Application of small-molecule Fibrillin-1 inhibitor in preparation of medicine for treating chronic kidney diseases

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of a small-molecule Fibrillin-1 inhibitor in preparation of a medicine for treating chronic kidney diseases. According to the application of the evodiamine or the pharmaceutically acceptable salt of the evodiamine in inhibiting the activity of the fibrillar protein-1 or preparing the fibrillar protein-1 inhibitor, the Evodiamine is subjected to a molecular interaction experiment and a cell phenotype experiment, and results show that the Evodiamine can be used as a small-molecule inhibitor of FBN1 and can effectively inhibit the phenotype of mesangial cell fibrosis induced by the FBN1. Furthermore, Evodiamine can significantly inhibit podocyte damage, mesangial cell activation and glomerular sclerosis progress, and can be used for effectively inhibiting CKD progress to achieve the purpose of treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of a small molecule Fibrillin-1 inhibitor in the preparation of drugs for treating chronic kidney disease. Background Technology

[0002] Fibrillin-1 (FBN1) is a large extracellular glycoprotein rich in cysteine, containing multiple epidermal growth factor (EGF)-like domains and a potential transforming growth factor β-binding protein (LTBP) domain. Studies have found that FBN1 is upregulated in decellularized kidney tissue scaffolds (KTS) prepared from CKD mouse kidneys. FBN1 secreted by renal tubular cells can promote endothelial cell apoptosis and lead to renal vascularization by activating the TGF-β1 / Smad3 signaling pathway. Research indicates that FBN1 is also mainly secreted by damaged podocytes and released extracellularly, acting on itself and surrounding mesangial cells, leading to podocyte damage and mesangial cell activation, massive proteinuria, and extracellular collagen deposition, ultimately resulting in glomerulosclerosis. However, there are currently few specific inhibitors that directly target fibrillin-1; most research focuses on its downstream pathways or related pathological mechanisms. There is an urgent need for a safe and cost-effective specific inhibitor targeting fibrillin-1.

[0003] Chronic kidney disease (CKD) affects over 800 million people worldwide, becoming a heavy burden on global public health. Chronic glomerular disease, characterized by proteinuria due to podocyte damage and glomerulosclerosis caused by mesangial cell activation, is the leading cause of CKD. However, currently, there are no effective drugs to slow the progression of CKD. Targeting the pathogenesis of chronic glomerular disease and finding drugs to effectively inhibit or delay CKD progression is undoubtedly a key focus in nephrology and a crucial strategic priority. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art, and to provide a small molecule compound as a novel and highly effective inhibitor of FBN1, thereby inhibiting the massive proteinuria caused by podocyte damage and the massive extracellular matrix production caused by mesangial cell activation in the progression of CKD, delaying or / and reversing the progression of chronic glomerular disease.

[0005] The first aspect of the present invention is to provide the use of evodiamine or a pharmaceutically acceptable salt thereof in the inhibition of fibrin-1 expression or in the preparation of fibrin-1 inhibitors.

[0006] A second aspect of the present invention aims to provide the use of evodiamine or a pharmaceutically acceptable salt thereof as a fibrin-1 inhibitor in the preparation of medicaments for the treatment or prevention of chronic kidney disease.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the invention provides the use of evodiamine or a pharmaceutically acceptable salt thereof in the inhibition of fibrin-1 activity or in the preparation of fibrin-1 inhibitors.

[0008] In some embodiments of the present invention, the pharmaceutically acceptable salt includes organic acid salts or inorganic acid salts.

[0009] In some embodiments of the present invention, the organic acid salts include acetate, maleate, fumarate, tartrate, succinate, lactate, p-toluenesulfonate, salicylate, or oxalate.

[0010] In some embodiments of the present invention, the inorganic acid salts include hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, or nitrate.

[0011] A second aspect of the invention provides the use of evodiamine or a pharmaceutically acceptable salt thereof as a fibrin-1 inhibitor in the preparation of a medicament for the treatment or prevention of chronic kidney disease.

