Patent Grant
12178792
The present invention belongs to the field of biomedicine, and in particular to a pharmaceutical composition and its use in the preparation of drugs for treatment of tumor multidrug resistance.
Cancer is a major public health problem needed to be resolved in China and even in all parts of the world. On average, millions of people are diagnosed with cancer every year, including more than 200 types of cancer. At present, cancer is one of the main diseases causing death. However, due to the poor selectivity of anti-cancer drugs, single drug treatment has the defects of high toxicity or poor treatment effect, so the research on new generation of low toxicity and high-efficiency drugs has become the top priority of malignant tumor treatment.
The chemical name of dacarbazine is 5-(3,3-dimethyl-1-triazenyl)-4-carboxamideimidazolium citrate. Decarbazine can decompose in vivo and release methyl cation (CH3)+, which plays an alkylation role; meanwhile, it can become a substance similar to the intermediate product of purine biosynthesis, and thus may interfere with purine biosynthesis.
Gemcitabine is a new cytosine nucleoside derivative. Like cytarabine, it is activated by deoxycytidine kinase and metabolized by cytidine deaminase. Gemcitabine is a pyrimidine anticancer drug with the same mechanism as cytarabine. Its main metabolite is incorporated into DNA in cells, and mainly acts on G1/S phase. In clinical practice, gemcitabine is effective for a variety of solid tumors.
OPDIVO® is a human programmed death receptor-1 (PD-1) that is suitable for blocking antibody therapy in following patients: (1) patients with unresectable and metastatic melanoma and ipilimumab, and if BRAF V600 mutation is positive, after administration of BRAF inhibitor, patients with disease progression. (2) After platinum-based chemotherapy, patients with advanced squamous non-small cell lung cancer.
Although dacarbazine, gemcitabine and OPDIVO® can be used to treat cancer, with the increase of medication time, drug resistance will also appear. Once drug resistance occurs, the role of drugs will obviously decline. Nowadays, drug resistance has become a major problem in the medical community. Therefore, it is urgent to develop new drugs to effectively reverse drug resistance.
Content of the Invention
In order to solve above problems, the present invention provides a pharmaceutical composition and its use in the preparation of a drug for treatment of tumor multidrug resistance.
The present invention provides a pharmaceutical composition, that contains chlorogenic acid and coumaroylquinic acid.
Further, the mass ratio of chlorogenic acid to coumaroylquinic acid is 100:0.01˜0.5.
Preferably, the mass ratio of chlorogenic acid to coumaroylquinic acid is 100:0.01˜0.1.
More preferably, the mass ratio of chlorogenic acid to coumaroylquinic acid is 100:0.05.
The present invention provides a method for preparing the pharmaceutical composition, in which chlorogenic acid and coumaroylquinic acid are used as active components, with the addition of pharmaceutically acceptable excipients, and so a commonly used pharmaceutical preparation is prepared.
Preferably, the preparation is an oral or injectable preparation.
The present invention provides the use of the pharmaceutical composition in the preparation of reversal agent of tumor multidrug resistance.
Wherein, said tumor is melanoma, lung cancer, liver cancer, kidney cancer, glioma, prostate cancer, gastric cancer, bladder cancer, colon cancer, breast cancer, ovarian cancer or cervical cancer.
Preferably, said tumor is melanoma or lung cancer.
Wherein, said pharmaceutical composition is used in combination with the antitumor drug, to prepare a reversal agent of tumor multidrug resistance.
Preferably, said antitumor drug is a chemotherapeutic drug or an immunotherapeutic drug.
More preferably, said chemotherapeutic drug is an imidazole or pyrimidine antitumor drug, preferably dacarbazine or gemcitabine.
Said immunotherapeutic drug is a PD-1 inhibitor, preferably OPDIVO®.
The present invention provides the use of the pharmaceutical composition in the preparation of PD-1/PD-L1 inhibitor.
Wherein, said PD-1/PD-L1 inhibitor is an anti-tumor drug.
Preferably, said anti-tumor drug is a drug against drug-resistant tumor.
More preferably, said drug against drug-resistant tumor is an anti-tumor immunosuppressant.
Further more preferably, said anti-tumor immunosuppressant is a PD-1 inhibitor except for the pharmaceutical composition of the present invention.
Further, except for the pharmaceutical composition of the present invention, said PD-1 inhibitor is OPDIVO®.
The present invention provides a drug combination, that comprises a pharmaceutical composition and an anti-tumor drug administered separately, as well as a pharmaceutically acceptable carrier; said pharmaceutical composition is the one containing chlorogenic acid and coumaroylquinic acid.
