Use of montelukast in the preparation of a drug for preventing and treating hepatolenticular degeneration

Abstract

The application discloses application of montelukast in preparation of a medicine for preventing and treating hepatolenticular degeneration and belongs to the technical field of biological medicines. The application finds that montelukast can cut off a CysLTs-mediated inflammatory cascade by specifically blocking cysteinyl leukotriene (CysLTs) receptors, and significantly inhibit the expression of a chemotactic factor (such as CCL2) and subsequent pathological recruitment of macrophages, thereby protecting the liver from secondary immune inflammatory damage induced by copper toxicity. The finding proves that montelukast can be used as a new strategy for anti-inflammatory liver protection that is independent of copper excretion, can solve the problem of inflammatory damage that cannot be improved by existing copper excretion drugs, and avoids side effects that may be caused by strong copper excretion. In addition, since montelukast is a marketed drug (old drug) that has been widely used in clinical application for many years, has clear pharmacokinetics and high safety, and is used as a medicine for preventing and treating hepatolenticular degeneration, the medicine has great clinical transformation potential and application prospect.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to the application of montelukast in the preparation of drugs for the prevention and treatment of Wilson's disease. Background Technology

[0002] Wilson's disease is an autosomal recessive inherited disorder of copper metabolism, primarily caused by... ATP7B Gene mutations lead to impaired copper excretion, resulting in excessive copper deposition in vital organs such as the liver, brain, and cornea. This causes cirrhosis, neuropsychiatric symptoms, and severe organ failure, making it a highly fatal and disabling disease. Although advancements in modern medicine have improved the prognosis of some patients through early diagnosis and lifelong copper chelation therapy, the clinical drug options for Wilson's disease remain very limited. Currently, treatment mainly relies on traditional copper chelators (such as penicillamine and tricornidine) and zinc preparations. However, clinical application faces numerous serious problems and challenges. For example, many patients cannot tolerate penicillamine due to severe side effects (nephrotoxicity, bone marrow suppression, autoimmune reactions, etc.); some patients experience paradoxical exacerbations of neurological symptoms after initiating treatment; and the need for lifelong medication leads to poor adherence. Therefore, there is an urgent need to develop novel drugs that are highly effective, low in toxicity, and have organ-protective effects for the prevention and treatment of Wilson's disease.

[0003] Montelukast is a potent and highly selective cysteine ​​leukotriene receptor 1 (CysLTR1) antagonist widely used in the treatment of asthma and allergic rhinitis in adults and children, with a good safety profile. In addition to its applications in the respiratory system, montelukast also possesses potential biological effects such as antioxidant, anti-inflammatory, anti-fibrotic, and neuroprotective properties. Current research indicates that montelukast has a wide range of pharmacological effects, improving various inflammatory and degenerative diseases by modulating leukotriene pathways and non-leukotriene-dependent pathways. Currently, its research in neurodegenerative diseases, liver fibrosis, and cardiovascular diseases is receiving considerable attention. However, to date, there are no published reports on the therapeutic effects and applications of montelukast in Wilson's disease (especially in cases of copper overload-induced organ damage). Summary of the Invention

[0004] The purpose of this invention is to provide the application of montelukast in the preparation of drugs for the prevention and treatment of Wilson's disease, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the application of montelukast in the preparation of drugs for the prevention and treatment of Wilson's disease.

[0006] The second technical solution of the present invention is a drug for preventing and treating Wilson's disease, including montelukast.

