Antitumor drug composition containing azvudine and chemotherapy agents

Abstract

This invention discloses a pharmaceutical composition comprising azvudine and a chemotherapy agent. The pharmaceutical composition of this invention exhibits a good synergistic effect in anti-tumor activity and can reduce the dosage of chemotherapy agents, improve efficacy and safety, thereby achieving the goal of prolonging patient survival.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, and specifically relates to an antitumor drug composition containing azvudine. Background Technology

[0002] Deoxycytidine kinase (DCK) is a broadly substrate-specific enzyme that phosphorylates pyrimidine and purine deoxynucleosides. It is a key enzyme in the salvage pathway of deoxynucleotide biosynthesis, maintaining normal DNA metabolism and phosphorylating various antiviral and anticancer nucleoside analogues. These drugs are activated only after phosphorylation, thereby inhibiting tumor growth. In recent decades, apoptosis has been extensively studied, and radiotherapy strategies targeting apoptosis have become an important means of cancer treatment.

[0003] Azvudine (FNC) is a broad-spectrum RNA virus inhibitor. As a synthetic nucleoside analog of viral RNA-dependent RNA polymerase (RdRp), it is metabolized intracellularly into an antiviral 5'-triphosphate metabolite (azvudine triphosphate). Its target is the viral RdRp, and it can block RNA chain synthesis and replication in host cells by inhibiting RdRp activity. In July 2021, azvudine tablets were approved for marketing in my country for the treatment of adult HIV-1 infected patients with high viral loads.

[0004] Patent document CN201010506595.X discloses the use of azvudine for the treatment of tumors, such as colon cancer, liver cancer, stomach cancer, esophageal cancer, lung cancer, breast cancer, cervical cancer, leukemia, and lymphoma. It was found that azvudine has a significant inhibitory effect on various human cancer cells and transplanted tumors in animals.

[0005] Among traditional cancer treatments, chemotherapy is currently the most widely used clinical treatment due to its strong therapeutic effect and high efficacy. However, most chemotherapy drugs used clinically have poor selectivity, causing significant damage to normal tissues while treating tumor tissue, leading to severe toxic side effects.

[0006] Monotherapy has drawbacks such as poor physicochemical properties, low bioavailability, and poor efficacy. However, if chemotherapy drugs can be combined with azvudine, the tumor response can be improved. Based on the complementary synergy between the two drugs in their anti-tumor mechanisms, chemotherapy-immune synergy can be achieved by dual-targeting tumor cells to enhance the anti-tumor efficacy. Summary of the Invention

[0007] This disclosure provides a pharmaceutical composition of azvudine (FNC) and a chemotherapy agent, and the use of the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of tumor diseases.

[0008] The pharmaceutical compositions of the present invention have the following advantages compared to individual drugs:

[0009] 1. Combination therapy enhanced the tumor-inhibiting effects of each individual drug;

[0010] 2. It delays the development of drug resistance, improves efficacy and safety, thereby achieving the goal of prolonging patient survival.

[0011] To address the technical problem of this invention, this invention provides a pharmaceutical composition comprising:

[0012] (i) Azvudine or its pharmaceutically acceptable salts, stereoisomers or isotopic derivatives;

[0013] (ii) Chemotherapy reagents.

[0014] In a preferred embodiment of the present invention, the chemotherapy reagent is selected from 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, leucovorin, and doxorubicin lipids. Plastosomes, daunorubicin liposomes, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paritaxetine, pemetrexed, pentostatin, procarbazine, raltitrexed, saplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thioguanine, topotecan, treoxantrone, vincristine, vinblastine, vindesine, vinorelbine, or any combination thereof.

[0015] In a preferred embodiment of the present invention, the chemotherapeutic agent is selected from capecitabine, cyclophosphamide, dacarbazine, paclitaxel, or any combination thereof.

[0016] In addition, the present invention provides another pharmaceutical composition comprising:

[0017] (i) Azvudine or its pharmaceutically acceptable salts, stereoisomers or isotopic derivatives;

[0018] (ii) Avastin.

[0019] In a preferred embodiment of the present invention, wherein (i) and (ii) are administered simultaneously, separately, or sequentially, or wherein (i) and (ii) are present in the same dosage form.

[0020] In a preferred embodiment of the present invention, it is used to treat tumor-related diseases.

