Therapeutic use of longacting conjugate of triple agonist having activities to all of glucagon / glp-1 / gip receptors for lung disease

Abstract

The present invention relates to a preventive or therapeutic use of a triple agonist and / or a conjugate thereof against lung disease, the triple agonist and / or conjugate thereof having activity with respect to all of glucagon and the GLP-1 and GIP receptors.

Description

Use of a long-acting triple activator conjugate having activity at all glucagon, GLP-1 and GIP receptors for the treatment of lung diseases

[0001] The present invention relates to the preventive or therapeutic use of a triple activator having activity at all of the glucagon, GLP-1 and GIP receptors and / or a combination thereof for lung diseases.

[0002]

[0003] The lungs are the primary organs responsible for respiration. Lung diseases can be caused by harmful substances, viruses, and immune disorders. Because lung diseases cause decreased lung function and respiratory distress, appropriate treatment based on the underlying cause is required.

[0004] Diseases related to the lungs include interstitial lung disease, progressive fibrosing interstitial lung disease, idiopathic interstitial pneumonia, nonspecific interstitial pneumonia, pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, alveolitis, pneumonia, emphysema, bronchitis, chronic obstructive pulmonary disease (COPD), combined pulmonary fibrosis and emphysema (CPFE), asthma, and respiratory infections (e.g., coronavirus disease 2019 (COVID-19)). These lung diseases are known to be interrelated and present with mixed symptoms, so caution is required in selecting treatments. The main pathogenic mechanisms of lung diseases include lung damage and inflammatory response, and fibrosis.

[0005] Specifically, when an inflammatory response occurs in the lungs due to viruses, microorganisms, or harmful substances, it is known that the secretion of inflammatory cytokines (e.g., IL-1, IL-6, TNF-α) by macrophages in the alveoli, attraction of neutrophils, and secretion of proteases occur, and the secretion of proteases (e.g., elastase) that break down the fibers that make up the lung tissue damages the elasticity and structure of the lung tissue, leading to pulmonary fibrosis. Since inflammation and fibrosis of the lung precede the progression of many lung diseases, they can be considered a fundamental mechanism for the prevention and treatment of lung diseases.

[0006] Pulmonary fibrosis is a representative example of a lung disease. Fibrosis is a condition characterized by the formation of excessive fibrous connective tissue in organs or tissues. Fibrosis refers to a condition in which tissues within the body are damaged by various stressors (such as infection, chemical irritants, and radiation), and the wound healing process becomes uncontrollable. Fibrosis occurs in various organs, including the lungs, heart, and liver, and as a fundamental treatment has not yet been developed, it remains a field with high unmet needs.

[0007] Idiopathic pulmonary fibrosis (IPF), a type of pulmonary fibrosis of unknown etiology in which fibrous interstitial pneumonia of unknown etiology progresses chronically, is a disease in which fibrosis progresses due to continuous damage to alveolar epithelial cells. Treatment methods using drugs such as pirfenidone and nintedanib have been studied, but side effects such as decreased appetite, weakness, digestive side effects, possible hepatotoxicity, and photosensitivity rash are known.

[0008] Chronic obstructive pulmonary disease (COPD), another representative example of lung disease, is a disease in which the airways narrow and gradually become obstructed due to an abnormal inflammatory response in the lungs caused by cigarette smoke, air pollution, or toxic inhaled substances. It is broadly divided into chronic bronchitis and emphysema. Smoking is known to be a major cause of COPD. Smoking acts as a potent irritant in lung tissue, increasing the production of various pro-inflammatory factors, growth factors, oxidants, and chemotactic factors. It also activates inflammatory signaling pathways, promoting the migration of numerous inflammatory cells, including neutrophils and macrophages, which further aggravates pulmonary inflammation. This ultimately leads to abnormal changes in lung tissue, such as airway wall thickening and pulmonary fibrosis, and deterioration of lung function. Therefore, alleviating lung inflammation is understood as one of the treatment methods for preventing and treating COPD.

[0009] Another prominent example of lung disease is the recent outbreak of the novel coronavirus (2019-nCoV or SARS-CoV-2), which causes lung damage and the resulting acute respiratory illness (COVID-19). The novel coronavirus, which spreads through the respiratory tract, penetrates cells primarily through ACE2 and TMPRSS2, which are expressed on type II alveolar epithelial cells, making the lungs a major vulnerable organ. Major symptoms include fever and cough, and healthy adults are likely to recover over time. However, if the initial immune system is weakened and the virus is severely infected in the lungs, it can trigger a severe inflammatory response due to lung damage, accelerating the progression of fibrosis, leading to symptoms such as acute respiratory distress syndrome (ARDS) and sepsis. Currently, COVID-19 treatments primarily involve drugs that suppress viral infection and proliferation or control lung inflammation. Therefore, improving lung inflammation and fibrosis is considered one of the treatment options for preventing and treating COVID-19.

[0010] As lung inflammation and fibrosis are major causes of the onset and development of lung diseases, improvement of lung inflammation and fibrosis has been studied as a treatment mechanism for various lung diseases.

[0011]

[0012] Meanwhile, GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) are representative gastrointestinal hormones and neurohormones that are involved in regulating blood sugar levels in response to food intake. Glucagon is a peptide hormone secreted by the pancreas and, together with the two aforementioned substances, participates in the regulation of blood sugar levels. Therapeutic agents are being developed using drugs that can exhibit activity on GLP-1 receptors, GIP receptors, and glucagon receptors, either individually or simultaneously (US 10,370,426, US 10,400,020).

[0013]

[0014] Although various studies on lung diseases have been conducted to date, the development of practical and effective treatments is still insufficient, and there is a need for continued development of treatments.

[0015]

[0016] One object of the present invention is to provide a pharmaceutical composition for preventing or treating lung diseases, comprising a peptide having activity against a glucagon receptor, a GLP-1 receptor, and a GIP receptor, or a long-acting conjugate of such a peptide.

[0017] Another object of the present invention is to provide a method for preventing or treating lung disease, comprising administering to a subject in need thereof a composition comprising the peptide or a sustained-release conjugate of the peptide.

[0018] Another object of the present invention is to provide a use of a composition comprising the peptide or a sustained-release conjugate of the peptide in the manufacture of a medicament for the prevention or treatment of lung diseases.

[0019]

[0020] One embodiment of the present invention is a pharmaceutical composition for preventing or treating lung disease, comprising a peptide having activity against a glucagon receptor, a GLP-1 (Glucagon-like peptide-1) receptor, and a GIP (Glucose-dependent insulinotropic polypeptide) receptor.

[0021] As one specific example, the pharmaceutical composition for preventing or treating the lung disease is characterized by comprising a pharmaceutically acceptable excipient and a pharmaceutically effective amount of a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 102.

[0022] A pharmaceutical composition according to any one of the preceding specific examples, wherein the peptide is in the form of a sustained-release conjugate, and the sustained-release conjugate is characterized by being represented by the following chemical formula 1:

[0023] [Chemical Formula 1]

[0024] X - L - F

[0025] However, at this time, X is a peptide having an amino acid sequence of any one of sequence numbers 1 to 102;

[0026] L is a linker containing ethylene glycol repeating units,

[0027] F is the immunoglobulin Fc region,

[0028] - indicates a covalent bond between X and L, and between L and F.

[0029] A composition according to any one of the preceding specific examples, wherein the peptide is characterized in that its C-terminus is amidated.

[0030] A composition according to any one of the preceding specific examples, wherein the peptide is characterized in that its C-terminus is amidated or has a free carboxyl group (-COOH).

[0031] A composition according to any one of the preceding specific examples, wherein the peptide comprises an amino acid selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97 and 100.

[0032] A composition according to any one of the preceding specific examples, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77 and 96.

[0033] A composition according to any one of the preceding specific examples, wherein the peptide sequence is characterized in that amino acids 16 and 20 from the N-terminus form a ring.

[0034] A composition according to any one of the preceding specific examples, characterized in that the chemical formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

[0035] A composition according to any one of the preceding specific examples, wherein F is an IgG Fc region.

[0036] A composition according to any one of the preceding specific examples, wherein the lung disease is interstitial lung disease (ILD), progressive fibrosing interstitial lung disease (PF-ILD), idiopathic interstitial pneumonias (IIP), non-specific interstitial pneumonia (NSIP), pulmonary fibrosis, fibrosing interstitial lung diseases (FILD), idiopathic pulmonary fibrosis (IPF), alveolitis, pneumonia, emphysema, bronchitis, chronic obstructive pulmonary disease, combined pulmonary fibrosis and emphysema (CPFE), asthma, or respiratory infection. It is characterized by being a disease.

[0037] A composition according to any one of the preceding specific examples, wherein the respiratory infectious disease is a respiratory viral, bacterial, mycoplasma, or fungal infectious disease.

[0038] A composition according to any one of the preceding specific examples, wherein the respiratory virus is any one selected from the group consisting of adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella Zoster virus, measles virus, respiratory syncytial virus, dengue virus, human immunodeficiency virus (HIV), influenza virus, coronavirus, severe acute respiratory syndrome associated virus (SARS-associated virus), and middle east respiratory syndrome coronavirus (MERS-CoV).

[0039] A composition according to any one of the preceding specific examples, wherein the coronavirus is SARS-CoV-2.

[0040] A composition according to any one of the preceding specific examples, wherein the pharmaceutical composition is characterized in that, upon administration, (i) inhibits macrophage activity, and / or (ii) reduces the expression of IL-1β, IL-6, IL-12, or TNF-α.

[0041] A composition according to any one of the preceding specific examples, wherein the pharmaceutical composition is characterized in that it has one or more of the following properties upon administration:

[0042] (i) Inhibition of myofibroblast differentiation;

[0043] (ii) decreased expression of α-SMA, collagen1α1, or fibronectin; or

[0044] (iii) inhibition of epithelial mesenchymal transition (EMT) of alveolar epithelial cells; and

[0045] (iv) decreased expression of collagen1α1 or collagen1α3

[0046] A composition according to any one of the preceding specific examples, wherein the pharmaceutical composition is characterized in that a mucolytic agent or a pharmaceutically acceptable salt thereof is additionally administered.

[0047] A composition according to any one of the preceding specific examples, wherein the mucolytic agent comprises ambroxol, N-acetylcysteine, N-acetylin, carbocysteine, domiodol, fudosteine, bromhexine, erdosteine, letostine, lysozyme, mesna, sobrerol, stepronin, tiopronin, tyloxapol, carbocysteine, dornase alfa, eprazinone, letosteine, neltenexine, and mecysteine. It is characterized by one or more selected from the military.

[0048] A composition according to any one of the preceding specific examples, wherein the peptide and the mucolytic agent or a pharmaceutically acceptable salt thereof are administered simultaneously, sequentially, or in reverse order.

[0049] A composition according to any one of the preceding specific examples, wherein the lung disease may be characterized as lung inflammation and fibrosis caused by coronavirus disease-19 (COVID-19).

[0050] A composition according to any one of the preceding specific examples, wherein said region F is a dimer composed of two polypeptide chains, characterized in that one end of L is linked to only one of said two polypeptide chains.

[0051] Another embodiment of the present invention is a method for preventing or treating a lung disease, comprising administering the peptide or a composition comprising the peptide to a subject in need thereof.

[0052] Another embodiment of the present invention is the use of the peptide or a composition comprising the same in the manufacture of a medicament for preventing or treating lung diseases.

[0053] Another embodiment of the present invention is the use of the peptide or a composition comprising the same for the prevention or treatment of lung diseases.

[0054]

[0055] The triple active agent or its sustained-release conjugate according to the present invention has activity against glucagon receptors, GLP-1 (Glucagon-like peptide-1) receptors, and GIP (Glucose-dependent insulinotropic polypeptide) receptors, and can exhibit a preventive or therapeutic effect on lung diseases.

[0056]

[0057] Figure 1 is a diagram showing in vivo changes in the level of inflammatory cytokine expression in lung tissue following treatment with a triple-activated, sustained-release conjugate.

[0058] Figure 2 is a diagram showing the in vivo improvement effect of emphysema according to treatment with a triple-active agent sustained-release complex.

[0059] Figure 3 is a diagram confirming the change in the expression level of myofibroblast differentiation markers (α-SMA, collagen1α1, fibronectin) in lung fibroblasts (MRC5 cells) according to treatment with a triple-activator sustained-release complex.

[0060] Figure 4 is a diagram confirming the change in the expression level of epithelial mesenchymal transition (EMT) markers (collagen1α1, collagen1α3) in lung alveolar epithelial cells (A549 cells) according to treatment with a triple-activated persistent complex.

[0061] Figure 5 is a diagram confirming in vivo the effect of improving fibrosis in lung tissue of BLM mice according to treatment with a triple-active agent sustained-release complex.

[0062] Figure 6 is a diagram confirming the change in survival rate of BLM mice according to treatment with a triple-activated persistent complex.

[0063]

[0064] Hereinafter, the present invention will be described in more detail.

[0065] Meanwhile, each description and embodiment disclosed herein can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the specific descriptions described below.

[0066]

[0067] Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as the generally accepted three-letter codes for other amino acids, such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine), and α-methyl-glutamic acid. Additionally, amino acids referred to herein by abbreviation are described according to the IUPAC-IUB nomenclature.

[0068]

[0069] Alanine Ala, A arginine Arg,R

[0070] Asparagine Asn, N Aspartic acid Asp, D

[0071] Cysteine ​​Cys, C Glutamic acid Glu, E

[0072] Glutamine Gln, Q Glycine Gly, G

[0073] Histidine His, H Isoleucine Ile, I

[0074] Leucine Leu, L Lysine Lys, K

[0075] Methionine Met, M Phenylalanine Phe, F

[0076] Proline Pro, P Serine Ser, S

[0077] Threonine Thr, T Tryptophan Trp, W

[0078] Tyrosine Tyr, Y Valine Val, V

[0079]

[0080] One embodiment of the present invention is a pharmaceutical composition for preventing or treating lung disease, comprising a peptide having activity against a glucagon receptor, a GLP-1 (Glucagon-like peptide-1) receptor, and a GIP (Glucose-dependent insulinotropic polypeptide) receptor.

