Application of combination of AKR1B1 inhibitor and immune checkpoint inhibitor in preparation of medicine for treating tumors and medicine composition of AKR1B1 inhibitor and immune checkpoint inhibitor

Abstract

The invention relates to the technical field of tumor immunotherapy, in particular to application of an AKR1B1 inhibitor and an immune checkpoint inhibitor in preparation of antitumor drugs and a pharmaceutical composition containing the AKR1B1 inhibitor and ICIs. After the AKR1B1 inhibitor is combined with the immune checkpoint inhibitor, the following technical effects are achieved: 1, the tumor growth is obviously inhibited: in a mouse breast cancer model and a lung cancer model, the tumor growth inhibition rate of the epalrestat single drug is about 30%; the inhibition rate of a PD-1 single drug is about 30%-40%; the inhibition rate of combined treatment is remarkably improved to 65%, and an obvious synergistic anti-tumor effect is shown. 2, the anti-tumor immune response is enhanced, and the activity of CD8 positive T cells can be remarkably improved by the epalrestat single drug; the combined treatment can improve the proportion of cytokines (such as granzyme B and interferon g) secreted by CD8 positive T cells in tumors. 3, the safety is high: under the administration dosage, no obvious drug toxicity is observed in the heart, liver, spleen, kidney and lung of the mouse;

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, specifically to the application of an Aldo-Keto Reductase Family 1 Member B1 (AKR1B1) inhibitor in combination with immune checkpoint inhibitors (ICIs) in the preparation of antitumor drugs, and to pharmaceutical compositions comprising the AKR1B1 inhibitor and ICIs. Background Technology

[0002] Tumor immunotherapy, especially immune checkpoint inhibitors such as programmed death receptor 1 (PD-1) / programmed death ligand 1 (PD-L1), has achieved great success in the treatment of various malignancies. However, this therapy still faces many challenges, including key bottlenecks such as low overall response rates, limited effective targets, heavy dependence on PD-1 / PD-L1 drugs, and the proliferation of PD-1 / PD-L1 drugs. The overall efficacy of ICIs alone is typically less than 25%, with only a small percentage of patients benefiting. Even in patients sensitive to ICIs, primary and acquired resistance exist.

[0003] Previous studies have primarily focused on the role of AKR1B1 in the metabolic dominance of tumor cells, but its key functions in immune cells have not been explored in depth. Therefore, there is an urgent need to develop novel combination therapy strategies to overcome resistance to ICIs, improve the efficacy of immunotherapy, and thus achieve more significant anti-tumor activity.

[0004] Current immune checkpoint inhibitor monotherapy has low response rates, especially in lung and breast cancer, where over 60% of patients do not respond to PD-1 / PD-L1 inhibitors. Existing methods fail to adequately reverse the immunosuppressive tumor microenvironment. Furthermore, current AKR1B1 targeting studies primarily focus on tumor cells themselves or drug resistance issues. For example, Chinese patent CN201610004380.5 discloses the application of an aldehyde-ketone reductase 1B1 inhibitor in the preparation of an anti-breast cancer drug; Chinese patent CN202010498449.0 discloses the application of AKR1B inhibitors in combination with lung cancer targeted drugs in the preparation of drugs for treating drug-resistant lung cancer. However, these existing technologies do not explore the function of AKR1B1 inhibitors in immune cells. Therefore, existing technologies cannot effectively address the problems of poor immunotherapy response and drug resistance. Summary of the Invention

[0005] The first primary objective of this invention is to overcome the shortcomings of the prior art and provide an application of AKR1B1 inhibitor combined with immune checkpoint inhibitors in the preparation of drugs for treating tumors, aiming to enhance the anti-tumor immune effect, improve the response rate of ICIs, and solve their drug resistance problem.

[0006] Another primary objective of this invention is to provide a kit or combination of drugs consisting of an AKR1B1 inhibitor and an immune checkpoint inhibitor for the treatment of patients who are insensitive to or resistant to immunotherapy, particularly those with lung cancer and breast cancer.

