Application of targeted CCR1 inhibitor in medicine for treating acute respiratory system inflammation diseases

Abstract

The invention discloses an application of a targeted CCR1 inhibitor in a medicine for treating acute respiratory system inflammatory diseases, which clarifies the interaction relationship among important inflammatory cells in the pathological process of inflammatory lung injury, and clarifies the interaction pathway among the important inflammatory cells of lung in the pathological process of inflammatory lung injury. Therefore, an inflammatory cell interaction pathway is taken as a target spot, the BX471 is selected as a targeted CCR1 inhibitor, and the BX471 blocks a CCL5-CCR1 pathway in a targeted manner to inhibit neutrophil chemotaxis, so that the inflammatory lung injury is effectively treated.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for treating inflammatory lung injury, and in particular to the use of a CCR1-targeted inhibitor in drugs for treating acute respiratory inflammatory diseases. Background Art

[0002] Acute respiratory distress syndrome (ARDS) refers to acute diffuse lung injury and subsequent acute respiratory failure caused by various intrapulmonary and extrapulmonary pathogenic factors such as pneumonia, gastric content aspiration, and large-area trauma. Its essence is a lung inflammatory response mediated by a variety of inflammatory cells and epithelial / endothelial cells and the inflammatory mediators they release. Among them, neutrophils are the first inflammatory cells to respond. Tissue-resident macrophages can recruit neutrophils to the site of inflammation by secreting chemokine (CXC motif) ligand 1 (CXCL1), chemokine (CXC motif) ligand 2 (CXCL2), interleukin-1 (IL-1), etc. Activated neutrophils produce a large number of neutrophil extracellular traps (NETs) through the NETosis pathway, which cause alveolar septal edema, alveolar hemorrhage, and destruction of alveolar walls. In ARDS, the ability of macrophages to phagocytize NETs and apoptotic neutrophils is significantly reduced, and they can prolong the lifespan of neutrophils by secreting granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), and tumor necrosis factor-α (TNF-α). At the same time, NETs can regulate macrophage polarization to the pro-inflammatory M1 phenotype. After being phagocytosed by alveolar macrophages, NETs can trigger macrophage caspase-1-dependent pyroptosis, and the large amount of cytokines released after cell pyroptosis further causes neutrophil infiltration. The vicious cycle caused by these phenomena promotes the continued progression of ARDS inflammation. In the pathological process of ARDS, there is a complex regulatory network between tissue-resident macrophages and neutrophils, but the interaction between macrophages and neutrophils remains unclear. Therefore, exploring potential therapeutic drugs targeting the interaction pathway between macrophages and neutrophils can provide promising ideas and strategies for the precise targeted treatment of ARDS. Summary of the invention

[0003] The purpose of the present invention is to provide a method for treating acute respiratory inflammatory diseases by using a targeted CCR1 inhibitor. The present invention uses a targeted CCR1 inhibitor to prepare a drug for treating acute respiratory inflammatory diseases, which can alleviate the pathological state of ARDS.

[0004] The technical solution provided by the present invention is as follows: application of a targeted CCR1 (CC motif chemokine receptor type 1 (CC chemokinereceptor type 1, CCR1)) inhibitor in a drug for treating acute respiratory inflammatory diseases.

[0005] In the above application, the targeted CCR1 inhibitor is BX471, CAS: 217645-70-0, and has the following structural formula:

[0006]

[0007] In the aforementioned application, the drug for treating acute respiratory inflammatory diseases comprises BX471 as an active ingredient and a pharmaceutically acceptable carrier.

[0008] The active ingredient is in the form of liquid and / or solid inhalable particles;

[0009] The pharmaceutically acceptable carrier includes a propellant, a dispersant, a surfactant, a liquid carrier and / or a solid carrier.

[0010] In the aforementioned application, the subject to which the drug for treating acute respiratory inflammatory diseases is applied includes humans or non-human mammals, preferably humans, mice or rats.

[0011] In the aforementioned application, the targeted CCR1 inhibitor alleviates the pathological state of acute respiratory inflammatory diseases by regulating the interaction pathway between macrophages and neutrophils.

[0012] In the aforementioned application, the interaction pathway is the CCR5 (CC motif chemokineligand 5 (CCL5))-CCR1 pathway.

[0013] In the aforementioned application, the targeted CCR1 inhibitor reduces the activity of neutrophils by preventing the release of calcium ions in neutrophils and reducing the changes in calcium ions in neutrophils.

