CN112585159A - Neuregulin polypeptide fragments and uses thereof - Google Patents

Abstract

The present invention provides specific neuregulin polypeptides that can be used in methods and compositions for preventing, treating, or delaying various diseases or disorders. The invention relates to application of neuregulin polypeptide in preparing a medicament for preventing, treating or delaying heart failure of mammals, and a using method of the medicament for preventing, treating or delaying heart failure of the mammals. In particular, the present invention provides a method for the prevention, treatment or delay of heart failure in a mammal by administering a medicament comprising a neuregulin polypeptide fragment in a specific individual suffering from or at risk of heart failure.

Description

Neuregulin polypeptide fragments and uses thereof Technical Field

The invention relates to application of neuregulin polypeptide in preparing a medicament for preventing, treating or delaying heart failure of mammals, and a using method of the medicament for preventing, treating or delaying heart failure of the mammals. In particular, the present invention provides a method for the prevention, treatment or delay of heart failure in a mammal by administering a medicament comprising a neuregulin polypeptide fragment in a specific individual suffering from or at risk of heart failure.

Background

Neuregulin (neuregulin, NRG; heregulin, HRG), also known as Glial Growth Factor (GGF), Neu Differentiation Factor (NDF), are glycoproteins with a molecular weight of about 44KD, which transmit signals between cells and are ligands 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 also evidence that NRG1 signaling plays a role in the development and function of several other organ systems and in the pathogenesis of human diseases including schizophrenia and breast cancer. There are many isomers of NRG 1. Studies on genetically mutated mice (knockout mice) have shown that the isoforms differ in the N-terminal region or Epidermal Growth Factor (EGF) -like region, and also differ in their function in vivo. The present invention is based on neuregulin 1 beta (NRG1 beta).

Neuregulin 1 β is a transmembrane protein (Holmes et al, Science 256,1205-1210, 1992). The membrane-external portion is N-terminal, and includes immunoglobulin-like region (Ig-like domain) and EGF-like region (EGF-like domain), and the membrane-internal portion is C-terminal. Under the action of metalloprotease in extracellular matrix, the extracellular part of neuregulin can be cut off by enzyme and is in free state, thus facilitating the binding with ErbB receptor on the surface of surrounding cells and activating corresponding cell signaling.

The ErbB receptor family is also classified into four classes, ErbB1, ErbB2, ErbB3 and ErbB4, which are transmembrane proteins with molecular weights around 180-185 KD. Except ErbB2, they all contain a ligand binding region at the membrane N-terminus; except for ErbB3, they all contain protein tyrosine kinase activity at the C-terminus of the membrane. Wherein ErbB1 is the receptor of epidermal growth factor, and ErbB3 and ErbB4 are the receptors of neuregulin. Among 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-.

When neuregulin binds to the extracellular portion of ErbB3 or ErbB4, it causes ErbB3, ErbB4 to form heterodimers with other ErbB receptors (often including ErbB2), or ErbB4 itself to form homodimers, which then leads to phosphorylation of the intracellular portion of the receptor (Yarden et al, Nat Rev Mol Cell Biol,2: 127-. The phosphorylated intramembrane fraction can further bind to various signaling proteins within the cell, thereby activating downstream ERK or AKT signaling pathways, resulting in a series of cellular responses: including stimulating or inhibiting cell proliferation, apoptosis, cell migration, cell differentiation, or cell adhesion.

Neuregulins are particularly important for cardiac development (WO0037095, CN1276381, WO03099300, WO9426298, US6444642, WO9918976, WO0064400, Zhao et al, j.biol.chem.273,10261-10269,1998). In early embryonic development, neuregulin expression is primarily localized to the endocardium and subsequently released to peripheral cardiomyocytes via the paracrine pathway and binds to the extracellular portion of the protein tyrosine kinase receptor ErbB4 on the cell membrane, which in turn forms heterodimers with ErbB2 in ErbB 4. Formation and activation of the ErbB4/ErbB2 complex is essential for early spongiform trabeculoplasty. Deletion of any of the three protein genes neuregulin, ErbB4 and ErbB2 resulted in embryos lacking trabeculae and dying of the uterus early in development. WO0037095 shows that a certain concentration of neuregulin can continuously activate an ERK signal pathway, promote growth and differentiation of cardiomyocytes, guide reconstruction of sarcomere and cytoskeleton at adhesion of the cardiomyocytes and cells, improve the structure of the cardiomyocytes, and enhance contraction of the cardiomyocytes. WO0037095 and WO003099300 also indicate that neuregulin can be used for the detection, diagnosis and treatment of various cardiovascular diseases.