[0007] The technical solution of this invention to solve the technical problem is as follows:

[0008] In a first aspect of the invention, the use of an AKR1B1 inhibitor in combination with an immune checkpoint inhibitor in the preparation of a therapeutic drug for tumors is provided, wherein the treatment is for cases of poor response to immunotherapy and drug resistance.

[0009] Further, the AKR1B1 inhibitor is selected from sorbinil, zopolrestat, or epalrestat, or a pharmaceutically acceptable salt of any one of them. Preferably, the AKR1B1 inhibitor is epalrestat or a pharmaceutically acceptable salt thereof.

[0010] Further, the immune checkpoint inhibitor is a blocker targeting T-cell inhibitory receptors, such as programmed death receptor 1 (PD-1) inhibitors, programmed death ligand 1 (PD-L1) inhibitors, and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors. Preferably, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. More preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0011] In some preferred embodiments, the anti-PD-1 antibody is selected from one or more of the following anti-PD-1 monoclonal antibodies: nivolumab, pembrolizumab, cimiplimab, toripalimab, sintilimab, camrelizumab, or equivalent antibodies thereof.

[0012] In some other preferred embodiments, the anti-PD-L1 antibody is selected from one or more of the following anti-PD-L1 monoclonal antibodies: atezolizumab, durvalumab, benmelstobart, or an equivalent antibody thereof.

[0013] Furthermore, the tumor is preferably lung cancer or breast cancer. AKR1B1 inhibitors combined with immune checkpoint inhibitors can treat patients who are insensitive to or resistant to immunotherapy, especially those with lung cancer and breast cancer.

[0014] The combination therapy described in this application refers to the simultaneous, separate, or sequential administration of an AKR1B1 inhibitor and an immune checkpoint inhibitor during patient treatment, with the administration routes being the same or different. In one embodiment of the invention, the combination therapy involves simultaneously administering epalrestat and an immune checkpoint inhibitor within the same treatment cycle, with both the AKR1B1 inhibitor and the immune checkpoint inhibitor being administered at therapeutically effective doses.

[0015] In a second aspect of the invention, the use of an AKR1B1 inhibitor in the preparation of a medicament for enhancing the therapeutic efficacy of immune checkpoint inhibitors in treating tumors is provided. Both the AKR1B1 inhibitor and the immune checkpoint inhibitor are used at therapeutically effective doses.

[0016] Further, the AKR1B1 inhibitor is selected from sorbinil, zopolrestat, or epalrestat, or a pharmaceutically acceptable salt of any one of them. Preferably, the AKR1B1 inhibitor is epalrestat or a pharmaceutically acceptable salt thereof.

[0017] Further, the immune checkpoint inhibitor is a blocker targeting T-cell inhibitory receptors, such as programmed death receptor 1 (PD-1) inhibitors, programmed death ligand 1 (PD-L1) inhibitors, and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors. Preferably, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. More preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0018] In some preferred embodiments, the anti-PD-1 antibody is selected from one or more of the following anti-PD-1 monoclonal antibodies: nivolumab, pembrolizumab, cimiplimab, toripalimab, sintilimab, camrelizumab, or equivalent antibodies thereof.

[0019] In some other preferred embodiments, the anti-PD-L1 antibody is selected from one or more of the following anti-PD-L1 monoclonal antibodies: atezolizumab, durvalumab, benmelstobart, or an equivalent antibody thereof.

[0020] Furthermore, the tumor is preferably lung cancer or breast cancer.

[0021] In a third aspect of the invention, an antitumor kit is provided, the kit comprising: a formulation containing an AKR1B1 inhibitor; and a formulation containing an immune checkpoint inhibitor. Both the AKR1B1 inhibitor and the immune checkpoint inhibitor are administered at therapeutically effective doses.

