Application of luliconazole as PD-1 inhibitor in preparation of medicine for treating colon cancer

Abstract

The invention discloses an application of luliconazole as a PD-1 inhibitor in preparation of a medicine for treating colon cancer. The invention also discloses an application of luliconazole in preparation of a PD-1 inhibitor. The luliconazole disclosed by the invention can be used for treating the colon cancer by promoting infiltration of T lymphocytes in tumor tissues and secretion of granzyme B. The luliconazole disclosed by the invention can inhibit the growth of colon cancer, so that the colon cancer can be treated or improved. In-vivo experiments prove that luliconazole treats or improves colon cancer by promoting tumor T lymphocyte infiltration and enhancing T lymphocyte functions.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular, relates to the use of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer. Background Art

[0002] Colorectal cancer (CRC) refers to cancer in which the lesions occur in the colon or rectum. The main clinical symptoms are blood in the stool, changes in bowel habits and changes in stool characteristics. There are many pathogenic factors of colon cancer, including viral and bacterial infections, alcohol, smoking, aging, ulcerative colitis and sedentary lifestyles, which can all lead to the occurrence of colorectal cancer. The conventional treatments for colorectal cancer are surgery, chemotherapy and radiotherapy. For localized cancer, surgical resection is generally used, while for cancer that has already metastasized, adjuvant chemotherapy or radiotherapy is required. Since chemotherapy or radiotherapy is non-specific and cytotoxic to any cell, it produces many side effects; in addition, many patients relapse even after adjuvant treatment. Therefore, it is crucial to have more alternative and effective treatments to treat CRC patients. Immunotherapy is a new treatment for colon cancer. Compared with conventional treatments, immunotherapy has the advantages of a wide range of treatment periods, good tolerance and few side effects. Therefore, immunotherapy drugs are the most promising drugs for the treatment of colorectal cancer, the most famous of which are programmed death-1 (PD-1) / programmed cell death ligand 1 (PD-L1) inhibitors.

[0003] PD-1 is an immune checkpoint in the process of T cell activation. Under normal circumstances, the combination of PD-L1 and PD-1 prevents T cells from being overactivated and causing damage to the body. However, tumor cells take advantage of this feature and interact with the upregulated PD-1 on the surface of T cells through their own overexpressed PD-L1, causing T cell function exhaustion, thereby achieving immune escape. In the tumor microenvironment, after PD-L1 interacts with PD-1, it transmits inhibitory signals to T cells, inhibiting T cell downstream signaling pathways, including PI3K / AKT, Ras–MEK–ERK, thereby affecting the biological functions of T cells, such as: inhibiting T cell proliferation and activation, causing tumor-specific T cell exhaustion and apoptosis, reducing the secretion of cytokines IL-2 and IFN-γ, and inhibiting the production of granzyme B (Gzm B), thereby achieving immune escape. PD-1 / PD-L1 inhibitors can effectively block the interaction between PD-L1 and PD-1 on tumor cells, thereby restoring the immune response of T cells to tumor antigens.

[0004] PD-1 / PDL-1 inhibitors include antibody inhibitors and non-antibody inhibitors. Monoclonal antibody drugs have made great progress in the clinical treatment of various cancers, but they also have certain limitations, such as low response rate, high price, inconvenient storage and transportation, etc. In contrast, small molecule inhibitors have a short half-life and can avoid the occurrence of serious immune-related adverse events. In addition, small molecule inhibitors are easy to transport and store, have good stability, and have excellent membrane permeability, which are more conducive to future clinical treatment. At present, the research on PD-1 / PD-L1 small molecule inhibitors is relatively lagging, and there is an urgent need to find and develop new PD-1 / PD-L1 small molecule inhibitors.

[0005] Luliconazole (LCZ) is an imidazole drug used to treat fungal skin infections. In vitro and in vivo studies have shown that luliconazole has excellent efficacy against Aspergillus and Candida. Currently, there are no reports on the anti-colon cancer effect of luliconazole. Summary of the invention

[0006] The purpose of the present invention is to provide a use of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a use of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer.

[0009] The structural formula of luliconazole (Yang J, et al. A sensitive and rapid bioanalytical method for the quantitative determination of luliconazole in rabbit eye tissues using UPLC-MS / MS assay. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Apr 1; ​​1194: 123173.) is shown below:

[0010]

[0011] The present invention provides an application of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer, wherein luliconazole is the sole active ingredient.

[0012] The drug for treating colon cancer is a drug for inhibiting the growth of CT26 cells.