Some prior art documents relating to the present invention are listed below: WO 0037095; 2. new application of growth factor neuregulin and analogues thereof: CN 1276381; neuredulin based methods and compositions for manipulating cardiac diseases WO 03099300; zhao YY, Sawyer DR, Balia RR, Opel DJ, Han X, Marchionni MA and Kelly RA.Neureegulin proton Survival and Growth of Cardiac Myocytes.J.biol.chem.273,10261-10269 (1998); methods for manipulating muscle diseases and disorders WO 9426298; methods of creating mythogenic format or subvalval or muscle cell mitogens, differentiation or subvalucing a neurogenin, US6444642.7.therapeutic Methods comprising use of a neurogenin, WO 9918976; methods for manipulating a consistent diagnostic heart failure, WO 0064400; 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, specific activator p185erbb2.science 256,1205-1210 (1992); metals DL, Neuregis, functions, Experimental Cell Research,284,14-30(2003), 11, Yarden Y, Sliwkowski X, Untingling the ErbB signaling network Nature Reviews, Molecular Cell Biology, 2127-.

Heart Failure (HF) is a syndrome of cardiac insufficiency caused by various heart diseases, including Systolic Heart Failure (SHF) and Diastolic Heart Failure (DHF). In 2008, the guidelines for diagnosis and treatment of acute/chronic Heart Failure issued by the European Heart Association (ESC) defined the latter as Heart Failure with preserved ejection fraction (HF-PEF). Systolic heart failure refers to the condition that the heart muscle contraction force is reduced, so that the heart blood output cannot meet the metabolic needs of the organism, the blood flow of organs and tissues is insufficient, and the pulmonary circulation and/or the systemic circulation congestion appear at the same time. Heart failure with preserved ejection fraction (HF-PEF) is often referred to as diastolic heart failure, which is the heart failure due to impaired left ventricular diastolic active relaxation capacity and decreased myocardial compliance, with increased stiffness due to hypertrophy of the myocardial cells with interstitial fibrosis, resulting in impaired left ventricular filling during diastole, decreased stroke volume, and increased left ventricular end-diastolic pressure. Epidemiological data from the american heart and lung institute in 2006 show that heart failure or diastolic heart failure with preserved ejection fraction accounts for over 50% of the total heart failure population. Heart failure with preserved ejection fraction can occur alone or in combination with systolic dysfunction. Heart failure with preserved ejection fraction is often seen in elderly women with hypertension, diabetes, 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 hypertrophic stimulus is mitochondrial synthesis and myofibrillar expansion (thickening of the wall of the chamber) accompanied by a proportional increase in single cell size, but no (or very little) increase in cell number.

When the ventricles are stressed, the initial response is an increase in the length of the muscle segment. This is followed by an increase in overall muscle mass. When the load is too severe, the myocardial contractility will weaken. In the mildest state, this attenuation is manifested as a decrease in the rate of contraction of the unloaded myocardium or a decrease in the rate of force development upon isometric contraction. As myocardial contractility further diminishes, a greater decrease in the rate of shortening of unloaded myocardium occurs, accompanied by a decrease in isometric muscle strength development and contraction length. At this point, circulatory compensation may still be provided by the heart enlargement and the increase in myocardial mass, which tends to maintain ventricular wall stress at normal levels. As contractility continues to decline, overt congestive heart failure occurs, manifested as a decline in cardiac output or work, and/or an increase in ventricular end-diastolic volume and diastolic pressure.

The transition from hypertrophy to heart failure is characterized by changes in several cell tissues. For example, normal hypertrophied cells are of a larger size, with enhanced and ordered contractile units and stronger cell-cell adhesion. Conversely, pathologically hypertrophic cells, which are also large in size and aggregated with proteins, exhibit disordering contractile proteins (disorganization of the sarcomere) and poor cell-cell adhesion (disorganization of the myofibers). Thus, in pathological hypertrophy, an increase in cell size and aggregation of contractile proteins are associated with the disorganized assembly of muscle sarcomere structures and the loss of robust cell-cell interactions.