Wherein, said antitumor drug is a chemotherapeutic drug or an immunotherapeutic drug.
Preferably, said chemotherapeutic drug is an imidazole or pyrimidine antitumor drug, preferably dacarbazine or gemcitabine.
Said immunotherapeutic drug is a PD-1 inhibitor, preferably OPDIVO®.
Wherein, said tumor is melanoma, lung cancer, liver cancer, kidney cancer, glioma, prostate cancer, gastric cancer, bladder cancer, colon cancer, breast cancer, ovarian cancer or cervical cancer.
Preferably, said tumor is melanoma or lung cancer.
Wherein, the mass ratio of said pharmaceutical composition and the antitumor drug is 1:1.
The mass ratio of chlorogenic acid to coumaroylquinic acid is 100:0.01˜0.5.
Preferably, the mass ratio of chlorogenic acid to coumaroylquinic acid is 100:0.01˜0.1.
More preferably, the mass ratio of chlorogenic acid to coumaroylquinic acid is 100:0.05.
The present invention provides a pharmaceutical composition comprising chlorogenic acid and coumaroylquinic acid, which can be used to prepare reversal agents of tumor multidrug resistance and PD-1/PD-L1 inhibitors. The experimental results of the present invention indicate that the combined use of chlorogenic acid and coumaroylquinic acid can play a synergistic effect. In particular, the combined use of chlorogenic acid and coumaroylquinic acid has a good reversal effect on tumor cell lines with multi-drug resistance to chemotherapeutic drugs and immunotherapeutic drugs, and can effectively solve the drug resistance of melanoma cell line B16 caused by dacarbazine or Lewis lung cancer induced by gemcitabine, and can effectively inhibit the expression of PD-1/PD-L1 in the transplanted tumor tissues of B16 melanoma and Lewis lung cancer in mice, and can effectively reverse the drug resistance of B16 melanoma cell lines and Lewis lung cancer cell lines caused by OPDIVO®, as well as can effectively act on the proliferation of CD4+T and CD8+T cells in B16 melanoma mice and Lewis lung cancer mice having drug-resistance.
The pharmaceutical composition of the present invention can be used to prepare PD-1/PD-L1 inhibitors, and both of them can play a synergistic effect to reverse the drug resistance.
Obviously, based on above content of the present invention, according to the common technical knowledge and the conventional means in the field, without department from above basic technical spirits, other various modifications, alternations or changes can further be made.
By following specific examples of said embodiments, above content of the present invention is further illustrated. But it should not be construed that the scope of above subject of the present invention is limited to following examples. The techniques realized based on above content of the present invention are all within the scope of the present invention.
The raw materials and equipment used in the specific embodiment of the present invention are all known products, which can be obtained by purchasing those commercially available.
1. Formula 1
1000 g chlorogenic acid, 1 g coumaroylquinic acid.
Preparative method: chlorogenic acid and coumaroylquinic acid were aseptically weighed according to the formula, thoroughly mixed, and aseptically dispensed as pulvis.
2. Formula 2
1000 g chlorogenic acid, 1 g coumaroylquinic acid, 500 g bulking agent, and 5 g binding agent.
Preparative method: chlorogenic acid, coumaroylquinic acid, bulking agent, and binding agent were aseptically weighed according to the formula, granulated, broke, and packaged as granules.
3. Formula 3
1000 g chlorogenic acid, 1 g coumaroylquinic acid, 500 g bulking agent, 5 g binding agent, and 3 g lubricant.
Preparative method: chlorogenic acid, coumaroylquinic acid, bulking agent, and binding agent were weighed according to the formula, granulated, and broke, then lubricant was added. The mixture was pressed to provide tablets.
Above-mentioned bulking agent is one or more of mannitol, lactose, starch, microcrystalline cellulose and dextrin; the binding agent is sodium carboxymethyl cellulose, PVP; the lubricant is magnesium stearate, talc, micronized silica gel.
1. Formula 1
1000 g chlorogenic acid, 1 g coumaroylquinic acid.
Preparative method (1): chlorogenic acid and coumaroylquinic acid were aseptically weighed according to the formula, thoroughly mixed, and aseptically packaged as powder injection.
Preparative method (2): chlorogenic acid and coumaroylquinic acid were weighed according to the formula, dissolved in injectable water, filtered and sterilized, then freeze-dried to obtain freeze-dried powder injection.
