Use of semaglutide in the preparation of a medicament for the prevention or treatment of autoimmune hepatitis

Abstract

The application belongs to the technical field of medicine. More particularly, it relates to application of semaglutide in preparation of a medicine for preventing or treating autoimmune hepatitis. The application proves for the first time through animal experiments that semaglutide can significantly reduce the serum ALT and AST levels of a mouse model of autoimmune hepatitis, effectively reduce the necrotic area of liver tissue and reduce inflammatory cell infiltration, and significantly down-regulate the expression of pro-inflammatory cytokines in serum, thereby confirming the effect of semaglutide in treating autoimmune hepatitis and effectively overcoming the defect of impaired immune function caused by non-specific immunosuppression in traditional therapy. Semaglutide has high safety and no hepatotoxicity, is suitable for long-term maintenance therapy, and as an old drug with new use, semaglutide has clear pharmacology and safety data, can greatly shorten the research and development cycle and reduce the conversion risk, and has wide clinical feasibility and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. More specifically, it relates to the use of smegglutide in the preparation of drugs for the prevention or treatment of autoimmune hepatitis. Background Technology

[0002] Autoimmune hepatitis (AIH) is a chronic hepatitis caused by the immune system's abnormal attack on liver cells. It can lead to liver inflammation, fibrosis, and even cirrhosis. Untreated AIH can cause liver failure and death in most patients within five years. Its incidence and prevalence are increasing globally. Because the pathogenesis is not fully understood, current clinical treatment for AIH mainly relies on glucocorticoids and immunosuppressive drugs. However, this approach is essentially broad-spectrum, non-specific immunosuppression. Long-term use not only easily leads to significant adverse reactions (such as glucocorticoid-related osteoporosis and increased risk of infection, and azathioprine-induced bone marrow suppression), but also discontinuation or dose reduction can cause significant adverse reactions and dependence, increasing the likelihood of AIH relapse. Therefore, there is an urgent clinical need to explore new treatment strategies with higher safety.

[0003] Smegglutide is a GLP-1R agonist commonly used clinically for obesity, diabetes, and liver-associated metabolic syndrome. Recent studies have demonstrated its therapeutic potential for various metabolic diseases. GLP-1Rs are widely distributed on the surface of various tissues and cells. Smegglutide, by specifically activating this receptor, exhibits significant efficacy in regulating glucose and lipid metabolism homeostasis, reducing oxidative stress, and improving insulin sensitivity. Recent research has further confirmed that smegglutide can effectively improve metabolic dysfunction-associated steatohepatitis (MASH), showing great promise in the prevention and treatment of metabolic liver diseases.

[0004] Although both AIH and MASH present as chronic liver injury and inflammatory response, they differ fundamentally in their disease nature, core driving mechanisms, and clinical medication logic, leading to completely different treatment options. The exact pathogenesis of AIH is not fully understood, but it is characterized by T-cell infiltration, interface hepatitis, and autoantibody production, with immunosuppression being the core of its treatment. MASH, on the other hand, is driven by insulin resistance, lipid metabolism disorders, and secondary metabolic inflammation, with metabolic regulation and weight loss being the core of its treatment. Due to these drastically different pathological mechanisms, clinical drug intervention strategies for the two are entirely different: AIH primarily uses glucocorticoids and immunosuppressants, while MASH utilizes metabolic regulators such as GLP-1R agonists, insulin sensitizers, and lipid-lowering drugs. Current technologies generally strictly limit the scope of action of semaglutide to the field of metabolic diseases.

[0005] Currently, there are no publicly available reports on the use of smegglutinin for autoimmune hepatitis in existing technologies, and its therapeutic effects and potential mechanisms of action in this disease remain a technological gap in this field. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of existing clinical treatment drugs for autoimmune hepatitis and to provide the application of smegglutinin in the preparation of drugs for the prevention or treatment of autoimmune hepatitis.

[0007] The first objective of this invention is to provide the use of smegglutinin in the preparation of drugs for the prevention or treatment of autoimmune hepatitis.

[0008] A second objective of this invention is to provide the use of smegglutinin in the preparation of medicaments for the prevention or treatment of symptoms associated with autoimmune hepatitis.

[0009] A third objective of this invention is to provide a drug for treating autoimmune hepatitis.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides the use of smegglutinin in the preparation of drugs for the prevention or treatment of autoimmune hepatitis.

[0011] As a specific feasible implementation, the present invention provides the use of smegglutinin as the sole active ingredient in the preparation of a drug for the prevention or treatment of autoimmune hepatitis.

