Application of nintedanib in preparation of medicine for treating migration plant mineralization

Abstract

The invention discloses application of nintedanib in preparation of a medicine for treating plant migration and plant mineralization, and belongs to the technical field of biological medicine. The research shows that the nintedanib has the effects of reducing deposition of extracellular matrix of an allogeneic kidney transplantation model mouse, improving renal tubular atrophy, relieving mouse CD206 + macrophage infiltration and reducing rejection injury degree and immune infiltration degree of kidney grafts after being administered under the administration dosage without generating liver and kidney toxicity and intestinal side effects; the lesion process of mouse chronic transplanted kidney rejection is obviously slowed down, and the damage of excessively activated inflammatory cells to the transplanted kidney is reduced. The results show that nintedanib can delay the progress of chronic transplantation renal fibrosis.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to the application of nintedanib in the preparation of drugs for treating graft fibrosis. Background Technology

[0002] Kidney transplantation is the best treatment for end-stage renal disease. In recent years, with the improvement of medical standards, including the development of transplantation surgery techniques and the combined use of new immunosuppressants, the short-term survival rate of transplanted kidneys has improved, but the long-term survival rate has not yet improved significantly.

[0003] Kidney fibrosis after transplantation is the leading cause of long-term transplant kidney failure. Studies have shown that approximately 40% of patients develop transplant interstitial fibrosis (IF) 3-6 months after kidney transplantation, and about 65% of patients develop IF 2 years later. The etiology of transplant kidney fibrosis is not fully understood, including theories such as caveolin-1 and SHROOM3 gene polymorphisms driving fibrosis formation, interleukin (IL)-33 potentially influencing fibrosis formation through multiple pathways, aldosterone playing an important role in promoting interstitial fibrosis, and tacrolimus inducing fibrosis.

[0004] Currently, there are no effective targeted drugs to slow the progression of transplanted kidney failure (IF). Clinically, the only way to prolong transplant survival is through the appropriate combination of immunosuppressants and the use of other drugs to minimize drug-induced damage. The lack of targeted drugs for IF in transplanted kidneys means that some patients inevitably experience recurrent kidney failure after their initial transplant, necessitating a second transplant. This undoubtedly increases the economic, psychological, and physiological burden on transplant patients. Furthermore, due to the extreme shortage of kidney donors, inhibiting the progression of IF in transplanted kidneys and extending their lifespan is a crucial issue that urgently needs to be addressed in the field of kidney transplantation.

[0005] Nintedanib (BIBF1120) is a multi-PTK inhibitor that blocks PDGFR, VEGER, FGFR, and Src family kinases. It has an anti-fibrotic effect on idiopathic pulmonary fibrosis and is currently approved by the FDA for the treatment of idiopathic pulmonary fibrosis. For example, it is marketed under the brand name Ofev, and its main ingredient is nintedanib ethoxylate soft capsules (generic name). Idiopathic pulmonary fibrosis begins with damage to alveolar epithelial cells. Damaged epithelial cells secrete cytokines and chemokines, attracting inflammatory cell aggregation, releasing pro-fibrotic factors, activating lung fibroblasts to transform into myofibroblasts, and synthesizing large amounts of extracellular matrix, thus initiating fibrosis. In animal models of pulmonary fibrosis induced by bleomycin or silica particles, nintedanib can inhibit extracellular matrix deposition and reduce the transdifferentiation of fibroblasts into myofibroblasts.

[0006] However, no studies have reported the pharmacological effects of nintedanib in chronic transplant kidney fibrosis, either in animal models or clinical trials. Summary of the Invention

[0007] 1. Purpose of the invention

[0008] The purpose of this application is to provide the use of nintedanib in the preparation of drugs for the treatment of graft fibrosis, especially transplanted kidney fibrosis. The applicant's research found that nintedanib can be used to treat allogeneic chronic transplanted kidney fibrosis.

[0009] 2. Technical Solution To achieve the aforementioned objectives, the technical solution adopted in this application is as follows: This application provides the use of nintedanib or its pharmaceutically acceptable salts, acids, esters, and pharmaceutical compositions thereof in the preparation of medicaments for the prevention or treatment of graft fibrosis.

[0010] Furthermore, the aforementioned graft fibrosis is chronic graft fibrosis.

