Recombinant human neuregulin derivatives and use thereof

Abstract

To provide optimized or improved neuregulin (NRG) for use in preventing, treating, or reducing cardiovascular diseases.SOLUTION: Disclosed is a use of recombinant human neuregulin derivatives in preparing a medicine for preventing, treating, or reducing the progression of cardiovascular diseases in mammals. In particular, the present invention relates to a novel recombinant human NRG-FC protein and a use thereof in the treatment of cardiovascular diseases. The protein has a prolonged half-life and enhanced biological activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for the manufacture of a medicament for preventing, treating, or alleviating cardiovascular disease in a mammal. In particular, the present invention relates to the use of recombinant human neuregulin derivatives for the treatment of cardiovascular disease. The present invention relates to a novel recombinant human NRG-Fc protein and its use for treating [Background technology]

[0002] BACKGROUND OF THE INVENTION Neuregulin (NRG; heregulin, HRG) is a glial growth factor (GGF) or novel differentiation factor (NDF) It is a glycoprotein with a molecular weight of approximately 44 kDa, and is a receptor of the ErbB family. Transmits signals between cells as a ligand for receptor tyrosine kinase. NRG family It consists of four members, including NRG1, NRG2, NRG3, and NRG4 (Falls et al., Exp Cel Res. 284:14-30, 2003). NRG1 plays important roles in the nervous system, heart, and breast. It has also been shown that NRG1 signaling is involved in the development and function of several other organ systems, as well as in schizophrenia and other neurodegenerative disorders. NRG1 has been shown to play a role in the development of human diseases, including breast cancer. Studies on mutant mice (knockout mice) have revealed that the N-terminal region Alternatively, isomers that differ in the epidermal growth factor (EGF)-like domain may exhibit different functions in vivo. The present invention is based on NRG1β.

[0003] NRG1β is a transmembrane protein (Holmes et al., Science 256, 1205-1210, 1992). The outer part of the membrane is the N-terminus and contains the Ig-like and EGF-like domains. The part is the C-terminus. Action of metalloproteinases (MMPs) in the extracellular matrix (ECM) Under this condition, the outer part of the membrane of the NRG is cleaved by the enzyme and becomes free, thereby This promotes the binding of ErbB receptors to peripheral cell surfaces, resulting in the associated cellular signaling pathways. They become activated.

[0004] The ErbB receptor family is divided into four categories, including ErbB1, ErbB2, ErbB3, and ErbB4. They are divided into three groups, all of which are transmembrane proteins with molecular weights of 180-185 kD. All but ErbB2 contain a ligand-binding domain at the N-terminus outside the membrane; Of these, all but ErbB3 contain protein tyrosine kinases at their C-termini on the inner membrane side. ErbB1 is an EGF receptor, whereas both ErbB3 and ErbB4 are NRG receptors. Among NRG receptors, only ErbB2 and ErbB4 were highly expressed in the heart (Yarden et al. Literature, Nat Rev. Mol Cell Biol, 2:127-137, 2001).

[0005] When NRG binds to the outer membrane portion of ErbB3 or ErbB4, ErbB3 or ErbB4 binds to other E It forms heterodimers with ErbB receptors (usually with ErbB2) or ErbB4 forms homodimers alone. They form dimers, resulting in phosphorylation of the inner membrane part of the receptor (Yarden et al., 2004). (Nat Rev. Mol Cell Biol, 2:127-137, 2001). The phosphorylated inner membrane It can further bind to various signaling proteins within the vacuole, thereby downstream ER Activation of the K or AKT signaling pathway leads to cell proliferation, cell apoptosis, cell migration, and cell It triggers a series of cellular responses including differentiation, or stimulation or inhibition of cell adhesion.

[0006] NRG is particularly important for cardiac development (WO0037095, CN1276381, WO03099300, WO9426298, U S6444642, WO9918976, WO0064400, Zhao et al., J. Biol. Chem. 273, 10261-10269, 1 During early embryonic development, NRG expression is primarily restricted to the endocardium, and subsequently spreads via the paracrine pathway. It is released into peripheral cardiomyocytes via the duct and binds to the outer membrane of the PTK receptor ErbB4 on the cell membrane. Furthermore, ErbB4 forms a heterodimer with ErbB2. The structure of the ErbB4 / ErbB2 complex The formation and activation of endothelial cells is essential for trabecular formation in the sponge-like heart at an early stage. Three protein genes, NRG, ErbB4, and ErbB2, are deleted. This leads to the removal of the embryonic trabeculae, causing embryonic lethality in utero at an early stage of development. In 095, a given concentration of NRG continuously activated the ERK signaling pathway, leading to proliferation of cardiomyocytes. promotes proliferation and differentiation, and induces sarcomere and cytoskeletal reorganization at cardiomyocyte adhesion sites. It has been shown that it can improve the structure of cardiomyocytes and enhance the contraction of cardiomyocytes. In addition, WO0037095 and WO003099300 disclose the use of NRG to detect various cardiovascular diseases. It has been pointed out that the disease can be diagnosed and treated.

[0007] Some prior art documents related to the present invention list the following: 1. Myocardial Function and operation: WO0037095; 2. Novel applications of growth factors, neuregulins, and their analogs 3. Neuregulin-based methods and compositions for treating cardiovascular disease: WO03099300; 4.Zhao YY, Sawyer DR, Baliga RR, Opel DJ, Han X, Marchionni MA, and and Kelly RA, "Neuregulin promotes cardiomyocyte survival and growth" ns Promote Survival and Growth of Cardiac Myocytes)” J. Biol. Chem.273, 10261-10 269 ​​(1998); 5. Methods for treating muscle diseases and disorders: WO9426298; 6. Methods for treating muscle diseases and disorders using neuregulin Increase myotube formation or survival, or muscle cell mitogenesis, differentiation, or survival Methods: US6444642. 7. Therapeutics involving the use of neuregulin: WO9918976; 8. Congestive heart failure Treatment methods: WO0064400; 9. Holmes WE, Sliwkowski MX, Akita RW, Henzel WJ, Lee J, P Mark JW, Yansura D, Abadi N, Raab H, Lewis GD, et al., "A specific activator of heme kinase Identification of regulin, a specific activator p185erbB2 bB2),” Science 256, 1205-1210 (1992); 10. Falls DL, “Neuregulins: Function, Neuregulins: functions, forms and signaling strategies s),” Experimental Cell Research, 284, 14-30 (2003). 11. Yarden Y, Sliwkowski X. "Untangling the ErbB signaling network" ” Nature Reviews: Molecular Cell Biology, 2127-137 (2001).

[0008] A promising new treatment involves the application of neuregulins in patients with cardiovascular disease ( (Hereinafter referred to as "NRG"). Existing research has shown that the EGF-like domain of NRG1 contains approximately 50 to 64 amino acids. that there are sufficient ATP-dependent agonists present that are capable of binding to and activating these receptors. Previous studies have shown that NRG-1β directly binds to ErbB3 and ErbB4 with high affinity. It has been shown that the orphan receptor ErbB2 can interact with ErbB3 or ErbB4. It can form heterodimers, the affinity of which is higher than that of ErbB3 or ErbB4 homodimers. Studies of neurogenesis have shown that the formation of the sympathetic nervous system requires the complete integration of NRG-1β, ErbB2, and It has been shown that the signal transduction pathways of NRG-1β, ErbB2, or ErbB4 are required. After targeted disruption of the nucleus, embryos die due to defects in cardiac development. Research has shown that NRG-1β and ErbB2 are involved in the development of the cardiovascular system and in maintaining normal cardiac function in adults. The important roles of NRG-1β and ErbB4 have been highlighted. Recombinant NRG-1β EGF-like domain has been shown to enhance sarcomere organization. Short-term administration of significantly worsened myocardial function in three different animal models of heart failure More importantly, NRG-1β can improve or prevent the progression of heart failure in animals. However, there is no evidence that cardiovascular disease can be prevented, treated, or alleviated. To find more effective neuromodulatory polypeptide proteins that can be used to This requires further optimization or improvement of the NRG. Summary of the Invention

[0009] (Summary of the Invention) The present invention relates to a method for the manufacture of a medicament for preventing, treating, or alleviating cardiovascular disease in a mammal. In particular, the present invention relates to the use of recombinant human neuregulin derivatives for the treatment of cardiovascular disease. The present invention relates to a novel recombinant human NRG-Fc protein and its use for treating cancer. In some embodiments, the mammal is a human. do.

