IL-27 receptor micromolecule agonist and application thereof

Abstract

The invention belongs to the field of medicines, and relates to application of balsalazide sodium in preparation of an IL-27 receptor small molecule agonist. Compared with the prior art, the balsalazide sodium provided by the invention can be combined with an IL-27 receptor, and has a good effect in treatment of IL-27 receptor mediated diseases such as obesity, hepatic fibrosis, pulmonary fibrosis and the like. Balsalazide sodium as a micromolecule agonist has the advantages of convenience in oral administration, good stability, low immunogenicity, strong penetrability, low production cost and the like, and a better treatment effect can be achieved by administration modes such as oral administration, subcutaneous injection, caudal vein injection, atomization and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to an IL-27 receptor small molecule agonist and application thereof. BACKGROUND

[0002] With the development of social economy, the change of people's living standards and dietary structure, the incidence of metabolic diseases is increasing, such as obesity, diabetes, insulin resistance, hypertension, hyperlipidemia, etc. Liver is an important organ for regulating the metabolism of glucose, fat, protein, bile acid, etc. in the body, and is also the central organ for maintaining the balance of the body. If the liver is damaged, the liver cell vitality decreases, the detoxification function decreases, the sensitivity to oxidative stress and other damage factors increases, which further leads to inflammation, fibrosis, etc. Liver damage caused by metabolic diseases mainly manifests as non-alcoholic fatty liver (NAFLD) caused by intrahepatic lipid accumulation and liver damage caused by bile acid accumulation. When the liver fat exceeds 10-15% of the liver wet weight, it is defined as fatty liver. NAFLD is a chronic and progressive process, which can be divided into simple fatty liver, fatty hepatitis (NASH), cirrhosis, and even liver cancer. Liver damage caused by bile accumulation can progress to liver fibrosis and even cirrhosis.

[0003] In terms of treatment of metabolic diseases such as obesity and metabolic fatty liver, the current main drug is GLP-1 receptor agonist drugs such as semaglutide, which can improve metabolic imbalance by inhibiting the feeding center to limit diet. However, it can easily cause depression, muscle loss, and gastrointestinal adverse reactions such as diarrhea, and cannot be used for patients with severe gastrointestinal diseases or sensitive to drug components. Currently, there is only one drug, Resmetirom (thyroid hormone receptor b agonist), approved by FDA last year for liver fibrosis, but its effect is limited and may affect the normal regulation of thyroid hormones. Therefore, it is of great clinical significance to develop drugs for treating metabolic diseases, fibrotic diseases and liver diseases, etc.

[0004] Studies have shown that IL-27 protein can directly target brown adipose tissue in the body, bind to IL-27Ra (IL-27 receptor) on the surface of adipocytes, and activate the p38 MAPK-PGC-1a signaling pathway, thereby promoting the expression of uncoupling protein UCP1, a key protein for thermogenesis, thereby increasing energy consumption and improving metabolic diseases caused by obesity. Therefore, IL-27 receptor is a potential therapeutic target for obesity and other diseases.

[0005] In addition, the prior art discloses that in Alzheimer's disease, IL-27 protein activates the downstream p38 signaling pathway by acting on the IL-27 receptor on the cognitive function neurons of the hippocampal dentate gyrus area of the brain, promotes the regeneration of neuron axons, and repairs nerve function, thereby playing a role in improving the cognitive and behavioral abilities of senile dementia. The prior art also discloses that IL-27 protein can help CD8+ cytotoxic T cells (CTL) maintain metabolic fitness and cytotoxic function, so that they maintain anti-tumor activity in the tumor microenvironment, and activation of the corresponding receptor can make IL-27 play a role in synergistic effect with existing immunotherapy. In addition, in the study of patients with advanced lung cancer (OAK study) and uroepithelial cancer (IMvigor210 / 211 study) receiving PD-L1 inhibitor monotherapy, high expression of IL27 and EBI3 genes is associated with good response and good prognosis of patients to immunotherapy, suggesting that IL-27 can be used as a marker for screening patient population suitable for immunotherapy. Therefore, IL-27 receptor is also a potential therapeutic target for Alzheimer's disease and tumors.