[0011] Furthermore, the aforementioned graft fibrosis includes fibrosis of transplanted kidneys, transplanted livers, transplanted hearts, transplanted lungs, transplanted pancreas, and / or transplanted small intestines.

[0012] Furthermore, the aforementioned graft fibrosis refers to transplanted kidney fibrosis. As a further explanation of this application, this application found that nintedanib administration to allogeneic kidney transplant model mice can alleviate renal tubular atrophy and reduce extracellular fibrous matrix deposition in the transplanted kidney; simultaneously, it can reduce inflammatory infiltration of immune cells and macrophages in the transplanted kidney, and decrease the transdifferentiation rate of CD68+CD206+ double-positive macrophages. Furthermore, serum creatinine and blood urea nitrogen measurements showed that nintedanib improved transplanted kidney function. Even further, no hepatotoxicity was observed at the dosage concentrations that achieved the above therapeutic effects.

[0013] Furthermore, the aforementioned transplanted kidney fibrosis is allogeneic chronic transplanted kidney fibrosis.

[0014] Furthermore, the aforementioned nintedanib or its pharmaceutically acceptable salts, acids, esters, and pharmaceutical compositions thereof include nintedanib ethanesulfonate.

[0015] Furthermore, the dosage forms of the aforementioned drugs include liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms. As a further explanation of this application, this application does not limit the specific dosage form of the drug; the specific dosage form of a drug has theoretical diversity, and its final dosage form design and corresponding pharmaceutical excipient selection need to be determined based on actual research and development and production needs.

[0016] Furthermore, the aforementioned liquid dosage forms include solutions, injections, etc.

[0017] Furthermore, the aforementioned gaseous dosage forms include aerosols, sprays, etc.

[0018] Furthermore, the aforementioned solid dosage forms include powders, tablets, etc.

[0019] Furthermore, the aforementioned semi-solid dosage forms include ointments, pastes, etc.

[0020] Furthermore, the above-mentioned solution is an oral solution.

[0021] Furthermore, the solvent for the above-mentioned oral solution includes polyethylene glycol (PEG300).

[0022] 3. Technical Effects Compared with the prior art, the advantages of this application are as follows: This application discloses the use of nintedanib in the preparation of drugs for treating chronic graft fibrosis, particularly transplanted kidney fibrosis. Studies in this application show that, at doses without hepatotoxicity, nephrotoxicity, or intestinal side effects, nintedanib reduces extracellular matrix deposition in allogeneic kidney transplant model mice, significantly slows the progression of transplanted kidney lesions in mice with chronic rejection, inhibits the allogeneic immune response of mouse macrophages, reduces the degree of rejection damage and immune infiltration in kidney transplants, reduces inflammatory foci, decreases damage to the transplanted kidney from overactivated inflammatory cells, and alleviates inflammation. These results indicate that nintedanib can delay the progression of chronic transplanted kidney fibrosis and can be used as a novel immunosuppressant for treating transplanted immune rejection of kidneys, especially inhibiting chronic fibrotic immune rejection of the kidneys. Attached Figure Description

[0023] Figure 1 HE and PAS staining results for the SYN group, CAN group, CAN solubilizer intervention group, CAN nintedanib 30 mg / kg / d group, CAN nintedanib 60 mg / kg / d group and CAN nintedanib 90 mg / kg / d group, as well as statistical bar charts comparing Banff scores for interstitial fibrosis (ci) and renal tubular atrophy (ct).

[0024] Figure 2 Representative immunohistochemical results and semi-quantitative analysis of collagen I in the SYN group, CAN group, CAN solubilizer intervention group, CAN nintedanib 30 mg / kg / d group, CAN nintedanib 60 mg / kg / d group and CAN nintedanib 90 mg / kg / d group.

[0025] Figure 3Bar charts showing creatinine and blood urea nitrogen monitoring in the SYN group, CAN group, CAN solubilizer intervention group, CAN nintedanib 30 mg / kg / d group, CAN nintedanib 60 mg / kg / d group, and CAN nintedanib 90 mg / kg / d group.

[0026] Figure 4 HE staining of the livers of the SYN group, CAN group, CAN solubilizer intervention group, CAN nintedanib 30 mg / kg / d group, CAN nintedanib 60 mg / kg / d group and CAN nintedanib 90 mg / kg / d group.

