Application of a small molecule compound in the preparation of anti-liver injury or anti-liver fibrosis drugs

Abstract

The present invention relates to the use of a small molecule compound in the preparation of an anti-liver injury or anti-liver fibrosis drug. The compound has a specific compound structure shown in formula I: Cell experiments confirm that the compound can inhibit the secretion of type III procollagen N-terminal peptide (PIIINP) of LX-2 cells and the inhibitory effect is stable, can improve acetaminophen (APAP)-induced HepG2 cell liver injury and inhibit the expression of liver fibrosis-related genes and proteins, inhibit the migration of LX-2 cells, etc. In addition, the compound exhibits low toxicity to hepatocytes; therefore, the compound of formula I structure has potential as a component of an anti-liver fibrosis drug.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to the use of a small molecule compound in the preparation of an anti-liver injury or anti-liver fibrosis drug. Background Art

[0002] The occurrence of liver fibrosis is due to the activation of the inflammatory process under the action of various causes, inducing changes in the activity of the cytokine network, and then promoting the activation and proliferation of HSC under the action of platelet-derived growth factor; further under the action of transforming growth factor, HSC secretes more extracellular matrix components such as collagen, leading to the occurrence of liver fibrosis and cirrhosis. In this process, normal liver tissue is gradually replaced by scar tissue, resulting in changes in liver structure and function.

[0003] Common causes of liver fibrosis include chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), autoimmune liver disease, drug or toxin exposure, genetic diseases, circulatory system problems, and parasitic infections. If liver fibrosis is not controlled, it may progress to cirrhosis, a more serious liver disease state that may lead to liver failure. LX-2 cells are a type of human hepatic stellate cells (HSCs) that play an important role in liver physiology and pathology. In chronic liver diseases, such as viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease, LX-2 cells change from a quiescent state to an activated state and begin to synthesize a large amount of extracellular matrix proteins, such as collagen, which leads to the aggravation of liver fibrosis. In addition, LX-2 cells are not only involved in the fibrosis process, but also can secrete a variety of pro-inflammatory factors, such as cytokines and chemokines, to promote inflammatory responses. Cell migration can bring these factors to the site of inflammation, exacerbating the local inflammatory response.

[0004] GLT25D1-mediated Glc α1, 2Galβ1-glycosylation modification is the most important post-translational modification of collagen and is a key target for controlling the assembly and secretion quality of collagen fibrils. Based on the applicant's previous research conclusions, by inhibiting GLT25D1-mediated collagen peptide Glc1, 2Gal1-glycosylation modification, the assembly of collagen fibrils and the secretion of type I and III collagen can be inhibited, and the occurrence of liver fibrosis can be partially blocked. Therefore, GLT25D1 can be used as a therapeutic target for anti-liver fibrosis. Finding a low-toxic and highly effective GLT25D1 inhibitor will hopefully be used in the development of anti-liver fibrosis drugs. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an application of a small molecule compound in the preparation of an anti-liver injury or anti-liver fibrosis drug. The small molecule compound can inhibit the expression of GLT25D1 and liver fibrosis-related proteins, and has no obvious toxicity to HepG2 cells, and has application prospects in anti-liver fibrosis drugs and treatment plans.

[0007] (II) Technical solution

[0008] A small molecule compound is used in the preparation of an anti-liver injury or anti-liver fibrosis drug, wherein the compound has a structure shown in Formula I: .

[0009] According to a preferred embodiment of the present invention, the compound of formula I achieves resistance to liver damage or liver fibrosis by targeting GLT25D1 expression.

[0010] According to a preferred embodiment of the present invention, the liver injury includes acetaminophen-induced liver injury.

[0011] According to a preferred embodiment of the present invention, the drug uses the compound of the structure shown in Formula I as the sole active ingredient or is prepared into a compound drug with other anti-liver injury or anti-liver fibrosis active ingredients.

[0012] According to a preferred embodiment of the present invention, the medicine further comprises pharmaceutically acceptable excipients.