[0081] In one embodiment, the peptide may comprise an amino acid sequence of any one of SEQ ID NOs: 1 to 102.

[0082] In another embodiment, the pharmaceutical composition for preventing or treating the lung disease may be a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a pharmaceutically effective amount of a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 102.

[0083]

[0084] The above “peptide having activity against glucagon receptors, GLP-1 receptors, and GIP receptors” may also be used interchangeably as a “triple activator” in the present invention.

[0085] These peptides include a variety of substances, such as various peptides, that have significant levels of activity against glucagon, GLP-1, and GIP receptors.

[0086] Although not particularly limited thereto, the triple activator having a significant level of activity against the glucagon, GLP-1, and GIP receptors has an in vitro activity against one or more of the glucagon, GLP-1, and GIP receptors, specifically two or more receptors, and more specifically all three receptors, of about 0.001% or more, about 0.01% or more, about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, compared to the native ligand of the corresponding receptor (native glucagon, native GLP-1, and native GIP). It can represent approximately 90% or more, approximately 100% or more, but the range of significant increases is included without limitation.

[0087] Here, the activity for the receptor may be, for example, a case where the in vitro activity for the receptor is 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or about 200% or more compared to the native type. However, it is not limited thereto.

[0088] In the present invention, the term "about" includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges of values ​​equal to or similar to the value following the term "about," but is not limited thereto.

[0089]

[0090] The method for measuring the in vitro activity of this triple activator can refer to Experimental Example 1 of the present specification, but is not particularly limited thereto.

[0091]

[0092] Meanwhile, the peptide is characterized by possessing one or more, two or more, specifically three, activities among the following i) to iii), and specifically by possessing significant activities:

[0093] i) activation of GLP-1 receptors; ii) activation of glucagon receptors; and iii) activation of GIP receptors.

[0094] Here, activating a receptor means, for example, that the in vitro activity for the receptor is about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more compared to the native type. However, the present invention is not limited thereto.

[0095]

[0096] Additionally, the peptide may have an increased half-life in the body compared to any one of natural GLP-1, natural glucagon, and natural GIP, but is not particularly limited thereto.

[0097]

[0098] Although not particularly limited thereto, such peptides may be non-naturally occurring.

[0099] The above peptide may be an analog of native glucagon, but is not particularly limited thereto. Specifically, the native glucagon analog includes a peptide having one or more differences in amino acid sequence compared to native glucagon, a peptide modified through modification of the native glucagon sequence, and a mimic of native glucagon.

[0100] Meanwhile, although not particularly limited thereto, native glucagon may have the following amino acid sequence:

[0101] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 118)

[0102] Specifically, the peptide may be an analog of native glucagon, but is not particularly limited thereto, in which at least one amino acid in the native glucagon sequence has been modified by a modification selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0103] Additionally, the substitution of the above amino acid includes both substitution with an amino acid or substitution with a non-natural compound.

[0104] Additionally, the addition may be made to the N-terminus and / or C-terminus of the peptide. Meanwhile, the length of the added amino acids is not particularly limited, and 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more amino acids may be added, broadly including, but not particularly limited to, the addition of a polypeptide.

[0105] More specifically, the peptide may be a peptide in which 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 amino acids are substituted with other amino acids selected from the group consisting of 1, 2, 3, 7, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 amino acids in the natural glucagon amino acid sequence, and may also independently or additionally have 1 or more, 2 or more, 3 or more amino acids substituted at the C-terminus thereof. It may have 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more amino acids added, but is not particularly limited thereto.

[0106] More specifically, the peptide may be a peptide in which 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 amino acids are substituted with other amino acids selected from the group consisting of 1, 2, 3, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 amino acids in the natural glucagon amino acid sequence, and may also independently or additionally have 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, It may have 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more amino acids added, but is not particularly limited thereto.

[0107] More specifically, the peptide may be a peptide in which 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 amino acids are substituted with other amino acids selected from the group consisting of 1, 2, 3, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 28, and 29 amino acids in the natural glucagon amino acid sequence, and may also independently or additionally have 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, Or it may have 11 or more amino acids added, but is not particularly limited to this.

[0108] More specifically, the peptide may be one in which 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or 14 amino acids selected from the group consisting of 1, 2, 13, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 amino acids in the natural glucagon amino acid sequence are substituted with other amino acids, and may also have 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more amino acids added to the C-terminus thereof independently or additionally, but is not particularly limited thereto.

[0109] The amino acids introduced above may be selected from the group consisting of tyrosine, alpha-methyl-glutamic acid, Aib, methionine, glutamic acid, histidine, lysine, leucine, isoleucine, glutamine, valine, glycine, alanine, cysteine, serine, alanine, aspartic acid, and arginine, but are not particularly limited thereto.

[0110] For example, the added amino acid sequence may be one or more amino acid sequences derived from a native GLP-1, native GIP, or native exendin-4 amino acid sequence.

[0111] These peptides may contain an intramolecular bridge (e.g., a covalent bridge or a non-covalent bridge), and may be in the form of a ring, for example, but not limited to, a ring formed between amino acids 16 and 20 of the peptide.

[0112] Non-limiting examples of the above rings may include lactam bridges (or lactam rings).

[0113] Additionally, the above peptides include all those modified to include an amino acid capable of forming a ring at a desired position, such that the peptide includes a ring.

[0114] For example, the 16th and 20th amino acid pairs of the peptide may be substituted with glutamic acid or lysine, respectively, which can form a ring, but are not limited thereto.

[0115] Such rings may be formed between amino acid side chains within the peptide, for example, a lactam ring may be formed between the side chain of lysine and the side chain of glutamic acid, but is not particularly limited thereto.

[0116] Examples of peptides produced by a combination of these methods include, but are not limited to, peptides having at least one amino acid sequence different from that of native glucagon and having the alpha-carbon of the N-terminal amino acid residue removed, and having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor, and the like, and a combination of various methods for producing analogs can produce peptides applicable to the present invention.

[0117] In addition, but not particularly limited thereto, the peptide of the present invention may have some amino acids substituted with other amino acids or non-natural compounds to avoid recognition by enzymes that decompose the active substance in order to increase the half-life in the body.

[0118] Specifically, it may be a peptide that increases the half-life in the body by avoiding the recognition action of a decomposing enzyme through a substitution of the second amino acid sequence in the amino acid sequence of the peptide, but amino acid substitutions or changes for avoiding the recognition action of a decomposing enzyme in the body are included without limitation.

[0119] Furthermore, such modifications for peptide production include modifications using L- or D-amino acids, and / or non-natural amino acids; and / or modifications of the native sequence, for example, modification of side chain functional groups, intramolecular covalent bonds, such as ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexanoic acid oxidation, biotinylation, etc.

[0120] Additionally, it includes all those in which one or more amino acids are added to the amino and / or carboxyl termini of native glucagon.

[0121] The amino acids substituted or added above can include the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides containing these amino acids and their typical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company or Bachem in the United States, or Anygen in Korea.

[0122] Amino acid derivatives can also be obtained in a similar manner, examples of which include 4-imidazoacetic acid.

[0123] In addition, the peptide according to the present invention may be modified in such a way that its N-terminus and / or C-terminus, etc. are chemically modified or protected with an organic group, or an amino acid is added to the peptide terminus, etc., to protect it from protein cleavage enzymes in the body and increase its stability.

[0124] In particular, in the case of chemically synthesized peptides, since the N- and C-terminals are charged, the N-terminus may be acetylated and / or the C-terminus amidated to remove the charge, but is not particularly limited thereto.

[0125] Additionally, the peptide according to the present invention includes the peptide itself, a salt thereof (e.g., a pharmaceutically acceptable salt of the peptide), or a solvate thereof. Furthermore, the peptide may be in any pharmaceutically acceptable form.

[0126] The type of the above salt is not particularly limited. However, it is preferable that it be in a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.

[0127] The above term, “pharmaceutically acceptable” means a substance that can be effectively used for the intended purpose without causing excessive toxicity, irritation, or allergic reaction within the scope of pharmaceutical judgment.

[0128] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0129] Additionally, the term "solvate" used in the present invention refers to a peptide according to the present invention or a salt thereof formed in a complex with a solvent molecule.

[0130]

[0131] As a specific example, the peptide may comprise an amino acid sequence represented by the following general formula 1.

[0132] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16- Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO: 103)

[0133] In the above general formula 1,

[0134] Xaa1 is histidine, 4-imidazoacetyl, or tyrosine,

[0135] Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib,

[0136] Xaa3 is glutamic acid or glutamine,

[0137] Xaa7 is threonine or isoleucine,

[0138] Xaa10 is leucine, tyrosine, lysine, cysteine, or valine,

[0139] Xaa12 is lysine, serine, or isoleucine,

[0140] Xaa13 is glutamine, tyrosine, alanine, or cysteine,

[0141] Xaa14 is leucine, methionine, or tyrosine,

[0142] Xaa15 is cysteine, aspartic acid, glutamic acid, or leucine,

[0143] Xaa16 is glycine, glutamic acid, or serine,

[0144] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine,

[0145] Xaa18 is alanine, glutamine, arginine, or histidine,

[0146] Xaa19 is alanine, glutamine, cysteine, or valine,

[0147] Xaa20 is lysine, glutamine, or arginine,

[0148] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid,

[0149] Xaa23 is isoleucine or valine,

[0150] Xaa24 is alanine, glutamine, cysteine, asparagine, aspartic acid, or glutamic acid,

[0151] Xaa27 is valine, leucine, or lysine,

[0152] Xaa28 is cysteine, lysine, alanine, asparagine, or aspartic acid,

[0153] Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine,

[0154] Xaa30 is cysteine, glycine, lysine, or histidine, or is absent;

[0155] R1 is cysteine, GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent;

[0156] Here,

[0157] m is -Cys-, -Pro-, or -Gly-Pro-,

[0158] n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or absent.

[0159] Examples of the triple activator include, but are not limited to, one comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 102, one comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, 13 to 102, and one (essentially) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, 13 to 102.

[0160] In another specific example, the triple activator may be (essentially) composed of, but is not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97 and 100.

[0161] In another specific example, the triple activator may be (essentially) composed of, but is not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 66, 67, 77, 96, 97 and 100.

[0162] In another specific example, the triple activator may be (essentially) composed of, but is not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77 and 96.

[0163] In addition, even if a peptide is described as being 'composed of' a specific sequence number in the present application, if it has the same or corresponding activity as a peptide composed of the amino acid sequence of the sequence number, it does not exclude meaningless sequence additions before and after the amino acid sequence of the sequence number, mutations that may occur naturally, or silent mutations thereof, and it is clear that even if it has such sequence additions or mutations, it falls within the scope of the present application.

[0164] The above may be applied to other specific examples or other aspects of the present invention, but is not limited thereto.

[0165] Specifically, in the general formula 1, Xaa14 may be leucine or methionine, and Xaa15 may be cysteine, aspartic acid, or leucine.

[0166] Examples of such peptides include, but are not limited to, peptides comprising or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, 14 to 17, and 21 to 102.

[0167] These peptides may significantly activate one or more of the glucagon receptor, the GLP-1 receptor, and the GIP receptor, but are not limited thereto. Specifically, they may significantly activate GLP-1, or additionally significantly activate the glucagon receptor and / or the GIP receptor, but are not limited thereto.

[0168] More specifically,

[0169] In the above general formula 1,

[0170] Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib,

[0171] Xaa7 is threonine,

[0172] Xaa10 is tyrosine, cysteine, or valine,

[0173] Xaa12 is lysine or isoleucine,

[0174] Xaa13 is tyrosine, alanine, glutamine, or cysteine,

[0175] Xaa14 is leucine, cysteine, or methionine,

[0176] Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid,

[0177] Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine,

[0178] Xaa18 is alanine, glutamine, arginine, or histidine,

[0179] Xaa19 is alanine, glutamine, valine, or cysteine,

[0180] Xaa20 is lysine, arginine, or glutamine,

[0181] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid,

[0182] Xaa23 is isoleucine or valine,

[0183] Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid,

[0184] Xaa27 may be a peptide, including, but not limited to, leucine or lysine.

[0185] More specifically,

[0186] In the above general formula 1,

[0187] Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib,

[0188] Xaa7 is threonine,

[0189] Xaa10 is tyrosine, cysteine, or valine,

[0190] Xaa12 is lysine or isoleucine,

[0191] Xaa13 is tyrosine, alanine, or cysteine,

[0192] Xaa14 is leucine or methionine,

[0193] Xaa15 is cysteine ​​or aspartic acid,

[0194] Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine,

[0195] Xaa18 is alanine, arginine, or histidine,

[0196] Xaa19 is alanine, glutamine, or cysteine,

[0197] Xaa20 is lysine or glutamine,

[0198] Xaa21 is glutamic acid, cysteine, or aspartic acid,

[0199] Xaa23 is valine,

[0200] Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid,

[0201] Xaa27 may be, but is not limited to, leucine or lysine.

[0202] More specifically,

[0203] In the above general formula 1,

[0204] Xaa2 is alpha-methyl-glutamic acid or Aib,

[0205] Xaa7 is threonine,

[0206] Xaa10 is tyrosine or cysteine,

[0207] Xaa12 is lysine or isoleucine,

[0208] Xaa13 is tyrosine, alanine, or cysteine,

[0209] Xaa14 is leucine or methionine,

[0210] Xaa15 is cysteine ​​or aspartic acid,

[0211] Xaa16 is glutamic acid,

[0212] Xaa17 is arginine, isoleucine, cysteine, or lysine,

[0213] Xaa18 is alanine, arginine, or histidine,

[0214] Xaa19 is alanine, glutamine, or cysteine,

[0215] Xaa20 is lysine or glutamine,

[0216] Xaa21 is glutamic acid or aspartic acid,

[0217] Xaa23 is valine,

[0218] Xaa24 is glutamine, asparagine, or aspartic acid,

[0219] Xaa27 is leucine,

[0220] Xaa28 can be cysteine, alanine, asparagine, or aspartic acid.

