CN112585159B - Neuregulin polypeptide fragments and uses thereof - Google Patents

Abstract

The present invention provides specific newbornalin polypeptides that can be used in methods and compositions for preventing, treating, or delaying various diseases or disorders. The invention relates to the use of a newbornalin polypeptide for the preparation of a medicament for preventing, treating or delaying heart failure in a mammal, and a method of use of said medicament for preventing, treating or delaying heart failure in a mammal. In particular, the present invention provides methods of preventing, treating or delaying heart failure in a mammal by administering a medicament comprising a fragment of a newbornalin polypeptide in a particular individual suffering from or at risk of heart failure.

Description

Neuregulin polypeptide fragments and uses thereof

Technical Field

The invention relates to the use of a newbornalin polypeptide for the preparation of a medicament for preventing, treating or delaying heart failure in a mammal, and a method of use of said medicament for preventing, treating or delaying heart failure in a mammal. In particular, the present invention provides methods of preventing, treating or delaying heart failure in a mammal by administering a medicament comprising a fragment of a newbornalin polypeptide in a particular individual suffering from or at risk of heart failure.

Background

Neuregulin (NRG; heregulin, HRG), also known as glial growth factor (glial growth factor, GGF), neu differentiation factor (new differentiation factor, NDF), is a glycoprotein with a molecular weight around 44KD, which transmits signals between cells, and is a ligand of the ErbB family of tyrosine kinase receptors. The neuregulin family contains 4 members: NRG1, NRG2, NRG3, NRG4 (Falls et al, exp Cell Res.284:14-30,2003). NRG1 plays an important role in the nervous system, heart and breast, and there is evidence that NRG1 signaling plays a role in the development, function of other organ systems, and pathogenesis of human diseases including schizophrenia and breast cancer. NRG1 has many isomers. Studies in genetically mutated mice (knockout mice) indicate that the N-terminal region or the Epidermal Growth Factor (EGF) analog differs in its isoforms, which also differ in their body functions. The present invention is based on neuregulin 1 beta (NRG 1 beta).

Neuregulin 1β is a transmembrane protein (Holmes et al, science 256,1205-1210,1992). The outer membrane portion is N-terminal, including immunoglobulin-like domain (Ig-like domain) and EGF-like domain (EGF-like domain), and the inner membrane portion is C-terminal. Under the action of metalloproteinase in extracellular matrix, the outer membrane part of neuregulin can be cut off by enzyme to be in free state, so as to be favorable for combining with ErbB receptor on the surface of surrounding cells and activating corresponding cell signaling.

The ErbB receptor family also falls into four classes, erbB1, erbB2, erbB3 and ErbB4, which are transmembrane proteins with molecular weights around 180-185 KD. Except ErbB2, they all contain ligand binding regions at the N-terminus outside the membrane; except ErbB3, they all contain protein tyrosine kinase activity at the C-terminus within the membrane. Wherein ErbB1 is a receptor for an epidermal growth factor and ErbB3 and ErbB4 are both receptors for a neuregulin. Of the receptors for neuregulin, only ErbB2 and ErbB4 were expressed in higher amounts in the heart (Yarden et al, nat Rev Mol Cell Biol,2:127-137,2001).

When neuregulin binds to the extramembranous portions of ErbB3 or ErbB4, it will cause ErbB3, erbB4 to form heterodimers with other ErbB receptors (often including ErbB 2), or ErbB4 itself to form homodimers, which in turn will cause the intramembranous portions of the receptor to be phosphorylated (Yarden et al, nat Rev Mol Cell Biol,2:127-137,2001). The phosphorylated intramembrane moiety can further bind to various signaling proteins within the cell, thereby activating downstream ERK or AKT signaling pathways, leading to a series of cellular responses: including stimulation or inhibition of cell proliferation, apoptosis, cell migration, cell differentiation, or cell adhesion.

Neuregulin is particularly important for heart development (WO 0037095, CN1276381, WO03099300, WO9426298, US6444642, WO9918976, WO0064400, zhao et al, J.biol. Chem.273,10261-10269,1998). In early embryonic development, the expression of neuregulin is primarily restricted to the endocardium, which is then released by the paracrine pathway into surrounding cardiomyocytes and binds to the extramembranous portion of the protein tyrosine kinase receptor ErbB4 on the cell membrane, erbB4 in turn forming a heterodimer with ErbB2. The formation and activation of the ErbB4/ErbB2 complex is essential for early spongiform heart trabecular formation. Deletion of any of the three protein genes for neuregulin, erbB4 and ErbB2 will leave the embryo trabecular-free and die of the uterus early in development. WO0037095 shows that a concentration of neuregulin can continuously activate ERK signaling pathway, promote growth and differentiation of cardiomyocytes, guide reconstruction of sarcomere and cytoskeleton at the adhesion site of cardiomyocytes and cells, improve the structure of cardiomyocytes, and enhance contraction of cardiomyocytes. WO0037095 and WO003099300 also indicate that neuregulin can be used for the detection, diagnosis and treatment of various cardiovascular diseases.

The following are examples of some prior art documents relating to the present invention: 1.Cardiac muscle function and manipulation:WO0037095;2. novel uses of growth factor neuregulin and analogs thereof: CN1276381;3.Neuregulin based methods and composition for treating cardiovascular diseases:WO03099300;4.Zhao YY,Sawyer DR,Baliga RR,Opel DJ,Han X,Marchionni MA and Kelly RA.Neuregulins Promote Survival and Growth of Cardiac Myocytes.J.Biol.Chem.273,10261-10269 (1998); 5.Methods for treating muscle diseases and disorder:WO9426298;6.Methods of increasing myotube formation or survival or muscle cell mitogenesis,differentiation or survival using a neuregulin:US6444642.7.Therapeutic methods comprising use of a neuregulin:WO9918976;8.Methods for treating congestive heart failure:WO0064400;9.Holmes WE,Sliwkowski MX,Akita RW,Henzel WJ,Lee J,Park JW,Yansura D,Abadi N,Raab H,Lewis GD,et al.Identification of heregulin,a specific activator p185erbB2.Science 256,1205-1210 (1992); 10.Falls DL.Neuregulins:functions,forms and signalingstrategies.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).

Heart Failure (HF) is a syndrome of heart failure caused by various heart diseases, including systolic heart failure (systolic heart failure, SHF) and diastolic heart failure (diastolic heart failure, DHF). In 2008, the "diagnosis and treatment of acute/chronic heart failure" issued by the European cardiology Congress (ESC) defines the latter as heart failure with preserved ejection fraction (Heart Failure with preserved ejection fraction, HF-PEF). Systolic heart failure refers to the manifestation of heart muscle contraction force decreasing to make heart discharge unable to meet the metabolic needs of the body, organ and tissue blood flow perfusion insufficiency, and pulmonary circulation and/or systemic circulation congestion. Heart failure with preserved ejection fraction (HF-PEF) is often referred to as diastolic heart failure, which is caused by impaired active relaxation capacity and reduced myocardial compliance in the left ventricle, and myocardial hypertrophy with increased stiffness due to interstitial fibrosis, resulting in impaired filling of the left ventricle in diastole, reduced stroke volume, and increased end-diastolic pressure in the left ventricle. Epidemiological data from the American heart and lung institute in 2006 have shown that heart failure or diastolic heart failure with preserved ejection fraction accounts for over 50% of the total heart failure population. Heart failure with retained ejection fraction may exist alone or may occur simultaneously with contractile dysfunction. Heart failure with retained ejection fraction is most common in elderly women with hypertension, diabetes mellitus, and left ventricular hypertrophy.

