Application of Ulixertinib in treating lung tissue inflammation caused by novel coronavirus

Abstract

The invention provides application of Ulixertinib in treatment of lung tissue inflammation caused by novel coronavirus, and the Ulixertinib can effectively relieve the lung inflammatory infiltration degree after infection of the novel coronavirus, reduce the expression level of cytokines, chemotactic factors or NETs related proteins and inhibit formation of NETs. The invention provides a brand new treatment strategy for novel coronavirus infection, especially severe lung tissue inflammation caused by multiple infection of novel coronavirus.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of Ulixertinib in treatment of lung tissue inflammation caused by novel coronavirus. BACKGROUND

[0002] The lung tissue inflammation caused by novel coronavirus is a pandemic caused by coronavirus. China has included it as an acute respiratory infectious disease in the Law of the People's Republic of China on Prevention and Control of Infectious Diseases, and has controlled it as a class B infectious disease according to class A. Since the pandemic of the novel coronavirus, a variety of mutant strains have been produced. The Omicron variant frequently causes breakthrough infections and reinfections due to its rapid antigen mutation. With the emergence of the novel coronavirus variant, the clinical symptoms of viral infection may change, and the continuous emergence of mutants brings new challenges to the research on the spread, prevention and treatment of the novel coronavirus.

[0003] At present, most of the patients infected with the novel coronavirus have relatively mild clinical symptoms, and often show fever, cough or sore throat. Most of the infected persons will spontaneously relieve symptoms within a week. However, a part of the patients infected with the novel coronavirus (including patients with reinfection of the novel coronavirus) will further develop into severe or critical cases as the disease worsens. The severe lung tissue inflammation caused by the novel coronavirus leads to restrictive ventilation dysfunction, impaired gas exchange and severe respiratory failure due to extensive damage to alveoli and destruction of lung structure, resulting in high mortality. However, there is still a lack of drugs that can effectively treat severe and critical lung tissue inflammation caused by the novel coronavirus. SUMMARY

[0004] In view of this, in order to make up for the shortcomings of the prior art, the present application is proposed.

[0005] The first aspect of the present application provides the application of any one of the following:

[0006] (1) The application of an ERK1 / 2 signaling pathway inhibitor in the preparation of a drug for treating and / or preventing novel coronavirus infection;

[0007] (2) The application of an ERK1 / 2 signaling pathway inhibitor in the preparation of a drug for treating and / or preventing diseases or conditions caused by novel coronavirus infection.

[0008] In the present invention, the term "treatment and / or prevention" can refer to therapeutic treatment or prophylactic measures, wherein the goal is to prevent or slow down (alleviate) an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable. Treatment can include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival without treatment.

[0009] In the present invention, the ERK1 / 2 is extracellular signal-regulated kinase 1 / 2 (ERK1 / 2).

[0010] In some embodiments, the ERK1 / 2 signaling pathway inhibitor includes, but is not limited to, a small nucleic acid drug, a ribozyme, a small molecule compound, a peptide, a peptide mimetic, a matrix analog, and / or an aptamer.

[0011] In some embodiments, the small nucleic acid drug includes, but is not limited to, an antisense oligonucleotide, an miRNA, an siRNA, an shRNA.

[0012] In the present invention, an antisense nucleotide refers to a sequence that interferes with the flow of genetic information from DNA to protein by binding (hybridizing) to the complementary nucleotide sequence of DNA, immature mRNA, or mature mRNA. In addition, since the antisense nucleotide is a long chain of monomer units, it can be easily synthesized to target the RNA sequence.

[0013] In the present invention, the siRNA refers to a short double-stranded RNA that can induce RNA interference by cleaving a specific mRNA. In addition, the siRNA is not limited to the region where the double-stranded RNA is completely paired, but can include a region where the strands are not paired due to a mismatch (corresponding nucleotides are not complementary) or a bulge (there are no nucleotides corresponding to one strand). The siRNA end structure can have a blunt end or a overhanging end, as long as the expression of the target gene can be inhibited by the RNA interference effect, and the adhering end structure can be a 3' end overhanging structure and a 5' end overhanging structure.

