WO2014075580A1

WO2014075580A1 - Use of aurintricarboxylic acid in preparation of drug targeting chemokine receptor

Info

Publication number: WO2014075580A1
Application number: PCT/CN2013/086679
Authority: WO
Inventors: 谢欣; 张菲菲; 魏巍
Original assignee: 中国科学院上海药物研究所
Priority date: 2012-11-15
Filing date: 2013-11-07
Publication date: 2014-05-22
Also published as: CN103800309A

Abstract

Disclosed is a use of aurintricarboxylic acid (ATA) as a selective chemokine receptor inhibitor. Also disclosed is a use of ATA in the preparation of a drug for prevention and/or treatment of autoimmune diseases comprising multiple sclerosis, rheumatoid arthritis, lupus erythematosus and inflammatory bowel disease.

Description

Use of aurintricarboxylic acid in the preparation of a medicament for targeting a chemokine receptor

3⁄4b ^ field

The present invention relates to the field of biomedicine, and in particular to the use of aurintricarboxylic acid in the preparation of a medicament for targeting a chemokine receptor, and further relates to the preparation of a preventive and/or therapeutic autoimmune disease by aurintricarboxylic acid. Use of the drug. Background technique

Multiple sclerosis (MS) is an autoimmune disease that occurs in the central nervous system and is characterized by demyelination and neurodegenerative diseases of the central nervous system. The experimental autoimmune encephalomyelitis (EEE) has similar pathological features to MS and is an ideal animal model for studying MS. It is widely accepted that excessive activation of CD4 ⁺ T cells, especially T helper-1 cell (Th-l ) or T helper 17 cell ( Th-17 ) Overactivation of the two subpopulations is the direct cause of the disease. In the EAE animal model, the integrity of the blood-brain barrier is disrupted, peripheral inflammatory CD4 ⁺ T cells infiltrate into the central nervous system, cause aggregation of other immune cells, activate microglia, and ultimately lead to neuromyelin shedding. , axonal injury, affecting the conduction of nerve impulses and can cause paralysis.

The activation and differentiation of T cells requires the involvement of antigen-presenting cells (APCs). Dendritic cells (DCs) are full-time antigen-presenting cells that play a very important role in the pathogenesis of EAE. Immature DCs in peripheral tissues, especially those in contact with the surrounding environment, like DCs in the skin and mucous membranes, will encounter them when they encounter antigens, and immature DCs will capture them and process them into small peptides. Capacitive complex molecules present it to the cell surface. At the same time, signals from pathogens or inflammatory factors initiate the maturation process of DCs, further enhancing their antigen-presenting capacity. The mature DC carries the antigen, moves out of the tissue, and reaches the secondary lymphatic organ. Here, DC stimulates the proliferation and differentiation of T cells by cell-to-cell contact and secretion of cytokines, thereby initiating an antigen-specific immune response.

The migration of mature DC cells to secondary lymphoid organs and the migration of pathogenic T cells to the central nervous system are largely regulated by chemokines. Chemokines can be broadly divided into two broad categories: chemokines that induce expression and chemokines that are constitutively expressed. The constitutively expressed chemokine induces basic leukocyte migration and transport, forming a system of secondary lymphoid organs, and the chemokines that induce expression mainly recruit leukocytes to respond to physiological stress and stimulate the migration of inflammatory cells to inflammatory tissues. Many studies have shown that the expression of multiple chemokines is up-regulated during EAE. After interfering with its function by specific antibodies to chemokines, it can well inhibit or alleviate the condition of EAE. Chemokines are classified into four classes based on their very conserved cysteine residues at the N-terminus: CXC, due to an amino acid residue between its two adjacent cysteine residues; CC, two conserved Cysteine residue in close proximity; CX3C, two conserved cysteine The acid residues are separated by 3 amino acid residues; C, such chemokines have only one conserved cysteine residue at the N-terminus. Among them, CXC and CC are two of the most important families of chemokines. Therefore, the present inventors took the receptors of these two types of chemokines as the main research object. Chemokines function through chemokine receptors, one chemokine can be a ligand for multiple chemokine receptors, and one chemokine receptor can also have multiple chemokine ligands. Chemokine receptors are a class of G-protein coupled receptors with seven transmembrane structures, and most are coupled to _Gai , and their transduced signals are inhibited by pertussis toxin. Chemokine receptors, expressed in many cells, such as T cells, neutrophils, monocytes, monocyte-derived macrophages, natural killer cells, dendritic cells, eosinophils, and alkaloids Cells involved in inflammation and immune response, such as neutrophils, are also expressed in cells of the central nervous system such as microglia, astrocytes, neurons, and endothelial cells. Different immune cells have different surface-specific high expression of chemokine receptors. For example, mature DCs express CCR7, Thl cells express CCR5 and CXCR3, and Th17 cells express CCR6. Therefore, by interfering with the function of these chemokine receptors, the migration of the corresponding cells to the site of inflammation can be inhibited, thereby achieving the purpose of alleviating the condition of EAE.