Cardiac (ventricular) hypertrophy is an important adaptive physiological response to increased cardiac operating pressure or demand. One of the early cellular changes that occurs after the action of the mast stimulus is mitochondrial synthesis and myofibril expansion (thickening of the chamber wall) with a proportional increase in single cell size, but no (or very little) increase in cell number.

When the ventricles are subjected to pressure, the initial response is an increase in the length of the muscle segments. Followed by an increase in total muscle mass. When the load is too severe, the myocardial contractility will decrease. In the slightest state, this decrease is manifested by a decrease in the rate of unloaded myocardial contractility or a decrease in the rate of force development during isometric contraction. As the myocardial contractility further decreases, a greater decrease in the rate of unloaded myocardial shortening occurs, accompanied by development of isometric contractile muscle strength and a decrease in contraction length. At this point, circulatory compensation may still be provided by the expansion of the heart and the increase in myocardial mass, which tends to maintain ventricular wall stress at normal levels. As the contractile force continues to decline, there may be apparent congestive heart failure, manifested as a decline in cardiac output or effort, and/or an increase in end-diastolic volume and diastolic pressure of the ventricles.

The transition from hypertrophy to heart failure is characterized by several changes in cell tissue. For example, normal mast cells have larger sizes with enhanced and ordered contractile units and stronger cell-cell adhesion. Conversely, the hypertrophic cells in pathological conditions, which are also large in size and have protein aggregates, exhibit disordering of contractile proteins (disorder of the sarcomere structure) and poor cell-cell adhesion (disorder of the myofibers). Thus, in hypertrophy of pathological states, an increase in cell size and aggregation of contractile proteins is associated with disordered assembly of the sarcomere structure and loss of firm cell-cell interactions.

Currently, over 2600 thousands of people worldwide have heart failure, and more than 55 ten thousand new patients are added in the United states each year, and heart failure is one of important and serious public health problems. Current drugs for treating heart failure are mainly focused on Angiotensin Converting Enzyme (ACE) inhibitors, which cause vasodilation, lower blood pressure and reduce the workload of the heart. Although the percent reduction in mortality is statistically different, the actual reduction in mortality after ACE inhibitors is only 3% -4% on average, and there are several potential side effects.

ACE inhibitors have also been used in combination with other drugs, such as digitalis, to increase the strength of systole; and/or some diuretic, to help reduce heart workload by causing the kidneys to reject more sodium and water in the blood. However, at least one study demonstrated that there was little difference in survival rate between digitalis and placebo in patients with grade II-III heart failure. Furthermore, diuretics improve certain symptoms of heart failure, but are not suitable for monotherapy.

Other options for preventing or treating heart failure have corresponding limitations. For example, heart transplantation is obviously more expensive and invasive than drug therapy and is further limited by the presence or absence of a donor heart. The use of mechanical devices, such as biventricular cardiac pacemakers, is also invasive and relatively expensive. Thus, new therapeutic measures are needed due to the shortfall of current therapeutic approaches.

One promising new therapeutic approach involves the administration of neuregulin (hereinafter "new york-green") to patients with or at risk of heart failure. Previous studies have shown that nivalgreen-1β (NRG-1β) can bind ErbB3 and ErbB4 directly with high affinity. The orphan receptor ErbB2 forms a heterodimer with ErbB3 or ErbB4 and has a higher affinity than ErbB3 or ErbB4 homodimers. The results of the development of nerves suggest that the formation of the sympathetic nervous system requires the complete NRG-1 beta, erbB2 and ErbB3 signaling systems. Targeted disruption of NRG-1 beta, or ErbB2 or ErbB4 results in embryonic lethality due to defects in cardiac development. Recent studies have also highlighted that NRG-1 beta, erbB2 and ErbB4 have important roles in cardiovascular development and maintenance of normal cardiac function in adults. Studies have shown that NRG-1 beta enhances the tissue architecture of the sarcomere of adult cardiomyocytes. Short term administration of a recombinant NRG-1βEGF analog significantly ameliorates or prevents deterioration of myocardial function in three different heart failure animal models. More importantly, NRG-1 beta can significantly prolong the survival of heart failure animals. These effects make NRG-1 beta a promising broad-spectrum therapeutic or lead compound for the treatment of heart failure caused by various common diseases. However, different newland green polypeptide fragments have different half-lives, different tissue distributions in vivo, and even different therapeutic effects, so there is still a need to optimize the length of the NRG polypeptide fragment to find a more effective neuromodulation polypeptide fragment that can be used clinically to prevent, treat or delay heart failure and/or cardiac hypertrophy.

Summary of The Invention

The invention relates to the use of a newbornalin polypeptide for the preparation of a medicament for preventing, treating or delaying heart failure in a mammal, and a method of use of said medicament for preventing, treating or delaying heart failure in a mammal. In particular, the present invention provides methods of preventing, treating or delaying heart failure in a mammal by administering a medicament comprising a fragment of a newbornalin polypeptide in a particular individual suffering from or at risk of heart failure. In certain embodiments, the mammal is a human. In certain embodiments, the subject is a human.

In a first aspect, the present invention provides fragments of a number of newland green polypeptides. In certain embodiments, the newbornalin polypeptide comprises an EGF domain of a neuregulin. In certain embodiments, the newbornalin polypeptide comprises the EGF domain of the β2 isomer of human neuregulin. In certain embodiments, the Neellin polypeptide comprises the amino acid sequence of SEQ ID NO. 1. In certain embodiments, the Neellin polypeptide comprises the amino acid sequence of SEQ ID NO. 2.

Certain newland green polypeptides contain the following amino acid sequences: ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln (SEQ ID NO: 1), i.e., human NRG-1, amino acid sequence 177-237.

Certain newland green polypeptides contain the following amino acid sequences: val Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys (SEQ ID NO: 2).

Preparation of the newbornalin polypeptide may be according to any of the related techniques well known in the art. Typical techniques for preparing a newland green polypeptide are provided herein. In certain embodiments, the newbornalin polypeptide may be recombinant. In certain embodiments, the newbornalin polypeptide is synthesized, e.g., by liquid phase or solid phase peptide synthesis.

Another aspect of the invention provides nucleic acids, vectors, and host cells related to the New land Grifola polypeptides. The nucleic acid or its complement encodes a newbornalin polypeptide or a fragment thereof. The nucleic acid may be double-stranded or single-stranded DNA or RNA, which can be inserted into a suitable vector for propagation and expression of the Neellin polypeptide. The transformed vector is transferred into a suitable host cell, such as a host cell capable of expressing the recombinant newbornalin polypeptide.

In another aspect, the invention provides the use of the new york polypeptide in therapeutic and non-therapeutic applications. In particular to the application of the New Zealand polypeptide in preventing, treating or delaying various heart diseases and disorders. Accordingly, the present invention provides pharmaceutical formulations comprising a new and improved therapeutic methods.

In another aspect, the invention provides a method of treating heart failure in a mammal. In certain embodiments, the method comprises injecting the newbornalin polypeptide into a mammal.