Currently, over 2600 million people worldwide suffer from heart failure, and over 55 million new patients are present in the united states alone each year, which is one of the important and serious public health problems. Current drugs for the treatment of 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 percentage decrease in mortality is statistically different, the actual decrease in mortality with ACE inhibitors averages only 3% to 4%, and there are several potential side effects.

ACE inhibitors have also been used in combination with other drugs, such as digitalis, to increase the force of cardiac contraction; and/or some diuretic that helps to reduce the workload of the heart by causing the kidneys to expel more sodium and water from the blood. However, at least one study demonstrated that there was no difference in survival between patients with II-III heart failure when using digitalis compared to placebo. In addition, diuretics improve some symptoms of heart failure, but are not suitable for use as a monotherapy.

Other options for preventing or treating heart failure have corresponding limitations. For example, heart transplantation is significantly 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 pacemakers, is also invasive and expensive. Therefore, new therapeutic measures are needed due to the deficiencies of current therapeutic measures.

One promising new therapeutic approach involves the administration of neuregulin (hereinafter "neuregulin") to patients with or at risk of heart failure. Previous studies have shown that neuregulin-1 beta (NRG-1 beta) binds directly to ErbB3 and ErbB4 with high affinity. The orphan receptor ErbB2 is capable of forming heterodimers with ErbB3 or ErbB4 and has higher affinity than ErbB3 or ErbB4 homodimers. Results of neurodevelopment studies suggest that the formation of the sympathetic nervous system requires the entire NRG-1 β, ErbB2, and ErbB3 signaling systems. Targeted disruption of NRG-1 β, or ErbB2 or ErbB4 results in embryonic lethality due to defects in cardiac development. Recent studies have also highlighted that NRG-1 β, ErbB2 and ErbB4 have important roles in cardiovascular development and maintenance of normal adult cardiac function. Research has shown that NRG-1 β enhances the organization of the sarcomere of adult cardiomyocytes. Short term administration of a recombinant NRG-1 β EGF-like domain significantly improved or prevented deterioration of myocardial function in three different animal models of heart failure. More importantly, NRG-1 β significantly prolongs the survival of heart failure animals. These effects make NRG-1 β promising as a broad-spectrum therapeutic agent or lead compound for the treatment of heart failure caused by a variety of common diseases. However, different neuregulin 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 in order to find a more effective neuregulin 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 application of neuregulin polypeptide in preparing a medicament for preventing, treating or delaying heart failure of mammals, and a using method of the medicament for preventing, treating or delaying heart failure of the mammals. In particular, the present invention provides a method for the prevention, treatment or delay of heart failure in a mammal by administering a medicament comprising a neuregulin polypeptide fragment in a specific 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 of the invention, neuregulin polypeptide fragments are provided. In certain embodiments, the neuregulin polypeptide comprises an EGF domain of a neuregulin. In certain embodiments, the neuregulin polypeptide comprises the EGF domain of a β 2 isoform of human neuregulin. In certain embodiments, the neuregulin polypeptide comprises the amino acid sequence of SEQ ID NO. 1. In certain embodiments, the neuregulin polypeptide comprises the amino acid sequence of SEQ ID NO. 2.

Certain neuregulin polypeptides contain the following amino acid sequence: 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 neuregulin polypeptides contain the following amino acid sequence: 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).

The neuregulin polypeptide can be prepared according to any of the relevant techniques well known in the art. Typical techniques for preparing neuregulin polypeptides are provided herein. In certain embodiments, the neuregulin polypeptide can be recombinant. In certain embodiments, the neuregulin polypeptide is synthetic, for example, by liquid phase or solid phase peptide synthesis.

In another aspect, the invention provides neuregulin polypeptide-related nucleic acids, vectors, and host cells. The nucleic acid or the complement thereof encodes neuregulin polypeptide or a fragment thereof. The nucleic acid may be double-stranded or single-stranded DNA or RNA, and can be inserted into a suitable vector for propagation and expression of neuregulin polypeptide. The modified vector is transferred into a suitable host cell, such as a host cell capable of expressing the recombinant neuregulin polypeptide.

In another aspect of the invention, neuregulin polypeptides are provided for therapeutic and non-therapeutic use. In particular to the application of neuregulin polypeptide in preventing, treating or delaying various heart diseases and disorders. Accordingly, the present invention provides pharmaceutical formulations comprising neuregulin polypeptides and related methods of treatment.

