Medicine for treating nephritis

Abstract

The invention discloses a medicine for treating nephritis, which belongs to the technical field of biological medicine and comprises a CX3CL1-CX3CR1 axial blocker. The drug can reduce mononuclear cell migration; inhibiting proliferation and activation of mesangial cells; cD45 positive cells in the glomerulus are reduced, the mRNA expression level of TNF alpha, IL-6 and IL-1beta is reduced, and immune cell infiltration and inflammatory response in the glomerulus are relieved; the pharmaceutical composition can be used for treating mesangial proliferative glomerulonephritis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a medicine for treating nephritis. Background Art

[0002] Mesangial proliferative glomerulonephritis (MsPGN) is the most common pathological type of primary glomerulonephritis worldwide, among which the most typical primary glomerulonephritis is IgA nephropathy (IgAN). MsPGN is characterized by inflammatory cell infiltration, mesangial cell proliferation, and increased extracellular matrix (ECM) in the glomeruli. Without timely and effective treatment, it can further lead to glomerular sclerosis and interstitial fibrosis, and eventually develop into end-stage renal disease, leading to an irreversible outcome. More and more studies believe that regional immune damage in the glomerulus is the key point to promote the progression of MsPGN disease. Various immune cells in the glomerulus interact with kidney intrinsic cells to jointly maintain the immune microenvironment in the glomerulus, further damage kidney cells, and promote cell proliferation and inflammatory response.

[0003] The pathogenesis and progression of MsPGN have not been fully and clearly explained, resulting in the lack of targeted and effective treatment options. Traditional treatment options mainly focus on renin-angiotensin-aldosterone system (RAAS) blockers and immunosuppressive therapies based on glucocorticoids and steroids. These treatments have the characteristics of large side effects, poor specificity, and low targeting. Therefore, it is necessary to develop a drug for the treatment of nephritis, especially for the treatment of mesangial proliferative glomerulonephritis, to reduce immune damage in the glomerular region and slow down the progression of the disease. Summary of the invention

[0004] In view of the above technical problems existing in the prior art, the present invention provides a drug for treating nephritis, which improves the problems of cell proliferation, extracellular matrix increase, cell activation, immune cell infiltration and inflammatory response in glomeruli in nephritis.

[0005] The invention discloses a medicine for treating nephritis, comprising a CX3CL1-CX3CR1 axis blocker.

[0006] Preferably, the CX3CL1-CX3CR1 axis blocker is selected from: quemolimab, AZD8797, JMS-17-2, JMS-17-2 hydrochloride, E6130, and alirocumab.

[0007] Preferably, the drug is administered by injection or oral administration.

[0008] Preferably, the final concentration of the blocker is 2-20 mM; or the dosage is 2-10 mg / kg / day.

[0009] Preferably, the final concentration of AZD8797 is 5-20 mM or the dosage is 1-10 mg / kg / day; The dosage of quemolimab is 1-8 mg / kg / day.

[0010] Preferably, the drug further comprises any one of the following ingredients or a combination thereof: Excipients, solvents, and unavoidable impurities.

[0011] Preferably, the drug is used to reduce the number of nucleated cells, the number of PCNA-positive cells, and the expression level of αSMA in the glomerulus, thereby alleviating the cell proliferation and activation effect in the glomerulus.

[0012] Preferably, the drug is used to reduce CD45-positive cells in glomeruli, reduce the mRNA expression levels of TNFα, IL-6, and IL-1β, and alleviate immune cell infiltration and inflammatory response in glomeruli.

[0013] Preferably, the drug is used to reduce monocyte migration; to inhibit mesangial cell proliferation, and to alleviate the induced inflammatory response.

[0014] Preferably, the drug is used to reduce the levels of CX3CL1 and CX3CR1, and reduce the infiltration of CX3CR1-positive monocytes; It is also used to reduce the phosphorylation levels of PI3K and AKT.

[0015] Preferably, the medicament is used to treat mesangial proliferative glomerulonephritis.

