Composition of carboxamidotriazole and chemotherapeutic drug and application of combination in solid tumor treatment

Abstract

The invention discloses application of carboxamidotriazole combined chemotherapeutic drugs in preparation of drugs for treating solid tumors, which mainly aims at the chemotherapeutic drugs such as gemcitabine, docetaxel or albumin paclitaxel to achieve the technical effects of effect enhancement and toxicity reduction, and the solid tumors are lung cancer, breast cancer, liver cancer and kidney cancer. In-vivo and in-vitro researches prove that the combination of carboxamidotriazole and gemcitabine, docetaxel or albumin paclitaxel can achieve a remarkable combined synergistic effect, and does not increase toxic and side effects, so that the carboxamidotriazole can benefit more patients through clinical combined medication.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composition of carboxyamide triazole and chemotherapeutic drugs and a combined use thereof for treating solid tumors. Background Art

[0002] Carboxyamide triazole is a non-cytotoxic anti-tumor drug. Its chemical name is 5-amino-1-[3,5-dichloro-4(4-chlorobenzoyl chloride)benzyl]-1H-1,2,3-triazole-4-benzamide, and its chemical structure is:

[0003]

[0004] Current studies have shown that CAI has a good effect on a variety of tumors. CAI can inhibit the proliferation of endothelial cells and a variety of tumor cell lines such as MDA-MB-231, OVCAR-3, and MCF-7. In addition, it can also inhibit the invasion of kidney cancer, bladder cancer, breast cancer, liver cancer, glioblastoma, etc. The results of early clinical studies have shown that carboxyamide triazole has low toxicity and is suitable for a wide range of people. Therefore, the development of its combined use can expand the range of people and indications, and has a high development value.

[0005] Gemcitabine is a representative of anti-metabolism anti-tumor drugs. It is widely used in the treatment of various solid tumors, such as non-small cell lung cancer, pancreatic cancer, breast cancer and ovarian cancer. At present, gemcitabine is still one of the important first-line chemotherapy drugs in clinical practice. However, its short half-life, bone marrow toxicity, hepatotoxicity, nephrotoxicity and drug resistance seriously limit the application and efficacy of gemcitabine.

[0006] Docetaxel is a taxane anti-tumor drug with strong cytotoxicity and anti-tumor effects. However, the use of docetaxel can cause many side effects, including strong bone marrow suppression and immunosuppression.

[0007] Pemetrexed is a folic acid antimetabolite anti-tumor agent that can inhibit the enzymatic conversion of folic acid metabolic derivatives. Pemetrexed combined with platinum chemotherapy has become the first choice chemotherapy for advanced non-small cell lung cancer due to its good efficacy and low toxicity. This regimen can prolong the patient's survival and improve the quality of life, but the effective rate is only 30% to 40%, and the median survival time is about 10 months. Almost all patients face the problem of drug resistance and recurrence. Some patients even have disease progression in the first efficacy evaluation after 1-2 cycles of chemotherapy, showing primary resistance to the chemotherapy regimen.

[0008] Paclitaxel is a natural product extracted from yew, which inhibits tumor cell mitosis by acting on microtubules. So far, paclitaxel is the best natural anticancer drug discovered so far. It belongs to a broad-spectrum chemotherapy drug and has good clinical efficacy in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, gastroesophageal cancer and other tumors. Because traditional paclitaxel is highly lipophilic and the active ingredient is difficult to dissolve in water, anhydrous ethanol, polyoxyethylene castor oil, etc. are used in the preparation process to help it dissolve. These organic solvents are prone to allergic reactions in patients, and severe cases may cause anaphylactic shock and endanger life. The incidence of allergic reactions is as high as 20%-40%. In order to effectively prevent, pretreatment with corticosteroids and antihistamines must be performed before medication. Paclitaxel needs to be diluted in large quantities before intravenous injection, and the infusion time must be maintained for at least 3 hours. In order to overcome this defect, albumin paclitaxel came into being. Albumin paclitaxel uses unique nanotechnology to combine hydrophobic paclitaxel with albumin, avoiding the use of solvents such as anhydrous ethanol and polyoxyethylene castor oil, and reducing toxicity. However, sensory symptoms are more common in peripheral neuropathy associated with taxanes, so it is still necessary to study how to ensure efficacy while further reducing toxicity.

[0009] Therefore, exploring combination therapy can, on the one hand, achieve complementarity between drugs, improve drug efficacy, and achieve a toxicity reduction effect. It is also expected to expand the applicable population, increase the scope of drug application, and bring hope to more patients. Summary of the invention

[0010] The purpose of the present invention is to provide the use of carboxyamide triazole combined with chemotherapy drugs in the preparation of drugs for treating solid tumors, mainly targeting chemotherapy drugs such as gemcitabine, docetaxel, pemetrexed or albumin paclitaxel, so as to achieve the technical effect of enhancing efficacy and reducing toxicity.

[0011] In some embodiments of the present invention, the solid tumor is lung cancer, breast cancer, liver cancer or kidney cancer.

[0012] More preferably, the solid tumor is lung cancer.

[0013] Furthermore, the solid tumor is non-small cell lung cancer.

[0014] Furthermore, the dosage of the carboxyamide triazole is 16 to 200 mg per square meter of body surface area.

[0015] Furthermore, the dosage of the carboxyamide triazole is 16 mg, 33 mg, 50 mg, 67 mg, 83 mg, 100 mg, 133 mg, 167 mg, and 200 mg per square meter of body surface area.

[0016] Furthermore, the dosage of gemcitabine is 800-1250 mg per square meter of body surface area.

