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WO2015021741A1 - Utilisation de doxycycline - Google Patents

Utilisation de doxycycline Download PDF

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Publication number
WO2015021741A1
WO2015021741A1 PCT/CN2014/000721 CN2014000721W WO2015021741A1 WO 2015021741 A1 WO2015021741 A1 WO 2015021741A1 CN 2014000721 W CN2014000721 W CN 2014000721W WO 2015021741 A1 WO2015021741 A1 WO 2015021741A1
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WIPO (PCT)
Prior art keywords
doxycycline
cells
group
growth
human
Prior art date
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Ceased
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PCT/CN2014/000721
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English (en)
Chinese (zh)
Inventor
饶子和
孙涛
杨诚
周红刚
郭宇
刘慧娟
刘艳荣
王静
张博
李珊
翟佳黛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOTECHNOLOGY AND MEDICINE
TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOTECHNOLOGY AND MEDICINE CO LTD
XUHE (TIANJIN) YIYAO KEJI YOUXIAN GONGSI
Original Assignee
TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOTECHNOLOGY AND MEDICINE
TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOTECHNOLOGY AND MEDICINE CO LTD
XUHE (TIANJIN) YIYAO KEJI YOUXIAN GONGSI
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Publication of WO2015021741A1 publication Critical patent/WO2015021741A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the present invention relates to the field of medicinal chemistry and, in particular, to the use of doxycycline. Background technique
  • Doxycycline (also known as doxycycline), a tetracycline antibiotic, is a well-known and widely used antibiotic. Tetracyclines mainly act on the 30S subunit of the bacterial ribosome, interfering with the amino acid tRNA binding to the site of action on the 30S subunit, blocking the binding of the aminoacyl tRNA to the ribosome-mRNA complex, inhibiting protein synthesis, and also altering The permeability of the cell membrane exposes the nucleotides of important substances in the cell, inhibits DNA synthesis, and thus achieves an antibacterial effect.
  • Doxycycline is a potent and widely used type of tetracycline (Adimora AA., 2002; Kovacova E, et al., 2002). Doxycycline is currently used clinically for the treatment of upper respiratory tract infections, biliary tract infections, urinary tract infections, chronic bronchitis, acute and chronic bronchitis, pneumonia, bronchitis, cellulitis and other symptoms.
  • human metastatic malignant tumors such as adenocarcinoma, cervical cancer, human poorly differentiated colon cancer, and human high metastatic gastric cancer has not been reported. Since the above-mentioned malignant tumors generally have the ability to metastasize and invade, once the above diseases occur, they are difficult to cure and cause great harm to human health.
  • the object of the present invention is to provide a drug for preparing a drug for treating high metastatic malignant tumor.
  • the molecular structural formula of the doxycycline is
  • the high metastatic malignant tumor includes human invasive choroidal melanoma, human malignant melanoma, human breast cancer, human chronic myeloid leukemia, promyelocytic leukemia, human large cell lung cancer, human small cell lung cancer, human high metastatic liver cancer.
  • the use of the medicament for treating a highly metastatic malignant tumor includes use as a drug for inhibiting tumor growth, application as a drug for inhibiting tumor metastasis, and application for inhibiting tumor invasion.
  • the medicament for treating a highly metastatic malignant tumor comprises doxycycline, doxycycline in a pharmaceutically acceptable salt, an ester, a hydrate or a combination thereof and an adjuvant.
  • the dosage form of the medicament for treating a highly metastatic malignant tumor is selected from the group consisting of a tablet, a capsule, a pill, a suppository, an aerosol, a liquid preparation, a granule, a powder, an injection, a syrup, and a wine. , tinctures, lotions, films or combinations thereof.
  • the administration of the drug for treating a highly metastatic malignant tumor includes oral administration, injection, implantation, external use, spraying, inhalation or a combination thereof.
  • the anti-tumor drug provided by the invention has the advantages that: the drug patient is not only easy to accept, but also low in price, widely available, easy to obtain, and easy to understand the patient's reaction to the drug.
  • doxycycline will change the market pattern of existing cancer chemotherapy drugs, becoming a clinical drug that can be taken for a long time and effectively inhibits tumor metastasis, invasion and recurrence.
  • the doxycycline used in the present invention is doxycycline hydrochloride, yellow powder, manufactured by Kaifeng Pharmaceutical Co., Ltd.
  • Figure 1A is a dose-response curve of the inhibitory effect of doxycycline on Mum-2C cells (pre-experimental results);
  • Figure 1B is a dose-response curve of the inhibition of Mum-2C cells by doxorubicin hydrochloride (pre-test results);
  • Figure 2A is a dose-response curve of doxycycline inhibition on Mum-2C cells (formal experimental results);
  • Figure 2B is a dose-response curve of doxorubicin hydrochloride inhibition on Mum-2C cells (official results);
  • Figure 3A is a dose-response curve of doxycycline inhibition on Mum-2B cells (pre-experimental results);
  • Figure 3B is a dose-response curve of doxorubicin hydrochloride inhibition on Mum-2B cells (pre-experimental results);
  • Figure 4A is a dose-response curve of doxycycline inhibition on Mum-2B cells (formal experimental results);
  • Figure 4B is a dose-response curve of doxorubicin hydrochloride inhibition on Mum-2B cells (official results);
  • Figure 5A is a dose-response curve of doxycycline inhibition on A875 cells (pre-experimental results);
  • Figure 5B is a dose-response curve of the inhibition of A875 cells by doxorubicin hydrochloride (pre-experimental results);
  • Figure 6A is a dose-response curve of doxycycline inhibition on A875 cells (formal experimental results);
  • Figure 6B is a dose-response curve of inhibition of A875 cells by doxorubicin hydrochloride (formal experiment) Result);
  • Figure 7A is a dose-response curve of doxycycline inhibition on A375 cells (pre-experimental results);
  • Figure 7B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on A375 cells (pre-experimental results);
  • Figure 8A is a dose-response curve of doxycycline inhibition on A375 cells (formal experimental results);
  • Figure 8B is a dose-response curve of the inhibition of A375 cells by doxorubicin hydrochloride (formal experimental results);
  • Figure 9A is the effect of doxycycline on the growth curve of Mum-2C cells (first test results).
  • a in Fig. 9C shows the cell morphology of the control group observed under an inverted microscope; and B in Fig. 9C shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope
  • Figure 10A is the effect of doxycycline on the growth curve of Mum-2B cells (first test result);
  • Figure 10B is the effect of doxycycline on the growth curve of Mum-2B cells (second test result);
  • Figure 10C is the effect of doxycycline on the growth morphology of Mum-2B cells
  • Figure 11A is the effect of doxycycline on the growth curve of A875 cells (first test result).
  • Fig. 11C is the effect of doxycycline on the growth morphology of ⁇ 875 cells;
  • Fig. ⁇ shows the cell morphology of the control group observed under an inverted microscope
  • Fig. B shows the cell morphology of the doxycycline (Doxy) administration group observed under an inverted microscope).
  • Figure 12C is the effect of doxycycline on the growth morphology of ⁇ 375 cells. ;
  • Fig. ⁇ shows the cell morphology of the control group observed under an inverted microscope
  • Fig. B shows the cell morphology of the doxycycline (Doxy) administration group observed under an inverted microscope
  • Figure 13A shows the results of microscopic observation under the microscope of microscopic staining of doxanthin and doxorubicin for inhibiting the invasive ability of Mum-2C cells for 24 h;
  • Figure 13B is a statistical result of inhibition of invasion of Mum-2C cells by doxycycline and doxorubicin hydrochloride for 24 h.
  • the concentration of doxycycline in Figure 13A and Figure 13B is: 1 ⁇
  • the concentration of doxorubicin hydrochloride is: 0.781 ⁇
  • **: compared with the control group ⁇ ⁇ 0.01 ( ⁇ 3 ) .
  • Figure 14A shows the results of microscopic observation under the microscope of microscopic staining of doxycycline and doxorubicin for inhibiting the invasion of human melanoma cells Mum-2B for 24 h;
  • Figure 14B is a statistical result of the inhibitory effect of doxycycline and doxorubicin on the invasion of human melanoma cells Mum-2B for 24 h;
  • Figure 15A shows the results of microscopic observation under the microscope of microscopic staining of doxycycline and doxorubicin for inhibiting the invasive ability of ⁇ 875 cells for 24 h;
  • Figure 15B shows the statistical results of the inhibitory effect of doxycycline and doxorubicin on the invasive ability of A875 cells for 24 h;
  • FIG. 16A is the result of inhibition of the invasive ability of doxycycline and doxorubicin hydrochloride on human melanoma cell ⁇ 375 after 24 h;
  • Figure 16B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasive ability of human melanoma cell A375 after 24 h;
  • Figure 17A is a dose-response curve of the inhibitory effect of doxycycline on MCF-7 cells (pre-experimental results);
  • 17 B is the dose-response curve of doxorubicin hydrochloride on MCF-7 cells (pre-experimental results);
  • Figure 18A is a dose-response curve of doxycycline inhibition on MCF-7 cells (formal experimental results);
  • Figure 18B is a dose-response curve of doxorubicin hydrochloride on MCF-7 cells (official experimental results);
  • Figure 19A is a graph showing the dose response curve of doxycycline on MDA-MB-231 cells (pre-experimental results).
