Application of CPTH2 to improvement of liver fatty degeneration caused by LXR agonist

Abstract

The invention discloses application of CPTH2 to improvement of liver fatty degeneration caused by an LXR agonist. LXR activation has been demonstrated to have both an ideal result (increasing cholesterol reverse transport) and a bad result (increasing liver adipogenesis) for rodents and primates. The research shows that the combined use of the CPTH2 and the LXR agonist can relieve the increase of triglyceride (TG) in the liver and glutamic oxalacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) indexes of liver injury in serum. Therefore, the CPTH2 can be applied to preparation of the medicine for improving liver fatty degeneration caused by the LXR agonist. The combination of the CPTH2 and the LXR agonist can be applied to preparation of medicines for treating liver fatty degeneration.

Description

Technical Field

[0001] The invention belongs to the field of small molecule chemical drugs and relates to the application of CPTH2 in improving liver fatty degeneration induced by LXR agonists. Background Art

[0002] Hepatic steatosis refers to the appearance of fat droplets in the cytoplasm due to imbalanced fat metabolism in hepatocytes. The mechanism of fatty degeneration is relatively complex, including excessive fat synthesis or input, impaired fat oxidation and utilization, and impaired lipoprotein formation and export. Hepatic nuclear receptors (LXRs) are the main regulators of the reverse cholesterol transport pathway. LXR agonists (such as the first-generation synthetic LXR agonists, T0901317 and GW3965 are full agonists of LXRα and LXRβ, and have been shown to induce the expression of LXR target genes in vitro and in vivo) can promote reverse cholesterol transport in mouse and human cells, which is of great significance in the fight against cardiovascular disease. However, the lipogenic effect of LXR can cause an adverse increase in liver triglyceride levels, leading to hepatic steatosis, which has become a major obstacle to the development of LXR agonists as cardiovascular disease drugs. However, scientists are still looking for selective LXR modifiers, but this work is complicated by the fact that the ligand binding domains of LXRα and LXRβ have highly similar amino acid sequences. For example, LXR-623 and CS-8080 that have entered clinical trials have been terminated due to adverse reactions and unknown safety. This suggests that it may be possible to improve the side effects of LXR agonists by combining drugs without affecting their effects.

[0003]

[0004] CPTH2, cyclopentylmethylene-[4-(4′-chlorophenyl)thiazol-2-yl)hydrazone, with a chemical structure as shown in formula (I), is a potent histone acetyltransferase (HAT) inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5. It has been used to inhibit histone acetyltransferase in cortical cultures, bud cells, and natural killer T cells (iNKT).

[0005] Histone acetylation is regulated by histone acetyltransferases (HATs). HATs include multiple families such as the MYST family, the GCN5-related N-acetyltransferase (GNATs) family, and the p300 / CREB binding protein family. Different types of HATs may acetylate different sites of different histones, such as H3K9, H3K14, H4K8, H4K16, etc., thereby regulating the expression of different genes and playing different roles in different diseases. For example, GCN5, a member of the GNATs family, can significantly promote the acetylation of H3K9 in the promoter region of vascular endothelial growth factor, restore the function of endogenous bone marrow stem cells, and alleviate the defect of angiogenesis in the bone of osteoporotic mice; Tip60, a member of the MYST family, regulates the acetylation levels of H3K9 and H3K27 in the promoter region of SETDB1, inhibiting the invasion and migration ability of lung adenocarcinoma cells; p300 / CBP, a member of the p300 / CREB binding protein family, mediates the acetylation levels of H3K9 and H3K14 in the promoter histones of PAI-1 and p21, increasing the expression of PAI-1 and p21 and aggravating renal fibrosis.

[0006] GCN5, also known as KAT2A, is a member of the GNATs family. It has a HAT catalytic domain and its catalytic activity is highly conserved throughout eukaryotes. It can play a regulatory role by promoting histone H3 lysine 9 acetylation (H3K9ac) and H3 lysine 14 acetylation (H3K14ac). The role of GCN5 in the pathogenesis of different types of diseases has been widely studied, such as cancer, diabetes, multidrug resistance, and neurological diseases. However, the role of GCN5 in liver lipid metabolism is still unclear, and there are no reports on studies using GCN5 as a target for intervention to improve liver steatosis. Summary of the invention

[0007] The purpose of the present invention is to provide the use of CPTH2 in the preparation of a drug for improving liver fatty degeneration induced by LXR agonists in view of the above-mentioned deficiencies in the prior art.

