Application of semeglutide in preparation of medicine for treating or preventing copper death related diseases

Abstract

The invention relates to the technical field of medicines, in particular to application of semeglutide in preparation of medicines for treating or preventing copper death related diseases. The research finds that the semeglutide can be used for inhibiting copper death of adipose tissues, livers and macrophages, and the semeglutide can be used for searching novel medicine research for targeted treatment of copper death.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to application of semaglutide in preparing medicines for treating or preventing copper-related death diseases. Background Art

[0002] Copper death is different from apoptosis, necrosis, and ferroptosis. When copper concentration is overloaded, it can lead to oxidative stress damage and cell dysfunction, mitochondrial respiratory dysfunction, and even cell death. Copper death is achieved by intracellular copper targeting lipoylated proteins and mediating the damage of iron-sulfur cluster proteins, the aggregation of lipoylated proteins, and protein toxic stress, which ultimately leads to cell death. Studies have shown that serum copper levels in obese patients are increased, which is closely related to copper homeostasis disorders. Studies have shown that the accumulation of intracellular copper is associated with the inflammatory state of macrophages. When the body develops metabolic diseases, it will lead to the infiltration of macrophages. The infiltration of macrophages is associated with copper death, indicating that copper death plays an important role in the inflammatory process of macrophages.

[0003] Semaglutide, a novel glucagon-like peptide-1 receptor agonist, has been approved in recent years for the treatment of obesity and has been shown to treat type 2 diabetes. While glucagon-like peptide-1 receptor agonists can improve adipose tissue inflammation, it remains unclear whether the pathogenesis of obesity is related to copper death, and whether semaglutide improves adipose tissue inflammation and copper death.

[0004] Therefore, this application is of great significance for developing more effective therapeutic drugs for diseases related to copper death and copper overload. Summary of the Invention

[0005] The object of the present invention is to provide the use of semaglutide in preparing a medicament for treating or preventing copper-related death diseases.

[0006] The present invention solves the technical problem by adopting the following technical solutions: In a first aspect, the present invention provides use of semaglutide in the preparation of a medicament for treating or preventing copper-related death diseases.

[0007] In one or more embodiments of the present invention, semaglutide is used as a copper death inhibitor in the preparation of a drug for treating or preventing copper death-related diseases.

[0008] In one or more embodiments of the present invention, the copper death-related disease is diabetic cardiomyopathy, Wilson's disease, Menkes disease, or metabolic dysfunction-related fatty liver disease.

[0009] Copper death and neoplastic and cancerous diseases. Tumor cells of neoplastic diseases require copper ion carriers, that is, inducing tumor cells or cancer cells to induce copper death, in order to achieve a therapeutic effect. Copper ion carriers (i.e., copper death inducers) such as elisimol and disulfiram (DSF) have been shown to increase intracellular copper ion levels and exert anti-cancer effects. The mechanism of action of copper ion carriers is to increase the production of reactive oxygen species and inhibit proteasomes, thereby producing cytotoxic effects on cancer cells and exerting a therapeutic effect. The new copper ion carrier elisimol can transport copper to mitochondria, thereby increasing oxidative stress and leading to cellular copper death. As a candidate drug, elisimol can enhance the efficacy of paclitaxel in patients with refractory solid tumors and stage IV metastatic melanoma, induce cell copper death and exert a therapeutic effect.

[0010] Copper death and non-tumor diseases. If copper death occurs in non-tumor diseases, copper ion chelators, also known as copper death inhibitors, or drugs are needed to reduce copper death in order to achieve a therapeutic effect. Therefore, the treatment directions of the two are opposite.