[0007] Based on the above technical solution, the present invention has the following technical effects: This invention is achieved through Atp7b Gene knockout mice ( Atp7b - / - Model experiments revealed that montelukast treatment significantly reduced liver damage caused by copper overload in animal models of Wilson's disease (manifested as decreased serum transaminase levels, reduced liver necrosis foci, and reduced inflammatory cell infiltration). Crucially, this invention found that the therapeutic effect of montelukast does not depend on reducing the copper load in the liver, but rather on specifically blocking cysteyl leukotrienes (CysLTs) receptors, interrupting the CysLTs-mediated inflammatory cascade, and significantly inhibiting the expression of chemokines (such as CCL2) and subsequent pathological recruitment of macrophages, thereby protecting the liver from copper-induced secondary immune-inflammatory damage. This discovery confirms that montelukast can serve as a novel, non-copper-dependent anti-inflammatory and hepatoprotective strategy, addressing inflammatory damage that existing copper-chelating drugs cannot improve, while avoiding the potential side effects of potent copper chelation. Furthermore, as montelukast is a marketed drug that has been widely used clinically for many years (an established drug), with well-defined pharmacokinetics and high safety profile, it possesses significant clinical translational potential and application prospects as a treatment for Wilson's disease. Attached Figure Description

[0008] Figure 1 The results of the study on changes in serum leukotriene C4 (LTC4) levels in patients with Wilson's disease in Example 1 are shown. In this study, A represents the population grouping and study design, and B represents the serum LTC4 levels in patients with Wilson's disease and healthy volunteers. Patients with Wilson's disease (n=31) and healthy volunteers (n=30) were included. The data are presented as medians (interquartile range). The statistical method used was the Mann-Whitney U test. **P<0.01.

[0009] Figure 2 This is a schematic diagram of the experimental procedure for Example 2.

[0010] Figure 3 This document presents the experimental results of changes and significance of cysteyl leukotrienes (CysLTs) in a Wilson's disease animal model in Example 2. A represents the copper content detection results in mouse liver tissue; B represents the serum alanine aminotransferase (ALT) level in mice; C represents the serum aspartate aminotransferase (AST) level in mice; D represents the leukotriene C4 (LTC4) level in mouse liver tissue; E represents typical images of HE staining and immunohistochemical staining (ATP7B, F4 / 80, and Ly6G) in liver tissue. All data are expressed as mean ± standard deviation. The control group (…) Atp7b + / -n = 6-8 mice / group; model group ( Atp7b - / - n = 6-8 mice / group; statistical method: unpaired Student's t-test; *P<0.05, **P<0.01, ***P<0.001; scale bar = 50μm.

[0011] Figure 4 This is a schematic diagram of the experimental procedure for Example 3.

[0012] Figure 5 The results of the efficacy evaluation of montelukast sodium in an animal model of Wilson's disease in Example 3 are shown below; where A represents the changes in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice after treatment with montelukast sodium 3 mg / kg; B represents the copper content in mouse liver tissue; C represents typical images of HE staining and F4 / 80 immunohistochemical staining of liver tissue after treatment with montelukast sodium 3 mg / kg; DF represents the protein concentrations of inflammatory factors (TNF-α, IL-6) and chemokines (CCL2) in liver tissue; the above data are expressed as mean ± standard deviation; control group ( Atp7b + / - +Vehicle) n=6-8 mice / group; model group ( Atp7b - / - +Vehicle) n=6-8 mice / group; treatment group ( Atp7b - / - +Montelukast) n=6-8 mice / group; statistical method: one-way ANOVA; *P<0.05, **P<0.01 and ***P<0.001; ns: no significant difference; scale bar=50μm. Detailed Implementation

[0013] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0014] This invention provides the application of montelukast in the preparation of drugs for the prevention and treatment of Wilson's disease.

[0015] In some specific implementation schemes, montelukast treats Wilson's disease by reducing serum ALT and AST levels, thereby reducing liver inflammatory factor levels and interrupting the secondary immune inflammatory damage caused by copper toxicity.

[0016] In some specific implementations, the liver inflammatory factors include TNF-α, IL-6, and CCL2.

[0017] This invention also provides a drug for preventing Wilson's disease, including montelukast.

[0018] In some specific implementations, the montelukast is montelukast sodium, montelukast free acid, or other pharmaceutically acceptable salt forms thereof.

[0019] In some specific implementation plans, pharmaceutically acceptable excipients are also included.

[0020] In some specific implementations, the dosage form of the drug is a solid dosage form, a semi-solid dosage form, a liquid dosage form, or a gaseous dosage form.