  • Figure 19B is a dose-response curve of inhibition of MDA-MB-231 cells by doxorubicin hydrochloride
  • Figure 20A is a graph showing the dose-response curve of doxycycline on MDA-MB-231 cells (positive experiment results);
  • Figure 20B is a dose-response curve of inhibition of MDA-MB-231 cells by doxorubicin hydrochloride
  • Figure 21A shows the effect of doxycycline on the growth curve of MCF-7 cells (first test results);
  • F 57950.806, * with the control group Compared with P ⁇ 0.01, # compared with the doxycycline 0.65625 ⁇ group, ⁇ 0.01, ⁇ compared with the doxycycline 1.3125 ⁇ group, P ⁇ 0.01, +P ⁇ 0.01 compared with the doxycycline 2.625 ⁇ group.
  • ⁇ 0.01 compared with the doxycycline 5.25 ⁇ group
  • Figure 21C is the effect of doxycycline on the morphology of MCF-7 cells;
  • Figure ⁇ shows the cell shape of the control group observed under an inverted microscope.
  • B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope.
  • Figure 22A is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasive ability of human breast cancer cell MCF-7 after 24 h;
  • Figure 22B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasive ability of human breast cancer cell MCF-7.
  • Figure 23 is the inhibition effect of doxycycline and doxorubicin on the invasive ability of human breast cancer cell MDA-MB-231. The results of microscopic observation under the microscope after 24 h;
  • Figure 23B shows the inhibitory effect of doxycycline and doxorubicin on the invasive ability of human breast cancer cell MDA-MB-231 24 h later;
  • Figure 24 is the dose-response curve of doxycycline inhibition on ⁇ 562 cells (pre-experimental results).
  • Figure 24 is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on ⁇ 562 cells (pre-experimental results);
  • Figure 25 is a dose-response curve of doxycycline inhibition on ⁇ 562 cells (formal experimental results);
  • Figure 25 is a dose-response curve of doxorubicin hydrochloride on ⁇ 562 cells (formal test results);
  • Figure 26 is the dose-response curve of doxycycline inhibition on HL60 cells (pre-experimental results).
  • Figure 26 is a dose-response curve of the inhibition of HL60 cells by doxorubicin hydrochloride (pre-experimental results);
  • Figure 27 ⁇ is the dose-response curve of doxycycline inhibition on HL60 cells (formal experimental results);
  • Figure 27B is a dose-response curve of the inhibition of HL60 cells by doxorubicin hydrochloride (formal test results);
  • Figure 28 is the effect of doxycycline on the growth curve of ⁇ 562 cells (the second test result).
  • Figure 28C is the effect of doxycycline on the morphology of human leukemia cells ⁇ 562;
  • Figure ⁇ shows the cell morphology of the control group observed under an inverted microscope
  • Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope
  • Figure 29A is the effect of doxycycline on the growth curve of human leukemia cell line HL60 (first test result);
  • Figure ⁇ shows the cell morphology of the control group observed under an inverted microscope
  • Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope
  • Figure 30A is a dose-response curve of the inhibitory effect of doxycycline on NCI-H460 cells (pre-experimental results);
  • Figure 30B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on NCI-H460 cells (pre-experimental results);
  • Figure 31A is a dose-response curve of doxycycline inhibition on NCI-H460 cells (formal experimental results);
  • Figure 31B is a dose-response curve of doxorubicin hydrochloride on NCI-H460 cells (positive experiment results);
  • Figure 32A is a dose response curve of doxycycline on NCI-H446 cells (pre-test results);
  • Figure 32B is a dose-response curve of the inhibition of doxorubicin hydrochloride on NCI-H446 cells (pre-experimental results);
  • Figure 33A is a dose response curve of doxycycline on NCI-H446 cells (official experimental results);
  • Figure 33B is a dose-response curve of doxorubicin hydrochloride on NCI-H446 cells (positive experiment results);
  • Figure 34A is the effect of doxycycline on the growth curve of NCI-H460 cells (first test result);
  • Figure 34 is the effect of doxycycline on the growth curve of human lung cancer cell line NCI-H460 (the second test result);
  • Figure 34C is the effect of doxycycline on the morphology of human lung cancer cell line NCI-H460;
  • Figure 35A is the effect of doxycycline on the growth curve of NCI-H446 cells (first test results);
  • Figure 35 is the effect of doxycycline on the growth curve of NCI-H446 cells (second test result).
  • Figure 35C is the effect of doxycycline on the morphology of human lung cancer cell line NCI-H446;
  • Figure ⁇ shows the cell morphology of the control group observed under an inverted microscope
  • Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope.
  • Figure 36A shows the inhibitory effect of doxycycline and doxorubicin on the invasion of NCI-H460 cells. The results of microscopic observation under a microscope after 24 h;
  • Figure 36B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasion of NCI-H460 cells after 24 h;
  • Figure 37 Inhibition of invasion of human lung cancer cell line NCI-H446 by doxycycline and doxorubicin hydrochloride. Results observed under microscope microscopy after 24 h;
  • Figure 37B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasion of human lung cancer cell line NCI-H446.
  • Figure 38 ⁇ is the dose-response curve of doxycycline inhibition on HCCLM3 cells (pre-experimental results);
  • Figure 38 B is a dose-response curve of inhibition of HCCLM3 cells by doxorubicin hydrochloride (pre-experimental results);
  • Figure 39 A is the dose-response curve of doxycycline inhibition on HCCLM3 cells (official experimental results);
  • Figure 39 B is the dose-response curve of doxorubicin hydrochloride on HCCLM3 cells (normal experimental results);
  • Figure 40A is the effect of doxycycline on the growth curve of HCCLM3 (human hepatoma cells) (first test result);
  • Figure 40 is the effect of doxycycline on the growth curve of HCCLM3 (human hepatoma cells) (second test result)
  • Figure 40C is the effect of doxycycline on the morphology of HCCLM3 cells
  • Figure ⁇ shows the cell morphology of the control group observed under an inverted microscope
  • Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope
  • Figure 41A is a dose response curve of doxycycline on pancreatic cancer ASPC-1 cells (pre-experimental results);
  • Figure 41B is a dose-response curve of inhibition of doxorubicin hydrochloride on pancreatic cancer ASPC-1 cells (pre-experimental results);
  • Figure 42A is a dose response curve of doxycycline on pancreatic cancer ASPC-1 cells (normal experimental results);
  • Figure 42B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on pancreatic cancer ASPC-1 cells (formal experimental results);
  • Figure 43A is the effect of doxycycline on the growth curve of ASPC-1 cells (first test results)
  • F 200.330, * compared with the control group P ⁇ 0.01, # ⁇ 0.01 compared with the doxycycline 0.977 ⁇ group, ⁇ 0.01 compared with the doxycycline 1.954 ⁇ group, ⁇ 0.01, compared with the doxycycline 3.909 ⁇ group.
  • Figure 43C is the effect of doxycycline on the growth morphology of human pancreatic cancer cell line ASPC-1;
  • Figure ⁇ shows the cell morphology of the control group observed under an inverted microscope
  • Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope
  • Figure 44A shows the inhibitory effect of doxycycline and doxorubicin on the invasion of pancreatic cancer cell ASPC-1. The results of microscopic observation under the microscope after 24 h;
  • Figure 44B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasion of pancreatic cancer cell ASPC-1.
  • Figure 45 is a dose-response curve of inhibition of doxycycline on cervical cancer cell line Hela (pre-experimental results);
  • Figure 45B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on cervical cancer cell line Hela (pre-experimental results);
  • Figure 46A is a dose-response curve of inhibition of doxycycline on cervical cancer cell line Hela (normal experimental results);
  • Figure 46B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on cervical cancer cell line Hela (normal experimental results);
  • Figure 47C is the effect of doxycycline on the growth morphology of Hela cells
  • Figure 48A shows the inhibitory effect of doxycycline and doxorubicin on human cervical cancer Hela invasion 24 h after crystal violet staining under microscope;
  • Figure 48B shows the inhibitory effect of doxycycline and doxorubicin on human cervical cancer Hela invasion 24 h later;
  • Figure 49 ⁇ is the dose-response curve of doxycycline inhibition on colon cancer cell line S W620 (pre-experimental results);
  • Figure 49B is a dose-response curve of inhibition of colon cancer cell line SW620 by doxorubicin hydrochloride
  • Figure 50A is a dose-response curve of doxycycline inhibiting colon cancer cell line SW620 (normal experimental results);
  • Figure 50B is a dose-response curve of inhibition of colon cancer cell line SW620 by doxorubicin hydrochloride (formal experimental results);
  • Figure 51A shows the inhibitory effect of doxycycline and doxorubicin on the invasion of colon cancer cell line SW620.