[0011] (1) Copper death and diabetic cardiomyopathy (DCM) Epidemiological studies have revealed a significant association between copper overload and diabetes risk. In patients with diabetes and diabetic cardiomyopathy (DCM), elevated circulating and extracellular copper levels are accompanied by decreased intracellular copper and increased systemic and cardiac total copper content, which is associated with the accumulation of reactive oxygen species. This suggests that dysregulated copper homeostasis induces copper death, which is involved in the progression of DCM. Tetrathiomolybdate (TTM) restores copper homeostasis in the heart of DCM by restoring the expression and localization of copper transporters and copper-binding proteins, promoting copper excretion and improving cardiac function in DCM rats. This drug can promote the excretion of circulating and extracellular copper ions, ameliorating copper death caused by dysregulated copper homeostasis. However, it has significant side effects, necessitating the development and identification of new inhibitors of copper death.

[0012] (2) Copper death and genetic diseases Genetic diseases include Wilson's disease and Menkes disease. Wilson's disease is the most typical example of newly discovered copper death. Wilson's disease is an autosomal recessive inherited disorder primarily caused by abnormal accumulation of copper ions in various tissues, leading to copper metabolism dysfunction. This in turn causes copper toxicity in multiple organs, including the liver and brain, and ultimately copper death in multiple organs and tissues. Menkes disease is a more typical example of copper death. Menkes disease is an X-linked recessive inherited copper metabolism disorder caused by mutations in the ATP7A gene, resulting in copper absorption and transport defects and subsequent copper death. This in turn causes severe neurodegeneration, connective tissue abnormalities, and growth and development disorders.

[0013] Copper mortality and metabolic dysfunction-associated fatty liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) Mitochondrial oxidative stress and inflammation triggered by excess lipids in hepatocytes are the pathogenesis of MASLD. Copper homeostasis plays a crucial role in lipid metabolism, redox processes, and other processes. Consequently, copper homeostasis imbalance triggers cytotoxic responses through oxidative stress and inflammation, and these mechanisms are closely associated with the development and progression of MASLD. Copper-induced cell death leads to MASLD, which progresses to metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and liver cancer. Therefore, targeted regulation of copper homeostasis offers a promising strategy for slowing the progression of MASLD. The role of copper death in MASLD requires understanding how copper overload interacts with dyslipidemia and insulin resistance—two hallmark features of MASLD—highlighting the potential of copper regulation as a therapeutic avenue.

[0014] In one or more embodiments of the present invention, the semaglutide is used in the preparation of a drug for inhibiting copper death in adipose tissue.

[0015] The purpose of this study is to provide a new approach for the treatment of semaglutide and to prepare drugs for inhibiting copper death in adipose tissue. The present invention experimentally verifies that copper death is involved in the obesity process and that semaglutide alleviates obesity-induced copper death.

[0016] In this study, the copper death mentioned above refers to copper death induced by a high-fat diet or copper death induced by palmitic acid (PA).

[0017] During the study, it was found that semaglutide reduces weight, and its effect is mainly through reducing subcutaneous adipose tissue and visceral white adipose tissue.

[0018] The present invention has found that semaglutide improves the function of adipose tissue mitochondria, increases the activity of mitochondrial complexes, and increases tissue heat production. It produces heat by increasing the activity of mitochondrial complex I and mitochondrial complex II in subcutaneous adipose tissue.

[0019] The present study found that semaglutide has the effect of inhibiting copper death in adipose tissue. A high-fat diet induces copper death in obese adipose tissue, reducing the expression levels of copper death protein ferredoxin 1 (FDX1) and dihydrolipoamide S-acetyltransferase (DLAT), while semaglutide increases the expression levels of copper death-related proteins FDX1 and DLAT in adipose tissue, thereby inhibiting the occurrence of copper death.

[0020] In one or more embodiments of the present invention, the semaglutide is used in the preparation of a drug for resisting macrophage copper apoptosis.

[0021] The present study found that semaglutide reduces the phenotype of pro-inflammatory macrophages, mainly manifested by reducing the expression level of M1 macrophage marker CD86.

