Application of CK2 inhibitor CX4945 in preparation of medicine for preventing and / or treating lupus nephritis

Abstract

The invention discloses an application of a CK2 inhibitor CX4945 in preparation of a medicine for preventing and / or treating lupus nephritis. The CK2 inhibitor CX4945 provided by the invention can significantly reduce the deposition of immune complex and complement C3 in the kidney of a lupus animal model, and effectively reduce the ratio of urine protein / creatinine. And the medicine has no obvious side effect, can be prepared into a lupus immunotherapy medicine, and provides a new combined treatment mode. The medicine can be prepared into an oral medicine, treatment is convenient, and patient compliance is high.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of the CK2 inhibitor CX4945 in the preparation of drugs for the prevention and / or treatment of lupus nephritis. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by dysregulation of immune tolerance, production of autoantibodies, and formation and deposition of immune complexes, leading to tissue and organ damage. The incidence of SLE is gradually increasing. There are over one million SLE patients in my country, primarily affecting women of childbearing age. SLE has a low clinical remission rate, high disability and mortality rates, with a cumulative 15-year mortality rate reaching as high as 20%, thus imposing a severe economic burden on families and society. SLE presents with diverse clinical phenotypes, affecting multiple and severe organs, commonly including the kidneys, cardiovascular system, skin, central nervous system, hematologic system, and respiratory system. The incidence of SLE in my country is approximately three times that of European and American populations. Furthermore, Chinese SLE patients experience earlier onset, more complex and diverse clinical symptoms, and are more prone to developing lupus nephritis (LN). LN is one of the most serious and common complications of SLE and a major risk factor for morbidity and mortality, potentially leading to end-stage renal disease (ESRD) and chronic kidney disease (CKD). SLE is highly heterogeneous and usually requires the use of immunosuppressants and hormones. These drugs may cause a series of side effects and risks, such as infection, osteoporosis, and hypertension. Moreover, the condition is prone to fluctuations, so there is an urgent need for new treatment strategies that specifically target the pathogenesis.

[0003] Routine induction therapy for severe lymphadenopathy (LN) involves high-dose corticosteroids combined with cyclophosphamide or mycophenolate mofetil (MMF). Cyclophosphamide is associated with myelosuppression and bladder toxicity. Higher cumulative doses of cyclophosphamide increase the risk of malignancy and infertility. Asians, who are generally lower in body weight, have a higher risk of infection when using MMF. However, due to the difficulty in quantitatively monitoring MMF, its use is generally not recommended for Asian patients at risk of toxicity.

[0004] The guidelines recommend rituximab as the primary salvage therapy for refractory lymphoma. Targeted therapies are expensive, have stringent usage requirements, require strict infection control, and have a high relapse rate.

[0005] Glucocorticoid use is one of the most significant factors contributing to organ damage in SLE. Long-term glucocorticoid use is associated with adverse effects on infection, metabolism, cardiovascular function, psychological well-being, and musculoskeletal health. Because infectious complications remain a leading cause of death in SLE, efforts have been made to minimize glucocorticoid use to control LN activity.

[0006] Evidence for the use of conventional calcineurin inhibitors in lymphoma (LN) is primarily derived from Asian studies, but data on the long-term nephrotoxicity induced and maintained by calcineurin inhibitors are lacking. The risk of infectious complications is particularly high when calcineurin inhibitors are used in combination with high doses of glucocorticoids, and caution is advised when using them.

[0007] The drugs mentioned above are currently recommended by clinical guidelines for the treatment and remission of LN. They mainly control the pathological progression of LN by inhibiting immune cells and their function, but they have significant side effects.

