WO2015021704A1 - Uses of doxycycline for preparing antitumor drugs - Google Patents

Abstract

The present invention provides uses of doxycycline for preparing antitumor drugs. As disclosed in the present invention, antitumor activities of doxycycline mainly comprise effects of inhibiting tumor growth and activities for resistance against tumor metastasis and tumor invasion. By means of experiments at in-vitro cell and animal levels, it is proved that doxycycline has effects on inhibiting growth and metastasis of malignant tumors such as breast cancer, liver cancer, melanoma, lung cancer, pancreatic cancer and colon cancer, and doxycycline also shows good antitumor effects on a tumor-bearing mouse model. In addition, doxycycline of the present invention has incomparable advantages which other drugs do not have, patients are more willing to receive treatment and reactions of the patients on the drugs can be conveniently obtained. Doxycycline will change the market pattern of existing tumor chemotherapy drugs, and become a clinical drug which can be used for a long time and can effectively inhibit metastasis, invasion and recurrence of tumors.

Description

 Application of doxycycline in the preparation of antitumor drugs

Technical field

 The present invention relates to the field of medicinal chemistry and, in particular, to the use of doxycycline for the preparation of antineoplastic agents. Background technique

 Doxycycline "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). About 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.

 So far, the application of doxycycline in malignant tumors such as liver cancer, breast cancer, lung cancer, pancreatic cancer, colon cancer, and melanoma has not been reported. Malignant tumors such as liver cancer, breast cancer, lung cancer, pancreatic cancer, colon cancer, and melanoma are high-risk diseases. Because these malignant tumors generally have the ability to metastasize and invade, it is difficult to cure once the above diseases occur, which may cause human health. The 4 big hazards. Existing anti-tumor drugs against liver cancer, breast cancer, lung cancer, pancreatic cancer, colon cancer, melanoma and other malignant tumors are not only low in anti-tumor activity, but also expensive, and patients are often unbearable, and even some patients cannot afford expensive prices. And gave up the treatment opportunity. Therefore, there is an urgent need to develop new anti-tumor growth, metastasis or invasion drugs. Summary of the invention

In view of the defects in the prior art that the antitumor drug has low antitumor activity and high cost, the object of the present invention is to provide an application of doxycycline in the preparation of an antitumor drug. In order to achieve the above object, the present invention provides the use of doxycycline for the preparation of an antitumor drug, wherein the doxycycline

 In the preparation of an antitumor drug, the tumor involved comprises: breast cancer, liver cancer, melanoma, lung cancer, pancreatic cancer, colon or a combination thereof.

 In the preparation of antitumor drugs, the use of antitumor drugs, including as a cytotoxic drug in the inhibition of tumor growth, as a drug against tumor metastasis, or as a drug against tumor invasion Applications.

 In use in the preparation of an anti-tumor drug, the anti-tumor agent comprises a therapeutically effective amount of doxycycline, or a pharmaceutically acceptable salt, or ester thereof, and an adjuvant.

In the preparation of antitumor drugs, the effective therapeutic amount of the antitumor drug is from 100 m _g / k _g to 300 mg / kg.

 In the preparation of the antitumor drug, the antitumor drug is selected from the group consisting of a tablet, a capsule, a pill, a liquid preparation, a granule, a powder, an injection, or a combination thereof.

 In the preparation of an antitumor drug, the administration of the antitumor drug includes oral administration, injection, implantation, external application, spraying, inhalation, or a combination thereof.

 The anti-tumor drug provided by the present invention has the advantage that the patient is easy to accept and easy to understand the patient's response to the drug, is inexpensive, and is easily available. In addition, 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, which was purchased from Shanghai Shengong (production batch number DB0889-25g). The chemical structural formula of doxycycline hydrochloride is:

DRAWINGS

 Figure 1 shows the inhibitory effect of cetcycline on the human breast cancer cell line MCF-7; Figure 2 shows the inhibitory effect of '''xicycline on the human hepatoma cell line HepG-2;

 Figure 3 shows the inhibitory effect of cetcycline-doxcycline on mouse melanoma B-16 cells. Figure 4 shows the inhibitory effect of ''sicycline on human small cell lung cancer cell NCI-H446; Figure 5 shows the inhibitory effect of '''xicycline on pancreatic cancer cell line PC-3;

 Figure 6 shows the inhibitory effect of '''xicycline on human colon cancer cell LOVO;

