Application of dauricine in preparation of myeloperoxidase inhibitor and medicine for treating acute lung injury

Abstract

The invention relates to application of dauricine in preparation of myeloperoxidase inhibitors and products for treating / preventing / improving diseases, inflammatory diseases and oxidative damage caused by abnormal myeloperoxidase activity, and provides a more effective treatment mode for diseases such as acute lung injury.

Description

Technical Field

[0001] The present invention relates to the field of pharmacotherapy, and in particular to the application of dauroxaline in the preparation of myeloperoxidase inhibitors and drugs for treating acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a critical disease that affects the respiratory system. The development of ALI includes a series of chain reactions in which neutrophils, macrophages, endothelial cells, and epithelial cells release inflammatory substances, which eventually lead to acute hypoxic respiratory insufficiency 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 146kDa glycosylated protein composed of two monomers, which accounts for about 5% of the dry weight of neutrophils and is mainly stored in the azurophilic granules of neutrophils. Activated neutrophils can release MPO in various ways such as degranulation or neutrophil extracellular traps. Therefore, MPO is also regarded as a marker of neutrophil activation. In the body's innate immunity, MPO can mediate the activation of H2O2 and Cl - The reaction produces a strong oxidant, hypochlorous acid (HOCl), which kills pathogens. However, HOCl can also react with the body's own biomacromolecules, causing oxidative stress and inflammatory reactions, damaging body cells, and promoting the occurrence and 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 skeletons) into the extracellular space or tissues in the form of biological spider webs. These structures, known as neutrophil extracellular traps (NETs), serve as a source of self-antigens that can trigger inflammatory cascades in local tissues and the bloodstream, and are therefore involved in the pathogenesis of ALI.

[0005] In normal innate immune responses, this process enables neutrophils to capture various pathogenic microorganisms and plays an important role. However, in some special cases, excessive and unbalanced immune responses can lead to dysregulated NETs release, which further aggravates the damage of inflammation to the body, thereby causing host tissue damage beyond the protective function. Studies have shown that excessive NETs release can aggravate lung inflammation damage, and myeloperoxidase, as an important component of NETs and related enzymes, inhibiting the release and function of NETs by inhibiting myeloperoxidase has become our research focus, and we have carried out research on myeloperoxidase inhibitors for the same therapeutic purpose.

[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 and the industry is actively developing it, there are no drugs targeting MPO on the market.

[0007] Radix Sophorae Fructus is a traditional Chinese medicine with the effects of clearing away heat and detoxifying, relieving cough and removing phlegm. It is clinically used to treat throat, swelling and pain, tonsils and chronic bronchitis. One of its main components is daurine, 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 daurine as a myeloperoxidase inhibitor and in improving acute lung injury. Summary of the invention

[0008] In view of the above problems existing in the prior art, the present invention selects dauberine as the active ingredient for the development of myeloperoxidase inhibitors, 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 with abnormal myeloperoxidase activity such as acute lung injury.

[0009] The first aspect of the present invention provides the use of daurine in the preparation of a myeloperoxidase inhibitor product.

[0010] The second aspect of the present invention provides the use of daurine in the preparation of products for treating / preventing / improving diseases caused by abnormal myeloperoxidase activity.

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

[0012] The third aspect of the present invention provides the use of dauroxaline in the preparation of products 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 invention provides the use of daurine in the preparation of products for treating / preventing / improving oxidative damage.

[0015] In the first, second, third and fourth aspects of the present invention, the daurisoline is also known as Daurisoline, CAS No.: 70553-76-3, molecular formula C 37 H 42 N2O6, chemical structure is:

[0016]

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

[0018] In some embodiments, the drug is an injection, tablet, powder, granule, pill, capsule, oral solution, ointment, cream or spray.

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

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

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

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

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

[0024] In some embodiments, the drug is administered orally, enterally, by injection, spraying, physical or chemical mediation, or is administered after being mixed or encapsulated with other substances.

[0025] Beneficial Effects

[0026] The present invention proves for the first time that daubersuline 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 THE DRAWINGS

[0027] Figure 1 This is the docking diagram of existing MPO inhibitors and MPO.

