A triple-active form exhibiting activity against glucagon, GLP-1, and GIP receptors is used for the treatment of sequelae of respiratory infections.

Abstract

Development of therapeutic agents for the prevention or treatment of sequelae of respiratory infectious diseases. [Solution] The present invention relates to the preventive or therapeutic use of a triplicate and / or conjugate having activity against glucagon, GLP-1, and GIP receptor for the sequelae of respiratory infection diseases.

Description

Technical Field

[0001] The present invention relates to the prophylactic or therapeutic use of a triple activator having activity against all of the glucagon, GLP-1 and GIP receptors and / or its conjugate for sequelae of respiratory infectious diseases.

Background Art

[0002] Respiratory infectious diseases are respiratory diseases caused by infection with pathogens (viruses, bacteria, mycoplasma, fungi, etc.). Representative respiratory infectious diseases include respiratory viral infectious diseases caused by pathogenic virus infections. Respiratory infectious diseases can cause severe lower respiratory infections accompanied by pneumonia or bronchitis from mild upper respiratory infections, and are fatal to people with reduced cardiopulmonary function.

[0003] Among respiratory viruses, the novel coronavirus (2019-nCoV or SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) not only has high transmissibility but also various mutant SARS-CoV-2 viruses with high infectivity have been confirmed, resulting in a worldwide pandemic. The novel coronavirus transmitted from the respiratory tract invades cells mainly through ACE2 and TMPRSS2 mainly expressed in type II alveolar epithelial cells, so the lungs are known to be the main vulnerable organs. The main symptoms include fever, cough, etc. Although healthy adults often recover over time, in some patients, extreme immune reactions such as cytokine release syndrome may induce lung injury, promote progressive fibrosis, and be accompanied by symptoms such as acute respiratory distress syndrome (ARDS) and sepsis.

[0004] In patients with respiratory infectious diseases such as the novel coronavirus, sequelae often remain even after complete recovery, causing difficulties in daily life. These sequelae are often caused by cytokine storms and resulting lung injuries.

[0005] In the case of the SARS-CoV-2 virus, which caused a global pandemic, it has been reported that an overimmune response occurs after infection, inducing not only severe pneumonia and acute respiratory distress, but also leaving behind sequelae even after complete recovery.

[0006] A typical mechanism that causes a hyperimmune response is that, upon infection with the SARS-CoV-2 virus, the inflammatory complex, a cytoplasmic polyprotein oligomer responsible for activating the inflammatory response, excessively secretes pro-inflammatory cytokines. This activates macrophages, which then strongly exhibit inflammatory macrophage characteristics, causing tissue damage. In addition, other major organs besides the lungs can also be damaged by hyperimmune inflammatory responses caused by cytokine storms.

[0007] Due to this hyperimmune inflammatory response, various sequelae remain even after complete recovery from respiratory viral infections, making appropriate treatment and management crucial even after treatment for respiratory infectious diseases. For example, in patients with COVID-19, various sequelae such as shortness of breath, cough, chest tightness, severe fatigue, heart disease, loss of lung function, and kidney damage appear even after a diagnosis of complete recovery. It is known that the risk of developing sequelae is particularly high in high-risk groups for COVID-19 and in patients who exhibit severe symptoms (Non-Patent Literature 1).

[0008] Post-COVID-19 pulmonary fibrosis, one of the long-term effects of COVID-19, is known to be primarily caused by lung damage due to viral infection, and one of the factors that causes lung damage is an excessive immune response (cytokine storm) due to viral infection.

[0009] Pulmonary fibrosis that appears as a sequela of COVID-19 is characterized by a much faster onset and progression of symptoms than commonly observed pulmonary fibrosis. Therefore, unlike conventional pulmonary fibrosis treatments that focus on symptom relief and progression delay from a long-term perspective, a therapeutic approach that simultaneously targets both pneumonia and fibrosis, which are the causes of pulmonary fibrosis, is required, but no effective drug has been identified to date. For example, the possibility of treatment with pirfenidone and nintedanib, which are known as antifibrotic drugs, has been suggested, but they do not have a significant effect on pneumonia itself, and their efficacy is limited. In addition, most COVID-19 patients experience liver dysfunction, but antifibrotic drugs, broadly classified as pirfenidone and nintedanib, are likely to cause hepatotoxicity, making it difficult to prescribe commonly known antifibrotic drugs to patients who have developed sequelae after COVID-19 (Non-Patent Literature 1).

[0010] In other words, while various drugs have been developed to treat infectious diseases caused by respiratory viruses, there are not enough drugs that can suppress inflammatory responses, minimize lung damage such as pulmonary fibrosis, and prevent or treat sequelae after respiratory infections.

[0011] On the other hand, GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) are representative gastrointestinal hormones, as well as neurohormones involved in regulating blood glucose levels in response to food intake. Glucagon is a peptide hormone secreted from the pancreas and, along with the two substances mentioned above, is involved in regulating blood glucose concentration. Therapeutic agents using drugs that exhibit activity on GLP-1 receptors, GIP receptors, and glucagon receptors, either individually or simultaneously, are being developed (Patent Documents 1 and 2).

[0012] Although various studies are currently being conducted on the sequelae of respiratory infectious diseases, the development of effective and practical treatments is still insufficient, and sustained development of such treatments is needed. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] U.S. Patent No. 10370426 [Patent Document 2] U.S. Patent No. 10400020 [Patent Document 3] International Publication No. 97 / 034631 [Patent Document 4] International Publication No. 96 / 032478 [Patent Document 5] International Publication No. 2017 / 116204 [Patent Document 6] International Publication No. 2017 / 116205 [Non-patent literature]

[0014] [Non-Patent Document 1] Deependra Kumar Rai et al., Indian J Tuberc. 2020 Nov 10 [Non-Patent Document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-Patent Document 3] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-Patent Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-Patent Document 5] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Summary of the Invention

Problems to be Solved by the Invention

[0015] There is a demand for the development of a therapeutic agent for preventing or treating sequelae of respiratory infectious diseases.

Means for Solving the Problems

[0016] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of sequelae of respiratory infectious diseases, comprising a peptide having activity against glucagon receptors, GLP-1 receptors, and GIP receptors, or a sustained-release conjugate thereof.

[0017] Furthermore, the present invention aims to provide a method for preventing or treating sequelae of respiratory infections, comprising the step of administering a composition containing the peptide or a persistent conjugate of the peptide to an individual who requires such a composition.

[0018] Furthermore, the present invention aims to provide a use for a composition containing the peptide or a sustained-release conjugate thereof in the manufacture of a drug for the prevention or treatment of sequelae of respiratory infectious diseases.

[0019] Furthermore, the present invention aims to provide uses for compositions containing the peptide or a persistent conjugate of the peptide for the prevention or treatment of sequelae of respiratory infectious diseases. [Effects of the Invention]

[0020] The triple-active compound or its sustained-release conjugate according to the present invention is active against glucagon receptors, GLP-1 (Glucagon-like peptide-1) receptors, and GIP (Glucose-dependent insulinotropic polypeptide) receptors, and exerts preventive or therapeutic effects against sequelae of respiratory infectious diseases. [Brief explanation of the drawing]

[0021] [Figure 1] Figure A shows the decrease in lung cytokine expression in SARS-CoV-2 infected hamsters after administration of the sustained-release conjugate of Sequence ID No. 42, and Figure B shows the change in the pneumonia score. [Figure 2] This figure shows the change in lung fibrosis area in hamsters that experienced a cytokine storm after administration of the sustained-release conjugate of SEQ ID NO: 42. [Modes for carrying out the invention]

[0022] One embodiment of the present invention is a pharmaceutical composition for the prevention or treatment of sequelae of respiratory infectious diseases, 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.

[0023] As a specific example, a pharmaceutical composition for the prevention or treatment of sequelae of respiratory infectious diseases is characterized by comprising a pharmaceutically acceptable excipient and a peptide having any of the amino acid sequences of SEQ ID NOs: 1 to 102 in a pharmaceutically effective amount.

[0024] In a pharmaceutical composition according to any of the above-described examples, the peptide is in the form of a sustained-release conjugate, and the sustained-release conjugate is represented by chemical formula (1).

[0025] XLF···(1)

[0026] Here, X is a peptide having one of the amino acid sequences of SEQ ID NOs: 1 to 102, L is a linker containing an ethylene glycol repeating unit, F is an immunoglobulin Fc region, and - indicates a covalent bond between X and L, and between L and F.

[0027] In a composition according to any of the above-mentioned specific examples, the respiratory infection disease is characterized in that it is a respiratory viral infection disease.

[0028] In a composition according to any of the above-mentioned specific examples, the respiratory viral infection disease is characterized by being an infection disease caused by a respiratory virus selected from the group consisting of adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella zoster virus, measle virus, respiratory syncytial virus, dengue virus, HIV (human immunodeficiency virus), influenza virus, coronavirus, severe acute respiratory syndrome associated virus (SARS-associated virus), and middle east respiratory syndrome coronavirus (MERS-CoV).

[0029] In a composition according to any of the above-mentioned specific examples, the coronavirus is characterized by being SARS-CoV-2.

[0030] In a composition according to any of the above-mentioned specific examples, the respiratory virus is characterized by being a mutant virus.

[0031] In a composition according to any of the above-mentioned specific examples, the mutant respiratory virus is characterized in that it induces the same sequelae as the respiratory virus.

[0032] In a composition according to any of the above-mentioned specific examples, the mutant respiratory virus is characterized by being selected from the group consisting of SARS-CoV-2 alpha mutation (B.1.1.7 lineage), SARS-CoV-2 beta mutation (B.1.351 lineage), SARS-CoV-2 gamma mutation (P.1 lineage), and SARS-CoV-2 delta mutation (B.1.617.2 lineage).

[0033] In a composition according to any of the above-mentioned specific examples, the sequelae of the respiratory infection disease are characterized by being at least one selected from the group consisting of fever, dyspnea, cough, pneumonia, pulmonary fibrosis, pain, muscle pain, fatigue, inflammation, and nervous system disorders.

[0034] In a composition according to any of the above-mentioned specific examples, the sequelae of the respiratory infection disease are characterized by being due to tissue damage caused by excessive secretion of cytokines.

[0035] In a composition according to any of the above-mentioned specific examples, the sequela of the respiratory infection disease is characterized by post-COVID-19 pulmonary fibrosis.

[0036] In a composition according to any of the above-mentioned specific examples, the pharmaceutical composition is characterized in that, upon administration, it exhibits at least one of the following properties. (i) Reduction in lung inflammation score (ii) Decreased expression or secretion of pro-inflammatory cytokines (iii) Reduction of pulmonary fibrosis area (iv) Suppression of inflammasome complex formation

[0037] In a composition according to any of the above-mentioned specific examples, the cytokine is characterized by being at least one selected from the group consisting of interleukin, tumor necrosis factor, and interferon.

[0038] In a composition according to any of the above-mentioned specific examples, the cytokine is characterized by being IL-1β, TNF-α, or IFN-γ.

[0039] In a composition according to any of the above-mentioned specific examples, the pharmaceutical composition is characterized in that it is administered to an individual in a state of cytokine storm syndrome, sepsis, or organ failure due to respiratory viral infection.

[0040] In a composition according to any of the above-mentioned specific examples, the peptide is characterized in that its C-terminus is amidated.

[0041] In a composition according to any of the above-described examples, the peptide is characterized in that its C-terminus is amidated or has a free carboxyl group (-COOH).

[0042] In a composition according to any of the above-mentioned specific examples, the peptide is characterized by having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96.

[0043] In a composition according to any of the above-mentioned specific examples, the peptide is characterized by the formation of a ring between amino acid residues.

[0044] In a composition according to any of the above-mentioned specific examples, the peptide sequence is characterized in that the 16th amino acid and the 20th amino acid from the N-terminus form a ring with each other.

