Antitumor pharmaceutical composition comprising azvudine and chemotherapeutic agent

Abstract

To provide a pharmaceutical composition comprising azvudine and a chemotherapeutic agent for preventing or treating tumor diseases.SOLUTION: A pharmaceutical composition comprises: (i) azvudine or a pharmaceutically acceptable salt, a stereoisomer or an isotopic derivative thereof; and (ii) a chemotherapeutic agent represented by capecitabine, cyclophosphamide, dacarbazine, or paclitaxel, or avastin. The pharmaceutical composition of the present invention shows a superior synergistic effect in antitumor applications, and can reduce the dosage of chemotherapeutic agent, and improve the therapeutic effect and safety, thereby achieving the goal of prolonging the survival of patients.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of medicine, specifically to an antitumor pharmaceutical composition containing azuvudine. [Background technology]

[0002] Deoxycytidine kinase (DCK) is an enzyme with broad substrate specificity that can phosphorylate pyrimidine and purine deoxynucleosides, and is a key enzyme in the salvage pathway of deoxynucleoside biosynthesis, maintaining normal DNA metabolism and phosphorylating various antiviral and anticancer nucleoside analogue drugs, which are activated only after phosphorylation, thereby inhibiting tumor growth. Radiotherapy strategies for apoptosis, which have been widely studied in the past decades, have become one of the important tools in tumor therapy.

[0003] Azuvudine (FNC) is a broad-spectrum RNA virus inhibitor, which, as an artificially synthesized nucleoside analogue of viral RNA-dependent RNA polymerase (RdRp), is metabolized to a 5'-triphosphate metabolite (azuvudine triphosphate) with antiviral activity in cells, and can act specifically on the novel coronavirus polymerase (RdRp), targeting the viral RdRp and inhibiting the activity of RdRp in host cells, thereby blocking the synthesis and replication of RNA chains. In July 2021, Azuvudine tablets were approved for sale in China to treat adult HIV-1-infected patients with high viral loads. In July 2022, Azuvudine was also approved as a treatment for COVID-19.

[0004] Patent document CN201010506595.X discloses the use of azuvidine to treat tumors such as colon cancer, liver cancer, gastric cancer, esophageal cancer, lung cancer, breast cancer, cervical cancer, leukemia, lymphoma, etc. It has been found that azuvidine has obvious inhibitory effects on various human cancer cells and transplanted tumors in animals.

[0005] Among conventional tumor treatment methods, chemotherapy has become the most widely used clinical tumor treatment method due to its strong therapeutic effect and high efficacy. However, most of the chemotherapeutic drugs currently used in clinical practice have low selectivity, and while treating tumor tissue, they may also cause significant damage to normal tissues in the human body, leading to serious toxic side effects.

[0006] Monotherapy has disadvantages such as poor physical and chemical properties, low bioavailability, and low efficacy. Therefore, if chemotherapy drugs can be combined with azuvidine, it may enhance the antitumor effect. Based on the complementary synergistic effect between the two drugs in the antitumor mechanism, both drugs target tumor cells, so that chemotherapy and immunity work together to enhance the antitumor effect. Summary of the Invention

[0007] The present disclosure provides a pharmaceutical composition of azuvudine (FNC) and a chemotherapeutic agent, and the use of this pharmaceutical composition in the preparation of a medicament for preventing or treating a tumor disease.

[0008] The pharmaceutical composition of the present invention has the following advantages compared to each single agent. 1. After combined use, the tumor-suppressing effect of each single agent was improved. 2. The development of drug resistance is delayed, improving efficacy and safety, thereby extending patient survival.

[0009] In order to solve the technical problem of the present invention, the present invention comprises: (i) azuvidine or a pharma- ceutically acceptable salt, stereoisomer or isotopic derivative thereof; (ii) a chemotherapeutic agent.

[0010] In a preferred technical embodiment of the present invention, the chemotherapeutic agent is capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofazimine, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, paclitaxel, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, folinic acid, doxorubicin liposomal, daunorubicin liposomal The agent is selected from the group consisting of cyclosporine, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paliittazine, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, triosphane, vinblastine, vincristine, vindesine, vinorelbine, and any combination thereof.

[0011] In a preferred technical embodiment of the present invention, the chemotherapeutic agent is selected from the group consisting of capecitabine, cyclophosphamide, dacarbazine, paclitaxel, and any combination thereof.

