Use of a gcn2 kinase inhibitor for the preparation of a medicament for the treatment of alcoholic liver disease

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a GCN2 kinase inhibitor in preparation of a medicine for treating alcoholic liver disease. The GCN2 kinase inhibitor is an ATP competitive inhibitor GCN2iB-1; the chemical formula of the GCN2iB-1 is C 18 H 12 ClF2N5O3S, the inhibitor can effectively inhibit the activity of GCN kinase, and exhibits significant therapeutic effect in a disease model: can effectively reduce liver lipid deposition, improve liver function indexes and dyslipidemia, and relieve oxidative stress and inflammatory response. Experimental results show that the inhibitor has clear treatment effect, and no obvious toxic side effects are observed. The application provides a new target and an effective candidate drug scheme for treatment of alcoholic fatty liver.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an application of a GCN2 kinase inhibitor in preparation of a medicine for treating alcoholic liver disease. BACKGROUND

[0002] Excessive alcohol intake is one of the primary risk factors for chronic liver disease worldwide. Alcoholic liver disease (ALD) is a progressive liver disease caused by long-term heavy drinking, which progresses from early alcoholic fatty liver disease (AFLD) to alcoholic steatohepatitis, and in severe cases, to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Compared with the general population, the risk of cardiovascular disease and cancer death in ALD patients is significantly increased, which has a heavy burden on global public health.

[0003] From the pathogenesis, the pathological damage process of ALD is closely related to the metabolism of alcohol in the liver: after alcohol enters the human body, it is mainly oxidized to acetaldehyde through the ethanol dehydrogenase (ADH) pathway and the cytochrome CYP2E1 pathway in the liver. In this process, on the one hand, the metabolite acetaldehyde has clear hepatotoxicity, which can directly damage the structure and function of hepatocytes; on the other hand, the metabolic process is accompanied by the generation of a large amount of reactive oxygen species (ROS), of which CYP2E1 pathway contributes more than 70% of ROS in the liver, and excessive ROS will destroy the redox homeostasis of the liver, and then trigger lipid metabolism disorder (manifested as abnormal accumulation of triglycerides in hepatocytes), persistent inflammatory response (such as high expression of inflammatory factors TNF-α, IL-6) and oxidative stress damage, ultimately driving the pathological progression of ALD.

[0004] Current clinical treatment methods for ALD have significant limitations, which are difficult to meet the clinical needs:

[0005] Limitations of basic treatment methods: the core way of current ALD treatment is strict abstinence from drinking, but the patient compliance is poor;

[0006] Limitations of drug treatment: although glucocorticoid drugs (such as prednisolone) for severe alcoholic hepatitis can alleviate some symptoms in the short term, the side effects are significant when used for a long time;

[0007] Limitations of end-stage treatment: liver transplantation is only suitable for end-stage patients, but is limited by donor shortage, etc.

[0008] In recent years, although candidate drugs targeting inflammation, oxidative stress and other mechanisms have entered clinical trials, no breakthrough has been made in the field of ALD drug treatment since the application of prednisolone. No new breakthrough therapeutic drug has been approved for marketing in this field, and alcohol abstinence is still a key factor affecting the long-term prognosis of patients.

[0009] Therefore, developing an ALD treatment drug with high safety, clear efficacy and the ability to specifically improve liver lipid deposition is a technical problem to be solved by the present application. SUMMARY

[0010] The purpose of the present application is to provide a GCN2 kinase inhibitor for use in the preparation of a medicament for treating or preventing alcoholic liver disease. The inhibitor provided by the present application effectively reduces liver lipid deposition, improves liver function indicators and dyslipidemia, and relieves oxidative stress and inflammatory response by efficiently inhibiting the activity of GCN kinase.

[0011] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0012] The purpose of the present application is to provide a GCN2 kinase inhibitor for use in the preparation of a medicament for treating or preventing alcoholic liver disease. The inhibitor provided by the present application effectively reduces liver lipid deposition, improves liver function indicators and dyslipidemia, and relieves oxidative stress and inflammatory response by efficiently inhibiting the activity of GCN kinase.