  • Figure 52 ⁇ is the dose-response curve of doxycycline on gastric cancer cell ⁇ 28 cells (pre-experimental results);
  • Figure 52 is a dose-response curve of doxorubicin hydrochloride on gastric cancer cell ⁇ 28 cells (pre-experimental results);
  • Figure 53 is a dose-response curve of doxycycline on gastric cancer cell ⁇ 28 cells (official experimental results);
  • Figure 53 is a dose-response curve of doxorubicin hydrochloride on gastric cancer cell ⁇ 28 cells (formal experimental results);
  • Figure 54C is the effect of doxycycline on the growth morphology of MKN28 cells;
  • Figure A shows the cell morphology of the control group observed under an inverted microscope
  • Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope
  • Fig. 55A is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasion of gastric cancer cell MKN28 after 24 h;
  • Fig. 55B is a statistical result of inhibition of invasion of gastric cancer cell MKN28 by doxycycline and doxorubicin hydrochloride for 24 hours;
  • Figure 56 is a dose-response curve of inhibition of NIH-3T3 cells by doxycycline and doxorubicin hydrochloride (first experimental results);
  • Figure 57 is a dose-response curve of inhibition of NIH-3T3 cells by doxycycline and doxorubicin hydrochloride (second experiment results);
  • Figure 58 is a dose-response curve of inhibition of HaCaT cells by doxycycline and doxorubicin hydrochloride (first experimental results);
  • Figure 59 is a dose-response curve of inhibition of HaCaT cells by doxycycline and doxorubicin hydrochloride (second experimental results);
  • Figure 60 is a dose-response curve of the inhibitory effect of doxycycline (Doxy) and doxorubicin hydrochloride (ADR) on HEK 293 cells and HEK 293T cells (first experimental results);
  • Doxy doxycycline
  • ADR doxorubicin hydrochloride
  • Figure 61 is a dose-response curve of the inhibitory effect of doxycycline (Doxy) and doxorubicin hydrochloride (ADR) on HEK 293 cells and HEK 293T cells (the results of the second experiment);
  • Doxy doxycycline
  • ADR doxorubicin hydrochloride
  • Figure 62 is the effect of doxycycline on the growth of mouse B16 melanocytes xenografts (first trial);
  • Figure 63 is the effect of doxycycline on the gross morphology of melanoma xenografts;
  • Figure 64 is the effect of doxycycline on body weight of B16 melanoma xenograft mice (first trial);
  • Figure 65 is the effect of doxycycline on the growth of mouse B16 melanoma xenografts (repeated test); 66 is the effect of doxycycline on the body weight of B16 melanoma xenograft mice (repeated test); (Note: * P ⁇ 0.05 compared with the control group; # compared with the cyclophosphamide group, P ⁇ 0.05)
  • Figure 67 Is the effect of doxycycline on the growth of mouse Lewis lung cancer xenografts (first trial);
  • Figure 68 is the effect of doxycycline on the body weight of Lewis lung cancer xenografts (first trial); (Note: * with the control group In comparison, P ⁇ 0.05; # compared with the cyclophosphamide group, P ⁇ 0.05)
  • Figure 69 is the effect of
  • Figure 73 is the effect of doxycycline on the growth of transplanted mouse MCF-7 breast cancer (repeated test);
  • FIG 74 is a bearing for doxycycline breast cancer MCF-7 xenografted mice body weight trees (replications); (Note: compared with control group * P ⁇ 0.05; # compared with cyclophosphamide group P ⁇ 0.05
  • Figure 75 is the effect of doxycycline on the growth of transplanted mouse H446 small cell lung cancer (first trial);
  • Figure 76 is the effect of doxycycline on the body weight of transplanted H446 small cell lung cancer xenografts (first test);
  • Figure 77 is the effect of doxycycline on the growth of transplanted mouse H446 small cell lung cancer (repeated test).
  • Figure 78 is the effect of doxycycline on the body weight of transplanted H446 small cell lung cancer xenografts (repeated test);
  • Figure 79 is the effect of doxycycline on the survival of tumor-bearing mice (first trial).
  • Figure 80 is the effect of doxycycline on the survival of tumor-bearing mice (repeated trial).
  • Figure 81 shows the effect of doxycycline on migration of MCF-7 cells
  • Figure 82 shows the effect of doxycycline on migration of HCCLM3 cells
  • Figure 83 shows the effect of doxycycline on B16 cell migration
  • Figure 84 shows the effect of doxycycline on the migration of human small cell lung cancer cell line NCI-H446;
  • Figure 85 shows the effect of doxycycline on the migration of pancreatic cancer cell ASPC-1;
  • Figure 86 shows the effect of doxycycline on migration of human colon cancer cells SW620. detailed description
  • the inventors have confirmed the anti-tumor activity of doxycycline having a tetracycline structure after extensive experimental studies.
  • the specific embodiment is as follows.
  • test materials used and their sources include:
  • Human melanoma cell line Mum-2C (human invasive choroidal melanoma cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • A875 human malignant melanoma: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.
  • A375 human malignant melanoma: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.
  • human breast cancer cell line
  • MCF-7 human breast cancer cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • MDA-MB-231 human breast cancer cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • Human leukemia cell line Human leukemia cell line:
  • K562 human chronic myeloid leukemia cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • HL60 human promyelocytic leukemia cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • NCI-H460 human large cell lung cancer cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • NCI-H446 human small cell lung cancer cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • Human liver cancer cell line Human liver cancer cell line:
  • HCCLM3 human high metastatic liver cancer cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • Human pancreatic cancer cell line Human pancreatic cancer cell line:
  • ASPC-1 human metastatic pancreatic adenocarcinoma cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • Human cervical cancer cell line Human cervical cancer cell line
  • Hela human cervical cancer cell line
  • SW620 human high metastatic colon cancer cell line
  • MKN28 human gastric cancer high metastatic cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • NIH-3T3 mouse embryo fibroblast cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • HaCaT human normal skin cells: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.
  • HEK 293 human embryonic kidney cells: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;
  • HEK 293T human embryonic kidney cell derivative
  • Doxycycline hydrochloride yellow powder, supplied by Kaifeng Pharmaceutical (Group) Co., Ltd., batch number: 201301011, purity: 91.0% (in accordance with the Chinese Pharmacopoeia 2010 edition two).
  • Doxorubicin hydrochloride Orange-red powder, purchased from Beijing Enoch Technology Co., Ltd., manufacturer: Admas-beta, article number: 41701A, batch number: P11099, purity 98%+.
  • Tetramethyl azozolium salt (MTT) Shanghai Shenggong Bioengineering Co., Ltd.
  • DMSO Dimercaptosulfoxide
  • DMSO dimercaptosulfoxide
  • RPMI 1640 medium purchased from Thermo Fisher Scientific, Hyclone brand, article number: SH30809.01B, lot number: NYG0920, specification: 500 mL.
  • Trypsin purchased from Shanghai Shenggong Bioengineering Co., Ltd., Item No.: T0458-10, Lot No.: 0301C314, Level: USP, Specification: 10 g.
  • Matrigel Matrigel Manufacturer BD Medical Devices, Inc. No. 356234, Lot No.: 2229979.
  • C0 2 incubator Shidukai Instrument Equipment (Shanghai) Co., Ltd., Model: STIK IL-161HI.
  • Automated enzyme-linked immunosorbent assay Thermo Fisher Scientific, model Multiscan FC.
  • Inverted phase contrast microscope Manufacturer: Olympus, Model: CKX41.
  • 96-well plate purchased from Thermo Fisher Scientific, Inc., Thermo Nunc brand, article number 167008.
  • 24-well plate purchased from Thermo Fisher Scientific, Inc., Thermo Nunc brand, article number 167008.
  • Transwell Chamber (BD-Falcon): Manufacturer BD Medical Devices, Catalog No. 353097, Lot No.: 3042760).
  • doxycycline hydrochloride solution Preparation of doxycycline hydrochloride solution: Weigh 0.01 g of doxycycline hydrochloride and dissolve it in 1 mL of PBS solution to prepare a 10 mg/mL solution. After it is fully dissolved, filter it with a 0.22 ⁇ M filter. It is used after sterilization, and it is used now when it is used. Dilute to the desired concentration with cell culture medium during use. The formulation and use of the solution should be performed in a sterile biosafety refusal.
  • doxorubicin hydrochloride solution Weigh 0.005 g of doxorubicin hydrochloride and dissolve it in 0.5 mL of DMSO to prepare a 10 mg/mL solution, which is ready for use every time. Dilute to the desired concentration with cell culture medium at the time of use. The formulation and use of the solution should be performed in a sterile biosafety refusal.
  • MTT stock solution Dissolve 1 g MTT in 200 mL PBS solution (concentration: 5 mg/mL), mix MTT completely, filter and sterilize with 0.22 ⁇ ⁇ filter, dispense, avoid The light was stored in a refrigerator at -20 °C. The formulation and use of the solution should be performed in a sterile biosafety refusal.
  • the experimental method is MTT colorimetry: the detection principle is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT to water-insoluble blue-violet crystal sputum (Formazan) and deposit in cells, while dead cells have no this function.
  • Dimercaptosulfoxide (DMSO) is capable of lysing sputum in cells, and its absorbance is measured at 490 nm by an enzyme-linked immunosorbent assay, which indirectly reflects the number of viable cells. Within a certain number of cells, the amount of strontium crystal formation is proportional to the number of cells. This method has been widely used for the detection of activity of some biologically active factors, large-scale anti-tumor drug screening, cytotoxicity tests, and tumor radiosensitivity assays.
  • the frozen melanoma cells were taken out from the liquid nitrogen and immediately placed in a 37 ° C water bath to melt the cells. Biosafety Rejection The cell suspension was pipetted into a centrifuge tube containing appropriate medium and centrifuged at 800 rpm/min for 5 minutes. The supernatant was discarded, and the cells were suspended in 1 mL of medium, and the cells were filled with appropriate medium. In the culture dish, the cells were cultured under the conditions of 37 ° C, 5% CO 2 , and saturated humidity.