In another aspect, the invention provides a method of inducing phosphorylation of an ErbB receptor in a cell. In certain embodiments, the method comprises contacting the cell with a newland green polypeptide.

Another aspect of the invention is to provide methods for inducing and maintaining activation of AKT signaling pathways in heart cells. In certain embodiments, the method comprises contacting the heart cells with newland grid Lin Duotai.

Another aspect of the invention is to provide for the induction and maintenance of activation of ERK signaling pathways within heart cells. In certain embodiments, the method comprises contacting the heart cells with newland grid Lin Duotai.

Detailed Description

A. Paraphrasing meaning

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications, and other publications mentioned herein are incorporated by reference in their entirety. The definitions set forth in this section control if they are contrary or inconsistent with the definitions set forth in the patents, applications, published applications and other publications incorporated by reference herein.

As used herein, unless otherwise indicated, "a" means "at least one" or "one or more than one".

As used herein, an "EGF-like domain" or "EGF-like domain" refers to a polypeptide fragment encoded by a neurogenin gene that binds to and activates ErbB2, erbB3, erbB4, or a heterologous or homodimer thereof, and has structural similarity to EGF receptor binding regions described in the following references: WO 00/64400; holmes et al, science,256:1205-1210 (1992); U.S. Pat. nos. 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 (1997); higashiyama et al, J.biochem.,122:675-680 (1997); and WO 97/09425, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the EGF-like domain binds to and activates ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers. In certain embodiments, the EGF-like domain comprises a receptor binding region amino acid of NRG-1. In certain embodiments, EGF-like domain refers to amino acids 177-226, 177-237 or 177-240 of NRG-1. In certain embodiments, the EGF-like domain comprises a receptor binding region amino acid of NRG-2. In certain embodiments, the EGF-like domain comprises a receptor binding region amino acid of NRG-3. In certain embodiments, the EGF-like domain comprises a receptor binding region amino acid of NRG-4.

An "effective dose" of an active ingredient for treating a particular disease, as used herein, is a dose sufficient to ameliorate, or in some way reduce, the symptoms associated with the disease. This dose may be effective in curing the disease but is typically used to ameliorate the symptoms of the disease.

As used herein, an "active ingredient" is any substance that is used to diagnose, cure, alleviate, treat or prevent a disease in a human or other animal, or to enhance physical or mental health.

As used herein, "amelioration" of symptoms of a particular disorder refers to permanent or temporary, sustained or transient alleviation of symptoms by administration of a particular active agent, which alleviation can be attributed to or associated with administration of the agent.

As used herein, "treatment" or "treatment" refers to any means by which the symptoms of an malaise, disorder or disease can be ameliorated or redirected. The effect may be prophylactic, such as to completely or partially prevent the occurrence of a disease or symptom thereof, or therapeutic, such as to partially or completely cure a disease and/or the adverse effects caused by the disease. Treatment also includes any pharmaceutical use of the compositions described herein.

As used herein, "vector (or plasmid)" refers to a dispersion member used to introduce heterologous DNA into a cell for expression or replication therein. The selection and use of these vectors is well known to the skilled person. Expression vectors include vectors capable of expressing DNA linked to regulatory sequences, such as promoter regions, capable of effecting expression of such DNA fragments. Thus, expression vector refers to a recombinant DNA or RNA component, such as a plasmid, phage, recombinant virus, or other vector, that when introduced into an appropriate host cell results in expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those that replicate in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.

As used herein, "cardiomyocyte differentiation" refers to a state characterized by a 10% reduction in DNA synthesis, greater than 10% inhibition of other factor-stimulated DNA synthesis, ordered sarcomere binding and cell-cell adhesion, sustained activation of MAP kinase, and p21 ^Cip1 Is described. For further discussion see WO00/37095, the contents of which are incorporated herein by reference in their entirety.

As used herein, "ejection fraction (ejection fraction)" or "EF" refers to the proportion of blood pumped from the full left ventricle by a heart beat. The following formula may be used to define: (left ventricular end diastole volume-left ventricular end systole volume)/left ventricular end diastole volume.

As used herein, "systolic fraction (fractional shortening)" or "FS" refers to the ratio of the diameter changes in the systolic and diastolic states of the left ventricle. The following formula may be used to define: (left ventricular end diastole inner diameter-left ventricular end systole inner diameter)/left ventricular end diastole inner diameter.

As used herein, "heart failure" or "heart failure" refers to a heart dysfunction in which the heart is unable to pump blood at the rate required by the metabolized tissue. Heart failure includes a variety of disease states such as congestive heart failure, myocardial infarction, tachyarrhythmia, familial cardiomyopathy, ischemic heart disease, congenital dilated cardiomyopathy, myocarditis, and the like. Heart failure may be caused by a variety of factors including, but not limited to: ischemic, congenital, rheumatic, or primary forms. Chronic cardiac hypertrophy is a distinct disease state that is a precursor to congestive heart failure and cardiac arrest.

As used herein, "myocardial infarction" refers to plaque necrosis of a portion of the myocardium caused by severe and sustained ischemia due to a blockage of a coronary artery or interruption of blood flow.

As used herein, "myoganglion or ordered, enhanced arrangement of myoganglion structures" refers to a state characterized by the ordered arrangement of contractile proteins exhibited by immunofluorescent staining of alpha-actin in cardiomyocytes. The alignment of alpha-actin in cells can be recognized by microscopy and its associated camera equipment. As used herein, "disorder or irregularity of the sarcomere or sarcomere structure" is in contrast to "ordered, enhanced arrangement of the sarcomere or sarcomere structure".

As used herein, "ordered or enhanced arrangement of cytoskeletal structures" refers to a state in cardiomyocytes characterized by an ordered arrangement of actin fibers as revealed by phalloidin (phaslloidin) staining. The alignment of actin filaments in cells can be identified by microscopy and its associated camera equipment, as exemplified in the present figures. As used herein, "disorder or irregularity of the cytoskeletal structure" refers to the situation as opposed to "ordered, or enhanced arrangement of the cytoskeletal structure".

As used herein, "protein" is synonymous with "polypeptide" or "peptide" unless the context clearly indicates otherwise.

As used herein, "sustained activation of MAP kinase" refers to a maintenance of the phosphorylation state of MAP kinase, p42/44, in a cell for at least 21 hours. WO00/37095 is further discussed and incorporated herein by reference.

"synergistic," "synergistic effect," or similar terms are used herein to describe an improved therapeutic effect obtained by combining one or more therapeutic agents and one or more retinoic acid compounds. Although synergistic effects mean effects greater than the sum (e.g., 1+1=3) in certain fields, in the medical field, one additive (1+1=2) or less additive (1+1=1.6) effect may also be synergistic. For example, if one of the two drugs alone inhibits 50% of the expansion of ventricular myocyte hypertrophy, it cannot be expected that the combination of the two drugs would completely stop the development of ventricular myocyte hypertrophy. In many cases, the two drugs cannot be administered together due to unacceptable side effects. In other cases, the drugs antagonize each other, slowing the progression of ventricular myocyte hypertrophy by less than 50% when used in combination. Thus, a synergistic effect is obtained if the combination of the two drugs slows the progression of ventricular myocyte hypertrophy by more than 50% without increasing unacceptable side effects.

