Use of bay 11-7085 in the prevention and treatment of pneumonia

Abstract

The application discloses application of BAY 11-7085 in preventing and treating pneumonia. In the application, it is found through actual administration test of a model mouse that BAY 11-7085 can significantly reduce expression of inflammatory factors and protein content in alveolar lavage fluid, reduce a lung pathology score, thereby treating pneumonia, and effectively relieving or inhibiting alveolar protein deposition, pulmonary edema and death caused by pneumonia.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of BAY 11-7085 in preventing and treating pneumonia. BACKGROUND

[0002] Pneumonia is a representative disease in pulmonary organic diseases, which generally refers to infectious inflammation occurring in alveoli, distal airways and lung interstitium, which can be caused by factors such as bacteria, viruses and other pathogens, among which bacterial and viral pneumonia is the most common. Of course, in a broader sense, physicochemical factors, immune damage, allergy and drugs can also cause pneumonia. Severe pneumonia can cause irreversible lung damage in the lungs, such as causing ground-glass nodules, thereby causing the patient's lung function to decline, and even the possibility of sequelae.

[0003] Viral pneumonia (also known as viral infectious pneumonia) is a common type of pneumonia in clinical practice, which can progress from local pneumonia to systemic infection, sepsis, septic shock and multiple system dysfunction. With the diversification and complexity of respiratory infection pathogen spectrum, the incidence and mortality of severe pneumonia are on the rise. According to statistics, there were 50 million cases of influenza-related lower respiratory tract infections worldwide in 2017, of which 810,000 were severe cases and 140,000 died. As can be seen, the harm of viral pneumonia cannot be underestimated. Highly pathogenic influenza A virus is a very common respiratory virus that can invade human lower respiratory tract epithelial cells and alveolar epithelial cells, causing diffuse lung damage, which can easily develop into severe pneumonia and acute respiratory distress syndrome. Respiratory syncytial virus pneumonia, also known as syncytial virus pneumonia, is a common interstitial pneumonia that often occurs in children. Severe cases have obvious respiratory distress, wheezing, cyanosis and three concave signs, and a small number of severe cases can also be complicated by heart failure and respiratory failure, which is more harmful to infants. However, there is still a lack of clear and effective treatment for pneumonia caused by highly pathogenic viruses, especially severe pneumonia. SUMMARY

[0004] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the purpose of the present application is to provide the application of BAY 11-7085 in preventing and treating pneumonia. In the present application, the inventors first found that BAY 11-7085 can effectively prevent or treat pneumonia or severe pneumonia caused by influenza virus and / or respiratory syncytial virus, which can rapidly reduce the expression of inflammatory factors and reduce the lung pathology score, thereby effectively relieving or curing pneumonia.

[0005] In a first aspect of the present application, the application of BAY 11-7085 in the preparation of a drug for preventing and / or treating pneumonia is provided.

[0006] In some embodiments of the application, the pneumonia comprises alveolar proteinosis, pulmonary edema, and death caused by the pneumonia.

[0007] In some embodiments of the application, the pneumonia comprises pneumonia caused by a pathogen.

[0008] In some embodiments of the application, the pathogen comprises bacteria, fungi, viruses, mycoplasma, and chlamydia.

[0009] In some embodiments of the application, there is provided a use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating viral pneumonia.

[0010] In some embodiments of the application, the viral pneumonia comprises alveolar proteinosis, pulmonary edema, and death caused by the viral pneumonia.

[0011] In some embodiments of the application, the virus comprises influenza virus (influenza virus).

[0012] In some embodiments of the application, there is provided a use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating influenza virus pneumonia.

[0013] In some embodiments of the application, the influenza virus comprises influenza virus type A, B, C, and D.

[0014] In some embodiments of the application, the influenza virus is influenza virus type A.

[0015] In some embodiments of the application, there is provided a use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating pneumonia caused by influenza virus type A.

[0016] In some embodiments of the application, the influenza virus type A is influenza virus subtype H1, H2, H3.

[0017] In some embodiments of the application, the influenza virus type A is influenza virus subtype H1, H2, H3 and N1, N2, such as H1N1, H2N2, H3N2.

[0018] In some embodiments of the application, the influenza virus type A is influenza virus subtype H5, H6, H7, H9, H10, such as H5N1, H5N6, H6N1, H7N7, H7N2, H7N3, H7N9, H9N2, H10N8, etc.