[0017] More preferably, the dosage of gemcitabine is 1000 mg per square meter of body surface area.

[0018] Furthermore, the dosage of docetaxel is 35-100 mg per square meter of body surface area.

[0019] More preferably, the dose of docetaxel is selected from 35 mg, 45 mg, 65 mg, 75 mg, and 100 mg per square meter of body surface area.

[0020] The dosage of albumin paclitaxel is 80 to 312 mg per square meter of body surface area.

[0021] The dose of nab-paclitaxel is 260 mg per square meter of body surface area.

[0022] Furthermore, the weight ratio of the carboxyamide triazole to gemcitabine is selected from 1:2 to 50.

[0023] Preferably, the weight ratio of the carboxyamide triazole to gemcitabine is selected from 1:2, 1:5, 1:10, 1:20, 1:32, and 1:50.

[0024] Preferably, the weight ratio of the carboxyamide triazole to docetaxel is selected from 1:0.17-4.

[0025] Preferably, the weight ratio of the carboxyamide triazole to docetaxel is selected from 1:1.167, 1:1.5, 1:2, 1:3, 1:4.

[0026] Preferably, the weight ratio of the carboxyamide triazole to albumin paclitaxel is selected from 1:0.5-12.

[0027] Preferably, the weight ratio of the carboxyamide triazole to albumin paclitaxel is selected from 1:0.69, 1:1, and 1:12.

[0028] A pharmaceutical composition comprises carboxyamide triazole, and one or more of gemcitabine, docetaxel or albumin paclitaxel; and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0029] In an embodiment of the present invention, the route of administration is selected from oral administration, parenteral administration, and transdermal administration, wherein the parenteral administration includes but is not limited to intravenous injection, subcutaneous injection, and intramuscular injection, preferably oral administration. In a preferred embodiment of the present invention, carboxyamide triazole is administered orally, and gemcitabine, docetaxel, and albumin paclitaxel are administered by injection. In a preferred embodiment of the present invention, carboxyamide triazole is administered in the form of soft capsules or tablets.

[0030] The present invention also relates to a pharmaceutical composition containing carboxyamide triazole and one or more of the chemotherapy drugs gemcitabine, docetaxel, and albumin paclitaxel, comprising one or more optional pharmaceutical carriers, excipients, and / or diluents. The pharmaceutical composition can be made into any pharmaceutically acceptable dosage form. For example, a pharmaceutical preparation containing carboxyamide triazole and gemcitabine, docetaxel, or albumin paclitaxel can be formulated as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, sterile powders for injection, and concentrated solutions for injection), suppositories, inhalants, or sprays. The carboxyamide triazole and gemcitabine, docetaxel, or albumin paclitaxel of the present invention can be administered alone or used in combination with one or more therapeutic agents.

[0031] The present invention is about "combination" is a mode of administration, which refers to the administration of at least one dose of carboxyamide triazole and at least one dose of gemcitabine, docetaxel or albumin paclitaxel within a certain time period, wherein both substances show pharmacological effects. The combined administration mode of the present invention is selected from simultaneous administration, independently formulated and co-administered, or independently formulated and administered sequentially. The combination of the present invention may refer to the simultaneous administration of carboxyamide triazole and one of gemcitabine, docetaxel or albumin paclitaxel, and the "simultaneous" used herein refers to the administration of carboxyamide triazole and one of gemcitabine, docetaxel or albumin paclitaxel at least partially overlapping in time. Therefore, simultaneous administration includes a dosing regimen in which the administration of one drug continues after the administration of another drug is stopped. For example, the administration of carboxyamide triazole continues after the administration of one of gemcitabine, docetaxel or albumin paclitaxel is stopped.

[0032] The term "effective amount" refers to an amount of a drug that is effective in treating a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of a tumor; inhibit (i.e., slow down to some extent and preferably prevent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to some extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the condition. Depending on the extent to which a drug can prevent the growth of existing cancer cells and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a line graph showing the tumor volume trend of the test substance in Example 2 in the A549 xenograft tumor model.

[0034] Figure 2 This is a line graph showing the relative tumor volume trend of the test substance in Example 2 in the A549 xenograft tumor model.

[0035] Figure 3This is a bar graph of tumor weight of the test substance in Example 2 in the A549 xenograft tumor model.

[0036] Figure 4 This is a comparison chart of the actual tumor size of the test substance in Example 2 in the A549 xenograft tumor model.

[0037] Figure 5 This is a body weight trend chart of the test substance in Example 2 in the A549 xenograft tumor model.

[0038] Figure 6 This is a line graph of the body weight change rate of the test substance in Example 2 in the A549 xenograft tumor model.

[0039] Figure 7 This is a line graph showing the tumor volume trend of the test substance in Example 3 in the NCI-H1650 xenograft tumor model.

[0040] Figure 8 This is a line graph showing the relative tumor volume trend of the test substance in Example 3 in the NCI-H1650 xenograft tumor model.

[0041] Fig. 9 This is a bar graph of tumor weight of the test substance in Example 3 in the NCI-H1650 xenograft tumor model.

[0042] Fig.10 This is a comparison chart of the actual tumor size of the test substance in Example 3 in the NCI-H1650 xenograft tumor model.

[0043] Fig.11 This is a graph showing the body weight trend of the test substance in Example 3 in the NCI-H1650 xenograft tumor model.

[0044] Fig.12 This is a line graph of the body weight change rate of the test substance in Example 3 in the NCI-H1650 xenograft tumor model. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described here are only used to explain the present invention and do not limit the scope of protection of the present invention. Example 1

[0046] In vitro experimental study of carboxyamide triazole combined with chemotherapeutic drugs.

