Application of ruliconazole as a PD-1 inhibitor in the preparation of drugs for treating colon cancer

Abstract

This invention discloses the use of luriconazole as a PD-1 inhibitor in the preparation of drugs for treating colon cancer. This invention also discloses the use of luriconazole in the preparation of PD-1 inhibitors. The luriconazole of this invention can treat colon cancer by promoting the infiltration of T lymphocytes and the secretion of granzyme B in tumor tissue. The luriconazole of this invention can inhibit the growth of colon cancer, thereby treating or improving colon cancer. In vivo experiments have confirmed that luriconazole treats or improves colon cancer by promoting tumor T lymphocyte infiltration and enhancing T lymphocyte function.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically, it relates to the use of ruliconazole as a PD-1 inhibitor in the preparation of drugs for treating colon cancer. Background Technology

[0002] Colorectal cancer (CRC) refers to cancer that occurs in the colon or rectum. The main clinical symptoms are rectal bleeding, changes in bowel habits, and changes in stool consistency. Many factors contribute to the development of colorectal cancer, including viral and bacterial infections, alcohol, smoking, aging, ulcerative colitis, and sedentary lifestyles. Conventional treatments for colorectal cancer include surgery, chemotherapy, and radiotherapy. For localized cancers, surgical resection is generally used, while for metastatic cancers, adjuvant chemotherapy or radiotherapy is required. Because chemotherapy and radiotherapy are non-specific and cytotoxic to all cells, they produce many side effects; furthermore, many patients relapse even after adjuvant therapy. Therefore, having more alternative and effective treatment methods for CRC patients is crucial. Immunotherapy is a novel treatment for colorectal cancer. Compared with conventional treatments, immunotherapy has advantages such as a wider treatment duration, better tolerability, and fewer side effects. Therefore, immunotherapy drugs are the most promising drugs for treating colorectal cancer, among which the most well-known are programmed death-1 (PD-1) / programmed cell death ligand 1 (PD-L1) inhibitors.

[0003] PD-1 is an immune checkpoint in the T cell activation process. Normally, the binding of PD-L1 to PD-1 prevents excessive T cell activation that could lead to organ damage. However, tumor cells exploit this characteristic, interacting their overexpressed PD-L1 with PD-1 regulated on the T cell surface, causing T cell exhaustion and thus achieving immune escape. In the tumor microenvironment, after PD-L1 interacts with PD-1, it transmits inhibitory signals into T cells, inhibiting downstream signaling pathways, including PI3K / AKT and Ras–MEK–ERK, thereby affecting T cell biological functions, 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 granzyme B (Gzm B) production, thus 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 T cell immune response to tumor antigens.

[0004] PD-1 / PD-L1 inhibitors include antibody-based and non-antibody-based inhibitors. Monoclonal antibody drugs have made significant progress in the clinical treatment of various cancers, but they also have certain limitations, such as low response rates, high cost, and inconvenient storage and transportation. In contrast, small molecule inhibitors have short half-lives and can avoid serious immune-related adverse events. Furthermore, small molecule inhibitors are easier to transport and store, have better stability, and superior membrane permeability, which is more beneficial for future clinical treatment. Currently, 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 antibiotic used to treat fungal skin infections. In vitro and in vivo studies have shown that luliconazole is highly effective against Aspergillus and Candida. Currently, there are no reports regarding the anti-colon cancer effects of luliconazole. Summary of the Invention

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

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides the use of ruliconazole as a PD-1 inhibitor in the preparation of a medicament 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 eyetissues 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 the application of ruliconazole as a PD-1 inhibitor in the preparation of a drug for treating colon cancer, wherein ruliconazole is the sole active ingredient in the application.

[0012] The drug used to treat colon cancer is a drug that inhibits 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; it is a drug that promotes the secretion of granzyme B in CT26 cells.

[0014] The present invention also provides a pharmaceutical formulation made of ruliconazole and medically acceptable excipients.

[0015] The dosage forms of the pharmaceutical preparations include injections, capsules, tablets, granules, pills, microcapsule preparations, microsphere preparations, and nano-preparations.

