CN107412736B - Anti-tumor combined medicine and application thereof in preparing anti-cancer medicine - Google Patents
Anti-tumor combined medicine and application thereof in preparing anti-cancer medicine Download PDFInfo
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- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
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- 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
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
Abstract
The invention discloses an anti-tumor combined medicament and application thereof in preparing an anti-cancer medicament, wherein the combined medicament comprises a first preparation and a second preparation, and the anti-tumor combined medicament comprises 10-40 parts of the first preparation and 30-50 parts of the second preparation in parts by weight. The invention also discloses application of chlorogenic acid in preparing an anti-cancer drug toxic and side effect inhibitor, wherein the anti-cancer drug is an angiogenesis inhibitor. The technical scheme of the invention can improve the targeting of the angiogenesis inhibitor to non-small cell lung cancer, gastric cancer and liver cancer, reduce the toxic and side effects of the angiogenesis inhibitor and reverse the drug resistance of the angiogenesis inhibitor.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to an anti-tumor combined medicine and application thereof in preparing anti-cancer medicines.
Background
Chlorogenic acid is a phenylpropanoid substance synthesized by pentose phosphate pathway intermediate product in the process of aerobic respiration of plants. The extraction and synthesis technology is mature, and the chlorogenic acid has been developed and applied to a plurality of fields such as food, health care products, cosmetics, medicines and the like. The current research results show that the chlorogenic acid has the effects of protecting the cardiovascular system, resisting oxidation, resisting ultraviolet, resisting radiation, resisting cancer, resisting bacteria, resisting virus, regulating immunity and the like.
The malignant tumor seriously harms the health of people, and according to WHO statistics, 700 million people die of the malignant tumor in the world in average each year. Cancer deaths will continue to rise worldwide, with an expectation that by 2030, there will likely be over 1310 thousands. Tumor invasion and metastasis are the main reasons for failure of tumor therapy. According to the tumor angiogenesis theory, when the diameter of a solid tumor is larger than 2mm, new angiogenesis needs to be induced to obtain blood supply, otherwise, the tumor is necrotized due to ischemia and hypoxia, so the angiogenesis inhibitor has good curative effect. According to the action target of the angiogenesis inhibitor, the angiogenesis inhibitor is divided into the following 8 types: directly acting on a receptor tyrosine kinase (PTK) inhibitor; (ii) inhibitors acting directly on nucleic acids (RNA); ③ inhibitors acting directly on VEGF; fourthly, acting on the matrix metalloproteinase inhibitor; inhibitor directly acting on the endothelial cells of new vessels; sixthly, antagonists acting on integrin proteins; seventhly, a non-specific angiogenesis inhibitor; the natural medicine and the components thereof have substances which can potentially inhibit angiogenesis.
The recombinant human endostatin injection (rh-endostatin, Endostar, code YH-16, trade name: Endoter) is a novel human endostatin and anti-tumor angiogenesis medicine which are independently and innovatively developed by Chinese scholars. It is often used in combination with other cytotoxic drugs in non-small cell lung cancer, gastric cancer, breast cancer, bone and soft tissue sarcomas. It has limited curative effect when used alone, is easy to generate drug resistance, and has adverse reactions of different degrees.
Fumagiline (TNP-470) is a derivative of fumagillin, TNP-470 is one of the most intense preparations for inhibiting the growth of blood vessels reported so far, is also the 1 st anti-tumor angiogenesis medicament which enters clinical phase III experiments, but does not have cytotoxic effect per se, but shows more effect of inhibiting the growth of tumor endothelial cells, so that the drug resistance is easily generated due to insufficient targeting. Vandetanib and vartanib, which are vascular endothelial growth factor inhibitors, also have the problems of drug resistance and toxic and side effects in clinical use.
Disclosure of Invention
In view of the above, the invention provides an anti-tumor combined drug and an application thereof in preparing an anti-cancer drug, and also discloses an application of chlorogenic acid in preparing an anti-cancer drug toxic and side effect inhibitor, wherein the anti-cancer drug is an angiogenesis inhibitor. The technical scheme of the invention can improve the targeting of the angiogenesis inhibitor to non-small cell lung cancer, gastric cancer, liver cancer and colon cancer, reduce the toxic and side effects of the angiogenesis inhibitor and reverse the drug resistance of the angiogenesis inhibitor.