[0021] In a preferred embodiment of the present invention, the tumor-related diseases are selected from 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, or choriocarcinoma.

[0022] In some embodiments, the dose of azvudine is selected from 1-100 mg, and the dose of the chemotherapeutic agent is selected from 1-500 mg.

[0023] In some embodiments, the dose of azvudine is selected from 1-100 mg, and the dose of bevacizumab (Avastin) is selected from 1-500 mg.

[0024] The dosage of azvudine described in this disclosure is selected from 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, etc. 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.

[0025] The dosage of the chemotherapy reagents described in this disclosure is selected from 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, 55m g, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74 mg, 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, 110mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg , 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290mg, 300mg, 310mg, 320mg, 330mg, 340mg, 35 0mg, 360mg, 370mg, 380mg, 390mg, 400mg, 410mg, 420mg, 430mg, 440mg, 450mg, 460mg, 470mg, 480mg, 490mg, 500mg.

[0026] In some embodiments, the dose of the azvudine is selected from 1-100 mg, and the administration frequency may be once, twice or three times a day; the dose of the chemotherapy reagent is selected from 1-500 mg, and the administration frequency may be once, twice or three times a day.

[0027] In some embodiments, the dose of azvudine is selected from 1-50 mg, and the administration frequency may be once or twice a day; the dose of the chemotherapy reagent is selected from 1-100 mg, and the administration frequency is once a day.

[0028] In some embodiments, the dose of azvudine is selected from 1-20 mg, and the administration frequency may be once or twice a day; the dose of the chemotherapy reagent is selected from 1-40 mg, and the administration frequency is once a day.

[0029] In some embodiments, the dose of azvudine is selected from 1-10 mg, and the administration frequency may be once or twice a day; the dose of the chemotherapy reagent is selected from 1-10 mg, and the administration frequency is once a day.

[0030] In some embodiments, the dosage of the chemotherapy reagent is selected from 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, 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, and 50 mg, and the administration frequency is once or twice a day. Alternatively, the dosage of the chemotherapy reagent is selected from 10 mg, 20 mg, 40 mg, and 60 mg, and the administration frequency is once a day.

[0031] In some embodiments, the dosage of azvudine is selected from 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, and 20 mg, and the dosing frequency is once or twice a day. The dosage of azvudine is selected from 1 mg, 2 mg, 4 mg, and 6 mg, and the dosing frequency is once a day.

[0032] In some embodiments, the dosage of the chemotherapy reagent is selected from 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, and 10 mg, and the administration frequency is once or twice a day. The dosage of the chemotherapy reagent is selected from 10 mg, 20 mg, 40 mg, and 60 mg, and the administration frequency is once a day.

[0033] In some embodiments, the dosage of the chemotherapy reagent is selected from 1 mg, 2 mg, 4 mg, 6 mg, 8 mg, and the administration frequency is once or twice a day; the dosage of the chemotherapy reagent is selected from 1 mg, 2.5 mg, 5 mg, 10 mg, and the administration frequency is once a day.

[0034] The combined routes of administration described in this 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, with oral administration being preferred.

[0035] The present invention also provides a pharmaceutical composition comprising the above-mentioned azvudine, a chemotherapy reagent, and one or more pharmaceutical carriers, excipients, and diluents. The pharmaceutical composition can be formulated into any pharmaceutically acceptable dosage form. For example, it can be formulated as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), suppositories, inhalers, or sprays; the pharmaceutical composition can also be formulated into the same dosage form, for example, azvudine and chemotherapy reagents can be formulated as compound tablets, compound capsules, compound pills, compound granules, compound solutions, compound suspensions, compound syrups, compound injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), compound suppositories, compound inhalers, or compound sprays.

[0036] The present invention also provides a method for treating tumor diseases, comprising administering to a patient an effective amount of the above-mentioned azvudine and an effective amount of the above-mentioned chemotherapy reagent.

[0037] The present invention also provides a pharmaceutical kit for use in the treatment of tumor diseases, wherein the pharmaceutical composition of azvudine and chemotherapy reagents as described in this disclosure is packaged.

[0038] This invention combines azvudine with chemotherapy reagents to enhance the efficacy of drugs for treating tumor diseases.