[0014] In the present invention, the shRNA refers to an RNA sequence that produces a strong hairpin bend, which can be used to silence gene expression by RNA interference. In addition, the shRNA can be delivered into cells using a vector for cell introduction, and such a vector is always delivered into daughter cells, so that gene silencing can be inherited. The shRNA hairpin structure is degraded into siRNA by intracellular mechanisms and binds to the RNA-induced silencing complex, which binds to the mRNA corresponding to the siRNA to degrade it.

[0015] In the present invention, the ribozyme refers to an enzyme RNA molecule capable of catalyzing specific RNA cleavage. The specific hybridization of the molecular sequence of the ribozyme and the complementary target RNA can induce endonucleolytic cleavage. The ribozyme can include known sequences responsible for cleaving one or more sequences complementary to the target RNA or functionally equivalent sequences. In addition, the ribozyme can be a hammerhead ribozyme or a Cech-type ribozyme, i.e., a ribonucleic acid endonuclease RNA, and can be formed from a modified oligonucleotide to improve safety and targeting. At the same time, the ribozyme can be distributed in cells in which the target gene is expressed in vivo. A DNA construct encoding the ribozyme under the control of a strong constitutive polymerase III or polymerase II promoter can be used, so that the transfected cells can destroy the endogenous target messenger and produce a sufficient amount of ribozyme to inhibit translation. Since the ribozyme has catalytic activity, unlike other antisense molecules, it can have to be maintained at a low concentration in the cell.

[0016] In the present invention, the small molecule compound includes, but is not limited to, a compound that can bind to the ERK1 / 2 kinase domain, a compound that can prevent the substrate from binding to ERK1 / 2, and a compound that directly inhibits the activity of ERK1 / 2.

[0017] In some embodiments, the small molecule compound includes, but is not limited to, Ulixertinib, ASN007, GDC-0994, MK-8353, LY3214996, KO-947, LTT462, HRS2543, HMPL-295S1, AZD0364, HH2710, JSI-1187.

[0018] In some embodiments, the small molecule compound is selected from Ulixertinib.

[0019] In the present invention, Ulixertinib can be Ulixertinib itself (also referred to as a compound below), and can also be a pharmaceutically acceptable salt, hydrate, solvate, or crystalline form of Ulixertinib.

[0020] In the present application, the pharmaceutically acceptable salt refers to acid salt formed with inorganic acid and / or organic acid. The pharmaceutically acceptable salt of the compound can be formed, for example, by reacting the compound with an amount of acid in a medium, for example, a medium in which the salt is precipitated or an aqueous medium (lyophilized after reaction).

[0021] Specific pharmaceutically acceptable salts include those which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and / or organic acids.

[0022] Examples of pharmaceutically acceptable salts are salts of inorganic acids, for example hydrochloric, hydrobromic, phosphoric, sulfuric or perchloric acid, or of organic acids, for example acetic, oxalic, maleic, tartaric, citric, succinic or malonic acid. Salts formed with conventional methods, for example ion exchange methods, are also included. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate.

[0023] In the present application, the hydrate refers to a compound combined with water.

[0024] In the present application, the solvate refers to a form of a compound or its salt combined with a solvent, usually formed by solvolysis reaction. This physical association can include hydrogen bonding. Conventional solvents include methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. In some cases, the solvate will be capable of isolation, for example, where one or more solvent molecules are incorporated in the crystal lattice of the solid state form of the compound. Solvates include both solution and isolated solvates.

[0025] In the present application, the crystalline form refers to a crystalline form of a compound with a specific crystal packing arrangement. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. The recrystallization solvent, crystallization rate, storage temperature, and other factors can lead to one crystalline form to be dominant. Various polymorphs of a compound can be prepared by crystallization under different conditions. In the present application, the crystalline form also includes special crystal state, such as amorphous, etc.

[0026] In the present invention, peptides and peptide mimetics can inhibit the activity of ERK1 / 2 protein by inhibiting the binding of ERK1 / 2 protein to other proteins. Non-hydrolyzable peptide mimetics can be prepared using a β-turn dipeptide core, a keto methylene pseudopeptide, an azepine, a benzodiazepine, a β-amino alcohol, or a substituted γ-lactam ring as the main residue.