[0022] The present study found that semaglutide has the ability to protect macrophages from the toxic effects of copper ions, mainly by reducing cellular copper ion levels. The copper death inhibitor (TTM) can reverse copper death, and the copper death inducer (ES) verifies that semaglutide reduces copper death.

[0023] The present invention has the effect of resisting macrophage copper death. In this study, it was found that palmitic acid (PA) significantly induced macrophage copper death, mainly by reducing the expression levels of FDX1 and DLAT. Semaglutide can increase the expression levels of FDX1 and DLAT, and has a significant effect of resisting macrophage copper death.

[0024] In one or more embodiments of the present invention, the semaglutide is used for preparing a drug for reducing copper death in fatty liver disease associated with metabolic dysfunction.

[0025] The present invention also demonstrates that semaglutide reduces copper death in metabolic dysfunction-associated fatty liver disease (MASLD), mainly by increasing the expression of copper death proteins FDX1 and DLAT, thereby inhibiting the occurrence of liver copper death and improving MASLD.

[0026] Therefore, the present application provides the pharmaceutical use of semaglutide to inhibit copper death in adipose tissue and macrophages, which is used to find new drugs for the targeted treatment of copper death.

[0027] In one or more embodiments of the present invention, the copper death is high-fat diet-induced copper death or palmitic acid-induced copper death.

[0028] In one or more embodiments of the present invention, the copper death is palmitic acid-induced copper death.

[0029] The present invention includes the following beneficial effects: This study discovered the medicinal use of semaglutide in inhibiting copper death in adipose tissue, liver and macrophages, and is used for research on finding new drugs for the targeted treatment of copper death. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the body weight change curves of the three groups of mice during 21 weeks.

[0031] Figure 2 The changes in adipose tissue weight in mice.

[0032] Figure 3 is the expression level of lipid metabolism factors in mouse adipose tissue.

[0033] Figure 4ELISA results of mitochondrial activity in mouse adipose tissue.

[0034] Figure 5 Changes in the expression level of copper-induced death proteins in adipose tissue.

[0035] Figure 6 Changes in the expression levels of M1 macrophage markers in adipose tissue.

[0036] Figure 7 is the expression level of copper death protein in liver tissue.

[0037] Figure 8 It is a copper death inhibitor and inducer that intervenes in the copper ion level of macrophages.

[0038] Figure 9 The expression levels of copper death proteins and copper ion levels in macrophages.

[0039] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0040] As used herein, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease. DETAILED DESCRIPTION

[0041] While the present invention specification describes specific embodiments in detail, those skilled in the art should recognize that the following embodiments are illustrative and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that improvements and modifications to the present invention, without departing from the principles of the present invention, will be made, and that the resulting technical solutions will fall within the scope of the claims. The following examples illustrate the beneficial effects of the present invention.

[0042] Example 1 Animal experiment on semaglutide alleviating copper death in adipose tissue

[0043] This study investigated the effect of semaglutide on adipose tissue copper death in vivo. 1. Experimental Animals Sixty male C57BL / 6J mice, weighing approximately 19 g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. Light and dark cycles alternated every 12 h, with free access to food and water. All experiments were approved by the Ethics Committee of Shihezi University School of Medicine.

[0044] 2. Experimental Grouping The mice were randomly divided into three groups: normal group (NC group, n=20), high-fat diet group (HFD group, n=20), and semaglutide group (HFD+sema group, n=20), where sema was semaglutide.

[0045] ① Normal group (NC group): After being fed with a normal diet for 16 weeks, the rats were subcutaneously injected with normal saline every three days for 5 weeks.

[0046] ② High-fat diet group (HFD group): After being fed a high-fat diet (Research Diets, USA) for 16 weeks, normal saline was injected subcutaneously every three days for 5 weeks.

[0047] ③Semaglutide group (HFD+sema group): After being fed with a high-fat diet for 16 weeks, semaglutide was subcutaneously injected for 5 weeks (10 noml / kg / 3d).