  • the adherent cells in the logarithmic growth phase are trypsinized, dispersed into individual cells, and suspended in the corresponding cell culture medium.
  • the cells were seeded on 96-well plates at 100 cell suspensions per well, 3000-4000 cells/well.
  • the 96-well culture plate was placed in a carbon dioxide (5%) incubator for 24 hours at 37 ° C to allow the cells to adhere.
  • the test group was added with cell culture medium containing different concentrations of doxycycline.
  • the positive control group was added with different concentrations of doxorubicin hydrochloride cell culture solution, and no drug was added.
  • Control wells with only the corresponding drug solvent doxcycline solvent is PBS, doxorubicin hydrochloride solvent is DMSO), and only the medium-free zero-free wells are added.
  • IC 5 was performed using Graphpad Prism 5 software. The calculation of the value.
  • the dose-response curve of the drug to the cells was plotted using Excel software, and each index was expressed by mean ⁇ standard deviation (X soil S ).
  • doxycycline grows on Mum-2C cells. At low doses, it showed significant inhibition. As the concentration of doxycycline increased, the inhibitory effect on Mum-2C cells increased gradually, showing a dose-dependent manner.
  • doxycycline significantly inhibited the growth of A875 cells at low doses, as the concentration of doxycycline increased, on A875 cells.
  • the inhibitory effect is gradually increased, showing a significant dose dependency.
  • the preliminary results showed that the half-inhibitory concentration (IC 50 value) of doxycycline and the positive drug doxorubicin hydrochloride on A875 cells were 3.0987 ⁇ 0.3297 ⁇ and 0.5440 ⁇ 0.0399 ⁇ , respectively.
  • the results of the formal experiment showed that doxycycline and the positive drug doxorubicin hydrochloride had an IC 5 on ⁇ 875. Values are 2.5683+0.0946 ⁇ and 0.1271 ⁇ 0.0070 ⁇ .
  • doxycycline significantly inhibited the growth of A375 cells at low doses, and increased the concentration of doxycycline on A375 cells.
  • the inhibitory effect is gradually increased, showing a significant dose dependency.
  • IC 50 value half-inhibitory concentration of doxycycline and the positive drug doxorubicin hydrochloride on A375 cells were 2.4153 ⁇ 0.1415 ⁇ and 0.1287 ⁇ 0.0073 ⁇ , respectively.
  • Cyclulin has a good inhibitory effect on the growth of the above four cells and shows a significant dose-dependent, and IC 5 . Values are below 10 ⁇ (approx. 5 g/mL).
  • the frozen cells were removed from the liquid nitrogen and immediately placed in a 37 ° (water bath to melt the cells. Biosafety Rejection)
  • the cell suspension was pipetted into a centrifuge tube containing the appropriate amount of medium and centrifuged at 800 rpm for 5 minutes. Discard the supernatant, suspend the cells in 1 mL medium, and pipette the cells in the appropriate amount of medium.
  • the cells were cultured at 37 ° C, 5% CO 2 , and saturated humidity.
  • the adherent cells in the logarithmic growth phase are trypsinized, dispersed into individual cells, and suspended in the corresponding cell culture medium.
  • the cells were seeded on a 24-well culture plate, 500 cells per well, 5000 cells/well, and 8 plates were inoculated.
  • the above 24-well culture plates were placed in a 37 ° C carbon dioxide (5%) incubator for 24 hours to allow the cells to adhere.
  • the doubling time of the cells in the control group and each of the drug-added groups was calculated.
  • the doxycycline significantly prolonged the doubling time of Mum-2C cells at a concentration of 0.3975 ⁇ , 0.795 ⁇ , 1.59 ⁇ ,
  • the doubling times of Mum-2C cells were 21.63, 22.37, 22.72, 24.04 and 35.56 hours, respectively, compared with the control group. Compared with 17.93 hours, they were extended by 20.67%, 24.75%, 26.73%, 34.12% and 98.32% respectively.
  • Mum-2C is a clone of choroidal melanoma liver metastasis.
  • the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on the doubling time of Mum-2B cells, and the experimental concentrations are 0.674 ⁇ , 1.35 ⁇ , 2.7 ⁇ , After 5.4 ⁇ and 10.8 ⁇ of doxycycline, the cell doubling time of Mum-2B cells was 14.37, 16.09, 21.78, 26.34 and 29.87 hours, respectively, which was 16.30% and 30.23%, respectively, compared with 12.35 hours in the control group. 76.29%, 113.25% and 141.74%.
  • doxycycline inhibited the growth of ⁇ 875 cells, and the statistical results showed that the effect of doxycycline on the growth of ⁇ 875 cells was dose dependent.
  • doxorubicin hydrochloride was used as a positive control in the experiment.
  • the doubling time of the cells in the control group and each drug-added group was calculated.
  • the doxycycline had a significant prolongation effect on the doubling time of ⁇ 875 cells.
  • the experimental concentrations were 0.602 ⁇ , 1.204 ⁇ , 2.408 ⁇
  • the doubling time of ⁇ 875 cells was 37.85, 40.07, and 44.28 hours, respectively, which was extended by 23.05%, 30.26%, and 43.95% (4.86 ⁇ M and 9.632 ⁇ , respectively, compared with 30.76 hours in the control group).
  • the inhibition of ⁇ 875 cells is too strong, Causes the inability to calculate cell doubling time).
  • A875 is a human skin melanoma cell line. Under normal conditions, the cells have polygonal cells with slightly pseudopods. They tend to grow like clones, have clear cell contours, have good diopter in the nuclear region, and contain small amounts of particles in the cells. After administration of doxycycline (4.816 ⁇ ) for 2 days, the number of cells decreased, some cells shrunk, some cells showed a flat aging morphology, and the number of suspended cells increased, and a large number of particles were observed in the cells (see Fig. 11C).
  • doxycycline inhibited the growth of ⁇ 375 cells, and the statistical results showed that the effect of doxycycline on the growth of ⁇ 375 cells was dose dependent.
  • doxorubicin hydrochloride was used as a positive control in the experiment.
  • the doubling time of the cells in the control group and each drug-added group was calculated.
  • the doxycycline significantly prolonged the doubling time of A 375 cells.
  • the concentration of the drug in the experiment was 1.03875 ⁇ , 2.0775 ⁇ , 4.155 ⁇ . 8.31 ⁇ and 16.62 ⁇ doxycycline
  • the doubling time of ⁇ 375 cells was 25.89, 26.92, 30.12, 34.49 and 48.67 hours, respectively, compared with 24.18 hours of the control group, respectively, extended by 7.07%, 11.30%, 24.55%. , 42.64% and 101.26%.
  • ⁇ 375 cells are classical human skin melanoma cell lines, polygonal, filopodia, with more melanin particles in the cells.
  • docetaxel 16.62 ⁇
  • the cells shrunk, the outline was unclear, the nuclear diopter disappeared, and the cells were fragmented and suspended (see Figure 12C).
  • the frozen cells were removed from the liquid nitrogen and immediately placed in a 37 ° (water bath to melt the cells. Biosafety Rejection)
  • the cell suspension was pipetted into a centrifuge tube containing the appropriate amount of medium and centrifuged at 800 rpm for 5 minutes. Discard the supernatant, suspend the cells in 1 mL medium, and pipette the cells in the appropriate amount of medium.
  • the cells were cultured at 37 ° C, 5% CO 2 , and saturated humidity.
  • Matrigel was diluted 1:1 with serum-free medium for the upper chamber of the bottom membrane of the Transwell chamber coated with a 24-well plate. Add 50 of the above culture solution per well and incubate in a 37 °C carbon dioxide incubator. 30 min-1 h.
  • Hydration basement membrane Aspirate the residual liquid in the culture plate, add 50 serum-free medium containing 10 mg/mL BSA to each well, incubate at 37 °C for 30 min.
  • the cells in the logarithmic growth phase were trypsinized, the digestion was terminated, the culture was discarded by centrifugation, washed 1-2 times with PBS, and resuspended in serum-free medium containing BSA. Adjust the cell density to 5 X 10 5 .
  • the matrigel and the cells in the upper chamber were wiped off with a cotton swab; the migrated cells were stained with 0.1% crystal violet, and the cells were counted under a microscope, and the results of the experiment were statistically calculated to calculate the migration inhibition rate (experimental method reference: Wei Wei, Wu Ximei, etc. The Pharmacological Experimental Methodology, Fourth Edition, Beijing: People's Medical Publishing House, 2010: 1627-1628).
  • Fig. 13A shows the results of microscopic observation under the microscope of microscopy after treatment with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. , can be seen that compared with the control group, doxycycline attack on Mum-2C cells Ability has a strong inhibitory effect.
  • the statistical results showed that the inhibitory rate of doxycycline on Mum-2C cell migration at a concentration of 1 ⁇ reached 45.12% (P ⁇ 0.01, which was significantly different from the control group).
  • the doxorubicin hydrochloride was 0.781 ⁇
  • the inhibition rate of Mum-2C cell migration was 38.09% (P ⁇ 0.01, which was significantly different from the control group).
  • Figure 14A shows the results of microscopic observation under microscopic microscope after treatment of cells with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. It can be seen that the inhibitory effect of doxycycline on the invasive ability of Mum-2B cells is weak compared with the control group.
  • the statistical results showed no significant inhibition of the invasive ability of doxycycline on Mum-2B cells at 1 ⁇ . (P>0.05, there was no significant difference compared with the control group).
