Application of composition in preparation of medicine for preventing and / or treating lung diseases

Abstract

The invention discloses application of a composition in preparation of a medicine for preventing and / or treating lung diseases. The composition comprises a substance capable of activating or enhancing an IL-4 / IL-4RA signal channel, lung diseases comprise pulmonary arterial hypertension, idiopathic pulmonary fibrosis and asthma, and by activating or enhancing the specific signal channel IL-4 / IL-4RA, the pulmonary arterial pressure can be greatly reduced, vascular remodeling can be reduced, and inflammatory response can be inhibited, so that the lung diseases can be effectively prevented and / or treated, and the lung diseases can be effectively prevented and treated. Especially pulmonary arterial hypertension.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and particularly relates to the application of a composition in the preparation of a drug for preventing and / or treating lung diseases. Background Art

[0002] Pulmonary Hypertension (PAH) is a malignant pulmonary vascular disease with high morbidity and mortality. Clinically, it is divided into 5 categories, characterized by elevated pulmonary artery blood pressure, which ultimately leads to right heart failure. The pathogenesis of PAH is complex, involving multiple aspects such as pulmonary vascular smooth muscle cell proliferation, endothelial dysfunction, and immune inflammatory responses. Current treatment methods mainly include vasodilators, endothelin receptor type A (RA) antagonists, phosphodiesterase 5 inhibitors, etc. These drugs can relieve symptoms and improve hemodynamics to a certain extent, but they usually cannot fundamentally change the course of the disease, and long-term use may cause certain side effects. Summary of the Invention

[0003] An embodiment of this application provides the application of a composition in the preparation of a drug for preventing and / or treating lung diseases, which can effectively treat lung diseases.

[0004] In a first aspect, this application provides the application of a composition in the preparation of a drug for preventing and / or treating lung diseases. The composition includes a substance that can activate or enhance the IL-4 / IL-4RA signaling pathway; the lung diseases include pulmonary hypertension, idiopathic pulmonary fibrosis, and asthma.

[0005] In any embodiment of this application, the substance that can activate or enhance the IL-4 / IL-4RA signaling pathway includes at least one of the following: (1) IL-4 protein; (2) a substance that can increase the activity and / or content of IL-4 protein; (3) a receptor agonist of IL-4 protein; (4) a substance that can increase the activity and / or content of the receptor agonist of IL-4 protein.

[0006] In any embodiment of this application, the IL-4 protein includes at least one of IL-4 recombinant protein, IL-4 protein truncation, IL-4 protein mutant, and IL-4 fusion protein.

[0007] In any embodiment of this application, the IL-4 fusion protein includes at least one of IL-4-Fc fusion protein, IL-4-anti-inflammatory factor fusion protein, and IL-4-targeting peptide fusion protein.

[0008] In any embodiment of the present application, the substances that can increase the activity and / or content of IL-4 protein include at least one of proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, polypeptides, peptide mimetics, nucleic acid molecules, and small molecule compounds that can increase the activity and / or content of IL-4 protein.

[0009] In any embodiment of the present application, the substances that can increase the activity and / or content of IL-4 protein include at least one of gene delivery vectors, protein delivery vectors, nucleic acid molecules, proteins, and fusion proteins that can increase the activity and / or content of IL-4 protein; preferably, the substances that can increase the activity and / or content of IL-4 protein include at least one of vectors expressing IL-4 protein, mRNA nucleic acid molecules encoding IL-4 protein, lipid nanoparticles carrying the IL-4 gene, viral vectors carrying the IL-4 gene, PEG-modified proteins encapsulating the IL-4 gene or protein, protein microspheres encapsulating the IL-4 gene or IL-4 protein, liposomes encapsulating the IL-4 gene or IL-4 protein, and extracellular vesicles encapsulating the IL-4 gene or IL-4 protein; the viral vectors include at least one of adeno-associated virus vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, and herpesvirus vectors.

[0010] In any embodiment of the present application, the receptor agonists of IL-4 protein include agonists of IL-4RA protein; the agonists of IL-4RA protein include at least one of ligands of IL-4RA protein, ligands of truncated IL-4RA protein, ligands of mutant IL-4RA protein, and ligands of IL-4RA fusion protein; preferably, the agonists of IL-4RA protein include ligands of IL-4RA protein.

[0011] In any embodiment of the present application, substances that enhance the receptor agonist activity and / or content of IL-4 protein include substances that can enhance the agonist activity and / or content of IL-4RA protein; preferably, substances that enhance the agonist activity and / or content of IL-4RA protein include at least one of proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, polypeptides, peptide mimetics, nucleic acid molecules, and small molecule compounds that can enhance the ligand activity and / or content of IL-4RA protein; preferably, substances that enhance the agonist activity and / or content of IL-4RA protein include at least one of a vector expressing the ligand of IL-4RA protein, an mRNA nucleic acid molecule encoding the ligand of IL-4RA protein, a lipid nanoparticle carrying the ligand gene of IL-4RA protein, a viral vector carrying the ligand gene of IL-4RA protein, a PEG-modified protein encapsulating the ligand gene of IL-4RA protein or the ligand of IL-4RA protein, a protein microsphere encapsulating the ligand gene of IL-4RA protein or the ligand of IL-4RA protein, a liposome encapsulating the ligand gene of IL-4RA protein or the ligand of IL-4RA protein, and an extracellular vesicle encapsulating the ligand gene of IL-4RA protein or the ligand of IL-4RA protein; the viral vector includes at least one of an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a retroviral vector, a poxviral vector, and a herpesviral vector.