[0008] CK2 is a serine / threonine kinase. The CK2 holoenzyme is a tetramer composed of two catalytic subunits, CK2α and CK2α', and two regulatory subunits, CK2β. The two catalytic subunits are encoded by CSNK2A1 (encoding CK2α) and CSNK2A2 (encoding CK2α'), respectively, while the regulatory subunit CK2β is encoded by CSNK2B. The regulatory subunits confer enzyme stability while influencing the substrate specificity of the catalytic subunits. CK2 can regulate various cellular processes, such as transcription and translation. CK2 expression and activity are abnormal in many tumors and are associated with tumor progression and poor prognosis. Although most of our current understanding of CK2 comes from the field of oncology, increasing evidence suggests that CK2 plays an important role in the activation, proliferation, and differentiation of immune cells. CX4945 is the first and only orally administered bioavailable small molecule inhibitor of CK2 to enter human clinical trials (structural formula shown). Figure 1 It has few side effects. In January 2022, the structurally optimized CK2 inhibitor CX-4945 (trade name silverasertib) was designated as an orphan drug by the FDA for the treatment of cholangiocarcinoma. Compared with normal cells, various tumor cells highly express CK2 and become addicted to CK2 in a "non-oncogene addiction" manner, making CK2 a potential anti-tumor target.

[0009] In human and mouse SLE, lupus nephritis (LN) is characterized by progressive inflammation triggered by immune complexes deposited in the kidneys, followed by complement activation, inflammatory cell recruitment, and renal function loss. The presence of proteinuria is a strong indicator of glomerular damage, reflecting the progression of kidney disease. Reducing or preventing proteinuria is often a key measure to prevent or delay the progression of CKD.

[0010] Currently, there are no reports on the use of the CK2 inhibitor CX4945 in the prevention and / or treatment of lupus nephritis. Summary of the Invention

[0011] The purpose of this invention is to provide a pharmaceutical use for the CK2 inhibitor CX4945.

[0012] The pharmaceutical uses of the CK2 inhibitor CX4945 provided by this invention are as described in any of the following (a1)-(a3):

[0013] 1) Use in the preparation of drugs for the prevention and / or treatment of lupus nephritis (LN);

[0014] 2) Application in the preparation of drugs to alleviate lupus nephritis (LN);

[0015] 3) Application in the preparation of drugs to delay the progression of lupus nephritis (LN);

[0016] 4) Use in the preparation of drugs for the prevention and / or treatment of proteinuria in lupus nephritis (LN).

[0017] Furthermore, the prevention and / or treatment of lupus nephritis embodies at least one of the following aspects:

[0018] 1) Significantly reduces glomerular immune complex deposition and complement C3 deposition;

[0019] 2) Significantly improves inflammatory pathological damage in the glomeruli and renal tubules.

[0020] 3) Significantly reduces the urine protein / creatinine ratio;

[0021] 4) Reduce proteinuria.

[0022] The CAS No. of the CX4945 is 1009820-21-6, and its structural formula is as follows: Figure 1 As shown.

[0023] The present invention also provides a pharmaceutical composition.

[0024] The pharmaceutical composition comprises CX4945 as its active ingredient.

[0025] The pharmaceutical composition has at least one of the following effects:

[0026] 1) Significantly reduces glomerular immune complex deposition and complement C3 deposition;

[0027] 2) Significantly improves inflammatory pathological damage in the glomeruli and renal tubules.

[0028] 3) Significantly reduces the urine protein / creatinine ratio;

[0029] 4) Reduce proteinuria.

[0030] Furthermore, a pharmaceutically acceptable carrier material may be added to the pharmaceutical composition.

[0031] The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems.

[0032] To formulate unit-dose dosage forms into tablets, a wide variety of carriers known in the art can be used. Examples of carriers include, for instance, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfonate, methylcellulose, and ethylcellulose. For preparing unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. For preparing unit-dose dosage forms into injectable formulations such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Additionally, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation.

[0033] The present invention also provides methods for treating and / or preventing diseases or conditions in a subject.