[0021] "Pharmaceutical acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with the receptor.

[0022] Example 1 Changes in cysteine ​​leukotrienes (CysLTs) levels in patients with Wilson's disease 1.1 Establish a cohort of patients with Wilson's disease and collect serum samples. Inclusion criteria for patients with Wilson's disease: ① Age ≥ 14 years, gender not limited; ② Diagnosed with Wilson's disease according to the Leipzig scoring system (score ≥ 4 points); ③ Have not undergone liver transplantation.

[0023] Exclusion criteria: ① Comorbid viral hepatitis, autoimmune liver disease, or other liver diseases; ② Comorbid severe heart, lung, or kidney failure or malignant tumors; ③ Incomplete data or lack of informed consent.

[0024] Clinical data collection mainly included the following: basic information (age, sex, BMI), liver function indicators (ALT, AST, total bilirubin), copper metabolism indicators (serum ceruloplasmin, 24-hour urinary copper), and Leipzig score. Simultaneously, age- and sex-matched healthy volunteers were recruited as a control group, and their basic information was collected. Peripheral blood (5 mL each time) was collected from patients with Wilson's disease at diagnosis and during treatment follow-up. After centrifugation, the supernatant was collected and immediately stored at -80°C. All subjects obtained approval from the hospital's ethics committee, followed the Declaration of Helsinki, and signed written informed consent forms.

[0025] 1.2 Detection of Cysteine ​​Leukotrienes (CysLTs) in Serum Detection of serum leukotriene C4 (LTC4) Cysteinyl leukotrienes (CysLTs) include LTC4, LTD4, and LTE4, among which LTC4 is the parent material and key precursor in this synthetic pathway. This embodiment uses an enzyme-linked immunosorbent assay (ELISA) kit to specifically detect the level of serum leukotriene C4 (LTC4) to reflect the activation status of the CysLTs pathway. The specific operation steps are as follows: (1) Sample processing: Place the frozen sample at room temperature to thaw. If there is precipitation, centrifuge at 3000 rpm for 20 minutes and take the supernatant for testing. (2) Measurement steps: Equilibrate all components of the kit and the sample to room temperature. Prepare the washing buffer according to the instructions. Set up blank wells, standard wells, and sample wells respectively. Except for the blank wells, add enzyme-labeled reagent to the standard wells and sample wells. After sealing with sealing film, incubate at 37°C for 60 minutes. Discard the liquid in the wells and wash 5 times with washing buffer. Add chromogenic reagent A and chromogenic reagent B to each well sequentially, gently shake to mix, and incubate at 37°C in the dark for 15 minutes to develop color. Add 50 μL of stop solution to each well to terminate the reaction. Preheat the microplate reader, adjust the wavelength to 450 nm, and zero the absorbance (OD value) of each well using a blank well. Finally, calculate the sample concentration based on the standard curve, which is the actual content of LTC4.

[0026] 1.3 Experimental Results like Figure 1 As shown, this invention included patients with Wilson's disease (n=31) and healthy volunteers (n=30). By measuring serum LTC4 levels and comparing them with healthy volunteers, it was found that serum LTC4 levels were significantly elevated in patients with Wilson's disease. As a key initiating molecule in CysLTs biosynthesis, the significantly elevated LTC4 level suggests abnormal overactivation of the cysteyl leukotrienes (CysLTs) synthesis pathway. It is hypothesized that abnormal activation of the CysLTs pathway may be involved in the liver damage process of Wilson's disease. Although montelukast primarily acts as an antagonist of the LTD4 receptor (CysLT1), the rapid conversion of LTC4 to LTD4 in vivo indirectly confirms an increase in the substrate of its downstream product, LTD4. Therefore, montelukast, as a specific blocker of this pathway, may improve disease progression by blocking this inflammatory cascade.

