Application of atropine combined with immune checkpoint inhibitors in antitumor drugs

Abstract

The application belongs to the technical field of biological medicine, and provides application of atropine combined with an immune checkpoint inhibitor in an antitumor drug. The application uses an RPM transgenic mouse to construct an orthotopic small cell lung cancer model, and adopts a dosing regimen of atropine pretreatment and sequential administration of an anti-PD-1 inhibitor. In vivo experiments prove that atropine can specifically block the cholinergic inhibition pathway of the tumor microenvironment, improve intratumoral CD8 + T cell infiltration level, and produce a synergistic tumor inhibition effect with the immune checkpoint inhibitor, and the tumor load of the combination group is significantly reduced compared with the single-drug group, and in vitro experiments verify that atropine can directly inhibit small cell lung cancer cell proliferation. The application solves the problems of low efficiency of the existing immune checkpoint inhibitor single drug for small cell lung cancer, the immune desert type of the tumor microenvironment, and insufficient effector CD8 + T cell infiltration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of atropine combined with immune checkpoint inhibitors in antitumor drugs. Background Technology

[0002] Small cell lung cancer is highly malignant and its tumor microenvironment exhibits significant immunosuppressive characteristics, often classifying it as an immune-cold tumor. Clinically, the efficacy of immune checkpoint inhibitor (ICI) monotherapy is relatively low. ICI activates the body's anti-tumor immunity by relieving immunosuppression of the PD-1 / PD-L1 pathway; however, it is constrained by the local inhibitory tumor microenvironment, often resulting in limited effector CD8+. + Problems such as insufficient T-cell infiltration and lymphocyte rejection by the tumor matrix limit the clinical benefits of drugs.

[0003] To enhance the anti-tumor efficacy of ICI (intracytoplasmic renal replacement therapy), current clinical protocols often combine chemotherapy drugs or anti-angiogenic agents. While chemotherapy can shrink tumors to some extent, its non-specific killing effect can easily cause adverse reactions such as bone marrow suppression and gastrointestinal damage. Combining multiple drugs further exacerbates systemic toxicity. Anti-angiogenic drugs only target tumor angiogenesis and are unlikely to fundamentally improve the tumor's inherent immunosuppressive state, thus having limited effect on enhancing T-cell recruitment in immune desert tumors.

[0004] Recent studies have confirmed that the neuro-tumor-immune axis plays a crucial regulatory role in the progression of small cell lung cancer. Within the tumor microenvironment, cholinergic nerves continuously release acetylcholine, which activates cholinergic receptors, inducing the activation of immunosuppressive cells and inhibiting effector lymphocyte infiltration, thus being a significant contributing factor to ICI resistance. However, current combination strategies for improving ICI efficacy rarely target cholinergic signaling pathways, and there is a lack of combination regimens based on neuroimmunological regulation.

[0005] Atropine is a clinically mature M-cholinergic receptor blocker that has been widely used in emergency medicine and for antispasmodic treatment. Its pharmacological data are comprehensive and its safety profile is well-established. However, its potential use in combination with intracellular immunotherapy (ICI) to reshape the tumor immune microenvironment and combat small cell lung cancer has not yet been explored. Given the limitations of existing technologies, such as lack of pathway targeting, limited synergistic effects, and significant toxic side effects from combination therapy, developing novel antitumor regimens combining atropine with ICI has significant clinical value in overcoming the bottlenecks in ICI treatment of small cell lung cancer. Summary of the Invention

[0006] The purpose of this invention is to provide an application of atropine combined with an immune checkpoint inhibitor in antitumor drugs. By pre-blocking cholinergic inhibitory signals in the tumor microenvironment and relieving local immunosuppression with atropine, and then sequentially combining it with an immune checkpoint inhibitor, the invention synergistically activates the body's antitumor immune response. This addresses the challenges posed by existing immune checkpoint inhibitor monotherapy for small cell lung cancer, including low efficacy, an immune desert-like tumor microenvironment, and limited effector CD8+.+ Technical problems such as insufficient T-cell infiltration and the high incidence of immunotherapy resistance in clinical practice.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: the application of atropine combined with an immune checkpoint inhibitor in the preparation of an anti-small cell lung cancer drug.

[0008] Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0009] Preferably, the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody.

[0010] Preferably, the atropine is an M-cholinergic receptor antagonist.

[0011] Preferably, the atropine and the immune checkpoint inhibitor are administered sequentially, with atropine administered first, followed by the immune checkpoint inhibitor, and the first administration of the immune checkpoint inhibitor is 14 days apart from the first administration of the atropine.

[0012] Preferably, the atropine is administered via intraperitoneal injection, and the immune checkpoint inhibitor is administered via tail vein injection; both are administered once every 3 days.

