Use of small-molecule compound NX-1607 in preparation of product for treating liver fibrosis

Abstract

The present invention relates to the technical field of biopharmaceuticals and particularly relates to use of a small-molecule compound, NX-1607, in the preparation of a product for treating liver fibrosis. NX-1607 has a highly efficient anti-liver fibrosis effect, exhibits a significant anti-fibrosis effect in multiple mouse models of liver fibrosis, and has wide adaptability. NX-1607 has also been found to up-regulate the expression of a key inhibitory factor of the TGF-β signaling pathway, SMAD7, and reduce the activation of hepatic stellate cells, thereby effectively alleviating liver fibrosis. This mechanism is different from existing anti-liver fibrosis drugs and provides new technical support for the treatment of liver fibrosis. NX-1607 exhibits no significant toxicity or side effects in an effective dose range (3-8 mg / kg / d), indicating a favorable safety profile. It can be seen that NX-1607 is a safe and effective anti-liver fibrosis drug and can alleviate the problems of the efficacy being limited and the side effects being relatively significant at present.

Description

Application of the small molecule compound NX-1607 in the preparation of products for treating liver fibrosis Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the small molecule compound NX-1607 in the preparation of products for treating liver fibrosis. Background Technology

[0002] Currently, liver fibrosis is a widespread pathological process caused by chronic liver injury, which may eventually develop into cirrhosis or even liver cancer. Although studies have shown that the TGF-β signaling pathway promotes the occurrence and development of liver fibrosis, there are still no effective anti-fibrotic drugs targeting the TGF-β signaling pathway in clinical practice.

[0003] Existing treatments for liver fibrosis mainly include: (1) Anti-inflammatory therapy: such as glucocorticoids, but long-term use may cause side effects, such as immunosuppression. (2) Antioxidant stress therapy: such as S-adenosylmethionine, but its efficacy is limited. (3) Anti-fibrotic drugs: including some experimental TGF-β pathway inhibitors, but most of them have not yet entered clinical use, and some drugs have problems such as poor pharmacokinetic properties and large side effects.

[0004] Therefore, there is an urgent need for a safe and effective anti-liver fibrosis drug to improve the shortcomings of current treatment methods. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of the small molecule compound NX-1607 in the preparation of products for treating liver fibrosis. This invention reveals that NX-1607 can upregulate the expression of SMAD7, a key inhibitor of the TGF-β signaling pathway, and reduce the activation of hepatic stellate cells (HSCs), thereby effectively alleviating liver fibrosis. It is a safe and effective anti-liver fibrosis drug that can improve upon the current limitations in efficacy and the significant side effects associated with traditional treatments.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of the small molecule compound NX-1607 in the preparation of products for treating liver fibrosis.

[0008] Preferably, the product includes a drug.

[0009] This invention provides a drug for treating liver fibrosis, the active ingredient of which includes the small molecule compound NX-1607; the unit dose of the drug is 3~8 mg / kg body weight in mice.

[0010] Preferably, the unit dose of the drug is 5 mg / kg body weight, based on the mouse's body weight.

[0011] Preferably, the drug further includes excipients; the excipients include a 5 g / L sodium carboxymethyl cellulose solution.

[0012] Beneficial effects

[0013] This invention provides the application of the small molecule compound NX-1607 in the preparation of products for treating liver fibrosis. This invention discovers that NX-1607 possesses highly effective anti-liver fibrosis activity, exhibiting significant anti-fibrotic effects in multiple mouse models of liver fibrosis (MCD diet model and CCL4-induced model), demonstrating broad applicability and promising potential as a novel anti-liver fibrosis drug candidate. This invention also discovers that NX-1607 can upregulate the expression of SMAD7, a key inhibitor of the TGF-β signaling pathway, and reduce the activation of hepatic stellate cells (HSCs), thereby effectively alleviating liver fibrosis. This mechanism differs from existing anti-liver fibrosis drugs, providing new technical support for liver fibrosis treatment. Furthermore, NX-1607 is a commercially available drug that has entered the clinical trial stage, and no significant toxic side effects were found within the effective dose range (3~8 mg / kg / d), demonstrating good safety. In summary, this invention discovers that NX-1607 is a safe and effective anti-liver fibrosis drug that can improve the current problems of limited efficacy and significant side effects. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0015] Figure 1 shows the Masson staining results of the MCD model after treatment with NX-1607.

[0016] Figure 2 shows the Sirius red staining results after treatment of the MCD model with NX-1607.

