Application of GHRH-A in the preparation of drugs for treating non-alcoholic fatty liver disease

Abstract

The present invention belongs to the field of biomedicine and mainly relates to the use of the growth hormone-releasing hormone receptor agonist MR-409 in the preparation of non-alcoholic fatty liver disease (NAFLD) drugs. Currently, there are no targeted drugs for the treatment of NAFLD, and lipid-lowering drugs are usually used in combination with liver-protecting drugs. MR-409 is a new type of GHRHR-A. GHRHR is widely expressed in the liver and pancreas, and literature reports that GHRHR-A has the effects of improving islet function, promoting the survival of transplanted islets, and alleviating diabetic eye complications. In the present invention, the study found that MR409 can improve the imaging, serum enzymology, serum metabolism, and histological indicators of non-alcoholic fatty liver disease in diabetic mice, and is expected to become a therapeutic drug for non-alcoholic fatty liver disease. The present invention provides a new use of MR-409 for the treatment of non-alcoholic fatty liver disease, provides a new strategy for the treatment of non-alcoholic fatty liver disease, has important clinical therapeutic significance, and provides a scientific basis for its future development into a drug for the treatment of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and mainly relates to the application of growth hormone-releasing hormone receptor agonist MR-409 in the preparation of diabetic non-alcoholic fatty liver disease medicine. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) refers to a clinical and pathological syndrome characterized by diffuse macrovesicular steatosis in hepatocytes, excluding liver damage caused by alcohol or other known liver-damaging factors. Patients without a history of excessive alcohol consumption may present with pathological changes similar to those of alcoholic liver disease (ALD), with the disease spectrum encompassing nonalcoholic simple fatty liver disease, nonalcoholic steatohepatitis, cirrhosis, and liver cancer. Due to increased social pressure, poor dietary habits, and emotional factors, the incidence of NAFLD has increased annually by 30%. Currently, there are no specific treatments for NAFLD. Clinically, symptomatic treatment with a combination of lipid-lowering and liver-protective medications is typically used to delay or arrest the progression of NAFLD. Therefore, the development of drugs that can better block and reverse NAFLD liver damage is an urgent challenge in the field of NAFLD treatment.

[0003] MR-409 is a member of a class of growth hormone-releasing hormone (GHRH) analogs. The addition of noncoding amino acids to its peptide chain resists protease degradation, resulting in a longer half-life and higher binding affinity. GHRH-A competitively inhibits the binding of endogenous growth hormone-releasing hormone (GHRH) to its receptor, thus avoiding the many side effects of endogenous GHRH production, such as tumors and acromegaly. Experimental studies and literature reports indicate that the GHRH receptor (GHRHR) is widely expressed in the liver and pancreas, and that GHRHR-A improves islet function, promotes transplanted islet survival, and alleviates diabetic eye complications. However, there are no reports on the therapeutic effect of MR-409 on NAFLD. We found that in a mouse model of diabetes-induced nonalcoholic fatty liver disease, treatment with MR409 significantly improved ultrasound imaging, histopathology, serum enzymes, and lipid profiles of the fatty liver.

[0004] Currently, the main treatment for NAFLD is non-specific symptomatic treatment, such as a combination of lipid-lowering and liver-protective medications. These treatments may delay but cannot reverse the liver damage caused by NAFLD. Therefore, the development of new drugs to treat NAFLD is urgently needed. Summary of the Invention

[0005] The present invention aims to provide a composition containing MR-409 for the treatment of nonalcoholic fatty liver disease (NAFLD). This composition offers a novel treatment strategy for NAFLD and has important clinical therapeutic implications. By promoting neuronal regeneration, it also provides an important scientific basis for the development of drugs targeting ischemic stroke.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: On the one hand, the present invention provides an application of GHRH-A in the preparation of a drug for treating non-alcoholic fatty liver disease, the technical key point of which is that the GHRH-A is MR409.

[0007] Furthermore, the main dosage form of the drug is injection.

[0008] On the other hand, the present invention also provides a composition containing GHRH-A for treating NAFLD, the technical key point of which is that the GHRH-A is MR409.

