Use of ccl6 / 15 / 23 in the diagnosis of allergic airway inflammation

Abstract

The present application relates to the field of medicine, in particular to the application of CCL6 / 15 / 23 in the diagnosis and treatment of allergic airway inflammation. Specifically, the present application provides a combination reagent and its use, the combination reagent comprising: (S1) an inhibitor targeting CCL15 and / or an inhibitor targeting CCL23; and (S2) an inhibitor targeting CCR1. And the combination reagent can be used for preparing a composition or preparation for (a) inhibiting the differentiation of eosinophils, (b) inhibiting airway inflammation, and / or (c) asthma.

Description

Technical Field

[0001] This invention relates to the field of medicine, and more specifically to the application of CCL6 / 15 / 23 in the diagnosis of allergic airway inflammation. Background Technology

[0002] Asthma is one of the most common chronic diseases, with its incidence rising in populations across various countries, accounting for 1-18% of the total population. Eosinophil count is a predictive risk factor and biomarker for acute asthma exacerbations. Eosinophils, along with HSCs and maturing in the bone marrow, are recruited to sites of inflammation and release a range of cytokines, chemokines, or granules to mediate airway pathological responses, excessive mucus secretion, airway remodeling, and airway hyperresponsiveness. Directly targeting the eosinophil differentiation process is an effective therapeutic strategy for controlling asthma clinical symptoms and mitigating disease exacerbations. Summary of the Invention

[0003] The purpose of this invention is to provide a combination reagent that targets and controls the granulocyte differentiation process, thereby helping to treat asthma. Specifically, this invention provides a combination reagent containing (S1) an inhibitor targeting CCL15 and / or an inhibitor targeting CCL23; and (S2) an inhibitor targeting CCR1, said combination reagent targeting and inhibiting the formation of the CCL6 / 15 / 23-CCR1 complex, thereby helping to reduce or prevent aging.

[0004] In a first aspect of the invention, a combination reagent is provided, the combination reagent comprising:

[0005] (S1) Inhibitors targeting CCL15 and / or inhibitors targeting CCL23; and

[0006] (S2) Inhibitors targeting CCR1.

[0007] In a second aspect of the invention, a use of a combination reagent is provided, the combination reagent comprising:

[0008] (S1) Inhibitors targeting CCL15 and / or inhibitors targeting CCL23; and

[0009] (S2) Inhibitors targeting CCR1;

[0010] The combined reagents are used to prepare compositions or formulations for (a) inhibiting eosinophil differentiation, (b) inhibiting airway inflammation, and / or (c) asthma.

[0011] In another preferred embodiment, the inhibitor targeting CCL15 includes small molecule compounds, antibodies, miRNAs, gene editing reagents, or combinations thereof.

[0012] In another preferred embodiment, the inhibitor targeting CCL23 includes small molecule compounds, antibodies, miRNAs, gene editing reagents, or combinations thereof.

[0013] In another preferred embodiment, the inhibitor targeting CCR1 includes small molecule compounds, antibodies, miRNAs, or combinations thereof.

[0014] In another preferred embodiment, the CCR1-targeting inhibitor includes BX471.

[0015] In another preferred embodiment, the CCL15, CCL23 and / or CCR1 are derived from mammals, preferably from humans.

[0016] In another preferred embodiment, the inhibitors targeting CCL15 and / or CCL23 can inhibit the formation of the “CCL6 / 15 / 23-CCR1 complex”.

[0017] In another preferred embodiment, the CCR1-targeting inhibitor can inhibit the formation of the "CCL6 / 15 / 23-CCR1 complex".

[0018] In another preferred embodiment, the combined reagent is also used to prepare a medicament for treating and / or relieving asthma.

[0019] In another preferred embodiment, the reagent is an oral or non-oral formulation.

[0020] In another preferred embodiment, the formulation includes: powder, granules, capsules, injections, inhalers, tinctures, oral liquids, tablets, lozenges, or drops.

[0021] In a third aspect of the invention, a kit is provided, the kit comprising:

[0022] (K1) A detection reagent, said detection reagent being used to detect CCL6 / 15 / 23 ligands and / or CCL6 / 15 / 23-CCR1 complexes; and

[0023] (K2) A therapeutic agent comprising: (S1) an inhibitor targeting CCL15 and / or an inhibitor targeting CCL23; and (S2) an inhibitor targeting CCR1.

[0024] In another preferred embodiment, the detection reagent is used to detect whether the object is a high-expressing object of CCL15 and / or CCL23.

[0025] In another preferred embodiment, “high expression” means that the ratio of the mRNA level M1 of CCL15 and / or CCL23 in the subject to the mRNA level M0 of CCL15 and / or CCL23 in the normal population (i.e., M1 / M0) is ≥ 1.5, preferably ≥ 2, and more preferably ≥ 4.

[0026] In another preferred embodiment, the CCL15 or CCL23 is of human origin.

[0027] In a fourth aspect of the invention, a pharmaceutical composition is provided, comprising:

[0028] (i) a first pharmaceutical composition comprising: an inhibitor targeting CCL15 and a pharmaceutically acceptable carrier; and

[0029] (ii) A second pharmaceutical composition comprising: an inhibitor targeting CCL23 and a pharmaceutically acceptable carrier.

[0030] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are the same pharmaceutical composition.

[0031] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions.

[0032] In a fifth aspect of the invention, a method for inhibiting the formation of the "CCL6 / 15 / 23-CCR1 complex" in vitro is provided, comprising the steps of:

[0033] (a) In the presence of an inhibitor targeting CCL15 and / or an inhibitor targeting CCL23, contacting CCL15 and / or CCL23 with CCR1 thereby inhibiting the formation of the “CCL6 / 15 / 23-CCR1 complex”.

[0034] In another preferred embodiment, in step (a), the CCR1 is on the cell membrane.

