A drug for treating nephritis

Abstract

The present invention discloses a drug for treating nephritis, belonging to the field of biomedicine. The drug comprises a CX3CL1-CX3CR1 axis blocker. The drug can reduce monocyte migration; inhibit mesangial cell proliferation and activation; 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. It can be used to treat 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, with IgA nephropathy (IgAN) being the most typical form. MsPGN is characterized by inflammatory cell infiltration within the glomeruli, mesangial cell proliferation, and increased extracellular matrix (ECM). Without timely and effective treatment, it can progress to glomerular sclerosis and interstitial fibrosis, ultimately leading to irreversible end-stage renal disease. A growing body of research suggests that immune damage within the glomerular region is a key driver of MsPGN disease progression. Multiple immune cells within the glomerulus interact with resident renal cells to maintain the glomerular immune microenvironment, further damaging renal cells and promoting cell proliferation and inflammatory responses.

[0003] The pathogenesis and progression of MsPGN remain unclear, resulting in the lack of effective, targeted treatment options. Traditional treatments primarily focus on renin-angiotensin-aldosterone system (RAAS) blockers and immunosuppressive therapies based on glucocorticoids and steroids. These treatments are associated with significant side effects, poor specificity, and limited targeting. Therefore, there is a need to develop drugs to treat nephritis, particularly mesangial proliferative glomerulonephritis, to mitigate immune damage in the glomerular region and slow disease progression. Summary of the Invention

[0004] In response to 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 present invention discloses a medicine for treating nephritis, comprising a CX3CL1-CX3CR1 axis blocker.

[0006] Preferably, the CX3CL1-CX3CR1 axis blocker is selected from:

[0007] quimolimab, AZD8797, JMS-17-2, JMS-17-2 hydrochloride, E6130, and alirocumab.

[0008] Preferably, the drug is administered by injection or oral administration.

[0009] Preferably, the final concentration of the blocker is 2-20 mM; or the dosage is 2-10 mg / kg / day.

[0010] Preferably, the final concentration of AZD8797 is 5-20 mM or the dosage is 1-10 mg / kg / day;

[0011] The dosage of quemolimab is 1-8 mg / kg / day.

[0012] Preferably, the drug further comprises any one of the following ingredients or a combination thereof:

[0013] Excipients, solvents, and inevitable impurities.

[0014] 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 glomeruli, thereby alleviating cell proliferation and activation in the glomeruli.

[0015] Preferably, 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.

[0016] Preferably, the drug is used to reduce monocyte migration; to inhibit mesangial cell proliferation, and to alleviate the inflammatory response caused.

[0017] Preferably, the drug is used to reduce the levels of CX3CL1 and CX3CR1, and reduce the infiltration of CX3CR1-positive monocytes;

[0018] It is also used to reduce the phosphorylation levels of PI3K and AKT.

[0019] Preferably, the medicament is used to treat mesangial proliferative glomerulonephritis.

[0020] Compared with the existing technology, 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

[0021] Figure 1A This is the molecular structure diagram of AZD8797;

[0022] Figure 1B Figure 1 is the PAS staining, PCNA and αSMA detection results of AZD8797;

[0023] Figure 1C This is a comparison chart of the nucleated cell count detection of AZD8797;

[0024] Figure 1D This is a comparison chart of the PCNA-positive cell rate of AZD8797;

[0025] Figure 1E This is a comparison chart of the αSMA expression level of AZD8797;

[0026] Figure 2A This is a comparison of the mRNA expression levels of AZD8797 in the glomeruli;

[0027] Figure 2B This is the result of CD45 immunofluorescence assay of AZD8797;

[0028] Figure 2C This is a comparison chart of the CD45 positive cell rate of AZD8797;

[0029] Figure 3A This is the crystal violet staining result of AZD8797;

[0030] Figure 3B This is a comparison of monocyte migration of AZD8797;

[0031] Figure 4A This is a comparison chart of TNFα mRNA expression levels of AZD8797;

[0032] Figure 4B This is a comparison chart of IL-6 mRNA expression levels of AZD8797;

[0033] Figure 4C This is a comparison chart of IL-1β mRNA expression levels of AZD8797;

[0034] Figure 4D This is the detection result diagram of DAPI staining, EDU staining and fusion staining of AZD8797;