[0045] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized by being non-glycosylated.

[0046] In a composition according to any of the above-described examples, the immunoglobulin Fc region is characterized by being selected from the group consisting of (a) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, (b) a CH1 domain and a CH2 domain, (c) a CH1 domain and a CH3 domain, (d) a CH2 domain and a CH3 domain, (e) a combination of at least one of the CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain with an immunoglobulin hinge region or a part of a hinge region, and (f) a dimer of each domain of the heavy chain constant region and a light chain constant region.

[0047] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized in that the disulfide bond-forming site has been removed, some amino acids at the N-terminus of the natural Fc have been deleted, a methionine residue has been added to the N-terminus of the natural Fc, the complement-binding site has been removed, or the ADCC (antibody-dependent cell-mediated cytotoxicity) site has been removed.

[0048] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized by being derived from IgG, IgA, IgD, IgE, or IgM.

[0049] In a composition according to any of the above-mentioned specific examples, F is characterized in that it is an IgG Fc region.

[0050] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized in that it is an IgG4 Fc region.

[0051] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized by being a non-glycosylated Fc region derived from human IgG4.

[0052] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region has a structure in which two polypeptide chains are linked by a disulfide bond, and is characterized in that the linkage is via a nitrogen atom of only one of the two chains.

[0053] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized in that it is in a dimeric form.

[0054] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized by containing a monomer having the amino acid sequence of SEQ ID NO: 123.

[0055] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized by being a homodimer consisting of monomers of the amino acid sequence of SEQ ID NO: 123.

[0056] In a composition according to any of the above-described examples, the immunoglobulin Fc region is characterized in that it is linked by the nitrogen atom of its N-terminal proline.

[0057] In a composition according to any of the above-described examples, the immunoglobulin Fc region, F and X, is not glycosylated. In a composition according to any of the above-described examples, the immunoglobulin Fc region is a hybrid of domains having different origins, derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0058] In a composition according to any of the above-described examples, region F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

[0059] In a composition according to any of the above-described examples, the conjugate is characterized in that one end of L is linked to an amino group or thiol group of F, and the other end of L is linked to an amino group or thiol group of X by a covalent bond formed by the reaction of L to an amino group or thiol group of X, respectively.

[0060] In a composition according to any of the above-mentioned specific examples, L is characterized in that it is polyethylene glycol.

[0061] In a composition according to any of the above-described examples, the ethylene glycol repeating unit is [OCH2CH2]n, where n is a natural number, and is determined such that the average molecular weight of the [OCH2CH2]n portion in the peptide conjugate, for example, the number-average molecular weight, is 1 to 100 kDa.

[0062] In a composition according to any of the above-mentioned specific examples, the value of n is determined such that the average molecular weight of the [OCH2CH2]n moiety in the peptide conjugate, for example, the number-average molecular weight, is 10 kDa.

[0063] In a composition according to any of the above-mentioned specific examples, the chemical formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

[0064] In a composition according to any of the above-mentioned specific examples, the peptide is characterized by having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96.

[0065] Another embodiment of the present invention is a method for preventing or treating sequelae of respiratory infections, comprising the step of administering the peptide, its sustained-release conjugate, or a composition containing the same to an individual who requires the peptide, its sustained-release conjugate, or a composition containing the same.

[0066] A further embodiment of the present invention is the use of the peptide, its sustained-release conjugate, or a composition containing the same in the manufacture of a drug for the prevention or treatment of sequelae of respiratory infectious diseases.

[0067] A further embodiment of the present invention is the use of the peptide, its sustained-release conjugate, or a composition containing the same for the prevention or treatment of sequelae of respiratory infectious diseases.

[0068] The present invention will be described in more detail below.

[0069] Furthermore, each description and embodiment disclosed in this invention applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this invention is included. Moreover, this invention is not limited to the following specific descriptions.

[0070] Throughout this specification, in addition to the usual one- and three-letter codes for naturally occurring amino acids, generally accepted three-letter codes are used for other amino acids such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine), and α-methylglutamic acid. Furthermore, amino acids referred to by abbreviations in this specification are written according to IUPAC-IUB nomenclature. Alanine Ala,A Arginine Arg,R Asparagine Asn,N Aspartic acid (Asp,D) Cysteine ​​(Cys,C) Glutamic acid Glu,E Glutamine Gln,Q Glycine (Gly,G) Histidine His,H Isoleucine (Ile,I) Leucine Leu,L Lysine, K Methionine Met,M Phenylalanine Phe,F Proline Pro,P Serine Ser,S Threonine Thr,T Tryptophan Trp,W Tyrosine Tyr,Y Valin Val,V

[0071] In this specification, "Aib" is used interchangeably with "2-aminoisobutyric acid" or "aminoisobutyric acid," and 2-aminoisobutyric acid and aminoisobutyric acid are used interchangeably.

[0072] One embodiment of the present invention is a pharmaceutical composition for the prevention or treatment of sequelae of respiratory infectious diseases, 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.

[0073] In one embodiment, the peptide may have any of the amino acid sequences of SEQ ID NOs: 1 to 102.

[0074] In other embodiments, the pharmaceutical composition for the prevention or treatment of sequelae of respiratory infectious diseases may be a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a peptide having any of the amino acid sequences of SEQ ID NOs: 1 to 102 in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means a dose in which the peptide or its sustained-release conjugate exerts a therapeutic effect on sequelae of respiratory infectious diseases while being safe for the patient and without toxicity or side effects. Specifically, it means, but is not limited to, a dose in which effects such as suppression of inflammatory responses and suppression of fibrosis by reducing cytokine secretion and / or expression are obtained.

[0075] The respiratory infection disease of the present invention is a respiratory disease caused by infection with pathogens (viruses, bacteria, fungi, etc.), and a typical cause of infection is a respiratory virus. Among respiratory infectious diseases, respiratory viral infectious diseases refer to respiratory diseases caused by pathogenic viral infections. These respiratory viruses include, but are not limited to, adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella zoster virus, measles virus, respiratory syncytial virus, dengue virus, HIV (human immunodeficiency virus), influenza virus, coronavirus, severe acute respiratory syndrome virus (SARS-associated virus), or middle east respiratory syndrome coronavirus (MERS-CoV). Furthermore, in the present invention, the respiratory virus includes, but is not limited to, mutant viruses in which mutations have occurred in the genome sequence or traits. The mutant virus of the present invention means a virus that has mutations compared to the respiratory virus described above, but still induces the same sequelae.

[0076] Examples of the aforementioned coronavirus include SARS-CoV-2, but are not limited to it. SARS-CoV-2 infection causes coronavirus disease 2019 (COVID-19). In the present invention, SARS-CoV-2 includes mutant viruses. Specific examples of mutant viruses include alpha (B.1.1.7 lineage), beta (B.1.351 lineage), gamma (P.1 lineage), and delta (B.1.617.2 lineage). However, any virus that exhibits different characteristics compared to existing SARS-CoV-2 viruses, or has mutations that induce different characteristics, is acceptable.

[0077] The pharmaceutical compositions of the present invention can prevent, treat, or improve sequelae of respiratory viruses and their variants, without being limited to them.

[0078] In this invention, Coronavirus Infection-19 refers to a viral infectious disease caused by infection with a novel coronavirus (2019-nCoV or SARS-CoV-2). Although the source and transmission route of infection have not yet been clearly identified, it is highly transmissible and has caused a global pandemic.

[0079] The novel coronavirus (2019-nCoV or SARS-CoV-2) is an RNA virus with a gene size of 27-32kb that infects humans and various animals, and primarily presents with respiratory symptoms such as fever, cough, difficulty breathing, shortness of breath, and sputum. This coronavirus is known to attack ciliated epithelial cells of the bronchi and type II alveolar epithelial cells, which have large amounts of receptors such as "ACE2" and "TMPRSS2" that aid in intracellular entry.

[0080] Like other viruses, coronaviruses hijack the host cell's resources and systems to rapidly replicate and are released outside the infected cell. Once released, the exponentially multiplying virus rapidly invades surrounding healthy ciliated epithelial cells and type II alveolar epithelial cells. The infected cells trigger the infiltration, proliferation, and activation of various inflammatory cells, including macrophages that secrete cytokines that cause strong inflammation, leading to secondary symptoms (such as fever, cough, and difficulty breathing).

[0081] The aforementioned cytokines are proteins secreted by immune cells that participate in the immune response by inducing the proliferation of macrophages or promoting the differentiation of the secreting cells themselves. When cytokines are excessively secreted due to respiratory viral infection, an excessive immune response can cause damage to body tissues or organs, a condition known as cytokine storm syndrome. Such cytokine storms can leave behind sequelae such as tissue damage, tissue fibrosis, and tissue loss after respiratory viral infection.

[0082] In patients with COVID-19, SARS-CoV-2 infection triggers the activation of immune cells in lung tissue. In particular, activated mononuclear cells infiltrate the lung tissue via the bloodstream, leading to tissue damage due to an excessive immune response, separate from the elimination of the virus. Cytokine storms are considered a major cause of lingering after-effects even after complete recovery from COVID-19.

[0083] The cytokines may be pro-inflammatory cytokines, and specific examples include interleukins, tumor necrosis factors, interferons, TGFβ, GM-CSF, and G-CSF. Examples of interleukins include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and IL-8; examples of tumor necrosis factors include TNF-α and TNFβ; and examples of interferons include IFNα, IFNβ, and IFNγ, but are not limited to these.

[0084] In this invention, "sequelae of respiratory infection disease" refers to abnormal symptoms that appear independently in patients with respiratory infection disease after treatment for the respiratory infection disease. Specifically, in this invention, "sequelae of respiratory infection disease" refers to sequelae of respiratory viral infection disease, and more specifically, sequelae of coronavirus infection-19 (COVID-19), but is not limited to these.

[0085] The sequelae of respiratory infections in this invention refer to pathological conditions such as tissue deformation and functional impairment that remain even after the source of infection has been removed. Symptoms include fever, difficulty breathing, cough, pneumonia, pulmonary fibrosis, pain, muscle pain, fatigue, inflammation, and nervous system disorders. In this invention, the sequelae of respiratory infections are caused by tissue damage due to excessive cytokine secretion, but are not limited to this.

[0086] In this invention, "COVID-19 sequelae" refers to the sequelae that appear in patients after infection with SARS-CoV-2. In COVID-19, it is known that many patients who are diagnosed as fully recovered complain of persistent symptoms such as chronic fatigue, pain, and difficulty breathing. Such sequelae of COVID-19 are also called chronic COVID-19 (Long COVID), and in the United States, patients suffering from such sequelae of COVID-19 are called "long-haulers," indicating that the damage caused not only by COVID-19 but also by its sequelae is serious.

[0087] Specifically, long-term effects of COVID-19 include, but are not limited to, fever, difficulty breathing, cough, pneumonia, pulmonary fibrosis, pain, muscle aches, fatigue, inflammation, and neurological disorders.

[0088] In particular, the after-effects of coronavirus infection-19 manifest as impaired and / or damage to the respiratory system, especially the lungs. Specifically, in lungs with advanced inflammation and / or fibrosis, irreversible tissue damage occurs, leading to functional loss, and the shrinking of the lungs due to fibrosis results in symptoms such as shortness of breath, cough, and pain.

[0089] For the purposes of the present invention, the peptide or its sustained-release conjugate according to the present invention, apart from its antiviral activity, prevents abnormal organ damage that occurs in patients with respiratory viral infections or coronavirus infection-19, and / or aids in the improvement and recovery of damaged organs, before and / or after the diagnosis of complete recovery, or during the recovery period.