[0012] In any embodiment of the present application, pulmonary hypertension includes at least one of arterial pulmonary hypertension, pulmonary hypertension caused by chronic obstructive pulmonary disease, and pulmonary hypertension caused by pulmonary vascular lesions associated with autoimmune diseases; preferably, pulmonary hypertension caused by pulmonary vascular lesions associated with autoimmune diseases includes lupus erythematosus-related pulmonary hypertension.

[0013] In any embodiment of the present application, the drug further includes a pharmaceutically acceptable carrier, excipient, or salt; preferably, the dosage form of the drug includes an oral dosage form or a parenteral dosage form; preferably, the administration route of the drug includes oral administration or parenteral administration routes.

[0014] Use of the composition of the examples of the present application in the preparation of a drug for preventing and / or treating lung diseases, the composition includes a substance that can activate or enhance the IL-4 / IL-4RA signaling pathway, and the lung diseases include pulmonary hypertension, idiopathic pulmonary fibrosis, and asthma. Among them, by activating or enhancing the specific signaling pathway IL-4 / IL-4RA, the pulmonary artery pressure can be significantly reduced, vascular remodeling can be reduced, and the inflammatory response can be inhibited, thereby effectively preventing and / or treating lung diseases. Description of the Drawings

[0015] To more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the attached drawings required for the embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other attached drawings can also be obtained based on these attached drawings.

[0016] Figure 1 It is a schematic diagram of the IL-4 level in the peripheral circulation of patients with pulmonary arterial hypertension.

[0017] Figure 2 It is a schematic diagram showing that IL-4 deficiency promotes pulmonary arterial pressure and vascular remodeling in PAH mice (where, Figure 2 A is a statistical chart of the right ventricular systolic pressure measured after the establishment of 4 groups of mouse models in Example 2; Figure 2 B is a schematic diagram of the dynamic right heart pressure measured after the establishment of 4 groups of mouse models in Example 2; Figure 2 C is a schematic diagram of the ratio of lung weight to body weight measured after the establishment of 4 groups of mouse models in Example 2; Figure 2 D is a schematic diagram of the ratio of heart weight to body weight measured after the establishment of 4 groups of mouse models in Example 2; Figure 2 E is a schematic diagram of the ratio of right ventricular weight to left ventricular weight (Hilton index) measured after the establishment of 4 groups of mouse models in Example 2; Figure 2 F is a schematic diagram of the detection of pulmonary vascular remodeling by hematoxylin and eosin staining (H&E staining); Figure 2 G is a schematic diagram of the detection of pulmonary vascular remodeling by Masson staining.

[0018] Figure 3 It is a schematic diagram showing that IL-4RA deficiency promotes pulmonary arterial pressure and vascular remodeling in PAH mice (where, Figure 3 A is a statistical chart of the right ventricular systolic pressure measured after the establishment of 4 mouse models in Example 3; Figure 3 B is a schematic diagram of the dynamic right heart pressure measured after the establishment of 4 groups of mouse models in Example 3; Figure 3 C is a statistical chart of the ratio of lung weight to body weight measured after the establishment of 4 groups of mouse models in Example 3; Figure 3 D is a schematic diagram of the ratio of heart weight to body weight measured after the establishment of 4 groups of mouse models in Example 3; Figure 3 E is a schematic diagram of the ratio of right ventricular weight to left ventricular weight (Hilton index) measured after the establishment of 4 groups of mouse models in Example 3; Figure 3 F is a schematic diagram of the detection of pulmonary vascular remodeling by hematoxylin and eosin (H&E staining); Figure 3 G is a schematic diagram of the detection of pulmonary vascular remodeling by Masson staining.

[0019] Figure 4Schematic diagram showing that recombinant protein IL-4 injection via the tail vein can reduce pulmonary artery pressure and vascular remodeling in PAH mice. Among them, Figure 4 A is a statistical chart of right ventricular systolic pressure; Figure 4 B is a statistical chart of the ratio of lung weight to body weight measured after establishing the mouse model; Figure 4 C is a schematic diagram of the ratio of heart weight to body weight measured after establishing the mouse model; Figure 4 D is a schematic diagram of the ratio of right ventricular weight to left ventricular weight (Hilton index) measured after establishing the mouse model; Figure 4 E is a schematic diagram of hematoxylin and eosin (H&E) staining to detect pulmonary vascular remodeling in each group of mice; Figure 4 F is a schematic diagram of Masson staining to detect pulmonary vascular remodeling. Detailed implementation manners

[0020] In order to make the application purpose, technical solutions and beneficial technical effects of this application clearer, the following further elaborates on this application in combination with examples. It should be understood that the embodiments described in this specification are only for explaining this application and not for limiting this application.

[0021] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly recorded; and any lower limit can be combined with other lower limits to form a range not explicitly recorded, and similarly any upper limit can be combined with any other upper limit to form a range not explicitly recorded. In addition, although not explicitly recorded, each point or single value between the range endpoints is included in this range. Therefore, each point or single value can be used as its own lower limit or upper limit to be combined with any other point or single value or with other lower limits or upper limits to form a range not explicitly recorded.

[0022] It should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, the elements defined by the statement "including..." do not exclude the existence of additional identical elements in the process, method, article or device including the said elements.

[0023] The term "IL-4" refers to interleukin-4, which is a glycoprotein, also known as a pleiotropic cytokine, and is the ligand of the IL-4 receptor.

[0024] The term "IL-4RA" or "IL-4RA protein" refers to the IL-4 receptor alpha chain (IL-4RA) protein, which is a transmembrane protein that initiates a series of intracellular signal transduction processes by binding to IL-4, thereby regulating the activity of immune cells.