[0035] In another preferred embodiment, the CCR1 is located on the cell membrane of an eosinophil (Eos).

[0036] In another preferred embodiment, the eosinophils are living human eosinophils.

[0037] In another preferred embodiment, the cell is an EOS cell.

[0038] In a sixth aspect of the invention, a method for treating and / or preventing asthma is provided, comprising administering the combination reagents described in the first aspect to a subject in need.

[0039] In another preferred embodiment, the object is an object with high expression of CCL15 and / or CCL23.

[0040] In another preferred embodiment, “high expression” means that the ratio of the mRNA level M1 of CCL15 and / or CCL23 in the subject to the mRNA level M0 of CCL15 and / or CCL23 in the normal population (i.e., M1 / M0) is ≥ 1.5, preferably ≥ 2, and more preferably ≥ 4.

[0041] In another preferred embodiment, the object is a person.

[0042] In another preferred embodiment, the subject is an asthma patient.

[0043] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0044] Figure 1 The increased expression of hCCL23 and hCCL15 is shown in asthmatic patients. Figure a shows the relative mRNA expression of hCCL23 and hCCL15 in total white blood cells (WBCs) of asthmatic patients (n=16) and healthy controls (n=16). Figure b shows the concentration of hCCL23 in plasma (n=31) of asthmatic patients as measured by ELISA compared to healthy controls (n=30). Data in a and b are presented as a midline (median line), with the vertical line length representing differences not exceeding 1.5 times the interquartile range (boxes indicate differences between the 25th and 75th percentiles). Figure c shows the correlation between hCCL23 protein levels and the number of eosinophils in human peripheral blood (n=61, linear regression and Spearman rank correlation). Figure d is a schematic diagram of the method used to separate peripheral blood leukocytes from asthmatic patients. Immunofluorescence staining and Wright-Giemsa staining of peripheral blood total white blood cells, monocytes, neutrophils, and eosinophils, EPX (green), hCCL23 (red), hCCL15 (red), and DAPI (blue), scale bar 40 μm. A magnified image is shown in the upper right corner, scale bar 10 μm. **, P < 0.01.

[0045] Figure 2This shows that hCCL23 and hCCL15 are not expressed in human leukocytes except for eosinophils. Figure a shows the expression of hCCL23 and hCCL15 in peripheral blood cells by eosinophils, observed and photographed using confocal microscopy, using immunofluorescence co-staining with antibodies against hCCL23 / hCCL15 (red), MBP (green), and DAPI (blue). Figure b shows no expression of hCCL23 and hCCL15 in T lymphocytes. CD3 (green), hCCL23 / hCCL15 (red), and DAPI (blue), scale bar 40 μm. Magnified image (far right), scale bar 10 μm.

[0046] Figure 3 The diagram shows the secretion of mCCL6, a homolog of hCCL23 and hCCL15, by mouse eosinophils. Figure a shows the sequence alignment of mCCL6 with hCCL23 and hCCL15. Shaded letters indicate residues specifically identical to hCCL23 and hCCL15 at each alignment position, and dashes indicate gaps inserted to optimize alignment. Figure b shows a schematic diagram of the construction of an allergic asthma mouse model and subsequent sample processing. Figure ce shows the expression of mCCL6 in BALF supernatant (c), lung tissue (d), and serum (e) from mice in the NS or OVA groups, detected by ELISA. Each point represents one mouse. Data from 4–5 mice per group are presented as mean ± SEM. Figure f shows the correlation between mCCL6 protein levels in mouse BALF and eosinophil count (n = 17, some points overlap). Figure g shows the flow cytometry gating method for leukocytes in mouse peripheral blood. Eosinophils (Eos) are defined as CD45... + SiglecF + Gr1 int Mononuclear cells are defined as CD45 + Gr1 hi SiglecF - CX3CR1 + Neutrophils (Neu) are defined as CD45+ cells. + Gr1 hi SiglecF - CX3CR1 - h. The expression of mCCL6 in peripheral blood mononuclear cells, eosinophils, and neutrophils of NS group mice was obtained using graph g. i. T cells (CD45) in mouse peripheral blood + CD3 + ) and B cells (CD45) + B220 +The flow cytometry gating method was used to measure mCCL6 expression in Eos, T cells, and B cells in the peripheral blood of jNS mice. **, P<0.01; ***, P<0.001.

[0047] Figure 4 This study showed increased mCCL6 secretion by eoCre / R26-tdTomato mice during asthma inflammation. Immunofluorescence of lung tissue from eoCre / R26-tdTomato mice in the NS and OVA groups revealed mCCL6 antibody (green), DAPI (blue), and eosinophils (tdTomato). + (Red). Scale bar 40μm, 20μm. b BALF cell immunofluorescence of NS or OVA group mice, mCCL6 antibody (green), DAPI (blue), Eos (EPX). + (Red). Scale bar 40μm, 20μm. c. Flow cytometry gating method for detecting Eos in BALF cells. d. Compared with NS mice, the mean fluorescence intensity of mCCL6 in eoCre / R26-tdTomato mouse BALF was increased in the OVA group. e. Immunofluorescence was performed using bone marrow cells from eoCre / R26-tdTomato mice in the NS and OVA groups; mCCL6 antibody (green), DAPI (blue), eoCre / R26-tdTomato... + (Red), scale bar 40μm. f statistical e in tdTomato + mCCL6 in cells + Percentage (n=6 per group, 3 images per mouse). g Eos(3×10 6 After treatment with IL-5 (20 ng / ml) and / or U0126 (20 μM) for 6 h, the concentration of mCCL6 in the cell culture supernatant was detected by ELISA.

[0048] Figure 5 This study showed that mCCL6 expression was almost unobserved in an allergic inflammation model in eosinophil-null (Eos-null) mice. After staining with mCCL6 antibody (red) and DAPI (blue) in the NS or OVA groups, Eos(EPX) expression was significantly reduced in the lung tissue of WT and Eos-null mice. + A representative image (green), scale bar 40 μm. A 3x magnified image (rightmost panel), scale bar 20 μm.