[0012] Specifically, the aforementioned autoimmune hepatitis includes acute autoimmune hepatitis or chronic autoimmune hepatitis.

[0013] Specifically, acute autoimmune hepatitis (acute AIH) includes two main categories: ① autoimmune hepatitis with no history of chronic hepatitis, presenting with acute liver injury as the initial symptom; ② autoimmune hepatitis presenting as chronic hepatitis, which may be an acute exacerbation or worsening, or even progress to liver failure.

[0014] Specifically, the treatment of autoimmune hepatitis includes improving abnormal liver function, reducing liver tissue necrosis and inflammatory cell infiltration, and inhibiting cytokine storms.

[0015] Specifically, the inflammatory factors include tumor necrosis factor-α, interferon-γ, interleukin-2, interleukin-6, and interleukin-1β.

[0016] Specifically, the improvement of abnormal liver function refers to reducing the levels of alanine aminotransferase and aspartate aminotransferase.

[0017] This invention provides the use of smegglutinin in the preparation of drugs for the prevention or treatment of symptoms caused by autoimmune hepatitis.

[0018] Specifically, the related symptoms include fatigue, abdominal discomfort, joint pain, itching, jaundice, enlarged liver, and nausea.

[0019] This invention provides a drug for treating autoimmune hepatitis, wherein the sole active ingredient of the drug is smegglutinin.

[0020] As an alternative implementation, the aforementioned drug for treating autoimmune hepatitis also contains pharmaceutical excipients, which include any one or a combination of at least two of the following: carriers, diluents, excipients, fillers, binders, wetting agents, emulsifiers, solubilizers, surfactants, or buffers.

[0021] Optionally, the pharmaceutical excipients may further include any one or a combination of at least two of the following: colorants, pH adjusters, antioxidants, and antibacterial agents.

[0022] Optionally, the carrier includes liposomes, micelles, dendritic macromolecules, microspheres, or microcapsules.

[0023] The present invention has the following beneficial effects: This invention, through animal experiments, demonstrates for the first time that semaglutide can significantly reduce serum ALT and AST levels in mice with an autoimmune hepatitis (AIH) model, effectively reduce the area of ​​liver tissue necrosis and decrease inflammatory cell infiltration, while significantly downregulating the expression of key pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-2, IL-6, and IL-1β in serum. From the perspectives of biochemical indicators, histopathology, and systemic inflammatory response, this invention fully validates the therapeutic effect of semaglutide on autoimmune hepatitis, overcoming the shortcomings of traditional therapies that cause non-specific immunosuppression and resulting in impaired immune function.

[0024] Smegglutide, a commonly used hypoglycemic drug in clinical practice, has not shown significant adverse reactions. Experiments have also confirmed that smegglutide alone has no toxic effect on the liver of normal mice, and has not caused transaminase abnormalities or pathological damage. Smegglutide has superior safety profile and tolerability, making it more suitable for long-term maintenance therapy in AIH patients, and is expected to significantly improve clinical medication adherence.

[0025] Smegglutide has been approved for marketing by regulatory agencies in multiple countries, and its pharmacokinetic characteristics, long-term safety data, and mature manufacturing process have been clearly defined. This invention explores a new indication for smegglutide in autoimmune hepatitis, which can significantly shorten the new drug development cycle, reduce the risk of clinical trial failure and development costs, and has extremely high clinical translation feasibility and broad market application prospects. Attached Figure Description

[0026] Figure 1 A schematic diagram of the experimental design.

[0027] Figure 2The results show the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of mice in each group.

[0028] Figure 3 The results show the hematoxylin-eosin (H&E) staining images of liver tissue from each group of mice and the quantitative analysis of the percentage of necrotic area.

[0029] Figure 4 The results were obtained for detecting the serum levels of TNF-α, IFN-γ, IL-2, IL-6 and IL-1β in mice of each group using enzyme-linked immunosorbent assay (ELISA).

[0030] Data in the figure are expressed as mean ± standard deviation, n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; scale bar = 100 μm. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0033] Smegglutinin, CAS number 910463-68-2.

[0034] The mouse tumor necrosis factor α (TNF-α) enzyme-linked immunosorbent assay kit, brand name Elabscience, catalog number EE-EL-M3063.

[0035] Mouse interferon-gamma (IFN-γ) enzyme-linked immunosorbent assay kit, brand name Elabscience, catalog number E-EL-M0048.

[0036] Mouse interleukin-2 (IL-2) enzyme-linked immunosorbent assay kit, brand name Elabscience, catalog number E-EL-M0042.