[0025] The term "IL-4 / IL-4RA signaling pathway" means that both IL-4 protein and IL-4RA protein are expressed in humans or other animals (such as mice), and they interact with each other to effectively mediate downstream signal transduction and conduct normal IL-4 signal or IL-4RA signal. Among them, IL-4 is the ligand in the signaling pathway and IL-4RA is the receptor in the signaling pathway.

[0026] The full English name of the term "arterial pulmonary hypertension" is pulmonary arterial hypertension, and the English abbreviation is PAH.

[0027] The above application content of this application does not intend to describe every disclosed embodiment or implementation method in this application. The following description more specifically illustrates exemplary embodiments. Throughout the application, guidance is provided through a series of examples, and these examples can be used in various combinations. In each instance, the enumeration is only a representative group and should not be construed as exhaustive.

[0028] Lung diseases, especially PAH, are a serious cardio-pulmonary disease characterized by elevated pulmonary artery blood pressure, ultimately leading to right heart failure. The pathogenesis of PAH is complex and involves multiple aspects such as pulmonary vascular smooth muscle cell proliferation, endothelial dysfunction, and immune-inflammatory responses. Figure 1 The reduced level of IL-4 in the peripheral circulation of patients with pulmonary hypertension indicates that the concentration level of IL-4 in the human body is closely related to PAH disease. Current treatment methods mainly include vasodilators, endothelin receptor antagonists, phosphodiesterase 5 inhibitors, etc. These drugs can relieve symptoms and improve hemodynamics to a certain extent, but they usually cannot fundamentally change the course of the disease, and long-term use is often accompanied by significant side effects such as hypotension and headache.

[0029] In view of the above problems, the inventors have developed a new treatment method that acts on the IL-4 / IL-4RA signaling pathway to treat lung diseases through a large number of animal experiments.

[0030] The first aspect of the embodiments of this application provides the use of a composition in the preparation of a drug for preventing and / or treating lung diseases. The composition includes substances that can activate or enhance the IL-4 / IL-4RA signaling pathway; lung diseases include pulmonary hypertension, idiopathic pulmonary fibrosis, and asthma.

[0031] Substances that can activate or enhance the IL-4 / IL-4RA signaling pathway achieve the prevention and treatment of lung diseases, such as pulmonary arterial hypertension, idiopathic pulmonary fibrosis, and asthma, especially pulmonary arterial hypertension, mainly through three aspects: regulating immune responses, inhibiting vascular remodeling, and improving pulmonary function indicators. Among them, regulating immune responses means regulating Th2-type immune responses, inhibiting the production of inflammatory cytokines, which include but are not limited to IL-6, TNF-α, etc., and reducing immune cell infiltration, thereby alleviating lung inflammation; inhibiting vascular remodeling means inhibiting the proliferation and fibrosis process of pulmonary vascular smooth muscle cells through anti-fibrotic effects, preventing abnormal remodeling of blood vessels; improving pulmonary function means improving pulmonary artery pressure and pulmonary function indicators.

[0032] In some embodiments, substances that can activate or enhance the IL-4 / IL-4RA signaling pathway include at least one of the following: (1) IL-4 protein; (2) substances that increase the activity and / or content of IL-4 protein; (3) receptor agonists of IL-4 protein; (4) substances that increase the activity and / or content of receptor agonists of IL-4 protein.

[0033] In some embodiments, the IL-4 protein includes at least one of recombinant IL-4 protein, truncated IL-4 protein, mutant IL-4 protein, and IL-4 fusion protein.

[0034] In some embodiments, the IL-4 fusion protein includes at least one of IL-4-Fc fusion protein, IL-4 anti-inflammatory factor fusion protein, and IL-4 targeting peptide fusion protein.

[0035] As an example, in the design of truncated IL-4 protein, the non-essential domains of IL-4 protein can be removed to retain its core receptor-binding function, thereby enhancing biological stability and optimizing immunomodulatory function. For example, some non-critical amino acid residues at the N-terminus or C-terminus of IL-4 can be removed while retaining the IL-4RA receptor-binding site, so that it can still effectively activate the IL-4 signaling pathway, but has a longer serum half-life or higher tissue specificity. Through protein structure analysis, a series of truncated forms can be designed and their affinity and functional characteristics can be optimized to ensure their effectiveness in the treatment of PAH. Truncated IL-4 protein can not only improve the stability of IL-4 protein, but also enhance its lung-targeting effect.

[0036] As an example, in the development of mutant IL-4 proteins, strategies such as site-directed mutagenesis or directed evolution can be used to optimize the key residues of IL-4 proteins to improve their stability, receptor affinity, or immunomodulatory capacity. For example: (1) Enhancing the receptor affinity of IL-4 proteins: By mutating the key residues of IL-4 at the IL-4RA binding interface, the interaction between IL-4 proteins and their receptors can be improved, enhancing their biological activity, and thus achieving the same therapeutic effect at lower doses. (2) Optimizing the immunomodulatory properties of IL-4 proteins: By mutating the key sites that affect Th2-type immune responses, such as K77R or Y124D, the immunological response characteristics of IL-4 proteins can be adjusted to make them more targeted for the treatment of PAH. (3) Enhancing the stability of IL-4 proteins: Through protein engineering optimization, such as introducing stability-enhancing mutations into the IL-4 structure, disulfide bond rearrangement or misfolding can be reduced, and the half-life of the protein in vivo can be increased. In mutant IL-4 proteins, the binding affinity of IL-4 proteins to receptors can be optimized through amino acid substitutions such as Q116E and R121D, improving their biological efficacy.