[0034] The method includes administering CX4945 or a pharmaceutical composition containing CX4945 to a subject in need;

[0035] The disease described is lupus nephritis (LN);

[0036] The condition described is caused by lupus nephritis (LN), such as glomerular immune complex deposition, complement C3 deposition, membranoproliferative glomerulonephritis, tubulointerstitial inflammation, mesangial proliferative glomerulonephritis, or proteinuria.

[0037] This invention demonstrates the therapeutic effect of CX4945 on lupus nephritis (LN) through the following aspects:

[0038] 1) Spontaneous Lupus Mouse Model: MRL / Lpr mice, a representative animal model for studying lupus pathogenesis, spontaneously develop lupus symptoms similar to those in humans around 16 weeks of age. Starting from week 15, MRL / Lpr mice were treated with CX4945 via gavage once daily for one month. CX4945 significantly reduced glomerular immune complex and complement C3 deposition. Figure 2 The urine protein / creatinine ratio decreased significantly. Figure 3 ).

[0039] 2) Imiquimod (IMQ)-induced lupus model: IMQ cream was applied to the ears of 8- to 10-week-old WT and STING KO C57BL / 6 mice three times a week for five weeks to establish a lupus animal model with a significant lupus phenotype. From the third week onwards, CX4945 was administered via gavage once daily for three weeks. CX4945 significantly reduced glomerular immune complex and complement C3 deposition. Figure 4 It significantly alleviates glomerulonephritis and tubulointerstitial inflammation. Figure 5 The urine protein / creatinine ratio decreased significantly. Figure 6 ).

[0040] 3) In vitro cell experiments have shown that CX4945 can effectively reduce CK2 phosphorylation activity in neutrophils, reduce ROS production levels, reduce NETs production, and significantly inhibit the pro-inflammatory effect of NETs on macrophages.

[0041] The inventors of this application discovered through research that the CK2 protein level and activity in neutrophils of SLE patients were significantly higher than those in healthy controls. Figure 7 Furthermore, the role of neutrophils in the pathological process of LN has been confirmed by many studies, suggesting that neutrophil therapy targeting abnormally expressed CK2 is a feasible clinical strategy for LN.

[0042] The applicant first demonstrated in vitro that CX4945 can significantly reduce CK2 phosphorylation activity in neutrophils of SLE patients. Figure 8 Furthermore, it can significantly reduce neutrophil ROS levels. Figure 9 Suppress NETs generation ( Figure 10 ) and reduce the pro-inflammatory activity of generated NETs ( Figure 11 Secondly, in animal studies, the applicant found that CX4945 significantly reduced glomerular immune complex and complement C3 deposition, decreased the urine protein / creatinine ratio, and delayed the pathological progression of lupus nephritis (LN). According to relevant data, a urine protein-creatinine (UP:Cr) ratio of <0.5-0.7 at 12 months can predict good long-term outcomes for LN, and guidelines recommend it as a remission treatment target, indicating that patients with persistent hyperproteinuria are not necessarily associated with adverse outcomes. This application found that CX4945 can significantly reduce urine protein, which has important clinical significance for delaying the progression of lupus nephritis.

[0043] Furthermore, in previous animal models of CX4945 treatment for tumors, the dosage of CX4945 administered to animals was typically 25 mg / kg or 75 mg / kg, with good tolerability. The applicant used a dosage of 15 mg / kg to treat lupus nephritis, which is far lower than the dosage used in cancer patients, resulting in fewer side effects. Attached Figure Description

[0044] Figure 1 This invention relates to the structural formula of the CK2-specific inhibitor CX4945;

[0045] Figure 2 CX4945 treatment significantly reduced glomerular immune complex and complement C3 deposition in the kidney tissue of MRL / lpr mice.

[0046] Figure 3 CX4945 treatment significantly reduced the urinary protein / creatinine ratio in MRL / lpr mice.

[0047] Figure 4 CX4945 treatment significantly reduced glomerular immune complex and complement C3 deposition in the kidney tissue of a lupus mouse model induced by imiquimod.