[0017] Figure 3 shows the detection results of serum liver damage markers ALT and AST after treatment with the MCD model NX-1607;

[0018] Figure 4 shows the results of RT-qPCR detection after treatment of the MCD model NX-1607;

[0019] Figure 5 shows the Masson staining results of the CCL4 model after NX-1607 treatment;

[0020] Figure 6 shows the Sirius red staining results of the CCL4 model after NX-1607 treatment;

[0021] Figure 7 shows the detection results of serum liver damage markers ALT and AST after CCL4 model NX-1607 treatment;

[0022] Figure 8 shows the results of RT-qPCR detection after NX-1607 treatment of the CCL4 model;

[0023] Figure 9 shows the Western Blot results of human hepatic stellate cell line LX2 after stimulation with NX-1607;

[0024] Among them, NX1607 in Figures 3, 4, 7 and 8 refers to the NX-1607 treatment group. Detailed Implementation

[0025] This invention provides the application of the small molecule compound NX-1607 in the preparation of products for treating liver fibrosis. The small molecule compound NX-1607 described in this invention is a commercially available small molecule compound that has entered the clinical trial stage. No significant toxic side effects have been observed within the effective dose range (3~8 mg / kg / day), demonstrating good safety. As one embodiment, the small molecule compound NX-1607 described in this invention was purchased from Sellck, catalog number E1957.

[0026] As one implementation method, the product can be a drug.

[0027] This invention reveals that NX-1607 has a highly effective anti-liver fibrosis effect, exhibiting significant anti-fibrotic activity in multiple mouse models of liver fibrosis (MCD diet model and CCL4-induced model). It can inhibit αSMA gene expression and Col1a1 gene expression, and reduce serum alanine aminotransferase and aspartate aminotransferase levels. It has broad applicability and is expected to become a novel candidate drug for anti-liver fibrosis.

[0028] This invention also found that NX-1607 can upregulate the expression of SMAD7, a key inhibitor of the TGF-β signaling pathway, and reduce the activation of hepatic stellate cells (HSCs), thereby effectively alleviating liver fibrosis. This mechanism is different from existing anti-liver fibrosis drugs and provides new technical support for the treatment of liver fibrosis.

[0029] Based on the above advantages, this invention provides a drug for treating liver fibrosis, the active ingredient of which includes the small molecule compound NX-1607; the unit dose of the drug, based on mouse body weight, is 3-8 mg / kg body weight. The unit dose mentioned in this invention refers to the effective unit dose at which the drug exerts its anti-liver fibrosis effect; if applied to humans, it can be converted to an equivalent dose using common knowledge in the art.

[0030] As one implementation method, the unit dose of the drug is 5 mg / kg body weight, based on the mouse's body weight.

[0031] In one embodiment, the drug further includes excipients; the excipients include a 5 g / L sodium carboxymethyl cellulose solution.

[0032] To further illustrate the present invention, the application of the small molecule compound NX-1607 provided by the present invention in the preparation of products for treating liver fibrosis is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] The method described in the literature [Rao J, Wang H, Ni M, Wang Z, Wang Z, Wei S, Liu M, Wang P, Qiu J, Zhang L, Wu C, Shen H, Wang X, Cheng F, Lu L. FSTL1 promotes liver fibrosis by reprogramming macrophage function through modulating the intracellular function of PKM2. Gut. 2022 Dec;71(12):2539-2550. doi: 10.1136 / gutjnl-2021-325150. Epub 2022 Feb 9. PMID: 35140065; PMCID: PMC9664121.] was used to model liver fibrosis in C57BL / 6J mice, resulting in two mouse liver fibrosis models (MCD diet model and CCL4-induced model).

[0035] Example 2

[0036] NX-1607 (or NX1607, purchased from Sellck) was dissolved in a 5 g / L sodium carboxymethyl cellulose solution to obtain an NX-1607 solution.

[0037] The MCD diet model mice constructed in Example 1 were divided into two groups: a control group (denoted as Vehicle) and an NX-1607 treatment group, with 6 mice in each group. The two groups of mice were treated as follows:

[0038] Mice in the NX-1607 treatment group were administered NX-1607 solution by gavage daily, with a daily gavage volume of 5 mg / kg body weight based on the mass of NX-1607; mice in the control group were administered an equal volume of solvent (i.e., 5 g / L sodium carboxymethyl cellulose solution) by gavage.

[0039] Six weeks after treatment, Masson staining and Sirius red staining were performed on paraffin sections of the livers of both groups of mice. The results are shown in Figures 1 and 2. The results showed that the degree of liver fibrosis was reduced in the MCD diet model mice after NX-1607 treatment.