As used herein, "cardiac hypertrophy" refers to a condition characterized by: an increase in the size of individual ventricular myocytes is sufficient to result in a clinical diagnosis of the patient or to identify the cells as being larger (e.g., two or more times larger than non-mast cells). It may be accompanied by the accumulation of contractile proteins in individual cardiomyocytes and activation of embryo gene expression.

Methods for detecting ventricular myocyte hypertrophy include both in vitro and in vivo methods. Methods for detecting ventricular myocyte hypertrophy in vitro include those described in WO00/37095, such as an increase in cell size and an increase in Atrial Natriuretic Peptide (ANP) expression. The change in cell size was used in a scoring system to determine the extent of hypertrophy. These changes can be observed with an inverted phase contrast microscope, and the extent of hypertrophy is measured in terms of an artificial scale of 7-0, with 7 indicating fully hypertrophic cells and 3 indicating unstimulated cells. The states represented by points 3 and 7 can be seen in FIGS. 2A and B of Simpson et al (1982) Circulation Res.51:787-801, respectively.

As used herein, "cardiac hypertrophy inhibition" refers to a decrease in one of the parameters characterizing hypertrophy relative to a condition of hypertrophy or an increase in one of the parameters characterizing hypertrophy relative to normal conditions. For example, inhibition of ventricular myocyte hypertrophy may be indicated by measuring the decrease in cell size relative to the condition of hypertrophy. Inhibition of ventricular myocyte hypertrophy refers to a 10% or more reduction in cell size relative to the size observed under hypertrophic conditions.

Prevention of ventricular myocyte hypertrophy is determined by preventing an increase in cell size relative to normal cells at an inducer concentration sufficient to induce hypertrophy.

Hypertrophy may be from any factor responsive to retinoic acid, including congenital viral, congenital, cardiotrophic, muscle trophic factors, or as a result of ischemia or ischemic injury such as myocardial infarction. Typically, treatment is performed to prevent or slow down the progression of hypertrophy, particularly after cardiac injury, such as after ischemia has occurred. Preferably, for the treatment of myocardial infarction, the agent is administered immediately after myocardial infarction to prevent or reduce hypertrophy.

As used herein, "activity unit" or "1U" refers to the amount of standard product that will cause 50% of the maximum response. In other words, in order to determine the activity unit of a certain active agent, the EC50 must be determined. EC50 may be determined by any method known in the art, including the methods used by the inventors in the examples below. Determination of the activity units is important for quality control of genetically engineered products and clinically used drugs, so that different pharmaceuticals and/or different batches of products can be quantified with the same standard.

In certain examples, the units of newland green are determined by measuring the activity of newland green by kinase receptor activated enzyme-linked immunosorbent assay (KIRA-ELISA), such as WO03/099300 and Sadick et al, 1996,Analytical Biochemistry,235:207-14, the contents of which are incorporated herein by reference in their entirety. Briefly, this method measures the neolanugrange-induced activation and phosphorylation of ErbB2 of the adherent breast cancer cell line MCF-7. The membrane proteins are solubilized with Triton X-100 lysate, wherein the receptor is captured by an ErbB 2-specific antibody (e.g., H4) coated in ELISA wells that does not cross-react with ErbB3 or ErbB 4. The degree of receptor phosphorylation was determined by anti-phosphotyrosine antibody ELISA.

B. Neilangellin

The present invention provides certain fragments of the New Zealand green polypeptides. In certain embodiments, the newbornalin polypeptide comprises an EGF domain of a neuregulin. In certain embodiments, the newbornalin polypeptide comprises the EGF domain of the β2 isomer of human neuregulin. In certain embodiments, the Neellin polypeptide comprises the amino acid sequence of SEQ ID NO. 1. In certain embodiments, the Neellin polypeptide comprises the amino acid sequence of SEQ ID NO. 2.

In certain embodiments, the Neellin polypeptide comprises the amino acid sequence of SEQ ID NO. 1. In a more preferred embodiment, the Neellin polypeptide is the amino acid sequence of SEQ ID NO. 1.

In certain embodiments, the Neellin polypeptide comprises the amino acid sequence of SEQ ID NO. 2. In a more preferred embodiment, the Neellin polypeptide is the amino acid sequence of SEQ ID NO. 2.

Preparation of New land green polypeptides

The preparation of the newbornalin polypeptide may be according to any obvious relevant technique. Typical techniques for preparing a newland green polypeptide can be found, for example, in U.S. Pat. Nos. 7,226,907 and 5,367,060, WO94/026298 and WO03/099300, the contents of which are incorporated herein by reference in their entirety.

The preparation of the newbornalin polypeptides of the invention may be according to any of the related techniques well known in the art. In certain embodiments, the newland green polypeptide is synthesized, e.g., by liquid or solid phase peptide synthesis, see Merrifield,1963, j.am.chem.soc.85:2149; fields et al 1990,Int J Pept Protein Res.35:161-214; fields et al 1991,Pept Res.4:95-101, the contents of which are incorporated herein by reference in their entirety.

In an optimized embodiment, the newbornalin polypeptide may be obtained from natural sources, synthetically or commercially. In certain embodiments. In certain embodiments, the newbornalin polypeptide may be obtained by recombinant expression of the protein followed by purification.

The newbornalin polypeptide may be purified by any technique known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and the like. The person skilled in the art is familiar with the purification of the newbornalin polypeptide.

Use of newland green polypeptides

The newbornalin polypeptide may be administered at the discretion of a person skilled in the art. Examples of this include the methods described in the following documents: U.S. Pat. Nos. 7,226,907 and 5,367,060, WO94/026298 and WO03/099300, the contents of which are incorporated herein by reference in their entirety.

The new and green polypeptides are useful in the treatment of a range of diseases and disorders. Typical diseases and disorders include heart diseases such as heart failure, viral myocarditis, dilated (hyperemic) cardiomyopathy (DCM), cardiotoxicity or myocardial infarction.

In certain embodiments, the invention provides a method for treating heart failure by administering an effective dose of a newland polypeptide.

The new and green polypeptides may be administered in the form of a pharmaceutical formulation.

The mode of administration of the newbornalin polypeptide is within the discretion of one skilled in the art and includes, but is not limited to, oral, intravenous, intragastric, rectal, intra-abdominal (membranous) or ventricular injection.

In a preferred embodiment, the composition for administration is a pharmaceutical formulation. The pharmaceutical formulation may be a pharmaceutical formulation containing a prophylactic or therapeutic dose of one or more prophylactic or therapeutic agents (e.g., a complex containing a newland polypeptide and other prophylactic or therapeutic agents), and a pharmaceutically acceptable carrier or excipient. In one embodiment and herein, "pharmaceutically acceptable" means that they have been passed by the relevant national authorities or are useful in animals, particularly humans, as described in U.S. or other widely recognized pharmaceutical. "Carrier" refers to diluents, adjuvants (such as Freund's complete and incomplete), excipients, or other carriers that aid in the administration of the therapeutic agent. The pharmaceutical carrier may be a sterile liquid such as water and oils, including petroleum, animal, vegetable or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The best carrier for intravenous administration of the pharmaceutical formulation is water. In preparing the injectable formulation, saline, dextrose, and glycerol liquids may be employed. Examples of suitable pharmaceutical carriers are described in e.w. martin, remington's Pharmaceutical Sciences.

