and given drugs. The grouping information is shown in Table 5.
表5
注:
1.N:每组小鼠数目;
2.给药体积:根据小鼠体重10mL/kg,如果体重下降超过15%,动物停止给药;待
体重恢复至-10%以内,再恢复给药。table 5
Note:
1. N: number of mice in each group;
2. Dosing volume: 10mL/kg based on mouse body weight. If the body weight drops by more than 15%, stop dosing the animal; resume dosing when the body weight recovers to within -10%.
注:
1.N:每组小鼠数目;
2.给药体积:根据小鼠体重10mL/kg,如果体重下降超过15%,动物停止给药;待
体重恢复至-10%以内,再恢复给药。table 5
Note:
1. N: number of mice in each group;
2. Dosing volume: 10mL/kg based on mouse body weight. If the body weight drops by more than 15%, stop dosing the animal; resume dosing when the body weight recovers to within -10%.
FACS检测Panel 1设定见表6。FACS detection Panel 1 settings are shown in Table 6.
表6:
Table 6:
Table 6:
FACS检测Panel 2设定见表7。
FACS detection Panel 2 settings are shown in Table 7.
表7:
Table 7:
Table 7:
受试物的配制Preparation of test substance
详见表8。See Table 8 for details.
表8:受试物配制方法
Table 8: Preparation of test substances
Table 8: Preparation of test substances
实验动物日常观察Daily observation of experimental animals
本实验方案的拟定及任何修改均通过了云桥生物IACUC的评估核准。实验动物的使用及福利遵照AAALAC的规定执行。每天监测动物的健康状况及死亡情况,例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动,摄食摄水量(仅目测),体重变化,外观体征或其它不正常情况。基于各组动物数量记录了组内动物死亡数和副作用。
The formulation of this experimental plan and any modification have been evaluated and approved by Yunqiao Biotechnology's IACUC. The use and welfare of experimental animals are carried out in accordance with AAALAC regulations. The health status and mortality of animals are monitored daily. Routine examinations include observing the effects of tumor growth and drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake (visual inspection only), weight changes, physical signs or other abnormal conditions. The number of deaths and side effects of animals in each group were recorded based on the number of animals in each group.
实验终止Experiment termination
若动物健康状况持续恶化,或瘤体积超过3,000mm3,或有严重疾病,或疼痛,须处以安乐死。有以下情况者,通知兽医并处以安乐死:明显消瘦,体重降低大于20%;不能自由取食和饮水;对照组瘤体积平均值达到3,000mm3,实验终止。动物出现以下临床表现且持续恶化:立毛,弓背,耳、鼻、眼或足色发白,呼吸仓促,抽搐,连续腹泻,脱水,行动迟缓,发声。If the health of the animal continues to deteriorate, or the tumor volume exceeds 3,000mm 3 , or there is a serious disease or pain, euthanasia must be carried out. In the following cases, notify the veterinarian and euthanize: obvious weight loss, weight loss greater than 20%; cannot freely eat and drink water; the average tumor volume of the control group reaches 3,000mm 3 , and the experiment is terminated. Animals with the following clinical manifestations and continue to deteriorate: piloerection, arched back, pale ears, nose, eyes or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.
肿瘤测量和实验指标Tumor measurements and experimental parameters
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。用游标卡尺测量肿瘤直径,每周测量3次。肿瘤体积的计算公式为:V=0.5×a×b2,a和b分别表示肿瘤的长径和短径。The experimental index is to examine whether the tumor growth is inhibited, delayed or cured. The tumor diameter is measured with a vernier caliper three times a week. The calculation formula of tumor volume is: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor respectively.
化合物的抑瘤疗效用TGI(%)评价,反映肿瘤生长抑制率。参照分组后第一天的肿瘤体积,根据以下公式计算肿瘤生长抑制率TGI(%)。TGI(%)=[1-(某给药组的平均瘤体积-该给药组开始时平均瘤体积)/(溶剂对照组的平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]×100%。The anti-tumor efficacy of the compound was evaluated by TGI (%), which reflects the tumor growth inhibition rate. With reference to the tumor volume on the first day after grouping, the tumor growth inhibition rate TGI (%) was calculated according to the following formula: TGI (%) = [1-(average tumor volume of a certain medication group-average tumor volume at the beginning of the medication group)/(average tumor volume of the solvent control group-average tumor volume at the beginning of the solvent control group)] × 100%.
肿瘤细胞解离Tumor cell dissociation
将合适大小的肿瘤(约0.5g)剪成小块,加入美天旎消化试剂盒(2.35ml1640、100μl酶D、10μl酶R、12.5μL酶A),37℃,200rpm,孵育1h。随后4℃离心350g,5min,弃上清,加入适量D-PBS重悬细胞,70μm无菌过滤器过滤;视情况,加入3ml ACK Lysing Buffer(红细胞裂解液),室温孵育5min。加入9ml D-PBS终止反应,混匀,4℃离心350g,5min,弃上清。加入适量D-PBS重悬细胞,并进行细胞计数,取约2x 106每管的细胞至流式管中,进入之后的染色步骤。Cut the tumor of appropriate size (about 0.5g) into small pieces, add Miltenyi digestion kit (2.35ml1640, 100μl enzyme D, 10μl enzyme R, 12.5μL enzyme A), incubate at 37℃, 200rpm for 1h. Then centrifuge at 4℃ for 350g for 5min, discard the supernatant, add appropriate amount of D-PBS to resuspend the cells, filter with 70μm sterile filter; add 3ml ACK Lysing Buffer (red blood cell lysis buffer) as needed, incubate at room temperature for 5min. Add 9ml D-PBS to terminate the reaction, mix well, centrifuge at 4℃ for 350g for 5min, and discard the supernatant. Add appropriate amount of D-PBS to resuspend the cells, count the cells, take about 2x 106 cells per tube into the flow tube, and enter the subsequent staining step.
FACS染色FACS staining
取2M细胞悬液至流式管中,并准备好相应的non-staining、单染管、FMO管;Take 2M cell suspension into flow cytometry tubes and prepare corresponding non-staining tubes, single staining tubes, and FMO tubes;
DPBS洗2次,350g,4℃离心5min;Wash twice with DPBS and centrifuge at 350 g for 5 min at 4 °C;
进行死活染色(1:1000DPBS稀释),室温避光孵育15min;Perform live or dead staining (1:1000 dilution in DPBS) and incubate at room temperature in the dark for 15 min;
Staining buffer洗2次,350g,4℃离心5min;Wash twice with staining buffer, centrifuge at 350g, 4℃ for 5min;
FcR Blocking:加入50μl anti-mouse CD16/CD32进行封闭(Staining buffer 1:50稀释),4℃避光孵育15min;FcR Blocking: Add 50 μl anti-mouse CD16/CD32 for blocking (Staining buffer 1:50 dilution), incubate at 4°C in the dark for 15 min;
加入50μL表面抗体(Staining buffer 1:50稀释),4℃避光孵育30min;Add 50 μL surface antibody (Staining buffer 1:50 dilution) and incubate at 4°C in the dark for 30 min.
Staining buffer洗2次,350g,4℃离心5min;Wash twice with staining buffer, centrifuge at 350g, 4℃ for 5min;
Panel 1细胞重悬于固定破核膜液,室温避光45-60min。Panel 2细胞重悬于固定液,4℃避光30min;Panel 1: Resuspend cells in fixative and nuclear membrane disrupting solution and incubate at room temperature in the dark for 45-60 min. Panel 2: Resuspend cells in fixative and incubate at 4°C in the dark for 30 min.
固定破膜后,Panel 1用True-NuclearTM 1X Perm Buffer洗两遍,Panel 2用Staining buffer洗两遍后重悬于Staining buffer,400g离心5min;After fixation and membrane permeabilization, Panel 1 was washed twice with True-Nuclear TM 1X Perm Buffer, and Panel 2 was washed twice with Staining buffer and then resuspended in Staining buffer and centrifuged at 400 g for 5 min;
Panel 1加入核内抗体(True-NuclearTM 1X Perm Buffer 1:100稀释),4℃避光孵育30min;Panel 1: nuclear antibody (1:100 dilution in True-Nuclear TM 1X Perm Buffer) was added and incubated at 4°C in the dark for 30 min;
Panel 1用True-NuclearTM 1X Perm Buffer洗两遍,400g离心5min;Panel 1 was washed twice with True-Nuclear TM 1X Perm Buffer and centrifuged at 400 g for 5 min;
加入适量的Staining buffer重悬细胞,流式上机检测。Add appropriate amount of Staining buffer to resuspend the cells and detect on flow cytometry.
FACS数据分析FACS data analysis
所有经流式细胞仪产生的数据用FlowJo软件分析。All data generated by flow cytometry were analyzed using FlowJo software.
统计分析Statistical Analysis
统计分析基于试验结束时肿瘤体积运用Prism Graphpad软件进行分析。多组间比较,采用Two-way ANOVA,Fisher’s LSD test法检验进行分析。P<0.05认为有显著性差异。Statistical analysis was performed using Prism Graphpad software based on the tumor volume at the end of the experiment. Two-way ANOVA and Fisher’s LSD test were used for comparison among multiple groups. P<0.05 was considered to be significantly different.
流式数据的统计学分析,为比较不同处理组的肿瘤浸润淋巴细胞亚群,运用Prism Graphpad软件进行分析。多组间比较,采用One-way ANOVA,Kruskal Wallis检验法进行分析。P<0.05认为有显著性差异。
Statistical analysis of flow cytometry data was performed using Prism Graphpad software to compare tumor infiltrating lymphocyte subsets in different treatment groups. One-way ANOVA and Kruskal Wallis test were used for comparison among multiple groups. P < 0.05 was considered to be significantly different.