[0014] In the aforementioned application, the targeted CCR1 inhibitor inhibits the activation and aggregation of neutrophils by reducing the expression of CD11 antigen-like family member b (CD11b).

[0015] In the aforementioned application, the effective concentration of BX471 is 1 nM-10 nM.

[0016] In the aforementioned application, the administration of the drug for treating acute respiratory inflammatory diseases includes nebulized inhalation, oral administration, subcutaneous injection, tail vein injection or airway instillation; wherein the drug for treating acute respiratory inflammatory diseases can be an inhalation preparation, an oral preparation, an intravenous injection preparation, an intramuscular injection preparation or an airway instillation preparation; the oral preparation includes capsules, granules, and tablets; the inhalation preparation includes sprays, aerosols, dry powder inhalers or a combination thereof.

[0017] Compared with the prior art, the present invention uses a targeted CCR1 inhibitor as a drug for the treatment of acute respiratory inflammatory diseases, which can alleviate the pathological state of ARDS. Furthermore, the present invention also clarifies the interaction relationship between important inflammatory cells in the pathological process of inflammatory lung injury, and clarifies the interaction pathway between important inflammatory cells in the lung in the pathological process of inflammatory lung injury. Therefore, the present invention targets the inflammatory cell interaction pathway and selects BX471 as a targeted CCR1 inhibitor. BX471 has a reversible and competitive CCR1 inhibitory effect and is highly selective for CCR1. Compared with 28 different G protein-coupled receptors, BX471 is more than 10,000 times more selective for CCR1. This selectivity enables BX471 to specifically target CCR1 and reduce nonspecific effects on other receptors, thereby improving the accuracy and safety of treatment. The present invention selects BX471 as a targeted CCR1 inhibitor, which can inhibit a variety of biological effects mediated by CCR1, can prevent the release of calcium ions, reduce changes in intracellular calcium ions, and thus affect the activity and function of cells; can reduce the expression of CD11b, which is a molecule related to leukocyte adhesion and migration, thereby inhibiting the activation and aggregation of leukocytes; can reduce the migration of leukocytes to the site of inflammation and reduce local inflammatory responses by inhibiting the CCR1-mediated signaling pathway. Therefore, BX471 can significantly reduce the inflammatory response caused by CCR1-mediated immune cell activation and migration. This makes BX471 a potential drug that can alleviate the pathological state of ARDS by regulating the interaction pathway between macrophages and neutrophils, thereby providing a promising strategy for the precise targeted treatment of ARDS. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The cell clustering obtained from single-cell data is shown;

[0019] Figure 2 A pathway diagram showing enhanced signaling between macrophages and neutrophils in the inflammatory lung injury group compared with the control group was presented;

[0020] Figure 3This is a cell experiment diagram showing the chemotactic effect of CCL5 on neutrophils;

[0021] Figure 4 Mouse bronchoalveolar lavage fluid cell counts are shown;

[0022] Figure 5 This is a graph showing the detection of inflammatory factors in the supernatant of mouse bronchoalveolar lavage fluid;

[0023] Figure 6 HE staining of mouse lung tissue verifies the therapeutic effect of BX471. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments, and the examples given are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0025] Example: Application of CCR1-targeted inhibitors in drugs for the treatment of acute respiratory inflammatory diseases.

[0026] 1. Single-cell sequencing data analysis;

[0027] 1.1. Construction of animal model: Select 6-8 week old C57BL / 6 wild-type healthy male mice, intraperitoneally inject 1% sodium pentobarbital for anesthesia, keep the mice upright, use tweezers to make the tongue of the mice stick out of the mouth, use a pipette to absorb 15 mg / kg lipopolysaccharide (LPS), drip LPS into the airway, and then shake the mice upright slightly to induce acute lung injury. The same method was used for mice in the control group, and the same volume of phosphate buffered saline (PBS) was dripped into the trachea. The mice were placed on a warming blanket for anesthesia recovery. After 24 hours, the mice were killed with 1% sodium pentobarbital and the lung tissues were collected in tissue preservation solution and stored at 4°C.