[0048] 3. Index detection 3.1 During this process, the weight changes of mice were detected. Figure 1 After being fed with a normal diet and a high-fat diet, the HFD group had a significant increase in body weight, and after 5 weeks of semaglutide intervention, their body weight decreased significantly.

[0049] 3.2 The mice were sacrificed, their white adipose tissue was extracted, and the changes in the weight of adipose tissue (subcutaneous adipose tissue and visceral adipose tissue) were detected. Figure 2 After semaglutide intervention, the weight of subcutaneous adipose tissue and visceral adipose tissue was significantly reduced, and the weight loss effect was obvious.

[0050] 3.3 PCR detection of the expression levels of lipid metabolism factors carnitine palmitoyltransferase 1A (Cpt1a) and fatty acid synthase (FAS) in adipose tissue of mice. After high-fat induction, the expression levels of Cpt1a and Fas in adipose tissue increased significantly, and adipogenesis increased. After semaglutide intervention (HFD+sema group), the expression levels of Cpt1a and Fas decreased significantly, and adipogenesis decreased. The results are attached. Figure 3 .

[0051] 3.4 Elisa was used to detect changes in mitochondrial activity in adipose tissue. Figure 4 Compared with the NC group, the activity of mitochondrial complex I and mitochondrial II in the HFD group was decreased, reducing energy metabolism and heat production. Compared with the HFD group, the activity of mitochondrial complex I and II in the HFD+sema group was significantly increased, increasing energy metabolism and heat production.

[0052] 3.5 Western blot was used to detect the expression levels of copper death proteins FDX1 and DLAT in adipose tissue. Compared with the NC group, the expression levels of FDX1 and DLAT in the HFD group were decreased, indicating that obesity induced copper death. Compared with the HFD group, the expression levels of FDX1 and DLAT in the HFD+sema group were increased, indicating that the copper death process was inhibited and the occurrence of copper death was reduced. The results are attached. Figure 5 .

[0053] 3.6 Immunofluorescence analysis of the expression of CD86, a marker of adipose tissue macrophages. Compared with the HFD group, the HFD+sema group showed decreased expression of CD86, a marker of M1 macrophages. This suggests that the drug exerts an anti-inflammatory effect by reducing pro-inflammatory macrophages.

[0054] 4. Results Analysis Figure 1 The weight change curves of the three groups of mice over 21 weeks show that semaglutide has a weight-reducing effect. Compared with the HFD group, the weight of mice was significantly reduced after 5 weeks of semaglutide intervention. * represents P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0055] Figure 2 The changes in adipose tissue weight in mice. Compared with the NC group, the subcutaneous adipose tissue and visceral adipose tissue in the HFD group increased significantly; compared with the HFD group, the subcutaneous adipose tissue and visceral adipose tissue in the HFD+sema group decreased significantly. * represents P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0056] Figure 3 The expression levels of lipid metabolism factors in adipose tissue of mice. Compared with the NC group, the expression levels of carnitine palmitoyltransferase 1A (Cpt1a) and fatty acid synthase (FAS) in the HFD group increased; compared with the HFD group, the expression levels of carnitine palmitoyltransferase 1A (Cpt1a) and fatty acid synthase (FAS) in the HFD+sema group decreased. * represents P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0057] Figure 4 The results of ELISA in mouse adipose tissue showed that the activities of mitochondrial complex I and II in the HFD group were significantly decreased compared with the NC group, and significantly increased compared with the HFD+sema group. P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0058] Figure 5The expression levels of copper-induced death proteins in adipose tissue were changed. Compared with the NC group, the expression levels of copper-induced death proteins FDX1 and DLAT in the HFD group were decreased. Compared with the HFD group, the expression levels of copper-induced death proteins FDX1 and DLAT in the HFD+sema group were increased. * represents P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0059] Figure 6 The expression level of M1 macrophage markers in adipose tissue was changed. Compared with the HFD group, the expression of M1 macrophage marker CD86 in the HFD+sema group was significantly reduced. * represents P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0060] Example 2 Animal experiment on semaglutide alleviating liver copper death

[0061] 1. Experimental Animals Thirty-six male C57BL / 6J mice, weighing approximately 19 g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. Light and dark cycles alternated every 12 h, with free access to food and water. All experiments were approved by the Ethics Committee of Shihezi University School of Medicine.