[0090] In this invention, pneumonia refers to a condition in which inflammation occurs in the parenchymal tissue or alveoli of the lungs, and specifically refers to inflammation that appears in patients with or who have recovered from respiratory infectious diseases. For the purposes of this invention, the pneumonia refers to acute pneumonia caused by respiratory viral infection, and chronic inflammation of such a condition leads to damage to lung tissue and / or fibrosis.

[0091] One of the serious long-term effects of COVID-19 is post-COVID-19 pulmonary fibrosis.

[0092] Pulmonary fibrosis refers to a condition in which the lungs are damaged by various stresses (chemical irritation, radiation, etc.), and the wound healing process subsequently becomes uncontrolled, resulting in excessive fibrosis of the tissue. In individuals with a normal immune system, lung function can be expected to recover, but in patients with severe inflammation (pneumonia), bleeding and congestion may occur in both lungs, making recovery difficult and potentially leading to adhesions and fibrosis. In particular, in patients with respiratory infections, an excessive immune response to the infection often occurs, and fibrosis symptoms frequently appear as a sequela. It is known that pulmonary fibrosis is more frequently observed in patients with moderate to severe symptoms of COVID-19, along with lung damage and impaired lung function due to an excessive immune response.

[0093] In particular, in patients with COVID-19, macrophages that suppress lung inflammation are replaced by pro-inflammatory macrophages derived from blood mononuclear cells, and inflammatory complexes present in lung epithelial cells and immune cells are activated. This is known to lead to sequelae due to hyperimmune reactions such as severe pneumonia and cytokine storms. Therefore, to reduce sequelae in patients with COVID-19, it is important to suppress excessive immune responses and inhibit lung damage and fibrosis.

[0094] The aforementioned inflammatory complex (Inflammasome Complex or Inflammasome) is a multi-protein intracellular complex that is involved in immune responses, such as activating the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and inducing a form of cell death called pyroptosis. In patients with COVID-19, it is known that excessive cytokine secretion and cytokine storms occur due to the activation of the inflammatory complex, resulting in long-term complications.

[0095] The peptide or its conjugate of the present invention can suppress the formation and activity of the inflammatory complex and reduce the secretion and / or expression of IL-1β, TNF-α, and IFN-γ cytokines, which are inflammatory factors typically expressed in patients with coronavirus infection-19. This can prevent the cytokine storm that occurs due to viral infection in patients with coronavirus infection-19, and thereby improve sequelae such as lung damage and pulmonary fibrosis. In other words, the peptide or its conjugate of the present invention can simultaneously suppress inflammation and fibrosis by mitigating the excessive immune response caused by respiratory viral infection, thereby improving sequelae of respiratory viral infection diseases.

[0096] For the purposes of the present invention, a pharmaceutical composition containing the peptide of the present invention or a conjugate thereof may exhibit at least one of the following properties when administered to an individual having sequelae of a respiratory infectious disease. (i) Alleviation of pneumonia caused by coronavirus infection (ii) Relief of post-COVID-19 pulmonary fibrosis accompanied by pneumonia

[0097] Specifically, the pharmaceutical composition may be a pharmaceutical composition that, upon administration, exhibits at least one of the following properties. (i) Reduction in lung inflammation score (ii) Decreased expression or secretion of pro-inflammatory cytokines (iii) Reduction of pulmonary fibrosis area (iv) Suppression of inflammasome complex formation

[0098] The properties of the pharmaceutical composition include, but are not limited to, that using a pharmaceutical composition containing the peptide or its conjugate reduces the weight of the lungs increased by fibrosis and reduces the degree of fibrous deposition.

[0099] In the present invention, the cytokine is at least one selected from the group consisting of interleukin, tumor necrosis factor, and interferon. Specifically, the cytokine is IL-1β, TNF-α, or IFN-γ, but is not limited to these.

[0100] The properties of the peptide (triple-active form) of the present invention not only improve pneumonia, which is the main cause of post-COVID-19 pulmonary fibrosis, but also directly improve pulmonary fibrosis (dual inhibitory action). Therefore, it has an inhibitory and ameliorative effect on post-COVID-19 pulmonary fibrosis, which appears as a sequela of coronavirus infection-19, and this also suggests a preventive or therapeutic effect against the sequelae of coronavirus infection-19.

[0101] Therefore, the sequelae of respiratory infections according to the present invention are, but are not limited to, post-COVID-19 pulmonary fibrosis.

[0102] In this invention, "post-COVID-19 pulmonary fibrosis" refers to pulmonary fibrosis that appears in patients with coronavirus infection-19, and means a pathological state including tissue fibrosis observed in the lungs of coronavirus infection-19 patients during the recovery period from coronavirus infection-19. It is suspected that the cause is inflammation of lung tissue due to viral infection, an excessive immune response (cytokine storm), and damage due to fibrosis, but the clear pathogenesis has not been elucidated. Unlike general pulmonary fibrosis, which develops over several months to several years, post-COVID-19 pulmonary fibrosis develops rapidly within a few days to several months.

[0103] On the other hand, sequelae of respiratory infections include a variety of neurological abnormalities such as loss of taste, altered consciousness, seizures, stroke, cerebral hemorrhage, encephalitis, dementia, and delirium. Furthermore, it is known that patients with severe infections are at a higher risk of developing depression, obsessive-compulsive disorder, psychosis, Parkinson's disease, and Alzheimer's disease.

[0104] It is presumed that such neurological disorders occur either when the source of infection directly acts on the nervous system (direct infection), or due to oxygen deficiency caused by decreased lung function, or nerve damage caused by the body's inflammatory response.

[0105] For example, SARS-CoV-2, one of the sources of infection, binds to angiotensin-converting enzyme 2 (ACE2) and inhibits the conversion of angiotensin II to angiotensin I. This leads to an increase in angiotensin II, causing vasoconstriction and damage to organs such as the kidneys, heart, and brain.

[0106] Alternatively, an infectious agent (e.g., SARS-CoV-2) can trigger an excessive immune response in the body, activating cytokines and various inflammatory substances, typically inducing a cytokine storm. Specifically, this can lead to blood clotting, thrombosis and stroke, vasculitis in nerves causing nerve damage, and direct damage to the blood-brain barrier (BBB) ​​by cytokine storms. This damage allows various inflammatory substances to pass through the BBB, causing cerebral edema and brain injury. In other words, even without direct infection from the infectious agent, excessive inflammatory responses can result in damage to various organs in the body, including the brain. Ultimately, neurological disorders are known to appear as sequelae of respiratory infections.

[0107] The pharmaceutical composition containing the peptide or its conjugate according to the present invention can alleviate inflammation in individuals with sequelae of respiratory infectious diseases, thereby exerting the effect of suppressing, delaying, or restoring central nervous system damage caused by inflammation.

[0108] In the present invention, the pharmaceutical composition, when administered to an individual suffering from cytokine storm syndrome, sepsis, or organ failure due to respiratory viral infection, reduces cytokine secretion and / or expression and suppresses the immune response. The pharmaceutical composition is administered to, but is not limited to, individuals with symptoms of pneumonia or pulmonary fibrosis after respiratory infection.

[0109] The pharmaceutical composition of the present invention contains, but is not limited to, a pharmaceutically effective amount of a peptide having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor, specifically a peptide containing any of the amino acid sequences of SEQ ID NOs: 1 to 102, a peptide essentially composed of any of the amino acid sequences of SEQ ID NOs: 1 to 102, or a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 102.

[0110] The "peptide having activity against glucagon receptor, GLP-1 receptor, and GIP receptor" in this invention is used in combination with the "triple-active compound" or "peptide" in this invention.

[0111] The triplicate metabolites having significant activity for glucagon, GLP-1, and GIP receptors exhibit in vitro activity of approximately 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 50%, 60%, 70%, 8%, 70%, 80%, 80%, 90%, 10%, 100%, 150%, and 200% or more compared to the natural ligands of the receptors (natural glucagon, natural GLP-1, and natural GIP), but are not limited to these ranges as long as they exhibit significant activity.

[0112] Here, examples of receptor activity compared to the natural product include in vitro activity of 0.001% or more, 0.01% or more, 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, and approximately 200% or more. However, it is not limited to these examples.

[0113] In this invention, "approximately" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and any numerical value within a range equivalent to or similar to the numerical value following the term "approximately" is acceptable.

[0114] On the other hand, the peptide contained in the composition of the present invention is characterized in that it has one or more, two or more, and especially three of the following activities i) to iii), specifically having significant activity. i) Activation of GLP-1 receptors ii) Activation of glucagon receptors iii) Activation of the GIP receptor

[0115] Here, activating the receptor can be achieved, for example, by increasing the in vitro activity of the receptor by approximately 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 8%, 80%, 9%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% compared to the natural product. However, it is not limited to these. A method for measuring the in vitro activity of such a triple-active product is shown in Experimental Example 1 of the present invention, but it is not limited to this.

[0116] Furthermore, the peptides have an extended half-life in the body compared to natural GLP-1, natural glucagon, and natural GIP, but are not limited to these.

[0117] Such peptides may, but are not limited to, those that do not exist in nature.

[0118] Even if the present invention describes a peptide "composed of" a specific sequence number, it does not exclude meaningless sequence additions before or after the amino acid sequence of the sequence number, naturally occurring mutations, or silent mutations, as long as they have the same or equivalent activity as the peptide consisting of the amino acid sequence of the sequence number. It goes without saying that peptides with such sequence additions or mutations are also included in this invention. In other words, even if there are differences in some sequences, if they show homology above a certain level and exhibit activity toward the glucagon receptor, they are included in the present invention.

[0119] For example, the peptide of the present invention may contain any of the amino acid sequences of SEQ ID NOs: 1 to 102, or may consist of (essentially constitute) any of the amino acid sequences of SEQ ID NOs: 1 to 102, or may have sequence identity of 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more with any of the amino acid sequences of SEQ ID NOs: 1 to 102, and is not limited to a specific sequence as long as it has a preventive or therapeutic effect against sequelae of respiratory infectious diseases.

[0120] Examples of the triple-active compound include, but are not limited to, those having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 102, those having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11 and 13 to 102, and those consisting of (essentially composed of) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11 and 13 to 102.

[0121] Furthermore, as a specific example, the triple-active peptide may contain any of the amino acid sequences of SEQ ID NOs. 21-24, 28, 29, 31, 32, 37, 42, 43, 50, 51-54, 56, 58, 64-73, 75-79, 82, 83, 91 and 96-102, and SEQ ID NOs. 21-24, 28, 29, 31, 32, 37, 42, 43, 50, 51-54, 56, 58, 64-7 It may be composed of any of the amino acid sequences 3, 75-79, 82, 83, 91 and 96-102, and may consist of any of the amino acid sequences 21-24, 28, 29, 31, 32, 37, 42, 43, 50, 51-54, 56, 58, 64-73, 75-79, 82, 83, 91 and 96-102, but is not limited to these.

[0122] As an example, the triple-active peptide may contain any of the amino acid sequences of SEQ ID NOs: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97, and 100, or it may be essentially composed of any of the amino acid sequences of SEQ ID NOs: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97, and 100, but is not limited to these.

[0123] As another example, the peptide may contain any of the amino acid sequences of SEQ ID NOs. 21, 22, 42, 43, 50, 66, 67, 77, 96, 97, and 100, or may be essentially composed of any of the amino acid sequences of SEQ ID NOs. 21, 22, 42, 43, 50, 66, 67, 77, 96, 97, and 100, or may consist of any of the amino acid sequences of SEQ ID NOs. 21, 22, 42, 43, 50, 66, 67, 77, 96, 97, and 100, but is not limited to these.

[0124] In another specific embodiment, the triple-active compound may contain any amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 27, 30-32, 34, 36, 37, 42, 43, 50-56, 58, 64-79, 83, 86, 91, 93 and 96-102. It may be essentially composed of any amino acid sequence selected from the group consisting of 91, 93 and 96-102, and may consist of any amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 27, 30-32, 34, 36, 37, 42, 43, 50-56, 58, 64-79, 83, 86, 91, 93 and 96-102, but is not limited to these.