[0049] Figure 6 The study showed that CCL6 deficiency reduced eosinophilic airway inflammation induced by OVA. Figure a shows the Ccl6 deficiency model established using the CRISPR / Cas9 system. - / - A schematic diagram of mice. b. CCL6 knockout efficiency, WT and Ccl6 - / -Eosinophil proteins were extracted from mice for Western blotting experiments. cWT and Ccl6 were also analyzed. - / - Mouse BALF cells were counted after Wright-Giemsa staining. OVA asthma model as follows: Figure 3 Figure b. Combined data from 9–10 mice per group in two independent experiments are presented as mean ± SEM. Representative micrographs of lung sections stained with H&E (d), EPX (f), and PAS (h) at 24 hours after the last OVA nebulization. Scale bar, 100 μm. Histological inflammation score (e) and PAS score (i) analyzed from d and h. g. Analysis of EPX in total nucleated cells in f. + Percentage of cells (n=4-5 mice per group, 4 images per mouse). Data from 4-5 mice per group are expressed as mean ± SEM, 5-7 images per mouse. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0050] Figure 7 OVA group WT and Ccl6 were displayed. - / - Immunofluorescence of mouse BALF cells and quantitative RT-PCR of lung tissue. a) Mouse BALF cells were immunofluorescently stained with EPX (red), mCCL6 (green), and DAPI (blue) antibodies, and then observed using a confocal microscope. Scale bars: 40 μm, 20 μm. b) 24 hours after the last NS or OVA nebulization, the relative mRNA levels of Tslp, Epx, and Muc5ac in lung tissue were determined by quantitative RT-PCR. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0051] Figure 8 The study showed that mCCL6 deficiency reduces TH2 cell infiltration in asthma inflammation. Figure a illustrates the gating method for TH2 cells in lung tissue. Figure b shows the infiltration of WT and Ccl6 in the NS and OVA groups. - / - In mouse lung tissue, TH2 cells (CD3) + CD4 + ICOS + ST2 + Representative flow cytometry dot plots of cells. Absolute value of TH2 cell count in cb. CD4+ in lung tissue (d). + With CD8 +The proportion of T cells. e. The relative mRNA levels of Il-13 and Il-25 in lung tissue were determined by quantitative RT-PCR 24 hours after the last NS or OVA nebulization. f. The protein concentrations of IL-4 and IL-33 in lung tissue were determined by ELISA. Data were obtained from three independent experiments, 4–6 mice in each group, mean ± SEM. ns, no statistically significant difference; *, P<0.05; ***, P<0.001.

[0052] Figure 9 This study demonstrates that CCL6 deficiency eliminates the impairment of hematopoietic stem cell homeostasis in allergen-induced airway inflammation. a) Mouse eosinophil differentiation lineage. bc) Quantitative numbers of eosinophils in peripheral blood (b) and bone marrow (c) of NS or OVA-infected WT and Ccl6- / - mice. d) Bone marrow stem-progenitor cell gating protocol. ef) Representative flow cytometry plots (e) and absolute values ​​(f) for LSK. gh) Representative flow cytometry plots (g) and absolute values ​​(h) for CMP, GMP, and MEP. ij) Representative flow cytometry plots (i) and absolute values ​​(j) for EoP. 4–6 mice per group, three independent replicates; statistical data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0053] Figure 10This demonstrates that mCCL6 directly binds to and activates CCR1 and downstream signaling pathways. a) Schematic diagram of the GloSensor assay for CCR1 and CCL6. The adenylate cyclase activator (Forskol in) artificially increases cAMP levels by activating adenylate cyclase (AC), while CCR1 activation by CCL6 inhibits AC activity, leading to a decrease in cAMP levels. b) Dose-response curve of intracellular cAMP levels detected by the GloSensor assay. GloSensor-HEK293T cells transfected with empty vector plasmid or mCCR1 were treated with Forskol (1 μM), and mCCL6 was added at the indicated concentration gradient. The decrease in cAMP was recorded after 30 minutes. Data represent the mean ± SEM of three technical replicates. The median effective concentration (EC50) was calculated using nonlinear regression (three parameters). cd mCCL6 intervention at different time points in mCCR1-293T (c) or empty vector control 293T (d) cells, Western blot results of p-ERK1 / 2, ERK1 / 2, p-p38, p38 and GAPDH (loaded control). e Dose-response curves of normalized intracellular cAMP signaling as measured by GloSensor assay. GloSensor-HEK293T cells transfected with mCCR1 or mCCR1-Ncut30 were treated with Forskolin (1 μM) and treated with mCCL6 at the indicated concentration gradient. The decrease in cAMP was recorded after 30 min. Data represent the mean ± SEM of three technical replicates (error bars less than the sign are not shown). f Mouse primary eosinophils (lacking mCCL6) treated with control or 400 ng / mL mCCL6 at 30 s (arrows indicated) and intracellular calcium was rapidly detected, with recordings continuing for 150 s by flow cytometry. Primary mouse eosinophils (lacking mCCL6) were treated with either control or 400 ng / mL mCCL6 for 2 hours, and mCCR1 surface expression was analyzed by flow cytometry.

