CN110638820B - Application of compound in preparation of medicine for treating renal cell carcinoma - Google Patents

Abstract

The invention provides application of 1,2,3,4, 6-pentagalloylglucose, or an optical isomer, a racemate or a solvate or pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing renal cancer or prostate cancer. Experiments show that 1,2,3,4, 6-pentagalloyl glucose can obviously reduce the cell activity of Renca kidney cancer and RM-1 prostate cancer in vitro and can reduce the tumor weight of tumor-bearing mice in vivo, and the test proves that the pentagalloyl glucose has inhibition effect on 2 tumors, and the gene expression of PD-L1 is detected in the two tumor-bearing mice, so that the 1,2,3,4, 6-pentagalloyl glucose can be preliminarily presumed to play the anti-tumor effect mainly by blocking PD 1/PD-L1.

Description

Application of compound in preparation of medicine for treating renal cell carcinoma

Technical Field

The invention relates to the technical field of medicines, and in particular relates to application of a compound in preparation of a medicine for treating renal cell carcinoma.

Background

The kidney cancer is the most common malignant tumor of the urinary system in China, and is listed as the first ten malignant tumors of the prevalence rate in western countries. The occurrence of kidney cancer is a result of the combined action of genetic factors and environmental factors, kidney cancer cells have high resistance to radiotherapy and chemotherapy, and the traditional immunotherapy represented by interleukins and interferons has extremely low efficiency and great side effects in the process of treating kidney cancer, so that the prevention and treatment of kidney cancer become the key and difficult point of research in the tumor field. Renal cancer patients have cellular and humoral immunity abnormalities, including CD4+ T cell damage, CD8+ T lymphocyte and regulatory T cell deficiency, and abnormal cytokine secretion. Previous studies have shown that programmed death factor (PD-1) and programmed death factor ligand (PD-L1) are closely related to cellular and humoral immunity. Pardol research shows that PD-1 and PD-L1 can cause the formation of an immunosuppressive tumor microenvironment, so that tumor cells can escape from immune monitoring and killing of organisms, and the blocking of a PD-1/PD-L1 signal channel can reverse the immunosuppression of tumors and enhance the endogenous antitumor immune effect. PD-1 and PD-L1 inhibitors have shown therapeutic effects in the treatment of various malignancies, including renal cancer. Therefore, the search for effective PD-1 and PD-L1 inhibitors will become a new treatment method for kidney cancer.

Disclosure of Invention

In view of the above, the present invention aims to provide a drug for treating or preventing renal cancer or prostate cancer.

Specifically, the invention provides application of 1,2,3,4, 6-pentagalloylglucose, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing renal cancer or prostate cancer.

Specifically, the pharmaceutically acceptable salt includes, but is not limited to, hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salt of the compound.

Further, the medicament for treating or preventing kidney cancer or prostate cancer is an oral administration dosage form, an injection administration dosage form or an external administration preparation.

Specifically, the use according to claim 1, wherein the medicament for treating or preventing renal cancer or prostate cancer includes, but is not limited to, injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, spray, aerosol.

Further, the medicament for treating or preventing kidney cancer or prostate cancer may further include a chemotherapeutic agent or an immunotherapeutic agent. Wherein, the chemotherapeutic agent or the immunotherapeutic agent may be an active substance having a corresponding antitumor effect in addition to the 1,2,3,4, 6-pentagalloylglucose of the present invention.

The invention also provides application of the 1,2,3,4, 6-pentagalloylglucose, or an optical isomer, a racemate or a solvate or pharmaceutically acceptable salt thereof in preparing the PD-1/PD-L1 inhibitor.

The PD-1/PD-L1 inhibitor can be any one of the dosage forms in pharmacy, such as an oral administration dosage form, an injection administration dosage form or an external administration preparation.

Specifically, the PD-1/PD-L1 inhibitor comprises tablets, capsules, soft capsules, gels, oral agents, suspensions, dissolved medicines, patches, ointments, pills, powders, injections, infusion solutions, freeze-dried injections, intravenous emulsions, liposome injections, suppositories, sustained-release preparations or controlled-release preparations.

Further, the structure of 1,2,3,4, 6-pentagalloylglucose is as follows:

when the compound having the structure of formula I, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof is used as a medicament, it can be used as it is, or in the form of a pharmaceutical composition/preparation. The pharmaceutical composition comprises 0.1-99%, preferably 0.5-90% of the compound of the invention, the rest can be other active ingredients with similar or synergistic effect, and can also or further comprise pharmaceutically acceptable carriers and/or excipients which are nontoxic and inert to human and animals.

