Pharmaceutical composition and application
Through the drug combination of GPR81/PPARα inhibitor combined with PDL1 antibody, the problem of lung cancer cells activate the GPR81 pathway through lactic acid has been solved, and the effect of significantly alleviating lung cancer progression and prolonging survival was achieved.
Patent Information
- Application Number
- CN202510071672.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-04-08
AI Technical Summary
Lung cancer cells activate the GPR81 pathway by producing lactic acid, resulting in a low anti-tumor immune response. Using PDL1 antibodies alone cannot effectively inhibit tumor growth, resulting in a shortening of the patient's survival.
The drug combination of GPR81/PPARα inhibitor combined with PDL1 antibody is used to enhance the proliferation of anti-tumor CD4+ T cells by reducing the production of Breg cells and the secretion of anti-tumor molecule ROS, thereby slowing down the tumor growth rate.
It significantly alleviates the progression of lung cancer, prolongs the patient's survival, and makes up for the defects of anti-tumor failure caused by the use of PDL1 antibody alone.
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Figure CN119770647A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to a drug combination and application. Background Art
[0002] Tumor cells regulate the phenotype and function of infiltrating immune cells by producing a variety of metabolites. For example, aerobic glycolysis in tumors leads to the accumulation of lactate (LA) in the tumor microenvironment, and lactate cannot be further metabolized by tumor cells to generate adenosine triphosphate (ATP) and can only be transported out of the cell through monocarboxylate transporters. As a byproduct of aerobic glycolysis, recent studies have shown that lactate is involved in the regulation of tumor cell growth and immune cell responses. In tumor cells, lactate dehydrogenase (LDH) converts pyruvate into lactate, and human tumor cells mainly express the LDHA isoform. Reduced LDHA expression leads to limited LA production and impaired tumor cell growth. Recent studies have shown that LA stimulation can lead to the accumulation of PD-1 and CLTA-4 in tumor-infiltrating regulatory T cells, resulting in impaired antitumor immune responses in patients with colorectal cancer or acute myeloid leukemia. In addition, lactate induces the activation of tumor-associated fibroblasts and type 2 macrophages, promoting the progression of lung cancer. Under normal physiological conditions, GPR81 is mainly expressed in adipocytes and also in some immune cells including B cells. GPR81 acts as an autocrine sensor of lactate produced by aerobic glycolysis, inhibiting lipolysis through cAMP reduction. However, recent studies have shown that GPR81 is highly expressed in tumor and immune cells of many different types of solid tumors, and GPR81 knockdown reduces the growth of breast and pancreatic tumor xenografts in immunosuppressed mice. In addition, the loss of GPR81 in various cancer cells limits cancer cell growth, invasiveness, chemotherapy resistance, and programmed death-ligand 1 (PD-L1) expression. Recent studies have shown that lactate may be involved in the progression of lung cancer through GPR81. However, the mechanism by which GPR81 exerts these effects remains incompletely understood.
[0003] Peroxisome proliferator-activated receptors (PPARs) are a class of nuclear receptors. They exist in three different isoforms: PPARα (NR1C1), PPARβ / δ (NR1C2), and PPARγ (NR1C3). PPARa is known for its ability to regulate genes involved in lipid metabolism. Studies have shown that activation of PPARα promotes proliferation of MDA-MB-231 and MCF-7 breast cancer cells. Recently, it was found in glioblastoma stem cells and primary glioblastoma cells that inhibition of PPARα by specific antagonists (GW6471 and AA452) can lead to growth arrest, reduced expression of mevalonate pathway enzymes, and reduced cholesterol and cholesterol ester levels. GW6471 is a competitive PPARα antagonist that acts at nanomolar concentrations. Another research group demonstrated that GW6471 induces apoptotic death and cell cycle arrest in renal cancer cells and acts synergistically with glycolysis inhibition. In addition, the same research group reported that PPARα antagonists significantly reduced the growth of renal cancer in xenografted mice and inhibited the enhancement of glycolysis without adverse effects. Studies in paraglandular tumors found that the specific PPARα antagonist GW6471 reduced cell viability, interfered with the cell cycle, induced caspase-dependent apoptosis, and significantly impaired its clonogenicity in head and neck PGL cells, supporting PPARα inhibition as a new therapeutic target for this chemotherapy-resistant tumor. However, the role of PPARα inhibitors in lung cancer remains unclear.