[0013] The drug for treating colon cancer is a drug that promotes the infiltration of T lymphocytes in CT26 cells; and is a drug that promotes the secretion of granzyme B in CT26 cells.

[0014] The present invention also provides a pharmaceutical preparation made from luliconazole and medically acceptable auxiliary materials.

[0015] The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, nano preparation and the like.

[0016] Luliconazole directly binds to PD-1, inhibits the binding of PD-L1 to PD-1 in the tumor microenvironment, relieves the phosphorylation levels of related proteins AKT and ERK in the TCR downstream signaling pathway of T cells, and promotes the expression of cytokines IL-2 and IFN-γ; in addition, luliconazole increases the number of CD4 + With CD8 + The number of T cells was significantly increased, the expression of tumor cell killing factor granzyme B was significantly increased, and CD8 + T cell function, thereby playing a role in treating colon cancer.

[0017] The second aspect of the present invention provides a use of luliconazole in the preparation of a PD-1 inhibitor.

[0018] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0019] The present invention provides luliconazole as a novel PD-1 small molecule inhibitor, and detects the inhibitory effect of luliconazole on PD-1 / PD-L1 binding by homogeneous time-resolved fluorescence (HTRF), and then uses surface plasmon resonance (SPR) technology to confirm that luliconazole can specifically bind to PD-1 protein and inhibit the binding of PD-1 and PD-L1. In vitro, a co-culture cell model in which PD-1 / PD-L1 interaction leads to T cell immunosuppression was used to confirm that luliconazole can promote the expression of cytokines IL-2 and IFN-γ in T cells, activate related proteins AKT and ERK in the downstream signaling pathway of PD-1 / PD-L1, and reverse T cell immunosuppression caused by PD-1 / PD-L1 interaction. In vivo, a colon cancer transplant mouse model was used to confirm that luliconazole can increase the number of CD4 + T, CD8 + The infiltration of T lymphocytes promotes the secretion of granzyme B, thereby enhancing the function of T lymphocytes, thereby killing tumor cells and inhibiting the growth of colon cancer.

[0020] The luliconazole of the present invention can treat colon cancer by promoting the infiltration of T lymphocytes in tumor tissues and the secretion of granzyme B. The luliconazole of the present invention can inhibit the growth of colon cancer, and thus can treat or improve colon cancer. In vivo experiments have confirmed that luliconazole treats or improves colon cancer by promoting tumor T lymphocyte infiltration and enhancing T lymphocyte function. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the results of HTRF technology detection of the inhibitory effect of luliconazole on PD-1 / PD-L1 binding.

[0022] Figure 2 Schematic diagram of the SPR analysis results of the binding affinity of luliconazole to PD-1 protein.

[0023] Figure 3 Schematic diagram of the effect of luliconazole on the activity of Jurkat cells in the co-culture system.

[0024] Figure 4 This is a schematic diagram of the effect of luliconazole on tumor growth in colon cancer-bearing mice.

[0025] Figure 5 Schematic diagram of the effect of luliconazole on T lymphocyte subsets in tumors of colon cancer-bearing mice.

[0026] Figure 6 Schematic diagram of immunohistochemical detection of the effect of luliconazole on the expression level of granzyme B in tumors of colon cancer-bearing mice. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0028] Example 1

[0029] Luliconazole inhibits the interaction between PD-L1 and PD-1

[0030] Homogeneous time-resolved fluorescence (HTRF) is a method for detecting receptor-ligand binding in a homogeneous (liquid phase) system. The PD-1 / PD-L1 HTRF kit was used to detect the effect of luliconazole on the PD-1 / PD-L1 interaction. A 1mM luliconazole solution was prepared and diluted 3-fold to a minimum concentration of 0.05μM to form drug solutions with concentrations of 1000μM, 333μM, 111μM, 37μM, 12μM, 4.12μM, 1.37μM, 0.47μM, and 0.05μM. The sample addition reaction was carried out according to the instructions of the kit, and the absorbance of each well at 665nm and 620nm was detected by a microplate reader. The inhibitory effect of luliconazole on PD-1 / PD-L1 binding was calculated based on the values ​​of 665nm / 620nm. The results are shown in the figure. Figure 1 As shown, Figure 1 The schematic diagram of the results of HTRF technology detection of the inhibitory effect of luliconazole on PD-1 / PD-L1 binding, where A is the schematic diagram of the HTRF experimental detection principle. PDL1 protein carries Tag1, and PD1 protein carries Tag2. Anti-Tag1-Eu and Anti-Tag2-XL665 antibodies are used to bind to the two tags respectively. When the two proteins bind, the distance between Eu and XL665 is close, and the laser excites Eu, Eu emits light and transfers energy to XL665, causing it to emit light. Adding a compound, the compound blocks the binding of PD1 and PDL1, and the distance between Eu and XL665 is far, so the energy cannot be transferred, and XL665 does not emit light. Among them, the signal detected at 620nm is the signal emitted by Anti-Tag1-Eu connected to PD-L1, and the signal detected at 665nm is the signal after the energy resonance transfer after PD-L1 binds to PD-1. The signal ratio of 665nm / 620nm is calculated to reflect the binding of PD-L1 ligand to PD-1 receptor in the whole system. B is a schematic diagram of the HTRF test results, and the IC of luliconazole inhibiting PD-1 / PD-L1 binding 50 The value is 27.3μM. When the concentration of luliconazole is low, the fluorescence intensity in the whole system is the highest, indicating that the PD-L1 ligand in the solution is tightly bound to the PD-1 receptor; and as the concentration of luliconazole increases, the fluorescence intensity in the whole system continues to weaken, indicating that the binding of PD-L1 to PD-1 in the solution is inhibited, and this inhibitory effect is caused by the action of the drug. It can be concluded that luliconazole can inhibit the PD-1 / PD-L1 interaction.