In order to solve the technical problems, the technical scheme of the invention is to adopt an anti-tumor combined medicament which is characterized by comprising a first preparation formed by chlorogenic acid and a pharmaceutically acceptable carrier and a second preparation formed by an angiogenesis inhibitor and a pharmaceutically acceptable carrier.
Preferably, the first preparation is 10-40 parts, and the second preparation is 30-50 parts.
Preferably, the angiogenesis inhibitor comprises a neovascular endothelial cell inhibitor and a vascular endothelial growth factor inhibitor.
Preferably, the neovascular endothelial cell inhibitor comprises enrofloxacin, fumagiline.
Preferably, according to the combination drug, the preparation is in the form of injection or oral preparation.
The invention also provides application of the combined medicament in preparing an anti-cancer medicament.
Preferably, the cancer comprises non-small cell lung cancer, gastric cancer and liver cancer.
The invention also provides application of chlorogenic acid in preparing an anticancer drug toxic and side effect inhibitor.
Preferably, the anti-cancer drug is an angiogenesis inhibitor.
Because of insufficient targeting, single-drug antitumor effect is limited and drug resistance is easy to generate due to insufficient targeting. A large number of experiments at present prove that the chlorogenic acid has the effects of resisting mutagenesis and cancers. Chlorogenic acid as inhibitor of G6PT (glucose-6-phosphotransferase) can initiate apoptosis of neutrophil and promyelocyte HL-60, inhibit metallothionein (MMP) secretion of human Hep3B hepatocarcinoma cell, and inhibit migration of glioma. Chlorogenic acid can cause tumor cells to generate larger DNA fragments under an alkaline environment and cause nuclear agglutination; chlorogenic acid can inhibit 4-nitroquinoline-1-oxide (4-nitroquinoline-1-oxide, 4NQO) induced increase of 8-hydroxydeoxybird (8-OH-dG), which is an important substance in the process of canceration and cell oxidative stress reaction. The sensitizing and anti-drug resistance effects of chlorogenic acid on angiogenesis inhibitors may be related to the above mechanisms.
And secondly, the chlorogenic acid and the angiogenesis inhibitor are used together, so that the toxic and side effects of the medicine are reduced in many aspects. Chlorogenic acid molecule contains a certain amount of active hydroxyl, can form hydrogen free radical with antioxidant effect, can eliminate activity of free radical such as hydroxyl free radical and superoxide anion, etc., and protect tissue from damage due to oxidation. It is an effective phenolic antioxidant with antioxidant capacity higher than that of caffeic acid, p-hydroxybenzoic acid, ferulic acid, syringic acid, butyl hydroxy-diphenyl ether, tocopherol, etc. In addition, chlorogenic acid has a greater effect on both NF-. gamma.and TNF-. alpha.levels in supernatants of intraepithelial lymphocytes (IEL) as well as intestinal Lamina Propria Lymphocytes (LPL). In vitro studies have shown that chlorogenic acid can induce human lymphocytes and human peripheral blood leukocytes to produce IFN- γ and IFN- α, and these effects may also be associated with the toxic side effects of chlorogenic acid in the present application that can reduce vascular endothelial growth inhibitors.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described in detail with reference to the following embodiments.
Neovascularization is a necessary prerequisite for local invasive growth and distant metastasis of malignant tumors. Tumor cells secrete a variety of growth factors, disrupting the balance between angiogenic and angiostatic factors normally present in the body, increasing the concentration of the angiogenic and/or inhibitory factors, stimulating endothelial cell proliferation, division and capillary formation. The neovascularization can increase blood perfusion, promote tumor growth, inhibit tumor cell apoptosis and make the tumor easy to infiltrate and transfer. Inhibit the formation of tumor neovascularization, can directly block the supply of nutrition and growth factors to tumors, and also provides a new target for tumor treatment.