[0221] Specifically,

[0222] In the above general formula 1,

[0223] Xaa1 is histidine or 4-imidazoacetyl,

[0224] Xaa2 is alpha-methyl-glutamic acid or Aib,

[0225] Xaa3 is glutamine,

[0226] Xaa7 is threonine,

[0227] Xaa10 is tyrosine,

[0228] Xaa12 is isoleucine,

[0229] Xaa13 is alanine or cysteine,

[0230] Xaa14 is methionine,

[0231] Xaa15 is aspartic acid,

[0232] Xaa16 is glutamic acid,

[0233] Xaa17 is isoleucine or lysine,

[0234] Xaa18 is alanine or histidine,

[0235] Xaa19 is glutamine or cysteine,

[0236] Xaa20 is lysine,

[0237] Xaa21 is aspartic acid,

[0238] Xaa23 is valine,

[0239] Xaa24 is asparagine,

[0240] Xaa27 is leucine,

[0241] Xaa28 is alanine or asparagine,

[0242] Xaa29 is glutamine or threonine,

[0243] Xaa30 may be cysteine, lysine, or absent.

[0244] More specifically,

[0245] In the above general formula 1,

[0246] Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib,

[0247] Xaa3 is glutamine,

[0248] Xaa7 is threonine,

[0249] Xaa10 is tyrosine, cysteine, or valine,

[0250] Xaa12 is lysine,

[0251] Xaa13 is tyrosine,

[0252] Xaa14 is leucine,

[0253] Xaa15 is aspartic acid,

[0254] Xaa16 is glycine, glutamic acid, or serine,

[0255] Xaa17 is glutamine, arginine, cysteine, or lysine,

[0256] Xaa18 is alanine, arginine, or histidine,

[0257] Xaa19 is alanine or glutamine,

[0258] Xaa20 is lysine or glutamine,

[0259] Xaa21 is glutamic acid, cysteine, or aspartic acid,

[0260] Xaa23 is valine,

[0261] Xaa24 is alanine, glutamine, or cysteine,

[0262] Xaa27 is leucine or lysine,

[0263] Xaa29 may be, but is not limited to, glycine, glutamine, threonine, or histidine.

[0264] Such peptides may, but are not particularly limited to, those in which the degree of activation of GLP-1 receptors and glucagon receptors is significant and higher than the degree of activation of GIP receptors; those in which the degrees of activation of GLP-1 receptors, glucagon receptors, and GIP receptors are all significant; and those in which the degrees of activation of GLP-1 receptors and GIP receptors are significant and higher than the degree of activation of glucagon receptors.

[0265] Examples of such peptides include, but are not limited to, peptides comprising or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 21 to 37, 39, 42, 43, 49 to 61, 64 to 83, 85, 86, 88, 89, 91 to 93, 95 to 102.

[0266] In a specific embodiment, the peptide may include an amino acid sequence represented by the following general formula 2.

[0267] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa 17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31- Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General formula 2, SEQ ID NO: 104)

[0268] In the above formula,

[0269] Xaa1 is 4-imidazoacetyl, histidine, or tyrosine;

[0270] Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib;

[0271] Xaa10 is tyrosine or cysteine

[0272] Xaa13 is alanine, glutamine, tyrosine, or cysteine;

[0273] Xaa14 is leucine, methionine, or tyrosine;

[0274] Xaa15 is aspartic acid, glutamic acid, or leucine;

[0275] Xaa16 is glycine, glutamic acid, or serine;

[0276] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;

[0277] Xaa18 is alanine, glutamine, arginine, or histidine;

[0278] Xaa19 is alanine, glutamine, cysteine, or valine;

[0279] Xaa20 is lysine, glutamine, or arginine;

[0280] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

[0281] Xaa23 is isoleucine or valine;

[0282] Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

[0283] Xaa28 is lysine, cysteine, asparagine, or aspartic acid;

[0284] Xaa29 is glycine, glutamine, cysteine, or histidine;

[0285] Xaa30 is cysteine, glycine, lysine, or histidine;

[0286] Xaa31 is proline or cysteine;

[0287] Xaa40 is either cysteine ​​or absent.

[0288] More specifically, in the general formula 2 above,

[0289] Xaa13 is alanine, tyrosine, or cysteine;

[0290] Xaa15 is aspartic acid or glutamic acid,

[0291] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0292] Xaa18 is alanine, arginine, or histidine;

[0293] Xaa21 is cysteine, glutamic acid, glutamine, or aspartic acid;

[0294] Xaa23 is isoleucine or valine;

[0295] Xaa24 is cysteine, glutamine, or asparagine,

[0296] Xaa28 is cysteine, asparagine, or aspartic acid;

[0297] Xaa29 is glutamine, cysteine, or histidine;

[0298] Xaa30 can be cysteine, lysine, or histidine.

[0299]

[0300] Examples of such peptides include, but are not limited to, peptides comprising or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 77, and 95 to 102, more specifically, peptides comprising or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 77, and 96 to 102.

[0301] In a specific embodiment, the peptide may include an amino acid sequence of the following general formula 3.

[0302] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 3, SEQ ID NO: 105),

[0303] In the above general formula 3,

[0304] Xaa1 is histidine or tyrosine;

[0305] Xaa2 is alpha-methyl-glutamic acid or Aib;

[0306] Xaa13 is alanine, tyrosine, or cysteine;

[0307] Xaa17 is arginine, cysteine, or lysine;

[0308] Xaa18 is alanine or arginine;

[0309] Xaa19 is alanine or cysteine;

[0310] Xaa21 is glutamic acid or aspartic acid;

[0311] Xaa24 is glutamine or asparagine,

[0312] Xaa28 is cysteine ​​or aspartic acid;

[0313] Xaa29 is cysteine, histidine, or glutamine;

[0314] Xaa30 is cysteine ​​or histidine;

[0315] Xaa31 is proline or cysteine;

[0316] Xaa40 may be cysteine ​​or absent.

[0317] Examples of such peptides include, but are not limited to, peptides comprising or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 71, 75 to 77, and 96 to 102.

[0318] In addition, in the general formula 1, R1 may be cysteine, GKKNDWKHNIT (SEQ ID NO: 106), CSSGQPPPS (SEQ ID NO: 109), GPSSGAPPPS (SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111), PSSGAPPPS (SEQ ID NO: 112), PSSGAPPPSG (SEQ ID NO: 113), PSSGAPPPSHG (SEQ ID NO: 114), PSSGAPPPSS (SEQ ID NO: 115), PSSGQPPPS (SEQ ID NO: 116), or PSSGQPPPSC (SEQ ID NO: 117), or may be absent, but is not particularly limited thereto.

[0319] Additionally, the peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0320] Specifically, the peptides of the present invention can be prepared using standard synthetic methods, recombinant expression systems, or any other method known in the art. Accordingly, the peptides of the present invention can be synthesized by a number of methods, including, for example, the following:

[0321] (a) a method of synthesizing a peptide stepwise or by fragment assembly by means of a solid-phase or liquid-phase method, and isolating and purifying the final peptide product; or

[0322] (b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture; or

[0323] (c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

[0324] A method for obtaining fragments of a peptide by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0325]

[0326] In addition, the peptide having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor may be in the form of a sustained-release conjugate in which a biocompatible substance is conjugated to the peptide having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor to increase its in vivo half-life. In the present specification, the biocompatible substance may be used in combination with a carrier.

[0327] In the present invention, the conjugate of the peptide can exhibit increased duration of efficacy compared to the peptide not bound to the carrier, and in the present invention, such a conjugate is referred to as a “sustained conjugate.”

[0328] On the other hand, these combinations may be non-naturally occurring.

[0329]

[0330] In one specific example of the present invention, the persistent conjugate may be represented by the following chemical formula 1, but is not limited thereto:

[0331] [Chemical Formula 1]

[0332] X - L - F

[0333] However, at this time, X is a peptide comprising an amino acid sequence of any one of sequence numbers 1 to 102;

[0334] L is a linker containing ethylene glycol repeating units,

[0335] F is an immunoglobulin Fc fragment or a derivative thereof,

[0336] - indicates a covalent bond between X and L, and between L and F.

[0337]

[0338] In the above conjugate, F is a substance capable of increasing the half-life of a peptide having activity against X, i.e., a glucagon receptor, a GLP-1 receptor, and a GIP receptor, specifically a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 102, and corresponds to one component of a moiety constituting the conjugate of the present invention.

[0339] The above F may be bonded to X by a covalent chemical bond or a non-covalent chemical bond, and F and X may be bonded to each other through L by a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.

[0340] Specifically, the L may be a non-peptide linker, for example, a linker containing ethylene glycol repeating units.

[0341] In the present invention, a "non-peptide linker" includes a biocompatible polymer having two or more repeating units bonded together. The repeating units are linked to each other via any covalent bond other than a peptide bond. The non-peptide linker may be a component of a moiety of the conjugate of the present invention, and corresponds to L in the above chemical formula 1.

[0342] The non-peptide linker that can be used in the present invention may be any polymer that is resistant to in vivo proteolytic enzymes, without limitation. In the present invention, the non-peptide linker may be used in combination with a non-peptide polymer.

[0343] Although not particularly limited thereto, the non-peptide linker may be a linker containing ethylene glycol repeating units, for example, polyethylene glycol, and derivatives thereof already known in the art and derivatives that can be easily prepared within the skill level of the art are also included in the scope of the present invention.

[0344] The repeating unit of the above non-peptide linker may be an ethylene glycol repeating unit, and specifically, the non-peptide linker may include an ethylene glycol repeating unit and a functional group used in the production of a conjugate at the terminal. The sustained conjugate according to the present invention may be in a form in which X and F are linked through the functional group, but is not limited thereto. In the present invention, the non-peptide linker may include two or three or more functional groups, and each functional group may be the same or different from each other, but is not limited thereto.

[0345] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following chemical formula 2:

[0346] [Chemical Formula 2]

[0347]

[0348]

[0349] Here, n= 10 to 2400, n= 10 to 480, or n= 50 to 250, but is not limited thereto.

[0350] In the above persistent conjugate, the PEG moiety is -(CH2CH2O) n -Not only the structure but also the connecting elements and this -(CH2CH2O) n - may also include, but is not limited to, intervening oxygen atoms.

[0351] In addition, in one specific embodiment, the complex may be a structure in which a peptide (X) comprising an amino acid sequence of general formula 1 and an immunoglobulin Fc region (F) are covalently linked via a linker containing ethylene glycol repeating units, but is not limited thereto.

[0352] The above polyethylene glycol is a term encompassing, but not limited to, all forms of ethylene glycol homopolymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0353] The non-peptide linker that can be used in the present invention can be used without limitation as long as it is a polymer containing an ethylene glycol repeating unit that is resistant to in vivo proteolytic enzymes. The molecular weight of the non-peptide polymer is in the range of more than 0 to about 100 kDa, in the range of about 1 to about 100 kDa, specifically in the range of about 1 to about 20 kDa, or in the range of about 1 to about 10 kDa, but is not limited thereto. In addition, the non-peptide linker of the present invention that is bound to the polypeptide corresponding to F may use not only one type of polymer but also a combination of different types of polymers.

[0354] In one specific embodiment, both ends of the non-peptide linker can be bonded to an amine group or thiol group of F, e.g., an immunoglobulin Fc region, and an amine group or thiol group of X, respectively.

[0355] Specifically, the non-peptide polymer may include, but is not limited to, a reactive group capable of binding to F (e.g., an immunoglobulin Fc region) and X at both ends, specifically, a reactive group capable of binding to X, or an amine group located at the N-terminus of F (e.g., an immunoglobulin Fc region) or lysine, or a thiol group of cysteine.

[0356] Additionally, the reactive group of the non-peptide polymer capable of binding to F, for example, an immunoglobulin Fc region and X, may be selected from the group consisting of an aldehyde group, a maleimide group and a succinimide derivative, but is not limited thereto.

[0357] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0358] In the above, succinimidyl derivatives may include, but are not limited to, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxy succinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate.

[0359] Non-peptide linkers may be linked to X and F via these reactive groups, but are not particularly limited thereto.

[0360] Furthermore, the final product formed by reductive amination via an aldehyde bond is much more stable than that formed via an amide bond. The aldehyde reactive group reacts selectively at the N-terminus at low pH, and can form a covalent bond with lysine residues at high pH, ​​such as pH 9.0.

[0361] In addition, the reactive groups at both ends of the non-peptide linker may be the same or different from each other, for example, one end may have a maleimide group, and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group. However, as long as F, specifically an immunoglobulin Fc region and X, can be bound to each end of the non-peptide linker, it is not particularly limited thereto.

[0362] For example, one end of the non-peptide linker may include a maleimide group as a reactive group, and the other end may include an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like.

[0363] When polyethylene glycol having hydroxyl reactive groups at both terminals is used as a non-peptide polymer, the sustained protein conjugate of the present invention can be prepared by activating the hydroxyl groups into the various reactive groups through a known chemical reaction, or by using polyethylene glycol having a commercially available modified reactive group.

[0364] In one specific embodiment, the non-peptide polymer may be linked to a cysteine ​​residue of X, more specifically, but not limited to, a -SH group of the cysteine.