[0037] Mouse interleukin-6 (IL-6) enzyme-linked immunosorbent assay kit, brand name Elabscience, catalog number E-EL-M0044.

[0038] Mouse interleukin-1β (IL-1β) enzyme-linked immunosorbent assay kit, brand name Elabscience, catalog number E-EL-M0037.

[0039] C57BL / 6J mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0040] Example 1 I. Animal grouping and modeling methods Twenty-four male C57BL / 6J mice aged 8-10 weeks were housed in a standard SPF environment. The C57BL / 6 mice were randomly divided into four groups (n = 6): a blank control group (PBS+NS), a model group (ConA+NS), a semaglutide group (PBS+Semaglutide), and a semaglutide treatment group (ConA+Semaglutide).

[0041] Pretreatment phase: Mice in the semaglutide monotherapy group and treatment group were subcutaneously injected with semaglutide (30 nmol / kg, sc), while mice in the blank control group and model group were administered an equal volume of normal saline (NS) by gavage. Administration was repeated every 2 days for 10 consecutive days (5 times in total).

[0042] Modeling phase: Two hours after the last administration, mice in the model group and treatment group were injected via tail vein with concanavalin A (ConA, 15 mg / kg, iv) to induce an autoimmune hepatitis model; mice in the blank control group and single-drug group were injected via tail vein with an equal volume of phosphate-buffered saline (PBS).

[0043] Sample Collection and Observation: Twelve hours after modeling, mice were anesthetized by intraperitoneal injection of 3.5% sodium pentobarbital (40 mg / kg, ip). Blood was collected from the mice, and they were euthanized by cervical dislocation. Mice in each group were allowed free access to food and water during the experiment. Mice were weighed before each administration, and their condition and weight were recorded. A schematic diagram of the experimental design is shown below. Figure 1 As shown.

[0044] II. Serum ALT and AST Detection Methods The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in each group were detected by a fully automated biochemical analyzer (Shenzhen Mindray BS-330E).

[0045] III. Experimental Results Serum biochemical test results as follows Figure 2 As shown, the results indicated that serum ALT and AST levels in the model group (ConA+NS) mice were significantly higher than those in the blank control group. P <0.0001 indicates the occurrence of acute liver injury, indicating that ConA successfully induced an autoimmune hepatitis model. Compared with the model group, the serum ALT and AST levels in the semaglutide treatment group (ConA+Semaglutide) were significantly reduced ( P<0.0001). The transaminase levels in the semaglutide group (PBS+Semaglutide) were not significantly different from those in the blank control group, indicating that semaglutide itself has no toxic effect on normal liver.

[0046] Example 2 The animal grouping and modeling methods in this embodiment are the same as in Example 1. After sampling, the liver tissue of each group of mice was stained with hematoxylin and eosin (H&E) to observe the pathological morphological changes of the liver tissue and assess the degree of liver damage.

[0047] I. Histopathological examination (H&E paraffin section staining) (1) Conventional dewaxing: Place the paraffin slices in an oven and bake at 55~60℃ for 6 hours.

[0048] (2) Dewaxing and rehydration: Immerse in xylene I for 15 min → xylene II for 15 min → anhydrous ethanol for 5 min → 95% ethanol for 5 min → 90% ethanol for 5 min → 80% ethanol for 5 min → 70% ethanol for 5 min, and finally immerse in deionized water for 5 min.

[0049] (3) Immerse in hematoxylin staining solution for 6 min and observe the staining under a microscope.

[0050] (4) Soak in tap water for 10 minutes to wash away excess staining solution.

[0051] (5) Rinse again with distilled water (for a few seconds).

[0052] (6) Immerse in eosin staining solution for 1 min.

[0053] (7) Rinse twice with 70% ethanol, then transfer to distilled water, wait for dehydration, and seal.

[0054] (8) Dehydration: sequentially through 70% ethanol for 10 s → 80% ethanol for 10 s → 90% ethanol for 10 s → 95% ethanol for 10 s → anhydrous ethanol for 10 s → xylene I for 10 min → xylene II for 10 min.

[0055] (9) After adding an appropriate amount of neutral resin to seal the slide, place the slide in a humidified box and air dry it overnight in a fume hood (do not cover the humidified box with water, at room temperature). (10) Use an optical microscope to observe the morphology of liver tissue and acquire images.