[0035] Figure 4E It is a comparison chart of the EdU-positive cell ratio of AZD8797;

[0036] Figure 5A Figure 1 is the PAS staining, PCNA and αSMA detection results of quimolimab;

[0037] Figure 5B This is a comparison chart of the nucleated cell count detected by quemolimab;

[0038] Figure 5C This is a comparison chart of the αSMA expression level detected by quemolimab;

[0039] Figure 5D This is a comparison chart of the PCNA-positive cell rate detected by quimolimab;

[0040] Figure 6A Comparison of quemolimab mRNA expression levels in glomeruli

[0041] Figure 6B This is the CD45 immunofluorescence test result of quimolimab;

[0042] Figure 6C This is a comparison chart of the CD45 positive cell rate of quimolimab;

[0043] Figure 7A This is a graph showing the results of Western blot detection of CX3CL1-CX3CR1;

[0044] Figure 7B is a comparison of the relative fluorescence intensity of CX3CL1;

[0045] Figure 7C is a comparison of the relative fluorescence intensity of CX3CR1;

[0046] Figure 7D This is the fluorescence detection result of CX3CR1-positive monocytes;

[0047] Figure 7E is a comparison of the CD68-positive cell rates in glomeruli;

[0048] Figure 7F is a comparison of the CX3CR1-positive cell rates in glomeruli;

[0049] Figure 8A This is the Western blot detection result of PI3K / AKT;

[0050] Figure 8B This is a comparison chart of PI3K phosphorylation;

[0051] Figure 8C This is a comparison chart of AKT phosphorylation;

[0052] Figure 8D is a comparison of the relative fluorescence intensity of p-PI3K;

[0053] Figure 8E is a comparison of the relative fluorescence intensity of PI3K;

[0054] Figure 8F is a comparison of the relative fluorescence intensity of p-AKT;

[0055] Figure 8G This is a comparison chart of the relative fluorescence intensity of AKT. DETAILED DESCRIPTION

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying 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 making creative efforts shall fall within the scope of protection of the present invention.

[0057] The present invention is described in further detail below with reference to the accompanying drawings:

[0058] The present invention provides a medicine for treating nephritis, comprising a CX3CL1-CX3CR1 axis blocker.

[0059] Chemokine (C-X3-C motif) ligand 1 (CX3CL1), also known as fractalkine, is a large cytokine protein composed of 373 amino acids. Soluble CX3CL1 is present in serum, urine, cerebrospinal fluid, amniotic fluid, and synovial fluid. It is primarily expressed by vascular endothelial cells, smooth muscle cells, neurons, and dendritic cells, and is also expressed in various renal cell types, including mesangial cells. Inflammatory cytokines such as TNF-α and IFN-γ can induce the expression of most CX3CL1 proteins. CX3CL1 expression can be regulated by proinflammatory and profibrotic cytokines.

[0060] CX3CR1 is the only G protein-coupled receptor for CX3CL1, expressed most abundantly in mononuclear macrophages. It mediates the adhesion and recruitment of monocytes to local diseased 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, thereby blocking the CX3CL1-CX3CR1 pathway and presents a potential therapeutic strategy for MsPGN.

[0061] The CX3CL1-CX3CR1 axis blocker is selected from the group consisting of quemolimab, AZD8797, JMS-17-2, JMS-17-2 hydrochloride, E6130, and alirocumab.

[0062] 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 .

[0063] Quetmolimab is a novel humanized anti-CX3CL1 monoclonal antibody that neutralizes CX3CL1 in vivo.

[0064] E6130 is an orally active, highly selective CX3CR1 modulator. JMS-17-2 is a potent and selective CX3CR1 antagonist.

[0065] Glomerular cell proliferation and activation assay. Experimental methods: Wild-type Wistar rats (male, 6-8 weeks old, 200-250 g) were housed in a constant temperature (20°C), constant humidity (70%), and a day-night cycle. Six to eight rats were randomly selected as a normal control group. A MsPGN model was established by tail vein injection of an anti-Thy1 antibody at 2.5 mg / kg. The rats were randomly divided into a model group and a treatment group, with 6-8 rats in each group. On days 3-7 after model establishment, the treatment group received continuous tail vein injections of the CX3CR1 antagonist AZD8797 (2 mg / kg / day); the control and model groups received an equal volume of solvent. On the 7th day, the rats were anesthetized with pentobarbital and killed. The renal tissues were collected for PAS staining (PAS staining, periodic acid-Schiff staining), and immunohistochemistry was used to detect proliferating cell nuclear antigen (PCNA positive cells rate per glomerulus) and αSMA (α-smooth muscle actin staining area ratio) to evaluate the effect of AZD8797 in reducing cell proliferation and activation in the glomerulus.