[0010] In a first aspect, the prophylactic invention provides several NRG fusion polypeptides. In some embodiments, the NRG fusion polypeptide comprises the EGF-like domain of NRG. In an embodiment, the NRG fusion polypeptide comprises the EGF-like domain of the NRG1β2 isomer. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the NRG fusion polypeptide comprises an amino acid sequence of its SEQ ID NO:1 analog. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of Ig Fc. In some embodiments, the NRG fusion polypeptide comprises an amino acid sequence of its Ig Fc analog. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of IgG Fc. In some embodiments, the NRG fusion polypeptide comprises an IgG Fc analog thereof. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of In some embodiments, the NRG fusion polypeptide comprises an IgG4 Fc amino acid sequence. In some embodiments, the NRG fusion polypeptide comprises its IgG1 Fc sequence. In some embodiments, the NRG fusion polypeptide comprises an amino acid sequence similar to that of the I In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of a gG4 Fc analog. The amino acid sequence of the IL-2 signal peptide is The sequence is cleaved during extracellular secretion in recombinant production. The NRG fusion polypeptide contains the EGF-like domain of NRG and the amino acid sequence of IgG Fc. In one embodiment, the NRG fusion polypeptide comprises the amino acid sequence of a linker peptide: The EGF-like domain of NRG is fused to IgG Fc via a peptide linker. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of SEQ ID NO:2. In an embodiment, the NRG fusion polypeptide comprises the amino acid sequence of SEQ ID NO:3.

[0011] Some NRG fusion polypeptides have the following amino acid sequence: [ka] That is, it contains the human NRG-1 amino acid sequence 177-237.

[0012] Some NRG fusion polypeptides include the following amino acid sequence: [ka] .

[0013] Some NRG fusion polypeptides include the following amino acid sequence: [ka] .

[0014] NRG fusion polypeptides may be prepared according to any relevant technique known in the art. General techniques for preparing NRG fusion polypeptides are provided herein. In some embodiments, the NRG fusion polypeptide is recombinant.

[0015] In a second aspect, the invention provides nucleic acids, vectors, and hosts related to NRG fusion polypeptides. The nucleic acid or its complementary sequence encodes an NRG fusion polypeptide or a fragment thereof. The nucleic acid can be inserted into a vector suitable for propagation and expression of the NRG fusion polypeptide. The modified vector can be double-stranded or single-stranded DNA or RNA. For example, the recombinant NRG fusion polypeptide is introduced into a host cell capable of expressing the polypeptide.

[0016] In a third aspect, the present invention provides therapeutic and non-therapeutic applications of NRG fusion polypeptides. In particular, the present invention provides NRG fusion polypeptides for the prevention, treatment, or management of cardiac diseases and disorders. Thus, the present invention provides NRG fusion polypeptides and methods for their application in alleviating The present invention provides pharmaceutical preparations, including methods of treatment therefor.

[0017] In a fourth aspect, the present invention provides a method for treating heart failure in a mammal. In some embodiments, the method comprises injecting an NRG fusion polypeptide into a mammal. Includes the process.

[0018] In a fifth aspect, the present invention provides a method for inducing phosphorylation of an ErbB receptor in a cell. In some embodiments, the method comprises exposing a NRG fusion polypeptide to a cell. The process includes:

[0019] In a sixth aspect, the present invention provides a method for inducing activation of the AKT signaling pathway in cardiac cells. In some embodiments, the method comprises administering an NRG fusion polypeptide to a mammalian animal. The method includes exposing the peptide to cardiac cells.

[0020] In a seventh aspect, the present invention provides a method for inducing activation of the ERK signaling pathway in cardiac cells, In some embodiments, the method comprises maintaining a NRG fusion polypeptide. The method includes exposing the peptide to cardiac cells. DETAILED DESCRIPTION OF THE INVENTION

[0021] (Detailed Description of the Invention) (A. Description) Unless otherwise defined, all scientific and technical terms used herein are understood to be of ordinary skill in the art. All patent documents referred to herein have the same meaning as understood by those skilled in the art. All patent applications, patent publications, and other publications are incorporated herein by reference in their entireties. Any definitions covered in this section may be found in the patent documents, patent applications, A different meaning from that explained in either the literature, open patent documents, or other publications. If the product has a particular flavor, the explanations given in this section shall prevail.

[0022] Unless otherwise specified, as used herein, "a" / "an" means "at least one" means "one" or "one or more."

[0023] As used herein, the term "EGF-like domain" refers to any of ErbB2, ErbB3, ErbB4, or heterodimers thereof. It is capable of binding to and activating a dimer or homodimer, and is described in the following literature: WO 00 / 64400; Holmes et al., Science, 256: 1205-1210 (1992); U.S. Patent No. 5,530,109 and 5,716,930; Hijazi et al., Int. J. Oncol., 13: 1061-1067 (1998); Chang et al. Nature, 387: 509-512 (1997); Carraway et al., Nature, 387: 512-516 (199 7); Higashiyama et al., J. Biochem., 122: 675-680 (1997); and WO 97 / 09425 It is encoded by the neuregulin gene, which has structural similarity to the EGF receptor binding domain of The foregoing refers to a polypeptide segment that is encoded by the polypeptide. In some embodiments, the EGF-like domain is ErbB2 / ErbB4 or It binds to and activates ErbB2 / ErbB3 heterodimers. In some embodiments, the EGF-like domain comprises amino acids in the receptor binding domain of NRG-1. In the context of the invention, the EGF-like domain refers to amino acids 177-226, 177-237, or 177-240 of NRG-1. In some embodiments, the EGF-like domain is an amino acid sequence in the receptor binding domain of NRG-2. In some embodiments, the EGF-like domain comprises the receptor binding domain of NRG-3. In some embodiments, the EGF-like domain comprises amino acids in the NRG-4 receptor binding domain. Contains amino acids in the binding domain.

[0024] As used herein, the Fc amino acid sequence may be selected from the human IgG-1 heavy chain. Ison, JW et al., Nucleic Acids Research, 10:4071-4079 (1982), or any other reference in the art. Any Fc sequence known in the art (for example, but not limited to, IgG-2, IgG-3, and IgG-4, (See other IgG species, including other immunoglobulins). The Fc fragment of an antibody is a monomeric polypeptide. The monomeric polypeptide segments are linked by disulfide bonds or It is well known that they can be linked together through non-covalent bonds to form dimers or multimers. The disulfide bonds formed between the monomeric subunits of native Fc molecules include: The type (e.g., IgG, IgA, IgE) or subtype (e.g., IgG-1, IgG-2, There are one to four intramolecular disulfide bonds depending on the type of antibody (IgG-3, IgA-1, IgA-2). The term "Fc" as used herein may refer to monomers, dimers, and multimers of Fc molecules. The presence of tein residues prevents disulfide bond formation, thereby preventing dimer formation. It should be noted that Fc monomers spontaneously form dimers unless specific conditions exist that prevent this. Cysteines can usually form disulfide bonds in the Fc dimer, but even if Even if the amino acid residue is removed or replaced by another residue, the monomer chains usually remain non-covalently bonded to each other. As used herein, the term "Fc" refers to the following forms: native monoclonal antibodies; dimers, natural dimers (disulfide bond linked), engineered dimers (disulfide bond and / or non- covalently linked) and modified monomers (i.e., derivatives) do.