[0039] The term "combination" as used in this invention refers to a route of administration that involves administering at least one dose of azvudine and at least one dose of a chemotherapeutic agent within a specified time period, wherein both substances exhibit pharmacological effects. The time period can be within a dosing cycle, preferably within 4 weeks, 3 weeks, 2 weeks, 1 week, or within 24 hours, more preferably within 12 hours. Azvudine and the chemotherapeutic agent can be administered simultaneously or sequentially. This period includes treatments in which azvudine and the chemotherapeutic agent are administered via the same or different routes of administration. Attached Figure Description

[0040] Figure 1 Effects of azvudine, capecitabine alone or in combination on the volume of subcutaneous xenograft tumors in a human colon cancer COLO 205 tumor model

[0041] Figure 2 Effects of azvudine, capecitabine alone or in combination on the volume of subcutaneous xenograft tumors in a human colorectal cancer LoVo tumor model

[0042] Figure 3 Effects of azvudine, cyclophosphamide, alone or in combination, on the volume of subcutaneous xenograft tumors in a human Burkitt's lymphoma cell Daudi tumor model

[0043] Figure 4 Effects of azvudine, cyclophosphamide, alone or in combination, on the volume of subcutaneous xenograft tumors in a human acute lymphoblastic leukemia cell MOLT4 tumor model.

[0044] Figure 5 Effects of azvudine, paclitaxel, alone or in combination on the volume of subcutaneous xenograft tumors in a human ovarian cancer OVCAR-8 tumor model

[0045] Figure 6 Effects of avastin, alone or in combination, on the volume of subcutaneous xenograft tumors in a human ovarian cancer OVCAR-8 tumor model

[0046] Figure 7 Effects of azvudine, dacarbazine alone or in combination on the volume of subcutaneous xenograft tumors in a human melanoma A2058 tumor model Detailed Implementation

[0047] The present disclosure will be explained in more detail below with reference to the embodiments. The embodiments of the present disclosure are only used to illustrate the technical solutions of the present disclosure and are not intended to limit the substance and scope of the present disclosure.

[0048] Experimental materials

[0049] Laboratory animals and their living environment

[0050] laboratory animals

[0051] BALB / c Nude mice, female, 7-8 weeks old (at the time of tumor cell inoculation), weighing 15.7-20.7g, 68 mice (48 plus 20 surplus mice). Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., housing environment: SPF grade. All animals were housed in individually ventilated, temperature- and humidity-controlled enclosures. The enclosure temperature was 20-26℃, humidity 40-70%, with 10-20 air changes per hour and a 12h / 12h light / dark cycle. They were continuously provided with cobalt-60 radioactively sterilized complete pelleted rat feed, with unlimited access. Tap water (autoclaved) was provided via a continuous water bottle supply. The enclosures were polysulfone enclosures, autoclaved, and measured 325mm × 210mm × 180mm.

[0052] Example 1: Results and Discussion of the Study on the Antitumor Effect of Azvudine Combined with Capecitabine in a Colo 205 Human Colon Cancer Model

[0053] Cell culture

[0054] Colo 205 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. Colo 205 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and mixed 1:1 with Matrigel before being used for subcutaneous tumor inoculation in mice. 5 × 10⁶ cells were subcutaneously inoculated into the right side of female mice. 6 Colo 205 cells were inoculated, with day 0 defined as the day of inoculation. The average tumor volume was 110.26 mm. 3 At that time, patients were randomly grouped according to tumor size.

[0055] The relative tumor proliferation rate, T / C%, is the percentage of tumor volume or weight in the treatment group and the control group at a given time point. The calculation formula is as follows:

[0056] Efficacy evaluation criteria

[0057] T / C% = TRTV / CRTV × 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the solvent control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment);

[0058] Or T / C% = TTW / CTW × 100% (TTW: average tumor weight at the end of the experiment in the treatment group; CTW: average tumor weight at the end of the experiment in the solvent control group).

[0059] The relative tumor inhibition rate, TGI (%), is calculated as follows: TGI% = (1-T / C) × 100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).