[0027] Example 2 Changes and significance of cysteyl leukotrienes (CysLTs) in an animal model of Wilson's disease 2.1 Constructing a mouse model of Wilson's disease use Atp7b Gene knockout mice (Atp7b) - / - As a model of Wilson's disease, littermate heterozygous mice ( Atp7b + / -As a phenotypically normal control group, the knockout mice were all purchased from Cyagen Biosciences Co., Ltd. Figure 2 As shown.

[0028] Mice were fed under constant laboratory conditions (12-hour / 12-hour light-dark cycle, room temperature 23±2℃, relative humidity 55±10%), with free access to food and water. All animal handling and experimental procedures were approved by the ethics committee. Twenty-week-old mice (at which point the model mice were already in a significant liver injury stage) were selected. After collecting venous blood from the mice, they were sacrificed, and liver tissue was rapidly excised: one portion was stored at -80℃ for biochemical analysis, and the other portion was fixed in formalin for pathological analysis. Specific groupings were as follows: ① Control group ( Atp7b + / - ① n = 6-8 mice / group; ② Model group ( Atp7b - / - ): n = 6-8 mice / group.

[0029] 2.2 Serum biochemical indicators and detection of liver leukotriene C4 (LTC4) and copper content Blood was collected from the eyeballs, and serum was separated by centrifugation at 3000 rpm for 10 minutes at 4°C. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using an automated biochemical analyzer to assess the degree of liver inflammation and damage. Simultaneously, frozen liver tissue was collected, homogenized with pre-chilled PBS, and the supernatant was collected by centrifugation. The copper content (μmol / g) in the liver tissue was measured using a tissue copper assay kit to determine the degree of copper overload. The level of leukotriene C4 (LTC4) in the liver tissue homogenate (pg / mg protein) was specifically detected using an ELISA kit to reflect the activation status of the local CysLTs pathway.

[0030] 2.3 Tissue hematoxylin-eosin (H&E) staining and immunohistochemical analysis To assess the pathological changes in the liver during disease progression, H&E staining and immunohistochemical staining were performed.

[0031] (1) H&E staining: routine dehydration, embedding, sectioning, staining, and observation of liver tissue morphology and structure.

[0032] (2) Immunohistochemical staining: After dewaxing and antigen retrieval, the sections were incubated with the following primary antibodies: ① Anti-ATP7B: to verify the knockout efficiency of ATP7B protein; ② Anti-F4 / 80: to label Kupffer cells / macrophages and assess macrophage infiltration; ③ Anti-Ly6G: to label neutrophils and assess neutrophil infiltration. Subsequently, the sections were incubated with secondary antibodies and DAB for staining, and the cell nuclei were counterstained with hematoxylin.

[0033] 2.4 Experimental Results like Figure 3 As shown, compared with the control group, Atp7b - / - A mouse model of Wilson's disease exhibited significant pathophysiological changes. Biochemical tests showed a sharp increase in copper content in the liver tissue of the model group mice (p<0.001), accompanied by a significant increase in serum ALT and AST levels (p<0.01), indicating severe hepatocellular damage. ELISA results showed that the LTC4 content in the liver tissue of the model group was significantly higher than that of the control group (p<0.001), confirming the overactivation of the CysLTs synthesis pathway in the diseased liver. Pathological and immunohistochemical results not only confirmed the absence of ATP7B protein in the liver of the model group mice, but also directly revealed the accompanying severe liver inflammation, especially the infiltration of a large number of F4 / 80 positive macrophages. Conclusion: The above results indicate that with the occurrence of copper overload and liver damage, LTC4 levels significantly increase, suggesting that the cysteylleukotrienes (CysLTs) synthesis pathway is abnormally activated locally in the liver. This activation of the inflammatory cascade is highly consistent with the degree of liver pathological damage, suggesting that it may be a key factor driving the progression of Wilson's disease and a potential therapeutic target.