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

In another aspect of the invention, methods of inducing phosphorylation of an ErbB receptor in a cell are provided. In certain embodiments, the method comprises contacting the cell with neuregulin polypeptide.

Another aspect of the invention provides for inducing and maintaining activation of the AKT signaling pathway in cardiac cells. In certain embodiments, the method comprises contacting a cardiac cell with neuregulin polypeptide.

Another aspect of the invention provides for inducing and maintaining activation of the ERK signaling pathway in cardiac cells. In certain embodiments, the method comprises contacting a cardiac cell with neuregulin polypeptide.

Detailed Description

A. Explaining the 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 herein by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

As used herein, the terms "a" or "an" mean "at least one" or "one or more than one," unless expressly specified otherwise.

As used herein, "EGF-like domain" or "EGF-like domain" refers to a polypeptide fragment encoded by the neuregulin 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-; chang et al, Nature, 387: 509-; 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 the ErbB2/ErbB4 or ErbB2/ErbB3 heterodimer. In certain embodiments, the EGF-like domain comprises the receptor binding domain amino acids of NRG-1. In certain embodiments, the 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 the receptor binding domain amino acids of NRG-2. In certain embodiments, the EGF-like domain comprises the receptor binding domain amino acids of NRG-3. In certain embodiments, the EGF-like domain comprises the receptor binding domain amino acids of NRG-4.

As used herein, an "effective amount" of an active ingredient to treat a particular disease is an amount sufficient to ameliorate, or in some way reduce, the symptoms associated with the disease. This dose may cure the disease, but is typically used to ameliorate the symptoms of the disease.

As used herein, an "active ingredient" is any substance 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 relief of symptoms by administration of a particular active agent, which relief can be attributed to or associated with administration of the agent.

As used herein, "treating" or "treatment" refers to any means by which the symptoms of a disorder, condition or disease may be ameliorated or otherwise favorably directed. The effect may be prophylactic, such as completely or partially preventing a disease or a symptom thereof, or therapeutic, such as a partial or complete cure for a disease and/or adverse effects caused by a disease. Treatment also includes any pharmaceutical use of the compositions described herein.

As used herein, "vector (or plasmid)" refers to a discrete component 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 the expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA component, such as a plasmid, phage, recombinant virus, or other vector, which when introduced into an appropriate host cell results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those which replicate in eukaryotic and/or prokaryotic cells, as well as those which remain episomal or those which integrate into the genome of the host cell.

As used herein, "cardiomyocyte differentiation" refers to a state characterized by a 10% or greater reduction in DNA synthesis, greater than 10% inhibition of DNA synthesis stimulated by other factors, ordered myo-sarcomere binding and cell-cell adhesion, sustained activation of MAP kinase, and p21^Cip1Enhanced expression of (a). For further discussion see WO00/37095, the contents of which are incorporated herein by reference in their entirety.

As used herein, "ejection fraction" or "EF" refers to the proportion of blood pumped from a filled left ventricle with a heartbeat. Can be defined by the following equation: (left ventricular end-diastolic volume-left ventricular end-systolic volume)/left ventricular end-diastolic volume.

As used herein, "fractional contraction" or "FS" refers to the ratio of the change in diameter of the left ventricle in the systolic state to the diastolic state. Can be defined by the following equation: (left ventricular end-diastolic inner diameter-left ventricular end-systolic inner diameter)/left ventricular end-diastolic inner diameter.

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

As used herein, "myocardial infarction" refers to the massive and persistent ischemic plaque necrosis of portions of the myocardium resulting from blockage of coronary arteries or interruption of blood flow.

As used herein, "ordered, enhanced arrangement of sarcomere or sarcomere structure" refers to a state characterized by an ordered arrangement of contractile proteins as demonstrated by immunofluorescence staining for alpha-actinin in cardiomyocytes. The alignment of alpha-actinin in cells can be identified by microscopy and its associated photographic equipment. As used herein, "disorder or irregularity of the sarcomere or sarcomere structure" is the opposite of "ordered, enhanced arrangement of sarcomere or sarcomere structures".

