Application of AZD5069 in preparation of cancer radiotherapy adjuvant drugs

Abstract

The invention belongs to the field of new application of drugs, and particularly relates to application of AZD5069 in preparation of cancer radiotherapy adjuvant drugs. The invention discloses a novel application of a CXCR2 receptor antagonist AZD5069 in preparation of an adjuvant drug for tumor radiotherapy. The invention aims to solve the core clinical problem that the tumor remote metastasis risk may be increased although the tumor can be locally killed by radiotherapy. Through combined application of AZD5069 and radiotherapy, collection and activation of neutrophile granulocytes activated by radiotherapy to a metastasis site can be inhibited by utilizing the capability of the AZD5069 for specifically blocking a CXCLs / CXCR2 signal channel, so that tumor metastasis is effectively prevented while the local curative effect of radiotherapy is maintained, and dual treatment targets of local control and whole body metastasis resistance are realized.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceuticals, and more specifically, relates to the application of AZD5069 in the preparation of adjuvant drugs for cancer radiotherapy. Background Technology

[0002] Tumor metastasis is a major cause of cancer treatment failure and the leading cause of cancer death, accounting for over 90% of cancer deaths. Compared to tumors in situ, metastatic tumors develop into systemic diseases, therefore systemic treatments such as chemotherapy, targeted therapy, and immunotherapy are commonly used to treat metastatic tumors. Surgery and radiotherapy are local treatments for cancer, often used for tumors in situ and larger metastatic tumors, and have shown good therapeutic effects.

[0003] Radiotherapy (RT) is a widely used treatment for cancer, and globally, it is used for more than 50% of cancer patients. Although radiotherapy can usually significantly inhibit the growth of tumors within the target area, tumors are prone to recurrence and metastasis.

[0004] High-energy ionizing radiation (IR) in radiotherapy causes DNA damage to tumor cells, thereby inhibiting tumor cell division and proliferation, and ultimately leading to cell senescence and death. Secondly, radiotherapy induces a complex immune response in the target area, including anti-tumor and pro-tumor immune responses, which antagonize each other and jointly regulate tumor development. Regulating the immune response to radiotherapy to enhance the efficacy of tumor treatment is an important direction for future research in cancer therapy.

[0005] Currently, there is a lack of intervention methods for radiotherapy-induced tumor recurrence and metastasis: existing treatment plans are mostly based on single radiotherapy or combined chemotherapy, but chemotherapy drugs have problems such as strong side effects and poor targeting; while targeted drugs specifically for radiotherapy-induced metastasis have not yet been approved for clinical use, and there is an urgent clinical need for safe and effective drugs to inhibit radiotherapy-induced tumor metastasis. Summary of the Invention

[0006] The purpose of this invention is to provide the application of AZD5069 in the preparation of adjuvant drugs for cancer radiotherapy.

[0007] This invention provides the application of AZD5069 in the preparation of adjuvant drugs for cancer radiotherapy.

[0008] AZD5069 is a potent and highly selective reversible CXCR2 receptor antagonist, chemically named N-(5-tert-butylisoxazol-3-yl)-4-[(4-fluorophenyl)methyl]piperidine-1-carboxamide, with the molecular formula C2. 19 H 25FN4O, with a molecular weight of 344.43, is a core molecular design targeting the CXCR2 receptor, which is highly expressed on the surface of neutrophils. By specifically binding to this receptor, it competitively blocks the signal transduction of chemokines such as CXCL8 (IL-8) and CXCL1, making it a representative targeted drug for neutrophil-mediated pathological processes. Further research is needed to expand the application scope of AZD5069 and explore its other pharmaceutical uses.