The recombinant human endostatin (endostar) is a novel medicine which is independently innovated and researched by Chinese scholars and specifically inhibits the proliferation of tumor vascular endothelial cells and the growth of tumors. Phase I and II clinical studies show that the enidol has a certain anti-tumor effect, and phase III and IV clinical studies show that the enidol can improve the curative effect, improve the life quality of patients and prolong the survival time of the patients when the enidol is used for treating the advanced NSCLC in combination with chemotherapy.
Fumagiline is a specific angiogenesis inhibitor. Animal experiments and clinical experiments show that the compound has an inhibiting effect on the growth and metastasis of various tumor tissues. From the results of studies of the mechanism of action of different levels on fumagiline, it is believed that the fumagiline tumor suppressor mechanism is primarily associated with its inhibition of tumor angiogenesis.
The two medicines are easy to generate drug resistance due to limited antitumor effect and insufficient targeting of a single medicine, and are mainly clinically used as auxiliary medicines of a combined treatment scheme, so that the popularization and application of the medicines are influenced to a certain extent.
Secondly, angiogenesis inhibitors can also produce a series of side effects, common adverse reactions include the aspects of a blood system, a digestive system, a cardiovascular system and the like, the adverse reactions in the aspect of the blood system mainly include the reduction of leucocytes and neutrophils, the reduction of platelets and hemoglobin and the like, the clinical treatment is troublesome, and the clinical application of the medicines is also influenced to a certain extent.
In the method, the angiogenesis inhibitor has the problems of insufficient targeting, toxic and side effects and drug resistance, and combines the beneficial effects of chlorogenic acid in a plurality of systems of a human body, so that unexpected technical effects are generated by creatively combining the two.
The invention discloses an anti-tumor combined medicament and application thereof in preparing an anti-cancer medicament, wherein the combined medicament comprises a first preparation and a second preparation, and the anti-tumor combined medicament comprises 10-40 parts of the first preparation, preferably 10-30 parts, more preferably 10-20 parts and 30-50 parts of the second preparation in parts by weight. The invention also discloses application of chlorogenic acid in preparing an anti-cancer drug toxic and side effect inhibitor, wherein the anti-cancer drug is an angiogenesis inhibitor.
The in vitro cell experiment proves that the chlorogenic acid has no toxicity to normal human cells. After the chlorogenic acid, the Endok and the vartanib are used together, the toxicity of the Endok and the vartanib to normal cells of people can be reduced, the toxic and side effects of the medicines are reduced to a certain extent, and the targeting property is improved. Secondly, in the test of the inhibition effect of the angiogenesis inhibitor drug and the chlorogenic acid on the human non-small cell lung cancer HCC827 cell line and the human gastric cancer MNK-45 cell line, the chlorogenic acid is combined with the enrofloxacin and the vartanib to generate the synergistic effect.
In vitro cell experiments also show that chlorogenic acid can reverse the enrichment resistance of HCC827 non-small cell lung cancer, and in vitro animal experiments further confirm the result.
In vitro animal experiments also prove that the chlorogenic acid, vandetanib and fumagiline are combined to be used on an H22 liver cancer mouse and an MFC stomach cancer mouse, so that the effect of synergistic sensitization is achieved on the medicines, and the toxic and side effects of the medicines are reduced.
In conclusion, the technical scheme of the invention can improve the targeting of the angiogenesis inhibitor to non-small cell lung cancer, gastric cancer and liver cancer, reduce the toxic and side effects of the angiogenesis inhibitor and reverse the drug resistance of the angiogenesis inhibitor.
The above is a detailed analysis of the summary of the invention, and the following is an example of the invention.
EXAMPLE 1 in vitro cell assay with combination
1. Experimental equipment
1.1 cell lines
Human non-small cell lung cancer HCC827 cell line, human gastric cancer MNK-45 cell line, H22 mouse hepatoma cell line, human normal lung epithelial cell BEAS-2B, human normal stomach epithelial cell GES-1 and human normal liver cell L-02.
1.2 test drugs
Chlorogenic acid, Endol, Watalanib
2. Test method
2.1 preparation of coated plates
A96-well plate 2 was taken and 40. mu.l of artificial matrix Matrigel (1/3 diluted with serum-free medium) was added thereto, and the mixture was cultured in a 37 ℃ incubator for 30min to solidify it.