[0048] Figure 5 After CX4945 treatment, imiquimod-induced lupus mouse models showed reduced renal interstitial inflammatory cell infiltration and fibrosis; reduced endothelial and mesangial cell proliferation; reduced crescent formation of the parietal epithelium of the glomerular capsule; improved glomerular capsule stenosis; reduced glomerular capillary compression; and alleviated renal tubular epithelial cell swelling.

[0049] Figure 6 CX4945 treatment significantly reduced the urinary protein / creatinine ratio in imiquimod-induced lupus mouse models.

[0050] Figure 7The protein level and activity of CK2 in neutrophils of SLE patients were significantly upregulated; A) Protein level of CK2 in neutrophils of SLE patients; B) Significantly upregulated activity of CK2 in neutrophils of SLE patients; C) Significantly upregulated activity of CK2 in bone marrow neutrophils (PMN) of MRL / lpr mice; D) Flow cytometry showed a significant upregulation of mean fluorescence intensity (MFI) of CK2a in neutrophils of SLE patients.

[0051] Figure 8 CX4945 can significantly reduce the phosphorylation activity of CK2 in neutrophils in vitro;

[0052] Figure 9 CX4945 significantly reduced ROS levels in neutrophils from SLE patients and mouse bone marrow neutrophils in vitro.

[0053] Figure 10 CX4945 significantly reduced NET production in neutrophils of SLE patients in vitro;

[0054] Figure 11 CX4945 significantly reduced the pro-inflammatory capacity of NETs generated by neutrophils in SLE patients in vitro; A) NETs generated by bone marrow neutrophils pretreated with CX4945 significantly reduced the pro-inflammatory capacity of mouse bone marrow-derived macrophages (BMDM); B) NETs generated by neutrophils in SLE patients pretreated with CX4945 significantly reduced the pro-inflammatory capacity of peripheral monocyte-derived macrophages (MoDM). Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Example 1: Efficacy Test of CX4945

[0058] I. Experimental Methods

[0059] 1. MRL / lpr spontaneous lupus mouse model:

[0060] 1) MRL / lpr spontaneous lupus mice were obtained from Shanghai Slack Laboratory Animal Co., Ltd., and were bred and raised by Cyagen (Suzhou) Biotechnology Co., Ltd.

[0061] 2) At week 15, female MRL / lpr mice were divided into two groups (n=7 per group): the CX4945 group and the control group (Vehicle). The CX4945 group was treated with CX4945 by gavage at 15 mg / kg once daily for one month, while the control group was given an equal amount of saline. Both groups of mice were euthanized at the end of week 19.

[0062] 3) Kidney tissue and urine samples: Mice were anesthetized with isoflurane, and the kidneys were perfused with cold PBS via left-sided cardiac puncture. The kidneys were then frozen in tissue. In OCT compounds, the tissue sections were frozen at -80°C until the section thickness was 10 μm for immunofluorescence staining. Immune complex deposition was assessed by immunofluorescence staining of IgG and C3 on frozen kidney sections. Urine was collected, and urinary albumin and creatinine were quantified using a mouse Albuwell ELISA kit and a creatinine companion kit (Exocell, Philadelphia, PA), and the albumin / creatinine ratio was calculated.

[0063] 2. Imiquimod (IMQ)-induced lupus model

[0064] 1) Imiquimod (IMQ)-induced lupus model: The ears of 8- to 10-week-old wild-type C57BL / 6N female mice were treated with 0.5% imiquimod cream (Idalor) by applying the drug to the epidermis three times a week for five weeks.

[0065] 2) During the third week of imiquimod application, the mice were divided into two groups (n=5 per group): the CX4945 group and the control group (Vehicle). The CX4945 group was administered by gavage at a dose of 15 mg / kg once daily for three weeks. The control group was given an equal amount of saline. Both groups of mice were euthanized at the end of the fifth week.

