Anti-tumor pharmaceutical composition comprising azvudine and chemotherapeutic agent

Abstract

Provided in the present invention is a pharmaceutical composition comprising azvudine and a chemotherapeutic agent. The pharmaceutical composition shows a relatively good synergistic effect in combating tumors, can reduce the dosage of the chemotherapeutic agent, and improve the efficacy and safety, thereby achieving the purpose of prolonging the survival time of a patient.

Description

Antitumor pharmaceutical composition comprising azvudine and a chemotherapeutic agent Technical Field

[0001] The present disclosure belongs to the field of medicine, and particularly relates to an anti-tumor pharmaceutical composition containing azvudine. Background Art

[0002] Deoxycytidine kinase (DCK) is an enzyme with broad substrate specificity that phosphorylates pyrimidine and purine deoxynucleosides. It is a key enzyme in the salvage pathway of deoxynucleotide biosynthesis, maintaining normal DNA metabolism and phosphorylating a variety of antiviral and anticancer nucleoside analogs. These drugs are activated only upon phosphorylation, thereby inhibiting tumor growth. Over the past few decades, apoptosis has been extensively studied, and radiotherapy strategies targeting apoptosis have become an important approach to tumor 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 into a 5'-triphosphate metabolite (azvudine triphosphate) with antiviral activity in cells. Its target is the viral RdRp, which can block the synthesis and replication of RNA chains in host cells by inhibiting the activity of RdRp. In July 2021, azvudine tablets were approved for marketing in China for the treatment of adult HIV-1-infected patients with high viral loads. In addition, studies have found that azvudine has a significant inhibitory effect on a variety of human cancer cells and animal transplanted tumors.

[0004] Among traditional tumor treatments, chemotherapy is currently the most widely used clinical tumor treatment due to its strong therapeutic effects and high efficacy. However, most chemotherapy drugs currently used in clinical practice have poor selectivity. While treating tumor tissue, they can also cause significant damage to normal tissues in the body, leading to serious toxic side effects.

[0005] Single-drug treatment has defects such as poor physical and chemical properties, low bioavailability, and poor efficacy. If chemotherapy drugs can be combined with azithromycin, the tumor effect can be improved. Based on the complementary synergy between the two drugs in the anti-tumor mechanism of action, by dual-targeting tumor cells, chemotherapy-immune synergy can be achieved to enhance the anti-tumor efficacy.

[0006] Summary of the Invention

[0007] The present disclosure provides a pharmaceutical composition of azvudine (FNC) and a chemotherapy agent, and use of the pharmaceutical composition in preparing a medicament for preventing or treating tumor diseases.

[0008] Compared with each single drug, the pharmaceutical composition disclosed herein has at least the following advantages:

[0009] 1. The combined use enhanced the tumor-suppressing effect of each drug alone;

[0010] 2. Delay the development of drug resistance, improve efficacy and safety, and thus achieve the goal of prolonging patient survival.

[0011] The present disclosure provides a pharmaceutical composition comprising:

[0012] (i) Azithromycin or a pharmaceutically acceptable salt, stereoisomer or isotopic derivative thereof;

[0013] (ii) Chemotherapeutic agents.

[0014] In an optional technical solution of the present disclosure, the chemotherapy agent 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, folinic acid, doxorubicin Liposomes, daunorubicin liposomes, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paritacept, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or any combination thereof.

[0015] In an optional technical solution of the present disclosure, the chemotherapy agent is selected from capecitabine, cyclophosphamide, dacarbazine, paclitaxel or any combination thereof.

[0016] In addition, the present disclosure also provides another pharmaceutical composition: which comprises:

[0017] (i) Azithromycin or a pharmaceutically acceptable salt, stereoisomer or isotopic derivative thereof;

[0018] (ii) Avastin.

[0019] In an optional technical solution of the present disclosure, (i) and (ii) are administered simultaneously, separately, or sequentially, or (i) and (ii) are present in the same dosage form.

[0020] In an optional technical solution of the present disclosure, it is used to treat tumor-related diseases.

[0021] In an optional technical solution of the present disclosure, the tumor-related disease is 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 optional 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 optional embodiments, the dose of the azvudine is selected from 1-100 mg, and the dose of the bevacizumab (Avastin) is selected from 1-500 mg.