[0016] Luliconazole directly binds to PD-1, inhibiting the binding of PD-L1 to PD-1 in the tumor microenvironment, thereby relieving the phosphorylation levels of AKT and ERK proteins in the suppressed TCR downstream signaling pathway of T cells, and promoting the expression of cytokines IL-2 and IFN-γ. Furthermore, luliconazole increases CD4+ expression in tumor tissues of colon cancer-bearing mice. + With CD8 + The number of T cells was significantly increased, and the expression of the tumor cell killing factor granzyme B was enhanced, thus increasing CD8+. + T-cell function, thereby playing a role in the treatment of colon cancer.

[0017] A second aspect of the present invention provides the use of ruliconazole in the preparation of PD-1 inhibitors.

[0018] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0019] This invention provides luliconazole as a novel small molecule inhibitor of PD-1. The inhibitory effect of luliconazole on PD-1 / PD-L1 binding was detected by homogeneous time-resolved fluorescence (HTRF), and subsequently confirmed by surface plasmon resonance (SPR) technology. The specific binding of luliconazole to PD-1 protein and inhibition of PD-1 / PD-L1 binding were then demonstrated. In vitro, using a co-culture cell model of T cell immunosuppression induced by PD-1 / PD-L1 interaction, it was confirmed 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 induced by PD-1 / PD-L1 interaction. In vivo, using a mouse model of colon cancer xenografts, it was confirmed that luliconazole can increase CD4+ in tumor tissue of tumor-bearing mice. + T, CD8 + The infiltration of T lymphocytes promotes the secretion of granzyme B, thereby enhancing the function of T lymphocytes, killing tumor cells, and inhibiting the growth of colon cancer.

[0020] The luliconazole of this invention can treat colon cancer by promoting the infiltration of T lymphocytes and the secretion of granzyme B in tumor tissue. The luliconazole of this invention can inhibit colon cancer growth, thereby treating or improving 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. Attached Figure Description

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

[0022] Figure 2 This is a schematic diagram showing the results of SPR analysis of the binding affinity between ruliconazole and PD-1 protein.

[0023] Figure 3 This is a schematic diagram showing the effect of ruliconazole on the viability of Jurkat cells in a co-culture system.

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

[0025] Figure 5 This is a schematic diagram showing the effect of ruliconazole on T lymphocyte subsets in colon cancer-bearing mice.

[0026] Figure 6 A schematic diagram illustrating the effect of ruliconazole on granzyme B expression levels in colon cancer-bearing mice as detected by immunohistochemistry. Detailed Implementation

[0027] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting 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 homogeneous (liquid-phase) systems. This study used a PD-1 / PD-L1 HTRF kit to investigate the effect of luriconazole on PD-1 / PD-L1 interaction. A 1 mM luriconazole solution was prepared and serially diluted 3-fold to a minimum concentration of 0.05 μM, resulting in drug solutions of 1000 μM, 333 μM, 111 μM, 37 μM, 12 μM, 4.12 μM, 1.37 μM, 0.47 μM, and 0.05 μM. The reaction was performed according to the kit instructions, and the absorbance at 665 nm and 620 nm was measured using a microplate reader. The inhibitory effect of luriconazole on PD-1 / PD-L1 binding was calculated based on the 665 nm / 620 nm values. Results are shown below. Figure 1 As shown, Figure 1 This diagram illustrates the results of HTRF (High-Temperature Resonance Fluid Reduction) detection of the inhibitory effect of ruliconazole on PD-1 / PD-L1 binding, where A represents the principle of the HTRF experiment. The PDL1 protein is labeled with Tag1, and the PD1 protein with Tag2. Anti-Tag1-Eu and Anti-Tag2-XL665 antibodies bind to the two tags, respectively. After binding, the distance between Eu and XL665 decreases, and laser excitation of Eu causes it to emit light, transferring energy to XL665, thus causing it to glow. Adding a compound blocks the binding of PD1 and PDL1, increasing the distance between Eu and XL665, preventing energy transfer, and XL665 does not emit light. The signal detected at 620 nm is emitted by Anti-Tag1-Eu linked to PD-L1, while the signal detected at 665 nm is the signal after the energy resonance transfer following PD-L1 binding with PD-1. The signal ratio at 665 nm / 620 nm reflects the binding status of the PD-L1 ligand to the PD-1 receptor throughout the system. B is a schematic diagram of HTRF detection results, showing the IC50 of luteconazole inhibiting PD-1 / PD-L1 binding. 50 The fluorescence intensity was 27.3 μM. At lower concentrations of luriconazole, the fluorescence intensity was highest, indicating that the PD-L1 ligand in the solution bound tightly to the PD-1 receptor. However, as the concentration of luriconazole increased, the fluorescence intensity continuously decreased, indicating that the binding of PD-L1 to PD-1 in the solution was inhibited, and this inhibitory effect was caused by the drug. Therefore, it can be concluded that luriconazole can inhibit the PD-1 / PD-L1 interaction.