Summary of the invention

The present inventors focused on the causes and mechanisms of autoimmune diseases and the use of chemokine receptor inhibitors in the treatment of autoimmune diseases. In compound screening, the inventors found that Aurintricarboxylic acid (ATA) inhibits a variety of chemokine receptor-mediated cell signaling and cell migration, including CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5, CXCR6. ATA is an inhibitor of protein-nucleic acid interactions and is therefore capable of inhibiting many enzymes involved in DNA or RNA. Its ammonium salt is called an aluminum reagent and is used to detect aluminum in water, biological tissues and food. In addition, ATA was found to exert anti-HIV activity by blocking the binding of the HIV coat protein gpl20 and CD4 molecules. In the present invention, the inventors found that ATA can significantly alleviate the clinical symptoms and pathological changes of the mouse EAE model. Excessive activation of inflammatory cells and infiltration of tissues are common features of many autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, lupus erythematosus, inflammatory bowel disease, and the like. Mechanism studies have shown that ATA does not directly affect the differentiation of pathogenic Th1 or TW7 cells, but inhibits the migration and aggregation of antigen-presenting DC cells into the spleen, thereby reducing T cell activation and differentiation; ATA can also inhibit The migration of diseased T cells into the central nervous system, thereby alleviating the disease.

Accordingly, it is an object of the present invention to provide the use of a ATA or a pharmaceutical composition comprising ATA for the preparation of a medicament for targeting a chemokine receptor.

Another object of the present invention is to provide use of a ATA or a pharmaceutical composition containing ATA for the preparation of a medicament for preventing and/or treating an autoimmune disease.

In the present invention, preferably, the chemokine receptor comprises CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5, CXCR6.

In the present invention, preferably, the chemokine receptor is one or more selected from the group consisting of CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5 and CXCR6.

In the present invention, preferably, the spermidine tricarboxylic acid simultaneously targets three or more of the chemokine receptors.

In the present invention, preferably, the autoimmune diseases include multiple sclerosis, rheumatoid arthritis, erythema, and inflammatory bowel disease.

Further, the present invention provides a method for preventing and/or treating an autoimmune disease, which comprises administering a therapeutically effective amount of ATA or a pharmaceutical composition containing ATA to a subject in need thereof.

In the present invention, preferably, the ATA-containing pharmaceutical composition comprises a therapeutically effective amount of aurin tricarboxylic acid and optionally a pharmaceutically acceptable carrier.

The present invention has the following advantages over existing chemokine inhibitors;

ATA can simultaneously target multiple chemokines including CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5, CXCR6, etc., while inhibiting the migration of DC cells to secondary lymphoid organs and pathogenic T cells. The migration to the central nervous system plays an inhibitory role in many aspects of the pathogenesis of autoimmune diseases, and may have a better therapeutic effect than drugs targeting a single chemokine. DRAWINGS

Figure 1 shows the function of ATA to inhibit chemokine receptors. among them:

A is a diagram showing the molecular structural formula of ATA.

B is a graph showing the calcium flow response evoked by ATA inhibition chemokines. HEK293 or CHO cell line stably expressing Gal6 and each receptor (CCR6, CCR7, CXCR4, S1P1, KOR or β2ΑΙΟ), after incubation with the dye fluo4-AM for 45 minutes, pre-incubated with ATA for 10 minutes, then with the corresponding ligand (30 nM) stimulation, calcium signal was recorded. Data were from three independent experiments, three replicate wells per experiment, expressed as mean ± standard error (mean ± SEM).

C is a graph showing ATA inhibition of chemokine-induced cell chemotaxis. Spleen cells isolated from female C57/B6 mice were stimulated with 10 μM ΐ ΐ LPS for 48 hours and then added to the upper chamber of the Transwell® transmembrane compartment sandwich system. The upper and lower chambers were separated by microporous membranes; SDF Factors such as CCL19, CCL20 or SIP (both 30 nM) that induce cell migration are added to the lower chamber; different concentrations of ATA are added to the upper and lower chambers. After 3 hours, cells migrating from the upper chamber to the lower chamber were counted by flow cytometry. Data were obtained from three independent experiments, three replicate wells per experiment, expressed as mean ± standard error.

D is a graph showing endocytosis of ATA-inhibiting chemokine receptors. Expression of CXCR4, CCR6 or S1P1 receptors After the cells were stimulated with the corresponding ligands in the presence or absence of ATA, immunofluorescence staining of the receptors was performed to observe the localization. The tailless arrow indicates that the receptor is located on the membrane, and the tail arrow refers to the endocytosis point formed by the receptor after endocytosis.

Figure 2 shows that ATA reduces the clinical signs and tissue damage of EAE. among them:

EAE was induced by immunizing female C57/B6 mice with MOG ₃₅ _ ₅₅ , once daily from the third day (A), day 12 (C) after immunization until the end of the experiment; or from the third day, administration By day 12 (B) _{; the} control group was given phosphate buffer and the clinical score was recorded daily. Data are expressed as mean ± standard error (6 mice per group). ^### p <0.001 ^# p<0.05 (two-way ANOVA test); <0.05 * p<0.01 compared with the control group (Mann-Whitney test) D and E were H&E staining (D) and fast blue staining of paraffin sections of the lumbar spinal cord tissue of normal or immunized control group and ATA administration group (20 mg/kg, day 3 administration, to the end of the experiment). E) Picture. F is the immunofluorescence staining image of CD45+ cells in frozen sections of normal or immunized control group and ATA administration group (20 mg/kg, day 3, until the end of the experiment), in the lower left corner of the picture. The picture is a magnified image of the central box. GI is a quantitative analysis of the total number of cell infiltration, demyelination area, and number of CD45+ cell infiltration in DF, and the data are expressed as mean ± standard error. Three mice were taken from each group, and 10 sections of each mouse spinal cord were analyzed for comparison. Compared with the control group, ***ρ<0.001 (Student's ?-test) o

Figure 3 shows that ATA inhibits in vivo but does not affect T cell differentiation in vitro. among them:

A: On day 12 after EAE immunization, control group and ATA administration group (20 mg/kg/day, day 3 to day 12) CD4+ T cells, CD1 lc DC Thl (IFN-γ) in mouse spleen Flow cytometry analysis of cells and TW7 (IL-17 cell ratio; B: 12 days after EAE immunization, control group and ATA administration group (20 mg/kg/day, day 3 to day 12) murine splenocytes with MOG ₃₅ - ₅₅ weight supernatants were collected after 48 hours stimulation, measured by ELISA IL-17a IFN-γ, IL -6 and TNF-α content; data are expressed as mean ± standard error (each group 6 mice), compared with the control group, *p<0.05 * p<0.01, **p<0.001 (Stittton's t-test); C and D: in the presence of differentiation factors and various concentrations of ATA From the spleen of 6-8 weeks old mice, the differentiation of naive CD4 ⁺ T cells into Th1 (C) or TW7 (D) in vivo, data from three independent experiments, expressed as mean ± standard deviation error; : Add different concentrations of ATA or camptothecin (0-30 μΜ) to DCs isolated from the spleens of 8-10 weeks old mice, culture for 18 hours, annexin V/propidium iodide (PI) staining, flow analysis Its apoptotic situation. Independent experiments, each experiment triplicates, expressed as mean ± standard error.

Figure 4 shows that ATA inhibits chemokine-mediated DC migration but does not affect other DC functions. Where: A: DCs from the spleens of mice isolated on day 12 after EAE immunization were added to the Transwell® transmembrane compartment In the upper chamber of the culture system, the upper and lower chambers are separated by a membrane with micropores; 30 nM CCL19 is added to the lower chamber; ΙΟμΜ ΑΤΑ is added to the upper and lower chambers. After 3 hours, cells migrating from the upper chamber to the lower chamber were counted by flow cytometry. Data were obtained from three independent experiments, three replicate wells per experiment, expressed as mean ± standard error. Compared with the CCL19 untreated group, ^### ρ<0.001 _; compared with the ATA untreated group, ***ρ<0.001 (Stittton's t-test).

B and C: The separated mouse spleen CD4 ⁺ T cells labeled with CFSE without ATA treatment of EAE after 12 days from immunization, the MOG ₃₅ - the presence or absence of ₅₅ (25 _μ ^ _ηιΐ), CD4 lc ⁺ DC was co-cultured for 72 hours with the control group or the sputum administration group (20 mg/k^day, day 3 administration to day 12) after the autoimmunization on the 12th day, and the CD4 was detected by flow cytometry. ⁺ T cell proliferation (B) or ELISA method to detect the content of cytokines such as IL-17a, IFN-y, IL-6 and TNF-α in the supernatant (C).

DH: CD4 ⁺ T cells and CD1 lc ⁺ DC were isolated from the spleen of ADA-treated mice on day 12 after EAE immunization, and CD4 ⁺ T cells were labeled with CFSE after MOG ₃₅ _ ₅₅ (25 μ^ηιΐ) In the presence or absence of ATA (10 μΜ), 72 chambers were co-cultured, and the proliferation of CD4+ sputum cells by flow cytometry (D) or ELISA was used to measure IL-17a, IFN-γ, IL-6 and TNF- in the supernatant. The content of cytokines such as α (EH). Data were from three independent experiments and expressed as mean ± standard error.

Figure 5 shows that ATA blocks the infiltration of inflammatory T cells into the central nervous system. among them:

A: Central nervous system infiltration in mice (ATA 20 mg/kg/day, day 3 to day 18) on day 18 after EAE immunization with 37-70% silica colloidal suspension (Percoll®) Cells, and the number of CD4+ T cells, Th1 (IFN-y) cells, and Th17 (IL-17 ⁺ ) cells were analyzed by flow cytometry. Data are expressed as mean ± standard error (5 mice per group) compared to the control group, p < 0.05 (Stittton's t-test).

B: 18 days after EAE immunization (ATA 20 mg/kg/day, day 3 to day 18), blood and CD4 ⁺ T cells, CD1 lc ⁺ DC, Thl in the control and ATA-administered mice Proportional flow analysis of (IFN-y) cells and Th17 (IL-17 ⁺ ) cells. Data are expressed as mean ± standard error (6 mice per group) compared to the control group, p < 0.05 (Stittton's t-test).

C: Immunofluorescence staining of CD45+ cells in the choroid plexus of mice in the normal group or the 18th day after immunization and the ATA administration group (20 mg/k^day, day 3 to day 18).

D: Quantitative analysis of the number of CD45+ cell infiltration in the C map, data are expressed as mean ± standard error. Three mice were taken from each group, and 5 sections were taken from each mouse for analysis. Compared with the control group, p < 0.01 (Stittton's t-test). Specific lung

In the following, the invention will be described in more detail with reference to the accompanying drawings, by way of example only, Material method

Experimental animals:

C57BL/6 female rats were purchased from Shanghai Experimental Animal Center (Shanghai, China) and raised in the SPF laboratory of the Experimental Animals Room of Shanghai Institute of Materia Medica. The light-dark 12-hour cycle was maintained, giving sufficient food and clean drinking water to The experiment was started at 8 weeks of age. All experiments were approved and introduced in accordance with the guidelines of the Animal Management and Use Committee of the Shanghai Institute of Materia Medica.