[0027] Figure 5 Bar charts showing gastrointestinal transit time and fecal water content in mice in the SYN group, CAN group, CAN solubilizer intervention group, CAN nintedanib 30 mg / kg / d group, CAN nintedanib 60 mg / kg / d group and CAN nintedanib 90 mg / kg / d group.

[0028] Figure 6 Representative images of CD68 / CD206 co-staining in the SYN group, CAN group, CAN solubilizer intervention group, CAN nintedanib 30 mg / kg / d group, CAN nintedanib 60 mg / kg / d group, and CAN nintedanib 90 mg / kg / d group, along with a bar chart comparing the percentage of CD68+CD206+ co-stained positive cells. Detailed Implementation

[0029] The present application will be further described below with reference to specific embodiments.

[0030] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0031] 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.

[0032] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0033] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0034] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0035] Unless otherwise specified in this application, data analysis shall be performed in accordance with the following methods: Continuous variables were expressed as mean ± standard deviation (SD), and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant. Data analysis and graphing were performed using GraphPad Prism 10.1.2 software. Immunohistochemistry and immunofluorescence slides involved in the experiment were compared and semi-quantitatively analyzed using Caseviewer 2.0 and ImageJ. All HE, PAS, and immunohistochemical slides were examined under an optical microscope by two senior pathologists unaware of the grouping information. Interstitial fibrosis (ci) and renal tubular atrophy (ct) were semi-quantitatively scored (0–3 points) strictly according to the Banff 2019 criteria. The average score of the two experts was taken as the Banff score for the sample, and pathological damage was scored on HE-stained liver slides. Quantitative analysis of immunofluorescence images was performed using ImageJ software. Five slides were randomly selected from each sample. Figure 6 The total number of cells in the field of view shown was determined based on DAPI-labeled nuclear signals, along with the number of single-positive and double-positive cells. All counts were performed under blinded conditions, and the results are expressed as the percentage of positive cells out of the total number of cells.

[0036] In this application, nintedanib (CAS: 656247-17-5) was purchased from a biological reagent supplier under the brand name Aladdin, catalog number N129725. For use, nintedanib was dissolved in a solvent prepared from polyethylene glycol (PEG300) and physiological saline (in a 1:1 volume ratio) to prepare a 10 mg / mL suspension for gavage. The polyethylene glycol (CAS No: 25322-68-3) was purchased from a biological reagent supplier under the brand name KKL Med Inc, catalog number KM12352. The tacrolimus reagent used to construct the chronic transplant renal fibrosis model was purchased from a biological reagent supplier under the brand name Selleck, catalog number S500317.

[0037] Example 1 This embodiment provides the application of nintedanib in the preparation of a drug for treating chronic renal fibrosis in allogeneic transplant recipients.

[0038] (1) Establishment of mouse kidney transplantation model In this embodiment, male purebred BALB / c mice, weighing 20-25 g, were purchased from the Experimental Animal Center of Nanjing Medical University; male purebred C57BL / 6 background mice, weighing 20-25 g, were also purchased from the Experimental Animal Center of Nanjing Medical University.

[0039] Using the established mouse kidney transplantation model, BALB / c wild-type mice were selected as kidney donors, and C57BL / 6 wild-type mice as recipients. To avoid interference from sex factors, all mice used in the experiment were male. In the SYN group, both donors and recipients were C57BL / 6 mice.

[0040] Donor surgery: The left kidney of a BALB / c wild-type mouse was used as the donor kidney. During the operation, the donor kidney was perfused with 4°C hypertonic citrate purine solution through the abdominal aorta using a syringe until the left renal vein was filled with the perfusion fluid. After the kidney turned white, it was ligated at the junction of the left renal vein and the inferior vena cava. Subsequently, the renal vein was transected to the right of the ligation site, the renal artery was trimmed, and the kidney, blood vessels, and ureter were fully freed and preserved in 4°C citrate purine solution.