[0012] In some embodiments of the present invention, the pharmaceutically acceptable salt includes organic acid salts or inorganic acid salts.

[0013] In some embodiments of the present invention, the organic acid salts include acetate, maleate, fumarate, tartrate, succinate, lactate, p-toluenesulfonate, salicylate, or oxalate.

[0014] In some embodiments of the present invention, the inorganic acid salts include hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, or nitrate.

[0015] In some embodiments of the present invention, the chronic kidney disease includes kidney damage or renal fibrosis (such as glomerular fibrosis).

[0016] In some embodiments of the present invention, the chronic kidney disease includes glomerular injury kidney disease, podocyte injury kidney disease, glomerular basement membrane injury kidney disease, tubular kidney disease, or interstitial kidney disease.

[0017] In some embodiments of the present invention, the drug reduces the expression of urinary albumin and glomerular collagen deposition in patients with chronic kidney disease; and / or inhibits the activation of glomerular mesangial cells (e.g., inhibiting the expression of glomerular α-SMA and PDGFR-β).

[0018] In some embodiments of the present invention, the drug improves podocyte damage in patients with chronic kidney disease (e.g., improves the expression of nephrin and podocin in the glomeruli); and / or inhibits the activation of the EGFR signaling pathway (e.g., inhibits the expression of p-EGFR, p-PI3K, and p-AKT).

[0019] In some embodiments of the present invention, the effective therapeutic dose of evodiamine or a pharmaceutically acceptable salt thereof in the drug is 30-60 mg / kg (subject); further, 30-50 mg / kg (subject); and even further, 40 mg / kg (subject).

[0020] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0021] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, lubricants, binders, humectants, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0022] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose, etc.; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin, or polyvinylpyrrolidone, etc.; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, microcrystalline silica, talc, or magnesium stearate; and the suspending agent is selected from microcrystalline silica, beeswax, cellulose, solid polyethylene glycol, etc. Alcohol: The wetting agent is selected from glycerin, Tween-80, hydrogenated castor oil, or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, or vegetable oil, wherein the vegetable oil is selected from soybean oil, castor oil, peanut oil, blended oil, etc.; the surfactant is selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan, or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavoring, stevia, acesulfame potassium, citric acid, or sodium saccharin; the preservative is selected from at least one of methylparaben or propylparaben.

[0023] In some embodiments of the present invention, the dosage form of the drug is selected from tablets (such as ordinary tablets, bilayer tablets, multilayer tablets, sustained-release tablets, single-compartment controlled-release tablets, dual-compartment controlled-release tablets, microporous controlled-release tablets, sublingual tablets, orally disintegrating tablets, dispersible tablets, enteric-coated tablets), pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing microcapsules or small pieces, pH-dependent capsules containing microcapsules or small pieces, gastrointestinal compound capsules), suppositories, injections (such as injection solutions, solutions for injection, injection solutions for intravenous drip, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.), sprays, and injections.

[0024] The beneficial effects of this invention are: This invention conducted molecular interaction experiments and cell phenotype experiments on Evodiamine. The results showed that Evodiamine can act as a small molecule inhibitor of FBN1 and can effectively inhibit the FBN1-induced mesangial cell fibrosis phenotype.