Reagents:

CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5, CXCR6, S1P1, DOR, KOR, 2AR and Gal6 plasmids were purchased from the Missouri S&T cDNA Resource Center. Auchin tricarboxylic acid (ATA), lipopolysaccharide, Hochest33342, forskolin, GLP-1, U50488 and isoproterenol were purchased from Sigma-Aldricho anti-mouse CD3 (145-2C11) monoclonal antibody. Anti-mouse CD28 (37.51) monoclonal antibodies and anti-mouse IFN-γ (R4-6A2) monoclonal antibodies were purchased from BD Pharmingen. Phycoerythrin-cyanine 7, PE-Cy7 anti-mouse CD4 (RM4-5), Allophycocyanin (APC) anti-mouse CD8a (53-6.7), allophycocyanin (Allophycocyanin, APC) anti-mouse IFN-γ (XMG1.2), phycoerythrin (PE) anti-mouse CDllc (N418) and anti-mouse CD45 (30-F11) were purchased from eBiosciences. Anti-myc and anti-HA monoclonal antibodies are hooked from Cell Signaling Technology. Alexa Fluor 488 goat anti-mouse IgG antibody, Alexa Fluor 488 goat anti-rat IgG antibody and Fluo-4 AM were purchased from Invitrogen. Recombination MCP-l, TARC, RANTES, ΜΙΡ-3α, ΜΙΡ-3β, TECK, SDF-1, CXCL13 and Π CXCL16 were purchased from PeproTech. Recombinant murine IL-12, recombinant murine IL-6, recombinant human TGF-βΙ, recombinant murine IL-Ιβ, recombinant murine IL-23 and recombinant murine TNF-α were purchased from R&D Systems.

Cell line:

The various cell lines stably expressing the receptor and God6 involved in the experiment were constructed in-house. The specific steps are as follows: HEK293 or CHO cells are trypsinized, centrifuged, and 200 μM electroporation (ATP 200 g/L, MgCl ₂ · 6H ₂ 0 120 g/L, KH ₂ P0 ₄ 12 g/L, NaHC0 ₃₎ 1.2 g/L), add the receptor plasmid and God6 plasmid 2 μ _§ at a concentration of 100-200 million cells/200 μΐ electroporation, mix well, and use Scientz-2C gene importer (Ningbo Xinzhi Biotechnology Co., Ltd.) Make an electric shock. After the electric shock, the cells are transferred to the culture plate or the culture dish to continue to culture the cells, and the cells can be used after 24 hours. The calcium flow test was performed. After 24 hours in the culture dish, antibiotics were added for screening, and positive cloned cells were selected to establish a monoclonal cell line.

^m EAE induction and mouse administration

Female C57BL/6 mice were injected subcutaneously with 200 MOG35-55 (MEVGWYRSPFSRVVHLYRNGK, purchased from Jill Biochemistry) supplemented with complete Freund's adjuvant and 5 m^ml heat-killed Mycobacterium tuberculosis (H37Ra strain, purchased from Difco Laboratories). Day 0. Peripheral injection of pertussis toxin 200 ng per mouse on day 0 and day 2

(Calbiochem) o The mice were scored daily, and the scores were based on the "5-point scale" as follows: 0 points, no clinical symptoms; 1 point, tail sputum; 2 points, mild hind squats (unilateral or bilateral hind limb weakness, no Completely paralyzed); 3 points, paraplegia (complete paralysis of both hind limbs); 4 points, paraplegia and forelimb weakness or paralysis; 5 points, sudden death or death.

Mouse administration:

EAE administration group: ATA (10-20 mg/kg) was administered intraperitoneally from the 3rd day or the 12th day after immunization until the end of the experiment; or from the 3rd day after immunization (administered ATA by intraperitoneal injection) (10-20 mg/kg) ) to day 12. The solvent for dissolving ATA was phosphate buffered saline (PBS) containing 0.4% dimethyl sulfoxide (DMSO).

EAE control group: A solvent control, i.e., a phosphate buffer containing 0.4% dimethyl sulfoxide, was administered by intraperitoneal injection from the 3rd day or the 12th day after immunization.

Example 2: Histopathology and immunofluorescence analysis

Disease mice in the EAE administration group and the EAE control group in Example 1 were anesthetized, fixed by PBS perfusion and 4% paraformaldehyde perfusion. The removed spinal tissue samples were fixed overnight in 4% paraformaldehyde. After fixed spinal cord tissue samples were embedded in paraffin, inflammatory cell infiltration was analyzed by hematoxylin and eosin (H&E) staining, and demyelination of the spinal cord was analyzed by fast blue staining. Frozen sections of the spinal cord and brain were stained with anti-mouse CD45-antibody and the corresponding fluorescent secondary antibody; the nuclei were stained with Hoch _eS t 33342 for 10 minutes at room temperature, and the sections were mounted with fluorescent sealing tablets (Dako).

«Example 3: Central nervous system infiltration cell separation

The spinal cord and brain tissue of the diseased mice of the EAE administration group and the EAE control group in Example 1 were ground on a 40 μηη filter, and the obtained cell suspension was centrifuged at 500 g for 10 minutes at 4° C., and 8 ml was used. Resuspend 37% Percoll® Reagent, carefully add to 4 ml of 70% Percoll® Reagent, and centrifuge at 25 °C 780 g for 25 minutes. Collect 37% to 70% Percoll® intermediate cells and perform flow assays.

Money Example 4: Flow Cytometry

Spleen cells of disease mice taken from the EAE administration group and the EAE control group in Example 1 or from Example 3 Surface staining of cells infiltrating into the mouse central nervous system. Cells were incubated with fluorescently labeled anti-mouse CD4, CD1 lc antibodies for 30 minutes at 4 °C. For intracellular staining, cells were first treated with phorbol 12-myristate 13-acetate (50 ng/ml; Sigma) ionomycin (750 ng) /ml; Sigma) and brefeldin A (3.0 μ^ηιΐ; Sigma) were mixed and incubated at 37 ° C for 5 hours, the cells were resuspended in a fixed / permeate (Cytofix / Cytoperm kit; In BD Pharmingen), intracellular IL-17a and IFN-γ staining were performed as described in the product instructions. Results were analyzed using a Guava easyCyteTM 8HT flow cytometer and GuavaSoft software.

