Application of natural product hematoxychalcone as fibroblast activating protein inhibitor

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a natural product hematoxychalcone as a fibroblast activating protein inhibitor. The invention provides an application of a natural product hematoxychalcone as a fibroblast activating protein inhibitor, and also provides an application of the natural product hematoxychalcone SAP as an FAP inhibitor in preparation of drugs for treating FAP high expression diseases, and the FAP high expression diseases are fibrotic diseases (such as hepatic fibrosis) or tumors. The invention has the following beneficial effect: the invention provides a new application of the natural product molecule SAP in the FAP inhibitor. The SAP disclosed by the invention has a relatively high inhibition rate on the enzyme activity of the FAP, and has relatively high affinity on the FAP protein. The micromolecule provided by the invention is good in biocompatibility and small in damage to cells and living bodies. The micromolecule provided by the invention has a certain improvement effect on FAP high-expression diseases such as hepatic fibrosis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of a natural product, hematoxylin chalcone, as a fibroblast activation protein inhibitor. Background Art

[0002] Fibroblast Activation Protein (FAP) is a serine protease expressed by fibroblasts and tumor-related stromal cells in various tissues such as the liver, lungs, pancreas, and kidneys. It was first discovered in fibroblasts, hence the name. It is a two-subunit protein, including an α subunit with protease activity and a β subunit. The α subunit has protease activity and its main function is to cut collagen and other matrix proteins, promote fibrosis and inflammatory response. FAP is rarely expressed 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 usually a process in which normal tissues are destroyed and function abnormally due to the deposition of excess connective tissue, mainly 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. The activation of fibroblasts plays a central role in the process of tissue fibrosis. Fibrosis is the result of an imbalance in the process of tissue damage and repair, and fibroblasts, as the main matrix-producing cells, undergo significant functional changes in this process, leading to excessive extracellular matrix (ECM) deposition and changes in tissue structure. Under normal circumstances, fibroblasts exist in the interstitium and are in a quiescent state, mainly used to maintain tissue structure and participate in routine tissue repair. When tissues are 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 a large amount of extracellular matrix molecules such as collagen and fibronectin. This excessive ECM deposition destroys the normal structure of the tissue and forms scar-like tissue. Activated fibroblasts also begin to express FAP at high levels. The expression of FAP is closely related to the activation state of fibroblasts, and has become an important marker for identifying and studying activated fibroblasts. FAP has become a potential target for anti-fibrosis and anti-tumor therapy due to its specific expression in fibroblasts activated under pathological conditions. However, the development of inhibitors against FAP, especially natural small molecule inhibitors, is very limited. The development of FAP inhibitors can help reduce pathological tissue damage by regulating the activation of fibroblasts, and provide new therapeutic approaches for anti-fibrosis and anti-tumor. Summary of the invention

[0004] In view of the above-mentioned existing problems and according to the existing deficiencies, the purpose of the present invention is to provide a natural product Sappanchalcone (SAP) for use as a fibroblast activation protein inhibitor and in the preparation of anti-fibrosis drugs. The fibroblast activation protein inhibitor has strong FAP inhibitory activity and low biological toxicity, and is suitable for the development of drugs or lead compounds for diseases related to high FAP expression, such as fibrosis or tumors.

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

[0006] In the first aspect of the present invention, a use of the natural product hematoxylin chalcone as a fibroblast activation protein inhibitor is provided. The CAS registration number of hematoxylin chalcone is 94344-54-4. Hematoxylin chalcone has a structure as shown in Formula I:

[0007]

[0008] The inventors investigated the inhibitory effect and affinity of SAP on FAP through in vitro enzyme activity experiments and SPR experiments. In vitro, the inhibitory level of SAP on FAP was at the μM level, and the IC 50 The SPR experimental results show that SAP has a high affinity for FAP, and the KD value is 3.88 μM. Therefore, the present invention provides a new use of the natural product SAP as a FAP inhibitor.