[0054] Figure 11 This study demonstrates that inhibiting CCR1 can alleviate OVA-induced eosinophilic airway inflammation. a. Schematic diagram of BMDE extraction and culture, and the BX471 intervention method. b. As shown in a, in WT BMDE treated with BX471, SiglecF... + F4 / 80 +The number of eosinophils, characterized by their cellularity, is shown in the figure (n = 4 mice per independent culture). c. Asthma treatment model with BX471. d. Differential count of BALF cells. e. Immunohistochemical staining of lung tissue with EPX antibody after BX471 treatment. Scale bar 100 μm. f. Analysis of EPX in e. + The ratio of cells to total nucleated cells (n=4-5 mice per group, 4 images per mouse). The concentration of mCCL6 in serum (g), lung tissue (h), and BALF supernatant (i) in the BX471 treatment model was detected by ELISA. The relative mRNA levels of Il-13 and Il-25 and the protein levels of IL-4 and IL-33 in lung tissue were determined by quantitative RT-PCR and ELISA. 4-5 mice per group, mean ± SEM. ns, no statistically significant difference; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0055] Figure 12 The study showed that CCR1 deficiency inhibits eosinophil differentiation in vitro, and that CCR1 is expressed and activated in bone marrow stem and progenitor cells in an allergic inflammation model. (a WT and Ccr1) - / - During mouse BMDE culture, SiglecF + F4 / 80 + The number of eosinophils was measured (n=3). b) Quantitative RT-PCR was used to detect CCR1 mRNA expression in stem and progenitor cells of bone marrow cells from mice in the NS and OVA groups. c) Flow cytometry was used to detect CCR1 protein expression on the surface of stem and progenitor cells in bone marrow cells from mice in the NS and OVA groups. Data are presented as mean ± SEM, with 4 mice per group. *, P<0.05; **, P<0.01.

[0056] Figure 13 The diagram shows that both whole bone marrow transplantation and competitive bone marrow transplantation in CCR1-deficient mice resulted in reduced eosinophil differentiation capacity in the OVA model. A schematic diagram of constructing an OVA asthma model after whole bone marrow transplantation is shown. WT recipient mice underwent lethal irradiation followed by either WT or Ccr1 gene knockout (Ccr1...). - / - Whole bone marrow transplantation from donor mice. bc. Flow cytometry counts (b) and absolute values ​​(c) of CMP, GMP, and MEP in the bone marrow. de. Flow cytometry counts (d) and absolute values ​​(e) of EoP in an asthma model. 2-5 mice per group. f. Schematic diagram of an allergic asthma model in mice after competitive bone marrow transplantation, where recipient mice received a lethal dose of irradiation followed by WT (CD45.1). + ) and Ccr1 - / -(CD45.2 + Competitive bone marrow transplantation using bone marrow cells. g WT or Ccr1 - / - Percentages of LSK, GMP, and EoP produced in the bone marrow of OVA mice. WT or Ccr1 levels in BALF and peripheral blood of OVA mice. - / - Percentage of eosinophils. Statistical data are presented as mean ± SEM, with 4 mice per group. *, P < 0.05; **, P < 0.01. Detailed Implementation

[0057] Through extensive and in-depth research, the inventors unexpectedly discovered that inhibiting the formation of the "CCL6 / 15 / 23-CCR1 complex" helps in the treatment and prevention of asthma. Based on this, the present invention was completed.

[0058] Specifically, experiments of this invention showed that CC chemokine ligand 6 (CCL6) was increased in asthmatic mice, and its human orthologs CCL15 and CCL23 were highly expressed in asthmatic patients, both primarily derived from eosinophils. Further studies using Ccl6 gene knockout mice showed that asthma was alleviated in these mice. Furthermore, the inventors identified the CCL6-CCR1 regulatory axis in hematopoietic stem cells (HSCs). The specific CCR1 antagonist BX471 targeting this axis significantly reduced eosinophil differentiation and airway inflammation. Therefore, this study is the first to identify the CCL6-CCR1 axis as being involved in the link between eosinophils and HSCs during the development of allergic airway inflammation. Thus, this invention also reveals a potential therapeutic strategy targeting G protein-coupled receptors (GPCRs) for future clinical treatment of asthma.

[0059] the term

[0060] As used herein, “the combination reagent of the present invention” refers to a combination reagent containing (S1) an inhibitor targeting CCL15 and / or an inhibitor targeting CCL23; and (S2) an inhibitor targeting CCR1.

[0061] As used herein, the terms “CCL6 / 15 / 23,” “CCL6 / 15 / 23 ligand,” and “CCL6 / 15 / 23 protein” are used interchangeably to refer to mouse CC chemokine ligand 6, human CC chemokine ligand 15, human CC chemokine ligand 23, or their homologues. It should be understood that this term includes both wild-type and mutant CCL6 / 15 / 23 protein, provided that the mutant retains at least 30% of the ligand activity of the wild-type, particularly the function or activity of forming a “CCL6 / 15 / 23–CCR1 complex” with the CCR1 receptor.

[0062] As used herein, the terms “CCL6-CCR1 regulatory axis” and “CCL6 / 15 / 23-CCR1 regulatory axis” are used interchangeably, referring to the regulation of downstream signaling pathways or functions by the CCL6 / 15 / 23 ligand and CCR1 through the formation of a key “CCL6 / 15 / 23-CCR1 complex”.

[0063] CCL6 / 15 / 23 ligands

[0064] The literature largely reports that mouse CC chemokine ligand 6 (mCCL6) is mainly produced by macrophages and attracts macrophages and CD4+. + T cells and eosinophils play important roles in various inflammatory diseases. Human CC chemokine ligand 15 (hCCL1, also known as MPIF-1) and human CC chemokine ligand 23 (hCCL23, also known as MIP-5, MIP-1δ) are orthologous analogs of mCCL6, belonging to the NC6 chemokine subfamily. However, the exact function of mCCL6 secreted by eosinophils and its pathogenic role in allergic asthma are not fully understood; similarly, the roles of hCCL23 and hCCL15 in asthma patients remain to be explored.