[0031] Example 2

[0032] Luliconazole specifically binds to PD-1

[0033] The PD-1 protein was prepared with 10mM sodium acetate buffer solution of different pH (5.5, 5.0, 4.5) to a final concentration of 50μg / ml. The sample was injected for detection, and the optimal pH was screened based on the response value (RU) of the PD-1 protein at different pH, and the protein was coupled overnight. Luliconazole solution (concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25μM) was prepared with PBS solution (containing 5% DMSO) for measurement, in which PBS solution (containing 5% DMSO) was used as a buffer, the injection speed was set to 30μl / min, and the binding and dissociation times were set to 60s. The Biacore T200 software was used for data processing to obtain the binding affinity curve obtained by single-site interaction fitting, the interaction mode and kinetic constant (Kd=23.62μM) between luliconazole and PD-1. The results are shown in Figure 2 As shown, Figure 2 The following is a schematic diagram of the results of SPR analysis of the binding affinity of luliconazole and PD-1 protein. Among them, A is the sensor diagram of the interaction between luliconazole and PD-1 protein at different concentrations; B is a schematic diagram of the binding affinity curve of luliconazole and PD-1 obtained by fitting the single-site interaction. HTRF experiments have confirmed that luliconazole can hinder the binding of PD-1 and PD-L1 and inhibit the PD-1 / PD-L1 interaction. Then, through SPR experimental analysis, it was concluded that luliconazole can specifically bind to PD-1 protein. Therefore, luliconazole is a PD-1 inhibitor that can hinder the PD-1 / PD-L1 interaction. The following Real-time qPCR and Western blot experiments are used to analyze the effect of luliconazole as a PD-1 inhibitor on T cells.

[0034] Example 3

[0035] Luliconazole reverses T cell immunosuppression caused by PD-1 / PD-L1 interaction

[0036] Jurkat cells with stable and high expression of PD-1 and luciferase reporter gene with nuclear factor of activated T cell response element, and CHO-K1 cells with stable expression of human T cell activation CD3 monoclonal antibody (OKT3) and PD-L1 were constructed, and these two cells were co-cultured to form a co-culture cell model in which PD-1 / PD-L1 interaction leads to T cell immunosuppression. In the co-culture cell model, OKT3 can activate Jurkat cells and promote the synthesis of cytokines IL-2 and IFN-γ; and after PD-1 binds to PD-L1, it can inhibit the conduction of related signaling pathways in Jurkat cells, such as PI3K-AKT and RAS-MEK-ERK signaling pathways, leading to Jurkat cell dysfunction and inhibiting the synthesis of cytokines in T cells. However, when PD-1 / PD-L1 inhibitors are added to the co-culture cell model, the binding of PD-L1 to PD-1 is blocked, so PD-1 cannot transmit inhibitory signals to T cells, and the conduction of signaling pathways in T cells is restored. Therefore, in the co-culture cell model, the ability of the compound to inhibit the PD-1 / PD-L1 interaction can be reflected by analyzing the phosphorylation levels of AKT and ERK proteins in the PD-1 / PD-L1 downstream signaling pathway and the expression changes of cytokines IFN-γ and IL-2.