Experiments show that 1,2,3,4, 6-pentagalloyl glucose can obviously reduce the cell activity of Renca kidney cancer and RM-1 prostate cancer in vitro and reduce the tumor weight of tumor-bearing mice in vivo, and the test proves that the pentagalloyl glucose has an inhibition effect on 2 tumors, and the gene expression of PD-L1 is detected in the two tumor-bearing mice, so that the 1,2,3,4, 6-pentagalloyl glucose can be preliminarily presumed to play an anti-tumor effect mainly by blocking PD 1/PD-L1.

Drawings

FIG. 1 is a graph of the inhibition of PD1/PD-L1 binding by 1,2,3,4, 6-pentagalloylglucose at various concentrations.

Detailed Description

The following detailed description of the embodiments of the present invention will be given in conjunction with examples to better understand the aspects of the present invention and the advantages of its various aspects. However, the specific embodiments and examples described below are for illustrative purposes only and are not limiting of the invention.

Secondly, it is to be noted that the concentrations referred to in the present invention are in volume percent (v/v). All percentages, ratios, proportions, or parts are by weight unless otherwise specified. In addition, if the specific conditions are not indicated, the invention is carried out according to the conventional conditions or the conditions suggested by the manufacturer, and the used raw material drugs or auxiliary materials and the used reagents or instruments are the conventional products which can be obtained commercially.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

Example 1,2,3,4, 6-PentaGalloylglucose Effect on renal cancer cells, prostate cancer

1. Experimental materials

1.1 drugs and reagents: 1,2,3,4, 6-pentagalloylglucose (MCE); RPMI1640 (GIBCO), fetal bovine serum (GIBCO); MTT (sigma).

1.2 cells: renca (CRL-2947, purchased from ATCC); RM-1 cell line (Shanghai Ministry of Industrial Co., ltd.).

1.3 instruments and devices: electronic balance (sidoris); centrifuge (good in Anhui); flex station 3 calcium streaming workstation (MD); inverted microscope (OLYPUS).

2. Experimental methods

2.1 cell culture

Cell subculture in culture flask at 37 ℃ with 5% CO ₂ Culturing in an incubator with saturated humidity, discarding culture solution after the cells in the culture bottle grow up basically (the cells are kept in logarithmic growth phase), adding a proper amount of PBS solution for gentle washing, discarding, then adding 1ml of 0.25% trypsin solution for digestion for 2-3 minutes, adding 2ml of complete culture medium to stop the action of pancreatin, transferring the cells into a centrifuge tube, centrifuging for 5min at 1000r, discarding supernatant, adding the complete culture medium, and carefully blowing and beating into single cells. The cells were counted by an automatic cell counter at 1X 10 ⁵ Per mL, 100. Mu.L per well in a 96 well cell culture plate, 100. Mu.L sterile water added to the marginal wells, continued at 37 ℃ C. And 5% CO ₂ And carrying out molding and administration tests after culturing for 18-20 hours in an incubator.

2.2 grouping and administration

Centrifuging and removing supernatant after cell pancreatin digestion, inoculating cells into a 96-well plate according to 10000 cells/well after heavy suspension counting, adding 1,2,3,4, 6-pentagalloyl glucose with final concentration of 6.25, 12.5, 25, 50 and 100uM after 24h, setting an experiment control group at the same time, setting 5 multiple wells for each concentration, and incubating for 24h.

2.3 cell viability assay

Each set of experiments was completed 4h before the end of the time period, 10ul MTT was added to each well. After continuing culturing for 4h, carefully sucking up the supernatant, adding 150uL DMSO into each well to dissolve crystals, after the crystals are completely dissolved, measuring the absorbance value at the wavelength of 570nm under an enzyme-linked immunosorbent assay, and calculating the survival rate and the IC50.

3. Results of the experiment

The experimental results of the inhibitory effect of 1,2,3,4, 6-O-pentagalloylglucose on the proliferation of each tumor cell are shown in Table 1, and it can be seen from the table that 1,2,3,4, 6-O-pentagalloylglucose has a certain inhibitory effect on the proliferation of 2 tumor cells, and the activity of the tumor cells gradually decreases with the gradual increase of the concentration. The IC50 values of the compound are 19.46uM and 69.23uM for renal cancer cells and prostate cancer cells respectively according to the formula.

TABLE 1,2,3,4,6-Pentagalloylglucose Effect on Renca, RM-1 cell viability

P <0.01, p <0.05 compared to control

Experimental example 2,3,4, 6-Pentaglloylglucose Effect on PD-1/PD-L1 binding in vitro

1. Experimental Material

1.1 drugs and reagents: 1,2,3,4, 6-pentagalloylglucose (MCE); RPMI1640 (GIBCO), fetal bovine serum (GIBCO); PD1/PD-L1 Binding Assay Kits (cisbio).

1.2 instruments and devices: electronic balance (sidoris); microplate reader (PE).