[0012] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: (i) azuvidine or a pharma- ceutically acceptable salt, stereoisomer or isotopic derivative thereof; (ii) Avastin.

[0013] In a preferred technical embodiment of the present invention, (i) and (ii) are administered simultaneously, separately or sequentially, or (i) and (ii) are present in the same dosage form.

[0014] In a preferred technical embodiment of the present invention, it is used to treat tumor-related diseases.

[0015] In a preferred technical embodiment of the present invention, the tumor-related disease is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, melanoma, brain tumor, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, lung cancer, renal cancer, skin cancer, glioblastoma, neuroblastoma, sarcoma, liposarcoma, osteochondroma, osteoma, osteosarcoma, seminoma, testicular tumor, uterine cancer, head and neck tumor, multiple myeloma, malignant lymphoma, polycythemia vera, leukemia, thyroid tumor, ureter tumor, bladder tumor, gallbladder cancer, non-small cell lung cancer, bile duct carcinoma, and choriocarcinoma.

[0016] In some embodiments, the dose of the azuvudine ranges from 1 to 100 mg and the dose of the chemotherapeutic agent ranges from 1 to 500 mg.

[0017] In some embodiments, the dose of azuvudine ranges from 1 to 100 mg and the dose of bevacizumab (Avastin) ranges from 1 to 500 mg.

[0018] The dosages of azuvudine described in this disclosure include 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg , 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74mg, 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 82mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96mg, 97mg, 98mg, 99mg, 100mg.

[0019] Doses of chemotherapeutic agents described in this disclosure include 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg , 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43mg, 44mg, 45mg, 46mg, 47mg, 48mg, 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg , 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74mg , 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 82mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93m g, 94mg, 95mg, 96mg, 97mg, 98mg, 99mg, 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, and 500 mg.

[0020] In some embodiments, the azuvidine may have a dose in the range of 1-100 mg and may be administered once, twice, or three times a day, and the chemotherapeutic agent may have a dose in the range of 1-500 mg and may be administered once, twice, or three times a day.

[0021] In some embodiments, the azuvidine has a dose in the range of 1-50 mg and can be administered once a day or twice a day, and the chemotherapeutic agent has a dose in the range of 1-100 mg and is administered once a day.

[0022] In some embodiments, the azuvidine has a dose in the range of 1-20 mg and can be administered once or twice daily, and the chemotherapeutic agent has a dose in the range of 1-40 mg and is administered once daily.

[0023] In some embodiments, the azuvidine has a dose in the range of 1-10 mg and can be administered once a day or twice a day, and the chemotherapeutic agent has a dose in the range of 1-10 mg and is administered once a day.

[0024] In some embodiments, the chemotherapeutic agent is administered at a dose of 1 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, and the dosage is 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg and the administration frequency is once a day or twice a day, and the chemotherapy agent has a dosage of 10 mg, 20 mg, 40 mg, 60 mg and the administration frequency is once a day.

[0025] In some embodiments, the azumab is administered at a dose of 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg and at a frequency of once daily or twice daily; or the azumab is administered at a dose of 1 mg, 2 mg, 4 mg, or 6 mg and at a frequency of once daily.

[0026] In some embodiments, the chemotherapeutic agent has a dosage of 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg and an administration frequency of once daily or twice daily, and the chemotherapeutic agent has a dosage of 10 mg, 20 mg, 40 mg, 60 mg and an administration frequency of once daily.

[0027] In some embodiments, the chemotherapeutic agent has a dosage of 1 mg, 2 mg, 4 mg, 6 mg, or 8 mg and is administered once daily or twice daily; and the chemotherapeutic agent has a dosage of 1 mg, 2.5 mg, 5 mg, or 10 mg and is administered once daily.

[0028] The combined administration routes according to the present invention include oral administration, parenteral administration, and transdermal administration. The parenteral administration includes, but is not limited to, intravenous injection, subcutaneous injection, and intramuscular injection, and is preferably oral administration.

[0029] The present invention also provides a pharmaceutical composition comprising the above-mentioned azuvidine, a chemotherapeutic agent, and one or more pharma- ceutically acceptable carriers, excipients, and diluents. The pharmaceutical composition can be in any pharma- ceutically acceptable dosage form. For example, it can be formulated as a tablet, capsule, drop pill, granule, solution, suspension, syrup, injection (including infusion, sterile powder for injection, and concentrated solution for injection), suppository, inhalant, or spray. The pharmaceutical composition can also be in the same dosage form, for example, azuvidine and a chemotherapeutic agent can be formulated as a composite tablet, composite capsule, composite drop pill, composite granule, composite solution, composite suspension, composite syrup, composite injection (including infusion, sterile powder for injection, and concentrated solution for injection), composite suppository, composite inhalant, or composite spray.