[0047] The purpose of this study was to evaluate the effects of carboxyamidotriazole (CAI) in combination with pemetrexed (PEM), docetaxel (DOC), gemcitabine (GEM) on A498, MCF7, A549, and Huh-7 cell lines using the CCK-8 method. The cell viability after treatment with different drug concentrations was detected, and the drug combination index (CI) was calculated to observe the ability of single or combined drugs to inhibit the proliferation of different types of cancer cells.

[0048] 1. Experimental Design

[0049] A498, MCF7, A549, and Huh-7 cells were cultured to the logarithmic growth phase, and a blank solvent control and a negative control were set for the cells, CAI monotherapy group, pemetrexed (PEM) / docetaxel (DOC) / gemcitabine (GEC) monotherapy group, CAI+PEM, CAI+DOC, and CAI+GEC combined drug groups. Each drug had 6 concentrations and 3 replicates. After 72 hours, the cell viability was detected by CCK-8 method and the CI was calculated. The drug solvent was DMSO.

[0050] 2. Experimental Materials

[0051] Cell lines serial number Cell Name Organization Source Processing time 1 A498 Kidney 72h 2 MCF7 Breast 72h 3 A549 Lung 72h 4 HUH-7 Liver 72h

[0052] All cells will be cultured at 37°C, 5% CO2 and 95% humidity. The culture medium used for cell culture is Gibco, and 10% fetal bovine serum is added.

[0053] Test product information: CAI was manufactured by the company; PEM, DOC, and GEC were purchased from the market. serial number Test substance purity(%) Solvents Storage conditions Storage conditions after dissolution 1 CAI 99.00 DMSO -20℃ -20℃ 2 PEM 99.04 DMSO -20℃ -80℃ 3 DOC 99.40 DMSO 4℃ -80℃ 4 GEC 99.33 DMSO 4℃ -80℃

[0054] 3. Experimental Methods

[0055] Cell culture

[0056] The cells were revived and cultured in their respective culture medium.

[0057] Cell plating and activity assay

[0058] Cells in the logarithmic growth phase were harvested and counted using a cell counter. The cell concentration was adjusted by dilution with culture medium, and 100 μL of the cell suspension was added to a 96-well cell plate (including the cell control T0 on the day of drug treatment) to make the cell density reach the specified concentration. The cells in the 96-well plate were cultured overnight at 37°C, 5% CO2 and 95% humidity.

[0059] Remove the culture medium from the 96-well plate inoculated with cells, add complete culture medium containing different concentrations of drug solution, and set up 3 replicates for each drug concentration. Place the cells in the 96-well plate with drug added at 37°C, 5% CO2, and 95% humidity for 72 hours.

[0060] After three days of culture, add 10 μL of CCK-8 solution to each well of the 96-well plate, taking care not to create bubbles. Place the 96-well plate in a cell culture incubator and incubate for 1-2 hours. Measure the absorbance at 450 nm using an ELISA reader.

[0061] Data processing and analysis

[0062] GraphPad Prism 5.0 software was used to analyze the data, and nonlinear S-curve regression was used to fit the data to obtain the dose-effect curve, from which the IC 50 value.

[0063] Cell survival rate (%) = (OD 待测药 -OD 培养液对照 ) / (OD 细胞对照 -OD 培养液对照 )×100%.

[0064] The compound joint action index analysis is calculated using the median effect principle. The formula for the joint action index of two compounds is:

[0065]

[0066] Where:

[0067] (D)1——the concentration required for compound 1 to produce effect X when it works in combination with other compounds;

[0068] (D)2——the concentration required for compound 2 to produce effect X when it works in combination with other compounds;

[0069] (Dx)1——the concentration of compound 1 required to produce effect X when used alone;

[0070] (Dx)2 – the concentration of compound 2 required to produce effect X when used alone.

[0071] The CI value can be used to quantitatively determine the nature and strength of the interaction between different compounds (Table 2). The interaction is measured by the average CI value (Weighted Average CI, CIwt), and the formula is:

[0072]

[0073] Where:

[0074] CI50 – CI value at EC50 (concentration at which the effect reaches 50%);

[0075] The same applies to CI75, CI90, and CI95.

[0076] The final measured average CI value, i.e., the combination drug index, was comprehensively evaluated according to the evaluation levels in Table 1. Table 1 Average CI value intervals and interaction evaluation of drug combination effects

[0077] IV. Experimental Results

[0078] Cell culture observation

[0079] During the cell culture process, there was no contamination, drug precipitation or crystallization.

[0080] Test results Table 2. CI values ​​and interaction evaluation of CAI combined with chemotherapeutic drugs on A498 cells Table 3. CI values ​​and interaction evaluation of CAI combined with chemotherapy drugs on MCF7 cells

[0081] Table 4 CI values ​​and interaction evaluation of CAI combined with chemotherapy drugs on A549 cells A549 IC50(CAI) / μM IC50(chemical drug) / μM CI (Combined Index) Interaction evaluation CAI+PEM 1.84 0.14 <0.1 The synergy is very strong CAI+DOC 0.96 0.0008 <0.01 The synergy is very strong CAI+GEC 0.41 0.001 <0.01 The synergy is very strong

[0082] Table 5 CI values ​​and interaction evaluation of CAI combined with chemotherapeutic drugs on Huh-7 cells Huh-7 IC50(CAI) / μM IC50(chemical drug) / μM CI (Combined Index) Interaction evaluation CAI+PEM 5.9 0.05 0.08 The synergy is very strong CAI+DOC 5.88 0.08 0.67 Strong synergistic effect CAI+GEC 3.46 0.15 0.81 Moderate synergy