[0027] In some embodiments, the peptide comprises an antibody.

[0028] In the present invention, the antibody can be a monoclonal antibody, a polyclonal antibody or a recombinant antibody and an antigen-binding fragment. If the sequence of the protein is known, antibodies against a specific protein can be easily prepared using techniques well known in the art. In particular, antibodies can be prepared using hybridoma methods or phage antibody library technology. Typically, hybridoma cells secreting monoclonal antibodies can be prepared by fusing immune cells separated from an immunologically suitable host animal (such as a mouse) injected with an antigen protein with a cancer cell line. The fusion of these two groups of cells can be carried out using polyethylene glycol, and the cells producing the antibody can be propagated by standard culture methods. After obtaining a homogeneous cell population by using a limiting dilution method subclone, hybridoma cells capable of producing antigen-specific antibodies can be prepared by culturing in vitro or in vivo. The antibodies prepared by the above method can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography and affinity chromatography.

[0029] Polyclonal antibodies can be prepared by injecting a biomarker protein or a fragment thereof as an immunogen into an external host. The external host can be a mammal, such as a mouse, rat, sheep, or rabbit. When the immunogen is injected intramuscularly, intraperitoneally, or subcutaneously, it can be administered with an adjuvant to increase antigenicity. Thereafter, blood is collected regularly from the external host to obtain serum that shows increased titer and specificity for the antigen, from which antibodies can be isolated and purified.

[0030] A "monoclonal" antibody or antigen-binding fragment refers to a population of homogeneous antibodies or antigen-binding fragments that are involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment encompasses intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecules comprising an antigen recognition site.

[0031] In the present application, the aptamer refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) which has a stable tertiary structure by itself and can bind to a target molecule with high affinity and specificity. Since the aptamer can bind to organic matter, peptide, membrane protein, etc. and block its function, it can be regarded as a chemical antibody that replaces a single antibody. In addition, the aptamer can be obtained by using a library of oligonucleotides called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to isolate oligomers that bind to specific chemical molecules or biological molecules with high affinity and selectivity.

[0032] In some embodiments, the novel coronavirus infection includes a first infection with a novel coronavirus.

[0033] In some embodiments, the novel coronavirus infection includes multiple infections with a novel coronavirus after a first infection with a novel coronavirus.

[0034] In some embodiments, the number of multiple times is not limited, and can be two, three, four, or more.

[0035] In some embodiments, the number of multiple times is selected from two.

[0036] In some embodiments, the disease or disorder caused by the novel coronavirus infection includes a disease or disorder caused by multiple infections with a novel coronavirus after a first infection with a novel coronavirus.

[0037] In some embodiments, the first infection with a novel coronavirus and the multiple infections with a novel coronavirus belong to different lineages of the same variant or different variants.

[0038] In some embodiments, the first infection with a novel coronavirus and the multiple infections with a novel coronavirus belong to different lineages of the same variant.

[0039] In some embodiments, the variant includes but is not limited to the Alpha variant, the Beta variant, the Gamma variant, the Delta variant, the Omicron variant, other variants.

[0040] In some embodiments, the variant is selected from the Omicron variant.

[0041] In some embodiments, the Omicron variant includes but is not limited to BA.1, BA.2, BA.3, BA.4, BA.5, or a descendant lineage thereof.

[0042] In some embodiments, the BA.2 descendant lineage includes XBB.

[0043] In some embodiments, the Omicron variant is selected from BA.5, XBB.

[0044] In some embodiments, the first infection of SARS-CoV-2 is selected from BA.5.

[0045] In some embodiments, the multiple infection of SARS-CoV-2 is selected from XBB.

[0046] In some embodiments, the disease or disorder caused by the SARS-CoV-2 infection is selected from one or more of the following: respiratory infection, acute respiratory syndrome, lung tissue inflammation, gastroenteritis, cough, fever, chills, vomiting, headache, chills, shortness of breath, cytokine storm.

[0047] In some embodiments, the disease or disorder caused by the SARS-CoV-2 infection is selected from lung tissue inflammation.

[0048] In some embodiments, the lung tissue inflammation is severe lung tissue inflammation caused by SARS-CoV-2.