[0006] However, the natural agonist IL-27 protein of IL-27 receptor has the disadvantages of short half-life and easy degradation. Although recombinant IL-27 protein has good stability, it can be recognized as a foreign antigen by the immune system of the body, triggering an immune response and affecting its activity. Moreover, the production of recombinant IL-27 protein requires complex cell culture and purification processes, resulting in high production costs and expensive drug prices, which limits its widespread use in clinical practice. In addition, IL-27 protein and recombinant IL-27 protein are macromolecular agonists that need to be injected, which limits their application.

[0007] Small molecule agonists have the advantages of convenient oral administration, good stability, low immunogenicity, strong penetration, and low production cost, but so far, no literature has reported IL-27 receptor small molecule agonists. Therefore, the development of IL-27 receptor small molecule agonists has important potential clinical application value for metabolic diseases, fibrotic diseases, Alzheimer's disease, or IL-27 receptor-mediated tumors and other diseases. SUMMARY

[0008] Therefore, the purpose of the present application is to provide an IL-27 receptor small molecule agonist, which provides a new drug selection for the treatment of IL-27 receptor-mediated diseases.

[0009] The present application provides the use of sodium balsalazide in the preparation of an IL-27 receptor small molecule agonist.

[0010] Compared with the prior art, the sodium balsalazide can bind to the IL-27 receptor, and has achieved good effects in the treatment of diseases mediated by the IL-27 receptor, such as obesity, liver fibrosis, and pulmonary fibrosis. Compared with the recombinant IL-27 protein, the sodium balsalazide as a small molecule agonist has the advantages of convenient oral administration, good stability, low immunogenicity, strong penetration, and low production cost, and good treatment effects can be achieved by oral administration, subcutaneous injection, tail vein injection, and aerosolization.

[0011] The application further provides application of the sodium balsalazide in preparation of a medicine for preventing and / or treating diseases mediated by the IL-27 receptor.

[0012] In an embodiment, the disease mediated by the IL-27 receptor is a metabolic disease, a fibrosis disease, Alzheimer's disease, or a tumor mediated by the IL-27 receptor.

[0013] In an embodiment, the metabolic disease is obesity, diabetes, or metabolic fatty liver.

[0014] In an embodiment, the fibrosis disease is liver fibrosis or pulmonary fibrosis.

[0015] In an embodiment, the tumor mediated by the IL-27 receptor includes lung cancer, uroepithelial cancer.

[0016] In an embodiment, the dosage form of the medicine is injection, tablet, capsule, granule, aerosol, or oral liquid agent.

[0017] The application further provides application of the sodium balsalazide in preparation of a medicine for preventing and / or treating metabolic diseases, fibrosis diseases, and liver diseases.

[0018] In an embodiment, the medicine is a medicine for inhibiting weight gain, and / or improving glucose tolerance, and / or improving insulin tolerance, and / or inhibiting liver fat accumulation, and / or inhibiting back fat accumulation, and / or reducing collagen deposition.

[0019] In an embodiment, the metabolic disease is obesity, diabetes, or metabolic fatty liver.

[0020] In an embodiment, the fibrosis disease is liver fibrosis or pulmonary fibrosis.

[0021] In an embodiment, the liver disease is chronic liver injury or cirrhosis.

[0022] The application further provides a complex, which is a complex of the sodium balsalazide covalently connected to the IL-27 receptor.

[0023] For better understanding and implementation, the application is described in detail below with reference to the drawings. Attached Figure Description

[0024] Figure 1 A schematic diagram of the human IL-27 quaternary complex.

[0025] Figure 2 The effect of sodium balsalazide treatment on UCP-1 expression in adipocytes in Example 1.

[0026] Figure 3 This is the grouping processing information for Example 2.

[0027] Figure 4 The effect of oral administration of sodium balsalazide on the body weight of obese mice.

