Application of BAY 11-7085 in prevention and treatment of pneumonia

Abstract

The invention discloses an application of BAY 11-7085 in prevention and treatment of pneumonia. According to the application disclosed by the invention, an actual administration test of a model mouse shows that the BAY 11-7085 can be used for remarkably reducing the expression of inflammatory factors and the protein content in pulmonary alveolar lavage fluid and reducing the pulmonary pathology score, so that the pneumonia is treated, and pulmonary alveolar protein deposition, pulmonary edema and death caused by the pneumonia are effectively relieved or inhibited.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of BAY 11-7085 in preventing and treating pneumonia. Background Art

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

[0003] Viral pneumonia (also known as viral pneumonia) is a common type of pneumonia in clinical practice, which can progress from local pneumonia to systemic infection, sepsis, septic shock and multi-system dysfunction. With the diversification and complexity of the pathogen spectrum of respiratory infections, the incidence and mortality of severe pneumonia are on the rise. According to statistics, in 2017, there were 50 million cases of influenza-related lower respiratory tract infections worldwide, including 810,000 severe cases and 140,000 deaths. It can be seen that 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 rapidly into severe pneumonia and acute respiratory distress syndrome. Respiratory syncytial virus pneumonia, referred to as syncytial virus pneumonia, is a common interstitial pneumonia that occurs frequently in children. Moderate and severe cases have obvious dyspnea, wheezing, cyanosis of lips and concave signs. A few severe cases may also be complicated by heart failure and respiratory failure, which are more harmful to infants and young children. However, there is still a lack of clear and effective treatment for pneumonia caused by highly pathogenic viruses, especially severe pneumonia. Summary of the invention

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

[0005] The first aspect of the present invention provides the use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating pneumonia.

[0006] In some embodiments of the invention, the pneumonia comprises alveolar protein deposition, pulmonary edema, and death caused by pneumonia.

[0007] In some embodiments of the present invention, the pneumonia includes pneumonia caused by a pathogen.

[0008] In some embodiments of the present invention, the pathogens include bacteria, fungi, viruses, mycoplasmas and chlamydia.

[0009] In some embodiments of the present invention, 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 invention, the viral pneumonia includes alveolar protein deposition, pulmonary edema, and death caused by viral pneumonia.

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

[0012] In some embodiments of the present invention, 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 present invention, the influenza virus includes influenza virus type A (A), type B (B), type C (C) and type D (D).

[0014] In some embodiments of the present invention, the influenza virus is influenza A (A) virus.

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

[0016] In some embodiments of the present invention, the influenza A virus is an influenza A virus of the H1, H2, or H3 subtype.

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

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

[0019] In some embodiments of the invention, the influenza viruses include H1N1, H2N2, H3N2, H5N1, H7N9 and H9N2.

[0020] In some embodiments of the present invention, the influenza virus includes H1N1, H2N2, and H3N2.

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

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

[0023] In some embodiments of the present invention, there is provided a 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 invention, the bacteria include but are not limited to Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa.

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

[0026] In some embodiments of the present invention, the medicament does not contain other therapeutically active ingredients besides BAY 11-7085.

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

[0028] In the present invention, the term "therapeutically active ingredient" also refers to active pharmaceutical ingredients, which refers to any substance or mixture of substances used in the manufacture of drugs, which has pharmacological activity or other direct effects in the diagnosis, treatment, symptom relief, management or prevention of diseases, or can affect the function or structure of the body.

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

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

[0031] In the present invention, the diluent and excipient are used to adjust the density and fluidity of the drug to ensure uniform distribution of the drug, and include but are not limited to starch, dextrin, sucrose, mannitol, lactose, microcrystalline cellulose, and the like.

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

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

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

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

[0036] In the present invention, the surfactant is used to reduce the surface tension of the drug and improve the dispersibility and stability of the drug, including but not limited to hexadecanol, Tween 80, and sodium dodecyl sulfate.

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

[0038] In the present invention, the lubricant is used to reduce the friction during tableting and promote the formation of tablets, including but not limited to talc, calcium stearate, magnesium stearate, silica gel powder and polyethylene glycol.

