Pharmaceutical composition and use thereof

Abstract

Provided are a pharmaceutical composition and use thereof. The pharmaceutical composition comprises a PD-1 monoclonal antibody or a CTLA-4 monoclonal antibody, and further comprises a Rage inhibitor. The prepared composition, by means of blocking Rage in combination with the PD-1 or CTLA-4 monoclonal antibody, can significantly inhibit tumor growth or even lead to the disappearance of tumors, thereby providing a new research direction for preparing more efficient tumor drugs.

Description

A pharmaceutical composition and its application Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a pharmaceutical composition and its application. Background Technology

[0002] Immune checkpoint inhibitors (ICIs) are a type of immunotherapy with potentially high specificity and low side effects. They primarily regulate immune cell activity and kill tumor cells through a series of pathways, including co-inhibition and co-stimulatory signals. The most classic examples are the immunosuppressants PD-1 / PD-L1 monoclonal antibodies and CTLA-4 monoclonal antibodies, which can relieve the inhibitory signals on T lymphocytes. Since the successful introduction of immune checkpoint blockade (ICB) therapy as a treatment for unresectable or metastatic melanoma in 2011, ICB treatment has provided long-term clinical benefits to patients with various tumor types, including a cure for some patients. Therefore, the clinical success of ICB therapy has revolutionized the field of cancer immunotherapy. In September 2014, the FDA granted accelerated approval to the first PD-1 immune checkpoint inhibitor for second-line treatment of melanoma. To date, many immune checkpoint inhibitors have been approved worldwide for second- or third-line, or even first-line, treatment of various solid tumors such as non-small cell lung cancer and urothelial carcinoma, as well as all microsatellite instability-high (MSI-H) solid tumors. This has made ICB therapy a pillar of cancer treatment alongside traditional treatments such as surgery, chemotherapy, and radiotherapy.

[0003] While ICB therapy demonstrates the immense potential of the human immune system in fighting cancer, its effectiveness is limited against some "cold" tumors, and many cancer patients do not respond to ICB therapy. With monotherapy, the response rate of immune checkpoint inhibitors is only 10% to 35%, with approximately 70% of patients not benefiting, and resistance to ICB therapy is very common. Mechanistically, CTLA-4 blocks CD28-B7 interaction to regulate APC-induced T cell responses, while PD-1 operates in the effector phase of TCR signaling to regulate T cell responses. The primary function of PD-1 and CTLA-4 monoclonal antibodies is to deactivate signals that suppress T cell function to exert their anti-tumor effects, but they cannot avoid the accompanying progressive exhaustion and depletion death of tumor-reactive T cells. Therefore, ICB therapy cannot exert a sustained anti-tumor effect, thus affecting the overall treatment outcome.

[0004] For overall clinical treatment efficacy, it is necessary to improve the response rate and duration of the body's immune function to achieve better results. Therefore, specific small molecule inhibitors may not only inhibit key oncogenic signaling pathways but also maintain cell viability while enhancing immune cell responses, thus playing a more sustained role and serving as adjunctive therapy to existing immune checkpoint inhibitors. Current ICB therapy is only effective for a small number of cancer patients, and many patients cannot benefit from treatment long-term. Therefore, the scientific community urgently needs to find mechanisms to enhance immune cell viability and avoid immune cell depletion, thereby researching new target immune checkpoint inhibitors, combining immune checkpoint inhibitors with different targets, or combining immune checkpoint inhibitors with other treatment methods to enhance immune cell viability and reduce immune cell depletion.

[0005] Summary of the Invention

[0006] This application aims to solve at least one of the technical problems existing in the prior art described above. To this end, this application proposes a pharmaceutical composition.

[0007] This application also provides a method for preparing the above-mentioned pharmaceutical composition.

[0008] This application also proposes the use of the above-mentioned pharmaceutical composition.

[0009] According to one aspect of this application, a pharmaceutical composition is provided comprising a PD-1 monoclonal antibody or a CTLA-4 monoclonal antibody, and further comprising a Rage inhibitor.

[0010] In some embodiments of this application, the Rage inhibitor includes molecules capable of blocking the transcription or translation of the Rage gene, or molecules capable of specifically inhibiting the expression or activation of the Rage protein.