[0019] In some embodiments of the present application, the influenza virus comprises H1N1, H2N2, H3N2, H5N1, H7N9 and H9N2.

[0020] In some embodiments of the present application, the influenza virus comprises H1N1, H2N2, H3N2.

[0021] In some embodiments of the present application, the influenza virus is H1N1.

[0022] In some embodiments of the present application, the virus further comprises respiratory syncytial virus.

[0023] In some embodiments of the present application, there is provided use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating pneumonia caused by respiratory syncytial virus.

[0024] In some embodiments of the present application, the bacteria comprise, but are not limited to, Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa.

[0025] In some embodiments of the present application, the fungi comprise, but are not limited to, Candida, Aspergillus, Mucor, Cryptococcus.

[0026] In some embodiments of the present application, the medicament does not contain other therapeutically active ingredients in addition to BAY 11-7085.

[0027] In some embodiments of the present application, the BAY 11-7085 is the only therapeutically active ingredient in the medicament.

[0028] In the present application, the term "therapeutically active ingredient" also refers to an active pharmaceutical ingredient, which refers to any substance or mixture of substances used in the manufacture of a pharmaceutical product, which substance has a pharmacological activity or other direct effect in the diagnosis, treatment, alleviation, handling or prevention of diseases, or which substance can affect the function or structure of the body.

[0029] In some embodiments of the present application, the medicament further contains a pharmaceutically acceptable adjuvant or carrier.

[0030] In some embodiments of the present application, the pharmaceutically acceptable adjuvant or carrier comprises at least one of a diluent, an excipient, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a flavoring agent, a sweetener and a lubricant.

[0031] In the present application, the diluent and excipient are used to adjust the density and flowability of the medicament, and ensure uniform distribution of the medicament, which comprises, but is not limited to, starch, dextrin, sucrose, mannitol, lactose, microcrystalline cellulose, etc.

[0032] In the present application, the binder is used to bind the drug particles together to form granules or tablets suitable for tableting, which includes but is not limited to cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone.

[0033] In the present application, the wetting agent is used to help the drug better contact with the liquid medium, promote the dissolution and dispersion of the drug, which includes but is not limited to glycerol.

[0034] In the present application, the disintegrant is used to promote the dissolution of the tablet in the oral cavity or gastrointestinal tract, facilitate the release and absorption of the drug, which includes but is not limited to sodium methyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, etc.

[0035] In the present application, the absorption enhancer is used to improve the permeability of the drug through the cell membrane, accelerate the absorption of the drug, which includes but is not limited to quaternary ammonium compounds.

[0036] In the present application, the surfactant is used to reduce the surface tension of the drug, improve the dispersibility and stability of the drug, which includes but is not limited to cetyl alcohol, Tween 80, sodium dodecyl sulfate.

[0037] In the present application, the adsorption carrier is used to adsorb drug molecules, increase the stability and bioavailability of the drug, which includes but is not limited to kaolin and soap clay.

[0038] In the present application, the lubricant is used to reduce the friction force during tableting, promote the molding of the tablet, which includes but is not limited to talc, calcium stearate, magnesium stearate, silica micropowder and polyethylene glycol.

[0039] In some embodiments of the present application, the mass percentage of BAY 11-7085 in the drug is greater than or equal to 50%, for example, 50%, 60%, 70%, 80%, 90%, etc.

[0040] In some embodiments of the present application, the mass percentage of BAY 11-7085 in the drug is greater than or equal to 70%, for example, 70%, 85%, 95%, etc.

[0041] In some embodiments of the present application, the mass percentage of BAY 11-7085 in the drug is greater than or equal to 90%, for example, 90%, 92%, 97%, etc.

[0042] In some embodiments of the present application, the mass percentage of BAY 11-7085 in the drug is greater than or equal to 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0043] In some embodiments of the present application, the purity of BAY 11-7085 in the drug is above 70%, or above 80%, or above 90%, or above 95%, such as 96%, 97%, 98%, 99%, 100%, etc.

[0044] In some embodiments of the present application, the concentration of BAY 11-7085 used in the drug is above 0.1 mg / kg, such as 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, etc.

[0045] In some embodiments of the present application, the concentration of BAY 11-7085 used in the drug is above 1 mg / kg.

[0046] In some embodiments of the present application, the concentration of BAY 11-7085 used in the drug is above 2 mg / kg, such as 2 mg / kg-30 mg / kg, or 2 mg / kg-10 mg / kg, such as 2 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, etc.