[0034] Example 3 Evaluation of the therapeutic effect of montelukast sodium on liver injury in an animal model of Wilson's disease 3.1 Dosing regimen and animal grouping like Figure 4 As shown, 16-week-old infants were selected. Atp7b - / - Mice (at this point exhibiting obvious liver dysfunction and pathological changes in liver tissue) were fed the same conditions as described in Example 2.1. The experimental mice were randomly divided into 3 groups: ① Control group ( Atp7b + / - +Vehicle): n=6-8, administered an equal volume of solvent (0.5% CMC-Na) by gavage; ② Model group ( Atp7b - / - +Vehicle): n=6-8, administered an equal volume of solvent via gavage; ③ Montelukast sodium treatment group ( Atp7b - / - +Montelukast): n=6-8 birds, treated with montelukast sodium 3 mg / kg / day by gavage.

[0035] After 4 weeks of continuous treatment (until the mice reached 20 weeks of age), the mice were sacrificed. Serum and liver tissue were collected to evaluate the protective effect of montelukast sodium against liver injury in Wilson's disease mice.

[0036] 3.2 Evaluation Indicators and Methods (1) Serum biochemical detection: Serum ALT and AST levels were detected (method as in Example 2).

[0037] (2) Liver pathological assessment: H&E staining and immunohistochemical staining (methods are the same as in Example 2).

[0038] (3) Detection of liver copper content (method as in Example 2).

[0039] (4) ELISA detection of liver inflammatory factors: Frozen liver tissue was collected, homogenized in pre-chilled PBS containing protease inhibitors, centrifuged at 4°C, and the supernatant was collected to determine protein concentration. The protein levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and chemokine ligand 2 (CCL2 / MCP-1) in the liver tissue homogenate were detected using an ELISA kit. The procedure was strictly performed according to the kit instructions.

[0040] 3.3 Experimental Results like Figure 5 As shown, this embodiment evaluates the effect of montelukast sodium on... Atp7b - / - Therapeutic effects and potential mechanisms of montelukast sodium treatment in mouse liver injury. Experimental results showed that after 4 weeks of continuous treatment with montelukast sodium, liver pathological damage and liver failure in mice were significantly improved. First, serum biochemical tests showed that compared with the model group given the solvent, the serum ALT and AST levels in the treatment group were significantly reduced (P<0.05), indicating a significant reduction in hepatocyte necrosis. Second, liver copper content analysis showed that although the treatment group exhibited significant hepatoprotective effects, its liver copper content was not significantly different from that of the model group (ns), strongly confirming that montelukast sodium does not exert its effects through a chelation mechanism that promotes copper excretion, but rather through other protective pathways.

[0041] Further histopathological and immunohistochemical analyses directly confirmed the aforementioned protective effects. H&E staining showed that the liver lobule structure in the treatment group tended to be intact, the necrotic foci were reduced, and the inflammatory area was significantly decreased. F4 / 80 immunohistochemical staining showed that a large number of macrophages were aggregated in the liver of the model group, while the number of F4 / 80 positive macrophages infiltrating after montelukast sodium treatment showed a significant decreasing trend.

[0042] Finally, to elucidate the molecular mechanism of reduced macrophage infiltration, ELISA was performed to detect liver inflammatory factors. The results showed that copper overload induced a strong inflammatory response in the model group's liver, manifested as abnormally elevated levels of pro-inflammatory factors (TNF-α, IL-6) and chemokine (CCL2) proteins. After treatment with montelukast sodium, the expression of these key inflammatory mediators was significantly inhibited (P<0.05). In conclusion, montelukast sodium, by blocking the CysLTs signaling pathway and inhibiting the expression of key inflammatory factors such as CCL2 and the pathological recruitment of macrophages, effectively interrupted the secondary immune-inflammatory damage caused by copper toxicity without altering the liver's copper load.

[0043] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of montelukast in the preparation of drugs for the prevention and treatment of Wilson's disease.

2. The application according to claim 1, characterized in that, Montelukast treats Wilson's disease by reducing serum ALT and AST levels, lowering liver inflammatory factor levels, and interrupting secondary immune inflammatory damage caused by copper toxicity.

3. The application according to claim 2, characterized in that, The liver inflammatory factors include TNF-α, IL-6, and CCL2.

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