[0011] In some embodiments of this application, the Rage inhibitor is a nucleic acid molecule, an antibody, or a small molecule compound.

[0012] In some embodiments of this application, the nucleic acid molecule is selected from siRNA, shRNA, and sgRNA.

[0013] In some embodiments of this application, the small molecule compound is selected from one or both of FPS-ZM1 and aziregane. In some embodiments of this application, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0014] In some embodiments of this application, the pharmaceutically acceptable excipient is selected from one or more of carriers, diluents, binders, lubricants, and wetting agents.

[0015] In some embodiments of this application, the pharmaceutical composition is selected from one or more of the following forms: solution, injection, spray, nasal drops, aerosol, powder, tablet, capsule, and granule.

[0016] In some embodiments of this application, the pharmaceutical composition may be introduced into the body, such as into muscles, intradermal, subcutaneous, venous, or mucosal tissues, by means of injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediation; or it may be introduced into the body after being mixed or encapsulated with other substances.

[0017] According to a second aspect of this application, the use of the above-described pharmaceutical composition is proposed, wherein the use is in the preparation of a medicament for treating tumors.

[0018] In some embodiments of this application, the tumor includes solid tumors and non-solid tumors.

[0019] In some embodiments of this application, the tumor includes at least one of the following: intestinal cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, fallopian tube cancer, kidney cancer, melanoma, brain tumor, thyroid cancer, parathyroid cancer, leukemia, lymphoma, myeloma, sarcoma, prostate cancer, bladder cancer, bile duct cancer, gallbladder cancer, and head and neck squamous cell carcinoma.

[0020] In some embodiments of this application, the intestinal cancer includes rectal cancer, colorectal cancer, small bowel cancer, and large bowel cancer.

[0021] In some embodiments of this application, the drug is used as follows: inhibiting or blocking the expression of the Rage target in an organism, followed by treatment with PD-1 monoclonal antibody or CTLA-4 monoclonal antibody.

[0022] In some embodiments of this application, the inhibition of Rage target expression in an organism includes the use of a Rage inhibitor.

[0023] In some embodiments of this application, blocking the expression of the Rage target in an organism includes gene knockout of the Rage target.

[0024] In some embodiments of this application, the organism includes animals.

[0025] In some embodiments of this application, the animals include humans and mice.

[0026] In some embodiments of this application, when the animal is a mouse, the dosage of the PD-1 monoclonal antibody is from 8 mg / kg to 12 mg / kg.

[0027] In some embodiments of this application, when the animal is a mouse, the dosage of the CTLA-4 monoclonal antibody is 6 mg / kg to 15 mg / kg.

[0028] According to some embodiments of this application, at least the following beneficial effects are achieved: the composition prepared by the scheme of this application can significantly inhibit tumor growth and even cause tumor disappearance by combining Rage inhibitor with PD-1 monoclonal antibody or CTLA-4 monoclonal antibody, providing a new research direction for the preparation of more efficient tumor drugs. Attached Figure Description

[0029] The present application will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0030] Figure 1 shows the detection results of B16 melanoma in the test examples of this application. In the figure, the numbers 1-2, 1-4, 3-1, 3-2, 1-5, 1-6, 3-3, 1-8, 1-19, 3-6, and 3-8 represent the groups. C57 is C57 wild-type mouse, KO is Rage gene knockout mouse, isotype is isotype control IgG, anti-PD-1 is PD-1 monoclonal antibody, and anti-CTLA-4 is CTLA-4 monoclonal antibody.

[0031] Figure 2 shows the results of tumor size monitoring at different time points in the B16 melanoma in the test examples of this application, corresponding to the experiment in Figure 1. In the figures, C57-isotype represents wild-type mice given isotype control IgG, C57-anti PD-1 represents wild-type mice given PD-1 monoclonal antibody, C57-anti CTLA-4 represents wild-type mice given CTLA-4 monoclonal antibody, and Rage represents wild-type mice. - / - -isotype refers to Rage gene knockout mice given isotype control IgG. - / - -anti PD-1 refers to Rage gene knockout mice treated with PD-1 monoclonal antibody. - / - -antiCTLA-4 refers to Rage gene knockout mice given CTLA-4 monoclonal antibody;

[0032] Figure 3 shows the detection results of Scc7 head and neck squamous tumor in the test examples of this application. Among them, C57 is C57 wild-type mouse, KO is Rage gene knockout mouse, isotype is isotype control IgG, anti-PD-1 is PD-1 monoclonal antibody, and anti-CTLA-4 is CTLA-4 monoclonal antibody.