[0125] In yet another specific embodiment, the triple-active compound may contain any amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 31, 32, 37, 42, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 79, 96, 97, 98, 99, 100, 101, and 102, and SEQ ID NOs: 21, 22, 31, 32, 37, 42, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, It may be required to consist of any amino acid sequence selected from the group consisting of 77, 79, 96, 97, 98, 99, 100, 101, and 102, or it may consist of any amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 31, 32, 37, 42, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 79, 96, 97, 98, 99, 100, 101, and 102, but it is not limited to these.

[0126] Alternatively, peptides having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more sequence identity with the aforementioned amino acid sequence are also included, and are not limited to specific sequences as long as they have a preventive or therapeutic effect against sequelae of respiratory infectious diseases.

[0127] In this invention, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are related to each other, and can be expressed as a percentage.

[0128] Homology and identity are often used interchangeably.

[0129] Whether any two peptide sequences are homologous, similar, or identical can be determined, for example, using default parameters as described in Non-Patent Document 2 and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, and FASTA (Non-Patent Documents 6, 7, and 8)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information.

[0130] Peptide homology, similarity, or identity can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 4, as disclosed in Non-Patent Document 9, for example. In summary, the GAP program defines similarity as the number of similar sequence symbols (i.e., amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a unary comparison matrix (where identity is 1 and non-identity is 0) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 10, as in Non-Patent Document 11; (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5); and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this invention refers to the relevance between sequences.

[0131] The peptides having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor may include intramolecular bridges (e.g., covalent or non-covalent bridges), and specifically may be in a ring-like form. For example, a ring may be formed between the 16th and 20th amino acids of the peptide, but is not limited thereto.

[0132] Examples of the aforementioned ring include, but are not limited to, lactam bridges (or lactam rings).

[0133] Furthermore, the peptides include all those that have been modified to include a ring by adding an amino acid that forms a ring at the desired position.

[0134] For example, this includes, but is not limited to, peptides in which the 16th and 20th amino acid pairs are replaced with glutamic acid or lysine, respectively, to form a ring.

[0135] Such rings are formed between the amino acid side chains within the peptide, for example, in the form of a lactam ring formed between the side chain of lysine and the side chain of glutamic acid, but are not limited to these forms.

[0136] Examples of peptides produced by the above combinations include peptides that differ from natural glucagon in at least one amino acid sequence, with the α-carbon of the N-terminal amino acid residue removed, and that are active against glucagon receptors, GLP-1 receptors, and GIP receptors. However, the invention is not limited to these examples, and peptides used in the present invention can be produced by combining various methods for analog production.

[0137] Furthermore, the peptide of the present invention may have some amino acids substituted with other amino acids or non-natural compounds to avoid recognition by active degrading enzymes and extend its half-life in the body, but is not limited to these.

[0138] Specifically, the peptide has an extended half-life in the body by evading recognition by digestive enzymes through the substitution of the second amino acid in its amino acid sequence. However, any amino acid substitution or modification that evades recognition by digestive enzymes in the body is acceptable.

[0139] Furthermore, such modifications for peptide production include modifications using L-type or D-type amino acids and / or unnatural amino acids, as well as modifications of the natural sequence, such as modifications of side-chain functional groups, intramolecular covalent bonds, for example, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexanolysis, and biotinylation.

[0140] Furthermore, this includes all forms in which at least one amino acid is added to the N and / or C-terminus of the triple-active form.

[0141] As mentioned above, the amino acids that can be substituted or added include not only the 20 amino acids commonly found in human proteins, but also abnormal or non-natural amino acids. Suppliers of abnormal amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids, as well as typical peptide sequences, can be synthesized and purchased from private peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in South Korea.

[0142] Amino acid derivatives can also be obtained in a similar manner, with 4-imidazoacetic acid being one example.

[0143] Furthermore, the triple-active product according to the present invention may be in a form in which its N-terminus and / or C-terminus are chemically modified to protect it from protein-cutting enzymes in living organisms and improve its stability, or it may be in a form protected by an organic group, or it may be in a form modified by adding amino acids to the peptide terminus, etc.

[0144] In particular, in the case of chemically synthesized triple-active compounds, the N and C-terminuses are charged, so acetylation of the N-terminus and / or amidation of the C-terminus are performed to remove the charge, but the process is not limited to these methods.

[0145] Specifically, the N-terminus or C-terminus of the peptide of the present invention has an amino group (-NH2) or a carboxyl group (-COOH), but is not limited to these.

[0146] The peptide according to the present invention may be a peptide with an amidated C-terminus, a peptide having a free carboxyl group (-COOH), or a peptide with an unmodified C-terminus, but is not limited to these.

[0147] As a specific example, the peptide in question has an amidated C-terminus, but it is not limited to this.

[0148] As a specific example, the peptide is non-glycosylated, but is not limited to this.

[0149] The peptides of the present invention can be synthesized by solid-phase synthesis, produced by recombinant methods, or manufactured by commercial commissioning, but are not limited to these methods.

[0150] Furthermore, the peptides of the present invention can be synthesized by methods well known in this field, such as automated peptide synthesizers, depending on their length, and can also be produced by genetic engineering techniques.

[0151] Specifically, the peptides of the present invention can be produced by standard synthesis methods, recombinant expression systems, or any other methods of the art. Therefore, the peptides of the present invention can be synthesized by many methods, including, for example, (a) a method of synthesizing the peptide stepwise by solid-phase or liquid-phase method or by fragment assembly, and separating and purifying the final peptide product; (b) a method of expressing a nucleic acid preparation encoding the peptide in host cells and recovering the expression product from a host cell culture; (c) a method of expressing a nucleic acid preparation encoding the peptide in a cell-free test tube and recovering the expression product; or a method of obtaining peptide fragments by any combination of (a), (b), and (c), and then recovering the peptide by linking the fragments.

[0152] The above provisions apply to other specific examples or embodiments of the present invention, but are not limited thereto.

[0153] Furthermore, the peptides 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 bound to the peptides having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor to extend their in vivo half-life. The biocompatible substance in this specification is used in combination with a carrier. The peptides contained in the pharmaceutical composition of the present invention may be in the form of a sustained-release conjugate.

[0154] The peptide conjugate in the present invention has improved efficacy persistence compared to the peptide without a carrier attached, and in the present invention, such a conjugate is referred to as a "sustained-release conjugate" or simply a "conjugate."

[0155] In specific embodiments of the present invention, the sustained-release conjugate may be in a form in which a peptide active against glucagon receptors, GLP-1 receptors, and GIP receptors is linked to an immunoglobulin Fc domain, which is a biocompatible substance. Specifically, the conjugate is in which an immunoglobulin Fc domain is covalently linked to a peptide active against glucagon receptors, GLP-1 receptors, and GIP receptors via a linker, but is not limited thereto.

[0156] Furthermore, such compound forms may not exist naturally.

[0157] In one specific example of the present invention, the persistent conjugate is represented by chemical formula (1), but is not limited thereto.

[0158] XLF···(1)

[0159] Here, X is a peptide having one of the amino acid sequences of SEQ ID NOs: 1 to 102, L is a linker containing an ethylene glycol repeating unit, F is an immunoglobulin Fc region, and - indicates a covalent bond between X and L, and between L and F.

[0160] X in the sustained-release conjugate of chemical formula (1) is a peptide (triple-active form) that is active against the glucagon receptor, GLP-1 receptor, and GIP receptor as described above. Specifically, it is a peptide having any of the amino acid sequences of SEQ ID NOs: 1 to 102, a peptide essentially composed of any of the amino acid sequences of SEQ ID NOs: 1 to 102, or a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 102, but is not limited to these.

[0161] The sustained-release conjugate of chemical formula (1) is a form in which a peptide having one of the amino acid sequences of SEQ ID NOs: 1 to 102 and an immunoglobulin Fc region are linked to each other by a linker. This conjugate exhibits improved sustained efficacy compared to a peptide having one of the amino acid sequences of SEQ ID NOs: 1 to 102 without the immunoglobulin Fc region.

[0162] The conjugate of the present invention exhibits significant activity against glucagon receptors, GLP-1 receptors, and GIP receptors even in its conjugate form, and therefore exerts preventive or therapeutic effects against sequelae of respiratory infectious diseases.

[0163] Specifically, the conjugates of the present invention exhibit in vitro activity of 0.01% or more, 0.1% or more, 0.2% or more, 0.5% or more, 0.7% 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, 800% or more, and 100% or more compared to the natural conjugates, but are not limited to these values.

[0164] For the purposes of the present invention, the peptide or its conjugate has an activity of 0.1% or more, 0.2% or more, 0.5% or more, 0.7% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% 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, or 100% or more compared to the natural product.

[0165] The composition of the present invention may (i) contain peptides active against glucagon receptors, GLP-1 receptors, and GIP receptors, or (ii) contain a sustained-release conjugate of the peptides active against glucagon receptors, GLP-1 receptors, and GIP receptors, the sustained-release conjugate exhibits excellent preventive or therapeutic effects against sequelae of respiratory infectious diseases based on improved persistence in the body.

[0166] In the persistent conjugate of chemical formula (1), the linkage between peptide X, which has one of the amino acid sequences of sequence numbers 1 to 102, and the immunoglobulin Fc region is either a physical bond or a chemical bond, and is either a non-covalent bond or a covalent bond, specifically a covalent bond, but is not limited to these.

[0167] Furthermore, X may be formed by linking F via a linker L. More specifically, X and L, and L and F may be linked to each other by covalent bonds, where the compound is a compound in which X, L, and F are linked by covalent bonds in the order of chemical formula (1).

[0168] The aforementioned F is an immunoglobulin Fc region, and more specifically, the immunoglobulin Fc region is derived from IgG, but is not limited to these.

[0169] As a specific example of the present invention, the F (immunoglobulin Fc region) is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains, but the invention is not limited to this.

[0170] In the present invention, the "immunoglobulin Fc region" refers to the region of immunoglobulin excluding the variable regions of the heavy and light chains, and including the heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3). The immunoglobulin Fc region may be a component that forms part of the conjugate of the present invention.

[0171] In this specification, the Fc region includes not only the natural sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, such as modified sequences that differ from the natural one, for example, by deletion, insertion, non-conservative or conservative substitution, or combination thereof, of at least one amino acid residue of the natural sequence. It is assumed that the derivatives, substituted and modified sequences possess the ability to bind to FcRn. In the present invention, F is a human immunoglobulin region, but is not limited thereto. The F (immunoglobulin Fc region) is a structure in which two polypeptide chains are linked by a disulfide bond, and is linked only via the nitrogen atom of one of the two chains, but is not limited thereto. The linkage via the nitrogen atom may be linked by reductive amination to the ε-amino atom or N-terminal amino group of lysine.

[0172] Reductive amination is a reaction in which an amine group or amino group in one reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) in another reactant to produce an amine, which is then reduced to form an amine bond. This is a well-known organic synthesis reaction in the field.

[0173] As a specific example, the aforementioned F is linked via the nitrogen atom of the proline at its N-terminus, but it is not limited to this.

[0174] The immunoglobulin Fc region is a component that forms part of the compound of chemical formula (1) of the present invention, and specifically corresponds to F in chemical formula (1).

[0175] Such immunoglobulin Fc regions include, but are not limited to, a hinge region within the heavy chain constant region.

[0176] The immunoglobulin Fc region in the present invention may include a specific hinge sequence at its N-terminus.

[0177] In this invention, "hinge arrangement" refers to a site located in the heavy chain that forms a dimer of the immunoglobulin Fc region via an interdisulfide bond.