[0028] Figure 2 This is the virtual docking diagram of batroxoline and myeloperoxidase.

[0029] Figure 3 This is the standard curve of hypochlorous acid concentration.

[0030] Figure 4 The concentration of hypochlorous acid generated by each group in the cell experiment, where * indicates P < 0.05, and ** indicates P < 0.01.

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

[0032] 6A, 6B, and 6C in FIG6 are HE staining images of lung sections of mice in the blank control group, modeling group, and drug-treated group, respectively.

[0033] Figure 7 Figure 3 Lung injury scores of each mouse group.

[0034] Figure 8 The release level of myeloperoxidase in each group of mice, where ** indicates P < 0.01, **** indicates P < 0.0001. DETAILED DESCRIPTION

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

[0036] Example 1 Virtual docking to verify the inhibitory effect of daurine on myeloperoxidase

[0037] (1) Selection of active sites

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

[0039] (2) Selection of protein receptors

[0040] In the article published by AstraZeneca in 2022 (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.), the X-ray crystal structure of the MPO receptor ligand complex was uploaded to the PDB library, numbered 7NI1 and 7NI3. We selected 7NI1 as the protein receptor.

[0041] (3) Virtual docking results

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

[0043] It can be seen that dauberine has a good docking effect with myeloperoxidase, based on which it can be inferred that it has a good myeloperoxidase inhibitory effect.

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

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

[0046] The taurine chloramine assay (see formula below) is based on the reaction of hypochlorous acid with taurine (TauNH2) to form taurine chloramine (TauNHCl), which is measured by reaction with s-thio-2-nitrobenzoic acid (TNB). Yellow TNB is oxidized to colorless DTNB. This is an extremely sensitive assay that can accurately measure hypochlorous acid concentrations as low as 5uM.

[0047] HOCl+TauNH2→TauNHCl+H2O

[0048] TauNHCl+2TNB→DTNB+Cl - +TauNH2

[0049] We took a 96-well plate, added 100 μL of TNB solution to the corresponding wells, and then added 100 μL of 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 150 μM, and 200 μM hypochlorous acid solutions to the corresponding wells, and set one well as a blank control group with a corresponding concentration of 0. After reacting for 15 minutes in the dark, the absorbance at 412 nm was measured, and the corresponding hypochlorous acid concentration standard curve was prepared according to the absorbance (such as Figure 3 The results show that the DTNB method can accurately measure the concentration of hypochlorous acid.

[0050] Table 1 Standard curve data

[0051]

[0052] (2) Myeloperoxidase activity inhibition assay

[0053] Myeloperoxidase can spontaneously catalyze the production of hypochlorous acid in the presence of substrates such as hydrogen peroxide and chloride ions. HL-60 cells are human promyelocytes that can be induced to become neutrophil-like cells (dHL-60) in a 1.3% DMSO environment for at least 5 days, and neutrophil-like cells can be activated in the presence of PMA to release myeloperoxidase.

[0054] Therefore, we can set up the system with 10uM daurine, 10mM taurine and 2×10 6 dHL-60 cells (induced by HL-60 cells under 1.3% DMSO for 6 days) were incubated with 100ng / ml PMA at 37°C for 1h and then placed on ice to terminate the reaction. Excessive H2O2 enzyme was added to eliminate the effect of H2O2. After centrifugation, 100uL of the supernatant was reacted with 400uM TNB solution in the dark for 15min, and the absorbance was detected at 405nm. HBSS was used instead of natural product as the blank control group. The results (such as Figure 4 As shown in the present study, it was found that batryticatine can effectively inhibit the activity of myeloperoxidase.

[0055] Example 3 Effect of batroxoline on lung injury in mice induced by LPS acute lung injury

[0056] 1. Experimental Materials

[0057] Fifteen 6- to 8-week-old male C57 mice weighing 20-22 g were purchased from Beijing Weitonglihua Experimental Animal Co., Ltd.

[0058] 2. Experimental Methods

[0059] 1) Preparation of batracholine

[0060] Precisely weigh daurine and prepare it with DMSO, PEG300, Tween 80 and saline to a concentration of 15 mg / ml.