[0028] Figure 5 The effect of oral administration of sodium balsalazide on food intake in obese mice.

[0029] Figure 6 The effect of oral administration of sodium balsalazide on the weight of various tissues in obese mice was investigated. In this study, liver, heart, spleen, lung, and kidney were represented by the liver, heart, spleen, lung, and kidney, respectively.

[0030] Figure 7 The effect of oral balsalazide sodium treatment on insulin resistance and diabetes resistance in obese mice.

[0031] Figure 8 The effect of oral balsalazide sodium treatment on hepatic fat accumulation in obese mice.

[0032] Figure 9 This is the grouping processing information for Example 3.

[0033] Figure 10 The effect of subcutaneous injection of sodium balsalazide on the body weight of obese mice.

[0034] Figure 11 The effect of subcutaneous injection of sodium balsalazide on food intake in obese mice.

[0035] Figure 12 The effect of subcutaneous injection of sodium balsalazide on the weight of liver, heart, spleen and lungs in obese mice.

[0036] Figure 13 The effect of subcutaneous injection of sodium balsalazide on the weight of the kidney, brown adipose tissue, and epididymis in obese mice was investigated. In this study, BAT represents brown adipose tissue, SCW represents subcutaneous white adipose tissue, and EP represents epididymis.

[0037] Figure 14 Effects of subcutaneous balsalazide sodium treatment on insulin resistance and diabetes resistance in obese mice.

[0038] Figure 15 The effect of subcutaneous injection of sodium balsalazide on hepatic fat accumulation in obese mice.

[0039] Figure 16 This is the grouping processing information for Example 4.

[0040] Figure 17 The effects of sodium balsalazide on liver fibrosis in mice.

[0041] Figure 18 The effect of sodium balsalazide on body weight in mice with pulmonary fibrosis.

[0042] Figure 19 The effect of sodium balsalazide on body weight in mice with pulmonary fibrosis after removing dead samples.

[0043] Figure 20 The study investigated the effect of balsalazide sodium on the survival rate of mice with pulmonary fibrosis. The survival rate of mice injected with balsalazide sodium was higher than that of mice injected with recombinant IL-27 protein and mice treated with balsalazide sodium nebulization.

[0044] Figure 21 The effects of sodium balsalazide on fibrotic plaques and hemorrhages in the lung tissue of mice with pulmonary fibrosis.

[0045] Figure 22 The effect of balsalazide sodium on the expression of collagen-related genes in the lung tissue of mice with pulmonary fibrosis. Detailed Implementation

[0046] In order to overcome the shortcomings of IL-27 protein and recombinant IL-27 protein, this invention aims to find a small molecule agonist of IL-27 receptor and explore its therapeutic effect on IL-27 receptor-mediated diseases.

[0047] First, this invention uses computer molecular docking technology to identify candidate small molecules that can bind to the active site of the IL-27 receptor: see [link / reference] Figure 1 It is an IL-27 quaternary complex, in which IL-27Ra binds to IL-27 at site2a (Y73, R74, E123) and site2b (E146, P187, Q194). Molecular docking was performed using cryo-electron microscopy structure 7U7N, and more than ten potential candidate small molecules were screened from a database containing more than 2,400 marketed drugs.

[0048] The inventors' previous research found that IL-27 protein promotes UCP1 expression, and that UCP1 expression was significantly reduced in IL-27RαKO mice (IL-27Rα gene knockout). Therefore, this invention uses more than ten candidate small molecules to stimulate brown adipocytes of WT mice and IL27RαKO mice, and assesses whether the IL-27 receptor signaling pathway is activated by UCP1 expression levels. Ultimately, it was found that the candidate small molecule balsalazide significantly increased UCP1 expression levels in wild-type mouse brown adipocytes, but did not increase UCP1 expression levels in IL-27 receptor knockout mouse brown adipocytes. This demonstrates that balsalazide can activate the IL-27 receptor signaling pathway and has the potential to promote heat production, increase body temperature, and improve hypothermia.