[0065] Eosinophil differentiation, asthma, and airway inflammation

[0066] Eosinophils are a type of white blood cell that differentiate from hematopoietic stem cells (HSCs) in the bone marrow. They are formed through the activation of hematopoietic stem cells (Lineage cells). - Sca-1 + c-Kit + The cells, including common myeloid progenitor cells (CMPs), granulocyte-monocyte progenitor cells (GMPs), and eosinophilic progenitor cells (EoPs), eventually develop into mature eosinophils. This process is regulated by a series of transcription factors (GATA-1, PU.1, C / EBP) and cytokines (IL-5, IL-3, GM-CSF).

[0067] Asthma is a heterogeneous disease, typically characterized by chronic airway inflammation, accompanied by variable airflow limitation and airway hyperresponsiveness.

[0068] Chronic airway inflammation is a key pathological feature of asthma, involving various inflammatory cells and cytokines, including eosinophils, mast cells, lymphocytes, neutrophils, and airway epithelial cells. Inflammatory mediators include histamine, leukotrienes, prostaglandins, IL-4, IL-5, IL-13, and GM-CSF. The pathological features of airway inflammation in asthma include inflammatory cell infiltration, epithelial cell destruction, basement membrane changes, and hypersecretion of airway mucus.

[0069] Pharmaceutical Compositions and Applications

[0070] The present invention also provides compositions, formulations, or products containing the combination reagents of the present invention, which may be used to treat or prevent asthma.

[0071] A preferred composition is a pharmaceutical composition comprising:

[0072] (i) a first pharmaceutical composition comprising: an inhibitor targeting CCL15 and a pharmaceutically acceptable carrier; and

[0073] (ii) A second pharmaceutical composition comprising: an inhibitor targeting CCL23 and a pharmaceutically acceptable carrier.

[0074] In another preferred embodiment, the pharmaceutical composition further contains an inhibitor targeting CCR1.

[0075] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are the same pharmaceutical composition.

[0076] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions.

[0077] As used herein, the term “effective amount” or “effective dose” refers to an amount that is functional or active (i.e., anti-aging) in humans and / or animals and is acceptable to humans and / or animals.

[0078] As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.

[0079] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0080] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the active ingredient of this invention is administered daily at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (preferably 0.0001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0081] Typically, when the pharmaceutical composition of the present invention is administered orally, the average daily dose for a subject weighing 60 kg is usually 10-500 mg, preferably 20-300 mg, and more preferably 50-250 mg. The daily dose may be divided into one, two, or multiple administrations.

[0082] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0083] The main advantages of this invention include:

[0084] (a) A combination formulation containing an inhibitor targeting CCL15 and / or CCL23; and an inhibitor targeting CCR1 was first proposed, and the combination formulation was found to treat asthma.

[0085] (b) The combination reagents of the present invention can not only treat or prevent asthma, but also be used to prepare kits for detecting asthma or patients with high expression of CCL15 and / or CCL23.

[0086] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0087] Example 1

[0088] Elevated levels of hCCL23 and hCCL15 orthologs in asthmatic patients

[0089] To investigate the clinical relevance of hCCL23 / hCCL15 and eosinophil count in allergic asthma, the inventors analyzed blood samples from 31 patients with acute asthma exacerbations and 30 healthy controls. Table 1 summarizes the clinical characteristics of the subjects.

[0090] Table 1. Clinical characteristics of the samples.

[0091]

[0092] Mean ± SEM or number (%). *NA, not available.

[0093] The inventors extracted total mRNA from peripheral blood leukocytes (WBCs) and found that the relative mRNA expression levels of hCCL23 and hCCL15 in asthma patients were higher than those in the control group. Figure 1 a). Furthermore, the serum hCCL23 concentration in the asthma group was higher than that in the healthy control group ( Figure 1 b), and correlated with the number of peripheral blood eosinophils ( Figure 1 c). Then, the inventors isolated human eosinophils, monocytes, and neutrophils from the blood of asthma patients and performed immunofluorescence staining ( Figure 1 d). Cells were validated by Wright-Giemsa staining and immunofluorescence staining. Notably, hCCL23 and hCCL15 expression was predominantly found in eosinophils compared to other cell types in leukocytes. Figure 1 e and Figure 2 (a, b)

[0094] Therefore, this suggests that the upregulation of hCCL23 and hCCL15 in asthma patients indicates that these cytokines may be related to allergic airway inflammation.

[0095] Example 2

[0096] Eosinophil-derived mCCL6 increased in a mouse asthma model.

[0097] hCCL23 and hCCL15 are two of the four chemokines in the NC6 subfamily and are orthologs of mCCL6. Figure 3 a). To investigate the roles of hCCL23 and hCCL15 in allergic airway inflammation, the inventors established an ovalbumin (OVA)-induced mouse model of allergic asthma and first examined the concentration of mCCL6 ( Figure 3 b).

[0098] The results showed that the bronchoalveolar lavage fluid (BALF) supernatant of mice in the OVA group ( Figure 3 c), lung tissue ( Figure 3 d) and serum ( Figure 3 The mCCL6 levels in mice in e) were significantly higher than those in the saline (NS) control group. Furthermore, in the correlation analysis, mCCL6 levels were positively correlated with eosinophil counts in BALF (Figure 3f).

[0099] To determine the origin of mCCL6, the inventors analyzed mCCL6 from different sites in control and OVA mice. Intracellular mCCL6 levels in different cell types in peripheral blood were determined by flow cytometry (Figure 3g, i). The results showed that the mean fluorescence intensity (MFI) of mCCL6 in eosinophils was significantly higher than that in monocytes, neutrophils, and lymphocytes (…). Figure 3 h and Figure 3 j) indicates that eosinophils are the main source of mCCL6 in vivo under homeostatic conditions.

[0100] The inventors further confirmed the origin of mCCL6 by crossing eoCre mice with R26-tdTomato mice, which produced a mouse strain with eosinophil-specific tdTomato fluorescence. As expected, eosinophils are the main source of mCCL6 in inflamed lung tissue. Figure 4 a). Specific staining for EPX in BALF cells confirmed the co-localization of mCCL6 and EPX. Figure 4 b).