[0004] Studies have found that lung cancer cells produce a metabolite called lactic acid, which can bind to the receptor GPR81 expressed on the surface of immune cell membranes to activate downstream signaling pathways, inducing the transformation of anti-tumor immune B cells in the tumor microenvironment into regulatory B cells that inhibit immune responses, leading to low anti-tumor immune responses in the tumor microenvironment. The use of multiple anti-tumor measures (surgery / biological therapy) cannot effectively inhibit tumor growth, ultimately reducing patient survival. Summary of the invention
[0005] The purpose of the present invention is to provide a drug combination and application to solve the problems existing in the above-mentioned background technology.
[0006] To achieve the above objectives, this application is implemented through the following technical solutions:
[0007] A drug combination consisting of a GPR81 / PPARα inhibitor combined with a PDL1 antibody.
[0008] Furthermore, the GPR81 / PPARα inhibitor and PDL1 antibody are used as drugs or pharmaceutical compositions, respectively.
[0009] Furthermore, the PPARα inhibitor is one or a combination of GW6471 or AA452.
[0010] Furthermore, the GPR81 inhibitor includes 3-OBA.
[0011] Furthermore, the medicine or pharmaceutical composition also includes an acceptable carrier.
[0012] Furthermore, the drug or pharmaceutical composition also includes pharmaceutically acceptable excipients.
[0013] An application, use of any of the above-mentioned drug combinations in the treatment of lung cancer.
[0014] Furthermore, the pharmaceutical composition enhances the proliferation of anti-tumor CD4+ T cells by reducing the production of Breg cells and the secretion of anti-tumor molecules ROS in lung cancer.
[0015] Furthermore, the application method is: the drug or drug composition comprising the GPR81 / PPARα inhibitor is used once a day and continuously, and the drug or drug composition comprising the PDL1 antibody is used once every three days.
[0016] The beneficial effects of the present invention are:
[0017] This technical solution combines GPR81 / PPARα inhibitors with PDL1 antibodies for the treatment of lung cancer, which can reduce the production of Breg cells and the secretion of anti-tumor molecules ROS in tumors, enhance the proliferation of anti-tumor CD4+ T cells, slow down tumor growth, significantly alleviate lung cancer progression, prolong its survival, and make up for the defect of anti-tumor failure caused by the use of PDL1 antibodies alone in some lung cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Schematic diagram of the proportion of mouse B220+CD25+CD81+ B cells after 48 hours of lactate (10mM) treatment.
[0019] Figure 2 Schematic diagram of the results of co-culturing CD3 / 28-stimulated T cells with lactate-induced B220+CD25+CD81+ B cells at different ratios for 72 hours.
[0020] Figure 3 Schematic diagram of the lactate-induced enhancement of reactive oxygen species (ROS) production in mouse B cells by the simultaneous addition of the NADPH oxidase inhibitor DPI (10 μM) to the induction system to effectively reverse the lactate-induced increase in ROS.
[0021] Figure 4After lactate treatment, B220+CD25+CD81+B cells produced more NAPDH oxidase subunit products, such as p22phox, p40phox, p47phox, p67phox, and gp91phox, suggesting that lactate enhances ROS secretion by upregulating NAPDH oxidase activity in B220+CD25+CD81+B cells.
[0022] Figure 5 Schematic diagram of the fact that the addition of MCT1 inhibitor αCHCA (concentration) does not affect the secretion of ROS in B220+CD25+CD81+ B cells upregulated by lactate.
[0023] Figure 6 Schematic diagram of flow cytometry data showing that the expression of GPR81 in B cells was significantly increased after lactate treatment.
[0024] Figure 7 Schematic diagram of sorting B220-positive cells in naive mice. The proportion of B220+CD25+CD81+B cells increased significantly under the action of lactic acid, and the ability of GPR81 agonist DHBA (25mM) to induce B220+CD25+CD81+B cells was the same as that of lactic acid.
[0025] Figure 8 Schematic diagram of the fluorescence quantitative PCR results showing that DHBA promoted the increase in the subunit products and ROS expression of NADPH oxidase in B220+CD25+CD81+B cells, and the addition of NADPH oxidase inhibitor DPI (10 μM) could reverse this effect.
[0026] Fig. 9 Schematic diagram showing that B220+CD25+CD81+ B cells induced by DHBA stimulation can inhibit T cell proliferation, and this ability can be offset by the GPR81 antagonist OBA (30 mM).