[0083] 5. Experimental Conclusion

[0084] From the above experimental results, it can be seen that in A498, MCF-7, A549 and Huh-7 cell lines, carboxyamide triazole combined with pemetrexed, docetaxel and gemcitabine showed a very strong combined effect. Therefore, it is expected to increase the new indications for the combination of carboxyamide triazole to achieve the effect of increasing efficacy and reducing toxicity, such as for indications such as renal cancer, breast cancer, lung cancer, liver cancer, etc. Example 2

[0085] Study on the inhibitory effect of carboxyamidotriazole combined with docetaxel and albumin-paclitaxel on the growth of human lung cancer A549 cell xenografts in nude mice.

[0086] 1. Experimental Animals

[0087] 1.1 Animal information

[0088] Species and strain: BALB / c Nude mice

[0089] Gender and age: female, 8-10 weeks old

[0090] Body weight: 18-20g, with a deviation of approximately ±20% of the mean body weight.

[0091] Number of animals vaccinated: 80

[0092] Number of animals in the group: 60

[0093] Animal source: Jiangsu Jicui Yaokang Biotechnology Co., Ltd., production license number: SCXK(Su)2018-0008, animal certificate number: 202329657.

[0094] 1.2 Animal husbandry

[0095] Living conditions: SPF environment, IVC mouse cages, 5 mice per cage, temperature: 20-26℃, humidity: 40-70%, light: 12 hours day and night alternation. Feed: irradiated mouse feed, purchased from Beijing Keao Xieli Feed Co., Ltd., free access. Drinking water: city tap water, filtered and sterilized before drinking. Bedding: corn cobs, purchased from Keao Xieli, used after autoclave, changed once a week. Adaptive feeding: mice were given an adaptive feeding period of no less than 7 days before the experiment. Animal identification: Each mouse cage is hung with an experimental information label card, which includes mouse information, cell inoculation information, animal experiment information and experimenter information, etc. Mice are marked with ear tags.

[0096] 1.3 Laboratory Animal Operation and Management Standards

[0097] All experimental animals were kept in an SPF-level animal laboratory during the experiment.

[0098] All operations and management of experimental animals strictly complied with the Guiding Principles for the Use and Care of Laboratory Animals.

[0099] 2. Experimental Materials

[0100] 2.1 Test drugs

[0101] The information of the test substances is shown in the table below, among which carboxyamide triazole was produced by the company; docetaxel and albumin paclitaxel were purchased from the market. Table 6 Test substance information

[0102] 2.2 Solvent prescription and storage conditions of dosing solution

[0103] The solvent of CAI is 100% PEG400; the storage conditions of the dosing solution are: room temperature and dark, and the shelf life is 3 days. The solvent of Docetaxel is 0.9% Saline; the storage conditions of the dosing solution are: 4℃ and dark, and it is used immediately after preparation. The solvent of Abraxane is 0.9% Saline; the storage conditions of the dosing solution are: 4℃ and dark, and it is used immediately after preparation.

[0104] 2.3 Cell lines

[0105] Human lung cancer cell line A549 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

[0106] 2.4 Culture medium

[0107] F12K basal culture medium and fetal bovine serum were purchased from GIBCO (Grand Island, NY, USA); Matrigel was purchased from CORNING (Corning, NY, USA).

[0108] 3. Experimental Design

[0109] The experimental design is shown in Table 7. Table 7. Experimental design for in vivo efficacy evaluation

[0110] IV. Experimental Methods

[0111] 4.1 Model establishment

[0112] A549 cells were cultured in F12K medium containing 10% FBS and maintained in a 37°C saturated humidity cell culture incubator with 5% CO2. A549 cells in the logarithmic growth phase were collected and resuspended in F12K basal medium containing 50% Matrigel. The cell concentration was adjusted to 5×10 7 / mL. Under sterile conditions, 0.1mL of cell suspension was inoculated subcutaneously on the right side of the mouse, with an inoculation concentration of 5×10 6 / 0.1mL / mouse.

[0113] 4.2 Grouping and Dosing

[0114] When the average tumor volume reaches 150 mm 3 Around 200 μg / cm2, the animals were randomly divided into groups according to tumor volume, so that the difference in tumor volume between groups was less than 10% of the mean. The day of grouping was designated as Day 0, and drug administration was started according to the animal weight. During the drug administration period, if the body weight of individual animals decreased by more than 15% compared with Day 0 (BWL ≥ 15%), the drug treatment would be stopped until the animal weight recovered (BWL < 15%), and then drug administration would be resumed.

[0115] 4.3 Weighing and observation

[0116] Animal body weight and tumor volume were measured twice a week during the experiment. Tumor length and width were measured by digital calipers, and tumor volume was estimated by length and width measurements. Animal clinical symptoms were observed and recorded once a day during the experiment. Clinical observations included the overall health status of the animals, abnormal weight, abnormal behavior, and other adverse reactions related to administration.

[0117] 4.4 Experimental endpoint description

[0118] According to animal welfare regulations, if any of the following conditions are met during the experiment, the animal will be removed from the experimental group and euthanized. 1. The animal's body weight decreases by more than 20% compared to Day 0 (BWL ≥ 20%); 2. The animal has severe adverse reactions, such as blindness, paralysis, etc.; 3. The tumor volume is greater than 2000mm 3 ; 4. Open ulcers formed on the surface of the tumor. The experimental period was 21 days. After the last weighing at the end of the experiment, the remaining animals were euthanized with CO2, and the tumor was weighed and photographed before the experiment ended.