[0178] In the present invention, the hinge sequence is a mutated hinge sequence having the following amino acid sequence, but with a portion of it deleted, resulting in a single cysteine ​​residue. Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (Sequence ID 103)

[0179] The aforementioned hinge sequence may contain only one cysteine ​​residue, with the 8th or 11th cysteine ​​residue in the hinge sequence of Sequence ID No. 103 being deleted. The hinge sequence of the present invention consists of 3 to 12 amino acids and contains only one cysteine ​​residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequence. Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (Sequence ID 104) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (Sequence ID 105) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 106) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (Sequence ID 107) Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (Sequence ID 108) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 109) Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 110) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 111) Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 112) Pro-Ser-Cys-Pro (Sequence ID 113) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 114) Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (Sequence ID 115) Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 116) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 117) Lys-Tyr-Gly-Pro-Pro-Cys-Pro (Sequence ID 118) Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 119) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 120) Glu-Pro-Ser-Cys (SEQ ID NO: 121) Ser-Cys-Pro (Sequence ID 122)

[0180] More specifically, the hinge sequence has the amino acid sequence of SEQ ID NO: 113 (Pro-Ser-Cys-Pro) or SEQ ID NO: 122 (Ser-Cys-Pro), but is not limited to these.

[0181] The immunoglobulin Fc region of the present invention is in a form in which two molecules of immunoglobulin Fc chains form a dimer due to the presence of a hinge sequence, and the compound of chemical formula (1) of the present invention is in a form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited to these.

[0182] In this invention, "N-terminus" means the amino terminus of a protein or polypeptide, and includes the very end of the amino terminus, or one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids from the very end. The immunoglobulin Fc region of this invention includes a hinge sequence at the N-terminus, but is not limited to this.

[0183] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes some or all of the heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), excluding the variable regions of the heavy and light chains of the immunoglobulin, provided that it has substantially equivalent or improved effects to those of the natural immunoglobulin. In addition, it may be a region in which a very long partial amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0184] For example, the immunoglobulin Fc region of the present invention is 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 at least one of the CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge region (or a part of a hinge region); or 6) a dimer of each domain of the heavy chain constant region and a light chain constant region. However, it is not limited to these.

[0185] The immunoglobulin Fc region in the present invention is in the form of a dimer or polymer composed of single-chain immunoglobulins consisting of domains of the same origin, but is not limited to these forms.

[0186] As a specific example, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, where the Fc region dimer F and X are covalently linked via a single linker L containing an ethylene glycol repeating unit. In a 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 further specific example of this embodiment, of the two polypeptide chains of the Fc region dimer F to which X is linked, only one molecule of X is covalently linked via L. In the most specific example of this embodiment, F is a homodimer.

[0187] As another specific example, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains, but is not limited to this.

[0188] In other embodiments of the persistent conjugate of the present invention, two molecules of X may be symmetrically bound to one Fc region in the form of a dimer. Here, the immunoglobulin Fc region and X may be linked to each other by a linker L. However, the present invention is not limited to the above examples.

[0189] Furthermore, the immunoglobulin Fc region of the present invention includes not only natural amino acid sequences but also sequence derivatives thereof. An amino acid sequence derivative means one in which at least one amino acid residue of a natural amino acid sequence is deleted, inserted, non-conservative or conservatively substituted, or has a different sequence due to a combination thereof.

[0190] For example, in the case of IgG Fc, amino acid residues 214-238, 297-299, 318-322, or 327-331, which are known to be important for binding, are used as suitable modification sites.

[0191] Furthermore, various derivatives are used, such as derivatives in which the disulfide bond-forming site has been removed, derivatives in which several amino acids at the N-terminus of the natural Fc are deleted, and derivatives in which a methionine residue has been added to the N-terminus of the natural Fc. In addition, to eliminate the effector function, the complement binding site, such as the C1q binding site, may be removed, and the ADCC (antibody-dependent cell-mediated cytotoxicity) site may also be removed. Techniques for producing such immunoglobulin Fc region sequence derivatives are disclosed in Patent Documents 3 and 4, among others.

[0192] Amino acid exchanges in proteins and peptides that do not alter the overall molecular activity are well known in the art (Non-Patent Literature 12). 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, and Asp / Gly. Modifications may also be made by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0193] The aforementioned Fc derivative may exhibit biological activity equivalent to that of the Fc region of the present invention, and may also have improved structural stability against heat, pH, etc., of the Fc region.

[0194] Furthermore, such Fc regions may be obtained from natural sources isolated from living organisms such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, or they may be recombinants or derivatives obtained from transformed animal cells or microorganisms. Here, the method of obtaining from natural sources may involve isolating total immunoglobulins from living human or animal organisms and then treating them with proteolytic enzymes. Treatment with papain cleaves them into Fab and Fc, and treatment with pepsin cleaves them into pF'c and F(ab)2. These can then be separated into Fc or pF'c using methods such as size exclusion chromatography. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0195] Furthermore, the immunoglobulin Fc region may be a natural glycan, an increased glycan compared to the natural one, a decreased glycan compared to the natural one, or a form from which the glycans have been removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering techniques using microorganisms are used to increase, decrease, or remove the immunoglobulin Fc glycans. Here, the immunoglobulin Fc region from which the glycans have been removed has a significantly reduced binding affinity to complement (c1q), and antibody-dependent cell-mediated cytotoxicity or complement-dependent cell-mediated cytotoxicity is reduced or eliminated, thus not inducing unwanted immune responses in the body. For these reasons, immunoglobulin Fc regions from which the glycans have been removed or which have been deglycosylated are suitable for their original purpose as drug carriers.

[0196] In this invention, "deglycosylation" refers to the Fc region from which sugar has been removed by an enzyme, and "aglycosylation" refers to the Fc region that is produced in prokaryotes, and more specifically in Escherichia coli, and has not been glycosylated.

[0197] On the other hand, the immunoglobulin Fc region is of human origin, or of animal origin such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and in more specific embodiments, it is of human origin.

[0198] Furthermore, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, or an Fc region resulting from a combination thereof or a hybrid thereof. In a more specific embodiment, it may be derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, it may be derived from IgG, which is known to extend the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited to these.

[0199] Furthermore, in one specific embodiment, the immunoglobulin Fc region is the human IgG4 Fc region, and may be in the form of a homodimer (mp) in which two monomers are linked by a disulfide bond (inter-chain form) between the third amino acid cysteine ​​of each monomer. Here, each monomer of the homodimer independently has / may have an internal disulfide bond between the 35th and 95th cysteine ​​and an internal disulfide bond between the 141st and 199th cysteine, i.e., two internal disulfide bonds (intra-chain form). Each monomer consists of 221 amino acids, and the amino acids that form the homodimer consist of a total of 442 amino acids, but are not limited to these. Specifically, an immunoglobulin Fc fragment is formed when two monomers having the amino acid sequence of SEQ ID NO: 123 (consisting of 221 amino acids) form a homodimer through a disulfide bond between the third amino acid, cysteine, of each monomer. The monomers of this homodimer independently form an internal disulfide bond between the 35th and 95th cysteine ​​positions, and an internal disulfide bond between the 141st and 199th cysteine ​​positions, but are not limited to this.

[0200] In chemical formula (1), F contains a monomer having the amino acid sequence of SEQ ID NO: 123, and F is a homodimer of the monomer having the amino acid sequence of SEQ ID NO: 123, but is not limited to these.

[0201] For example, the immunoglobulin Fc region is a homodimer containing the amino acid sequence of SEQ ID NO: 124 (consisting of 442 amino acids), but it is not limited to this.

[0202] As a specific example, the immunoglobulin Fc region and X mentioned above are not glycosylated, but this is not the only example.

[0203] On the other hand, in the present invention, "combination" with respect to the immunoglobulin Fc region means that when forming a dimer or polymer, polypeptides encoding single-chain immunoglobulin Fc regions of the same origin bind to single-chain polypeptides of different origins. That is, a dimer or polymer can be produced from at least two regions selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc regions.

[0204] In this invention, "hybrid" means that within the constant region of a single-chain immunoglobulin, there are sequences corresponding to immunoglobulin Fc regions of at least two different origins. Various forms of hybrids are possible in this invention. That is, hybrids of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include a hinge.

[0205] On the other hand, IgG is also divided into subclasses IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations thereof or hybridization thereof are also possible. Specifically, these are IgG2 and IgG4 subclasses, and more specifically, Fc fragments of IgG4 that have little to no effector function, such as complement-dependent cytotoxicity (CDC).

[0206] Furthermore, the aforementioned conjugates exhibit improved efficacy and duration compared to natural GLP-1, GIP, or glucagon, or to X without F modification. Such conjugates include, but are not limited to, the forms described above, as well as forms encapsulated in biodegradable nanoparticles.

[0207] On the other hand, in chemical formula (1), L may be a non-peptide linker, such as a linker containing ethylene glycol repeating units.

[0208] The "non-peptide linker" in the present invention includes a biocompatible polymer in which at least two repeating units are bonded together. The repeating units are linked to each other by any covalent bond other than peptide bonds. The non-peptide linker is a component that forms part of the compound of the present invention and corresponds to L in chemical formula (1).

[0209] The non-peptide linker used in the present invention may be any polymer that is resistant to in vivo proteolytic enzymes. The non-peptide linker in the present invention is used in combination with the non-peptide polymer.

[0210] Furthermore, the non-peptide linker of the present invention, which is bound to the polypeptide corresponding to F, may be not only one type of polymer, but also a combination of different types of polymers.

[0211] In one specific embodiment, the conjugate may be formed in which F and X are covalently linked to each other via a non-peptide linker having a reactive group bound to F, specifically an immunoglobulin Fc domain, and X, specifically a triple-active compound, at both ends.

[0212] Specifically, the non-peptide linker in the present invention contains a reactive group at its terminus and can form a conjugate by reacting with other components constituting the conjugate. When a non-peptide linker having reactive functional groups at both ends forms a conjugate by bonding to X and F of chemical formula (1) via the respective reactive groups, the non-peptide linker or non-peptide polymer is also called a non-peptide polymer linker moiety or non-peptide linker linker moiety.

[0213] The non-peptide linker is a linker containing ethylene glycol repeating units, such as polyethylene glycol linker, but is not limited to these. Furthermore, derivatives of these linkers known in the art, as well as derivatives that can be easily produced by the art, are also included in the present invention.

[0214] The "polyethylene glycol linker" in the present invention includes a biocompatible polymer in which at least two ethylene glycol repeating units are bonded. The repeating units are linked to each other by any covalent bond other than peptide bonds. The polyethylene glycol linker is a component that forms part of the compound of the present invention and corresponds to L in chemical formula (1).

[0215] Specifically, L (polyethylene glycol linker) is a linker containing ethylene glycol repeating units, such as polyethylene glycol, but is not limited thereto. The polyethylene glycol used herein encompasses, but is not limited to, ethylene glycol homopolymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG). Furthermore, derivatives of these known in the art and derivatives readily available in the art are also included in the present invention.

[0216] The polyethylene glycol linker contains ethylene glycol repeating units and may also contain functional groups used in the formation of the conjugate at its terminals until it is configured as a conjugate. The persistent conjugate according to the present invention is in a form in which X and F are linked via the functional groups, but is not limited thereto. The non-peptide linker in the present invention contains two or more functional groups, and each functional group may be the same or different, but is not limited thereto.

[0217] Specifically, the linker is polyethylene glycol (PEG) represented by chemical formula (2), but is not limited to this.

[0218] JPEG2026091850000002.jpg2339...(2)

[0219] Here, n is 10 to 2400, n is 10 to 480, or n is 50 to 250, but is not limited to these ranges.

[0220] The PEG portion of the aforementioned persistent conjugate includes, but is not limited to, the -(CH2CH2O)n- structure as well as the oxygen atom interposed between the linking element and its -(CH2CH2O)n-.