[0013] Furthermore, the above-mentioned drug also includes a pharmaceutically acceptable carrier or excipient, additive; the carrier or excipient is preferably selected from one or more of a diluent, a binder, an adsorption carrier, a filler and a disintegrant; the additive is preferably selected from one or more of a stabilizer, a bactericide, a buffer, an isotonic agent, a chelating agent, a pH controller and a surfactant.

[0014] Furthermore, the administration route of the above-mentioned drug is oral, transdermal, intramuscular, subcutaneous or intravenous injection.

[0015] Furthermore, the dosage form of the drug can be tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, creams, sprays, drops or patches.

[0016] (III) Beneficial effects

[0017] The compound of formula I provided by the present invention is confirmed by cell experiments: the compound can significantly inhibit the secretion of type III procollagen N-terminal peptide (PIIINP) of LX-2 cells at lower concentrations (0.001mM and 0.1mM) and the inhibitory effect is stable; the compound can improve acetaminophen (APAP)-induced HepG2 cell liver injury at a concentration of 0.001mM; the compound can significantly inhibit the expression of liver fibrosis-related genes (Col 1A1, Col3A1 and ACTA2) at a concentration of 0.001mM, and inhibit the expression level of GLT25D1 and liver fibrosis-related proteins (GLT25D1, Collagen I and α-SMA); the compound inhibits the migration of LX-2 cells at a concentration of 0.001mM. The above conclusions show that the compound of formula I of the present invention has a good therapeutic effect of resisting liver injury or liver fibrosis.

[0018] The above compound was further evaluated by the ADMET Descriptors module of the Dcovery Studio 2019 software. The docking score of the compound with the target GLT25D1 reached -11.7525, which means that it has a strong interaction with the target receptor. In addition, cell experiments also confirmed that the compound has no obvious toxicity to HepG2 cells. In summary, the compound of formula I is a potential component of anti-liver fibrosis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figures show the secretion of type III procollagen N-terminal peptide (PIIINP) and type IV collagen (CIV) in LX-2 cells before and after treatment of LX-2 cells with the compound of formula I; Figure A shows the secretion of PIIINP in LX-2 cells after treatment of LX-2 cells with the compound of formula I; Figure B shows the secretion of CIV in LX-2 cells after treatment of LX-2 cells with the compound of formula I. ns, ** and **** indicate no statistical difference, P<0.01 and P<0.0001, respectively.

[0020] Figure 2 The effect of the compound of formula I on the viability of HepG2 cells; ** and **** indicate P<0.01 and P<0.0001, respectively.

[0021] Figure 3 The effect of the compound of formula I on the APAP-induced acute liver injury model; in Figure A, the ALT level in the supernatant of HepG2 cells after treatment with the compound of formula I in the APAP-induced acute liver injury model; in Figure B, the AST level in the supernatant of HepG2 cells after treatment with the compound of formula I in the APAP-induced acute liver injury model; **** indicates P<0.0001.

[0022] Figure 4 The effect of the compound of formula I on the expression of GLT25D1 and liver fibrosis-related genes; Figure AD shows the mRNA levels of GLT25D1 (A), Col 1A1 (B), Col3A1 (C) and ACTA2 (D) under three conditions: blank control, addition of human TGF-β1 recombinant protein (10 ng / mL), and addition of the compound of formula I (0.001 mM) after pre-stimulation with TGF-β1 recombinant protein (10 ng / mL). ns, *, **, *** and **** indicate no statistical difference, P<0.05, P<0.01, P<0.001 and P<0.0001, respectively.

[0023] Figure 5 The effect of the compound of formula I on the expression of GLT25D1 and liver fibrosis-related proteins; Figure A: The protein expression of GLT25D1, Collagen I and α-SMA under three conditions: blank control, addition of human TGF-β1 recombinant protein (10 ng / mL), and addition of the compound of formula I (0.001mM) after pre-stimulation with TGF-β1 recombinant protein (10 ng / mL); Figure BD: Gray value analysis of the left Western Blot band. GAPDH is the internal reference protein. *, ** and *** indicate P<0.05, P<0.01, and P<0.001, respectively.