[0025] Further experiments were conducted using ADR and 5 / 6NX models. Results showed that compared to the ADR and 5 / 6NX model groups, the Evodiamine gavage group exhibited significantly lower urinary albumin, reduced expression of mesangial cell activation markers, significantly reduced glomerular interstitial collagen deposition, and restored podocyte marker expression levels. This indicates that Evodiamine effectively inhibits glomerular podocyte injury, mesangial cell activation, and glomerulosclerosis in both ADR and 5 / 6NX models. Furthermore, Evodiamine effectively inhibited the EGFR signaling pathway in both ADR and 5 / 6NX models, suggesting that it inhibits mesangial cell fibrosis and podocyte injury while also antagonizing the EGFR signaling pathway. Simultaneously, Evodiamine showed no significant toxic side effects in mouse experiments. These results demonstrate that Evodiamine significantly inhibits podocyte injury, mesangial cell activation, and the progression of glomerulosclerosis without significant toxic side effects, making it suitable for developing drugs that effectively inhibit the progression of glomerular disease (CKD). Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1Figures show the molecular interaction experiments, surface plasmon resonance (SPR) results, and Western blot results of Evodiamine and FBN1. In Figure A, the molecular docking experiment results of Evodiamine and FBN1 are shown; blue represents the protein structure of FBN1, and pink represents the chemical structure of Evodiamine. Figure B shows the surface plasmon resonance (SPR) results of Evodiamine and FBN1. Figure C shows the Western blot results of Evodiamine inhibiting FBN1-induced mesangial cell fibrosis in vitro. Figure D is a statistical graph of the results in Figure C, where ** represents... P <0.01.

[0027] Figure 2 The figures show the urinary albumin to creatinine ratio (A) and kidney Masson and PAS staining (B) of mice in the doxorubicin mouse model. In this figure, Ctrl represents the sham treatment group; ADR represents the model control group; and ADR + EVO represents the drug-treated group. ** represents... P <0.01, scale bar is 25μM.

[0028] Figure 3 Immunofluorescence staining images to detect platelet-derived growth factor receptor β (PDGFR-β) and smooth muscle actin α (α-SMA), indicators of mesangial cell activation in a doxorubicin mouse model. In this figure, Ctrl represents the sham treatment group; ADR represents the model control group; and ADR +EVO represents the drug-treated group. The scale bar is 25 μM.

[0029] Figure 4 Immunostaining images of nephrin and podocin, podocyte markers, in various groups of the doxorubicin mouse model. In this image, Ctrl represents the sham treatment group; ADR represents the model control group; and ADR +EVO represents the drug-treated group. The scale bar is 25 μM.

[0030] Figure 5 Immunoblotting results of the EGFR signaling pathway in each group of mice in the ADR model are shown. In the figure, A represents the immunoblotting results of the EGFR signaling pathway in each group of mice; B represents the statistical results of A. In this figure, Ctrl represents the sham treatment group; ADR represents the model control group; and ADR +EVO represents the drug-treated group. **Represents... P <0.01.

[0031] Figure 6 This image shows the urinary albumin to creatinine ratio (A) and Masson and PAS staining of the kidneys in mice of the 5 / 6NX nephrectomy model (B). In this image, Ctrl represents the sham treatment group; 5 / 6NX represents the model control group; and 5 / 6NX +EVO represents the drug-treated group. **Representative P <0.01.

[0032] Figure 7 This is an immunofluorescence staining image of platelet-derived growth factor receptor β (PDGFR-β) and smooth muscle actin α (α-SMA), indicators of mesangial cell activation in different groups of mice in a 5 / 6NX nephrectomy model. In this image, Ctrl represents the sham treatment group; 5 / 6NX represents the model control group; and 5 / 6NX +EVO represents the drug treatment group. The scale bar is 25 μM.

[0033] Figure 8 This is an immunostaining image of nephrin and podocin, markers of podocytes, in different groups of mice in a 5 / 6NX nephrectomy model. In this image, Ctrl represents the sham treatment group; 5 / 6NX represents the model control group; and 5 / 6NX +EVO represents the drug treatment group. The scale bar is 25 μM.

[0034] Figure 9 This is an immunoblotting map of the EGFR signaling pathway in each group of mice in a 5 / 6NX nephrectomy model. In the figure, A represents the immunoblotting results of the EGFR signaling pathway in each group of mice, and B is a statistical graph of the results in A. In this figure, Ctrl represents the sham treatment group; 5 / 6NX represents the model control group; and 5 / 6NX +EVO represents the treatment group. *** represents... P <0.001.