[0037] In the in vitro co-culture system, after adding luliconazole at concentrations of 2.5, 5, and 10 μM for 24 hours, the Real-time qPCR method was used to detect the expression levels of IL-2 and IFN-γ mRNA in Jurkat cells, and Western blot was used to detect the expression levels of p-AKT and p-ERK proteins in Jurkat cells. Figure 3 As shown, Figure 3Figure 1 is a schematic diagram of the effect of luliconazole on the activity of Jurkat cells in the co-culture system; A is a schematic diagram of the changes in the expression levels of IL-2 and IFN-γ mRNA in Jurkat cells; it can be seen from the figure that under the action of luliconazole, the expression levels of IL-2 and IFN-γ mRNA in Jurkat cells in the co-culture system increased significantly (P<0.05). This suggests that luliconazole can reverse the immunosuppression of Jurkat cells caused by the interaction of PD-1 / PD-L1 and reactivate Jurkat cells. B is a schematic diagram of the changes in the expression levels of p-AKT and p-ERK proteins in Jurkat cells. The upper left figure in B is a typical figure of Western blot detection of the effect of different concentrations of luliconazole (2.5, 5, 10μM) on the expression level of p-AKT protein in Jurkat cells in the co-culture cell model. The protein expression level is judged by comparing the color depth of the band. As can be seen from the figure, the p-AKT protein band in the Jurkat cells of the model group is very light, indicating that after the interaction of PD-1 / PD-L1, the downstream cell signaling pathway of Jurkat cells is inhibited and the AKT protein cannot be phosphorylated. Compared with the model group, after the addition of luliconazole, the grayscale of the phosphorylated AKT protein band in Jurkat cells is enhanced, while there is no significant difference in the protein bands of total protein AKT and internal reference protein GAPDH, indicating that under the action of luliconazole, the expression level of p-AKT protein in Jurkat cells is increased. The upper right figure in B is a figure after the grayscale value analysis of the Western Blot experimental results using Image J, and then the results of three experiments are statistically analyzed. It can be seen from the figure that after the addition of luliconazole, the expression level of p-AKT protein in Jurkat cells is significantly increased compared with the model group. Similarly, from the lower left and lower right figures in B, it can be seen that compared with the model group, the expression level of p-AKT protein in Jurkat cells is significantly increased after the addition of luliconazole. The above results suggest that luliconazole can relieve the inhibition of Jurkat cell activity caused by PD-1 / PD-L1 interaction.

[0038] Example 4

[0039] Luliconazole inhibits tumor growth in colon cancer-bearing mice

[0040] Construction of colon cancer transplant mouse model: Take CT26 cells with good growth status and adjust the cell concentration to 1×10 7 200 μl of cell suspension was injected subcutaneously on the dorsal side of the right forelimb of Balb / c mice. The tumor growth was continuously observed after inoculation. When the tumor volume reached 50 mm 3At the time of the experiment, 42 mice were divided into the following groups according to their body weight: solvent control group (n=12), low-dose luliconazole group (10 mg / kg, n=12), high-dose luliconazole group (20 mg / kg, n=12), and positive control group (PD-L1 antibody 10 mg / kg, n=6). The low-dose luliconazole group and the high-dose luliconazole group were intraperitoneally injected with 10 mg / kg and 20 mg / kg of luliconazole at 0.2 ml / 30 g, and the solvent control group was intraperitoneally injected with the corresponding volume of the dosing solvent once a day for ten consecutive days. The positive control group was intraperitoneally injected with 10 mg / kg of PD-L1 antibody once every two days for ten days. After grouping, the mice were weighed and the tumor volume was measured every two days.

[0041] After treatment, the mouse tumors were isolated, weighed, and photographed. Figure 4 As shown, Figure 4 Figure 1 is a schematic diagram of the effect of luliconazole on tumor growth in mice bearing colon cancer; A is a schematic diagram of tumor images of tumor-bearing mice. It can be seen from the figure that compared with the solvent control group, the tumor volume of the low-dose luliconazole group and the high-dose luliconazole group was significantly smaller. B is a schematic diagram of tumor weight in tumor-bearing mice. Compared with the solvent control group, the tumor weight of the colon cancer-bearing mice in the low-dose luliconazole group (10 mg / kg), the high-dose luliconazole group (20 mg / kg), and the positive control group (PD-L1 antibody 10 mg / kg) was significantly lighter (P<0.05). C is a schematic diagram of the tumor growth curve of tumor-bearing mice. It can be seen from the figure that with the extension of the inoculation time, the tumor volume of the mice in the solvent control group gradually increased, and in the later stage, it showed exponential growth, while under the action of the drug, the tumor volume of the mice grew slowly. On day 16 of inoculation of CT26 cells, compared with the solvent control group, the tumor volume of the low-dose luliconazole group (10 mg / kg), high-dose luliconazole group (20 mg / kg), and positive control group (PD-L1 antibody 10 mg / kg) was significantly reduced (P<0.05). The above data all indicate that luliconazole can significantly inhibit the growth of colon cancer in mice and reduce the tumor weight of mice.