[0047] In some embodiments of the present application, the drug further contains a second therapeutically active ingredient.

[0048] In some embodiments of the present application, the second therapeutically active ingredient can be contained in the drug, such as by mixing, packaging, etc. to mix or package the second therapeutically active ingredient and BAY 11-7085 as the same drug, or by independent packaging to be independent of the drug.

[0049] In some embodiments of the present application, the second therapeutically active ingredient includes a BAY 11-7085 derivative or other pneumonia treatment drug.

[0050] In the present application, BAY 11-7085 is a chemical substance with CAS number 196309-76-9 and chemical name (2E)-3-[[4-tert-butylphenyl]sulfonyl]-2-propenoyl cyanide.

[0051] The derivatives of BAY 11-7085 include compounds obtained by chemical modification or change based on the structure of BAY 11-7085, which can have similar biological activity or different physical and chemical properties, or have better efficacy or lower toxicity.

[0052] In some embodiments of the present application, the BAY 11-7085 derivatives include salts, esters and amides of BAY 11-7085.

[0053] In some embodiments of the present application, examples of the salt of BAY 11-7085 include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferred examples of the metal salts include: alkali metal salts such as sodium salts, potassium salts, and the like; alkaline earth metal salts such as calcium salts, magnesium salts, barium salts, and the like; and aluminum salts. Preferred examples of the salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, and the like. Preferred examples of the salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Preferred examples of the salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Preferred examples of the salts with basic amino acids include salts with arginine, lysine, ornithine, and the like. Preferred examples of the salts with acidic amino acids include salts with aspartic acid, glutamic acid, and the like.

[0054] In some embodiments of the present application, the salt is a pharmaceutically acceptable salt. For example, when the active ingredient contains an acidic functional group, examples thereof include inorganic salts such as alkali metal salts (e.g., sodium salts, potassium salts, and the like), alkaline earth metal salts (e.g., calcium salts, magnesium salts, and the like), and the like, ammonium salts, and the like, and when the compound contains a basic functional group, examples thereof include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like, and salts with organic acids such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like.

[0055] In some embodiments of the present application, the other pneumonia treatment drug includes an antibiotic, an antifungal drug, an antiviral drug, and a hormone.

[0056] In some embodiments of the present application, the other pneumonia treatment drug includes a penicillin, a cephalosporin, a carbapenem, a macrolide, a quinolone, an enzyme inhibitor-containing drug, and a monobactam drug.

[0057] In some embodiments of the present application, the other pneumonia treatment drug includes penicillin, amoxicillin, cefuroxime, cefprozil, cefaclor, ceftriaxone, cefixime, cefoperazone / sulbactam, azithromycin, roxithromycin, erythromycin, ofloxacin, levofloxacin, vancomycin, moxifloxacin, teicoplanin, linezolid, oseltamivir, acetaminophen, and the like.

[0058] In some embodiments of the present application, the form of the drug includes oral preparations, injection or spray, such as capsules, tablets, pills, granules, and powders.

[0059] In some embodiments of the present application, the form of the drug is injection.

[0060] In some embodiments of the present application, the pneumonia is severe pneumonia.

[0061] In some embodiments of the present application, the pneumonia is severe pneumonia caused by the above-mentioned pathogens, such as severe pneumonia caused by influenza A virus and / or respiratory syncytial virus.

[0062] In the present application, severe pneumonia refers to pneumonia with a mortality rate of more than 50% when mice are used as the subject.

[0063] When humans are used as the subject, severe pneumonia refers to those who meet one of the following major criteria or ≥3 of the following secondary criteria:

[0064] 1. Major criteria:

[0065] (1) requiring endotracheal intubation for mechanical ventilation treatment;

[0066] (2) septic shock still requires vasopressor treatment after active fluid resuscitation.

[0067] 2. Secondary criteria:

[0068] (1) respiratory rate ≥30 / min;

[0069] (2) oxygenation index ≤250mmHg (1mmHg=0.133kPa);

[0070] (3) multiple lobe infiltration;

[0071] (4) disturbance of consciousness and / or disorientation;

[0072] (5) blood urea nitrogen ≥7.14mmol / L;

[0073] (6) systolic blood pressure <90mmHg requiring active fluid resuscitation.

[0074] In some embodiments of the present application, the subject of the drug is an animal, including mammals, such as humans.