实验结果Experimental Results
受试物CTX和IN10018的配伍治疗在乳腺癌4T1细胞BALB/c小鼠皮下同种移植瘤模型中的体内药效研究In vivo efficacy study of the combination of CTX and IN10018 in a subcutaneous BALB/c mouse breast cancer 4T1 cell transplant model
细胞接种后,每天观察肿瘤生长情况,接种后第7天根据肿瘤体积进行分组,入组平均肿瘤体积约为51mm3。由于FACS检测实验需要,所有组均在接种后的第20天,即分组给药之后的第13天安乐死,整个实验结束。分组给药后第13天,对照组的肿瘤体积为857.9±204.2mm3。CTX(10mg/kg)和IN10018(25mg/kg)单药组的肿瘤体积分别为759.5±73.6mm3和626.0±108.9mm3;CTX+IN10018(10+25mg/kg)联用组的肿瘤体积为319.3±86.3mm3。综合肿瘤体积与对照组进行比较,CTX(10mg/kg)和IN10018(25mg/kg)单药组的抑瘤率TGI分别为12.3%(p=0.0469)和28.8%(p<0.0001);CTX+IN10018(10+25mg/kg)联用组的TGI为66.8%(p<0.0001)。综合瘤体积与CTX+IN10018(10+25mg/kg)联用组相比较,进行统计学分析,对照组、CTX(10mg/kg)和IN10018(25mg/kg)单药组的P值分别为p<0.0001,p<0.0001和p<0.0001。各组的抑瘤效果评价详见表9,各剂量组在不同时间段的肿瘤体积如图1所示。After cell inoculation, tumor growth was observed every day. Groups were divided according to tumor volume on the 7th day after inoculation, and the average tumor volume was about 51 mm 3 . Due to the need for FACS detection experiments, all groups were euthanized on the 20th day after inoculation, that is, on the 13th day after group administration, and the entire experiment was terminated. On the 13th day after group administration, the tumor volume of the control group was 857.9±204.2 mm 3 . The tumor volumes of the CTX (10 mg/kg) and IN10018 (25 mg/kg) single-drug groups were 759.5±73.6 mm 3 and 626.0±108.9 mm 3 , respectively; the tumor volume of the CTX+IN10018 (10+25 mg/kg) combination group was 319.3±86.3 mm 3 . The comprehensive tumor volume was compared with the control group. The tumor inhibition rates TGI of CTX (10 mg/kg) and IN10018 (25 mg/kg) monotherapy groups were 12.3% (p=0.0469) and 28.8% (p<0.0001), respectively; the TGI of CTX+IN10018 (10+25 mg/kg) combination group was 66.8% (p<0.0001). The comprehensive tumor volume was compared with the CTX+IN10018 (10+25 mg/kg) combination group, and statistical analysis was performed. The P values of the control group, CTX (10 mg/kg) and IN10018 (25 mg/kg) monotherapy groups were p<0.0001, p<0.0001 and p<0.0001, respectively. The evaluation of the tumor inhibition effect of each group is shown in Table 9, and the tumor volume of each dose group at different time periods is shown in Figure 1.
表9:受试物对小鼠乳腺癌4T1细胞的BALB/c小鼠移植瘤模型的抑瘤效果评价(基于分组给药后第13天数据)
注:
1.按照分组给药后的天数来计算,数据为平均值±标准差(mean±SD);
2.TGI(%)=[1-(T13-T0)/(V13-V0)]×100%;
3.*:p<0.05,****:p<0.0001,vs.对照组,Two-way ANOVA;
4.****:p<0.0001,vs.CTX+IN10018(10+25mg/kg)组,Two-way ANOVA;Table 9: Evaluation of the tumor inhibition effect of the test substance on the BALB/c mouse transplanted tumor model of mouse breast cancer 4T1 cells (based on the data on the 13th day after group administration)
Note:
1. Calculated according to the number of days after group administration, the data are mean ± standard deviation (mean ± SD);
2. TGI (%) = [1 - (T 13 - T 0 ) / (V 13 - V 0 )] × 100%;
3.*: p<0.05, ****: p<0.0001, vs. control group, Two-way ANOVA;
4. ****: p<0.0001, vs. CTX+IN10018 (10+25 mg/kg) group, Two-way ANOVA;
注:
1.按照分组给药后的天数来计算,数据为平均值±标准差(mean±SD);
2.TGI(%)=[1-(T13-T0)/(V13-V0)]×100%;
3.*:p<0.05,****:p<0.0001,vs.对照组,Two-way ANOVA;
4.****:p<0.0001,vs.CTX+IN10018(10+25mg/kg)组,Two-way ANOVA;Table 9: Evaluation of the tumor inhibition effect of the test substance on the BALB/c mouse transplanted tumor model of mouse breast cancer 4T1 cells (based on the data on the 13th day after group administration)
Note:
1. Calculated according to the number of days after group administration, the data are mean ± standard deviation (mean ± SD);
2. TGI (%) = [1 - (T 13 - T 0 ) / (V 13 - V 0 )] × 100%;
3.*: p<0.05, ****: p<0.0001, vs. control group, Two-way ANOVA;
4. ****: p<0.0001, vs. CTX+IN10018 (10+25 mg/kg) group, Two-way ANOVA;
受试物CTX和IN10018的配伍治疗在乳腺癌4T1细胞BALB/c小鼠皮下同种移植瘤模型中的体重变化及临床状况研究Study on the changes in body weight and clinical status of the combined treatment of the test substances CTX and IN10018 in the subcutaneous homograft tumor model of breast cancer 4T1 cells in BALB/c mice
实验按照给药方案进行,实验过程中,每天观察动物摄食饮水等活动,每周记录3次动物体重。分组给药13天之后,对照组的平均体重从分组给药当天(Day0)的21.4g,变化至Day13的23.8g,体重增长率为11.5%;CTX(10mg/kg)和IN10018(25mg/kg)单药组的平均体重分别从Day0的21.9g,21.7g变化至Day13的23.0g和21.7g,其体重变化率分别为4.8%和-0.2%;CTX+IN10018(10+25mg/kg)联用组两药联用组的平均体重从Day0的21.7g变化至Day13的21.9g,体重变化率为0.8%。在整个给药周期中,各组组动物体重均无明显下降且状态良好。各组体重数据见表10。各剂量组在不同时间段的体重变化如图2所示。The experiment was carried out according to the dosing regimen. During the experiment, the animals' activities such as eating and drinking were observed every day, and the weight of the animals was recorded 3 times a week. After 13 days of group administration, the average weight of the control group changed from 21.4g on the day of group administration (Day0) to 23.8g on Day13, with a weight growth rate of 11.5%; the average weight of the CTX (10mg/kg) and IN10018 (25mg/kg) single drug groups changed from 21.9g and 21.7g on Day0 to 23.0g and 21.7g on Day13, respectively, with a weight change rate of 4.8% and -0.2%, respectively; the average weight of the CTX+IN10018 (10+25mg/kg) combination group changed from 21.7g on Day0 to 21.9g on Day13, with a weight change rate of 0.8%. During the entire dosing cycle, the weight of the animals in each group did not decrease significantly and was in good condition. The weight data of each group are shown in Table 10. The changes in body weight of each dosage group at different time periods are shown in FIG2 .
表10:受试物对小鼠乳腺癌4T1细胞的BALB/c小鼠移植瘤模型的体重变化评价(基于分组给药后第13天数据)
注:
1.按照分组给药后的天数来计算,第0天的动物存活数目/第21天的动物存活数目;
2.数据为平均值±标准差(mean±SD);
3.体重变化率=[(W13-W0)/W0]*100%;Table 10: Evaluation of the test substance on the body weight change of the BALB/c mouse transplanted tumor model of mouse breast cancer 4T1 cells (based on the data on the 13th day after group administration)
Note:
1. Calculated according to the number of days after group administration: the number of animals surviving on day 0/the number of animals surviving on day 21;
2. Data are mean ± standard deviation (mean ± SD);
3. Weight change rate = [(W 13 -W 0 )/W 0 ]*100%;
注:
1.按照分组给药后的天数来计算,第0天的动物存活数目/第21天的动物存活数目;
2.数据为平均值±标准差(mean±SD);
3.体重变化率=[(W13-W0)/W0]*100%;Table 10: Evaluation of the test substance on the body weight change of the BALB/c mouse transplanted tumor model of mouse breast cancer 4T1 cells (based on the data on the 13th day after group administration)
Note:
1. Calculated according to the number of days after group administration: the number of animals surviving on day 0/the number of animals surviving on day 21;
2. Data are mean ± standard deviation (mean ± SD);
3. Weight change rate = [(W 13 -W 0 )/W 0 ]*100%;
受试物CTX和IN10018的配伍治疗在乳腺癌4T1细胞BALB/c小鼠皮下同种移植瘤模型中治疗后的FACS数据研究FACS data study of the combined treatment of the test substances CTX and IN10018 in the subcutaneous homograft tumor model of breast cancer 4T1 cells in BALB/c mice
所有动物在分组给药13天时,均全部安乐死,剥离肿瘤,并按照实验方案使用流式细胞仪收集实验数据,并在数据收集完成后,使用Flowjo软件进行数据分析。All animals were euthanized on day 13 of group administration, and their tumors were removed. Flow cytometry was used to collect experimental data according to the experimental protocol. After data collection was completed, Flowjo software was used for data analysis.
调节性T细胞(Treg)占比的圈门策略为活的CD45+淋巴细胞中的CD3+T细胞中的CD4+T细胞中的CD25和FoxP3双阳性细胞所占CD4+T细胞的百分比。对照组的Treg细胞在CD4+T细胞的百分比为8.5%;CTX(10mg/kg)和IN10018(25mg/kg)单药组的Treg细胞在CD4+T细胞的百分比分别为7.0%和9.2%;CTX+IN10018(10+25mg/kg)两药联用组的Treg细胞在CD4+T细胞的百分比为3.4%。综合Treg细胞在CD4+T细胞的百分比与对照组进行比较,进行统计学分析,CTX(10mg/kg)和IN10018(25mg/kg)单药组的P值分别为p=0.8726和p=0.7083;CTX+IN10018(10+25mg/kg)两药联用组的P值为p=0.1646。The gating strategy for the proportion of regulatory T cells (Treg) is the percentage of CD25 and FoxP3 double-positive cells in CD4+T cells among CD3+T cells in live CD45+ lymphocytes. The percentage of Treg cells in CD4+T cells in the control group was 8.5%; the percentage of Treg cells in CD4+T cells in the CTX (10mg/kg) and IN10018 (25mg/kg) single-drug groups was 7.0% and 9.2%, respectively; the percentage of Treg cells in CD4+T cells in the CTX+IN10018 (10+25mg/kg) combination group was 3.4%. The percentage of comprehensive Treg cells in CD4+T cells was compared with that of the control group for statistical analysis. The P values of CTX (10 mg/kg) and IN10018 (25 mg/kg) single-drug groups were p=0.8726 and p=0.7083, respectively; the P value of CTX+IN10018 (10+25 mg/kg) combination group was p=0.1646.