[0033] Figure 4 shows the results of tumor size monitoring at different time points for Scc7 head and neck tumor cells in the test examples of this application, corresponding to the experiment in Figure 3. In this figure, C57-isotype represents wild-type mice given isotype control IgG, C57-anti PD-1 represents wild-type mice given PD-1 monoclonal antibody, C57-anti CTLA-4 represents wild-type mice given CTLA-4 monoclonal antibody, and Rage... - / - -isotype refers to Rage gene knockout mice given isotype control IgG. - / - -anti PD-1 refers to Rage gene knockout mice treated with PD-1 monoclonal antibody. - / - -antiCTLA-4 refers to Rage gene knockout mice given CTLA-4 monoclonal antibody;

[0034] Figure 5 shows the detection results of Lewis lung adenocarcinoma in the test examples of this application. Among them, WT is C57 wild-type mouse, Rage-KO is Rage gene knockout mouse, isotype is isotype control IgG, anti-PD-1 is PD-1 monoclonal antibody, and anti-CTLA-4 is CTLA-4 monoclonal antibody.

[0035] Figure 6 shows the results of monitoring tumor size at different time points in Lewis lung adenocarcinoma cells in the test examples of this application, corresponding to the experiment in Figure 5. C57-isotype represents wild-type mice given isotype control IgG, C57-anti PD-1 represents wild-type mice given PD-1 monoclonal antibody, C57-anti CTLA-4 represents wild-type mice given CTLA-4 monoclonal antibody, and Rage... - / - -isotype refers to Rage gene knockout mice given isotype control IgG. - / - -anti PD-1 refers to Rage gene knockout mice treated with PD-1 monoclonal antibody. - / - -anti CTLA-4 refers to Rage gene knockout mice given CTLA-4 monoclonal antibody. Detailed Implementation

[0036] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0037] Experimental materials: C57 mice, 6-8 weeks old, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.; Rage- / - mice were Rage gene knockout mice with a C57 background, and knockout mice and their littermate control mice, 6-8 weeks old (gifted from Southern Medical University; for specific construction and identification methods, refer to the literature "RAGE Control of Diabetic Nephropathy in a Mouse Model: Effects of RAGE Gene Disruption and Administration of Low-Molecular Weight Heparin").

[0038] Example 1

[0039] This embodiment provides a pharmaceutical composition comprising a Rage inhibitor and a PD-1 monoclonal antibody.

[0040] Example 2

[0041] This embodiment provides a pharmaceutical composition comprising a Rage inhibitor and a CTLA-4 monoclonal antibody.

[0042] Test case

[0043] 1. Application of the pharmaceutical composition in the treatment of B16 melanoma

[0044] Experimental methods:

[0045] Animal groups: WT-isotype group (wild-type mice C57-isotype given isotype control IgG), WT-anti-PD-1 group (wild-type mice C57-anti-PD-1 given PD-1 monoclonal antibody), WT-anti-CTLA-4 group (wild-type mice C57-anti-CTLA-4 given CTLA-4 monoclonal antibody), Rage - / - -isotype group (i.e., Rage gene knockout mice given isotype control IgG); Rage - / - -anti PD-1 group (i.e., Rage gene knockout mice given PD-1 monoclonal antibody), Rage - / - -anti CTLA-4 group (i.e., Rage gene knockout C57 background mice given CTLA-4 monoclonal antibody), with at least 2 mice in each group per experiment (3 mice in each group in this example).

[0046] The specific experimental steps are as follows:

[0047] (1) Shave the fur and prepare the skin on the left back of each group of mice one day in advance;

[0048] (2) Digestion of B16 melanoma cells: After washing with pre-cooled DPBS, centrifuge to remove the liquid phase. The centrifugation conditions were 4°C, 800 rpm, 5 minutes. The washing was repeated 3 times.