[0013] As shown in the formula.

[0014] Preferably, the GCN2 kinase inhibitor can improve liver function.

[0015] Preferably, the GCN2 kinase inhibitor can improve dyslipidemia.

[0016] Preferably, the GCN2 kinase inhibitor can reduce lipid deposition in the liver.

[0017] Preferably, the GCN2 kinase inhibitor can relieve oxidative stress in the liver.

[0018] Preferably, the GCN2 kinase inhibitor can relieve inflammation in the liver.

[0019] Preferably, the dosage form of the medicament is an oral dosage form or an injection dosage form.

[0020] The present application also provides a pharmaceutical composition for treating alcoholic liver disease. The active ingredient of the pharmaceutical composition includes the above-mentioned GCN2 kinase inhibitor GCN2iB-1, and further comprises a pharmaceutically acceptable excipient.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] (1) The GCN2 inhibitor does not act through a single pathway, but can intervene in the disease process from multiple key pathological links. Specifically, it can effectively reverse liver lipid deposition: directly reduce the abnormal accumulation of lipids in the liver, alleviate the core pathological feature of fatty liver from the root; improve liver function and metabolism comprehensively: can significantly reduce the level of serum transaminase (such as ALT, AST), indicating that it can effectively reduce liver cell damage, while improving dyslipidemia and restoring normal metabolic homeostasis; relieve oxidative stress and inflammation: can reduce the oxidative stress induced by alcohol metabolism and inhibit the subsequent inflammatory cascade, thereby preventing the progression of the disease to alcoholic hepatitis and liver fibrosis.

[0023] (2) Clear mechanism of action and strong specificity: as an ATP competitive inhibitor, the mechanism of action of the inhibitor is to directly bind to the ATP binding site of GCN2 kinase, thereby specifically and efficiently blocking its kinase activity. This is conducive to drug optimization and subsequent mechanism of action research.

[0024] (3) High safety, the present application has been verified by animal experiments, and no obvious toxic side effects have been observed at the dose producing significant therapeutic effect, showing good biological safety and tolerance. This key advantage lays a solid foundation for subsequent clinical development and application.

[0025] (4) Provides a new approach for drug screening, the causal relationship between GCN2 inhibition and the treatment of alcoholic fatty liver established by the present application provides a clear target and effective technical path for high-throughput screening and discovering more candidate drugs for treating the disease. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0027] Figure 1 Figure 1 is a schematic diagram of the improvement of liver damage and dyslipidemia in alcohol liquid feed mice by GCN2iB treatment, wherein A is an experimental flowchart; B-E are schematic diagrams for detecting the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC) in serum, respectively.

[0028] Figure 2Figure 2A-B shows that GCN2iB treatment reduces alcohol-induced liver lipid deposition in mice; A is a schematic diagram of liver tissue section staining of mice in each group; B-C are analysis graphs of detecting the content of triglyceride TG and total cholesterol TC in liver tissue of mice in each group.

[0029] Figure 3 Figure 3A-E shows that GCN2iB treatment alleviates alcohol-induced oxidative stress and inflammatory response in mouse liver; A is a schematic diagram of DHE staining of frozen sections and F4 / 80 immunohistochemical staining of paraffin sections of liver tissue of mice in each group; B is a statistic of the relative fluorescence intensity of DHE; C is a statistic of the relative number of F4 / 80 positive cells (macrophages); D-E are detection of 3-nitrotyrosine (3'-NT) (D) and 4-hydroxy nonenoic acid (4-HNE) (E) in mouse liver; 5 mice in each group; ** represents p<0.01, *** represents p<0.001. DETAILED DESCRIPTION

[0030] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0031] The production process, experimental method or detection method involved in the embodiments of the present application are all conventional methods in the prior art without special instructions, and their names and / or abbreviations are all conventional names in the field, which are very clear and explicit in the related application field, and the skilled person in the art can understand the conventional process steps and apply the corresponding equipment according to the conventional conditions or the conditions recommended by the manufacturer.

