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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
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  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
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  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Definitions

• the present disclosure belongs to the field of medicine, and in particular relates to an anti-tumor pharmaceutical composition containing azithromycin.
• DCK Deoxycytidine kinase
• Azvudine is a broad-spectrum RNA virus inhibitor.
• RdRp viral RNA-dependent RNA polymerase
• azvudine triphosphate 5'-triphosphate metabolite
• 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.
• azvudine tablets were approved for marketing in China for the treatment of adult HIV-1 infected patients with high viral loads.
• studies have found that azvudine has a significant inhibitory effect on a variety of human cancer cells and animal transplanted tumors.
• 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, chemotherapy-immunosynergy can be achieved through dual targeting of tumor cells to enhance the anti-tumor efficacy.
• the present disclosure provides a pharmaceutical composition of azithromycin (FNC) and a chemotherapeutic agent, and use of the pharmaceutical composition in preparing a medicament for preventing or treating tumor diseases.
• FNC azithromycin
• the pharmaceutical composition disclosed in the present invention has at least the following advantages:
• composition comprising:
• 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, doxorubicin Liposomes, daunorubicin liposomes, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paritacept, pemetrexed, pentostatin, proc
• the chemotherapy agent is selected from capecitabine, cyclophosphamide, dacarbazine, paclitaxel or any combination thereof.
• composition which comprises:
• 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.
• the dose of azvudine is selected from 1-100 mg, and the dose of the chemotherapeutic agent is selected from 1-500 mg.
• the dosage of Azvudine is selected from 1-100 mg, and the dosage of Bevacizumab (Avastin) is selected from 1-500 mg.
• the dosage of azithromycin described in the present disclosure can 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, 0mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56 mg, 57 mg, 58 mg, 59 mg, 0mg, 51mg, 52mg, 53mg, 54m
• the dosage of the chemotherapeutic agent described in the present disclosure can 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, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg,
• the dosage of the azithromycin 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 dosage of the chemotherapeutic agent is selected from 1-500 mg, and the administration frequency can be once a day, twice a day or three times a day.
• the dosage of the azithromycin is selected from 1-50 mg, and the administration frequency can be once a day or twice a day, and the dosage of the chemotherapeutic agent is selected from 1-100 mg, and the administration frequency is once a day.
• the dosage of the azvudine is selected from 1-20 mg, and the administration frequency can be once a day or twice a day, and the dosage of the chemotherapeutic agent is selected from 1-40 mg, and the administration frequency is once a day.
• the dosage of the azithromycin is selected from 1-10 mg, and the administration frequency can be once a day or twice a day, and the dosage of the chemotherapeutic agent is selected from 1-10 mg, and the administration frequency is once a day.
• the dosage of the chemotherapeutic 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, and the dosage of the chemotherapeutic agent is selected from 10 mg, 20 mg, 40 mg, 60 mg, and the frequency of administration is once a day.
• 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.
• the dosage of the chemotherapeutic 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 chemotherapeutic agent is selected from 10 mg, 20 mg, 40 mg, 60 mg, and the administration frequency is once a day.
• the dosage of the chemotherapeutic 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 chemotherapeutic agent is selected from 1 mg, 2.5 mg, 5 mg, 10 mg, and the administration frequency is once a day.
• 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.
• the present disclosure also provides a pharmaceutical composition of the above-mentioned azithromycin and a chemotherapeutic agent and 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 injections, 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 injections, sterile powders for injection and concentrated solutions for injection), composite suppositories, composite inhalants, or composite sprays.
• 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 chemotherapeutic agent to a patient.
• the present disclosure also provides a drug kit for use in treating tumor diseases, wherein the drug composition of the azvudine and a chemotherapeutic agent described in the present disclosure is packaged.
• the present disclosure administers azithromycin in combination with a chemotherapeutic agent, thereby enhancing the effect of the drug in treating neoplastic diseases.
• “Combination” as used in the present disclosure is a mode of administration, which means administering at least one dose of azvudine and at least one dose of a chemotherapeutic agent within a certain time period, wherein both substances show pharmacological effects.
• the time period may be within one administration cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours, more preferably within 12 hours.
• Azvudine and the chemotherapeutic agent may be administered simultaneously or sequentially. This period includes treatments in which azvudine and the chemotherapeutic agent are administered by the same route of administration or by different routes of administration.