[0009] This invention addresses the clinical bottleneck of existing radiotherapy in the treatment of solid tumors (such as lung cancer, colorectal cancer, pancreatic cancer, and breast cancer), which can only achieve local tumor killing but cannot intervene in the radiotherapy-induced pathological chain of "CXCLs release → CXCR2 receptor activation → neutrophil recruitment / activation → tumor metastasis," resulting in persistently high tumor recurrence rates and distant metastasis risks (clinical recurrence and metastasis rates of 35%-65%). It also addresses the current lack of safe and effective combination therapies that precisely target this pathway. This invention, through the synergistic innovation of AZD5069 and radiotherapy, leverages the potent and highly selective CXCR2 receptor antagonistic activity and proven clinical safety of AZD5069 to precisely block the radiotherapy-induced tumor metastasis pathway, achieving the dual therapeutic goals of "local control + long-term anti-metastasis," thus filling the clinical gap in precise intervention for radiotherapy-related recurrence and metastasis.

[0010] Furthermore, the radiotherapy includes X-ray irradiation therapy, gamma-ray irradiation therapy, and heavy ion irradiation therapy.

[0011] Furthermore, the cancers mentioned include pancreatic cancer and lung cancer.

[0012] Furthermore, the drug includes AZD5069 and pharmaceutically acceptable excipients.

[0013] Furthermore, the pharmaceutically acceptable excipients are natural, semi-natural, or fully synthetic.

[0014] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0015] Furthermore, the drug is administered orally or by injection.

[0016] Furthermore, the content of AZD5069 in the drug is 0.1wt% to 99wt%.

[0017] Furthermore, the drug is used to inhibit tumor metastasis caused by radiotherapy.

[0018] The beneficial effects of this invention are as follows: This invention is the first to disclose the innovative application of AZD5069 as an adjuvant drug in radiotherapy. By precisely blocking the CXCL2 / CXCR2 axis, it achieves targeted intervention against radiotherapy-related metastases, filling the technological gap in existing radiotherapy regimens that "only kill tumors but do not prevent metastasis" and lack precise target drugs. Existing anti-metastasis combination regimens mostly use non-specific anti-inflammatory drugs or broad-spectrum anti-angiogenic drugs, which suffer from poor targeting and numerous adverse reactions. This invention uses the potent and highly selective CXCR2 receptor antagonist AZD5069, targeting only the core metastatic pathway induced by radiotherapy, making it more precise and safer than non-specific intervention regimens. Existing radiotherapy combination targeted drugs are mostly newly developed drugs, resulting in long development cycles and high translational risks. This invention uses AZD5069, which has entered Phase I / II clinical trials, leveraging its proven clinical safety, oral bioavailability, and clear target activity, shortening the clinical translation cycle by more than 50% compared to traditional combination regimens, and reducing research and application costs. Existing technologies for studying the mechanisms of action of radiotherapy combined with drugs are mostly limited to single aspects. This invention elucidates the multi-dimensional, full-chain mechanism of AZD5069—"blocking CXCR2 signaling + inhibiting neutrophil recruitment + improving the immune microenvironment + synergistic radiotherapy"—which is more comprehensive than single-target intervention and offers greater assurance of clinical efficacy. AZD5069 is an oral formulation, convenient to administer, with no significant drug interactions with radiotherapy, requiring no adjustment of radiotherapy dosage or treatment protocol. The selection criteria for the applicable population are clearly defined; the beneficiaries can be quickly identified by detecting peripheral blood CXCL2 levels and the proportion of CXCR2-positive neutrophils, facilitating its widespread clinical application. Attached Figure Description

[0019] Figure 1 The figures show the effects of local radiotherapy on tumor growth and lung metastasis in spontaneous metastasis models of Panc02 pancreatic cancer and LLC lung cancer. A is a comparison of the tumor growth curves of Panc02 pancreatic cancer in the radiotherapy group and the control group (no radiotherapy); B is a comparison of the tumor growth curves of LLC lung cancer in the radiotherapy group and the control group (no radiotherapy); C is a visual observation of lung metastases in the radiotherapy group and the control group; D is a statistical analysis of the number of Panc02 pancreatic cancer metastases in the lungs of the radiotherapy group and the control group; and E is a statistical analysis of the number of LLC lung cancer metastases in the lungs of the radiotherapy group and the control group.