[0009] Furthermore, the amino acid sequence of MR-409 is: NMeTyr-D-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-NHCH3.

[0010] Furthermore, the main dosage form of the composition is injection.

[0011] Furthermore, when the composition is used, the dosage of the active ingredient MR-409 is 15 μg.

[0012] Beneficial effects of the present invention: The present invention discloses the use of a growth hormone-releasing hormone receptor agonist, MR-409, in the preparation of a drug for non-alcoholic fatty liver disease. Experiments have confirmed that: MR-409 can significantly improve the liver imaging results of diabetic mice with non-alcoholic fatty liver disease; MR-409 can significantly improve the serum enzyme indicators of diabetic mice with non-alcoholic fatty liver disease; MR-409 can significantly improve the serum metabolic indicators of diabetic mice with non-alcoholic fatty liver disease; MR-409 can significantly improve the liver histological test results of diabetic mice with non-alcoholic fatty liver disease; MR-409 can significantly improve liver lipid deposition in diabetic mice with non-alcoholic fatty liver disease. The preferred dose of MR-409 is 15 ug / mouse, and the preferred administration method is subcutaneous injection every other day. The above experimental results show that MR-409 can significantly improve non-alcoholic fatty liver disease in diabetic mice. The above new uses of MR-409 provide a new strategy for the treatment of non-alcoholic fatty liver disease, which has important clinical therapeutic significance and provides a scientific basis for its future development into a drug for the treatment of non-alcoholic fatty liver disease.

[0013] Our experimental results show that MR409 can improve the imaging, serum enzymology, serum metabolism, and histological indicators of non-alcoholic fatty liver disease in diabetic mice, and is expected to become a new therapeutic approach for non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram showing the imaging results of MR-409 improving non-alcoholic fatty liver disease in diabetic mice.

[0015] Figure 2 This is a schematic diagram showing that MR-409 improves serum enzyme indicators in diabetic mice with non-alcoholic fatty liver disease.

[0016] Figure 2 In the figure, A shows the changes in serum aspartate aminotransferase (AST) in diabetic mice with nonalcoholic fatty liver disease after administration of MR409; B shows the changes in serum alanine transaminase (ALT) in diabetic mice with nonalcoholic fatty liver disease after administration of MR409.

[0017] Figure 3 Schematic diagram showing that MR-409 improves serum metabolic indicators of non-alcoholic fatty liver disease in diabetic mice.

[0018] Figure 3In the figure, A shows the changes in serum triglyceride (TG) in diabetic mice with non-alcoholic fatty liver disease after administration of MR409; B shows the changes in serum total cholesterol (TC) in diabetic mice with non-alcoholic fatty liver disease after administration of MR409; C shows the changes in serum low-density lipoprotein (LDL) in diabetic mice with non-alcoholic fatty liver disease after administration of MR409; D shows the changes in serum high-density lipoprotein (HDL) in diabetic mice with non-alcoholic fatty liver disease after administration of MR409.

[0019] Figure 4 This is a schematic diagram showing that MR-409 can improve the liver histological test results of non-alcoholic fatty liver disease in diabetic mice.

[0020] Figure 5 Schematic diagram showing that MR-409 can improve hepatic lipid deposition in diabetic mice with non-alcoholic fatty liver disease. DETAILED DESCRIPTION

[0021] The following combination Figures 1 to 5 , the specific contents of the present invention are described in detail through specific embodiments.

[0022] The present invention relates to application of growth hormone releasing hormone receptor agonist MR-409 in preparing medicine for non-alcoholic fatty liver disease.

[0023] 1. Materials:

[0024] BKS-Leprem2Cd479 / Nju and C57BL / 6 male mice, 12 weeks old, animal certification number: SCXK (Su) 2018-0008, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. MR-409 was provided by the Andrew V. Schally group (University of Miami, USA) and prepared with 10% polyethylene glycol to the desired concentration.

