Application of APE1 inhibitor in preparation of medicine for treating asthma

Abstract

The invention belongs to the field of small molecule compound medicines, and relates to application of an APE1 inhibitor in preparation of a medicine for treating asthma. The invention provides a novel treatment strategy of asthma, discovers a new target APE1 for treating asthma, realizes an effective treatment effect on asthma by using an APE1 inhibitor C10 compound, can remarkably inhibit the inflammatory cell activity such as mast cells, eosinophilic granulocytes and lymphocytes of asthma, and can remarkably inhibit the inflammatory cell activity of mice lung pathology observation. The C10 compound disclosed by the invention has obvious relieving and relieving effects on lung pathological changes of asthma mice, so that the C10 compound can be clinically used for preventing or treating asthma, and has the industrial advantages of small molecular weight and simple synthesis process.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule compound medicine, and relates to the application of an APE1 inhibitor in the preparation of a medicine for treating asthma. Background Art

[0002] Apurinic apyrimidinic endonuclease / redox factor (APE-1) is an important intracellular bifunctional protein. Its C-terminus participates in base excision repair after DNA mismatch; its N-terminus contains redox activity, which can reduce the transcription factor with low oxidative activity to the transcription factor with high reduced activity. The active transcription factor can bind to the promoter sequence of downstream DNA to initiate the expression of downstream genes. Its downstream regulated transcription factors include HIF-1α, STAT3, Egr-1, AP-1, NF-κb, CREB, p53, etc. Studies have found that APE1 plays a vital role in regulating immune balance. Many of its substrates, such as HIF-1α, STAT3, and NF-κb, are closely related to human diseases caused by immune disorders. Therefore, we speculate that an APE1 redox inhibitor will alleviate or cure certain diseases related to immune disorders.

[0003] Bronchial asthma, or asthma for short, is a common chronic inflammatory airway disease characterized by recurrent wheezing, shortness of breath, chest tightness or coughing, which often occur or worsen at night or in the early morning. The onset of asthma is related to genetic and environmental factors, among which genetic factors mainly determine the patient's susceptibility to the disease, while environmental factors such as various allergens, air quality, smoking, exercise, etc. are specific triggers.

[0004] At present, there is no drug that can completely cure asthma in clinical practice. The first-line drugs for treating asthma are mostly drugs that dilate the bronchi and reduce airway inflammation, including glucocorticoids (such as budesonide), β2 receptor agonists (such as salbutamol), anticholinergics (such as ipratropium), theophyllines (such as aminophylline), antihistamines (such as ketotrienol) and leukotriene modifiers (such as montelukast). These drugs have a strong effect of inhibiting airway inflammation and relaxing airway smooth muscle, but long-term use will produce drug resistance and bring serious adverse reactions, including osteoporosis, growth restriction, arrhythmia, reduced immunity, neurotoxicity and other serious side effects.

[0005] Therefore, the existing asthma treatment drugs are still limited and far from meeting clinical needs. There is still a lack of long-term, safe, effective and economical small molecule oral drugs. It is urgent to develop new, safe, effective and suitable for long-term asthma treatment drugs. Summary of the invention

[0006] In view of the above technical problems, the present invention provides the use of APE1 inhibitors in the preparation of drugs for treating asthma. After tens of thousands of compound simulation screening technologies, computer simulation methods are used to screen compounds, and a number of compounds with potential efficacy against asthma are screened out by predicting the physical properties, structure and activity of molecules. Among them, small molecule compounds represented by C10 and its derivatives show excellent effects.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] Use of an APE1 inhibitor in the preparation of a drug for treating asthma, wherein the APE1 inhibitor is a C10 compound or E3330;

[0009] The C10 compound has a structure as shown in Formula 1:

[0010]

[0011] Wherein R1 and R2 are respectively a five-membered ring, a six-membered ring or a benzo parallel compound, the five-membered ring is furan, thiophene, pyrrole, thiazole or imidazole, the six-membered ring is pyridine, pyrazine, pyrimidine or pyridazine, and the benzo parallel compound is an indole or isoindole compound.