[0119] 4.5 Evaluation indicators

[0120] The tumor volume (TV) was calculated as follows: 1 / 2 × a × b 2 , where a and b are the measured length and width of the tumor, respectively.

[0121] Tumor growth inhibition rate (%TGI TV )The calculation formula is: (1-TV T / TV C )×100%,TV C is the average tumor volume of the negative control group, TV T is the average tumor volume of the treatment group.

[0122] The relative tumor volume (RTV) was calculated as follows: Vt / V0, where V0 was the tumor volume at the time of grouping and Vt was the tumor volume at each measurement.

[0123] Relative tumor proliferation rate (%T / C RTV ) is calculated as: RTV / C RTV × 100%, where T RTV RTV for the treatment group, C RTV RTV was the negative control group.

[0124] Tumor inhibition rate (%TGI TW )The calculation formula is: (TW C -TWT ) / TW C × 100%, of which TW C is the average tumor weight of the negative control group, TW T is the average tumor weight of the treatment group.

[0125] The calculation formula of animal body weight change rate (%BWC) is: (BWt-BW0) / BW0×100%, where BWt is the animal weight at each measurement and BW0 is the animal weight at grouping.

[0126] According to the China NMPA "Technical Guidelines for Non-clinical Studies of Cytotoxic Anti-tumor Drugs" (November 2006), %T / C RTV ≤40% and P<0.05 after statistical analysis was considered effective. If the number of drug-related animal deaths exceeded 20%, the drug dose was considered to be severely toxic.

[0127] 5. Statistical Analysis

[0128] In this study, experimental data were expressed as Mean ± SEM.

[0129] With time point as the X-axis, tumor volume (mm 3 ) as the Y axis to draw the tumor growth curve; the time point as the X axis, the animal weight (g) as the Y axis to draw the animal weight change curve. The two-tailed t-test was used for comparison between groups, P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference (Microsoft Excel 2007, Redmond, WA, USA).

[0130] 6. Experimental Results

[0131] A549 cells in the logarithmic growth phase were collected and inoculated into the right fat pad of mice. The actual inoculation time was 5×10 6 / 0.1mL / mouse. A total of 80 animals were inoculated in this experiment, and 60 animals were used for grouping. After grouping, the drugs were administered immediately. The CAI 40mg / kg group was orally administered once a day for 21 consecutive days (PO, QD×21), the Abraxane 20mg / kg group was administered once a week through the tail vein for 3 consecutive times (IV, QW×3wks), and the Docetaxel 7.5mg / kg group was administered once a week through the tail vein for 3 consecutive times (IV, QW×3wks). The remaining animals were killed on Day 21, and the tumors were weighed and photographed for record.

[0132] The experimental results are shown below;

[0133] ① Effect of the test substance on tumor size in the A549 xenograft tumor model Table 8. Antitumor activity of the test substances in the A549 xenograft tumor model (21 days)

[0134] Note: *: P<0.05 compared with the solvent control group, **: P<0.01 compared with the solvent control group, ***: P<0.001 compared with the solvent control group, $: P<0.05 compared with group 2, $$: P<0.01 compared with group 2, &: P<0.05 compared with group 3, #: P<0.05 compared with group 4, ##: P<0.01 compared with group 4, ###: P<0.001 compared with group 4.

[0135] Combination Figures 1 to 4 As shown in Table 8, on Day 21, the average tumor weight of the Docetaxel 7.5 mg / kg single-dose group was 0.444±0.049 g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 49.14%, which was significantly different from the average tumor weight of the vehicle control group (0.873±0.103g) (P<0.01). RTV It was 59.77% (P<0.001).

[0136] The average tumor weight of the Abraxane 20mg / kg group was 0.499±0.055g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 42.84%, which was significantly different from the average tumor weight of the vehicle control group (P<0.01). RTV It was 62.06% (P<0.001).

[0137] The average tumor weight of the CAI HD 40 mg / kg single-dose group was 0.538±0.066 g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 38.37%, which was significantly different from the average tumor weight of the vehicle control group (P<0.05). RTV 68.46% (P<0.01).

[0138] The average tumor weight of the CAI HD 40mg / kg and Docetaxel 7.5mg / kg combined group was 0.291±0.028g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 66.67%, which was significantly different from the average tumor weight of the vehicle control group (P<0.001). RTV It was 44.57% (P<0.001).

[0139] The average tumor weight of the CAI HD 40 mg / kg and Abraxane 20 mg / kg combined group was 0.311±0.055 g (N=9), and the tumor inhibition rate %TGI TW The relative tumor growth rate (%T / C) was 64.38%, which was significantly different from the average tumor weight of the vehicle control group (P<0.001). RTV It was 42.86% (P<0.001).

