Use of darapladib in the preparation of a medicament for preventing and treating metabolic dysfunction-associated steatohepatitis

Abstract

The application belongs to the field of new use of medicines, and more particularly relates to application of Darapladib in preparation of medicines for preventing and treating metabolic dysfunction related fatty hepatitis. The application firstly finds that after treatment by Darapladib, the liver weight, liver / body ratio, serum total cholesterol, steatosis, ballooning, inflammatory infiltration and collagen deposition of MASH model mice are significantly reduced. The application expands the traditional use of Darapladib in cardiovascular diseases, and provides a new treatment strategy for MASH.

Description

Technical Field

[0001] This invention belongs to the field of novel uses of pharmaceuticals, and more specifically, relates to the use of Darapladib in the preparation of drugs for the prevention and treatment of metabolic dysfunction-related steatohepatitis. Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MASH, formerly known as non-alcoholic steatohepatitis NASH), has become the most common chronic liver disease worldwide. During the course of MASH, oxidized phospholipids are significantly elevated in the liver and blood, and are a contributing factor to the disease. Using antibodies to prevent the phagocytosis of oxidized phospholipids by macrophages, thereby inhibiting pro-inflammatory responses, can significantly improve steatosis, inflammation, fibrosis, hepatocellular death, and liver cancer progression in MASH.

[0003] Darapladib is a reversible lipoprotein-associated phospholipase A2 (Lp-PLA2) specific inhibitor, which has previously been used primarily in clinical and basic research for the treatment of cardiovascular diseases such as atherosclerosis. However, the exact role of Lp-PLA2 in the process of inflammatory necrosis and stellate cell activation (leading to fibrosis) in the liver microenvironment has not been elucidated, and the therapeutic potential of Darapladib against MASH remains untapped. Summary of the Invention

[0004] The purpose of this invention is to provide the use of Darapladib in the preparation of drugs for the prevention and treatment of metabolic dysfunction-related steatohepatitis, wherein the structural formula of Darapladib is as follows: .

[0005] This invention provides the use of darapladib in the preparation of drugs for the prevention and treatment of metabolic dysfunction-related steatohepatitis.

[0006] This invention is the first to apply the known Lp-PLA2-specific inhibitor darapladib to the treatment of metabolic dysfunction-associated steatohepatitis (MASH). Using a MASH mouse model, darapladib intervention significantly reduced body weight, liver weight, and liver-to-body ratio in the model mice, as well as lowering serum total cholesterol, alleviating lipid deposition, ballooning degeneration, and inflammatory cell infiltration in hepatocytes, and effectively blocking and reversing the progression of liver fibrosis. This invention overcomes the traditional limitations of darapladib's use in cardiovascular diseases, directly linking its target to the key pathogenic factors of MASH (lipid metabolism disorder, inflammatory damage, and fibrosis), providing a novel drug strategy and application for the treatment of MASH.

[0007] Furthermore, the Darapladib is used to alleviate hepatomegaly in patients with metabolic dysfunction-related steatohepatitis.

[0008] Furthermore, the Darapladib is used to reduce serum total cholesterol levels in patients with metabolic dysfunction-related steatohepatitis.

[0009] Furthermore, the Darapladib is used to reduce lipid deposition in the liver of patients with metabolic dysfunction-related steatohepatitis.

[0010] Furthermore, the Darapladib is used to reduce liver inflammatory damage in patients with metabolic dysfunction-related steatohepatitis.

[0011] Furthermore, the Darapladib is used to reduce liver fibrosis in patients with metabolic dysfunction-related steatohepatitis.

[0012] Furthermore, the drug is made with Darapladib as the active ingredient, plus pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

[0014] The beneficial effects of this invention are as follows: (1) It has pioneered a new targeted therapy drug: This invention reveals for the first time the exact efficacy of the known drug Darapladib in the prevention and treatment of metabolic dysfunction-related steatohepatitis (MASH, i.e., non-alcoholic steatohepatitis NASH), providing a new and highly promising candidate drug for the treatment of MASH and liver fibrosis, which lack specific drugs in clinical practice.

[0015] (2) Effectively improves systemic obesity and liver enlargement: This invention has demonstrated that in vivo intervention with Darapladib can significantly reduce the absolute body weight, absolute liver weight and liver-to-body ratio of MASH individuals induced by a high-fat diet, effectively alleviating systemic metabolic obesity and abnormal liver enlargement caused by severe lipid accumulation.

[0016] (3) Significantly alleviates systemic lipid metabolism disorder: Darapladib can significantly reduce the abnormally elevated total cholesterol (TC) concentration in the blood of MASH mice, improve lipid metabolism balance at the system level, cut off the continuous transport of free fatty acids to the liver, and thus reduce lipotoxic damage.