[0012] Preferably, R1 and R2 are cyclohexyl or substituted cyclohexyl respectively.

[0013] Furthermore, the APE1 inhibitor is a C10 compound, and the C10 compound has a structure as shown in Formula 2 below:

[0014]

[0015] Wherein R is C, N, S or O element; R1, R2, R3, R4, R5 or R6 are hydrogen, or various saturated or unsaturated, cyclic or non-cyclic, substituted or unsubstituted alkyl compounds.

[0016] Furthermore, the C10 compound has a structure as shown in Formula 3 below:

[0017]

[0018] Furthermore, the application is the application of the C10 compound and a pharmaceutically acceptable salt thereof in the preparation of a drug for treating asthma.

[0019] Furthermore, the asthma includes drug-induced asthma, exercise-induced asthma and allergic asthma.

[0020] Furthermore, the application includes: the allergic inflammation of the lungs of asthmatic mice intervened by the C10 compound is reduced, including the reduction of the infiltration of inflammatory cells around the bronchus and the proliferation of goblet cells, as well as the reduction of eosinophils, lymphocytes and neutrophils in BAL Fs.

[0021] Furthermore, the asthma described in the present application is type II inflammatory asthma or allergic asthma.

[0022] Through mechanism exploration, it was found that APE1 regulates the DNA binding activity of a series of transcription factors during the occurrence and development of CRS, inducing ILC2 and Th2 cells to secrete type II inflammatory response cytokines IL-4, IL-5, IL-9 and IL-13, and promoting pleiotropic type II immune mechanisms; oxidative stress response induces APE1 nuclear translocation and promotes the production of type II inflammatory cytokines.

[0023] As an APE1 inhibitor, the C10 compound can inhibit the upregulation of type II cytokines interleukin (IL)-4, IL-5, and IL-13, thereby avoiding airway inflammation.

[0024] The beneficial effects of the present invention are as follows: the C10 compound of the present invention has an excellent therapeutic effect on asthma, and can significantly inhibit the activity of inflammatory cells such as mast cells, eosinophils, lymphocytes, etc. in asthma. Observation of lung pathology in mice shows that the C10 compound of the present invention has a significant effect of reducing and alleviating the pathological changes in the lungs of asthmatic mice.

[0025] The C10 compound of the present invention has a simple structure and excellent potential, belongs to a small molecule oral drug, has a simple synthesis method, and has good industrialization prospects.

[0026] Application prospects: In the treatment of asthma, C10 compound can relieve Th2 inflammation, reduce inflammatory cell infiltration, and effectively reduce the expression levels of IgE and OVA-sIgE in serum, indicating that C10 compound can improve the allergic reaction of AS mice. Animal experiments have confirmed that C10 compound effectively reduces collagen deposition in lung tissue and reduces lung tissue cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 This is a comparison chart of the total cell count and inflammatory cell (eosinophil, neutrophil) count in the bronchoalveolar lavage fluid of mice in the Example C10 treatment group and the AS mouse model group;

[0029] Figure 2This is a comparison chart of mouse serum IgE and OVA-sIgE in the C10 treatment group and the AS model group in the example;

[0030] Figure 3 This is a comparison chart of Th, Th2 and Th17 inflammatory factors in the bronchoalveolar lavage fluid of mice in the C10 treatment group and the AS model group in the example;

[0031] Figure 4 This is a comparison of HE staining of the lungs of mice in the C10 treatment group and the AS model group in the example;

[0032] Figure 5 This is a comparison of PAS staining of the lungs of mice in the C10 treatment group and the AS model group in the example;

[0033] Figure 6 This is a comparison of Masson staining of the lungs of mice in the C10 treatment group and the AS model group in the example;