[0035] Figure 10 This figure shows the drug safety results of evodiamine in mice, including serum functional indicators and HE staining images of organs from each group of mice. In this figure, ns represents no significant difference. The scale bar is 100 μM. Detailed Implementation The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0038] Evodiamine, CAS: 518-17-2; Molecular formula C 19 H 17 N3O; molecular weight is 303.4, molecular structure is as follows: .

[0039] The evodiamine used in the examples was purchased from Chengdu Purifa Technology Development Co., Ltd.

[0040] Example 1: Evodiamine is a novel inhibitor of FBN1 Molecular docking, surface plasmon resonance, and cell experiments were used to investigate whether Evodiamine can be used as an inhibitor of FBN1, as detailed below: Molecular docking: The 3D structure of FBN1 was downloaded from the PDB website (https: / / www2.rcsb.org / ), and the 3D structure of Evodiamine was downloaded from the PubChem website (https: / / pubchem.ncbi.nlm.nih.gov / ). The docking was performed using Discovery Studio, and the results were analyzed and displayed using PyMOL.

[0041] Surface plasmon resonance: The protein chip was pretreated with recombinant FBN1 protein. After chip preparation, Evodiamine was diluted to different concentration gradients using 1XPBS. The protein chip, different concentrations of Evodiamine, and regeneration solution were then placed in a biomolecular interaction instrument (PlexArray HT A100) for analysis. Signal changes in the binding of different concentrations of Evodiamine to the protein chip were detected. Finally, the corresponding binding and dissociation curves of Evodiamine and FBN1 were obtained, and the results were analyzed and displayed using GraphadPrism 9.0.

[0042] Cellular experiments: Rat glomerular mesangial cells (HBZY-1) were purchased from the China Type Culture Collection Center (Wuhan, China). HBZY-1 cells were stimulated with recombinant protein FBN1 (100 ng / mL) (10224-FI, R&D Systems), followed by incubation with different concentrations of Evodiamine (0.05 μM, 0.1 μM, and 0.2 μM) with or without the addition of HBZY-1. After incubation for 48 hours, HBZY-1 lysates were collected, and the expression levels of fibrosis-related markers in HBZY-1 cells from different treatment groups were detected by Western blot.

[0043] Experimental results 1) There is a direct binding between Evodiamine and FBN1. like Figure 1As shown in Figure A, molecular docking results demonstrate hydrogen bonding between FBN1 and Evodiamine, suggesting that Evodiamine may be a novel inhibitor of FBN1. Surface plasmon resonance experiments revealed a concentration-dependent binding and dissociation process of Evodiamine to FBN1. Figure 1 (B) indicates a direct binding between Evodiamine and FBN1.

[0044] 2) Evodiamne can inhibit FBN1-induced mesangial cell fibrosis. like Figure 1 As shown in Figure C, the results of Western blot suggest that Evodiamine can inhibit the expression of FBN1-induced mesangial cell fibrosis-related proteins, such as COL4A1, FN, and α-SMA. Figure 1 D in the middle is a statistical graph of the Western blot results.

[0045] Example 2: Inhibition of glomerular fibrosis in ADR mice by evodiamine The doxorubicin mouse model (ADR) is a classic animal model of focal segmental glomerulosclerosis (FSGS). The ADR model is characterized by rapidly and completely demonstrating different pathological stages and features of glomerular injury and renal fibrosis through induced oxidative stress damage to podocytes. These stages include podocyte foot process fusion, proteinuria, mesangial cell activation and matrix proliferation, segmental glomerular sclerosis, extensive tubulointerstitial fibrosis, and renal failure. Pathologically, Masson and PAS staining are commonly used to observe glomerular collagen deposition and sclerosis. More specific glomerular fibrosis and podocyte injury are often qualitatively and quantitatively observed and evaluated using immunostaining, Western blotting, and urine albumin kits for markers of mesangial cell activation such as smooth muscle actin α (α-SMA), platelet-derived growth factor receptor β (PDGFR-β), and podocyte markers nephrin and podocin. This example uses the doxorubicin mouse model to investigate the role of evodiamine in glomerular fibrosis, as detailed below: 1. Experimental animals: Male BALB / c mice, weighing 20–22g, SPF grade. The animals were weighed and numbered. Eighteen healthy mice weighing 20–22g were selected and randomly divided into three groups of six each. These included a sham treatment group, a model control group, and a drug treatment group.