[0037] As an example, IL-4 fusion proteins refer to proteins obtained by fusing the functional region of IL-4 with other proteins having immunomodulatory or lung-protective effects, thereby improving their pharmacokinetic properties and tissue targeting.

[0038] Optionally, IL-4-Fc fusion proteins refer to proteins obtained by fusing IL-4 proteins with the IgG Fc segment, thereby increasing the serum half-life of IL-4 proteins, prolonging the duration of drug efficacy, reducing the dosing frequency, while reducing their immunogenicity and improving the treatment stability.

[0039] As an example, IL-4 anti-inflammatory factor fusion proteins can be proteins obtained by fusing IL-4 proteins with IL-10 proteins, enabling them to have both anti-inflammatory and anti-fibrotic effects, thereby enhancing the therapeutic effect on PAH.

[0040] As an example, IL-4-targeting peptide fusion proteins refer to proteins obtained by fusing IL-4 proteins with lung vascular-specific targeting peptides, thereby enhancing the selective delivery of IL-4 proteins to the lungs, reducing systemic side effects, and improving the therapeutic effect.

[0041] Optionally, IL-4 proteins include recombinant IL-4 proteins.

[0042] As an important immunomodulatory factor, IL-4 protein can play a role in treating lung diseases by regulating Th2-type immune responses. In the above-mentioned lung diseases, excessive inflammatory responses and immune cell infiltration often lead to lung tissue damage and dysfunction. IL-4 protein can not only reduce lung inflammation by inhibiting the production of inflammatory cytokines such as IL-6 and TNF-α, but also further relieve inflammatory symptoms by reducing immune cell infiltration. At the same time, pulmonary vascular remodeling is an important pathological feature of various lung diseases, especially chronic lung diseases such as pulmonary arterial hypertension (PAH). Through its antifibrotic effect, IL-4 protein can not only slow down the thickening of the blood vessel wall by inhibiting the proliferation of pulmonary vascular smooth muscle cells, but also reduce the deposition of extracellular matrix such as collagen fibers by inhibiting the fibrotic process of blood vessels, thereby preventing abnormal remodeling of blood vessels. In addition, IL-4 protein can also significantly improve lung function. By activating or enhancing the IL-4 / IL-4RA signaling pathway, IL-4 protein can not only reduce pulmonary arterial pressure, but also improve lung function indexes such as vital capacity and peak expiratory flow in patients, improve the respiratory function of patients, and at the same time help delay the progression of lung diseases, especially pulmonary arterial hypertension, and improve the quality of life and prognosis of patients.

[0043] Optionally, the method for preparing IL-4 protein includes introducing the nucleic acid or vector encoding IL-4 protein into a host cell and then inducing its expression.

[0044] Optionally, the vector encoding IL-4 protein contains the nucleic acid encoding IL-4 protein.

[0045] Optionally, the vector encoding IL-4 protein can be expressed in vivo, in vitro or ex vivo. Preferably, the vector is a prokaryotic expression vector, a viral expression vector or a eukaryotic expression vector. For example, Escherichia coli series vectors, phages, etc.

[0046] Optionally, the host cell can be a eukaryotic cell or a prokaryotic cell.

[0047] As an example, eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells or CHO cells, etc.

[0048] As an example, prokaryotic cells include Escherichia coli, etc.

[0049] Optionally, the method for preparing IL-4 protein further includes purifying IL-4 protein by methods such as affinity chromatography and ion exchange chromatography.

[0050] By way of example, the sequences of IL-4 protein include, but are not limited to: NP_067258.1 (Mouse), NP_000580.1 (Human, isoform1), NP_001341919.1 (Human, isoform3), NP_758858.1 (Human, isoform2).

[0051] In some embodiments, the substances that enhance the activity and / or content of IL-4 protein include at least one of proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, polypeptides, peptide mimetics, nucleic acid molecules, and small molecule compounds that can enhance the activity and / or content of IL-4 protein.

[0052] In some embodiments, the substances that enhance the activity and / or content of IL-4 protein include at least one of gene delivery vectors, protein delivery vectors, nucleic acid molecules, proteins, and fusion proteins that can enhance the activity and / or content of IL-4 protein; preferably, the substances that enhance the activity and / or content of IL-4 protein include at least one of a vector expressing IL-4 protein, an mRNA nucleic acid molecule encoding IL-4 protein, a lipid nanoparticle carrying the IL-4 gene, a viral vector carrying the IL-4 gene, a PEG-modified protein encapsulating the IL-4 gene or IL-4 protein, a protein microsphere encapsulating the IL-4 gene or IL-4 protein, a liposome encapsulating the IL-4 gene or IL-4 protein, and an extracellular vesicle encapsulating the IL-4 gene or IL-4 protein; the viral vectors include at least one of adeno-associated virus vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, and herpesvirus vectors.

[0053] By way of example, the above-mentioned IL-4 gene refers to a gene capable of encoding IL-4 protein.

[0054] In some embodiments, the receptor agonists of IL-4 protein include agonists of IL-4RA protein; the agonists of IL-4RA protein include at least one of a ligand of IL-4RA protein, a ligand of a truncated IL-4RA protein, a ligand of a mutant IL-4RA protein, and a ligand of an IL-4RA fusion protein; preferably, the agonist of IL-4RA protein includes a ligand of IL-4RA protein.

[0055] Optionally, the IL-4RA protein includes a recombinant IL-4RA protein.

[0056] Optionally, the ligand of IL-4RA protein includes a ligand of recombinant IL-4RA protein.