[0034] Figure 7 This is a comparison of TUNEL staining of mouse lung tissues in the C10 treatment group and the AS model group in the example;

[0035] Figure 8 This is a comparison of ROS staining in the lung tissue of mice in the C10 treatment group and the AS model group in the example;

[0036] Fig. 9 This is a transmission electron microscopic comparison of the lung tissues of mice in the C10 treatment group and the AS model group in the example;

[0037] Fig.10 This is a comparison chart of apoptosis detected by Beas-2B flow cytometry between the C10 treatment group and the AS model group in the example;

[0038] Fig.11 This is a comparison of ROS in Beas-2B in the C10 treatment group and the AS model group in the example;

[0039] Fig.12 The expression of APE1 in the normal tissue adjacent to the tumor of the clinical sample lung cancer patient and the bronchial epithelial tissue of the AS patient is compared in the example. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the present application, the present application is further explained in conjunction with specific implementation methods.

[0041] In some embodiments of the present invention, it was confirmed that APE1 was highly expressed in the upper air tissues of asthma patients, and the therapeutic effect on asthma was achieved by using the known APE1 inhibitor E3330. At the same time, it was verified that the C10 compound exerted a better therapeutic effect in AS in vivo and in vitro models.

[0042] Unless otherwise specified, the raw materials or reagents used in the examples of the present invention are commercially available products.

[0043] The percentages used in the embodiments of the present invention are all by mass unless otherwise specified.

[0044] Example 1

[0045] Pharmacodynamic evaluation of C10 treatment in in vivo and in vitro AS models

[0046] Clinical samples: AS airway epithelial tissues were selected from asthma patients in the Department of Pulmonary Diseases, Qilu Hospital of Shandong University, and control samples were selected from normal tissues adjacent to tumors of lung cancer patients in the Department of Thoracic Surgery. In accordance with ethical requirements and with informed consent from patients, airway epithelial tissues obtained under bronchoscope were fixed or frozen for the next step of pathological histology testing.

[0047] Experimental animals: BALB / C mice, female, 6-8 weeks old, weighing 18-20 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The mice were housed in an SPF constant temperature animal room (temperature: 23+2°C, relative humidity: 55±10%, light-dark cycle 12 h), and the model was established after 1 week of isolation and adaptive feeding.

[0048] Experimental materials: ovalbumin (OVA, A5503, Sigma Aldrich), aluminum hydroxide adjuvant (239186, SigmaAldrich), phosphate buffered saline (PBS, B310KJ, source culture), C10 (Formula 3, synthesized by a biochemical company), E3330 (49581, Novus Biologicals), high glucose DMEM (L110KJ, Gibco), fetal bovine serum (FBS, 10099141, Gibco,), IL-13 recombinant protein (HY-P7033, MCE), LPS from Escherichia coli (L4391, Sigma-Aldrich), dimethyl sulfoxide DMSO (D8371, Solarbio), corn oil (C7030, Solarbio).

[0049] Reagent preparation: Sensitization solution: weigh 1 mg OVA and 112.5 mg Al(OH)3, add them into 10 ml PBS, place in a 4°C constant temperature oscillator and shake thoroughly to mix; 1% OVA stimulation solution (10 mg / ml OVA): weigh 100 mg OVA, add it into 10 ml PBS, dissolve and mix thoroughly, the stimulation solution must be freshly prepared.

[0050] 1. Construction of animal model:

[0051] 1.1 Animal grouping: The animals were randomly divided into 7 groups, with 6 animals in each group. Group A was the blank control group; Group B was the AS model group (nasal drops control, AS in); Group C was the C10 nasal drops group (C10 in); Group D was the E3330 nasal drops group (E3330i.n.); Group E was the AS model group (oral gavage control, AS po); Group F was the C10 gavage group (C10 po); and Group G was the E3330 gavage group (E3330 po).

[0052] The C10 treatment group included group C and group F.

