Use of natural product chalcone as a fibroblast activation protein inhibitor

Abstract

The present application relates to the technical field of biological medicine, in particular to application of natural product sappanone as fibroblast activation protein inhibitor. The present application provides application of natural product sappanone as fibroblast activation protein inhibitor, and also provides application of natural product sappanone SAP as FAP inhibitor in preparation of medicine for treating FAP high expression disease, wherein the FAP high expression disease is fibrosis disease (such as liver fibrosis) or tumor. The present application has the following beneficial effects: the present application provides a new use of natural product molecule SAP as FAP inhibitor. The SAP provided by the present application has high inhibition rate on enzyme activity of FAP and high affinity to FAP protein. The small molecule provided by the present application has good biocompatibility and small damage to cells and living bodies. The small molecule provided by the present application has certain improvement effect on FAP high expression disease such as liver fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a natural product, hematoxylin and eosinogen, as an inhibitor of fibroblast activating proteins. Background Technology

[0002] Fibroblast activation protein (FAP) is a serine protease expressed by fibroblasts and tumor-associated mesenchymal cells in various tissues, including the liver, lungs, pancreas, and kidneys. It was initially discovered in fibroblasts, hence its name. It is a two-subunit protein, consisting of an α-subunit with protease activity and a β-subunit. The α-subunit possesses protease activity and its main function is to cleave collagen and other matrix proteins, promoting fibrosis and inflammatory responses. FAP expression is low in normal tissues, but its expression level is significantly increased in tumor tissues of certain cancer types or in fibroblasts activated during fibrosis.

[0003] Tissue fibrosis is a process in which normal tissue is disrupted and its function becomes abnormal due to the deposition of excess connective tissue, primarily collagen. This pathological phenomenon is usually an abnormal repair response of tissues after injury or chronic damage. Fibrosis can occur in a variety of organs and tissues, including the lungs, liver, kidneys, heart, and skin. Fibrosis activation plays a central role in the process of tissue fibrosis. Fibrosis is the result of an imbalance in the tissue damage repair process, and fibroblasts, as the main matrix-generating cells, undergo significant functional changes in this process, leading to excessive extracellular matrix (ECM) deposition and alterations in tissue structure. Under normal circumstances, fibroblasts exist in the stroma, in a quiescent state, mainly used to maintain tissue structure and participate in routine tissue repair. When tissue is damaged or stimulated by inflammatory factors (such as TGF-β, IL-1, TNF-α, etc.), fibroblasts are activated and proliferate, transforming into myofibroblasts and producing large amounts of extracellular matrix molecules such as collagen and fibronectin. This excessive ECM deposition disrupts the normal structure of the tissue, forming scar-like tissue. Activated fibroblasts also begin to express FAP at high levels. FAP expression is closely related to the activation state of fibroblasts, making it an important marker for identifying and studying activated fibroblasts. Due to its specific expression in pathologically activated fibroblasts, FAP has become a potential target for anti-fibrotic and anti-tumor therapies. However, the development of FAP inhibitors, especially natural small molecule inhibitors, is currently very limited. Developing FAP inhibitors could help reduce pathological tissue damage by regulating fibroblast activation and provide new therapeutic avenues for anti-fibrotic and anti-tumor treatments. Summary of the Invention

[0004] To address the aforementioned problems and shortcomings, the present invention aims to provide a natural product, Sappanchalcone (SAP), as an inhibitor of fibroblast activating protein and its application in the preparation of anti-fibrotic drugs. This fibroblast activating protein inhibitor possesses strong FAP inhibitory activity and low biotoxicity, making it suitable for developing drugs or lead compounds for diseases associated with high FAP expression, such as fibrosis or tumors.

[0005] Specifically, the technical solution of the present invention is as follows:

[0006] In a first aspect of the invention, the use of the natural product hematoxylin and succinylcholine as an inhibitor of fibroblast activating proteins is provided. Hematoxylin and succinylcholine have CAS registration number 94344-54-4 and have the structure shown in Formula I:

[0007]

[0008] The inventors investigated the inhibitory effect and affinity of hematoxylin and eosin (SAP) on FAP using in vitro enzyme activity and SPR assays. In vitro, SAP showed inhibitory levels of μM and IC50 against FAP. 50 The concentration was 3.66 μM. SPR experiments showed that SAP has a high affinity for FAP, with a KD value of 3.88 μM. Therefore, this invention provides a novel use of the natural product SAP as an FAP inhibitor.