[0049] Furthermore, this invention has demonstrated good therapeutic effects in animal models of diseases mediated by the IL-27 receptor using sodium balsalazide, including obesity, diabetes, metabolic fatty liver, liver fibrosis, and pulmonary fibrosis.

[0050] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0051] Example 1 This embodiment utilizes the characteristic of IL-27R signaling (IL-27 receptor signaling) promoting UCP-1 expression in adipocytes to verify the cellular activity of potential molecules and identify sodium balsalazide, an active candidate molecule, from among the potential molecules. The specific steps include:

[0052] (1) Primary adipocyte culture: Under aseptic conditions, adipose tissue was taken from the groin of experimental animals (WT mice or IL-27 receptor knockout mice). The adipose tissue was washed several times with PBS to remove blood and impurities. The adipose tissue was minced and digested with a solution containing collagenase II (0.2 mg / ml), and incubated in a 37°C incubator for 20-30 minutes to decompose the extracellular matrix.

[0053] Add an equal volume of basal culture medium (containing FBS) to terminate the trypsin reaction. Then centrifuge at 600g for 5 minutes, discard the supernatant, and collect the precipitated adipocytes.

[0054] Dilute the adipocyte suspension to a suitable concentration (approximately 2 × 10⁻⁶). 5 (cells / mL) were seeded into cell culture dishes. A basal medium containing 10% FBS and 1% antibiotic was added, and the dishes were then placed in an incubator at 37°C and 5% CO2 for static incubation.

[0055] (2) Adipocyte passage: Observe the cell growth status, and passage when the cell density reaches about 80%-90%. Remove the culture medium and wash the cells twice with PBS. Add trypsin-EDTA solution and incubate in a 37°C incubator for 3-5 minutes until the cells detach from the bottom of the dish.

[0056] Add an equal volume of complete culture medium (containing FBS) to neutralize trypsin. Gently pipette to suspend the cells, transfer them to centrifuge tubes, and collect the cells by centrifugation.

[0057] Fractionation culture: Dilute the cell suspension to the appropriate concentration (approximately 2 × 10⁻⁶). 5 (cells / mL) were inoculated into new culture dishes or cell plates. Complete culture medium was added, and the cells were cultured at 37°C and 5% CO2.

[0058] (3) Adipocyte differentiation induction: When the primary adipocytes reached approximately 80% confluence, the culture medium was replaced with one containing insulin and a differential additive. The culture medium consisted of DMEM medium, 10% FBS, 1% antibiotics, 5 μg / mL insulin, 1 μM dexamethasone, 1 μM rosiglitazone, 250 μM 3-isobutyl-1-methylxanthine, and 500 μM indomethacin.

[0059] Observe the changes in cell morphology every day. Adipocytes begin to deposit lipid droplets and usually complete differentiation within 3-7 days.

[0060] The culture medium containing insulin and additives (1 μM dexamethasone, 1 μM rosiglitazone, 250 μM 3-isobutyl-1-methylxanthine, 500 μM indomethacin) was changed every 2-3 days until the experiment was completed, yielding pre-brown adipocytes.

[0061] Prebrown adipocytes were seeded into 24-well plates and cultured for 3 days after the cell density reached approximately 80% by replacing the medium with maintenance medium containing 5 μg / mL insulin, 1 nM triiodothyronine (T3), and 1 μM rosiglitazone.

[0062] (4) Drug intervention: Different concentration gradients of sodium salsaizumab (candidate small molecule sodium salsaizumab) were added for 24 hours. The compound was dissolved in DMSO, and the blank group was treated with DMSO.

[0063] (5) Preparation of protein samples: After collecting adipocyte samples, total cell protein was extracted using RIPA lysis buffer. RIPA lysis buffer containing protease inhibitors was added to the cell samples, mixed, and then placed on ice for 30 min for lysis. After sonication, the samples were centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was transferred to a new EP tube, 5× loading buffer was added, and the samples were mixed and then heat-denatured at 95 °C for a total of 10 min.