[0040] Six weeks after treatment, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of liver injury, were measured in the serum of both groups of mice. The results are shown in Figure 3. This indicates that P < 0.01. P < 0.001. The results showed that NX-1607 treatment reduced the levels of liver injury markers (ALT and AST) in the serum of MCD diet model mice.

[0041] Six weeks after treatment, the relative expression levels of the liver fibrosis molecular markers αSMA and Col1a1 were detected by RT-qPCR, with actin as an internal reference gene. The primer sequences used are shown in Table 1. The results are shown in Figure 4. This indicates that P < 0.01.

[0042] Table 1 RT-qPCR primers

[0043] ;

[0044] The results showed that the relative expression levels of the molecular markers of liver fibrosis, αSMA and Col1a1, decreased in MCD diet model mice after NX-1607 treatment.

[0045] Example 3

[0046] The method used was similar to that in Example 2, except that the MCD diet model mice constructed in Example 1 were replaced with the CCL4-induced model mice constructed in Example 1. The results are shown in Figures 5-8. This indicates that P < 0.01. This means P < 0.001.

[0047] The results showed that after NX-1607 treatment, the degree of liver fibrosis in CCL4-induced model mice was reduced, the levels of liver injury markers (ALT and AST) in serum were reduced, and the relative expression levels of the liver fibrosis molecular markers αSMA gene and Col1a1 gene were decreased.

[0048] Example 4

[0049] Human hepatic stellate cell line LX2 was stimulated for 24 h with transforming growth factor-β (TGF-β, stimulation concentration 10 ng / ml) and NX-1607 (stimulation concentration 100 nM, solvent: 5 g / L sodium carboxymethyl cellulose solution), respectively. Cells were then collected, and total protein was extracted for Western blot analysis. The specific Western blot analysis method is as follows:

[0050] 1. Cell lysis and protein extraction

[0051] Wash cells twice with pre-cooled PBS (phosphate-buffered saline) to remove culture medium residue. Add an appropriate amount of RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors), and incubate on ice for 30 min, mixing by pipetting every 10 min. Centrifuge at 12,000×g, 4℃ for 15 min, and collect the supernatant, which is the total protein. Determine the protein concentration using a BCA protein quantification kit. Take 15 μg of protein sample, add 5× protein loading buffer (SDS-PAGE Loading Buffer), and denature at 100℃ for 5 min.

[0052] 2. SDS-PAGE electrophoresis

[0053] Prepare a 10% SDS-PAGE gel and perform gel polymerization. Load the sample into the gel wells, add the protein molecular weight marker, and perform electrophoresis (80V for 30 min, 120V for 60 min) until the proteins are properly separated.

[0054] 3. Protein transfer

[0055] The gel protein was transferred to a PVDF membrane (0.22 μm) by wet transfer (400 mA, 60 min).

[0056] 4. Blocking and antibody incubation

[0057] Block the membrane with 5% skim milk powder (room temperature, 2 hours). Add SMAD7 and GAPDH primary antibodies (antibody dilution ratio 1:1000) and incubate overnight at 4°C. Wash three times with TBST for 10 minutes each time. Add HRP-labeled secondary antibody (1:5000) and incubate at room temperature for 1 hour. Wash the membrane three times with TBST for 10 minutes each time.

[0058] 5. ECL color development and signal detection

[0059] Add ECL luminescent substrate to the membrane and incubate in the dark for 1 min. Detect protein bands using a chemiluminescence imaging system or X-ray film. Perform normalization analysis of the target protein based on the band intensity of GAPDH.

[0060] The results are shown in Figure 9. The results show that NX-1607 promoted the expression of SMAD7 in hepatic stellate cells (0.91) compared with TGF-β stimulation (0.47).

[0061] In summary, this invention provides a safe and effective small molecule drug for treating liver fibrosis, NX-1607. NX-1607 can effectively alleviate liver fibrosis by promoting the expression of SMAD7, a key inhibitor of the TGF-β signaling pathway, and reducing the activation of hepatic stellate cells (HSCs), thereby improving the shortcomings of current treatment methods.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of the small molecule compound NX-1607 in the preparation of products for treating liver fibrosis.

2. The application according to claim 1, characterized in that, The products include pharmaceuticals.

3. A drug for treating liver fibrosis, characterized in that, The active ingredient includes the small molecule compound NX-1607; the unit dose of the drug is 3-8 mg / kg body weight in mice.

4. The drug according to claim 3, characterized in that, The unit dose of the drug is 5 mg / kg body weight, based on the mouse's body weight.

5. The drug according to claim 3 or 4, characterized in that, The drug also includes excipients; the excipients include a 5 g / L sodium carboxymethyl cellulose solution.

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