[0031] Example 2

[0032] Luliconazole specifically binds to PD-1

[0033] PD-1 protein was prepared using 10 mM sodium acetate buffer solutions at different pH values ​​(5.5, 5.0, 4.5) to a final concentration of 50 μg / ml. The optimal pH was determined based on the response value (RU) of PD-1 protein at different pH values, and the protein was coupled overnight. Luriconazole solutions (concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μM) were prepared using PBS solution (containing 5% DMSO) as the buffer. The injection rate was set to 30 μl / min, and the binding and dissociation time was set to 60 s. Data processing was performed using Biacore T200 software to obtain binding affinity curves fitted by single-site interactions, the interaction mode of luriconazole and PD-1, and the kinetic constant (Kd = 23.62 μM). Results are shown below. Figure 2 As shown, Figure 2 This diagram illustrates the binding affinity of luliconazole to PD-1 protein using SPR analysis. A shows the sensor plots of the interaction between different concentrations of luliconazole and PD-1 protein; B is a schematic diagram of the binding affinity curves of luliconazole and PD-1 obtained by fitting single-site interactions. HTRF experiments confirmed that luliconazole can inhibit the binding of PD-1 to PD-L1, thus suppressing the PD-1 / PD-L1 interaction. Subsequently, SPR analysis showed that luliconazole specifically binds to the PD-1 protein; therefore, luliconazole is a PD-1 inhibitor that can inhibit the PD-1 / PD-L1 interaction. The following analysis uses Real-time qPCR and Western blot experiments to analyze the effects of luliconazole as a PD-1 inhibitor on T cells.

[0034] Example 3

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

[0036] Jurkat cells stably expressing PD-1 and a luciferase reporter gene carrying an element for activating T cell nuclear factor responses, and CHO-K1 cells stably expressing a human T cell-activating CD3 monoclonal antibody (OKT3) and PD-L1, were co-cultured to create a co-culture cell model of T cell immunosuppression caused by PD-1 / PD-L1 interaction. In this co-culture model, OKT3 activated Jurkat cells, promoting the synthesis of cytokines IL-2 and IFN-γ. PD-1, upon binding to PD-L1, inhibited related signaling pathways in Jurkat cells, such as the PI3K-AKT and RAS-MEK-ERK signaling pathways, leading to Jurkat cell dysfunction and inhibiting cytokine synthesis in T cells. However, when a PD-1 / PD-L1 inhibitor was added to the co-culture cell model, the binding of PD-L1 to PD-1 was blocked, preventing PD-1 from transmitting inhibitory signals to T cells, thus restoring signaling pathways in T cells. Therefore, in co-culture cell models, by analyzing the phosphorylation levels of AKT and ERK proteins and the expression changes of cytokines IFN-γ and IL-2 in the downstream signaling pathway of PD-1 / PD-L1, the ability of compounds to inhibit PD-1 / PD-L1 interaction can be reflected.