[0075] In some embodiments of the present application, when humans are used as the subject of administration, the humans are selected from infants aged 0-6 years, children aged 6-12 years, adolescents aged 12-18 years, and adults aged 18 years or older (such as the elderly aged 60 years or older).

[0076] In some embodiments of the present application, there is provided a use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating severe pneumonia.

[0077] In some embodiments of the present application, there is provided a use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating viral severe pneumonia.

[0078] In some embodiments of the present application, there is provided a use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating severe pneumonia caused by influenza virus and / or respiratory syncytial virus.

[0079] In a second aspect of the present application, there is provided a method for preventing and / or treating pneumonia, comprising administering BAY 11-7085.

[0080] In some embodiments of the present application, the method for preventing and / or treating pneumonia, including the type of pneumonia (such as influenza virus pneumonia, respiratory syncytial virus pneumonia and severe pneumonia), the purity of BAY 11-7085, the concentration, the subject, etc. are as described in the first aspect.

[0081] The beneficial effects of the present application are:

[0082] The present application first discovers the preventive and therapeutic effects of BAY 11-7085 in pneumonia, especially viral pneumonia and severe pneumonia, which can effectively reduce the expression of inflammatory factors and reduce the lung pathology score, thereby effectively alleviating or inhibiting the death caused by severe pneumonia. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 Experimental procedure for modeling mice infected with different titers of influenza virus.

[0084] Figure 2 Body weight changes of mice infected with different titers of influenza virus.

[0085] Figure 3 Comparison of survival time of mice infected with different titers of influenza virus.

[0086] Figure 4 Experimental procedure for treating mice with influenza virus pneumonia with different concentrations of BAY 11-7085.

[0087] Figure 5 Body weight changes of mice treated with different concentrations of BAY 11-7085 for influenza virus pneumonia.

[0088] Figure 6 Lung tissue staining of mice treated with different concentrations of BAY 11-7085 for influenza virus pneumonia.

[0089] Figure 7 Lung pathological score of mice treated with different concentrations of BAY 11-7085 for influenza virus pneumonia.

[0090] Figure 8 Changes in protein concentration in the bronchoalveolar lavage fluid of mice treated with different concentrations of BAY 11-7085 for influenza virus pneumonia.

[0091] Figure 9 Changes in inflammatory factors in the lung tissue of mice treated with different concentrations of BAY 11-7085 for influenza virus pneumonia.

[0092] Figure 10 Flowchart of experiments in which mice with influenza virus pneumonia were treated with BAY 11-7085 and positive control drugs.

[0093] Figure 11 Changes in body weight of mice treated with BAY 11-7085 and positive control drugs for influenza virus pneumonia.

[0094] Figure 12 Lung pathological score of mice treated with BAY 11-7085 and positive control drugs for influenza virus pneumonia.

[0095] Figure 13 Changes in inflammatory factors of mice treated with BAY 11-7085 and positive control drugs for influenza virus pneumonia.

[0096] Figure 14 Flowchart of experiments in which mice with severe influenza pneumonia were treated with BAY 11-7085.

[0097] Figure 15 Changes in body weight of mice treated with BAY 11-7085 and infected control groups for severe influenza pneumonia.

[0098] Figure 16 Comparison of survival time of mice treated with BAY 11-7085 and infected control groups for severe influenza pneumonia.

[0099] Figure 17 Comparison of interleukin-6 mRNA levels at different time points of a respiratory syncytial virus pneumonia cell model treated with BAY 11-7085 and infected control groups.

[0100] Figure 18 Comparison of TNF-α expression levels at different time points of a respiratory syncytial virus pneumonia cell model treated with BAY 11-7085 and infected control groups. DETAILED DESCRIPTION

[0101] The present application will be further described in details by specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available or can be obtained by prior art methods unless otherwise specified. The test or test method is the conventional method in the art unless otherwise specified.

[0102] Example 1 Infection degree of wild type mice infected with different virus titers

[0103] In this example, the infection degree test of different virus titers was carried out using mice. The experimental animals were 8-10 weeks old C57BL / 6 mice (wild type mice) of specific pathogen free (SPF level) purchased from Shanghai Southern Model Organisms Technology Co., Ltd.