[0009] The inventors found through cytotoxicity experiments that SAP has low toxicity to stellate cells LX-2, parenchymal cells AML-12, and endothelial cells HUVEC at a certain dose, and has good cell safety. The results of animal acute and chronic toxicity experiments also showed that SAP had no obvious toxic side effects after administration to mice, indicating that SAP has sufficient safety.

[0010] In a second aspect of the present invention, provided is the use of the natural product sappanchalcone SAP having a structure as shown in Formula I as a FAP inhibitor in the preparation of a drug for treating a disease with high FAP expression, wherein the disease with high FAP expression is a fibrotic disease (such as liver fibrosis) or a tumor.

[0011] The inventors injected SAP intraperitoneally into a mouse liver fibrosis model induced by carbon tetrachloride, and confirmed the anti-fibrosis effect of SAP through Masson staining, Sirius Red staining, HYP content and other indicators.

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

[0013] 1) Applied to the preparation of various products of FAP inhibitors;

[0014] 2) It is used to treat various diseases with high expression of FAP;

[0015] 3) As a lead compound SAP, it is used to fight diseases such as fibrosis / tumor.

[0016] In specific applications,

[0017] The medicine of the present invention not only contains the active ingredient hematoxylin, but also includes pharmaceutically acceptable pharmaceutical excipients. When used as inactive substances, pharmaceutical excipients have important functions such as solubilization, solubilization, and release regulation in addition to excipients, carriers, and stability. According to the purpose, they can be divided into solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, etc. Specifically, it can include but is not limited to one or more of the following ingredients: lactose, microcrystalline cellulose, starch, mannitol, sucrose, calcium phosphate, calcium carbonate, methylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, magnesium stearate, talcum powder, silicon dioxide, and various types of colloidal silicon dioxide.

[0018] In the medicine of the present invention, the active ingredient sappanchalcone exists in the form of its pharmaceutically acceptable salt, which not only helps to improve the stability and solubility of the medicine, but also may enhance its bioavailability, thereby optimizing the therapeutic effect.

[0019] The drug of the present invention is suitable for a variety of common administration routes, including but not limited to oral administration, injection, and use by inhalation spray. The drug can be used alone or in combination with other drugs to enhance the therapeutic effect as needed. The drug of the present invention can be prepared into a variety of suitable dosage forms, such as capsules, tablets, pills, liquids, powders, granules or injections, to meet the needs of different patients.

[0020] When using the drug of the present invention, in order to ensure that the desired therapeutic effect can be effectively and safely achieved for a specific patient, different compositions and administration methods, the dosage of the active ingredient can be adjusted according to the specific situation. Many factors need to be considered to determine the appropriate dosage of the active ingredient, including but not limited to: route of administration, frequency of administration, drug metabolism rate, treatment cycle, combined use with other drugs, patient's age, gender, weight, overall health and medical history, etc. By comprehensively considering these factors, the dosage of the active ingredient can be accurately adjusted to achieve the best therapeutic effect without causing adverse effects on the patient.

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

[0022] 1) The present invention provides a new use of a natural product molecule SAP as a FAP inhibitor. The SAP of the present invention has a high inhibition rate on the enzyme activity of FAP and has a high affinity for the FAP protein.

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

[0024] 3) The small molecules provided by the present invention have a certain improvement effect on diseases with high expression of FAP, such as liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings constituting the description of the present invention are only used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute improper limitations on the present invention.

[0026] Figure 1 It is a curve diagram of the inhibition of FAP by small molecule SAP at different concentrations in Example 1.

[0027] Figure 2 It is a curve chart of the affinity determination between small molecule SAP and FAP at different concentrations in Example 1.

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

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

[0030] Figure 5 It is the result graph of relevant indicators in the acute toxicity experiment in Example 2 (wherein A, B, C, D, E and F are respectively the test graphs of body weight on different days in acute toxicity, ALT enzyme activity determination graph, AST enzyme activity determination graph, creatinine determination graph, urea nitrogen determination graph and total bilirubin determination graph).