[0016] Compared with the prior art, the beneficial effects of the present invention are: it can reduce monocyte migration; inhibit mesangial cell proliferation and activation; reduce CD45-positive cells in glomeruli, reduce the mRNA expression levels of TNFα, IL-6, and IL-1β, and alleviate immune cell infiltration and inflammatory response in glomeruli; and can be used to treat mesangial proliferative glomerulonephritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A This is the molecular structure diagram of AZD8797; Figure 1B This is the PAS staining, PCNA and αSMA detection results of AZD8797; Figure 1C This is a comparison chart of the nucleated cell count of AZD8797; Figure 1DThis is a comparison chart of the PCNA-positive cell rate of AZD8797; Figure 1E This is a comparison chart of the αSMA expression level of AZD8797; Figure 2A This is a comparison of the mRNA expression levels in the glomeruli of AZD8797; Figure 2B This is the result of CD45 immunofluorescence detection of AZD8797; Figure 2C This is a comparison chart of the CD45 positive cell rate of AZD8797; Figure 3A This is the crystal violet staining result of AZD8797; Figure 3B This is a comparison of monocyte migration of AZD8797; Figure 4A This is a comparison chart of the mRNA expression levels of TNFα of AZD8797; Figure 4B This is a comparison chart of the mRNA expression levels of IL-6 of AZD8797; Figure 4C This is a comparison chart of the mRNA expression levels of IL-1β of AZD8797; Figure 4D This is the detection result diagram of DAPI staining, EDU staining and fusion staining of AZD8797; Figure 4E It is a comparison chart of the ratio of EdU-positive cells of AZD8797; Figure 5A This is the PAS staining, PCNA and αSMA detection results of quimolimab; Figure 5B This is a comparison chart of the nucleated cell count detected by quimolimab; Figure 5C This is a comparison chart of the αSMA expression level of quemolimab; Figure 5D This is a comparison chart of the PCNA-positive cell rate of quimolimab; Fig. 6A Comparison of mRNA expression levels in glomeruli of quemolimab Figure 6B This is the result of CD45 immunofluorescence detection of quimolimab; Figure 6C This is a comparison chart of the CD45 positive cell rate of quimolimab; Fig. 7A This is a graph showing the results of Western blot detection of CX3CL1-CX3CR1; Figure 7Bis a comparison of the relative fluorescence intensity of CX3CL1; Figure 7C is a comparison of the relative fluorescence intensity of CX3CR1; Fig.7D This is the fluorescence detection result of CX3CR1-positive monocytes; Fig. 7E It is a comparison chart of CD68 positive cell rate in glomerulus; Figure 7F is a comparison of the CX3CR1-positive cell rates in glomeruli; Fig. 8A This is the Western blot test result of PI3K / AKT; Figure 8B This is a comparison chart of PI3K phosphorylation; Figure 8C This is a comparison chart of AKT phosphorylation; Fig.8D is a comparison of the relative fluorescence intensity of p-PI3K; Fig. 8E is a comparison chart of the relative fluorescence intensity of PI3K; Fig.8F is a comparison of the relative fluorescence intensity of p-AKT; Figure 8G This is a comparison chart of the relative fluorescence intensity of AKT. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings: The present invention provides a medicine for treating nephritis, comprising a CX3CL1-CX3CR1 axis blocker.

[0020] Chemokine (C-X3-C motif) ligand 1 (CX3CL1), also known as fractalkine, is a macromolecular cytokine protein composed of 373 amino acids. Soluble CX3CL1 is present in serum, urine, cerebrospinal fluid, amniotic fluid and synovial fluid. It is mainly expressed by vascular endothelial cells, smooth muscle, neurons and dendritic cells, and is also expressed in a variety of renal cells including mesangial cells. Inflammatory cytokines such as TNF-α and IFN-γ can induce the expression of most CX3CL1. The expression of CX3CL1 can be regulated by pro-inflammatory and pro-fibrotic cytokines.

[0021] CX3CR1 is the only G protein-coupled receptor of CX3CL1, which is most abundantly expressed in mononuclear macrophages and mediates the adhesion and recruitment of monocytes in local disease tissues. The CX3CL1-CX3CR1 signaling pathway may be involved in the interaction between mesangial cells and monocytes in MsPGN, promoting immune damage in the glomerular region. The present invention targets CX3CR1 through a blocker to block the CX3CL1-CX3CR1 pathway, which is a potential treatment strategy for MsPGN.

[0022] The CX3CL1-CX3CR1 axis blocker is selected from the group consisting of quemolimab, AZD8797, JMS-17-2, JMS-17-2 hydrochloride, E6130, and alirocumab.

[0023] Among them, AZD8797 is an isomeric non-competitive antagonist of CX3CR1, and its molecular formula is: C 19 H 25 N5OS2, molecular structure see Figure 1A .

[0024] Quetmolimab is a novel humanized anti-CX3CL1 monoclonal antibody that can neutralize CX3CL1 in vivo.

[0025] E6130 is an orally active, highly selective CX3CR1 modulator. JMS-17-2 is a potent and selective CX3CR1 antagonist.