肿瘤细胞上PD-L1的表达水平的圈门策略为活的CD45-肿瘤细胞群中看PD-L1的表达。对照组的肿瘤细胞PD-L1的表达水平为17.7%;CTX(10mg/kg)和IN10018(25mg/kg)单药组的肿瘤细胞PD-L1的表达水平分别为9.0%和5.2%;CTX+IN10018(10+25mg/kg)两药联用组的肿瘤细胞PD-L1的表达水平为6.5%。综合肿瘤细胞PD-L1的表达水平与对照组进行比较,进行统计学分析,CTX(10mg/kg)和IN10018(25mg/kg)单药组的P值分别为p=0.0692和p=0.0013;CTX+IN10018(10+25mg/kg)两药联用组的P值为p=0.0054。The gating strategy for the expression level of PD-L1 on tumor cells is to look at the expression of PD-L1 in the living CD45- tumor cell population. The expression level of PD-L1 in tumor cells of the control group was 17.7%; the expression levels of PD-L1 in tumor cells of the CTX (10 mg/kg) and IN10018 (25 mg/kg) monotherapy groups were 9.0% and 5.2%, respectively; the expression level of PD-L1 in tumor cells of the CTX+IN10018 (10+25 mg/kg) combination group was 6.5%. The expression level of PD-L1 in tumor cells was compared with that of the control group for statistical analysis. The P values of the CTX (10 mg/kg) and IN10018 (25 mg/kg) monotherapy groups were p=0.0692 and p=0.0013, respectively; the P value of the CTX+IN10018 (10+25 mg/kg) combination group was p=0.0054.
CD11b+CD11c+双阳DC细胞的圈门策略为活的CD45+总淋巴细胞中CD11b+CD11c+DC细胞所占CD45+总淋巴细胞的比例。对照组的CD11b+CD11c+DC细胞所占比例为5.2%;CTX(10mg/kg)和IN10018(25mg/kg)单药组的CD11b+CD11c+DC细胞所占比例分别为8.9%和4.9%;CTX+IN10018(10+25mg/kg)两药联用组的CD11b+CD11c+DC细胞所占比例为7.6%。综合CD11b+CD11c+DC细胞所占CD45+总淋巴细胞的比例与对照组进行比较,进行统计学分析,CTX(10mg/kg)和IN10018(25mg/kg)单药组的P值分别为p=0.1995和p=0.9149;CTX+IN10018(10+25mg/kg)两药联用组的P值为p=0.3924。The gating strategy for CD11b+CD11c+ double positive DC cells is the ratio of CD11b+CD11c+DC cells to the total CD45+ lymphocytes in the live CD45+ lymphocytes. The proportion of CD11b+CD11c+DC cells in the control group was 5.2%; the proportion of CD11b+CD11c+DC cells in the CTX (10mg/kg) and IN10018 (25mg/kg) single drug groups was 8.9% and 4.9%, respectively; the proportion of CD11b+CD11c+DC cells in the CTX+IN10018 (10+25mg/kg) combination group was 7.6%. The proportion of CD11b+CD11c+DC cells in the total CD45+ lymphocytes was compared with that in the control group for statistical analysis. The P values of the CTX (10 mg/kg) and IN10018 (25 mg/kg) single-drug groups were p=0.1995 and p=0.9149, respectively; the P value of the CTX+IN10018 (10+25 mg/kg) combination group was p=0.3924.
CD86和MHC II在CD11b+CD11c+DC细胞中的占比的圈门策略为活的CD45+淋巴细胞中CD11b+CD11c+DC细胞的CD86和MHC II的表达水平。对照组的CD86和MHC II的表达水平分别为24.2%和59.1%;CTX(10mg/kg)单药组的CD86和MHC II的表达水平分别为41.3%和72.6%;IN10018(25mg/kg)单药组的CD86和MHC II的表达水平分别为34.0%和67.5%;CTX+IN10018(10+25mg/kg)两药联用组的CD86和MHC II的表达水平分别为52.8%和80.2%。综合CD86的表达水平与对照组进行比较,进行统计学分析,CTX(10mg/kg)和IN10018(25mg/kg)单药组的P值分别为p=0.0303和p=0.1992;CTX+IN10018(10+25mg/kg)两药联用组的P值为p=0.0005。综合MHC II的表达水平与对照组进行比较,进行统计学分析,CTX(10mg/kg)和IN10018(25mg/kg)单药组的P值分别为p=0.0692和p=0.2189;CTX+IN10018(10+25mg/kg)两药联用组的P值为p=0.0046。The gating strategy for the proportion of CD86 and MHC II in CD11b+CD11c+DC cells is the expression level of CD86 and MHC II in CD11b+CD11c+DC cells in live CD45+ lymphocytes. The expression levels of CD86 and MHC II in the control group were 24.2% and 59.1%, respectively; the expression levels of CD86 and MHC II in the CTX (10mg/kg) monotherapy group were 41.3% and 72.6%, respectively; the expression levels of CD86 and MHC II in the IN10018 (25mg/kg) monotherapy group were 34.0% and 67.5%, respectively; the expression levels of CD86 and MHC II in the CTX+IN10018 (10+25mg/kg) combination group were 52.8% and 80.2%, respectively. The expression level of CD86 was compared with that of the control group, and statistical analysis was performed. The P values of the CTX (10 mg/kg) and IN10018 (25 mg/kg) single-drug groups were p=0.0303 and p=0.1992, respectively; the P value of the CTX+IN10018 (10+25 mg/kg) combination group was p=0.0005. The expression level of MHC II was compared with that of the control group, and statistical analysis was performed. The P values of the CTX (10 mg/kg) and IN10018 (25 mg/kg) single-drug groups were p=0.0692 and p=0.2189, respectively; the P value of the CTX+IN10018 (10+25 mg/kg) combination group was p=0.0046.
各组的Treg和CD11b+CD11c+DC细胞所占群的比例以及PD-L1、CD86和MHC II的表达水平详见表11及图3、图4、图5、图6和图7。The proportion of Treg and CD11b+CD11c+DC cells in each group and the expression levels of PD-L1, CD86 and MHC II are shown in Table 11 and Figures 3, 4, 5, 6 and 7.
表11:受试物对小鼠乳腺癌4T1细胞的BALB/c小鼠移植瘤模型治疗后的FACS数据
注:
1.数据为平均值±标准差(mean±SD)
2.*:p<0.05,**:p<0.01,***:p<0.001,vs.对照组,One-way ANOVA,Kruskal Wallis test;Table 11: FACS data of the test substance after treatment of BALB/c mouse transplanted tumor model of mouse breast cancer 4T1 cells
Note:
1. Data are mean ± standard deviation (mean ± SD)
2. *: p<0.05, **: p<0.01, ***: p<0.001, vs. control group, One-way ANOVA, Kruskal Wallis test;
注:
1.数据为平均值±标准差(mean±SD)
2.*:p<0.05,**:p<0.01,***:p<0.001,vs.对照组,One-way ANOVA,Kruskal Wallis test;Table 11: FACS data of the test substance after treatment of BALB/c mouse transplanted tumor model of mouse breast cancer 4T1 cells
Note:
1. Data are mean ± standard deviation (mean ± SD)
2. *: p<0.05, **: p<0.01, ***: p<0.001, vs. control group, One-way ANOVA, Kruskal Wallis test;
实验结论Experimental results
在本实验中,我们评价了受试物CTX和IN10018的配伍治疗在4T1小鼠乳腺癌细胞BALB/c小鼠皮下同种移植瘤模型中的体内药效。与空白对照组相比,CTX(10mg/kg)、IN10018(25mg/kg)单药治疗组和CTX+IN10018(10+25mg/kg)两药联用组均有明显肿瘤生长抑制作用,和对照组相比都有统计学差异。综合整个给药周期,CTX+IN10018(10+25mg/kg)两药联用组的肿瘤体积相对于其余各单药治疗组的肿瘤体积一直都更小,而且和CTX(10mg/kg)以及IN10018(25mg/kg)单药治疗组相比均具有统计学差异。以上数据均显示出CTX(10mg/kg)和IN10018(25mg/kg)两药联用相对于单药具有更好的药效,CTX+IN10018(10+25mg/kg)的配伍联合给药治疗具有更好的抑制肿瘤生长的效果。且该实验环磷酰胺通过口服给药,用量也有所降低,解决用量大,副作用大的问题。In this experiment, we evaluated the in vivo efficacy of the combined treatment of the test substances CTX and IN10018 in the BALB/c mouse subcutaneous homograft tumor model of 4T1 mouse breast cancer cells. Compared with the blank control group, the CTX (10 mg/kg), IN10018 (25 mg/kg) monotherapy group and the CTX + IN10018 (10 + 25 mg/kg) combination group all had significant tumor growth inhibition effects, and there were statistical differences compared with the control group. Comprehensively considering the entire dosing cycle, the tumor volume of the CTX + IN10018 (10 + 25 mg/kg) combination group was always smaller than the tumor volume of the other single-drug treatment groups, and there were statistical differences compared with the CTX (10 mg/kg) and IN10018 (25 mg/kg) monotherapy groups. The above data all show that the combination of CTX (10mg/kg) and IN10018 (25mg/kg) has better efficacy than single drugs, and the combination of CTX+IN10018 (10+25mg/kg) has a better effect in inhibiting tumor growth. In addition, the cyclophosphamide in this experiment is administered orally, and the dosage is also reduced, solving the problem of large dosage and severe side effects.
在持续13天的给药周期中,动物体重变化良好,整个给药周期的活动、饮水进食以及精神状况也未发现异常,说明动物对CTX+IN10018(10+25mg/kg)的配伍联合给药治疗耐受。During the 13-day dosing cycle, the animals' body weight changed well, and no abnormalities were found in their activities, drinking water, eating, and mental state throughout the dosing cycle, indicating that the animals tolerated the combined treatment of CTX+IN10018 (10+25 mg/kg).