[0060] On day 25 post-tumor inoculation (day 20 after grouping), the average tumor volume in the Vehicle group mice was 1465.47 mm. 3 In the monotherapy group of azvudine (1 mg / kg), the average tumor size was 985.14 mm. 3 There was no statistically significant difference between the control group and the control group (p = 0.540). The relative tumor inhibition rate (TGI) was 32.43%, and the mean tumor volume in the Capecitabine, 400 mg / kg treatment group was 264.96 mm. 3 The tumor inhibition rate (TGI) was 81.93% in the control group, showing a statistically significant difference compared to the control group (p<0.001). The average tumor volume in the combination group (azvudine 1 mg / kg and Capecitabine 400 mg / kg) was 144.76 mm. 3The tumor inhibition rate (TGI) was 90.14%, which was statistically significant compared to the control group (<0.001).

[0061] Table 1. Experimental design of antitumor effects of different doses of the test drug in the COLO 205 human colon cancer tumor model.

[0062]

[0063]

[0064] Table 2. Efficacy analysis of each group in the Colo 205 human colon cancer model.

[0065]

[0066] Tumor weight inhibition results

[0067] Table 3. Tumor weight analysis in each group of the Colo 205 human colon cancer model.

[0068]

[0069] Example 2: Pharmacodynamic study of azvudine combined with capecitabine in human colorectal cancer LoVo cells

[0070] Cell culture

[0071] Human colon cancer cells LoVo (catalog number: ECACC-87060101) were cultured in vitro in a monolayer under the following conditions: F12K medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0072] 0.1 mL (10 × 10) 6 LoVo cells were subcutaneously injected into the right posterior dorsal region of each mouse, resulting in an average tumor volume of approximately 147 mm². 3 Dosing will begin in groups at that time.

[0073] Table 4. Experimental design of antitumor effects of different doses of the test drug in the COLO 205 human colon cancer tumor model.

[0074]

[0075] The experimental results are shown in Table 5 and Table 6. The changes in tumor volume (21 days after administration) of Lovo cell subcutaneous xenograft tumor BALB / c nude mice after treatment with the test drug are shown in Table 5.

[0076] Table 5. Evaluation of the antitumor efficacy of the test drugs in the LoVo xenograft model (calculated based on tumor volume on day 21 after administration).

[0077]

[0078]

[0079] Table 6. Evaluation of the antitumor efficacy of the test drug on the LoVo xenograft model (calculated based on tumor size on day 21 after administration).

[0080]

[0081] In this experiment, the in vivo efficacy of the test substance in the LoVo xenograft tumor model was evaluated. Tumor volume and weight in each group after 21 days of administration are shown in Tables 5, 6, and 2, respectively. 21 days after the start of administration, the tumor volume of tumor-bearing mice in the blank control group reached 1481 mmHg. 3 The test substance azvudine (1 mg / kg) showed a smaller tumor-suppressing effect compared to the blank control group, with a tumor volume of 902 mm. 3 The T / C ratio was 62.00%, the TGI was 43.40%, and the p-value was 0.086. The test substance Capecitabine (400 mg / kg) showed a smaller tumor-suppressive effect compared to the blank control group, with a tumor volume of 919 mm². 3 The T / C ratio was 60.94%, the TGI ratio was 42.11%, and the p-value was 0.696.

[0082] The test substance combination group, azvudine + Capecitabine (1 + 400 mg / kg), showed a significant tumor-suppressing effect compared to the blank control group, with a tumor volume of 676 mm. 3 The T / C ratio was 448.18%; the TGI was 60.29%; and the p-value was 0.282. The combination of azvudine and Capecitabine enhanced the tumor-suppressive effect of Capecitabine monotherapy in this LoVo colorectal tumor, increasing the TGI from 42.1% to 60.3%.

[0083] Example 3: In vivo pharmacodynamics of azvudine combined with cyclophosphamide in a subcutaneous xenograft tumor model of human Burkitt's lymphoma cells (Daudi). Human Burkitt's lymphoma cells (Daudi, catalog number: DSMZ-ACC129) were cultured in suspension in vitro under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin, incubated at 37°C in a 5% CO2 incubator. When the cell saturation reached 80%-90% and the desired number was achieved, the cells were harvested, counted, and seeded.

[0084] Tumor cells will be inoculated with 0.2 mL (10 × 10⁻⁶) 6 Daudi cells (with matrix gel, volume ratio 1:1) were subcutaneously inoculated into the right posterior dorsal region of each mouse, resulting in an average tumor volume of approximately 100 mm². 3 Dosing will begin in groups at that time.