[0025] As used herein, Fc analogs include variants, analogs, or derivatives, but Fc analogs can be constructed, for example, by making multiple substitutions in residues or sequences.

[0026] Fc analogs include insertion, deletion, and substitution analogs.

[0027] Variant (or analog) polypeptides include insertion mutations, where one or more amino acid residues is added to the Fc amino acid sequence. The insertion site can be at either end of the protein or at both ends. or an internal domain of the Fc amino acid sequence. Insertional variants resulting from the addition of residues to both termini include, for example, fusion proteins and The protein may include a sequence and an amino acid marker.

[0028] In an Fc deletion variant (or analog), one or more amino acid residues in the Fc polypeptide Deletions may occur at one or both ends of the Fc polypeptide. and one or more residues may be removed from the Fc amino acid sequence. includes all segments of the Fc polypeptide sequence.

[0029] In Fc substitution variants (or analogs), one or more amino acid residues of the Fc polypeptide are It is removed and replaced with another residue, whereas autogenic substitutions are conservative, The present invention also includes non-conservative substitutions.

[0030] For example, to prevent some or all disulfide bond bridges in the Fc sequence, The cysteine ​​residues may be removed or substituted with other amino acids. Alternatively, one or more of these cysteine ​​residues may be replaced with another amino acid, e.g., arane. As shown in another embodiment, the amino substitution may be introduced with methylamino or serine. The modifications are: (1) to remove the Fc receptor binding site; (2) to remove the complement (C1q) binding site; and / or (3) removing antibody-dependent cell-mediated cytotoxicity (ADCC) sites. These sites are well known in the art and can be used to identify any of the known substitutions within the Fc range. For example, see Molecular Immunology, Vol. 29, Issue 5, 633-639 (1992), See ADCC sites on IgG1.

[0031] Similarly, one or more tyrosine residues can be substituted with phenylalanine residues. Additionally, variants with other amino acid insertions, deletions, and / or substitutions are also contemplated and are within the scope of the present invention. Conservative amino acid substitutions are usually preferred. can cause changes in the form of amino acids, such as D-amino acids.

[0032] Signal peptide: Also known as a leader peptide, it is located at the N-terminus of a protein molecule. It is a polypeptide segment, usually 15-30 amino acids in length, that The protein can be secreted. After the protein is secreted, the signal sequence is removed. will be done.

[0033] As used herein, signal peptide sequences are used in mammalian cells and insect cells / baculoviruses. Secretory signal peptides used in the expression of virus expression systems, such as those of melittin, IFN, and IL-2 Contains a signal peptide.

[0034] As used herein, the IL-2 signal peptide has the following amino acid sequence: [ka] or an amino acid sequence analogous thereto. Addition of peptide sequences improves secretion efficiency and simplifies downstream purification processes Furthermore, the signal peptide sequence contributes to maintaining the stability and activity of the fusion peptide. It plays a meaningful role in

[0035] Linker peptide: A linker peptide is a peptide that binds the fusion protein segment to the fusion protein. It is an array that connects the members.

[0036] Linker peptides as used herein are divided into two types: 1. Flexible linkers 2. Rigid linkers, such as (EAAAK)n (n≦6) or (XP)n (X is preferably Preferably, it is alanine, glutamic acid, or lysine.

[0037] As used herein, a linker peptide has the following amino acid sequence: [ka] or an amino acid sequence analogous thereto.

[0038] As used herein, an "effective dose" of an active ingredient for the treatment of a particular disease is defined as the amount of the active ingredient that is effective to treat the symptoms of the disease. This dose is effective enough to improve or alleviate in some way the disease. Although it may be effective, it is usually used to improve the symptoms of the disease.

[0039] As used herein, an "active ingredient" refers to a compound or compound that is effective in diagnosing, curing, mitigating, or treating a disease in humans or other animals. Any medical device used to treat or prevent or promote physical or mental health It is a substance.

[0040] As used herein, "amelioration" of a particular disorder refers to the persistence of symptoms with the use of a particular active agent. means relief, permanent or temporary, lasting or transient, which relief is attributable to or related to the use of the reagent. To connect.

[0041] As used herein, "treatment" means to improve or reverse the symptoms of discomfort, disorder, or disease. The effect may be preventative; for example, the disease or its symptoms may be completely alleviated. The effect may also be therapeutic, e.g., the treatment of a disease and / or its The treatment may involve the use of any of the compositions described herein, resulting in partial or complete cure of the adverse effects of the disease. This also includes use.

[0042] As used herein, a "vector (or plasmid)" refers to a vector that introduces heterologous DNA into a cell to cause the cell These vectors refer to the dispersible components used to express or replicate in a host. The use and application of expression vectors are well known to those skilled in the art. It is possible to express DNA linked to a promoter region that can affect expression of the A fragment. Thus, an expression vector includes a vector that can produce a cloned gene when introduced into an appropriate host cell. Recombinant DNA or RNA components that cause expression of cloned DNA, e.g., plasmids, vectors, refers to a bacteriophage, recombinant virus, or other vector; eukaryotic cells and / or Vectors that replicate in prokaryotic cells and remain free or integrated into the host cell genome Suitable expression vectors, including integrating vectors, are well known to those skilled in the art.

[0043] As used herein, "cardiomyocyte differentiation" refers to a greater than 10% decrease in DNA synthesis, as described below, and other factors. >10% inhibition of DNA synthesis stimulated by α-glucan, ordered sarcomere cohesion and cell-cell adhesion , a condition characterized by persistent activation of MAP kinases and enhanced expression of p21 Cip1 For further discussion, see WO00 / 37095, the entire contents of which are incorporated herein by reference. It is incorporated into the subsection.

[0044] As used herein, "ejection fraction" or "EF" refers to the total amount of blood ejected from the left ventricle with one heartbeat. The ejection fraction can be defined by the following formula: (left ventricular end-diastolic ejection fraction) volume (LVEDV)-left ventricular end-systolic volume (LVESV)) / LVEDV.

[0045] As used herein, "fractional shortening" or "FS" refers to the ratio of the diameter of the left ventricle in the systolic state to the diameter in the dilated state. The shortening fraction can be defined by the following formula: (left ventricular Left ventricular diastolic diameter (LVDD)-left ventricular systolic diameter (LVSD)) / LVDD.

[0046] As used herein, cardiovascular disease includes heart failure, myocardial infarction, coronary arteriosclerotic heart disease, arrhythmia, This refers to myocarditis, valvular heart disease, infective endocarditis, pericardial disease, ischemic heart disease, congenital heart disease, etc. These diseases tend to induce myocardial injury.

[0047] As used herein, "myocardial injury" refers to myocardial injury caused by pathological heart disease. Myocardial injury tends to induce cardiac dysfunction, thereby affecting human health. The development of myocardial injury involves the production of oxygen free radicals, calcium ion overload, and damage. Inflammatory response due to neutrophil infiltration in the injured area, apoptosis or necrosis of cardiomyocytes, energy -Tissue metabolic disorders due to impaired supply, abnormalities in cardiac electrical signal transmission, and cholesterol accumulation , the formation of atherosclerotic plaques, and some other pathophysiological changes are involved.

