CN110638820A - 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, 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 1,2,3,4, 6-pentagalloyl glucose is proved to have an inhibition effect on 2 tumors and to detect the gene expression of PD-L1 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.

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 particularly 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 malignant tumor with the first ten diseases rate in western countries. The occurrence of kidney cancer is the 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 interleukin and interferon has extremely low effective rate and great side effect 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. Pardoll research shows that PD-1 and PD-L1 can cause the formation of immunosuppressive tumor microenvironment, so that tumor cells can escape from immune monitoring and killing of organisms, and blocking a PD-1/PD-L1 signal channel can reverse the immunosuppression of tumors and enhance endogenous antitumor immune effects. PD-1 and PD-L1 inhibitors have shown therapeutic effects in the treatment of various malignancies, including kidney 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, a racemate 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, a solvate or a pharmaceutically acceptable salt thereof in preparation of 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, granules, patches, ointments, pills, powder, 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 can reduce the tumor weight of tumor-bearing mice in vivo, and the 1,2,3,4, 6-pentagalloyl glucose is proved to have an inhibition effect on 2 tumors and to detect the gene expression of PD-L1 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 11 Effect of 2,3,4, 6-Pentagalloylglucose on renal cancer cells and prostate cancer 1, Experimental Material

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 deg.C and 5% CO₂Culturing 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 10⁵one/mL, 100. mu.L per well was seeded in 96-well cell culture plates, 100. mu.L of sterile water was added to the marginal wells, and the incubation was continued at 37 ℃ with 5% CO₂And 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, setting 5 multiple wells for each concentration, and incubating for 24 h.

2.3 cell viability assay

Each set of experiments was completed 4h before the end of the timeframe, 10ul MTT was added to each well. After further culturing for 4h, carefully sucking up the supernatant, adding 150uL DMSO into each well to dissolve the 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 IC 50.

3. Results of the experiment

The experimental results of the inhibition effect of 1,2,3,4, 6-O-pentagalloyl glucose 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-pentagalloyl glucose has a certain inhibition effect on the proliferation of 2 tumor cells, and the activity of the tumor cells is gradually reduced along with the gradual increase of the concentration. The IC50 for renal and prostate cancer cells was calculated to be 19.46uM and 69.23uM, respectively.

TABLE 11 Effect of 2,3,4, 6-Pentaglloylglucose on Renca, RM-1 cell viability

P <0.01, p <0.05 compared to control

Experimental example 21 Effect of 2,3,4, 6-Pentagalloylglucose on in vitro binding of PD-1/PD-L1

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 in DMSO (dimethyl sulfoxide) to 50mM as a storage solution, diluting the drug to 30uM/mL by using a diluent in a kit before an experiment, taking the diluted drug as a high concentration, sequentially diluting the drug to 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 of Tag1-PD-L1 protein and 4uL of Tag2-PD1 protein according to the instruction sequence of the kit operation, standing the diluted compounds in a dark place at room temperature for 15min, adding 10uL of anti-Tag1-Eu3+ and anti-Tag2-XL which are uniformly mixed in advance into each hole, standing the diluted compounds in a dark place at room temperature 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 the in vitro PD1/PD-L1 combination is continuously increased along with the increase of the administration concentration, and the IC of the inhibition rate is calculated₅₀It was 16.01 uM.

Experimental example 31 inhibition of transplanted tumors in nude mice by 2,3,4, 6-pentagalloylglucose

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 Ministry of Industrial Co., Ltd.); nude mice (Yangzhou university center of comparative medicine, SCXK (su) 2017-.

1.3 instruments

Syringe (1mL, needle size 27); sterile surgical instruments (including scalpel, scissors, tweezers, needle holder, etc.)

2. Experimental methods

2.1 cell culture

Subculturing tumor cells in culture flask at 37 deg.C and 5% CO₂Culturing 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 mice disinfected the skin on the back, lifted the disinfected skin with the left thumb and forefinger, separated 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 100mm³When the tumor is taken, nude mice are randomly divided into a tumor model group, a low dose group (25mg/kg), a medium dose group (50mg/kg) and a high dose group (100mg/kg), at least 10 mice in each group are administrated according to the volume of 10mL/kg, the administration is carried out once a day for 15 days continuously, the model group is replaced by the equivalent amount of normal saline, 1 hour after the last administration, the mice are dislocated and killed, the tumor tissues are carefully stripped, and the mice are weighed and compared.

3. Results

The results of the experiments showed that the tumors were improved in weight average to some extent in comparison with 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 in comparison with the model group (. sup. < 0.05;. sup. sup. < 0.01). The specific data are shown in Table 2.

TABLE 21 Effect of 2,3,4, 6-Pentaglloylglucose on tumor cell tumor weight

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

According to experiments, 1,2,3,4, 6-pentagalloyl glucose can obviously reduce the cell activity of Renca kidney cancer and RM-1 prostate cancer in vitro, can reduce the tumor weight of tumor-bearing mice in vivo, proves that the 1,2,3,4, 6-pentagalloyl glucose has inhibition effect on 2 tumors, the gene expression of PD-L1 is detected in tumor cells of the two tumor-bearing mice, and the 1,2,3,4, 6-pentagalloyl glucose has better binding rate in the detection of the in vitro binding rate of PD-1/PD-L1, so that the 1,2,3,4, 6-pentagalloyl glucose is preliminarily presumed to play the anti-tumor effect mainly by blocking 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 (7)

1,2,3,4, 6-pentagalloylglucose, or an optical isomer, a racemate, a solvate or pharmaceutically acceptable salt thereof is applied to preparation of a medicament for treating or preventing renal cancer or prostate cancer.

2. The use according to claim 1, wherein the pharmaceutically acceptable salt comprises a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salt.

3. The use according to claim 1, wherein the medicament for treating or preventing kidney cancer or prostate cancer is in an oral administration form, an injection administration form, or an external administration form.

4. The use according to claim 1, wherein the medicament for treating or preventing kidney or prostate cancer is selected from the group consisting of injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, spray, aerosol.

5. The use according to claim 1, wherein the medicament for the treatment or prevention of renal or prostate cancer further comprises a chemotherapeutic or immunotherapeutic agent.

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

7. The use of claim 6, wherein the PD-1/PD-L1 inhibitor comprises a tablet, a capsule, a soft capsule, a gel, an oral preparation, a suspension, a granule, a patch, an ointment, a pill, a powder, an injection, an infusion solution, a lyophilized injection, an intravenous emulsion, a liposome injection, a suppository, a sustained release preparation or a controlled release preparation.

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