[0221] As a specific example, the ethylene glycol repeating unit is represented as [OCH2CH2]n, where the n value is a natural number and is determined such that the average molecular weight of the [OCH2CH2]n portion in the peptide conjugate, for example, the number-average molecular weight, is greater than 0 to approximately 100 kDa, but is not limited to this. As another specific example, the n value is a natural number, and the average molecular weight of the [OCH2CH2]n portion in the peptide conjugate, for example, the number-average molecular weight is approximately 1 to approximately 100 kDa, approximately 1 to approximately 80 kDa, approximately 1 to approximately 50 kDa, approximately 1 to approximately 30 kDa, approximately 1 to approximately 25 kDa, approximately 1 to approximately 20 kDa, approximately 1 to approximately 15 kDa, approximately 1 to approximately 13 kDa, approximately 1 to approximately 11 kDa, approximately 1 to approximately 10 kDa, approximately 1 to approximately 8 kDa, approximately 1 to approximately 5 kDa, approximately 1 to approximately 3.4 kDa, approximately 3 to approximately 30 kDa, approximately 3 to approximately 27 kDa, approximately 3 to approximately 25 kDa, approximately 3 to approximately 22 kDa, approximately 3 to approximately 20 kDa, approximately 3 to approximately 18 kDa, approximately 3 to approximately 16 kDa, approximately 3 to approximately 15kDa, approximately 3-13kDa, approximately 3-11kDa, approximately 3-10kDa, approximately 3-8kDa, approximately 3-5kDa, approximately 3-3.4kDa, approximately 8-30kDa, approximately 8-27kDa, approximately 8-25kDa, approximately 8-22kDa, approximately 8-20kDa, approximately 8-18kDa, approximately 8-16kDa The ranges are approximately 8-15kDa, 8-13kDa, 8-11kDa, 8-10kDa, 9-15kDa, 9-14kDa, 9-13kDa, 9-12kDa, 9-11kDa, 9.5-10.5kDa, or 10kDa, but are not limited to these ranges.

[0222] Furthermore, while the conjugate in one specific embodiment has a structure in which a peptide (X) and an immunoglobulin Fc region (F) are covalently linked via a linker containing ethylene glycol repeating units, it is not limited to this structure.

[0223] Furthermore, the persistent conjugate in one specific embodiment has a structure in which the peptide (X) and the immunoglobulin Fc region (F) of the present invention are covalently linked via a linker L containing ethylene glycol repeating units, but is not limited thereto.

[0224] The non-peptide linker used in the present invention may be any polymer that is resistant to in vivo proteolytic enzymes and contains ethylene glycol repeating units. The molecular weight of the non-peptide polymer is in the range of greater than 0 to about 100 kDa, about 1 to about 100 kDa, specifically about 1 to about 20 kDa, or about 1 to about 10 kDa, but is not limited to these ranges. Furthermore, the non-peptide linker of the present invention that is bound to the polypeptide corresponding to F may be not only one type of polymer, but also a combination of different types of polymers.

[0225] Specifically, the non-peptide linker may have reactive groups at both ends in a state where it is not bound to F and X, and may be bound to F and X via the reactive groups.

[0226] As a specific example, both ends of the linker are bonded to the thiol group, amino group, or hydroxyl group of the immunoglobulin Fc region, and to the thiol group, amino group, azide group, or hydroxyl group of peptide (X), but are not limited to these.

[0227] Specifically, the linker includes, but is not limited to, a reactive group whose two ends are bound to the immunoglobulin Fc region and peptide (X), respectively, a reactive group which is bound to the thiol group of cysteine ​​in the immunoglobulin Fc region and at least one selected from the group consisting of an amino group located at the N-terminus, lysine, arginine, glutamine and / or histidine, and a hydroxyl group located at the C-terminus, and a reactive group which is bound to the thiol group of cysteine ​​in peptide (X) and at least one selected from the group consisting of an amino group of lysine, arginine, glutamine and / or histidine, an azide group of azidrisine, and a hydroxyl group.

[0228] More specifically, the linker's reactive group is at least one selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited to these.

[0229] In the above, examples of aldehyde groups include, but are not limited to, propionaldehyde groups and butyraldehyde groups.

[0230] In the above, succinimide derivatives include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate.

[0231] The linker may be linked to F, which is an immunoglobulin Fc region, and X, which is a peptide (triply active form), via the above-mentioned reactive group, and converted into a linker linkage.

[0232] Furthermore, the final product generated by reductive amination via aldehyde bonds is far more stable than that linked by amide bonds. Aldehyde reactive groups selectively react at the N-terminus at low pH, and can form covalent bonds with lysine residues at high pH, ​​such as pH 9.0.

[0233] Furthermore, the reactive groups at both ends of the non-peptide linker may be the same or different. For example, it may have aldehyde groups at both ends, or one end may have a maleimide group and the other end may have an aldehyde group, propionaldehyde group, or butyraldehyde group. However, it is not limited to this, as long as F, specifically an immunoglobulin Fc region and X, are bound to each end of the non-peptide linker.

[0234] For example, one end of the non-peptide linker may contain a maleimide group as a reactive group, while the other end may contain an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like.

[0235] When polyethylene glycol having hydroxyl reactive groups at both ends is used as a non-peptidic polymer, the persistent protein conjugate of the present invention can be produced by activating the hydroxyl groups as various reactive groups as described above through known chemical reactions, or by using commercially available polyethylene glycol having modified reactive groups.

[0236] In one specific embodiment, the non-peptidic polymer is linked to a cysteine ​​residue of X, more specifically to the -SH group of cysteine, but is not limited to this.

[0237] For example, in the peptide corresponding to X, the non-peptidic polymer may be linked to the 10th, 13th, 15th, 17th, 19th, 21st, 24th, 28th, 29th, 30th, 31st, 40th, or 41st cysteine ​​residue, but is not limited to these.

[0238] Specifically, a reactive group of a non-peptidic polymer may be linked to the -SH group of the cysteine ​​residue. The reactive group is as described above.

[0239] Furthermore, in the aforementioned conjugate, the reactive group of the non-peptidic polymer may be linked to the -NH2 located at the N-terminus of the immunoglobulin Fc region, but this is merely one example.

[0240] When using maleimide-PEG-aldehyde, the maleimide group can be linked to the -SH group of the peptide via a thioether bond, and the aldehyde group can be linked to the -NH2 group of immunoglobulin Fc via a reductive alkylation reaction. However, these are not the only examples.

[0241] Through such reductive alkylation, the N-terminal amino group of the immunoglobulin Fc domain is linked to the oxygen atom at one end of PEG via a linker functional group having the structure -CH2CH2CH2-, forming a structure such as -PEG-O-CH2CH2CH2NH-immunoglobulin Fc, and a thioether bond can be formed to link one end of PEG to the sulfur atom located at the cysteine ​​of the peptide. The aforementioned thioether bond may have the structure of chemical formula (3).

[0242] JPEG2026091850000003.jpg3125...(3)

[0243] However, this is not limited to the above example; it is merely one example.

[0244] Furthermore, in the aforementioned conjugate, the linker's reactive group may be linked to the -NH2 located at the N-terminus of the immunoglobulin Fc region, but this is merely one example.

[0245] Furthermore, in the aforementioned conjugate, the peptide according to the present invention may be linked to a linker having a reactive group via its C-terminus, but this is merely one example.

[0246] In this invention, "C-terminus" refers to the carboxyl end of a peptide, and for the purposes of this invention, it refers to the position where the linker is attached. For example, although not limited to these, it includes not only the very last amino acid residue of the C-terminus, but also all amino acid residues surrounding the C-terminus, specifically the 1st to 20th amino acid residues from the very end.

[0247] Furthermore, the aforementioned conjugates exhibit improved efficacy persistence compared to X without F modification, and such conjugates include not only the aforementioned forms but also forms encapsulated in biodegradable nanoparticles, among others.

[0248] On the other hand, with regard to the aforementioned triple-active compound and its sustained-release conjugate, the full texts of Patent Documents 5 and 6 are incorporated herein by reference.

[0249] Unless otherwise specified herein, the detailed descriptions and claims of the “peptides” or “conjugates” in which such peptides are covalently linked to biocompatible substances according to the present invention apply to all categories that include not only such peptides or conjugates, but also salts of such peptides or conjugates (e.g., pharmaceutically acceptable salts of the peptide) or solvates thereof. Therefore, even if the specification only refers to “peptides” or “conjugates,” the description applies equally to specific salts thereof, specific solvates thereof, and specific solvates of specific salts thereof. These salt forms may be, for example, any pharmaceutically acceptable salt.

[0250] The type of salt is not particularly limited. However, it is preferable that it be in a form that is safe and effective for individuals, such as mammals, but is not particularly limited thereto.

[0251] The term "pharmaceutically acceptable" means a substance that can be used effectively for its desired purpose without inducing excessive toxicity, irritation, or allergic reactions, within the bounds of pharmaceutical judgment.

[0252] In this invention, "pharmaceutically acceptable salts" include salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromate, 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, and benzenesulfonic acid. Salts derived from suitable bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0253] Furthermore, in this invention, "solvate" means a peptide or salt thereof that has formed a complex with a solvent molecule according to the present invention.

[0254] The composition according to the present invention may contain a peptide (triply active form) or a conjugate thereof, and more specifically, it may contain a pharmacologically effective amount of a peptide or a conjugate thereof. It may also further contain a pharmaceutically acceptable carrier. The composition according to the present invention may have applications for the prevention or treatment of sequelae of respiratory infectious diseases.

[0255] In the present invention, "prevention" means any action that suppresses or delays the sequelae of respiratory infections by administering the peptide (for example, in the form of the peptide itself or a persistent conjugate to which a biocompatible substance is bound) or a composition containing the peptide. In the present invention, prevention of sequelae of respiratory infections means, independently of suppressing the source of infection, suppressing or delaying abnormal bodily reactions that may occur in an infected individual after infection (for example, organ dysfunction or damage).

[0256] In the present invention, "treatment" means any action that improves or favorably alters the symptoms of sequelae of respiratory infections by administering the peptide (for example, in the form of the peptide itself or a persistent conjugate to which a biocompatible substance is bound) or a composition containing the peptide. In the present invention, treatment of sequelae of respiratory infections means, but is not limited to, restoring or favorably altering abnormal bodily reactions (for example, organ dysfunction or damage) that may occur in an infected individual after infection, independently of suppressing the source of infection.

[0257] Specifically, the peptides, sustained-release conjugates, or compositions containing the same according to the present invention can prevent or treat sequelae of respiratory infections by improving excessive inflammatory responses and fibrosis, but are not limited to these.

[0258] In the present invention, the prevention or treatment of sequelae of respiratory infectious diseases is performed before and / or after the diagnosis of complete recovery from the respiratory infectious disease.

[0259] As used herein, "administration" means introducing a predetermined substance (e.g., a triple active substance or its sustained conjugate) into a patient by any suitable method, and the administration route of the composition is not particularly limited thereto, but can be administered by any general route capable of delivering the composition to a target in vivo, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, etc.

[0260] The use of a triple active substance or its sustained conjugate having activity against all of the glucagon, GLP-1 and GIP receptors of the present invention can reduce the number of administrations to chronic patients who must be administered daily due to an epochal improvement in blood half-life and in vivo efficacy sustaining effect, and thus has a great advantage in that it can improve the quality of life of patients.

[0261] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier or diluent. Such a pharmaceutically acceptable carrier or diluent may be non-naturally occurring.

[0262] "Pharmaceutically acceptable" as used herein means an amount sufficient to exert a therapeutic effect and no side effects, and can be easily determined by those skilled in the art based on known elements in the medical field such as the type of disease, the age, weight, health status, sex of the patient, sensitivity to the drug, administration route, administration method, number of administrations, treatment period, formulation, drugs used simultaneously, etc.