[0024] Figure 6 The effect of the compound of formula I on the migration of LX-2 cells; wherein: blank control, addition of human TGF-β1 recombinant protein (10 ng / mL) and pre-stimulation with TGF-β1 recombinant protein (10 ng / mL) followed by addition of the compound of formula I (0.001 mM), the migration of cells treated with the three methods were observed after 12 h, 24 h and 48 h of culture; * and ** represent P<0.05 and P<0.01, respectively. DETAILED DESCRIPTION

[0025] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0026] Example 1

[0027] This example proves that the compound of formula I can inhibit the secretion of collagen in LX-2 cells. The verification process is as follows:

[0028] 1. Experimental basis and methods

[0029] TGF-β1 is the most common hepatic stellate cell stimulating factor in the liver and the most commonly used hepatic stellate cell stimulating factor in vitro studies. This example uses human recombinant TGF-β1 (10ng / ml) to induce LX-2 cell activation in vitro to simulate the hepatic stellate cell activation process in vitro. In order to explore the effect of the compound of formula I on the secretion of collagen by LX-2 cells, TGF-β1 recombinant protein was used to induce activated LX-2 cells, and double antibody sandwich immunochromatography was used to determine the secretion of type III procollagen N-terminal peptide (PIIINP) and type IV collagen (CIV) in the supernatant of LX-2 cells after treatment with different concentrations of the compound of formula I (low concentration: 0.001mM; medium concentration: 0.01mM; high concentration: 0.1mM). The collected cell supernatant was used to measure the secretion of type III collagen precursor and IV collagen using collagen test strips.

[0030] The LX-2 used in this experiment came from the Institute of Infectious Diseases, Ditan Hospital, Beijing, and was identified by STR to be consistent with LX-2 related cell information. LX-2 adherent growth was cultured in a high-glucose medium containing a mixture of 10% FBS and 1% penicillin-streptomycin. Among them, the method of using human recombinant TGF-β1 (10ng / ml) to induce activation of LX-2 cells is as follows: prepare LX-2 cell suspension, add 10% complete medium to adhere to the wall for culture, culture for 12h, replace serum-free medium for starvation for 12h, and finally replace with 2% complete medium, add human TGF-β1 recombinant protein (10ng / mL) for pre-stimulation for 2h, then add the compound of formula I for culture for 24h.

[0031] 2. Experimental results

[0032] The results are as follows Figure 1 As shown, for type III procollagen N-terminal peptide (PIIINP), compared with the control group, the level of PIIINP secreted by LX-2 cells in the TGF-β1 group increased significantly, while after being treated with three concentrations of 0.001mM, 0.01mM, and 0.1mM of the compound of formula I, the level of PIIINP secreted by LX-2 cells decreased significantly, among which 0.001mM and 0.1mM concentrations had better effects on inhibiting PIIINP secretion (as shown in Figure 2). Figure 1 For type IV collagen (CIV), neither TGF-β1 stimulation nor treatment with three concentrations of 0.001mM, 0.01mM, and 0.1mM of the compound of formula I had a significant effect on the level of CIV secreted by LX-2 cells (e.g. Figure 1 B).

[0033] Example 2

[0034] This example proves that the compound of formula I has no obvious toxicity to HepG2 cells, and the verification process is as follows:

[0035] 1. Experimental basis and methods

[0036] In order to determine whether the compound of formula I has hepatotoxicity, the CCK8 experiment was used to detect the effects of different concentrations (low concentration: 0.001 mM; medium concentration: 0.01 mM; high concentration: 0.1 mM) of the compound of formula I on the viability of HepG2 cells, comprising the following steps:

[0037] (1) HepG2 cells were digested with trypsin to prepare a cell suspension, which was seeded into a 96-well plate and cultured in a DMEM medium containing a mixture of 10% FBS and 1% penicillin-streptomycin for 24 h. Different concentrations of the compound of formula I were then added and cultured for another 24 h.