[0064] (6) Western blot: After the protein sample was brought to room temperature, it was added to the SDS-PAGE gel wells in the same volume. Electrophoresis was performed at 60V constant voltage, and the membrane was transferred at 350mA constant current for 1.5h. It was blocked by shaking with 5% skim milk powder at room temperature for 1h. After washing, the primary antibody (HSP90, β-actin, UCP1, with bands of 90kDa, 43kDa, and 33kDa respectively) was incubated overnight at 4℃. The next day, after washing, the secondary antibody was incubated at room temperature for 2h. The developing solution was dropped onto the surface of the protein sample, and chemiluminescence imaging was performed.

[0065] Depend on Figure 2 As can be seen, using HSP90 / β-actin as an internal control for protein bands, 0.1 μM sodium balsalazide significantly upregulated the expression level of UCP1 protein in brown adipocytes of WT mice, which can improve the heat production and energy consumption capacity of brown adipocytes, but cannot improve the expression level of UCP1 in brown adipocytes of IL-27 receptor knockout mice. This indicates that sodium balsalazide can activate the IL-27 receptor-related pathway and has the potential to treat IL-27 receptor-related diseases. Therefore, in subsequent experiments, an animal model of IL-27 receptor-related diseases will be used to verify the effect of sodium balsalazide.

[0066] Example 2 This embodiment verifies the therapeutic effect of balsalazide sodium on obese model mice, specifically including the following steps: Mice were induced to develop an obesity model by long-term feeding with a high-fat diet (HFD) (≥12 weeks). Eight-week-old SPF-grade male C57BL / 6 mice were purchased from Jicui Pharmaceutical, and the HFD was purchased from Research Diets, Inc. (USA), consisting of the Rodent Diet with 60% kcal% fat, for a total of 20 weeks. The intervention method was gavage administration of balsalazide sodium in water / physiological saline solution (600 mg / kg) at 24-hour intervals. After 3 weeks, the mice were sacrificed, and serum was collected and frozen. Fat, liver, spleen, heart, kidney, lung, large intestine, and small intestine were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (HE). Grouping information is provided in [reference needed]. Figure 3 .

[0067] See Figure 4 , Figure 5 and Figure 6Oral administration of sodium balsalazide (600 mg / kg, once daily, as protected by this patent) to obese mice (6-week-old C57 mice induced by a 30-week high-fat diet, weighing >50 g) significantly reduced body weight, liver weight, and fat weight, including back adipose tissue weight. Simultaneously, it had no significant effect on other organs such as the spleen and heart, indicating that sodium balsalazide has good safety for other vital organs while exerting its weight-loss effect. Sodium balsalazide intervention did not affect the food intake of obese mice, suggesting that its weight-loss effect is not achieved through appetite suppression, but may exert its effect through other pathways such as regulating metabolism.

[0068] See Figure 6 Balsalazide sodium can reduce liver weight and back fat tissue weight.

[0069] See Figure 7 Treatment with balsalazide significantly improved insulin and glucose tolerance in mice, indicating that balsalazide helps improve insulin sensitivity and glucose metabolism in obese mice, and has potential value in preventing and improving obesity-related glucose metabolism disorders.

[0070] See Figure 8 Balsalazide treatment can improve hepatic fat accumulation. Hepatic fat accumulation is an important part of a series of health problems caused by obesity, and this effect of balsalazide helps to reduce the burden on the liver and lower the risk of obesity-related liver diseases (such as fatty liver).

[0071] In summary, sodium balsalazide showed significant weight loss in obese mouse models, effectively reducing liver and adipose tissue weight, improving glucose metabolism and liver fat accumulation, without significant adverse effects on other organs or affecting food intake. It has good safety and potential application value, providing new candidate drugs and research directions for the treatment of obesity and related metabolic diseases.