2. Formula 2
1000 g chlorogenic acid, 1 g coumaroylquinic acid, 2667 g scaffolding agent, 67 g antioxidant. Preparative method: chlorogenic acid, coumaroylquinic acid, scaffolding agent, and antioxidant were weighed according to the formula, dissolved in injectable water, filtered and sterilized, then freeze-dried to obtain freeze-dried powder injection.
The above-mentioned scaffolding agent are mannitol, lactose, and glucose; the antioxidants are sodium bisulfite, vitamin C, glutathione, and folic acid.
The following examples are used to elucidate the beneficial effects of the present invention.
1. Materials
1.1 Test drugs
A375 melanoma cell lines are those routinely cultured in the laboratory. Cell lines are passaged before use, and the cells that are in a good growth state and grow in the logarithmic phase are selected for use.
2. Experimental Method
2.1 Cultivation of Drug-Resistant Cell Lines
A375 melanoma cell lines were exposed to dacarbazine (400 μg/ml) for 3 months, and centrifuged, to culture dacarbazine-resistant A375 cells. The cells were observed every day, and taken through 3 passages for about 3 days, to ensure cell viability.
2.2 Determining the IC50 Values of Dacarbazine Against Cell Lines and Drug-Resistant Strains, and Calculating the Drug-Resistant Index
Above-mentioned cell lines and drug-resistant cell lines in the logarithmic growth phase were selected, the cell concentration was adjusted to 8×103 cells/well, and then cells were seeded in a 96-well plate. For the experiment, there were three groups: blank group, control group and dacarbazine group. In the blank group, only medium was added, without inoculating cells; in the control group, medium was added, and cells were inoculated; in the dacarbazine group, medium was added, cells were inoculated, and then dacarbazine was added at different concentrations. The plates were placed in an incubator and incubated for 48 h, then 5 mg/ml MTT (20 μl) was added to each well, and the plate was further incubated for 4 h. The upper liquid was removed, and 150 μl DMSO was added to each well, then the plate was allowed to stand for 30 min until the crystals are completely dissolved. the OD value of each well was measured at 570 nm with a microplate reader to calculate the inhibition rate of tumor cell growth.
The inhibition rate=(1-OD value(dacarbazine group-blank group)/OD value(control group−blank group))×100%.
Drug-resistant index=IC50 value of drug-resistant cells/IC50 value of sensitive cells
2.3 Determining the Concentration of Non-Cytotoxic Composition and its Single Compound by MTT Method
The culture and treatment methods of cells and drug-resistant cells were the same as above. For the experiment, there were eight groups: blank group, control group and test-drug groups. In the blank group, only medium was added, without inoculating cells; in the control group, medium was added, and cells were inoculated; in the test-drug groups, except for medium and cells, work solutions of test drugs were added at different concentrations, to make a final concentration of 1, 2, 4, 8, 16, 32, 64, and 128 μg/mL. The plates were placed in an incubator and incubated for 48 h, then 5 mg/ml MTT (20 μl) was added to each well, and the plate was further incubated for 4 h. The upper liquid was removed, and 150 μl DMSO was added to each well, then the plate was allowed to stand for 30 min until the crystals are completely dissolved. the OD value of each well was measured at 570 nm with a microplate reader to calculate the inhibition rate of tumor cell growth.
The inhibition rate=(1−OD value(test-drug group−blank group)/OD value(control group−blank group))×100%,
The concentration of each test drug with an inhibition rate of below 10% was regarded as the reversal concentration of the non-toxic dose.
2.4 the Effect of Chlorogenic Acid on Reversing Drug-Resistant Cell Lines
The culture and experimental methods of cells were the same as above.
In the experiment, there are following groups: drug-resistant cell negative group, drug-resistant cells+test drug group 1 (30 μg/ml), drug-resistant cells+test drug group 2 (30 μg/ml), drug-resistant cells+test drug group 3 (30 μg/ml), drug-resistant cells+test drug group 4 (30 μg/ml), drug-resistant cells+test drug group 5 (30 μg/ml), drug-resistant cells+test drug group 6 (30 μg/ml).
Each test drug group was added with different concentrations of dacarbazine, each concentration including 3 replicate wells, and the OD value of each well was measured, to observe whether the cytotoxicity was shown. Each test drug and dacarbazine were respectively used to act on drug resistant cells, so as to compare with the effects of dacarbazine administrated alone on drug-resistant cells, and the IC50 value of dacarbazine against drug-resistant cell lines was calculated, as well as the IC50 value of each test drug after reversing drug-resistant cell lines.