[0030] The present invention also provides a method for treating a tumor disease, which comprises administering to a patient an effective amount of the azuvidine and an effective amount of the chemotherapeutic agent.

[0031] The present invention also provides a pharmaceutical kit for use in medicine to treat tumor diseases, which contains a pharmaceutical composition of azuvudine and a chemotherapeutic agent as described in the present disclosure.

[0032] In the present invention, the combined administration of azuvudine and a chemotherapeutic agent enhances the efficacy of the drugs for treating tumor diseases.

[0033] "Combination" as described in the present invention refers to a method of administration, in which at least one dose of azuvidine and at least one dose of a chemotherapeutic agent are administered within a certain period of time, and both drugs exhibit pharmacological effects. The period may be within one administration cycle, and is preferably within 4 weeks, 3 weeks, 2 weeks, 1 week, or 24 hours, and more preferably within 12 hours. Azuvidine and a chemotherapeutic agent may be administered simultaneously or consecutively. This period includes treatment in which azuvidine and a chemotherapeutic agent are administered by the same administration route or different administration routes. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1. Effect of azuvidine and capecitabine, alone or in combination, on tumor volume of subcutaneous xenografts in the human colon cancer COLO 205 tumor model. [Diagram 2] FIG. 2. Effect of azuvidine and capecitabine alone or in combination on tumor volume of subcutaneous xenografts in the human colorectal cancer LoVo tumor model. [Diagram 3] FIG. 3 shows the effect of azuvudine and cyclophosphamide, alone or in combination, on the tumor volume of subcutaneous xenografts in a human Burkitt lymphoma cell Daudi tumor model. [Figure 4] FIG. 4 shows the effect of azuvudine and cyclophosphamide, alone or in combination, on the tumor volume of subcutaneous xenografts in a human acute lymphoblastic leukemia cell MOLT4 tumor model. [Diagram 5]FIG. 5. Effect of Azuvidine and Paclitaxel, alone or in combination, on tumor volume of subcutaneous xenografts in the human ovarian cancer OVCAR-8 tumor model. [Figure 6] FIG. 6. Effect of Avastin, alone or in combination, on subcutaneous xenograft tumor volume in the human ovarian cancer OVCAR-8 tumor model. [Figure 7] FIG. 7. Effect of Azuvuzine and Dacarbazine, alone or in combination, on subcutaneous xenograft tumor volume in a human melanoma A2058 tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present disclosure will be described in more detail below with reference to examples. The examples of the present disclosure are only used to describe the technical aspects of the present disclosure, and are not intended to limit the essence and scope of the present disclosure.

[0036] Experimental materials Experimental animals and breeding environment Experimental animals Sixty-eight (48 mice plus 20 remaining mice) female BALB / c nude mice, 7-8 weeks old (age of mice at the time of tumor cell inoculation), weighing 15.7-20.7 g, were used. Mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and kept in an SPF-grade breeding environment. Experimental animals were kept in an independent ventilation box with constant temperature and humidity, under conditions of a breeding room temperature of 20-26oC, 40-70% humidity, 10-20 times / hour ventilation, and a 12h / 12h light / dark alternation. Cobalt-60 radiation-sterilized mouse pellet food was continuously provided ad libitum and allowed to be consumed ad libitum, and high-pressure steam-sterilized tap water was always provided in a drinking water bottle and allowed to drink ad libitum. A high-pressure sterilized polysulfone mouse box with a standard size of 325mm x 210mm x 180mm was used as the breeding mouse box.

[0037] Example 1: Test results and discussion on the antitumor effect of a combination of azuvidine and capecitabine in the Colo 205 human colon cancer model cell culture Colo 205 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Exponentially growing Colo 205 cells were resuspended in PBS to the appropriate concentration and mixed with Matrigel at a 1:1 ratio, and then used for subcutaneous tumor inoculation in mice. Female mice were inoculated with 5 × 10 6 Colo 205 cells were inoculated, and the day of inoculation was designated as day 0. The average tumor volume was 110.26 mm 3 When they reached 18.5 years of age, they were randomly assigned to groups based on tumor size.