[0046] 2. Processing of each group 1) Model control group: On the day of modeling, accurately weigh each mouse and calculate the corresponding dose of adriamycin (ADR). The dose of adriamycin injected into the mice was 12 mg / kg. Beforehand, place the mice in a constant temperature metal heater, set the temperature to 38℃, and heat for about 10 minutes. Then, place the mice in a restraint device, ensuring unobstructed breathing, straighten their tail veins and disinfect them with alcohol swabs. Smoothly inject the adriamycin into the mouse's tail vein, remove the needle, press a small amount with a disposable cotton ball, verify the marking, and place them in the appropriate cage.

[0047] 2) Treatment group: Same as 1). Weigh the mice, administer doxorubicin, verify the label, and place them in the appropriate cages. On day 8 after the tail vein injection of doxorubicin, administer 40 mg / kg / day of Evodiamine by gavage for 14 consecutive days.

[0048] 3) Sham treatment group: Same as 1) Weigh the mice and give them an equal volume of physiological saline (operation as above), verify the label, and place them in the corresponding cages.

[0049] 3. Experimental Procedure Evodiamine was dissolved in DMSO to prepare a 10 mg / mL stock solution, which was then diluted to 1 mg / mL with a PEG300:Tween-80:physiological saline:drug ratio of 8:1:9:2. Mice in each group were housed in cages, and urine samples were collected at week 3. The sham treatment group and the model control group received only oral gavage with control solutions (10% DMSO, 40% PEG300, 5% Tween-80, and 45% physiological saline). The drug-treated group received oral gavage with a solution containing 40 mg / kg body weight of Evodiamine. After 3 weeks of housing, all mice were euthanized, and both kidneys were harvested and fixed in 10% neutral buffered formaldehyde and then cryopreserved in liquid nitrogen. After dehydration, embedding, sectioning, and slide preparation, formaldehyde-fixed tissues were stained with Masson and PAS, and immunostained with α-SMA, PDGFR-β, nephrin, and podocin, respectively. The expression of the EGFR signaling pathway and the ratio of urinary albumin to urinary creatinine (mouse urinary albumin kit, G-AEFI00544.96, Bethyl Laboratories; mouse urinary creatinine kit, AU480, Beckman Coulter) were detected.

[0050] 4. Experimental Results 1) Evodiamine reduces urinary albumin levels and glomerular collagen deposition in ADR mice. Experimental results are as follows Figure 2 As shown, the ratio of urinary albumin to urinary creatinine in the drug-treated mice was [data missing]. Figure 2 (A) and glomerular collagen deposition ( Figure 2The ratio of B in the model was significantly lower than that in the control group.

[0051] 2) Evodiamine inhibits mesangial cell activation in ADR mice Experimental results are as follows Figure 3 As shown, compared with the model control group, the levels of α-SMA and PDGFR-β in the glomeruli of mice in the drug-treated group were significantly reduced.

[0052] 3) Evodiamine inhibits podocyte damage in ADR mice. Experimental results are as follows Figure 4 As shown, compared with the model control group, the levels of nephrin and podocin in the glomeruli of mice in the drug-treated group were significantly restored.

[0053] 4) Evodiamine reduces the activation of the EGFR signaling pathway in ADR mice. Experimental results are as follows Figure 5 As shown, compared with the model control group, the activation level of the EGFR signaling pathway in the kidney tissue of mice in the drug-treated group was significantly reduced.

[0054] The above experimental results indicate that evodiamine can significantly reduce glomerular collagen deposition in ADR mice, significantly decrease urinary albumin levels and glomerular mesangial cell activation markers α-SMA and PDGFR-β, restore the expression levels of podocyte markers nephrin and podocin, and significantly inhibit the abnormally activated EGFR signaling pathway in CKD models. Therefore, evodiamine has a good inhibitory effect on glomerular fibrosis in ADR mice and can be considered a new drug for effectively inhibiting CKD progression.