[0085]

[0086] Table 7 Experimental Results

[0087] Tables 8 and 9 show the tumor volume and tumor changes in each group after treatment with the test drug in mice with CB 17SCID tumors transplanted subcutaneously using Daudi cells.

[0088] Table 8 Tumor volume 27 days after drug administration

[0089]

[0090] Table 9 Tumor weight 27 days after drug administration

[0091]

[0092] In this experiment, we evaluated the in vivo efficacy of the test substance in the Daudi xenograft tumor model. Tumor volumes at different time points for each group are shown in Tables 8 and 9. Twenty-seven days after the start of drug administration, the tumor volume in the blank control group of tumor-bearing mice reached 2,895 mmHg. 3 The test substance, azvudine (1 mg / kg), showed a significant tumor-suppressing effect compared to the blank control group, with tumor volumes of 909 mmHg. 3 The T / C ratio was 30.64%; the TGI was 71.09%; and the p-value was <0.0001. The test substance cyclophosphamide (50 mg / kg) showed significant tumor inhibition compared to the blank control group, with a tumor volume of 263 mm²; the T / C ratio was 8.83%; the TGI was 94.17%; and the p-value was <0.0001. The test substance combination group, azvudine + cyclophosphamide (1 + 50 mg / kg), showed significant tumor inhibition compared to the blank control group, with a tumor volume of 13 mm². 3 The T / C ratio was 0.42%; the TGI ratio was 103.15%; and the p-value was <0.0001.

[0093] In this experiment, the test substance azvudine (1 mg / kg) combined with cyclophosphamide enhanced the tumor-suppressive effect of single-agent CTX in human Burkitt's lymphoma cells Daudi, with the TGI increasing from 94.17% to 103.15%.

[0094] Example 4: In vivo pharmacodynamic study of azvudine combined with cyclophosphamide in a human acute lymphoblastic leukemia cell MOLT4 subcutaneous xenograft tumor model.

[0095] Cell culture

[0096] Human acute lymphoblastic leukemia cells MOLT4 (catalog number: ECACC-85011413) were cultured in suspension in vitro under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, incubated at 37°C in a 5% CO2 incubator. When the cell saturation reached 80%-90% and the desired number was achieved, the cells were harvested, counted, and seeded.

[0097] Tumor cell inoculation

[0098] 0.2 mL (10 × 10) 6 MOLT4 cells (with matrix gel, volume ratio 1:1) were subcutaneously inoculated into the right posterior dorsal region of each mouse, resulting in an average tumor volume of approximately 148 mm². 3 Dosing will begin in groups at that time.

[0099]

[0100] The changes in tumor volume and weight in each group after treatment with the test drug in SCID Beige mice with subcutaneous xenografted MOLT4 cells are shown in Tables 10 and 11.

[0101] Table 10 Tumor volume 28 days after drug administration

[0102]

[0103]

[0104] Table 11 Tumor weight 28 days after drug administration

[0105]

[0106] In this experiment, we evaluated the in vivo efficacy of the test substance in the MOLT4 xenograft tumor model. Tumor volumes at different time points in each group are shown in Tables 10 and 11. Twenty-eight days after the start of drug administration, the tumor volume in the blank control group of tumor-bearing mice reached 2,896 mmHg. 3 The test substance, azvudine (1 mg / kg), showed tumor-suppressive activity compared to the blank control group, with a tumor volume of 979 mm. 3 The T / C ratio was 33.89, the TGI was 69.73%, and the p-value was <0.0001. Cyclophosphamide (CTX) (50 mg / kg) showed a significant tumor-suppressive effect compared to the blank control group, with a tumor volume of 716 mm. 3The T / C ratio was 24.53%, the TGI was 79.34%, and the p-value was <0.0001. The test substance combination group (azivudine + cyclophosphamide 1 + 50 mg / kg) showed a significant tumor-suppressive effect compared to the blank control group, with a tumor volume of 253 mm². 3 The T / C ratio was 8.75%, the TGI was 96.14%, and the p-value was <0.001. The analysis and statistical results of tumor weight in the test substance combination group were basically consistent with the tumor volume data.