[0028] 1.2. Obtaining single cell suspension: After centrifugation, remove the tissue preservation solution, wash twice with Dulbecco's modified eagle medium (DMEM), and then quickly cut the lung into small tissue pieces, transfer to a centrifuge tube containing enzymatic solution, and digest in a 37°C water bath. Use a pipette to blow several times every 15-20 minutes. After 30 minutes, observe the tissue digestion status by trypan blue staining microscopy, and stop digestion until there are no obvious tissue pieces. Filter the cell suspension using a 40μm cell sieve, centrifuge at 4°C, 300g for 5 minutes, and remove the supernatant. Add red blood cell lysis solution to resuspend the cells. After 2-5 minutes of red blood cell lysis, add an equal amount of pre-cooled PBS containing 0.04% bovine serum albumin (BSA) to stop red blood cell lysis. Centrifuge as above to remove the supernatant. Add pre-cooled PBS (containing 0.04% BSA) to wash and resuspend the cells, and centrifuge again to remove the supernatant. Trypan blue staining and blood cell counting were used to observe the survival rate. Cells with a survival rate greater than 90% could be used for the next experiment.

[0029] 1.3.10X Genomics single-cell transcriptome sequencing: The prepared single-cell suspension is quickly used by the fully automatic instrument Chromium Controller to generate single-cell GEMs (Gel Bead in emulsion), and reverse transcription is used to generate cDNA. A demulsification reaction system is prepared, and PCR amplification is performed using cDNA as a template, followed by fragmentation, end repair, and "A" addition. After the adapter is connected, polymerase chain reaction (PCR) amplification is performed, and then the product is purified. The library is quality inspected, the PCR product is denatured into a single strand, and a circularization reaction system is prepared to digest the uncircularized linear DNA molecules. The single-stranded circular DNA molecule is replicated by rolling circles to form a DNA nanoball containing multiple copies, which is sequenced by combined probe anchor polymerization technology.

[0030] 1.4. Data reading and preprocessing: The original FASTQ format data were analyzed using Cell Ranger software downloaded from the 10X Genomics official website, and the data were converted and a gene expression matrix was generated. The Seurat (version 4.3.0.1) package of R (version 4.3.1) was used to read the generated files and construct a Seurat object.

[0031] 1.5. Filter low-quality cells and genes: The standard for cell quality control is to remove cells with gene expression levels below 200, and the standard for gene quality control is to remove genes expressed in less than 3 cells.

[0032] 1.6. Removal of mitochondrial genes and extreme values: First, cells with gene counts less than 200 and more than 2500 were filtered out. Cells with mitochondrial gene counts less than 10% and extreme values ​​were also filtered out.

[0033] 1.7. Standardization, dimensionality reduction, and clustering: SCTransform was used to standardize and batch the data. Linear dimensionality reduction was performed through PCA principal component analysis, and the first 30 principal components were selected for tSNE nonlinear dimensionality reduction. Then, the K nearest neighbor method was used for clustering, and each cell subpopulation was clustered using the cell markers on the CellMarker 2.0 website.

[0034] 1.8. Cell communication analysis: Cell communication analysis was performed using Cellchat (version 1.6.1). Standardized and cell-annotated single-cell sequencing data were imported into R to construct a cellchat object, and the mouse ligand-receptor database "CellChatDB.mouse" was imported. After preprocessing the data, the results were visualized using netVisual_bubble, netVisual_aggregate, etc.

[0035] 2. In vitro experiments have shown that CCL5 can attract neutrophils and this effect can be blocked by BX471;

[0036] 2.1. Cell culture and induction: Human myeloid leukemia cells (HL-60) were cultured in RPMI 1640 (Roswell Park Memorial Institute) medium containing 10% fetal bovine serum (FBS) and suspended in a 37°C, 5% CO2 incubator. The cell density was adjusted to 2×10 5 The cells were cultured in RPMI 1640 medium containing 10% FBS and 1.25% dimethyl sulfoxide (DMSO) to induce neutrophils for 5 days.

[0037] 2.2. Flow cytometry to detect neutrophil induction rate: Collect the cell suspension, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in PBS containing 1% BSA, and adjust the cell density to 1×10 7 Take 100 μl of the above resuspended cells and add them into the flow cytometry tube to make the cell count reach 1×10 6Each tube was divided into two groups, and the test sample and the control group were set up separately. CD11b antibody was added to each tube and mixed gently, and an equal amount of 1% BSA was added as a blank control. Incubate at 4°C in the dark for 30 minutes, centrifuge at 1000rpm for 5 minutes, and discard the supernatant. Wash twice with 4°C pre-cooled PBS, resuspend in PBS to a final volume of 200μl, and detect and analyze by flow cytometry within 1 hour. Cells with an induction rate greater than 80% were used for subsequent experiments.