[0057] Optionally, the method for preparing the ligand of the IL-4RA protein includes introducing the nucleic acid or vector encoding the ligand of the IL-4RA protein into a host cell and then inducing its expression.

[0058] Optionally, the vector encoding the ligand of the IL-4RA protein contains the nucleic acid encoding the ligand of the IL-4RA protein.

[0059] Optionally, the vector encoding the ligand of the IL-4RA protein can be expressed in vivo, in vitro or ex vivo. Preferably, the vector is a prokaryotic expression vector, a viral expression vector or a eukaryotic expression vector. For example, E. coli series vectors, phages, etc.

[0060] Optionally, the host cell can be a eukaryotic cell or a prokaryotic cell.

[0061] As an example, eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells or CHO cells, etc.

[0062] As an example, prokaryotic cells include E. coli, etc.

[0063] Optionally, the method for preparing the ligand of the IL-4RA protein further includes purifying the ligand of the IL-4RA protein by methods such as affinity chromatography and ion exchange chromatography.

[0064] As an example, the sequences of the IL-4RA protein include but are not limited to: NP_000409.1 (Human, interleukin-4 receptor subunit alpha isoform a precursor), NP_001244335.1 (Human, interleukin-4 receptor subunit alpha isoform a precursor), NP_001244336.1 (Human, interleukin-4 receptor subunit alpha isoform c), NP_001244926.1 (Human, interleukin-4 receptor subunit alpha isoform d), NP_001008700.1 (Mouse, interleukin-4 receptor subunit alpha isoform 1 precursor), NP_001350912.1 (Mouse, interleukin-4 receptor subunit alpha isoform 2).

[0065] In some embodiments, substances that enhance the receptor agonist activity and / or content of IL-4 protein include substances that can enhance the agonist activity and / or content of IL-4RA protein; preferably, substances that enhance the agonist activity and / or content of IL-4RA protein include at least one of proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, polypeptides, peptide mimetics, nucleic acid molecules, and small molecule compounds that can enhance the ligand activity and / or content of IL-4RA protein; preferably, substances that enhance the agonist activity and / or content of IL-4RA protein include at least one of a vector expressing the ligand of IL-4RA protein, an mRNA nucleic acid molecule encoding the ligand of IL-4RA protein, a lipid nanoparticle carrying the ligand gene of IL-4RA protein, a viral vector carrying the ligand gene of IL-4RA protein, a PEG-modified protein encapsulating the ligand gene of IL-4RA protein or the ligand of IL-4RA protein, a protein microsphere encapsulating the ligand gene of IL-4RA protein or the ligand of IL-4RA protein, a liposome encapsulating the ligand gene of IL-4RA protein or the ligand of IL-4RA protein, and an extracellular vesicle encapsulating the ligand gene of IL-4RA protein or the ligand of IL-4RA protein; the viral vector includes at least one of an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a poxvirus vector, and a herpesvirus vector.

[0066] As an example, the above-mentioned ligand gene of IL-4RA protein refers to a gene that can encode the ligand of IL-4RA protein.

[0067] In some embodiments, pulmonary hypertension includes at least one of pulmonary arterial hypertension (PAH), pulmonary hypertension caused by chronic obstructive pulmonary disease, and pulmonary hypertension caused by autoimmune disease-related pulmonary vascular lesions; preferably, pulmonary hypertension caused by autoimmune disease-related pulmonary vascular lesions includes lupus-related pulmonary hypertension.

[0068] As an example, the lung disease is selected from at least one of pulmonary arterial hypertension (PAH), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, and systemic lupus erythematosus-associated pulmonary arterial hypertension (SLE-PAH).

[0069] Preferably, the lung disease is pulmonary arterial hypertension (PAH).

[0070] The "drug" described in the present invention can be used to treat humans or non-human animals, such as non-human mammals. The drug can contain pharmaceutically acceptable carriers, excipients or salts commonly used in the prior art.

[0071] As an example, the excipient is selected from at least one of diluents, binders, lubricants and wetting agents.

[0072] Optionally, the drug can be in any suitable dosage form, such as a dosage form administered via the gastrointestinal tract or a dosage form administered non-gastrointestinally, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder aerosols, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, dripping pills, gels, etc. The various dosage forms of the drug can be prepared according to the conventional production methods in the pharmaceutical field.

[0073] As an example, the dosage form of the drug includes at least one of tablets, capsules, granules, suspensions, dripping pills, injections and aerosols.

[0074] The drug can be administered by any suitable route, such as gastrointestinal administration (e.g., oral) or non-gastrointestinal administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, infusion, intracerebral, intrathecal, transdermal, intrarectal, etc.) routes.

[0075] As an example, the administration route of the drug includes intravenous injection, subcutaneous injection or inhalation administration.

[0076] The drug can contain the ligand of the IL-4 protein and the IL-4RA protein in a weight ratio of 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%).

[0077] The administration dose of the drug is 180 μg / kg - 1000 μg / kg (for example, 180 μg / kg, 190 μg / kg, 200 μg / kg, 210 μg / kg, 220 μg / kg, 230 μg / kg, 240 μg / kg, 250 μg / kg, 300 μg / kg, 500 μg / kg, 800 μg / kg, 1000 μg / kg), and it can also be adjusted according to the results of clinical observations.

[0078] Use of the composition of the embodiments of the present application in the preparation of a drug for preventing and / or treating pulmonary diseases, the composition comprising a substance capable of activating or enhancing the IL-4 / IL-4RA signaling pathway, and the pulmonary diseases include pulmonary arterial hypertension, idiopathic pulmonary fibrosis, and asthma. Among them, by activating or enhancing the specific signaling pathway IL-4 / IL-4RA, the pulmonary arterial pressure can be significantly reduced, vascular remodeling can be reduced, and the inflammatory response can be inhibited, thereby effectively preventing and / or treating pulmonary diseases, especially pulmonary arterial hypertension PAH in pulmonary vascular-related diseases.