[0027] Fig.10 Schematic diagram showing that spleen B cells in tumor-bearing (lung cancer) mice can also inhibit T cell proliferation.
[0028] Fig.11 Schematic diagram of the increased expression of the nuclear transcription factor PPARα found in B cells stimulated with lactate or DHBA.
[0029] Fig.12 Schematic diagram of the increase in the proportion of ROS+PPARα+ B220+CD25+CD81+ B cells after lactate stimulation, and this effect was inhibited by OBA.
[0030] Fig.13Schematic diagram showing that the PPARα inhibitor GW6471 (20 μM) can also inhibit the secretion of ROS by B220+CD25+CD81+ B cells.
[0031] Fig.14 After knocking down the lactate dehydrogenase LDHA in the mouse lung cancer cell line D121 using lentiviral technology (LDHA-KO), 3×10 5 Wild-type D121 cells or LDHA-KO cells were inoculated subcutaneously into B6 mice. It was found that the tumor growth rate of mice slowed down after silencing LDHA. The results of pathological tissue staining showed that the Ki67 of LDHA-KO cells was reduced, indicating a decrease in cell proliferation ability.
[0032] Fig.15 Schematic diagram showing that compared with the group inoculated with wild-type D121 cells, the proportion of B220+CD25+CD81+B cells in the spleen and tumors of mice in the LDHA-KO group was reduced, and ROS expression decreased.
[0033] Fig.16 Schematic diagram of the significant decrease in the expression of ROS and PPARα in B220+CD25+CD81+ B cells in the spleen and tumors of LDHA-KO group mice, and the inhibition of lactate-induced increase in the proportion of B220+CD25+CD81+ B cells by OBA and GW6471.
[0034] Fig.17 The activation of GPR81 plays a role through the cAMP / PKA / TAZ pathway. Schematic diagram of the significant increase in cAMP, PRKAR1A (PKA key subunit), and TAZ expression in B220+CD25+CD81+B cells after lactate stimulation.
[0035] Fig.18 Schematic diagram of animal experimental results showing that compared with the wild-type D121 cell group, the expression of PRKAR1A and TAZ in B220+CD25+CD81+B cells in the spleen and tumors of LDHA-KO group mice was decreased.
[0036] Fig.19 Schematic diagram of mice treated with OBA (0 mg / kg, once a day, continuous injection) and / or PDL1 antibody intraperitoneally after subcutaneous inoculation of D121 cells for 7 days, compared with the group treated with PDL1 monoclonal antibody alone (10 mg / kg, once every three days, three times in total).
[0037] Fig. 20 Combined OBA treatment significantly inhibited tumor cell growth. Clinical results showed that the lactate level and the proportion of CD24+CD38+B cells in the peripheral blood of lung cancer patients were significantly increased, and the two were positively correlated.
[0038] Fig.21 This is a schematic diagram showing that high LDHA in lung cancer cells indicates a poor prognosis. DETAILED DESCRIPTION
[0039] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, but cannot be construed as limiting the technical solution of the present invention.
[0040] The technical solution is based on the fact that lactic acid produced by tumors can activate the PPARα pathway by binding to GPR81, induce anti-tumor B cells to transform into Breg cells that promote tumor growth, promote the secretion of reactive oxygen species (ROS) in Breg cells, and inhibit the activity of anti-tumor T cells.
[0041] The present application provides a drug combination consisting of a GPR81 / PPARα inhibitor combined with a PDL1 antibody.
[0042] In the present application, GPR81 / PPARα inhibitor and PDL1 antibody are used in drugs or pharmaceutical compositions respectively. In the present technical solution, GPR81 / PPARα inhibitor and PDL1 antibody are not prepared into a drug or pharmaceutical composition, but are still used in the form of their own drugs or pharmaceutical compositions.
[0043] In the present application, the PPARα inhibitor is one or a combination of GW6471 or AA452. In other embodiments of the present application, it may also be other PPARα inhibitors.
[0044] In the present application, the GPR81 inhibitor includes 3-OBA.
[0045] In the present application, the drug or pharmaceutical composition also includes an acceptable carrier.
[0046] In the present application, the drug or pharmaceutical composition also includes a pharmaceutically acceptable excipient.
[0047] The present application also provides an application, namely, application of any of the above-mentioned drug combinations in the treatment of lung cancer.