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

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

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

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

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

[0044] In some embodiments of the present invention, the concentration of BAY 11-7085 in the drug is 0.1 mg / kg or more, 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 invention, the concentration of BAY 11-7085 in the medicament is 1 mg / kg or more.

[0046] In some embodiments of the present invention, the concentration of BAY 11-7085 in the drug is 2 mg / kg or more, 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 invention, the medicament further contains a second therapeutically active ingredient.

[0048] In some embodiments of the present invention, the second therapeutically active ingredient may be included in the drug, for example, the second therapeutically active ingredient may be mixed with BAY 11-7085 or packaged as the same drug by mixing or packaging, or may be packaged independently from the drug by an independent packaging method.

[0049] In some embodiments of the invention, the second therapeutically active ingredient comprises a BAY 11-7085 derivative or other pneumonia therapeutic drug.

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

[0051] Derivatives of BAY 11-7085 include compounds obtained by chemical modification or alteration based on the structure of BAY 11-7085. These derivatives may have similar biological activities or different physicochemical properties, or may have better efficacy or lower toxicity.

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

[0053] In some embodiments of the present invention, examples of the salt of BAY 11-7085 include metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, salts formed with basic or acidic amino acids, and the like. Preferred examples of 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 salts formed with organic bases include salts formed with the following organic bases: trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, and the like. Preferred examples of salts formed with inorganic acids include: salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Preferred examples of the salt with an organic acid 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 salt with a basic amino acid include salts with arginine, lysine, ornithine, and the like. Preferred examples of the salt with an acidic amino acid include salts with aspartic acid, glutamic acid, and the like.

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

[0055] In some embodiments of the present invention, the other pneumonia treatment drugs include antibiotics, antifungal drugs, antiviral drugs and hormones.

[0056] In some embodiments of the present invention, the other pneumonia treatment drugs include penicillins, cephalosporins, carbapenems, macrolides, quinolones, enzyme inhibitors and monocyclic amide drugs.

[0057] In some embodiments of the present invention, the other pneumonia treatment drugs include penicillin, amoxicillin, cefuroxime, cefprozil, cefaclor, ceftriaxone, cefotaxime, cefoperazone / sulbactam, azithromycin, roxithromycin, erythromycin, ofloxacin, levofloxacin, vancomycin, moxifloxacin, teicoplanin, linezolid, oseltamivir, acetaminophen, etc.

[0058] In some embodiments of the present invention, the drug is in the form of an oral preparation, an injection or a spray, such as a capsule, a tablet, a pill, a granule, or an granule.

[0059] In some embodiments of the present invention, the drug is in the form of an injectable dosage form.

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

[0061] In some embodiments of the present invention, 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 invention, when mice are used as subjects, severe pneumonia refers to pneumonia with a mortality rate exceeding 50%.

[0063] When targeting humans, severe pneumonia refers to those who meet 1 of the following major criteria or ≥ 3 of the following minor criteria:

[0064] 1. Main criteria:

[0065] (1) Need endotracheal intubation and mechanical ventilation;

[0066] (2) Septic shock still requires vasoactive drug treatment after active fluid resuscitation.

[0067] 2. Secondary criteria:

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

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

[0070] (3) multilobar infiltration;

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

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

[0073] (6) Systolic blood pressure <90 mmHg requires active fluid resuscitation.

[0074] In some embodiments of the present invention, the drug is administered to animals, including mammals, such as humans.

[0075] In some embodiments of the present invention, when humans are used as the administration subjects, the humans are selected from infants aged 0-6 years, children aged 6-12 years, adolescents aged 12-18 years, and adults over 18 years old (eg, elderly people over 60 years old).

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

[0078] In some embodiments of the present invention, 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 A virus and / or respiratory syncytial virus.

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

[0080] In some embodiments of the present invention, 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, concentration, and subject of BAY 11-7085, are as described in the first aspect.

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

[0082] The present invention discovered for the first time the preventive and therapeutic effects of BAY 11-7085 in pneumonia, especially viral pneumonia and severe pneumonia. It can effectively reduce the expression of inflammatory factors and reduce lung pathology scores, thereby effectively alleviating or inhibiting death caused by severe pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Schematic diagram of the experimental process for modeling infection 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 Schematic diagram of the experimental process of treating mice with influenza virus pneumonia with different concentrations of BAY 11-7085.