[0038] (2) Aspirate and discard the supernatant. Add CCK-8 reagent to serum-free DMEM at a ratio of 1:10 to each well and tap gently to mix.

[0039] (3) After incubation in a 37°C incubator for 1 h, the culture plate was removed and the absorbance (OD value) at 450 nm was measured using an ELISA reader to calculate the viability of the HepG2 cells.

[0040] The HepG2 cell line used in this experiment was from the Institute of Infectious Diseases, Ditan Hospital, Beijing. After STR identification, it was consistent with the relevant cell information of HepG2. HepG2 adhered to the wall and was cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin mixture.

[0041] 2. Experimental results

[0042] The results are as follows Figure 2 As shown, the viability of HepG2 cells decreases with the increase of the concentration of the compound of formula I. When the concentration of the compound of formula I is 0.001 mM, it has no obvious effect on the viability of HepG2 cells, that is, no obvious liver toxicity. When the concentration of the compound of formula I is 0.1 mM, the viability of HepG2 cells is significantly reduced.

[0043] Example 3

[0044] This example demonstrates that the compound of formula I can improve acetaminophen (APAP)-induced liver injury in HepG2 cells. The verification process is as follows:

[0045] 1. Experimental basis and methods

[0046] In order to further observe whether the compound of formula I has a protective effect on HepG2 cells at a concentration of 0.001 mM, acetaminophen (APAP, concentration of 10 mM) was used to induce an acute liver injury model in vitro in HepG2 cells, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the supernatant of HepG2 cells were detected after treatment with 0.001 mM of the compound of formula I.

[0047] 2. Experimental results

[0048] The results are as follows Figure 3 As shown, compared with the control group, after acetaminophen APAP induction, the ALT and AST levels in the supernatant of HepG2 cells were significantly increased, while after treatment with 0.001 mM of the compound of formula I, the ALT and AST levels in the supernatant of HepG2 cells were significantly lower than those in the group treated with 10 mM APAP ( Figure 3 A, 3B). It can be seen that the compound of formula I can reduce the hepatocyte damage induced by acetaminophen (APAP).

[0049] Example 4

[0050] This example demonstrates that the compound of formula I can inhibit the expression of genes related to liver fibrosis, and the verification process is as follows:

[0051] 1. Experimental basis and methods

[0052] It has been demonstrated in Example 1 that the compound of formula I (0.001 mM) can inhibit the secretion of collagen in LX-2 cells. In order to further explore the effect of the compound of formula I (0.001 mM) on the mRNA expression levels of GLT25D1 and liver fibrosis-related genes in LX-2 cells, this experiment used TGF-β1 recombinant protein to induce activated LX-2 cells, and qRT-PCR was used to detect the relative levels of GLT25D1, Col 1A1, Col3A1 and ACTA2 gene mRNA in LX-2 cells after treatment with 0.001 mM of the compound of formula I.

[0053] 2. Experimental results

[0054] The results are as follows Figure 4 As shown in Figure 2, compared with the control group, the mRNA levels of GLT25D1, Col 1A1, Col3A1, and ACTA2 in LX-2 cells in the TGF-β group were significantly increased (Figure 2 Figure 4A to D); After treatment with 0.001 mM of the compound of formula I, the mRNA levels of Col 1A1, Col3A1 and ACTA2 in LX-2 cells were significantly decreased compared with the TGF-β group, while the mRNA level of GLT25D1 showed only a downward trend, with no significant difference (such as Figure 4 A to D).

[0055] Example 5

[0056] This example demonstrates that the compound of formula I can inhibit the expression of GLT25D1 and liver fibrosis-related proteins. The verification process is as follows:

[0057] 1. Experimental basis and methods

[0058] In order to further explore the effect of the compound of formula I (0.001 mM) on the expression levels of GLT25D1 and liver fibrosis-related proteins in LX-2 cells, this experiment used TGF-β1 recombinant protein to induce activated LX-2 cells, and used Western Blot to detect the relative expression levels of GLT25D1, Collagen I and α-SMA proteins in LX-2 cells after treatment with 0.001 mM of the compound of formula I.