[0048] In the present application, the pharmaceutical composition enhances the proliferation of anti-tumor CD4+ T cells by reducing the production of Breg cells and the secretion of anti-tumor molecules ROS in lung cancer.
[0049] The application method of the present application is: the drug or drug composition containing the GPR81 / PPARα inhibitor is used once a day and used continuously, and the drug or drug composition containing the PDL1 antibody is used once every three days.
[0050] Example
[0051] Intraperitoneal injection of GPR81 inhibitor (3-OBA, 80 mg / kg, once a day, continuous injection) in tumor-bearing mice combined with PDL1 antibody (10 mg / kg, once every three days, a total of three times) can reduce the production of Breg cells and the secretion of anti-tumor molecules ROS in tumors, enhance the proliferation of anti-tumor CD4+ T cells, slow down the growth rate of mouse tumors, significantly alleviate the progression of lung cancer in mice, prolong their survival, and make up for the defect of anti-tumor failure caused by the use of PDL1 antibodies alone in some lung cancer patients.
[0052] In the technical solution of the present application, those skilled in the art can conduct tests on the dosage of GPR81 inhibition and PDL1 antibodies based on actual clinical practice, with the focus on the dosage of GPR81 inhibition and PDL1 antibodies, i.e., the GPR81 inhibitor is used daily, and the PDL1 antibody is used at set intervals.
[0053] This technical solution discovered for the first time that lactic acid produced by tumors can induce the production of Breg cells through the GPR81 / PPARα pathway, providing a new theoretical basis for the production of Breg cells. Blocking the GPR81 / PPARα pathway with small molecule inhibitors can inhibit the production of tumor Breg cells and the secretion of ROS, and enhance the anti-tumor response of immune cells. Compared with the use of PDL1 antibodies alone, the use of GPR81 / PPARα inhibitors combined with PDL1 antibodies can effectively alleviate the growth of solid tumors such as lung cancer tumors and prolong the survival of tumor mice.
[0054] In the test conducted according to the above-mentioned embodiment, the in vitro data showed that the proportion of B220+CD25+CD81+ B cells in mice increased after 48 hours of lactic acid (10mM) treatment ( Figure 1 ); CD3 / 28-stimulated T cells were co-cultured with lactate-induced B220+CD25+CD81+ B cells at different ratios for 72 hours. The results showed that B220+CD25+CD81+ B cells significantly inhibited T cell proliferation ( Figure 2 ).
[0055] Lactate treatment enhanced the production of reactive oxygen species (ROS) in mouse B cells. Adding NADPH oxidase inhibitor DPI (10 μM) to the induction system effectively reversed the lactate-induced increase in ROS ( Figure 3 ).
[0056] After lactate treatment, B220+CD25+CD81+B cells produced more NAPDH oxidase subunit products, such as p22phox, p40phox, p47phox, p67phox, gp91phox ( Figure 4 ), suggesting that lactate enhances ROS secretion by upregulating NAPDH oxidase activity in B220+CD25+CD81+ B cells.
[0057] MCT1 is a lactate transporter expressed on the cell surface. The results showed that the addition of MCT1 inhibitor αCHCA (concentration) did not affect the secretion of ROS by lactate-upregulated B220+CD25+CD81+B cells ( Figure 5 ), indicating that lactate does not affect B cell function through MCT1.
[0058] Furthermore, flow cytometry data showed that the expression of GPR81 in B cells was significantly increased after lactate treatment ( Figure 6 ), sorting naive mouse B220 positive cells, the proportion of B220+CD25+CD81+B cells increased significantly under the action of lactic acid, and the ability of GPR81 agonist DHBA (25mM) to induce B220+CD25+CD81+B cells was the same as that of lactic acid ( Figure 7 ).
[0059] Fluorescence quantitative PCR results showed that DHBA promoted the increase of NAPDH oxidase subunit products and ROS expression in B220+CD25+CD81+B cells ( Figure 8 ), which was reversed by the addition of the NADPH oxidase inhibitor DPI (10 μM). Figure 8 ).
[0060] B220+CD25+CD81+ B cells induced by DHBA stimulation were able to inhibit T cell proliferation, and this ability could be offset by the GPR81 antagonist OBA (30 mM) ( Fig. 9 In addition, spleen B cells of tumor-bearing (lung cancer) mice can also inhibit T cell proliferation ( Fig.10 ).