[0026] Glomerular cell proliferation and activation test. Experimental methods: Wild-type Wistar rats (male, 6-8 weeks old, 200-250 g) were selected and kept in a constant temperature (20°C), constant humidity (70%), and day and night cycle environment. 6-8 rats were randomly selected as the normal control group. The MsPGN model was established by tail vein injection of anti-Thy1 antibody 2.5 mg / kg, and the rats were randomly divided into a model group and a treatment group, with 6-8 rats in each group. On the 3rd to 7th day after the model was established, the treatment group was continuously injected with CX3CR1 antagonist AZD8797 (2 mg / kg / d) through the tail vein; the control group and the model group were given an equal volume of solvent. On the 7th day, the rats were anesthetized with pentobarbital and killed. The renal tissues were obtained for PAS staining (PAS staining, periodic acid-Schiff staining), and the proliferating cell nuclear antigen PCNA (PCNA positive cells rate per glomerulus, PCNA positive cell rate) and αSMA (α-smooth muscle actin staining area rate) were detected by immunohistochemistry experiments to evaluate the effect of AZD8797 in reducing cell proliferation and activation in the glomerulus.

[0027] Experimental results: Figure 1B , Figure 1C , Figure 1D and Figure 1E Compared with the control group, the number of nucleated cells, PCNA-positive cell rate, and αSMA expression level in the glomeruli of the model group were significantly increased, while the number of nucleated cells, PCNA-positive cell rate, and αSMA expression level in the glomeruli of the AZD8797 treatment group were significantly decreased.

[0028] AZD8797 in the experimental method was replaced by quimolimab (3 mg / kg / d). Figure 5A , Figure 5B and Figure 5D The number of nucleated cells, PCNA-positive cell rate, and αSMA expression level in the glomerulus of the model group were significantly increased, while the number of nucleated cells, PCNA-positive cell rate, and αSMA expression level in the quemolimab treatment group were significantly decreased.

[0029] Glomerular immune cell infiltration and inflammation test. Experimental methods: Wild-type Wistar rats (male, 6-8 weeks old, 200-250 g) were selected and raised in a constant temperature (20℃), constant humidity (70%), and day and night cycle environment. 6-8 rats were randomly selected as the control group. The MsPGN model was established by tail vein injection of anti-Thy1 antibody 2.5 mg / kg, and the rats were randomly divided into model group and treatment group, with 6-8 rats in each group. On the 3rd to 7th day after the model was established, the treatment group was continuously injected with CX3CR1 antagonist AZD8797 (2 mg / kg / d) by tail vein, and the control group and model group were given an equal volume of solvent. On the 7th day, the rats were anesthetized with pentobarbital and killed, and the renal tissues were taken for immunofluorescence detection of CD45, and the mRNA expression levels of TNFα, IL-6, and IL-1β were detected by real-time quantitative PCR to evaluate the effect of AZD8797 on reducing glomerular immune cell infiltration and inflammatory response.

[0030] Experimental results: Figure 2A , Figure 2B and Figure 2C Compared with the control group, the number of CD45-positive cells in the glomeruli of the model group increased, and the mRNA levels of TNFα, IL-6, and IL-1β were significantly increased; compared with the model group, the number of CD45-positive cells in the glomeruli of the AZD8797 treatment group was significantly reduced, and the mRNA expression levels of TNFα, IL-6, and IL-1β were significantly decreased.

[0031] Replace AZD8797 in the experimental method with quimolimab (3 mg / kg / d). Fig. 6A , Figure 6B and Figure 6C Compared with the control group, the number of CD45-positive cells in the glomeruli of the model group increased, and the mRNA levels of TNFα, IL-6, and IL-1β were significantly increased; compared with the model group, the number of CD45-positive cells in the glomeruli of the quimolimab treatment group was significantly reduced, and the mRNA expression levels of TNFα, IL-6, and IL-1β were significantly decreased.

[0032] Induced monocyte migration test. Experimental methods: Primary human renal mesangial cells (HRMC) were cultured in medium supplemented with 10% fetal bovine serum, 1% MC growth factor, and 1% penicillin / streptomycin solution. The human monocytic cell line THP-1 was cultured in medium 1640 supplemented with 10% FBS, 100 U / mL P / S, and 0.05 mM β-mercaptoethanol. All cells were placed in a cell culture incubator at 37°C and 5% carbon dioxide. HRMC were seeded in 24-well plates, transfected with plasmids to overexpress CX3CL1, and co-cultured with THP-1 in a transwell system with an 8μm pore size, and samples were taken. 5mM AZD8797 was added to the co-culture system, and crystal violet staining was performed to observe the effect on monocyte migration.