[0022] Further, the AKR1B1 inhibitor is selected from sorbinil, zopolrestat, or epalrestat, or a pharmaceutically acceptable salt of any one of them. Preferably, the AKR1B1 inhibitor is epalrestat or a pharmaceutically acceptable salt thereof.

[0023] Further, the immune checkpoint inhibitor is a blocker targeting T-cell inhibitory receptors, such as programmed death receptor 1 (PD-1) inhibitors, programmed death ligand 1 (PD-L1) inhibitors, and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors. Preferably, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. More preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0024] In some preferred embodiments, the anti-PD-1 antibody is selected from one or more of the following anti-PD-1 monoclonal antibodies: nivolumab, pembrolizumab, cimiplimab, toripalimab, sintilimab, camrelizumab, or equivalent antibodies thereof.

[0025] In some other preferred embodiments, the anti-PD-L1 antibody is selected from one or more of the following anti-PD-L1 monoclonal antibodies: atezolizumab, durvalumab, benmelstobart, or an equivalent antibody thereof.

[0026] In a fourth aspect of the invention, a pharmaceutical composition is provided comprising an AKR1B1 inhibitor and an immune checkpoint inhibitor. In the composition, both the AKR1B1 inhibitor and the immune checkpoint inhibitor are used at therapeutically effective doses.

[0027] Further, the AKR1B1 inhibitor is selected from sorbinil, zopolrestat, or epalrestat, or a pharmaceutically acceptable salt of any one of them. Preferably, the AKR1B1 inhibitor is epalrestat or a pharmaceutically acceptable salt thereof.

[0028] Further, the immune checkpoint inhibitor is a blocker targeting T-cell inhibitory receptors, such as programmed death receptor 1 (PD-1) inhibitors, programmed death ligand 1 (PD-L1) inhibitors, and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors. Preferably, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. More preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0029] In some preferred embodiments, the anti-PD-1 antibody is selected from one or more of the following anti-PD-1 monoclonal antibodies: nivolumab, pembrolizumab, cimiplimab, toripalimab, sintilimab, camrelizumab, or equivalent antibodies thereof.

[0030] In some other preferred embodiments, the anti-PD-L1 antibody is selected from one or more of the following anti-PD-L1 monoclonal antibodies: atezolizumab, durvalumab, benmelstobart, or an equivalent antibody thereof.

[0031] Furthermore, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients are selected from one or more of diluents, binders, disintegrants, lubricants, suspending agents, emulsifiers, solubilizers, stabilizers, pH adjusters, isotonic agents, preservatives, flavoring agents, and coloring agents. The dosage form of the drug may be tablets, capsules, granules, or oral liquids suitable for oral administration, or injections, lyophilized powder injections, etc., suitable for parenteral administration.

[0032] In a fifth aspect of the invention, the use of AKR1B1 as a biomarker in the preparation of a diagnostic kit for predicting the efficacy of tumor immunotherapy is provided. The efficacy of tumor immunotherapy in patients can be predicted by detecting the expression level of AKR1B1 in subject samples. Patients with high AKR1B1 expression have more severe disease progression stages.

[0033] Furthermore, the tumor is preferably lung cancer or breast cancer.

[0034] This invention demonstrates through experiments that the combination of an AKR1B1 inhibitor and an immune checkpoint inhibitor exhibits the following significant synergistic antitumor effects:

[0035] 1. Significantly inhibits tumor growth: In mouse models of breast and lung cancer, epalrestat monotherapy showed a tumor growth inhibition rate of approximately 30%; PD-1 monotherapy showed an inhibition rate of approximately 30%-40%; while the combination therapy significantly increased the inhibition rate to 65%, demonstrating a clear synergistic anti-tumor effect (see reference). Figure 1 B Figure 2 B provides corroborating evidence.