[0024] The dosage of azivudine described in the present disclosure may be 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, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg 0mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74mg, 75m g, 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 chemotherapeutic agent of the present disclosure may be 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 optional embodiments, the dose of azvudine is selected from 1-100 mg, and the administration frequency can be once a day, twice a day or three times a day, and the dose of the chemotherapy agent is selected from 1-500 mg, and the administration frequency can be once a day, twice a day or three times a day.

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

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

[0029] In some optional embodiments, the dosage of the azvudine is selected from 1-10 mg, and the administration frequency can be once a day or twice a day, and the dosage of the chemotherapy agent is selected from 1-10 mg, and the administration frequency is once a day.

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

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

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

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

[0034] The combined administration route disclosed herein is oral administration, parenteral administration, and transdermal administration. The parenteral administration includes but is not limited to intravenous injection, subcutaneous injection, and intramuscular injection, and oral administration is preferred.

[0035] The present disclosure also provides a pharmaceutical composition comprising the above-mentioned azithromycin and a chemotherapeutic agent, as well as one or more pharmaceutical carriers, excipients, and diluents. The pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form. For example, it can be formulated into tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), suppositories, inhalants, or sprays. The pharmaceutical composition can also be prepared into the same dosage form, for example, azithromycin and a chemotherapeutic agent can be formulated into composite tablets, composite capsules, composite pills, composite granules, composite solutions, composite suspensions, composite syrups, composite injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), composite suppositories, composite inhalants, or composite sprays.

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

[0037] The present disclosure also provides a pharmaceutical kit for use in treating tumor diseases, wherein the pharmaceutical composition of azvudine and a chemotherapeutic agent described in the present disclosure is packaged.

[0038] The present disclosure administers azvudine in combination with a chemotherapeutic agent, thereby enhancing the efficacy of the drug in treating tumor diseases.

[0039] As used herein, "combination" refers to a regimen that includes administering at least one dose of azvudine and at least one dose of a chemotherapeutic agent within a timeframe, wherein both agents exhibit a pharmacological effect. The timeframe can be within a single 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 timeframe includes treatments in which azvudine and the chemotherapeutic agent are administered via the same route of administration or via different routes of administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG1 is a graph showing the effects of azvudine, capecitabine, or both alone or in combination on subcutaneous xenograft tumor volume in a human colon cancer COLO 205 tumor model, where FNC represents azvudine;

[0041] FIG2 is a graph showing the effects of azvudine, capecitabine, or both alone or in combination on subcutaneous xenograft tumor volume in the human colorectal cancer LoVo tumor model, where FNC represents azvudine;

[0042] FIG3 is a graph showing the effects of azvudine, cyclophosphamide alone or in combination on subcutaneous xenograft tumor volume in a human Burkitt's lymphoma cell Daudi tumor model, where CTX is cyclophosphamide;

[0043] FIG4 is a graph showing the effects of azvudine, cyclophosphamide, or both alone or in combination on subcutaneous xenograft tumor volume in a human acute lymphoblastic leukemia cell MOLT4 tumor model;

[0044] FIG5 is a graph showing the effects of azvudine, paclitaxel, or both alone or in combination on subcutaneous xenograft tumor volume in the human ovarian cancer OVCAR-8 tumor model;

[0045] FIG6 is a graph showing the effects of Azivudine, Avastin alone or in combination on subcutaneous xenograft tumor volume in the human ovarian cancer OVCAR-8 tumor model;

[0046] FIG7 is a graph showing the effects of azvudine, dacarbazine, or both alone or in combination on subcutaneous xenograft tumor volume in a human melanoma A2058 tumor model. DETAILED DESCRIPTION

[0047] The present disclosure will be explained in more detail below with reference to 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 essence and scope of the present disclosure.