在连续给药13天之后,安乐死所有动物,并剥离肿瘤,制备单细胞悬液并染色进行流式分析。与空白对照组相比,CTX+IN10018(10+25mg/kg)两药联用组的Treg细胞在CD4+T细胞中的占比数值最低,虽然统计学上没有差异,但是也能说明CTX+IN10018(10+25mg/kg)的配伍联合给药可以降低肿瘤微环境中Treg细胞比例的趋势。与空白对照组相比,CTX(10mg/kg)单药治疗组和CTX+IN10018(10+25mg/kg)两药联用组的CD11b+CD11c+DC细胞在CD45+淋巴细胞所占的比例最高,但是和空白对照组相比没有统计学差异,说明CTX在10mg/kg的单药剂量下或者和IN10018 25mg/kg联合治疗,有提高肿瘤微环境CD11b+CD11c+DC细胞比例的趋势。与空白对照组相比,CTX(10mg/kg)、IN10018(25mg/kg)单药治疗组和CTX+IN10018(10+25mg/kg)两药联用组的CD45-肿瘤细胞上的PD-L1表达水平都很低,其中和CTX(10mg/kg)单药组以及CTX+IN10018(10+25mg/kg)两药联用组相比均具有统计学意义,说明CTX+IN10018(10+25mg/kg)的配伍联合给药治疗可以显著的降低肿瘤微环境中CD45-肿瘤细胞的PD-L1的表达。与空白对照组相比,CTX(10mg/kg)、IN10018(25mg/kg)单药治疗组和CTX+IN10018(10+25mg/kg)两药联用组的CD11b+CD11c+DC细胞上的CD86和MHC II的表达水平也均呈现出升高的趋势,且CTX+IN10018(10+25mg/kg)两药联用组的数值相对最高且和空白对照组相比均有统计学差异。以上数据均显示出CTX+IN10018(10+25mg/kg)的配伍联合给药治疗具有显著提升肿瘤微环境中CD11b+CD11c+DC细胞CD86的MHC II的水平的效果。After 13 consecutive days of administration, all animals were euthanized, and the tumors were removed, and single cell suspensions were prepared and stained for flow cytometry analysis. Compared with the blank control group, the CTX+IN10018 (10+25 mg/kg) combination group had the lowest percentage of Treg cells in CD4+T cells. Although there was no statistical difference, it also showed that the combined administration of CTX+IN10018 (10+25 mg/kg) could reduce the trend of the proportion of Treg cells in the tumor microenvironment. Compared with the blank control group, the proportion of CD11b+CD11c+DC cells in CD45+ lymphocytes was the highest in the CTX (10 mg/kg) monotherapy group and the CTX+IN10018 (10+25 mg/kg) combination group, but there was no statistical difference compared with the blank control group, indicating that CTX at a single dose of 10 mg/kg or in combination with IN10018 25 mg/kg tends to increase the proportion of CD11b+CD11c+DC cells in the tumor microenvironment. Compared with the blank control group, the PD-L1 expression levels on CD45- tumor cells in the CTX (10 mg/kg), IN10018 (25 mg/kg) monotherapy groups and the CTX+IN10018 (10+25 mg/kg) combination group were very low, which was statistically significant compared with the CTX (10 mg/kg) monotherapy group and the CTX+IN10018 (10+25 mg/kg) combination group, indicating that the combined administration of CTX+IN10018 (10+25 mg/kg) can significantly reduce the expression of PD-L1 in CD45- tumor cells in the tumor microenvironment. Compared with the blank control group, the expression levels of CD86 and MHC II on CD11b+CD11c+DC cells in the CTX (10mg/kg), IN10018 (25mg/kg) monotherapy group and the CTX+IN10018 (10+25mg/kg) combination group also showed an upward trend, and the CTX+IN10018 (10+25mg/kg) combination group had the highest value and was statistically different from the blank control group. The above data all show that the combined administration of CTX+IN10018 (10+25mg/kg) has the effect of significantly increasing the level of MHC II of CD86 on CD11b+CD11c+DC cells in the tumor microenvironment.
实施例2:不同浓度的口服环磷酰胺和不同浓度的IN10018之间的配伍治疗在乳腺癌4T1细胞BALB/c小鼠皮下同种移植瘤模型中的体内抗肿瘤药效研究Example 2: In vivo antitumor efficacy study of different concentrations of oral cyclophosphamide combined with different concentrations of IN10018 in a subcutaneous BALB/c mouse breast cancer 4T1 cell homograft model
实验材料:Experimental Materials:
小鼠:7-8周龄的雌性BALB/c小鼠购自上海灵畅生物科技有限公司。动物到达后在实验环境适应性饲养后开始实验。动物在SPF级动物房以IVC(独立送风系统)笼具饲养(每笼4只)。所有笼具、垫料及饮水在使用前均需灭菌。所有实验人员在动物房操作时应穿着防护服和乳胶手套。笼具、饲料及饮水每周更换两次。饲养环境及光照情况如下:Mice: Female BALB/c mice aged 7-8 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The experiment began after the animals were adapted to the experimental environment upon arrival. The animals were kept in IVC (independent ventilation system) cages (4 per cage) in an SPF animal room. All cages, bedding and drinking water must be sterilized before use. All experimental personnel should wear protective clothing and latex gloves when operating in the animal room. Cages, feed and drinking water were changed twice a week. The breeding environment and lighting conditions are as follows:
温度:20-26℃Temperature: 20-26℃
湿度:40-70%Humidity: 40-70%
光照周期:12小时光照,12小时无光照
Photoperiod: 12 hours light, 12 hours no light
笼具:以聚碳酸酯制成,体积300mm×180mm×150mm。垫料为玉米芯,每周更换两次。Cage: Made of polycarbonate, 300mm×180mm×150mm. Bedding is corn cobs, changed twice a week.
食物:实验动物在整个实验阶段中可自由进食(辐照灭菌,干颗粒状食物)。Food: The experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.
饮水:实验动物可自由饮用灭菌水。Drinking water: Experimental animals were free to drink sterile water.
笼具标识:每笼动物信息卡应注明笼内动物数目,性别,品系,接收日期,给药方案,实验编号,组别以及实验开始日期。Cage identification: The animal information card for each cage should indicate the number of animals in the cage, gender, strain, receipt date, dosing regimen, experiment number, group and start date of the experiment.
动物标识:实验动物以耳标进行标识。Animal identification: Experimental animals were identified with ear tags.
供试品信息见表1。原辅料信息见表12。The information of the test products is shown in Table 1. The information of raw materials and auxiliary materials is shown in Table 12.
表12:
Table 12:
Table 12:
细胞培养Cell culture
乳腺癌细胞4T1(来源南京科佰生物科技有限公司,货号:CBP60352)由应世生物科技(南京)有限公司维持传代。细胞体外单层培养,培养条件为RPMI-1640培养基中加10%胎牛血清,37℃,5%CO2培养箱中培养。一周两到三次用胰酶-EDTA进行常规消化处理传代。当细胞处于指数生长期,汇合度为80%-90%时,收取细胞,计数后接种。Breast cancer cell 4T1 (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60352) was maintained and subcultured by Yingshi Biotechnology (Nanjing) Co., Ltd. The cells were cultured in a monolayer in vitro, and the culture conditions were RPMI-1640 medium with 10% fetal bovine serum, cultured in a 37°C, 5% CO2 incubator. Two to three times a week, routine digestion and subculture were performed with trypsin-EDTA. When the cells were in the exponential growth phase and the confluence was 80%-90%, the cells were harvested, counted, and inoculated.
细胞接种及分组Cell seeding and grouping
将0.1ml含有2×105个细胞的细胞悬液皮下接种于每只小鼠的右后背。当肿瘤体积达到~63mm3左右时(细胞接种后的第8天),根据肿瘤体积进行随机分组并给药,分组信息见表13。
0.1 ml of cell suspension containing 2×10 5 cells was subcutaneously inoculated on the right back of each mouse. When the tumor volume reached about 63 mm 3 (8th day after cell inoculation), the mice were randomly divided into groups according to the tumor volume and given drugs. The grouping information is shown in Table 13.
表13:体内药效实验动物分组及给药方案
注:
1.N:每组小鼠数目;
2.给药体积:根据小鼠体重10mL/kg;Table 13: Animal groups and dosing regimens for in vivo efficacy experiments
Note:
1. N: number of mice in each group;
2. Dosing volume: 10mL/kg based on mouse body weight;
注:
1.N:每组小鼠数目;
2.给药体积:根据小鼠体重10mL/kg;Table 13: Animal groups and dosing regimens for in vivo efficacy experiments
Note:
1. N: number of mice in each group;
2. Dosing volume: 10mL/kg based on mouse body weight;
受试物的配制Preparation of test substance
详见表14See Table 14 for details
表14:受试物配制方法
Table 14: Preparation of test substances
Table 14: Preparation of test substances
实验动物日常观察Daily observation of experimental animals
本实验方案的拟定及任何修改均通过了云桥生物IACUC的评估核准。实验动物的使用及福利遵照AAALAC的规定执行。每天监测动物的健康状况及死亡情况,例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动,摄食摄水量(仅目测),体重变化,外观体征或其它不正常情况。基于各组动物数量记录了组内动物死亡数和副作用。
The formulation of this experimental plan and any modification have been evaluated and approved by Yunqiao Biotechnology's IACUC. The use and welfare of experimental animals are carried out in accordance with AAALAC regulations. The health status and mortality of animals are monitored daily. Routine examinations include observing the effects of tumor growth and drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake (visual inspection only), weight changes, physical signs or other abnormal conditions. The number of deaths and side effects of animals in each group were recorded based on the number of animals in each group.