[0140] CAI HD 40mg / kg alone can significantly inhibit the growth and proliferation of human lung cancer A549 xenografts in Balb / c Nude mice (P<0.05). Docetaxel 7.5mg / kg and Abraxane 20mg / kg alone can extremely significantly inhibit the growth and proliferation of human lung cancer A549 xenografts in Balb / c Nude mice (P<0.01, P<0.01). CAI HD 40mg / kg and Docetaxel 7.5mg / kg combined administration can extremely significantly inhibit the growth and proliferation of human lung cancer A549 xenografts in Balb / c Nude mice (P<0.001). Compared with CAI HD 40 mg / kg alone, the combined administration of CAI HD 40 mg / kg and Docetaxel 7.5 mg / kg significantly inhibited the growth and proliferation of human lung cancer A549 xenografts in Balb / c Nude mice (P<0.01). Compared with Docetaxel 7.5 mg / kg alone, the combined administration of CAI HD 40 mg / kg and Docetaxel 7.5 mg / kg significantly inhibited the growth and proliferation of human lung cancer A549 xenografts in Balb / c Nude mice (P<0.05). The combined administration of CAI HD 40 mg / kg and Abraxane 20 mg / kg can significantly inhibit the growth and proliferation of human lung cancer A549 xenografts in Balb / c Nude mice (P<0.001). The combined administration of CAI HD 40 mg / kg and Abraxane 20 mg / kg significantly inhibited the growth and proliferation of human lung cancer A549 xenograft tumors in Balb / c Nude mice compared with the administration of CAI HD 40 mg / kg or Abraxane 20 mg / kg alone (P<0.05, P<0.05).

[0141] ② Effects of the test substances on the body weight of tumor-bearing animals

[0142] In this experiment, the effects of CAI, Abraxane and Docetaxel alone and in combination on the body weight of human lung cancer A549 animals are shown in Table 9. Figure 5 and Figure 6 . Table 9 Effects of the test substances on animal body weight in the A549 xenograft tumor model (21 days)

[0143] The experimental results showed that on Day 21, the vehicle control group, the Abraxane 20 mg / kg single administration group, the CAI HD 40 mg / kg single administration group, and the CAI HD 40 mg / kg and Docetaxel 7.5 mg / kg combined administration group had no inhibitory effect on the weight growth of animals, and the weight of animals increased by 0.56%-6.44% (0.10g-1.42g) compared with the beginning of the experiment on Day 0. The Docetaxel 7.5 mg / kg single administration group and the CAI HD 40 mg / kg and Abraxane 20 mg / kg combined administration group all had an inhibitory effect on the weight growth of animals, and the weight of animals decreased by 0.81%-1.13% (0.18g-0.28g) compared with the beginning of the experiment on Day 0.

[0144] In summary, the CAI HD 40mg / kg and Docetaxel 7.5mg / kg co-administration groups and the CAI HD 40mg / kg and Abraxane 20mg / kg co-administration groups all had a combined synergistic effect. During the experiment, no changes in animal body weight and toxic side effects related to the co-administration of CAI HD 40mg / kg and Docetaxel 7.5mg / kg were observed, indicating that the test substance was well tolerated at the administered dose. Example 3

[0145] This example investigates the inhibitory effect of combined administration of the test substances on the growth of human lung cancer NCI-H1650 cell xenograft tumors in nude mice.

[0146] 1. Experimental Materials

[0147] 1. Animal information

[0148] Species and strains: BALB / c Nude mice, gender and age: female, 8-10 weeks old, body weight: 18-20g, deviation is about ±20% of the mean body weight. Number of inoculated animals: 120, number of grouped animals: 90. Animal source: Jiangsu Jicui Yaokang Biotechnology Co., Ltd., production license number: SCXK (Su) 2018-0008, animal qualification certificate number: 202330296.

[0149] The animal husbandry and experimental animal operation management specifications are the same as in Example 2.

[0150] 2. Test drugs

[0151] The information of the test substances is shown in the table below, among which carboxyamide triazole is homemade by the company; pemetrexed and gemcitabine are commercially available products. Table 10 Test substance information

[0152] 3. Solvent prescription and storage conditions of dosing solution

[0153] CAI: Solvent: 100% PEG400, Storage conditions of dosing solution: Store at room temperature away from light, shelf life 3 days. Pemetrexed solvent: 0.9% Saline, Storage conditions of dosing solution: Store at 4℃ away from light, prepare and use immediately. Gemcitabine solvent: 0.9% Saline; Storage conditions of dosing solution: Store at 4℃ away from light, prepare and use immediately.

[0154] 4. Cell lines and culture medium

[0155] Human lung cancer cell line NCI-H1650 was purchased from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences. RPMI 1640 basal medium and fetal bovine serum were purchased from GIBCO (Grand Island, NY, USA); Matrigel was purchased from CORNING (Corning, NY, USA).

[0156] 2. Experimental Design

[0157] The experimental design is shown in Table 11. Table 11 Dosage regimen design

[0158] 3. Experimental Methods

[0159] 1. Model building

[0160] NCI-H1650 cells were cultured in RPMI 1640 medium containing 10% FBS and maintained in a 37°C saturated humidity cell culture incubator with 5% CO2. NCI-H1650 cells in the logarithmic growth phase were collected and resuspended in RPMI1640 basal medium containing 50% Matrigel, and the cell concentration was adjusted to 2×10 7 / mL. Under sterile conditions, 0.1mL of cell suspension was inoculated subcutaneously on the right side of the mouse, with an inoculation concentration of 2×10 6 / 0.1mL / mouse.

[0161] 2. Grouping and Dosing

[0162] When the average tumor volume reaches 150 mm 3 Around 200 μg / cm2, the animals were randomly divided into groups according to tumor volume, so that the difference in tumor volume between groups was less than 10% of the mean. The day of grouping was designated as Day 0, and drug administration was started according to the animal weight. During the drug administration period, if the body weight of individual animals decreased by more than 15% compared with Day 0 (BWL ≥ 15%), the drug treatment would be stopped until the animal weight recovered (BWL < 15%), and then drug administration would be resumed.

[0163] The weighing and observation, experimental endpoint description, evaluation indexes and statistical analysis methods are the same as those in Example 2.