  • concentration of doxorubicin hydrochloride was 0.5 ⁇ , the inhibition rate of Mum-2B cell migration was almost 100% (P ⁇ 0.01, which was significantly different from the control group).
  • Figures 15A and 15B The inhibitory effect of doxycycline on the invasive ability of A875 cells is shown in Figures 15A and 15B.
  • Figure 14A shows the results of crystal violet staining under the microscope after treatment with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. It was found that doxycycline had a strong inhibitory effect on the invasive ability of A875 cells compared with the control group.
  • the statistical results (Fig. 14B) showed that the inhibition rate of doxycycline on migration of ⁇ 875 cells at 0.5 ⁇ reached 65.69% ( ⁇ 0.01, which was significantly different from the control group).
  • the inhibition rate of doxorubicin hydrochloride on migration of ⁇ 875 cells reached 38.31% ( ⁇ 0.05, which was significantly different from the control group).
  • Figure 16A shows the results of crystal violet staining under the microscope after treatment with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. It was found that doxycycline had a strong inhibitory effect on the invasive ability of A375 cells compared with the control group.
  • the statistical results showed that the inhibitory effect of doxycycline on the migration of ⁇ 375 cells at 0.2 ⁇ reached 35.43% ( ⁇ ⁇ 0.01, which was significantly different from the control group).
  • the inhibition rate of ⁇ 375 cells migration by doxorubicin hydrochloride at 1 ⁇ reached 28.81% ( ⁇ ⁇ 0.01, which was significantly different from the control group).
  • Example 1 The method described under the corresponding MTT assay in Example 1 is the same except that the cell lines used in this example are human breast cancer cells MCF-7 and MDA-MB-231.
  • the inhibitory effect of MDA-MB-231 cells gradually increased, showing a dose-dependent effect of i.
  • the preliminary results showed that the half-inhibitory concentration (IC 50 value) of doxycycline and the positive drug doxorubicin hydrochloride on MDA-MB-231 were 1.0436 ⁇ 0.0845 ⁇ and 0.7248 ⁇ 0.2459 ⁇ , respectively.
  • doxycycline inhibited the growth of both breast cancer cells in a dose-dependent manner, IC 5 .
  • the values are all below 10 ⁇ (about 5 g/mL).
  • doxycycline has a certain inhibitory effect on the growth of MCF-7 cells.
  • Statistical results show that doxycycline has a dose-dependent effect on the growth of MCF-7 cells.
  • doxorubicin hydrochloride was used as a positive control in the experiment.
  • the doubling time of the cells in the control group and each drug-added group was calculated.
  • the doxorubicin had a significant prolongation effect on the doubling time of MCF-7 cells.
  • the concentration of the drug in the experiment was 0.65625 ⁇ 1.3125 ⁇ 2.625 ⁇ 5.25.
  • the doubling time of MCF-7 cells was 31.43 35.87 44.89 54.73 and 87.75 hours, respectively, which was extended by 6.54%, 21.59%, 52.17% and 85.52, respectively, compared with 29.50 hours of the control group.
  • MCF-7 is a highly metastatic breast cancer cell line with clonal growth of polygonal epithelial cells with intercellular bridges. After administration of doxycycline (10.5 ⁇ ) for three days, a large number of vacuoles appeared in the cells, the bridge disappeared, the contours were blurred, the cells were shrunk and suspended, and the number of cells was significantly reduced (see Figure 21C). The cell growth curve test was performed in vitro. It was found that doxycycline is fine for human breast cancer. The growth of MCF-7 has a certain inhibitory effect, which can significantly prolong the doubling time of cells. The statistical results show that the effect of doxycycline on the doubling time of MCF-7 cells is dose-dependent.
  • Example 1 The method described under the corresponding invasiveness test in Example 1 was the same except that the cell lines used in the present example were human breast cancer cells MCF-7 and MDA-MB-231.
  • Figure 22A shows crystal violet staining after treatment of cells with doxycycline and the positive drug doxorubicin hydrochloride for 24 h.
  • the results observed under the microscope showed that doxycycline had a strong inhibitory effect on the invasion ability of MCF-7 cells compared with the control group.
  • the statistical results Fig.
  • Fig. 23A shows the results of 24 hours of crystal violet staining after treatment of cells with doxycycline and the positive drug doxorubicin hydrochloride. It can be seen that doxycycline to MDA-MB-231 compared with the control group. The invasive ability of cells has a strong inhibitory effect.
  • the statistical results Fig.
  • Example 1 The method described in the corresponding MTT assay in Example 1 was the same except that the cell lines used in the present example were human leukemia cells K562 and HL60. At the same time, K562 and HL60 cells are suspension cells, which do not need to be digested with trypsin before passage and plating.
  • doxycycline significantly inhibited the growth of K562 cells at low doses, and increased the concentration of doxycycline on K562 cells.
  • the inhibitory effect is gradually increased, showing a significant dose dependency.
  • the preliminary results showed that the half-inhibitory concentration (IC 5 ) of doxycycline on K562 cells was 1.4483+0.0996 ⁇ .
  • the results of formal experiments indicate that doxycycline affects IC 5 in ⁇ 562 cells. The value is 2.9403 + 0.2778 ⁇ .
  • the experimental results showed that doxorubicin hydrochloride inhibited the growth of ⁇ 562 cells in vitro (1( 5 . value greater than 50 ⁇ 1).
  • IC 5 The effect of doxycycline on the growth of these two leukemia cells was found to be in a dose-dependent manner, IC 5 . Values are below 10 ⁇ (approx. 5 g/mL).
  • doxycycline has a certain inhibitory effect on the growth of K562 cells.
  • the statistical results show that the effect of doxycycline on the growth of K562 cells is dose dependent.
  • doxorubicin hydrochloride was used as a positive control in the experiment.
  • K562 is a chronic myeloid leukemia cell line in which cells are suspended and have a distinct concentric lens contour. After administration of doxycycline (11.76 ⁇ ), the cells shrunk, shattered, and the refractive index increased, the concentric contour disappeared, and the cell volume decreased and the number decreased (see Figure 28C).
  • doxycycline has a certain inhibitory effect on the growth of HL60 cells.
  • Statistical results show that doxycycline has a dose-dependent effect on the growth of HL60 cells.
  • doxorubicin hydrochloride was used as a positive control in the experiment.
  • Doxycycline has a significant prolongation effect on the doubling time of HL60 cells, and the experimental concentrations are 0.304 ⁇ , 0.607 ⁇ , 1.214 ⁇ , and 2.428.
  • the doubling time of HL60 cells was 32.42, 42.52, 42.83 and 52.82 hours, respectively, which was 8.14%, 41.82%, 42.86% and 76.19%, respectively, compared with 29.98 hours in the control group.
  • ⁇ ⁇ doxycycline inhibited HL60 cells too much, which made it impossible to calculate the doubling time).
  • HL60 is a promyelocytic leukemia cell line.
  • the cells are suspended and have a distinct concentric circular membrane contour, and some cells tend to adhere to a cluster. After doxycycline ( 1.214 ⁇ ), the cells shrunk, shattered, concentric contours disappeared, cell agglomeration was evident, cell volume decreased, and the number decreased (see Figure 29C).
  • Example 1 The method described in the corresponding sputum detection in Example 1 was the same except that the cell lines used in the present example were human lung cancer cells NCI-H460 and NCI-H446.
  • the preliminary results showed that the half-inhibitory concentration (IC 5() value) of doxycycline on NCI-H460 cells was 1.0638 ⁇ 0.1266 ⁇ .
  • the results of formal experiments indicate the IC 5 of doxycycline on NCI-H460.
  • the value is 1.9340 ⁇ 0.0286 ⁇ .
  • the experimental results showed that doxorubicin hydrochloride did not significantly inhibit the growth of NCI-H460 cells.
  • Doxycycline has a significant prolongation effect on the doubling time of NCI-H460 cells at a concentration of 0.5 ⁇ , 1.0 ⁇ , 2.0 ⁇
  • the doubling time of NCI-H460 cells after 4.0 ⁇ and 8.0 ⁇ of doxycycline was 48.56, 58.60, 81.13, 116.13 and 208.47 hours, respectively, which was 17.97%, 42.34% and 97.09%, respectively, compared with 41.17 hours in the control group. , 182.09% and 406.43%.
  • NCI-H460 cells are human large cell lung cancer cell lines, characterized by epithelial cells, clone-like growth, polygonal shape, and very few cells are fusiform. After administration of doxycycline ( 7.724 ⁇ ) for 3 days, the cell outline disappeared, the refractive index decreased, the intercellular bridge disappeared, and most of the cells were fragmented or condensed (see Figure 34C).
  • Doxycycline has a significant prolongation effect on cell doubling time at doses of 0.25 ⁇ , 0.5 ⁇ , 1 ⁇ , and 2 ⁇
  • the doubling time of NCI-H446 cells was 16.32, 20.03, 23.56 and 25.81 hours, respectively, which was 18.18%, 45.07%, 70.59% and 86.90%, respectively, compared with 13.81 hours in the control group.
  • NCI-H446 is a small cell lung cancer of neuroectodermal origin.
  • the cells are small, clone-like, some cells are suspended, and a small number of cells have pseudopods.
  • doxycycline 3.9404 ⁇
  • the cell volume became larger, the refractive index disappeared, the pseudopod decreased, the intracellular particles increased, and the suspended cells increased.