[0101] Furthermore, the inventors analyzed mCCL6 MFI in BALF eosinophils and found that the accumulation level of mCCL6 in eosinophils was more than twice that of the OVA group compared with the control group (NS). Figure 4 c and Figure 4d).

[0102] Immunofluorescence analysis of bone marrow cells showed that, under the airway inflammatory response induced by OVA, the level of mCCL6 secreted by eosinophils was elevated. Figure 4 e and Figure 4 f), in this case, IL-5 may promote the release of mCCL6 via the MAPK pathway. Figure 4 g). In eosinophil-free mice (Eos-null), mCCL6 and airway inflammation were hardly observed in the asthma model. Figure 5 ).

[0103] In summary, these results reveal that eosinophils are the main source of mCCL6 in both homeostatic and airway allergic inflammation, and that the level of mCCL6 secreted by eosinophils increases under conditions of allergic airway inflammation.

[0104] Example 3

[0105] CCL6-deficient mice exhibited reduced eosinophilic airway inflammation and a decreased eosinophil differentiation lineage.

[0106] To investigate the contribution of mCCL6 to asthma in vivo, the inventors constructed a Ccl6 gene knockout (Ccl6) system using the CRISPR / Cas9 system. - / - ) mice ( Figure 6 a). By comparing wild-type (WT) and Ccl6 - / - Western blot analysis of mCCL6 in eosinophils sorted from mouse bone marrow confirmed the knockout efficiency. Figure 6 b). Male and female Ccl6 - / - The mice were healthy, with no underlying defects in peripheral blood leukocyte differential count (Table 2).

[0107] Table 2. WT and Ccl6 - / - Mouse peripheral blood leukocyte differential count

[0108]

[0109] Mean ± SEM.

[0110] However, after establishing the OVA-induced asthma model, the inventors discovered that Ccl6... - / - A significant decrease in eosinophil count was observed in the BALF of mice, while no significant changes were observed in other cell types in the BALF. Figure 6 c). Pathological lung section analysis showed that the WT mice in the OVA group exhibited significant peribronchial inflammatory cell infiltration, while Ccl6... - / - In mice, inflammatory infiltration was significantly reduced. Figure 6d, e), especially on EPX staining, eosinophil infiltration is reduced ( Figure 6 f, g). Periodic acid-Schiff (PAS) staining further revealed Ccl6 - / - Mice showed a mild response to OVA-induced allergic airway inflammation, with reduced mucus secretion. Figure 6 h, i). Additionally, dual immunofluorescence staining of BALF cells with EPX and mCCL6 antibodies confirmed the presence of WT and Ccl6 in the OVA group. - / - These results in mice ( Figure 7 a).

[0111] Consistent with these findings, in the lung tissue of WT mice in the OVA asthma group, the mRNA expression of inflammation-related thymic stromal lymphopoietin (Tslp), Epx, and secreted airway mucin Muc5ac was increased, while in Ccl6... - / - The symptoms were significantly relieved in mice. Figure 7 b). Using flow cytometry in lung tissue ( Figure 8 a) Analysis of TH2 cells revealed that Ccl6 in the OVA asthma group... - / - mice ( Figure 8 (b, c) In lung tissue, TH2 cell infiltration was reduced, while CD4 cell infiltration was reduced. + / CD8 + The ratio did not change significantly. Figure 8 d). The inventors further evaluated the production of typical TH2 cytokines in lung tissue and found that, under OVA induction, the mRNA expression of Il-13 and Il-25 in the lung tissue of WT mice was upregulated ( Figure 8 e) and elevated concentrations of IL-4 and IL-33 proteins, while Ccl6 - / - Significant relief was observed in mice. Figure 8 f). These data indicate that OVA-induced airway inflammation is CCL6-dependent in vivo.

[0112] Eosinophil differentiation occurs through hematopoietic stem cells (HSCs), common myeloid progenitor cells (CMPs), granulocytic-monocytic progenitor cells (GMPs), and eosinophilic progenitor cells (EoPs). Figure 9 a). Ccl6 after OVA atomization - / - mouse peripheral blood ( Figure 9 b) and bone marrow ( Figure 9 c) The number of mature eosinophils was not increased, indicating a deficiency of mCCL6 accompanied by reduced eosinophil lineage differentiation. First, we assessed WT and Ccl6 levels in the OVA group by flow cytometry. - / - The number of eosinophil-associated stem and progenitor cells in mice ( Figure 9d). The proportion of HSCs represented by LSK was increased in WT mice in the OVA group, but not in Ccl6. - / - Only slight changes were observed in mice. Figure 9 e, f). By analyzing the lineages associated with eosinophilia, including CMP, GMP, megakaryocyte-erythroid progenitor (MEP) ( Figure 9 g, h) and EoP( Figure 9 In our studies (i, j), we found that mCCL6 deficiency suppressed the increase of hematopoietic stem and progenitor cells associated with eosinophil differentiation. These data suggest that mCCL6 is crucial for HSC eosinophil differentiation in allergic airway inflammation.

[0113] Example 4

[0114] CCR1 was identified as the mCCL6 receptor.

[0115] The exact receptor for mCCL6 has not yet been precisely defined. A previous study reported that CCR1, belonging to the G protein-coupled receptor (GPCR) superfamily, is the putative receptor for mCCL6 in an IL-13-induced lung inflammation and remodeling model. Using GloSensor assays... Figure 10 a) The inventors first discovered that mCCL6 can activate mCCR1, leading to the activation of Gαi and downregulation of the second messenger molecule cyclic AMP (cAMP), triggering a downstream signaling cascade. Figure 10 b, e). Furthermore, mCCL6 intervention in HEK293T cells transiently transfected with mCCR1 induced time-dependent expression of p-ERK1 / 2 and p-p38 (b, e). Figure 10 c, d), provide evidence of mCCR1 activation. Like classic GPCRs, CCR1 is internalized after ligand binding and activation. Similarly, in primary eosinophils, administration of CCL6 leads to rapid intracellular calcium influx and subsequent CCR1 internalization. Figure 10 f, g).