[0025] 2. Methods:

[0026] 2.1 Experimental groups and drug administration

[0027] 1) Twelve-week-old BKS-Leprem2Cd479 / Nju and C57BL / 6 mice were used as experimental animals and divided into a wild-type group (WT), a model group (BKS-Leprem2Cd479 / Nju, DB / DB), and a drug-treated group (MR409).

[0028] 2) Dosing: The MR-409 group received a single dose of 15 μg of the test drug [MR-409 was dissolved in DMSO to prepare a stock solution at a concentration of 100 mg / ml. Immediately before use, the solution was diluted with 10% polyethylene glycol to a working concentration of 0.1 mg / ml (final DMSO concentration <1%)]. The model group received an equal volume of normal saline.

[0029] 3) Administration: Subcutaneous injection.

[0030] 4) Administration schedule: Administer once every other day at the same time for a total of 8 weeks.

[0031] Experimental procedures

[0032] 1) Small Animal Imaging: After mice in each group were anesthetized with isoflurane vapor using a small animal anesthesia machine and reached a stable state, they were secured in a supine position on an operating table with medical tape. Abdominal hair was removed with depilatory cream. The ambient temperature was maintained at approximately 22-24°C. Ultrasound was performed at a frequency of 40 MHz, and the same person performed both the operation and the acquisition of images.

[0033] 2) Mouse Serum Enzyme Assay: After fasting for 8 hours, whole blood was collected from each group of mice and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected to prepare serum, which was aliquoted and stored at -80°C. Serum alanine aminotransferase (ALT / GPT) and aspartate aminotransferase (AST / GOT) levels were measured according to the kit instructions.

[0034] 3) Serum metabolic assay in mice: After fasting for 8 h, whole blood was collected from each group of mice and centrifuged at 3000 rpm for 15 min. Serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels were measured according to the kit and ELISA instructions.

[0035] 4) Mouse Liver Histological HE Staining: After anesthesia, mice in each group were perfused with normal saline and then 4% paraformaldehyde. The livers were removed and cut into small cubes (<8 mm cubes) and fixed in 4% paraformaldehyde. Prior to staining, tissue blocks were rinsed with tap water overnight, then dehydrated and permeabilized, embedded in paraffin, and sectioned (4 μm thickness), spread, baked, and dried. Staining began with a gradual hydration, hematoxylin staining, differentiation, eosin staining, dehydration, permeabilization, mounting, microscopic examination, and photography.

[0036] 5) Oil Red O staining of mouse liver histology: After anesthesia, mice in each group were perfused with normal saline and then 4% paraformaldehyde. The livers were removed and cut into small cubes (<8 mm in size) and fixed in 4% paraformaldehyde. Tissue blocks were placed in 20% and then 30% sucrose solutions until they sank to the bottom. The blocks were then frozen and embedded in OCT embedding medium. Sections (10 μm thickness) were then sliced at -20°C and stored at -80°C. Before staining, sections were briefly rinsed in distilled water, immersed in 60% isopropanol for 1-2 seconds, blocked and stained with Oil Red O staining solution (prepared fresh). The sections were then immersed in 60% isopropanol for 1-2 seconds, washed in distilled water for 1-2 seconds, and counterstained for nuclei with hematoxylin for 1-2 seconds. The sections were then dried with filter paper, mounted with glycerol-gelatin, and examined under a microscope.

[0037] 3. Experimental results:

[0038] 1) Effects of MR-409 on imaging findings of non-alcoholic fatty liver disease in diabetic mice.

[0039] Ultrasound imaging revealed that in the wild-type (WT) mice, echogenicity was faint, fine, and evenly distributed within the liver parenchyma, with no diffusely increased echogenicity. The intrahepatic ductal structures were distinct and well-defined. Ultrasound imaging revealed diffusely increased echogenicity (bright liver) in the transgenic diabetic (BKS-Leprem2Cd479 / Nju, DB / DB) diabetic mice near the liver (with unclear, less distinct, ductal structures), suggesting more severe fatty liver disease. Ultrasound imaging revealed diffusely increased echogenicity in a few areas of the liver near the liver, with more distinct, more clearly defined ductal structures, suggesting that MR409 significantly improved the symptoms of fatty liver in diabetic mice. These results suggest that MR-409 can significantly improve imaging findings in diabetic mice with non-alcoholic fatty liver disease.