[0033] Experimental results: see Figure 3A and Figure 3B Compared with the normal control group and the negative control group, the monocyte migration induced by the CX3CL1 overexpression group was significantly enhanced, and the addition of AZD8797 reduced the monocyte migration under this effect.

[0034] Mesangial cell proliferation and inflammation detection. Experimental method: HRMC were inoculated in a 6-well plate and treated with human recombinant 0-50ng / mL TNFα for 24 hours to simulate the inflammatory environment, and samples were taken as control samples. HRMC with simulated inflammation were co-cultured with THP-1 in a transwell system with a pore size of 0.4μm, and samples were taken as co-culture samples. 5mMAZD8797 was added to the co-culture system, and mesangial cells were collected as treatment samples for EDU (5-ethynyl-2-deoxyuridine) experiments. Real-time quantitative PCR was performed to detect the mRNA expression levels of TNFα, IL-6 and IL-1β, and the effects on mesangial cell proliferation, activation and inflammation levels were observed.

[0035] Experimental results: see Figure 4A , Figure 4B , Figure 4C and Figure 4D Compared with the control samples, the number of EDU-positive cells in cells co-cultured with monocytes increased, and the mRNA expression levels of TNFα, IL-6, and IL-1β increased; the relative EDU positive cell rate % of mesangial cells in the treated samples decreased, and the mRNA levels of TNFα, IL-6, and IL-1β decreased.

[0036] Based on the above tests, it can be concluded that CX3CL1-CX3CR1 axis blockers, especially AZD8797 and quimolimab, can reduce the number of nucleated cells, PCNA-positive cells, and αSMA expression levels in the glomeruli, reduce cell proliferation and activation in the glomeruli; reduce CD45-positive cells in the glomeruli, reduce the mRNA expression levels of TNFα, IL-6, and IL-1β, reduce immune cell infiltration and inflammatory response in the glomeruli; reduce monocyte migration; inhibit mesangial cell proliferation and reduce the inflammatory response caused by it. It can be seen that CX3CL1-CX3CR1 axis blockers can be used to treat nephritis, especially for the treatment of mesangial proliferative glomerulonephritis.

[0037] Blocking CX3CL1-CX3CR1 interaction detection in glomeruli. Experimental methods: Wild-type Wistar rats (male, 6-8 weeks old, 200-250 g) were selected and kept in a constant temperature (20°C), constant humidity (70%), and day and night cycle environment. 6-8 rats were randomly selected as the control group. The MsPGN model was established by tail vein injection of anti-Thy1 antibody 2.5 mg / kg, and the rats were randomly divided into model group and treatment group, with 6-8 rats in each group. On the 3rd to 7th day after the model was established, the treatment group (6-8 rats) was continuously injected with CX3CL1 monoclonal antibody quemolimab (3 mg / kg / d) by tail vein, and the control group and model group were given an equal volume of solvent. On the 7th day, the rats were killed by pentobarbital anesthesia, and the renal tissues were obtained for Western blot and immunofluorescence experiments to detect the expression levels of CX3CL1 and CX3CR1 and the infiltration of CX3CR1-positive monocytes in the glomeruli and to evaluate the blocking effect of quimolimab on the CX3CL1-CX3CR1 interaction.

[0038] Experimental results: Fig. 7A , Figure 7B and Figure 7C , compared with the control group, the levels of CX3CL1 and CX3CR1 in the glomeruli of the model group were increased, such as Fig.7D , Fig. 7E and Figure 7F , CX3CR1-positive monocyte infiltration increased; the levels of CX3CL1 and CX3CR1 in the glomeruli of the quemolimab-treated group were decreased, and the infiltration of CX3CR1-positive monocytes was reduced.

[0039] Inhibit PI3K / AKT signaling pathway detection in the glomerulus. PI3K (phosphatidylinositol kinase) is a dimer composed of the regulatory subunit p85 and the catalytic subunit p110. When it binds to growth factor receptors (such as EGFR), it can change the protein structure of AKT and activate it, and activate or inhibit a series of downstream substrates such as apoptosis-related proteins Bad and Caspase9 activity by phosphorylation, thereby regulating cell proliferation, differentiation, apoptosis, migration and other phenotypes. P-PI3K represents phosphorylated PI3K; p-AKT represents phosphorylated AKT, and β-Actin is used as an internal reference.