[0048] As used herein, "heart failure" or "HF" refers to a condition in which the heart is unable to pump blood at the rate required by metabolic tissues. Heart failure refers to a malfunction of the heart that makes it unable to pump blood. HF), myocardial infarction, tachyarrhythmia, familial myocardial hypertrophy, ischemic heart disease, congenital dilated cardiomyopathy, Heart failure includes many pathological conditions, such as myocarditis. Heart failure can be ischemic, congenital, rheumatic, and primary. Chronic cardiac hypertrophy can be caused by many factors, including sexual factors. This is a precursor to CHF and cardiac arrest.

[0049] As used herein, "myocardial infarction" refers to a severe and life-threatening cardiac arrest resulting from coronary artery blockage or interruption of blood flow. It refers to patchy necrosis of a portion of the myocardium induced by persistent ischemia.

[0050] As used herein, "ordered and reinforced arrangement of sarcomeres or sarcomere structures" means , an ordered array of contractile proteins represented by immunofluorescent staining of α-actinin The ordered arrangement of α-actinin is visible under a microscope and As used herein, the term "sarcomere" can be identified by the imaging device connected to the "Disorder or irregularity of the sarcomere structure" means "orderly structure of the sarcomere or It is the opposite of "enhanced array."

[0051] As used herein, "ordered or reinforced arrangement of cytoskeletal structures" refers to phalloides. Cardiomyocytes are characterized by the orderly arrangement of actin filaments, as revealed by chromosome staining. The orderly arrangement of actin filaments in cells is shown in the drawings of this invention. As shown in the figure, the identification can be performed using a microscope and a connected imaging device. The term "disorder or irregularity of the cytoskeleton structure" used here means "orderly reinforced cytoskeletal structure" It is the antonym of "array".

[0052] As used herein, "protein" means "polysaccharide" unless otherwise specified herein. It has the same meaning as "lipeptide" or "peptide."

[0053] As used herein, an "activity unit" or "1 U" is an amount that can produce a response that is 50% of the maximum. It refers to the dosage of the standard product. In other words, to determine the active unit of a certain active agent, the EC For example, if the EC50 of a product is 0.067 μg / ml, the dosage should be 1 Furthermore, if you use 1 μg of product, it means you will use 14.93 U (1 / 0.067). EC50 can be calculated using methods known in the art, including the method used by the inventors in the following embodiments. The determination of activity units may be performed using any method known in the art. It is important for the quality control of genetically engineered products and drugs, and as a result, different medicines and / or different Product batches can be quantified on the same basis.

[0054] In some embodiments, the NRG units are as described in WO 03 / 099300 and Sadick et al., 1996, Kinase receptors, as described in detail in Analytical Biochemistry, 235: 207-14. Determined by measuring the activity of NRG via a total activated enzyme-linked immunosorbent assay (KIRA-ELISA). No. 6,239,999, the contents of which are incorporated herein by reference in their entireties.

[0055] B. NRG Fusion Polypeptides The present invention provides several NRG fusion polypeptide fragments. In some embodiments, the NRG fusion polypeptide comprises the EGF-like domain of NRG. In some embodiments, the NRG fusion polypeptide contains the EGF-like domain of the human NRG-β2 isomer. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of SEQ ID NO:1. In an embodiment, the NRG fusion polypeptide comprises an amino acid sequence analogous to SEQ ID NO:1. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of an IgG Fc. In some embodiments, the NRG fusion polypeptide has the amino acid sequence of its IgG Fc analog. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of IgG Fc. In some embodiments, the NRG fusion polypeptide comprises an amino acid sequence of an IgG Fc analog. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of IgG1 Fc. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of IgG4 Fc. In some embodiments, the NRG fusion polypeptide comprises an amino acid sequence of an IgG1 Fc analog. In some embodiments, the NRG fusion polypeptide comprises an amino acid sequence of an IgG4 Fc analog. In some embodiments, the NRG fusion polypeptide comprises an IL-2 signaling peptide. The amino acid sequence of the IL-2 signal peptide is recombinantly produced. In some embodiments, the NRG fusion polypeptide is cleaved during extracellular secretion in the In some embodiments, the domain comprises the functional domain of EGF of NRG and the amino acid sequence of IgG Fc. In the present invention, the NRG fusion polypeptide comprises the amino acid sequence of a linker peptide and an EGF sequence of NRG. In some embodiments, the functional domains of are fused to the IgG Fc via a peptide linker. In some embodiments, the NRG fusion polypeptide comprises the amino acid sequence of SEQ ID NO:2. In one embodiment, the NRG fusion polypeptide comprises the amino acid sequence of SEQ ID NO:3.

[0056] In a more preferred embodiment, the NRG fusion polypeptide comprises an amino acid sequence of the EGF-like domain of NRG. and the amino acid sequence of IgG Fc or an IgG Fc analog thereof. In the present invention, the NRG fusion polypeptide contains the amino acid sequence of the IL-2 signal peptide, the EGF-like domain of NRG, and a main amino acid sequence and an amino acid sequence of IgG Fc or an IgG Fc analog thereof, and The EGF-like domain of G is fused to IgG Fc via a peptide linker, and the IL-2 signal peptide The amino acid sequence is cleaved during extracellular secretion in recombinant production. In such a case, the NRG fusion polypeptide may comprise the amino acid sequence of NRG SEQ ID NO:1, an IgG1 or IgG4 subunit. The NRG comprises the amino acid sequence of an Fc of the type I, and is fused to the IgG Fc via a peptide linker. In a more preferred embodiment, the NRG fusion polypeptide has the amino acid sequence of SEQ ID NO:2. In a more preferred embodiment, the NRG fusion polypeptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the present invention provides an NRG fusion polypeptide for use in The present invention provides a method for treating heart failure by administering an amount of

[0057] The NRG fusion polypeptides can be administered in the form of pharmaceutical formulations.

[0058] Methods of administration of NRG fusion polypeptides include, but are not limited to, oral administration, intravenous administration, and the like. injection, intragastric administration, rectal administration, intraperitoneal (intrapleural) administration, and intracerebroventricular injection, and may be performed by those skilled in the art. is determined.

[0059] In a more preferred embodiment, the composition for administration is a pharmaceutical formulation. or a therapeutic dose of one or more prophylactic or therapeutic agents (e.g., a compound comprising an NRG fusion polypeptide). compounds and other prophylactic or therapeutic agents) and pharmaceutically acceptable carriers or excipients. In one embodiment and herein, "pharmaceutically acceptable" means that the approved by the Department of Pharmacopoeia or recorded in the United States Pharmacopoeia or other widely recognized pharmacopeia As used herein, the term "medicament" means that the pharmaceutical preparation described can be used in animals, particularly humans. A "carrier" refers to a diluent, adjuvant (e.g., Freund's complete adjuvant and incomplete adjuvant), Drug carriers refer to adjuvants, excipients, or any other carriers that aid in the administration of a therapeutic agent. The liquid may be a sterile liquid such as water or oil. Oil may be petroleum, animal, vegetable, or synthetic oil, e.g. Examples include peanut oil, soybean oil, mineral oil, and sesame oil. The carrier of choice for intravenous infusion of pharmaceutical preparations is water. Saline, glucose, and glycerol are used to prepare the infusion mixture. An example of a suitable pharmaceutical carrier is provided in the book "Remington's Pharmaceutical Sciences" by E.W. Martin. This information is described in "Mington's Pharmaceutical Sciences."