As used herein, "ordered or enhanced arrangement of cytoskeletal structures" refers to a state characterized by ordered arrangement of actin fibers as revealed by phalloidin (phaseolin) staining in cardiomyocytes. The alignment of actin fibers in cells can be identified by a microscope and its associated camera, as exemplified in the present picture. As used herein, "disorder or irregularity of the cytoskeletal structure" refers to the opposite of "ordered, or enhanced, arrangement of the cytoskeletal structure".

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

As used herein, "sustained activation of MAP kinase" means that the phosphorylation state of P42/44, a MAP kinase, in cells is maintained for at least 21 hours. Further discussion is provided in WO00/37095, the contents of which are 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 with one or more retinoic acid compounds. Although in some areas a synergistic effect means a greater than additive effect (e.g. 1+1 ═ 3), in the medical area a additive (1+1 ═ 2) or less than additive (1+1 ═ 1.6) effect may also be synergistic. For example, if one of the two drugs is administered alone to inhibit the development of ventricular myocyte hypertrophy by 50%, it cannot be expected that the combination of the two drugs will 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 and, when used in combination, slow the progression of ventricular myocyte hypertrophy by less than 50%. Thus, if the combination of two drugs slows the progression of ventricular myocyte hypertrophy by more than 50% without increasing unacceptable side effects, a synergistic effect is obtained.

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

There are two methods used to detect ventricular myocyte hypertrophy, in vitro and in vivo. 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 degree of hypertrophy is measured on an artificial 7-0 scale, with 7 points representing fully hypertrophic cells and 3 points representing unstimulated cells. The status of 3 points and 7 points can be seen in FIGS. 2A and B of Simpson et al (1982) Circulation Res.51:787-801, respectively.

As used herein, "inhibition of cardiac hypertrophy" means a decrease in one of the parameters indicative of hypertrophy relative to hypertrophic conditions, or preventing an increase in one of the parameters indicative of hypertrophy relative to normal conditions. For example, inhibition of ventricular myocyte hypertrophy can be shown by measuring the decrease in cell size relative to hypertrophic conditions. Inhibition of ventricular myocyte hypertrophy means a 10% or greater reduction in cell size relative to the size observed under hypertrophic conditions.

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

Hypertrophy may result from any factor responsive to retinoic acid including congenital viral, congenital, cardiotrophic, sarcotrophic factors, or as a result of ischemia or ischemic injury such as myocardial infarction. Typically, treatment is performed to prevent or slow the progression of hypertrophy, particularly after cardiac injury, such as after ischemia. 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, to determine the unit of activity of an active agent, 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. The determination of the activity units is important for the quality control of genetically engineered products and clinically used drugs, so that different drugs and/or different batches of products can be quantified with the same standard.

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

B. Neuregulin

The invention provides neuregulin polypeptide fragments. In certain embodiments, the neuregulin polypeptide comprises an EGF domain of a neuregulin. In certain embodiments, the neuregulin polypeptide comprises the EGF domain of a β 2 isoform of human neuregulin. In certain embodiments, the neuregulin polypeptide comprises the amino acid sequence of SEQ ID NO. 1. In certain embodiments, the neuregulin polypeptide comprises the amino acid sequence of SEQ ID NO. 2.

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

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

Preparation of neuregulin polypeptide

The neuregulin polypeptide can be prepared according to any obvious relevant art. Exemplary techniques for preparing neuregulin polypeptides are described, 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 neuregulin polypeptide of the invention can be prepared according to any of the techniques known in the art. In certain embodiments, the neuregulin polypeptide is synthesized, e.g., by liquid phase 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 preferred embodiments, the neuregulin polypeptide is obtained from natural sources, synthetically, or commercially. In certain embodiments. In certain embodiments, the neuregulin polypeptide can be obtained by recombinant expression of the protein followed by purification.

Neuregulin polypeptide can 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. One skilled in the art is familiar with the purification of neuregulin polypeptides.

Use of neuregulin polypeptides

Neuregulin polypeptides may be administered at the discretion of those 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 hereby incorporated by reference in their entirety.

Neuregulin polypeptides are useful for treating a range of diseases and disorders. Typical diseases and disorders include heart diseases such as heart failure, viral myocarditis, dilated (congestive) cardiomyopathy (DCM), cardiotoxicity or myocardial infarction.

In certain embodiments, the present invention provides a method of treating heart failure by administering an effective amount of neuregulin polypeptide.

