Use of dauricine surine in preparing myeloperoxidase inhibitors and drugs for treating acute lung injury

Abstract

The present application relates to the application of Bauhinia Sulline in the preparation of myeloperoxidase inhibitors, and the treatment / prevention / improvement of diseases caused by abnormal myeloperoxidase activity, inflammatory diseases, oxidative damage products, and provides a more effective treatment for diseases such as acute lung injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmacotherapy, in particular to the use of daemoropsol in the preparation of myeloperoxidase inhibitors and drugs for treating acute lung injury. BACKGROUND

[0002] Acute lung injury (ALI) is a critical illness affecting the respiratory system. The development of ALI involves a series of chain reactions of inflammatory substances released by neutrophils, macrophages, endothelial cells and epithelial cells, eventually leading to acute hypoxic respiratory failure or respiratory failure. Due to the complex pathogenesis and diverse causes of ALI, there is currently a lack of effective and safe treatment methods in clinical practice.

[0003] Neutrophils are key players in innate immune defense and are among the first cells recruited to sites of inflammation. They fight pathogens through classical pathways such as phagocytosis and degranulation. Myeloperoxidase (MPO) is a heme peroxidase, a 146 kDa glycosylated protein composed of two monomers, accounting for about 5% of the dry weight of neutrophils, mainly stored in the azurophilic granules of neutrophils. Activated neutrophils can release MPO through various means such as degranulation or neutrophil extracellular traps, so MPO is also considered a marker of neutrophil activation. In the body's innate immunity, MPO can mediate the reaction of H2O2 and Cl - to produce strong oxidant hypochlorous acid HOCl, which kills pathogens. However, HOCl can also react with human biological macromolecules, causing oxidative stress and inflammatory reactions, damaging body cells, and thus promoting the development of diseases.

[0004] In addition, previous studies have shown that when exposed to lipopolysaccharide (LPS), neutrophils release their intact nuclear chromatin complexes (including histone-rich extracellular DNA and protein scaffolds) into the extracellular space or tissues in the form of biological spider webs. These structures are called neutrophil extracellular traps (NETs), which serve as a source of autoantigens and can trigger an inflammatory cascade in local tissues and blood flow, thus being related to the pathogenesis of ALI.

[0005] This process plays an important role in normal innate immune response, enabling neutrophils to capture a variety of pathogenic microorganisms, but in some special cases, excessive, unbalanced immune response can lead to NETs release disorder, further exacerbating inflammation damage to the body, thereby causing host tissue damage beyond the protective function. Studies have shown that excessive NETs release can exacerbate lung inflammation damage, and myeloperoxidase as an important component of NETs and related enzymes, by inhibiting myeloperoxidase to inhibit the release and function of NETs, has become the focus of our research, and the research work of myeloperoxidase inhibitors for the same therapeutic purpose has been carried out.

[0006] In summary, the role of MPO in inflammatory diseases is particularly noteworthy, whether in the pathological process of chronic or acute inflammatory diseases, studies have shown the role of MPO. However, although MPO has a promising prospect as a drug target, it is actively being developed in the industry, and there is no drug targeting MPO on the market.

[0007] North bean root is a traditional Chinese medicine with the effects of clearing heat and detoxifying, relieving cough and expelling sputum, and is clinically used for the treatment of sore throat, swollen pain, tonsillitis and chronic bronchitis. One of the main components is dauricine surin base, which has been found to have autophagy inhibition function and can be used as a protective agent for ischemia-reperfusion. However, there is no literature report on the role of dauricine surin base as a myeloperoxidase inhibitor and improvement of acute lung injury. SUMMARY

[0008] In view of the above problems existing in the prior art, the present application selects dauricine surin base as an active ingredient for developing a myeloperoxidase inhibitor, or as a treatment / prevention product for inflammatory diseases, or as a treatment / prevention product for oxidative damage-related diseases, and is particularly suitable as a treatment / prevention product for diseases caused by abnormal myeloperoxidase activity such as acute lung injury.

[0009] The first aspect of the present application provides the use of dauricine surin base in the preparation of a myeloperoxidase inhibitor product.

[0010] The second aspect of the present application provides the use of dauricine surin base in the preparation of a product for treating / preventing / improving diseases caused by abnormal myeloperoxidase activity.

[0011] In some embodiments, the diseases caused by abnormal myeloperoxidase activity include acute lung injury, atherosclerosis, acute coronary syndrome, Hashimoto's thyroiditis, vasculitis, preferably acute lung injury.

[0012] The third aspect of the present application provides the use of dauricine surin base in the preparation of a product for treating / preventing / improving inflammatory diseases.

[0013] In some embodiments, the inflammatory disease is acute inflammation or chronic inflammation.

[0014] The fourth aspect of the present application provides use of daurisoline in the preparation of a product for treating / preventing / ameliorating oxidative damage.