Reversal index=IC50 value before reversal/IC50 value after reversal.
2.5 Experimental Results
2.5.1 the Drug-Resistant Index of Dacarbazine and the Non-Cytotoxic Concentration of Each Test Drug for Drug-Resistant A375 Melanoma Cell Lines
According to above table, the IC50 value of dacarbazine against the drug-resistant A375 melanoma cell lines was 560 μg/ml, and the drug-resistant index calculated according to the formula was 6.22; for each test drug, the concentration without significant cytotoxicity (all inhibition rates <10%) was about less than 800 μg/ml.
2.5.2 Reversal Effect of Each Test Drug on Drug-Resistant A375 Melanoma Cell Lines.
After the non-cytotoxic concentration (30 μg/ml) of each test drug group acted on the drug-resistant A375 melanoma cell lines, the IC50 value of dacarbazine against the drug-resistant A375 cell lines was very different. Among them, the test drug groups of both chlorogenic acid and coumaroylquinic acid administrated alone had no significant reversal effect on the drug-resistant A375 cell lines, while the test drug groups of the combination of chlorogenic acid and coumaroylquinic acid showed significant reversal effects on drug-resistant A375 cell lines. Both of them had a synergistic effect, and when the ratio of two drugs was 100:0.01˜100:0.5, a synergistic effect was observed. With the increase of the proportion of coumaroylquinic acid in the composition, the reversal effect of the composition on the drug-resistant A375 cell lines presented a trend of first increase and then decrease, and thus the best ratio was 100:0.01˜100:0.1.
1. Materials
1.1 Test Drugs
A549 lung cancer cell lines are those routinely cultured in the laboratory. Cell lines are passaged before use, and the cells that are in a good growth state and grow in the logarithmic phase are selected for use.
2. Experimental Method
2.1 Cultivation of Drug-Resistant Cell Lines
A549 lung cancer cell lines were exposed to gemcitabine (2000 μg/ml) for 3 months, and centrifuged, to culture gemcitabine-resistant A549 cells. The cells were observed every day, and taken through 3 passages for about 3 days, to ensure cell viability.
2.2 Determining the IC50 Values of Gemcitabine Against Cell Lines and Drug-Resistant Strains, and Calculating the Drug-Resistant Index
Above-mentioned cell lines and drug-resistant cell lines in the logarithmic growth phase were selected, the cell concentration was adjusted to 8×103 cells/well, and then cells were seeded in a 96-well plate. For the experiment, there were three groups: blank group, control group and gemcitabine group. In the blank group, only medium was added, without inoculating cells; in the control group, medium was added, and cells were inoculated; in the gemcitabine group, medium was added, cells were inoculated, and then gemcitabine was added at different concentrations. The plates were placed in an incubator and incubated for 48 h, then 5 mg/ml MTT (20 μl) was added to each well, and the plate was further incubated for 4 h. The upper liquid was removed, and 150 μl DMSO was added to each well, then the plate was allowed to stand for 30 min until the crystals are completely dissolved. the OD value of each well was measured at 570 nm with a microplate reader to calculate the inhibition rate of tumor cell growth. The inhibition rate=(1−OD value (gemcitabine group−blank group)/OD value (control group−blank group))×100%. Drug-resistant index=IC50 value of drug-resistant cells/IC50 value of sensitive cells
2.3 Determining the Concentration of Non-Cytotoxic Composition and its Single Compound by MTT Method
The culture and treatment methods of cells and drug-resistant cells were the same as above. For the experiment, there were eight groups: blank group, control group and test-drug groups. In the blank group, only medium was added, without inoculating cells; in the control group, medium was added, and cells were inoculated; in the test-drug groups, except for medium and cells, work solutions of test drugs were added at different concentrations, to make a final concentration of 10, 20, 40, 80, 160, 320, 640, and 1280 μg/mL. The plates were placed in an incubator and incubated for 48 h, then 5 mg/ml MTT (20 μl) was added to each well, and the plate was further incubated for 4 h. The upper liquid was removed, and 150 μl DMSO was added to each well, then the plate was allowed to stand for 30 min until the crystals are completely dissolved. the OD value of each well was measured at 570 nm with a microplate reader to calculate the inhibition rate of tumor cell growth. The inhibition rate=(1-OD value (test drug group-blank group)/OD value (control group-blank group))×100%. The concentration of each test drug with an inhibition rate of below 10% was regarded as the reversal concentration of the non-toxic dose.