[0107] In this experiment, the test substance azvudine (1 mg / kg), cyclophosphamide (50 mg / kg), and azvudine + cyclophosphamide (1 + 50 mg / kg) significantly inhibited the growth of MOLT4 xenograft tumors at the tested doses. In this experiment, the combination of the test substance azvudine (1 mg / kg) and cyclophosphamide enhanced the tumor-suppressive effect of cyclophosphamide alone in MOLT4 human acute lymphoblastic leukemia tumors, increasing the TGI from 79.34% to 96.14%.

[0108] Example 5: Evaluation of the efficacy of azvudine combined with paclitaxel in a human ovarian cancer OVCAR-8 tumor model.

[0109] OVCAR-8 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. OVCAR-8 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and mixed 1:1 with Matrigel before being used for subcutaneous tumor inoculation in mice.

[0110] Female mice were subcutaneously injected with 1×10⁻⁶ on the right side. 7 OVCAR-8 cells. The average tumor volume was 174.25 mm. 3 At that time, patients were randomly grouped according to tumor size.

[0111] All experimental protocols used in this study were reviewed and approved by the Crown Bio IACUC committee. Animal procedures were performed according to AAALAC requirements. Post-tumor inoculation monitoring included monitoring tumor growth and the impact of treatment on normal animal behavior, specifically monitoring activity levels, food and water intake, weight gain or loss (measured twice weekly), and any abnormalities in the eyes, coat, or other areas. All clinical symptoms observed during the experiment were recorded in the raw data. Tumor volume calculation formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis).

[0112] The relative tumor proliferation rate, T / C%, is the percentage of tumor volume or weight in the treatment group and the control group at a given time point. The calculation formula is as follows:

[0113] T / C% = T RTV / C RTV ×100% (T) RTV : Mean RTV in the treatment group; C RTV : Average RTV of the solvent control group; RTV = V t / V0, where V0 is the tumor volume of the animal at the time of grouping, V t (This refers to the tumor volume in the animal after treatment);

[0114] Or T / C% = T TW / C TW ×100% (T) TW : Average tumor weight at the end of the trial in the treatment group; C TW (The average tumor weight at the end of the experiment in the solvent control group).

[0115] The relative tumor inhibition rate, TGI (%), is calculated as follows: TGI% = (1-T / C) × 100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).

[0116] To compare tumor volume on a given day across different treatment groups, we first used the Bartlett's test to verify the hypothesis of homogeneity of variance among all groups. When the p-value of the Bartlett's test is not less than 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 less than 0.05, we used the Tukey HSD test for pairwise comparisons between all groups, or Dunnett's t-test for pairwise comparisons between each treatment group and the control group. When the p-value of the Bartlett's test was less than 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 less than 0.05, we used the Conover test for pairwise comparisons between all groups or pairwise comparisons between each treatment group and the control group, and adjusted the p-value accordingly based on the number of groups in the multiple tests.

[0117] Furthermore, for the purpose of exploratory data analysis, we performed pairwise comparisons between all groups at any time point. Because this comparison only used tumor volume data from the two groups being compared at specific time points, multiple test corrections were unnecessary. First, we used the Bartlett test to verify the hypothesis of homogeneity of variance between the two groups. When the p-value of the Bartlett test was not less than 0.05, we used Welch's t-test to compare whether the means of the two groups were equal; when the p-value of the Bartlett test was less than 0.05, we used the Mann-Whitney U test to compare whether the medians of the two groups were equal.

[0118] All statistical analyses and graphs were performed in the R language environment (version 3.3.1). Unless otherwise specified, all tests are two-tailed tests, and a p-value less than 0.05 is considered statistically significant.

[0119] Experimental results

[0120] On day 34 after drug administration, the tumor volume in the solvent control group was 558.68 mm. 3 The mean tumor volume in the treatment groups treated with the test drug azvudine 1 mg / kg, the positive control drug Paclitaxel 15 mg / kg, and the positive control drug Avastin 10 mg / kg was 460.67 mm. 3 403.40mm 3 456.04mm 3 There were no statistically significant differences 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. The mean tumor volume in the treatment group treated with azvudine 1 mg / kg combined with Paclitaxel 15 mg / kg was 157.34 mm. 3 The tumor inhibition rate (TGI) was 71.57% in the control group, showing a statistically significant difference compared to the control group (p = 0.00707). The combination of avastin (1 mg / kg) and paplitaxel significantly enhanced the antitumor activity of paplitaxel alone (TGI = 26.5%). The mean tumor volume in the avastin 10 mg / kg treatment group was 294.36 mm. 3 The tumor growth rate (TGI) was 48.61%, showing a statistically significant difference compared to the control group (0.140). The combination of the test drug avastin and Avastin significantly enhanced the antitumor effect of Avastin monotherapy (TGI of 17.70%). Tumor growth in each treatment group and the control group is shown in Table 12.