[0013] Preferably, the atropine dosage is 0.4 mg / kg, and the immune checkpoint inhibitor dosage is 200 μg / animal.

[0014] Preferably, atropine reduces the activity of immunosuppressive cells and promotes CD8 expression by blocking the cholinergic signaling pathway in the tumor microenvironment. + T-cell infiltration reshapes the tumor immune microenvironment and produces a synergistic anti-tumor effect with immune checkpoint inhibitors.

[0015] The present invention further provides a method for enhancing the efficacy of immune checkpoint inhibitors in the treatment of small cell lung cancer.

[0016] Preferred methods include sequential administration of atropine and immune checkpoint inhibitors.

[0017] The beneficial effects of this invention are:

[0018] 1. Improve the tumor immune microenvironment: increase CD8 levels within the lesion. + The degree of T cell infiltration can alleviate the immune desertification state in small cell lung cancer.

[0019] 2. Promotes effector T cell infiltration: enhances CD8 + T cells are recruited to tumor tissues to enhance the anti-tumor immune response.

[0020] 3. Enhance the efficacy of ICI: By relieving neurotransmitter-mediated immunosuppression, it synergizes with immune checkpoint inhibitors to improve the treatment response rate.

[0021] 4. Clear mechanism and strong scalability: This strategy is based on the regulatory mechanism of the neuro-immune-tumor axis and can be extended to a variety of tumor types with neuroregulatory characteristics.

[0022] 5. High safety potential: Atropine is a widely used clinical drug with well-defined pharmacological properties, which is conducive to accelerating clinical translation. Attached Figure Description

[0023] Figure 1 In this invention, atropine inhibits the proliferation of tumor cells;

[0024] Figure 2 In this invention, atropine combined with immune checkpoint inhibitors can effectively inhibit tumor progression (A is a schematic diagram of the timeline of RPM transgenic mouse modeling and drug intervention. Lung tumors in situ were induced by intraventricular infusion of Adv-CGRP-Cre adenovirus at week 0. Atropine administration was initiated at week 3, and ICI administration was initiated at week 5, with administration every 3 days. Samples were collected and tested uniformly at week 7 of modeling; B shows a panoramic pathological section of complete lung tissue from four groups of mice and magnified images of the tumor area. The rows represent the blank control group, ICIs monotherapy group, atropine monotherapy group, and atropine combined with ICIs group, respectively. The boxes represent magnified areas of tumor lesions; C is a scatter plot of quantitative tumor scores for each group of mice. The vertical axis represents the percentage of tumor area to total lung area. Control represents the blank control group, ICIs represents the immune checkpoint inhibitor monotherapy group, Atrop represents the atropine monotherapy group, and A+I represents the atropine combined with immune checkpoint inhibitor group).

[0025] Figure 3 The images show the quantitative immunohistochemical staining of CD8-positive immune cells in mouse tumor tissues of each group in this invention (A is the immunohistochemical microscopic image of CD8 and NK1.1 in lung tumor tissues of each group, the first row is CD8-specific staining, the second row is NK1.1-specific staining, 10× is the low magnification field, and the boxed area is the 40× high magnification field; B is the quantitative statistical scatter plot of CD8-positive cells per unit area of ​​tumors in each group; C is the quantitative statistical scatter plot of NK1.1-positive cells per unit area of ​​tumors in each group; Control represents the PBS blank control group, ICI represents the anti-PD-1 monotherapy group, Atrop represents the atropine monotherapy group, and A+I represents the atropine combined with the anti-PD-1 inhibitor group). Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Example 1: In vitro cell experiments verified that atropine inhibits the proliferation of small cell lung cancer tumor cells.

[0030] 1. Method

[0031] Cells and reagents:

[0032] Primary mouse SCLC cell lines from Rb1 fl Trp53 fl Myc LSL Cell lines were obtained by isolating and culturing newly formed tumors in transgenic mouse models. Cell lines were cultured in RPMI-1640 (Gibco) cells (containing 10% fetal bovine serum and 1% penicillin and streptomycin) at 37°C in a 5% CO2 incubator, and routinely passaged according to the growth status and proliferation rate of each cell line.

[0033] CCK-8 proliferation assay: RPM primary tumor cells were divided into groups of 5 × 10⁻⁶. 3 Cells were seeded in 96-well plates and, after 6 hours of stabilization, treated with atropine. CCK-8 working solution was added to each well at designated time points, and the cells were incubated at 37 °C for 1–2 hours. The absorbance (OD value) at 450 nm was then measured using a microplate reader. The OD value reflects cell viability or proliferation level, and cell survival rate or inhibition rate was calculated according to the experimental design. Each experiment included three blank control wells and three replicate wells. Results are expressed as averages.