[0263] The pharmaceutical composition containing the peptide of the present invention may further contain a pharmaceutically acceptable excipient. In the case of oral administration, binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavors, etc. can be used for the excipient, and in the case of injections, buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. can be used, but are not particularly limited thereto.

[0264] The composition of the present invention can be manufactured in various forms by mixing it with pharmaceutically acceptable excipients as described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it can be manufactured in single-use ampoules or multi-dose forms. In addition, it can be manufactured in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0265] Examples of carriers, excipients, and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The formulation may also further contain fillers, anti-agglomerates, lubricants, wetting agents, fragrances, preservatives, and the like.

[0266] Furthermore, the pharmaceutical composition of the present invention may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0267] Furthermore, the composition may be formulated into a unit-dose type formulation suitable for intravenous administration to a patient by conventional methods in the pharmaceutical field, specifically into a formulation useful for the administration of protein pharmaceuticals, and may be administered orally, or by parenteral administration routes including, but not limited to, skin, intravenous, intramuscular, intraarterial, intramedullary, intrameningeal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, topical, sublingual, vaginal, or rectal routes using administration methods commonly used in the art.

[0268] Furthermore, the conjugate can be mixed with various drug-acceptable carriers such as physiological saline or organic solvents, and to improve stability and absorption, carbohydrates such as glucose, sucrose, and dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, low molecular weight proteins, and other stabilizers can be used as drugs.

[0269] Another aspect of the present invention provides a method for preventing or treating sequelae of respiratory infections, comprising the step of administering to an individual a pharmaceutical composition containing a pharmaceutically effective amount of a peptide having any of the amino acid sequences of SEQ ID NOs: 1 to 102 or a sustained-release conjugate thereof.

[0270] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of drug that is the active ingredient, along with various related factors such as the disease being 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 contains, but is not limited to, the aforementioned triple-active compound or a sustained-release conjugate containing the same in a pharmaceutically effective amount.

[0271] The statement that the peptide or sustained-release conjugate is present in a pharmaceutically effective amount means that it is present in an amount sufficient to obtain the desired pharmacological activity (e.g., prevention, improvement, or treatment of sequelae of respiratory infections) through the triple-active compound or sustained-release conjugate, and that it is present at a level that is pharmaceutically acceptable, with no toxicity or side effects in the administered individual, or only at a minimal level. However, it is not limited to these meanings. Such a pharmaceutically effective amount is determined by comprehensively considering factors such as the number of administrations, the patient, and the dosage form.

[0272] The sustained-release conjugate of the present invention is administered in doses of approximately 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.10 mg, 0.11 mg, 0.12 mg, 0.13 mg, 0.14 mg, 0.15 mg or more per kg, but is not limited to these doses.

[0273] The pharmaceutical composition of the present invention contains the above-mentioned components (active ingredients) in an amount of 0.01 to 99% by weight / volume, but is not particularly limited thereto.

[0274] The total effective dose of the composition of the present invention may be administered to a patient as a single dose, or as part of a fractionated treatment protocol involving multiple doses over a long period. The pharmaceutical composition of the present invention may vary in content of the active ingredient depending on the severity of the disease. Specifically, the total dose of the peptide or its sustained-release conjugate of the present invention is preferably about 0.0001 mg to 500 mg per kg of body weight per day. However, the effective dose of the peptide or its conjugate is determined by considering various factors such as the patient's age, weight, health status, sex, disease severity, diet, and excretion rate, as well as the route of administration and number of treatments of the pharmaceutical composition. Considering these factors, a person with ordinary skill in the art should be able to determine an appropriate effective dose of the composition of the present invention for a specific use. The pharmaceutical composition according to the present invention is not particularly limited in dosage form, route of administration, and method of administration, as long as it achieves the effects of the present invention.

[0275] Since the pharmaceutical composition of the present invention exhibits excellent in vivo persistence and potency, the number and frequency of administrations of the pharmaceutical formulation of the present invention can be significantly reduced.

[0276] For example, the pharmaceutical composition of the present invention is administered once a week, once every two weeks, once every four weeks, or once a month, but is not limited to these doses.

[0277] Another embodiment of the present invention provides a method for preventing or treating sequelae of respiratory infections, comprising the step of administering the triple-active peptide and / or triple-active sustained-release conjugate, or a composition containing the same, to an individual requiring the triple-active peptide and / or triple-active sustained-release conjugate, or a composition containing the same.

[0278] The triple-active compound and / or the sustained-release triple-active compound, or compositions containing the same, and the sequelae, prevention, and treatment of respiratory infections are as described above.

[0279] In the present invention, the individual referred to is an individual suspected of having sequelae of a respiratory infection. The individual suspected of having sequelae of a respiratory infection means a mammal, including humans, mice, and livestock, that has a respiratory infection, has an abnormal physical condition despite having recovered from a respiratory infection, or is at risk of having one. However, any individual that can be treated with the triple-active compound and / or conjugate of the present invention, or the composition containing the same, is acceptable. Administration of the triple-active compound, its sustained-release conjugate, or a composition containing the same can suppress excessive immune responses (cytokine storms) and improve pneumonia and fibrosis by inhibiting cytokine secretion and / or expression. Therefore, the individual referred to is an individual with a cytokine storm, sepsis, or organ failure, and is particularly an individual with pneumonia and pulmonary fibrosis symptoms after a respiratory infection, but is not limited to these.

[0280] In the present invention, "administration" means introducing a predetermined substance to a patient by any appropriate method, and the administration route of the composition is not limited to these, but can be any common route that can deliver the composition to a target in the body, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc.

[0281] The method of the present invention may include the step of administering a pharmaceutical composition containing the triple active substance or its sustained conjugate in a pharmaceutically effective amount. A suitable total daily dosage is determined by the attending physician within the scope of sound medical judgment and can be administered once or in several divided doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient depends on the type and degree of response to be achieved, whether other formulations are used in some cases, the specific composition, the patient's age, weight, general health status, sex, diet, administration time, administration route, secretion rate of the composition, treatment period, various factors including drugs administered with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical field, and preferably varies in amount according to these factors.

[0282] The pharmaceutical composition of the present invention is administered once a week, once every two weeks, once every four weeks, or once a month, but is not limited thereto.

[0283] Another aspect of realizing the present invention is the use of a composition containing the triple active substance or its sustained conjugate in the manufacture of a medicament for the prevention or treatment of sequelae of respiratory infectious diseases. For the purposes of the present invention, the triple active substance, its sustained conjugate, or the composition containing it has the effect of suppressing the cytokine storm, but is not limited thereto.

[0284] The triple active substance and / or its conjugate, or the composition containing it, for the sequelae, prevention and treatment of respiratory infectious diseases are as described above.

[0285] Another aspect of realizing the present invention provides the use of the triple active substance or its sustained conjugate, or a composition containing it, for the prevention or treatment of sequelae of respiratory infectious diseases. For the purposes of the present invention, the triple active substance, its sustained conjugate, or the composition containing it has the effect of suppressing the cytokine storm, but is not limited thereto.

[0286] The triple-active compound and / or its conjugate, or compositions containing the same, and the sequelae, prevention, and treatment of respiratory infectious diseases are as described above.

[0287] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention, and the present invention is not limited to them. [Examples]

[0288] Preparation of a triple-active compound A triple-active metabolite exhibiting activity against GLP-1, GIP, and the glucagon receptor was constructed. Its sequences are shown in Table 1.

[0289] [Table 1] JPEG2026091850000005.jpg142150 JPEG2026091850000006.jpg140150 JPEG2026091850000007.jpg142150 JPEG2026091850000008.jpg143150 JPEG2026091850000009.jpg139150 JPEG2026091850000010.jpg72150

[0290] In the sequences shown in Table 1, the amino acid denoted by X is the non-natural amino acid Aib (2-aminoisobutyric acid), and the underlined amino acids indicate that they form a ring with each other. Also in Table 1, CA represents 4-imidazoacetyl. The triple-active peptide can be used as a triple-active form with an amidated C-terminus, as needed. [Examples]

[0291] Fabrication of a triple-active sustained-release conjugate To pegyrate a 10kDa PEG having a maleimide group and an aldehyde group at each end, i.e., maleimide-PEG-aldehyde (10kDa, NOF, Japan), to the cysteine ​​residue of the triple-active product of Example 1 (SEQ ID NOs. 21, 22, 42, 43, 50, 77, and 96), the molar ratio of the triple-active product to maleimide-PEG-aldehyde was set to 1:1 to 3, the protein concentration to 1 to 5 mg / ml, and the reaction was carried out at low temperature for 0.5 to 3 hours. The reaction was carried out in an environment with 20-60% isopropanol added to 50 mM Tris buffer (pH 7.5). After the reaction was complete, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the triple-active product mono-pegylated to cysteine.

[0292] Next, as described above, the molar ratio of the purified monopegated triple-active compound to immunoglobulin Fc (homodimer of SEQ ID NO: 123) was set to 1:1 to 5, and the protein concentration was set to 10 to 50 mg / ml. The reaction was carried out at 4 to 8°C for 12 to 18 hours. The reaction was carried out in an environment where 100 mM potassium phosphate buffer (pH 6.0) was supplemented with the reducing agents 10 to 50 mM sodium borohydride and 10 to 30% isopropanol. After the reaction was complete, the reaction solution was applied to a butylcellulose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify the conjugate containing the triple-active compound and immunoglobulin Fc. The purified persistent conjugate has a structure in which the triple-active peptide, polyethylene glycol (PEG) linker, and Fc dimer are covalently linked in a 1:1:1 molar ratio within the molecule, with the PEG linker linked to only one of the two polypeptide chains of the Fc dimer.

[0293] On the other hand, immunoglobulin Fc is formed when two monomers having the amino acid sequence of SEQ ID NO: 123 (consisting of 221 amino acids) form a homodimer via a disulfide bond between the third amino acid, cysteine, of each monomer. The monomers of the homodimer each independently have an internal disulfide bond between the 35th and 95th cysteine ​​positions, and an internal disulfide bond between the 141st and 199th cysteine ​​positions.

[0294] After preparation, the purity analyzed by reverse-phase chromatography, size exclusion chromatography, and ion exchange chromatography was over 95%.

[0295] Here, the conjugate formed by linking the triple-active form of SEQ ID NO: 21, with its C-terminus amidated, and immunoglobulin Fc via a PEG linker is named "the conjugate containing SEQ ID NO: 21 and immunoglobulin Fc" or "the persistent conjugate of SEQ ID NO: 21." These are used interchangeably in the present invention.

[0296] Here, the conjugate formed by linking the triple-active form of SEQ ID NO: 22, with its C-terminus amidated, and immunoglobulin Fc via a PEG linker is named "the conjugate containing SEQ ID NO: 22 and immunoglobulin Fc" or "the persistent conjugate of SEQ ID NO: 22." These are used interchangeably in the present invention.

[0297] Here, the conjugate formed by linking the triple-active form of SEQ ID NO: 42, with its C-terminus amidated, and immunoglobulin Fc via PEG is named "the conjugate containing SEQ ID NO: 42 and immunoglobulin Fc" or "the persistent conjugate of SEQ ID NO: 42." These are used interchangeably in the present invention.

[0298] Here, the conjugate formed by linking the triple-active form of SEQ ID NO: 43, with its C-terminus amidated, and immunoglobulin Fc via PEG is named "the conjugate containing SEQ ID NO: 43 and immunoglobulin Fc" or "the persistent conjugate of SEQ ID NO: 43." These are used interchangeably in the present invention.