2.1.1 culture with addition of drugs
Preparing single cell suspension from human non-small cell lung cancer HCC827 cell line, human gastric cancer MNK-45 cell line, human normal lung epithelial cell BEAS-2B and human normal stomach epithelial cell GES-1 at logarithmic production phase, inoculating 50 μ l (5 × 10) per well in 96-well plate4One/hole).
Dissolving chlorogenic acid, Endonic acid and Watalanib in water, diluting with culture medium to obtain chlorogenic acid solution, Endonic solution, Watalanib solution, culture medium Endonic + chlorogenic acid solution, Watalanib + chlorogenic acid solution, adding 50 μ l of the medicinal solution into each inoculated well, and adding 50 μ l of culture medium diluent into each well of control group; and 3 multiple holes are inoculated in each test group, medicine is added according to groups after the cells adhere to the wall, and standing culture is carried out for 12 hours.
2.1.2 determination
The above test groups were examined under the microscope (x 40) randomly for 5 fields from the top, bottom, left, right, center, counting the number of tubular structures, and averaging each field.
2.2 data processing
(1) Calculating GI (growth inhibition ratio) — (1-number)Drug groupNumber ofControl group)×100％
(2) The formula Q ═ E (a + b)/(Ea + Eb-Ea × Eb) is used to calculate the presence or absence of synergy. Wherein E (a + b) is the inhibition rate of the combination of the two medicines, namely the actual combination effect, Ea and Eb are the inhibition rates of the combination of the two medicines, the denominator (Ea + Eb-Ea multiplied by Eb) is the expected combination effect, and Q is the ratio of the two. When the Q value is 0.85-1.15, the combination effect of the two medicines is additive (+), the Q value is synergistic (++) when the Q value is 1.15-20, the Q value is more than 20 and is obvious synergistic (+++), the Q value is antagonistic when the Q value is 0.05-0.85, and the Q value is less than 0.05 and is obvious antagonism.
3. Test results
3.1 Effect of Individual and Combined drugs on Normal cell lines
TABLE 1 Effect of drug alone on human Normal cells
TABLE 2 Effect of combination on human Normal cells
The experimental results show that when the chlorogenic acid is independently applied to the human normal lung epithelial cell BEAS-2B and the human normal stomach epithelial cell GES-1, the IC50 values are 1383.8ug/ml and 1288.6ug/ml respectively, and the chlorogenic acid has no toxicity to normal cells. After the chlorogenic acid, the Endok and the Watalnib are used together, the IC50 value is higher than the IC50 value when the Endok and the Watalnib are used separately, and the chlorogenic acid is proved to be capable of reducing the toxicity of the Endok and the Watalnib to normal cells of people, reducing the toxic and side effects of the medicines to a certain extent and improving the targeting property.
3.2 Effect of Individual and Combined drugs on tumor cell lines
TABLE 3 Effect of drug alone on tumor cells
TABLE 4 Effect of combination on human NSCLC HCC827 cell line
TABLE 5 Effect of combination on human gastric cancer MNK-45 cell line
The result shows that in the test of the inhibition effect of the combination of the angiogenesis inhibitor and the chlorogenic acid on the HCC827 cell line of the human non-small cell lung cancer, the Q value of the combination of 20 mu g/ml of the enicity and 10-50 mu g/ml of the chlorogenic acid is 1.15-20, and the combination effect of the two medicines is synergistic (+ +); the Q value of the combined medicine of 35 mu g/ml vartanib and 10-50 mu g/ml chlorogenic acid is 1.15-20, and the combined effect of the two medicines is synergistic (+ +). In the experiment of the inhibitory effect of the angiogenesis inhibitor drug and the chlorogenic acid on the MNK-45 cell strain of human gastric cancer, the Q value of the 40 mu g/ml of the combined drug of the chlorogenic acid and 10-50 mu g/ml of the angiogenesis inhibitor drug is 1.15-20, and the combined effect of the two drugs is synergy (+ +); the Q value of the 15 mu g/ml vartanib and 10-50 mu g/ml chlorogenic acid combined drug is 1.15-20, and the combined effect of the two drugs is synergistic (+ +).