[0032] The various instruments, equipment, raw materials or reagents used in the embodiments of the present application do not have special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional methods well known to those skilled in the art.

[0033] Liquid feed: alcohol-free calorie-matched type (item number XSYT-ED-088), alcohol type feed (item number XSYT-ED-087), both purchased from Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.;

[0034] Example 1

[0035] 1.1 Experimental animals and grouping

[0036] 8-10 week-old wild-type C57BL / 6N mice were selected and randomly divided into 4 groups (5 mice in each group, with no significant difference in body weight), and the specific grouping is as follows:

[0037] The control group was given alcohol-free, calorie-matched liquid feed for whole course feeding;

[0038] The alcohol modeling group was first adapted for 1 week (including 1% (v / v) alcohol in liquid feed), and the alcohol concentration was gradually increased to 5% (v / v) for 6 weeks. Then the alcohol modeling group mice were injected intraperitoneally with 0 mg / kg (only olive oil), 1 mg / kg, 3 mg / kg of drug (GCN2iB) in olive oil solution every 3 days, for 7 times, and the 5% (v / v) alcohol liquid feed was continuously fed during the administration period.

[0039] 1.2 Experimental method

[0040] Each group of mice was fed with liquid feed according to the above grouping scheme, and the feed consumption was recorded daily, and the mice were weighed once a week to monitor the growth status. After the experiment, whole blood was collected by eyeball blood collection method, and placed in a centrifuge tube, and the upper serum was separated for serum index detection; after blood collection, the mice were sacrificed, the liver tissue was washed with pre-cooled physiological saline, and the water was absorbed with filter paper, then a part of the liver tissue was fixed in 4% neutral formaldehyde fixing solution for subsequent histological analysis; another part of the liver tissue was quickly placed in liquid nitrogen for freezing, and then transferred to a-80℃ refrigerator for storage for subsequent biochemical index detection, and the experimental process is shown in Figure 1 A.

[0041] Example 2: Serum index detection

[0042] According to the kit instructions, the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) content and total cholesterol (TC) content in the serum of each group of mice were detected by automatic biochemical analyzer. Statistical analysis: the data are expressed as "mean ± standard deviation (x ± s)", single factor analysis of variance was performed using SPSS 22.0 software, LSD-t test was used for comparison between groups, **P<0.01, ***P<0.001, the difference is statistically significant.

[0043] The results of serum index detection are shown in Figure 1 B-E.

[0044] According to the experimental data of Figure 1 B-E, compared with the control group, the ALT activity, AST activity, TG content and TC content in the serum of the alcohol modeling group (only injected with olive oil) mice were significantly increased, which indicated that long-term alcohol exposure had successfully induced liver function damage and blood lipid metabolism disorder in mice;

[0045] However, after treatment with GCN2iB at 1 mg / kg and 3 mg / kg, the activities of ALT and AST in the serum of mice were significantly reduced, and the contents of TG and TC were also significantly decreased, and the improvement effect of the high-dose group (3 mg / kg) was better than that of the low-dose group (1 mg / kg).

[0046] The results of this example demonstrate that the drug GCN2iB can improve alcohol-induced liver function damage and dyslipidemia

[0047] Example 3 Drug GCN2iB reduces alcohol-induced lipid deposition in the liver of mice

[0048] A portion of the liver tissue was placed in 4% paraformaldehyde fixing solution and fixed at 4°C for 48 h. Then the tissue was sequentially placed in 50%, 70%, 80%, 95%, and 100% ethanol for 1 h. Subsequently, the tissue was transferred into a mixture of 100% ethanol / dimethylbenzene (1:1, v / v) for 30 min, and then placed in pure dimethylbenzene for continuous transparency until the tissue was completely transparent under naked eye observation. After transparency, the tissue was transferred into an embedding box together with dimethylbenzene, and an equal volume of molten paraffin was added, and the tissue was immersed in a constant temperature oven at 62°C for 2 h. Then, fresh molten paraffin was added twice, each time for 2 h. After the end of the immersion, paraffin embedding was completed, and after the wax block solidified, a 5 μm thick section was cut with a paraffin sectioning machine, and the section was placed in a 37°C oven for overnight drying and was ready for use.