[0072] Example 3 Building upon Example 2, this example further investigates the impact of different administration methods on the therapeutic effect of balsalazine. (See also...) Figure 9 An obesity model was induced in mice by long-term feeding with a high-fat diet (HFD) for ≥12 weeks. Eight-week-old SPF-grade male C57BL / 6 mice were purchased from Jicui Pharmaceutical. The HFD diet (Rodent Diet with 60% kcal% fat) was purchased from Research Dietes, USA, and the mice were fed for 20 weeks. The intervention method was subcutaneous injection (600 mg / kg) of sodium balsalazine / physiological saline solution at 24-hour intervals. Mice were sacrificed after 3 weeks, and serum was collected and frozen. Fat, liver, spleen, heart, kidneys, lungs, large intestine, and small intestine were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (HE).

[0073] See Figure 10 to Figure 15 Subcutaneous injection of sodium balsalazide showed a more significant effect on weight loss in obese mice, indicating that this route of administration may be more conducive to the effective action of sodium balsalazide in vivo, more efficiently regulating the body's energy balance and metabolic processes, thereby more effectively promoting weight loss in obese mice. The organ function, glucose tolerance, and insulin tolerance of mice treated with subcutaneous sodium balsalazide were similar to those treated with oral administration, with a more pronounced effect, fully demonstrating that subcutaneous sodium balsalazide, while exerting its therapeutic effects such as weight loss, has good safety for vital organs and does not cause significant toxic side effects. This provides stronger experimental evidence for the application of sodium balsalazide in the treatment of obesity and related metabolic diseases, suggesting that subcutaneous injection may be a more ideal method of administration for sodium balsalazide.

[0074] Example 4 Obesity is one of the causes of non-alcoholic fatty liver disease (NAFLD). The disease initially presents as simple fatty liver (steatohepatitis), progressing to non-alcoholic steatohepatitis (NASH), and further deteriorating into liver fibrosis and eventually cirrhosis. The pathological features of NASH are hepatocellular damage, inflammation, and fibrosis, which can lead to cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Since balsalazine can improve obesity and reduce liver weight, this example further explores the therapeutic effect of balsalazine on liver fibrosis.

[0075] See Figure 16 MASH liver fibrosis in mice was induced using the WD+CCl4 method: Mice were fed a Western diet (WD: high fat, high cholesterol, high sucrose, and fructose) for a long period, followed by repeated intraperitoneal injections of hepatotoxic carbon tetrachloride (CCl4) at a volume of 20 ml / kg for 8 weeks, creating a chronic liver injury-induced liver fibrosis model. Starting from week 9 of modeling, mice were administered the drug via daily intraperitoneal injection. The positive control group received Rezdiffra (resimeltiro), the treatment group received recombinant IL-27 protein or balsalazine, and the negative control group received an equal volume of physiological saline. Mice were sacrificed after 3 weeks, and relevant indicators were measured.

[0076] See Figure 17Balsalazine significantly improved liver fibrosis, and its effect in reducing collagen deposition was superior to Rezdiffra (resimeltiro) and recombinant IL-27 protein. Collagen deposition is one of the important characteristics of liver fibrosis; excessive collagen accumulation leads to liver structural damage and functional impairment. Balsalazine effectively reduced abnormal collagen deposition in the liver, helping to maintain normal liver tissue structure and function. Its effect was more significant than that of Rezdiffra, which is currently known to have some anti-fibrotic effects, suggesting that balsalazine may have greater potential in the treatment of liver fibrosis. These results provide strong experimental evidence for balsalazine as a potential anti-MASH liver fibrosis drug, and its mechanism of action and clinical application prospects deserve further in-depth research.

[0077] Example 5 Based on Example 4, this example further explores the therapeutic effect of balsalazide sodium on other fibrotic diseases, namely pulmonary fibrosis, specifically including the following steps.

[0078] Bleomycin-induced pulmonary fibrosis: Mice were anesthetized with tribromoethanol-tert-amyl alcohol and administered a single tracheotomy injection of 1 mg / kg bleomycin (MCE, HY-108345) (solvent: sterile PBS). Control mice were injected with an equal volume of sterile PBS.