2.4 the Effect of Chlorogenic Acid on Reversing Drug-Resistant Cell Lines
The culture and experimental methods of cells were the same as above. In the experiment, there are following groups: drug-resistant cell negative group, drug-resistant cells+test drug group 1 (30 μg/ml), drug-resistant cells+test drug group 2 (30 μg/ml), drug-resistant cells+test drug group 3 (30 μg/ml), drug-resistant cells+test drug group 4 (30 μg/ml), drug-resistant cells+test drug group 5 (30 μg/ml), drug-resistant cells+test drug group 6 (30 μg/ml). Each test drug group was respectively added with different concentrations of gemcitabine, each concentration including 3 replicate wells, and the OD value of each well was measured, to observe whether the cytotoxicity was shown. Each test drug and gemcitabine were respectively used to act on drug resistant cells, so as to compare with the effects of gemcitabine administrated alone on drug-resistant cells, and the IC50 value of gemcitabine against drug-resistant cell lines was calculated, as well as the IC50 value of each test drug after reversing drug-resistant cell lines.
Reversal index=IC50 value before reversal/IC50 value after reversal.
2.5 Experimental Results
2.5.1 the Drug-Resistant Index of Gemcitabine and the Non-Cytotoxic Concentration of Each Test Drug for Drug-Resistant A549 Lung Cancer Cell Lines
According to above table, the IC50 value of gemcitabine against the drug-resistant A549 lung cancer cell lines was 3620 μg/ml, and the drug-resistant index calculated according to the formula was 4.63; for each test drug, the concentration without significant cytotoxicity (all inhibition rates <10%) was about less than 1000 g/ml.
2.5.2 Reversal Effect of Each Test Drug on Drug-Resistant A549 Lung Cancer Cell Lines.
After the non-cytotoxic concentration (30 μg/ml) of each test drug group acted on the drug-resistant A549 lung cancer cell lines, the IC50 value of gemcitabine against the drug-resistant A549 cell lines was very different. Among them, the test drug groups of both chlorogenic acid and coumaroylquinic acid administrated alone had no significant reversal effect on the drug-resistant A549 cell lines, while the test drug groups of the combination of chlorogenic acid and coumaroylquinic acid showed significant reversal effects on drug-resistant A549 cell lines; and when the ratio of two drugs was 100:0.01˜100:0.5, both of them reached a synergistic effect. With the increase of the proportion of coumaroylquinic acid in the composition, the reversal effect of the composition on the drug-resistant A549 cell lines presented a trend of first increase and then decrease, and thus the best ratio was 100:0.01˜100:0.1.
Summary: according to the experimental results of Examples 1 and 2 above, the combination of chlorogenic acid and coumaroylquinic acid has a good effect on reversing drug resistance of tumor cell lines with chemical drug resistance. Chlorogenic acid and coumaroylquinic acid have synergistic effect, and the combination of chlorogenic acid: coumaroylquinic acid with a ratio of 100:0.01˜100:0.1 shows the most significant effect.
1 Experimental Materials
1.1 Test Drugs
B16 melanoma cell lines were induced by the increased gradient concentration of dacarbazine, and established by clone screening and cultured without drug before the experiment. Lewis lung cancer cell lines were induced by the increased gradient concentration of gemcitabine, and established by clone screening and cultured without drug before the experiment.
1.3 Test Animals
After the drug was removed from the drug-resistant cell lines, the cell concentration was adjusted to 1×107/ml with the culture medium. 1×107/ml of cells were subcutaneously injected into the right armpit of mice, 0.1 ml per mouse.
2.2 Administration Method
After the average diameter of tumor reached 100 mm, the mice were randomly divided into test drug group 1, test drug group 2, test drug group 3, test drug group 4, test drug group 5, test drug group 6, positive drug group, and negative group, respectively.
Test drug group: the test drug group was first intraperitoneally injected, once a day, 30 mg/kg/time, for 5 consecutive days; administration was stopped, and the positive drug was given by intraperitoneal injection on the next day; wherein the positive drug dacarbazine was administrated once every other day, 60 mg/kg/time; wherein the positive drug gemcitabine was given once every other day, 300 mg/kg/time.
Positive drug group: positive drug dacarbazine was given once every other day, 60 mg/kg/time; positive drug gemcitabine was given once every other day, 300 mg/kg/time.
Negative group: normal saline was intraperitoneally injected, once a day, for 15 consecutive days.