[0040] Experimental methods: Wild-type Wistar rats (male, 6-8 weeks old, 200-250 g) were selected and kept in a constant temperature (20℃), constant humidity (70%), and day and night cycle environment. 6-8 rats were randomly selected as the control group. The MsPGN model was established by tail vein injection of anti-Thy1 antibody 2.5 mg / kg, and the rats were randomly divided into model group and treatment group, with 6-8 rats in each group. On the 3rd to 7th day after the model was established, the treatment group was continuously injected with CX3CL1 monoclonal antibody quimolimab (3 mg / kg / d) by tail vein, and the control group and model group were given equal volumes of solvents. On the 7th day, the rats were anesthetized with pentobarbital and killed, and the renal tissues were taken to extract proteins, and the expression and phosphorylation levels of PI3K and AKT were detected to evaluate the inhibitory effect of quimolimab on the PI3K / AKT signaling pathway.

[0041] Experimental results: Fig. 8A , Figure 8B , Figure 8C , Fig.8D , Fig. 8E , Fig.8F and Figure 8G Compared with the control group, the PI3K / AKT signaling pathway was activated in the glomeruli of the model group, and the phosphorylation levels of PI3K and AKT were increased; the PI3K / AKT signaling pathway was inhibited in the glomeruli of the quimolimab treatment group, and the phosphorylation levels of PI3K and AKT were decreased. Figure 8B In the figure, the ordinate is the relative value of PI3K phosphorylation (p-PI3K / PI3K ratio); Figure 8C In the figure, the vertical axis is the relative value of AKT phosphorylation (p-AKT / AKT ratio).

[0042] The present invention discloses for the first time that a blocking agent is used to improve the problems of cell proliferation, extracellular matrix increase, cell activation, immune cell infiltration and inflammatory response in glomeruli in nephritis.

[0043] The CX3CL1-CX3CR1 axis blocker of the present invention can be prepared as an injection or oral agent, with a final concentration of 2-20mM or a dosage of 2-10 mg / KG / day, and the solvent can be DMSO, ethanol or water. Before use, the working solution can be prepared by using the AZD8797 stock solution. Taking 1 mL of the working solution as an example, take 50μL of the clarified DMSO stock solution and add it to 400μL of PEG300 and mix evenly; add 50μL of Tween80 to the above mixed solution, mix evenly to clarify it; then continue to add 500μLddH2O to obtain the working solution. The final concentration of AZD8797 is 5-20mM or the dosage is 1-10 mg / kg / day. Quemolizumab can be used in an aqueous solution or a phosphate solution, etc., and the dosage is 1-8 mg / kg / day.

[0044] The drug can be in the form of powder or mixed solution. It should be noted that the preparation or form of the drug is not limited thereto and can be prepared and adjusted according to actual needs, such as adding appropriate excipients, etc. In general, the drug for treating nephritis utilizes a CX3CL1-CX3CR1 axis blocker.

[0045] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A drug for treating nephritis, characterized in that: These include CX3CL1-CX3CR1 axis blockers.

2. The drug according to claim 1, characterized in that The CX3CL1-CX3CR1 axis blocker is selected from: quemolimab, AZD8797, JMS-17-2, JMS-17-2 hydrochloride, E6130, and alirocumab.

3. The drug according to claim 1, characterized in that The drug is given by injection.

4. The drug according to claim 3, characterized in that It also includes any of the following ingredients or their combination: Excipients and solvents; The final concentration of the blocking agent is 2-20 mM; or the dosage is 2-10 mg / kg / day.

5. The drug according to claim 4, characterized in that The final concentration of AZD8797 is 5-20 mM or the dosage is 1-10 mg / kg / day; The dosage of quemolimab is 1-8 mg / kg / day.

6. The drug according to claim 1, characterized in that The drug is used to reduce the number of nucleated cells, the number of PCNA-positive cells, and the expression level of αSMA in the glomerulus, and to alleviate the cell proliferation and activation effect in the glomerulus.

7. The drug according to claim 1, characterized in that The drug is used to reduce CD45 positive cells in glomeruli, lower the mRNA expression levels of TNFα, IL-6, and IL-1β, and alleviate immune cell infiltration and inflammatory response in glomeruli.

8. The drug according to claim 1, characterized in that The drug is used to reduce monocyte migration; it is also used to inhibit mesangial cell proliferation and alleviate the induced inflammatory response.

9. The drug according to claim 1, characterized in that The drug is used to reduce the levels of CX3CL1 and CX3CR1 and reduce the infiltration of CX3CR1-positive monocytes; It is also used to reduce the phosphorylation levels of PI3K and AKT.

10. The drug according to claim 1, characterized in that The medicine is used for treating mesangial proliferative glomerulonephritis.

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