[0036] 2. Enhanced anti-tumor immune response: Epalrestat monotherapy can significantly increase the activity of CD8-positive T cells. Combination therapy can increase the proportion of cytokines (such as granzyme B and interferon g) secreted by CD8-positive T cells within the tumor (see reference). Figure 1 F, Figure 2 (F provides corroboration). The above results demonstrate that epalrestat can serve as a potentiator for tumor immunotherapy, enhancing the efficacy of immune checkpoint inhibitors (such as PD-1 antibodies) in treating tumors.

[0037] 3. High safety profile: At the dosage used, no significant drug toxicity was observed in the heart, liver, spleen, kidneys, and lungs of mice (see reference). Figure 1 G provides corroborating evidence. Attached Figure Description

[0038] Figure 1 The effect of the combination of epalrestat and PD-1 antibody on tumor progression in a mouse orthotopic breast cancer model. Specifically:

[0039] A is a schematic diagram of the grouping and drug administration process in an orthotopic mouse model of breast cancer.

[0040] B shows a comparison of tumor growth size in orthotopic breast cancer xenografts from 4T1 cell homologous mice in different treatment groups.

[0041] C represents the weight comparison of orthotopic breast cancer xenografts from 4T1 cells in different treatment groups.

[0042] Image D shows tumors of orthotopic breast cancer xenografts from 4T1 cell-derived mice in different treatment groups.

[0043] E represents a comparison of body weight in mice with different treatment groups and in situ mammary gland models derived from 4T1 cells.

[0044] F represents a comparison of the levels of granzyme B and interferon g secreted by CD8-positive T cells infiltrating the tumors of 4T1 cell homologous mouse orthotopic breast cancer xenografts in different treatment groups.

[0045] G is a representative immunohistochemical image of the heart, lungs, liver, spleen, and kidneys in an orthotopic mouse model of breast cancer treated with epalrestat and in a solvent control group, demonstrating that the drug has no therapeutic toxicity.

[0046] Figure 2 The effect of the combination of epalrestat and PD-1 antibody on tumor progression in a mouse lung adenocarcinoma model. Specifically:

[0047] A is a schematic diagram of the grouping and drug administration process in a mouse model of lung adenocarcinoma.

[0048] B shows a comparison of tumor size in LLC cell-derived mouse lung adenocarcinoma xenografts from different treatment groups.

[0049] C represents a comparison of tumor weight in LLC cell-derived mouse lung adenocarcinoma xenografts from different treatment groups.

[0050] Image D shows tumors of LLC cell-derived mouse lung adenocarcinoma xenografts from different treatment groups.

[0051] E represents a comparison of body weight in mice with LLC cell homologous lung adenocarcinoma models from different treatment groups.

[0052] F represents a comparison of the levels of granzyme B secreted by CD8-positive T cells infiltrating the tumors of LLC cell-derived mouse lung adenocarcinoma xenografts in different treatment groups.

[0053] Figure 3 Correlation analysis of AKR1B1 with disease progression stage and prognosis in patients. Among them:

[0054] A is a bar chart showing the proportion of patients in different clinical stages (stage I, stage II, stage III, and stage IV) after grouping them into low-expression and high-expression AKR1B1 groups in a breast cancer patient cohort. This chart is used to show the relationship between AKR1B1 expression levels and tumor stage distribution.

[0055] Figure B is a comparative analysis of AKR1B1 expression levels in normal and tumor tissues of lung cancer patients, used to illustrate the abnormal expression characteristics of AKR1B1 in tumor tissues.

[0056] C represents the distribution of the proportion of patients in different clinical stages (stage I, stage II, stage III, and stage IV) in the lung cancer patient cohort after being divided into low-expression and high-expression groups of AKR1B1, to verify the correlation between AKR1B1 expression level and disease stage.

[0057] D represents the Kaplan-Meier survival analysis curve for overall survival (OS) of breast cancer patients, stratified according to the high and low AKR1B1 expression groups. The horizontal axis represents the follow-up time (months), and the vertical axis represents the survival probability, used to assess the relationship between AKR1B1 expression level and overall survival outcome.