[0062] 2. Experimental Grouping The above mice were randomly divided into three groups: normal group (NCD group, n=12), metabolic dysfunction-associated fatty liver disease group (MASLD group, n=12), and semaglutide group (MASLD+sema group, n=12), where sema was semaglutide.

[0063] ① Normal group (NCD group): After being fed with a normal diet for 18 weeks, the rats were subcutaneously injected with normal saline every three days for 5 weeks.

[0064] ② Metabolic dysfunction-associated fatty liver disease group (MASLD group): After being fed a high-fat diet (Research Diets, USA) for 18 weeks, the metabolic dysfunction-associated fatty liver disease model was successfully induced, and then normal saline was subcutaneously injected every three days for 5 weeks.

[0065] ③Semaglutide group (MASLD+sema group): After 18 weeks of high-fat diet feeding, the metabolic dysfunction-related fatty liver disease model was successfully induced, and then semaglutide was subcutaneously injected for 5 weeks (10 noml / kg / 3d).

[0066] 3.Indicator detection: 3.1 Oil red staining was used to detect the lipid content in the liver. Compared with the NCD group, the lipid content in the liver of the MASLD group was significantly increased. Compared with the MASLD group, the lipid content in the liver of the MASLD+sema group was significantly decreased. Semaglutide reduced liver lipid deposition.

[0067] 3.2 Western blot was used to detect the expression levels of liver copper death proteins FDX1 and DLAT. Compared with the NCD group, the expression levels of FDX1 and DLAT in the MASLD group were decreased, inducing the occurrence of copper death. Compared with the MASLD group, the expression levels of FDX1 and DLAT in the MASLD+sema group were increased, the copper death process was inhibited, and the occurrence of copper death was reduced.

[0068] 4. Results Analysis Figure 7 Compared with the NCD group, the liver lipid content in the MASLD group was significantly increased, and the expression levels of FDX1 and DLAT were decreased; compared with the MASLD group, the liver lipid content in the MASLD+sema group was significantly decreased, and the expression levels of FDX1 and DLAT were increased. * represents P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0069] Example 3 Cell experiment on semaglutide alleviating copper-induced death

[0070] 1. Experimental Materials Mouse RAW264.7 macrophages were purchased from Wuhan Sewell Technology Co., Ltd. RAW264.7 macrophages were cultured with 90% culture medium and 10% fetal bovine serum, streptomycin, and penicillin in a 37° incubator and passaged for subsequent experiments.

[0071] 2. Intervention with copper death inhibitors and inducers The cells were divided into NC group, PA (palmitic acid) group, PA+TTM group, PA+sema (50 nmol / L) group, and PA+sema+ES group. TTM (tetrathiomolybdate), a classic copper-induced cell death inhibitor, was administered at a concentration of 50 μmol / L; ES (elisimol), a classic copper-induced cell death inducer, was administered at a concentration of 10 nmol / L; and sema (semaglutide) was administered.

[0072] After the intervention, the cell supernatant of each group was collected for subsequent copper ion detection. Figure 8 Copper ion levels in the NC group, PA group, PA+TTM group, PA+sema (50 nmol / L) group, and PA+sema+ES group.

[0073] 3. Drug intervention The cells were divided into NC group, PA group, PA+sema (10 nmol / L) group, PA+sema (50 nmol / L) group, and PA+sema (100 nmol / L) group.

[0074] After cell culture, the original culture medium was removed and the cells were washed three times with phosphate buffer. Complete culture medium was added to the NC group, palmitic acid solution (100 umol / L) was added to the PA group, and different concentrations of the drug (10 nmol / L, 50 nmol / L, and 100 nmol / L) were added to the PA+sema groups for pretreatment for 24 h.