[0121] Table 12. Efficacy analysis of avastin combined with paclitaxel or avastin in the OVCAR-8 humanized ovarian cancer model.

[0122]

[0123]

[0124] Example 6: In vivo pharmacodynamic study of azvudine combined with dacarbazine against a subcutaneous xenograft tumor model of human melanoma A2058 cells. Human melanoma cells A2058 (catalog number: CRL-11147) were cultured in vitro in a monolayer under the following conditions: DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, in a 37°C CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0125] Tumor cells will be inoculated with 0.2 mL (5 × 10⁻⁶) 6 A2058 and Matrigel cells were subcutaneously injected into the right posterior dorsal region of each mouse, resulting in an average tumor volume of approximately 136 mm². 3 Dosing will begin in groups at that time.

[0126] Experimental Results Analysis

[0127] Table 13. Evaluation of the antitumor efficacy of azvudine combined with dacarbazine in the A2058 xenograft tumor model (calculated based on tumor volume on day 14 after administration).

[0128]

[0129] In this embodiment, we evaluated the in vivo efficacy of the test substance in the A2058 xenograft tumor model. Tumor volumes at different time points in each group are shown in Table 13. 14 days after the start of drug administration, the tumor volume in the blank control group of tumor-bearing mice reached 2724 mmHg. 3 The test substance, azvudine (1 mg / kg), showed a smaller tumor-suppressing effect compared to the blank control group, with a tumor volume of 2253 mmHg. 3 The T / C ratio was 79.67%, the TGI was 18.22%, and the p-value was 0.518. Dacarbazine (60 mg / kg) showed tumor-suppressive activity compared to the blank control group, with a tumor volume of 1255 mmHg. 3 The T / C ratio was 44.77%, the TGI was 56.74%, and the p-value was 0.002. The test substance combination group (azvudine + Dacarbazine 1 + 60 mg / kg) showed a significant tumor-suppressing effect compared to the blank control group, with a tumor volume of 910 mmHg. 3 The T / C ratio was 31.52%, the TGI ratio was 70.07%, and the p-value was <0.001.

[0130] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises (i) and (ii): (i) 1-100 mg of azvudine or a pharmaceutically acceptable salt thereof; (ii) 1-500mg chemotherapy reagent; The chemotherapy reagents mentioned are capecitabine, cyclophosphamide, dacarbazine, or paclitaxel.

2. A pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises (i) and (ii): (i) 1-100 mg of azvudine or a pharmaceutically acceptable salt thereof; (ii) 1-500mg bevacizumab.

3. The pharmaceutical composition according to claim 1 or 2, wherein (i) is taken in doses of 1-50 mg; and (ii) is taken in doses of 1-100 mg.

4. The pharmaceutical composition according to claim 1 or 2, wherein (i) is taken in a dose of 1-20 mg; and (ii) is taken in a dose of 1-40 mg.

5. The pharmaceutical composition according to claim 1 or 2, wherein (i) is taken in a dose of 1-10 mg; and (ii) is taken in a dose of 1-10 mg.

6. The pharmaceutical composition according to claim 1 or 2, wherein (i) the dosage is 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 (ii) The dosage is 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, 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, or 50 mg.

7. The pharmaceutical composition according to claim 1 or 2, wherein, The administration frequency for (i) and (ii) is once a day.

8. The pharmaceutical composition according to claim 1 or 2, wherein (i) and (ii) are administered simultaneously or sequentially.

9. The pharmaceutical composition according to claim 1 or 2, wherein (i) and (ii) are present in the same dosage form.

10. The pharmaceutical composition according to any one of claims 1 to 9, for treating tumor-related diseases; said tumor-related diseases are selected from ovarian cancer, melanoma, colorectal cancer, malignant lymphoma, and leukemia.

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