Example 2 chlorogenic acid Reversal of Endof resistance in HCC827 non-Small cell Lung cancer in vitro
1 Material
The tested medicines are: chlorogenic acid and degree of enfane
Cell lines: human non-small cell lung carcinoma HCC827 cell line.
2 test method
2.1 cultivation of drug-resistant cell lines
Inducing HCC827 human non-small cell lung cancer cell line by using an saturation concentration gradient increasing method, cloning, screening and establishing, and removing drug before experiment and culturing.
2.2 detecting the IC50 of the Endoth on the cell strain and the drug-resistant strain, and calculating the drug-resistant times
Collecting HCC827 cells and drug-resistant cell strain in logarithmic growth phase, and adjusting cell concentration to 8 × 103The method comprises the steps of inoculating the cells in a 96-well plate, inoculating the cells in a control group, inoculating the cells in the control group, adding the culture medium in the test group, inoculating the cells in the test group, adding different concentrations of saturation working solution in the test group, placing the test group in an incubator, incubating for 48 hours, adding 5mg/ml MTT20 mu l in each well, incubating for 4 hours, sucking the supernatant, adding 150 mu l of DMSO in each well, standing for 30 minutes, completely dissolving crystals, detecting the absorbance value OD (OD) of each well at 570nm by using a microplate reader, calculating the growth inhibition rate of the tumor cells, calculating the inhibition rate (1-OD570 (test group-blank group)/570 (control group-blank OD group)) × 100% and calculating the drug resistance times (drug resistance IC50 value/sensitive cell IC50 value).
2.3MTT method for determining concentration of non-cytotoxic chlorogenic acid
HCC827 cells and drug-resistant cells were cultured and treated as described above. The experiments were divided into 3 groups: blank group, control group and experimental group. The blank group is only added with culture medium without inoculating cells; adding culture medium to the control group and inoculating cells; adding culture medium into the experimental group, inoculating cells, adding chlorogenic acid working solution with different concentrations to enable the final concentrations to be 1, 2, 4, 8, 16, 32, 64 and 128 mu g/mL respectively, placing the experimental group in an incubator for incubation for 48 hours, adding 5mg/mL MTT20 mu l into each hole, continuing the incubation for 4 hours, sucking the supernatant, adding DMSO150 mu l into each hole, standing for 30min, and waiting for the crystals to be completely dissolved. And detecting the light absorption value OD value of each hole at 570nm by using an enzyme-labeling instrument, and calculating the growth inhibition rate of the tumor cells. The inhibition rate is (1-OD570 (experimental group-blank group)/OD 570 (control group-blank group)) × 100%, and the concentration of chlorogenic acid with an inhibition rate of less than 10% is taken as the reversal concentration of the nontoxic dose.
2.4 action of chlorogenic acid to reverse drug-resistant HCC827 cell line
The cell culture and experimental methods were as above. The experimental groups were as follows: drug-resistant HCC827 non-small cell lung cancer group, drug-resistant HCC827 non-small cell lung cancer + chlorogenic acid 10ug/ml group, drug-resistant HCC827 non-small cell lung cancer + chlorogenic acid 20ug/ml group, adding different concentrations of entitlements into the experimental groups respectively, measuring OD value of each well, observing the change of cell killing effect of entitlements on drug-resistant HCC827 non-small cell lung cancer and entitlements on drug-resistant HCC827 non-small cell lung cancer group, calculating IC50 of entitlements on drug-resistant HCC827 non-small cell lung cancer and IC50 after drug-resistant HCC827 non-small cell lung cancer is reversed by 10ug/ml and 20 ug/ml. Fold reversal-IC 50 value before/IC 50 value after reversal.
3 results of the test
3.1 fold of drug resistance
The IC50 of the enrichments for HCC827 non-small cell lung cancer and drug-resistant HCC827 non-small cell lung cancer are 21.8 mu g/ml and 120 mu g/ml respectively. The drug resistance multiple is calculated to be 5.5 according to a formula.