[0079] Treatment with recombinant IL-27 protein or balsalazide: On day 7 of bleomycin treatment, mice were grouped according to body weight and percentage of weight loss, and then treated daily with either recombinant IL-27 protein or balsalazide. The balsalazide tail vein injection group (BLM + balsalazide IV) received 300 mg / kg balsalazide via tail vein injection daily; the recombinant IL-27 treatment group (BLM + IL-27 IV) received 300 μg / kg recombinant IL-27 via tail vein injection daily; control mice were treated with the same dose of saline via tail vein injection. The balsalazide nebulization treatment group (BLM + balsalazide INh) received 300 mg / L balsalazide via nebulization for 20 minutes daily starting on day 7 of bleomycin treatment, while control mice were treated with saline nebulization.

[0080] The actual weight of each mouse was measured and recorded daily. The difference between the weight and the initial weight was divided by the initial weight to obtain the percentage of weight loss. Mice were sacrificed 13 days after bleomycin administration. The condition of the mice was observed daily, and the number of surviving and dying mice was recorded, and survival curves were plotted. The fresh weight of the mouse lungs was weighed and photographed. Lung density = fresh weight of lungs / mouse body weight * 100%.

[0081] RNA was extracted from the accessory lobes of the lungs and the expression of fibrosis-related genes was detected, with Hprt as an internal control.

[0082] See Figure 18 to Figure 20Balsalazide sodium and recombinant IL-27 protein showed good therapeutic effects on bleomycin-induced pulmonary fibrosis, both alleviating weight loss, improving survival rate, and reducing lung damage and collagen expression in model mice. Furthermore, both tail vein injection and nebulization were effective.

[0083] See Figure 21 The group receiving sodium balsalazine via tail vein injection had fewer fibrotic plaques and fewer bleeding points in their lung tissue.

[0084] See Figure 22 In the group receiving sodium balsalazide via tail vein injection, the expression of collagen-related genes in lung tissue was reduced, and its inhibitory effect on Col1a1, Col3a1, and Fn1 was superior to that of recombinant IL-27 protein.

[0085] In summary, balsalazine demonstrated multifaceted and favorable therapeutic effects in a bleomycin-induced mouse model of pulmonary fibrosis. It not only improved the overall condition and survival of mice and reduced lung damage, but also regulated the expression of collagen and related genes at the molecular level, with effectiveness across different administration routes. These results provide strong experimental evidence for balsalazine as a potential anti-pulmonary fibrosis drug, warranting further in-depth research into its mechanism of action and clinical application value.

[0086] Compared with existing technologies, this invention provides the application of sodium balsalazepam in animal models of various diseases such as obesity, diabetes, metabolic fatty liver, liver fibrosis, and pulmonary fibrosis, and has the following advantages.

[0087] (1) Balsalazine can bind to the IL-27 receptor and has achieved good results in the treatment of diseases mediated by the IL-27 receptor, such as obesity, liver fibrosis, and pulmonary fibrosis. Based on the similarity of receptor effects and the correlation of signaling pathways, it is speculated that it has the potential to alleviate other diseases mediated by the IL-27 receptor.

[0088] (2) Balsalazine sodium can be administered via various routes such as oral, subcutaneous injection, tail vein injection and nebulization. All of these routes can achieve good therapeutic effects, providing a variety of options for clinical application. The appropriate route of administration can be selected according to the patient's specific condition.

[0089] (3) Balsalazide sodium has no other effects on the spleen, heart, kidneys, etc. of mice, nor does it affect food intake. This indicates that balsalazide sodium does not seriously interfere with the normal physiological functions of the body while exerting its therapeutic effect, and has high safety.

[0090] (4) In a liver fibrosis model, balsalazide sodium was more effective than resmetiro and recombinant IL-27 protein in reducing collagen deposition; in a pulmonary fibrosis model, both tail vein injection and nebulization of balsalazide sodium effectively alleviated symptoms in the model mice, with effects comparable to recombinant IL-27 protein. This indicates that balsalazide sodium has unique advantages and good efficacy in the treatment of fibrotic diseases, providing a new and powerful option for the treatment of fibrotic diseases. Lung cancer is also one of the important complications in patients with pulmonary fibrosis. Balsalazide sodium can improve pulmonary fibrosis, indicating that it may be able to alleviate lung cancer and pulmonary fibrosis combined with lung cancer.