[0365]

[0366] For example, the non-peptide polymer may be linked to cysteine ​​residue 10, cysteine ​​residue 13, cysteine ​​residue 15, cysteine ​​residue 17, cysteine ​​residue 19, cysteine ​​residue 21, cysteine ​​residue 24, cysteine ​​residue 28, cysteine ​​residue 29, cysteine ​​residue 30, cysteine ​​residue 31, cysteine ​​residue 40, or cysteine ​​residue 41 in the peptide corresponding to X, but is not particularly limited thereto.

[0367] Specifically, a reactive group of a non-peptide polymer can be linked to the -SH group of the cysteine ​​residue, and all of the above-described reactive groups are applicable. If maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X via a thioether bond, and the aldehyde group can be linked to F, specifically, the -NH2 group of immunoglobulin Fc, via a reductive amination reaction, but is not limited thereto, and this is one example.

[0368] Additionally, in the above conjugate, the reactive group of the non-peptide polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, but this is only one example.

[0369]

[0370] Meanwhile, the above F may be an immunoglobulin Fc region, and more specifically, the immunoglobulin Fc region may be derived from IgG, but is not particularly limited thereto.

[0371] In the present invention, the "immunoglobulin Fc region" refers to a region including the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3) portion, excluding the heavy chain and light chain variable regions of an immunoglobulin. The immunoglobulin Fc region may be a component forming a moiety of the complex of the present invention. The immunoglobulin Fc region may be used interchangeably with "immunoglobulin Fc fragment."

[0372] In this specification, the term Fc region includes not only the native sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, for example, sequences in which one or more amino acid residues in the native sequence are altered by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, thereby becoming different from the native sequence.

[0373] The above F is a structure in which two polypeptide chains are linked by a disulfide bond, and may be a structure in which only the nitrogen atom of one of the two chains is linked, but is not limited thereto. The linkage via the nitrogen atom may be linked through reductive amination to the epsilon amino atom of lysine or the N-terminal amino group.

[0374] A reductive amination reaction is a reaction in which an amine group or amino group of a reactant reacts with an aldehyde of another reactant (i.e., a functional group capable of reductive amination) to produce an amine, and then an amine bond is formed through a reduction reaction. It is an organic synthesis reaction widely known in the relevant technical field.

[0375] In one specific example, the F may be linked via the nitrogen atom of the N-terminal proline, but is not limited thereto.

[0376] The above immunoglobulin Fc region is a component forming a moiety of the complex of the chemical formula 1 of the present invention, and specifically, may correspond to F in the chemical formula 1.

[0377]

[0378] Such immunoglobulin Fc regions may include, but are not limited to, a hinge region in the heavy chain constant region.

[0379] In the present invention, the immunoglobulin Fc region may include a specific hinge sequence at the N-terminus.

[0380] The term "hinge sequence" of the present invention refers to a region located in the heavy chain that forms a dimer of the immunoglobulin Fc region through an inter disulfide bond.

[0381] In the present invention, the hinge sequence may be mutated to have only one cysteine ​​residue by deleting a portion of the hinge sequence having the following amino acid sequence, but is not limited thereto:

[0382] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 119).

[0383] The above hinge sequence may be one in which the 8th or 11th cysteine ​​residue of the hinge sequence of SEQ ID NO: 119 is deleted, thereby containing only one cysteine ​​residue. The hinge sequence of the present invention may be composed of 3 to 12 amino acids, including only one cysteine ​​residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequences: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 120), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 121), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 122), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 123), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 124), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 125), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 126), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 127), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 128), Pro-Ser-Cys-Pro (SEQ ID NO: 129), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 130), Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 131), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 132), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 133), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 134), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 135), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 136), Glu-Pro-Ser-Cys (SEQ ID NO: 137), Ser-Cys-Pro (SEQ ID NO: 138).

[0384] More specifically, the hinge sequence may include, but is not limited to, the amino acid sequence of SEQ ID NO: 129 (Pro-Ser-Cys-Pro) or SEQ ID NO: 138 (Ser-Cys-Pro).

[0385] The immunoglobulin Fc region of the present invention may be in the form of two immunoglobulin Fc chain molecules forming a dimer due to the presence of a hinge sequence, and further, the conjugate of chemical formula 1 of the present invention may be in the form of one end of a linker being connected to one chain of the immunoglobulin Fc region of the dimer, but is not limited thereto.

[0386] The term "N-terminus" of the present invention refers to the amino terminus of a protein or polypeptide, and may include the most amino acid, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the most amino acid. The immunoglobulin Fc region of the present invention may include a hinge sequence at the N-terminus, but is not limited thereto.

[0387]

[0388] In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region including part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1) of an immunoglobulin, excluding only the heavy and light chain variable regions, as long as it has substantially the same or improved effects as the native type. It may also be a region in which a relatively long portion of the amino acid sequence corresponding to CH2 and / or CH3 is removed.

[0389] For example, the immunoglobulin Fc region of the present invention may be a dimer of 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains from among the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a portion of a hinge region), or 6) a heavy chain constant region and a light chain constant region. However, the present invention is not limited thereto.

[0390] In addition, as one embodiment of the sustained-release complex of the present invention, the immunoglobulin Fc region F is a dimer composed of two polypeptide chains, wherein the Fc region dimer F and X are covalently linked via one and the same linker L containing an ethylene glycol repeating unit. In one specific example of this embodiment, X is covalently linked via linker L to only one of the two polypeptide chains of the Fc region dimer F. In a more specific example of this embodiment, only one molecule of X is covalently linked via L to one of the two polypeptide chains of the Fc region dimer F to which X is linked. In the most specific example of this embodiment, the F is a homodimer.

[0391] In another embodiment of the sustained binding agent of the present invention, it is also possible for two molecules of X to symmetrically bind to one Fc region in a dimeric form. In this case, the immunoglobulin Fc and X may be linked to each other by a non-peptide linker. However, the present invention is not limited to the examples described above.

[0392] In addition, the immunoglobulin Fc region of the present invention includes not only a native amino acid sequence but also a sequence derivative thereof. An amino acid sequence derivative means a sequence having a different sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues in the native amino acid sequence.

[0393] For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322 or 327 to 331, which are known to be important for binding, can be used as suitable sites for modification.

[0394] In addition, various types of derivatives are possible, such as those in which the site capable of forming disulfide bonds is removed, several amino acids at the N-terminus of the native Fc are removed, or a methionine residue is added to the N-terminus of the native Fc. In addition, the complement binding site, for example, the C1q binding site, or the ADCC (antibody dependent cell mediated cytotoxicity) site may be removed to eliminate the effector function. Techniques for producing such sequence derivatives of the immunoglobulin Fc region are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478, etc.

[0395] Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in the art (H.Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation may also occur.

[0396] The Fc derivative described above exhibits biological activity equivalent to the Fc region of the present invention and may have increased structural stability of the Fc region against heat, pH, etc.

[0397] In addition, such Fc region may be obtained from a natural type isolated from an animal such as a human, cow, goat, pig, mouse, rabbit, hamster, rat or guinea pig, or may be a recombinant or a derivative thereof obtained from a transformed animal cell or microorganism. Here, the method for obtaining from a natural type may be a method of isolating the entire immunoglobulin from a human or animal body and then treating it with a protease to obtain it. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated using size-exclusion chromatography or the like. In a more specific embodiment, the Fc region of human origin is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0398] In addition, the immunoglobulin Fc region may have native sugar chains, sugar chains with increased sugar chains compared to the native form, sugar chains with decreased sugar chains compared to the native form, or sugar chains removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or remove the sugar chains of the immunoglobulin Fc. Here, the immunoglobulin Fc region from which the sugar chains have been removed has a significantly reduced binding affinity to complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, so it does not induce unnecessary immune responses in vivo. In this respect, the form that is more suitable for the original purpose as a drug carrier is an immunoglobulin Fc region from which the sugar chains have been removed or deglycosylated.

[0399] In the present invention, “deglycosylation” refers to an Fc region from which sugars have been removed by an enzyme, and aglycosylation refers to an Fc region that has not been glycosylated and produced in a prokaryotic animal, or in a more specific embodiment, in E. coli.

[0400] Meanwhile, the immunoglobulin Fc region may be of animal origin such as human or cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., and in a more specific embodiment, is of human origin.

[0401] Additionally, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to enhance the half-life of a ligand binding protein. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0402] In addition, in one specific embodiment, the immunoglobulin Fc fragment is a fragment of human IgG4 Fc, and may be in the form of a homodimer in which two monomers are linked via a disulfide bond (inter-chain form) between cysteines at amino acid position 3 of each monomer, wherein each monomer of the homodimer independently has / can have an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, i.e., two internal disulfide bonds (intra-chain form). The number of amino acids in each monomer may be composed of 221 amino acids, and the amino acids forming the homodimer may be composed of a total of 442 amino acids, but is not limited thereto. Specifically, the immunoglobulin Fc fragment is a homodimer formed by two monomers having an amino acid sequence of SEQ ID NO: 139 (consisting of 221 amino acids) through a disulfide bond between cysteine, which is the 3rd amino acid of each monomer, and the monomers of the homodimer may independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, but are not limited thereto.

[0403] Meanwhile, in the present invention, the term "combination" in relation to the immunoglobulin Fc region means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of different origin. That is, it is possible to produce a dimer or multimer from two or more fragments selected from the group consisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE.

[0404] In the present invention, the term "hybrid" means that a sequence corresponding to two or more different origin immunoglobulin Fc fragments exists within a single-chain immunoglobulin constant region. In the present invention, various types of hybrids are possible. That is, a hybrid of domains consisting of one to four domains from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc is possible, and may include a hinge.

[0405] Meanwhile, IgG can also be divided into subclasses of IgG1, IgG2, IgG3, and IgG4, and combinations or hybridizations of these are also possible in the present invention. Specifically, the subclasses are IgG2 and IgG4, and most specifically, the Fc fragment of IgG4, which has almost no effector function such as complement-dependent cytotoxicity (CDC).

[0406] In addition, the above-described conjugate may have an increased duration of effect compared to natural GLP-1, GIP, or glucagon, or compared to X in which F is not modified, and such conjugate includes, but is not limited to, not only the above-described forms but also forms encapsulated in biodegradable nanoparticles.

[0407]

[0408] In addition, the above-described conjugate may have an increased duration of effect compared to natural GLP-1, GIP, or glucagon, or compared to a form in which F is not modified, and such conjugate includes not only the above-described form but also a form encapsulated in a biodegradable nanoparticle.

[0409]

[0410] A composition comprising the peptide (e.g., the peptide itself or a sustained-release conjugate thereof to which a biocompatible material is bound) may be used for the prevention or treatment of lung diseases.

[0411] In the present invention, the term "prevention" means any act of inhibiting or delaying the onset of a lung disease by administering the peptide (e.g., the peptide itself or a sustained-release conjugate form to which a biocompatible material is bound) or a composition containing the same, and "treatment" means any act of improving or benefiting the symptoms of a lung disease by administering the peptide (e.g., the peptide itself or a sustained-release conjugate form to which a biocompatible material is bound) or a composition containing the same.

[0412] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any appropriate method, and the route of administration of the composition is not particularly limited thereto, but the composition may be administered through any general route that can reach the target in the body, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0413]

[0414] The use of the triple activator or long-acting conjugate thereof, which is active at all of the glucagon, GLP-1, and GIP receptors of the present invention, has the great advantage of reducing the number of administrations required for chronic patients who require daily administration due to a dramatic increase in blood half-life and in vivo sustained effect, thereby improving the quality of life of patients, and thus is of great help in the treatment of pulmonary diseases. Furthermore, the triple activator or long-acting conjugate thereof has a preventive effect on pulmonary diseases, such as delaying the recurrence of symptoms of pulmonary diseases, and / or has an effect of significantly reducing symptoms of pulmonary diseases, and thus is of great help in the prevention and / or treatment of pulmonary diseases.

[0415]

[0416] In the present invention, the term "pulmonary disease" means all diseases in which abnormalities occur in the tissue or function of the lung, and specifically, since the triple active agent of the present invention or a long-acting combination thereof can inhibit inflammation and fibrosis reactions, the lung disease for the purpose of the present invention is a disease accompanied by lung inflammation and fibrosis or a disease according to the progression of fibrosis, for example, interstitial lung disease (ILD), progressive fibrosing interstitial lung disease (PF-ILD), idiopathic interstitial pneumonias (IIP), non-specific interstitial pneumonia (NSIP), pulmonary fibrosis, fibrosing interstitial lung diseases (FILD), idiopathic pulmonary fibrosis (IPF), alveolitis, pneumonia, These may include, but are not limited to, emphysema, bronchitis, chronic obstructive pulmonary disease, combined pulmonary fibrosis and emphysema (CPFE), asthma, or respiratory tract infections.

[0417]

[0418] The triple activator of the present invention may exhibit a preventive or therapeutic effect on lung diseases by inhibiting the activity of macrophages, which are the main mechanism of lung inflammation, and inhibiting the differentiation of fibroblasts into myofibroblasts and the epithelial-mesenchymal transition of alveolar epithelial cells, which are the main mechanisms of pulmonary fibrosis, but is not limited thereto.

[0419] Specifically, the pharmaceutical composition of the present invention may suppress inflammation by (i) inhibiting macrophage activity and / or (ii) reducing the expression of IL-1β, IL-6, IL-12, or TNF-α upon administration, but is not limited thereto. In addition, but not limited thereto, the pharmaceutical composition of the present invention may have one or more of the following properties upon administration:

[0420] (i) Inhibition of myofibroblast differentiation;

[0421] (ii) decreased expression of α-SMA, collagen1α1, or fibronectin;

[0422] (iii) Inhibition of epithelial mesenchymal transition (EMT) of alveolar epithelial cells;

[0423] (iv) Reduction in expression of collagen1α1 or collagen1α3. Through this, the pharmaceutical composition of the present invention may inhibit fibrosis, but is not limited thereto.