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

[0032] Figure 7 It is the result graph of relevant indicators in the chronic toxicity experiment in Example 2 (wherein A, B, C, D, E and F are respectively the test graphs of body weight on different days in chronic toxicity, ALT enzyme activity determination graph, AST enzyme activity determination graph, creatinine determination graph, urea nitrogen determination graph and total bilirubin determination graph).

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

[0034] Fig. 9 It is a graph showing the serum biochemistry results of different groups in the efficacy study in Example 3 (Figure A is a graph showing the ALT enzyme activity assay, and Figure B is a graph showing the AST enzyme activity assay), and a graph showing the results of tissue hydroxyproline content assay (Figure C).

[0035] Fig.10 It is the Masson staining diagram of different groups in the efficacy study in Example 3.

[0036] Fig.11 This is the Sirius Red staining picture of different groups in the efficacy study in Example 3. DETAILED DESCRIPTION

[0037] In order to help those skilled in the art better understand the content of the present invention, an embodiment of the present invention is described in detail below. This embodiment is implemented based on the technical solution of the present invention, and a detailed implementation method and specific operation process are given. However, the content of the present invention is not limited to the following examples.

[0038] The sources of reagents, materials and instruments involved in the examples of the present invention are as follows: Fibroblast activation protein FAP was purchased from Beijing Sino Biological, 10464-H07H; SAP was purchased from Wuhan Zhongbiao Technology Co., Ltd.; CCK-8 was purchased from Bio-Tech, C0037; LDH-Glo TM J2380 was purchased from Promega, USA; HEPES buffer was purchased from Bio-Tech; isoflurane was purchased from Smart, E00548; fluorescence microplate reader, Thermo Fisher Scientific, USA; molecular interaction instrument Biacore S200, Sterfan, USA; microplate reader, BioTek, USA; small animal living visible light 3D imaging system, Tianneng ABLX6; Visualsonic Vevo2100LAZER system was purchased from Fuji VisualSonics. Human hepatic stellate cells (LX-2), mouse hepatocytes (AML-12), human umbilical vein endothelial cells (HUVEC) and DMEM culture medium were purchased from Keygen Biotechnology.

[0039] Example 1: Determination of the inhibitory rate and affinity of SAP to FAP activity

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

[0041] The inhibition rate test uses an in vitro fluorescence method to determine the enzyme activity. The enzyme activity determination material Cs-FAP uses the material proposed by the applicant's previous patent application (202311842514.7). Specifically: FAP protein solution (98μL, 2μg / mL) diluted in HEPES buffer was added to a 96-well plate, followed by adding SAP solution (100μl) of different concentrations, incubated at 37°C for 1h, and then adding Cs-FAP solution (2μl, 1mM), incubated again at 37°C for 30min, and then detected using a fluorescent microplate reader. Figure 1 The results showed that the inhibitory level of SAP on FAP was at μM level, and the IC 50 The inhibitory level was 3.66 μM, which was high.

[0042] The affinity level was measured using an SPR molecular interaction instrument. Specifically: After FAP was fixed on the surface of the instrument's dedicated chip, SAP solutions of different concentrations were prepared, and the affinity between SAP and FAP was measured using a machine program. Figure 2 The results showed that SAP had a high affinity for FAP, with a KD value of 3.88 μM.