[0041] Recipient surgery: After receiving C57BL / 6 anesthesia, the right kidney of the recipient mouse was transcribed along the midline of the abdomen. The suture site of the recipient's abdominal aorta and inferior vena cava was separated, with the upper and lower edges of the suture site and the branch vessels between them encircled by 9-0 atraumatic sutures for subsequent blood flow occlusion. After occlusion, the renal artery and vein were anastomosed end-to-end with 11-0 atraumatic sutures within 45 minutes; ice chips were used during the anastomosis to prevent the donor kidney from overheating. After the anastomosis was completed and checked for leakage, renal blood flow was restored. Within seconds, the transplanted kidney parenchyma became bright red, uniform, and clear, the renal vein was full, the anastomosis was full, and the renal artery pulsation was obvious. Clear urine was seen flowing from the ureter within 3-5 minutes. The ureter was repositioned, and the donor and recipient ureters were intermittently anastomosed with 9-0 atraumatic sutures, followed by the transection of the recipient's left kidney.

[0042] Postoperative management: After abdominal closure, mice were subcutaneously injected with cefazolin sodium (0.2 g / kg) and placed on heated cages for resuscitation. From the first postoperative day, tacrolimus dissolved in corn oil was administered intraperitoneally at a dose of 1 mg / kg / day for 14 consecutive days. Successful model establishment was considered achieved when mice could survive solely on the transplanted kidney. With the completion of tacrolimus administration, the initial model establishment was complete, and subsequent experimental interventions could be performed.

[0043] (2) Group intervention The recipient mice were randomly divided into 6 groups, and the groups and treatments are as follows: SYN: Both donor and recipient mice are C57BL / 6 wild-type mice; CAN: No drug intervention was performed after the above transplantation model was constructed; CAN vehicle control: After the above transplantation model was established, the patient was treated with 200 μL / d of the prepared nintedanib solubilizer (PEG300: sterile saline volume ratio 1:1). CAN (30 mg / kg / d nintedanib): Nintedanib was administered by gavage at a dose of 30 mg / kg / d after the transplantation model was established. CAN (60 mg / kg / d nintedanib): Nintedanib was administered by gavage at a dose of 60 mg / kg / d after the transplantation model was established; CAN (90 mg / kg / d Nintedanib): After the transplantation model was established, nintedanib was administered by gavage at a dose of 90 mg / kg / d.

[0044] (3) Physiological and pathological analysis Sample Collection: Following the previously described dosing regimen, samples were collected after 8 weeks of tacrolimus administration. Mice were placed in a small animal anesthesia induction box and anesthesia was induced using isoflurane gas (3.5%). After the mice lost consciousness, they were transferred to a maintenance mask (1.5–2%). (i) Inferior vena cava blood collection method: The mice were fixed in a prone position with their abdomen elevated. The abdominal skin and peritoneum were cut open to expose the inferior vena cava. Using a sterile 1 mL syringe (with a 25G needle), the syringe was inserted at approximately a 30-degree angle along the course of the inferior vena cava, and 0.8–1.0 mL of whole blood was collected by slow aspiration. The collected whole blood was slowly injected into a 1.5 mL sterile centrifuge tube and left at room temperature at an angle for approximately 2 hours to allow it to coagulate naturally. Subsequently, the tube was centrifuged at 3000 rpm for 15 minutes in a 4°C centrifuge. After centrifugation, the supernatant (i.e., serum) was carefully aspirated using a 200 μL micropipette, avoiding aspiration of basal cells or flocculent precipitates. Serum was aliquoted into pre-chilled 200 μL centrifuge tubes (approximately 100 μL per tube) and immediately transferred to a -80°C cryogenic freezer for long-term storage for subsequent renal function testing. (ii) A section of the transplanted kidney, perpendicular to the longitudinal circumference of the kidney, was taken for pathological sectioning and immediately immersed in a sufficient volume (more than 10 times the volume of the tissue block) of 4% paraformaldehyde phosphate buffer (PFA, pH 7.4). Ensure the tissue is completely covered by the liquid. Fix at 4°C for 24 hours for subsequent paraffin embedding and pathological analysis.

[0045] (a) Histopathological analysis The primary antibodies COL-1, CD206, and CD68 used in this experiment were purchased from Cell Signaling Technology, with catalog numbers 72026T, 24595T, and 97778T, respectively; the secondary antibody kit was purchased from Absin Biotech, with catalog number abs50012-20T.