[0424]

[0425] In the inflammatory response in the lungs, alveolar macrophages are involved, and the inflammatory cytokines (IL-1, IL-6, TNF-α) secreted by these macrophages attract neutrophils and secrete proteases. In particular, elastase is secreted, which breaks down elastin, a type of fiber that constitutes lung tissue and is involved in elasticity. It is known that elastin, the elastic tissue of the alveoli, is damaged by elastase, which ultimately damages the alveoli and leads to tissue fibrosis. Inflammation and fibrosis in the lungs are lung diseases in themselves, but the progression and aggravation of inflammation and fibrosis can lead to other lung diseases. Therefore, suppressing inflammation and fibrosis is required for the prevention and treatment of lung diseases.

[0426]

[0427] The properties of the triple active agent of the present invention as described above mean that it has an effect of inhibiting and improving inflammation and fibrosis of the lungs, and this also suggests a preventive or therapeutic effect on lung diseases accompanied by inflammation or fibrosis of the lungs.

[0428]

[0429] In the present invention, the term chronic obstructive pulmonary disease (COPD) refers to a disease that exhibits irreversible airway obstruction, and is known to exhibit destruction of lung parenchyma and pulmonary fibrosis due to chronic inflammation and damage. Infiltration of various inflammatory cells is observed in the airways of patients with COPD, and the number of macrophages, in particular, is known to increase with the severity of the disease, suggesting that inflammation plays a key role in the pathogenesis of COPD. When harmful substances are inhaled, the innate immune system responds, causing epithelial cells to secrete numerous inflammatory mediators, which activate alveolar macrophages and neutrophils. Therefore, controlling the inflammatory response is a crucial factor in the treatment of COPD. COPD is generally categorized into two types: emphysema and chronic bronchitis. However, it is known that emphysema and chronic bronchitis often coexist in patients.

[0430]

[0431] In the present invention, the term chronic bronchitis refers to a respiratory disease in which chronic inflammation occurs in the bronchial tubes, causing hypertrophy and increase in the mucus glands in the airway or structural damage due to inflammation, resulting in thickening of the mucus layer and decreased ciliary movement, thereby blocking the flow of air and causing breathing difficulties, etc.

[0432]

[0433] In the present invention, the term emphysema refers to a disease in which inflammation occurs in the bronchi or lungs due to various causes, which causes protease to be secreted, the basic skeleton of the alveoli to be destroyed, and the blood vessel structure to be damaged, resulting in loss of gas exchange function.

[0434]

[0435] The term "alveolitis" in this invention refers to a disease in which the alveoli, responsible for oxygen exchange, become inflamed. Mold, dust, petroleum, toluene, acetone, chemicals, etc. can cause macrophages to accumulate within the alveoli, thickening the alveolar septa, which can progress to alveolitis. As alveolitis progresses, the alveoli are destroyed, scarred, hardened, and breathing difficulties occur.

[0436]

[0437] As used herein, the term "asthma" refers to an inflammatory airway obstructive disease that causes breathing difficulties due to airway inflammation, unlike chronic obstructive pulmonary disease (COPD). The airways of asthmatic patients are known to secrete large amounts of cytokines such as IL-4, IL-5, and IL-13, and to exhibit significant proliferation and hypertrophy of bronchial smooth muscle. As with COPD, controlling the inflammatory response is crucial for treatment.

[0438]

[0439] The term "pneumonia" in the present invention refers to a disease in which inflammation occurs in the parenchyma or alveoli of the lungs. It is a disease with a high risk of various complications, and is caused by bacterial infection, viruses, protozoa, fungi, and chemicals. It is known that pneumonia occurs when the immune mechanism against pathogens in the respiratory organs (typically alveolar macrophages) does not function properly, or when the level of pathogens exceeds the limit that can be defended by the normal immune mechanism. It is known that patients with chronic lung diseases such as asthma, chronic obstructive pulmonary disease, emphysema, and bronchiectasis are more likely to develop pneumonia. In the present invention, pulmonary inflammation or pneumonia may include, but is not limited to, pulmonary inflammation or pneumonia caused by or accompanying other lung diseases.

[0440]

[0441] As used herein, the term "respiratory infectious disease" refers to a respiratory disease caused by infection with a pathogen (such as a virus, bacteria, or fungus). Respiratory infectious diseases can be categorized based on the pathogen causing them. Representative causes of respiratory infections include respiratory viruses, bacteria, mycoplasma, and fungi. Respiratory infectious diseases caused by the above pathogens are accompanied by inflammation, and therefore, treatment effects can be expected through improvement of inflammation.

[0442] The term "respiratory viral infection" in the present invention refers to a respiratory disease caused by a pathogenic viral infection, which can cause anything from mild upper respiratory tract infection to severe lower respiratory tract infection with pneumonia and bronchitis. Respiratory viral infections are known to be fatal in people with compromised cardiopulmonary function. Respiratory viral infection causes inflammation in the respiratory tract, including the lungs, and if this inflammation is not suppressed for a long period of time, it can lead to fibrosis, which can lead to more serious lung diseases. Therefore, it is important to treat respiratory viral infection while suppressing inflammation and fibrosis.

[0443] The respiratory virus causing the respiratory viral infection disease may be, but is not limited to, adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella Zoster virus, measles virus, respiratory syncytial virus, dengue virus, human immunodeficiency virus (HIV), influenza virus, coronavirus, severe acute respiratory syndrome associated virus (SARS-associated virus), or middle east respiratory syndrome coronavirus (MERS-CoV).

[0444] A non-limiting example of the above coronavirus is SARS-CoV-2, infection with which can cause coronavirus disease 2019 (COVID-19).

[0445] The term coronavirus disease 2019 (COVID-19) in the present invention refers to a viral infectious disease caused by infection with a coronavirus (2019-nCoV or SARS-CoV-2). Although the exact source and route of infection have not yet been identified, its highly contagious nature has caused a global pandemic. Coronaviruses are RNA viruses with a genetic size of 27 to 32 kb that can infect humans and various animals, and are known to mainly present with respiratory symptoms such as cough accompanied by fever, difficulty breathing, shortness of breath, and phlegm.

[0446] In particular, the reason severe pneumonia accompanies COVID-19 is because the coronavirus attacks ciliated epithelial cells in the bronchial tract and Type II alveolar epithelial cells (Type II epithelial cells within the alveoli). These cells contain numerous enzyme receptors that facilitate the virus's attachment. Receptors such as ACE2 and TMPRSS2 enhance the virus's ability to penetrate cells.

[0447] Foreign substances and pathogens that penetrate during breathing attach to the mucous membrane of the ciliated epithelial cells of the bronchial tubes. As the name suggests, ciliated epithelial cells possess numerous cilia, which expel pathogens, including coronaviruses, from the mucous membrane toward the mouth and nose. However, ciliary movement alone cannot expel many viruses simultaneously. Furthermore, smoking, dust, dry weather, and low temperatures impair ciliary movement. Because ciliary movement is impaired during dry, cold winters, the risk of influenza virus infections, including COVID-19, increases.

[0448] Like other viruses, coronaviruses hijack the resources and systems of their host cells, multiplying rapidly and releasing themselves from the infected cell. These exponentially multiplying viruses then escape and rapidly invade surrounding healthy ciliated epithelial cells and Type II alveolar epithelial cells. These infected cells secrete cytokines that induce strong inflammation and transform into inflammatory cells.

[0449] The primary function of Type II alveolar epithelial cells is to secrete surfactant, maintaining alveoli turgor while facilitating gas exchange through Type I alveolar epithelial cells. When attacked by the coronavirus, Type II alveolar epithelial cells transform into inflammatory cells, losing their primary function. This causes inflammation (pneumonia) in the lungs, which in turn causes secondary symptoms (fever, cough, shortness of breath, etc.).

[0450]

[0451] The triple activator of the present invention can suppress inflammatory responses by inhibiting macrophage activity and / or reducing the expression level of inflammatory cytokines (IL-1β, IL-6, IL-12, or TNF-α) in lung tissue, and thus can exhibit a preventive or therapeutic effect on lung diseases caused by or accompanied by inflammation (e.g., alveolitis, pneumonia, emphysema, bronchitis, chronic obstructive pulmonary disease, asthma, or respiratory infection diseases).

[0452]

[0453] Meanwhile, fibrosis is a disease that forms excessive fibrous connective tissue in an organ or tissue. Fibrosis refers to a condition in which normal wound healing fails to control itself after tissues are damaged by an inflammatory response caused by various factors (such as infection, chemical irritation, or radiation). In the lungs, in particular, prolonged inflammation that remains untreated often leads to fibrosis, which prevents tissue regeneration and hardens, leading to serious lung disease. Therefore, treating fibrosis requires suppressing inflammation to prevent the progression of fibrosis. Therefore, immunosuppressants (e.g., steroids, cytotoxic agents) are also used. In this study, the term "fibrosis" may be used interchangeably with "fibrosis."

[0454]

[0455] In the present invention, the term pulmonary fibrosis refers to a condition in which proliferation of fibrous connective tissue occurs in the lungs, resulting in destruction of normal lung structure, hardening and devastation of lung tissue. There are parenchymal, interstitial and mixed types, but interstitial pulmonary fibrosis is particularly problematic, in which proliferation of fibrous connective tissue occurs around the alveolar walls and bronchioles. In general, transforming growth factor-β (TGF-β) is produced by various cells such as alveolar macrophages, activated alveolar epithelial cells, fibroblasts and myofibroblasts, and induces fibroblast proliferation and migration of macrophages and fibroblasts, and is known to stimulate the expression of inflammatory and fibrotic cytokines such as TNF-α, PDGF, IL-1β and IL-13, thereby further enhancing the fibrotic response (Proc Am Thorac Soc., 9(3):111-116 (2012)). The pulmonary fibrosis of the present invention may include, but is not limited to, pulmonary fibrosis caused by or accompanying other pulmonary diseases.

[0456] In the present invention, the term combined pulmonary fibrosis and emphysema (CPFE) is a representative disease in which fibrosis coexists with emphysema.

[0457]

[0458] The term interstitial lung disease (ILD) as used herein, also known as diffuse parenchymal lung disease (DPLD), refers to a group of diseases characterized by proliferation of the interstitial compartment of the lung, infiltration of inflammatory cells, and fibrosis, which result in abnormal collagen deposition. The interstitial lung diseases are classified into occupational, environmental, iatrogenic, connective tissue disease, or idiopathic, depending on their cause.

[0459] In the present invention, the term Progressive Fibrosing Interstitial Lung Disease (PF-ILD) refers to a chronic fibrosing interstitial lung disease exhibiting a progressive phenotype, and may include, but is not limited to, autoimmune interstitial lung disease, sclerosis-associated interstitial lung disease, mixed connective tissue disease-associated interstitial lung disease, nonspecific idiopathic interstitial pneumonia, and unclassified idiopathic interstitial pneumonia.

[0460] Among the above interstitial lung diseases, idiopathic interstitial pneumonias (IIP) are lung diseases of unknown cause and are distinguished by their histological form invading the pulmonary interstitium. Representative examples include non-specific interstitial pneumonia (NSIP) and idiopathic pulmonary fibrosis (IPF).

[0461] In the present invention, the term, idiopathic pulmonary fibrosis (IPF), is the most common disease among idiopathic interstitial pneumonias, and is defined as pulmonary fibrosis of unknown cause. It is developed when alveolar epithelial cells are damaged by repeated inflammatory responses due to various exposures, and the wounds do not heal normally, causing fibrosis. It is known that pulmonary fibrosis progresses through a complex interaction among genetic predisposition, environmental factors, and lung infections. Specifically, the pathogenesis is known to include proliferation of fibroblasts / myofibroblasts in the lungs due to various factors secreted from damaged alveolar epithelial cells and infiltrated inflammatory cells, resulting in secretion and accumulation of collagen, and excessive deposition of extracellular matrix (ECM).

[0462]

[0463] The triple active agent of the present invention has one or more of the following properties: (i) inhibition of myofibroblast differentiation; (ii) reduction in expression of α-SMA, collagen1α1, or fibronectin; (iii) inhibition of epithelial mesenchymal transition (EMT) of alveolar epithelial cells; and (iv) reduction in expression of collagen1α1 or collagen1α3, and thus can inhibit and improve fibrosis and exhibit a preventive or therapeutic effect on lung diseases caused by or accompanied by fibrosis (e.g., interstitial lung disease, progressive fibrosing interstitial lung disease, idiopathic interstitial pneumonia, nonspecific interstitial pneumonia, pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, mixed pulmonary fibrosis and emphysema).

[0464]

[0465] Meanwhile, the triple activator according to the present invention can not only exhibit a therapeutic effect on COVID-19, but also, since COVID-19 is known to leave pulmonary fibrosis symptoms as a sequela even after cure, and the triple activator of the present invention exhibits an effect of improving pulmonary fibrosis, the triple activator can exhibit efficacy on pulmonary inflammation and / or pulmonary fibrosis caused by COVID-19.

[0466]

[0467] The pharmaceutical composition of the present invention may be administered additionally with a mucolytic agent or a pharmaceutically acceptable salt thereof, but is not limited thereto. For the purposes of the present invention, the pharmaceutical composition is intended for combined administration of a triple activator and a mucolytic agent, and the triple activator and the mucolytic agent may be administered simultaneously, sequentially, or in reverse order, but is not limited thereto.

[0468] When the term "combination" is used herein, it should be understood to refer to simultaneous, separate, or sequential administration. If sequential or separate administration is used, the interval between administrations of the secondary components should be such that the beneficial effects of the combination are not lost. The combined administration of a triple activator and a mucolytic agent may take the following forms, but is not limited to:

[0469] a) administered as a mixture comprising (i) a triple active agent or a combination thereof and (ii) a mucolytic agent or a pharmaceutically acceptable salt thereof; or

[0470] b) (i) the triple active agent or a combination thereof and (ii) the mucolytic agent or a pharmaceutically acceptable salt thereof are administered in separate forms.