[0048] Experimental Materials

[0049] Experimental animals and breeding environment

[0050] experimental animals

[0051] Nude female BALB / c mice, 7-8 weeks old (age at time of tumor cell inoculation), weighing 15.7-20.7 g, were obtained from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Sixty-eight mice (48 plus 20 surplus mice) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The animals were housed in SPF-grade enclosures at a constant temperature and humidity of 20-26°C, 40-70% humidity, 10-20 air changes per hour, and a 12-hour light / dark cycle. They were continuously provided with cobalt-60-irradiated sterilized complete mouse pellets, with ad libitum access. Tap water (sterilized after autoclaving) was also available, and the animals were kept in autoclaved polysulfone enclosures measuring 325 mm × 210 mm × 180 mm.

[0052] Example 1 Testing the Anti-tumor Effect of Azivudine Combined with Capecitabine in the Colo 205 Human Colon Cancer Model Study Results and Discussion

[0053] Cell culture

[0054] Colo 205 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Colo 205 cells in the exponential growth phase were collected, resuspended in PBS to an appropriate concentration, and mixed with Matrigel 1:1 for subcutaneous tumor inoculation in mice. 5×10 6 For Colo 205 cells, the day of inoculation was defined as day 0. When the average tumor volume was 110.26 mm 3 Patients were randomly divided into groups according to tumor size.

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

[0056] Efficacy evaluation criteria

[0057] T / C%=TRTV / CRTV×100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV=Vt / V0, 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 vehicle control group).

[0059] The relative tumor inhibition rate (TGI) was 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 the 25th day after tumor inoculation (the 20th day after grouping), the average tumor volume of mice in the Vehicle group was 1465.47 mm 3 The average tumor size of the test drug monotherapy group, azvudine (1 mg / kg), was 985.14 mm 3 There was no statistically significant difference compared with the control group (p = 0.540), the relative tumor inhibition rate TGI (%) was 32.43%, and the average tumor volume of the Capecitabine, 400 mg / kg treatment group was 264.96 mm 3 Compared with the control group, there was a statistically significant difference (p < 0.001), the relative tumor inhibition rate TGI (%) was 81.93%, and the average tumor volume of the combined group of azithromycin, 1 mg / kg, and capecitabine, 400 mg / kg, was 144.76 mm 3Compared with the control group, there was a statistically significant difference (<0.001), and the relative tumor inhibition rate TGI (%) was 90.14%.

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

[0062] Table 2. Efficacy analysis of each group in the Colo 205 human colon cancer model

[0063] Tumor weight inhibition results

[0064] Table 3. Tumor remodeling analysis of each group in the Colo 205 human colon cancer model

[0065] Example 2 Pharmacodynamic Study of Azivudine Combined with Capecitabine in Human Colon Cancer LoVo Cells

[0066] Cell culture

[0067] Human colon cancer cells, LoVo (Cat. No. ECACC-87060101), were cultured as monolayers in F12K medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged twice weekly using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0068] 0.1 mL (10 × 10 6 ) LoVo cells were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 147 mm 3 The group dosing started at 14:00.

[0069] Table 4. Experimental design of the antitumor effect of different doses of test drugs in the COLO 205 human colon cancer tumor model

[0070] Experimental results

[0071] After treatment of BALB / c nude mice bearing subcutaneous Lovo cell xenograft tumors with the test drugs, the changes in tumor volume (21 days after administration) in each group are shown in Table 5, and the changes in tumor weight are shown in Table 6.

[0072] Table 5 Evaluation of the antitumor efficacy of the test drugs in the LoVo xenograft tumor model (calculated based on the tumor volume on day 21 after administration)

[0073] Table 6 Evaluation of the anti-tumor efficacy of the test drugs in the LoVo xenograft tumor model (calculated based on the tumor size on day 21 after administration)

[0074] In this study, the in vivo efficacy of the test substance was evaluated in a LoVo xenograft tumor model. The tumor volume and weight of each group 21 days after administration are shown in Tables 5, 6, and 2, respectively. 21 days after the start of administration, the tumor volume of the tumor-bearing mice in the blank control group reached 1481 mm 3 The test substance azvudine (1 mg / kg) had a smaller tumor inhibition effect than the blank control group, and the tumor volume was 902 mm 3 , T / C was 62.00%, TGI was 43.40%, and p value was 0.086. Capecitabine (400 mg / kg) had a smaller tumor inhibition effect than the blank control group, with a tumor volume of 919 mm 3 , T / C was 60.94%, TGI was 42.11%, and the p value was 0.696.