[0053] 1.2 Establishment of animal model: OVA was used to establish the AS mouse model. In the basic sensitization stage, groups B, C, D, E, F, and G were sensitized by intraperitoneal injection of 200 μL of sensitizing drug on days 1, 8, and 15, respectively, and the blank control group was intraperitoneally injected with the same dose of PBS on days 1, 8, and 15. One week later, that is, from day 22, groups B, C, D, E, F, and G were given aerosol inhalation of 1% OVA provocation solution for 20 minutes each time, once a day, for 1 week.

[0054] 1.3 Animal administration: After successful modeling, mice in groups C and D were administered with C10 nasal drops (10 mg / kg) 1 hour before OVA challenge, 20 μl each time (10 μl each in the left and right nasal cavities), once a day, for 2 consecutive weeks; mice in group B (i.e., AS model nasal drop control group) were administered with an equal volume of PBS + DMSO nasal drops, once a day, for 2 consecutive weeks. Mice in groups F and G were administered with C10 intragastric solution 1 hour before OVA challenge (10 mg / kg in group F and 20 mg / kg in group G), 200 μl each time, once a day, for 2 consecutive weeks; mice in group E (i.e., AS model intragastric gavage control group) were administered with an equal volume of DMSO + corn oil intragastric gavage, once a day, for 2 consecutive weeks. Mice in the blank group were given an equal volume of PBS nasal drops and intragastric gavage at the same time. Among them, the preparation method of C10 nasal drops is: dissolve 28.6mg C10 powder in 0.286ml DMSO, add 2.574ml PBS to dilute after complete dissolution, that is, the drug concentration is 10mg / ml; the preparation method of E3330 nasal drops is: dissolve 28.6mg E3330 powder in 1.83ml DMSO, add 1.03ml PBS to dilute after complete dissolution, that is, the drug concentration is 10mg / ml. The preparation method of C10 intragastric solution is: dissolve 30mg C10 powder in 1.2ml DMSO, add 28.8ml corn oil to dilute after complete dissolution, that is, the drug concentration is 1mg / ml; the preparation method of E3330 nasal drops is: dissolve 57.2mg E3330 powder in 0.572ml DMSO, add 28.028ml corn oil to dilute after complete dissolution, that is, the drug concentration is 2mg / ml.

[0055] 2. Mouse serum collection: After 24 hours of drug intervention, mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital. Eyeballs were removed and peripheral blood was collected in 1.5 ml EP tubes, stored at room temperature for 0.5 hours, and then centrifuged (3000 rpm / 15-30 min / 4°C) after chromatography, and the supernatant was frozen at -80°C.

[0056] 3. Obtaining and testing bronchoalveolar lavage fluid: Mice were killed by cervical dislocation and disinfected with 75% alcohol. The mouse neck was split open to fully expose the trachea, and 1 mL of pre-cooled PBS was used to gently perfuse the lung cavity from the trachea. The lung cavity was rinsed repeatedly for 3 times. The bronchoalveolar lavage fluid was collected and centrifuged (1200 rpm / 5 min / 4 ° C), and the supernatant was frozen at -80 ° C for ELISA detection. 100 ul PBS was added to the cell pellet for re-suspending, and the cell was counted using a cell counter and spun at 1000 rpm for 5 min. The cell pellet was fixed with paraformaldehyde for subsequent Dif staining to evaluate the state of lung inflammation.

[0057] 4. Histopathological examination: The mice were fixed in a clean bench, the chest cavity of the mice was opened, and part of the mouse lung tissue was immediately placed in a 4% neutral paraformaldehyde solution for fixation for 24 hours. Subsequently, the tissue was decalcified with EDTA decalcifier, dehydrated, waxed, embedded, and sectioned. HE, PAS, Masson, and Sirius Red staining were performed to observe the structural integrity of the mouse lung tissue and inflammatory infiltration.

[0058] 5. Transmission electron microscopy: Take out the mouse lungs and quickly put them into the transmission electron microscopy fixative.