[0038] Relative tumor growth rate was expressed as T / C%, which is the percentage value of the relative tumor volume or tumor weight between the treatment and control groups at a given time point and was calculated according to the following formula: Efficacy Evaluation Criteria T / C%=TRTV / CRTV×100% (where TRTV is the mean RTV of the treatment group, CRTV is the mean RTV of the vehicle control group, and RTV=Vt / V0, where V0 is the tumor volume of the animals when grouped, and Vt is the tumor volume of the animals after treatment). Or, T / C%=TTW / CTW×100%, where TTW is the mean tumor weight at the end of the experiment in the treatment group and CTW is the mean tumor weight at the end of the experiment in the vehicle control group.

[0039] The relative tumor inhibition rate was expressed as TGI (%) and calculated using the following formula: TGI% was calculated as (1-T / C) x 100%, where T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and control group, respectively, at a given time point.

[0040] On the 25th day after tumor inoculation (20th day after grouping), the mean tumor volume of mice in the vehicle group was 1465.47 mm 3 In the azuvidine 1 mg / kg treatment group, the mean tumor volume was 985.14 mm 3There was no statistically significant difference compared to the control group (p=0.540), and the relative tumor inhibition rate (TGI) (%) was 32.43%. In the capecitabine 400 mg / kg treatment group, the mean tumor volume was 264.96 mm 3 The mean tumor volume in the azure 1 mg / kg and capecitabine 400 mg / kg combination groups was 144.76 mm2, which was statistically significant compared to the control group (p<0.001), with a relative tumor inhibition rate (TGI) of 81.93%. 3 There was a statistically significant difference (<0.001) compared with the control group, and the relative tumor inhibition rate (TGI) (%) was 90.14%.

[0041] [Table 1]

[0042] [Table 2]

[0043] Tumor weight suppression results

[0044] [Table 3]

[0045] Example 2 Pharmacodynamic study of the combination of azuvidine and capecitabine in human colon cancer LoVo cells cell culture Human colon cancer cells LoVo (Cat. No.: ECACC-87060101) were cultured in vitro as a monolayer. The culture conditions were F12K medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37oC in a 5% CO2 incubator. Routine trypsin-EDTA digestion was performed twice a week for subculture. When the cells reached 80%-90% saturation and the required number was reached, the cells were collected, counted, and inoculated.

[0046] LoVo cells 0.1mL (10×10 6 ) was inoculated subcutaneously into the right back of each mouse, resulting in a mean tumor volume of approximately 147 mm 3 When this was reached, the subjects were divided into groups and treatment was started.

[0047] [Table 4]

[0048] Experimental Results Table 5 shows the change in tumor volume (21 days after administration) in each group after administration of the test drug to BALB / c nude mice bearing subcutaneous xenografts of Lovo cells, and Table 6 shows the change in tumor weight.

[0049] [Table 5]

[0050] [Table 6]

[0051] In this experiment, the in vivo efficacy of the test drug was evaluated in the LoVo xenograft tumor model. The tumor volume and tumor weight of each group after 21 days of administration are shown in Tables 5, 6, and 2, respectively. After 21 days of administration, the tumor volume of the tumor-bearing mice in the blank control group was 1481 mm 3 The test drug azuvidine (1 mg / kg) showed a slight antitumor effect compared to the blank control group, with the tumor volume decreasing to 902 mm 3 The T / C was 62.00%, the TGI was 43.40%, and the p value was 0.086. The test drug capecitabine (400 mg / kg) showed a slight antitumor effect compared to the blank control group, with the tumor volume decreasing to 919 mm 3 , T / C was 60.94%, TGI was 42.11%, and p value was 0.696.

[0052] The combination of the test drugs, azuvidine and capecitabine (1 + 400 mg / kg), showed a clear antitumor effect compared to the blank control group, with the tumor volume decreasing to 676 mm3 , T / C was 48.18%, TGI was 60.29%, and p-value was 0.282. The combination of azuvudine and capecitabine could enhance the antitumor effect of single-agent capecitabine in these LoVo colorectal tumors, increasing the TGI from 42.1% to 60.3%.