[0066] 3) Kidney tissue and urine samples: Mice were anesthetized with isoflurane, and the kidneys were perfused with cold PBS via left-sided cardiac puncture. The kidneys were then frozen in tissue. In OCT compounds, the tissue sections were frozen at -80°C until the section thickness was 10 μm for immunofluorescence staining. Immune complex deposition was assessed by immunofluorescence staining of IgG and C3 on frozen kidney sections. Urine was collected, and urinary albumin and creatinine were quantified using a mouse Albuwell ELISA kit and a creatinine companion kit (Exocell, Philadelphia, PA), and the albumin / creatinine ratio was calculated.

[0067] 3. In vitro cell experiments

[0068] 1) Peripheral EDTA-anticoagulated blood samples were collected from SLE patients. Peripheral neutrophils were purified using Ficoll-pague combined with Dextron sedimentation. Lysis was performed on ice for 30 minutes using RIPA high-efficiency protein lysis buffer (Solarbio, with added protease and phosphatase inhibitors). After centrifugation at 10,000 rpm for 15 minutes, the supernatant was collected as the neutrophil protein sample. Protein quantification was performed using a BCA kit (Thermo). Before protein electrophoresis, 5* protein loading buffer (Solarbio) was added, and the sample was boiled in a 100°C metal bath for 15 minutes.

[0069] 2) Load the sample at a total protein content of 15 micrograms into a 12% precast gel (ACE) and electrophoresis at 150V for 40 minutes; transfer the membrane using the Bio-rad rapid transfer system and a pre-activated PVDF membrane, following the manufacturer's instructions; block the successfully transferred PVDF membrane in 5% skim milk (BD) for 1 hour; incubate overnight at 4°C with primary antibody (antibody sourced from Abcam and Proteintech); wash three times with TBST buffer, then incubate at room temperature with secondary antibody (CST) for 1 hour; wash three times with TBST buffer, then add developing substrate (Millipore) for development.

[0070] 3) Collect neutrophils from SLE patients and healthy controls according to the method in 1). Perform lysis on ice with high-efficiency protein lysis buffer (with added protease inhibitors). After centrifugation at 10,000 rpm for 15 minutes, collect the supernatant and measure CK2 protein activity according to the instructions of the CK2 activity assay kit (MBL, CY-1170).

[0071] 4) ROS generation experiment: Neutrophils from SLE patients were collected according to the method in 1). The cells were resuspended in HBSS and pretreated with CX4945 (5μM) for 30 minutes in a cell incubator at 37°C. The probe was labeled according to the instructions of DHR123 (Invitrogen). Fluorescence values ​​were recorded under PMA or LPS conditions every 10 minutes, and the ROS generation curve was plotted.

[0072] 5) Flow cytometry detection of the MFI value of the CK2a subunit: Neutrophils from SLE patients were collected according to method 1). The cells were resuspended in 1 ml of the permeabilization buffer from the Foxp3 permeabilization kit (Invitrogen) and gently homogenized. The cells were incubated overnight at 4°C. 2 ml of the permeabilization wash buffer (10*, diluted 10 times with ddH2O) from the kit was added. The cells were centrifuged at 1000 rpm for 5 minutes at 4°C. After centrifugation, the cells were stained with primary antibody (CSNK2A, Proteintech, 1:400 dilution) on ice for 30 minutes. After washing once with wash buffer, the cells were stained with secondary antibody (Donkey anti-Rabbit IgG (H+L) Alexa Fluor). TM 555 (Invitrogen, 1:500), stained on ice for 30 minutes, washed once with washing buffer, and resuspended in 200 μL PBS buffer. Detected using a BD FACSymphony-A1 flow cytometer, and data were analyzed using FlowJo software.