实验终止Experiment termination
若动物健康状况持续恶化,或瘤体积超过3,000mm3,或有严重疾病,或疼痛,须处以安乐死。有以下情况者,通知兽医并处以安乐死:明显消瘦,体重降低大于20%;不能自由取食和饮水;对照组瘤体积平均值达到3,000mm3,实验终止。动物出现以下临床表现且持续恶化:立毛,弓背,耳、鼻、眼或足色发白,呼吸仓促,抽搐,连续腹泻,脱水,行动迟缓,发声。If the health of the animal continues to deteriorate, or the tumor volume exceeds 3,000mm 3 , or there is a serious disease or pain, euthanasia must be carried out. In the following cases, notify the veterinarian and euthanize: obvious weight loss, weight loss greater than 20%; cannot freely eat and drink water; the average tumor volume of the control group reaches 3,000mm 3 , and the experiment is terminated. Animals with the following clinical manifestations and continue to deteriorate: piloerection, arched back, pale ears, nose, eyes or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.
肿瘤测量和实验指标Tumor measurements and experimental parameters
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。用游标卡尺测量肿瘤直径,每周测量2~3次。肿瘤体积的计算公式为:V=0.5×a×b2,a和b分别表示肿瘤的长径和短径。The experimental index is to examine whether the tumor growth is inhibited, delayed or cured. The tumor diameter is measured with a vernier caliper 2 to 3 times a week. The calculation formula of tumor volume is: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor respectively.
化合物的抑瘤疗效用TGI(%)评价,反映肿瘤生长抑制率。参照分组后第一天的肿瘤体积,根据以下公式计算肿瘤生长抑制率TGI(%)。TGI(%)=[1-(某给药组的平均瘤体积-该给药组开始时平均瘤体积)/(溶剂对照组的平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]×100%。The anti-tumor efficacy of the compound was evaluated by TGI (%), which reflects the tumor growth inhibition rate. With reference to the tumor volume on the first day after grouping, the tumor growth inhibition rate TGI (%) was calculated according to the following formula: TGI (%) = [1-(average tumor volume of a certain medication group-average tumor volume at the beginning of the medication group)/(average tumor volume of the solvent control group-average tumor volume at the beginning of the solvent control group)] × 100%.
统计分析Statistical Analysis
统计分析基于试验结束时肿瘤体积运用Prism Graphpad软件进行分析。多组间比较,采用Two-way ANOVA,Fisher’s LSD test法检验进行分析。P<0.05认为有显著性差异。Statistical analysis was performed using Prism Graphpad software based on the tumor volume at the end of the experiment. Two-way ANOVA and Fisher’s LSD test were used for comparison among multiple groups. P<0.05 was considered to be significantly different.
实验结果Experimental Results
受试物CTX(10mg/kg)和不同剂量的IN10018的配伍治疗在乳腺癌4T1细胞BALB/c小鼠皮下同种移植瘤模型中的体内药效研究In vivo efficacy study of the combination of CTX (10 mg/kg) and different doses of IN10018 in a subcutaneous BALB/c mouse model of breast cancer 4T1 cells
细胞接种后,每天观察肿瘤生长情况,接种后第8天根据肿瘤体积进行分组,入组平均肿瘤体积约为63mm3。由于肿瘤负荷,所有组别在接种后的第32天,即分组给药之后的第24天安乐死,整个实验结束。
After cell inoculation, tumor growth was observed every day. Groups were divided according to tumor volume on the 8th day after inoculation, and the average tumor volume was about 63 mm 3 . Due to tumor burden, all groups were euthanized on the 32nd day after inoculation, which was the 24th day after group administration, and the entire experiment was terminated.
分组给药后第24天,对照组的肿瘤体积为2795.1±1412.0mm3。CTX(10mg/kg)单药组的肿瘤体积为1991.3±1011.1mm3;CTX+IN10018(10+10mg/kg)和CTX+IN10018(10+20mg/kg)联用组的肿瘤体积分别为1625.1±323.3mm3和1268.4±190.7mm3。综合肿瘤体积与对照组进行比较,CTX(10mg/kg)单药组的抑瘤率TGI为29.4%(p<0.0001);CTX+IN10018(10+10mg/kg)联用组的抑瘤率TGI为42.8%(p<0.0001);CTX+IN10018(10+20mg/kg)联用组的抑瘤率TGI为55.9%(p<0.0001),均有显著的统计学差异。On the 24th day after group administration, the tumor volume of the control group was 2795.1±1412.0 mm 3 , the tumor volume of the CTX (10 mg/kg) monotherapy group was 1991.3±1011.1 mm 3 , and the tumor volumes of the CTX+IN10018 (10+10 mg/kg) and CTX+IN10018 (10+20 mg/kg) combination groups were 1625.1±323.3 mm 3 and 1268.4±190.7 mm 3 , respectively. Compared with the control group based on the comprehensive tumor volume, the tumor inhibition rate TGI of the CTX (10 mg/kg) monotherapy group was 29.4% (p<0.0001); the tumor inhibition rate TGI of the CTX+IN10018 (10+10 mg/kg) combination group was 42.8% (p<0.0001); the tumor inhibition rate TGI of the CTX+IN10018 (10+20 mg/kg) combination group was 55.9% (p<0.0001), all of which showed significant statistical differences.
综合瘤体积与CTX+IN10018(10+20mg/kg)联用组相比较,进行统计学分析,CTX(10mg/kg)单药组和CTX+IN10018(10+10mg/kg)联用组的P值分别为p<0.0001、p=0.0239。即CTX+IN10018(10+20mg/kg)联用组与CTX(10mg/kg)单药组和CTX+IN10018(10+10mg/kg)联用组均有显著的统计学差异。The tumor volume was compared with the CTX+IN10018 (10+20 mg/kg) combination group, and statistical analysis was performed. The P values of the CTX (10 mg/kg) monotherapy group and the CTX+IN10018 (10+10 mg/kg) combination group were p<0.0001 and p=0.0239, respectively. That is, the CTX+IN10018 (10+20 mg/kg) combination group had significant statistical differences with the CTX (10 mg/kg) monotherapy group and the CTX+IN10018 (10+10 mg/kg) combination group.
各组的抑瘤效果评价详见表15,剂量组在不同时间段的肿瘤体积如图8所示。The evaluation of the tumor inhibition effect of each group is shown in Table 15, and the tumor volume of the dosage group at different time periods is shown in Figure 8.
表15:受试物对小鼠乳腺癌4T1细胞的BALB/c小鼠移植瘤模型的抑瘤效果评价(基于分组给药后第24天数据)
注:
1.按照分组给药后的天数来计算,数据为平均值±标准差(mean±SD);
2.TGI(%)=[1-(T24-T0)/(V24-V0)]×100%;
3.****:p<0.0001,vs.对照组,Two-way ANOVA,Fisher’s LSD test;
4.*:p<0.05,****:p<0.0001,vs.CTX+IN10018(10+20mg/kg)组,Two-way ANOVA,Fisher’s LSD test; Table 15: Evaluation of the tumor inhibition effect of the test substance on the BALB/c mouse transplanted tumor model of mouse breast cancer 4T1 cells (based on the data on the 24th day after group administration)
Note:
1. Calculated according to the number of days after group administration, the data are mean ± standard deviation (mean ± SD);
2. TGI (%) = [1 - (T24 - T0) / (V24 - V0)] × 100%;
3. ****: p<0.0001, vs. control group, Two-way ANOVA, Fisher's LSD test;
4. *: p<0.05, ****: p<0.0001, vs. CTX+IN10018 (10+20 mg/kg) group, Two-way ANOVA, Fisher's LSD test;
注:
1.按照分组给药后的天数来计算,数据为平均值±标准差(mean±SD);
2.TGI(%)=[1-(T24-T0)/(V24-V0)]×100%;
3.****:p<0.0001,vs.对照组,Two-way ANOVA,Fisher’s LSD test;
4.*:p<0.05,****:p<0.0001,vs.CTX+IN10018(10+20mg/kg)组,Two-way ANOVA,Fisher’s LSD test; Table 15: Evaluation of the tumor inhibition effect of the test substance on the BALB/c mouse transplanted tumor model of mouse breast cancer 4T1 cells (based on the data on the 24th day after group administration)
Note:
1. Calculated according to the number of days after group administration, the data are mean ± standard deviation (mean ± SD);
2. TGI (%) = [1 - (T24 - T0) / (V24 - V0)] × 100%;
3. ****: p<0.0001, vs. control group, Two-way ANOVA, Fisher's LSD test;
4. *: p<0.05, ****: p<0.0001, vs. CTX+IN10018 (10+20 mg/kg) group, Two-way ANOVA, Fisher's LSD test;
受试物不同剂量的CTX和IN10018不同剂量之间的配伍治疗在乳腺癌4T1细胞BALB/c小鼠皮下同种移植瘤模型中的体重变化及临床状况研究Study on the changes in body weight and clinical status of different doses of CTX and IN10018 combined with different doses in the subcutaneous transplantation tumor model of breast cancer 4T1 cells in BALB/c mice
实验按照给药方案进行,实验过程中,每天观察动物摄食饮水等活动,每周记录2~3次动物体重。分组给药后的第24天,对照组体重从分组给药当天(Day 0)的21.0g变化至25.1g,体重变化率19.4%;CTX(10mg/kg)单药组的平均体重从Day 0的20.2g变化至21.2g,体重变化率为5.2%;CTX+IN10018(10+10mg/kg)联用组的平均体重从Day 0的21.0g变化至20.9g,体重变化率为-0.2%;CTX+IN10018(10+20mg/kg)联用组的平均体重从Day 0的20.7g变化至21.4g,体重变化率为3.3%;治疗组动物的精神状况良好,摄食运动正常。各组体重数据见表16,各剂量组在不同时间段的体重变化如图9所示。The experiment was carried out according to the dosing regimen. During the experiment, the animals' activities such as eating and drinking were observed every day, and the weight of the animals was recorded 2 to 3 times a week. On the 24th day after group administration, the weight of the control group changed from 21.0g on the day of group administration (Day 0) to 25.1g, with a weight change rate of 19.4%; the average weight of the CTX (10mg/kg) monotherapy group changed from 20.2g on Day 0 to 21.2g, with a weight change rate of 5.2%; the average weight of the CTX+IN10018 (10+10mg/kg) combination group changed from 21.0g on Day 0 to 20.9g, with a weight change rate of -0.2%; the average weight of the CTX+IN10018 (10+20mg/kg) combination group changed from 20.7g on Day 0 to 21.4g, with a weight change rate of 3.3%; the animals in the treatment group were in good mental state, and their eating and movement were normal. The body weight data of each group are shown in Table 16, and the changes in body weight of each dosage group at different time periods are shown in Figure 9.