[0058] E represents the Kaplan-Meier survival analysis curve for overall survival (OS) of lung cancer patients, stratified according to AKR1B1 high expression group and low expression group. The horizontal axis represents follow-up time (days), and the vertical axis represents survival probability, used to assess the relationship between AKR1B1 expression level and overall survival outcome of patients. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0060] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields.

[0061] The source of the experimental materials involved in this invention is explained as follows:

[0062] BALB / C and C57BL / 6 mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd.; the mouse breast cancer cell line 4T1 and mouse lung cancer cell line LLC were purchased from the Cell Bank of the Chinese Academy of Sciences; Epalrestat was purchased from Selleck Chemicals with a purity of 99.9%; the anti-mouse PD-1 antibody was purchased from BioX Cell, product name InVivoMAb anti-mouse PD-1 (CD279); for solvent controls, dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, PEG300 from Selleck Chemicals, and Tween-80 from Aladdin Reagent Co., Ltd.; flow cytometry-related antibodies (CD8, granzyme B, interferon g) and erythrocyte lysis buffer were purchased from Biolegend; routine animal experimental and data analysis equipment, such as electronic balances, were provided by the Shanghai Jiao Tong University School of Medicine platform.

[0063] Example 1: Effect of the combination of epalrestat and PD-1 antibody on tumor progression in a mouse orthotopic breast cancer model.

[0064] 1.1 Reagents and Materials

[0065] BALB / c female mice, 7 weeks old, weighing 18–23 g; triple-negative breast cancer cell line 4T1; Matrigel; epalrestat (AKR1B1 inhibitor); anti-mouse PD-1 monoclonal antibody; solvent controls (5% DMSO, 5% PEG300, 2% Tween 80, 88% water); vernier calipers; mouse tumor tissue digestion kit (McTeinen Biotech GmbH, Germany); flow cytometry-related antibodies (CD8, granzyme B, interferon g); electronic balance and other routine animal experimental and data analysis equipment.

[0066] 1.2 Experimental methods and data processing methods:

[0067] Construction of a mouse orthotopic breast cancer model: Seven-week-old female BALB / c mice, weighing 18-23 grams, were selected. The 4T1 triple-negative breast cancer cell line in logarithmic growth phase was used, and the cell number was adjusted to 2 × 10⁻⁶ cells / mL. 5 (Each mouse) was then mixed with matrix gel at a 1:1 ratio. After anesthetizing the mice, 0.05 mL of the cell mixture was in situ injected into the fourth pair of fat pads on the right side of the mice. Approximately 7 days after inoculation, mice with successfully established tumors of about 4 mm in diameter were selected for subsequent experiments. The mice were randomly divided into groups of 5.

[0068] Grouping and dosing regimen:

[0069] Group 1: Solvent Control Group. A solvent control was provided, which consisted of 5% (v / v) dimethyl sulfoxide (DMSO), 5% (v / v) PEG300, 2% (v / v) Tween 80 and 88% (v / v) water.

[0070] Group 2: Epalrestat monotherapy group (75 mg / kg, once daily, orally).

[0071] Group 3: Anti-mouse PD-1 antibody monotherapy group (200 μg / mouse, twice a week, intraperitoneal administration).

[0072] Group 4: Combined administration of epalrestat and anti-mouse PD-1 antibody (epalrestat and anti-mouse PD-1 antibody were administered simultaneously during the same administration cycle, with epalrestat at a dose of 75 mg / kg, once daily, orally; and anti-mouse PD-1 antibody at a dose of 200 μg / mouse, twice weekly, intraperitoneally).

[0073] Tumor growth monitoring and volume calculation: The day of tumor cell inoculation was designated as day 0. Every three days thereafter, the major and minor diameters of the tumor were measured using calipers. The tumor volume was calculated using the following formula. Tumor volume data at each time point were summarized and statistically compared using repeated measures two-way ANOVA.