[0471] When the triple active agent and the mucolytic agent or a pharmaceutically acceptable salt thereof are in separate forms, the triple active agent and the mucolytic agent or a pharmaceutically acceptable salt thereof may be formulated as separate preparations and administered simultaneously, separately, sequentially, or in reverse order.

[0472] In the present invention, combined administration should be understood not only to mean simultaneous administration, but also to mean an administration form in which the triple active agent and the mucolytic agent or a pharmaceutically acceptable salt thereof act together on an individual so that each substance can perform a level equivalent to or higher than its original function.

[0473] As a specific example, the triple activator and the mucolytic agent or a pharmaceutically acceptable salt thereof may be mixed and administered in combination as a single formulation, or the triple activator and the mucolytic agent or a pharmaceutically acceptable salt thereof may be formulated separately and administered in combination simultaneously, sequentially, or in reverse order, but is not limited thereto. When formulated separately and administered in combination, each formulation may be administered via different routes, but is not limited thereto. In addition, but is not limited thereto, the triple activator and the mucolytic agent or a pharmaceutically acceptable salt thereof may be formulated separately and included in a single kit. The pharmaceutically acceptable salt of the mucolytic agent includes a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0474] The term "mucolytic agents" in the present invention refers to drugs that promote the secretion, liquefaction, or discharge of sputum, phlegm, or mucus from the respiratory tract, and in particular, refers to drugs that only function to break down mucus in the lungs and thin respiratory secretions. In the present invention, mucolytic agents may be used interchangeably with expectorants.

[0475] Specific examples of the mucolytic agent of the present invention include any one selected from the group consisting of ambroxol, N-acetylcysteine, N-acetylin, carbocysteine, domiodol, fudosteine, bromhexine, erdosteine, letostine, lysozyme, mesna, sobrerol, stepronin, tiopronin, tyloxapol, carbocysteine, dornase alfa, eprazinone, letosteine, neltenexine, and mecysteine. There may be more than one, but it is not limited to these.

[0476]

[0477] Meanwhile, combined administration of a triple activator and a mucolytic agent may exhibit therapeutic effects on respiratory infectious diseases (e.g., coronavirus disease 2019 (COVID-19)). Since the triple activator of the present invention exhibits effects of improving lung inflammation and pulmonary fibrosis, combined administration of a triple activator and a mucolytic agent may exhibit efficacy on respiratory infectious diseases (e.g., coronavirus disease 2019 (COVID-19)) or pulmonary inflammation and pulmonary fibrosis caused by them.

[0478]

[0479] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier or diluent. Such pharmaceutically acceptable carrier or diluent may be non-naturally occurring.

[0480] In the present invention, the term "pharmaceutically acceptable" means a sufficient amount to exhibit a therapeutic effect and not causing side effects, and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to drugs, administration route, administration method, number of administrations, treatment period, and drugs used in combination or simultaneously.

[0481] A pharmaceutical composition comprising the peptide of the present invention may include pharmaceutically acceptable excipients. The excipients are not particularly limited thereto, but may include binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. for oral administration, and may include a mixture of buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections, and may include a base, excipients, lubricants, preservatives, etc. for topical administration.

[0482] The composition of the present invention can be prepared in various forms by mixing it with the pharmaceutically acceptable excipients described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be prepared in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0483] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, preservatives, and the like may be additionally included.

[0484] In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquids, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, lyophilized preparations, and suppositories.

[0485] In addition, the composition is formulated into a unit dosage form suitable for administration into a patient's body according to a conventional method in the pharmaceutical field, specifically, into a form of a preparation useful for administration of a protein drug, and can be administered by a parenteral route including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal, or rectal route using an administration method conventionally used in the art.

[0486] Additionally, the above complex may be used in combination with various carriers acceptable as pharmaceuticals, such as saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers may be used as pharmaceuticals to increase stability or absorbability.

[0487] In another aspect of the present invention, a method for preventing or treating a lung disease is provided, comprising administering to a subject a pharmaceutical composition containing a pharmaceutically effective amount of a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 102 or a sustained-release conjugate thereof.

[0488]

[0489] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of drug as the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, and weight, and the severity of the disease. Specifically, the composition of the present invention may contain a pharmaceutically effective amount of the triple active agent or a sustained-release conjugate comprising the same, but is not limited thereto.

[0490] Including the above peptide or sustained-release conjugate in a pharmaceutically effective amount means the extent to which the desired pharmacological activity (e.g., prevention, improvement, or treatment of lung disease) can be achieved due to the triple active substance or sustained-release conjugate, and may also mean a pharmaceutically acceptable level in which no or minimal toxicity or side effects occur in the administered subject, but is not limited thereto. Such a pharmaceutically effective amount may be determined comprehensively considering the number of administrations, patients, formulation, etc.

[0491]

[0492] Although not particularly limited thereto, the pharmaceutical composition of the present invention may contain the above-mentioned ingredient (active ingredient) in an amount of 0.01 to 99% by weight to volume.

[0493]

[0494] The total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Specifically, a preferred total dose of the triple active agent or the sustained-release conjugate thereof of the present invention may be about 0.0001 mg to 500 mg per kg of patient body weight per day. However, the dose of the triple active agent or the conjugate thereof is determined by taking into consideration various factors such as the route of administration and the number of treatments of the pharmaceutical composition, as well as the patient's age, weight, health status, sex, severity of the disease, diet, and excretion rate. Therefore, considering these points, a person having ordinary skill in the art will be able to determine an appropriate effective dosage according to a specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, or administration method as long as it exhibits the effects of the present invention.

[0495] The pharmaceutical composition of the present invention has excellent in vivo persistence and potency, and thus can significantly reduce the number and frequency of administration of the pharmaceutical preparation of the present invention.

[0496]

[0497] Another embodiment of the present invention provides a method for preventing or treating a lung disease, comprising administering to a subject in need thereof the triple activator (peptide) and / or a sustained-release conjugate of the triple activator, or a composition comprising the same.

[0498] The triple activator and / or the sustained-release combination of the triple activator, or the composition containing the same, for lung diseases, prevention and treatment are as described above.

[0499] In the present invention, the subject is a subject suspected of having a lung disease, and the subject suspected of having a lung disease means a mammal including a human, a mouse, a livestock, etc. that has developed or may develop the disease, but any subject treatable with the triple active agent and / or combination of the present invention, or the composition containing the same, is included without limitation. In particular, the subject may be a subject with inflammation and / or fibrosis in the lung, or a subject with a lung disease caused by or accompanied by inflammation and / or fibrosis, but is not limited thereto.

[0500] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any appropriate method, and the route of administration of the composition is not particularly limited thereto, but the composition may be administered through any general route that can reach the target in the body, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0501]

[0502] The method of the present invention may include administering a pharmaceutical composition comprising the triple active agent or a sustained-release conjugate thereof in a pharmaceutically effective amount. An appropriate total daily dosage may be determined by a treating physician within the scope of sound medical judgment, and may be administered once or in several divided doses. However, for the purposes of the present invention, it is preferable that a specific therapeutically effective amount for a specific patient be applied differently depending on various factors including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field.

[0503] Although not limited thereto, the pharmaceutical composition of the present invention may be administered once a week, once every two weeks, or once every four weeks, but is not limited thereto.

[0504] A specific example of the method for preventing or treating lung diseases of the present invention includes, but is not limited to, a method of simultaneously, sequentially, or in reverse order co-administering a triple activator (peptide) and / or a sustained-release conjugate of the triple activator, or a composition containing the same, and a mucolytic agent or a pharmaceutically acceptable salt thereof. The co-administration is as described above.

[0505]

[0506] Another embodiment of the present invention is the use of a composition comprising the triple active agent or a sustained-release conjugate thereof in the manufacture of a medicament for the prevention or treatment of lung diseases.

[0507] The triple active agent and / or combination thereof, or compositions containing the same, for lung diseases, prevention and treatment are as described above.

[0508]

[0509] Another embodiment of the present invention provides a use of the triple active agent or a sustained-release combination thereof, or a composition comprising the same, for the prevention or treatment of lung diseases.

[0510] The triple active agent and / or combination thereof, or compositions containing the same, for lung diseases, prevention and treatment are as described above.

[0511]

[0512] The use of the present invention for the prevention or treatment of lung diseases may be, but is not limited to, the use of a combination of a triple activator (peptide) and / or a sustained-release conjugate of the triple activator, or a composition containing the same, with a mucolytic agent or a pharmaceutically acceptable salt thereof. The combination is as described above.

[0513]

[0514] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0515]

[0516] Example 1: Preparation of a triple activator

[0517] A triple activator exhibiting activity at all GLP-1, GIP, and glucagon receptors was prepared, and its sequence is shown in Table 1 below.

[0518]

[0519] 서열번호서열정보1H X Q G T F T S D V S S Y L D G Q A A K E F I A W L V K G C2H X Q G T F T S D V S S Y L D G Q A Q K E F I A W L V K G C3H X Q G T F T S D V S S Y L L G Q A A K Q F I A W L V K G G G P S S G A P P P S C4H X Q G T F T S D V S S Y L L G Q Q Q K E F I A W L V K G C5H X Q G T F T S D V S S Y L L G Q Q Q K E F I A W L V K G G G P S S G A P P P S C6H X Q G T F T S D V S S Y L D G Q A A K E F V A W L L K G C7H X Q G T F T S D V S K Y L D G Q A A K E F V A W L L K G C8H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L K G C9H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L A G C10H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L A G G G P S S G A P P P S C11CA G E G T F T S D L S K Y L D S R R Q Q L F V Q W L K A G G P S S G A P P P S H G12CA G E G T F I S D L S K Y M D E Q A V Q L F V E W L M A G G P S S G A P P P S H G13CA G E G T F I S D Y S I Q L D E I A V Q D F V E W L L A Q K P S S G A P P P S H G14CA G Q G T F T S D Y S I Q L D E I A V R D F V E W L K N G G P S S G APPPSH G15CA GQGTFTSDLSKQMDEEAVRLF IEWLKNGGPSSGAPPPSH G16CA GQGTFTSDLSKQMDSEAQQLF IEWLKNGGPSSGAPPPSH G17CA GQGTFTSDLSKQMDEERAREF IEWLLAQKPSSGAPPPSH G18CA GQGTFTSDLSKQMDSERAREF IEWLKNTGPSSGAPPPSH G19CA GQGTFTSDLSIQYDSEHQRDF IEWLKDTGPSSGAPPPSH G20CA GQGTFTSDLSIQYEEEAQQDF VEWLKDTGPSSGAPPPSH G21Y XQGTFTSDYSKYL DEC R AKE FVQWLLDHHPSSGQPPPS고리 형성22Y XQGTFTSDYSKCL DEC R AKE FVQWLLDHHPSSGQPPPS고리 형성23Y XQGTFTSDYSKYL DEC R AKE FVQWLLAQKGKKNDWKHNIT고리 형성24Y XQGTFTSDYSKYL DEC R AKE FVQWLKNGGPSSGAPPPS고리 형성25H XQGTFTSDCSKYLDERAAQDF VQWLLDGGPSSGAPPP S26H XQGTFTSDCSKYLDSRAAQDF VQWLLDGGPSSGAPPP S27HXQGTFTSDYSKYLDERACQDF VQWLLDQGGPSSGAPPP S28H XQGTFTSDYSHYL DEK A AKE FVQWLLNTC고리 형성30H XQGTFTSDYYKYL DEK A QKE FVQWLLDTC고리 형성31H XQGTFTSDYSKYL DEK A CKE FVQWLLAQ고리 형성32H XQGTFTSDYSKYL DEK A CKD FVQWLLDGGPSSGAPPPS고리 형성33H XQGTFTSDYSIAM DEI H QKD FVNWLLAQKC고리 형성34H XQGTFTSDYSKYL DEK R QKE FVNWLLAQKC고리 형성35H XQGTFTSDYSIAM DEI H QKD FVNWLLNTKC고리 형성36H XQGTFTSDYSKYL CEK R QKE FVQWLLNGGPSSGAPPPSG고리 형성37H XQGTFTSDYSKYL DEC R QKE FVQWLLNGGPSSGAPPPSG고리 형성38CA XQGTFTSDKSSYLDERAAQDF VQWLLDGGPSSGAPPPS S39H XQGTFTSDYSKYLDGQHAQCF VAWLLAGGGPSSGAPPP S40H XQGTFTSDKCREDITORACQDF VQWLLDGGPSSGAPPP S41H XQGTFTSDKSHYLDECAAQDF VQWLLDGGPSSGAPPP S42Y XQGTFTSDYSHYL TIRE R AKE FVQWLLDHHPSSGQPPPSC고리 형성43Y XQGTFTSDYSHYL TIRE R AKE FVQWLLDHHCSSGQPPPS고리 형성44H GQGTFTSDCSKQLDGQAAQEF VAWLLAGGPSSGAPPP S45H GQGTFTSDCSKYMDGQAAQDF VAWLLAGGPSSGAPPPS 46H GQGTFTSDCSKYLDEQHAQEF VAWLLAGGPSSGAPPP S47H GQGTFTSDCSKYLDGQRAQEF VAWLLAGGPSSGAPPP S48H GQGTFTSDCSKYLDGQRAQDF VNWLLAGGPSSGAPPP S49CA XQGTFTSDYSICM DEI H QKD FVNWLLNTK고리 형성50H XQGTFTSDYSKYL DEK R AKE FVQWLLDHHPSSGQPPPSC고리 형성51H XQGTFTSDYSKYL DECK R QKE FVQWLLNTC고리 형성52H XQGTFTSDYSKYL DEK R QKE FVQWLLDTC고리 형성53H XEGTFTSDYSIAM DEI H QKD FVNWLLAQC 고리 형성54H XEGTFTSDYSIAM DEI H QKD FVDWLLAEC NUMBER55H XQGTFTSDYSIAM DEI H QKD FVNWLLAQC고리 형성56H XQGTFTSDYSKYL DEK R QKE FVNWLLAQC고리 형성57H XQGTFTSDYSIAM DEI H QKD FVNWLLNTC고리 형성58H XQGTFTSDYSKYL DECK R QKE FVQWLLNTKC고리 형성59CA XQGTFTSDYSICM DEK H QKD FVNWLLNTK고리 형성60CA XQGTFTSDYSIAM DEK H CKD FVNWLLNTK고리 형성61CA XQGTFTSDYSIAM DEI A CKD FVNWLLNTK고리 형성62CA XQGTFTSDKSKYLDERAAQDF VQWLLDGGPSSGAPPP S63CA XQGTFTSDCSKYLDERAAQDF VQWLLDGGPSSGAPPP S64Y XQGTFTSDYSKYL DEC A AKE FVQWLLDHHPSSGQPPPS고리 형성 65H XQGTFTSDYSKCL DEK R AKE FVQWLLDHHPSSGQPPPS고리 형성66Y XQGTFTSDYSKYL DEC R AKD FVQWLLDHHPSSGQPPPS고리 형성67Y XQGTFTSDYSKYL DEC A AKD FVQWLLDHHPSSGQPPPS고리 형성68Y XQGTFTSDYSKCL DECK A AKE FVQWLLDHHPSSGQPPPS고리 형성69Y XQGTFTSDDISKCL DER A AKE FVQWLLDHHPSSGQPPPS고리 형성70Y XQGTFTSDDISKCL DEK R AKD FVQWLLDHHPSSGQPPPS고리 형성71Y XQGTFTSDSCYL DER A CKD FVQWLLDHHPSSGQPPPS고리 형성72Y XQGTFTSDSCYL DER A AKD FVQWLLDHHPSSGQPPPS고리 형성73CA XQGTFTSDSYSYL DEC R AKE FVQWLLDHHPSSGQPPPS고리 형성74CA XQGTFTSDYSKCL DEK R AKE FVQWLLDHHPSSGQPPPS고리 형성75Y XQGTFTSDYSKYL DEK A AKE FVQWLLDHHPSSGQPPPSC고리 형성76Y XQGTFTSDYSKYL DECK R AKD FVQWLLDHHPSSGQPPPSC고리 형성77Y XQGTFTSDYSKYL DECK A AKD FVQWLLDHHPSSGQPPPSC고리 형성78H XQGTFTSDYSKYL DECK R QKE FVQWLLDTKC고리 Page 79H XEGTFTSDYSIAM DEI H QKD FVNWLLAQKC고리 형성80H XEGTFTSDYSIAM DEI H QKD FVDWLLAEKC고리 형성81CA XQGTFTSDYSKYL DEK R QKE FVQWLLNTC고리 형성82CA XQGTFTSDYSKYL DEK R QKE FVQWLLDTC고리 형성83CA XEGTFTSDYSIAM DEI H QKD FVNWLLAQC고리 형성84CA XEGTFTSDYSIAM DEI H QKD FVDWLLAEC고리 형성85CA XQGTFTSDYSIAM DEI H QKD FVNWLLAQC고리 형성86CA XQGTFTSDYSKYL DEK R QKE FVNWLLAQC고리 형성87CA XQGTFTSDYSIAM DEI H QKD FVNWLLNTC 고리 형성88CA XQGTFTSDYSKYL DEK R QKE FVQWLLNTKC고리 형성89CA XQGTFTSDYSKYL DEK R QKE FVQWLLDTKC고리 형성90CA XEGTFTSDYSIAM DEI H QKD FVNWLLAQKC FUCKING 91CA XEGTFTSDYSIAM DEI H QKD FVDWLLAEKC FUCKING 92CA XQGTFTSDYSIAM DEI H QKD FVNWLLAQKC FUCKING93CA XQGTFTSDYSKYL DEK R QKE FVNWLLAQKC고리 형성94CA XQGTFTSDYSIAM DEI H QKD FVNWLLNTKC고리 형성95Y XQGTFTSDYSKYL DEK R AKE FVQWLLCHHPSSGQPPPS고리 형성96Y XQGTFTSDYSKYL DEK R AKE FVQWLLDHCPSSGQPPPSRing Formation97Y XQGTFTSDYSKYL DEK R AKE FVQWLLDCHPSSGQPPPSRing Formation98Y XQGTFTSDYSKAL DEK A AKE FVNWLLDHHPSSGQPPPSCRing Formation99Y XQGTFTSDYSKAL DEK A AKD FVNWLLDHHPSSGQPPPSCRing Formation100Y XQGTFTSDYSKAL DEK A AKE FVQWLLDQHPSSGQPPPSCRing Formation101Y XQGTFTSDYSKAL DEK A AKE FVNWLLDQHPSSGQPPPSCRing Formation102Y XQGTFTSDYSKAL DEK A AKD FVNWLLDQHPSSGQPPPSCRing Formation