[0053] Example 3 In vivo pharmacodynamics of the combination of azuvudine and cyclophosphamide in a subcutaneous xenograft tumor model of human Burkitt's lymphoma Daudi cells. Human Burkitt's lymphoma cells, Daudi (catalog number: DSMZ-ACC129), were cultured in suspension in vitro. The culture conditions were RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37oC in a 5% CO2 incubator. When the cells reached 80%-90% saturation and the required number was reached, the cells were collected, counted, and inoculated.

[0054] Tumor cell inoculation Daudi cells (Matrigel added, volume ratio 1:1) 0.2 mL (10 × 10 6 ) was inoculated subcutaneously into the right back of each mouse, and the average tumor volume was approximately 100 mm 3 When this was reached, the subjects were divided into groups and medication was started.

[0055] [Table 7]

[0056] Experimental Results Tables 8 and 9 show the changes in tumor volume and tumor weight in each group after administration of the test drug to SCID mice bearing subcutaneous xenografted Daudi cell tumors CB 17.

[0057] [Table 8]

[0058] [Table 9-1]

[0059] In this study, the in vivo efficacy of the test drug was evaluated in the Daudi xenograft tumor model. The tumor volumes of each group at different time points are shown in Table 8 and Table 9-1. 27 days after the start of treatment, the tumor volume of the tumor-bearing mice in the blank control group was 2,895 mm 3 The test drug azuvidine (1 mg / kg) showed a clear antitumor effect compared to the blank control group, with the tumor volume reaching 909 mm 3 The T / C was 30.64%, the TGI was 71.09%, and the p value was <0.0001. The test drug cyclophosphamide (50 mg / kg) showed a clear antitumor effect compared to the blank control group, with the tumor volume decreasing to 263 mm 3 The T / C was 8.83%, the TGI was 94.17%, and the p value was <0.0001. The combination of the test drugs, azuvudine + cyclophosphamide (1 + 50 mg / kg), showed a clear antitumor effect compared to the blank control group, with the tumor volume decreasing by 13 mm 3 , T / C was 0.42%, TGI was 103.15%, and p value was <0.0001.

[0060] In this experiment, the combination of the test drug azuvudine (1 mg / kg) and cyclophosphamide enhanced the antitumor effect of single-agent CTX in the human Burkitt's lymphoma Daudi cells, increasing the TGI from 94.17% to 103.15%.

[0061] Example 4 In vivo pharmacodynamic study of the combination of azuvudine and cyclophosphamide on a subcutaneous xenograft tumor model of human acute lymphoblastic leukemia cells MOLT4 cell culture Human acute lymphoblastic leukemia cells MOLT4 (catalog number: ECACC-85011413) were cultured in suspension in vitro. The culture conditions were RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37oC in a 5% CO2 incubator. When the cells reached 80%-90% saturation and the required number was reached, the cells were collected, counted, and inoculated.

[0062] Tumor cell inoculation MOLT4 cells (with Matrigel, volume ratio 1:1) 0.2 mL (10 × 10 6 ) were inoculated subcutaneously into the right back of each mouse, and the average tumor volume was approximately 148 mm 3 When this was reached, the subjects were divided into groups and treatment was started. [Table 9-2]

[0063] Tables 10 and 11 show the changes in tumor volume and weight in each group after administration of the test drug to SCID Beige mice with subcutaneous MOLT4 cell xenograft tumors.

[0064] [Table 10]

[0065] [Table 11]

[0066] In this study, the in vivo efficacy of the test drug was evaluated in a MOLT4 xenograft tumor model. The tumor volumes of each group at different time points are shown in Tables 10 and 11. 28 days after the start of treatment, the tumor volume of the tumor-bearing mice in the blank control group was 2,896 mm 3 The test drug azuvidine (1 mg / kg) showed an antitumor effect compared to the blank control group, with the tumor volume decreasing to 979 mm 3The T / C was 33.89, the TGI was 69.73%, and the p value was <0.0001. Cyclophosphamide CTX (50 mg / kg) showed a clear antitumor effect compared to the blank control group, with the tumor volume decreasing to 716 mm 3 The T / C was 24.53%, the TGI was 79.34%, and the p value was <0.0001. The combination of the test drugs, azuvudine + cyclophosphamide (1 + 50 mg / kg), showed a clear antitumor effect compared to the blank control group, with the tumor volume decreasing to 253 mm 3 , T / C was 8.75%, TGI was 96.14%, and p value was <0.001. The analysis and statistical results of tumor weight in the test drug combination group were basically consistent with the tumor volume data.