[0049] Before HE staining, the section was deparaffinated and rehydrated in the following order: dimethylbenzene for 10 min x 3 times, anhydrous ethanol for 3 min x 3 times, 95% ethanol for 3 min, 85% ethanol for 3 min, 70% ethanol for 3 min, and after 70% ethanol, an immunohistochemical recording pen was used to circle the tissue area. Then, HE staining was performed: the section was placed in hematoxylin staining solution for 1 min, and after washing with slow-flowing water for about 10 min, it was placed in eosin staining solution for 5 min. After staining, the section was dehydrated in 80% ethanol, 95% ethanol, and anhydrous ethanol for 3 min each, and then transparentized with dimethylbenzene for 5 min x 3 times, and neutral resin was added dropwise for mounting, and it was placed overnight under ventilation conditions. After the resin was completely solidified, the results were observed and photographed under a light microscope, as shown in Figure 2 .

[0050] The results of liver tissue section staining are shown in Figure 2 A, the number of lipid droplets in the liver tissue of the alcohol modeling group mice was large, and the volume was large; and the number of lipid droplets in the GCN2iB treatment group was significantly reduced, and the volume was reduced; H&E staining showed that the liver of the modeling group had balloon-like lesions and fuzzy cell boundaries; and the drug treatment group had reduced fat vacuoles and reduced tissue damage.

[0051] The results of liver tissue biochemical detection are shown inFigure 2 B-C show: compared with the control group, the alcohol modeling group liver tissue TG content (Figure 2B), TC content (Figure 2C) significantly increased (***P<0.001), after GCN2iB treatment, the liver TG, TC content of the two drug groups were significantly decreased ( **P<0.01, ***P<0.001).

[0052] It is proved that GCN2iB can inhibit alcohol-induced abnormal deposition of liver lipids.

[0053] Example 4 Drug GCN2iB alleviates alcohol-induced oxidative stress and inflammatory response in mouse liver

[0054] 4.1 The level of liver oxidative stress was detected using DHE fluorescent probe.

[0055] After the mice were sacrificed, the liver tissue was quickly taken and immediately placed in OCT embedding agent and stored at -80°C. Before staining, the frozen tissue was transferred to -20°C for about 2 h, then installed in the freezing microtome, prepared 5 μm thick sections and attached to cationic glass slides. During staining, the glass slides were taken out from -80°C and sequentially rewarmed at -20°C to 4°C for about 30 min. The residual OCT on the surface of the section was washed with distilled water, the water around the section was absorbed, and the tissue area was circled with an immunohistochemical pen.

[0056] According to the reagent instruction, 40 μmol / L DHE working solution was prepared with 1×PBS, and was added to the circled area to completely cover the tissue, and was placed in a wet box at 37°C for 30 min. After incubation, wash with 1×PBS for 3 times, add anti-quenching mounting medium for mounting, observe under fluorescence microscope and collect images for evaluation of ROS level in liver tissue, see Figure 3 A-B.

[0057] As Figure 3 A-B shows that DHE staining results show that the level of superoxide anion (O2 - ) in the liver tissue of the alcohol modeling group is increased, and the fluorescence intensity is significantly increased; while after GCN2iB treatment, the fluorescence intensity is significantly reduced, which indicates that the drug can reduce oxidative stress.

[0058] 4.2 Liver inflammatory response was detected by F4 / 80 immunohistochemical staining of macrophage infiltration.