[0049] (3) Cell counting: B16 melanoma cells were adjusted to 100 μL of DBPS containing 1 × 10⁻⁶ cells. 6 cell;

[0050] (4) Within 1 hour of cell collection, inject 1×10-1 cells subcutaneously into the back near the left leg of each mouse. 6 One B16 melanoma cell;

[0051] (5) On the day of and day 2 of subcutaneous injection of B16 melanoma cells, the WT-anti-PD-1 group and Rage group... - / - -The anti-PD-1 group received intraperitoneal injections of 10 mg / kg PD-1 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / - -isotype groups were intraperitoneally injected with 10 mg / kg of isotype control IgG;

[0052] (6) Day 3 after subcutaneous injection of B16 melanoma cells, WT-anti-CTLA-4 group and Rage - / - -The anti-CTLA-4 group received intraperitoneal injections of 8 mg / kg CTLA-4 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / - -isotype groups were intraperitoneally injected with 8 mg / kg of isotype control IgG;

[0053] (7) Subcutaneous injection of B16 melanoma cells on days 6 and 9, WT-anti-CTLA-4 group and Rage - / - -The anti-CTLA-4 group received intraperitoneal injections of 4 mg / kg CTLA-4 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / - -isotype groups were intraperitoneally injected with 4 mg / kg of isotype control IgG;

[0054] (8) Samples were taken and tested 14 days after the mold was made.

[0055] The experimental results are shown in Figures 1 and 2. As can be seen from the figures, the pharmaceutical composition of this application can significantly inhibit the growth of B16 melanoma and even kill the tumor.

[0056] 2. Application of the drug composition in the treatment of Scc7 head and neck squamous cell carcinoma

[0057] Experimental methods:

[0058] Animal groups: WT-isotype group, WT-anti-PD-1 group, WT-anti-CTLA-4 group, Rage - / - -isotype group, Rage - / - -anti PD-1 group, Rage - / - -anti CTLA-4 group, with at least 2 mice in each group for each experiment (3 mice in each group in this example).

[0059] The specific experimental steps are as follows:

[0060] (1) Shave the fur and prepare the skin on the left back of the mouse one day in advance;

[0061] (2) Digestion of Scc7 head and neck squamous cell line: After washing with pre-cooled DPBS, centrifuge to remove the liquid phase. The centrifugation conditions were 4°C, 800 rpm, 5 minutes. The washing was repeated 3 times.

[0062] (3) Cell counting: Scc7 cells were adjusted to contain 1×10⁶ cells per 100 μL DBPS. 6 One cell;

[0063] (4) Within 1 hour of cell collection, inject 1×10-1 cells subcutaneously into the back near the left leg of each mouse. 6 One Scc7 head and neck squamous cell carcinoma;

[0064] (5) On the day of and the second day after subcutaneous injection of Scc7 cells, the WT-anti-PD-1 group and Rage... - / - -The anti-PD-1 group received intraperitoneal injections of 10 mg / kg PD-1 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / - -isotype groups were intraperitoneally injected with 10 mg / kg of isotype control IgG;

[0065] (6) On day 3 after subcutaneous injection of Scc7 cells, the WT-anti-CTLA-4 group and Rage - / - -The anti-CTLA-4 group received intraperitoneal injections of 8 mg / kg CTLA-4 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / - -isotype groups were intraperitoneally injected with 8 mg / kg of isotype control IgG;

[0066] (7) Subcutaneous injection of Scc7 cells on days 6 and 9, WT-anti-CTLA-4 group and Rage - / - -The anti-CTLA-4 group received intraperitoneal injections of 4 mg / kg CTLA-4 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / --isotype groups were intraperitoneally injected with 4 mg / kg of isotype control IgG;

[0067] (8) Samples were taken and tested 14 days after the mold was made.

[0068] The experimental results are shown in Figures 3 and 4. As can be seen from the figures, the drug composition of this application can significantly inhibit the growth of Scc7 head and neck squamous tumors.

[0069] 3. Application of the pharmaceutical composition in the treatment of Lewis lung adenocarcinoma

[0070] Experimental methods:

[0071] Animal groups: WT-isotype group, WT-anti-PD-1 group, WT-anti-CTLA-4 group, Rage - / - -isotype group, Rage - / - -anti PD-1 group, Rage - / - -anti CTLA-4 group, with at least 2 mice in each group for each experiment (3 mice in each group in this example).