[0066] 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.

[0067] AZD8797 in the experimental method was replaced with 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 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 quimolimab treatment group were significantly decreased.

[0068] Glomerular immune cell infiltration and inflammation were assessed. Wild-type Wistar rats (male, 6-8 weeks old, 200-250 g) were housed in a constant temperature (20°C), constant humidity (70%), and a day-night cycle. Six to eight rats were randomly selected as a control group. A MsPGN model was established by tail vein injection of an anti-Thy1 antibody at 2.5 mg / kg. The rats were then randomly divided into a model group and a treatment group, with 6-8 rats in each group. From day 3 to 7 after model establishment, the treatment group received continuous tail vein injection of the CX3CR1 antagonist AZD8797 (2 mg / kg / day), while the control and model groups received an equal volume of solvent. On day 7, the rats were anesthetized with pentobarbital and sacrificed. Renal tissues were collected for immunofluorescence analysis of CD45 and real-time quantitative PCR analysis of TNFα, IL-6, and IL-1β mRNA expression to assess the efficacy of AZD8797 in reducing glomerular immune cell infiltration and inflammation.

[0069] 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.

[0070] The AZD8797 in the experimental method was replaced by quimolimab (3 mg / kg / d). Figure 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-treated group decreased significantly, and the mRNA expression levels of TNFα, IL-6, and IL-1β were significantly decreased.

[0071] Induced Monocyte Migration Assay. Experimental Methods: Primary human renal mesangial cells (HRMC) were cultured in a 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 maintained in a cell culture incubator at 37°C with 5% carbon dioxide. HRMC were seeded in 24-well plates, transfected with a plasmid to overexpress CX3CL1, and co-cultured with THP-1 cells in an 8μm pore transwell system. Samples were collected. 5 mM AZD8797 was added to the co-culture system, and the effect on monocyte migration was observed by crystal violet staining.

[0072] 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.

[0073] Mesangial cell proliferation and inflammation assays. Human human fibroblasts (HRMCs) were seeded in 6-well plates and treated with 0-50 ng / mL human recombinant TNFα for 24 hours to simulate an inflammatory environment. Samples were collected as control samples. HRMCs with simulated inflammation were co-cultured with THP-1 cells in a 0.4 μm pore transwell system, and samples were collected as co-culture samples. 5 mM AZD8797 was added to the co-culture system, and mesangial cells were collected as treatment samples for EDU (5-ethynyl-2-deoxyuridine) assays. Real-time quantitative PCR was performed to measure TNFα, IL-6, and IL-1β mRNA expression levels to observe the effects on mesangial cell proliferation, activation, and inflammation.

[0074] 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 were elevated; 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 were reduced.

[0075] Based on the above tests, it can be concluded that CX3CL1-CX3CR1 axis blockers, especially AZD8797 and quemolimab, can reduce the number of nucleated cells and PCNA-positive cells in the glomeruli, as well as the expression level of αSMA, reducing cell proliferation and activation within the glomeruli. They also reduce CD45-positive cells in the glomeruli and the mRNA expression levels of TNFα, IL-6, and IL-1β, alleviating glomerular immune cell infiltration and inflammatory responses. They also reduce monocyte migration and inhibit mesangial cell proliferation, alleviating the inflammatory response caused by mesangial cells. Therefore, CX3CL1-CX3CR1 axis blockers can be used to treat nephritis, especially mesangial proliferative glomerulonephritis.

[0076] Blockade of CX3CL1-CX3CR1 interaction in glomeruli was tested. Experimental Methods: Wild-type Wistar rats (male, 6-8 weeks old, 200-250 g) were housed in a constant temperature (20°C), constant humidity (70%), and a day-night cycle. Six to eight rats were randomly selected as a control group. A MsPGN model was established via tail vein injection of an anti-Thy1 antibody at 2.5 mg / kg. The rats were randomly divided into a model group and a treatment group, with 6-8 rats in each group. On days 3-7 after model establishment, the treatment group (6-8 rats) received continuous tail vein injections of the CX3CL1 monoclonal antibody quimolimab (3 mg / kg / day). The control and model groups received equal volumes of solvent. On the 7th day, the rats were anesthetized with pentobarbital and killed, and renal tissues were collected for Western blot and immunofluorescence assays 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 quemolimab on the CX3CL1-CX3CR1 interaction.