[0104] The specific experimental method is as follows:

[0105] After the 8-10 weeks old C57BL / 6 mice were anesthetized by isoflurane tracheal anesthesia, they were divided into PBS uninfected group, low lethal dose infection group (450 TCID50), medium dose infection group (600 TCID50) and lethal dose infection group (900 TCID50). Each group was dropped 50 μL of influenza virus (H1N1 influenza virus strain: HK / 415742 / 09-Mut) through the nose to obtain different concentration gradient of influenza virus pneumonia mouse models. This modeling method was used to determine the degree of virus infection, which was helpful for the subsequent drug screening. At the same time, a blank control (dropping the same amount of normal saline) was set. The body weight and survival time of the mice were recorded within 14 days of modeling. The specific experimental process is shown in Figure 1

[0106] The body weight and survival time of the mice infected with the virus are shown in Figure 2 and Figure 3 The group with mortality more than 50% without drug administration was regarded as the severe group. It can be found from the figure that the mortality of the mice in the 900 x TCID50 infection group was more than 50%, and the trend of body weight decrease was the most obvious. It was indicated that the infection of 900 x TCID50 H1N1 influenza virus PR8 strain could cause viral severe pneumonia in mice, and the infection of 450 TCID50 H1N1 influenza virus PR8 strain could cause viral pneumonia in mice.

[0107] Example 2 Treatment effect of different concentrations of BAY 11-7085 on viral pneumonia

[0108] ​In this embodiment, the therapeutic effect in the influenza virus pneumonia mouse model was compared using different concentrations of BAY 11-7085. The experimental animals were 8-9-week-old C57BL / 6 mice (wild-type mice) obtained from Shanghai Southern Model Animals, Co., Ltd. and were specific pathogen free (SPF level).

[0109] The modeling method of the influenza virus pneumonia mouse model was the same as that in Embodiment 1 described above. In this embodiment, the influenza virus used was the H1N1 influenza A virus strain (HK / 415742 / 09-Mut) with a titer of 450 x TCID50 (half the number of tissue culture infectious doses, which can be determined by the Reed-Muench method, interpolation method or Karber method). A blank control (the same amount of normal saline was dropped) was also set up. Part of the mice in each group were sacrificed on the seventh day after modeling, and the alveolar lavage fluid and lung tissue samples were obtained. The specific experimental procedure is shown in Figure 4

[0110] Specifically, the experimental mice were modeled by nasal instillation of the virus on day 0 of the experiment. Drug administration was performed according to the grouping on days 1-5 of the experiment. Among them, the experimental group was administered different concentrations (2 mg / kg and 10 mg / kg) of BAY 11-7085 by intraperitoneal injection; the model group was administered DMSO. The number of mice in each group was 10, and the body weight of the mice in each group was observed and recorded every day.

[0111] The lung tissue samples of each group were taken and stained with hematoxylin and eosin, and then subjected to pathological scoring.

[0112] The specific method is as follows:

[0113] ​The lung tissue samples of each group were perfused with physiological saline through the heart to remove the blood in the lungs, and then the lung tissue was perfused and fixed with 4% paraformaldehyde (PFA) buffer solution. The lung was taken out and immersed in 4% PFA solution for 4-24 hours for fixation. The fixed lung tissue was dehydrated with gradient ethanol (from 70% to 100%), and then treated with xylene for transparency. The transparent lung tissue was embedded in paraffin, and after the paraffin was hardened, it was ready for use. The paraffin-embedded lung tissue was cut into thin sections with a thickness of 3-5 microns using a microtome, and attached to a glass slide. The paraffin sections were deparaffinized and rehydrated in xylene and gradient ethanol solutions, respectively, and then stained with hematoxylin-eosin for 5-10 minutes to stain the cell nuclei. The tissue sections were washed with tap water and differentiated for 1-2 seconds with hydrochloric acid ethanol. They were then returned to an alkaline environment (such as ammonia water) for blueing, washed with tap water, and then stained with eosin staining solution for 1-3 minutes to stain the cytoplasm. After staining, the sections were dehydrated with gradient ethanol and then transparentized with xylene, followed by neutral gum mounting. Then, pathological scoring was performed, in which the following points were evaluated: inflammatory cell infiltration: the type and number of inflammatory cells around the alveoli and bronchi were observed; alveolar hemorrhage: the presence or absence of red blood cell extravasation in the alveolar lumen was evaluated; interstitial edema: whether the alveolar septum was thickened and whether it was accompanied by edema was examined; and hyaline membrane formation: whether there was a hyaline membrane in the alveoli (one of the characteristics of acute respiratory distress syndrome) was observed.