[0072] The specific experimental steps are as follows:

[0073] (1) Shave the fur and prepare the skin on the left back of the mouse one day in advance;

[0074] (2) Digest the Lewis lung adenocarcinoma cell line, wash with pre-cooled DPBS, centrifuge to remove the liquid phase. Centrifugation conditions were 4°C, 800 rpm, 5 minutes. Washing was repeated 3 times.

[0075] (3) Cell counting: Adjust the cell concentration to 1×10⁻⁶ cells per 100 μL DBPS. 6 cell;

[0076] (4) Within 1 hour of cell collection, inject 1×10-1 cells subcutaneously into the back near the left leg of each mouse. 6 One Lewis lung adenocarcinoma cell;

[0077] (5) On the day of and day 2 of subcutaneous injection of Lewis lung adenocarcinoma cells, the WT-anti-PD-1 group and the Rage group... - / - -The anti-PD-1 group received intraperitoneal injections of 10 mg / kg PD-1 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / - -isotype groups were intraperitoneally injected with 10 mg / kg of isotype control IgG;

[0078] (6) Day 3 after subcutaneous injection of Lewis lung adenocarcinoma cells, WT-anti-CTLA-4 group and Rage - / --The anti-CTLA-4 group received intraperitoneal injections of 8 mg / kg CTLA-4 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / - -isotype groups were intraperitoneally injected with 8 mg / kg of isotype control IgG;

[0079] (7) Subcutaneous injection of Lewis lung adenocarcinoma cells on days 6 and 9, WT-anti-CTLA-4 group and Rage - / - -The anti-CTLA-4 group received intraperitoneal injections of 4 mg / kg CTLA-4 monoclonal antibody, corresponding to the WT-isotype group and Rage. - / - -isotype groups were intraperitoneally injected with 4 mg / kg of isotype control IgG;

[0080] (8) Samples were taken and tested 14 days after the mold was made.

[0081] The experimental results are shown in Figures 5 and 6. As can be seen from the figures, the pharmaceutical composition of this application can significantly inhibit the growth of Lewis lung adenocarcinoma tumors.

[0082] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the protection of this application. Furthermore, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes a PD-1 monoclonal antibody or a CTLA-4 monoclonal antibody, and also includes a Rage inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that, Rage inhibitors include molecules that can block the transcription or translation of the Rage gene, or molecules that can specifically inhibit the expression or activation of the Rage protein.

3. The pharmaceutical composition according to claim 2, characterized in that, The Rage inhibitor is a nucleic acid molecule, antibody, or small molecule compound.

4. The pharmaceutical composition according to claim 3, characterized in that, The nucleic acid molecule is selected from siRNA, shRNA and sgRNA; and / or, the small molecule compound is selected from one or two of FPS-ZM1 and azirreg.

5. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition further includes pharmaceutically acceptable excipients; preferably, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings.

6. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is selected from one or more of the following forms: solution, injection, spray, nasal drops, aerosol, powder, tablet, capsule, and granule.

7. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition may be introduced into the body, such as into muscles, intradermal, subcutaneous, venous, or mucosal tissues, by means of injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediation; or it may be introduced into the body after being mixed or encapsulated with other substances.

8. Use of the pharmaceutical composition according to any one of claims 1 to 7 in the preparation of a medicament for treating tumors.

9. The application according to claim 8, characterized in that, The tumor includes solid tumors and non-solid tumors; preferably, the tumor includes at least one of intestinal cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, fallopian tube cancer, kidney cancer, melanoma, brain tumor, thyroid cancer, parathyroid cancer, leukemia, lymphoma, myeloma, sarcoma, prostate cancer, bladder cancer, bile duct cancer, gallbladder cancer, and head and neck squamous cell carcinoma; more preferably, the intestinal cancer includes rectal cancer, colorectal cancer, small bowel cancer, and large bowel cancer.

10. The application according to claim 8, characterized in that, The method of using the drug is as follows: after inhibiting or blocking the expression of the Rage target in the organism, treatment is performed with PD-1 monoclonal antibody or CTLA-4 monoclonal antibody.

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