[0059] 2. Experimental results

[0060] The results are as follows Figure 5 As shown in Figure 2, compared with the control group, the relative expression levels of GLT25D1, Collagen I and α-SMA proteins in LX-2 cells in the TGF-β group were significantly increased (as shown in Figure 2). Figure 5 A to D); After treatment with 0.001 mM of the compound of formula I, the relative expression levels of GLT25D1, Collagen I and α-SMA proteins in LX-2 cells were significantly decreased compared with those in the TGF-β group (e.g. Figure 5 A to D).

[0061] Example 6

[0062] This example proves that the compound of formula I can inhibit the migration of LX-2 cells. The verification process is as follows:

[0063] 1. Experimental basis and methods

[0064] In this experiment, the scratch test was used to evaluate the migration of hepatic stellate cells. Scratch test method: LX-2 cells that had been pre-stimulated with human TGF-β1 recombinant protein (10 ng / mL) for 2 hours were added with different concentrations of Formula I compounds and cultured for 20 hours. Parallel horizontal lines were drawn on the back of a 12-well plate with a marker pen, with an interval of 0.3-0.5 cm. At least 3 straight lines per well were used as the baseline of the scratch; after the scratch was completed, the cells were washed 3 times with PBS to wash away the non-adherent cells, and different concentrations of Formula I compounds were added and placed in an incubator for culture. The cells were taken out after 0, 12, 24, 48, and 72 hours, and observed and photographed under a microscope. Image J software was used to analyze and measure the migration rate of the scratch.

[0065] 2. Experimental results

[0066] The results are as follows Figure 6 As shown, at 12h and 24h, there was no significant difference in the migration rate of LX-2 cells among the control group, TGF-β group and 0.001mM compound of formula I treatment group; at 48h, compared with the control group, the migration rate of LX-2 cells in the TGF-β group was significantly increased, while after treatment with 0.001mM compound of formula I, the migration rate of LX-2 cells was significantly inhibited compared with that of LX-2 cells activated by TGF-β without administration.

[0067] The above examples prove that the compound of formula I can delay the progression of liver fibrosis, inhibit the expression of liver fibrosis-related genes and proteins, improve drug-induced liver cell damage, and have no obvious toxicity to HepG2 cells after co-incubation with liver cells. In addition, the ADMET Descriptors module of Dcovery Studio 2019 software was used to evaluate the compound of formula I, and the docking score S between it and the receptor GLT25D1 was -11.7525, indicating that the compound has a strong interaction with the receptor.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a small molecule compound in the preparation of an anti-liver injury or anti-liver fibrosis drug, characterized in that: The compound has a compound structure shown in Formula I: The liver injury is acetaminophen-induced liver injury.

2. The use according to claim 1, characterized in that: The compound of formula I achieves resistance to liver damage or liver fibrosis by targeting GLT25D1 expression.

3. The use according to claim 1, characterized in that: The drug uses the compound of the structure shown in formula I as the sole active ingredient or is prepared into a compound drug with other active ingredients for resisting liver damage or liver fibrosis.

4. The use according to claim 1 or 3, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

5. The use according to claim 1 or 3, characterized in that: The drug also includes a pharmaceutically acceptable carrier, excipient or additive; the carrier or excipient is selected from one or more of a diluent, a binder, an adsorption carrier, a filler and a disintegrant; the additive is selected from one or more of a stabilizer, a bactericide, a buffer, an isotonic agent, a chelating agent, a pH controller and a surfactant.

6. The use according to claim 1 or 3, characterized in that: The drug is administered orally.

7. The use according to claim 1 or 3, characterized in that: The dosage form of the medicine is tablet, capsule, oral liquid, lozenge, granule, granule, pill, powder, paste, pill, suspension, powder and solution.

Images