[0037] In an in vitro co-culture system, after treatment with 2.5, 5, and 10 μM ruliconazole for 24 h, the expression levels of IL-2 and IFN-γ mRNA in Jurkat cells were detected by Real-time qPCR, and the expression levels of p-AKT and p-ERK proteins in Jurkat cells were detected by Western blot. Results are as follows: Figure 3 As shown, Figure 3The first image shows the effect of ruliconazole on Jurkat cell viability in a co-culture system. Image A shows the changes in IL-2 and IFN-γ mRNA expression levels in Jurkat cells. As can be seen, ruliconazole significantly increased the expression levels of IL-2 and IFN-γ mRNA in Jurkat cells in the co-culture system (P<0.05). This suggests that ruliconazole can reverse the immunosuppression of Jurkat cells caused by PD-1 / PD-L1 interaction and reactivate Jurkat cells. Image B shows the changes in p-AKT and p-ERK protein expression levels in Jurkat cells. The upper left image in B is a typical example of the effect of different concentrations of ruliconazole (2.5, 5, 10 μM) on p-AKT protein expression levels in Jurkat cells detected by Western blot in a co-culture cell model. Protein expression levels were determined by comparing the intensity of the band colors. As shown in the figures, the p-AKT protein band in the Jurkat cells of the model group was very faint, indicating that downstream cell signaling pathways were inhibited after PD-1 / PD-L1 interaction, preventing AKT protein phosphorylation. Compared to the model group, the grayscale of the phosphorylated AKT protein band in Jurkat cells increased after the addition of luliconazole, while there was no significant difference in the protein bands of total AKT and the internal control protein GAPDH, indicating that the expression level of p-AKT protein in Jurkat cells increased under the treatment of luliconazole. The upper right figure in B is a figure obtained by analyzing the grayscale values ​​of the Western Blot experimental results using ImageJ and then statistically analyzing the results of the three experiments. As shown in the figure, the expression level of p-AKT protein in Jurkat cells increased significantly after the addition of luliconazole compared to the model group. Similarly, the lower left and lower right figures in B show that the expression level of p-AKT protein in Jurkat cells increased significantly after the addition of luliconazole compared to the model group. The above results suggest that ruliconazole 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 a mouse model of colon cancer xenograft: CT26 cells in good growth condition were collected and the cell concentration was adjusted to 1×10⁻⁶ cells with sterile PBS. 7 Cells / ml. 200 μl of cell suspension was subcutaneously injected into the dorsal side of the right forelimb of Balb / c mice. Tumor growth was continuously observed after inoculation, and the tumor was monitored until it reached 50 mm². 3Forty-two mice were divided into the following groups based on their body weight: solvent control group (n=12), low-dose luriconazole group (10 mg / kg, n=12), high-dose luriconazole group (20 mg / kg, n=12), and positive control group (PD-L1 antibody 10 mg / kg, n=6). The low-dose and high-dose luriconazole groups received intraperitoneal injections of 10 mg / kg and 20 mg / kg luriconazole (0.2 ml / 30 g), respectively. The solvent control group received an intraperitoneal injection of the corresponding volume of the solvent. Administration was once daily for ten consecutive days. The positive control group received an intraperitoneal injection of 10 mg / kg PD-L1 antibody every two days for ten days. After grouping, mouse body weight and tumor volume were measured every two days.

[0041] After treatment, the mouse tumors were isolated, weighed, and photographed for recording. The results are as follows: Figure 4 As shown, Figure 4 The diagram illustrates the effect of luriconazole on tumor growth in colon cancer-bearing mice. A shows an image of the tumor in the mice, indicating that the tumors in the low-dose and high-dose luriconazole groups were significantly smaller than those in the solvent control group. B shows the tumor weight in the mice; compared to the solvent control group, the tumor weights in the low-dose (10 mg / kg), high-dose (20 mg / kg), and positive control groups (PD-L1 antibody 10 mg / kg) colon cancer-bearing mice were significantly lighter (P<0.05). C shows the tumor growth curve in the mice; as the inoculation time increased, the tumor volume in the solvent control group gradually increased, exhibiting exponential growth in the later stages, while the tumor volume under drug treatment increased slowly. Sixteen days after CT26 cell inoculation, compared with the solvent control group, the tumor volume of the low-dose luriconazole group (10 mg / kg), the high-dose luriconazole group (20 mg / kg), and the positive control group (PD-L1 antibody 10 mg / kg) was significantly reduced (P<0.05). These data all indicate that luriconazole can significantly inhibit the growth of colon cancer in mice and reduce tumor weight.