[0074] Tumor volume (mm) 3 = Tumor long diameter (mm) × Tumor short diameter 2 (mm) 2 ) / 2

[0075] Flow cytometry analysis of immune infiltration: Tumor tissue samples were collected from mice at the experimental endpoint. After removing visible fat, fibrous tissue, and necrotic areas, the tumor tissue was cut into small pieces. A commercially available tumor tissue dissociation kit was used to enzymatically digest the tumor tissue, followed by mechanical dissociation to prepare a tumor-derived single-cell suspension.

[0076] After digestion, the resulting cell suspension was filtered and washed, and erythrocyte components were removed via erythrocyte lysis. The obtained cells were resuspended in flow cytometry staining buffer, the cell concentration was adjusted, and fluorescently labeled primary antibody was added. The cells were then incubated at low temperature in the dark. After incubation, the cells were washed and resuspended in buffer for flow cytometry analysis.

[0077] During flow cytometry data acquisition, CD45 cells are first delineated from the live cell population. + Immune cell populations, further at CD45 + CD3 + Analysis of CD8 in T cell population +The proportion and quantity of cytokines secreted by T cells were analyzed. Data from each group were summarized and compared using statistical methods to evaluate the effects of different drug treatments on intratumoral CD8+. + The influence of T cell secretion of cytokines.

[0078] 1.3 Experimental Results:

[0079] Table 1-1 shows the changes in tumor volume (mm) in each group of mice. 3 (, Mean ± SD).

[0080]

[0081] Table 1-2 shows the endpoint tumor weight (Mean ± SD) for each group of mice.

[0082]

[0083] As shown in Table 1-1 and Figure 1 As shown in B, compared with the solvent control group, both the epalrestat monotherapy group and the anti-PD-1 antibody monotherapy group could inhibit tumor volume growth to some extent, while the combined administration group had the most significant inhibitory effect on tumor growth. Statistical analysis showed that the combined administration had a significantly better anti-tumor effect than the single drug (*p < 0.01).

[0084] Endpoint tumor weight analysis results (see Table 1-2) Figure 1 C and Figure 1 D) Consistent with the trend of tumor volume change, the tumor weight was significantly reduced in the combined treatment group.

[0085] Further tumor tissue analysis showed (see reference) Figure 1 F), epalrestat monotherapy significantly enhances intratumoral CD8. + The ability of T cells to secrete granzyme B and interferon g; based on this, the combination therapy of epalrestat and anti-PD-1 antibody further significantly enhanced intratumoral CD8. + The levels of granzyme B and interferon g secretion in T cells suggest that combined therapy can more effectively enhance the function of cytotoxic T cells in the tumor microenvironment.

[0086] Toxicity assessment results show (for reference) Figure 1 (E and 1G), at the above-mentioned dosages, the weight changes of mice in each group were stable, and no obvious drug toxicity was observed in the heart, lungs, liver, spleen and kidneys.

[0087] The efficacy of epalrestat combined with anti-PD-1 antibody in a mouse model of breast cancer was evaluated based on the Coefficient of Drug Interaction (CDI). Using tumor weight as the endpoint, and after normalization with the solvent control group, the CDI value of the combined treatment group was calculated to be approximately 0.78 (<1), indicating that epalrestat and anti-PD-1 antibody exhibited a significant synergistic antitumor effect in the mouse model of breast cancer.

[0088] The above examples demonstrate that the combined use of the AKR1B1 inhibitor epalrestat and a PD-1 antibody can significantly inhibit tumor progression in a mouse orthotopic breast cancer model without increasing significant toxicity, and by enhancing CD8. + T-cell-mediated anti-tumor immune responses exert synergistic therapeutic effects.

[0089] Example 2: Effect of the combination of epalrestat and PD-1 antibody on tumor progression in a mouse lung cancer model.

[0090] 2.1 Reagents and Materials

[0091] C57BL / 6J female mice (7 weeks old); mouse lung cancer cell line LLC; PBS buffer; epalrestat; anti-mouse PD-L1 antibody; injection and gavage needles, vernier calipers, electronic balance.