[0060] Typical pharmaceutical formulations and dosage forms include one or more excipients. Suitable excipients include, but are not limited to, starch, glucose, lactose, , sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate Sodium, Glyceryl Monostearate, Mica, Sodium Chloride, Dried Skim Milk, Propane Some excipients may be used in pharmaceutical formulations or Whether or not a compound can be incorporated into a dosage form depends on, but is not limited to, the nature of the compound or compound(s) that will be administered to the patient. The method of administration and the particular active ingredient in the dosage form are well known in the art. If necessary, the drug or unit dosage form may contain trace amounts of wetting agents, emulsifying agents, or pH buffering agents. A buffer may be included.

[0061] Pharmaceutical formulations may be prepared using excipients known in the art or as described in, for example, the United States Pharmacopoeia (USP) SP(XXI). In general, lactose-free drug products contain the active ingredient, the binder, and the excipients disclosed in NF(XVI). Contains acceptable amounts of fillers / fillers and pharmaceutically compatible lubricants. Typical lactose The free drug contains the active ingredient, microcrystalline cellulose, pregelatinized starch, and maize stearate. Contains magnesium.

[0062] The pharmaceutical formulations and dosage forms provided by the present invention reduce the rate of decomposition of the active ingredient. The compounds referred to herein as "stabilizers" include, but are not limited to, one or more compounds capable of The present invention does not include, but is not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0063] Pharmaceutical formulations and unit dosage forms include solutions, suspensions, emulsions, tablets, capsules, powders, and Oral dosage forms may be administered in standard carriers, such as pharmaceutical grade mannitol. , lactose, starch, magnesium stearate, sodium saccharin, cellulose Pharmaceutical preparations and dosage forms may contain the precise prophylactic or therapeutic dose. These include preventative or therapeutic agents formulated to provide a specific therapeutic effect in patients. The pharmaceutical formulation should be suitable for the method of administration. In an optimized embodiment, the pharmaceutical formulation or single unit dosage form is sterile and preferably animal-free. and most preferably to a mammal, and most preferably to a human. It should be.

[0064] The form of the pharmaceutical formulation containing the NRG fusion polypeptide should be suitable for its mode of administration. Modes of administration include, but are not limited to, injection (e.g., intravenous injection, intramuscular injection, subcutaneous or intradermal injection), oral administration, sublingual administration, inhalation, intranasal administration, transdermal administration, topical administration These include intramuscular, intratumoral, intrasynovial, and rectal administration. Therefore, the pharmaceutical preparations may be administered by certain conventional procedures, such as intravenous injection, intramuscular / subcutaneous injection, oral administration, Refers to conventional procedures used to produce pharmaceutical formulations for intranasal or topical administration. In one embodiment, the pharmaceutical formulation can be produced by conventional subcutaneous administration. Compatible with infusion administration modes. Typically, drugs administered by intravenous infusion are sterile, etc. If necessary, the drug may contain a solubilizing agent and an anti-inflammatory agent to relieve pain at the injection site. The formulation may contain a local anesthetic such as lignocaine for pain relief.

[0065] Dosage forms include, but are not limited to, tablets, caplets, capsules, e.g., soft gelatin capsules, and the like. Latin capsules, cachets, pills, medicinal drops, dispersions, suppositories, ointments, and patches (poultices) , pastes, powders, bandages, emulsions, salves, solutions, patches, aerosols (e.g., nasal liquid dosage forms suitable for oral or mucosal administration Liquid dosage forms include suspensions (e.g., aqueous or non-aqueous suspensions, oil-in-water emulsions, or oil Includes injectable emulsions, solutions and panaceas; liquid dosage forms are suitable for injectable administration; sterile solid The body (e.g., crystalline or amorphous body) can be reconstituted into a liquid dosage form suitable for administration by injection. can.

[0066] Depending on the various applications, there are differences in the shape and formulation among different drugs containing NRG fusion polypeptides. For example, a dosage form used for an acute disorder may be more effective than a dosage form used for the long-term treatment of the same disease. Similarly, between dosage forms effective for treating various cancers, Similarly, the amount of active ingredient contained in an injectable dosage form may vary depending on the dosage form used to treat the same disease or disorder. As will be apparent to those skilled in the art, the amount of active ingredient contained in an oral dosage form for treating Thus, the formulations described above, as well as other specific dosage forms encompassed by the present invention, differ from one another. Remington's Pharmaceutical Sciences, 18th edition, Mack Press, Easton, Pe. See nnsylvania (1990).

[0067] The NRG fusion polypeptides can be administered by, but not limited to, oral administration, intravenous injection, intragastric administration, or the like. administration, intraduodenal administration, intraperitoneal administration, and intracerebroventricular injection, any method recognized by those skilled in the art. It can be administered by

[0068] C. Dosage and Route of Administration The dosage of the NRG fusion polypeptides provided by the present invention will depend on the nature and severity of the disease or The discomfort and the route of administration of the active ingredient may vary. Also, the frequency and dosage of administration may vary. The specific treatment (e.g., therapeutic or preventative), the severity of the impairment, disease, or discomfort Specific individual factors depending on the dose, route of administration, age, weight, response, and medical history of the patient Effective doses may be derived from in vitro or animal model test systems. The dose-response curve can be selected according to the

[0069] Repeatable dosages of NRG fusion polypeptides are given in milligrams per kilogram of body weight. Contains gram or microgram amounts of NRG (e.g., about 1 microgram per kilogram of body weight) grams to about 500 milligrams per kilogram of body weight, about 100 micrograms per kilogram of body weight gram to approximately 5 milligrams per kilogram of body weight, or 1 microgram per kilogram of body weight (Lamb ~ approximately 50 micrograms per kilogram of body weight). For example, 0.0 micrograms per kilogram of body weight The active peptide is used in an amount of 0.01 mg / kg to 15 mg / kg. The preferred dosage is 0.001 mg / kg to 15 mg / kg. / kg, 0.005mg / kg~10mg / kg, 0.01mg / kg~5mg / kg, 0.001mg / kg~4mg / kg, 0.005mg / kg~3mg / kg, 0.01mg / kg~2mg / kg, 0.001mg / kg~1mg / kg, 0.005mg / kg~0.5mg / kg, 0.010mg / kg~0. 2 mg / kg, and 0.005 mg / kg to 0.050 mg / kg.

[0070] Additionally, repeatable dosages of NRG fusion polypeptides are used per kilogram of body weight. The number of units (U) or unit amount of NRG (e.g., about 1 U per kilogram of body weight to about 1 U per kilogram of body weight) Approximately 5,000 U per lamb, approximately 10 U per kilogram of body weight to 1,000 U per kilogram of body weight, or (This ranges from about 100 U per kilogram of body weight to 500 U per kilogram of body weight.) For example, The active peptide is used at 10 U / kg to 1,000 U / kg per day. The preferred dosage is 1 U / kg to 10,000U / kg, 1U / kg~5,000U / kg, 10U / kg~5,000U / kg, 10U / kg~1,000U / kg, 50U / kg~2,00 0U / kg, 50U / kg~1,000U / kg, 50U / kg~500U / kg, 100U / kg~1,000U / kg, 100U / kg~500U / kg , and may contain 100 U / kg to 200 U / kg.