[0067] In this study, the test drugs azuvudine (1mg / kg), cyclophosphamide (50mg / kg) and azuvudine + cyclophosphamide (1+50mg / kg) showed significant inhibitory effects on the growth of MOLT4 xenograft tumors at the doses tested.In this study, the combination of the test drug azuvudine (1mg / kg) and cyclophosphamide could enhance the antitumor effect of single-agent cyclophosphamide in MOLT4 human acute lymphoblastic leukemia tumors, increasing the TGI from 79.34% to 96.14%.

[0068] Example 5. Efficacy evaluation of the combination of azuvidine and paclitaxel in the human ovarian cancer OVCAR-8 tumor model OVCAR-8 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Exponentially growing OVCAR-8 cells were harvested, resuspended in PBS to the appropriate concentration, and mixed 1:1 with Matrigel before being used for subcutaneous tumor inoculation in mice.

[0069] 1 × 10 subcutaneous injection into the right side of female mice 7 OVCAR-8 cells were inoculated. The mean tumor volume was 174.25 mm 3 When they reached 18.5 years of age, they were randomly assigned to groups based on tumor size.

[0070] The experimental protocol for the animal experiments used in this study was reviewed and approved by the CrownBio IACUC Committee. During the experiment, animal experiments were performed in accordance with AAALAC requirements. After tumor inoculation, regular monitoring was performed, including tumor growth and the effect of treatment on the normal behavior of the animals, specifically monitoring the activity, feeding and drinking status, weight gain and loss (weight measurement twice a week), eyes, hair coat, and other abnormalities of the experimental animals. Clinical symptoms observed during the experiment were recorded as raw data. Tumor volume was calculated as tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the long diameter and b represents the short diameter).

[0071] Relative tumor growth rate was expressed as T / C%, which is the percentage value of the relative tumor volume or tumor weight between the treatment and control groups at a given time point and was calculated according to the following formula: T / C%=T RTV / C RTV ×100% (T RTV is the mean RTV of the treatment group, and C RTV is the mean RTV of the vehicle control group, and RTV = V t / V0, V0 is the tumor volume of the animals when grouped, V t is the tumor volume of the animal after treatment.) Or, T / C%=T TW / C TW ×100% (T TW is the mean tumor weight at the end of the study in the treatment group, and C TW is the mean tumor weight at the end of the experiment in the vehicle control group.)

[0072] The relative tumor inhibition rate was expressed as TGI (%) and calculated using the following formula: TGI% was calculated as (1-T / C) x 100%, where T and C are the relative tumor volume (RTV) or tumor weight (TW) at a specific time point in the treatment group and control group, respectively.

[0073] To compare tumor volumes on a given day in different treatment groups, we first verified the assumption of equal variance between all groups with Bartlett's test. If the p-value of the Bartlett's test was ≥ 0.05, one-way ANOVA was used to test whether the means of all groups were equal. If the p-value of the one-way ANOVA was < 0.05, pairwise comparisons between all groups were performed with Tukey's HSD test or pairwise comparisons between each treatment group and the control group with Dunnett's test. If the p-value of the Bartlett's test was < 0.05, the Kruskal-Wallis test was used to test whether the medians of all groups were equal. If the p-value of the Kruskal-Wallis test was < 0.05, pairwise comparisons between all groups were performed with Conover test or pairwise comparisons between each treatment group and the control group, with correction of the corresponding p-values ​​based on the number of groups for multiple tests.

[0074] In addition, for the purpose of exploratory data analysis, pairwise comparisons between all groups at any given time point were performed. This comparison only used the tumor volume data of the two groups being compared at a given time point, so no correction for multiple testing was necessary. First, the assumption of equal variance between the two groups was verified using the Bartlett test, and if the p-value of the Bartlett test was ≥ 0.05, the mean values ​​of the two groups were compared for equality using the Welch's t test, whereas if the p-value of the Bartlett test was < 0.05, the medians of the two groups were compared for equality using the Mann-Whitney U test.

[0075] All statistical analyses and graphing were performed in the R language environment (version 3.3.1). Unless otherwise stated, all tests were two-sided and p values ​​less than 0.05 were considered statistically significant.