[0046] (i) Immunohistochemistry Paraffin-embedded kidney tissue sections were dewaxed, hydrated, had antigens removed, and blocked before undergoing immunohistochemical staining using a two-step ultrasensitive method. The specific steps are as follows: Slicing and baking: Place the paraffin slices in a baking machine and bake at 60~70°C for 30 minutes to promote full adhesion of the tissue to the glass slide.

[0047] Dewaxing and hydration: The sections were placed in xylene (twice, 10 min each time), 100%, 95%, 90%, 85%, and 75% ethanol (10 min each), and finally placed in distilled water for 5 min to complete the dewaxing and hydration process.

[0048] PBS washing: Wash the slides three times with 1×PBS solution for 5 min each time.

[0049] Antigen retrieval: Place the slides in sodium citrate buffer at pH 6.0, microwave on high until boiling, then reduce to medium heat and heat for 15 minutes. Remove the slides after the retrieval solution has cooled to room temperature.

[0050] PBS washing: Wash the slides three times with 1×PBS solution, 5 min each time.

[0051] Blocking endogenous peroxidase activity: The slices were placed in a 3% hydrogen peroxide (H2O2) solution and incubated at room temperature for 10 min to eliminate endogenous peroxidase activity.

[0052] PBS washing: Wash the slides three times with 1×PBS solution for 5 min each time.

[0053] Blocking nonspecific binding: Add 10% goat serum (same species as the secondary antibody) blocking solution and incubate at 37°C for 1 h.

[0054] Primary antibody incubation: Add an appropriate amount of primary antibody working solution (diluted according to the instructions) and incubate overnight at 4°C. Set up a negative control group (using PBS instead of primary antibody).

[0055] PBS washing: Wash the slides three times with 1×PBS solution for 5 min each time.

[0056] Secondary antibody incubation: Follow the instructions of the ultrasensitive two-step immunohistochemistry kit, add reagent 1 and reagent 2 in sequence, and incubate each at 37°C for 20 min.

[0057] PBS washing: Wash the slides three times with 1×PBS solution for 5 min each time.

[0058] DAB colorimetric reaction: Add DAB working solution and observe the colorimetric reaction under a microscope. When a brownish-yellow precipitate appears on the target tissue, immediately terminate the reaction with PBS solution.

[0059] Counterstaining: Counterstain with hematoxylin solution for 30 s to 1 min, adjusting the time as needed according to the staining depth.

[0060] Dehydration, clearing and mounting: After rinsing with tap water for 30 seconds, the sections were dehydrated in sequence with ethanol, cleared with xylene, and finally mounted with neutral resin.

[0061] (ii) HE staining Tissue specimens were fixed in 4% paraformaldehyde, routinely dehydrated, and embedded in paraffin, with sections 3–4 μm thick. After dewaxing in xylene and rehydration with graded ethanol, sections were stained with hematoxylin-eosin (HE). Hematoxylin-stained nuclei were rinsed with tap water, differentiated with hydrochloric acid alcohol, and then blued with alkaline water. Subsequently, cytoplasm was stained with eosin, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin.

[0062] (iii) PAS staining Paraffin sections were dewaxed with xylene and rehydrated with graded ethanol, then stained with periodic acid-Schiff (PAS) reaction. After cyclic acid oxidation, the sections were stained with Schiff's reagent, rinsed with tap water, counterstained with hematoxylin, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin.

[0063] (b) Measurement of serum creatinine and blood urea nitrogen Serum creatinine (Cr) was measured in mice using a creatine oxidase assay kit (microplate method) (purchased from Nanjing Jiancheng Bioengineering Institute, catalog number C011-2-1). Detection principle: Creatinine is converted to creatine by creatine amide hydrolase. Creatine is then hydrolyzed to creatine and urea by creatine aminohydrolase. Creatine is further catalyzed by creatine oxidase to produce glycine, formaldehyde, and hydrogen peroxide. Hydrogen peroxide reacts with 2,4-(6-triiodo-3-hydroxybenzoic acid) and 4-aminoantipyrine under the catalysis of peroxidase to form a purple-red compound. The compound can be detected colorimetrically at a wavelength of 546 nm.