[0056] II. Experimental Results H&E staining pathological examination results as follows: Figure 3As shown, in the blank control group (PBS+NS) and the semaglutide group (PBS+Semaglutide), the liver lobule structure was intact, the hepatocyte cords were neatly arranged, and there was no necrosis or inflammatory cell infiltration. In the model group (ConA+NS), large areas of hepatocyte necrosis were observed, accompanied by a large amount of inflammatory cell infiltration. In the semaglutide treatment group (ConA+Semaglutide), the necrosis area was significantly reduced, with only scattered punctate necrosis, and the liver tissue structure remained basically intact. Quantitative analysis of liver necrosis area showed that compared with the model group, the degree of liver tissue necrosis in the semaglutide treatment group was significantly reduced. P <0.01).

[0057] Example 3 The animal grouping and modeling methods in this embodiment are the same as in Embodiment 1.

[0058] I. Enzyme-linked immunosorbent assay (ELISA) The ELISA kits (catalog numbers: EE-EL-M3063, E-EL-M0048, E-EL-M0042, E-EL-M0044, E-EL-M0037, purchased from Elabscience) were used to conduct experiments according to the instructions in each kit. The concentrations of tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the serum samples of mice in each group were measured.

[0059] Specific experimental steps: (1) After the whole blood sample was placed at room temperature for 1 hour, it was centrifuged at 3500 rpm for 15 minutes at 4°C, and the supernatant was taken for testing.

[0060] (2) Set up standard wells, blank wells and sample wells respectively. Add 100 μL of serially diluted standard to the standard wells, add 100 μL of sample diluent to the blank wells, and add 100 μL of the sample to be tested to the remaining wells to cover the microplate. Incubate at 37°C for 90 minutes.

[0061] (3) Discard the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the plate with a membrane, and incubate at 37°C for 1 hour.

[0062] (4) Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing buffer to each well, soak for 1 minute, aspirate or shake off the liquid in the microplate, pat dry, and repeat this washing step 3 times.

[0063] (5) Add 100 μL of HRP enzyme conjugate working solution to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes.

[0064] (6) Shake off the liquid in the hole and wash the plate 5 times, using the same method as step (3).

[0065] (7) Add 90 μL of substrate solution (TMB) to each well, cover the microplate with a membrane, and incubate at 37°C in the dark for about 15 minutes.

[0066] (8) Add 50 μL of stop solution to each well to terminate the reaction. Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader. Substitute the absorbance into the standard curve to obtain the concentration of inflammatory factors.

[0067] II. Experimental Results The ELISA results of serum cytokine levels in each group of mice are as follows: Figure 4 As shown, the results indicated that the serum levels of TNF-α, IFN-γ, IL-2, IL-6, and IL-1β in the model group (ConA+NS) mice were significantly higher than those in the blank control group (PBS+NS). P <0.0001 or P <0.05 indicates that ConA induced a typical cytokine storm. Compared with the model group, the levels of all pro-inflammatory factors in the semaglutide treatment group (ConA+Semaglutide) were significantly reduced ( P <0.05 or P <0.01 or P <0.0001).

[0068] In summary, smegglutinin can effectively alleviate ConA-induced autoimmune hepatitis, significantly reduce serum transaminase levels, improve liver pathological damage, and inhibit systemic inflammatory responses.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of smegglutide in the preparation of drugs for the prevention or treatment of autoimmune hepatitis.

2. The application according to claim 1, characterized in that, The autoimmune hepatitis mentioned includes acute autoimmune hepatitis or chronic autoimmune hepatitis.

3. The application according to claim 1, characterized in that, The treatment for autoimmune hepatitis includes improving abnormal liver function, reducing liver tissue necrosis and inflammatory cell infiltration, and inhibiting cytokine storms.

4. The application according to claim 3, characterized in that, The inflammatory factors include tumor necrosis factor-α, interferon-γ, interleukin-2, interleukin-6, and interleukin-1β.

5. The application according to claim 3, characterized in that, The improvement of abnormal liver function refers to reducing the levels of alanine aminotransferase and aspartate aminotransferase.

6. Application of smegglutide in the preparation of drugs for the prevention or treatment of symptoms caused by autoimmune hepatitis.

7. A drug for treating autoimmune hepatitis, characterized in that, The only active ingredient in the drug is smegglutinin.

8. The drug according to claim 7, characterized in that, It also contains pharmaceutical excipients, which include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, emulsifier, cosolvent, surfactant or buffer.

9. The drug according to claim 8, characterized in that, Pharmaceutical excipients also include any one or a combination of at least two of the following: colorants, pH adjusters, antioxidants, and antibacterial agents.

10. The drug according to claim 8, characterized in that, The carrier includes liposomes, micelles, dendritic macromolecules, microspheres, or microcapsules.

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