[0038] 2.3. Neutrophil migration experiment Transwell: 3 μm Transwell chamber was used. 100 μl 1×10 6 10 ng / ml cell suspension, BX471 was added to the inhibitor group to make the final concentrations 1nM, 5nM, and 10nM, 10 ng / ml CCL5 in 0.1% FBS RPMI 1640 medium was added to the lower chamber, and a blank control group was set up and cultured in a 37°C incubator for 4 hours. The cells in the lower chamber were collected, centrifuged at 1000 rpm and 4°C for 5 minutes, and the cells were resuspended in PBS and counted.

[0039] 3. BX471 treatment of lung injury mouse model;

[0040] 3.1. Preparation of BX471 solution: Weigh an appropriate amount of sulfobutylether-β-cyclodextrin (SBE-β-CD) and dissolve it in physiological saline to prepare a 40% solution, filter it through a 0.45 μm filter and place it at 4°C overnight. Add BX471 and filter it through a 0.45 μm filter again after it is completely dissolved.

[0041] 3.2. Construction of mouse model of lung injury: The method of constructing the mouse model is the same as that mentioned above.

[0042] 3.3. Injection of BX471 to treat lung injury in mice: After establishing the mouse lung injury model, 20 mg / kg BX471 solution was intraperitoneally injected at 0, 8, and 16 hours, and the mouse abdomen was gently massaged to promote drug absorption. After 24 hours, the mice were killed by intraperitoneal injection of 1% sodium pentobarbital. Half of the lung lobe was ligated with cotton thread, and the unligated lung lobe was lavaged with 0.40 ml PBS. This was repeated 3 times and the alveolar lavage fluid was collected. An appropriate amount of 4% paraformaldehyde was injected for internal fixation, and the lung lobe was removed and placed in a centrifuge tube containing 4% paraformaldehyde for external fixation and HE staining. The other half of the lung lobe was placed in a cryovial and stored at -80°C for subsequent experiments.

[0043] 3.4. Evaluation of the effect of BX471 in treating lung injury in mice: The total cell density of bronchoalveolar lavage fluid (BALF) was counted under a microscope using a blood cell counting plate, and then centrifuged at 4°C 1000rpm for 5 minutes. The supernatant was collected for enzyme-linked immunosorbent assay (ELISA) to detect the concentration of inflammatory factors in the BALF supernatant. The precipitated cells were resuspended in PBS and stained with Giemsa, and counted under an optical microscope. Neutrophils, macrophages and lymphocytes were counted separately to obtain the percentage of each type of cell. Formaldehyde-fixed lung tissue was stained with HE to compare lung tissue edema, inflammatory exudation and alveolar destruction.

[0044] 4. Experimental results:

[0045] Figure 1 Cell clustering obtained from single-cell data is shown. Figure 1 After dimensionality reduction, clustering and grouping, the single-cell data were divided into 15 cell subsets, namely B cells, basophils, dendritic cells, endothelial cells, alveolar epithelial cells, fibroblasts, M1 macrophages, M2 macrophages, monocytes, myofibroblasts, neutrophils, NK cells, pericytes, regulatory T cells and helper T cells. Figure 1 Different colors represent different cell subpopulations, and each dot represents a cell. The more dots there are, the greater the number of cell subpopulations.

[0046] Figure 2The signaling pathway between macrophages and neutrophils in inflammatory lung injury compared with the control group is shown. Compared with the control group, the communication intensity of the CCL5-CCR1 axis between macrophages and neutrophils in the inflammatory lung injury group of mice increased most significantly (the redder the color of the bubble, the stronger the increase in communication intensity compared with the control group, the bluer the color, the weaker the increase in communication intensity compared with the control group, and the size of the bubble represents the p value, the larger the bubble, the smaller the p value). It can be seen from the circle diagram that in the control group, only neutrophils, NK cells, basophils and regulatory T cells are involved in the CCL5-CCR1 pathway, while in the inflammatory lung injury group, each cell interacts in this pathway, but compared with the control group, the increased communication intensity between macrophages and neutrophils in the CCL5-CCR1 pathway is the most obvious.

[0047] Figure 3 This is a cell experiment diagram showing the chemotactic effect of CCL5 on neutrophils. Figure 3 The Transwell experiment in the results showed that CCL5 can chemotaxis neutrophils, and the chemotaxis increased with the increase of CCL5 concentration, and its minimum effective concentration was 10ng / ml (p<0.05). 10ng / ml CCL5 was added to the lower chamber, and 1nM, 5nM, and 10nM BX471 were added to the experimental group. The results showed that the chemotaxis could be inhibited by the CCR1 inhibitor BX471, and its minimum effective concentration was 1nM (p<0.01). When the concentration of BX471 increased to 5nM, the number of chemotactic neutrophils was only slightly higher than that of the control group, indicating that this concentration can have a greater inhibitory effect. When the concentration of BX471 continued to increase to 10nM, the inhibitory effect did not increase significantly. (*p<0.05, **p<0.01, ***p<0.001).