[0073] 6) NETs generation experiment: Neutrophils were collected from SLE patients according to method 1), and cultured in serum-free RPMI 1640 medium at a concentration of 1*10⁻⁶ cells / mL. 6 Resuspend cells at a density of / ml; add poly-L-L pretreated cell slides to 24-well cell culture plates (Corning); carefully drop 50 μL of cell suspension into the middle of each cell slide, and incubate at 37°C for 15 minutes to allow cell sedimentation; carefully aspirate and discard the supernatant, add 50 μL of CX4945 (5 μM) or serum-free RPMI 1640 medium, and pretreat at 37°C for 30 minutes; carefully aspirate and discard the supernatant, add 50 μL of PMA (50 nM) and LPS (1 Incubate with 0.00 ng / ml or A23187 (4 μM) at 37°C for 4 hours to stimulate NET generation; add 500 μL of 4% tissue fixative (Solarbio) and fix at room temperature for 20 minutes, then wash three times with PBS buffer, block with 0.2% Gelatin (Solarbio) at room temperature for 1 hour, then stain with primary antibody (anti-NE antibody, Millipore, 1:400) at room temperature for 90 minutes; wash three times with PBS buffer, then stain with secondary antibody (Donkey anti-Rabbit IgG (H+L) Alexa Fluor) TMStain with Hoechst 33342 (Invitrogen, 1:500) at room temperature for 60 minutes; wash three times with PBS buffer and then stain with Hoechst 33342 (Invitrogen, 1:1000) at room temperature in the dark for 5 minutes; wash three times with PBS buffer and then mount with Prolong (Invitrogen). After the slides are dry, they are photographed and counted under a fluorescence microscope.

[0074] 7) Method for obtaining animal bone marrow neutrophils: When MRL / lpr mice or C57BL / 6N mice were euthanized at the planned experimental time point, femurs were taken. After gently rubbing away the attached muscle and connective tissue on the surface of the femur with gauze, the joint ridges at both ends were cut off with scissors. The bone marrow tissue was gently blown out with a 22G syringe needle and aspirated back and forth to form a single-cell suspension. After filtration, neutrophils were isolated using a mouse neutrophil separation magnetic bead kit (Mittenia) for CK2 protein activity and ROS level generation experiments.

[0075] 8) Culture method of animal bone marrow-derived macrophages (BMDM): Prepare bone marrow single-cell suspension according to the method in 7), resuspend the cells in IMDM medium (supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin complex (Gibco) and 50 ng / ml mouse M-CSF (Biolegend)), and culture at 2*10 6 The cells were seeded at a density of 1 / ml onto 12-well cell culture plates (Corning) and incubated at 37°C for 5 days. The culture medium was changed on the third day. On the fifth day, the cell status was observed under a microscope. When the cells reached 90% confluence, stimulation tests could be performed.

[0076] 9) Culture method of human peripheral monocyte-derived macrophages (MoDM): EDTA-anticoagulated peripheral blood was used to separate the upper layer of PBMCs by Ficoll density gradient centrifugation. The cells were resuspended in RPMI medium (supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin complex (Gibco), and 50 ng / ml human M-CSF (Biolegend)). The cells were cultured at a rate of 2*10-1. 6 The cells were seeded at a density of 1 / ml onto 12-well cell culture plates (Corning) and incubated at 37°C for 5 days. The culture medium was changed on the third day. On the fifth day, the cell status was observed under a microscope. When the cells reached 90% confluence, stimulation tests could be performed.

[0077] 10) Detection of the pro-inflammatory capacity of NETs: Following the experimental method for generating NETs in 6), 1*10 6Cells were seeded into 48-well Corning cell culture plates and pretreated with CX4945 to stimulate NET production as described in step 6). After 4 hours, the supernatant was carefully discarded, and 100 μL of pre-warmed MNase (Invitrogen, 30 U / ml) was added. The plates were incubated at 37°C for 30 minutes. The supernatant was collected and centrifuged at 5000 rpm for 5 minutes. The collected supernatant was the NET sample. Protein quantification of CX4945 pretreated and untreated NET samples was performed using a BCA kit (Thermo). Mouse BMDM and human MoDM were stimulated at a protein concentration of 1 μg / ml. After incubation at 37°C for 4 hours, cells were collected, lysed with TRIzol, and RNA was extracted (ZYMO Tissue Cell RNA Extraction Kit). The RNA was reverse transcribed into cDNA (TAKARA Reverse Transcription Kit) and then subjected to qRRCR (PowerTrack SYBR Green). KIT (Invitrogen), using GAPDH (human) / Gapdh (mouse) as internal controls, calculated the relative expression levels of inflammatory cytokines using the ΔΔCT method.