[0071] Generally, in the methods provided by the present invention for the various diseases described herein, The recommended daily dosage of NRG fusion polypeptides is approximately 0.001 mg to 1,000 mg (NRG-containing). In certain cases, the total daily dosage ranges from 0.001 mg to 15 mg, 0.005 mg to 15 mg. mg~10mg, 0.01mg~5mg, 0.001mg~4mg, 0.005mg~3mg, 0.01mg~2mg, 0.001mg~1mg, 0. The dose may be 0.005 mg to 0.5 mg, or 0.010 mg to 0.2 mg. For case treatment, the patient's systematic response Depending on the patient's condition, a low dose may be administered initially, for example, about 0.1 μg to 1 μg per day, or about 20 μg to 1,000 μg per day. g can be used in either a single dose or multiple doses as needed. It may be necessary to increase the dosage of the active ingredient beyond the ranges set forth herein. This dosage will be apparent to one skilled in the art. Furthermore, the clinician or treating specialist will determine the response of each individual patient. Know how and when to interrupt, adjust, or terminate treatment as needed. It should be noted that in some embodiments, the dosage of NRG In some embodiments, the dosage of NRG is about 1 U / day to 5,000 U / day. In some embodiments, the dosage of NRG is about 10 U / day to 2,000 U / day. In some embodiments, the dosage of NRG is about 10 U / day to 1,000 U / day. In this embodiment, the dosage of NRG is about 100 U / day to 200 U / day.

[0072] NRG fusion polypeptides may also be administered according to a dosing schedule or "treatment cycle." The daily dosage for treatment is listed in detail above. Treatment can be administered for 2 days, 5 days, or The treatment may last for 1, 7, 10 days, 2, 3, 4, 5, or 6 weeks.

[0073] In some embodiments, the NRG fusion polypeptide is administered daily during a treatment cycle. In some embodiments, the NRG fusion polypeptide is administered 3, 4, 5, 6, 7, or 8 times during the treatment cycle. In some embodiments, the administration is continued for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. The NRG fusion polypeptide is administered on day 1 of the treatment cycle and on one or more of the remaining days of the treatment cycle. In some embodiments, the NRG fusion polypeptide is not used in a treatment cycle. Use daily for 3, 5, 7, or 10 days during the treatment cycle and not use on the remaining days of the treatment cycle. During a treatment cycle, NRG fusion polypeptides should be administered at regular intervals. Intervals are 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days The period may be 12 days, 13 days, or 2 to 6 weeks. [Brief explanation of the drawings]

[0074] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 is a schematic diagram of an expression vector. [Figure 2] FIG. 2 is a schematic diagram of the NRG-IgG1 / IgG4-Fc fusion protein. [Figure 3] FIG. 3 shows the results of SDS-PAGE / Western blot of the expression of IFN / IL2-eGFP in 293F. [Figure 4] FIG. 4 shows the results of SDS-PAGE / Western blot of the expression in IL2-NRG-IgG1-Fc 293F. [Figure 5] FIG. 5 shows the results of SDS-PAGE of purified NRG-IgG1-Fc fusion polypeptide (reducing conditions). [Figure 6] FIG. 6 shows the echocardiographic results of all rats with heart failure before and after drug administration. [Example]

[0075] (Implementation) (Embodiment 1) Construction of a vector for expression of a fusion polypeptide Human NRG (SEQ ID NO: 1), a linker, and a human immunoglobulin (IgG1 or IgG4) Fc fragment. The full-length DNA sequence containing the nucleotide sequence nucleotides ... A Kozak sequence and a sequence containing the melittin signal peptide were introduced at the 5' end, while an EcoRI site was introduced. After appropriate sequencing, the DNA was purified by CaCl2 to The plasmid was introduced into E. coli, amplified, and stored.

[0076] The DNA sequences containing IL-2-eGFP and IFN-eGFP were subcloned into the pcDNA3.1(+) vector. A sequence containing a restriction enzyme Hind III site and a Kozak sequence was introduced at the 5' end, while an EcoRI site was introduced at the 5' end. After appropriate sequencing, the DNA was transformed into E. coli using CaCl2. The plasmid was introduced, amplified, and stored.

[0077] Human NRG (SEQ ID NO: 1), a linker, and a human immunoglobulin (IgG1 or IgG4) Fc fragment. The full-length DNA sequence containing the nucleotide sequence nucleotides ... A sequence containing a Kozak sequence and an IL-2 signal peptide or an IFN signal peptide sequence is added to the 5' end. A sequence containing an EcoRI site was introduced at the 3' end, while a sequence containing an EcoRI site was introduced at the 4' end. The DNA was introduced into E. coli using CaCl2 and amplified, and the plasmid was stored.

[0078] For example, a peptide containing NRG, a linker peptide, and a human immunoglobulin (IgG1 or IgG4) Fc fragment. The full-length DNA sequence, including the above, has the corresponding amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3. Figure 2 shows the schematic of the NRG-IgG1 / IgG4-Fc fusion protein. Figure.

[0079] (Embodiment 2: Protein Expression and Detection) The appropriately sequenced plasmid was transiently transfected into 293F cells. Preparation before transfection was as follows: 293F cells in logarithmic growth phase with over 95% viability were used. Freshly prepared SMM 293-TII culture medium mixed with 1% penicillin / streptomycin mixture Inoculate the soil at a density of 1.2–1.5 × 10 6 The concentration was adjusted to 100 cells / ml and cultured for 24 hours. On the day of incubation, cell viability should be greater than 90% and the cell density should be 2.0–2.5 × 10 6 cells / mL and the volume should be 20 mL.

[0080] Transfection of Melittin-NRG-IgG1 / IgG4-Fc and IFN-eGFP / IL-2-eGFP vectors: 30 μg of the plasmid was taken and transfected with transfection reagent and Sinofection® (100 μL Transfection was performed using the method of Sino Biological Co., Ltd. The feed was replenished 24 hours after the first incubation and every 48 hours thereafter. The culture conditions were 37°C, 8% CO2, 120 rpm. Cultures were performed on an orbital shaker, and samples were collected every 24 hours. Expression of target protein in supernatant and cell lysis buffer was analyzed by SDS-PAGE and Western blot. The target protein was detected by mitigating cell lysis in the melittin-NRG-IgG1 / IgG4-Fc expression system. It was not detected in the buffer or supernatant, and therefore melittin was used as the signal peptide. In this case, it was shown that the NRG-IgG1 / IgG4-Fc fusion polypeptide was not expressed in 293F cells.

[0081] In contrast, when IFN / IL-2 was used as the signal peptide, the eGFP fusion polypeptide was 29 The IL2 signal peptide was found to be more efficient than IFN in terms of extracellular secretion of eGFP. was also significantly better (see Figure 3).

[0082] Transfection of IL-2-NRG-IgG1 / IgG4-Fc vector: Plasmid DNA (30 μg and 45 μg) g) was mixed with 150 mM NaCl solution to make 0.5 ml, and then allowed to stand for 5 to 10 minutes. The solution was mixed with 150 mM NaCl solution to make 0.5 mL, and then allowed to stand for 5-10 minutes. The mixture was mixed with the PEI solution and incubated at room temperature for 20 to 30 minutes to form the DNA-PEI complex. The transfection solution was added dropwise to the cell culture solution while gently shaking the scoop. The cultures were then returned to the shaker and further cultured. 24 hours after transfection The feed was replenished every 48 hours thereafter. The culture medium was incubated at 37°C in 8% CO2 on an orbital shaker. The culture was carried out and samples were taken every 24 hours to detect the transfection efficiency of the cells. Depending on the expression characteristics of different proteins, the experiments were carried out for 6 to 10 days after transfection. The supernatant was collected by centrifugation and stored at -20°C.

[0083] Samples with different DNA / PEI mass ratios were collected at different time points and analyzed by SDS-PAGE and Western blotting. Expression was detected by analysis (see Figure 4 for results) and transfection and the optimal conditions for sample collection were determined.

[0084] (Embodiment 3: Purification of fusion proteins) An equal volume of binding buffer (0.02 M disodium hydrogen phosphate, pH 7.0) was added to the supernatant to adjust the pH value to 7.0 The sample can also be pretreated with a dialysis system or a desalting column. The supernatant was then filtered first through a 1 um filter head, followed by a 0.45 um filter membrane. The mixture was filtered through a centrifuge tube and finally through an affinity column. Samples at 100°C were collected and examined by SDS-PAGE (see Figure 5 for results).