  • Most, most cells have aging performance (see Figure 35C).
  • Example 1 The method described in the corresponding invasiveness test in Example 1 was the same except that the cell lines used in the present example were human lung cancer cells NCI-H460 and NCI-H446.
  • Figure 36A shows the crystal violet staining fluoroscopy after 24 h treatment with doxycycline and the positive drug doxorubicin hydrochloride.
  • Fig. 37 ⁇ and 37 ⁇ The inhibitory effect of doxycycline on the invasive ability of NCI-H446 is shown in Fig. 37 ⁇ and 37 ⁇ : Fig. 37 ⁇ Analysis of cells treated with doxycycline and the positive drug doxorubicin hydrochloride after 24 h, crystal violet staining As a result, it can be seen that compared with the control group, doxycycline has a strong inhibitory effect on the invasion ability of NCI-H446 cells.
  • the statistical results Fig.
  • Example 1 The method described in the corresponding MTT assay in Example 1 is the same except that the cell line used in this example is human hepatoma cell HCCLM3.
  • doxycycline significantly inhibited the growth of HCCLM3 cells at low doses. As the concentration of doxycycline increased,
  • HCCLM3 cells gradually increased in a dose-dependent manner.
  • doxycycline has a certain inhibitory effect on the growth of HCCLM3 cells.
  • the statistical results show that the effect of doxycycline on the growth of HCCLM3 cells is dose dependent.
  • doxorubicin hydrochloride was used as a positive control in the experiment.
  • Doxycycline has a significant prolongation effect on cell doubling time at doses of 0.7805 ⁇ , 1.561 ⁇ , 3.122 ⁇ , 6.244 ⁇ , and 12.488.
  • the doubling time of HCCLM3 cells was 22.72, 24.12, 25.91, 27.27, and 35.18 hours, respectively, which was 6.52%, 13.07%, 21.46%, and 27.82%, respectively, compared with 21.33 hours in the control group. And 64.92%.
  • HCCLM3 cells are human metastatic liver cancer, clone-like growth, and clones are often aggregated. After administration of doxycycline (12,488 ⁇ ) for 3 days, it was observed that the cells formed small clones, or the clones were poorly formed, and there were a large number of cell suspensions, and the fusion ability between the clones was weakened, and the total number of cells was reduced (see Fig. 40C).
  • Doxy-2 storage rate AD -2 Survival inhibition rate concentration ( ⁇ ⁇ ⁇ (OD : r 3 ⁇ 4i i.) %) (concentration ' ⁇ ) ( OD50 inn .)
  • doxycycline inhibited the growth of ASPC-1 cells in a dose-dependent manner.
  • the statistical results showed that the effect of doxycycline on the doubling time of ASPC-1 cells was dose-dependent.
  • doxorubicin hydrochloride was used as a positive control in the experiment.
  • Doxycycline has a significant prolongation effect on cell doubling time.
  • ASPC-1 cells When administered at concentrations of 0.977 ⁇ , 1.954 ⁇ , 3.909 ⁇ , 7.818 ⁇ and 15.636 ⁇ of doxycycline, the doubling times of ASPC-1 cells were 26.51, 27.70, 29.44, 29.83 and 42.94 hours, respectively, compared with 20.89 hours of the control group. In comparison, they were extended by 26.95%, 32.64%, 40.97%, 42.85% and 105.61%, respectively.
  • ASPC-1 is a human metastatic pancreatic cancer cell line with bidirectional differentiation potential, and the cells are mostly clonal epithelial-like growth, and some cells have extraneural nerves.
  • the germ layer features a filamentous pseudopod.
  • doxycycline (15.636 ⁇ )
  • the cells were suspended, and most of them were epithelial-like, with suspension and apoptosis of filopodia.
  • Epithelial-like cells age and the cell outline is unclear (see Figure 43C).
  • Example 2 The same procedure as described in the corresponding invasiveness test in Example 1 was carried out except that the cell line used in the present example was human pancreatic cancer cell ASPC-1. Experimental results:
  • Example 1 The method described in the corresponding MTT assay in Example 1 was the same except that the cell line used in the present example was human cervical cancer cell Hela.
  • doxycycline has a certain inhibitory effect on the growth of Hela cells.
  • the statistical results show that the effect of doxycycline on the growth of Hela cells is dose dependent.
  • doxorubicin hydrochloride was used as a positive control in the experiment. Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on the doubling time of Hela cells at doses of 0.3325 ⁇ , 0.665 ⁇ , 1.33 ⁇ , 2.66 ⁇ M.
  • the doubling time of Hela cells was 21.31, 22.52, 23.56, 24.42, and 27.11 hours, respectively, which was 3.21%, 9.07%, and 14.13%, respectively, compared with 20.64 hours of the control group. , 18.28% and 31.34%.
  • Hela cells are cervical cancer epithelial cell lines and are classical epithelial tumor cell lines with epithelial-like growth. After administration of doxycycline (5.32 ⁇ ), the cell outline disappeared, most cells aged, cytoplasmic particles increased, and some cells shattered (see Figure 47C).
  • Example 1 The method described in the corresponding invasiveness test in Example 1 was the same except that the cell line used in the present example was human cervical cancer cell Hela.
  • Figure 48A shows the crystal violet staining after 24 h treatment with doxycycline and the positive drug doxorubicin hydrochloride.
  • the results observed under the microscope showed that doxycycline had a strong inhibitory effect on the invasion ability of Hela cells compared with the control group.
  • the statistical results (Fig. 48B) showed that the inhibitory effect of doxycycline on Hela cell migration at a concentration of 0.391 ⁇ reached 34.1% (P ⁇ 0.01, which was significantly different from the control group), and the doxorubicin hydrochloride concentration was 0.2 ⁇ .
  • the inhibition rate of Hela cell migration was 65.8% (P ⁇ 0.01, which was significantly different from the control group).
  • Example 1 The method described in the corresponding MTT assay in Example 1 is the same except that the cell line used in this example is human colon cancer cell S W620.
  • MTT assay for the inhibitory effect of doxecycline on the growth of colon cancer cell line SW620 is shown in Table 27 and Figure 49 and Table 28 and Figure 50.
  • the growth of SW620 on SW620 is evident at low doses. Inhibition, and exhibit a dose response.
  • concentration of doxycycline increased, the inhibitory effect on SW620 cells increased gradually, showing a dose-dependent manner.
  • Example 1 The method described under the corresponding invasiveness test in Example 1 was the same except that the cell line used in the present example was human colon cancer cell S W 620.
  • Fig. 51A shows the inhibitory effect of doxycycline on the invasion ability of colon cancer cell line SW620.
  • Fig. 51A shows the results of microscopic observation under the microscope of microscopic staining of cells treated with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. It can be seen that compared with the control group, doxycycline has a strong inhibitory effect on the invasion ability of SW620 cells.
  • the statistical results Fig.
  • Example 1 The method described in the corresponding MTT assay in Example 1 was the same except that the cell line used in the present example was human gastric cancer cell MKN28.
  • MTT assay for the inhibitory effect of doxycycline on the growth of gastric cancer cell line MKN28 As shown in Table 29 and Figure 52 and Table 30 and Figure 53, doxycycline significantly inhibited MKN28 cells at low doses. , as the concentration of doxycycline increases, it is fine for MKN28 The inhibition of o is gradually increased in a dose-dependent manner.
  • the half-inhibitory concentration (IC 5 ) of doxycycline on MKN28 was 2.4517 ⁇ 0.25 o 91 ⁇ as measured in the pre-experiment.
  • the IC 50 value of doxycycline for ⁇ 28 was 1.6640+0.1853 ⁇ H - ⁇ .
  • the experimental results show that doxorubicin hydrochloride almost grows on ⁇ 28 cells.
  • Table 30 The inhibition of doxycycline and doxorubicin hydrochloride on ⁇ 28 cells.
  • Doxycycline has a significant prolongation effect on cell doubling time at doses of 0.692 ⁇ , 1.384 ⁇ , 2.768 ⁇ , 5.536 ⁇ , and 11.072.
  • the doubling time of ⁇ 28 cells was 31.48, 35.79, 45.93, 56.52 hours and 87.50 hours, respectively, which was 0.37%, 14.09%, 46.44%, 80.23%, and 31.36 hours, respectively. 178.96%.
  • ⁇ 28 is a human metastatic gastric cancer cell line with epithelial-like features and a common divisional phase. After administration of doxycycline (11.07 ⁇ ) for 3 days, the cells shrunk into spheres, most of which were suspended, showing anoikis, and few adherent cells remained (see Figure 54C).
  • Example 1 The method described under the corresponding invasiveness test in Example 1 was the same except that the cell line used in the present example was human gastric cancer cell ⁇ 28.
  • Fig. 55 ⁇ and 55 ⁇ The inhibitory effect of doxycycline on the invasion of ⁇ 28 cells is shown in Fig. 55 ⁇ and 55 ⁇ : Fig. 55 ⁇
  • Fig. 55 ⁇ The results of observation under the microscope of crystal violet staining after treatment of cells with doxycycline and the positive drug doxorubicin hydrochloride for 24 h, can be seen Compared with the control group, the inhibitory effect of doxycycline on MKN28 cells was not obvious at low concentrations.
  • the statistical results (Fig. 55B) showed that doxycycline had no significant effect on the invasion of ⁇ 28 cells at 1 ⁇ concentration ( ⁇ >0.05, no significant difference compared with the control group), and the apoptosis of ⁇ 28 cells by doxorubicin hydrochloride was also observed.