[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 CD4 in the tumor tissues of mice in each group in Example 4. + T cells, CD8 +Changes in T cell count were observed. CD4 / CD8 antibody (prepared with PBS containing 10% goat serum at a dilution of 1:100) was added to paraffin sections and incubated overnight. HRP secondary antibody was then added and incubated at room temperature for 30 minutes. DAB staining was performed under a microscope to stain the cell nuclei in the tissue sections. Finally, images were acquired and observed under a fluorescence microscope. The staining area of ​​positive regions in the images was observed, and ImageJ was used to quantitatively analyze the positive regions, obtaining the percentage of positive areas. The results were then summarized and statistically analyzed to observe CD4+ in tumors of tumor-bearing mice. + T, CD8 + Changes in T cell count, results as follows Figure 5 As shown, Figure 5 This diagram illustrates the effect of ruliconazole on T lymphocyte subsets in colon cancer-bearing mice. In Figure A, the left image shows typical results of CD4 and CD8 immunohistochemical staining, while the right image shows the quantitative analysis results after immunohistochemical staining. As shown in the figure, compared with the solvent control group, ruliconazole treatment significantly increased the number of CD8+ T cells in mouse tumor tissue (P<0.05). The number of CD4+ T cells also increased, but not to a statistically significant degree. Flow cytometry was used to detect CD4+ T cells in tumors of tumor-bearing mice. + T, CD8 + Changes in T cell count, results as follows Figure 5 As shown in Figure B, the left image is a typical graph of CD4 and CD8 flow cytometry results, with the percentage of CD4 / CD8 cells indicated by numbers. The right image is a statistical analysis graph of the percentage of CD4 / CD8 cells. The graph shows that, compared with the solvent control group, after ruliconazole treatment, CD4 levels in the tumor tissue of colon cancer-bearing mice were significantly reduced. + T, CD8 + The number of T cells increased significantly (P<0.05). These data indicate that ruliconazole can significantly increase the number of T lymphocytes in tumor tissue.

[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. The results are as follows: Figure 6 As shown, Figure 6This diagram illustrates the effect of immunohistochemical detection of granzyme B expression levels in tumors of colon cancer-bearing mice. The diagram includes a typical graph showing changes in granzyme B protein content in tumors of tumor-bearing mice as detected by immunohistochemistry, and a statistical graph showing the results after quantitative analysis. As can be seen from the graph, compared with the solvent control group, the low-dose (10 mg / kg) and high-dose (20 mg / kg) groups of luriconazole showed significantly more positive areas of granzyme B staining in the pathological sections. Quantitative analysis of the staining results showed that, compared with the solvent control group, the expression level of granzyme B in tumor tissues of colon cancer-bearing mice was significantly increased in the low-dose (10 mg / kg) and high-dose (20 mg / kg) groups (P < 0.05). This indicates that luriconazole can enhance CD8 expression. + T cell function plays an anti-tumor role.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Use of luliconazole as a PD-1 inhibitor in the manufacture of a medicament for treating colon cancer.

2. Use of luliconazole as a PD-1 inhibitor for the manufacture of a medicament for the treatment of colon cancer according to claim 1, characterized in that, The structure of the luliconazole is shown as follows:

3. Use of luliconazole as a PD-1 inhibitor for the manufacture of a medicament for the treatment of colon cancer according to claim 1, characterized in that, The luliconazole is used as the only active ingredient in the use.

4. Use of luliconazole as a PD-1 inhibitor for the manufacture of a medicament for the treatment of colon cancer according to claim 1, characterized in that, The medicament for treating colon cancer is a medicament for inhibiting the growth of CT26 cells.

5. Use of luliconazole as a PD-1 inhibitor for the manufacture of a medicament for the treatment of colon cancer according to claim 1, characterized in that, The medicament for treating colon cancer is a medicament for promoting the infiltration of T lymphocytes in CT26 cells; a medicament for promoting the secretion of granzyme B in CT26 cells.

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