Use of sulfur-containing amino acids or metabolites thereof for the preparation of a medicament for the treatment of wilson's disease or for the improvement of hepatic copper accumulation

Abstract

The application discloses application of sulfur-containing amino acids or metabolites thereof in preparation of a medicine for treating Wilson disease or improving liver copper accumulation, and relates to the technical field of biological medicines. The application specifically discloses that the sulfur-containing amino acids or metabolites thereof inhibit copper death, maintain the function of an iron-sulfur cluster assembly system, maintain the stability and / or expression level of Fe-S proteins, and correct metabolic disorders of sulfur-containing amino acids caused by copper overload, and can improve liver function indexes and liver pathological damage of Wilson disease patients. The application discloses, for the first time, a new mechanism that copper death can be rescued by supplementing sulfur-containing amino acids and metabolites thereof, directly aims at a core link of liver damage of Wilson disease, serves as a dietary nutrient component, has no accumulation toxicity when taken for a long time, avoids serious adverse reactions of existing medicines such as penicillamine and trientine, and provides a new technical platform for research and development of medicines for treating Wilson disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of sulfur-containing amino acids or their metabolites in the preparation of drugs for treating Wilson's disease or improving hepatic copper accumulation. Background Technology

[0002] Wilson's disease (WD) is an autosomal recessive genetic disorder caused by a mutation in the ATP7B gene, leading to impaired copper transport and abnormal accumulation of copper in tissues such as the liver and brain. Current clinical treatment primarily relies on copper chelators, but these treatments suffer from limited efficacy, significant side effects, and unclear long-term safety. For example, D-Penicillamine, as a first-line treatment, reduces the body's copper load by promoting urinary copper excretion, but its clinical application is severely limited: approximately 30% of patients are forced to discontinue the drug due to adverse reactions such as rash, fever, bone marrow suppression, and proteinuria; more seriously, 10%-50% of patients with neurological symptoms experience irreversible worsening of neurological symptoms in the early stages of treatment. Trientine, as an alternative drug, has a relatively lower incidence of serious adverse reactions, but it still has side effects such as bone marrow suppression and gastrointestinal reactions, and its accessibility is poor and its price is high.

[0003] Recent studies have revealed that copper overload can induce a novel form of cell death—copper death—characterized by oligomerization of mitochondrial lipoyl-modified proteins and loss of iron-sulfur (Fe-S) proteins. This provides new insights into elucidating the pathogenesis of disease-related liver disease (WD) and developing novel, safe, and effective therapeutic drugs. Therefore, developing novel drugs that can directly promote copper excretion, improve liver pathology, and regulate copper death-related pathways has significant clinical implications. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of the prior art by providing the use of sulfur-containing amino acids or their metabolites in the preparation of medicaments for treating Wilson's disease or improving hepatic copper accumulation.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of sulfur-containing amino acids or their metabolites in the preparation of medicaments for treating Wilson's disease or improving hepatic copper accumulation, wherein the sulfur-containing amino acids include methionine and / or cysteine, and the metabolites of the sulfur-containing amino acids include S-adenosylmethionine.

[0007] Preferably, the drug works by inhibiting copper death.

[0008] Preferably, the inhibition of copper death includes:

[0009] (a) To maintain the functionality of the iron-sulfur cluster assembly system;

[0010] (b) Maintain the stability and / or expression level of Fe-S protein;

[0011] (c) Correct the metabolic disorder of sulfur-containing amino acids caused by copper overload.

[0012] Preferably, copper overload leads to metabolic disorders of sulfur-containing amino acids, which in turn causes the loss of iron-sulfur proteins; the sulfur-containing amino acids or their metabolites maintain the function of the iron-sulfur cluster assembly system by supplementing the sulfur source, thereby rescuing copper death and thus treating Wilson's disease or improving liver copper accumulation.

[0013] Preferably, the drug is used to improve liver function indicators in patients with Wilson's disease, including reducing serum ALT and AST levels.

[0014] Preferably, the drug is used to improve liver pathological damage in patients with Wilson's disease, including improving liver tissue structure and reducing liver fibrosis.

[0015] In a second aspect, the present invention provides a pharmaceutical composition for treating Wilson's disease or improving hepatic copper accumulation, comprising an effective amount of at least one selected from sulfur-containing amino acids, their metabolites or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier; wherein the sulfur-containing amino acid is selected from methionine and / or cysteine, and the metabolite is S-adenosylmethionine.

[0016] Thirdly, the present invention provides a method for screening candidate substances for treating Wilson's disease, the method comprising the following steps:

[0017] Under copper overload conditions, the candidate material was brought into contact with cells, and then the expression level of the iron-sulfur cluster assembly system protein or the stability of the Fe-S protein in the cells was detected.