[0017] (4) Targeted repair of liver micropathological damage: Histological evidence shows that Darapladib can directly improve the core lesions of MASH, significantly reduce the accumulation of macrovesicular lipid droplets in hepatocytes, and greatly reduce the degree of hepatocyte ballooning degeneration and inflammatory cell infiltration, promoting the restoration of liver lobule structure to normal morphology.

[0018] (5) Definite blocking and reversal of liver fibrosis: The present invention has achieved anti-fibrotic effects. Darapladib can significantly destroy and degrade the dense “bridging fibrosis” network formed under the pathological state of MASH, greatly reduce the collagen deposition area in liver tissue, and achieve effective blocking and reversal of the incurable process of liver fibrosis in the middle and late stages. Attached Figure Description

[0019] Figure 1 The charts show the statistical results of macroscopic physical indicators of mice. In the charts, A is the weight of mice, B is the liver weight of mice, and C is the liver-to-body ratio of mice. * indicates P < 0.05 and ** indicates P < 0.01.

[0020] Figure 2 The graph shows a comparison of the statistical results of total cholesterol (TC) levels in the blood of mice in each group, where *** indicates P < 0.001.

[0021] Figure 3 The images show pathological sections of mouse liver tissue. In this image, A shows the hematoxylin-eosin (H&E) staining results of mouse liver tissue, B shows the quantitative analysis data of fatty degeneration, C shows the quantitative analysis data of ballooning degeneration, and D shows the quantitative analysis data of dense inflammatory cell infiltration. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0022] Figure 4 The images show fibrosis-specific staining and collagen area quantitative analysis of mouse liver tissue. In the image, A is the fibrosis-specific staining of mouse liver tissue, and B is the collagen area quantitative analysis. * indicates P < 0.05. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0024] Darapladib is an orally active Lp-PLA2 inhibitor with a molecular weight of 666.77 and the molecular formula C2. 36 H 38 F4N4O2S, CAS number 356057-34-6.

[0025] Example 1: The effect of Darapladib intervention on improving obesity and abnormal liver enlargement in MASH mice.

[0026] I. Experimental Methods.

[0027] Darapladib treatment group: A working solution of darapladib with a concentration of 5 mg / mL was prepared using 6% (v / v) dimethyl sulfoxide (DMSO), 40% (v / v) polyethylene glycol (PEG300), 5% (v / v) polysorbate 80 (Tween80), and 49% (v / v) physiological saline as blank solvent carriers.

[0028] Eight-week-old C57BL / 6J mice were fed a high-fat, high-fructose, high-cholesterol diet (AMLN diet) for 30 weeks to induce the MASH model. Starting from week 24 of AMLN induction, the mice were administered the drug via gavage at a dose of 50 mg / kg once daily for 6 consecutive weeks.

[0029] AMLN high-fat model group: Using the same MASH mouse model and administration regimen as the treatment group, blank solvent carriers were administered by gavage according to the equivalent volume, once daily for 6 consecutive weeks.

[0030] Normal control group: Eight-week-old C57BL / 6J mice were fed a routine diet. During weeks 24-30 of normal feeding, blank solvent carriers were administered via gavage at the same regimen as the treatment group, according to the equivalent volume, once daily for 6 consecutive weeks.

[0031] At 38 weeks of age, the mice in the above three groups were weighed, their whole livers were dissected and collected, weighed, and subjected to routine pathological analysis.

[0032] II. Experimental Results.

[0033] Figure 1 The macroscopic changes in mice in the normal control group (Control), the AMLN high-fat model group (NTC), and the Darapladib treatment group (Darapladib) are presented using statistical bar charts. The charts show the absolute body weight, absolute liver weight, and the liver-to-body weight ratio (the ratio of liver weight to body weight) of the mice at sacrifice in each group. Figure 1As shown, after induction with a high-fat, high-fructose diet using AMLN, the body weight, liver weight, and liver-to-body ratio of the model group mice were all significantly increased compared to the normal control group, indicating that the model mice not only developed systemic obesity, but their livers also became abnormally enlarged due to severe lipid accumulation. However, after 6 weeks of treatment with Darapladib, the liver weight and liver-to-body ratio of the treatment group mice significantly decreased compared to the model group, and their body weight also decreased. This result conclusively demonstrates that Darapladib can effectively intervene in systemic metabolic obesity induced by a high-fat diet and can significantly promote the metabolic excretion of accumulated lipids in the liver, effectively reversing abnormal liver hypertrophy under NASH pathological conditions.

[0034] Example 2: The alleviating effect of Darapladib intervention on systemic lipid metabolism disorder in MASH mice.

[0035] I. Experimental Methods.