3.2MTT method for determining the concentration of chlorogenic acid without cytotoxicity to drug-resistant cells
TABLE 6 reversion of chlorogenic acid on HCC827 non-small cell lung cancer resistant cells
The test result shows that the IC50 of the enrichments on the drug-resistant cell strain HCC827 non-small cell lung cancer is reduced after the non-cytotoxic concentration (10 mug/ml and 20 mug/ml) of the chlorogenic acid acts on the HCC827 non-small cell lung cancer drug-resistant cell HCC827 non-small cell lung cancer. When 10 mu g/ml chlorogenic acid is used as a reversal agent, the IC50 of the enrofloxacin to the drug-resistant HCC827 non-small cell lung cancer is 44 mu g/ml, and the reversal multiple is 2.73, and when 20 mu g/ml chlorogenic acid is used as the reversal agent, the IC50 of the enrofloxacin to the drug-resistant HCC827 non-small cell lung cancer is 13.4 mu g/ml, and the reversal multiple is 8.96.
Example 3 Combined antitumor animal experiments
1. Test equipment
1.1 animals
Female parent and body weight of Kunming mouse 16-21 g.
1.2 cell lines
H22 mouse hepatoma cell line and MFC mouse gastric cancer cell line.
1.3 test drugs
Chlorogenic acid, vandetanib, fumagiline
2. Test method
Taking cells in logarithmic growth phase, digesting with trypsin until the cells are detached from the wall, adding physiological saline to prepare cell suspension, and mixing the prepared cell suspension according to 0.2ml-1(number of cells about 1 × 106Respectively), inoculating the mixture under the left front axilla of a mouse, randomly grouping according to the weight, and respectively obtaining a chlorogenic acid group, vandetanib, fumagiline, a chlorogenic acid + vandetanib combined medicine group, a chlorogenic acid + fumagiline combined medicine group and a negative group, wherein each group comprises 6 patients, and the administration is carried out on the second day after inoculation.
The chlorogenic acid group is administered by intraperitoneal injection for 15 times once a day; the vandetanib group is administrated by continuous gavage for 15 times once a day; the fumagiline group is administrated by intraperitoneal injection once every other day for 7 times; the combined medicine combination of chlorogenic acid and vandetanib is injected into the abdominal cavity once a day and vandetanib is injected into the stomach once a day, and the administration is continuously carried out for 15 times; the chlorogenic acid and fumagiline combined medicine group is injected intraperitoneally once a day for 15 times continuously, and simultaneously injected intraperitoneally once every other day for fumagiline; the negative group was administered 15 times continuously by intraperitoneal injection of physiological saline once a day. Observing the weight of the animals each day after the administration; the tumor volume of the group to be negative is about 0.5cm3Stopping the experiment, removing eyeball, collecting blood, measuring WBC, HBC and HGB content, removing cervical vertebra, killing mouse, and weighingThe tumors were stripped and weighed.
3. Data processing
(1) Tumor inhibition rate [% 1- (mean tumor weight in administration group/mean tumor weight in negative group) ] × 100%;
(2) the two medicines are combined together, Q is E (a + b)/(Ea + Eb-Ea multiplied by Eb), wherein E (a + b) is the inhibition rate of the two medicines when used together, namely the actually measured combination effect, Ea and Eb are the inhibition rates of the two medicines when used together, the denominator (Ea + Eb-Ea multiplied by Eb) is the expected combination effect, and Q is the ratio of the two. When the Q value is 0.85-1.15, the combination effect of the two medicines is additive (+), the Q value is synergistic (++) when the Q value is 1.15-20, the Q value is more than 20 and is obvious synergistic (+++), the Q value is antagonistic when the Q value is 0.05-0.85, and the Q value is less than 0.05 and is obvious antagonism.