[0059] Select the liver paraffin section prepared by the foregoing method, deparaffinized with xylene and gradiently rehydrated with ethanol to 75% ethanol according to the same procedure of HE staining. Immerse the section in 0.01 mol / L sodium citrate buffer and heat to micro-boiling in a microwave oven for about 15 min for antigen repair. After natural cooling to room temperature, immerse in PBS for 3 times. Add 3% hydrogen peroxide solution and incubate at room temperature for about 10 min to block the endogenous peroxidase activity. After PBS washing, add 1% BSA for blocking for 30 min. Discard the blocking solution, dilute the Proteintech F4 / 80 antibody (28463-1-AP) to prepare a working solution of the primary antibody at a dilution of 1:4000, drop it on the tissue surface and incubate in a 4°C humidified box overnight. The next day, immerse in PBS for 3 times according to the Zhongshan Golden Bridge Rabbit Two-step HRP immunohistochemical detection kit instruction. Then, develop the color according to the same company's DAB color reagent kit instruction. After gradiently dehydrating with ethanol and clearing with xylene, drop neutral resin for mounting, observe and take photos under an optical microscope, analyze the distribution characteristics and number of F4 / 80 positive cells (macrophages), and evaluate the degree of liver inflammatory response. The results are shown in Figs. Figure 3 A、 Figure 3 C.

[0060] The F4 / 80 immunohistochemical staining results show that the number of F4 / 80 positive macrophages in the liver of the alcohol modeling group is significantly increased, and the number of positive cells is significantly reduced after drug treatment, and the inflammatory infiltration is reduced.

[0061] 4.3 Detection of 3-NT and 4-HNE contents in liver tissue (ELISA method)

[0062] The 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) contents in the liver tissue were detected by the ELISA kit of Beijing Lv Yuanbo Biological Technology Co., Ltd.

[0063] Weigh about 20 mg of liver tissue into 200 μL of pre-cooled PBS, homogenize thoroughly under ice bath conditions, and centrifuge at 4°C and 13000 rpm for 15 min to take the supernatant as the sample to be tested, and part of the supernatant is used to determine the protein concentration by BCA kit. Then, according to the ELISA kit instruction, sample, incubation, washing, color development and reaction termination are performed, and the absorbance value is measured at 450 nm wavelength. According to the standard curve, the 3-NT or 4-HNE content is calculated, and combined with the protein quantification result, the index level under the unit protein content is converted, and the results are shown in Figs. Figure 3 D-E.

[0064] The levels of oxidative stress markers 3'-NT (Figure 3D) and 4-HNE (Figure 3E) in liver tissues were significantly increased in the alcohol modeling group, and were significantly decreased after GCN2iB treatment. Further indicated that GCN2iB can effectively inhibit alcohol-induced liver oxidative stress response and inflammatory infiltration.

[0065] In summary, the serum index detection, liver tissue biochemical analysis and histological detection confirmed that the drug GCN2iB can play a protective role on alcoholic liver injury by improving alcohol-induced liver function damage, regulating lipid metabolism, reducing liver lipid deposition, inhibiting oxidative stress and inflammatory response, and has a dose-dependent effect, providing a new drug selection for the treatment of alcoholic liver disease.

[0066] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which belong to the protection scope of the present application.

Claims

1. Use of a GCN2 kinase inhibitor for the manufacture of a medicament for the treatment or prevention of alcoholic liver disease, characterized in that, The inhibitor is an ATP-competitive inhibitor, the GCN2 kinase inhibitor is GCN2iB-1; the chemical formula of the GCN2iB-1 is C 18 H 12 ClF2N5O3S, the chemical structural formula is as shown in formula 1: As shown in the formula.

2. Use according to claim 1, characterized in that, The GCN2 kinase inhibitor can improve liver function.

3. Use according to claim 1, characterized in that, The GCN2 kinase inhibitor can improve dyslipidemia.

4. The use according to claim 1, characterized in that, The GCN2 kinase inhibitor can reduce lipid deposition in the liver.

5. The use according to claim 1, characterized in that, The GCN2 kinase inhibitor can alleviate oxidative stress in the liver.

6. The use according to claim 1, characterized in that, The GCN2 kinase inhibitor can alleviate inflammation in the liver.

7. Use according to claim 1, characterized in that, The dosage form of the drug is an oral dosage form or an injection dosage form.

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