2.3 Evaluation of Antitumor Effect
After completion of administration, the experiment was stopped, and the mice were killed by cervical dislocation and weighed. The tumor was stripped and weighed, to calculate the tumor inhibition rate.
Tumor inhibition rate %=[1−(the average tumor weight in the drug group/the average tumor weight in the negative group)]×100%.
2.4 Expression of PD-1/PD-L1
The positive expression rate of PD-1/PD-L1 in tumor tissues was detected by immunohistochemical SP method.
2.5 Determination of the Number of CD4+T and CD8+T Lymphocytes
The number of CD4+T and CD8+T lymphocytes was analyzed by immunofluorescence staining, and the average number of CD4+T and CD8+T cells infiltrated in 6 high power fields was counted.
3 Experimental Results
3.1 Effect of Each Experimental Group on the Inhibition Rate of Drug-Resistant Transplanted Tumor
From Table 5 and
From Table 6 and
The results showed that the combination of chlorogenic acid and coumaroylquinic acid could effectively inhibit the expression of PD-1/PD-L1 in the transplanted tumor tissues of drug-resistant B16 melanoma and Lewis lung cancer in mice, and both of them had synergistic effect. Among them, the composition of chlorogenic acid: coumarinic acid in the ratio of 100:0.01˜100:0.05 was the best.
3.3 the Number of CD4+T and CD8+ T Cells in Drug-Resistant Transplanted Tumor of Each Experimental Group
The results showed that by comparing the experimental group of the combination of chlorogenic acid and coumaroylquinic acid with the positive drug group (dacarbazine and gemcitabine), the number of CD4+T and CD8+ T cells was significantly increased, indicating that the combination of chlorogenic acid and coumaroylquinic acid can effectively promote the proliferation of CD4+T and CD8+ T cells in mice with drug-resistant B16 melanoma and Lewis lung cancer, and chlorogenic acid and coumaroylquinic acid have synergistic effect.
1 Experimental Materials
1.1 Test Drugs
B16 melanoma cell lines; Lewis lung cancer cell lines.
1.3 Test Animals
The concentration of cell lines was adjusted to 1×107/ml with the culture medium. 1×107/ml of cells were subcutaneously injected into the right armpit of mice, 0.1 ml per mouse. On the second day after inoculation, the model group was intraperitoneally injected with OPDIVO®, 30 mg/kg, once every other day; in the blank group, the same amount of saline was intraperitoneally injected on the second day after inoculation, once every other day; the tumor volume was measured before each administration, and the administration of model group was stopped after the tumor volume exponentially increased and there was no significant difference from the tumor volume of blank group.
2.2 Administration Method
The model mice were randomly divided into groups, eight for one group, and included test drug group 1, test drug group 2, test drug group 3, test drug group 4, positive drug OPDIVO® group, single drug chlorogenic acid group, test drug 1+OPDIVO® group, test drug 2+OPDIVO® group, test drug 3+OPDIVO® group, test drug 4+OPDIVO® group, and negative group, respectively.
Test drug group: from the second day after stopping administration to the model, the group received drug by intraperitoneal injection, once a day, 30 mg/kg.
Single drug group: from the second day after stopping administration to the model, the group received drug by intraperitoneal injection, once a day, 30 mg/kg.
Test drug+OPDIVO® group: from the second day after stopping administration to the model, the group received the test drug by intraperitoneal injection, once a day, and the test drug was stopped after administrating for 5 consecutive days; on the next day, OPDIVO® was intraperitoneally injected, once every other day, with the dosage of 30 mg/kg.
Positive drug group: once every other day, 30 mg/kg.
Negative group: normal saline was intraperitoneally injected, once a day, the same amount of saline.
Before each administration, the tumor volume was measured, and the experiment was stopped when the average tumor volume of negative group was greater than 1.5 cm3.
2.3 Evaluation of Antitumor Effect
After completion of administration, the experiment was stopped, and the mice were killed by cervical dislocation and weighed. The tumor was stripped and weighed, to calculate the tumor inhibition rate.
Tumor inhibition rate %=[1−(the average tumor weight in the drug group/the average tumor weight in the negative group)]×100%.
2.4 Expression of PD-1/PD-L1
The positive expression rate of PD-1/PD-L1 in tumor tissues was detected by immunohistochemical SP method.
2.5 Determination of the Number of CD4+T and CD8+T Lymphocytes
The number of CD4+T and CD8+T lymphocytes was analyzed by immunofluorescence staining, and the average number of CD4+T and CD8+T cells infiltrated in 6 high power fields was counted.