Example 5: CD4+ T cell isolation and in vitro differentiation

Isolation of naive CD4+ T cells from spleens of 6-8 weeks old normal female C57BL/6 mice using magnetic beads (Dynal® Mouse CD4 Cell Negative Isolation kit; Invitrogen), and the isolated CD4+ T cells were added with anti-CD3 (2 g/ml). 145-2C11; BD Pharmingen) and anti-CD28 (2 μ^ηιΐ; 37.51; BD Pharmingen) antibody were added with IL-12 (10 ng/ml; R&D) and anti-IL-4 (10 μ^ηιΐ; 11B11; BD Pharmingen) induced differentiation into Th1 cells, or added anti-IL-4, anti-IFN-y (10 μ^ηιΐ; BD Pharmingen) and TGF-βΙ (3 ng/ml) ^ IL-6 (30 Ng/ml; eBioscience), tumor necrosis factor (10 ng/ml; R&D), IL-23 (10 ng/ml; R&D) and IL-Ιβ (10 ng/ml; R&D) Th- 17 "Differentiation factor combination" to induce differentiation into Th-17 cells. During the above differentiation, various concentrations (0-30 μΜ) of ATA were simultaneously added to evaluate their effects on the differentiation of sputum cells. Three days later, cells were collected for staining of intracellular IFN-y and IL-17a.

Example 6: DC separation and DC-T cell co-culture experiment

The mice from the EAE control group (12 days after immunization) or the EAE-administered group (ATA, 20 mg/k^day, day 3 administration) were sorted by MACS magnetic beads (Miltenyi Biotec). After CD1 lc ⁺ DC cells in the spleen on day 12, CD4 ⁺ T cells in the spleen of CFSE-labeled EAE-controlled mice (12 days after immunization) were mixed at a ratio of 3:1 in MOG. ₃₅ _ ₅₅ ( 25 _μ§ /ιη1) in the presence or absence of culture for 72 hours; or CD4 ⁺ T cells and DC cells were isolated from EAE control mice (12 days after immunization) in co-culture system ΑΤΑ (10 μΜ) was added, and after 72 hours of culture, the proliferation of CD4 ⁺ Τ cells was detected by flow cytometry. The cytokines IFN-γ, IL-17a, IL-6 and TNF- were detected by ELISA. The content of α (Dakewe, Shenzhen, China).

Example 7: DC Apoptosis Experiment

CDl lc ⁺ DCs from spleens of 8-10 week old normal C57BL/6 mice were incubated with ATA (0-30 μΜ) or camptothecin (0-30 μΜ) for 18 hours at 37 °C. Cells were harvested for Annexin V/propidium iodide staining. Early apoptosis was positive for Annexin V, propidium iodide was negative, and late apoptosis was positive for Annexin V and propidium iodide. Detected by flow cytometry.

^m 8: Chemotaxis experiment

In vitro chemotaxis experiments Whole spleen cells or DC cells were extracted from EAE control mice (Day 12 post-immunization) in Example 1 using a 5 μηη pore size penetrating chamber (Transwell®, Corning) ₀ and used for the cells. RPMI-1640 medium containing ₁₀ μ§ / ιη1 LPS 48 hours of culture, cells were washed and diluted with RPMI-1640 medium containing 0.5% BSA to a density of l xlO ⁷ cells / ml suspension. The corresponding ligands of various chemokine receptors (SDF-1, CCL19, CCL20 or SIP (both 30 nM)) were added to the lower chamber and 100 μM cell suspension was added to the upper chamber. For chemotaxis retardation experiments, various concentrations of cesium (0-100 μΜ) were added to the lower chamber and the upper chamber. Splenocytes that migrated from the upper chamber to the lower chamber after 3 hours of normal culture were counted by flow cytometry.

Example 9: Calcium flow experiment

HEK293 cell lines stably constructed by various G-protein coupled receptors (including CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5 and CXCR6) and Gal6 (constructed by the laboratory as described above) were inoculated In a 96-well plate, after 24 hours of incubation, incubate the cells with 2 μΜ ί1ιιο 4-ΑΜ dye for 45 minutes at 37 ° C, remove the dye, and add 50 μ with different concentrations of ATA (0-100 μΜ) or 1% DMSO ( The negative control) of HBSS was incubated for 10 min at room temperature, and then stimulated with the corresponding ligands (SDF-1, CCL19, CCL20, SIP, etc. (both 30 nM)) as agonists to record calcium signals. Read with a Flex Station 3 microplate reader (Molecular Devices). At the specified time point, the detector can automatically add 25 μΐ agonist (final concentration 30 ηΜ) to the reaction system, and simultaneously stimulate the intensity of dye fluorescence induced by intracellular calcium concentration change at 525 nm with 485 nm light. The change.

u 10: Receptor endocytosis experiment

HEK293 cell lines stably expressing Myc-CXCR4, EGFP-CCR6 or HA-S1P1 (constructed by the laboratory as described above) were inoculated on coverslips and cultured overnight. Cells were pre-incubated with ATA (0-30 μΜ) for 10 minutes and then stimulated with the corresponding ligand (SDF-1, CCL20 or SIP) for 30 min at 30 nM. Then, the cells were fixed with 4% paraformaldehyde, 0.3% Triton X-100 was disrupted, and then incubated with anti-myc or HA-labeled antibody at 4 ° C overnight, and the corresponding Alexa Fluor 488 secondary antibody was incubated for 1 hour at room temperature, Hochest 33342 stained. After the nucleus, a photograph was taken with a fluorescence confocal microscope (Olympus FVlOi confocal microscope).