[0114] Each indicator was scored according to a 0-4 scoring standard: 0 points: normal, no damage. 1 point: slight damage, less than 25% of the affected area. 2 points: moderate damage, 25%-50% of the affected area. 3 points: severe damage, 50%-75% of the affected area. 4 points: extremely severe damage, more than 75% of the affected area. The lung injury scores of each mouse were compared and summarized.

[0115] Further detection of protein content in the alveolar lavage fluid was performed to determine the therapeutic effect of the drug on lung edema caused by pneumonia. The specific detection method was as follows: after the mouse lung tissue was dissected at the neck, the trachea was cannulated for bilateral lavage. The PBS lavage volume was 0.8 mL, and the number of lavages was 2-3 times. The recovered lavage fluid was centrifuged at 1500 r for 10 minutes at 4°C, and the supernatant was recovered for BCA protein concentration detection.

[0116] The lung tissue supernatant (obtained by centrifugation after grinding the lung tissue) of the experimental mice was detected using flow cytometry multi-factor detection (Cytometric Bead Array, CBA) to analyze the expression of IL-6 (interleukin-6).

[0117] The results are as follows: Figures 5-9As shown.

[0118] As shown. Figure 5 It can be found that the body weight of the model group mice decreased significantly after nasal feeding of the influenza virus, while the body weight decrease was significantly reduced after administration of different concentrations of BAY 11-7085, and the mice in the administration group were able to recover to the pre-infection weight level in a shorter time, especially the high-dose group had a sustained and significant recovery trend compared with the model group. Thus it can be shown that the therapeutic effect of BAY 11-7085 can effectively improve the decrease in body weight caused by influenza virus infection as the dose increases.

[0119] As shown. Figure 6 and Figure 7 As shown in the results, compared with the model group, the tissue necrosis in the case slices of the BAY-117085 treatment group mice was lower, the lung tissue pathological score of the BAY-117085 treatment group mice was lower, the lung injury of the BAY-117085 treatment group mice was lighter, and there was a significant difference compared with the influenza virus pneumonia modeling group.

[0120] As shown. Figure 8 and Figure 9 As shown in the results, the protein content and inflammatory cytokines in the bronchoalveolar lavage fluid of the BAY 11-7085 treatment group mice were significantly decreased compared with the influenza virus pneumonia modeling group. Thus it can be shown that administration of different concentrations of BAY 11-7085 can effectively control lung inflammation and alveolar protein deposition and pulmonary edema caused by pneumonia.

[0121] Example 3 BAY 11-7085 and positive control oseltamivir administration group for the treatment and prevention effect of viral pneumonia

[0122] In this embodiment, the prevention and prevention effect of BAY 11-7085 and positive control oseltamivir administration group for viral pneumonia is verified, and the experimental animals are 8-10 week old C57BL / 6 mice (wild type mice) of specific pathogen free (SPF level) purchased from Shanghai South Model Organism Technology Co., Ltd.

[0123] The specific experimental method is as follows:

[0124] After 8-10 week old C57BL / 6 mice were anesthetized by isoflurane airway anesthesia, 50 μL of non-lethal dose of influenza virus (H1N1 influenza A virus strain: HK / 415742 / 09-Mut) was dropped into the nasal cavity to obtain an influenza virus pneumonia mouse model. At the same time, a blank control (dropping the same amount of normal saline, PBS group) was set. Part of the mice in each group were randomly sacrificed on the 7th day and the 14th day after modeling, and the bronchoalveolar lavage fluid and lung tissue samples were obtained. The specific experimental process is as follows Figure 10Results are shown in Figure 6.

[0125] Specifically, the experimental mice were modeled by intranasal instillation of virus on day 0 of the experiment. On days 1-5 of the experiment, the mice were administered drugs according to the grouping. Among them, the positive group was administered 30 mg / kg of Oseltamivir by intraperitoneal injection (OSEL group), the experimental group was administered 10 mg / kg of BAY 11-7085 (CAS: 196309-76-9, BAY group) by intraperitoneal injection; the model group was administered DMSO (DMSO group). The number of mice in each group was 10-12, and the body weight of the mice in each group was observed and recorded every day.

[0126] The lung tissue samples of each group were taken, and the lung injury scores of the mice in different groups were compared, in the same manner as in Example 2.

[0127] The lung tissue supernatant (obtained by centrifugation after grinding the lung tissue) of the experimental mice was detected by flow cytometry (Cytometric Bead Array, CBA) to analyze the expression of anti-inflammatory factors (IL-27, IL-10) and interferons (IFN-γ, IFN-β).