[0087] Figure 5 Figure 3. Body weight changes of mice infected with influenza virus pneumonia treated with different concentrations of BAY 11-7085.

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

[0089] Figure 7 Figure 3 Lung pathology scores of mice infected with influenza virus pneumonia treated with different concentrations of BAY 11-7085.

[0090] Figure 8 To investigate the changes in protein concentration in bronchoalveolar lavage fluid of mice infected with influenza virus pneumonia and treated with different concentrations of BAY 11-7085.

[0091] Fig. 9 The changes of inflammatory factors in lung tissue of mice with influenza virus pneumonia treated with different concentrations of BAY 11-7085.

[0092] Fig.10 Schematic diagram of the experimental process of administering BAY 11-7085 and positive control drugs to treat mice with influenza virus pneumonia.

[0093] Fig.11 Figure 3. Body weight changes of mice treated with BAY 11-7085 and positive control drugs in influenza virus pneumonia mice.

[0094] Fig.12 Lung histopathological scores of mice treated with BAY 11-7085 and positive control drugs in influenza virus pneumonia mice.

[0095] Fig.13 The changes in inflammatory factors in mice infected with influenza virus pneumonia treated with BAY 11-7085 and positive control drugs.

[0096] Fig.14 Schematic diagram of the experimental process of administering BAY 11-7085 to treat mice with severe influenza pneumonia.

[0097] Fig.15 Figure 3. Body weight changes of mice treated with BAY 11-7085 and the infected control group.

[0098] Fig.16 To compare the survival time of mice with severe influenza pneumonia treated with BAY 11-7085 and the infected control group.

[0099] Fig.17 To compare the interleukin-6 mRNA levels at different time points in the respiratory syncytial virus pneumonia cell model treated with BAY 11-7085 and the infection control group.

[0100] Fig.18 To compare the TNF-α expression levels at different time points in the respiratory syncytial virus pneumonia cell model treated with BAY 11-7085 and the infection control group. DETAILED DESCRIPTION

[0101] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

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

[0103] In this example, mice were used to conduct infection degree tests at different virus titers. The experimental animals were 8-10 week old C57BL / 6 mice (wild-type mice), specific pathogen-free (SPF grade), purchased from Shanghai Model Organisms Technology Co., Ltd.

[0104] The specific experimental method is:

[0105] After anesthetizing 8-10 week old C57BL / 6 mice with isoflurane airway anesthesia, they were divided into PBS uninfected group, low lethal dose infection group (450TCID50), medium dose infection group (600TCID50) and lethal dose infection group (900TCID50). Each group was instilled with 50 μL of influenza virus (H1N1 influenza A virus strain: HK / 415742 / 09-Mut) through the nasal cavity to obtain influenza virus pneumonia mouse models with different concentration gradients. Through this modeling method, we can determine the degree of viral infection, which is helpful for subsequent drug screening. At the same time, a blank control (instillation of an equal amount of normal saline) was set up. The weight of the mice and the time of survival and death were recorded within 14 days of modeling. The specific experimental process is as follows. Figure 1 shown.

[0106] The weight and survival time of mice infected with the virus Figure 2 and Figure 3 The group with a mortality rate of more than 50% when no drug was administered was taken as the severe group. It can be found from the figure that the mortality rate of mice in the 900×TCID50 infection group was greater than 50%, and the weight loss trend of mice was the most obvious, indicating that 900×TCID50 of H1N1 influenza virus PR8 strain infection can cause severe viral pneumonia in mice, while 450TCID50 of H1NI influenza virus PR8 strain infection can cause viral pneumonia in mice.

[0107] Example 2 Therapeutic effects of different concentrations of BAY 11-7085 on viral pneumonia

[0108] In this example, different concentrations of BAY 11-7085 were used to compare the therapeutic effects in the influenza virus pneumonia mouse model. The experimental animals were 8-9 week old C57BL / 6 mice (wild-type mice), specific pathogen-free (SPF grade), purchased from Shanghai Model Organisms Technology Co., Ltd.