 Figure 7 shows the effect of '''xicycline on migration of MCF-7 cells;

 Figure 8 shows the effect of cycline on the migration of HepG2 cells;

 Figure 9 shows the effect of cyclulin on the migration of B16 cells;

 Figure 10 shows the effect of %ccycline on migration of human small cell lung cancer cells NCI-H446; Figure 11 shows the effect of cilin on the migration of pancreatic cancer cell line PC-3;

 Figure 12 shows the effect of %ccycline on LOVO migration in human colon cancer cells;

 Figure 13 shows the inhibitory effect of cyclulin on the tumor size of tumor-bearing mice;

 Figure 14 shows the effect of cyclulin on the tumor growth state of tumor-bearing mice. detailed description

 The technical solutions in the embodiments of the present invention are clearly and completely described in the following with reference to the accompanying drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of the present invention.

 The inventors have confirmed, through extensive experimental studies, the antitumor activity of tetracycline-structured doxycycline. The specific embodiment is as follows.

In the following examples, the test materials used and their sources include: Doxycycline hydrochloride, purchased from Shanghai Shengong (production batch number: DB0889-25g); high sugar DMEM medium, RIPM 1640 medium, fetal bovine serum FBS (Hy clone brand) purchased from Thermo company; Thiazole blue (MTT) was purchased from Sigma, USA; 96-well plate (Nunc brand) was purchased from Thermo; Hochest 33342 was purchased from Biyuntian Biotechnology Co., Ltd.; DMSO was purchased from Sigma, USA; C57/BL mice were purchased from Shanghai Lake Laboratory Animals Ltd. Example 1 Inhibition of doxycycline on the growth of different tumor cells

 Doxycycline was formulated into 30 mM mother liquor in DMSO and stored. The medium was diluted with the culture solution to the use concentration.

 The cell line was selected as: human breast cancer cell line MCF-7 (medium: RIPM 1640 plus 10% FBS, purchased from Beijing Jin Zijing Biomedical Technology Co., Ltd.); human hepatoma cell line HepG-2 (medium: DMEM high) Sugar medium plus 10% FBS, purchased from Beijing Jin Zijing Biomedical Technology Co., Ltd.); mouse melanoma B-16 cells (medium: RIPM 1640 plus 10% FBS, purchased from Beijing Jin Zijing Biomedical Technology Co., Ltd.); Human small cell lung cancer cell NCI-H446 (medium: RIPM 1640 plus 10% FBS, purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.); Pancreatic cancer cell PC-3 (medium: RIPM 1640 plus 10% FBS, Purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.); human colon cancer cell LOVO (medium: RIPM 1640 plus 10% FBS, purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.).

 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 specific experimental steps are as follows: After the cells are trypsinized, the cells are dispersed into individual cells and suspended in the corresponding medium. The cells were seeded on 96-well plates at 4000 cells/well. The cells were cultured overnight in a 37 ° C carbon dioxide (5%) incubator to allow the cells to adhere. The next day, discard the culture solution and add a cell culture medium containing a series of doxycycline concentrations (specific concentrations in each cell) The results are described in the results) and control wells without drug concentration were set. After incubating for 48 hours in a 37 ° C carbon dioxide ( 5 % ) incubator, 20 μM TT mother liquor (5 mg/mL mother liquor concentration) was added to each well and incubation was continued for 4 h. Then, the culture solution was aspirated, 150 μl of DMSO was added to each well as a solvent to dissolve hydrazine, and after dissolution, the absorbance at 490 nm was measured with a microplate reader (Multiscan FC, Thermo company), and the cell survival rate was calculated by the following formula:

 (Experimental group OD/control group OD) x l 00 %

Experimental results 1 Inhibition of doxycycline on human breast cancer cell line MCF-7

 The dose response curve of doxycycline on the cytotoxicity of human breast cancer cell line MCF-7 is shown in Figure 1. The concentrations of doxycycline used were: 0 μΜ, 0.39063 μΜ, 0.78125 μΜ, 1.5625 μΜ, 3.125 μΜ, 6.25 μΜ, 12.5 μΜ, 25 μΜ, 50 μΜ, 100 μΜ. It can be seen from Figure 1 that the survival rate of MCF-7 cells is 20% at 100 μΜ. The inhibition of proliferation of MCF-7 cells by doxycycline reached 80%. It indicated that doxycycline had a good inhibitory effect on the proliferation of MCF-7 cells.