[0059] Afterwards, the resin blocks were formed after PBS bleaching, 1% osmium acid fixation, gradient ethanol, infiltration, and embedding. After trimming the surface of the resin block, ultrathin sections with a thickness of 70 nm were made and the sections were picked up and placed on two aromatic film grids. The sections were stained with 2% uranyl acetate solution for 30 minutes, and then stained with lead citrate staining solution for 5 minutes, and then washed thoroughly with water. The sections were baked under infrared light for 10 minutes, and after drying, the ultrastructure was observed under a transmission electron microscope at 80KV, and photographed at different magnifications.

[0060] 6.ELISA test: Take out the mouse serum and pulmonary lavage fluid supernatant samples and thaw them on ice. According to the reagent instructions, add the standard and sample in sequence, and then detect the absorbance on the machine after adding enzyme, incubation, washing, color development, termination and other steps to calculate the sample concentration.

[0061] 7. Cell culture: Beas-2B cell line was obtained from American Type Culture Collection, and high-glucose DMEM medium with 10% FBS was added and cultured in an incubator at 37°C and 5% CO2. When the cells were 50-60% confluent, the cells were treated with C10 (20 μm) or E3330 (40 μm) for 1 hour, and then stimulated with 20 ng / mL IL-13 recombinant protein combined with 2 ug / ml LPS for 48 hours.

[0062] 8. Flow cytometry: Open the chest cavity, carefully dissect the mouse lungs, remove the mouse lungs and place them in 1640 culture medium containing collagenase D, DNaseⅠ and DSpaseⅡ, cut them into pieces and incubate them in a 37℃ water bath for 40-50min. The digested lungs were ground and filtered with a 70μm filter to obtain a single cell suspension. After 48 hours of Beas-2B intervention, the cell suspension was digested and stained according to the instructions of the apoptosis kit and tested on the machine.

[0063] 9. TUNEL assay: After dewaxing, lung tissue sections were incubated with 20 μg / ml DNase-free proteinase K (#ST532, Beyotime) at 37°C for 30 min, and then incubated with 50 μl of TUNEL assay solution at 37°C for 1 hour. Samples were examined using a fluorescence microscope.

[0064] 11. Statistical analysis: All experimental research data were analyzed by SPSS25.0 software, and the quantitative data in the data were expressed as (x±S). If the comparison between three or more groups of samples conformed to the normal distribution and the variance was homogeneous, one-way ANOVA was used; if the data did not conform to the normal distribution, the non-parametric Kruskal-Wallis test and Dunnetts's pairwise comparison were used. P<0.05 indicated that the difference was statistically significant, P<0.01 was considered to be significantly different, P<0.001 was considered to be significantly different, and NS indicated that the difference was not statistically significant. All statistical graphs were generated using GraphPad Prism 8.0.

[0065] The experimental results are as follows Figure 1-Figure 11 shown.

[0066] pass Figure 1 It can be seen that compared with the AS model group, C10 can effectively reduce the total number of exfoliated cells and the number of eosinophils and neutrophils in the bronchoalveolar lavage fluid, indicating that the inflammatory state of the nasal cavity of AS mice has been improved.

[0067] pass Figure 2It can be seen that compared with the AS model group, C10 can effectively reduce the expression levels of IgE and OVA-sIgE in serum, indicating that C10 can improve the allergic reaction of AS mice.

[0068] IL-4, IL-5, and IL-13 are mainly inflammatory mediators produced by Th2 cells. The levels of IL-4, IL-5, and IL-13 are significantly increased in asthma patients. IL-4 can promote B cell proliferation and differentiation and induce IgE synthesis. IL-4 can also attract eosinophils to the airways, increasing airway responsiveness. Figure 3 It can be seen that compared with the AS model group, the compound C10 of the present invention has a significant inhibitory effect on the levels of IL-4, IL-5, and IL-13 in OVA-induced asthma mice. C10 can upregulate the expression levels of Th1 (IFN-γ) type and downregulate the expression levels of Th2 type (IL-4, IL-5, IL-13) and Th17 type (IL-17A) inflammatory factors, indicating that C10 can regulate the immune balance of local tissues.