[0020] Figure 2The image shows the results of single-cell RNA sequencing (scRNA-Seq) analysis of the effect of radiotherapy on neutrophil activation-related signaling (CXCR2) in CD45⁺ immune cells of Panc02 pancreatic cancer-bearing mice. In the image, A is a verification image of the specific expression of the CXCR2 gene in the neutrophil subset, and B is a comparison of the expression level of the CXCR2 gene in the neutrophil subset between the control group (control group, not receiving radiotherapy) and the radiotherapy group (IR group).

[0021] Figure 3 The image shows the flow cytometry results of the effect of radiotherapy on the expression of CXCR2 molecules on the surface of neutrophils in the lung microenvironment before lung metastasis in Panc02 pancreatic cancer-bearing mice. In the image, A is the flow cytometry result of lung tissue from Panc02 pancreatic cancer-bearing mice, and B is the statistical graph of CXCR2 gene transcriptional expression on neutrophils.

[0022] Figure 4 The image shows the treatment regimen and efficacy verification results of radiotherapy combined with the CXCR2 specific inhibitor AZD5069 in a spontaneous metastasis model of Panc02 pancreatic cancer. In the image, A is the tumor volume statistics, B is the lung tissue metastatic nodule statistics, and C is the lung tissue staining image. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0024] Example 1 I. Establishment of the experimental model.

[0025] The spontaneous metastasis model in mice was established using the following method: All animal experiments were approved by the Biomedical Ethics Committee of the School of Medicine, Xi'an Jiaotong University. A suspension of Panc02 pancreatic cancer cells in logarithmic growth phase (1×10⁻⁶) was used. 6 (each / animal) and LLC lung cancer cell suspension (1×10) 6 (Number of mice per group) were injected subcutaneously into the right back of C57BL / 6 mice (n=7~10 mice per group, with a radiotherapy group and a control group). When the tumor volume grew to 100 mm³ (day 10 after inoculation), the mice in the IR group received a single 15 Gy X-ray local tumor radiotherapy (avoiding the lungs and normal tissues), while the control group received sham irradiation under the same conditions. All other feeding conditions were the same.

[0026] II. Monitoring of local tumor growth.

[0027] Starting from day 10 after inoculation, the long diameter (a) and short diameter (b) of the tumor were measured every 5 days using vernier calipers. The tumor volume was calculated using the formula V=ab² / 2, and a tumor growth curve was plotted.

[0028] III. Detection of lung metastasis.

[0029] Mice were sacrificed on day 40 after inoculation, and lung tissue was completely dissected. After fixation with 4% paraformaldehyde solution, the number of metastatic lesions on the surface was counted by gross observation, and paraffin sections were prepared for HE staining. The infiltration of metastatic lesions was observed under a microscope.

[0030] IV. Experimental Results.

[0031] like Figure 1 As shown, 15 Gy local radiotherapy can effectively inhibit the growth of primary lesions in Panc02 pancreatic cancer and LLC lung cancer. There was no statistically significant difference in the number of lung metastases between the radiotherapy group and the control group (P>0.05), indicating that although 15 Gy local radiotherapy can inhibit the growth of primary tumors, it has no significant effect on controlling spontaneous lung metastasis in Panc02 pancreatic cancer and LLC lung cancer.

[0032] The above experimental results confirm that existing local radiotherapy regimens suffer from a key technical bottleneck in tumor treatment: "locally effective but ineffective against metastasis." Radiotherapy can only inhibit the proliferation of tumor cells at the primary lesion, but it cannot block the distant metastasis process of tumor cells. Metastasis is the core reason for treatment failure and poor prognosis in cancer patients. This technical deficiency directly reflects the limitations of existing radiotherapy technology and provides clear experimental evidence and problem guidance for the technical solution of this invention, which aims to solve the problem of "radiotherapy combined with anti-metastasis strategy."

[0033] Example 2 I. Single-cell RNA sequencing (scRNA-Seq).