[0164] IV. Experimental Results

[0165] NCI-H1650 cells in logarithmic growth phase were collected and inoculated into the right fat pad of mice. The actual inoculation time was 2×10 6 / 0.1mL / mouse. A total of 120 animals were inoculated in this experiment, and 90 animals were used for grouping. After grouping, the drugs were given immediately. The CAI 40mg / kg and 20mg / kg dose groups were orally administered once a day for 21 consecutive days (PO, QD×21), the Pemetrexed 60mg / kg dose group was intraperitoneally administered once a day for 21 consecutive days (IP, QD×21), and the Gemcitabine 15mg / kg dose group was intraperitoneally administered twice a week for 6 consecutive times (IP, BIW×3wks). The remaining animals were killed on Day 21, and the tumors were weighed and photographed for record.

[0166] 1. Evaluation of anti-tumor activity of test substances

[0167] In this experiment, the effects of CAI, Pemetrexed and Gemcitabine alone and in combination on the tumor volume of human lung cancer NCI-H1650 are shown in Table 12. Figures 7-10 shown.

[0168] Day 21, the average tumor weight of the Pemetrexed 60mg / kg group was 0.534±0.094g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 48.16%, which was significantly different from the average tumor weight of the vehicle control group (1.030±0.214g) (P<0.05). RTV It was 60.64% (P>0.05).

[0169] The average tumor weights of the CAI HD 40 mg / kg and CAI LD 20 mg / kg groups were 0.753±0.157 g and 0.859±0.109 g, respectively. The tumor inhibition rate was %TGI TWThe relative tumor growth rate (%T / C) was 26.89% and 16.60%, respectively, which had no significant difference compared with the average tumor weight of the vehicle control group (P>0.05, P>0.05). RTV The percentages were 77.90% (P>0.05) and 91.25% (P>0.05) respectively.

[0170] The average tumor weight of the CAI HD 40mg / kg and Pemetrexed 60mg / kg combined group was 0.487±0.086g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 52.72%, which was significantly different from the average tumor weight of the vehicle control group (P<0.05). RTV The average tumor weight of the CAI LD 20mg / kg and Pemetrexed 60mg / kg combined group was 0.642±0.113g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 37.67%, which was not significantly different from the average tumor weight of the vehicle control group (P>0.05). RTV It was 66.90% (P>0.05).

[0171] The average tumor weight of the group treated with Gemcitabine 15 mg / kg alone was 0.372±0.065 g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 63.88%, which was significantly different from the average tumor weight of the vehicle control group (P<0.01). RTV It was 42.55% (P<0.01).

[0172] The average tumor weight of the CAI HD 40mg / kg and Gemcitabine 15mg / kg combined group was 0.274±0.051g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 73.40%, which was significantly different from the average tumor weight of the vehicle control group (P<0.01). RTV The average tumor weight of the CAI LD 20mg / kg and Gemcitabine 15mg / kg combined group was 0.368±0.065g, and the tumor inhibition rate was %TGI TW The relative tumor growth rate (%T / C) was 64.27%, which was significantly different from the average tumor weight of the vehicle control group (P<0.01). RTV It was 45.74% (P<0.01).

[0173] The experimental results showed that Pemetrexed 60mg / kg alone, CAI HD 40mg / kg and Pemetrexed 60mg / kg combined administration can significantly inhibit the growth and proliferation of human lung cancer NCI-H1650 xenograft tumors in Balb / c Nude mice (P<0.05, P<0.05). CAI HD 40mg / kg and Gemcitabine 15mg / kg combined administration can effectively inhibit the growth and proliferation of human lung cancer NCI-H1650 xenograft tumors in Balb / c Nude mice (% T / C RTV ≤40%, P<0.05). Gemcitabine 15mg / kg alone and CAI LD 20mg / kg and Gemcitabine 15mg / kg combined administration can significantly inhibit the growth and proliferation of human lung cancer NCI-H1650 xenograft tumors in Balb / c Nude mice (P<0.01, P<0.01). Compared with CAI HD 40mg / kg alone, the combined administration of CAI HD 40mg / kg and Gemcitabine 15mg / kg significantly inhibited the growth and proliferation of human lung cancer NCI-H1650 xenograft tumors in Balb / c Nude mice (P<0.01), and there was no significant difference compared with Gemcitabine 15mg / kg alone (P>0.05). The combined administration of CAI LD 20 mg / kg and Gemcitabine 15 mg / kg significantly inhibited the growth and proliferation of human lung cancer NCI-H1650 xenograft tumors in Balb / c Nude mice compared with CAI LD 20 mg / kg alone (P<0.01), but there was no significant difference compared with Gemcitabine 15 mg / kg alone (P>0.05). Table 12 Antitumor activity of the test substance in the NCI-H1650 xenograft tumor model (Day 21)

[0174] Note: *: P<0.05 compared with the solvent control group, **: P<0.01 compared with the solvent control group, &: P<0.05 compared with group 3, &&: P<0.01 compared with group 3, &&&: P<0.001 compared with group 3, ##: P<0.01 compared with group 4.

[0175] 2. Effects of the test substance on the body weight of tumor-bearing animals

[0176] In this experiment, the effects of CAI, Pemetrexed and Gemcitabine alone and in combination on the body weight of human lung cancer NCI-H1650 animals are shown in Table 13. Figures 11-12 .