• FIG1 is a graph showing the effects of azithromycin, capecitabine alone or in combination on the volume of subcutaneous xenograft tumors in the human colon cancer COLO 205 tumor model, where FNC is azithromycin;
• FIG2 is a graph showing the effects of azithromycin, capecitabine alone or in combination on subcutaneous xenograft tumor volume in the human colorectal cancer LoVo tumor model, FNC is azithromycin;
• FIG3 is a graph showing the effects of azithromycin, cyclophosphamide alone or in combination on subcutaneous xenograft tumor volume in a human Burkitt's lymphoma cell Daudi tumor model, where CTX is cyclophosphamide;
• FIG4 is a graph showing the effects of azithromycin, cyclophosphamide alone or in combination on subcutaneous xenograft tumor volume in a human acute lymphoblastic leukemia cell MOLT4 tumor model;
• FIG5 is a graph showing the effects of azithromycin, paclitaxel alone or in combination on subcutaneous xenograft tumor volume in the human ovarian cancer OVCAR-8 tumor model;
• FIG6 is a graph showing the effect of azithromycin, Avastin alone or in combination on subcutaneous xenograft tumor volume in the human ovarian cancer OVCAR-8 tumor model;
• FIG. 7 is a graph showing the effects of azithromycin, dacarbazine, or both alone or in combination on subcutaneous xenograft tumor volume in a human melanoma A2058 tumor model.
• mice Female, 7-8 weeks (age of mice at the time of tumor cell inoculation), weighing 15.7-20.7g, 68 mice (48 mice plus 20 surplus mice). Purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., breeding environment: SPF grade. The experimental animals were all raised in independent ventilation boxes with constant temperature and humidity. The temperature of the breeding room was 20-26°C, the humidity was 40-70%, the ventilation was 10-20 times/hour, and the light and dark alternation time was 12h/12h during the day and night; Cobalt 60 radiation sterilized mouse complete granular feed was continuously supplied, and unlimited free intake was allowed.
• the mouse breeding box was a polysulfone mouse box, which was used after high-pressure sterilization, with a specification of 325mm ⁇ 210mm ⁇ 180mm.
• Colo 205 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Colo205 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and mixed with Matrigel 1:1 for subcutaneous tumor inoculation in mice. 5 ⁇ 10 6 Colo 205 cells were subcutaneously inoculated on the right side of female mice, and the day of inoculation was defined as day 0. When the average tumor volume reached 110.26 mm 3 , the mice were randomly divided into groups according to tumor size.
• Relative tumor proliferation rate is the percentage of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point.
• the calculation formula is as follows:
• 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).
• 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).
• the average tumor volume of mice in the Vehicle group was 1465.47 mm 3
• the average tumor volume of the Capecitabine 400 mg/kg treatment group was 264.96 mm 3 , which was statistically significantly different from the control group (p ⁇ 0.001), and the relative tumor inhibition rate TGI (%) was 81.93%
• the average tumor volume of the combination group, the azithromycin 1 mg/kg combined with the Capecitabine 400 mg/kg group was 144.76 mm 3 , which was statistically significantly different from the control group ( ⁇ 0.001), and the relative tumor inhibition rate TGI (%) was 90.14%.
• Human colon cancer cells LoVo (Cat. No.: ECACC-87060101) were cultured in a monolayer in vitro in F12K medium with 10% fetal bovine serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, and cultured in a 37°C 5% CO 2 incubator. The cells were routinely digested and passaged with trypsin-EDTA twice a week. When the cell saturation was 80%-90% and the number reached the required level, the cells were harvested, counted, and inoculated.
• LoVo cells 0.1 mL (10 ⁇ 10 6 ) were subcutaneously inoculated on the right back of each mouse, and group dosing began when the average tumor volume reached about 147 mm 3 .
• the in vivo efficacy of the test substance in the LoVo xenograft tumor model was evaluated.
• the tumor volume and tumor weight of each group after 21 days of administration are shown in Table 5, Table 6 and Table 2, respectively. 21 days after the start of administration, the tumor volume of the tumor-bearing mice in the blank control group reached 1481mm 3.
• the test substance azithromycin (1mg/kg) had a smaller tumor inhibition effect than the blank control group, with a tumor volume of 902mm 3 , a T/C of 62.00%, a TGI of 43.40%, and a p value of 0.086.
• test substance capecitabine 400mg/kg had a smaller tumor inhibition effect than the blank control group, with a tumor volume of 919mm 3 , a T/C of 60.94%, a TGI of 42.11%, and a p value of 0.696.
• the combined group of Azvudine + 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 ; T/C of 448.18%; TGI of 60.29%; and p value of 0.282.