[0034] Panc02 pancreatic cancer cells in logarithmic growth phase were harvested and their concentration adjusted to 1×10⁻⁶ cells / year. 6Single-cell suspensions were injected subcutaneously into the right back of C57BL / 6 mice at a dose of 100 μL. Three mice were used in each group for biological replicates. When the tumor volume reached 100 mm³ (day 10 post-inoculation), mice in the IR group received a single 15 Gy local tumor radiotherapy (avoiding the lungs and normal tissue), while the Control group received sham irradiation under the same conditions. Mice were sacrificed on day 3 post-irradiation. Intact lung tissue was rapidly dissected, rinsed with pre-cooled PBS buffer to remove blood and impurities, and blotted dry with filter paper. The tissue was then chopped, digested with 1 mg / ml collagenase IV (Sigma) and 0.2 mg / ml DNase I (Sigma) at 37°C for 1 h, and filtered to obtain a single-cell suspension. 10 μL of Live / Dead fluorescent dye (BioLegend) was added to each 100 μL of the single-cell suspension, and the cells were incubated at room temperature in the dark for 20 minutes. The cells were then washed with flow beffer (PBS containing 1% serum) and centrifuged at 1900 rpm for 5 minutes. Surface staining: APC-Cy7 labeled anti-mouse CD45 antibody (Biolegend), incubated at 4°C in the dark for 30 minutes; washed with flow beffer and centrifuged at 1900 rpm for 5 minutes. Resuspended in flow beffer and sorted using a BD flow cytometer: dead cells were first removed through the Live / Dead channel, then CD45 cells were selected. + Positive cell population, sorted CD45 +Cells were collected in enzyme-free centrifuge tubes containing 1 mL of flow beffer. Immediately after sorting, the cells were centrifuged at 4°C and 1900 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 200 μL of enzyme-free water. Cell viability was assessed (viable cell rate ≥80%). Cell concentration was determined using a Countess II automated cell counter (target concentration 500–1000 cells / μL). Qualified single-cell suspensions were used for subsequent experiments. Single-cell sequencing was performed using the 10X Genomics platform. The specific steps included: single-cell suspension quality control, generation of GEMs (Gel Bead-In-Emulsions), reverse transcription to cDNA, cDNA recovery and amplification, cDNA quality control, library construction, library quality control, and sequencing. The sequencing process was controlled and real-time data analysis was performed using Illumina Data Collection Software to obtain raw sequencing data. Data processing was performed using CellRanger (version 7.0) from 10X Genomics: based on the gene expression matrix output by CellRanger, data standardization (NormalizeData function), normalization (ScaleData function), and principal component analysis (PCA) were performed using Seurat software (version 4.0). Dimensionality reduction clustering analysis was performed using t-SNE or UMAP algorithms, and cell subpopulations were divided according to the clustering results. For the DEGs of each cell subpopulation, GO and KEGG enrichment analysis were performed using clusterProfiler software, and significantly enriched items with an Adjusted P value < 0.05 were screened. Genes related to tumor metastasis, inflammatory response, and neutrophil activation were analyzed in detail.

[0035] II. Flow cytometry detection.

[0036] Intact lung tissue was rinsed with pre-cooled PBS buffer to remove surface blood and impurities, and the tissue was blotted dry with filter paper. The tissue was then chopped and digested with 1 mg / ml collagenase IV (Sigma) and 0.2 mg / ml DNase I (Sigma) at 37°C for 1 hour. The resulting single-cell suspension was obtained by filtration. 10 μL of Live / Dead fluorescent dye (biolegend) was added to every 100 μL of the single-cell suspension, and the mixture was incubated at room temperature in the dark for 20 minutes. The cells were then washed with a flow beffer and centrifuged at 1900 rpm for 5 minutes. Surface staining was performed using APC-Cy7 labeled anti-mouse CD45 antibody, FITC labeled anti-mouse CD11b antibody (biolegend), PE labeled anti-mouse Ly6C antibody (biolegend), PE-Cy7 labeled anti-mouse Ly6G antibody (biolegend), and APC labeled anti-mouse CXCR2 antibody (biolegend) was incubated at 4°C in the dark for 30 minutes. The cells were then washed with a flow beffer and centrifuged at 1900 rpm for 5 minutes. The flow beffer was resuspended and analyzed using a BD flow cytometer, followed by analysis using Flowjo v10 software.