[0015] In the first, second, third, and fourth aspects of the present application, the daurisoline is also known as Daurisoline, with a CAS number of 70553-76-3, a molecular formula of C 37 H 42 N2O6, and a chemical structure of:

[0016]

[0017] In some embodiments, the product is in the form of a medicine, food, or functional health product.

[0018] In some embodiments, the medicine is in the form of an injection, tablet, powder, granule, pill, capsule, oral liquid, ointment, cream, or spray.

[0019] In some embodiments, the food or functional health product is in the form of an oral liquid, tea, lozenge, capsule, drink, or effervescent tablet.

[0020] In some embodiments, the food or functional health product is used for daily health care or treatment assistance for the diseases mentioned above.

[0021] In some embodiments, the daurisoline is the only active ingredient in the product.

[0022] In some embodiments, the medicine further comprises one or more pharmaceutically acceptable excipients.

[0023] In one embodiment, the excipients include diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, and sustained-release agents commonly used in the pharmaceutical field.

[0024] In some embodiments, the medicine is administered orally, intragastrically, by injection, spray, physical or chemical mediation, or is administered after being mixed or coated with other substances.

[0025] Advantages

[0026] The present application first proves that daurisoline has the function of a myeloperoxidase inhibitor, can effectively inhibit the activity of MPO and the release of NETs, and has a significant therapeutic or preventive effect on diseases such as acute lung injury and lung inflammation. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Docking diagram of existing MPO inhibitors with MPO.

[0028] Figure 2 Docking diagram of daurisoline with myeloperoxidase.

[0029] Figure 3 Hypochlorous acid concentration standard curve.

[0030] Figure 4 Concentration of hypochlorous acid generated in each group in cell experiment, wherein * indicates P<0.05, and ** indicates P<0.01.

[0031] Figure 5 IL-1 β concentration in the bronchoalveolar lavage fluid of each group in mouse experiment.

[0032] Fig. 6A, 6B, 6C are HE staining diagrams of lung sections of mice in blank control group, modeling group, and administration group, respectively.

[0033] Figure 7 Lung injury score diagram of each group of mice.

[0034] Figure 8 Myeloperoxidase release level diagram of each group of mice, wherein ** indicates P<0.01, and **** indicates P<0.0001. DETAILED DESCRIPTION

[0035] The following examples can enable those skilled in the art to more fully understand the present application, but the present application is not limited in the scope of the described examples.

[0036] Example 1 Virtual docking verifies the inhibitory effect of daurisoline on myeloperoxidase

[0037] (1) Selection of active site

[0038] According to literature review and comparison with AstraZeneca inhibitors, the heme pocket at the positions of Arg 239 and His 336 in the C chain of MPO is the catalytic site of MPO, see Figure 1 .

[0039] (2) Selection of protein receptor

[0040] In the article (Discovery of AZD4831, a Mechanism-Based Irreversible Inhibitor of Myeloperoxidase, As a Potential Treatment for Heart Failure with Preserved Ejection Fraction. J. Med. Chem. 2022, 65, 11485-11496.) published by AstraZeneca in 2022, the X-ray crystal structure of the MPO receptor ligand complex was uploaded to the PDB library, with the numbers 7NI1 and 7NI3, and we selected 7NI1 as the protein receptor.

[0041] (3) Virtual docking results

[0042] Molecular docking was performed using the professional commercial software Schrodinger 13.4, which is widely used in the prior art for molecular docking. The selected docking method was semi-flexible docking, and the mode was selected as SP. Generally, for natural products, a docking score less than -6 can be considered as good docking effect. The docking score of daurisoline B is -7.969, and the results are shown in Figure 2

[0043] It can be seen that daurisoline B and myeloperoxidase have good docking effect, and it can be inferred that it has good myeloperoxidase inhibitory effect.

[0044] Example 2 In vitro cell verification of the inhibitory effect of daurisoline B on myeloperoxidase

[0045] (1) Verification of taurine chloramine assay (DTNB method)

[0046] Taurine chloramine assay (see formula below) is based on the reaction of hypochlorous acid with taurine (TauNH2) to generate taurine chloramine (TauNHCl), which is determined by reaction with s-thio-2-nitrobenzoic acid (TNB) to oxidize yellow TNB to colorless DTNB. This is an extremely sensitive determination method, which can accurately measure the concentration of hypochlorous acid as low as 5 uM.

[0047] HOCl + TauNH2→ TauNHCl + H2O

[0048] TauNHCl + 2TNB→ DTNB + Cl - + TauNH2

[0049] ​We take 96-well plate, in which the corresponding hole to join 100 μL of TNB solution, and then in the corresponding hole to join 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM of hypochlorous acid solution of 100 μL, set a hole as blank control group corresponding concentration is 0. After 15 min in the dark, take out the absorbance at 412 nm, according to the absorbance to make the corresponding hypochlorous acid concentration standard curve (as shown in Figure 3 The results show that DTNB method can accurately measure the concentration of hypochlorous acid.