4. Test results
4.1 Effect of combination on tumor inhibition Rate
TABLE 7 Effect of combination on the tumor suppression Rate of transplanted tumors in HCC827 non-Small cell Lung cancer mice
TABLE 8 Effect of combination on the tumor suppression rate of MFC gastric cancer mouse transplants
The results show that in the tumor inhibition rate test of the combined drug on the transplanted tumor of the HCC827 non-small cell lung cancer mouse, the combination of the chlorogenic acid and the vandetanib, the chlorogenic acid and the fumagillin has better tumor inhibition rate, and has significant difference compared with the single use. The Q value calculation result of the combined medication indicates that the combination of the chlorogenic acid and the angiogenesis inhibitor has synergistic effect on the tumor inhibition effect of the HCC827 non-small cell lung cancer mouse transplanted tumor.
In the tumor inhibition rate test of the combined drug for the transplanted tumor of the MFC gastric cancer mouse, the combination of the chlorogenic acid and the vandetanib and the combination of the chlorogenic acid and the fumagiline has better tumor inhibition rate, and has significant difference compared with the single use. The calculation result of the Q value of the combined drug shows that the combined use of the chlorogenic acid and the angiogenesis inhibitor has synergistic effect on the tumor inhibition effect of the MFC gastric cancer mouse transplanted tumor.
4.2 Effect of combination on mouse blood routine
TABLE 9 Effect of combination on HCC827 non-small cell Lung cancer mice transplantation tumor mice blood routine
P < 0.05, p < 0.01 compared to negative group; compared with the positive group, delta p is less than 0.05, and delta p is less than 0.01
TABLE 10 Effect of combination on MFC gastric carcinoma mouse transplantation tumor mouse blood conventions
P < 0.05, p < 0.01 compared to negative group; compared with the positive group, delta p is less than 0.05, and delta p is less than 0.01
The results show that: chlorogenic acid (20mg/kg) can remarkably improve the reduction of WBC, RBC and HGB in the post-hemorrhage routine of vandetanib and fumagiline in an HCC827 non-small cell lung cancer mouse transplantation tumor mouse; meanwhile, the reduction phenomena of WBC, RBC and HGB in the conventional blood of a mouse with the MFC gastric cancer transplantation tumor, namely the mouse with the post-use vandetanib and the fumagiline can be obviously improved; the chlorogenic acid can relieve the bone marrow suppression effect generated by the angiogenesis inhibitor, the increase of the platelet count can reduce the occurrence of hemorrhagic adverse reactions, and the increase of the hemoglobin can prevent anemia and anemia-related adverse reactions, so that the chlorogenic acid can reduce the toxic and side effects of the angiogenesis inhibitor from many aspects.
Example 4 chlorogenic acid in animal experiments to combat multidrug resistance
1 materials and methods
The tested medicines are: chlorogenic acid, Endok, Framazon
Test cell lines: drug-resistant HCC827 non-small cell lung cancer cell line is induced by using an saturation gradient increasing method on the HCC827 non-small cell lung cancer cell line, constructed by cloning, screening and cultured without drugs before an experiment. The MNK-45 cell strain for human stomach cancer is induced by the gradient increasing method of fumagillin concentration to MNK-45 stomach cancer cell line, and is cloned, screened and cultured before experiment.
The test animals were: BABL/C-nu mice are female and 18-22 g in weight;
2 test method
2.1 establishment of tumor model of Experimental animal
Adjusting the cell concentration of the drug-resistant cell strain to 1 × 10 with culture solution7M1, injected subcutaneously in the right axilla of mice to 1 × 1070.1ml of cells/m 1.
2.2 methods of administration
The average diameter of the tumor to be detected reaches 100mm3The mice are respectively an entitle continuous medicine group, a chlorogenic acid + entitle group, a chlorogenic acid group and a negative group.
The drug-resistant MNK-45 gastric cancer cell transplantation tumor mice are respectively a fumagillin sustained use medicine group, a chlorogenic acid + fumagillin group, a chlorogenic acid group and a negative group.
The group of the medicine for continuous use of the medicine is as follows: the preparation is administered by intraperitoneal injection once a day at a dose of 50mg/kg for 15 days.
Chlorogenic acid + degree group: firstly, injecting chlorogenic acid into abdominal cavity once a day at a dose of 20mg/kg, and continuously administering for 5 days; after the administration of chlorogenic acid is stopped, intraperitoneal injection is started the next day, once a day, 50mg/kg is administered continuously for 10 days.