3⁄4M processing

Data were analyzed by GraphPad Prism software. Nonlinear regression analysis yielded a dose-dependent curve and calculated the half-inhibitory concentration (IC ₅₍₎ ). Data were expressed as mean±standard error. Statistical differences between EAE mice treated groups were analyzed by two-way ANOVA test. Statistical differences in EAE scores at time points were used by Man-Hui. Mann-Whitney test analysis. Other data analyses (eg, gene expression, pathological statistical analysis after cytokine production) were analyzed by the Stittton's t-test. p < 0.05 was considered statistically significant. result

ATA inhibits the function of various chemokine receptors

Chemokine receptors are a class of G-protein coupled receptors (GPCRs) that play a very important role in cell migration and contribute to the pathogenesis of EAE. When screening chemokine receptor antagonists, the inventors discovered ATA

(Fig. 1A) It can inhibit cell signal transduction and migration caused by various chemokines. First, in Example 9, the inventors utilized stable expression of Gal6 and various GPCRs (including chemokine receptors including: CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5 or CXCR6; non-chemokine receptors) Including: S1P1, DOR, KOR, GLP-1R and β2ΑΡ0 cell lines detect the inhibitory effect of guanidine on GPCR-mediated calcium flux response. Gal6 is a type of pan-host G protein, often associated with G protein-coupled receptors. Coupling, initiates a downstream calcium signaling pathway. The results of Example 9 show that ATA dose-dependently inhibits most chemokine receptor-mediated calcium flux responses, except CCR2 only

(Figure 1B, Table 1 below). However, for other non-chemokine receptors, ATA showed no inhibitory effect (Fig. 1B, Table 1 below).

In Example 8, the inventors subsequently examined the effect of ATA on ligand activity of several different chemokines, such as CCL19/CCR7, CCL20/CCR6 or SDF-1/CXCR4, spleen cell migration. The results of Example 8 show that ATA dose-dependently inhibits the migration of spleen cells induced by CCL19, CCL20 or SDF-1 with IC50 of 0.24, 3.36, 11.46 μΜ, respectively (Fig. 1C, Table 1 below); S1P1 receptor It plays an important role in the migration of lymphocytes from secondary lymphoid organs, but ATA does not affect S1P1-mediated cell chemotaxis (Fig. 1C, Table 1 below).

Receptor endocytosis is a common phenomenon in which GPCRs are stimulated by their ligands. In Example 10, the inventors also examined the effect of ATA on the endocytosis effects of CXCR4, CCR6 and S1P1 receptors after ligand stimulation. Cells expressing CXCR4, CCR6 or S1P1 receptors were preincubated with ATA for 10 minutes and then stimulated with the corresponding ligands SDF-1, CCL20 or S1P for 30 minutes, followed by immunofluorescence staining of the receptor. The results of Example 10 showed that the receptors were localized on the cell membrane without ligand stimulation, and the receptor was endocytosed into the cells after stimulation of the ligand (Fig. 1D); Endocytosis of CXCR4 and CCR6 receptors was significantly reduced; however, ATA did not affect endocytosis of the S1P1 receptor (Fig. 1D). In summary, ATA inhibits the function of many chemokine receptors, but does not affect the function of other GPCRs.

ATA alleviates the clinical symptoms and pathological features of EAE

In Example 1, immunization with MOG ₃₅ _ ₅₅ 8-9 week old female C57BL mice induced EAE / 6, from day 3 or day 12 by intraperitoneal injection of ATA (10 or 20 mg / kg) to the end of the experiment, Or dosing from day 3 to day 12. The solvent control was PBS containing 0.4% DMSO. The clinical score data for EAE are shown in Figures 2A-2C and Table 2 below. ATA dose-dependently ameliorated the clinical score of EAE (Fig. 2A) and significantly reduced the highest clinical score and cumulative clinical score of the disease (Fig. 2A, Table 2 below). When administered only from day 3 to day 12, ATA was also able to reduce its severity at the onset and peak of EAE disease, but after 20 days, the disease score increased, indicating that drug removal resulted in The recurrence of the disease (Figure 2B). After the start of the disease (starting from the 12th day), 20 mg/kg ATA was still effective in reducing the severity of EAE (Fig. 2C, Table 2), indicating that the drug not only has a preventive effect, but also achieves the purpose of treating the disease.

In Example 2, the inventors analyzed pathological sections of the spinal cord 21 days after immunization. The results of the study of Example 2 showed that ATA administration significantly reduced leukocyte infiltration of the spinal cord compared to the control group (Fig. 2D and 2G); fast blue staining showed extensive demyelination of the white matter of the EAE mouse in the control group. Phenomenon, after deprivation of ATA, demyelination was significantly reduced (Figures 2E and 2H); immunofluorescence staining of frozen sections showed that the number of CD45+ cells in the spinal cord of EAE mice was significantly reduced after ATA administration (Figures 2F and 21). ).