[0009] The inventors discovered through cytotoxicity experiments that SAP exhibits low toxicity to LX-2 stellate cells, AML-12 parenchymal cells, and HUVEC endothelial cells at certain doses, demonstrating good cellular safety. Animal acute and chronic toxicity experiments also showed that SAP had no significant toxic side effects after administration to mice, indicating that SAP possesses sufficient safety.

[0010] In a second aspect of the invention, the use of the natural product hematoxylin and acetylcholine SAP having the structure shown in Formula I as an FAP inhibitor in the preparation of a medicament for treating FAP-overexpressing diseases, wherein the FAP-overexpressing diseases are fibrotic diseases (such as liver fibrosis) or tumors.

[0011] The inventors confirmed the anti-fibrotic effect of SAP by injecting it intraperitoneally into a carbon tetrachloride-induced mouse liver fibrosis model and using indicators such as Masson staining, Sirius Red staining, and HYP content.

[0012] The application of SAP as a FAP inhibitor includes, but is not limited to, any one of the following aspects (1) to (3):

[0013] 1) Applied to various products prepared as FAP inhibitors;

[0014] 2) Applied to the treatment of various diseases with high FAP expression;

[0015] 3) SAP is used as a lead compound for treating diseases such as fibrosis and tumors.

[0016] In practical applications

[0017] The drug of this invention not only contains the active ingredient hematoxylin and acetamipridone, but also includes pharmaceutically acceptable excipients. When used as inactive substances, pharmaceutical excipients, in addition to acting as shape enhancers, carriers, and improving stability, also possess important functions such as solubilization, co-solubilization, and release regulation. According to their uses, they can be classified as solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, and stabilizers. Specifically, they may include, but are not limited to, one or more of the following components: lactose, microcrystalline cellulose, starch, mannitol, sucrose, calcium phosphate, calcium carbonate, methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, magnesium stearate, talc, silica, and various types of colloidal silica.

[0018] In the medicament of this invention, the active ingredient, hematoxylin and acetamipridone, exists in the form of its pharmaceutically acceptable salt. This not only helps to improve the stability and solubility of the drug, but may also enhance its bioavailability, thereby optimizing its therapeutic effect.

[0019] The medicament of this invention is suitable for use via a variety of common routes of administration, including but not limited to oral, injection, and inhalation spray. It can be used alone or in combination with other medications to enhance efficacy, as needed. The medicament of this invention can be formulated into various suitable dosage forms, such as capsules, tablets, pills, liquids, powders, granules, or injections, to meet the needs of different patients.

[0020] When using the medicament of this invention, in order to ensure that the expected therapeutic effect is achieved effectively and safely for a specific patient, different compositions, and routes of administration, the dosage of the active ingredient can be adjusted according to the specific circumstances. Determining the appropriate dosage of the active ingredient requires consideration of numerous factors, including but not limited to: route of administration, frequency of administration, drug metabolism rate, treatment duration, combination with other drugs, patient's age, sex, weight, overall health status, and medical history. By comprehensively considering these factors, the dosage of the active ingredient can be precisely adjusted to achieve the best therapeutic effect without causing adverse effects on the patient.

[0021] The beneficial effects of one or more of the above technical solutions of the present invention are:

[0022] 1) This invention provides a novel use of the natural product molecule SAP as an FAP inhibitor. The SAP described in this invention exhibits a high inhibition rate against the enzymatic activity of FAP and a high affinity for the FAP protein.

[0023] 2) The small molecules provided by this invention have good biocompatibility and cause little damage to cells and living organisms.

[0024] 3) The small molecule provided by this invention has a certain ameliorative effect on diseases with high FAP expression, such as liver fibrosis. Attached Figure Description

[0025] The accompanying drawings constituting this invention are only used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention, but do not constitute an improper limitation of the invention.

[0026] Figure 1 This is a graph showing the inhibition of FAP by different concentrations of small molecule SAP in Example 1.

[0027] Figure 2 This is a graph showing the affinity of different concentrations of small molecule SAP and FAP in Example 1.