[0075] The combined group of Azivudine + Capecitabine (1+400 mg / kg) had a significant tumor inhibition effect compared with 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. Combination therapy of azvudine and capecitabine enhanced the tumor inhibition effect of capecitabine alone in LoVo colorectal tumors, increasing the TGI from 42.1% to 60.3%.

[0076] Example 3 In vivo pharmacodynamics of azithromycin combined with cyclophosphamide in a human Burkitt's lymphoma cell Daudi subcutaneous xenograft tumor model

[0077] Human Burkitt's lymphoma Daudi cells (Cat. No. DSMZ-ACC129) were cultured in suspension in RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator. When cell saturation reached 80%-90% and the required number of cells was reached, the cells were harvested, counted, and inoculated.

[0078] 0.2 mL (10 × 10 6 Daudi cells (with Matrigel, volume ratio of 1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 100 mm 3 The group dosing started at 14:00.

[0079] Experimental results

[0080] The tumor volume and tumor changes of each group in SCID mice bearing Daudi cell subcutaneous xenograft tumors after treatment with the test drugs are shown in Figures 8 and 9.

[0081] Table 8 Tumor volume after 27 days of administration

[0082] Table 9 Tumor weight after 27 days of administration

[0083] In this study, we evaluated the in vivo efficacy of the test compound in a Daudi xenograft tumor model. The tumor volumes of each group at different time points are shown in Tables 8 and 9. 27 days after the start of administration, the tumor volume of the tumor-bearing mice in the blank control group reached 2,895 mm 3 The test substance azithromycin (1 mg / kg) had a significant tumor inhibition effect compared with the blank control group, and the tumor volume was 909 mm 3 ; T / C was 30.64%; TGI was 71.09%; p value was <0.0001. The test substance cyclophosphamide (50 mg / kg) had a significant tumor inhibition effect compared with the blank control group, with a tumor volume of 263 mm; T / C was 8.83%; TGI was 94.17%; p value was <0.0001. The test substance combination group azvudine + cyclophosphamide (1 + 50 mg / kg) had a significant tumor inhibition effect compared with the blank control group, with a tumor volume of 13 mm 3 ; T / C was 0.42%; TGI was 103.15%; p value was <0.0001.

[0084] In this experiment, the test substance azvudine (1 mg / kg) combined with cyclophosphamide can enhance the tumor inhibitory effect of single-agent CTX in the human Burkitt's lymphoma Daudi cell line, and the TGI is increased from 94.17% to 103.15%.

[0085] Example 4 In vivo pharmacodynamic study of azithromycin combined with cyclophosphamide on human acute lymphoblastic leukemia MOLT4 cell subcutaneous xenograft tumor model

[0086] Cell culture

[0087] Human acute lymphoblastic leukemia MOLT4 cells (Cat. No. ECACC-85011413) were cultured in suspension in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator. When cell saturation reached 80%-90% and the required number of cells was reached, the cells were harvested, counted, and inoculated.

[0088] 0.2 mL (10 × 10 6 MOLT4 cells (with Matrigel, volume ratio of 1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 148 mm 3 The group dosing started at 14:00.

[0089] The changes in tumor volume and weight of each group in SCID Beige mice bearing MOLT4 cell subcutaneous xenograft tumors after treatment with the test drugs are shown in Figures 10 and 11.

[0090] Table 10 Tumor volume after 28 days of administration

[0091] Table 11 Tumor weight after 28 days of administration

[0092] In this study, we evaluated the in vivo efficacy of the test compound 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 dosing, the tumor volume of the tumor-bearing mice in the blank control group reached 2,896 mm 3 The test substance azithromycin (1 mg / kg) had an anti-tumor effect compared with the blank control group, and the tumor volume was 979 mm 3 , T / C was 33.89, TGI was 69.73%, p value <0.0001. Cyclophosphamide CTX (50 mg / kg) had a significant tumor inhibition effect compared with the blank control group, with a tumor volume of 716 mm 3 , T / C was 24.53%, TGI was 79.34%, and p value was <0.0001. The combined group of azithromycin + cyclophosphamide (1+50mg / kg) had a significant tumor inhibition effect compared with the blank control group, with a tumor volume of 253mm 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 substance combination group were basically consistent with the tumor volume data.