[0076] Experimental Results On the 34th day after administration, the tumor volume in the vehicle control group was 558.68 mm 3 The mean tumor volume was 460.67 mm in the group treated with the test drug azuvudine 1 mg / kg, the group treated with the active drug paclitaxel 15 mg / kg, and the group treated with the active drug Avastin 10 mg / kg. 3 , 403.40mm 3 , 456.04mm 3 There was no statistically significant difference compared to the control group (p = 0.952, 0.769, and 0.957), and the relative tumor inhibition rates (TGI) (%) were 18.09%, 26.49%, and 17.70%, respectively. In the treatment group that combined the test drug azuvudine 1 mg / kg and paclitaxel 15 mg / kg, the mean tumor volume was 157.34 mm 3 This was a statistically significant difference compared to the control group (p = 0.00707), with a relative tumor inhibition rate (TGI) of 71.57%. When the test drug azuvidine was administered in combination with paclitaxel, the antitumor effect of paclitaxel alone was significantly improved (TGI was 26.5%). In the treatment group administered the test drug azuvidine 1 mg / kg in combination with Avastin 10 mg / kg, the mean tumor volume was 294.36 mm 3 There was a statistically significant difference compared to the control group (p = 0.140), and the relative tumor inhibition rate TGI (%) was 48.61%. When the test drug Azvidin was administered in combination with Avastin, the antitumor effect of Avastin alone was significantly improved (TGI was 17.70%). The tumor growth status of each treatment group and the control group is shown in Table 12.

[0077] [Table 12]

[0078] Example 6 In vivo pharmacodynamic study of a combination of azuvidine and dacarbazine on a subcutaneous xenograft tumor model of human melanoma A2058 cells Human melanoma cells A2058 (catalog number: CRL-11147) were cultured in monolayer in vitro. The culture conditions were DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37oC in a CO2 incubator. Routine digestion with trypsin-EDTA was performed twice a week for subculture. When the cells reached 80%-90% saturation and the required number was reached, the cells were collected, counted, and inoculated.

[0079] Tumor cell inoculation Matrigel-added A2058 cells 0.2mL (5 x 10 6 ) was inoculated subcutaneously into the right back of each mouse, and the average tumor volume was approximately 136 mm 3 When this was reached, the subjects were divided into groups and treatment was started. Analysis of experimental results

[0080] [Table 13]

[0081] In this example, the in vivo efficacy of the test drug was evaluated in the A2058 xenograft tumor model. The tumor volumes of each group at different time points are shown in Table 13. 14 days after the start of administration, the tumor volume of tumor-bearing mice in the blank control group was 2724 mm 3 The test drug azuvidine (1 mg / kg) showed a slight antitumor effect compared to the blank control group, with the tumor volume decreasing to 2253 mm 3 The T / C was 79.67%, the TGI was 18.22%, and the p value was 0.518. Dacarbazine (60 mg / kg) showed an antitumor effect compared to the blank control group, with the tumor volume decreasing to 1255 mm 3 The T / C was 44.77%, the TGI was 56.74%, and the p value was 0.002. The combination of the test drugs, azuvidine and dacarbazine (1 + 60 mg / kg), showed a clear antitumor effect compared to the blank control group, with the tumor volume decreasing to 910 mm 3 , T / C was 31.52%, and TGI was 70.07%, with a p value of <0.001.

[0082] Although specific embodiments of the present invention have been described above, it should be understood by those skilled in the art that these are merely examples, and various changes and modifications can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. (i) Azubudine or a pharmaceutically acceptable salt, stereoisomer, or isotope derivative thereof, (ii) A pharmaceutical composition comprising a chemotherapeutic agent or Avastin® (bevacizumab), A pharmaceutical composition in which the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, paclitaxel, and combinations thereof.

2. The pharmaceutical composition according to claim 1, wherein (i) and (ii) are administered simultaneously, separately, or sequentially, or (i) and (ii) exist in the same dosage form.

3. Used to treat tumor-related diseases, The pharmaceutical composition according to any one of claims 1 to 2, wherein the tumor-related disease is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, melanoma, brain tumor, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, lung cancer, kidney cancer, skin cancer, glioblastoma, neuroblastoma, sarcoma, liposarcoma, osteochondroma, osteoma, osteosarcoma, seminoma, testicular tumor, uterine cancer, head and neck tumor, multiple myeloma, malignant lymphoma, polycythemia vera, leukemia, thyroid tumor, ureteral tumor, bladder tumor, gallbladder cancer, non-small cell lung cancer, bile duct cancer, and choriocarcinoma.

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