  • Example 1 The method described in the corresponding MTT assay in Example 1 was the same except that the cell lines used in this example were NIH-3T3, HEK 293, HEK 293T, HaCaT cells.
  • Doxy-l xis rate of ownership rate ADR-1 Survival inhibition rate concentration ( ⁇ ) (OD 570 mn ) concentration ' ⁇ ) COD57M dish) (3 ⁇ 4 3 ⁇ 4)
  • doxycycline inhibited the growth of HaCaT cells very weakly, and the concentration of doxycycline at 50 ⁇ had almost no inhibitory effect on cell growth.
  • Doxorubicin hydrochloride inhibited cells, and the inhibition of cells at 0.1953 ⁇ reached 90.77%. The results showed that the inhibitory effect of doxycycline on the growth of HaCaT cells was weaker than that of the positive drug doxorubicin hydrochloride, indicating that doxycycline was less toxic to HaCaT cells.
  • Doxycycline has a certain inhibitory effect on the growth of HEK 293 cells.
  • concentration is 25 ⁇
  • the inhibition rate is about 60%.
  • Doxorubicin hydrochloride achieved a 70% inhibition rate of 293 293 cells at a concentration of 0.78125 ⁇ M.
  • the results showed that the inhibitory effect of doxycycline on the growth of ⁇ 293 cells was weaker than that of the positive drug doxorubicin hydrochloride. This indicates that doxycycline is less toxic to ⁇ 293 cells.
  • doxycycline has a strong inhibitory effect on the growth of HEK 293 cells. At a concentration of 3.125 ⁇ , the inhibition rate is 71.05%. The inhibitory rate of azin hydrochloride to ⁇ 293 cells at a concentration of 0.3906 ⁇ reached 71.96%.
  • the inhibitory effect of doxycycline on HEK 293 cells was stronger than that on 293 cells. This is mainly because the HEK 293 ⁇ cell line is a high transfection efficiency derivative produced by the ⁇ 293 cell line inserted into the temperature-sensitive gene of SV40 ⁇ -antigen, and the transduction of S V40 ⁇ -antigen induces malignant transformation of cells. This indicates that doxycycline has a strong inhibitory effect on the growth of malignant transformed cells, and that doxycycline has a certain selectivity for the inhibition of normal cells and malignant transformed cells.
  • Doxy-i P" ⁇ rate cotton rate ADR-i activity inhibition concentration ( ⁇ > OD-- 70 ⁇ ; ': .; concentration ⁇ : : ⁇ ) OD- ;7 .;;, imi) ⁇ : )
  • Doxy-2 survival rate ADR-2 ⁇ viability rate of concentration ⁇ ) (OD mil ) , ⁇ %) (%) ( ) (%)
  • test materials used and their sources include:
  • B16-F10 mouse melanoma cell line purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.
  • Lewis lung cell line purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.
  • human breast cancer cell line MCF-7 purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.
  • Human small cell lung cancer cell line H446 purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.
  • B16-BL6 mouse melanoma cell line purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.
  • the animal strains used in Examples 11 and 12 were C57 mice, SPF grade. A total of 120 C57 mice aged 4-6 weeks and 16-18 g (60 for the first trial and repeated trials) were introduced in this test. Females and animals were introduced in strict accordance with the Standard Operating Procedures for the Introduction of Test Animals (SOPA). -001-V00 ) and recorded in the "Test Animal Introduction Record Form" (BG-015-V00), provided by the Experimental Animal Center of the Chinese Academy of Military Medical Sciences and Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. : 0001631 (first test), 11400700018994 (repeated test).
  • SOPA Standard Operating Procedures for the Introduction of Test Animals
  • Example 13 The animal strain was selected as BABc/nu mice, and the SPF grade was used. In this trial, a total of 78 C57 mice with a mouse age of 5-6 weeks and 18-20 g were introduced (39 for the first trial and repeated trials respectively). Females and animals were introduced in strict accordance with the Standard Operating Procedures for the Introduction of Test Animals (SOPA-001). -V00 ) and recorded in the "Introduction Record of Test Animals" (BG-015-V00), provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the People's Liberation Army, with the certificate number: 0000804 (first test), 0022032 (repeated test).
  • Example 14 The animal strain was selected as BABc/nu mouse, SPF grade. In this trial, a total of 78 C57 mice with a mouse age of 5-6 weeks and 18-20 g were introduced (39 for the first trial and repeated trials respectively). Females and animals were introduced in strict accordance with the Standard Operating Procedures for the Introduction of Test Animals (SOPA-001). -V00 ) and recorded in the "Introduction Record of Test Animals" (BG-015-V00), provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the People's Liberation Army, with the certificate number: 0000804 (first test), 0022032 (repeated test).
  • Example 15 Animal strain was selected as C57 mouse, and SPF grade was introduced in this experiment. A total of 120 C57 mice of 4-6 weeks and 16-18 g were injected (60 for the first trial and repeated trials), female, The introduction of animals is strictly in accordance with the "Standard Operating Procedures for the Introduction of Test Animals” (SOPA-001-V00) and recorded in the "Introduction Record of Test Animals” (BG-015-V00), by the Experimental Animal Center of the Chinese Academy of Military Medical Sciences and Beijing. Provided by Vitallihua Laboratory Animal Technology Co., Ltd., certificate number: 000804 (first test), 11400700020415 (repeated test).
  • the person After the animal arrives, the person receives the animal in the double-corridor barrier environment mouse breeding room 2, fill in
  • Test Animal Receiving Record Form (BG-017-V00) was used to observe the general condition of the animals at the time of receiving, and the animals were randomly selected for weighing to ensure that the test animals were basically in agreement with the introduction standards.
  • Test sample Doxycycline hydrochloride: Yellow powder, supplied by Kaifeng Pharmaceutical (Group) Co., Ltd., batch number: 201301011, Purity: 91.0% (in accordance with the Chinese Pharmacopoeia 2010 edition 2).
  • Positive control drug Cyclophosphamide: white granules, Alfa-Aesa, article number: L11508, lot number: K17X007, purity: 97+%.
  • Solvent 0.9% normal saline.
  • Test sample and positive control drug preservation 4 ° C
  • doxycycline hydrochloride solution Preparation of doxycycline hydrochloride solution: Weigh 720 mg of doxycycline and dissolve it in 60 mL of 0.9% physiological saline solution to prepare 12 mg/mL doxycycline hydrochloride solution. After that, it was suction filtered through a 0.22 ⁇ M filter. The diluted 0.9% physiological saline was separately diluted to 6 mg/ml and 3 mg/ml in a clean bench.
  • the test consisted of a negative control group, a positive control group, and a treatment group.
  • the negative control group consisted of 20 rats in each group, and the positive control group, the high dose group, the middle dose group and the low dose group each had 10 rats.
  • the treatment group was set in three dose groups: high, medium and low. The dose is set at 4:2:1. According to the clinical maximum dose, this experiment will prepare three different concentrations (high, medium and low) of doxycycline solution at concentrations of 12mg/ml, 6mg/ml and 3mg/ml, respectively, at a dose of 60mg/kg. , 30mg/kg and 15mg/kg.
  • the positive control group was treated with cyclophosphamide (4 mg/ml) at a dose of 20 mg/kg.
  • the negative control group used physiological saline.
  • the frozen cells were removed from the liquid nitrogen and immediately placed in a 37 ° (water bath to melt the cells. Biosafety Rejection)
  • the cell suspension was pipetted into a centrifuge tube containing the appropriate amount of medium and centrifuged at 800 rpm for 5 minutes. The supernatant was discarded and 10 ml of RPMI1640 containing 10% calf serum was added.
  • the cells were cultured at 37 ° C, 5% CO 2 , saturated humidity to 80% contact confluence, and 0.2% trypsin was digested into single cell suspension. The cells were counted under a light microscope using a counting plate, and the number of cells was adjusted to lx 10 7 /ml with sterile physiological saline.
  • the operator wears sterile gloves, a hat, and a mask.
  • Single-handed mice, 75% alcohol cotton balls were used to disinfect the right groin, each subcutaneously injected with 0.1 ml of B 16 malignant melanoma cell suspension. After the injection, the small tweezers pinch the pinhole for a while to prevent the liquid from overflowing.
  • the vital signs and tumor growth of the mice were observed daily.
  • C57BL/6 mice with melanoma B 16-F10 with strong tumor growth and no ulceration were selected and sacrificed by cervical dislocation.
  • the skin of the animal was disinfected with alcohol, the skin was cut, and the tumor was removed.
  • the tumor tissue was cut into 1.5 mm 3 to prepare B 16 melanoma cell suspension, and then sterile saline was added in a certain proportion and inoculated subcutaneously into the mouse murine.
  • the number of tumor cells per mouse was 1 X 10 . 6 , each 0.1ml.
  • Animals were randomized the next day after tumor inoculation.
  • the intragastric administration was started when the tumor grew to 40 mm 3 .
  • the negative control group received 0.1 ml of normal saline per day, and the positive control group received 0.1 ml of cyclophosphamide (4 mg/ml) per day.
  • the high dose group, the middle dose group and the low dose group were respectively 0.1 ml of different concentrations (12 mg/ml, 6 mg/ml and 3 mg/ml) of doxycycline were administered.