[0093] In this experiment, the test substances azvudine (1 mg / kg), cyclophosphamide (50 mg / kg), and azvudine + cyclophosphamide (1 + 50 mg / kg) had a significant inhibitory effect on the growth of MOLT4 xenograft tumors at the tested doses. In this experiment, the test substance azvudine (1 mg / kg) combined with cyclophosphamide can enhance the tumor inhibitory effect of cyclophosphamide alone in MOLT4 human acute lymphoblastic leukemia tumors, and the TGI increased from 79.34% to 96.14%.

[0094] Example 5 Evaluation of the therapeutic effect of azvudine combined with paclitaxel in the human ovarian cancer OVCAR-8 tumor model

[0095] OVCAR-8 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. OVCAR-8 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and mixed with Matrigel at a 1:1 ratio for subcutaneous tumor inoculation in mice.

[0096] Female mice were inoculated subcutaneously on the right side with 1×10 7 OVCAR-8 cells. The average tumor volume was 174.25 mm 3 Patients were randomly divided into groups according to tumor size.

[0097] The experimental protocols for animal experiments during this experiment were reviewed and approved by the CrownBio IACUC Committee. During the experiment, animal experimental operations were in accordance with the requirements of AAALAC. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of animals, including the activity of experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Clinical symptoms observed during the experiment were recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter).

[0098] The relative tumor growth rate, T / C%, is the percentage of the tumor volume or weight of the treatment group and the control group at a certain time point. The calculation formula is as follows:

[0099] T / C%=T RTV / C RTV ×100%(T RTV : Average RTV of treatment group; C RTV : Average RTV of vehicle control group; RTV = V t / V0, V0 is the tumor volume of the animal when grouped, V t is the tumor volume of the animal after treatment);

[0100] Or T / C% = T TW / C TW ×100%(T TW : Average tumor weight at the end of the experiment in the treatment group; C TW : Average tumor weight at the end of the experiment in the vehicle control group).

[0101] The relative tumor inhibition rate (TGI) was 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).

[0102] To compare tumor volumes on a given day across treatment groups, we first used the Bartlett test to verify the assumption of homogeneity of variance across all groups. When the p-value from the Bartlett test was ≥0.05, one-way ANOVA was used to test the equality of the means across all groups. If the p-value from the one-way ANOVA was less than 0.05, we used the Tukey HSD test for pairwise comparisons between all groups or the Dunnett's t-test for pairwise comparisons between each treatment group and the control group. When the p-value from the Bartlett test was less than 0.05, the Kruskal-Wallis test was used to test the equality of the medians across all groups. If the p-value from the Kruskal-Wallis test was less than 0.05, we used the Conover test for pairwise comparisons between all groups or between each treatment group and the control group, adjusting the p-value for multiple testing.

[0103] In addition, for exploratory data analysis purposes, we performed pairwise comparisons between all groups at any time point. Because these comparisons only used tumor volume data from the two groups at the specific time point, no correction for multiple testing was required. We first used the Bartlett test to verify the assumption of homogeneity of variance between the two groups. When the p-value of the Bartlett test was ≥0.05, we used the Welch's t-test to compare the equality of the means between the two groups. When the p-value of the Bartlett test was less than 0.05, we used the Mann-Whitney U test to compare the equality of the medians between the two groups.

[0104] All statistical analyses and graphics were performed in the R language environment (version 3.3.1). Unless otherwise specified, all tests were two-tailed, and p values ​​less than 0.05 were considered statistically significant.