[0008] Another object of the present invention is to provide the use of CPTH2 combined with LXR agonist in the preparation of a drug for treating hepatic fatty degeneration.

[0009] Another object of the present invention is to provide a pharmaceutical composition for treating fatty degeneration of the liver and its application.

[0010] The purpose of the present invention can be achieved through the following technical solutions:

[0011] Application of CPTH2 in the preparation of drugs for improving liver fatty degeneration induced by LXR agonists.

[0012] As a preferred embodiment of the present invention, the LXR agonist is selected from T0901317 or GW3965.

[0013] LXR activation has been shown to have both desirable (increased reverse cholesterol transport) and undesirable (increased hepatic lipogenesis) outcomes in rodents and primates. Our study demonstrated that the combination of CPTH2 and LXR agonists alleviated increases in hepatic triglycerides (TG) and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), markers of liver injury.

[0014] Application of CPTH2 combined with LXR agonist in the preparation of drugs for treating hepatic steatosis.

[0015] As a preferred embodiment of the present invention, the LXR agonist is selected from T0901317 or GW3965.

[0016] As a preferred embodiment of the present invention, CPTH2 combined with LXR agonist can reduce serum TG, TC, LDL-c, ALT, AST and liver TG, TC levels in patients with hepatic steatosis and reduce lipid accumulation.

[0017] A pharmaceutical composition for treating liver fatty degeneration, comprising CPTH2 and LXR agonist as effective ingredients.

[0018] As a preferred embodiment of the present invention, the LXR agonist is selected from T0901317 or GW3965.

[0019] As a preferred embodiment of the present invention, it also comprises auxiliary materials for preparing the active ingredients into pharmaceutical preparations.

[0020] As a preferred embodiment of the present invention, the dosage form of the pharmaceutical preparation is tablets, capsules, or injections.

[0021] The invention discloses an application of the drug combination in preparing a drug for treating fatty degeneration of the liver.

[0022] Beneficial effects:

[0023] The experiment of the present invention found that the liver weight and liver weight to body weight ratio of mice given LXR agonist T0901317 increased, and compared with the model group, the levels of liver TG, serum liver injury indicators ALT and AST were abnormally increased. However, the TG, TC, LDL-c content in the serum and the TC content in the liver of mice treated with LXR agonist T0901317 were significantly lower than those in the model group. In contrast, in mice treated with LXR agonist T0901317 and CPTH2, we found that their serum TG, TC, LDL-c, ALT, AST and liver TG and TC levels were further reduced. Finally, liver hematoxylin and eosin (H&E) and Oil Red O staining showed that the co-administration of LXR agonist T0901317 and CPTH2 significantly reduced lipid accumulation in HFD-fed mice. In summary, these data indicate that the combined use of LXR agonists and CPTH2 may exert a synergistic lipid-lowering effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The experimental results of food intake of mice;

[0025] Figure 2 The experimental results are for the weight of mice;

[0026] Figure 3 The experimental results are for mouse liver weight;

[0027] Figure 4 This is the experimental result of the ratio of liver weight to body weight of mice;

[0028] Figure 5 The experimental results of triglyceride (TG) content in mouse serum;

[0029] Figure 6 The experimental results of total cholesterol (TC) content in mouse serum;

[0030] Figure 7 This is the experimental result of the content of high-density lipoprotein (HDL-c) in mouse liver;

[0031] Figure 8 This is the experimental result of the low-density lipoprotein (LDL-c) content in mouse liver;

[0032] Fig. 9 The experimental results of triglyceride (TG) content in mouse serum;

[0033] Fig.10 The experimental results of total cholesterol (TC) content in mouse serum;

[0034] Fig.11 The experimental results of aspartate aminotransferase (AST) content in mouse serum;

[0035] Fig.12 The experimental results of alanine aminotransferase (ALT) content in mouse serum;

[0036] Fig.13 The experimental results of hematoxylin-eosin staining (H&E staining) and Oil Red O staining of mouse liver tissue;