[0116] Example 5

[0117] Targeted inhibition or absence of CCR1 impairs sustained differentiation of eosinophils and reduces airway inflammation.

[0118] The inventors have demonstrated that the interaction between mCCL6 and CCR1 promotes eosinophilia and allergic inflammation. The inventors utilized bone marrow-derived eosinophils (BMDE) and treated them with BX471 (a potent and specific CCR1 antagonist). Figure 11 a). Administration of BX471 on days 0, 4, and 8, or days 4 and 8, resulted in eosinopenia on days 8, 9, and 10. Figure 11b). In the knockout of the Ccr1 gene (Ccr1 - / - Similar results were also confirmed in the differentiation of BMDEs in mice. Figure 12 a) This indicates that inhibition of CCR1 can reduce eosinophil differentiation.

[0119] Next, the inventors analyzed the expression and activation of CCR1 in bone marrow-derived stem and progenitor cells in an asthma model. Although no difference in CCR1 expression was found between NS and OVA mice, the cell surface CCR1 level was significantly reduced in the OVA group, representing ligand recognition and CCR1 internalization. Figure 12 b, c). The inventors conditionally knocked out CCR1 in the hematopoietic system of mice through whole bone marrow transplantation and established an OVA asthma model to analyze eosinophil differentiation ( Figure 13 a). Bone marrow cells lacking CCR1 exhibit reduced eosinophil differentiation ( Figure 13 (be). In addition, a system with WT and Ccr1 was established. - / - A chimeric model of bone marrow cells in which stem cells and progenitor cells share the same microenvironment under differentiation stress induced by OVA-stimulated mCCL6. Figure 13 f). Compared to WT cells, CCR1-deficient cells showed a significantly reduced differentiation rate from stem cells, progenitor cells (including GMP and EoP) to mature eosinophils. Figure 13 gi).

[0120] The inventors further explored the role of specific CCR1 inhibition in in vivo allergic airway inflammation. During NS or OVA nebulization, mice were subcutaneously injected every 8 hours with BX471 (20 mg / kg) or the carrier. Figure 11 c). In BALF cells, BX471 treatment completely reversed OVA-induced eosinophilia ( Figure 11 d). Peribronchial EPX after BX471 treatment of lung tissue + Eosinophil infiltration decreased to levels comparable to those in the NS group mice. Figure 11 e, f). Analysis of mCCL6 levels showed that BX471 treatment significantly reduced mCCL6 levels in lung tissue and BALF. Figure 11 The data (gi) indicate that the decreased mCCL6 level is consistent with changes in eosinophils. The relative mRNA levels of Il-13 and Il-25 in lung tissue (gi) Figure 11 j) and the protein levels of IL-4 and IL-33 ( Figure 11Analysis of the data also showed that BX471 treatment inhibited the increase in TH2 cytokines induced by OVA. These data suggest that blocking mCCL6-CCR1 signaling can prevent eosinophilic inflammation in the lungs.

[0121] discuss

[0122] Eosinophils produce a variety of cellular mediators that participate in the occurrence and development of allergic asthma. For example, active eosinophils produce cytokines such as interleukins (IL-4, IL-13), CC chemokine ligand 5 (CCL5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulate type 2 (TH2) effector cells in dendritic cells and T helper lung immune responses.

[0123] Eosinophils also secrete granules, including eosinophil peroxidase (EPX), eosinophil major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxins, which directly contribute to asthma pathology. These factors form an immune regulatory network in allergic asthma, revealing dynamic interactions between eosinophils and other immune cells.

[0124] The inventors have discovered that eosinophils disrupt HSC maintenance and mobilization primarily through eosinophil-derived mCCL6, thereby disrupting HSC homeostasis. The effects of eosinophils on stem cells suggest that eosinophils may be involved in the pathogenesis of asthma. However, the complex roles of eosinophils and active agents in allergic asthma remain not fully understood.

[0125] Studies have shown that mCCL6 (also known as C10) is mainly produced by macrophages and primarily attracts macrophages and CD4+. + T cells and eosinophils. mCCL6 plays an important role in inflammatory processes, including pulmonary fibrosis, allergic bronchopulmonary aspergillosis, sepsis, and experimental demyelinating diseases.

[0126] Mouse CCL6, along with human CCL23 (also known as MPIF-1) and CCL15 (also known as MIP-5, MIP-1δ), may activate CCR1. Previous studies by the inventors have demonstrated the potential role of eosinophil-secreted mCCL6 in HSC damage during inflammatory airway diseases.

[0127] However, prior to this invention, the exact function of mCCL6 was not fully understood, and its pathogenic role in allergic asthma was unclear; similarly, the roles of hCCL23 and hCCL15 in asthma patients were also unclear.

[0128] In this invention, the inventors explored the role of eosinophil-secreted mCCL6 in the pathology of allergic inflammation. mCCL6 interacts with CCR1, forming a positive feedback loop that exacerbates asthma. In asthma patients, the expression of human orthologs of mCCL6, hCCL23 and hCCL15, is also increased, supporting the clinical relevance of the current findings.

[0129] Specifically, in the research of this invention, the inventors found elevated hCCL23 and hCCL15 levels in asthmatic patients. Animal studies showed similar results, including increased expression of eosinophil-derived mCCL6, which is responsible for eosinophilic airway inflammation. Using Ccl6... - / - In mice, the inventors found that CCL6 deficiency significantly reduced OVA-induced eosinophilia, excessive mucus secretion, and airway TH2 response. Specifically, the inventors provided direct evidence that mCCL6 activates CCR1 and induces downstream p-ERK1 / 2 and p-p38 expression. In vitro and in vivo treatment with the specific CCR1 antagonist BX471 significantly alleviated eosinophil differentiation and OVA-induced eosinophilic airway inflammation. Therefore, the inventors conclude that the CCL6-CCR1 axis is an important regulatory mechanism in the pathogenesis of asthma and a potential target for further therapeutic research.