[0078] II. Results

[0079] 1) In a mouse model of spontaneous lupus erythematosus (MLL / LPR), MRL / LPR significantly reduced glomerular immune complex and complement C3 deposition. Figure 2 The urine protein / creatinine ratio decreased significantly. Figure 3 ).

[0080] 2) In an imiquimod (IMQ)-induced lupus model trial, CX4945 significantly reduced glomerular immune complex and complement C3 deposition. Figure 4 It significantly alleviates glomerulonephritis and tubulointerstitial inflammation. Figure 5 The urine protein / creatinine ratio decreased significantly. Figure 6 ).

[0081] 3) In vitro cell experiments have shown that CX4945 can effectively reduce CK2 phosphorylation activity in neutrophils, reduce ROS production levels, reduce NETs production, and significantly inhibit the pro-inflammatory effect of NETs on macrophages.

[0082] from Figure 7 The results showed that the CK2 protein level and activity in neutrophils of SLE patients were significantly higher than those in healthy controls.

[0083] In vitro experiments have confirmed that CX4945 can significantly reduce CK2 phosphorylation activity in neutrophils of SLE patients. Figure 8 Furthermore, it can significantly reduce neutrophil ROS levels. Figure 9Suppress NETs generation ( Figure 10 ) and reduce the pro-inflammatory activity of generated NETs ( Figure 11 ).

[0084] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The pharmacological uses of the CK2 inhibitor CX4945 are as described in any of the following (a1)-(a3): 1) Use in the preparation of drugs for the prevention and / or treatment of lupus nephritis; 2) Application in the preparation of drugs to relieve lupus nephritis; 3) Application in the preparation of drugs to delay the progression of lupus nephritis; 4) Use in the preparation of drugs for the prevention and / or treatment of proteinuria in lupus nephritis; The CAS No. of the CX4945 is 1009820-21-6.

2. The application according to claim 1, characterized in that: The prevention and / or treatment of lupus nephritis embodies at least one of the following aspects: 1) Significantly reduces glomerular immune complex deposition and complement C3 deposition; 2) Significantly improves inflammatory pathological damage to the glomeruli and renal tubules; 3) Significantly reduces the urine protein / creatinine ratio; 4) Reduce proteinuria.

3. The application according to claim 1 or 2, characterized in that: The CX4945 was administered to mice with lupus nephritis at a dose of 15 mg / kg for the treatment of lupus nephritis.

4. A pharmaceutical composition wherein the active ingredient comprises CX4945 as described in claim 1; The pharmaceutical composition has at least one of the following effects: 1) Significantly reduces glomerular immune complex deposition and complement C3 deposition; 2) Significantly improves inflammatory pathological damage to the glomeruli and renal tubules; 2) Significantly reduced the urine protein / creatinine ratio; 3) Reduce proteinuria.

5. The composition according to claim 4, characterized in that: The pharmaceutical composition also contains a pharmaceutically acceptable carrier material.

6. A method of treating and / or preventing a disease or condition in a subject, comprising administering CX4945 or the pharmaceutical composition of claim 4 or 5 to a subject in need; in, The disease is lupus nephritis; the symptoms are those caused by lupus nephritis.

7. The method according to claim 6, characterized in that: The symptoms caused by lupus nephritis include: glomerular immune complex deposition, complement C3 deposition, membranoproliferative glomerulonephritis, tubulointerstitial inflammation, mesangial proliferative glomerulonephritis, or proteinuria.

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