Experimental results 2 The inhibitory effect of doxycycline on human hepatoma cell line HepG-2.

 The dose response curve of cytotoxicity of doxycycline on human hepatoma cell line HepG-2 is shown in Figure 2. The concentrations of the drugs used were: 0 μΜ, 0.39063 μΜ, 0.78125 μΜ, 1.5625 μΜ, 3.125 μΜ, 6.25 μΜ, 12.5 μΜ, 25 μΜ, 50 μΜ, 100 μΜ. As can be seen from Figure 2, the survival rate of HepG-2 cells was 80% when the concentration of doxycycline was 100 μΜ. The inhibitory effect of doxycycline on HepG-2 cell proliferation was 20%. It indicated that doxycycline had a certain inhibitory effect on the proliferation of HepG-2 cells.

Experimental results 3 inhibitory effect of doxycycline on mouse melanoma B-16 cells

 The results of doxycycline on mouse melanoma B-16 cytotoxicity test are shown in Figure 3. The concentrations of the drugs used were: 0 μΜ, 0.78 μΜ, 1.56 μΜ, 3.125 μΜ, 12.5 μΜ, 25 μΜ, 50 μΜ, 100 μΜ. As can be seen from the figure, the cell survival rate was 0.04% at a drug concentration of 100 μΜ. The inhibitory rate of doxycycline on B-16 cell proliferation was 99.6%. This indicates that doxycycline has a very good inhibitory effect on the proliferation of B-16 cells.

Experimental results 4 inhibitory effect of doxycycline on human small cell lung cancer cell line NCI-H446

The test results of doxycycline on human small cell lung cancer cells NCI-H446 are shown in Fig. 4. The concentrations of doxycycline used were: 0 μΜ, 0.78 μΜ, 1.5625 μΜ, 3.125 μΜ, 6.25 μΜ, 12.5 μΜ, 25 μΜ, 50 μΜ, 100 μΜ, 200 μΜ. As can be seen from the figure, the cell survival rate was 29% at a drug concentration of 200 μΜ. The inhibition rate of doxycycline on the proliferation of human small cell lung cancer cell line NCI-H446 reached 71%. It indicated that doxycycline had a good inhibitory effect on the proliferation of human small cell lung cancer cell line NCI-H446.

Experimental results 5 Inhibition of panax cancer cell line PC-3 by doxycycline

 The toxicity test results of doxycycline on human pancreatic cancer cell line PC-3 are shown in Fig. 5. The drug concentrations used were: 0 μΜ, 1.563 μΜ, 3.125 μΜ, 6.25 μΜ, 12.5 μΜ, 25 μΜ, 100 μΜ, 200 μΜ. As can be seen from the figure, the cell survival rate was 74% at a drug concentration of 200 μΜ. The inhibition rate of doxycycline on the proliferation of human pancreatic cancer cell line PC-3 reached 26%. This indicates that doxycycline has a certain inhibitory effect on the proliferation of human pancreatic cancer cell line PC-3.

Experimental results 6 Inhibition of doxycycline on human colon cancer cell line LOVO

 The toxicity test results of doxycycline on human colon cancer cell LOVO are shown in Figure 7. The concentrations of the drugs used were: 0 μΜ, 0.781 μΜ, 1.563 μΜ, 3.125 μΜ, 6.25 μΜ, 12.5 μΜ, 25 μΜ, 100 μΜ, 200 μΜ. As can be seen from the figure, the cell survival rate was 79% at a drug concentration of 200 μΜ. The inhibition rate of doxycycline on LOVO proliferation in human colon cancer cells reached 21%. It indicates that doxycycline has a certain inhibitory effect on the proliferation of human colon cancer fine LOVO. Example 2 In vitro cell level detection of the effect of doxycycline on cell migration

 experimental method:

 After trypsinization, the cells are dispersed into individual cells and suspended in the corresponding medium. The cells were seeded on 96-well plates at 4000 cells/well. The cells were cultured overnight in a 37 ° (carbon dioxide (5%) incubator. The next day, the culture solution was discarded and a medium containing a series of concentrations of the test substance (doxycycline) was added. Days, aspirate the culture solution, wash the cells twice with PBS, add Hochest 33342 (dissolved in a solution of pH 7.4 in a solution of 5 g/mL). The staining solution is 50 μ! 7 well, 37 ° C carbon dioxide (5%) Incubate for 20 min in the incubator. Aspirate the staining solution, wash twice with PBS, add the medium containing a certain concentration of the drug to be tested, and then place it on the high-content analysis platform ArrayScanVTI (manufactured by Thermo) for real-time dynamic monitoring. , test the effect of drugs on cell migration.