[0061] 2) Animal grouping and drug administration

[0062] After the mice were adaptively fed for 3-5 days, they were fasted at 6 pm the day before the experiment and were allowed to drink water freely. During the experiment, the mice were randomly divided into groups according to their weight, with 5 mice in each group. The drug was administered by intraperitoneal injection, with 0.1 ml given for every 10g of mouse body weight, that is, the dosage was 15 mg / kg. The control group and the modeling group were given the corresponding blank solvent. One hour after the administration, the modeling group and the drug administration group performed pulmonary aerosol administration of mice, and gave a dose of 10 mg / kg of LPS to model acute lung injury. After 24 hours, the mouse lung tissue was taken, and the alveolar lavage fluid was used for relevant tests.

[0063] Determination indicators:

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

[0065] 2) Mouse lung section injury and scoring

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

[0067] 3. Experimental results

[0068] (1) Elisa assay for IL-1 in bronchoalveolar lavage fluid β The concentration, according to Figure 5 It can be seen that the expression level of the inflammatory factor IL-1β in the model group was significantly increased compared with the blank control group, while the expression level of the inflammatory factor IL-1β in the drug-treated group was significantly lower than that in the model group and was equivalent to that in the blank control group.

[0069] (2) Based on Fig. 6A , 6B , 6C shows that the modeling group ( Figure 6B ) compared with the blank control group ( Fig. 6A ), the lungs showed obvious damage, while the drug group ( Figure 6C ) had significantly better lung damage than that in the modeling group.

[0070] (3) The lung damage of each group was scored based on the following pathological conditions: alveolitis, interstitial inflammation, alveolar wall widening, vascular dilation, congestion, interstitial loosening and edema, hemorrhage, emphysema, pulmonary edema, hyaline membrane formation, and inflammatory exudation in the bronchial cavity. The results are as follows: Figure 7 As shown, the modeling group showed almost all the above symptoms, and its lung injury scores were significantly higher than those of the blank control group and the drug-treated group, indicating that the acute lung injury model in mice was successfully established. At the same time, batroxobin can significantly alleviate the acute lung injury in mice caused by LPS.

[0071] (4) The expression of myeloperoxidase in the lungs of each group of mice was detected by WB experiment. The results are as follows Figure 8 As shown, the expression level of myeloperoxidase in the modeling group was significantly increased compared with the blank control group, while the expression level of myeloperoxidase in the drug-treated group was significantly reduced compared with the modeling group.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of batroxoline in the preparation of myeloperoxidase inhibitor products.

2. The use of batroxoside in the preparation of products for treating / preventing / improving diseases caused by abnormal myeloperoxidase activity.

3. The use according to claim 2, characterized in that: The diseases caused by abnormal myeloperoxidase activity include acute lung injury, atherosclerosis, acute coronary syndrome, Hashimoto's thyroiditis, and vasculitis, preferably acute lung injury.

4. Use of batroxoline in the preparation of products for treating / preventing / improving inflammatory diseases. Preferably, the inflammatory disease is acute inflammation or chronic inflammation.

5. Application of batroxoline in the preparation of products for treating / preventing / improving oxidative damage.

6. The use according to any one of claims 1 to 5, characterized in that: The product is in the form of medicine, food or functional health care product. Preferably, the food or functional health care product is used for daily health care and therapeutic assistance for any disease described in claims 1-5.

7. The use according to claim 6, characterized in that: The medicine is in the form of injection, tablet, powder, granule, pill, capsule, oral solution, ointment, cream or spray.

8. The use according to claim 6, characterized in that: The food or functional health product is oral liquid, tea, lozenge, capsule, drink or effervescent tablet.

9. The use according to claim 6 or 7, characterized in that The drug also includes one or more pharmaceutically acceptable excipients. Preferably, the excipients include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and sustained-release agents in the pharmaceutical field.

10. Use according to claim 6, 7 or 9, characterized in that The drug is administered orally, enterally, by injection, spraying, physical or chemical mediation, or is administered after being mixed or encapsulated with other substances.

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