[0109] The method for establishing the influenza virus pneumonia mouse model is the same as in Example 1 above. In this example, the influenza virus used is the H1N1 influenza A virus strain (HK / 415742 / 09-Mut), and the titer used is 450×TCID50 (half tissue culture infection dose, which can be determined by the Reed-Muench method, interpolation method or Karber method). A blank control is also set up (an equal amount of saline is dripped in). On the seventh day after the modeling is completed, some mice in each group are killed and alveolar lavage fluid and lung tissue samples are obtained. The specific experimental process is as follows Figure 4 shown.

[0110] Specifically, the experimental mice were subjected to virus nasal instillation modeling on day 0 of the experiment. The drugs were administered according to the groups on days 1-5 of the experiment. Among them, the experimental group was intraperitoneally injected with different concentrations (2 mg / kg and 10 mg / kg) of BAY 11-7085; the model group was administered with DMSO. There were 10 mice in each group, and the weight of the mice in each group was observed and recorded every day.

[0111] Lung tissue samples from each group were taken out and stained with hematoxylin and eosin, and then pathological scores were calculated.

[0112] The specific method is as follows:

[0113] Each group of lung tissue samples was perfused with 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. The lungs were removed 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 transparentized with xylene. The transparent lung tissue was embedded in paraffin and used after the paraffin hardened. The paraffin-embedded lung tissue was cut into thin slices with a thickness of 3-5 microns using a slicer and attached to a slide. The paraffin sections were placed in xylene and gradient ethanol solutions for dewaxing and rehydration in turn, and hematoxylin-eosin staining was performed. The cells were stained in hematoxylin solution for 5-10 minutes to color the nuclei. The tissue sections were rinsed with tap water and decolorized with hydrochloric acid ethanol for 1-2 seconds after differentiation. They were placed in an alkaline environment (such as ammonia water) to return to blue, the tissue sections were rinsed 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, transparentized with xylene, and then sealed with neutral gum. Pathological scoring was then performed, in which the following points were evaluated: Inflammatory cell infiltration: observe the type and number of inflammatory cells around the alveoli and bronchi; alveolar hemorrhage: evaluate whether there is red blood cell extravasation in the alveolar cavity; interstitial edema: check whether the alveolar septum is thickened and accompanied by edema; hyaline membrane formation: observe whether there is hyaline membrane in the alveoli (one of the characteristics of acute respiratory distress syndrome).

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

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

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

[0117] The results are as follows Figure 5-Figure 9shown.

[0118] Depend on Figure 5 It can be found that after nasogastric administration of influenza virus, the weight of mice in the model group decreased significantly, while after administration of different concentrations of BAY 11-7085, the weight loss was significantly reduced, and the mice in the drug-treated group were able to recover to the weight level before infection in a shorter time, especially the high-dose group had a sustained and significant recovery trend compared with the model group. This shows that the therapeutic effect of BAY 11-7085 can effectively improve the weight loss caused by influenza virus infection as the dose increases.

[0119] Depend on Figure 6 and Figure 7 The results shown show that compared with the model group, the degree of tissue necrosis in the case sections of mice in the BAY-117085 treatment group was lower, the lung tissue pathology score of mice in the BAY-117085 treatment group was lower, and compared with the influenza virus pneumonia modeling group, the lung damage of mice in the BAY-117085 treatment group was lighter and there was a significant difference.

[0120] Depend on Figure 8 and Fig. 9 The results show that the protein content and inflammatory cytokines in the alveolar lavage fluid of mice treated with BAY 11-7085 were significantly lower than those in the influenza virus pneumonia modeling group. This shows 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 Therapeutic and preventive effects of BAY 11-7085 and positive control oseltamivir on viral pneumonia

[0122] In this example, the prevention and prophylactic effects of viral pneumonia were verified by BAY 11-7085 and the positive control oseltamivir administration group. The experimental animals were 8-10 weeks old C57BL / 6 mice (wild-type mice), specific pathogen-free (SPF grade), purchased from Shanghai Model Organisms Technology Co., Ltd.