[0520] In the sequence described in Table 1 above, the amino acid indicated by X is Aib (2-aminoisobutyric acid), an unnatural amino acid, and the underlined amino acids indicate that the underlined amino acids form a ring with each other. In addition, in Table 1 above, CA indicates 4-imidazoacetyl, and Y indicates tyrosine.

[0521]

[0522] Example 2: Preparation of a sustained-release conjugate of a triple-activator

[0523]

[0524] In order to pegylate the cysteine ​​residues of the triple activator (SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96) of Example 1 with 10 kDa PEG having a maleimide group and an aldehyde group at each of the two terminals, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), the molar ratio of the triple activator and maleimide-PEG-aldehyde was 1:1 to 3, and the protein concentration was 1 to 5 mg / mL, and the reaction was performed at low temperature for 0.5 to 3 hours. At this time, the reaction was performed under an environment in which 20 to 60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP Sepharose HP (GE healthcare, USA) to purify the tri-activated product mono-PEGylated on cysteine.

[0525] Next, the purified mono-PEGylated tri-active substance and immunoglobulin Fc (homodimer of SEQ ID NO: 139) were reacted at a molar ratio of 1:1 to 5 and a protein concentration of 10 to 50 mg / mL at 4 to 8°C for 12 to 18 hours. The reaction was performed in an environment in which 100 mM potassium phosphate buffer (pH 6.0) was added with 10 to 50 mM sodium cyanoborohydride as a reducing agent and 10 to 30% isopropanol. After the reaction was completed, the reaction solution was applied to a butyl Sepharose FF purification column (GE healthcare, USA) and a Source ISO purification column (GE healthcare, USA), and the conjugate containing the tri-active substance and immunoglobulin Fc was purified. This purified sustained-release conjugate is a structure in which a trifunctional peptide, a polyethylene glycol (PEG) linker, and an Fc dimer are covalently linked in a molar ratio of 1:1:1 within the molecule, with the PEG linker being linked to only one of the two polypeptide chains of the Fc dimer.

[0526] Meanwhile, the immunoglobulin Fc is a homodimer formed by two monomers having an amino acid sequence of SEQ ID NO: 139 (consisting of 221 amino acids) through a disulfide bond between cysteine, the 3rd amino acid of each monomer, and the monomers of the homodimer independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199.

[0527] The purity analyzed by reverse phase chromatography, size exclusion chromatography and ion exchange chromatography after manufacturing was over 95%.

[0528] Here, the complex in which the trifunctional agent of sequence number 21 and the immunoglobulin Fc are linked via a PEG linker is referred to as a 'complex comprising sequence number 21 and immunoglobulin Fc' or a 'persistent complex of sequence number 21', and these may be used interchangeably herein.

[0529] Here, the complex in which the trifunctional agent of sequence number 22 and the immunoglobulin Fc are linked via a PEG linker is referred to as a 'complex comprising sequence number 22 and immunoglobulin Fc' or a 'persistent complex of sequence number 22', and these may be used interchangeably herein.

[0530] Here, the conjugate in which the triple active agent of sequence number 42 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 42 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 42', and these may be used interchangeably herein.

[0531] Here, the conjugate in which the trifunctional agent of sequence number 43 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 43 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 43', and these may be used interchangeably herein.

[0532] Here, the complex in which the triple active agent of sequence number 50 and immunoglobulin Fc are linked via PEG is named as 'a complex comprising sequence number 50 and immunoglobulin Fc' or 'a persistent complex of sequence number 50', and these may be used interchangeably herein.

[0533] Here, the conjugate in which the trifunctional agent of sequence number 77 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 77 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 77', and these may be used interchangeably herein.

[0534] Here, the conjugate in which the triple active agent of sequence number 96 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 96 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 96', and these may be used interchangeably herein.

[0535]

[0536] Experimental Example 1: In vitro activity measurement of a triple activator and its sustained-release conjugate

[0537]

[0538] In order to measure the activity of the triple activator and its sustained-release conjugate prepared in Examples 1 and 2 above, a method of measuring cell activity in vitro was used using cell lines each transformed with a GLP-1 receptor, a glucagon (GCG) receptor, and a GIP receptor.

[0539]

[0540] Each of the above cell lines is transformed to express the human GLP-1 receptor, human GCG receptor, and human GIP receptor genes, respectively, in CHO (Chinese hamster ovary), and is suitable for measuring the activity of GLP-1, GCG, and GIP. Therefore, the activity for each part was measured using each transformed cell line.

[0541]

[0542] In order to measure the GLP-1 activity of the triple activator and its sustained conjugate prepared in Examples 1 and 2, human GLP-1 was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple activator and its sustained conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GLP-1 receptor, and 5 μl of each serially diluted substance was added to the cells, followed by adding 5 μl of a buffer containing a cAMP antibody and incubating at room temperature for 15 minutes. Then, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the above reaction was completed was applied to the LANCE cAMP kit (PerkinElmer, USA) to measure EC through the accumulated cAMP. 50 After calculating the values, they were compared with each other. The relative titers compared to human GLP-1 are shown in Tables 2 and 3 below.

[0543]

[0544] In order to measure the GCG activity of the triple activator and its sustained conjugate prepared in Examples 1 and 2, human GCG was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple activator and its sustained conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GCG receptor, and 5 μl of each serially diluted substance was added to the cells, followed by 5 μl of a buffer containing cAMP antibody and incubation at room temperature for 15 minutes. Then, 10 μl of a detection mix containing cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the reaction was completed was applied to the LANCE cAMP kit (PerkinElmer, USA) to measure the EC through the accumulated cAMP. 50 After calculating the values, they were compared with each other. The relative titers compared to human GCG are shown in Tables 2 and 3 below.

[0545]

[0546] In order to measure the GIP activity of the triple activator and its sustained conjugate prepared in Examples 1 and 2, human GIP was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple activator and its sustained conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GIP receptor, and 5 μl of each serially diluted substance was added to the cells, followed by 5 μl of a buffer containing cAMP antibody and incubation at room temperature for 15 minutes. Then, 10 μl of a detection mix containing cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the reaction was completed was applied to the LANCE cAMP kit (PerkinElmer, USA) to measure the EC through the accumulated cAMP. 50 After calculating the values, they were compared with each other. The relative titers compared to human GIP are shown in Tables 2 and 3 below.

[0547]

[0548] Relative potency ratio of triple active agentIn vitro activity (%) compared to natural peptideSequence number vs GLP-1 vs Glucagon vs GIP13.2<0.1<0.125.9<0.1<0.131.8<0.1<0.148.5<0.1<0.1542.1<0.1<0.1617.0<0.1<0.1713.7<0.1<0.1814.20.10<0.1932.10.13<0.11046.0<0.1<0.1111.4<0.1<0.1120.4<0.1<0.113< 0.1< 0.1< 0.11428.0< 0.1< 0.11579.2<0.1<0.1162.1< 0.1< 0.1170.2< 0.1< 0.118<0.1<0.1<0.119<0.1<0.1<0.120<0.1<0.1<0.12117.826722.72220.114059.7234.019.3<0.12441.29.3< 0.12582.60.1<0.12664.50.2<0.12783.10.80.92817.21.6<0.12938.56.0<0.1301420.70.8311352.22.4321511.78.83324.5<0.110.43419.10.920.6357 .5<0.11.33637.40.390.2372366.212.2382.3--3913.90.53<0.14075.2<0.1<0.14134.3<0.1<0.14233.9205.87.84312.688.43.70441.3<0.1<0.1456.6< 0.1< 0.1461.4< 0.1< 0.1472.4< 0.1< 0.1481.5< 0.1< 0.14929.8<0.13.35067.450.52.75114.42.00.15244.17.50.3531618.41.35430.61.40.15527.10.72.45657.94.90.85711.7<0.10.35839.12.60.25940.3<0.14.060106.2<0 .18.26159.8<0.12.8625.2<0.1<0.16315.3<0.1<0.16464.660.192.96595.425.211.66615.817217.26728.546.239.86827.98.81076924.39.662.87015.171.364.47190.112.794.77211.51.01.67322.65.43.07412.90.91.07535.18.518.07610.347.611.77738.712.235.57851.014.00.127941.54.91.4808.10.00.1817.80.3<0.1829.51.1<0.18347.31.30.4844.2<0.1<0.1854.3<0.10.38628.40.40.2870.9<0.1< 0.1889.60.3<0.1897.10.7<0.1907.4<0.1<0.19131.916.80.3920.8<0.10.4935.70.30.7940.5<0.1<0.1952.10.4<0.19634.4194.85.29710.562.82.69828.18.247.19920.914.957.710042.212.7118.510123.213.940.110223.329.558.0.

[0549] Relative potency ratio of the triple active long-acting conjugate Long-acting conjugate compared to the native peptide In vitro activity (%) vs GLP-1 vs Glucagon vs GIP2 10.11.6 0.22 20.10.9 0.54 23.12 3.11.24 32.11 3.5 0.65 015.46.9 0.77 76.71.76.69 60.3 4.0 0.3

[0550] The novel triple activator sustained-release conjugate manufactured above functions as a triple activator capable of activating all of the GLP-1 receptor, GIP receptor, and glucagon receptor, and thus can be used as a therapeutic agent for lung diseases.