[0075] After 24 hours, the culture medium was washed away and complete culture medium was added to the NC group, while palmitic acid (100 μmol / L) was added to the PA group and the PA+sema group. Cells and supernatants were collected for subsequent copper death protein detection. Figure 9 Figure 3. Expression levels of copper-induced death proteins and copper ion levels in the NC, PA, and PA+sema groups. Compared with the NC group, the PA group showed decreased expression of FDX1 and DLAT, increased copper ion levels, and induced copper death. Compared with the PA group, the PA+sema groups showed increased expression of FDX1 and DLAT, decreased copper ion levels, and inhibited copper death.

[0076] 4. Experimental Results Figure 8 The copper concentration levels of macrophages after the addition of copper death inhibitors and inducers. Compared with the NC group, the copper ion level in the PA group increased; compared with the PA group, the copper ion levels in the PA+TTM group and the PA+sema group decreased; compared with the PA+sema group, the copper ion level in the PA+sema+ES group increased. * represents P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0077] Figure 9 The expression levels of copper death proteins and copper ion levels in macrophages. Compared with the NC group, the expression levels of copper death proteins FDX1 and DLAT in the PA group were decreased, and the copper ion level was increased. Compared with the PA group, the expression levels of copper death proteins FDX1 and DLAT in the PA+sema groups were increased, and the copper ion level was decreased. * represents P <0.05, ** represents P <0.01, *** represents P Value < 0.001.

[0078] As shown in the figure, decreased expression of macrophage copper death proteins, FDX1 and DLAT, indicates copper death, while increased FDX1 and DLAT alleviate copper death. Copper ion levels were also measured in the NC, PA, and PA+sema groups. Copper ion levels were elevated in the PA group, while decreased in the PA+sema group.

[0079] In this experiment, the NC group, PA group, PA+TTM group, PA+sema group, and PA+sema+ES group were compared. The PA+TTM group was compared with the PA+sema group. TTM is a copper death inhibitor, and PA is an inducer of copper death. After adding TTM and semaglutide, the copper ion level was significantly reduced compared with the PA group. In fact, it was a comparison of the effects of the copper death inhibitor and semaglutide. After adding ES (ilisimol) copper death inducer, the PA+sema and PA+sema+ES groups were compared. The copper ion concentration in the PA+sema+ES group increased, which was actually a reverse (bidirectional) verification of the effect of semaglutide.

[0080] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principles of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of semaglutide in the preparation of drugs for treating or preventing copper-related death diseases.

2. The use according to claim 1, characterized in that The invention relates to the use of semaglutide as a copper death inhibitor in the preparation of drugs for treating or preventing copper death-related diseases.

3. The use according to claim 1 or 2, characterized in that The copper death-related disease is diabetic cardiomyopathy, Wilson's disease, Menkes disease or metabolic dysfunction-related fatty liver disease.

4. The use according to claim 1, characterized in that The application of semaglutide in preparing a drug for inhibiting copper death in adipose tissue.

5. The use according to claim 1, characterized in that The application of semaglutide in preparing a drug for resisting macrophage copper death.

6. The use according to claim 1, characterized in that The application of semaglutide in the preparation of a drug for reducing copper death in fatty liver disease associated with metabolic dysfunction.

7. The use according to claim 4, characterized in that The copper death is copper death induced by a high-fat diet or copper death induced by palmitic acid.

8. The use according to claim 5 or 6, characterized in that The copper death is palmitic acid-induced copper death.

9. The use according to claim 4 or 5, characterized in that The semaglutide inhibits tissue copper death by increasing the expression of FDX1 and DLAT in tissues.

10. The use according to claim 6, characterized in that Semaglutide inhibits copper death in liver tissue by increasing the expression of FDX1 and DLAT in liver tissue.

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