[0130] Previous and current research by the inventors has highlighted the function of a key chemokine, mCCL6, primarily derived from eosinophils, which promotes eosinophil development and triggers allergic airway inflammation by directly acting on bone marrow HSCs. Eosinophils are multifunctional leukocytes, and evidence supports that the interaction between eosinophils and progenitor cells has a broader role, potentially acting as effective effector cells in the initiation and promotion of allergic inflammation. Blocking progenitor cells or inhibiting HSC differentiation at the source of HSCs has been considered an effective therapeutic strategy for asthma in animal studies. Here, the inventors have identified mCCL6 as a key mediator involved in crosstalk between eosinophils and progenitor cells in a mouse model of allergic inflammation. Increased HSC and EoP populations depended primarily on the upregulation of mCCL6 expression, which may be involved in the development of allergic inflammation, suggesting a potential feedback role for eosinophils and their progenitor cells in lung pathology.

[0131] Hematopoietic stem cells (HSCs) are responsible for the lifelong production of blood cells. Furthermore, HSCs must respond to acute or chronic demands, such as injury or inflammation. Pro-inflammatory cytokines, such as IL-1, tumor necrosis factor-α (TNF-α), and interferon (IFN), are known to promote HSC proliferation and increase cellular output from bone marrow cells. During the chronic inflammatory cycle, the inventors noted that high levels of mCCL6, compared to homeostasis, lead to an imbalance in HSC differentiation. Therefore, the inventors hypothesize that the continuous production of mCCL6 by eosinophils is a trigger for HSC dysfunction and the chronic inflammatory regulatory cycle. The inventors' research provides new insights into how chronic inflammatory signaling influences HSCs' maintenance of airway pathological responses.

[0132] Chemokines are small proteins that act as immunomodulators by activating GPCRs to mediate the transport of immune cells. Chemokines and their receptors have become major players and key therapeutic targets in a wide range of immune and inflammatory diseases. However, the biology and biochemistry of inflammatory chemokines and their receptors are highly complex, partly due to the mixed ligand binding exhibited by the receptors. Conversely, chemokines bind to multiple chemokine receptors. mCCL6, hCCL15, and hCCL23 belong to the NC6 subfamily with an N-terminal extension. hCCL23 has been reported to bind to the cell surface receptor CCR1 and is associated with total IgE in children with asthma.

[0133] Previous studies have shown that hCCL15 binds to the cell surface receptor CCR1 and may contribute to asthma severity and airflow limitation by affecting airway smooth muscle cells. Furthermore, the inventors provide evidence that mCCL6 activates CCR1 downstream of Gαi protein and related phosphorylated signaling proteins. Activation of HSCs and promotion of HSC differentiation provide compelling evidence for the CCL6-CCR1 axis. Many CCR1 antagonists have recently been identified and investigated in inflammatory diseases, and these antagonists have shown potential therapeutic effects in clinical trials. These data suggest that targeting the CCL6-CCR1 axis may be a promising strategy for prevention or treatment, alleviating allergic inflammation. The inventors' findings contribute to a new theory of chemokine receptor function and suggest that NC6 family chemokines and related receptors may be potential biomarkers and targets for eosinophilic airway inflammation.

[0134] In summary, this study demonstrates elevated levels of hCCL23 and hCCL15 orthologs in asthmatic patients and illustrates the crucial role of eosinophil-derived mCCL6 in a murine model of allergic asthma and in allergen-induced eosinophils. Airway inflammation is mediated by mCCL6 and is alleviated in mice with mCCL6 deficiency or specific CCR1 inhibition. Therefore, a better understanding of the novel functional role of CCL6-CCR1 interactions in eosinophil differentiation during allergic airway inflammation may lead to the development of new therapeutic targets for allergic asthma. The inventors' findings also contribute to a new theory of chemokine receptor function as biomarker and suggest that targeting NC6 family chemokines and related receptors with potent neutralizing antibodies or specific inhibitors may be a potential therapeutic strategy for eosinophilic airway inflammation.

[0135] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A combination agent, characterized by, The combined reagents comprise: (S1) Inhibitors targeting CCL6; and (S2) Inhibitors targeting CCR1; The inhibitor targeting CCL6 is selected from gene editing reagents; the inhibitor targeting CCR1 is selected from BX471. The CCL6-targeting inhibitor inhibits the formation of the "CCL6-CCR1 complex"; The aforementioned CCR1-targeting inhibitor inhibits the formation of the "CCL6-CCR1 complex".

2. A reagent kit, characterized in that, The kit includes: (K1) A detection reagent, said detection reagent being used to detect CCL6 ligands and / or CCL6-CCR1 complexes; and (K2) A therapeutic agent comprising: (S1) an inhibitor targeting CCL6; and (S2) an inhibitor targeting CCR1; The inhibitor targeting CCL6 is selected from gene editing reagents; the inhibitor targeting CCR1 is selected from BX471.

3. The kit according to claim 2, characterized in that, The detection reagent is used to detect whether the object is a CCL6 highly expressed object.

4. A method for inhibiting the formation of the "CCL6-CCR1 complex" in vitro, characterized in that, Including the following steps: (a) In the presence of an inhibitor targeting CCL6, CCL6 is contacted with CCR1, thereby inhibiting the formation of the "CCL6-CCR1 complex"; The CCR1 is located on the cell membrane; The inhibitor targeting CCL6 is selected from gene editing reagents.

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