Typical pharmaceutical formulations and dosage forms contain one or more excipients. Suitable excipients are well known to those skilled in the art of pharmaceutical technology and include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, mica, sodium chloride, dried skim milk, propylene, glycol, water, alcohol and the like. Whether a certain excipient is suitable for incorporation into a pharmaceutical formulation or dosage form depends on many factors well known in the art, including, but not limited to, the manner in which the dosage form is administered to a patient and the particular active ingredient in the dosage form. If desired, the formulation or single dosage form may contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents.

Pharmaceutical formulations contain excipients well known in the art or which are published, for example, on the United States Pharmacopeia (USP) SP (XXI)/NF (XVI). Generally, lactose-free formulations contain an active ingredient, a binder/filler, and a pharmaceutically compatible and dose acceptable lubricant. Typical lactose-free dosage forms contain an active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

The pharmaceutical formulations and dosage forms of the present invention contain one or more compounds that reduce the rate of decomposition of the active ingredient. The compound is referred to herein as a "stabilizer" and includes, but is not limited to, antioxidants such as ascorbic acid, pH buffers or salt buffers.

Pharmaceutical formulations and single dosage forms may take the following forms: solutions, suspensions, emulsions, tablets, capsules, powders, sustained release forms, and the like. Formulations for oral administration contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. The agents and dosage forms contain a prophylactic or therapeutic dose of the purified prophylactically or therapeutically effective agent, which is admixed with a quantity of carrier to be shaped for better administration to a patient. The dosage form should be suitable for the mode of administration. In an optimized embodiment, the pharmaceutical formulation or single dosage form should be sterile and administered in a suitable form, preferably the subject is an animal, more preferably the subject is a mammal, most preferably the subject is a human.

The form of the pharmaceutical formulation containing the new yoelin should be adapted to the mode of administration. Modes of administration include, but are not limited to, injection (e.g., intravenous, intramuscular, subcutaneous, or intradermal), oral, buccal (e.g., sublingual), inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, intrasynovial, and rectal administration. In a particular embodiment, formulation may be formulated with reference to certain conventional procedures, such as those used to prepare pharmaceutical formulations for administration to humans by intravenous or subcutaneous or intramuscular injection, oral, intranasal or topical administration. In a certain embodiment, the pharmaceutical formulation is in a form that conforms to the conventional manner of subcutaneous administration. Typically, the formulation for intravenous administration is a sterile isotonic solution. If desired, the formulation can also contain a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site.

Dosage forms include, but are not limited to, the following forms: tablets, caplets, capsules such as soft elastic gelatin capsules, cachets, tablets, troches, dispersions, suppositories, ointments, cataplasms (creams), pastes, powders, dressings, emulsions, plasters, solutions, drug patches, aerosol (e.g., nasal sprays or inhalers), colloids; liquid dosage forms suitable for oral or mucosal administration to patients include suspensions (e.g., aqueous or non-aqueous suspensions, oil-in-water emulsions, or water-in-oil emulsions), solutions, and prodrugs; the liquid dosage form is suitable for patients taking injection; sterile solids (e.g., crystalline or amorphous) can be reconstituted to a liquid dosage form suitable for administration by injection to a patient.

Depending on the application, the formulation, shape and type of dosage form of the newbornaline polypeptide will also vary. For example, a dosage form for acute treatment of a disorder may contain more of the newbornalin polypeptide than a dosage form for long-term treatment of the same disease. Likewise, dosage forms that have therapeutic effects against different cancers are also different. Similarly, an injectable dosage form will contain less of the active ingredient than an oral dosage form for the treatment of the same disease or disorder. Those skilled in the art will appreciate that the manner of formulation described above and other specific dosage forms encompassed by the present invention will vary. See, for details, lemmington, pharmacology, 18 th edition, mack Press, iston, pa (1990).

The administration of the newbornalin polypeptide can be carried out in any way according to the discretion of a person skilled in the art, including but not limited to these: oral, intravenous, intragastric, duodenal, intraperitoneal or ventricular administration.

C. Dosage and route of administration

The amount of newbornaline used in the present invention will vary with the nature and severity of the disease or condition, and the route of administration of the active ingredient. The frequency and dosage of administration will also vary with the particular factors of each patient, depending on the particular treatment (e.g., therapeutic or prophylactic agent), the severity of the disorder, disease, or condition, the route of administration, and the age, weight, response, and prior history of the patient. The effective dose can be extrapolated from dose-response curves obtained in vitro or in animal model test systems.

Exemplary dosages of newland for use include how much milligrams or micrograms of newland are administered per kilogram of body weight of the subject (e.g., about 1 microgram per kilogram of body weight to about 500 milligrams per kilogram of body weight, about 100 micrograms per kilogram of body weight to about 5 milligrams per kilogram of body weight, or about 1 microgram per kilogram of body weight to about 50 micrograms per kilogram of body weight). Such as a dosage to be administered to a patient, typically the weight of active peptide used per kilogram of body weight of the patient is from 0.001mg/kg to 15mg/kg. Suitable amounts are also: 0.001mg/kg-15mg/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-0.2mg/kg, 0.005mg/kg-0.050mg/kg.

Exemplary dosages of N.sub.He also include how many units (U) or unit amounts of N.sub.He.He are administered per kilogram of body weight (e.g., about 1U per kilogram of body weight to about 5,000U per kilogram of body weight, about 10U per kilogram of body weight to about 1,000U per kilogram of body weight, or about 100U per kilogram of body weight to about 500U per kilogram of body weight). Such as a dosage to be administered to a patient, typically in the range of 10U/kg to 1,000U/kg of active peptide per kilogram of body weight of the patient. Suitable amounts are also: 1U/kg-10,000U/kg, 1U/kg-5,000U/kg, 10U/kg-1,000U/kg, 50U/kg-2,000U/kg, 50U/kg-1,000U/kg, 50U/kg-500U/kg, 100U/kg-1,000U/kg, 100U/kg-500U/kg, 100U/kg-200U/kg.

In general, for the various diseases described herein, the range of amounts of newland green recommended in the methods of the invention per day is: about 0.001mg to 1000mg per day. In certain cases, the total daily dosage may range from: 0.001mg-15mg, 0.005mg-10mg, 0.01mg-5mg, 0.001mg-4mg, 0.005mg-3mg, 0.01mg-2mg, 0.001mg-1mg, 0.005mg-0.5mg, 0.010mg-0.2mg. The patient may be initially treated with a low dose, such as about 0.1 μg to about 1 μg per day, and if necessary up to about 20 μg to about 1,000 μg per day, either as a single dose or in divided doses, depending on the overall patient response. In some cases, it may be necessary to use dosages of the active ingredient outside the ranges taught herein, as will be apparent to those of ordinary skill in the art. Furthermore, it should be noted that the clinician or treating physician should know how and when to interrupt, adjust or terminate the treatment based on the response of the patient's individual. In some embodiments, the amount of N.sub.He is about 1U/day to about 10,000U/day. In some specific cases, the amount of newland used is about 1U/day to about 5,000U/day. In some specific cases, the amount of N.sub.N.E. is from about 10U/day to about 2,000U/day. In some specific cases, the amount of N.sub.He is about 10U/day to about 1,000U/day. In some embodiments, the amount of newland used is about 100U/day to about 200U/day.