[0079] "Pharmaceutically acceptable" as used in the present invention means that it neither significantly stimulates the organism nor inhibits the biological activities and characteristics of the active substances of the administered product.

[0080] "Treatment" as used in the present invention means slowing down, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, disease, or illness after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, illnesses, or disorders.

[0081] "Prevention" as used in the present invention means a manner implemented to prevent or delay the occurrence of a disease, disorder, or symptom in an organism.

[0082] Examples

[0083] The following examples more specifically describe the content disclosed in the present application. These examples are only for illustrative purposes, because various modifications and changes within the scope of the present application disclosure are obvious to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on mass, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further treatment, and the instruments used in the examples are all commercially available.

[0084] Example 1 Treatment of PAH mouse model with IL-4 recombinant protein

[0085] (1) Establishment of an animal model, specifically including the following steps.

[0086] S1, animal grouping;

[0087] Healthy C57BL / 6J mice, weighing 20 - 25 g and aged 8 - 10 weeks, were selected and divided into 4 groups of 8 mice each. The grouping is as follows:

[0088] Group A: Normal mice + blank control (PBS);

[0089] Group B: PAH mice + blank control (PBS);

[0090] Group C: PAH mice + treatment with IL-4 recombinant protein.

[0091] S2. Establishment of the PAH model;

[0092] Mice were treated in a 10% hypoxic environment (10% O2) for 28 days to induce PAH.

[0093] (II) Preparation of the IL-4 recombinant protein, which specifically includes the following steps:

[0094] Commercially available IL-4 recombinant protein (manufactured by Novaprotein, model CX88) was used and prepared into an IL-4 recombinant protein solution with a concentration of 50 μg / mL using phosphate buffer for use.

[0095] (III) Experimental methods, which specifically include the following steps:

[0096] S1. Group C mice were intravenously injected with IL-4 recombinant protein once every three days for 4 consecutive weeks;

[0097] S2. Group A mice and Group B mice were intravenously injected with the blank control solution PBS once every three days for 4 consecutive weeks;

[0098] S3. Group B mice and Group C mice were placed in a 10% O2 environment for hypoxic exposure treatment for 4 weeks to induce PAH;

[0099] S4. Group A was placed in a conventional air environment with 21% O2 and cultured for 4 weeks.

[0100] (IV) Detection and analysis, which specifically include the following steps:

[0101] S1. Pulmonary artery pressure measurement;

[0102] After the experiment, the pulmonary artery pressure (mPAP) of the mice was measured using the right heart catheterization method. By analyzing the changes in the pulmonary artery pressure of the mice in each group above, the therapeutic effect of the IL-4 recombinant protein was evaluated.

[0103] S2. Assessment of vascular remodeling;

[0104] The lung tissues of the mice in each of the above groups were subjected to HE staining, Masson trichrome staining, and immunohistochemical analysis. The therapeutic effect of the IL-4 recombinant protein was evaluated by analyzing the wall thickness of the pulmonary blood vessels, vascular smooth muscle hyperplasia, and fibrosis conditions.

[0105] (V) Experimental Results

[0106] 1. The IL-4 recombinant protein can reduce pulmonary artery pressure

[0107] After measurement, the average pulmonary artery pressure of the mice in group A was 27.26 mmHg, that of the mice in group B was 41.24 mmHg, and that of the mice in group C was 37.14 mmHg. The pulmonary artery pressure of the mice in group B was significantly higher than that of the mice in group A; compared with the mice in group B, the pulmonary artery pressure of the mice in group C was significantly reduced.

[0108] 2. The IL-4 recombinant protein can inhibit vascular remodeling

[0109] 2.1 The IL-4 recombinant protein can reduce the degree of pulmonary vascular smooth muscle hyperplasia and the thickness of the vascular wall

[0110] The results of HE staining showed that compared with group B, the degree of pulmonary vascular smooth muscle hyperplasia in the mice of group C was significantly reduced, and the thickness of the vascular wall decreased significantly.

[0111] 2.2 The IL-4 recombinant protein can reduce collagen deposition in the adventitial region of the pulmonary blood vessels

[0112] The results of Masson staining showed that compared with group B, the collagen deposition in the adventitial region of the pulmonary blood vessels of the mice in group C decreased, indicating that the fibrosis process was alleviated and the degree of vascular wall remodeling was significantly reduced.

[0113] In summary, the IL-4 recombinant protein showed a significant therapeutic effect in the PAH mouse model (see Figure 4 ). The treatment with the IL-4 recombinant protein can effectively reduce the pulmonary artery pressure, alleviate the smooth muscle hyperplasia and vascular wall fibrosis of the pulmonary blood vessels, and show a good anti-PAH effect.

[0114] Example 2 Experiment of the IL-4 gene knockout mouse model in hypoxia-induced PAH

[0115] (I) Establishment and grouping of the animal model, which specifically includes the following steps,

[0116] S1, animal grouping;

[0117] The IL-4 gene knockout mice were purchased from Shanghai Model Organisms Center, Inc., and the catalog number was NM-KO-190503.

[0118] Thirty IL-4 gene knockout mice (IL-4- / -) and thirty wild-type mice (WT) were selected, with a body weight of about 20 - 25 g and an age of 8 - 10 weeks.

[0119] S2, Establishment of the PAH model;

[0120] The PAH model was induced by the chronic hypoxia (10% O2) environment method and continuously exposed for 4 weeks.