[0061] The expression of the nuclear transcription factor PPARα was increased in B cells stimulated with lactate or DHBA. Fig.11 ). Furthermore, the proportion of ROS+PPARα+B220+CD25+CD81+B cells increased after lactate stimulation, and this effect was inhibited by OBA ( Fig.12 ). The use of PPARα inhibitor GW6471 (20 μM) can also inhibit the secretion of ROS in B220+CD25+CD81+ B cells ( Fig.13 ).
[0062] After knocking down the lactate dehydrogenase LDHA in the mouse lung cancer cell line D121 using lentiviral technology (LDHA-KO), 3×10 5Wild-type D121 cells or LDHA-KO cells were inoculated subcutaneously into B6 mice. It was found that the tumor growth rate of mice slowed down after silencing LDHA. Pathological tissue staining results showed that the Ki67 of LDHA-KO cells decreased, indicating that the cell proliferation ability decreased ( Fig.14 ). Compared with the wild-type D121 cell-inoculated group, the proportion of B220+CD25+CD81+B cells in the spleen and tumor of mice in the LDHA-KO group was reduced, and the expression of ROS was decreased ( Fig.15 ).
[0063] In addition, the expression of ROS and PPARα in B220+CD25+CD81+ B cells in the spleen and tumors of LDHA-KO mice was significantly reduced, and OBA and GW6471 inhibited the increase in the proportion of B220+CD25+CD81+ B cells induced by lactate ( Fig.16 ). The activation of GPR81 works through the cAMP / PKA / TAZ pathway. After lactate stimulation, the expression of cAMP, PRKAR1A (PKA key subunit), and TAZ in B220+CD25+CD81+B cells increased significantly ( Fig.17 ).
[0064] The results of animal experiments showed that compared with the wild-type D121 cell group, the expression of PRKAR1A and TAZ in B220+CD25+CD81+B cells in the spleen and tumor of LDHA-KO mice was decreased ( Fig.18 ).
[0065] Mice were subcutaneously inoculated with D121 cells for 7 days and then intraperitoneally treated with OBA (0 mg / kg, once a day, continuous injection) and / or PDL1 antibody. Compared with the group treated with PDL1 monoclonal antibody alone (10 mg / kg, once every three days, three times in total) ( Fig.19 ), combined with OBA treatment significantly inhibited tumor cell growth. Clinical results showed that the lactic acid level and CD24+CD38+B cell ratio in the peripheral blood of lung cancer patients were significantly increased, and the two were positively correlated ( Fig. 20 High LDHA in lung cancer cells indicates a poor prognosis ( Fig.21 ).
[0066] The above results indicate the importance of blocking the lactate / GPR81 pathway in the treatment of lung cancer. Combined OBA / PDL1 antibody treatment significantly alleviates the progression of lung cancer in mice and prolongs their survival, making up for the defect of anti-tumor failure caused by the use of PDL1 antibody alone in some lung cancer patients.
[0067] The basic principles and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which shall fall within the scope of the present invention to be protected. The scope of protection of the present invention shall be defined by the attached claims and their equivalents.
Claims
1. A drug combination, characterized in that It consists of a GPR81 / PPARα inhibitor combined with a PDL1 antibody.
2. The drug combination according to claim 1, characterized in that The GPR81 / PPARα inhibitor and PDL1 antibody are used in medicine or pharmaceutical composition respectively.
3. The drug combination according to claim 1, characterized in that The PPARα inhibitor is one or a combination of GW6471 or AA452.
4. The pharmaceutical combination according to claim 1, characterized in that The GPR81 inhibitors include 3-OBA.
5. The pharmaceutical combination according to claim 2, characterized in that: The medicament or pharmaceutical composition further includes an acceptable carrier.
6. The pharmaceutical combination according to claim 1, characterized in that The medicament or pharmaceutical composition further includes pharmaceutically acceptable excipients.
7. An application, characterized in that: Use of the drug combination according to any one of claims 1 to 6 in the treatment of lung cancer.
8. The use according to claim 7, characterized in that: The pharmaceutical composition enhances the proliferation of anti-tumor CD4+ T cells by reducing the generation of Breg cells and the secretion of anti-tumor molecules ROS in lung cancer.
9. The use according to claim 7, characterized in that: The application method is: the drug or drug composition containing the GPR81 / PPARα inhibitor is used once a day and continuously, and the drug or drug composition containing the PDL1 antibody is used once every three days.