[0064] (c) Immunofluorescence Dewaxing and rehydration: Immerse the sample twice in xylene, each time for 10 minutes; immerse the sample twice in anhydrous ethanol, each time for 5 minutes; immerse the sample in 95% ethanol for 5 minutes; immerse the sample in 75% ethanol for 5 minutes; immerse the sample in distilled water for 5 minutes.

[0065] Antigen retrieval: Prepare antigen retrieval solution (TDTA) with pure water at a ratio of 1:50; place the sample in the solution and heat at 95~98°C for 15~20 minutes (microwave / water bath are both acceptable); allow to cool naturally to room temperature (at least 30 minutes).

[0066] Permeabilization: Prepare with PBS + 0.2% Triton X-100; soak the sample in room temperature for 10 minutes; wash once with PBS solution.

[0067] Sealing: 5% BSA sealed at room temperature for 1 hour.

[0068] CD68 primary antibody incubation (overnight): CD68 primary antibody is diluted at a ratio of 1:200 and used: incubate overnight (12~16 h) in a humidified chamber at 4°C.

[0069] Secondary antibody incubation: Wash 3 times with PBS, 10 minutes each time; incubate with universal rabbit secondary antibody for 30 minutes; wash 3 times with PBS, 10 minutes each time.

[0070] Fluorescent dye staining: TYR-520 Plus fluorescent dye was diluted 1:1; 20 μl of each slide was stained for 10 minutes; the slides were washed with PBS 3 times, 10 minutes each time.

[0071] Blocking: Block with blocking peroxidase solution for 10 minutes; then block with protein blocking solution for 10 minutes.

[0072] CD206 staining: The previous procedure of overnight incubation with primary antibody and secondary antibody followed by staining with fluorescent dye was repeated using CD206 primary antibody and TYR-570Plus fluorescent dye.

[0073] Nuclear staining: Wipe the area around the tissue with gauze; DAPI: PBS solution prepared at a ratio of 1:100 and stained for 30 seconds.

[0074] Mounting: After rinsing once with TBST solution, wipe dry and mount the slide.

[0075] (d) Mouse gastrointestinal function test (i) Whole intestinal transit time Mice were fasted for 12 hours prior to the experiment but had free access to water. On the day of the experiment, mice were placed individually in clean cages for 30 minutes to acclimatize. Each mouse was administered 0.2 mL of charcoal suspension via gavage. Timing began immediately after gavage. Mice defecation was continuously observed, and the time of the first passage of black feces was recorded. The time from gavage to the first passage of black feces was defined as the total intestinal transit time (min).

[0076] (ii) Fecal moisture content Mice were housed individually for 24 hours, and fresh feces were collected. The wet weight of the feces was immediately measured. The feces were then dried in a 60°C oven for 24 hours. The dry weight was measured again. The moisture content was calculated using the following formula: (wet weight - dry weight) / wet weight × 100%.

[0077] (4) Results Analysis Figure 1The upper and middle figures show the comparison of HE and PAS staining between the groups. It can be seen that after nintedanib intervention, the inflammatory infiltration of immune cells in the transplanted kidneys of mice was reduced. Based on the Banff 2019 semi-quantitative scoring system, the scores of interstitial fibrosis (ci) and renal tubular atrophy (ct) in the transplanted kidney tissue were significantly reduced, indicating that chronic structural injury was significantly reduced.

[0078] Figure 2 Immunohistochemical staining of collagen I in paraffin sections of mouse transplanted kidneys revealed semi-quantitative results indicating that nintedanib reduced collagen I deposition. The difference between the nintedanib 30 mg / kg / d group and the no-drug group was significant (CAN group vs. CAN 30 mg / kg / d group, P < 0.0001), and the degree of fibrosis was further reduced with increasing intervention concentration (CAN 30 mg / kg / d group vs. CAN 60 mg / kg / d group, P < 0.0001). There was no difference between the solubilizer intervention group and the no-drug group (P = 0.68), suggesting that solubilizers do not constitute a variable affecting fibrosis.

[0079] Figure 3 For comparison of serum creatinine and blood urea nitrogen in mice, the nintedanib intervention group reduced serum creatinine and blood urea nitrogen levels in mice (BUN: CAN group vs. CAN 30 mg / kg / d group, P=0.0025; Scr: CAN group vs. CAN 30 mg / kg / d group, P=0.0196), but there was no statistically significant difference between the treatment groups.