[0034] 2. Results Analysis

[0035] To evaluate the effect of atropine on the proliferation of SCLC cells, the CCK8 assay was used to detect cell viability after treatment with different concentrations of atropine. The results showed that ( Figure 1 Compared with the control group, atropine treatment significantly reduced cell viability, and the inhibitory effect gradually increased with increasing drug concentration, showing a dose-dependent trend (P < 0.05). These results suggest that blocking cholinergic receptor signaling can inhibit SCLC cell proliferation.

[0036] Example 2: Efficacy test of atropine combined with an anti-PD-1 inhibitor in vivo for the treatment of small cell lung cancer in mice.

[0037] 1. Construction of a transgenic animal model for small cell lung cancer

[0038] The experimental animals used were Jackson laboratory RPM gene-modified mice (standard genotype Igs2, catalog number #029971). tm1(CAG-Myc*T58A / luc)Wrey Trp53 tm1Brn Rb1 tm3Tyj / OlvrJ The genetic background of the mice is FVB / 129 hybrid strain. The mice were anesthetized with isoflurane inhalation and sprayed with Adv-CGRP-Cre adenovirus carrying Cre recombinase via airway nebulization to induce gene recombination in lung tissue target cells, spontaneously forming in situ small cell lung cancer. The mice were then fed in a conventional SPF-grade environment.

[0039] Drug intervention was initiated on day 21 of modeling. Mice were randomly divided into 4 groups: blank control group (PBS solvent), atropine monotherapy group (Atrop), anti-PD-1 inhibitor monotherapy group (ICI), and atropine + anti-PD-1 combination group (A+I), with 5 mice in each group.

[0040] Administration method:

[0041] Atropine administration: Starting from day 21, atropine injection is administered intraperitoneally every three days at a dose of 0.4 mg / kg.

[0042] Anti-PD-1 antibody administration (ICI): Starting from day 35, administer anti-mouse PD-1 monoclonal antibody via tail vein injection, once every three days, at a dose of 200 μg / mouse;

[0043] The control group received an equal volume of sterile PBS via intraperitoneal injection simultaneously, and continued administration until 49 days after model establishment.

[0044] Tumor pathological analysis:

[0045] Forty-nine days after modeling, mice were sacrificed, and their lungs were dissected, fixed in 4% paraformaldehyde, and embedded in paraffin. The largest lung section was selected for HE staining, and the tumor score (tumor area / total lung tissue area * 100%) was calculated. Simultaneously, continuous sections of tissue were selected for immunohistochemical staining (CD8 and NK1.1), and the density of positive cells in the tumor area (number of positive cells / tissue area) was calculated.

[0046] 2. Results Analysis

[0047] 1) In a mouse model of RPM small cell lung cancer, atropine monotherapy inhibited tumor growth to some extent compared with the control group, while the combination therapy group showed the most significant anti-tumor effect. The combination therapy significantly reduced tumor burden and delayed tumor progression, suggesting a synergistic anti-tumor effect between atropine and immune checkpoint inhibitors. Figure 2 ).

[0048] 2) Immunohistochemical staining showed that, compared with the control group and the immune checkpoint inhibitor monotherapy group, atropine and atropine combined with immune checkpoint inhibitor treatment resulted in a decrease in CD8+ in tumor tissue. + T cell infiltration was significantly increased. These results suggest that atropine may enhance the antitumor efficacy of immune checkpoint inhibitors by improving the tumor immune microenvironment and promoting effector T cell infiltration and activation. Figure 3 ).

[0049] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The application of atropine combined with an immune checkpoint inhibitor in the preparation of an anti-small cell lung cancer drug.

2. The application according to claim 1, characterized in that, The immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

3. The application according to claim 2, characterized in that, The immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody.

4. The application according to claim 1, characterized in that, Atropine is an M-cholinergic receptor antagonist.

5. The application according to claim 1, characterized in that, The atropine and the immune checkpoint inhibitor are administered sequentially, with atropine administered first, followed by the immune checkpoint inhibitor, and the first administration of the immune checkpoint inhibitor is 14 days apart from the first administration of the atropine.

6. The application according to claim 5, characterized in that, The atropine was administered via intraperitoneal injection, and the immune checkpoint inhibitor was administered via tail vein injection; both were administered once every 3 days.

7. The application according to claim 6, characterized in that, The atropine dosage was 0.4 mg / kg, and the immune checkpoint inhibitor dosage was 200 μg / animal.

8. The application according to claim 1, characterized in that, Atropine reduces the activity of immunosuppressive cells and promotes CD8 expression by blocking cholinergic signaling pathways in the tumor microenvironment. + T-cell infiltration reshapes the tumor immune microenvironment and produces a synergistic anti-tumor effect with immune checkpoint inhibitors.

9. A method for enhancing the efficacy of immune checkpoint inhibitors in the treatment of small cell lung cancer, characterized in that, This includes sequential administration of atropine and immune checkpoint inhibitors.

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