[0037] III. Experimental Results.

[0038] like Figure 2 As shown, CXCR2 is specifically highly expressed in a subset of neutrophils in lung tissue, confirming that CXCR2 can serve as a specific molecular marker of neutrophil activation. Compared with the control group, the expression level of CXCR2 gene in neutrophils in the IR group was significantly increased, directly confirming that radiotherapy can induce activation phenotypic transformation of neutrophils in the pre-metastatic lung microenvironment, and that CXCR2 is the core molecular target of this activation process.

[0039] To confirm the regulatory effect of radiotherapy on the neutrophil activation phenotype at the protein level, directly supporting the core mechanism and technical solution of this invention, flow cytometry results of lung tissue from Panc02 pancreatic cancer-bearing mice were obtained as follows: Figure 3 As shown, the radiotherapy group (IR group) showed increased CXCR2 expression on neutrophils compared to the control group (no radiotherapy). This is completely consistent with the transcriptional expression trend of the CXCR2 gene, directly confirming that radiotherapy can upregulate CXCR2 expression on the neutrophil surface at the protein level. This further confirms that CXCR2 is a core molecular marker of radiotherapy-induced neutrophil activation and provides direct phenotypic evidence for intervention strategies targeting CXCR2.

[0040] Example 3 I. Drug administration modeling.

[0041] The drug AZD5069 used in this invention was purchased from MCE (MedChemExpress), catalog number HY-19855.

[0042] A mouse spontaneous metastasis model was established by: 1×10⁻⁶ Panc02 pancreatic cancer cell suspension in logarithmic growth phase. 6 C57BL / 6 mice were subcutaneously injected with 150 AZD5069 spores (n=7-10 per group, with separate radiotherapy and control groups). When the tumor reached 100 mm³ (day 10 post-inoculation), local radiotherapy (15 Gy, precisely covering the primary tumor lesion, avoiding lung irradiation) was administered to both the IR and IR+AZD5069 groups. Drug intervention was initiated in the IR+AZD5069 group 24 hours post-radiotherapy, using AZD5069 via gavage at a dose of 10 mg / kg, once daily, for 30 days post-tumor. The control group received no radiotherapy or drug intervention, only an equal volume of PBS via gavage. The IR group received an equal volume of corn oil via gavage starting day 2 post-radiotherapy, with the same frequency and duration as the IR+AZD5069 group, eliminating interference from solvent and gavage procedures and ensuring the scientific validity of the inter-group comparison. All three groups of mice were raised under the same SPF-grade feeding conditions. The experimental endpoint was uniformly measured by the local tumor volume, the size and number of lung tissue metastases, and the core efficacy indicators.

[0043] II. Experimental Testing.

[0044] The steps for monitoring tumor growth and detecting lung metastasis are the same as in Example 1.

[0045] III. Experimental Results and Discussion.

[0046] Neutrophils, the most numerous white blood cells in the human immune system, are traditionally recognized for their core functions of fighting infection and clearing foreign substances. However, recent studies have confirmed that neutrophils play a crucial "cancer-promoting role" in the occurrence, development, and metastasis of tumors. Especially after anti-tumor treatments such as radiotherapy, their abnormal activation has become a core effector driving tumor recurrence and metastasis. In the tumor microenvironment and pre-metastatic microenvironment of solid tumors (such as lung cancer, colorectal cancer, and breast cancer), neutrophils are among the most numerous infiltrating immune cells. Their infiltration density is significantly positively correlated with tumor stage, metastasis risk, and poor patient prognosis. Clinical data show that the incidence of distant metastasis in tumor patients with high neutrophil infiltration is 2 to 3 times higher than that in ordinary patients, and the metastasis-free survival is shortened by more than 40%.

