Application of compound CA in preparation of antitumor drugs

Abstract

The invention discloses application of a compound CA in preparation of antitumor drugs, and belongs to the technical field of biological medicines. It is found for the first time that the compound CA can down-regulate the expression level of tumor cells PD-L1, improve infiltration of immune cells in tumor tissues and significantly inhibit growth of glioma and breast cancer tumors, and has the potential of being further developed into anti-glioma and anti-breast cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of compound CA in the preparation of anti-tumor drugs. Background Art

[0002] Immune suppression plays a key role in the development and progression of tumors. Programmed cell death protein 1 (PD-1), a member of the CD28 / CTLA-4 superfamily, is a transmembrane protein widely expressed on the surface of activated immune cells, including T cells, B cells, and monocytes. PD-1 binds to its ligands, PD-L1 or PD-L2, to transmit negative regulatory signals to T cells, thereby inhibiting T cell function and maintaining immune homeostasis. PD-L1, a key member of the B7 family, is expressed in various tissues and cell types, with particularly high expression in antigen-presenting cells and some solid organs. Under normal physiological conditions, the PD-1 / PD-L1 pathway helps prevent autoimmune responses. However, numerous studies in recent years have demonstrated that PD-L1 expression is significantly upregulated in various malignancies, particularly in solid tumors such as gliomas and breast cancer. By overexpressing PD-L1, tumor cells bind to PD-1 on the surface of T cells, significantly inhibiting T cell activation and effector function, thereby evading recognition and clearance by the immune system.

[0003] Although a variety of immune checkpoint inhibitors have been approved for clinical use, therapeutic strategies targeting the PD-1 / PD-L1 pathway still face challenges such as unstable efficacy, high rates of drug resistance, and non-response in some patients. Therefore, developing new therapeutic approaches that can effectively regulate PD-L1 expression or stability has become an important area of ​​anti-tumor immunity research. Summary of the Invention

[0004] 1. Purpose of the Invention

[0005] The present invention aims to provide a method for treating tumors by using a compound CA for the preparation of anti-tumor drugs, especially anti-glioma and / or anti-breast cancer drugs. The compound CA can inhibit the expression of PD-L1 in tumor cells, thereby treating tumors.

[0006] 2. Technical solution

[0007] In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

[0008] The present invention provides the use of compound CA in the preparation of anti-tumor drugs. The chemical name of compound CA is 5-(4-{[5-(2-carboxyphenyl)furan-2-yl]methylidene}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-2-chlorobenzoic acid, and its molecular structure is shown in Formula 1:

[0009]

[0010] As a further illustration of the present application, compound CA, as a PD-L1 inhibitor, can inhibit the expression of PD-L1 in tumor cells.

[0011] Furthermore, the above-mentioned tumors include glioma and / or breast cancer.

[0012] Furthermore, the above-mentioned tumors include gliomas.

[0013] Furthermore, the above-mentioned tumor includes breast cancer.

[0014] Furthermore, the above-mentioned tumors include glioma and breast cancer.

[0015] The present invention also provides a pharmaceutical composition, which includes compound CA, the molecular structure of which is shown in Formula 1; the pharmaceutical composition can inhibit the expression of PD-L1, thereby treating tumors.

[0016] 3. Beneficial effects

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention discloses the use of a small molecule immunomodulatory compound, CA, as a PD-L1 inhibitor. The invention first discovered that this compound can downregulate PD-L1 expression in tumor cells, increase immune cell infiltration in tumor tissue, and significantly inhibit the growth of gliomas and breast cancer. This compound has the potential to be further developed as an anti-glioma and anti-breast cancer drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results are the results of Western Blot analysis of PD-L1 protein expression in mouse glioma cells GL261-Luc and mouse breast cancer cells 4T1 after treatment with compound CA.

[0020] Figure 2 The results are the results of flow cytometry detection of PD-L1 protein expression in mouse glioma cells GL261-Luc and mouse breast cancer cells 4T1 after treatment with compound CA.

[0021] Figure 3 This figure shows the inhibitory effect of compound CA on the growth of intracranial glioma in C57BL / 6J mice.

[0022] Figure 4 This is a graph showing the inhibitory effect of compound CA on the growth of subcutaneous breast cancer tumors in BALB / c mice.

[0023] Figure 5 This is a diagram showing the changes in tumor microenvironment infiltration in subcutaneous breast cancer tumor tissue of BALB / c mice before and after treatment with compound CA. DETAILED DESCRIPTION

[0024] The present application is further described below with reference to specific embodiments.

[0025] 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 to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0026] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0027] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0028] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0029] Concentration, amount and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0030] The compound CA used in the present invention was purchased from ChemDiv, USA.