[0049] In some embodiments, the severe lung tissue inflammation caused by SARS-CoV-2 exhibits one or several of the following symptoms compared to mild lung tissue inflammation caused by SARS-CoV-2: increased degree of inflammatory infiltration in the lungs, increased cytokines, increased chemokines, increased NETs formation, increased expression level of NETs-related proteins.

[0050] In some embodiments, the NETs-related proteins include, but are not limited to, citH3, NE, MPO-DNA, PAD4.

[0051] The second aspect of the present application provides a pharmaceutical composition for treating and / or preventing SARS-CoV-2 infection or a disease or disorder caused by the infection, the pharmaceutical composition being selected from the ERK1 / 2 signaling pathway inhibitor described in the first aspect of the present application.

[0052] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0053] The pharmaceutically acceptable carrier and / or adjuvant according to the present application includes any substance suitable for use in humans and / or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergic reaction), i.e., with a reasonable benefit / risk ratio. The pharmaceutically acceptable carrier and / or adjuvant is used as necessary to aid formulation stability or to help improve its activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration. The pharmaceutical composition thus formulated can administer the drug according to any appropriate administration mode known to those skilled in the art as necessary, and the pharmaceutical composition is used by administering a safe and appropriate dose of the pharmaceutical composition according to the present application to a subject.

[0054] Further, the pharmaceutically acceptable carrier and / or adjuvant includes, but is not limited to, a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, and / or a disintegrant. Among them, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, water; the binder includes, but is not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginic acid salt, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; the surfactant includes, but is not limited to, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, stearic acid monoglyceride, cetyl alcohol; the wetting agent includes, but is not limited to, glycerol, starch; the adsorption carrier includes, but is not limited to, starch, lactose, bentonite, silica gel, kaolin, soap clay; the lubricant includes, but is not limited to, zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauryl sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate.

[0055] Further, the pharmaceutical composition can be administered in any convenient pharmaceutical dosage form.

[0056] Further, the dosage form includes, but is not limited to, a gastrointestinal administration dosage form, a non-gastrointestinal administration dosage form.

[0057] Further, the gastrointestinal administration dosage form includes, but is not limited to, a solution, a drop, a tablet, a capsule, a granule, a film, a gel, a powder, an emulsion, a suspension, a drop pill, a suppository, an aerosol, a spray, a powder spray, a patch, an ointment, or a cream.

[0058] Further, the non-gastrointestinal administration dosage form includes, but is not limited to, an injection administration dosage form, a respiratory administration dosage form, a cavity administration dosage form, a mucosal administration dosage form, a skin administration dosage form.

[0059] Further, the injection administration forms include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and intracavity injection, etc.; the respiratory administration forms include, but are not limited to, spray, aerosol, powder spray, etc.; the cavity administration forms include, but are not limited to, suppository, aerosol, effervescent tablet, drop, drop pill, etc., for rectum, vagina, urethra, nasal cavity, ear canal, etc.; the mucous membrane administration forms include, but are not limited to, eye drops, nose drops, eye ointment, gargle, sublingual tablet, sticking tablet, film, etc.; the skin administration forms include, but are not limited to, external solution, lotion, liniment, ointment, plaster, paste, patch, etc.

[0060] In the present application, the pharmaceutical composition is administered to the patient in a pharmaceutically effective dose. "Pharmaceutically effective dose" means a dose sufficient to produce the desired effect for the condition it is administered. The exact dose depends on the activity of the compound, the mode of administration, the nature and severity of the condition, the age and weight of the patient, and different doses can be required. The dose administration can be implemented by a single administration in the form of individual dose units (otherwise, several smaller dose units) and also by multiple administrations of subdivided doses at specific time intervals.

[0061] In some embodiments, the pharmaceutical composition can be administered before infection, or after infection.

[0062] When the infection is more than once, the pharmaceutical composition can be administered before the first infection, after the first infection, before multiple infections, or after multiple infections, or before the first infection, after the first infection, before multiple infections, and after multiple infections.

[0063] When the infection is more than twice, the pharmaceutical composition can also be administered between multiple infections and other multiple infections.

[0064] In some embodiments, the pharmaceutical composition is administered immediately after the first infection of the new coronavirus.