The group of continuous use drugs of fumagiline: the preparation is administered by intraperitoneal injection, once every other day, 30mg/kg, and 7 times.
Chlorogenic acid + fumagoxin group: firstly, injecting chlorogenic acid into abdominal cavity once a day at a dose of 20mg/kg, and continuously administering for 5 days; after the administration of chlorogenic acid was stopped, the next day, i.e., the next day, was administered with fumagiline, 30mg/kg, 7 times every other day.
Chlorogenic acid group: the preparation is administered by intraperitoneal injection once a day at a dose of 20mg/kg for 5 days, and then is administered by gavage once a day for 10 days.
Negative group: the normal saline is injected into the abdominal cavity once a day for 5 days continuously, and the normal saline is injected into the abdominal cavity once a day for 10 days continuously.
2.3 evaluation of antitumor Effect
After the administration, the experiment was stopped, the mice were sacrificed by removing the cervical vertebrae and weighed, the tumors were stripped and weighed, and the tumor inhibition rate was calculated.
2.4 data processing
Tumor inhibition rate [% 1- (mean tumor weight in administration group/mean tumor weight in negative group) ] × 100%;
3 results of the test
3.1 Effect of Each test group on the tumor inhibition Rate of drug-resistant transplantable tumors
TABLE 11 influence of the groups on the tumor weight and tumor suppression rate of drug-resistant HCC827 non-small cell lung cancer transplantable tumors
P < 0.05, p < 0.01 compared to negative group; compared with the degree of en, delta p is less than 0.05 and delta p is less than 0.01
The results show that the chlorogenic acid group and the enidegree group have small tumor inhibition rate on drug-resistant HCC827 non-small cell lung cancer transplantable tumors and have no obvious tumor inhibition effect, while the chlorogenic acid + the enidegree group has obvious tumor inhibition rate on the drug-resistant HCC827 non-small cell lung cancer transplantable tumors, which indicates that the chlorogenic acid can effectively solve the drug resistance of the HCC827 non-small cell lung cancer caused by the enidegree.
TABLE 12 influence of the test groups on the tumor weight and tumor inhibition rate of drug-resistant MNK-45 gastric cancer transplantable tumor
P < 0.05, p < 0.01 compared to negative group; Δ p < 0.05 and Δ p < 0.01 in comparison with fumagiline
The results show that the tumor inhibition rate of the chlorogenic acid group and the fumagillin group to the drug-resistant MNK-45 gastric cancer transplantation tumor is low, no obvious tumor inhibition effect is realized, the tumor inhibition rate of the chlorogenic acid + fumagillin group to the drug-resistant MNK-45 gastric cancer transplantation tumor is obvious, and the chlorogenic acid can effectively solve the MNK-45 gastric cancer drug resistance caused by fumagillin.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (6)
1. An anti-tumor combined medicine is characterized by comprising a combined medicine of 20 mu g/ml of enrofloxacin and 10-50 mu g/ml chlorogenic acid, or a combined medicine of 40 mu g/ml enrofloxacin and 10-50 mu g/ml chlorogenic acid.
2. An anti-tumor combined medicine is characterized by comprising a drug combination of 15 mu g/ml vartanib and 10-50 mu g/ml chlorogenic acid, or a drug combination of 35 mu g/ml vartanib and 10-50 mu g/ml chlorogenic acid.
3. An anti-tumor combined medicine is characterized by comprising 50mg/kg of vandetanib and 20mg/kg of chlorogenic acid combined medicine, 30mg/kg of fumagillin and 20mg/kg of chlorogenic acid combined medicine, or 50mg/kg of enrofloxacin and 20mg/kg of chlorogenic acid combined medicine.
4. The combination according to any one of claims 1 to 3, wherein the dosage form of the enrobent, vartanib, vandetanib, fumagiline, chlorogenic acid in the combination is an injection or an oral preparation.
5. Use of a combination according to any one of claims 1 to 3 in the manufacture of an anti-cancer medicament.
6. The use of claim 5, wherein the cancer comprises non-small cell lung cancer, gastric cancer, liver cancer.
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