[0177] The experimental results showed that the vehicle control group, Pemetrexed 60 mg / kg single-administration group, CAI HD 40 mg / kg single-administration group, CAI LD 20 mg / kg single-administration group, Gemcitabine 15 mg / kg single-administration group, and CAI LD 20 mg / kg and Gemcitabine 15 mg / kg combined administration group had no inhibitory effect on animal weight growth. The average animal weight increased by 0.55%-5.28% (0.13g-1.22g) compared with the beginning of the experiment on Day 0. The average animal weight in the CAI HD 40 mg / kg and Pemetrexed 60 mg / kg combined administration group, CAI LD 20 mg / kg and Pemetrexed 60 mg / kg combined administration group, and CAI HD 40 mg / kg and Gemcitabine 15 mg / kg combined administration group was 2.5477 g / cm2 and 1.822 g / cm2, respectively. Compared with the beginning of the experiment, the concentration of 0 was reduced by 1.02%-5.82% (0.26g-1.36g). No drug-related animal deaths were observed during the experiment. Table 13 Effects of the test substances on animal body weight in the NCI-H1650 xenograft tumor model

[0178] In summary, the combined administration of CAI HD 40mg / kg and Pemetrexed 60mg / kg can significantly inhibit the growth and proliferation of human lung cancer NCI-H1650 xenograft tumors in Balb / c Nude mice. During the experiment, the weight loss of animals associated with the combined administration of CAI HD40mg / kg and Pemetrexed 60mg / kg was observed. In summary. The low-dose group of CAI combined with Pemetrexed had no obvious effect, and although the high-dose group had obvious effects, the toxic effects also increased, and the effect of increasing efficacy and reducing toxicity could not be achieved.

[0179] The combined administration of CAI HD 40mg / kg and Gemcitabine 15mg / kg can effectively inhibit the growth and proliferation of human lung cancer NCI-H1650 xenograft tumors in Balb / c Nude mice, and is significantly better than the CAI HD 40mg / kg single administration group. During the experiment, the weight loss of animals associated with the combined administration of CAI HD 40mg / kg and Gemcitabine 15mg / kg was observed. The combined administration of CAI LD 20mg / kg and Gemcitabine 15mg / kg group can significantly inhibit the growth and proliferation of human lung cancer NCI-H1650 xenograft tumors in Balb / c Nude mice, and is significantly better than the CAI LD20mg / kg single administration group. During the experiment, no animal weight changes and toxic side effects related to the combined administration of CAI LD 20mg / kg and Gemcitabine 15mg / kg were observed, indicating that the test substance was well tolerated at the administered dose.

[0180] The combination of carboxyamide triazole with a variety of chemotherapy drugs shows that not all chemotherapy drugs are suitable for combination therapy, and even if the in vitro activity is significant, it still needs to be combined with in vivo animal experiments for comprehensive evaluation. In this application, albumin paclitaxel, docetaxel and gemcitabine were selected as the preferred combination drug candidates to improve the application direction and popularity of carboxyamide triazole and benefit more patients.

Claims

1. Use of carboxyamide triazole combined with chemotherapeutic drugs in the preparation of drugs for treating solid tumors, characterized in that: The chemotherapy drug is gemcitabine, docetaxel or albumin-paclitaxel.

2. The use according to claim 1, characterized in that The solid tumor is lung cancer, breast cancer, liver cancer or kidney cancer.

3. The use according to claim 2, characterized in that: The solid tumor is lung cancer.

4. The use according to claim 3, characterized in that The solid tumor is non-small cell lung cancer.

5. The use according to claim 1, characterized in that: The dosage of the carboxyamide triazole is 16 to 200 mg per square meter of body surface area.

6. The use according to claim 1, characterized in that: The dosage of the carboxyamide triazole is 16 mg, 33 mg, 50 mg, 67 mg, 83 mg, 100 mg, 133 mg, 167 mg, and 200 mg per square meter of body surface area.

7. The use according to claim 1, characterized in that The dosage of gemcitabine is 800-1250 mg per square meter of body surface area.

8. The use according to claim 7, characterized in that The dose of gemcitabine was 1000 mg per square meter of body surface area.

9. The use according to claim 1, characterized in that The dosage of docetaxel is 35-100 mg per square meter of body surface area.

10. The use according to claim 9, characterized in that The dose of docetaxel is selected from 35 mg, 45 mg, 65 mg, 75 mg, and 100 mg per square meter of body surface area.

11. The use according to claim 1, characterized in that The dosage of albumin paclitaxel is 80 to 312 mg per square meter of body surface area.

12. The use according to claim 11, characterized in that The dose of nab-paclitaxel is 260 mg per square meter of body surface area.

13. The use according to claim 1, characterized in that The weight ratio of the carboxyamide triazole to gemcitabine is selected from 1:2 to 50.

14. The use according to claim 13, characterized in that The weight ratio of the carboxyamide triazole to gemcitabine is selected from 1:2, 1:5, 1:10, 1:20, 1:32, and 1:

50.

15. The use according to claim 1, characterized in that The weight ratio of the carboxyamide triazole to docetaxel is selected from 1:0.17-4.

16. The use according to claim 1, characterized in that The weight ratio of the carboxyamide triazole to docetaxel is selected from 1: 1.167，1：1.5，1：2，1：3，1：4。 17. The use according to claim 1, characterized in that The weight ratio of the carboxyamide triazole to albumin paclitaxel is selected from 1:0.5-12.

18. The use according to claim 17, characterized in that The weight ratio of the carboxyamide triazole to nab paclitaxel is selected from 1: 0.69，1:1，1:12。 19. A pharmaceutical composition comprising carboxyamide triazole, and one or more of gemcitabine, docetaxel or nab-paclitaxel; and a pharmaceutically acceptable carrier, excipient and / or diluent.

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