[0043] Example 2: Cell / animal toxicity test of SAP

[0044] The MTT / LDH method was used in the study of cytotoxicity. The specific test process was as follows: First, the MTT method was used to test the effect of SAP on the viability of different cells. Different cells were plated at 1×10 4 Cells were inoculated into 96-well plates, with 150 μL of DMEM culture medium per well, and cultured overnight in an incubator (37°C, 5% CO) to adhere to the plate. 2 ). The culture medium was aspirated, and 100 μL of SAP cell culture medium containing different concentration gradients was added respectively, and the cells were incubated in the incubator for another 24 h. After the incubation, the incubation medium was aspirated, and MTT working solution was added. After 4 h, the MTT solution was aspirated, and 150 μL of DMSO was added to each well. After shaking, the absorbance at 490 nm was measured by an ELISA reader. The results are shown in Figure 3 As shown in the figure, the activity of SAP on stellate cells (LX-2) gradually increased with the increase of concentration, but it had no significant effect on the cell viability of parenchymal cells (AML-12) and endothelial cells (HUVEC). The LDH method was then used to determine whether SAP would be toxic to LX-2. The results ( Figure 4 ) showed that the cytotoxicity of SAP at different concentrations to LX-2 was less than 10%, indicating that it had no obvious toxicity to LX-2, and the specific inhibition of LX-2 cell activity was also conducive to its anti-fibrotic effect.

[0045] At the animal level, acute and chronic toxicity experiments were used to test the toxicity of SAP to whole animals. Specifically: In the acute toxicity experiment, a single intraperitoneal injection of 200 mg / kg SAP solution was given, and the weight of the mice was recorded every two days. 14 days after the injection, blood and major organs were collected, serum indicators related to liver and kidney damage were tested, and H&E staining of organs was performed. In the chronic toxicity experiment, SAP was administered at a concentration of 20 mg / kg, and intraperitoneal injections were given every other day for 15 consecutive times. The collection and testing items were the same as those in the acute toxicity experiment. In the acute experiment, the body weight-related biochemical indicators of each group are as follows: Figure 5 As shown in the figure, the results showed that there were no significant differences in the six indicators of mouse body weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), urea nitrogen (BUN) and bilirubin (TBIL) between the acute toxicity group (A-Tox) and the control group (Control). H&E staining results ( Figure 6 ) showed that a single high-concentration administration did not cause significant damage to multiple organs. The results of chronic toxicity experiments are as follows Figure 7 and 8 , indicating that the chronic toxicity group (C-Tox) had no significant difference in all indicators compared with the control group, and had no obvious toxicity to mice. The above experimental results all indicate that SAP has good biosafety.

[0046] Example 3: Pharmacodynamics study of SAP

[0047] The liver fibrosis model induced by carbon tetrachloride was established in mice by intraperitoneal injection of 25% CCl 4 (1mL / kg; CCl 4 :Oil, v:v), twice a week for six weeks. After four weeks of injection, different concentrations of SAP solution (LSAP: 1mg / kg; MSAP: 5mg / kg; HSAP: 10mg / kg) were injected every two days for two weeks. 24h after the last administration, blood and liver tissue were collected to detect related indicators and perform Masson and Sirius Red staining. The results of serum ALT, AST and liver hydroxyproline content (HYP) are as follows Fig. 9 , indicating that SAP can improve carbon tetrachloride-induced liver fibrosis in a dose-dependent manner, and the section staining results further confirmed the efficacy of SAP. Fig.10 The Masson staining results showed that the area of ​​the blue region (collagen) was significantly reduced after SAP administration. Fig.11 The Sirius Red staining results were the same as those of Masson staining. The red area (collagen) was significantly reduced after administration, indicating that it can effectively improve liver fibrosis.

[0048] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. The use of the natural product hematoxylin chalcone as a fibroblast activation protein inhibitor, characterized in that: The hematoxylin chalcone has a structure as shown in Formula I: 。 2. Use of the natural product sappanchalcone having a structure as shown in Formula I as a FAP inhibitor in the preparation of drugs for treating diseases with high FAP expression.

3. The use according to claim 2, characterized in that: The FAP high expression disease is a fibrotic disease or a tumor.

4. The use according to claim 3, characterized in that: The disease is liver fibrosis.

5. The use according to claim 2, characterized in that: The medicine comprises sappanchalcone and pharmaceutically acceptable pharmaceutical excipients.

6. The use according to claim 2, characterized in that: The hematoxylinchalcone exists in the form of a pharmaceutically acceptable salt thereof.

7. The use according to claim 2, characterized in that: The dosage form of the drug includes one or more of capsule, tablet, pill, liquid, powder, granule and injection.

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