[0105] Experimental results

[0106] On day 34 after administration, the tumor volume of the vehicle control group was 558.68 mm 3 The average tumor volumes of the test drug Azithromycin 1mg / kg treatment group, the positive drug Paclitaxel 15mg / kg treatment group, and the sexual drug Avastin 10mg / kg treatment group were 460.67mm 3 , 403.40mm 3 , 456.04mm 3There was no statistically significant difference compared with the control group (p = 0.952, 0.769 and 0.957). The relative tumor inhibition rate TGI (%) was 18.09%, 26.49% and 17.70%, respectively. The average tumor volume of the test drug azithromycin 1mg / kg combined with paclitaxel 15mg / kg treatment group was 157.34mm 3 , there was a statistically significant difference compared to the control group (p = 0.00707), and the relative tumor inhibition rate TGI (%) was 71.57%. After the test drug Azivudine was co-administered with Paclitaxel, the anti-tumor effect of Paclitaxel alone was significantly improved (TGI was 26.5%). The average tumor volume of the test drug Azivudine 1mg / kg combined with Avastin 10mg / kg treatment group was 294.36mm 3 , with a statistically significant difference (0.140) compared to the control group, and a relative tumor inhibition rate (TGI) of 48.61%. The combined administration of the test drug azithromycin with Avastin significantly enhanced the anti-tumor effect of Avastin alone (TGI of 17.70%). Tumor growth in each treatment group and control group is shown in Table 12.

[0107] Table 12. Efficacy analysis of each group in the OVCAR-8 human ovarian cancer model of azithromycin combined with paclitaxel or Avastin

[0108] Example 6 In vivo pharmacodynamic study of azithromycin combined with dacarbazine on human melanoma A2058 cell subcutaneous xenograft tumor model

[0109] Human melanoma A2058 cells (Cat. No. CRL-11147) were cultured as monolayers in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C CO2 incubator. Cells were routinely digested and passaged twice weekly using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0110] Tumor cell inoculation

[0111] 0.2 mL (5 × 10 6 A2058 cells plus Matrigel were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 136 mm 3 The group dosing started at 14:00.

[0112] Experimental results analysis

[0113] Table 13. Evaluation of the anti-tumor efficacy of azvudine combined with dacarbazine in the A2058 xenograft tumor model (calculated based on the tumor volume on day 14 after administration)

[0114] In this example, we evaluated the in vivo efficacy of the test compound in an 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 the tumor-bearing mice in the blank control group reached 2724 mm 3 The test substance azithromycin (1 mg / kg) had a smaller tumor inhibition effect than the blank control group, and the tumor volume was 2253 mm 3 , T / C was 79.67%, TGI was 18.22%, and p value was 0.518. Dacarbazine (60 mg / kg) had an anti-tumor effect compared with the blank control group, and the tumor volume was 1255 mm 3 , T / C was 44.77%, TGI was 56.74%, and p value was 0.002. The combined group of Azivudine + Dacarbazine (1+60 mg / kg) had a significant tumor inhibition effect compared with the blank control group, with a tumor volume of 910 mm 3 , T / C was 31.52%, TGI was 70.07%, and the p value was <0.001.

[0115] Although the specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims. Industrial Applicability

[0116] The pharmaceutical composition disclosed herein exhibits synergistic effects in anti-tumor treatment, can reduce the dosage of chemotherapy preparations, improve efficacy and safety, thereby achieving the goal of prolonging patient survival, and has broad industrial implementation prospects.

Claims

1. A pharmaceutical composition comprising: (i) azithromycin or a pharmaceutically acceptable salt, stereoisomer or isotopic derivative thereof; (ii) Chemotherapeutic agents.

2. The pharmaceutical composition according to claim 1, wherein The chemotherapeutic agent 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, folinic acid, liposomal doxorubicin, daunorubicin lipid Any one of plasmids, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paritacept, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof, or any combination thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein The chemotherapeutic agent is selected from capecitabine, cyclophosphamide, dacarbazine, paclitaxel or any combination thereof.

4. A pharmaceutical composition comprising: (i) azithromycin or a pharmaceutically acceptable salt, stereoisomer or isotopic derivative thereof; (ii) Avastin.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein said (i) and (ii) are administered simultaneously, separately, sequentially or said (i) and (ii) are present in the same dosage form.

6. The pharmaceutical composition according to any one of claims 1 to 5, for use in treating tumor-related diseases.

7. The pharmaceutical composition according to claim 6, wherein The tumor-related disease is 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 carcinoma or choriocarcinoma.

8. A method for treating a tumor-related disease, comprising the step of administering the pharmaceutical composition of any one of claims 1 to 6 to an individual in need thereof.

9. The method of claim 8, wherein the tumor-related disease is 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 carcinoma or choriocarcinoma.