[0065] In some embodiments, the pharmaceutical composition is administered 1-29 days after the first infection of the new coronavirus.

[0066] In some embodiments, the pharmaceutical composition is administered 29 days after the first infection.

[0067] In some embodiments, the pharmaceutical composition is administered in a pharmaceutically effective amount.

[0068] The third aspect of the present application provides any one of the following uses:

[0069] (1) The use of ulixertinib in the preparation of a drug for reducing the degree of inflammatory infiltration in the lung;

[0070] (2) Use of ulixertinib in the preparation of a drug for reducing the expression level of a cytokine or chemokine;

[0071] In some embodiments, the cytokine comprises IL-1β, TNF-α;

[0072] In some embodiments, the chemokine comprises CCL2, CCL3, CCL4, CCL7, CXCL1, CXCL10;

[0073] (3) Use of ulixertinib in the preparation of a drug for inhibiting the formation of NETs;

[0074] (4) Use of ulixertinib in the preparation of a drug for reducing the expression level of a NETs-related protein.

[0075] In some embodiments, the NETs-related protein comprises citH3, NE, MPO-DNA, PAD4.

[0076] The fourth aspect of the present application provides any one of the following methods:

[0077] (1) A method for reducing the expression level of a cytokine or chemokine in vitro, the method comprising administering ulixertinib;

[0078] (2) A method for inhibiting the formation of NETs in vitro, the method comprising administering ulixertinib;

[0079] (3) A method for reducing the expression level of a NETs-related protein in vitro, the method comprising administering ulixertinib.

[0080] The fifth aspect of the present application provides a method for treating and / or preventing a novel coronavirus infection or a disease or disorder caused by the infection, the method comprising administering ulixertinib.

[0081] The present application has the advantages and beneficial effects:

[0082] The present application provides the use of ulixertinib in the treatment of lung tissue inflammation caused by a novel coronavirus, which can effectively reduce the degree of pulmonary inflammatory infiltration after a novel coronavirus infection, reduce the expression level of a cytokine, a chemokine or a NETs-related protein, and inhibit the formation of NETs. The present application provides a novel treatment strategy for novel coronavirus infection, especially severe lung tissue inflammation caused by multiple infections of a novel coronavirus. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1Figure 6 is a graph of the results of the degree of inflammatory infiltration in the lungs of mice;

[0084] Figure 2 Figure 7 is a graph of the expression levels of cytokines IL-1β, TNF-α, chemokines CCL2, CCL3, CCL4, CCL7, CXCL1, and CXCL10;

[0085] Figure 3 Figure 8 is a graph of the results of NETs formation;

[0086] Figure 4 Figure 9 is a graph of the expression levels of NETs-related proteins. DETAILED DESCRIPTION

[0087] The present application is further illustrated by the following examples. The following description is merely exemplary in nature and is not intended to limit the present application, as described, any modifications that come within the scope of the present application are intended to be within the scope of the present application. Any skilled person, based in the technical content disclosed above, may make any simple modification or equivalent change to the following examples, as long as it does not deviate from the technical scheme of the present application.

[0088] Example 1

[0089] 1. Experimental materials

[0090] 1.1 Experimental virus

[0091] BA.5 strain: SARS-CoV-2 / human / CHN / GD-5 / 2022 (Gene Bank: OP678016) Omicron XBB.1 (Genbank ID: PQ312715).

[0092] 1.2 Experimental animals and cells

[0093] 6-8-week-old female BALB / c mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0094] 1.3 Experimental instruments

[0095] Immunofluorescence microscope (Leica, USA), cell counter (DeNOVIX, USA), centrifuge (Eppendorf, Germany).

[0096] 1.4 Experimental reagents and consumables

[0097] Ulixertinib hydrochloride (MCE, HY-15816), antibody diluent (Abeam, abs9299), four-color multiplex fluorescent immunohistochemical staining kit (Abeam, abs50028) cith3 ELISA detection kit (citrullinated histone 3, Shanghai Keaibo Company, CB11403-Mu), NE (neutrophil elastase, Shanghai Keaibo Company, CB10847-Mu), PAD4 ELISA detection kit (peptidyl arginine deiminase 4, Shanghai Keaibo Company, CB11404-Mu), MPO-DNA ELISA detection kit (myeloperoxidase-DNA complex, Shanghai Keaibo Company, CB14718-Mu), LEGENDplex™ Proinflammatory Chemokine Panel (13-plex) (Mouse) (Biolegend, 740446), EPX360-26092-901 MO CYTO / CHEMO PANEL 1A kit (ThermoFisher Scientific, EPX360-26092-901).