表16:受试物对小鼠乳腺癌4T1细胞的BALB/c小鼠移植瘤模型的体重变化评价(基于分组给药后第24天数据)
注:
1.按照分组给药后的天数来计算,第0天的动物存活数目/第24天的动物存活数目;
2.数据为平均值±标准差(mean±SD);
3.体重变化率=[(W24-W0)/W0]*100%;Table 16: Evaluation of the test substance on the body weight change of the BALB/c mouse transplant tumor model of mouse breast cancer 4T1 cells (based on the data on the 24th day after group administration)
Note:
1. Calculated according to the number of days after group administration: the number of animals surviving on day 0/the number of animals surviving on day 24;
2. Data are mean ± standard deviation (mean ± SD);
3. Weight change rate = [(W 24 -W 0 )/W 0 ]*100%;
注:
1.按照分组给药后的天数来计算,第0天的动物存活数目/第24天的动物存活数目;
2.数据为平均值±标准差(mean±SD);
3.体重变化率=[(W24-W0)/W0]*100%;Table 16: Evaluation of the test substance on the body weight change of the BALB/c mouse transplant tumor model of mouse breast cancer 4T1 cells (based on the data on the 24th day after group administration)
Note:
1. Calculated according to the number of days after group administration: the number of animals surviving on day 0/the number of animals surviving on day 24;
2. Data are mean ± standard deviation (mean ± SD);
3. Weight change rate = [(W 24 -W 0 )/W 0 ]*100%;
实验结论Experimental results
在本实验中,我们分别评价了受试物不同浓度的CTX和不同浓度的IN10018之间的配伍治疗在4T1小鼠乳腺癌细胞BALB/c小鼠皮下同种移植 瘤模型中的体内药效。与空白对照组相比,CTX和IN10018不同剂量之间的联用配伍治疗均有明显肿瘤生长抑制作用,且和对照组相比都有统计学差异。综合整个给药周期,CTX+IN10018(10+20mg/kg)联用组的肿瘤体积要小于CTX+IN10018(10+10mg/kg)联用组、CTX(10mg/kg)单药组的肿瘤体积,且具有统计学差异。以上数据说明CTX在10mg/kg的剂量下,和IN1001810mg/kg和20mg/kg之间的配伍治疗具有剂量依赖作用,IN10018可以更好的抑制肿瘤生长,同时,IN10018在高浓度(20mg/kg)下相比在低浓度(10mg/kg),可以更好的增效CTX(10mg/kg)的治疗水准。In this experiment, we evaluated the in vivo efficacy of different concentrations of CTX and different concentrations of IN10018 in the BALB/c mouse subcutaneous transplant tumor model of 4T1 mouse breast cancer cells. Compared with the blank control group, the combination therapy of CTX and IN10018 at different doses had a significant tumor growth inhibitory effect, and there were statistical differences compared with the control group. Comprehensively considering the entire dosing cycle, the tumor volume of the CTX+IN10018 (10+20mg/kg) combination group was smaller than that of the CTX+IN10018 (10+10mg/kg) combination group and the CTX (10mg/kg) monotherapy group, and there was a statistical difference. The above data show that CTX at a dose of 10 mg/kg and the combination treatment with IN10018 between 10 mg/kg and 20 mg/kg have a dose-dependent effect, and IN10018 can better inhibit tumor growth. At the same time, IN10018 at a high concentration (20 mg/kg) can better enhance the therapeutic level of CTX (10 mg/kg) than at a low concentration (10 mg/kg).
综合整个给药周期,CTX和不同剂量IN10018之间的配伍治疗的相关组别,动物的体重无异常且精神状况良好,摄食运动正常,表现出对CTX(10mg/kg)、和IN10018(10mg/kg)、IN10018(20mg/kg)之间的配伍治疗的耐受。Comprehensively considering the entire dosing cycle, in the relevant groups treated with the combination of CTX and different doses of IN10018, the animals had no abnormalities in body weight and were in good mental state, with normal eating and movement, showing tolerance to the combination treatment of CTX (10 mg/kg), IN10018 (10 mg/kg), and IN10018 (20 mg/kg).
实施例3:环磷酰胺和IN10018之间的配伍治疗在卵巢癌ID8-Luc细胞C57BL/6小鼠腹腔移植腹水模型中的体内抗肿瘤药效研究Example 3: In vivo anti-tumor efficacy study of the combination of cyclophosphamide and IN10018 in the ascites model of ovarian cancer ID8-Luc cells transplanted into the peritoneal cavity of C57BL/6 mice
实验材料:Experimental Materials:
小鼠:7-8周龄的雌性C57BL/6小鼠购自上海灵畅生物科技有限公司。动物到达后在实验环境适应性饲养后开始实验。动物在SPF级动物房以IVC(独立送风系统)笼具饲养(每笼4只)。所有笼具、垫料及饮水在使用前均需灭菌。所有实验人员在动物房操作时应穿着防护服和乳胶手套。笼具、饲料及饮水每周更换两次。饲养环境及光照情况如下:Mice: 7-8 week old female C57BL/6 mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The experiment started after the animals were adapted to the experimental environment upon arrival. The animals were kept in IVC (independent ventilation system) cages (4 per cage) in an SPF animal room. All cages, bedding and drinking water must be sterilized before use. All experimenters should wear protective clothing and latex gloves when operating in the animal room. Cages, feed and drinking water were changed twice a week. The breeding environment and lighting conditions are as follows:
温度:20-26℃Temperature: 20-26℃
湿度:40-70%Humidity: 40-70%
光照周期:12小时光照,12小时无光照Photoperiod: 12 hours light, 12 hours no light
笼具:以聚碳酸酯制成,体积300mm×180mm×150mm。垫料为玉米芯,每周更换两次。Cage: Made of polycarbonate, 300mm×180mm×150mm. Bedding is corn cobs, changed twice a week.
食物:实验动物在整个实验阶段中可自由进食(辐照灭菌,干颗粒状食物)。
Food: The experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.
饮水:实验动物可自由饮用灭菌水。Drinking water: Experimental animals were free to drink sterile water.
笼具标识:每笼动物信息卡应注明笼内动物数目,性别,品系,接收日期,Cage identification: Each cage animal information card should indicate the number of animals in the cage, gender, strain, receipt date,
给药方案,实验编号,组别以及实验开始日期。Dosage regimen, experiment number, groups and experiment start date.
动物标识:实验动物以耳标进行标识。Animal identification: Experimental animals were identified with ear tags.
供试品信息见表1。The information of the test products is shown in Table 1.
原辅料信息见表12。The information of raw materials and auxiliary materials is shown in Table 12.
细胞培养Cell culture
卵巢癌细胞ID8-Luc(由广东医科大学改造构建)由应世生物科技(南京)有限公司维持传代。细胞体外单层培养,培养条件为DMEM培养基中加10%胎牛血清和1ug/ml的Puromycin,37℃,5%CO2培养箱中培养。一周两到三次用胰酶-EDTA进行常规消化处理传代。当细胞处于指数生长期,汇合度为80%-90%时,收取细胞,计数后接种。Ovarian cancer cell ID8-Luc (constructed by Guangdong Medical University) was maintained and subcultured by Inshi Biotechnology (Nanjing) Co., Ltd. The cells were cultured in monolayer in vitro, and the culture conditions were DMEM medium with 10% fetal bovine serum and 1ug/ml Puromycin, cultured in a 37°C, 5% CO2 incubator. Two to three times a week, routine digestion and subculture were performed with trypsin-EDTA. When the cells were in the exponential growth phase and the confluence was 80%-90%, the cells were harvested, counted, and inoculated.
细胞接种及分组Cell seeding and grouping
将0.1ml含有5×106个细胞的细胞悬液腹腔注射接种于每只小鼠。当在细胞接种后的第14天,根据动物的体重进行随机分组并给药,分组信息,分组信息见表17。Each mouse was inoculated intraperitoneally with 0.1 ml of a cell suspension containing 5×10 6 cells. On the 14th day after cell inoculation, the animals were randomly divided into groups and given medications according to their body weights. The grouping information is shown in Table 17.
表17:体内药效实验动物分组及给药方案
注:
1.N:每组小鼠数目;
2.给药体积:根据小鼠体重10mL/kg,如果体重下降超过15%,动物停止给药;待
体重恢复至初始值,再恢复给药; Table 17: Animal groups and dosing regimens for in vivo efficacy experiments
Note:
1. N: number of mice in each group;
2. Dosing volume: 10mL/kg based on the mouse body weight. If the body weight drops by more than 15%, stop dosing the animal; resume dosing when the body weight returns to the initial value;
注:
1.N:每组小鼠数目;
2.给药体积:根据小鼠体重10mL/kg,如果体重下降超过15%,动物停止给药;待
体重恢复至初始值,再恢复给药; Table 17: Animal groups and dosing regimens for in vivo efficacy experiments
Note:
1. N: number of mice in each group;
2. Dosing volume: 10mL/kg based on the mouse body weight. If the body weight drops by more than 15%, stop dosing the animal; resume dosing when the body weight returns to the initial value;
受试物的配制Preparation of test substance
详见表18。See Table 18 for details.
表18:受试物配制方法
Table 18: Test substance preparation method
Table 18: Test substance preparation method
实验动物日常观察Daily observation of experimental animals
本实验方案的拟定及任何修改均通过了云桥生物IACUC的评估核准。实验动物的使用及福利遵照AAALAC的规定执行。每天监测动物的健康状况及死亡情况,例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动,摄食摄水量(仅目测),体重变化,外观体征或其它不正常情况。基于各组动物数量记录了组内动物死亡数和副作用。The formulation of this experimental plan and any modification have been evaluated and approved by Yunqiao Biotechnology's IACUC. The use and welfare of experimental animals are carried out in accordance with AAALAC regulations. The health status and mortality of animals are monitored daily. Routine examinations include observing the effects of tumor growth and drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake (visual inspection only), weight changes, physical signs or other abnormal conditions. The number of deaths and side effects of animals in each group were recorded based on the number of animals in each group.