[0077] Experimental results: Figure 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, e.g. Figure 7D 、 Figure 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.

[0078] Inhibition of the PI3K / AKT signaling pathway in glomeruli. PI3K (phosphatidylinositol kinase) is a dimer composed of the regulatory subunit p85 and the catalytic subunit p110. Binding to growth factor receptors (such as EGFR) alters the protein structure of AKT, leading to its activation. Phosphorylation activates or inhibits the activity of a series of downstream substrates, such as the apoptosis-related protein Bad and Caspase 9, thereby regulating phenotypes such as cell proliferation, differentiation, apoptosis, and migration. P-PI3K represents phosphorylated PI3K; p-AKT represents phosphorylated AKT. β-Actin serves as an internal control.

[0079] Experimental Methods: Wild-type Wistar rats (male, 6-8 weeks old, 200-250 g) were housed in a constant temperature (20°C), constant humidity (70%), and a day-night cycle. Six to eight rats were randomly selected as the control group. A MsPGN model was established by tail vein injection of an anti-Thy1 antibody at 2.5 mg / kg. The rats were randomly divided into a model group and a treatment group, with 6-8 rats in each group. From day 3 to 7 after model establishment, the treatment group received continuous tail vein injections of the CX3CL1 monoclonal antibody quimolimab (3 mg / kg / day), while the control and model groups received an equal volume of solvent. On day 7, the rats were anesthetized with pentobarbital and sacrificed. Renal tissue was harvested for protein extraction, and the expression and phosphorylation levels of PI3K and AKT were measured to evaluate the inhibitory effect of quimolimab on the PI3K / AKT signaling pathway.

[0080] Experimental results: Figure 8A 、 Figure 8B 、 Figure 8C 、 Figure 8D 、 Figure 8E 、 Figure 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 middle, the vertical axis 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).

[0081] The present invention discloses for the first time that a blocker is used to improve cell proliferation, extracellular matrix increase, cell activation, immune cell infiltration and inflammatory response in glomeruli in nephritis.

[0082] The CX3CL1-CX3CR1 axis blocker of the present invention can be prepared as an injectable or oral formulation, with a final concentration of 2-20 mM or a dosage of 2-10 mg / kg / day. The solvent can be DMSO, ethanol, or water. Before use, a working solution can be prepared from the AZD8797 stock solution. For 1 mL of working solution, add 50 μL of the clarified DMSO stock solution to 400 μL of PEG300 and mix thoroughly. Add 50 μL of Tween80 to this mixture, mix thoroughly to clarify, and then add 500 μL of ddH2O to obtain a working solution. The final concentration of AZD8797 is 5-20 mM, or the dosage is 1-10 mg / kg / day. Quemolizumab can be used in aqueous or phosphate solutions, for example, at a dosage of 1-8 mg / kg / day.

[0083] The drug can be in the form of a powder or a mixed solution. It should be noted that the preparation and form of the drug are not limited to this and can be prepared and adjusted according to actual needs, such as by adding appropriate excipients. In general, drugs for treating nephritis utilize CX3CL1-CX3CR1 axis blockers.

[0084] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of a CX3CL1-CX3CR1 axis blocker in the preparation of a medicament for treating mesangial proliferative glomerulonephritis, wherein the CX3CL1-CX3CR1 axis blocker is quemolimab.

2. The use according to claim 1, characterized in that The drug is given as an injection.

3. The use according to claim 2, characterized in that Medications also include: excipients and solvents; The final concentration of the blocking agent is 2-20 mM; or the dosage is 2-10 mg / kg / day.

4. The use 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 glomeruli, thereby alleviating cell proliferation and activation effects in the glomeruli.

5. The use 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.

6. The use according to claim 1, characterized in that The drug is used to reduce monocyte migration and inhibit mesangial cell proliferation, thereby alleviating the inflammatory response caused.

7. The use 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.

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