The new glanglin may also be administered via a dose schedule or "treatment cycle". The daily dose during the treatment cycle is detailed above. The treatment period may last for 2 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks.

In some embodiments, the new and improved therapeutic regimen is administered daily during the treatment cycle. In certain embodiments, the duration of the new york elin administration during a treatment cycle is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, the new york is administered on the first day of a treatment cycle, and the new york is not administered for the remaining day or days of the treatment cycle. In certain embodiments, the new york is administered daily for 3, 5, 7 or 10 days during a treatment cycle, with no administration remaining during the cycle.

Drawings

Fig. 1: nellagreen or NLF102 protein content in serum at different time points

Fig. 2: efficacy experiment cardiac hyper-results of long-term administration of NRG, NLF-102 pump tail vein for treating rat heart failure

Examples

EXAMPLE 1 NLF102 protein expression purification

1. Construction of expression vector, transformation and screening of expression engineering bacteria and preservation of bacteria

The target DNA fragment was amplified by PCR using cDNA of human normal liver tissue as a template and KOD high-fidelity amplification enzyme (TOYOBO Co.). The PCR product is purified by a gel kit (Tiangen) and recovered, and then is subjected to double digestion by restriction enzyme, and then is connected with an expression vector pET-22b (+) through T4 ligase (NEB company), so that a corresponding recombinant expression plasmid is constructed. Subsequently, we transformed competent cells BL21 (DE 3) with the correct sequencing expression plasmid by CaCl2 method to construct recombinant long fragment human N-New land green NLF102 expression engineering bacteria. A series of single colonies were randomly selected and inoculated into 2mL of LB-Amp liquid medium, cultured overnight at 37℃and 250rpm, inoculated into LB-Amp liquid medium, cultured at 37℃and 250rpm until OD 600.0, and then protein expression was induced by adding IPTG to a final concentration of 1mM, and after 4 hours, the cells were collected by centrifugation. SDS-PAGE electrophoresis and Coomassie brilliant blue staining analysis were performed to determine the correctness of the molecular weight of the protein. The engineering bacteria of the recombinant long fragment human new york green with correct and stable expression are obtained after screening, glycerol is added, and then the engineering bacteria are mixed evenly and packaged into a freezing tube, and frozen and stored for a long time at the temperature of minus 80 ℃ or in liquid nitrogen.

2. Fermentation of expression engineering bacteria, protein expression, bacterial breaking and obtaining of coarse inclusion body

And (3) inoculating a proper amount of frozen glycerinum into LB medium containing ampicillin (final concentration of 50 ug/mL), shaking the strain in a test tube at a proper rotating speed at 37 ℃ for overnight, and activating the strain. The next day, the activated bacterial liquid is inoculated into 250mL of LB culture medium containing 50ug/mL of ampicillin final concentration, the bacterial liquid is cultured in a triangular flask at 37 ℃ in a shaking way, after the OD600 of the bacterial liquid reaches 1.0, IPTG is added, and after the bacterial liquid is continuously induced at 37 ℃ at 250rpm for 4 hours, the bacterial liquid is collected by centrifugation at 8000rpm for 30 min.

The bacterial cells were prepared according to the procedure 1:10 (m/v) PBS was added, and after homogenization by a homogenizer, the bacteria were broken in a high pressure breaker. And centrifuging and collecting the precipitate to obtain the crude inclusion body.

3. Washing, denaturation and renaturation of inclusion bodies

Washing, denaturing and renaturation are carried out on the crude inclusion body, and inclusion body renaturation liquid is obtained.

4. Protein purification

NLF102 was purified by anion exchange (Q column) -enterokinase cleavage-cation exchange (SP column) -size exclusion chromatography.

HPLC detection of the purity of target proteins

6. Detection of endotoxin content

And (3) referring to the four general rules 1143 of the pharmacopoeia of the people's republic of China of 2015 edition, and detecting the endotoxin content of the obtained protein.

Bca method for detecting the concentration of protein samples

Reference is made to Pierce BCA Protein Assay Kit specification (Thermo Fisher, cat. No. 23225). And detecting the protein concentration of the protein sample obtained by the experiment.

NLF102 is here a recombinant N.sub.New Yoghurt polypeptide with the amino acid sequence SEQ ID NO. 2. The NLF102 protein with satisfactory endotoxin content obtained by the above experiment is used in the subsequent experiment.

Example 2 binding of receptor to New land green polypeptide

MCF-7 cells were collected, counted, centrifuged and resuspended in DMEM (10% serum, 9. Mu.g/ml insulin) at a cell density of 5X 10 ⁴ /ml. A96-well plate was plated, 100. Mu.l of suspension was added to each well, and the mixture was allowed to stand at 37℃overnight. The following day, the cells were washed three times with PBS and the whole was incubated with serum-free DMEM for 24 hours.

With coating buffer (50 mM Na ₂ CO ₃ -NaHCO ₃ pH 9.6) was diluted to 6. Mu.g/ml of ErbB2 antibody H4 (ErbB 2 monoclonal antibody, zeta-stratospheric) and added to a 96-well plate at 50. Mu.l per well. The antibodies were allowed to bind to the plate overnight at 4 ℃.

The DMEM medium was aspirated from the MCF-7 cells, and NRG and NLF102 were serially diluted with DMEM and added to wells at 100. Mu.l per well. Here, NRG is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 1, NLF102 is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 2. The blank was only DMEM. Incubation was carried out at 37℃for 20 min, washed once with PBS buffer, and 100. Mu.l/Kong Liejie buffer (50mM Hepes,pH8.0, 150mM NaCl,2mM sodium orthovanadate, 0.01% mercuric powder, 1% Triton X-100 and 1 tablet protease inhibitor mixed partial/25 ml) was added, lysed at 4℃for 30 min, after which the cells were detached from the plate by shaking the plate gently, and centrifuged at 15,000rpm for 15 min. Here, NRG is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 1, NLF102 is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 2.

Antibody coated plates were rinsed 5 times with wash (10mM PBS,pH7.4,0.05%Tween 20), 200 μl of 5% skim milk wash was added to each well, incubated for 2 hours at 37℃and washed 3 more times with wash.

Adding 90 μl of the lysed cell fluid into corresponding coating plate, incubating at 37deg.C for 1 hr, washing with washing solution for 5 times, and adding 100 μl of horseradish hydrogen peroxide with proper concentrationEnzyme (HRP) biphospate tyrosine antibody (san krus biotechnology), incubated for 1 hour at 37 ℃. Washing was performed 5 times, and fresh prepared HRP substrate solution (50 mM citric acid, 100mM Na was added ₂ HPO ₄ pH5.0,0.2mg/ml Tetramethylbenzidine (TMB), 0.003% H ₂ O ₂ ) Incubate for 10 min at 37 ℃. Finally 50 μl of 2M H is added to each well ₂ SO ₄ The HRP activity was destroyed to terminate the reaction. The OD value of 450nm per well was measured on a microplate reader (BIORAD Model 550), and the EC50 was the concentration of the New land Grifolin polypeptide reaching half the maximum absorbance. The lower the EC50 value, the higher the affinity of the receptor for the new york polypeptide.

EC50 values of NRG, NLF102 are shown in table 1, with the EC50 value of NLF102 being lower than that of NRG.