[0037] Fig.14 These are the results of the quantitative Oil Red O staining of mouse liver. DETAILED DESCRIPTION

[0038] Experimental methods:

[0039] After one week of acclimatization, 7-week-old male C57BL / 6J mice were fed HFD (US Research Diets, catalog number D12492, 60% fat content) to induce liver fat accumulation. Six weeks after modeling, the mice were divided into four groups according to their weight: control group, T0901317 (5 mg / kg) group, CPTH2 (20 mg / kg) group, and T0901317 and CPTH2 (20 mg / kg) group. The model group mice were orally given solvent as a control, and the mice in the drug group were orally given T0901317 (5 mg / kg), CPTH2 (20 mg / kg) or both. The weight and food intake of mice in each group were recorded for later data statistics, and HFD was continued for 2 weeks. After that, the mice were fasted overnight, blood was collected after anesthesia, and then they were killed and liver tissue was obtained by dissection.

[0040] Example 1: Effect of combined administration of T0901317 and CPTH2 on hepatic steatosis

[0041] Experimental method: The body weight and average food intake of mice were recorded at the same time every week after grouping (marking the tail with a marker). After the experiment, all graphs and statistics were performed by GraphPad Prism8.0, and the data were expressed as mean standard error (SE). Statistical analysis was performed by t-test, and differences with p<0.05 were considered statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant).

[0042] Experimental results: Figure 1-2 As shown in Figure 2, the weight of mice gradually increased with HFD feeding, but there were no significant differences in body weight and food intake among the groups. Figure 3-4As shown in the figure, compared with the model group mice, the liver weight (LW) and liver weight to body weight ratio (LW / BW) of the T0901317 alone group mice were statistically significantly increased, more than double the model group. CPTH2 administration, T0901317 and CPTH2 combined administration significantly downregulated the increase in liver weight and liver weight to body weight ratio induced by HFD, HFD and T0901317. This shows that T0901317 can induce lipid accumulation in liver cells, and CPTH2 can reverse the adverse effects of T0901317 on the liver.

[0043] Example 2: Changes in the levels of total cholesterol (TC), total triglycerides (TG), high-density lipoprotein (HDL-c) and low-density lipoprotein (LDL-c) in serum

[0044] Experimental method: Separation of serum: Blood was collected from the eye sockets of anesthetized mice, and centrifuged after standing at low temperature for 4 hours. The upper serum was transferred to a new centrifuge tube and stored at -80°C. The levels of total cholesterol (TC), total triglycerides (TG), high-density lipoprotein (HDL-c) and low-density lipoprotein (LDL-c) in serum were detected according to the instructions of the conventional kit.

[0045] Experimental results: Figure 5-8 As shown, compared with the model group, the TG content in the serum of mice in the T0901317-treated group decreased by 50%, the TC content decreased by 26%, the LDL-c content decreased by 21.6%, and there was no statistical difference in HDL-c. These indicators jointly maintain the balance of serum lipids, indicating that T0901317 is of great significance in regulating lipid metabolism. On the other hand, the levels of TG, TC, and LDL-c were further decreased in mice co-administered with T0901317 and CPTH2, suggesting that the combined use of T0901317 and CPTH2 may play a synergistic lipid-lowering role.

[0046] Example 3: Changes in the content of total triglycerides (TG) and total cholesterol (TC) in the liver

[0047] Experimental method: Extraction of lipid components in liver tissue: Weigh 25-30 mg of liver tissue and homogenize it in 0.5 ml PBS. After standing for 30 minutes, centrifuge and absorb 60 ul of supernatant for BCA quantitative detection. Add 1 ml of organic extraction reagent (chloroform: methanol = 2:1 volume fraction) to the remaining sample, vortex evenly, and shake for 3 hours. Then add 0.5 ml of 0.9% saline, vortex evenly, and centrifuge. Transfer the lower organic phase to a new centrifuge tube and evaporate it in a fume hood overnight. Add 80 ul of 0.1% Triton X-100 (prepared with anhydrous ethanol) to dissolve the lipids at the bottom of the tube and store at 4°C. Detect the content of total cholesterol (TC) and total triglycerides (TG) in liver tissue according to the instructions of the conventional kit.