• the combined use of Azvudine + Capecitabine can enhance the tumor inhibition effect of Capecitabine alone in the LoVo colorectal tumor, with TGI increasing from 42.1% to 60.3%.
• Example 3 In vivo pharmacodynamics of azithromycin combined with cyclophosphamide in a human Burkitt's lymphoma cell Daudi subcutaneous xenograft tumor model
• Human Burkitt's lymphoma Daudi cells (Cat. No.: DSMZ-ACC129) were cultured in vitro in suspension in RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, and cultured in a 37°C 5% CO 2 incubator. When the cell saturation reached 80%-90% and the number reached the required level, the cells were collected, counted, and inoculated. Tumor cell inoculation
• 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 of 8.83%, TGI of 94.17%, and p value of ⁇ 0.0001.
• test substance azithromycin (1 mg/kg) combined with cyclophosphamide can enhance the tumor-suppressing effect of single-agent CTX in the human Burkitt's lymphoma cell line Daudi, and the TGI is increased from 94.17% to 103.15%.
• Example 4 In vivo pharmacodynamic study of azithromycin combined with cyclophosphamide on human acute lymphoblastic leukemia MOLT4 cell subcutaneous xenograft tumor model
• MOLT4 cells Human acute lymphoblastic leukemia MOLT4 cells (Cat. No.: ECACC-85011413) were cultured in vitro in suspension in RPMI1640 medium with 10% fetal bovine serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, and cultured in a 37°C 5% CO 2 incubator. When the cell saturation reached 80%-90% and the number reached the required level, the cells were collected, counted, and inoculated. Tumor cell inoculation
• MOLT4 cells with Matrigel, volume ratio of 1:1
• Cyclophosphamide CTX (50 mg/kg) had a significant inhibitory effect compared with the blank control group, with a tumor volume of 716 mm 3 , a T/C of 24.53%, a TGI of 79.34%, and a p value of ⁇ 0.0001.
• the combined group of test substance, 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 of 8.75%, TGI of 96.14%, and p value of ⁇ 0.001.
• the analysis and statistical results of tumor weight in the combined group of test substance were basically consistent with the tumor volume data.
• 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.
• 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%.
• OVCAR-8 cells were cultured in RPMI1640 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 with Matrigel 1:1 for subcutaneous tumor inoculation in mice.
• mice Female mice were subcutaneously inoculated with 1 ⁇ 10 7 OVCAR-8 cells on the right side. When the average tumor volume reached 174.25 mm 3 , the mice were randomly divided into groups according to tumor size.
• Relative tumor proliferation rate is the percentage of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point.
• the calculation formula is as follows:
• T/C% TTW /C TW ⁇ 100% ( TTW : average tumor weight of the treatment group at the end of the experiment; C TW : average tumor weight of the vehicle control group at the end of the experiment).
• 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).
• the tumor volume of the vehicle control group was 558.68 mm 3
• the average tumor volume of the test drug Azivudine 1mg/kg combined with Avastin 10mg/kg treatment group was 294.36mm 3 , which was statistically significantly different from the control group (0.140), and the relative tumor inhibition rate TGI (%) was 48.61%.
• the tumor growth of each treatment group and the control group is shown in Table 12.
• Example 6 In vivo pharmacodynamic study of azithromycin combined with dacarbazine on human melanoma A2058 cell subcutaneous xenograft tumor model
• Human melanoma cells A2058 (Cat. No.: CRL-11147) were cultured in a monolayer in vitro in DMEM medium with 10% fetal bovine serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, and cultured in a 37°C CO 2 incubator. The cells were routinely digested and passaged with trypsin-EDTA twice a week. When the cell saturation reached 80%-90% and the number reached the required level, the cells were harvested, counted, and inoculated.
• the in vivo efficacy of the test substance in the A2058 xenograft tumor model was shown in Table 13. 14 days after the start of administration, the tumor volume of tumor-bearing mice in the blank control group reached 2724mm 3 .
• the test substance azithromycin (1mg/kg had a smaller tumor inhibition effect compared with the blank control group, with a tumor volume of 2253mm 3 , a T/C of 79.67%, a TGI of 18.22%, and a p value of 0.518.
• the pharmaceutical composition disclosed in the present invention exhibits synergistic effects in anti-tumor aspects, can reduce the dosage of chemotherapy preparations, improve efficacy and safety, thereby achieving the goal of prolonging the survival of patients, and has broad prospects for industrial implementation.

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