The neuregulin polypeptide may be administered in the form of a pharmaceutical formulation.

The mode of administration of neuregulin polypeptide will be determined by one skilled in the art and includes, but is not limited to, oral, intravenous, intragastric, rectal, intraperitoneal or intraventricular administration.

In a preferred embodiment, the composition for administration is a pharmaceutical formulation. The pharmaceutical formulation can be a composition comprising a prophylactic or therapeutic amount of one or more prophylactic or therapeutic agents (e.g., a complex comprising neuregulin polypeptide and other prophylactic or therapeutic agents), and a pharmaceutically acceptable carrier or excipient. In one embodiment and as used herein, "pharmaceutically acceptable" means that the compound is one that has been approved by the relevant national authorities or is otherwise documented as being useful in the art of pharmaceutical use in animals, particularly humans. "carrier" refers to a diluent, adjuvant (e.g., Freund's complete adjuvant and incomplete adjuvant), excipient, or other carrier that aids in the administration of the therapeutic agent. The pharmaceutical carrier can 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 most preferred carrier for intravenous injection of pharmaceutical preparations is water. In preparing injectable formulations, saline, dextrose and glycerol liquids may be employed. Examples of suitable Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, written by E.W. Martin.

Typical pharmaceutical formulations and dosage forms contain one or more excipients. Suitable excipients are well known to those skilled in the pharmaceutical arts 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, powdered 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 unitary 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 published on, for example, 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. A typical lactose-free dosage form contains the active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

Pharmaceutical formulations and dosage forms of the 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.

The pharmaceutical formulations and single dosage forms may take the form of: solutions, suspensions, emulsions, tablets, capsules, powders, sustained release forms, and the like. Oral formulations contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. The pharmaceutical agents and dosage forms contain a prophylactic or therapeutic amount of a purified prophylactically or therapeutically effective agent which is admixed with an amount of a carrier to be shaped for better administration to a patient. The dosage form should be adapted to the mode of administration. In a preferred 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, and most preferably the subject is a human.

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

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

Depending on the application, the formulation, shape and type of formulation of neuregulin polypeptide will vary. For example, a dosage form for acute treatment of a disorder may contain more neuregulin polypeptide than a dosage form for chronic treatment of the same disease. Similarly, the dosage forms for different cancers have different therapeutic effects. Similarly, injectable dosage forms contain smaller amounts of the active ingredient than oral dosage forms that treat the same disease or disorder. It is well known to those skilled in the art that the above formulation and other specific dosage forms included in the present invention are different. See Remington pharmacy, 18 th edition, Mack Press, Iston, Pa.1990.

Administration of neuregulin polypeptides may be carried out by any route, including but not limited to, according to the judgment of those skilled in the art: oral, intravenous, intragastric, duodenal, intraperitoneal or intraventricular injection.

C. Dosage and route of administration

The amount of neuregulin 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), disorder, disease, or severity of discomfort, route of administration, and age, weight, response, and prior medical history of the patient. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

Exemplary dosages for neuregulin include administering to a subject milligrams or micrograms of neuregulin per kilogram of body weight (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). For example, the amount of active peptide administered to a patient will typically be in the range of 0.001mg/kg to 15mg/kg per kg of body weight of the patient. 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-0.050 mg/kg.

Exemplary dosages for neuregulin also include how many units (U) or unit amounts of neuregulin per kilogram body weight is administered to a subject (e.g., about 1U per kilogram body weight to about 5,000U per kilogram body weight, about 10U per kilogram body weight to about 1,000U per kilogram body weight, or about 100U per kilogram body weight to about 500U per kilogram body weight). As the dose to be administered to a patient, the unit of active peptide used per kg of body weight of the patient is typically from 10U/kg to 1,000U/kg. 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 recommended daily dosage range for neuregulin in the methods of the present invention is: about 0.001mg to 1000mg per day. In particular instances, the total amount administered per day 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-0.2 mg. When a patient is scheduled for treatment, a low dose, such as about 0.1 μ g to about 1 μ g per day, may be used initially, and if necessary increased to about 20 μ g to about 1,000 μ g per day, either in single or divided doses, depending on the patient's overall response. In some cases, it may be necessary to use dosages of the active ingredient which are outside the ranges set forth 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 therapy depending on the individual patient response. In some embodiments, neuregulin is used in an amount of about 1U/day to about 10,000U/day. In some embodiments, neuregulin is used in an amount of about 1U/day to about 5,000U/day. In some embodiments, neuregulin is used in an amount of about 10U/day to about 2,000U/day. In some embodiments, neuregulin is used in an amount of about 10U/day to about 1,000U/day. In some embodiments, neuregulin is used in an amount of about 100U/day to about 200U/day.