[0080] Figure 4 HE staining of the livers of mice in each group showed that the hepatocytes in each group were in good morphology, with no hepatocyte swelling, vacuolation or inflammation. The pathological score of liver injury showed no liver injury, indicating that the chronic transplanted kidney fibrosis model and drug intervention did not cause liver injury.

[0081] Figure 5 In the experiment to test the gastrointestinal function of mice, the difference between the CAN 90 mg / kg / d group and the CAN group was statistically analyzed. There was no statistically significant difference, indicating that no intestinal damage side effects occurred in mice within the effective therapeutic dose range.

[0082] Figure 6 The images show representative regions of CD68 and CD206 immunofluorescence co-staining across all mouse experimental groups. Nintedanib intervention was observed to reduce inflammatory infiltration of macrophages, particularly M2 macrophages marked by CD206 (percentage of double-staining positive cells: CAN group vs. CAN 30 mg / kg / d group, P < 0.0001; CAN group vs. CAN 60 mg / kg / d group, P < 0.0001), indicating that nintedanib can affect macrophage transdifferentiation.

[0083] In summary, HE and PAS staining showed that the degree of inflammatory cell infiltration in the transplanted kidney tissue of the nintedanib treatment group was significantly reduced compared with the CAN model group alone. Furthermore, based on the Banff 2019 semi-quantitative scoring system, the scores for interstitial fibrosis (CI) and tubular atrophy (CT) in the treated transplanted kidney were significantly lower than those in the control group. This suggests that nintedanib intervention alleviates chronic structural damage. Further immunohistochemical staining and semi-quantitative analysis of collagen I in mouse transplanted kidneys confirmed the reduction in chronic transplanted kidney fibrosis in the treated mice. Simultaneously, immunofluorescence co-staining showed that nintedanib could reduce the proportion of CD68+CD206+ double-positive macrophages. Further supplementary analysis, including HE staining of liver sections, gastrointestinal transit time, fecal water content detection, and serum creatinine and urea nitrogen concentration measurements, confirmed that at the dosage concentration that exerts its anti-fibrotic effect and protects renal function, nintedanib has no toxic effects on the liver, kidney, or gastrointestinal tract in the context of kidney transplantation. This indicates that nintedanib can significantly delay the development of chronic transplanted kidney fibrosis in mice and can be used as a drug for the treatment of chronic transplanted kidney fibrosis in mice.

[0084] It should be noted that although the above embodiments use mice as the treatment subjects, the present invention is not limited to treating mouse kidney transplants. Nintedanib can also be used as a protective agent to prolong the survival time of transplanted kidneys in other mammals.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. Use of nintedanib or its pharmaceutically acceptable salts, acids, esters, and pharmaceutical compositions thereof in the preparation of medicaments for the prevention or treatment of graft fibrosis.

2. The application according to claim 1, characterized in that, The graft fibrosis is chronic graft fibrosis.

3. The application according to claim 2, characterized in that, The graft fibrosis is allogeneic chronic graft fibrosis.

4. The application according to claim 3, characterized in that, The graft fibrosis includes fibrosis of transplanted kidney, transplanted liver, transplanted heart, transplanted lung, transplanted pancreas and / or transplanted small intestine.

5. The application according to claim 4, characterized in that, The graft fibrosis refers to transplanted kidney fibrosis.

6. The application according to any one of claims 1-5, characterized in that, The dosage forms of the drug include liquid dosage forms, gas dosage forms, solid dosage forms, and semi-solid dosage forms.

7. The application according to claim 6, characterized in that, The liquid dosage forms include solutions and injections; the gaseous dosage forms include aerosols and sprays; the solid dosage forms include powders and tablets; and the semi-solid dosage forms include ointments and pastes.

8. The application according to claim 7, characterized in that, The solution is an oral solution.

9. The application according to claim 8, characterized in that, The solvent for the oral solution includes polyethylene glycol.

10. The application according to claims 1-9, characterized in that, The nintedanib or its pharmaceutically acceptable salts, acids, esters, and pharmaceutical compositions thereof include nintedanib ethanesulfonate.

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