[0123] The specific experimental method is:

[0124] After 8-10 weeks of C57BL / 6 mice were anesthetized with isoflurane airway anesthesia, 50 μL of non-lethal doses of influenza virus (H1N1 influenza A virus strain: HK / 415742 / 09-Mut) were dripped into the nasal cavity to obtain an influenza virus pneumonia mouse model. At the same time, a blank control group (an equal amount of saline was dripped, PBS group) was set up. On the 7th and 14th days after the modeling was completed, some mice in each group were randomly killed and alveolar lavage fluid and lung tissue samples were obtained. The specific experimental process is as follows. Fig.10shown.

[0125] Specifically, the experimental mice were subjected to virus nasal instillation modeling on day 0 of the experiment. The drugs were administered according to the groups on days 1-5 of the experiment. Among them, the positive group was intraperitoneally injected with 30 mg / kg of oseltamivir (OSEL group), and the experimental group was intraperitoneally injected with 10 mg / kg of BAY 11-7085 (CAS: 196309-76-9, BAY group); the model group was administered with DMSO (DMSO group). The number of mice in each group was 10-12, and the weight of mice in each group was observed and recorded every day.

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

[0127] The supernatant of the lung tissue of experimental mice (obtained by grinding the lung tissue and centrifuging it) was detected using 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] The results are as follows Figure 11-13 shown.

[0129] Depend on Fig.11 It can be found that after nasogastric administration of influenza virus, the weight of infected mice in each group decreased significantly. Among them, the weight loss trend of the drug-administered groups (OSEL group and BAY group) after infection was significantly less than that of the control model group (DMSO), indicating that the drugs in each group can effectively resist the weight loss caused by viral infection. Among them, the weight of mice in the oral oseltamivir group (OSEL group) began to recover on the 9th day after virus inoculation, and the effect of intraperitoneal injection of BAY 11-7085 (BAY group) was basically similar to that of intraperitoneal injection of three times the dose of oseltamivir. The above results show that BAY 11-7085 can effectively improve the weight loss caused by influenza virus infection pneumonia like the known influenza A and B and influenza A and B pneumonia treatment drugs, and even better.

[0130] Depend on Fig.12 The results show that compared with the model group (DMSO), BAY 11-7085 (BAY) can effectively reduce the lung pathology score, and the effect is close to that of the positive control (Oseltamivir) with three times the dosage, indicating that BAY 11-7085 can effectively improve the lung pathological changes manifested in pneumonia caused by influenza virus.

[0131] Depend on Fig.13The results showed that compared with the model group (infection solvent control group), the administration of BAY 11-7085 was able to significantly increase the expression of anti-inflammatory factors and interferon, thereby effectively controlling lung inflammation. The effect was consistent with that of the positive drug oseltamivir at three times the dosage, showing a surprising therapeutic effect.

[0132] In addition, the inventors also adopted a method substantially the same as that of this example, advancing the administration time to day 0 of the experiment, and similarly observed the improvement effect of BAY 11-7085 on lung pathological scores and pro-inflammatory factors, thereby effectively preventing pneumonia.

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

[0134] The experiment was conducted according to the method in Example 2. In this example, the influenza virus used was the strain of H1N1 influenza A virus: HK / 415742 / 09-Mut, and a lethal dose of 900×TCID50 was used to model severe pneumonia in mice, and a BAY 10 mg / kg administration group (BAY 11-7085 group or WT+H1N1+BAY group), and a model group was administered with DMSO (solvent control group or WT+H1N1+DMSO group). A blank control was also set up (an equal amount of saline was dripped in - WT+PBS+Saline group). There were 10 mice in each group, and the weight and mortality rate of mice in each group were observed and recorded every day. The experimental process is as follows: Fig.14 .

[0135] The results are as follows Figure 15-16 shown.

[0136] Depend on Fig.15 and Fig.16 The results showed that in the case of lethal dose virus infection, treatment with 10 mg / kg BAY11-7085 inhibitor can effectively improve the weight loss of mice infected with severe pneumonia, and the mortality rate of mice in the BAY 11-7085 treatment group was reduced, effectively preventing deaths caused by severe pneumonia infection caused by respiratory viruses.