[0128] Results are shown in Figure 6. Figures 11-13

[0129] As can be seen from the results shown in Figure 6, compared with the model group (DMSO), BAY 11-7085 (BAY) can effectively reduce the pathological score of the lung, and the effect is close to that of three times the amount of the positive control (Oseltamivir), indicating that BAY 11-7085 can effectively improve the pathological changes of the lung caused by influenza virus-induced pneumonia. Figure 11 As can be seen from the results shown in Figure 6, compared with the model group (DMSO), BAY 11-7085 (BAY) can effectively reduce the pathological score of the lung, and the effect is close to that of three times the amount of the positive control (Oseltamivir), indicating that BAY 11-7085 can effectively improve the pathological changes of the lung caused by influenza virus-induced pneumonia.

[0130] Figure 12 As can be seen from the results shown in Figure 6, compared with the model group (DMSO), BAY 11-7085 (BAY) can effectively reduce the pathological score of the lung, and the effect is close to that of three times the amount of the positive control (Oseltamivir), indicating that BAY 11-7085 can effectively improve the pathological changes of the lung caused by influenza virus-induced pneumonia.

[0131] As can be seen from the results shown in Figure 6, compared with the model group (DMSO), BAY 11-7085 (BAY) can effectively reduce the pathological score of the lung, and the effect is close to that of three times the amount of the positive control (Oseltamivir), indicating that BAY 11-7085 can effectively improve the pathological changes of the lung caused by influenza virus-induced pneumonia. Figure 13 ​​The results show that, compared with the model group (infection solvent control group), the expression of anti-inflammatory factors and interferon can be significantly improved after administration of BAY 11-7085, thereby effectively controlling lung inflammation, and the effect is consistent with that of the positive drug oseltamivir at three times the dosage, which shows a surprising effect.

[0132] In addition, the inventors also used a method basically the same as the present embodiment to advance the administration time to the 0th day of the experiment, and the improvement effect of BAY 11-7085 on lung pathological score and pro-inflammatory factors can also be observed, thereby effectively preventing pneumonia.

[0133] Example 4 Treatment effect of BAY 11-7085 on severe viral pneumonia

[0134] The experiment was performed according to the method in Example 2. In the present embodiment, the influenza virus used was the H1N1 influenza virus strain: HK / 415742 / 09-Mut, and the lethal dose of 900xTCID50titer was used to model severe pneumonia mice, and the administration group of BAY 10mg / kg (BAY 11-7085 group or WT+H1N1+BAY group), and the model group was administered with DMSO (solvent control group or WT+H1N1+DMSO group). At the same time, a blank control (dropping the same amount of saline-WT+PBS+Saline group) was set. The number of mice in each group was 10, and the body weight and mortality of mice in each group were observed and recorded every day, and the experimental procedure was as follows Figure 14 .

[0135] The results are shown in Figures 15-16 .

[0136] From Figure 15 and Figure 16 The results show that, in the case of lethal dose of virus infection, the treatment of 10mg / kg BAY 11-7085 inhibitor can effectively improve the weight loss of mice infected with severe pneumonia, and the mortality of mice in the BAY 11-7085 treatment group is reduced, effectively preventing death caused by respiratory virus-induced severe pneumonia infection.

[0137] In summary, influenza virus-induced pneumonia can cause obvious damage and inflammatory response in the lungs of mice, and administration of BAY-117085 can effectively reduce lung inflammation and lung damage in mice, to some extent, the weight loss and disease progression of influenza virus pneumonia in mice can be rescued. Especially in the severe pneumonia mouse model induced by a lethal dose, the treatment of BAY 11-7085 can effectively reduce the number of dead mice, which provides a new idea for the treatment of influenza virus severe pneumonia.