The effect of doxycycline on cells can be divided by the software that comes with the high content analysis platform. Analysis. The effect of the drug on cell migration rate is primarily indicated by the slope of the curve in the resulting average distance-time plot. The higher the slope, the faster the cell migration rate, and the smaller the slope, the slower the cell migration rate.

Experimental results 1 inhibition of migration of MCF-7 cells by doxycycline

 The inhibitory effect of doxycycline on MCF-7 cell migration is shown in Figure 7. In the figure, D7 is the control group, D8 and D9 are the dosing group, the D8 group has a drug concentration of 20 μΜ, and the D9 group has a drug concentration of 4 μΜ. As can be seen from the figure, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. It was proved that doxycycline significantly inhibited the migration of MCF-7 cells.

Experimental results 2. Inhibition of HepG-2 cell migration by doxycycline

 The inhibitory effect of doxycycline on HepG-2 cell migration is shown in Figure 8. In the figure, C6 is the control group, C7 and C8 are the doxycycline group, and the C7 and C8 groups are all 20 μΜ. It can be seen from the figure that the slope of the motion curve of the cells in the dosing group is significantly smaller than that of the control group. It was demonstrated that doxycycline inhibited the migration of HepG-2 cells.

Experimental results 3 Inhibition of B16 cell migration by doxycycline

 The inhibitory effect of doxycycline on B16 cell migration is shown in Figure 9. In the figure, B6 is the control group, and B7 and B8 are the forces. In the drug group, the drug concentration in the B7 group was 4 μΜ, and the drug concentration in the Β8 group was 20 μΜ. As can be seen, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. It was demonstrated that doxycycline significantly inhibited the migration of B16 cells.

Experimental results 4 inhibition of migration of NCI-H446 cells by doxycycline

 The inhibitory effect of doxycycline on NCI-H446 cell migration is shown in Figure 10. In the figure, D11 is the control group, D9 and D10 are the dosing group, the D9 group has a drug concentration of 4 μΜ, and the D10 group has a drug concentration of 20 μΜ. As can be seen, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. It was proved that doxycycline significantly inhibited the migration of NCI-H446 cells.

Experimental results 5 inhibition of PC-3 cell migration by doxycycline

 The inhibitory effect of doxycycline on PC-3 cell migration is shown in Figure 11. In the figure, C2 is the control group, C3, C4, and C5 are the dosing group, and the drug concentration is 20 μΜ. As can be seen, the slope of the motion curve of the cells in the dosing group was smaller than that of the control group. It was proved that doxycycline has a certain inhibitory effect on the migration of PC-3 cells.

Experimental results 6 inhibition of LOVO cell migration by doxycycline The inhibitory effect of doxycycline on LOVO cell migration is shown in Figure 12. D2 was the control group, C9, CIO, and C11 were in the dosing group. The C9 drug concentration was 20 μΜ, the C10 drug concentration was 10 μΜ, and the Cl l drug concentration was 4 μΜ. As can be seen, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. It was proved that doxycycline has a certain inhibitory effect on the migration of LOVO cells. Example 3 Effect of doxycycline on a melanoma mouse model

Experimental method for establishing a tumor-bearing model in mice:

1. Preparation of B-16 cell suspension: After trypsinization, the cells were centrifuged at 800 rpm, the cells were resuspended in PBS, centrifuged again, and the remaining medium was washed away. After the cells were counted, the cells were made into a cell suspension of lx10 ⁷ cells/mL.

2. Establishment of tumor-bearing mouse model: 10 C57/BL mice were randomly divided into 2 groups. Melanoma cells were inoculated subcutaneously into C57BL/6 mice, and each mouse was inoculated with 100 μ ₀

 3. After inoculation of the cells, observe the growth time of the tumor in mice and the growth characteristics of the tumor in mice.

 The mouse tumor is allowed to grow until it is ready to be administered.