[0018] Among them, candidate substances that upregulate the expression of iron-sulfur cluster assembly system proteins or improve the stability of Fe-S proteins suggest that they may be used to treat Wilson's disease.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention reveals for the first time that metabolic disorders of sulfur-containing amino acids are a key link in copper overload-induced cell death, and based on this discovery, provides new applications for sulfur-containing amino acids and their metabolites in the preparation of drugs for treating Wilson's disease and improving hepatic copper accumulation. Experimental results show that S-adenosylmethionine intervention can significantly reduce serum transaminase levels in Wilson's disease model mice, improve liver function, and reduce hepatic copper accumulation. Liver histopathological staining further confirms that it can improve liver tissue structural disorders and reduce the degree of liver fibrosis. At the mechanistic level, this invention demonstrates that sulfur-containing amino acids and their metabolites block the occurrence and development of copper death at its source by maintaining the function of the iron-sulfur cluster assembly system and stabilizing Fe-S protein expression. This mechanism of action is not possessed by existing copper chelators. Compared with existing therapeutic drugs, the sulfur-containing amino acids and their metabolites used in this invention are normal dietary nutrients, which have advantages such as long-term use, convenient administration, and low price. They avoid the serious side effects such as bone marrow suppression and neurological deterioration commonly caused by drugs such as penicillamine, significantly improving drug safety and patient compliance.

[0021] In summary, this invention provides a novel mechanism and a safe and effective intervention strategy for the treatment of Wilson's disease, and has significant clinical translational value. Attached Figure Description

[0022] Figure 1 This is the result of the rescue effect of methionine on copper death.

[0023] Figure 2 The results show the rescue effects of S-adenosylmethionine (SAM) on copper death and maintenance of Fe-S clusters. In A, different concentrations of AG-270 promote copper death induced by Es / Cu in Huh-7 cells; in B, the intracellular SAM content decreases after AG-270 treatment; in C, the rescue effect of exogenous SAM (0.5, 1, 2 mM) on copper death is not blocked by AG-270; in D, both copper overload and AG-270 downregulate the expression of proteins FXN, NFS1, and NFU1 required for iron-sulfur cluster assembly; and in E, SAM maintains the protein expression levels of FXN, NFS1, and NFU1 in copper overload-treated cells.

[0024] Figure 3 This is the result of the salvage effect of cysteine ​​on copper death.

[0025] Figure 4 SAM vs ATP7B - / - Effects of rhodamine staining on liver function and liver copper content in Wilson's disease mice, where A represents serum ALT levels in each group; B represents serum AST levels in each group; C represents liver tissue copper content in each group; and D represents rhodamine staining results.

[0026] Figure 5 SAM vs ATP7B - / -The ameliorative effect on mouse liver histopathology, where A is the result of HE staining of liver tissue (×200) and B is the result of Masson trichrome staining (×200).

[0027] Figure 6 SAM vs ATP7B - / - Effects of the iron-sulfur cluster assembly system and Fe-S proteins on mouse liver, where A represents the expression level of key proteins (FXN, NFS1, NFU1) in the iron-sulfur cluster assembly system, and B represents the expression level of Fe-S proteins (FDX1, ACO2, PLOD1). Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] This invention uses ATP7B gene knockout (ATP7B) as an example. - / - Mice were used as a Wilson's disease (WD) model and administered oral SAM via gavage daily for 4 weeks, with a saline gavage group serving as a control. The results showed that SAM intervention significantly reduced serum transaminase (ALT and AST) levels, promoted liver function recovery, and reduced hepatic copper accumulation, suggesting a copper excretion-promoting effect. HE staining of liver tissue showed that SAM treatment improved liver tissue structural disorder; Masson staining further indicated that SAM reduced collagen deposition and alleviated liver fibrosis. In addition, SAM treatment upregulated the expression of iron-sulfur cluster assembly system-related proteins in the liver of WD mice and increased Fe-S protein levels.

[0030] The following is a detailed description. In the embodiments of the present invention, unless otherwise specified, all materials are commercially available.

[0031] Example 1: Sulfur-containing amino acids and S-adenosylmethionine rescue copper death

[0032] Copper accumulation is the pathological basis of WD, and copper overload is the direct cause of cellular copper death. Therefore, methods to salvage cellular copper death have reference and guiding significance for the treatment of WD. In this embodiment, the Huh-7 liver cancer cell line, which is common in this field, was used as a model to detect the effects of sulfur-containing amino acids and metabolites on copper death.

[0033] 1.1 Methionine rescues copper death

[0034] In methionine-free medium, copper-induced cell death in Huh-7 cells was induced by treatment with a equiproportionate mixture of 250 nM elesclomol (Es) and copper chloride (CuCl2, Cu), with DMSO as the solvent control. Different concentrations of methionine were added as intervention conditions. The standard cell culture medium contained 30 mg / L of methionine. Cell viability was assessed by CCK8 assay after 72 hours of culture in methionine-free medium with different concentrations of methionine added, observing the effects of methionine deprivation and addition on cell viability.

[0035] The results are as follows Figure 1 As shown, compared with the 30 mg / L methionine group, methionine deprivation promoted copper death, while 60 mg / L methionine significantly rescued copper death.