[0091] (5) Balsalazide sodium can not only improve liver damage caused by metabolic diseases such as obesity, but also effectively improve liver fibrosis. Liver damage and liver fibrosis are important risk factors for the development of liver cancer. Balsalazide sodium, by alleviating liver damage and liver fibrosis, suggests that it may reduce the risk of liver cancer, providing a new strategy for the prevention and treatment of liver cancer. In addition, for some patients with metabolic diseases who are not suitable for GLP-1 receptor agonists, balsalazide sodium provides a new treatment option, expanding the patient population for the treatment of metabolic diseases.

[0092] (6) Compared with biological macromolecular drugs (such as IL-27 protein and recombinant IL-27 protein), balsalazine sodium, as a small molecule agonist, has the advantages of convenient oral administration, good stability, low immunogenicity, strong penetration and low production cost. It overcomes the limitations of biological macromolecular drugs such as easy degradation, high production cost and need for injection administration, and has better clinical application prospects.

[0093] (7) IL-27 protein helps CD8+ cytotoxic T cells (CTLs) maintain metabolic fitness and cytotoxic function, enabling them to maintain anti-tumor activity in the tumor microenvironment. Activation of the corresponding receptor can allow IL-27 protein to exert its effects and synergistically enhance existing immunotherapies. Furthermore, in studies of advanced lung cancer (OAK study) and urothelial carcinoma patients receiving PD-L1 inhibitor monotherapy (IMvigor210 / 211 study), high expression of IL27 and EBI3 genes was associated with good response to immunotherapy and good prognosis, suggesting that IL-27 can serve as a biomarker for screening suitable patient populations for immunotherapy. Since both balsalazine and IL-27 protein can act on the IL-27 receptor, balsalazine has the potential to treat IL-27 receptor-mediated tumors.

[0094] (8) IL-27 protein activates the downstream p38 signaling pathway by acting on IL-27 receptors on cognitive neurons in the dentate sulcus region of the hippocampus, promoting neuronal axon regeneration and repairing nerve function, thereby improving cognitive and behavioral abilities in Alzheimer's disease. Based on this, it is speculated that balsalazine sodium provided by this invention also has the potential to treat Alzheimer's disease.

[0095] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of this application.

[0096] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of sodium balsalazide in the preparation of small molecule agonists of IL-27 receptors.

2. Use of sodium balsalazepam in the preparation of drugs for the prevention and / or treatment of diseases mediated by IL-27 receptors.

3. The application according to claim 2, characterized in that, The diseases mediated by the IL-27 receptor are metabolic diseases, fibrotic diseases, Alzheimer's disease, or IL-27 receptor-mediated tumors.

4. The application according to claim 3, characterized in that, The metabolic diseases mentioned are obesity, diabetes, or metabolic fatty liver.

5. The application according to claim 3, characterized in that, The fibrotic disease referred to is liver fibrosis or pulmonary fibrosis.

6. The application according to claim 3, characterized in that, The tumors mediated by the IL-27 receptor include lung cancer and urothelial carcinoma.

7. The application according to claim 2, characterized in that, The dosage form of the drug is injection, tablet, capsule, granule, aerosol or oral liquid.

8. Use of sodium balsalazide in the preparation of drugs for the prevention and / or treatment of metabolic diseases, fibrotic diseases and liver diseases.

9. The application according to claim 8, characterized in that, The drug is a drug that inhibits weight gain and / or improves glucose tolerance, and / or improves insulin tolerance, and / or inhibits hepatic fat accumulation, and / or inhibits back fat accumulation, and / or reduces collagen deposition.

10. A complex, characterized in that, This complex is a complex of sodium balsalazide covalently linked to the IL-27 receptor.

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