[0098] 2. Experimental methods

[0099] Animal experiments:

[0100] In the presence of initial infection of Omicron BA.5 strain, XBB variant re-infection induced more severe inflammatory response, leading to aggravated pathological damage of lung tissue. In terms of pathological manifestations, significant body weight loss, increased neutrophil infiltration in lung tissue, phenotype of aggravated inflammatory infiltration in lung tissue with abnormal deposition of citH3 / MPO-DNA, excessive release of NETs and aggravated lung injury could be observed. Therefore, the mouse model infected with BA.5 first and XBB strain second was selected as the model of severe lung tissue inflammation caused by novel coronavirus to study the therapeutic effect of drugs.

[0101] 6-8 week-old female BALB / c mice (n=5 per group) were anesthetized and inoculated with 50 μL (10 5 TCID 50) BA.5 virus. Each mouse was monitored daily for signs of disease, body weight trends until 29 dpi, the Ulixertinib group was injected intraperitoneally with Ulixertinib hydrochloride (MCE, HY-15816) 40 mg / kg, 2 h later, the mice were infected again with the XBB strain (the same amount as the BA.5 virus), and 3 dpi, each group of mice was executed by cervical dislocation, and fresh lung tissue was collected for subsequent analysis of neutrophil trap (NETs) related indicators such as citH3, MPO-DNA complex, etc. The Model group was the same as the Ulixertinib group except that it was not injected with Ulixertinib hydrochloride.

[0102] All experimental operations related to infection with the novel coronavirus (SARS-CoV-2 virus) in this study were carried out in ABSL-3. Animal experiments were reviewed by the Animal Ethics Committee, and the approval number was BLL23007.

[0103] Immunofluorescence staining

[0104] 1) The sections were soaked in xylene I / II for 10 minutes each; gradient ethanol (100% — 95% — 80% — 70%) was hydrated for 5 minutes each, and PBS was washed for 5 minutes x 3 times;

[0105] 2) The sections were immersed in sodium citrate buffer (pH 6.0) and heated at 95°C for 20 minutes, and then naturally cooled to room temperature;

[0106] 3) 0.5% Triton X-100 (prepared with PBS) was permeated for 10 minutes, PBS was washed for 5 minutes x 3 times, and 5% goat serum (prepared with PBS) was blocked at room temperature for 60 minutes;

[0107] 4) Diluted primary antibodies were added, MPO (abcam, ab134132, 1; 1000), citH3 (abcam, ab281584, 1; 2000), PAD4 (abcam, ab96758, 1:1000), and the wet box was incubated at 4°C overnight;

[0108] 5) The next day, it was warmed at room temperature for 45 minutes, PBS was washed for 5 minutes x 5 times, and the corresponding species fluorescent secondary antibody (such as Alexa Fluor 488 / 594 labeled) was added, and incubated at 37°C for 60 minutes., PBS was washed for 5 minutes x 5 times;

[0109] 6) DAPI (1 μg / mL) was added to stain the nucleus for 10 minutes, and PBS was washed;

[0110] 7) Anti-fluorescence quenching mounting agent was mounted, and dried at room temperature in the dark;

[0111] 8) Images were acquired using a fluorescence microscope (Leica DMi8) under 20x / 40x objective.

[0112] ELISA

[0113] 1) Bronchoalveolar lavage fluid samples were centrifuged at 12,000 x g for 15 min at 4°C, and the supernatant was collected for use;

[0114] 2) The standard / sample supernatant was added to the pre-coated 96-well plate according to the kit requirements, 100 μL per well, and incubated at 37°C for 60 min. After discarding the liquid, the wells were washed 5 times with washing buffer (PBS containing 0.05% Tween-20);

[0115] 3) 100 μL of biotinylated detection antibody was added to each well, and incubated at 37°C for 60 min, and washed 5 times;

[0116] 4) 100 μL of horseradish peroxidase (HRP)-labeled streptavidin was added, and incubated at 37°C for 30 min in the dark, and washed 5 times;

[0117] 5) 100 μL of TMB substrate solution was added, and developed at room temperature in the dark for 15 min, and the reaction was stopped by adding 50 μL of 2M H2SO4;

[0118] 6) The absorbance (OD value) was measured at 450 nm using a microplate reader.