[0047] Neutrophils release pro-inflammatory factors and proteases that can induce the formation of a "pre-metastatic microenvironment" in metastatic target organs. This microenvironment recruits immunosuppressive cells such as regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), suppressing the body's anti-tumor immune surveillance function. Simultaneously, it promotes angiogenesis and lymphangiogenesis, providing a "suitable environment" for the distant colonization and proliferation of circulating tumor cells, significantly increasing the success rate of tumor cell metastasis. The tumor microenvironment releases a large number of chemokines (such as CXCL1, CXCL2, and CXCL8). These chemokines specifically bind to the CXCR2 receptor, which is highly expressed on the surface of neutrophils, mediating the directed chemotactic migration of neutrophils from peripheral blood to the tumor microenvironment and potential metastatic target organs (lung, liver, bone, etc.), providing a "cellular basis" for metastasis initiation.

[0048] Although the mechanism of neutrophil-mediated tumor metastasis is well understood, current clinical practice lacks precise and safe targeted interventions, mainly due to the following technical shortcomings: Limited efficacy of non-specific interventions: Existing regimens mostly use broad-spectrum anti-inflammatory drugs (such as glucocorticoids) or non-selective chemotherapy drugs, which can mildly inhibit neutrophil function but have poor targeting and suppress normal immune function, leading to increased risk of infection and adverse reactions. Furthermore, they cannot block the CXCR2-mediated specific chemotactic recruitment pathway, resulting in poor anti-metastasis efficacy. Insufficient clinical translation of specific target drugs: Drugs targeting neutrophil-specific targets such as CXCR2 and NE are mostly in the preclinical research stage, lacking validated clinical safety data. Their development cycle is long, and the translation risk is high, failing to meet the urgent clinical needs for post-radiotherapy metastasis intervention. In summary, neutrophil abnormal activation-mediated tumor metastasis has become a major obstacle to improving clinical efficacy after radiotherapy.

[0049] Experimental results are as follows Figure 4 As shown, compared with the Control group, although the IR group could significantly inhibit local tumor growth (P<0.05), it could not effectively inhibit lung metastases, fully exposing the inherent technical defects of radiotherapy alone. The IR+AZD5069 group not only achieved a local tumor inhibition effect comparable to the IR group, but also the size and number of lung metastases were significantly lower than those in the IR group, and even approached the level of the Control group. This indicates that the "radiotherapy combined with CXCR2 inhibitor AZD5069" regimen of the present invention can, on the basis of ensuring local tumor control, inhibit radiotherapy-induced pancreatic cancer lung metastases by specifically blocking the CXCR2 pathway, successfully solve the core bottleneck of existing radiotherapy technology, and achieve the dual treatment goals of "local tumor control" and "anti-metastasis".

[0050] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0051] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0052] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of AZD5069 in the preparation of a medicament for assisting radiotherapy of cancer.

2. Use according to claim 1, characterized in that, The radiotherapy includes X-ray irradiation therapy, γ-ray irradiation therapy and heavy ion irradiation therapy.

3. Use according to claim 1, characterized in that, The cancer includes pancreatic cancer and lung cancer.

4. Use according to claim 1, characterized in that, The medicament includes AZD5069 and pharmaceutically acceptable excipients.

5. Use according to claim 4, characterized in that, The pharmaceutically acceptable excipients are natural products, semi-natural products or total synthetic products.

6. Use according to claim 4, characterized in that, The medicament further includes a pharmaceutically acceptable carrier.

7. Use according to claim 4, characterized in that, The medicament is administered in the form of oral administration preparations or injection administration preparations.

8. Use according to claim 4, characterized in that, The content of AZD5069 in the medicament is 0.1wt%-99wt%.

9. Use according to claim 4, characterized in that, The medicament is used for inhibiting tumor metastasis caused by radiotherapy.

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