[0137] In summary, influenza virus-induced pneumonia can cause significant damage and inflammation in the lungs of mice, and administration of BAY-117085 can effectively reduce lung inflammation and lung damage in mice, and to a certain extent can save the weight loss and disease progression of influenza virus pneumonia in mice. In particular, in the mouse model of severe pneumonia induced by a lethal dose, treatment with BAY 11-7085 can effectively reduce the number of deaths in mice, providing a new idea for the treatment of severe pneumonia caused by influenza virus.

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

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

[0140] A549 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO. 2 Under the conditions, the plate was cultured to a confluency 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). The RSV virus strain infected 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 10μM BAY 11-7085 (treated group) after infection, respectively. At 24 hours and 36 hours after infection, the total RNA of each group of cells was collected and extracted using TRIzol reagent. The extracted RNA concentration was quantified, and the A260 / A280 value was measured using a spectrophotometer to ensure the quality of the RNA (A260 / A280 was approximately 1.8-2.0). The extracted RNA was reverse transcribed using a commercially available reverse transcription kit. After reverse transcription was completed according to the instruction manual, the cDNA concentration was determined and the cDNA was stored.

[0141] The SYBR Green real-time quantitative PCR method was used to detect the mRNA expression levels of the following inflammatory factors: TNF-α (tumor necrosis factor α), IL-6 (interleukin-6), and β-actin was used as the internal reference gene to analyze the relative mRNA expression differences among the 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-α-F: 5'-CCTCTCTCTAATCAGCCCTCTG-3' (SEQ ID NO: 3);

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

[0148] The results are as follows Figure 17-Figure 18 shown.

[0149] The results show that after respiratory syncytial virus infection modeling pneumonia, the levels of TNF-α (tumor necrosis factor α) and IL-6 (interleukin-6) increased significantly. After treatment with BAY 11-7085, the levels of TNF-α (tumor necrosis factor α) and IL-6 (interleukin-6) decreased significantly, proving the preventive and therapeutic effects of BAY 11-7085 on respiratory syncytial virus pneumonia.

[0150] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of BAY 11-7085 in the preparation of a medicament for preventing and / or treating pneumonia; Preferably, the pneumonia includes pneumonia caused by pathogens; Preferably, the pathogens include bacteria, fungi, viruses, mycoplasmas and chlamydia.

2. The use according to claim 1, characterized in that: The viruses include influenza virus and respiratory syncytial virus; The influenza virus includes influenza A virus; Preferably, the influenza virus includes influenza A viruses of subtypes H1, H2, H3, H5, H6, H7, H9, and H10; Preferably, the influenza viruses include H1N1, H2N2, H3N2, H5N1, H7N9 and H9N2.

3. The use according to claim 1 or 2, characterized in that: The pneumonia is severe pneumonia.

4. The use according to claim 1 or 2, characterized in that: The drug also contains a pharmaceutically acceptable adjuvant or carrier; Preferably, the pharmaceutically acceptable adjuvant or carrier includes at least one of a diluent, an excipient, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a flavoring agent, a sweetener and a lubricant.

5. The use according to claim 1 or 2, characterized in that: The mass percentage of BAY 11-7085 in the drug is greater than or equal to 50%, 70% or 90%.

6. The use according to claim 1 or 2, characterized in that: The concentration of BAY 11-7085 in the drug is 0.1 mg / kg or more.

7. The use according to claim 1 or 2, characterized in that: The drug also contains a second therapeutically active ingredient; The second therapeutically active ingredient includes a BAY 11-7085 derivative or other pneumonia treatment drug; Preferably, the BAY 11-7085 derivatives include salts, esters and amidates of BAY 11-7085; or, the other pneumonia therapeutic drugs include antibiotics, antifungal drugs, antiviral drugs and hormones.

8. The use according to claim 1 or 2, characterized in that: The medicine may be in the form of capsules, tablets, pills, granules, granules, injections or sprays.

9. The use according to claim 1 or 2, characterized in that: The drug is used by humans.

10. 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.

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