[0048] Figure 4 Shown is the cell count of mouse bronchoalveolar lavage fluid. Figure 4 The results showed that compared with the control group, the absolute values ​​of total inflammatory cells and neutrophils in the bronchoalveolar lavage fluid of mice in the inflammatory lung injury group increased significantly (p < 0.001). After BX471 treatment of mice with inflammatory lung injury, the absolute values ​​of total inflammatory cells and neutrophils in the bronchoalveolar lavage fluid decreased significantly (p < 0.01), while there was no statistical difference in the absolute values ​​of macrophages and lymphocytes between the inflammatory lung injury group and the control group and the BX471 group (*p < 0.05, **p < 0.01, ***p < 0.001).

[0049] Figure 5This is a graph showing the detection of inflammatory factors in the alveolar lavage fluid supernatant of mice. Compared with the control group, the expression of inflammatory factors such as IL-1β and IL-17a in the alveolar lavage fluid in mice with inflammatory lung injury was significantly increased (p < 0.001), and the expression of IL-1β and IL-17a was significantly decreased after BX471 treatment (p < 0.001). (*p < 0.05, **p < 0.01, ***p < 0.001).

[0050] Figure 6 HE staining of mouse lung tissue verifies the therapeutic effect of BX471. Figure 5 HE staining results of lung tissue showed that compared with the control group, the number of neutrophils infiltrated in the alveolar cavity of mice with inflammatory lung injury increased significantly, the alveolar septum edema increased significantly, the alveolar septum widened, the alveolar wall was severely damaged, and alveolar hemorrhage occurred. After BX471 treatment, the number of neutrophils in the alveolar cavity decreased significantly, lung tissue edema and alveolar hemorrhage were alleviated, the alveolar structure was relatively intact, and the treatment effect was obvious.

[0051] In summary, the present invention, based on single-cell sequencing analysis, concluded that macrophages secrete CCL5 to act on the CCR1 receptor of neutrophils to cause the recruitment of neutrophils to the lungs of mice with inflammatory lung injury. By intraperitoneally injecting the CCR1 inhibitor BX471 into mice with inflammatory lung injury, the recruitment of neutrophils in the lungs was reduced, thereby reducing their release of NSTs, phagocytosis and degranulation, etc., effectively alleviating lung tissue edema, alveolar hemorrhage and destruction of alveolar structure in mice with inflammatory lung injury, thereby achieving a therapeutic effect. The above experiments confirm that BX471 can become a targeted CCR1 inhibitor, and then become a drug with the potential to treat acute respiratory inflammatory diseases, providing a promising strategy for the precise targeted treatment of ARDS.

[0052] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. Application of CCR1-targeted inhibitors in the treatment of acute respiratory inflammatory diseases.

2. The use according to claim 1, characterized in that: The targeted CCR1 inhibitor is BX471, CAS: 217645-70-0, and has the following structural formula:

3. The use according to claim 2, characterized in that: The drug for treating acute respiratory inflammatory diseases comprises BX471 as an active ingredient and a pharmaceutically acceptable carrier.

4. The use according to claim 2, characterized in that: The targeted CCR1 inhibitor alleviates the pathological state of acute respiratory inflammatory diseases by regulating the interaction pathway between macrophages and neutrophils.

5. The use according to claim 4, characterized in that: The interaction pathway is the CCL5-CCR1 pathway.

6. The use according to claim 2, characterized in that: The targeted CCR1 inhibitor reduces the activity of neutrophils by preventing the release of calcium ions in neutrophils and reducing the changes of calcium ions in neutrophils.

7. The use according to claim 2, characterized in that: The targeted CCR1 inhibitor inhibits the activation and aggregation of neutrophils by reducing the expression of CD11b.

8. The use according to claim 2, characterized in that: The targeted CCR1 inhibitor reduces the migration of neutrophils to the inflammatory site and reduces the local inflammatory response by inhibiting the CCR1-mediated signaling pathway.

9. The use according to claim 3, characterized in that: The effective concentration of BX471 is 1 nM-10 nM.

10. The use according to claim 1, characterized in that: The administration of the drug for treating acute respiratory inflammatory diseases includes aerosol inhalation, oral administration, subcutaneous injection, tail vein injection or airway drip.

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