[0055] Example 3: Inhibition of glomerular fibrosis in 5 / 6NX mice by evodiamine The 5 / 6NX nephrectomy model (5 / 6NX) is a commonly used animal model of chronic kidney disease, characterized by significant progressive decline in renal function and its complications due to the loss of numerous healthy nephrons. These complications are mainly manifested as a significant increase in serum creatinine and blood urea nitrogen, glomerular sclerosis, and tubulointerstitial fibrosis. This example uses the 5 / 6NX nephrectomy model to investigate the inhibitory effect of evodiamine on glomerular fibrosis. Details are as follows: 1. Experimental animals: CD-1 mice, male, weighing 25–30g, SPF grade. The animals were weighed and numbered. Eighteen healthy mice weighing 25–30g were selected and randomly divided into three groups of six each. These included a sham treatment group, a model control group, and a drug treatment group.

[0056] 2. Processing of each group 1) Model control group: Mice were anesthetized at room temperature with 50 mg / kg of 1% sodium pentobarbital. The mice were placed in a dorsal position. After confirming the basic location of the left kidney, the skin was prepared and disinfected. A 1.0 cm incision was made in the skin to expose the fascia layer and the left kidney. The kidney was squeezed out and covered with a sterile drape. The renal capsule was peeled off, and the upper and lower poles of the kidney were quickly removed with a scalpel. Disposable gelatin sponges were quickly placed at both wound sites to stop bleeding. After removing the drape, the remaining 1 / 3 of the kidney, along with the gelatin sponges, was placed into the abdominal cavity and sutured layer by layer. On day 8, the mice were placed in the dorsal position as before. After preparation and disinfection, the right kidney was squeezed out, and the perirenal fat, connective tissue, and renal capsule were peeled off. The renal pedicle was tightly ligated with surgical sutures, and the right kidney was completely removed with tissue scissors and then sutured layer by layer.

[0057] 2) Sham treatment group: The operation time was the same as the model control group, but only the skin, subcutaneous tissue, muscle layer and peritoneum were cut, and the kidneys were not treated.

[0058] 3) Medication group: The operation time was the same as the model control group. Five weeks after complete removal of the right nephrology, Evodiamine was administered by gavage at a dose of 40 mg / kg / d.

[0059] 3. Experimental Procedure Mice in each group were housed and treated in cages. Urine samples were collected at week 8. After 8 weeks of housing, mice in each group were euthanized, and the left kidneys of all mice were harvested. The kidneys were fixed in 10% neutral buffered formaldehyde and then frozen in liquid nitrogen. After dehydration, embedding, sectioning, and slide preparation, the formaldehyde-fixed tissues were stained with Masson and PAS, and immunostained with α-SMA, PDGFR-β, nephrin, and podocin. The expression of the EGFR signaling pathway and the level of urinary albumin were also detected (experimental procedure as in Example 2).

[0060] 4. Experimental Results 1) Evodiamine reduced urinary albumin to creatinine levels and glomerular collagen deposition in 5 / 6NX mice. Experimental results are as follows Figure 6 As shown, the urinary albumin level and glomerular collagen deposition in the drug-treated mice were significantly lower than those in the model control group.

[0061] 2) Evodiamine inhibits the activation of mesangial cells in 5 / 6NX mice. Experimental results are as follows Figure 7 As shown, compared with the model control group, the levels of α-SMA and PDGFR-β in the glomeruli of mice in the drug-treated group were significantly reduced.

[0062] 3) Evodiamine inhibits damage to podocytes in 5 / 6NX mice. Experimental results are as follows Figure 8 As shown, compared with the model control group, the levels of nephrin and podocin in the glomeruli of mice in the drug-treated group were significantly restored.