[0119] Cytokine detection

[0120] The mouse bronchoalveolar lavage fluid sample was collected and centrifuged at 10,000 rpm at 4°C for 20 min, and 30 μL of supernatant was prepared for sample preparation.

[0121] 35 μL of the treated sample (diluted serum or lung sample) was taken, and LEGENDplex™ magnetic capture beads (corresponding to Proinflammatory Chemokine or Inflammation Panel) were added. Incubate at room temperature in the dark for 2 hours (the specific time is according to the kit instructions), and shake well to ensure complete binding.

[0122] Use a magnetic stand to separate the capture beads, and discard the supernatant. Add an appropriate amount of washing buffer, and gently resuspend the magnetic beads. Repeat the washing 2-3 times (according to the kit requirements).

[0123] Add biotin-labeled detection antibody, and incubate at room temperature in the dark for 1 hour, and shake well to mix. After removing the unbound antibody by washing, add streptavidin-phycoerythrin (SA-PE), and incubate at room temperature in the dark for 30 min, and shake well to mix.

[0124] Wash to remove unbound SA-PE, resuspend the magnetic beads with buffer, and transfer to flow tube. Detect PE fluorescence signal on Beckman Cytoflex flow cytometer, and record signal intensity of each cytokine.

[0125] Synchronously detect gradient dilution standard, generate standard curve. Compare sample signal intensity with standard curve, and quantitatively calculate concentration of each cytokine (unit: pg / ml).

[0126] Use LEGENDplex™ data analysis software to automatically process flow data.

[0127] 3. Experimental results

[0128] Ulixertinib (Ulixertinib) reduces the inflammation of the severe lung tissue caused by reinfection by blocking the Syk-ERK1 / 2-PAD4 signal axis.

[0129] Two hours before reinfection, Ulixertinib was injected intraperitoneally to block ERK1 / 2, and the intervention effect of Ulixertinib on the pathological damage of the severe lung tissue caused by BA.5-XBB reinfection was evaluated. As shown in the results Figures 1-4 , compared with the Model (BA.5-XBB) group, the degree of inflammatory infiltration in the lungs of mice treated with Ulixertinib inhibitor was significantly reduced Figure 1 ). The expression levels of cytokines IL-1β, TNF-α, chemokines CCL2, CCL3, CCL4, CCL7, CXCL1, and CXCL10 in the Ulixertinib treatment group were significantly down-regulated Figure 2 ). MPO and citH3 are direct morphological markers of NETs, and their secretion is positively correlated with the degree of lung tissue inflammatory damage. We used immunofluorescence staining to co-localize MPO+citH3+ double-positive cells in lung tissue, and found that the number of MPO+citH3+ co-localized positive cells in the Ulixertinib treatment group decreased by 87.67%, indicating that Ulixertinib treatment can effectively inhibit the formation of NETs Figure 3 ). ELISA detection results showed that Ulixertinib treatment significantly reduced the expression levels of four key NETs-related proteins (citH3, NE, MPO-DNA complex, and PAD4) in lung tissue Figure 4 ). The above results show that Ulixertinib treatment can effectively inhibit the formation of NETs and alleviate the lung tissue inflammation induced by BA.5-XBB reinfection.

[0130] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. Any of the following applications: (1) Use of ERK1 / 2 signaling pathway inhibitors in the preparation of drugs for treating and / or preventing novel coronavirus infection; (2) The use of ERK1 / 2 signaling pathway inhibitors in the preparation of drugs for treating and / or preventing diseases or symptoms caused by novel coronavirus infection.

2. The use according to claim 1, characterized in that The ERK1 / 2 signaling pathway inhibitors include small nucleic acid drugs, ribozymes, small molecule compounds, peptides, peptide mimetics, matrix analogs and / or aptamers.