[0036] 1.2 SAM Saves Copper from Death

[0037] Different concentrations (0.5, 1, 2, and 4 μM) of the MAT2A inhibitor AG-270 were added to normal culture medium. A SAM intervention group was established, receiving 500 μM SAM in addition to induction. A combined SAM+AG-270 intervention group received both 500 μM SAM and 20 μM AG-270. After 24 hours of treatment, cell viability was assessed using the CCK-8 assay, and intracellular SAM content was detected using liquid chromatography-mass spectrometry. The results were then statistically analyzed.

[0038] The results are as follows Figure 2 As shown. Figure 2 The A in the figure indicates that AG-270 promotes copper death, while Figure 2 The B in the figure indicates that AG-270 reduces intracellular SAM content. Figure 2 The "C" in the figure indicates that SAM rescues copper from death, and its rescue effect is not blocked by AG-270.

[0039] 1.3 Cysteine ​​rescues copper death

[0040] Huh-7 cells were treated for 24 h with 250 nM Es / Cu, 200 μM cysteine ​​(cys), or a combination of both. Cell viability was determined by the CCK-8 assay.

[0041] The results are as follows Figure 3 As shown, exogenous cysteine ​​supplementation can significantly inhibit copper-induced cell death.

[0042] Example 2: Investigation into the therapeutic effects and mechanisms of S-adenosylmethionine on Wilson's disease

[0043] Twenty-four ATP7B mice, 12 weeks old, half male and half female, weighing 25-35 g, were selected.- / - Mice (provided by the Animal Center of Hangzhou Normal University) were randomly divided into two groups as a Wilson's disease model: the experimental group was administered SAM (50 mg / kg and 100 mg / kg body weight) orally daily, while the control group was administered an equal volume of physiological saline by gavage, for 4 consecutive weeks. After the intervention, serum samples were collected to detect ALT and AST; liver tissue was taken to determine copper content; HE staining and Masson staining were performed to observe pathological changes; Western blotting was used to detect the expression of proteins such as FDX1, LIAS, DLAT, FXN, NFS1, and NFU1; and immunohistochemical analysis was used to analyze the expression and localization of ACO2 and PLOD1.

[0044] The results are as follows Figure 4 As shown, SAM reduced the levels of transaminases ALT and AST, indicating improved liver function. Simultaneously, SAM reduced hepatic copper accumulation, suggesting that SAM promotes copper excretion through an ATP7B-independent mechanism.

[0045] The results are as follows Figure 5 As shown, SAM improved liver tissue morphology and reduced liver fibrosis. These results clarify, at the whole-animal level, that SAM has the function of rescuing copper death.

[0046] like Figure 6 As shown, SAM treatment upregulated the protein levels of iron-sulfur cluster assembly proteins FXN, NFS1, and NFU1. Immunohistochemical results showed that SAM treatment increased the expression of Fe-S proteins ACO2 and PLOD1. These results at the whole-animal level indicate that SAM plays a role in rescuing copper death by restoring iron-sulfur cluster assembly and maintaining Fe-S protein homeostasis.

Claims

1. The use of sulfur-containing amino acids or their metabolites in the preparation of drugs for treating Wilson's disease or improving hepatic copper accumulation, characterized in that, The sulfur-containing amino acids include methionine and / or cysteine, and the metabolites of the sulfur-containing amino acids include S-adenosylmethionine.

2. The application according to claim 1, characterized in that, The drug works by inhibiting copper death.

3. The application according to claim 2, characterized in that, The inhibition of copper death includes: (a) To maintain the functionality of the iron-sulfur cluster assembly system; (b) Maintain the stability and / or expression level of Fe-S protein; (c) Correct the metabolic disorder of sulfur-containing amino acids caused by copper overload.

4. The application according to claim 3, characterized in that, Copper overload leads to metabolic disorders of sulfur-containing amino acids, which in turn causes the loss of iron-sulfur proteins. The sulfur-containing amino acids or their metabolites maintain the function of the iron-sulfur cluster assembly system by supplementing the sulfur source, thereby rescuing copper death and treating Wilson's disease or improving hepatic copper accumulation.

5. The application according to claim 1, characterized in that, The drug is used to improve liver function indicators in patients with Wilson's disease, including reducing serum ALT and AST levels.

6. The application according to claim 1, characterized in that, The drug is used to improve liver pathological damage in patients with Wilson's disease, including improving liver tissue structure and reducing liver fibrosis.

7. A pharmaceutical composition for treating Wilson's disease or improving hepatic copper accumulation, characterized in that, It contains an effective amount of at least one selected from sulfur-containing amino acids, their metabolites, or pharmaceutically acceptable salts thereof; wherein the sulfur-containing amino acid is selected from methionine and / or cysteine, and the metabolite is S-adenosylmethionine.

8. The pharmaceutical composition according to claim 7, characterized in that, It also includes pharmaceutically acceptable carriers.

9. A method for screening candidate substances for treating Wilson's disease, characterized in that, The method includes the following steps: Under copper overload conditions, candidate substances were added to cell culture medium, and then the expression levels of iron-sulfur cluster assembly system proteins or the stability of Fe-S proteins in the cells were detected. Among them, candidate substances that upregulate the expression of iron-sulfur cluster assembly system proteins or improve the stability of Fe-S proteins suggest that they may be used to treat Wilson's disease.

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