ATA reduces the proportion of DCs and effector T cells in the spleen at the onset of disease

Activation, proliferation and differentiation of CD4+ T cells are prerequisites for the development of EAE disease. In Example 4, the inventors examined the proportion of each subpopulation of cells in the spleen of mice on the 12th day after EAE immunization by flow cytometry. The ratio of the two major effector T cells Th1 and TW7 cells in CDl lc ⁺ DC and EAE of the ATA-administered mice was significantly lower than that of the control group ( FIG. 3A ). This result is consistent with the administration group mouse splenocytes in vitro with MOG ₃₅ - ₅₅ weight stimulation, supernatant cytokines (including IFN-y, IL17, IL6 Wo Π TNF-α) have also significantly reduced (FIG. 3B ).

The inventors then further verified whether ATA reduced the proportion of these two cells in the spleen by directly affecting the differentiation of Th1 or TW7 cells. In Example 5, naive CD4 ⁺ T cells were sorted from 6-8 week old female C57BL/6 mouse spleen cells by immunomagnetic beads, activated with anti-CD3 and anti-CD28 antibodies, and then added with different differentiation factors and Cells were induced to differentiate into Th1 or TW7 cells at various concentrations of ATA. Three days later, cells were collected for staining of intracellular IFN-γ, IL-17a. Flow analysis revealed that ATA did not directly affect the in vitro differentiation of Th1 (Fig. 3C) or Thl7 (Fig. 3D). The differentiation of Th1 or Thl7 in vivo is largely influenced by DC. Since ATA does not directly affect the differentiation of Th1 or TW7, the reduction of these cells in vivo is most likely due to the decrease in CD1 lc ⁺ DC (Fig. 3A). In Example 7, the inventors examined whether ATA promotes apoptosis of DC, thereby reducing the amount of DC in the spleen. Figure 3E shows that the type I topoisomerase inhibitor camptothecin dose-dependently promotes apoptosis in DC, but ATA does not have this effect, indicating that ATA does not promote DC apoptosis.

ATA suppresses the migration of DC, does not affect his function.

The DC captures, processes, and presents antigen to MHC molecules in peripheral tissues. DC then to secondary lymphoid organs Migration, and in these organs, present antigens to naive T cells, causing them to differentiate into Th1 or Thl7, thereby initiating an antigen-specific immune response. Because ATA does not promote DC apoptosis, it is likely to inhibit DC migration to the spleen. In Example 8, the inventors used chemotaxis experiments to study the effect of ATA on DC migration. CCR7 is a major chemokine receptor that is highly expressed on mature DCs that mediates DC migration. The results showed that ATA (10 μΜ) was able to completely inhibit the chemotactic effect of the obtained DC isolated from the spleen of mice on day 12 after the induction of CCR7 ligand CCL19 (Fig. 4A).

In Example 6, the inventors designed a DC-CD4+ T cell co-culture experiment in order to further verify whether ATA affects the antigen presentation of DCs to CD4 ⁺ T cells and the secretion of cytokines. CD4 ⁺ T cells were isolated from control mice, labeled with CFSE, and co-cultured with CD1 lc ⁺ DC isolated from control EAE or ATA-administered EAE mice for 72 hours. DCs from ATA-administered EAE mice had the same stimulation of CD4 ⁺ T cell proliferation (Fig. 4B) and their ability to secrete cytokines (Fig. 4C). At the same time, the inventors designed another experiment in which CD4 ⁺ T cells isolated from control EAE mice were co-cultured with CDl lc ⁺ DC, and ATA (10 μΜ) was added to the culture system, and flow was used 72 hours later. The proliferation of CD4+ sputum cells (Fig. 4D) and their cytokine secretion were examined by ELISA and ELISA (Fig. 4Ε-4Η). The results showed that co-culture of CD4 ⁺ T cells and CD1 lc ⁺ DC significantly enhanced the proliferation of CD4 ⁺ T cells and the production of cytokines. MOG ₃₅ — ₅₅ stimuli can further enhance this effect. However, ATA does not affect the proliferation of CD4 ⁺ T cells and the secretion of their cytokines in this system. In summary, the protective effect of ATA on EAE is to inhibit the migration and aggregation of DC into the spleen, thereby reducing the activation and differentiation of T cells.

ATA blocks the infiltration of pathogenic lymphocytes into the central nervous system

ATA also showed a certain therapeutic effect after the onset of the disease (Fig. 2C). Therefore, the inventors further verified whether ATA affects the process of pathogenic T cells infiltrating into the central nervous system. Flow cytometry was used to detect CNS leukocyte infiltration on day 18 after immunization. After ATA administration, the number of CD4 ⁺ T cells and the two major pathogenic cells ThH and TW7 cells was significantly reduced (Fig. 5A). However, at the same time, the proportion of TW7 cells in the blood was found to be significantly increased in the ATA administration group as compared with the control group (Fig. 5B). This interesting phenomenon suggests that ATA may inhibit the infiltration of inflammatory T cells into the CNS, resulting in the accumulation of TW7 cells in the blood. The choroid plexus forms part of the blood-brain barrier, and the passage of T cells through the choroid plexus into the central nervous system is a critical step in the initiation of EAE. It has been reported in the literature that CCL20/CCR6-mediated infiltration of Th1 cells into the CNS triggers the onset of EAE. CD45+ cells of CCR6 knockout mice cannot cross the blood-brain barrier and accumulate in the choroid plexus. Figures 5C and 5D show that there is more accumulation of CD45+ cells in the choroid plexus of EAE mice compared to the control group, which indicates that ATA also inhibits the infiltration of pathogenic T cells into the CNS. Thereby alleviating the condition of EAE. discuss

Chemokines are a class of cytokines associated with leukocyte migration and inflammatory responses that can be divided into two large families (CXC and CC) and two small families (C and CX3C) depending on their structural properties. The chemokine receptor downstream signal is delivered by the heterotrimeric G protein. G protein can regulate a variety of signaling pathways, including intracellular calcium, mitogen-activated protein kinases (MAPK), PLCP, PI3K, Ras and Rho GTPases. These transduction signals are thought to be responsible for the movement of cells and the transport of immune cells.