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

In some particular cases, neuregulin is used daily during the treatment period. In certain embodiments, the neuregulin is administered for a duration of 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days during a treatment cycle. In some particular cases, neuregulin is administered on the first day of a treatment cycle and no neuregulin is administered for the remaining day or days of the treatment cycle. In some embodiments, neuregulin is administered daily for 3, 5,7, or 10 days during a treatment cycle, with no drug being administered for the remainder of the cycle.

Drawings

FIG. 1: content of neuregulin or NLF102 protein in serum at different time points

FIG. 2: drug effect experiment heart super-junction fruit for treating rat heart failure by long-time administration of NRG, NLF-102 pump tail vein

Examples

Example 1NLF102 protein expression purification

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

The target DNA fragment was amplified by PCR using human normal liver tissue cDNA as a template with KOD high fidelity amplification enzyme (TOYOBO Co.). After the PCR product was purified by a gel kit (Tiangen), it was subjected to double digestion with restriction enzymes, and then ligated with an expression vector pET-22b (+) by T4 ligase (NEB Corp.), thereby constructing a corresponding recombinant expression plasmid. Subsequently, we transformed competent cells BL21(DE3) with the expression plasmid with correct sequencing by the CaCl2 method to construct recombinant long-fragment human neuregulin NLF102 expression engineering bacteria. And randomly selecting a series of single colonies, inoculating the single colonies into 2mL LB-Amp liquid culture medium, culturing at 37 ℃ and 250rpm overnight, inoculating the single colonies into the LB-Amp liquid culture medium, culturing at 37 ℃ and 250rpm until OD 6001.0, adding IPTG (isopropyl-beta-thiogalactoside) to a final concentration of 1mM to induce protein expression, and centrifuging after 4 hours, and collecting thalli. SDS-PAGE electrophoresis and Coomassie blue staining analysis are carried out to determine the correctness of the molecular weight of the protein. Screening to obtain engineering bacteria of recombinant long-fragment human neuregulin with correct stability and high expression, adding glycerol, mixing, subpackaging into a freezing tube, and freezing at-80 ℃ or in liquid nitrogen for a long time.

2. Expression engineering bacteria fermentation, protein expression, bacteria breaking and coarse inclusion body obtaining

A proper amount of the frozen glycerol strain is inoculated into LB culture medium containing ampicillin (the final concentration is 50ug/mL), and the strain is activated by shaking the strain in a test tube at 37 ℃ at a proper rotating speed overnight. Inoculating the activated bacterial liquid into 250mL LB culture medium containing 50ug/mL of ampicillin, shaking at 37 ℃ in a triangular shaking flask for culture, adding IPTG after the OD600 of the bacterial liquid reaches 1.0, continuing to induce at 37 ℃ and 250rpm for 4h, then 8000rpm, and centrifuging for 30min to collect the bacteria.

The thalli is prepared according to the following steps of 1: adding PBS (10 m/v), uniformly mixing by a homogenizer, and breaking the bacteria in a high-pressure bacteria breaker. Centrifuging and collecting the precipitate to obtain the crude inclusion body.

3. Washing and denaturation and renaturation of Inclusion bodies

And washing, denaturing and renaturing the crude inclusion body to obtain an inclusion body renaturation solution.

4. Protein purification

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

HPLC detection of target protein purity

6. Detection of endotoxin content

The content of endotoxin in the obtained protein was determined by referring to 1143, a general rule in four parts of pharmacopoeia of the people's republic of China, 2015 edition.

BCA assay for detecting protein sample concentration

Refer to Pierce BCA Protein Assay Kit Specification (Thermo Fisher, Cat. No. 23225). And (4) detecting the protein concentration of the protein sample obtained in the experiment.

NLF102 is herein a recombinant neuregulin polypeptide having the amino acid sequence of SEQ ID NO. 2. The NLF102 protein with the endotoxin content meeting the requirement obtained by the experiment is used for subsequent experiments.