[0069] pass Figure 4 It can be seen that compared with the AS model group, C10 can effectively improve the structural integrity of lung tissue, alleviate the infiltration of mixed inflammatory cells around lung blood vessels and bronchi, and bronchial mucus exudation, indicating that the pathological changes in the lungs of AS mice were alleviated.

[0070] pass Figure 5 It can be seen that compared with the AS model group, C10 can effectively reduce the number of goblet cells and improve the proliferation and hypertrophy of goblet cells.

[0071] pass Figure 6 It can be seen that compared with the AS model group, C10 can effectively reduce collagen deposition in lung tissue.

[0072] pass Figure 7 It can be seen that compared with the AS model group, C10 inhibited the apoptosis of lung tissue cells in AS mice.

[0073] pass Figure 8 It can be seen that compared with the AS model group, C10 effectively reduced the production of ROS in the lung tissue of AS mice, indicating that C10 reduced oxidative damage in the lung tissue of AS mice.

[0074] pass Fig. 9 It can be seen that compared with the AS model group, C10 effectively improved the mitochondrial morphology in the lung tissue of AS mice.

[0075] pass Fig.10 It can be seen that in the in vitro model of AS induced by Beas-2B stimulated by IL-13 recombinant protein combined with LPS, C10 inhibited epithelial cell apoptosis and improved cell activity.

[0076] pass Fig.11 It can be seen that in the in vitro model of AS induced by Beas-2B stimulated by IL-13 recombinant protein combined with LPS, C10 effectively reduced the production of ROS in epithelial cells.

Claims

1. Use of an APE1 inhibitor in the preparation of a drug for treating asthma, characterized in that: The APE1 inhibitor includes a C10 compound; The C10 compound has a structure as shown in Formula 1: Wherein R1 and R2 are respectively a five-membered ring, a six-membered ring or a benzo parallel compound, the five-membered ring is furan, thiophene, pyrrole, thiazole or imidazole, the six-membered ring is pyridine, pyrazine, pyrimidine or pyridazine, and the benzo parallel compound is an indole or isoindole compound.

2. Use of the APE1 inhibitor according to claim 1 in the preparation of a medicament for treating asthma, characterized in that: The R1 and R2 are cyclohexyl or substituted cyclohexyl.

3. Use of the APE1 inhibitor according to claim 1 in the preparation of a medicament for treating asthma, characterized in that: The APE1 inhibitor is a C10 compound, and the C10 compound has a structure as shown in Formula 2 below: Wherein R is C, N, S or O element; R1, R2, R3, R4, R5 or R6 are hydrogen, or various saturated or unsaturated, cyclic or non-cyclic, substituted or unsubstituted alkyl compounds.

4. Use of the APE1 inhibitor according to claim 3 in the preparation of a medicament for treating asthma, characterized in that: The C10 compound has a structure as shown in Formula 3 below:

5. Use of the APE1 inhibitor according to any one of claims 1 to 4 in the preparation of a medicament for treating asthma, characterized in that: The application is the application of the C10 compound and a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating asthma.

6. The use according to claim 5, characterized in that The asthma includes type II inflammatory asthma, drug-induced asthma, exercise-induced asthma and infectious asthma.

7. The use according to claim 6, characterized in that The asthma includes type II inflammatory asthma and allergic asthma.

8. The use according to claim 7, characterized in that Asthmatic mice treated with C10 compound showed reduced allergic inflammation in the lungs, including decreased infiltration of peribronchial inflammatory cells and goblet cell proliferation, as well as decreased eosinophils, lymphocytes, and neutrophils in BALFs.

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