Table 1 EC50 values of NRG, NRG102

Sample of	EC50(nM)
		NRG	1.69
NLF102	1.40

Example 3 analysis of New Zealand long fragment NLF102 on primary cardiomyocyte therapy

1 isolation of primary cardiomyocytes from neonatal rat

After successful isolation, the cardiomyocytes were cultured, and after the waiting cell state was stretched and stabilized, the cardiomyocytes were seen to spread and spontaneously contract rhythmically.

2 starvation of cells and administration

The DMEM complete medium was discarded, washed twice with PBS, and the control, NRG and NLF102 groups were supplemented with fresh DMEM medium without FBS, whereas the Sham group was supplemented with DMEM complete medium (10% FBS), and each was treated for 24 hours. After that, the DMEM medium was discarded, and DMEM complete medium was added. Wherein, 10nM NRG and NLF102 were added to NRG and NLF102 groups, respectively, and equal volumes of PBS was added to Control and Sham groups, and the treatment was performed for 24h. Here, NRG is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 1, NLF102 is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 2.

3 staining of sarcomere

The complete medium was discarded and the cell slide or cell culture well was washed with PBS. The climbing sheet cells face upwards, put into a clean cell culture plate and fixed by methanol. Rinsing the fixed climbing slices or cell holes on a PBST shaker. Blocking solution was blocked at room temperature, 1% ngs was added to the slide or cell well to dilute Alpha Actinin primary antibody (ab 9465, abcam), and incubated at room temperature for 2h or overnight at 4 ℃. PBST room temperature rinse. Anti-murine secondary Alexa Flour 555 (ab 4409, abcam) was diluted with 1% NGS and incubated at room temperature in the dark. PBST room temperature rinse. DAPI stains the nucleus. A drop of anti-quenching sealing tablet is added on a clean glass slide, and the glass slide can be stored for a long time at 4 ℃ after sealing. Cells were covered with an appropriate amount of PBS and photographed under a fluorescence microscope.

4 statistical analysis

The distribution of muscle segments in cells was observed and divided into three classes according to the area in the occupied cardiomyocytes: 2/3, preferably (Well); 1/3-2/3, medium (modelate); <1/3, is the difference (Poor)

All experimental data are expressed as ± SD, single factor analysis of variance using GraphPad Prism 6 software, p <0.05 indicates significant differences compared to Control group, and p <0.01 indicates very significant differences compared to Control group.

5 analysis of experimental results

Statistics of the percentage of the area level of the muscle segments of each group to the whole cells are shown in Table 2:

table 2: different grades of muscle small area grade account for the percentage of whole cells

By analyzing the proportion of the muscle segments to the total cell area, we can understand the primary cardiomyocyte status. The addition of NRG or NLF102 with fewer sarcomere than Sham group indicates that NRG or NLF102 has the function of promoting sarcomere reconstruction. In addition, the NLF102 has a more remarkable effect than NRG.

EXAMPLE 4 Elisa assay for intravenous NRG, NLF102 half-life in rats

1 laboratory animal

Strain and source: wistar rats supplied by Shanghai Sipuler-BiKai laboratory animal Co., ltd

Sex, body weight: male, 210-250 g

2 experimental procedure

2.1 sample preparation

2.1.1 preparation of sample to be tested

30ug/kg of recombinant human New Yougelin (NRG) or New Yougelin long fragment NLF102 was tail-injected into rats and orbital blood collection was performed at different time points after administration, respectively. Standing at room temperature for 2h, and centrifuging to separate supernatant after blood is coagulated. Rat serum containing newland green or newland green long fragment NLF102 was diluted 1:1 with dilution buffer for use. Here, NRG is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 1, NLF102 is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 2.

2.1.2 preparation of standard: the standard samples of the N.sub.Heterol and N.sub.Heterol long fragment NLF102 were prepared with rat serum at concentrations ranging from 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml and 0ng/ml, followed by dilution with dilution buffer at a 1:1 ratio.

2.2 experimental procedure

2.2.1 plate coating and blocking: the NRG1-EGF domain antibody was diluted with coating buffer and 50uL of 4 degree coating was added to each reaction well in a 96-well plate overnight. The next day the coating buffer was discarded, the plates were washed, blocking buffer was added and blocked at room temperature.

2.2.2 loading: washing the flat plate, drying the flat plate on absorbent paper, adding 50uL of corresponding standard sample or sample to be detected into each reaction hole, and incubating for 2 hours at room temperature.

2.2.3 addition of detection antibody: plates were washed and incubated at room temperature with biotin-labeled Chicken IgY anti-New land Greenlin antibodies.

2.2.4 addition of enzyme-labeled antibodies: the plates were washed and incubated with strepavidin-HRP antibody at room temperature.

2.2.5 color development: the plate was washed and a temporarily prepared TMB substrate solution was added to each reaction well, and after 20 minutes of reaction at 37℃in the absence of light, 50uL of 2M sulfuric acid was added to terminate the reaction.

2.2.6 detection: absorbance at 450nM was measured for each reaction well and the amount of newland and protein in the samples was calculated from the standard curve.

2.2.7 data were analyzed using GraphPad Prism 5.0 software.

3 experimental results and discussion

We injected 30ug/kg of recombinant human new york-line (NRG) and new york-line long fragment NLF102, respectively, through rat tail vein, and after orbital blood collection and serum separation, respectively, were performed after dosing, and then the new york-line or NLF102 protein content in serum at different time points was detected, and the results are shown in table 3, fig. 1:

table 3: half-life results statistics

EXAMPLE 5 Long-term intravenous sustained drug efficacy experiment of recombinant New Zealand Lin Duotai against rat heart failure

1. Purpose of experiment

On a rat model with heart failure caused by left coronary ligation, recombinant human new york green (NRG) and recombinant neuregulin NLF102 are continuously and intravenously instilled by an insulin injection pump with Medun force, and the therapeutic effects of the NRG and NLF102 on the rat heart failure model are compared. Here, NRG is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 1, NLF102 is a recombinant N.sub.He-He polypeptide having the amino acid sequence of SEQ ID NO. 2.

2. Experimental materials

2.1 laboratory animals

2.1.1 lines, sources: wistar rats supplied by Shanghai Sipuler-BiKai laboratory animal Co., ltd

2.1.2 sex, body weight: male, 210-250 g

2.2 reagent medicine

2.2.1 excipients: developed by Shanghai Zesheng technology development Co., ltd., dosage form: freeze-dried powder, specification: 2 mgAlb/bottle

2.2.2NLF102 solution: developed by Shanghai Zesheng technology development Co., ltd., dosage form: solution, specification: 0.485mg/ml

2.2.3 recombinant human New land green (NRG) finished product: developed by Shanghai Zesheng technology development Co., ltd., dosage form: freeze-dried powder, specification: 250 mug/count

2.2.4 isoflurane: rui Ward Life technologies Co., ltd., specification: 100 ml/bottle

3. Experimental apparatus and equipment

3.1 anesthesia machine (isoflurane evaporator): MSS INTERNATIONAL LTD

3.2 cardiac ultrasound detector: vivid E95, probe model: 12S-D

3.5 insulin pump: meidun force, model: MMT-712EWS, MMT-722NAS/L

4. Experimental method

4.1 establishment of rat coronary artery ligation-induced heart failure model

Rats were anesthetized with 4% strength isoflurane by a gas anesthesia machine, fixed supine after anesthesia, and sterilized with 75% alcohol after chest dehairing. After the left front skin of the chest is cut, the chest muscle is blunt separated, the 4 th rib and the 5 th rib are exposed, the intercostal muscles of the 4 th rib and the 5 th rib are blunt separated by using a hemostatic forceps, and the two hands are matched for extrusion, so that the heart is extruded from the chest, the heart is fully exposed, the pulmonary inflation and heartbeat condition are observed, the left auricle and the pulmonary artery cone are fully exposed, and the left anterior descending branch of the coronary artery is ligated by using a 6-0 surgical suture between the left auricle and the pulmonary artery cone. Then the chest is pressed hard to exhaust, then the chest muscle and skin are sutured, the operation is carried out in a cage for feeding, and the condition of the rat is closely observed, if the occurrence of acute arrhythmia is found, the heart massage is carried out urgently for 3-5 minutes.