[0040] 2) Effects of MR-409 on serum enzyme indicators of non-alcoholic fatty liver disease in diabetic mice.

[0041] The experimental results showed that compared with the normal WT group, the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model DB / DB mice were significantly increased. After 8 weeks of drug administration, the serum ALT and AST levels in the MR409 group mice were significantly decreased. These results suggest that MR409 can significantly improve serum enzyme indicators in diabetic mice with non-alcoholic fatty liver disease. p <0.05, ** p <0.01 vs. WT group, #p<0.05 vs. DB / DB group.

[0042] 3) Effects of MR-409 on serum metabolic parameters in diabetic mice with non-alcoholic fatty liver disease.

[0043] Experimental results showed that compared with the normal WT group, serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL) levels were significantly increased in the DB / DB model mice. After 8 weeks of drug administration, serum TG, TC, and LDL levels were significantly decreased in the MR409 group. Compared with the normal WT group, serum high-density lipoprotein (HDL) levels were significantly decreased in the DB / DB model mice. After 8 weeks of drug administration, serum HDL levels were significantly increased in the MR409 group. These results suggest that MR409 can significantly improve serum metabolic markers in diabetic mice with non-alcoholic fatty liver disease. *p<0.05, **p<0.01, ***p<0.001 vs. WT group, #p<0.05, ##p<0.01, ###p<0.001 vs. DB / DB group.

[0044] 4) Effect of MR-409 on liver histological examination results of non-alcoholic fatty liver disease in diabetic mice.

[0045] Experimental results showed that HE staining of paraffin sections of livers from the normal WT group revealed uniform cytoplasm staining of hepatocytes, normal nuclei of normal size and morphology, intact and clear hepatic lobules, hepatocytes with no degeneration or necrosis and radial arrangement around the central vein, no fibrous tissue proliferation, and neatly arranged hepatic plates. In the model DB / DB group, hepatocytes showed pale and loose cytoplasm staining, severe hepatic lobules destruction, and disorganized arrangement. Hepatocytes showed swelling, necrosis, hydropic changes, ballooning, and predominantly bullous steatosis (indicated by red arrows). After 8 weeks of drug administration, the MR409 group showed partial hepatic lobules destruction, mild hepatocyte degeneration and necrosis, and significant improvement in liver architecture, with the disappearance of ballooning and bullous hepatocytes. These results suggest that MR409 can significantly improve liver histological findings in diabetic mice with non-alcoholic fatty liver disease.

[0046] 5) Effects of MR-409 on hepatic lipid deposition in diabetic mice with non-alcoholic fatty liver disease.

[0047] The experimental results showed that Oil Red O staining of frozen liver sections in the normal WT group revealed neatly arranged hepatocytes with minimal lipid deposition. In the model DB / DB group, hepatocytes showed ballooning and bullous degeneration, with strong positive lipid staining. In the MR409-treated group, hepatocytes were neatly arranged, with no ballooning and bullous degeneration, and lipid deposition significantly improved. This suggests that MR409 can significantly improve hepatic lipid deposition in diabetic mice with non-alcoholic fatty liver disease.

[0048] The above examples demonstrate that MR-409 can improve liver imaging findings in diabetic mice with non-alcoholic fatty liver disease; improve serum enzyme indicators in diabetic mice with non-alcoholic fatty liver disease; improve serum metabolic indicators in diabetic mice with non-alcoholic fatty liver disease; improve liver histological findings in diabetic mice with non-alcoholic fatty liver disease; and improve liver lipid deposition in diabetic mice with non-alcoholic fatty liver disease. Therefore, MR-409 has great clinical application prospects.

Claims

1. A use of GHRH-A in the preparation of a medicament for treating non-alcoholic fatty liver disease, characterized in that: The GHRHA is MR409.

2. The use according to claim 1, characterized in that: The medicine is an injection.

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