[0031] Example 1

[0032] This example provides an in vitro experiment on the inhibition of PD-L1 protein expression in tumor cells by compound CA.

[0033] In this embodiment, the tumor cells are glioma GL261-Luc cells.

[0034] In this embodiment, the detection of PD-L1 protein expression level includes Western blot and flow cytometry.

[0035] Specifically, the experiment includes the following steps:

[0036] (1) Glioma GL261-Luc cells cultured to the logarithmic growth phase were trypsinized and resuspended, seeded into a 6-well plate at a density of 150,000 cells per well, and cultured in a cell culture incubator in DMEM complete medium containing 10% calf serum for 24 hours;

[0037] (2) Compound CA was diluted to 10 μM, 20 μM, 40 μM, and 60 μM using dimethyl sulfoxide (DMSO) and added to the above cells. The DMSO-treated group (no CA added) served as the control group and cultured for another 24 h (the drug CA action time is 24 h);

[0038] (3) Detection of PD-L1 protein expression level

[0039] (a) Western blot analysis of PD-L1 protein expression levels

[0040] After treatment, cells were gently rinsed with pre-chilled PBS buffer to remove culture medium and residues, and 100 μl of lysis buffer was added to each well to lyse the cells. Cells were sonicated for 1 minute in an ice bath and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected to obtain total cell protein.

[0041] The protein concentration was determined by the BCA method, and equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane;

[0042] The PVDF membrane was blocked with a blocking solution prepared with 5% skim milk powder. After blocking, primary antibodies for PD-L1 and β-actin were added, and the membrane was incubated on a shaker at 4°C overnight. The next day, the membrane was washed three times and then a secondary antibody containing HRP was added and incubated at room temperature for 1 hour. The protein bands were detected by chemiluminescence to analyze changes in PD-L1 protein expression levels.

[0043] The results are as follows Figure 1 As shown in (a), the higher the added CA concentration, the lower the PD-L1 protein expression level, proving that compound CA can inhibit the PD-L1 protein expression in glioma cells.

[0044] (b) Detection of PD-L1 protein expression level by flow cytometry

[0045] After the treatment, the cells were collected and washed twice with PBS, incubated with the antibody PD-L1-PE (PE is a fluorescent labeled protein) in the dark for 30 minutes, centrifuged at 1000 rpm at 4°C for 5 minutes to discard the excess antibody, and detected by flow cytometry.

[0046] The results are as follows Figure 2 As shown in (a), the higher the added CA concentration, the lower the PD-L1 protein expression level, proving that compound CA can inhibit the PD-L1 protein expression in glioma cells.

[0047] Example 2

[0048] This example provides an in vitro experiment on the inhibition of PD-L1 protein expression in tumor cells by compound CA.

[0049] In this embodiment, the tumor cells are breast cancer 4T1 cells.

[0050] In this embodiment, the detection of PD-L1 protein expression level includes Western blot and flow cytometry.

[0051] Specifically, the experiment includes the following steps:

[0052] (1) Breast cancer 4T1 cells cultured to the logarithmic growth phase were trypsinized and resuspended, seeded into a 6-well plate at a density of 150,000 cells per well, and cultured in a cell culture incubator with DMEM complete medium containing 10% calf serum for 24 hours;

[0053] (2) Compound CA was diluted to 5 μM, 10 μM, 20 μM, and 30 μM using DMSO and added to the above cells. The DMSO-treated group served as the control group and the cells were cultured for 24 h (the action time of drug CA was 24 h).

[0054] (3) Detection of PD-L1 protein expression level

[0055] (a) Western blot analysis of PD-L1 protein expression levels

[0056] After treatment, cells were gently rinsed with pre-chilled PBS buffer to remove culture medium and residues, and 100 μl of lysis buffer was added to each well to lyse the cells. Cells were sonicated for 1 minute in an ice bath and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected to obtain total cell protein.

[0057] The protein concentration was determined by the BCA method, and equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane;

[0058] The PVDF membrane was blocked with a blocking solution prepared with 5% skim milk powder. After blocking, primary antibodies for PD-L1 and β-actin were added, and the membrane was incubated on a shaker at 4°C overnight. The next day, the membrane was washed three times and then a secondary antibody containing HRP was added and incubated at room temperature for 1 hour. The protein bands were detected by chemiluminescence to analyze changes in PD-L1 protein expression levels.

[0059] The results are as follows Figure 1 As shown in (b), the higher the added CA concentration, the lower the PD-L1 protein expression level, proving that compound CA can inhibit the PD-L1 protein expression in breast cancer cells.