[0121] S3, Experimental grouping;

[0122] Group A (Normoxia_WT): Wild-type mice + normal environment;

[0123] Group B (Normoxia_IL-4KO): IL-4 knockout mice + normal environment;

[0124] Group C (Hypoxia_WT): Wild-type mice + hypoxic environment;

[0125] Group D (Hypoxia_IL-4KO): IL-4 knockout mice + hypoxic environment.

[0126] (II) Experimental methods

[0127] S1, Mice in Group A and Group B were placed in a conventional air environment with 21% O2 and cultured for 4 weeks

[0128] S2, Mice in Group C and Group D were placed in an environment with 10% O2 for hypoxic environment exposure treatment for 4 weeks to induce PAH.

[0129] (III) Detection and analysis, the same as in Example 1

[0130] (IV) Experimental results

[0131] 1. Knocking out the IL-4 gene increases pulmonary artery pressure

[0132] From the measurement results, the average pulmonary artery pressure of mice in Group A was 27.01 mmHg, that of mice in Group B was 26.58 mmHg, that of mice in Group C was 38.38 mmHg, and that of mice in Group D was 41.81 mmHg. The pulmonary artery pressure of mice in Group D was significantly higher than that of mice in Group A, Group B, and Group C.

[0133] 2. Knocking out the IL-4 gene promotes vascular remodeling

[0134] 2.1 Knocking out the IL-4 gene increases the degree of pulmonary vascular smooth muscle hyperplasia and the thickness of the vascular wall

[0135] See Figure 2F. Compared with groups A, B, and C, the smooth muscle hyperplasia and vascular wall thickness of the pulmonary vessels in group D mice were significantly increased, and the degree of fibrosis of the vascular wall was more severe.

[0136] 2.2 Knockout of the IL-4 gene increases collagen deposition in the adventitial region of the pulmonary vessels

[0137] See Figure 2 G. Masson staining showed that compared with groups A, B, and C, there was obvious collagen deposition in the adventitial region of the pulmonary vessels in group D mice, indicating the exacerbation of the fibrosis process.

[0138] In summary, the IL-4 gene knockout mouse model showed obvious pathological changes during the process of hypoxia-induced PAH, including significantly increased pulmonary artery pressure and significantly aggravated vascular remodeling (see Figure 2 ).

[0139] Example 3 Experiment of the IL-4RA gene knockout mouse model in hypoxia-induced PAH

[0140] (1) Establishment and grouping of the animal model, which specifically includes the following steps.

[0141] S1. Animal grouping;

[0142] IL-4RA gene knockout mice were purchased from Cyagen Biosciences (Suzhou) Inc., with the model number S-KO-16195.

[0143] Healthy IL-4RA gene knockout mice (IL-4RA- / -) and wild-type mice (WT), both with a C57BL / 6J background, weighing 20-25 g and aged 8-10 weeks, were grouped as follows.

[0144] Group A (Normoxia_WT; WT control): Wild-type mice were raised in a normoxic (21% O2) environment.

[0145] Group B (Normoxia_IL-4ra; IL-4RA- / - control): IL-4RA knockout mice were raised in a normoxic (21% O2) environment.

[0146] Group C (Hypoxia_WT; WT + hypoxia exposure): Wild-type mice were exposed to a 10% O2 hypoxic environment for 28 days.

[0147] Group D (Hypoxia_IL-4ra; IL-4RA- / - + hypoxia exposure): IL-4RA knockout mice were exposed to a 10% O2 hypoxic environment for 28 days.

[0148] (2) Experimental method

[0149] S1. Place the mice in Group A and Group B in a conventional air environment with 21% O2 and culture them for 4 weeks.

[0150] S2. Place the mice in Group C and Group D in an environment with 10% O2 for hypoxic environmental exposure treatment for 4 weeks to induce PAH.

[0151] (3) Detection and analysis are the same as in Example 1.

[0152] (4) Experimental results

[0153] 1. Knocking out the IL-4RA gene increases pulmonary artery pressure.

[0154] After measurement, the average pulmonary artery pressure of the mice in each group is as follows.

[0155] Group A (Normoxia_WT; WT control): 24.87 mmHg;

[0156] Group B (Normoxia_IL-4ra; IL-4RA- / - control): 26.81 mmHg;

[0157] Group C (Hypoxia_WT; WT + hypoxia): 39.76 mmHg;

[0158] Group D (Hypoxia_IL-4ra; IL-4RA- / - + hypoxia): 43.09 mmHg.

[0159] It can be seen that the pulmonary artery pressure of the mice in Group D is significantly higher than that in Group C and much higher than that in Group A and Group B.

[0160] 2. Knocking out the IL-4RA gene promotes vascular remodeling.

[0161] 2.1 Knocking out the IL-4RA gene increases the thickness of the vascular wall.

[0162] See Figure 3 Figure F. The HE staining results show that the thickness of the vascular wall of the small pulmonary arteries in Group D mice is significantly increased compared with that in Group A, Group B, and Group C. It shows that blocking the IL-4 signal receptor will exacerbate vascular remodeling during pulmonary hypertension.

[0163] 2.2 Knocking out the IL-4RA gene increases collagen deposition in the adventitial region of the pulmonary vessels.

[0164] See Figure 3 Figure G. The Masson staining results show that there is obvious collagen deposition in the adventitial region of the pulmonary vessels in Group D mice, and the degree of fibrosis is high, compared with Group A, Group B, and Group C.