4.2 Experimental grouping and administration

4.2.1 Experimental grouping cases

Heart function of rats is detected by using a B ultrasonic machine Vivid E95 in 2, 3 and 4 weeks after operation, and rats with EF values in the range of 28.6-42.4% are selected to enter the next experiment after the 4 th week heart ultrasonic detection. The rats were randomly divided into 3 groups, an excipient group and NRG 6. Mu.g/kg group, NLF 102.6. Mu.g/kg group, EF values of the excipient group and NRG group were 37.3%, EF values of the NLF102 group were 37.4%, 12 animals each group were continuously administered by insulin pump for 8 hours on a daily basis for 10 days, NRG dose was 0.75. Mu.g/kg/h, dose volume was 5ml/kg, dose concentration was 1.2. Mu.g/ml, dose NLF102 dose was 1.2. Mu.g/kg/h, dose volume was 5ml/kg, dose concentration was 1.92. Mu.g/ml, and cardiac superdetection was performed again on day 1 after the end of dose. The false operation group only wires and does not ligate the coronary artery, and 13 false operation groups do not do administration treatment.

4.2.2 dispensing methods

1) Excipient: adding 1ml of physiological saline into each bottle to prepare mother liquor, adding 49.76ml of physiological saline into 0.24ml of mother liquor to dilute into 9.6 mug/ml of Alb solution

2) NRG:250 μg NRG/bottle, 1ml physiological saline is added into each bottle to prepare mother liquor, 0.24ml mother liquor is added into 49.76ml physiological saline to dilute into 1.2 μg/ml NRG solution

3) NLF102:0.485mg/ml, 0.20ml of solution is taken and added with 49.80ml of physiological saline to dilute into 1.92 mug/ml of NLF102 solution

4.3 Observation index

4.3.1 cardiac function detection

After the rats were anesthetized with 4% isoflurane by a gas anesthesia machine, the left lateral recumbent position was fixed on the surgical plate. The rat head was fixed in the respiratory mask of the gas anesthesia machine, and isoflurane concentration of 2% was maintained. Chest dehairing, sterilizing with 75% alcohol, smearing couplant, and detecting left ventricular echo with rat cardiac ultrasonic probeA signal. Selecting a B-mode, placing a super-cardiac probe beside the sternum, directing a probe mark at 2-3 o' clock, cutting the heart vertically by sound beams and the long axis direction of the heart, adjusting the probe to the level of two papillary muscles, obtaining a transverse short axis section of the papillary muscles of the left ventricle, and collecting and storing a section of dynamic image of the transverse plane of the papillary muscles of the left ventricle. Selecting an M-mode, keeping the probe on the short axis surface of the left ventricle of the papillary muscle, adjusting an M-shaped sampling line to pass through the weakest point of the pulsation of the anterior wall, adjusting the focal length, collecting a section of M-shaped curve (the left chamber cavity and the anterior and posterior walls of the left chamber should be clearly displayed), measuring the inner diameter (D) of the left ventricle at the end diastole and the end systole, and adopting a Teichholtz formula V=7/(2.4+D) for the measurement of the inner diameter (D) ³ Left ventricular end diastole and end systole volumes EDV, ESV were calculated and the outgoing blood fraction (Ejection Fraction, EF) values were calculated, ef= (EDV-ESV)/EDV 100%.

4.4 data processing

All experimental data are expressed as x±sd, single factor analysis of variance was performed using GraphPad Prism 6 software, P <0.05 indicated significant differences between groups, and P <0.01 indicated very significant differences between groups.

5. Experimental results

5.1 cardiac superconductivity results

The LVEDd and LVEDs of the model control group before grouping and administration are respectively 0.956+/-0.076 cm and 0.806 +/-0.065 cm, and 0.991+/-0.075 cm and 0.851+/-0.068 cm after administration; the pre-administration LVEDd and LVEDs of NRG administration group are respectively 0.952+ -0.086 cm, 0.802+ -0.076 cm, and 0.969+ -0.075 cm and 0.810+ -0.078 cm after administration; the LVEDd and LVEDs before and after the NLF102 administration group are respectively 0.930+/-0.059 cm, 0.783+/-0.054 cm, and 0.936+/-0.060 cm and 0.781+/-0.075 cm after the administration; the EF value of the model control group before the group administration is 37.3+/-3.2%, and the EF value after the group administration is 33.9+/-4.9%; the EF value before administration of the NRG administration group is 37.3+/-3.8 percent, and the EF value after administration is 38.9+/-4.8 percent; the EF values for NLF102 dosing groups were 37.4±2.9% before dosing and 39.2±6.6% after dosing. There was a significant increase in EF values in both NRG and 1NLF102 treated heart failure rats following dosing, and there was a significant difference (p < 0.05) between both groups compared to the model control group. The results are shown in Table 4 and FIG. 2.

6. Conclusion and discussion

The NRG freeze-dried powder and the NLF102 solution have good therapeutic effects on heart failure rats, and the therapeutic effect of NLF102 is better than NRG.

Table 4 NRG, NLF-102 pump tail vein long time administration for treating heart failure of rats efficacy experiment cardiac super results (x+ -SD)

^** : post-treatment groups were p compared to vehicle group<0.01； ^* : post-treatment groups were p compared to vehicle group<0.05

The scope of the present invention is not limited to the description of the embodiments. It will be apparent to those skilled in the art that many modifications and variations can be made to the present invention without departing from the spirit or scope thereof. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Sequence listing

<110> Shanghai SpA's GmbH for Zeta science and technology development

<120> neuregulin polypeptide fragments and uses thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 61

<212> PRT

<213> Homo sapiens

<400> 1

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln

50 55 60

<210> 2

<211> 102

<212> PRT

<213> Homo sapiens

<400> 2

Val Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu Gly

1 5 10 15

Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu

20 25 30

Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser

35 40 45

His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly

50 55 60

Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu

65 70 75 80

Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val

85 90 95

Met Ala Ser Phe Tyr Lys

100

Claims (3)

1. A new york green polypeptide is shown in SEQ ID NO. 2.

2. Use of a newland polypeptide for the preparation of a medicament for preventing, treating or delaying heart failure in a mammal; wherein the Neulogreen polypeptide is the amino acid sequence of SEQ ID NO. 2.

3. A pharmaceutical formulation comprising a newbornalin polypeptide, which has the amino acid sequence shown in SEQ ID No. 2, and a pharmaceutically acceptable carrier, excipient or diluent.