[0028] Figure 3 This is a graph showing the viability of different cells at different concentrations of small molecule SAP as described in Example 2.

[0029] Figure 4 This is a graph showing the toxicity assay of different concentrations of small molecule SAP on LX-2 cells in Example 2.

[0030] Figure 5 The results of relevant indicators in the acute toxicity test in Example 2 are shown in the figure (where A, B, C, D, E and F are the test figures of body weight at different days in the acute toxicity test, ALT enzyme activity test, AST enzyme activity test, creatinine test, blood urea nitrogen test and total bilirubin test, respectively).

[0031] Figure 6 This is a diagram showing the H&E staining results of different tissues in the acute toxicity experiment of Example 2.

[0032] Figure 7 The results of relevant indicators in the chronic toxicity experiment in Example 2 are shown in the figure (where A, B, C, D, E and F are the test figures of body weight at different days in the chronic toxicity experiment, ALT enzyme activity assay, AST enzyme activity assay, creatinine assay, blood urea nitrogen assay and total bilirubin assay, respectively).

[0033] Figure 8This is a diagram showing the H&E staining results of different tissues in the chronic toxicity experiment of Example 2.

[0034] Figure 9 The figures shown are the serum biochemical results of different groups in the efficacy study of Example 3 (Figure A is the ALT enzyme activity assay, Figure B is the AST enzyme activity assay), and the tissue hydroxyproline content assay results (Figure C).

[0035] Figure 10 These are Masson staining images of different groups in the efficacy study of Example 3.

[0036] Figure 11 These are Sirius Red staining images of different groups in the efficacy study of Example 3. Detailed Implementation

[0037] To enable those skilled in the art to better understand the content of this invention, the embodiments of this invention are described in detail below. These embodiments are implemented based on the technical solution of this invention, and provide detailed implementation methods and specific operation processes. However, the content of this invention is not limited to the following examples.

[0038] The reagents, materials, and instruments used in this invention are sourced as follows: Fibroblast activating protein FAP was purchased from Beijing Yiqiao Shenzhou, 10464-H07H; SAP was purchased from Wuhan Zhongbiao Technology Co., Ltd.; CCK-8 was purchased from Beyotime, C0037; LDH-Glo TM All microplates were purchased from Promega (J2380), Beyotime Biotechnology (HEPES buffer), Smart Biotech (E00548), Thermo Fisher Scientific (fluorescent microplate reader), Topvan Biotech (molecular interaction analyzer), BioTek Microplate reader, Tianneng ABLX6 small animal in vivo visible light 3D imaging system, and Fuji VisualSonic Vevo2100 LAZER system. Human hepatic stellate cells (LX-2), mouse hepatocytes (AML-12), human umbilical vein endothelial cells (HUVEC), and DMEM culture medium were purchased from KGI Biotechnology.

[0039] Example 1: Determination of the inhibition rate and affinity of SAP for FAP activity

[0040] In this embodiment, the inhibition rate and affinity of the probe SAP to FAP were determined through in vitro experiments.

[0041] Inhibition rate was measured using an in vitro fluorescence assay. The enzyme activity assay material, Cs-FAP, was the material proposed in the applicant's previous patent application (202311842514.7). Specifically: FAP protein solution diluted in HEPES buffer (98 μL, 2 μg / mL) was added to a 96-well plate, followed by the addition of different concentrations of SAP solution (100 μL), incubation at 37°C for 1 h, then the addition of Cs-FAP solution (2 μL, 1 mM), incubation again at 37°C for 30 min, and detection using a fluorescence microplate reader. Figure 1 The results showed that SAP inhibited FAP at the μM level, IC50 50 The concentration was 3.66 μM, indicating a high level of inhibition.

[0042] Affinity levels were determined using an SPR molecular interaction analyzer. Specifically, FAP was immobilized on the surface of the instrument's dedicated chip, and SAP solutions of different concentrations were prepared. The affinity between SAP and FAP was then determined using the machine's program. Figure 2 The results showed that SAP has a high affinity for FAP, with a KD value of 3.88 μM.