[0138] Example 5 Therapeutic effect of BAY 11-7085 on respiratory syncytial virus (RSV virus)-induced pneumonia

[0139] In this example, the therapeutic effect of BAY 11-7085 on respiratory syncytial virus-induced pneumonia was verified, and the titer of RSV used was (3.5 x 10 5 FFU / mL), and human lung adenocarcinoma cell line A549 was used for the experiment. The specific method is as follows:

[0140] A549 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and plated at 37°C, 5% CO2, to a confluence of 70-80%. The cells were grouped into: uninfected group (negative control), RSV-infected group (RSV), and RSV-infected group treated with BAY 11-7085 (RSV+BAY). RSV strain was used to infect A549 cells at a concentration of MOI = 1, and the infection time was 24 and 36 hours. The RSV-infected group and the BAY 11-7085-treated group were treated with DMSO (RSV-infected group) and 10 μM BAY 11-7085 (treated group) after infection, respectively. At 24 hours and 36 hours after infection, total RNA was collected from the cells in each group, and total RNA was extracted using TRIzol reagent. The concentration of the extracted RNA was quantified, and the A260 / A280 value was measured using a spectrophotometer to ensure the quality of the RNA (A260 / A280 was about 1.8-2.0). The extracted RNA was reverse transcribed using a commercially available reverse transcription kit, and after reverse transcription according to the instructions, the cDNA concentration was determined and the cDNA was stored.

[0141] The mRNA expression levels of the following inflammatory factors were detected using SYBR Green real-time quantitative PCR method: TNF-α (tumor necrosis factor α), IL-6 (interleukin-6), and β-actin was used as an internal reference gene to analyze the relative mRNA expression differences between groups.

[0142] Among them, the primers corresponding to IL-6 are:

[0143] IL-6-F: 5'-ACTCACCTCTTCAGAACGAATTG-3' (SEQ ID NO: 1);

[0144] IL-6-R: 5'-CCATCTTTGGAAGGTTCAGGTTG-3' (SEQ ID NO: 2).

[0145] The primers corresponding to TNF-α are:

[0146] TNF-a-F: 5'-CCTCTCTCTAATCAGCCCTCTG-3' (SEQ ID NO: 3);

[0147] TNF-a-R: 5'-GAGGACCTGGGAGTAGATGAG-3' (SEQ ID NO: 4).

[0148] The results are shown in Table 1. Figures 17-18

[0149] As can be seen from the results, after modeling pneumonia by respiratory syncytial virus infection, the levels of TNF-a (tumor necrosis factor a) and IL-6 (interleukin-6) increased significantly, and after treatment with BAY 11-7085, the levels of TNF-a (tumor necrosis factor a) and IL-6 (interleukin-6) decreased significantly, proving the prophylactic and therapeutic effects of BAY 11-7085 on respiratory syncytial virus pneumonia.

[0150] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.​

Claims

1. Use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating pneumonia. The BAY 11-7085 is (2E)-3-[[4-tert-butylphenyl]sulfonyl]-2-propenenitrile. The pneumonia is viral pneumonia. The virus is influenza virus or respiratory syncytial virus.

2. The use according to claim 1, wherein The influenza virus comprises influenza A virus.

3. Use according to claim 2, characterized in that, The influenza A virus comprises H1, H2, H3, H5, H6, H7, H9, H10 subtypes.

4. Use according to claim 2, characterized in that, The influenza virus comprises H1N1, H2N2, H3N2, H5N1, H7N9 and H9N2.

5. Use according to any one of claims 1 to 4, characterized in that, The pneumonia is severe pneumonia.

6. Use according to any one of claims 1 to 4, characterized in that, The medicament further comprises a pharmaceutically acceptable adjuvant or carrier.

7. Use according to claim 6, characterized in that, The pharmaceutically acceptable adjuvant or carrier comprises at least one of diluent, binder, wetting agent, disintegrant, absorption enhancer, surfactant, flavoring agent, sweetening agent and lubricant.

8. Use according to claim 6, characterized in that, The mass percentage of BAY 11-7085 in the medicament is greater than or equal to 50%, 70% or 90%.

9. Use according to claim 6, characterized in that, The concentration of BAY 11-7085 in the medicament is greater than or equal to 0.1 mg / kg.

10. Use according to claim 1 or 2, characterized in that, The medicament further comprises a second therapeutically active ingredient. The second therapeutically active ingredient is a salt of BAY 11-7085 or other pneumonia treatment drug. The other pneumonia treatment drug comprises antibiotic, antifungal drug, antiviral drug and hormone.

11. Use according to any one of claims 1 to 4, characterized in that, The form of the medicament comprises capsule, tablet, pill, granule, injection or spray.

12. Use according to any one of claims 1 to 4, characterized in that, The subject of the medicament is human.

13. Use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating severe pneumonia caused by influenza A virus and / or respiratory syncytial virus. The BAY 11-7085 is (2E)-3-[[4-tert-butylphenyl]sulfonyl]-2-propenenitrile.

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