[0092] 2.2 Experimental Methods and Data Processing

[0093] The experimental protocol (grouping, dosage, and administration method) was similar to that of Example 1, but the model was 7-week-old female C57BL / 6J mice subcutaneously inoculated with the LLC lung cancer cell line (5 × 10⁻⁶). 5 Lung cancer model constructed (dosing procedure as follows) Figure 2 (As shown in A).

[0094] 2.3 Experimental Results:

[0095] Table 2-1 shows the tumor volume changes in each group of mice (Mean ± SD):

[0096]

[0097] Table 2-2 shows the endpoint tumor weight (Mean ± SD) for each group of mice:

[0098]

[0099] like Figure 2 As shown in Figure A, an LLC lung cancer mouse model was successfully established and drug intervention was carried out according to the predetermined schedule.

[0100] Tumor volume monitoring results show (see Table 2-1 and...) Figure 2 (B) Compared with the solvent control group, both the AKR1B1 inhibitor monotherapy group and the anti-PD-L1 antibody monotherapy group showed a certain degree of tumor growth inhibition. However, the combined administration group showed the most significant tumor growth inhibition effect, with its tumor volume being significantly lower than that of either monotherapy group, and the difference was statistically significant.

[0101] Tumor weight was analyzed at the experimental endpoint (see Table 2-2). Figure 2 (C and 2D) The results showed that the tumor weight of mice in the combined administration group was significantly reduced, further validating the superiority of the combined treatment in reducing tumor burden.

[0102] At the above-mentioned dosage, the body weight of mice in each group remained stable. Figure 2 (E) No obvious drug toxicity was observed.

[0103] The efficacy of combined administration of epalrestat and anti-PD-1 antibody in a mouse model of lung cancer was evaluated based on the Coefficient of Drug Interaction (CDI). Using tumor weight as the endpoint, and after normalization with the solvent control group, the CDI value of the combined administration group was calculated to be approximately 0.90 (<1), indicating that epalrestat and anti-PD-1 antibody exhibited synergistic anti-tumor effects in this mouse tumor model.

[0104] Analysis of the tumor immune microenvironment showed (for reference) Figure 2 F), epalrestat alone can promote the production of granzyme B in tumor CD8⁺ T cells; in contrast, epalrestat combined with immune checkpoint inhibitors can further increase the overall expression level of granzyme in tumor CD8⁺ T cells, suggesting that combination therapy more effectively activates the cytotoxic effect of tumor-infiltrating CD8⁺ T cells.

[0105] The above results indicate that the combined use of AKR1B1 inhibitors and PD-L1 inhibitors can significantly inhibit tumor growth in an LLC lung cancer model and by enhancing intratumoral CD8. + T cell infiltration and cytotoxic function play a synergistic role in anti-tumor activity.

[0106] Example 3: Clinical cohort and patient prognostic analysis of AKR1B1 as a molecular marker

[0107] 3.1 Reagents and Materials

[0108] 1. Biological samples: Tumor tissue sample chips and corresponding normal tissue sample chips from triple-negative breast cancer or lung adenocarcinoma patients. The sample chips were sourced from Changsha Yaxiang Biotechnology Co., Ltd., with ethical inquiry number (E6KJ0RQY3QVIKU).

[0109] 2. Main reagents: tissue fixative and embedding reagents; antigen retrieval solution; blocking solution; primary antibody: anti-AKR1B1 antibody; fluorescently labeled secondary antibody; nuclear dyes.

[0110] 3. Major Instruments and Equipment: Tissue microtome; confocal microscope; image acquisition and analysis software (Qupath)

[0111] 3.2 Experimental Methods

[0112] Images were acquired for each experimental group under the same imaging conditions. Image analysis software was used to separate and quantify different fluorescence signals, and the number and proportion of AKR1B1-positive cells were statistically analyzed. All data are presented as mean values, and statistical methods were used for inter-group comparisons to assess the correlation between AKR1B1 expression levels and the tumor immune microenvironment and clinical outcomes.