[0063] 4) Evodiamine reduces the activation of the EGFR signaling pathway in 5 / 6NX mice. Experimental results are as follows Figure 9 As shown, compared with the model control group, the activation level of the EGFR signaling pathway in the kidney tissue of mice in the drug-treated group was significantly reduced.

[0064] The above results indicate that Evodiamine can significantly reduce glomerular collagen deposition in 5 / 6NX mice, significantly decrease urinary albumin levels and glomerular mesangial cell activation markers α-SMA and PDGFR-β, restore the expression levels of podocyte markers nephrin and podocin, and significantly inhibit the abnormally activated EGFR signaling pathway in CKD models. Therefore, Evodiamine could be a novel drug for effectively inhibiting CKD progression.

[0065] Example 4: Evodiamine showed no significant toxic side effects in mice. 1. Experimental animals: Male BALB / c mice, weighing 20-22g, SPF grade. The animals were weighed and numbered. Eighteen healthy mice weighing 20-22g were selected and randomly divided into two groups of six each: a sham treatment group and a drug treatment group.

[0066] 2. Processing of each group 1) Drug treatment group: Each mouse was administered Evodiamine by gavage at a dose of 40 mg / kg / d for 2 weeks, after which the mice were sacrificed, and serum and tissues from the heart, liver, spleen, lungs and kidneys were collected for instrumental analysis and HE staining.

[0067] 2) Sham treatment group: Mice were administered an equal volume of physiological saline by gavage daily for 2 weeks, after which the mice were euthanized. Serum and tissues from the heart, liver, spleen, lungs and kidneys were collected for analysis of various functional serum indicators (Beckman Council AU480 fully automated biochemical analyzer; all kits were purchased from Beckman Council) and HE staining of each organ tissue.

[0068] 3. Experimental Results 1) Evodiamine does not cause changes in serum functional markers in mice. Experimental results are as follows Figure 10 As shown, the renal function, liver function, fatty acid metabolism, bilirubin metabolism, and serum albumin of the mice in the drug-treated group were not significantly different from those in the control group.

[0069] 2) Evodiamine does not cause morphological changes in any organ in mice. Experimental results are as follows Figure 10HE staining showed that, compared with the control group, the mice in the drug-treated group had no obvious morphological changes in the heart, lungs, liver, spleen and kidneys.

[0070] The above results indicate that Evodiamine, within its effective dose range, did not significantly alter any functional serological parameters in mice, nor did it cause any morphological changes in any of the mouse organs. Therefore, Evodiamine has no significant toxic side effects in mice.

[0071] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The use of evodiamine or a pharmaceutically acceptable salt thereof in the inhibition of fibrin-1 activity or in the preparation of fibrin-1 inhibitors.

2. The use of evodiamine or a pharmaceutically acceptable salt thereof as a fibrin-1 inhibitor in the preparation of drugs for the treatment or prevention of chronic kidney disease.

3. The application according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts include organic acid salts or inorganic acid salts.

4. The application according to claim 3, characterized in that, The organic acid salts include acetate, maleate, fumarate, tartrate, succinate, lactate, p-toluenesulfonate, salicylate, or oxalate; the inorganic acid salts include hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, or nitrate.

5. The application according to claim 2, characterized in that, The chronic kidney disease includes kidney damage or kidney fibrosis.

6. The application according to claim 2, characterized in that, The chronic kidney disease includes glomerular injury kidney disease, podocyte injury kidney disease, glomerular basement membrane injury kidney disease, tubular kidney disease, or interstitial kidney disease.

7. The application according to claim 5 or 6, characterized in that, The drug reduces the expression of urinary albumin and glomerular collagen deposition in patients with chronic kidney disease; and / or inhibits the activation of glomerular mesangial cells.

8. The application according to claim 7, characterized in that, The drug improves podocyte damage in patients with chronic kidney disease; and / or inhibits the activation of the EGFR signaling pathway.

9. The application according to claim 8, characterized in that, The effective therapeutic dose of evodiamine or its pharmaceutically acceptable salt in the drug is 30–60 mg / kg.

10. The application according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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