3. The use according to claim 2, characterized in that The small molecule compounds include Ulixertinib, ASN007, GDC-0994, MK-8353, LY3214996, KO-947, LTT462, HRS2543, HMPL-295S1, AZD0364, HH2710, and JSI-1187; Preferably, the small molecule compound is selected from Ulixertinib or a pharmaceutically acceptable salt, hydrate, solvate or crystalline form thereof; Preferably, the pharmaceutically acceptable salt of Ulixertinib includes an acid salt formed with an inorganic acid and / or an organic acid; Preferably, the inorganic acid comprises hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid or perchloric acid; Preferably, the organic acid comprises acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid; Preferably, the solvent of the solvate includes methanol, ethanol, acetic acid, dimethyl sulfoxide, tetrahydrofuran, and diethyl ether.

4. The use according to claim 1, characterized in that The novel coronavirus infection includes the first infection with the novel coronavirus; Preferably, the novel coronavirus infection includes multiple infections with the novel coronavirus after the first infection.

5. The use according to claim 4, characterized in that The novel coronavirus that is infected for the first time and the novel coronavirus that is infected multiple times belong to different lineages or different variants of the same variant; Preferably, the novel coronavirus that is infected for the first time and the novel coronavirus that is infected multiple times belong to different lineages of the same variant strain; Preferably, the variants include Alpha variants, Beta variants, Gamma variants, Delta variants, Omicron variants, and other variants; Preferably, the variant is selected from the Omicron variant; Preferably, the Omicron variants include BA.1, BA.2, BA.3, BA.4, BA.5 or their descendant lineages; Preferably, the BA.2 progeny lineage comprises XBB; Preferably, the Omicron variant is selected from BA.5 and XBB; Preferably, the novel coronavirus that causes the first infection is selected from BA.5; Preferably, the novel coronavirus that is infected multiple times is selected from XBB.

6. The use according to any one of claims 1 to 5, characterized in that The disease or condition caused by the novel coronavirus infection is selected from one or more of the following: respiratory system infection, acute respiratory syndrome, lung tissue inflammation, gastroenteritis, cough, fever, chills, vomiting, headache, chills, shortness of breath, and cytokine storm; Preferably, the disease or condition caused by the novel coronavirus infection is selected from lung tissue inflammation; Preferably, the lung tissue inflammation is severe lung tissue inflammation caused by the new coronavirus; Preferably, the severe lung tissue inflammation caused by the novel coronavirus exhibits one or more of the following symptoms compared to the mild lung tissue inflammation caused by the novel coronavirus: increased degree of lung inflammatory infiltration, increased cytokines, increased chemokines, increased NETs formation, and increased expression levels of NETs-related proteins; Preferably, the NETs-related proteins include citH3, NE, MPO-DNA, and PAD4.

7. A pharmaceutical composition for treating and / or preventing novel coronavirus infection or diseases or conditions caused by infection thereof, characterized in that: The pharmaceutical composition is selected from the ERK1 / 2 signaling pathway inhibitor according to any one of claims 1 to 3.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

9. Any of the following applications: (1) Use of Ulixertinib in the preparation of a drug for reducing the degree of pulmonary inflammatory infiltration; (2) Use of Ulixertinib in the preparation of drugs for reducing the expression level of cytokines or chemokines; Preferably, the cytokines include IL-1β and TNF-α; Preferably, the chemokines include CCL2, CCL3, CCL4, CCL7, CXCL1, and CXCL10; (3) Application of Ulixertinib in the preparation of drugs for inhibiting NETs formation; (4) The use of Ulixertinib in the preparation of drugs for reducing the expression level of NETs-related proteins; Preferably, the NETs-related proteins include citH3, NE, MPO-DNA, and PAD4.

10. Any of the following methods: (1) A method for reducing the expression level of cytokines or chemokines in vitro, characterized in that: The method comprises administering Ulixertinib; (2) A method for inhibiting NETs formation in vitro, characterized in that the method comprises administering Ulixertinib; (3) A method for reducing the expression level of NETs-related proteins in vitro, characterized in that the method comprises administering Ulixertinib.

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