After the antigen enters the peripheral tissues, the immature DCs slowly mature under the induction of pathogens or inflammatory factors. Mature DCs carrying antigen migrate out of peripheral tissues to secondary lymphoid organs where they stimulate T cell proliferation and differentiation. During the maturation of DC, the type of chemokine receptors expressed on the surface changes. It is determined that the expression of the chemokine receptor CCR7, which is accumulated in the T cell-rich region, is gradually up-regulated by mature DCs carrying antigen. Recent studies have also shown that CXCR4 and CCR7-mediated signaling synergistically regulate DC migration to the spleen white pulp. Drugs that inhibit DC migration are used to treat autoimmune diseases. For example, cyclosporin A (CsA) is a very important and effective immunosuppressive agent for the treatment of organ transplants, allergic disorders, autoimmune diseases and acute inflammation. CsA mainly interferes with the expression of chemokine receptors by inhibiting the production of PGE ₂ under LPS stimulation, and impairs the migration ability of DCs. The inventors' data show that ATA non-specifically inhibits the function of chemokine receptors and that it is also best for inhibiting CCL19/CCR7-mediated DC chemotaxis. This suggests that inhibiting the homing of DCs to secondary lymphoid organs may be an effective method for treating autoimmune diseases.

The infiltration of lymphocytes into CNS is another key step in the development of EAE, a neurodegenerative disease, which is also regulated by chemokine receptors. Leukocyte exudation from the blood to the extravascular is a multi-step process. A key step in this cascade is the binding of chemokine receptors expressed on the surface of leukocytes in the circulation to chemokines on the surface of vascular endothelial cells, triggering intracellular signaling, leading to integrin activation, leukocyte arrest and extravasation . Recent evidence suggests that TW7 cells expressing IL-17 are the most important pro-inflammatory T cells in EAE. TW7 cells highly express the chemokine receptor CCR6, and its ligand CCL20 is highly expressed on choroid plexus cells of healthy and EAE mice. Reboldi et al reported that the process of CCR6+ Thl7 cells entering the CNS through choroid plexus epithelial cells is very important for the initiation of EAE. CCR6 knockdown can block the infiltration of TW7 cells and alleviate the condition of EAE. The inventors have also demonstrated that ATA inhibits CCL20/CCR6-mediated chemotaxis of splenocytes and reduces the infiltration of inflammatory T cells into CNS.

The role of many other chemokine receptors in the pathogenesis of EAE has also been reported, and ATA as a non-specific inhibitor of multiple chemokine receptors is responsible for its therapeutic effect on EAE. It is noteworthy that ATA has been reported to inhibit HIV entry by inhibiting the binding of CD4 to the viral envelope glycoprotein gpl20. It is well known that CCR5 and CXCR4 are co-receptors of HIV invasion. According to the inventor's data, ATA may also pass inhibition CCR5 and CXCR4 block HIV entry into CD4 ⁺ T cells.

The present invention discloses the activity of ATA to inhibit the function of chemokine receptors, and discloses the inhibition of ATA by

CCL19/CCR7-mediated homing of DCs to secondary lymphoid organs and CCL20/CCR6-mediated TW7 cell entry

The process of CNS can achieve the purpose of treating EAE.

Table 1: ATA inhibits chemokine receptor-mediated calcium flux response and cell migration. among them:

The inhibitory activity of ATA on various chemokine receptors and other GPCRs was examined by calcium flow and chemotaxis assays. The data was obtained from three independent experiments, three replicate wells per experiment, expressed as mean ± standard error.

【Table 1】

^a data is expressed as mean ± standard error;

NA: not detected Table 2: ATA reduces EAE clinical score. among them:

A statistical table of the effects of different doses of ATA and the mode of administration on the clinical score of EAE. 【Table 2】

^a mean standard error;

***p<0.001, **p<0.01, *p<0.05 (Stittton's t-test).

Claims

Rights request

1. The use of aurin tricarboxylic acid or a pharmaceutical composition containing aurin tricarboxylic acid in the preparation of drugs targeting chemokine receptors.

2. The use as claimed in claim 1, wherein the drug targeting chemokine receptors is a chemokine receptor inhibitor.

3. The use according to claim 1 or 2, wherein the chemokine receptors include CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5, CXCR6.

4. The use according to claim 3, wherein the chemokine receptor is one or more selected from the group consisting of CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR4, CXCR5 and CXCR6.

5. The use according to claim 3, characterized in that aurin tricarboxylic acid targets more than 3 chemokine receptors at the same time.

6. The use of aurintricarboxylic acid or pharmaceutical compositions containing aurintricarboxylic acid in the preparation of drugs for preventing and/or treating autoimmune diseases.

7. The use according to claim 6, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, lupus erythematosus and inflammatory bowel disease.

8. The use according to any one of claims 1 to 7, wherein the pharmaceutical composition containing aurin tricarboxylic acid contains a therapeutically effective amount of aurin tricarboxylic acid and optionally a pharmaceutically acceptable carrier.

9. A method for preventing and/or treating autoimmune diseases, the method comprising administering a therapeutically effective amount of aurintricarboxylic acid or a pharmaceutical composition containing aurintricarboxylic acid to a subject in need thereof.

10. The method of claim 9, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, lupus erythematosus and inflammatory bowel disease.