[0043] Example 2: Cell / Animal Toxicity Tests of SAP

[0044] The cytotoxicity study used the MTT / LDH method, and the specific testing procedure is as follows: First, the MTT method was used to test the effect of SAP on different cell viability. Different cells were stored at 1×10⁶ cells per well. 4 Cells were seeded into 96-well plates with 150 μL of DMEM culture medium per well and incubated overnight at 37°C, 5% CO2. The culture medium was then removed, and 100 μL of SAP cell culture medium containing different concentration gradients was added to each well. Incubation was continued for 24 h. After incubation, the incubation medium was removed, and MTT working solution was added. After 4 h, the MTT solution was removed, and 150 μL of LDMSO was added to each well. After shaking, the absorbance at 490 nm was measured using a microplate reader. The results are as follows: Figure 3 As shown, the viability of SAP on stellate cells (LX-2) gradually increased with increasing concentration, but had no significant effect on the viability of parenchymal cells (AML-12) and endothelial cells (HUVEC). Subsequently, the LDH method was used to determine whether SAP was toxic to LX-2 cells, and the results were... Figure 4 The results showed that different concentrations of SAP had less than 10% cytotoxicity to LX-2 cells, indicating that it had no significant toxicity to LX-2 cells. The specific inhibition of LX-2 cell activity also helped it to exert its anti-fibrotic effect.

[0045] At the animal level, the toxicity of SAP to whole animals was tested through acute and chronic toxicity experiments. Specifically: In the acute toxicity experiment, a single intraperitoneal injection of SAP solution at a concentration of 200 mg / kg was administered, and mouse body weight was recorded every two days thereafter. Fourteen days post-injection, blood and major organs were collected, and serum markers related to liver and kidney injury were detected. Organs were stained with Hematoxylin and eosin (H&E). In the chronic toxicity experiment, SAP was administered at a concentration of 20 mg / kg, via intraperitoneal injection every other day for 15 consecutive times. The collection and detection methods were the same as in the acute toxicity experiment. The biochemical indicators related to body weight in each group during the acute experiment are as follows: Figure 5 As shown, the results indicated that there were no significant differences in body weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), blood urea nitrogen (BUN), and bilirubin (TBIL) between the acute toxicity group (A-Tox) and the control group (Control). H&E staining results ( Figure 6 This indicates that a single high-concentration dose did not cause significant damage to multiple organs. The results of chronic toxicity studies are as follows: Figure 7 and 8 The results indicate that there were no significant differences in any indicators between the chronic toxicity group (C-Tox) and the control group, and that it also showed no significant toxicity to mice. All of these experimental results demonstrate that SAP has good biocompatibility.

[0046] Example 3: Pharmacodynamic Study of SAP

[0047] A carbon tetrachloride-induced mouse liver fibrosis model was established at the animal level. 25% CCl4 (1 mL / kg; CCl4:Oil, v:v) was administered intraperitoneally twice weekly for six weeks. Four weeks later, different concentrations of SAP solutions (LSAP: 1 mg / kg; MSAP: 5 mg / kg; HSAP: 10 mg / kg) were injected every two days for two weeks. Twenty-four hours after the last administration, blood and liver tissue were collected to detect relevant parameters and perform Masson and Sirius Red staining. Serum ALT, AST, and hepatic hydroxyproline (HYP) levels are shown below. Figure 9 The results indicate that SAP can improve carbon tetrachloride-induced liver fibrosis in a dose-dependent manner, and the results of tissue section staining further confirm the efficacy of SAP. Figure 10 Masson staining results showed that SAP administration significantly reduced the area of ​​the blue region (collagen). Figure 11 The Sirius Red staining results were the same as those of Masson staining, and the area of ​​the red region (collagen) was significantly reduced after administration, both indicating that it can effectively improve liver fibrosis.

[0048] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. The application of hematoxylin and acetylcholine, a natural product having the structure shown in Formula I, as an FAP inhibitor in the preparation of drugs for treating liver fibrosis: Equation I.

2. The application according to claim 1, characterized in that, The drug includes succinolone and pharmaceutically acceptable excipients.

3. The application according to claim 1, characterized in that, The hematoxylin exists in the form of its pharmaceutically acceptable salt.

4. The application according to claim 1, characterized in that, The dosage form of the drug includes one or more of capsules, tablets, pills, granules, and injections.

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