[0050] Table 1 standard curve data

[0051]

[0052] (2) Myeloperoxidase activity inhibition detection

[0053] Myeloperoxidase can spontaneously catalyze the generation of product hypochlorous acid in the presence of hydrogen peroxide, chloride ion and other substrates. HL-60 cells are human promyelocytes, which can be induced to become neutrophil-like cells (dHL-60) in 1.3% DMSO environment for at least 5 days, and the neutrophil-like cells can be activated in the presence of PMA, thereby releasing myeloperoxidase.

[0054] Thus we can set the system 10 uM of Bauhinia Suline, 10 mM of taurine and 2 x 10 6 dHL-60 cells (obtained by inducing HL-60 cells in 1.3% DMSO for 6 days), 100 ng / ml of PMA is applied for 1 h at 37℃, and then cooled in ice to terminate the reaction, and an excess of H2O2 enzyme is added to eliminate the effect of H2O2, and after centrifugation, 100 uL of supernatant is reacted with 400 uM of TNB solution in the dark for 15 min, and the absorbance is detected at 405 nm. HBSS is used as a blank control group instead of natural products. The results (as shown in Figure 4 It is found that Bauhinia Suline can effectively inhibit the activity of myeloperoxidase.

[0055] Example 3 Effect of Bauhinia Suline on lung injury of LPS acute lung injury modeling mice in vivo

[0056] 1. Experimental materials

[0057] 6-8 week old male C57 mice, 15, body weight 20-22 grams, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0058] 2. Experimental method

[0059] 1) Preparation of Bauhinia Suline

[0060] Precisely weigh the daemoropsidesurine, and prepare it into a concentration of 15mg / ml with DMSO, PEG300, Tween 80 and normal saline

[0061] 2) Animal grouping and administration

[0062] After the mice are adaptively fed for 3-5 days, they are fasted at 6 o'clock the day before the experiment, and are allowed to drink water freely. At the time of the experiment, the mice are randomly grouped according to weight, with 5 mice in each group. Intraperitoneal injection is performed for administration, with 0.1 ml per 10 g of mouse weight, i.e. the administration dose is 15 mg / kg, and the control group and the model group are given the corresponding blank solvent. One hour after administration, the model group and the administration group are given pulmonary aerosol administration to the mice, with a dose of 10 mg / kg of LPS for acute lung injury modeling. The lung tissue of the mice is taken 24 hours later, and the alveolar lavage fluid is subjected to relevant detection.

[0063] Index for determination:

[0064] 1) Concentration of inflammatory factors in the alveolar lavage fluid

[0065] 2) Lung section injury and score of the mice

[0066] 3) WB verification of the release of myeloperoxidase in the lungs of the mice

[0067] 3. Experimental results

[0068] (1) Elisa determination of the concentration of IL-1 in the alveolar lavage fluid β According to Figure 5 , the expression level of the inflammatory factor IL-1β of the model group is significantly higher than that of the blank control group, and the expression level of the inflammatory factor IL-1β of the administration group is significantly lower than that of the model group, and is equivalent to that of the blank control group.

[0069] (2) According to Figure 6A , 6B , 6C, the lung of the model group ( Figure 6B ) has obvious damage relative to the blank control group ( Figure 6A ), and the lung damage of the administration group ( Figure 6C ) is obviously better than that of the model group.

[0070] (3) The lung damage of each group is scored according to the following pathological conditions: alveolitis, interstitial inflammation, alveolar wall widening, vascular dilation, hyperemia, interstitial edema, hemorrhage, pulmonary emphysema, pulmonary edema, hyaline membrane formation, and inflammatory exudation in the bronchial lumen. The results are shown in Figure 7 . The model group has almost all of the above symptoms, and the lung injury score is significantly higher than that of the blank control group and the administration group, indicating that the mouse acute lung injury modeling is successful, and daemoropsidesurine can significantly alleviate the acute lung injury of the mice caused by LPS.

[0071] (4) WB experiment was used to detect the expression of myeloperoxidase in the lungs of mice in each group. The results are shown in Figure 6. The expression level of myeloperoxidase in the model group was significantly higher than that in the blank control group, while the expression level of myeloperoxidase in the administration group was significantly lower than that in the model group. Figure 8

[0072] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.​

Claims

1. Use of dauricine surine in the preparation of a medicament for treating / preventing acute lung injury.

2. Use according to claim 1, characterized in that, The medicament is an injection, a tablet, a powder, a granule, a pill, a capsule, an oral liquid, a paste, a cream or a spray.

3. Use according to claim 1 or 2, characterized in that, The medicament further comprises one or more pharmaceutically acceptable adjuvants.

4. Use according to claim 3, characterized in that, The adjuvants include diluents, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and sustained-release agents commonly used in the pharmaceutical field.

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