  • the diameter of the transplanted tumor was measured daily with a vernier caliper.
  • Tumor volume was calculated using the method of measuring the tumor diameter.
  • TRTV treatment group RTV; CRTV: negative control group RTV.
  • mice B16 melanoma xenograft model established in this experiment was the first after tumor cell inoculation.
  • a nodular mass of about 40 mm 3 was reached subcutaneously in the mouse sac.
  • the tumor growth curve see Table 39 and Figure 62
  • the tumor volume of the cyclophosphamide group and the doxycycline high-dose group were smaller than the control group, and the difference was statistically significant ( P ⁇ 0.05).
  • the tumor volume of the cyclophosphamide group and the doxycycline high, medium and low dose groups were smaller than the control group, the difference was statistically significant (P ⁇ 0.05).
  • the tumor volume in the middle and low dose groups of doxycycline was smaller than that in the control group, and the tumor volume in the low dose group of doxycycline on day 14 was smaller than that in the cyclophosphamide group. Significance (P ⁇ 0.05).
  • the transplanted tumor after administration of doxycycline showed a decrease in blood supply vessels and easy exfoliation of the tumor when the tumor was dissected, suggesting that doxycycline inhibits tumor angiogenesis and infiltration of surrounding normal tissues.
  • the role see Figure 63).
  • the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 36%; high dose group: 49%; medium dose group: 65%; low dose group: 63% (see Table 39).
  • Tumor inhibition rate Note: * P ⁇ 0.05 compared with the control group; # P ⁇ 0.05 compared with the cyclophosphamide group. 2, repeat the test
  • the mouse B 16 melanin xenograft model established in this experiment can also reach about 40 mm 3 of nodular mass in the mouse sac on the 8th day after tumor cell inoculation, which is consistent with the first test results.
  • the results of this trial showed (Table 40 and Figure 65) that on the 9th day of melanoma xenograft growth, the tumor volume in the high-dose and middle-dose groups was significantly smaller than that in the control group, about 1/2 of the control group; On the 10th day of growth, the tumor volume of the cyclophosphamide group and the doxycycline high and middle dose groups were smaller than the control group; on the 11th day of tumor growth, only the doxycycline high dose group had a smaller tumor volume than the control group.
  • the tumor volume of the cyclophosphamide group, doxycycline high, and low dose groups was smaller than that of the control group; at 13, 14, 15 and 19, 20 days of tumor growth At 5 time points, the tumor volume of the cyclophosphamide group and the doxycycline high, medium and low dose groups were smaller than those of the control group. In addition, on the 19th day of tumor growth, the tumor volume of the high dose group of doxycycline was smaller than that of the middle dose group; the above results were statistically significant (P ⁇ 0.05).
  • the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 59%; high dose group: 73%; medium dose group: 28%; low dose group: 56% (see Table 40).
  • the operation method is the same as that under the corresponding cell culture in Example 11, except that The cultured cells were mouse Lewis lung cancer cells.
  • C57BL/6 mice bearing Lewis lung cancer with strong tumor growth and no ulceration were selected and sacrificed by cervical dislocation.
  • the skin of the animal was disinfected with alcohol, the skin was cut, and the tumor was peeled off.
  • the tumor tissue was cut into 1.5 mm 3 to prepare B 16 melanoma cell suspension, then sterile saline was added in a certain proportion, and inoculated into the back of the mouse.
  • the number of tumor cells per mouse was 1 10 6 .
  • mice in the negative control group were given 0.1 ml of normal saline per day, and the mice in the positive control group were given 0.1 ml of cyclophosphamide (4 mg/ml) per day.
  • the high-dose group, the middle-dose group and the low-dose group were given 0.1 ml of different concentrations each day (12 mg). /ml, 6mg/ml and 3mg/ml) of doxycycline.
  • the diameter of the transplanted tumor was measured daily with a vernier caliper.
  • the mouse Lewis lung cancer xenograft model constructed in this experiment can reach about 40 mm 3 tumor subcutaneously about 11 days after tumor cell inoculation.
  • the tumor growth curve obtained after measuring the tumor size can be seen ( See Table 41, Figure 67), when the tumor grew to the 15th day, the tumor volume of the cyclophosphamide group and the doxycycline high, medium and low dose groups were smaller than the control group; subsequently, when the tumor grew to the 16th day, The tumor volume of the middle and low dose groups of doxycycline was smaller than that of the control group; on day 17, the tumor volume of the high, medium and low dose groups of doxycycline was smaller than that of the control group; on the 18th day, the doxycycline was low.
  • the tumor volume of the dose group was smaller than that of the control group and the cyclophosphamide group; on the 19th day, the tumor volume of the high and middle dose groups of doxycycline was smaller than that of the control group; the above results were statistically significant (P ⁇ 0.05).
  • the inhibition rate of each group at the terminal time point was: cyclophosphamide group: 41%; High dose group: 68%; medium dose group: 68%; low dose group: 27% (see Table 41).
  • the weight of the high, middle and low dose groups was greater than that of the control group, the difference was statistically significant (P ⁇ 0.05); the doxycycline high, medium and low dose compared with the cyclophosphamide group.
  • the body weight of the group was greater than that of the cyclophosphamide group, and the difference was statistically significant (P ⁇ 0.05) (see Figure 68).
  • Tumor inhibition rate Note: * P ⁇ 0.05 compared with the control group; # P ⁇ 0.05 compared with the cyclophosphamide group.
  • the mouse Lewis lung cancer xenograft model constructed in this experiment can reach about 40 mm 3 of tumor under the skin about 8 days after tumor cell inoculation, and measure the tumor size.
  • the tumor growth curves obtained after i r- are shown in Fig. 69 and Table 42.
  • the tumor volume of the cyclophosphamide group, the doxycycline high, medium and low dose groups was smaller than that of the control group; on the 13th day of tumor growth, doxycycline was high, The tumor volume of the middle dose group was smaller than that of the control group; on the 14th day, the tumor volume of the cyclophosphamide group, the doxycycline high and low dose groups was smaller than that of the control group; on the 16th and 17th days, cyclophosphamide and doxycycline
  • the tumor volume of the high-dose group was smaller than that of the control group; on the 20th day, the tumor volume of the high dose group of doxycycline was smaller than that of the control group.
  • the above results were statistically significant (P ⁇ 0.05).
  • the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 30%; high dose group: 61%; medium dose group: 53%; low dose group: 43% (see Table 42).
  • the test consisted of a negative control group, a positive control group, and a treatment group.
  • the negative control group consisted of 15 rats in each group, and the positive control group, the high dose group, the middle dose group and the low dose group each had 6 rats.
  • the positive control group used cyclophosphamide (4 mg/ml).
  • the treatment group was set in three dose groups: high, medium and low. The dose is set at 4:2:1. According to the clinical maximum dose, three different concentrations (high, medium and low) of doxycycline solution will be prepared in this experiment. The concentrations are: 12mg/ml 6mg/ml and 3mg/ml, respectively, the dose is 60mg/kg 30mg /kg and 15mg/kg.
  • the positive control group was treated with cyclophosphamide (4 mg/ml) at a dose of 30 mg/kg.
  • the procedure was the same as in the corresponding cell culture in Example 11, except that the cells cultured were MCF-7 breast cancer cells.
  • mice bearing MCF-7 breast cancer with vigorous growth and no ulceration were selected and sacrificed by cervical dislocation.
  • the skin of the animal was disinfected with alcohol, the skin was cut, and the tumor was removed.
  • the tumor tissue was cut into 1.5 mm 3 to prepare MCF-7 breast cancer cell suspension, then sterile saline was added in a certain proportion, and inoculated into the back of the mouse.
  • the number of tumor cells per mouse was 1 10 6 , each 0.1ml. Animals were randomized the next day after tumor inoculation.
  • the intragastric administration was started after the tumor was grown to about 40 mm 3 .
  • mice in the negative control group were given 0.1 ml of normal saline per day, and the mice in the positive control group were given 0.1 ml of cyclophosphamide (4 mg/ml) per day.
  • the high-dose group, the middle-dose group and the low-dose group were given 0.1 ml of different concentrations each day (12 mg). /ml, 6mg/ml and 3mg/ml) of doxycycline.
  • the diameter of the transplanted tumor was measured every other day using a vernier caliper.
  • the mouse MCF-7 breast cancer xenograft model constructed in this experiment can reach about 40 mm 3 of tumor nodules subcutaneously on the 16th day after tumor inoculation.
  • the tumor volumes in the high, medium and low dose groups of doxycycline were smaller than those in the control group on days 20, 24, 28 and 30 of tumor growth.
  • the tumor volume of the high and middle dose groups of doxycycline was smaller than that of the control group; in addition, on days 20 and 22 of tumor growth, the doxycycline high, medium, and low dose groups The tumor volume was smaller than that of the cyclophosphamide group; on the 28th day of tumor growth, the tumor volume of the high and medium dose groups of doxycycline was smaller than that of the cyclophosphamide group.
  • the above results were statistically significant (P ⁇ 0.05)
  • the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 45%; high dose group: 67%; medium dose group: 75%; low dose group: 72% (see Table 43).

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Abstract

La présente invention concerne une utilisation de doxycyclique dans la préparation d'un médicament pour le traitement d'une tumeur maligne hautement métastatique. La doxycycline peut inhiber la croissance, la métastase et l'invasion de cellules tumorales.
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