[0085] (Embodiment 4) Binding of a receptor to an NRG polypeptide MCF-7 cells were harvested, counted, centrifuged, and resuspended in DMEM (10% serum, 9 μg / ml insulin). Resuspend cells to a density of 5 x 10 4 A 96-well plate was spread. 100 μL of the solution was added to each well. The cells were then placed in a suspension of 1 L and left to stand overnight at 37°C. The next day, the cells were washed three times with PBS and incubated for 24 hours in serum-free DMEM. The mixture was cultured for 1 hour.

[0086] ErbB2 antibody H4 was diluted in coating buffer (50 mM Na2CO3-NaHCO3, pH 9.6) to 4 μg / ml. Then, 50 μl of each well was placed in a 96-well plate. The bodies were allowed to bind to the plates.

[0087] The DMEM medium for MCF-7 cells was removed by aspiration. Then, NRG and NRG-IgG1-Fc were diluted sequentially with DMEM. Then, 100 μl was added to each well, where NRG is the amino acid sequence of SEQ ID NO: 1. The recombinant NRG polypeptide has the amino acid sequence of SEQ ID NO: 2, and NRG-IgG1-Fc has the amino acid sequence of SEQ ID NO: 2. The blank control contained only DMEM. After incubation for 20 minutes in PBS buffer, the cells were washed with 100 μl / well of Lysis buffer (50 mM Hepes, pH 8.0, 150 mM NaCl, 2 mM sodium orthovanadate, 0.01% Thimerosal, 1% Triton X-100, and 25 ml of protease inhibitor cocktail) were added to each well. The plate was then gently shaken to remove the lysate from the plate. The cells were detached from the suspension and centrifuged at 15,000 rpm for 15 minutes.

[0088] Wash the antibody-coated plate with washing solution (10 mM PBS, pH 7.4, 0.05% Tween 20) for 5 min. 200 μL of washing solution containing 5% skim milk was added to each well and incubated at 37°C for 2 hours. This was then washed three times with washing solution.

[0089] 90 μl of lysed cells per well was added to the coated plate and incubated at 37°C for 1 hour. The plates were then washed five times with washing solution. 100 μl of human hemoglobin peroxidase (HRP) was added to the mixture, and the mixture was incubated at 37° C. for 1 hour. After washing, the plate was filled with freshly prepared HRP substrate solution (50 mM citric acid, 100 mM NaHPO, pH 5.0, 0.2 mg / ml Tetramethylbenzidine (TMB, 0.003% H2O2) was added and incubated at 37°C for 10 minutes. Finally, 50 μL of 1M H2SO4 was added to each well to quench the HRP activity and terminate the reaction. The OD value of each well was measured at 450 nm on a microplate reader. The EC50 was determined as the absorbance The EC50 value was the concentration of NRG or fusion protein at which the maximum value was reached. The affinity of the receptor to NRG or the fusion protein was increased.

[0090] Tables 1 and 2 show the EC50 values ​​of NRG, NRG-IgG1-Fc, and NRG-IgG4-Fc. The EC50 value of G-IgG4-Fc is slightly higher than that of NRG. Table 1. EC50 values ​​of NRG and NRG-IgG1-Fc [Table 1] Table 2. EC50 values ​​of NRG and NRG-IgG4-Fc [Table 2]

[0091] (Embodiment 5) Intravenously or subcutaneously injected NRG-IgG1-Fc and NRG-IgG4-Fc fusions in rats ELISA method used to detect peptide half-life) Rats were injected with 250 μg / kg of NRG-IgG1-Fc via the tail vein or 500 μg / kg of NRG-IgG1-Fc intradermally. 250 μg / kg of NRG-IgG4-Fc was injected into the rats via the tail vein. At this time, blood was collected from the jugular vein and allowed to stand at room temperature for at least 30 minutes. The rat serum containing NRG-IgG1-Fc was diluted and collected for further use. The solution was diluted 1:1 with dilution buffer.

[0092] Standard NRG-IgG1-Fc samples were prepared using rat serum at concentrations of 5,000 ng / ml, 2,500 ng / ml, and 1,000 ng / ml. , 200ng / ml, 40ng / ml, 8ng / ml, 1.6ng / ml, 0.32ng / ml, 0.064ng / ml, and 0ng / ml concentration ranges The solution was prepared at room temperature and subsequently diluted 1:1 with dilution buffer.

[0093] Plate coating and blocking: Coat the plate with human NRG1 / HRG1-β1 EGF domain antibody. The mixture was diluted with the HCl buffer and added to each well of a 96-well plate at a concentration of 50 μL. The mixture was then incubated at 4°C overnight. The next day, discard the coating buffer while washing the plate. Blocking buffer was added to the plate at room temperature. The plate was then dried with a piece of absorbent paper. After incubation, 50 μL of the corresponding standard sample or test sample was added to each reaction well and incubated at room temperature for 2 hours. The plate was washed and then anti-human IgG1 Fc (HRP) antibody was added. The plate was then washed and incubated at room temperature for 1 hour. The MB substrate solution was added to each reaction well. After 20 minutes of incubation at 37°C in the dark, 50 μL of 1 M sulfuric acid was added. The absorbance of each reaction well was measured at 450 nm to determine the NRG and protein content in the sample. Protein content was calculated according to the standard curve. Data were analyzed using GraphPad Prism 5.0. analyzed.

[0094] The results are shown in Tables 3, 4 and 5, respectively: Table 3: Half-life results of intravenously injected NRG-IgG1-Fc [Table 3] Table 4: Half-life results of subcutaneously injected NRG-IgG1-Fc [Table 4] Table 5: Half-life results of intravenously injected NRG-IgG4-Fc [Table 5]

[0095] Experimental data show a half-life of 10 minutes for intravenously injected NRG and 1.5 minutes for subcutaneously injected NRG. Compared with the half-life of 5 hours, the fusion peptides NRG-IgG1-Fc and NRG-IgG4-Fc were immunoglobulin-dependent in rats. It has been shown that the half-life of intravenously and subcutaneously injected NRG fragments can be significantly prolonged. there was.

[0096] (Embodiment 6) Intravenously injected NRG-IgG1-Fc fusion in the treatment of rats with heart failure. Pharmacodynamics experiments of polypeptides (6.1 Experimental Purpose) In a rat model of heart failure induced by left coronary artery ligation, To compare the therapeutic effects of four administration methods on insulin resistance, a Medtronic insulin infusion pump was used. Rats were intravenously infused once daily with NRG-IgG1-Fc fusion protein via a 24-well platelet-free donor (PWD) and recombinant human NRG (rhNRG) ) was infused intravenously in a continuous manner.