 4. Make doxycycline into 10 mg/ml with normal saline. One group of mice was used as an experimental group, and doxycycline was intraperitoneally administered, and each mouse was administered with 50 mg/kg. Another group of mice was used as a control group, and normal saline was intraperitoneally injected as a control. The daily administration was performed, and the volume of the tumor of the administration group and the control group was measured every day. Six days after the administration, the mice were sacrificed, and tumor tissues were taken for sectioning for histomorphometric analysis.

 Experimental results:

 The inhibitory effect of doxycycline on tumor size in tumor-bearing mice is shown in Figure 13 (C: control group, no administration, only saline; DOX: doxycycline administration group), doxycycline The tumor volume of the mice in the drug group was significantly smaller than that in the control group, and the tumor volume of the mice in the doxycycline group was relatively uniform, which showed that doxycycline had a good inhibitory effect on the growth of mouse melanoma.

The effect of doxycycline on tumor growth status in tumor-bearing mice is shown in Figure 14 (C: control group, no administration, only saline; DOX: doxycycline administration group): The blood vessels are rich, the peritoneum is invaded, and the necrosis is less. The doxycycline-administered group has small tumor volume, few blood vessels, easy to peel off the invading peritoneum, and a large amount of necrosis in the middle. Doxycycline administration compared to the control group The tumor metastasis of the group was not obvious. It is shown that doxycycline has a certain ability to resist tumor metastasis.

 It can be seen from Example 1 that when the concentration of doxycycline is 100 μΜ, the inhibition rate of doxycycline on MCF-7 cell proliferation is 80%, and the inhibition rate on HepG-2 cell proliferation is 20%. The inhibition rate of B-16 cell proliferation reached 99.6%. When doxycycline concentration was 200 μΜ, doxycycline inhibited the proliferation of human small cell lung cancer cell line NCI-H446 by 71% and inhibited the proliferation of human pancreatic cancer cell line PC-3 by 26%. The inhibition rate of LOVO proliferation in human colon cancer cells reached 21%. It can be seen from Example 2 that the slope of the movement curve of the cells in the drug-added group is smaller than that of the control group, and the migration of doxycycline to MCF-7 cells, HepG-2B16 cells, NCI-H446 cells, PC-3 cells, and LOVO cells was confirmed. There is a certain inhibition. This indicates that doxycycline has different inhibitory effects on the proliferation and migration of malignant tumor cells of breast cancer, liver cancer, melanoma, lung cancer, pancreatic cancer and colon cancer. It can be seen from Example 3 that the volume of d and mouse tumors in the doxycycline-administered group was smaller than that in the control group, and the tumor size of the d and the rats in the doxycycline-administered group was relatively uniform, and no malignant growth occurred; It was found that doxycycline can significantly inhibit tumor invasion of the peritoneum.

 The use of doxycycline in the preparation of antitumor drugs can be seen from the above experiments. At the same time, the doxycycline of the present invention has advantages unmatched by other drugs, and the patient is easy to accept and easy to understand the patient's response to the drug, and is inexpensive and easy to obtain. In addition, doxycycline will change the market pattern of existing cancer chemotherapy drugs, and gradually become a clinical drug that can be taken for a long time and effectively inhibits tumor metastasis, invasion and recurrence.

 The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalents, improvements, etc., which are included in the spirit and scope of the present invention, should be included in the present invention. Within the scope of protection.

Claims

Claim

The use of doxycycline in the preparation of an antitumor drug, wherein the doxilin sub-knot

 2. The use according to claim 1, characterized in that the tumor involved comprises: breast cancer, liver cancer, melanoma, lung cancer, pancreatic cancer, colon cancer or a combination thereof.

 3. The use according to claim 1, wherein the application of the antitumor drug comprises the use as a cytotoxic drug in an inhibitor against tumor growth, as a drug against tumor metastasis, or as Application in anti-tumor drugs.

 4. The use according to claim 1, wherein the antitumor drug comprises doxycycline, a pharmaceutically acceptable salt of doxycycline, an ester or a combination thereof, and an adjuvant.

 The use according to claim 1, wherein the effective therapeutic amount of the antitumor drug is from 100 mg/kg to 300 mg/kg.

 The use according to claim 5, wherein the antitumor drug is selected from the group consisting of a tablet, a capsule, a pill, an oral liquid preparation, a granule, a powder, an injection, or a combination thereof.

 7. The use according to claim 1, wherein the administration of the antitumor drug comprises oral administration, injection, implantation, topical application, spraying, inhalation or a combination thereof.