3 Experimental Results
3.1 Tumor Volume of Drug-Resistant Model Group and Blank Group
From Tables 11 and 12 as well as
3.2 Effect of Each Experimental Group on Tumor Volume of Drug-Resistant Model Group
Experimental results showed that the continuous treatment effect of positive drug OPDIVO® on drug-resistant B16 melanoma cell line mice was not obvious, and the effect of single drug chlorogenic acid on tumor volume control was not obvious; the tumor volume in the combination group of chlorogenic acid and coumaroylquinic acid was different from that of positive drug OPDIVO® group and negative group (P<0.05), indicating that the control effect on tumor volume was obvious. In the experimental group of the combination of chlorogenic acid and coumaroylquinic acid+OPDIVO®, the effect of continuous treatment on drug-resistant mice is very significant, and compared with the positive drug OPDIVO® group and the negative group, there was an extremely significant difference (P<0.01), indicating that the combination of chlorogenic acid and coumaroylquinic acid could effectively reverse the drug resistance of B16 melanoma mice induced by OPDIVO®, so that OPDIVO® could continue to play an anti-tumor effect.
Experimental results showed that the continuous treatment effect of positive drug OPDIVO® on drug-resistant Lewis lung cancer mice was not obvious, and the effect of single drug chlorogenic acid on tumor volume control was not obvious; the tumor volume in the combination group of chlorogenic acid and coumaroylquinic acid was different from that of positive drug OPDIVO® group and negative group (P<0.05), indicating that the control effect on tumor volume was obvious. In the experimental group of the combination of chlorogenic acid and coumaroylquinic acid+OPDIVO®, the effect of continuous treatment on drug-resistant mice is very significant, and compared with the positive drug OPDIVO® group and the negative group, there was an extremely significant difference (P<0.01), indicating that the combination of chlorogenic acid and coumaroylquinic acid could effectively reverse the drug resistance of Lewis lung cancer mice induced by OPDIVO®, so that OPDIVO® could continue to play an anti-tumor effect.
3.3 Effect of Each Experimental Group on the Tumor Inhibition Rate of Drug-Resistant Model Group
From Table 15 and
From Table 16 and
3.4 the Expression of PD-1/PD-L1 in Drug-Resistant Tumors of Each Experimental Group
Experimental results showed that the expression of PD-1/PD-L1 in tumor tissues of drug-resistant B16 melanoma and Lewis lung cancer mice was not significantly inhibited by positive drug OPDIVO® group and single drug chlorogenic acid group, but inhibited by the combination of chlorogenic acid and coumaroylquinic acid, while the inhibitory effect in the test group of the combination of chlorogenic acid and coumaroylquinic acid+OPDIVO® was significant, indicating that the combination of chlorogenic acid and coumaroylquinic acid can effectively inhibit the expression of PD-1/PD-L1 in the tumor tissues of OPDIVO®-resistant B16 melanoma and Lewis lung cancer mice.
3.5 the Number of CD4+T and CD8+T Cells in Drug-Resistant Transplanted Tumor of Each Experimental Group
Experimental results showed that the proliferation of CD4+T and CD8+T cells in drug-resistant B16 melanoma and Lewis lung cancer mice was not obvious in positive drug OPDIVO® group and single drug chlorogenic acid group, but some proliferation was observed in the combination group of chlorogenic acid and coumaroylquinic acid, while in the test group of the combination of chlorogenic acid and coumaroylquinic acid+OPDIVO®, the increase in the number of D4+T and CD8+ T cells was extremely significant, indicating that the combination of chlorogenic acid and coumaroylquinic acid can effectively promote the proliferation of D4+T and CD8+ T cells in OPDIVO®-resistant B16 melanoma and Lewis lung cancer mice.
1 Experimental Materials
1.1 Test Drugs
Lewis lung cancer cell lines were induced by the increased gradient concentration of dacarbazine and gemcitabine, and established by clone screening, then cultured without drug before the experiment.
1.3 Test Animals
After the drug was removed from the drug-resistant cell lines, the cell concentration was adjusted to 1×107/ml with the culture medium. 1×107/ml of cells were subcutaneously injected into the right armpit of mice, 0.1 ml per mouse.
2.2 Administration Method
After the average diameter of tumor reached 100 mm, the mice were randomly divided into test drug group 1, test drug group 2, test drug group 3, test drug group 4, test drug group 5, test drug group 6, positive drug group, and negative group, respectively.