实验终止Experiment termination
若动物健康状况持续恶化,或有严重疾病,或疼痛,须处以安乐死。有以下情况者,通知兽医并处以安乐死:明显消瘦,体重降低大于20%;不能自由取食和饮水,实验终止。动物出现以下临床表现且持续恶化:立毛,弓背,耳、鼻、眼或足色发白,呼吸仓促,抽搐,连续腹泻,脱水,行动迟缓,发声。If the health of the animal continues to deteriorate, or if there is a serious illness or pain, it must be euthanized. If the following conditions occur, notify the veterinarian and euthanize the animal: obvious weight loss, weight loss greater than 20%; cannot freely eat and drink, the experiment is terminated. If the animal shows the following clinical manifestations and continues to deteriorate: piloerection, arched back, pale ears, nose, eyes or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.
肿瘤测量和实验指标Tumor measurements and experimental parameters
腹围:待动物重新分组(分组给药30天之后),每周记录动物的腹围一次,使用柔软的皮尺,对动物的腹围进行测量,即动物腹部最大的周长值。发病率:以动物腹围大于6.5cm,作为动物有腹水发生,来计算动物的发病率,发病率=最终发病动物只数/该组动物总只数*100%。Abdominal circumference: After the animals are regrouped (30 days after group administration), the abdominal circumference of the animals is recorded once a week. The abdominal circumference of the animals is measured using a soft tape measure, which is the maximum circumference of the animal's abdomen. Morbidity: The animal's abdominal circumference is greater than 6.5 cm, which is considered to be ascites. The morbidity of the animals is calculated. Morbidity = the final number of animals with the disease / the total number of animals in the group * 100%.
体重:每周记录1~2次动物的体重,观察动物体重随着时间以及疾病发生的变化情况。Body weight: Record the animal's body weight 1 to 2 times a week and observe changes in the animal's body weight over time and with the occurrence of disease.
统计分析Statistical Analysis
统计分析基于试验结束时运用Prism Graphpad软件进行分析。体重即腹围数据的多组间比较,采用Two-way ANOVA,Fisher’s LSD test法检验进行分析,P<0.05认为有显著性差异。Statistical analysis was performed using Prism Graphpad software at the end of the experiment. The comparison of body weight and abdominal circumference data among multiple groups was analyzed using Two-way ANOVA and Fisher’s LSD test. P < 0.05 was considered to be significantly different.
实验结果Experimental Results
受试物CTX和IN10018之间的配伍治疗在卵巢癌ID8-Luc细胞C57BL/6小鼠腹水模型中的发病率的研究Study on the incidence of the combination therapy between the test substances CTX and IN10018 in the ascites model of ovarian cancer ID8-Luc cells C57BL/6 mice
细胞接种后,每天观察肿瘤生长情况,接种后第14天根据动物体重进行分组,观察动物的体重变化、发病率、腹围变化。并在接种后第63天,即分组给药之后的第49天,G1、G2和G3组安乐死所有存活动物。在持续63天的观察之中,对照组在接种后第44天,即分组给药之后的第30天开始初次有动物出现腹水的症状,并在分组给药之后的第33天,发病率到达100%,所有动物均表现出腹水的症状;CTX(3mg/kg)单药治疗组在分组给药后的第30天开始发病,随后在分组给药之后的第36天达到最高发病率100%;CTX+IN10018(3+12.5mg/kg)两药联用给药组在给药后的第30天开始发病,并均在给药后的第46天发病率达到最高85.7%。各组的发病率评价详见表19。各剂量组在不同时间段的发病率如图10所示。After cell inoculation, tumor growth was observed every day. Animals were grouped according to their weight on the 14th day after inoculation to observe changes in weight, morbidity, and abdominal circumference. On the 63rd day after inoculation, i.e., the 49th day after group administration, all surviving animals in groups G1, G2, and G3 were euthanized. During the 63-day observation, the control group began to show symptoms of ascites for the first time on the 44th day after inoculation, i.e., the 30th day after group administration, and the morbidity reached 100% on the 33rd day after group administration, and all animals showed symptoms of ascites; the CTX (3 mg/kg) monotherapy group began to develop the disease on the 30th day after group administration, and then reached the highest morbidity of 100% on the 36th day after group administration; the CTX+IN10018 (3+12.5 mg/kg) two-drug combination administration group began to develop the disease on the 30th day after administration, and the morbidity reached a maximum of 85.7% on the 46th day after administration. The evaluation of the morbidity of each group is shown in Table 19. The incidence rates of each dose group at different time periods are shown in FIG10 .
表19:受试物对小鼠卵巢癌ID8-Luc细胞的C57BL/6小鼠腹水模型的发病率和生存率评价(基于分组给药后第49天数据)
注:
1.初始发病天数:即该组第一只动物腹围达到6.5cm的天数,数值以分组给药后的天数来显示;
2.最高发病天数:即该组最后一只动物腹围达到6.5cm的天数,数值以分组给药后的天数来显示;
3.发病率=最终发病动物只数/该组动物总只数*100%;Table 19: Evaluation of the morbidity and survival rate of the C57BL/6 mouse ascites model of mouse ovarian cancer ID8-Luc cells by the test substance (based on the data on day 49 after group administration)
Note:
1. Initial onset day: the day when the abdominal circumference of the first animal in the group reaches 6.5 cm. The value is displayed as the number of days after grouping and drug administration;
2. Days of maximum onset: the day when the abdominal circumference of the last animal in the group reached 6.5 cm. The value is displayed as the number of days after grouping and drug administration;
3. Incidence rate = final number of animals with disease/total number of animals in the group*100%;
注:
1.初始发病天数:即该组第一只动物腹围达到6.5cm的天数,数值以分组给药后的天数来显示;
2.最高发病天数:即该组最后一只动物腹围达到6.5cm的天数,数值以分组给药后的天数来显示;
3.发病率=最终发病动物只数/该组动物总只数*100%;Table 19: Evaluation of the morbidity and survival rate of the C57BL/6 mouse ascites model of mouse ovarian cancer ID8-Luc cells by the test substance (based on the data on day 49 after group administration)
Note:
1. Initial onset day: the day when the abdominal circumference of the first animal in the group reaches 6.5 cm. The value is displayed as the number of days after grouping and drug administration;
2. Days of maximum onset: the day when the abdominal circumference of the last animal in the group reached 6.5 cm. The value is displayed as the number of days after grouping and drug administration;
3. Incidence rate = final number of animals with disease/total number of animals in the group*100%;
受试物CTX和IN10018之间的配伍治疗在卵巢癌ID8-Luc细胞C57BL/6小鼠腹水模型中的腹围和体重变化的研究Study on the changes of abdominal circumference and body weight in the ascites model of ovarian cancer ID8-Luc cells C57BL/6 mice by the combination therapy of the test substance CTX and IN10018
在持续63天的观察之中,小鼠由于发生腹水,导致其体重和腹围出现生长。在接种之后的第63天,即分组给药之后的第49天,安乐死G1、G2和G3组的所有动物,对CTX和IN10018之间的配伍治疗的疗效观察结束。在分组给药之后的第49天,对照组动物的体重由分组给药之后的20.7g变化至32.7g,体重变化率为58.4%;CTX(3mg/kg)单药治疗组的体重由分组给药之后的20.3g变化至36.1g,体重变化率为80.2%;CTX+IN10018(3+12.5mg/kg)两药联用给药组的体重由分组给药之后的20.9g变化至28.9g,体重变化率为37.2%。综合平均体重与对照组进行统计学分析,CTX(3mg/kg)单药治疗组和CTX+IN10018(3+12.5mg/kg)两药联用给药组的P值分别为p=0.0129和p=0.0049。在分组给药之后的第42天,对照组动物的腹围是8.3±1.1cm;CTX(3mg/kg)单药治疗组的腹围是8.5±1.1cm;CTX+IN10018(3+12.5mg/kg)两药联用给药组的腹围是7.2±0.9cm。在分组给药之后的第46天,对照组动物的腹围是9.3±1.5cm;CTX(3mg/kg)单药治疗组的腹围是10.1±1.0cm;CTX+IN10018(3+12.5mg/kg)两药联用给药组的腹围是8.1±1.6cm。在第49天,对照组动物的腹围是9.9±1.7cm;CTX(3mg/kg) 单药治疗组的腹围是10.8±0.7cm;CTX+IN10018(3+12.5mg/kg)两药联用给药组的腹围是9.1±1.6cm。综合腹围数据与对照组进行统计学分析,CTX(3mg/kg)单药治疗组和CTX+IN10018(3+12.5mg/kg)两药联用给药组在第42天、46天和49天的P值分别为p=0.6444&p=0.0273、p=0.1148&p=0.0202和p=0.0702&p=0.1248。各组的体重和腹围评价详见表20和表21。各剂量组在不同时间段的体重和腹围如图11和图12所示。During the 63-day observation, the mice developed ascites, which resulted in an increase in their weight and abdominal circumference. On the 63rd day after inoculation, i.e., the 49th day after group administration, all animals in the G1, G2, and G3 groups were euthanized, and the observation of the efficacy of the combination treatment between CTX and IN10018 ended. On the 49th day after group administration, the body weight of the control group animals changed from 20.7g after group administration to 32.7g, with a body weight change rate of 58.4%; the body weight of the CTX (3mg/kg) monotherapy group changed from 20.3g after group administration to 36.1g, with a body weight change rate of 80.2%; the body weight of the CTX+IN10018 (3+12.5mg/kg) combination administration group changed from 20.9g after group administration to 28.9g, with a body weight change rate of 37.2%. The average body weight was statistically analyzed with the control group. The P values of the CTX (3 mg/kg) monotherapy group and the CTX+IN10018 (3+12.5 mg/kg) combination therapy group were p=0.0129 and p=0.0049, respectively. On the 42nd day after group administration, the abdominal circumference of the control group animals was 8.3±1.1 cm; the abdominal circumference of the CTX (3 mg/kg) monotherapy group was 8.5±1.1 cm; the abdominal circumference of the CTX+IN10018 (3+12.5 mg/kg) combination therapy group was 7.2±0.9 cm. On the 46th day after group administration, the abdominal circumference of the control group animals was 9.3±1.5 cm; the abdominal circumference of the CTX (3 mg/kg) monotherapy group was 10.1±1.0 cm; and the abdominal circumference of the CTX+IN10018 (3+12.5 mg/kg) combination therapy group was 8.1±1.6 cm. On the 49th day, the abdominal circumference of the control group animals was 9.9±1.7cm; the abdominal circumference of the CTX (3mg/kg) monotherapy group was 10.8±0.7cm; the abdominal circumference of the CTX+IN10018 (3+12.5mg/kg) dual-drug combination group was 9.1±1.6cm. The comprehensive abdominal circumference data were statistically analyzed with the control group. The P values of the CTX (3mg/kg) monotherapy group and the CTX+IN10018 (3+12.5mg/kg) dual-drug combination group on the 42nd, 46th and 49th days were p=0.6444&p=0.0273, p=0.1148&p=0.0202 and p=0.0702&p=0.1248, respectively. The weight and abdominal circumference evaluation of each group are shown in Tables 20 and 21. The weight and abdominal circumference of each dose group at different time periods are shown in Figures 11 and 12.