[0048] Experimental results: Figure 9-10 As shown in the figure, compared with the model group, T0901317 treatment downregulated 42.8% of liver TC content, indicating that T0901317 plays a key role in liver cholesterol metabolism. However, administration of T0901317 can increase liver TG content by 2.7 times, which will burden the liver with excessive lipid accumulation and is also a major obstacle to the development of LXR agonists as cardiovascular disease drugs. In the liver tissue of mice given CPTH2 and T0901317 at the same time, TG content decreased significantly by 58%, showing the great advantage of combined administration.

[0049] Example 4: Changes in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels

[0050] Experimental method: Serum was obtained as described in Example 2. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were detected according to the instructions of conventional kits.

[0051] Experimental results: T0901317-induced hepatic lipid accumulation can cause certain liver damage, such as Figure 11-12 As shown in the results, serum aspartate aminotransferase (AST) increased by 1.4 times, and serum alanine aminotransferase (ALT) levels increased by 1.5 times. In addition, CPTH2 administration can not only alleviate the increase in serum liver damage indicators caused by the HFD diet, but also further reduce AST and ALT levels when used in combination with T0901317. This once again shows that the combination of T0901317 and CPTH2 can play a synergistic lipid-lowering effect.

[0052] Example 5: HE and Oil Red O pathological sections of liver tissue

[0053] Experimental methods: The liver was fixed in 4% paraformaldehyde at 4 °C overnight, embedded in paraffin, and cut into 5 μm thick sections. Liver sections were stained with hematoxylin and eosin (H&E) to evaluate morphology. Liver sections were stained with Oil Red O and counterstained with hematoxylin to evaluate lipid accumulation. Sections were observed and captured using an upright fluorescence microscope (Olympus, BX53, Tokyo, Japan). Lipid droplets were quantified using ImageJ (Version 1.5a). GraphPad Prism 8.0 was used for graphing and statistics, and data are expressed as mean standard error (SE). Statistical analysis was performed by t-test, and differences with p < 0.05 were considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant).

[0054] Experimental results: Fig.13The liver tissue pathological sections shown in the figure show that obvious lipid droplets appeared in the liver of mice fed with HFD, and the liver steatosis of mice in the T0901317 group was further aggravated. CPTH2 reversed liver steatosis and exerted a better improvement effect when used in combination with T0901317. The relative quantitative results of the red area representing neutral lipids in the Oil Red O staining image showed consistency, as shown in Figure 2. Fig.14 shown.

[0055] In summary, the combined use of T0901317 and CPTH2 may exert a synergistic lipid-lowering effect and significantly improve hepatocyte degeneration in C57BL / 6J mice fed a high-fat diet.

Claims

1. Application of CPTH2 in the preparation of drugs for improving liver fatty degeneration induced by LXR agonists.

2. The use according to claim 1, characterized in that , the LXR agonist is selected from T0901317 or GW3965.

3. The use according to claim 1 or 2, characterized in that The LXR agonist can alleviate the increase of triglycerides in the liver and liver damage indicators in the serum caused by the LXR agonist; the liver damage indicators in the serum are aspartate aminotransferase AST and alanine aminotransferase ALT.

4. Application of CPTH2 combined with LXR agonists in the preparation of drugs for the treatment of hepatic fatty degeneration.

5. The use according to claim 4, characterized in that , the LXR agonist is selected from T0901317 or GW3965.

6. The use according to claim 4, characterized in that ,CPTH2 combined with LXR agonists can reduce serum TG, TC, LDL-c, ALT, AST and liver TG, TC levels in patients with hepatic steatosis and reduce lipid accumulation.

7. A pharmaceutical composition for treating fatty degeneration of the liver, characterized in that: It contains CPTH2 and LXR agonists as active ingredients.

8. The pharmaceutical composition according to claim 7, characterized in that The LXR agonist is selected from T0901317 or GW3965.

9. The pharmaceutical composition according to claim 8, characterized in that It also contains auxiliary materials for preparing the active ingredients into pharmaceutical preparations.

10. Use of the drug combination according to any one of claims 7 to 9 in the preparation of a drug for treating fatty degeneration of the liver.

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