[0551]

[0552] Experimental Example 2: Confirmation of the anti-inflammatory effect of a triple-active, sustained-release conjugate in the lungs.

[0553]

[0554] It was confirmed using mice administered elastase (ELA) whether the triple active substance sustained-release conjugate according to the present invention manufactured in the above example exhibited an anti-inflammatory effect in the lungs.

[0555]

[0556] Specifically, C57BL / 6 mice (ORIENT Bio, Busan, Korea) were treated with ELA 0.2 U / mice. The mice were divided into a vehicle control group, a long-acting conjugate of SEQ ID NO: 42 (hereinafter, the triple-active long-acting conjugate; 6.5 nmol / kg, Q2D, subcutaneously), and a group administered roflumilast (10 mg / kg, QD, orally), an anti-inflammatory agent and COPD treatment, and repeated administration was performed for 3 weeks. Afterwards, the degree of change in the expression of inflammatory cytokines in lung tissue according to administration of the vehicle, the triple-active long-acting conjugate, and roflumilast was confirmed using qPCR. 1-way ANOVA was used for statistical analysis to evaluate the efficacy of the triple-active long-acting conjugate (* to **p<0.05 to 0.01).

[0557]

[0558] As a result, it was confirmed that when the triple-active long-acting conjugate was repeatedly administered, the expression of IL-1β, IL-6, IL-12, and TNF-α in lung tissue was significantly and equally reduced compared to the excipient control group and the roflumilast administration group (Fig. 1).

[0559]

[0560] Through this, it was confirmed that the triple active substance sustained-release conjugate of the present invention has the effect of suppressing and improving inflammation in the lungs.

[0561]

[0562] Experimental Example 3: Confirmation of the therapeutic effect of a triple-active, sustained-release conjugate on emphysema and chronic obstructive pulmonary disease.

[0563]

[0564] The present inventors sought to determine whether a triple-active long-acting conjugate could exhibit in vivo therapeutic effects on emphysema and chronic obstructive pulmonary disease (COPD), which are representative lung diseases and diseases caused by lung inflammation.

[0565]

[0566] To this end, we used an animal model in which chronic obstructive pulmonary disease was induced by lung tissue damage through intratracheal administration of elastase. Specifically, C57BL / 6 mice (ORIENT Bio, Busan, Korea) were treated with 0.2 U / mice of elastase. The COPD mice were divided into a vehicle control group, a triple-active long-acting conjugate (6.5 nmol / kg, Q2D, subcutaneously), and a roflumilast (10 mg / kg, QD, oral) group, and repeated administration was performed for 3 weeks. After 3 weeks of repeated administration, lung tissues were collected from each mouse through necropsy, and the degree of emphysema in the lung tissues was evaluated through H&E staining.

[0567]

[0568] As a result, when the triple active substance sustained-release combination was repeatedly administered, it was confirmed that it had an excellent emphysema improvement effect compared to the excipient control group and the roflumilast group, which is known as a COPD treatment (* ~ **p<0.05 ~ 0.01) (Fig. 2).

[0569]

[0570] Through this, it was confirmed that the triple active substance sustained-release conjugate of the present invention exhibits an effect of improving inflammation and emphysema in lung tissue, thereby having excellent therapeutic efficacy for chronic obstructive pulmonary disease.

[0571]

[0572] Experimental Example 4: Confirmation of the effect of a triple-activated sustained-release conjugate on improving pulmonary fibrosis.

[0573]

[0574] In addition to the anti-inflammatory effect in lung tissue confirmed in the above examples, we sought to determine whether the triple-active long-acting conjugate according to the present invention also has an anti-pulmonary fibrosis effect. To this end, we used the MRC5 cell line, a lung fibroblast, and the A549 cell line, a lung epithelial cell, to determine the effects on the differentiation of lung fibroblasts into myofibroblasts and the epithelial mesenchymal transition (EMT) of alveolar epithelial cells, both of which are known to be important in the process of pulmonary fibrosis.

[0575]

[0576] Experimental Example 4-1: Confirmation of the inhibitory effect of lung fibroblast differentiation into myofibroblasts.

[0577] First, MRC5 cells, which are lung fibroblasts, were divided into a vehicle-treated group and a triple-activator long-acting complex-treated group, and TGF-β1 was simultaneously treated to induce differentiation into myofibroblasts. A group that was not treated with TGF-β1 or the triple-activator long-acting complex was used as a negative control group.

[0578]

[0579] After 48 to 72 hours of simultaneous treatment, the expression levels of myofibroblast differentiation markers α-SMA, collagen1α1, and fibronectin in each group were confirmed via quantitative PCR. Statistical analysis was performed using a one-way ANOVA to evaluate the efficacy of the triple-activator sustained-release complex. (* to **p<0.05 to 0.01)

[0580]

[0581] As a result, it was confirmed that when the triple-activator sustained-release complex was treated simultaneously with TGF-β1, the expression of myofibroblast differentiation markers decreased compared to the excipient-treated group (Fig. 3).

[0582]

[0583] Experimental Example 4-2: Confirmation of the Inhibitory Effect of Epithelial-Mesenchymal Transition (EMT) in Alveolar Epithelial Cells

[0584] Next, A549 cells were divided into a vehicle-treated group and a triple-activator sustained-release complex-treated group, pretreated, and then sequentially treated with TGF-β1 and LPS to induce EMT. A group that was not treated with the triple-activator sustained-release complex, TGF-β1, or LPS was used as a negative control group.

[0585]

[0586] After 48 hours, the expression levels of EMT markers collagen1α1 and collagen3α1 were determined in each group using quantitative PCR. Statistical analysis was performed using a one-way ANOVA to evaluate the efficacy of the triple-activator sustained-release complex. (* ~ ***p<0.05 ~ 0.001)

[0587]

[0588] As a result, it was confirmed that the expression of EMT markers in alveolar epithelial cells was significantly reduced compared to the excipient-treated group when pretreated with the triple-activator sustained-release complex (Fig. 4).

[0589]

[0590] Experimental Example 4-3: In vivo Effects of Fibrosis Improvement

[0591] To confirm the efficacy of the triple-active long-acting conjugate manufactured in the above examples in improving pulmonary fibrosis, particularly idiopathic pulmonary fibrosis (IPF), a representative pulmonary fibrosis, in vivo, bleomycin (BLM) mice were used. Bleomycin is known to induce idiopathic fibrosis by damaging the DNA of alveolar epithelial cells when administered intratracheally, thereby causing epithelial-mesenchymal transition. In addition, it is known that when the BLM treatment dose exceeds a certain level, the survival rate of the mice is reduced due to severe lung damage caused by idiopathic fibrosis.

[0592]

[0593] Accordingly, we confirmed the effect of the triple-active long-acting conjugate on improving pulmonary fibrosis in mice treated with BLM, and further confirmed whether the survival rate could be increased through improvement of pulmonary fibrosis.

[0594]

[0595] IPF mice treated with BLM 1.5 U / head in C57BL / 6 mice (ORIENT Bio, Busan, Korea) were divided into the vehicle control group, the triple-active agent long-acting conjugate (3.9 nmol / kg, Q2D, subcutaneously), the pirfenidone (300 mg / kg, QD, orally), and the ambroxol (45 mg / kg, BID, intraperitoneally) group, and repeated administration was performed for 2 weeks. After 2 weeks of repeated administration, lung tissues were collected from each mouse by necropsy, and the degree of fibrosis in the lung tissues was evaluated using Masson's trichrome staining. Statistical analysis was performed using a 1-way ANOVA to evaluate the efficacy of the triple-active agent long-acting conjugate (* ~ ***p < 0.05 ~ 0.001).

[0596]

[0597] As a result, when repeatedly administered with the triple-active, sustained-release combination, we were able to confirm excellent efficacy in reducing the masson trichrome staining positive area in lung tissue compared to the excipient control group and the pirfenidone group, known as an IPF treatment group (* ~ ***p<0.05 ~ 0.001). In addition, it was confirmed that ambroxol, known as an expectorant, showed an efficacy equivalent to that of the triple-active, sustained-release combination (Fig. 5).

[0598]

[0599] In addition, IPF mice treated with BLM 3.0 U / head in C57BL / 6 mice (ORIENT Bio, Busan, Korea) were divided into the vehicle control group, the triple-active long-acting combination (3.9 nmol / kg, Q2D, subcutaneously) administration group, the pirfenidone (300 mg / kg, QD, orally) administration group, and the amboroxol (45 mg / kg, BID, intraperitoneally) administration group, and repeated administration was performed for 2 weeks, and the survival rate during repeated administration was confirmed.

[0600]

[0601] As a result, it was confirmed that when the sustained-release combination of triple activators was repeatedly administered, it showed an excellent survival rate compared to the excipient control group, the pirfenidone administration group, and the ambroxol administration group (Figure 6).

[0602]

[0603] Through this, it was confirmed that the sustained-release combination of triple activators has a therapeutic effect on pulmonary fibrosis or idiopathic pulmonary fibrosis (IPF) and is effective in improving the survival rate accordingly.

[0604]

[0605] It was confirmed that the triple-active long-acting complex according to the present invention can improve pulmonary fibrosis by inhibiting myofibroblast differentiation of pulmonary fibroblasts and EMT of alveolar epithelial cells, and it was also confirmed that it can be effective in preventing and treating pulmonary fibrosis, particularly idiopathic pulmonary fibrosis, through its effect of improving pulmonary fibrosis in vivo.

[0606]

[0607] In summary, the above results suggest that the triple active agent conjugates of the present invention can effectively prevent or treat related lung diseases by improving lung inflammation and pulmonary fibrosis, and thus can be provided as new lung disease treatments.

[0608]

[0609] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A pharmaceutical composition for preventing or treating lung disease, Pharmaceutically acceptable excipients and A pharmaceutical composition comprising a pharmaceutically effective amount of a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 102.

2. In the first paragraph, the peptide is in the form of a sustained conjugate, and the sustained conjugate is a pharmaceutical composition represented by the following chemical formula 1: [Chemical Formula 1] X - L - F However, at this time, X is a peptide having an amino acid sequence of any one of sequence numbers 1 to 102; L is a linker containing ethylene glycol repeating units, F is the immunoglobulin Fc region, - indicates a covalent bond between X and L, and between L and F.

3. A pharmaceutical composition according to claim 1 or 2, wherein the C-terminus of the peptide is amidated.

4. A pharmaceutical composition according to claim 1 or 2, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97 and 100.

5. A pharmaceutical composition according to claim 4, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96.

6. A pharmaceutical composition according to claim 1 or 2, wherein the peptide sequence comprises amino acids 16 and 20 from the N-terminus forming a ring.

7. A pharmaceutical composition in claim 2, wherein the chemical formula of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

8. A pharmaceutical composition in the second paragraph, wherein F is an IgG Fc region.

9. In the first or second paragraph, the lung disease is interstitial lung disease (ILD), progressive fibrosing Interstitial Lung Disease (PF-ILD), idiopathic interstitial pneumonias (IIP), non-specific interstitial pneumonia (NSIP), pulmonary fibrosis, fibrosing interstitial lung diseases (FILD), idiopathic pulmonary fibrosis (IPF), alveolitis, pneumonia, emphysema, bronchitis, chronic obstructive pulmonary disease, combined pulmonary fibrosis and emphysema (CPFE), asthma, or respiratory infection. A pharmaceutical composition for treating a disease.

10. A pharmaceutical composition according to claim 9, wherein the respiratory infectious disease is a respiratory virus, bacterial, mycoplasma, or fungal infectious disease.

11. A pharmaceutical composition according to claim 10, wherein the respiratory virus is any one selected from the group consisting of adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella Zoster Virus, measles virus, respiratory syncytial virus, dengue virus, human immunodeficiency virus (HIV), influenza virus, coronavirus, severe acute respiratory syndrome associated virus (SARS-associated virus), and middle east respiratory syndrome coronavirus (MERS-CoV).

12. A pharmaceutical composition according to claim 11, wherein the coronavirus is SARS-CoV-2.

13. A pharmaceutical composition according to claim 1 or 2, which, when administered, (i) inhibits macrophage activity, or (ii) reduces the expression of IL-1β, IL-6, IL-12, or TNF-α.

14. A pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition has at least one of the following properties when administered: (i) Inhibition of myofibroblast differentiation; (ii) decreased expression of α-SMA, collagen1α1, or fibronectin; (iii) inhibition of epithelial mesenchymal transition (EMT) of alveolar epithelial cells; and (iv) Decreased expression of collagen1α1 or collagen1α3.

15. A pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is additionally administered with a mucolytic agent or a pharmaceutically acceptable salt thereof.

16. In paragraph 15, the mucolytic agent is selected from the group consisting of ambroxol, N-acetylcystein, N-acetylin, carbocysteine, domiodol, fudosteine, bromhexine, erdosteine, letostine, lysozyme, mesna, sobrerol, stepronin, tiopronin, tyloxapol, carbocysteine, dornase alfa, eprazinone, letosteine, neltenexine, and mecysteine. A pharmaceutical composition, wherein one or more of the following are selected:

17. A pharmaceutical composition according to claim 14, wherein the peptide and the mucolytic agent or a pharmaceutically acceptable salt thereof are administered simultaneously, sequentially, or in reverse order.

18. A pharmaceutical composition according to claim 9, wherein the lung disease is pneumonia (pulmonary inflammation) or pulmonary fibrosis caused by coronavirus infection-19 (COVID-19).

19. A pharmaceutical composition in claim 2, wherein the region F is a dimer composed of two polypeptide chains, and one terminal of L is linked to only one of the two polypeptide chains.

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