[0165] In summary, knocking out the IL-4RA gene significantly promotes the increase in pulmonary artery pressure and vascular remodeling in PAH mice (seeFigure 3 ) After the deletion of IL-4RA, the pulmonary artery pressure increased significantly, the abnormal proliferation of vascular smooth muscle cells occurred, and the degree of fibrosis was aggravated.

[0166] As described above, the above is only the specific implementation manner of the present application. Those skilled in the art can clearly understand that for the convenience and brevity of description, the specific working process described above can refer to the corresponding process in the foregoing method embodiments and will not be repeated here. It should be understood that the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of various equivalent modifications or replacements, and these modifications or replacements should be covered within the protection scope of the present application.

Claims

1. Use of a composition in the preparation of a medicament for the prevention and / or treatment of lung diseases, characterized in that the composition comprises a substance capable of activating or enhancing the IL-4 / IL-4RA signaling pathway; the lung diseases include pulmonary arterial hypertension, idiopathic pulmonary fibrosis, and asthma.

2. The application according to claim 1, wherein The substance capable of activating or enhancing the IL-4 / IL-4RA signaling pathway comprises at least one of the following: (1) IL-4 protein; (2) A substance that increases the activity and / or content of IL-4 protein; (3) An agonist of the IL-4 protein receptor; (4) A substance that increases the activity and / or content of the agonist of the IL-4 protein receptor.

3. The application according to claim 2, wherein The IL-4 protein comprises at least one of an IL-4 recombinant protein, an IL-4 protein truncation, an IL-4 protein mutant, and an IL-4 fusion protein.

4. The application according to claim 3, characterized in that, The IL-4 fusion protein comprises at least one of an IL-4-Fc fusion protein, an IL-4 anti-inflammatory factor fusion protein, and an IL-4 targeting peptide fusion protein.

5. The application according to claim 2, wherein The substance that increases the activity and / or content of IL-4 protein comprises at least one of a protein, an antibody, an antibody mimetic, a fusion protein, a protein analog, a gene delivery vector, a protein delivery vector, a polypeptide, a peptide mimetic, a nucleic acid molecule, and a small molecule compound that can increase the activity and / or content of IL-4 protein.

6. The application according to claim 2, wherein The substance that increases the activity and / or content of IL-4 protein comprises at least one of a gene delivery vector, a protein delivery vector, a nucleic acid molecule, a protein, and a fusion protein that can increase the activity and / or content of IL-4 protein; Preferably, the substance that increases the activity and / or content of IL-4 protein comprises at least one of a vector expressing IL-4 protein, an mRNA nucleic acid molecule encoding IL-4 protein, a lipid nanoparticle carrying the IL-4 gene, a viral vector carrying the IL-4 gene, a PEG-modified protein encapsulating the IL-4 gene or IL-4 protein, a protein microsphere encapsulating the IL-4 gene or IL-4 protein, a liposome encapsulating the IL-4 gene or IL-4 protein, and an extracellular vesicle encapsulating the IL-4 gene or IL-4 protein; The viral vector comprises at least one of an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a poxvirus vector, and a herpesvirus vector.

7. The application according to claim 2, characterized in that, The agonist of the IL-4 protein receptor comprises an agonist of the IL-4RA protein; The agonist of the IL-4RA protein comprises at least one of a ligand of the IL-4RA protein, a ligand of the IL-4RA protein truncation, a ligand of the IL-4RA protein mutant, and a ligand of the IL-4RA fusion protein; Preferably, the agonist of the IL-4RA protein comprises a ligand of the IL-4RA protein.

8. The application according to claim 2, characterized in that The substance that increases the activity and / or content of the agonist of the IL-4 protein receptor comprises a substance that can increase the activity and / or content of the agonist of the IL-4RA protein; Preferably, the substance for increasing the agonist activity and / or content of the IL-4RA protein includes at least one of a protein, an antibody, an antibody mimetic, a fusion protein, a protein analog, a gene delivery vector, a protein delivery vector, a polypeptide, a peptide mimetic, a nucleic acid molecule, and a small molecule compound that can increase the agonist activity and / or content of the IL-4RA protein; Preferably, the substance for increasing the agonist activity and / or content of the IL-4RA protein includes at least one of a vector expressing the ligand of the IL-4RA protein, an mRNA nucleic acid molecule encoding the ligand of the IL-4RA protein, a lipid nanoparticle carrying the ligand gene of the IL-4RA protein, a viral vector carrying the ligand gene of the IL-4RA protein, a PEG-modified protein encapsulating the ligand gene of the IL-4RA protein or the ligand of the IL-4RA protein, a protein microsphere encapsulating the ligand gene of the IL-4RA protein or the ligand of the IL-4RA protein, a liposome encapsulating the ligand gene of the IL-4RA protein or the ligand of the IL-4RA protein, and an extracellular vesicle encapsulating the ligand gene of the IL-4RA protein or the ligand of the IL-4RA protein; The viral vector includes at least one of an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a poxvirus vector, and a herpesvirus vector.

9. The application according to claim 1, characterized in that, The pulmonary hypertension includes at least one of arterial pulmonary hypertension, pulmonary hypertension caused by chronic obstructive pulmonary disease, and pulmonary hypertension caused by autoimmune disease-related pulmonary vascular lesions; Preferably, the pulmonary hypertension caused by autoimmune disease-related pulmonary vascular lesions includes lupus erythematosus-related pulmonary hypertension.

10. The application according to claim 1, wherein The drug further includes a pharmaceutically acceptable carrier, excipient, or salt; Preferably, the dosage form of the drug includes an oral dosage form or a parenteral dosage form; Preferably, the administration route of the drug includes enteral administration or parenteral administration route.

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