[0042] Example 5

[0043] Luliconazole increases the number of T lymphocytes in tumors of colon cancer-bearing mice

[0044] Immunohistochemistry and flow cytometry were used to detect the CD4 + T cells, CD8 +Changes in the number of T cells. Add CD4 / CD8 antibodies (the antibodies are prepared in PBS containing 10% goat serum, with a dilution ratio of 1:100) to the paraffin sections and incubate overnight; then add HRP secondary antibody and incubate at room temperature for 30 minutes; perform DAB color development under a microscope to stain the cell nuclei of the tissue sections. Finally, observe under a fluorescence microscope and collect images. By observing the staining area of ​​the positive area in the staining map, and using Image J to quantitatively analyze the positive area, the area percentage of the positive area is obtained, and the results are summarized and statistically analyzed to observe the CD4 in the tumor of the tumor-bearing mouse. + T, CD8 + The changes in the number of T cells, the results are as follows Figure 5 As shown, Figure 5 The following is a schematic diagram of the effect of luliconazole on T lymphocyte subsets in tumors of mice bearing colon cancer. The left figure in A is a typical picture of CD4 and CD8 immunohistochemical staining results, and the right figure is a quantitative analysis result after immunohistochemical staining. As can be seen from the figure, compared with the solvent control group, luliconazole treatment significantly increased the number of CD8+T cells in mouse tumor tissues (P<0.05), and the number of CD4+T cells also increased, but did not reach a statistically significant difference. Flow cytometry was used to detect the number of CD4+T cells in tumors of tumor-bearing mice. + T, CD8 + Changes in the number of T cells, such as Figure 5 As shown in B, the left figure is a typical figure of CD4 and CD8 flow cytometry test results, the percentage of CD4 / CD8 cells has been marked with numbers, and the right figure is a statistical analysis of the percentage of CD4 / CD8 cells. It can be seen from the figure that compared with the solvent control group, after luliconazole treatment, the CD4 in the tumor tissue of colon cancer-bearing mice was + T, CD8 + The number of T cells increased significantly (P<0.05). The above data show that luliconazole can significantly increase the number of T lymphocytes in tumor tissues.

[0045] Example 6

[0046] Luliconazole enhances the function of tumor-infiltrating immune cells in colon cancer-bearing mice

[0047] CD8 + T cells can secrete granzyme B to kill tumor cells. Therefore, immunohistochemistry was used to detect the expression level of granzyme B in tumor tissues of mice after luliconazole treatment. Figure 6 As shown, Figure 6This is a schematic diagram of the effect of luliconazole on the expression level of granzyme B in tumors of colon cancer-bearing mice detected by immunohistochemistry. The figure includes a typical graph of changes in the content of granzyme B protein in tumors of tumor-bearing mice detected by immunohistochemistry and a statistical result graph after quantitative analysis; it can be seen from the figure that compared with the solvent control group, the pathological sections of the low-dose luliconazole group (10 mg / kg) and the high-dose luliconazole group (20 mg / kg) have significantly more positive areas of granzyme B staining; after quantitative statistics of the staining results, it was found that compared with the solvent control group, the expression level of granzyme B in the tumor tissues of colon cancer-bearing mice in the low-dose luliconazole group (10 mg / kg) and the high-dose luliconazole group (20 mg / kg) was significantly increased (P<0.05). This shows that luliconazole can enhance CD8 + T cell function plays an anti-tumor role.

[0048] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A use of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer.

2. The use of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer according to claim 1, characterized in that: The structural formula of luliconazole is shown below:

3. The use of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer according to claim 1, characterized in that: Luliconazole is the only active ingredient in the application.

4. The use of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer according to claim 1, characterized in that: The drug for treating colon cancer is a drug for inhibiting the growth of CT26 cells.

5. The use of luliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer according to claim 1, characterized in that: The drug for treating colon cancer is a drug that promotes the infiltration of T lymphocytes in CT26 cells; and is a drug that promotes the secretion of granzyme B in CT26 cells.

6. A pharmaceutical preparation, characterized in that It is a pharmaceutical preparation made of luliconazole and medically acceptable excipients.

7. The pharmaceutical preparation according to claim 6, characterized in that The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, and nano preparation.

8. A use of luliconazole in the preparation of PD-1 inhibitors.

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