表20:受试物对小鼠卵巢癌ID8-Luc细胞的C57BL/6小鼠腹水模型的体重变化评价(基于分组给药后第49天数据)
注:
1.数据为平均值±标准差(mean±SD);
2.体重变化率=[(Wi-W0)/W0]*100%,i为第i天该组平均体重;
3.*:p<0.05,**:p<0.01,vs.对照组,Two-way ANOVA;Table 20: Evaluation of the body weight changes of the C57BL/6 mouse ascites model of mouse ovarian cancer ID8-Luc cells by the test substance (based on the data on day 49 after group administration)
Note:
1. Data are mean ± standard deviation (mean ± SD);
2. Weight change rate = [(W i - W 0 )/W 0 ]*100%, i is the average weight of the group on the i-th day;
3.*: p<0.05, **: p<0.01, vs. control group, Two-way ANOVA;
注:
1.数据为平均值±标准差(mean±SD);
2.体重变化率=[(Wi-W0)/W0]*100%,i为第i天该组平均体重;
3.*:p<0.05,**:p<0.01,vs.对照组,Two-way ANOVA;Table 20: Evaluation of the body weight changes of the C57BL/6 mouse ascites model of mouse ovarian cancer ID8-Luc cells by the test substance (based on the data on day 49 after group administration)
Note:
1. Data are mean ± standard deviation (mean ± SD);
2. Weight change rate = [(W i - W 0 )/W 0 ]*100%, i is the average weight of the group on the i-th day;
3.*: p<0.05, **: p<0.01, vs. control group, Two-way ANOVA;
表21:受试物对小鼠卵巢癌ID8-Luc细胞的C57BL/6小鼠腹水模型的腹围变化评价(基于分组给药后第42/46/49天数据)
注:
1.数据为平均值±标准差(mean±SD);
2.*:p<0.05,vs.对照组,Two-way ANOVA; Table 21: Evaluation of abdominal circumference changes of the test substance in the C57BL/6 mouse ascites model of mouse ovarian cancer ID8-Luc cells (based on data on days 42/46/49 after group administration)
Note:
1. Data are mean ± standard deviation (mean ± SD);
2.*: p<0.05, vs. control group, Two-way ANOVA;
注:
1.数据为平均值±标准差(mean±SD);
2.*:p<0.05,vs.对照组,Two-way ANOVA; Table 21: Evaluation of abdominal circumference changes of the test substance in the C57BL/6 mouse ascites model of mouse ovarian cancer ID8-Luc cells (based on data on days 42/46/49 after group administration)
Note:
1. Data are mean ± standard deviation (mean ± SD);
2.*: p<0.05, vs. control group, Two-way ANOVA;
实验结论Experimental results
在本实验中,我们分别评价了受试物CTX和IN10018之间的配伍治疗在ID8-Luc小鼠卵巢癌细胞C57BL/6小鼠腹水模型中的体内药效。和空白对照组相比,CTX+IN10018(3+12.5mg/kg)的配伍治疗,可以明显的推迟动物的腹水的发病率,且到实验结束,发病率也没达到100%,组内仍然有动物没有发病;CTX+IN10018(3+12.5mg/kg)的配伍治疗,在动物的体重和腹围数据上也是有明显的优势,在持续63天的观察之中,CTX+IN10018(3+12.5mg/kg)的配伍治疗组的体重数据和腹围数据均要小于空白对照组和CTX(3mg/kg)单药治疗组,且和空白对照组相比均具有统计学差异。以上数据均说明CTX在3mg/kg和IN10018在12.5mg/kg的剂量下的配伍治疗,具有更好的疗效。In this experiment, we evaluated the in vivo efficacy of the combination therapy of the test substance CTX and IN10018 in the ID8-Luc mouse ovarian cancer cell C57BL/6 mouse ascites model. Compared with the blank control group, the combination therapy of CTX+IN10018 (3+12.5mg/kg) can significantly delay the incidence of ascites in animals, and by the end of the experiment, the incidence rate did not reach 100%, and there were still animals in the group that did not develop the disease; the combination therapy of CTX+IN10018 (3+12.5mg/kg) also had obvious advantages in the weight and abdominal circumference data of animals. During the observation for 63 days, the weight data and abdominal circumference data of the combination therapy group of CTX+IN10018 (3+12.5mg/kg) were both smaller than those of the blank control group and the CTX (3mg/kg) monotherapy group, and there were statistical differences compared with the blank control group. The above data indicate that the combination therapy of CTX at a dose of 3 mg/kg and IN10018 at a dose of 12.5 mg/kg has a better therapeutic effect.
通过引用将本发明中所提及的所有参考文献均完整合并入本文,就如同每一篇文献均单独列出一样。应理解,在阅读了本发明的公开内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落入本申请所附权利要求书所限定的范围内。
All references mentioned in the present invention are fully incorporated herein by reference, just as if each document was listed separately. It should be understood that after reading the disclosure of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to the application.
Claims (11)
- IN10018或其药学上可接受的盐和环磷酰胺在制备用于在对象中治疗肿瘤的药物中的用途,所述IN10018结构如下:
Use of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide in the preparation of a medicament for treating a tumor in a subject, wherein the structure of IN10018 is as follows:
- IN10018或其药学上可接受的盐和环磷酰胺的药物组合产品,其用于在对象中治疗肿瘤,所述IN10018结构如下:
A pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide for treating a tumor in a subject, wherein the structure of IN10018 is as follows:
- 一种治疗肿瘤的方法,该方法包括向有需要的对象施用治疗有效量的IN10018或其药学上可接受的盐和环磷酰胺,所述IN10018结构如下:
A method for treating a tumor, comprising administering a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and cyclophosphamide to a subject in need thereof, wherein the structure of IN10018 is as follows:
- 如权利要求1-3任一项所述的用途、药物组合产品或者方法,其中所述IN10018药学上可接受的盐为酒石酸盐。The use, pharmaceutical combination or method according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt of IN10018 is a tartrate.
- 如权利要求1-4任一项所述的用途、药物组合产品或者方法,其中所述IN10018或其药学上可接受的盐和所述环磷酰胺被同时或依次施用于所述对象。The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the cyclophosphamide are administered to the subject simultaneously or sequentially.
- 如权利要求1-5任一项所述的用途、药物组合产品或者方法,其中所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。The use, pharmaceutical combination or method according to any one of claims 1 to 5, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, Salivary gland cancer, metastatic tumors caused by spindle cell cancer, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML); preferably, the tumor is ovarian cancer, liver cancer, lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, gastric cancer, pancreatic cancer or colon cancer; more preferably, the tumor is ovarian cancer or breast cancer.
- 一种试剂盒或药学上可接受的组合物,其包括:A kit or pharmaceutically acceptable composition comprising:(a)IN10018或其药学上可接受的盐;和(a) IN10018 or a pharmaceutically acceptable salt thereof; and(b)环磷酰胺,(b) cyclophosphamide,所述IN10018结构如下:
The structure of IN10018 is as follows:
- 如权利要求7所述的试剂盒或组合物,其用作药物。The kit or composition according to claim 7, for use as a medicine.
- 如权利要求7-8任一项所述的试剂盒或组合物,所述IN10018药学上可接受的盐为酒石酸盐。The kit or composition according to any one of claims 7 to 8, wherein the pharmaceutically acceptable salt of IN10018 is tartrate.
- 如权利要求7-9任一项所述的试剂盒或组合物,其用于在对象中治疗肿瘤。The kit or composition of any one of claims 7 to 9, for use in treating a tumor in a subject.
- 如权利要求10所述的试剂盒或组合物,其中所述肿瘤选自膀胱癌、乳腺癌、子宫颈癌、结肠癌(包括结直肠癌)、食管癌、食管鳞状细胞癌、头颈癌、肝癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、黑色素瘤、骨髓瘤、横纹肌肉瘤、炎性肌纤维母细胞瘤、成神经细胞瘤、胰腺癌、前列腺癌、肾癌、肾细胞癌、肉瘤(包括骨肉瘤)、皮肤癌(包括鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌、子宫癌、间皮瘤、胆管癌、平滑肌肉瘤、脂肪肉瘤、鼻咽癌、神经内分泌癌、卵巢癌、唾液腺癌、梭形细胞癌引起的转移瘤、间变性大细胞淋巴瘤、甲状腺未分化癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、神经胶质瘤以及恶性血液病,例如急性髓细胞性白血病(AML)、急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)、慢性淋巴细胞性白血病(CLL)和慢性粒细胞白血病(CML);优选的,其中所述肿瘤为卵巢癌、肝癌、肺癌、乳腺癌、神经胶质瘤、食管癌、头颈癌、胃癌、胰腺癌或结肠癌;更优选的,所述肿瘤为卵巢癌或乳腺癌。 The kit or composition of claim 10, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland cancer, Metastatic tumors caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML); preferably, the tumor is ovarian cancer, liver cancer, lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, gastric cancer, pancreatic cancer or colon cancer; more preferably, the tumor is ovarian cancer or breast cancer.
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