[0036] At the experimental endpoint (week 30), mice in each group were fasted overnight (16 hours, with free access to water) to eliminate the direct interference of diet on recent blood lipids. The following day, whole blood was collected from mice using the enucleation method and placed in sterile centrifuge tubes. After allowing the blood to clot naturally at room temperature for 30 minutes, the tubes were centrifuged at 4000 rpm for 5 minutes at 4°C. The clear, pale yellow liquid at the top was carefully aspirated; this was the serum sample, aliquoted, and stored at -80°C for later use. The frozen serum samples were thawed on ice. The procedure was strictly followed according to the instructions of the tissue / serum total cholesterol (TC) enzymatic biochemical assay kit. After mixing an appropriate amount of serum sample with the working solution, the absorbance (OD value) of each well was measured using a microplate reader at a specific wavelength (500 nm). The absolute concentration of triglycerides in the serum of each group of mice (unit: mmol / L) was calculated based on the standard curve.

[0037] II. Experimental Results.

[0038] An abnormally high level of total cholesterol (TC) in the blood is one of the most typical features of lipid metabolism disorder in NAFLD / MASH patients. Figure 2 The concentration levels of total cholesterol (TC) in the blood of the three groups of mice were compared using a bar chart (vertical axis unit: mmol / L). The chart clearly shows that the serum TC level in the model group mice was abnormally high due to a long-term high-fat diet, far exceeding that of the normal control group; while the TC level in the Darapladib treatment group was significantly reduced, with the bar height being significantly lower than that in the model group. This result demonstrates that Darapladib has excellent total cholesterol-lowering effects, can improve lipid metabolism disorders at the systemic level, and interrupt the continuous transport of free fatty acids to the liver.

[0039] Example 3: The pathological repair effect of Darapladib intervention on lipid deposition and inflammatory damage in hepatocytes of MASH mice.

[0040] I. Experimental Methods.

[0041] Left lobe liver tissue from mice in each group was fixed, embedded, and sectioned, and then stained with hematoxylin and eosin (H&E). Lipid deposition and pathological morphological changes in hepatocytes were observed under a microscope.

[0042] II. Experimental Results.

[0043] H&E staining results are as follows Figure 3 As shown, the normal control group exhibited intact liver lobule structure with no significant lipid droplet accumulation; the model group showed a large accumulation of clear lipid droplet vacuoles in the liver tissue, exhibiting typical macrovesicular steatosis, accompanied by significant hepatocyte ballooning degeneration and inflammatory cell infiltration; while the Darapladib treatment group showed a significant reduction in lipid accumulation, a significant decrease in the number and staining area of ​​lipid droplet vacuoles, and a significant alleviation of ballooning degeneration and inflammatory infiltration. This indicates that Darapladib can effectively repair and improve lipid deposition and inflammatory damage in hepatocytes induced by a high-fat diet.

[0044] Example 4: The blocking and reversal effect of Darapladib intervention on the progression of liver fibrosis in MASH mice.

[0045] I. Experimental Methods.

[0046] Left lobe liver tissue from mice in each group was fixed, embedded, and sectioned. Further fibrosis-specific staining with Sirius Red was performed to observe collagen fiber deposition under a microscope, and the percentage of collagen-positive area was quantitatively calculated using image analysis software (Image J).

[0047] II. Experimental Results.

[0048] Liver fibrosis staining and quantitative analysis results are as follows: Figure 4 As shown, in the normal control group, only a small amount of normal collagen was distributed in the blood vessel walls; in the model group, a large number of dense, dark collagen fibers were deposited around the hepatic sinusoids and portal areas, intertwining to form a bridging fibrotic network, and the percentage of collagen-positive area was significantly increased; while in the Darapladib treatment group, this dense collagen network was significantly disrupted, the collagen strands became thinner and fewer in number, and the percentage of collagen-positive area decreased significantly. This indicates that Darapladib treatment can directly block and effectively reverse the liver fibrosis process under MASH pathological conditions.

[0049] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0050] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0051] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of darapladib in the preparation of drugs for the prevention and treatment of metabolic dysfunction-related steatohepatitis, characterized in that, The structural formula of Darapladib is as follows: .

2. The application according to claim 1, characterized in that, The Darapladib is used to alleviate hepatomegaly in patients with metabolic dysfunction-related steatohepatitis.

3. The application according to claim 1, characterized in that, The Darapladib is used to reduce total cholesterol levels in the blood of patients with metabolic dysfunction-related fatty liver disease.

4. The application according to claim 1, characterized in that, The Darapladib is used to reduce lipid deposition in the liver of patients with metabolic dysfunction-related steatohepatitis.

5. The application according to claim 1, characterized in that, The Darapladib is used to reduce liver inflammatory damage in patients with metabolic dysfunction-related steatohepatitis.

6. The application according to claim 1, characterized in that, The Darapladib is used to reduce liver fibrosis in patients with metabolic dysfunction-related steatohepatitis.

7. The application according to claim 1, characterized in that, The drug is made with Darapladib as the active ingredient and pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

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