[0113] 3.3 Experimental Results and Illustrations

[0114] Table 3-1 shows the statistical results of the disease progression stage ratio in the AKR1B1 high and low expression groups of breast cancer patients.

[0115]

[0116] Table 3-2 shows the statistical results of the disease progression stage ratio in the AKR1B1 high and low expression groups of lung cancer patients.

[0117]

[0118] This invention presents a systematic analysis of a clinical patient cohort. The results show that, in patients with triple-negative breast cancer and lung adenocarcinoma, after grouping according to AKR1B1 expression levels, patients with high AKR1B1 expression showed a significant trend towards advanced disease progression, with a significantly higher proportion of stage III and IV patients compared to patients with low AKR1B1 expression. This suggests that AKR1B1 expression levels are closely related to tumor clinical stage (see Tables 3-1 and 3-2). Figure 3 A and Figure 3 C) Further analysis showed that the expression level of AKR1B1 in tumor tissue was significantly higher than that in corresponding normal tissue, further supporting the abnormal expression characteristics of AKR1B1 during tumorigenesis and development (see reference). Figure 3B). Furthermore, survival analysis stratified by AKR1B1 expression levels showed that patients with high AKR1B1 expression had significantly shorter overall survival, indicating that high AKR1B1 expression is associated with poor clinical prognosis (see reference). Figure 3 D、 Figure 3 E). These results collectively demonstrate that high expression of AKR1B1 is significantly associated with worsening tumor progression and poor patient prognosis. These data suggest that AKR1B1 may be a potential molecular marker of tumor immunotherapy failure, and inhibiting this target may enhance the efficacy of immune checkpoint inhibitors, providing a new biomarker for tumor immunotherapy.

[0119] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Claims

1. Application of AKR1B1 inhibitors combined with immune checkpoint inhibitors in the preparation of drugs for treating tumors.

2. Application of AKR1B1 inhibitors in the preparation of drugs to enhance the efficacy of immune checkpoint inhibitors in the treatment of tumors.

3. The application according to claim 1 or 2, characterized in that, The AKR1B1 inhibitor is selected from Sorbinil, Zopolrestat, or Epalrestat, or a pharmaceutically acceptable salt of any one of them; The immune checkpoint inhibitor is a blocker targeting T-cell inhibitory receptors, selected from programmed death receptor 1 (PD-1) inhibitors, programmed death ligand 1 (PD-L1) inhibitors, or cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors.

4. The application according to claim 1 or 2, characterized in that, The AKR1B1 inhibitor is epalrestat or its pharmaceutically acceptable salt; The immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

5. The application according to claim 1 or 2, characterized in that, The tumors are lung cancer and breast cancer.

6. An antitumor medicine box, characterized in that, The kit includes: a formulation containing an AKR1B1 inhibitor; and a formulation containing an immune checkpoint inhibitor.

7. The antitumor drug kit according to claim 6, characterized in that, The AKR1B1 inhibitor is selected from Sorbinil, Zopolrestat, or Epalrestat, or a pharmaceutically acceptable salt of any one of them; The immune checkpoint inhibitor is a blocker targeting T-cell inhibitory receptors, selected from programmed death receptor 1 (PD-1) inhibitors, programmed death ligand 1 (PD-L1) inhibitors, or cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors.

8. The antitumor drug kit according to claim 6, characterized in that, The AKR1B1 inhibitor is epalrestat or its pharmaceutically acceptable salt; The immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

9. A pharmaceutical composition, characterized in that, It includes therapeutically effective doses of AKR1B1 inhibitors and therapeutically effective doses of immune checkpoint inhibitors.

10. The application of AKR1B1 as a biomarker in the preparation of a diagnostic kit for predicting the efficacy of tumor immunotherapy, wherein the tumors are lung cancer and breast cancer.

Images