2. Experimental methods

Dissolving a drug into 50mM DMSO (dimethyl sulfoxide) as a storage solution, diluting the drug into 30uM/mL by using a diluent in a kit before an experiment, then taking the diluted drug as a high concentration, sequentially diluting the drug into 5 concentrations of 30, 10, 3.33, 1.11 and 0.37uM/mL according to three times of dilution, respectively adding 2uL of diluted compounds with different concentrations, 4uL Tag1-PD-L1 protein and 4uL Tag2-PD1 protein according to the instruction sequence of the kit operation, standing the mixture at room temperature in a dark place for 15min, adding 10uL of anti-Tag1-Eu3+ and anti-Tag2-XL665 which are uniformly mixed in advance into each hole, standing the mixture at room temperature in a dark place for 2h, and detecting the fluorescence value of each hole under a fluorescence microplate reader.

3. Results of the experiment

As shown in figure 1, the inhibition rate of 1,2,3,4, 6-pentagalloylglucose on in vitro PD1/PD-L1 binding is continuously increased along with the increase of the administration concentration, and the IC of the inhibition rate is calculated ₅₀ It was 16.01uM.

Experimental example 3,2,3,4,6-Pentagalloylglucose inhibition of transplanted tumors in nude mice

1. Experimental Material

1.1 drugs and reagents

1,2,3,4, 6-pentagalloylglucose (MCE); RPMI1640 (GIBCO), fetal bovine serum (GIBCO); PD1/PD-L1 Binding Assay Kits (cisbio).

1.2 animals and cells

Renca (CRL-2947, purchased from ATCC); RM-1 cell line (Shanghai Mingsheng industries, ltd.); nude mice (Yangzhou university center of comparative medicine, SCXK (su) 2017-0007).

1.3 Instrument

Syringe (1 mL, needle size 27); sterile surgical instruments (including scalpels, scissors, forceps, needle holders, etc.).

2. Experimental methods

2.1 cell culture

Subculturing tumor cells in culture flasks at 37 ℃ with 5% CO ₂ Culturing in an incubator with saturated humidity, discarding culture solution after the cells in the culture bottle grow up basically (the cells are kept in logarithmic growth phase), adding a proper amount of PBS solution for gentle washing, discarding, then adding 1ml of 0.25% trypsin solution for digestion for 2-3 minutes, adding 2ml of complete culture medium to stop the action of pancreatin, transferring the cells into a centrifuge tube, centrifuging for 5min at 1000r, discarding supernatant, adding the complete culture medium, and carefully blowing and beating into single cells. Counting by an automatic cell counter, adjusting the cell concentration to be suspension of the required cell concentration, and storing on ice for later use.

2.2 preparation of tumor model

The nude mouse disinfected the skin on the back, lifted the disinfected skin with the thumb and forefinger of the left hand to separate the skin from the underlying muscles, and injected 20ul of fully vortexed cell suspension into the lifted skin folds. If resistance is met during injection, the injection angle is not correct, and the needle insertion direction is required to be changed in time.

2.3 Observation and Experimental groups

Tumor growth was observed daily after inoculation, after several weeks, tumor growth was visible, the longest longitudinal and widest transverse diameters of the tumors were measured with a vernier caliper, and tumor volume was calculated using the following formula: 0.5x length x width 2 until the tumor volume reaches 100mm ³ When the mice are randomly divided into a tumor model group, a low dose group (25 mg/kg), a medium dose group (50 mg/kg) and a high dose group (100 mg/kg), at least 10 mice in each group,the drug was administered once a day in a volume of 10mL/kg for 15 days, the model group was replaced with an equal amount of physiological saline, and 1 hour after the last administration, the mice were killed by dislocation, tumor tissues were carefully dissected, and weighed for comparison.

3. As a result, the

The results of the experiments showed that the tumors were improved to some extent in weight average compared to the model control group after administration of different concentrations of 1,2,3,4, 6-pentagalloylglucose, wherein the medium and high dose groups had statistical differences (p < 0.05;. P < 0.01) compared to the model group. Specific data are shown in table 2.

TABLE 2 influence of 1,2,3,4,6-Pentagalloylglucose on tumor cell tumor weight

P <0.05, P <0.01 compared to model control.

According to experiments, 1,2,3,4, 6-pentagalloylglucose can obviously reduce the cell activity of Renca kidney cancer and RM-1 prostate cancer in vitro and reduce the tumor weight of tumor-bearing mice in vivo, and proves that the pentagalloylglucose has an inhibition effect on 2 tumors, the gene expression of PD-L1 is detected in the tumor body cells of the two tumor-bearing mice, and the 1,2,3,4, 6-pentagalloylglucose has a better binding rate in the detection of the in vitro PD-1/PD-L1 binding rate, so that the 1,2,3,4, 6-pentagalloylglucose can be preliminarily presumed to play an anti-tumor effect mainly by blocking the PD 1/PD-L1.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (1)

1.1,2,3,4, 6-pentagalloylglucose for use as a PD-1/PD-L1 binding inhibitor in vitro.

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