[0097] (6.2 Experimental Materials) 6.2.1 Experimental Animals 6.2.1.1 Strain and Source: Viruses provided by Shanghai Sippe-Bk Lab Animal Co., Ltd. Talat) (6.2.1.2 Sex, weight, and number: Male, 200-250g, 150 animals) 6.2.2 Reagents (6.2.2.1 Excipients: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.; Dosage form: Lyophilized powder; (2mg Alb / bottle) (6.2.2.2 Recombinant Human NRG: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd., Dosage Form: Lyophilized Powder; Specification: 250μg / piece) (6.2.2.3 Recombinant human NRG-IgG1-Fc fusion protein: Provided by Zensun (Shanghai) Sci & Tech Co., Ltd. (developed by FDA, dosage form: injection) (6.2.2.4 Isoflurane: RWD Life Technology)

[0098] (6.3 Experimental equipment) (6.3.1 Anesthesia equipment (isoflurane evaporator): MSS INTERNATIONAL) (6.3.2 Ultrasound cardiac detector: Vivid E95; probe type: 12S-D) (6.3.3 Insulin Pump: Medtronic; Models: MMT-712EWS, MMT-722NAS / L) 6.3.4 PowerLab multi-channel physiological recorder: ADInstruments (Shanghai); Model: ML-8 45)

[0099] (6.4 Experimental Method) 6.4.1 Establishment of a rat model of coronary artery ligation-induced heart failure Rats were anesthetized with 4% isoflurane in a gas anesthesia system, and then fixed in a supine position. The chest was shaved and then disinfected with 75% alcohol. After incising the skin on the left anterior chest, the pectoral muscle was bluntly cut. The muscle between the fourth and fifth ribs was blunted using a hemostat. The lungs were then cut open and compressed with both hands until the heart was forced out of the chest cavity and completely exposed. The left atrial appendage and the pulmonary artery cone were completely exposed and their During this time, the left anterior descending coronary artery was ligated with a No. 6-0 suture. The air was evacuated, and then the pectoral muscles and skin were sutured. After surgery, the rats were returned to their cages and The patient was placed under observation. In the case of acute arrhythmia, cardiac massage was performed urgently for 3 to 5 minutes. After surgery, Each rat was given an intramuscular injection of 80,000 U of penicillin sodium for two days.

[0100] 6.4.2 Grouping and Drug Administration (6.4.2.1 Grouping) The results of grouping are shown in Table 6. Two and four weeks after surgery, the cardiac function of the rats was measured by B-ultrasound. After cardiac ultrasound examination at 4 weeks, rats with EF values ​​between 28% and 45% were selected for the next experiment. Selected. Table 6. Grouping of experimental animals and administration schedule [Table 6]

[0101] The rats were randomly divided into four groups according to the results of echocardiography. The rats were given intravenous injections and were divided into a vehicle group, a NRG-IgG1-Fc group at 30 μg / kg four times a day, and a NRG-IgG group. Mice were divided into two groups and administered 1-Fc 6 μg / kg four times a day by intravenous injection daily for 10 days according to Table 1. The administration volume was set at 2 ml / kg / dose, while the administration concentrations were set at 5 μg / ml and 15 μg / ml, respectively. It was determined.

[0102] The NRG tail vein group was treated with an insulin pump for 8 hours per day for 10 days. The administration volume was 5 ml / kg, the dose of NRG was 6 μg / kg, and the concentration was 1.2 μg / ml.

[0103] In the sham-operated group, only suture was performed without coronary artery ligation or medication.

[0104] (6.4.2.2 Distribution method) 1) Vehicle: 2 mg Alb / bottle, add 1 ml of normal saline to each bottle to prepare the mother solution. 0.24 ml of the mother solution was diluted with 49.76 ml of normal saline to give a 9.6 μg / ml Alb solution. 2) NRG-IgG1-Fc: NRG-IgG1-Fc stock solution of 0.4 mg to 0.8 mg / ml was diluted with normal saline and The antibody was NRG-IgG1-Fc with a constant working concentration. 3) NRG: 250 μg NRG / bottle, add 1 ml of normal saline to each bottle to prepare the mother solution. 0.24 ml of the mother solution was diluted with 49.76 ml of normal saline to give a 1.2 μg / ml NRG solution.

[0105] (6.4.3 Observation Indicators) (6.4.3.1 Cardiac Function Tests) After anesthesia with 4% isoflurane in a gas anesthesia system, the rats were placed in a left lateral position with a surgical stent. The rat's head was fixed to the breathing apparatus of a gas anesthesia device while maintaining the isoflurane concentration at 2%. The rats were fixed to a mask. The chest was shaved and disinfected with 75% alcohol. The rat heart was then examined for left ventricular echo signals using an ultrasound probe. In B-mode, the cardiac ultrasound probe is placed near the left side of the sternum, and the probe is positioned between 2 and 3 o'clock. The acoustic beam was directed across the heart in a direction perpendicular to the long axis of the heart. Adjust the probe until it is parallel to both papillary muscles in the horizontal plane, and then adjust the probe until it is parallel to both papillary muscles in the horizontal plane. A short-axis view of the ventricle was obtained, and dynamic images of the left ventricular papillary muscles were collected and stored. In the "M-mode" mode, the probe is held in the short axis of the left ventricle and the sampling line of the M-mode is adjusted. The focal length was adjusted to allow it to pass through the weakest point of the heartbeat on the anterior wall. Use curved lines (the left ventricular cavity and the anterior and posterior walls of the left ventricle should be clearly visible) The left ventricular end-diastolic diameter (D) and left ventricular end-systolic diameter (D) were measured. End-systolic volume (ESV) was calculated using the Teichholtz formula: V = 7 / (2.4 + D) * D3. Ejection fraction (EF) was also calculated. ) was calculated as follows: EF = (EDV - ESV) / EDV × 100%.

[0106] (6.4.3.2 Cardiac Hemodynamics) A physiological recorder is used to record hemodynamic indices, such as carotid artery pressure, intraventricular pressure, +dp / dt and -dp / The dt was recorded. Main procedure: Rats were given an intraperitoneal injection of 20% urethane at an injection volume of 6 ml / kg. The right common carotid artery was isolated and its distal end was ligated. Its proximal end was clamped with an arterial clamp. The vein was then blocked, and a small opening was made between the two ends. A PE50 catheter connected to the probe was inserted. After stabilization, the carotid artery pressure was recorded, and then the catheter was further inserted into the left ventricle. After stabilization, the LVSP, LVEDP, +dp / dt, and -dp / dt indices were measured. LabChart7 was used for analysis.

[0107] (6.4.4 Data Processing) All experimental data [ka] One-way ANOVA analysis was performed using GraphPad 6. P<0.05 indicates significant differences between groups. P<0.01 indicated a highly significant difference between groups.

[0108] (6.5 Experimental Results) The results of the cardiac ultrasound examination are shown in Table 7 and Figure 6. The results of this experiment were obtained by randomly dividing rats with heart failure into After grouping, cardiac ultrasound data showed no significant differences between the groups before administration. There was a clear difference between the groups after the 10-day infusion of NRG-IgG1-Fc (30ug / kg / day). The EF and FS values ​​of the rat hearts were significantly increased (P<0.001), and the LVESV (P<0.001) and LVEDV (P<0.05 ), LVDs (P<0.001), and LVDd (P<0.05) were significantly reduced. These results suggest that α-Fc can significantly improve cardiac function in rats with heart failure. After 8 hours of continuous infusion for 10 days, the EF, LVESV, FS, and LVDs values ​​in the NRG group improved. The equimolar amount of NRG-IgG1-Fc (30 μg / kg) significantly reduced cardiac damage in treated rats compared to the NRG group. Furthermore, NRG-IgG1-Fc was shown to have a superior therapeutic effect against N It was easier to use than the RG. Table 7. Cardiac ultrasound examination results for rats in each group before and after treatment [Table 7]

[0109] The scope of the present invention is not limited to the embodiments. As will be apparent to those skilled in the art, the present invention is not limited to the embodiments. The present invention can be modified and varied in many ways without departing from its spirit and scope. The embodiments described herein are provided by way of example only and the present invention is not to be construed as limiting the scope of the present invention. Subject only to the scope of the appended claims and their equivalents.

Claims

1. A neuregulin (NRG) fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

2. A pharmaceutical composition comprising the NRG fusion polypeptide described in claim 1.

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