Test drug group: the test drug group was first intraperitoneally injected, once a day, 30 mg/kg/time, for 5 consecutive days; administration was stopped, and the positive drug was given by intraperitoneal injection on the next day; wherein the positive drug dacarbazine was administrated once every other day, 60 mg/kg/time; wherein the positive drug gemcitabine was given once every other day, 300 mg/kg/time.
Positive drug group: positive drug dacarbazine was given once every other day, 60 mg/kg/time; positive drug gemcitabine was given once every other day, 300 mg/kg/time.
Negative group: normal saline was intraperitoneally injected, once a day, for 15 consecutive days.
2.3 Evaluation of Antitumor Effect
After completion of administration, the experiment was stopped, and the mice were killed by cervical dislocation and weighed. The tumor was stripped and weighed, to calculate the tumor inhibition rate.
Tumor inhibition rate %=[1−(the average tumor weight in the drug group/the average tumor weight in the negative group)]×100%.
2.4 Expression of PD-1/PD-L1
The positive expression rate of PD-1/PD-L1 in tumor tissues was detected by immunohistochemical SP method.
2.5 Determination of the Number of CD4+T and CD8+T Lymphocytes
The number of CD4+T and CD8+T lymphocytes was analyzed by immunofluorescence staining, and the average number of CD4+T and CD8+T cells infiltrated in 6 high power fields was counted.
3 Experimental Results
3.1 Effect of Each Experimental Group on the Inhibition Rate of Drug-Resistant Transplanted Tumor
From Table 21 and
3.2 Expression of PD-1/PD-L1 in Drug-Resistant Transplanted Tumor of Each Experimental Group
Experimental results showed that the combination group of chlorogenic acid and coumaroylquinic acid could effectively inhibit the expression of PD-1/PD-L1 in the transplanted tumor tissues of B16 cancer mice with multidrug resistance, and both of chlorogenic acid and coumaroylquinic acid had synergistic effect, in which the combination of chlorogenic acid and coumaroylquinic acid in the ratio of 100:0.01˜100:0.05 was the best.
3.3 the Number of CD4+T and CD8+T Cells in Multidrug-Resistant Transplanted Tumor of Each Experimental Group
Experimental results showed that the number of CD4+T and CD8+T cells in the experimental group of the combination of chlorogenic acid and coumaroylquinic acid was significantly higher than that in the positive drug groups (dacarbazine and gemcitabine), indicating that the combination of chlorogenic acid and coumaroylquinic acid can effectively promote the proliferation of CD4+T and CD8+T cells in multidrug-resistant Lewis lung cancer mice, and chlorogenic acid and coumaroylquinic acid have a synergistic effect.
In summary, the present invention provides a pharmaceutical composition, which comprises chlorogenic acid and coumaroylquinic acid, and the combination can be used to prepare reversal agents of tumor multidrug resistance and PD-1/PD-L1 inhibitors. The experimental results of the present invention show that the combined use of chlorogenic acid and coumaroylquinic acid can play a synergistic effect. In particular, the combined use of chlorogenic acid and coumaroylquinic acid has a good reversal effect on tumor cell lines with multi-drug resistance to chemotherapeutic drugs and immunotherapeutic drugs, and can effectively solve the drug resistance of B16 melanoma cell lines caused by dacarbazine and the drug resistance of Lewis lung cancer induced by gemcitabine, as well as can effectively inhibit the expression of PD-1/PD-L1 in the transplanted tumor tissues of B16 melanoma mice and Lewis lung cancer mice with drug resistance, and effectively reverse the drug resistance of B16 melanoma cell lines and Lewis lung cancer cell lines caused by OPDIVO®, and can effectively promote the proliferation of CD4+T and CD8+T cells in drug-resistant B16 melanoma mice and Lewis lung cancer mice.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201810206147.4 | Mar 2018 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/077826 | 3/12/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/174571 | 9/19/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 10265289 | Zhang | Apr 2019 | B2 |
| 10960042 | Lavaud | Mar 2021 | B2 |
| 20030229140 | Bandyopadhyay | Dec 2003 | A1 |
| Number | Date | Country |
|---|---|---|
| 1646112 | Jul 2005 | CN |
| 104758277 | Jul 2015 | CN |
| 106890169 | Jun 2017 | CN |
| 108159038 | Jun 2018 | CN |
| 108498497 | Sep 2018 | CN |
| Number | Date | Country | |
|---|---|---|---|
| 20210085630 A1 | Mar 2021 | US |