[0060] (b) Detection of PD-L1 protein expression level by flow cytometry

[0061] After the treatment, the cells were collected and washed twice with PBS, incubated with the antibody PD-L1-PE (PE is a fluorescent labeled protein) in the dark for 30 minutes, centrifuged at 1000 rpm at 4°C for 5 minutes to discard the excess antibody, and detected by flow cytometry.

[0062] The results are as follows Figure 2 As shown in (b), the higher the added CA concentration, the lower the PD-L1 protein expression level, proving that compound CA can inhibit the PD-L1 protein expression in breast cancer cells.

[0063] Example 3

[0064] This example provides an experiment on the inhibition of intracranial glioma growth in mice by compound CA.

[0065] The specific steps include:

[0066] (1) Glioma GL261-Luc cells in the logarithmic growth phase were digested with 0.25% trypsin, collected and centrifuged at 3000 rpm to remove the supernatant, washed twice with PBS buffer, and resuspended in sterile PBS;

[0067] (2) 6-8 week old male C57BL / 6J mice were selected and anesthetized with 0.8% sodium pentobarbital intraperitoneally. They were placed in a prone position and fixed on a stereotaxic apparatus. 2 μl of cell suspension (containing 2.0×10 5 cells) to establish an intracranial glioma model;

[0068] (3) The general condition of the mice was monitored daily after surgery, including mental state, food intake, activity, and weight changes. Starting from the first week after inoculation, the growth of the intracranial tumor in the mice was monitored using a small animal in vivo imaging system. Images were taken every 7 days, and the fluorescence signal intensity was recorded to evaluate the changes in tumor volume.

[0069] (4) Three weeks after tumor cell inoculation, mice with similar tumor sizes were randomly divided into groups according to tumor growth for drug intervention experiments: mice in the experimental group (CA) were treated with compound CA via tail vein injection, 0.3 mg / kg, once every 3 days; mice in the control group (DMSO) were injected with an equal volume of DMSO dilution;

[0070] (5) The treatment lasted until the sixth week, and the mice were euthanized at the end of the experiment.

[0071] The fluorescence signal intensity results are as follows Figure 3 As shown, the relative fluorescence intensity of mice in the experimental group (CA) was significantly lower than that in the control group, indicating that compound CA can inhibit glioma growth.

[0072] Example 4

[0073] This example provides an experiment on the inhibition of subcutaneous breast cancer tumor growth in mice by compound CA.

[0074] The specific steps include:

[0075] (1) Breast cancer 4T1 cells in the logarithmic growth phase were digested with 0.25% trypsin, harvested, centrifuged at 3000 rpm, and the supernatant removed. The cells were washed twice with PBS buffer and then resuspended in sterile PBS.

[0076] (2) 6-8 week old female BALB / c mice were subcutaneously inoculated with 0.1 ml of 4T1 cell suspension in the right axilla of each mouse to establish a subcutaneous tumor model;

[0077] (3) After inoculation, the general condition of the mice was observed daily, including mental state, diet, activity, body weight, and tumor growth at the inoculation site. 3At the same time, the mice were randomly divided into groups for drug intervention test: the mice in the experimental group were injected with compound CA (0.2 mg / kg) through the tail vein once every 3 days; the mice in the control group were injected with an equal volume of DMSO dilution;

[0078] (5) During the treatment period, the long and short diameters of the tumor were measured using an electronic caliper every 3 days for 12 consecutive days. The results were as follows: Figure 4 As shown in the figure, the tumor volume of mice in the experimental group was significantly smaller than that in the control group, indicating that compound CA can inhibit the growth of breast cancer.

[0079] (6) After the experiment, the mice were killed, and the tumor tissues were removed and weighed;

[0080] (7) Tumor tissue was minced and placed in a digestion solution containing collagenase IV and deoxyribonuclease I, and digested at 37°C for 3 hours to obtain a single-cell suspension of tumor cells;

[0081] (8) Count the cells and take 1×10 6 The cells were incubated with the antibody mixture (including CD45, CD3 and CD8 antibodies) at room temperature for 30 minutes in the dark. After staining, the cells were washed with PBS, resuspended and subjected to flow cytometry to detect CD3. + CD8 + T cell expression changes. Experimental results are as follows Figure 5 As shown, after treatment with compound CA, the tumor microenvironment of breast cancer tumor tissues had more CD3 + CD8 + T cell infiltration, combined with the above experimental results, is very likely to reduce immune escape by inhibiting the expression of PD-L1 in tumor cells.

Claims

1. The use of compound CA in the preparation of anti-tumor drugs, characterized in that: The molecular structure of the compound CA is as follows:

2. The use according to claim 1, characterized in that The tumor includes glioma and / or breast cancer.

3. The use according to claim 2, characterized in that The tumor includes a glioma.

4. The use according to claim 2, characterized in that The tumor includes breast cancer.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound CA as claimed in claim 1.

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