[0299] Here, the conjugate formed by linking the triple-active form of SEQ ID NO: 50, with its C-terminus amidated, and immunoglobulin Fc via PEG is named "conjugate containing SEQ ID NO: 50 and immunoglobulin Fc" or "persistent conjugate of SEQ ID NO: 50." These are used interchangeably in the present invention.

[0300] Here, the conjugate formed by linking the triple-active form of SEQ ID NO: 77, with its C-terminus amidated, and immunoglobulin Fc via PEG is named "the conjugate containing SEQ ID NO: 77 and immunoglobulin Fc" or "the persistent conjugate of SEQ ID NO: 77." These are used interchangeably in the present invention.

[0301] Here, the conjugate formed by linking the triple-active form of SEQ ID NO: 96, with its C-terminus amidated, and immunoglobulin Fc via PEG is named "the conjugate containing SEQ ID NO: 96 and immunoglobulin Fc" or "the persistent conjugate of SEQ ID NO: 96." These are used interchangeably in the present invention.

[0302] Experimental Example 1: Measurement of in vitro activity of the triple-active compound and its sustained-release conjugate. To measure the activity of the triple-active compounds and their sustained-release conjugates produced in Examples 1 and 2, in vitro cell activity measurements were performed using cell lines transformed with GLP-1 receptor, glucagon (GCG) receptor, and GIP receptor, respectively.

[0303] Each of the aforementioned cell lines was transformed from CHO (Chinese hamster ovary) to express the human GLP-1 receptor, human GCG receptor, and human GIP receptor genes, respectively, making them suitable for measuring the activity of GLP-1, GCG, and GIP. Therefore, the activity of each part was measured using each transformed cell line.

[0304] To measure the GLP-1 activity of the triple-active compounds and their sustained-release conjugates prepared in Examples 1 and 2, human GLP-1 was serially diluted fourfold from 50 nM to 0.000048 nM, and the triple-active compounds and their sustained-release conjugates prepared in Examples 1 and 2 were serially diluted fourfold from 400 nM to 0.00038 nM. The culture medium was removed from the CHO cells expressing the human GLP-1 receptor that had been cultured as described above, and 5 μl of each of the serially diluted substances was added to the cells. Then, 5 μl of buffer containing cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Next, 10 μl of detection mix containing cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysates after the above reaction were applied to the LANCE cAMP kit (PerkinElmer, USA), and EC was detected from the accumulated cAMP. 50 The values ​​were calculated and compared with each other. The relative titers to human GLP-1 are shown in Tables 2 and 3.

[0305] To measure the GCG activity of the triple-active compounds and their sustained-release conjugates prepared in Examples 1 and 2, human GCG was serially diluted fourfold from 50 nM to 0.000048 nM, and the triple-active compounds and their sustained-release conjugates prepared in Examples 1 and 2 were serially diluted fourfold from 400 nM to 0.00038 nM. The culture medium was removed from the CHO cells expressing the human GCG receptor that had been cultured as described above, and 5 μl of each serially diluted substance was added to the cells. Then, 5 μl of buffer containing cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Next, 10 μl of detection mix containing cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysates after the above reaction were applied to the LANCE cAMP kit (PerkinElmer, USA), and EC was detected from the accumulated cAMP. 50 The values ​​were calculated and compared with each other. The relative titers to human GCG are shown in Tables 2 and 3.

[0306] To measure the GIP activity of the triple-active compounds and their sustained-release conjugates prepared in Examples 1 and 2, human GIP was serially diluted fourfold from 50 nM to 0.000048 nM, and the triple-active compounds and their sustained-release conjugates prepared in Examples 1 and 2 were serially diluted fourfold from 400 nM to 0.00038 nM. The culture medium was removed from the CHO cells expressing the human GIP receptor that had been cultured as described above, and 5 μl of each of the serially diluted substances was added to the cells. Then, 5 μl of buffer containing cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Next, 10 μl of detection mix containing cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysates after the above reaction were applied to the LANCE cAMP kit (PerkinElmer, USA), and EC was detected from the accumulated cAMP. 50 The values ​​were calculated and compared with each other. The relative titers to human GIP are shown in Tables 2 and 3.

[0307] [Table 2] JPEG2026091850000012.jpg142150 JPEG2026091850000013.jpg142150 JPEG2026091850000014.jpg142150 JPEG2026091850000015.jpg55150

[0308] [Table 3]

[0309] As mentioned above, the novel triple-active sustained-release conjugate we created has the function of a triple-active compound that activates all three receptors: GLP-1 receptor, GIP receptor, and glucagon receptor, and can therefore be used as a therapeutic agent for the sequelae of respiratory infectious diseases.

[0310] Experimental Example 2: Improvement of acute pneumonia by inducing a cytokine storm due to SARS-CoV-2 infection. To confirm the efficacy of the persistent conjugate of Sequence ID No. 42, prepared in the above example, in improving lung inflammation caused by SARS-CoV-2 infection, a hamster model (SYRIAN HAMSTER, RjHAN:aura, Central Animal Research Center) was used in which acute lung inflammation was induced by infection with SARS-CoV-2.

[0311] First, the subcultured SARS-CoV-2 was 10 3 TCID 50 The virus solution was prepared in mL (PBS), and 120 μL of the solution was administered intranasally to hamsters to induce viral infection by inhalation. The hamster models infected with SARS-CoV-2 were divided into an excipient control group and a sustained-release conjugate group of SEQ ID NO: 42, and repeated administration was performed at 2-day intervals.

[0312] In the initial trial, lung tissue was collected from each hamster by necropsy after two repeated administrations, and the expression levels of the inflammation-related cytokines IL-1β, TNF-α, and IFN-γ were evaluated by quantitative PCR.

[0313] As a result, administration of the sustained-release conjugate of Sequence ID No. 42 was found to reduce the expression of inflammation-related cytokines in lung tissue compared to the excipient control group (Figure 1A, ††p <0.01 vs. SARS-CoV-2, vehicle by unpaired t-test).

[0314] In the second trial, lung tissue was collected from hamster models infected with SARS-CoV-2 after four repeated administrations of the excipient or the sustained-release conjugate of SEQ ID NO: 42. The degree of inflammation in the tissue was evaluated by H&E staining. The negative control group consisted of hamsters not infected with SARS-CoV-2 and not administered the sustained-release conjugate of SEQ ID NO: 42.

[0315] As a result, as shown in Figure 1A, a significant reduction in lung inflammation score was observed in the sustained-release conjugate group of SEQ ID NO: 42 compared to the excipient control group (Figure 1B, †††p <0.001 vs. SARS-CoV-2, vehicle by unpaired t-test).

[0316] These results confirm that the persistent conjugate of SEQ ID NO: 42 lowers the levels of cytokines involved in inflammatory responses in the body, thereby improving inflammation. This indicates that the persistent conjugate according to the present invention can effectively improve and regulate acute pneumonia, known as a sequela of COVID-19, by suppressing cytokine storms and inhibiting excessive immune responses.

[0317] Experimental Example 3: Improvement of pulmonary fibrosis by inducing cytokine storms Using an LPS (lipopolysaccharide) hamster model in which pulmonary fibrosis was induced by excessive acute pneumonia, the efficacy of the sustained-release conjugate of SEQ ID NO: 42 in improving pulmonary fibrosis was evaluated.

[0318] First, 100-200 μg of LPS was injected into hamsters via intratracheal, intraperitoneal, and intratracheal injections on days 0, 1, and 3. The hamsters that received LPS were divided into an excipient control group and a sustained-release conjugate group of Sequence ID No. 42, and repeated administration was performed at 2-day intervals. After 6 repeated administrations, on day 11, lung tissue was collected from each hamster by necropsy, and the degree of tissue fibrosis was evaluated by MT (Masson-trichrom) staining. The negative control group consisted of hamsters that did not receive LPS or the sustained-release conjugate of Sequence ID No. 42. As a result, it was confirmed that administration of the sustained-release conjugate of Sequence ID No. 42 significantly reduced the fibrosis area in the lung tissue compared to the excipient control group (Figure 2, †~††p <0.05 ~ 0.01 vs. LPS, vehicle by unpaired t-test).

[0319] These results confirm that the persistent conjugate of sequence number 42 suppresses pulmonary fibrosis.

[0320] In summary, the experimental results of the above examples show that the persistent conjugates according to the present invention, represented by the persistent conjugate of SEQ ID NO: 42, improve the excessive pneumonia inflammatory response caused by SARS-CoV-2 infection and effectively improve even pulmonary fibrosis, a typical sequela of COVID-19.

[0321] From the above description, those skilled in the art in which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A pharmaceutical composition for the prevention or treatment of sequelae of respiratory infectious diseases, Pharmacologically acceptable excipients, A pharmaceutical composition comprising a peptide having one of the amino acid sequences of SEQ ID NOs: 1 to 102 in a pharmaceutically effective amount.

2. The pharmaceutical composition according to claim 1, wherein the peptide is in the form of a sustained-release conjugate, and the sustained-release conjugate is represented by the following chemical formula (1). 【Chemistry 1】 Here, X is a peptide with one of the amino acid sequences from SEQ ID NOs: 1 to 102. L is a linker containing ethylene glycol repeating units, F is the immunoglobulin Fc region, The dash (-) indicates a covalent bond between X and L, and between L and F.

3. The pharmaceutical composition according to claim 1 or 2, wherein the respiratory infection disease is an infectious disease caused by a respiratory virus.

4. The pharmaceutical composition according to claim 3, wherein the respiratory virus is selected from the group consisting of adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella zoster virus, measle virus, respiratory syncytial virus, dengue virus, HIV (human immunodeficiency virus), influenza virus, coronavirus, severe acute respiratory syndrome associated virus (SARS-associated virus), and middle east respiratory syndrome coronavirus (MERS-CoV).

5. The pharmaceutical composition according to claim 3, wherein the respiratory virus is SARS-CoV-2.

6. The pharmaceutical composition according to claim 3, wherein the respiratory virus is a mutant virus.

7. The pharmaceutical composition according to claim 6, wherein the mutant respiratory virus induces the same sequelae as the respiratory virus.

8. The pharmaceutical composition according to claim 6, wherein the mutant respiratory virus is selected from the group consisting of SARS-CoV-2 alpha mutant virus (B.1.1.7 lineage), SARS-CoV-2 beta mutant virus (B.1.351 lineage), SARS-CoV-2 gamma mutant virus (P.1 lineage), and SARS-CoV-2 delta mutant virus (B.1.617.2 lineage).

9. The pharmaceutical composition according to claim 1 or 2, wherein the sequelae of the respiratory infection disease are at least one selected from the group consisting of fever, dyspnea, cough, pneumonia, pulmonary fibrosis, pain, muscle pain, fatigue, inflammation, and nervous system disorders.

10. The pharmaceutical composition according to claim 9, wherein the sequelae of the respiratory infection disease are due to tissue damage caused by excessive secretion of cytokines.

11. The pharmaceutical composition according to claim 1 or 2, wherein the sequela of the respiratory infection disease is post-COVID-19 pulmonary fibrosis.

12. The pharmaceutical composition according to claim 1 or 2, wherein, upon administration, it exhibits at least one of the following properties. (i) Reduction in lung inflammation score (ii) Decreased expression or secretion of pro-inflammatory cytokines (iii) Reduction in pulmonary fibrosis area (iv) Suppression of inflammasome complex formation

13. The pharmaceutically acceptable composition according to claim 12, wherein the cytokine is at least one selected from the group consisting of interleukin, tumor necrosis factor, and interferon.

14. The pharmaceutical composition according to claim 12, wherein the cytokine is IL-1β, TNF-α, or IFN-γ.

15. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is administered to an individual in a state of cytokine storm syndrome, sepsis, or organ failure due to respiratory viral infection.

16. The pharmaceutical composition according to claim 1 or 2, wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96.

Images