Example 2 binding of receptor to neuregulin 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 10⁴And/ml. The plates were plated in 96-well plates and 100. mu.l of the suspension was added to each well overnight at 37 ℃. The cells were washed three times with PBS the next day, and the medium was replaced with serum-free DMEMFor 24 hours.

Using coating buffer (50mM Na)₂CO ₃-NaHCO ₃pH9.6) diluted ErbB2 antibody H4(ErbB2 monoclonal antibody, Zesheng) to 6. mu.g/ml and added to a 96-well plate at 50. mu.l per well. The antibody was allowed to bind to the plate overnight at 4 ℃.

The MCF-7 cells were aspirated and NRG, NLF102 were serially diluted with DMEM and added to the wells at 100. mu.l per well. Here, NRG is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 1 and NLF102 is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 2. The blank was DMEM only. After incubation at 37 ℃ for 20 minutes, washed once with PBS buffer, 100. mu.l/well lysis buffer (50mM Hepes, pH8.0, 150mM NaCl, 2mM sodium orthovanadate, 0.01% Thimersaxon, 1% Triton X-100 and 1 protease inhibitor cocktail/25 ml) was added, lysed at 4 ℃ for 30 minutes, and the plates were gently shaken to detach the cells from the plates and centrifuged at 15,000rpm for 15 minutes. Here, NRG is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 1 and NLF102 is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 2.

The antibody-coated plate was washed 5 times with a wash solution (10mM PBS, pH7.4, 0.05% Tween 20), 200. mu.l of a 5% skim milk wash solution was added to each well, incubated at 37 ℃ for 2 hours, and washed 3 times with the wash solution.

The lysed cell sap was added to the corresponding coated plate at 90. mu.l per well and incubated for 1 hour at 37 ℃ followed by 5 washes with wash solution, 100. mu.l of horseradish catalase (HRP) bisphosphate tyrosine antibody (St. crus Biotechnology) at the appropriate concentration was added and incubated for 1 hour at 37 ℃. The wash was washed 5 times and fresh prepared HRP substrate solution (50mM citric acid, 100mM Na) was added₂HPO ₄0.2mg/ml of Tetramethylbenzidine (TMB), 0.003% H, pH5.0₂O ₂) Co-incubation was performed at 37 ℃ for 10 minutes. Finally 50. mu.l of 2MH was added to each well₂SO ₄HRP activity was destroyed to stop the reaction. OD values were measured at 450nm per well on a microplate reader (BIORADModel550) and EC50 was the concentration of neuregulin polypeptide that reached half the maximum absorbance. The lower the EC50 value, the lower the receptorThe higher the affinity for neuregulin polypeptide.

The EC50 values of NRG and NLF102 are shown in Table 1, and the EC50 value of NLF102 is lower than the EC50 value of NRG.

TABLE 1 EC50 values for NRG, NRG102

Sample (I)	EC50(nM)
NRG	1.69
NLF102	1.40

Example 3 analysis of treatment of primary cardiomyocytes with neuregulin long fragment NLF102

1 isolation of neonatal rat Primary cardiomyocytes

After separation, the cells were cultured, and when the cells were in a stable state, the cells were seen to spread and spontaneously contract rhythmically.

2 starvation of cells and administration

DMEM complete medium was discarded, PBS was washed twice, and the control group, the NRG and NLF102 group were supplemented with fresh DMEM medium without FBS, and the Sham group was supplemented with DMEM complete medium (10% FBS), all treated for 24 h. After that, DMEM medium was discarded and DMEM complete medium was added. Of these, NRG and NLF102 were added at 10nM in the NRG and NLF102 groups, while PBS was added at equal volume in the Control group and the Sham group, and treated for 24 h. Here, NRG is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 1 and NLF102 is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 2.

3 dyeing of sarcomere

Discard complete medium, PBS wash cell slide or cell culture wells. The slide cells face up and are placed in a clean cell culture plate and fixed with methanol. The fixed slide or well is rinsed on the PBST shaker. The blocking solution is blocked at room temperature, and Alpha actin primary antibody (ab9465, Abcam) is diluted with 1% NGS in the slide or cell well and incubated at room temperature for 2h or overnight at 4 ℃. PBST was rinsed at room temperature. Anti-mouse secondary antibody Alexa flow 555(ab4409, Abcam) was diluted with 1% NGS and incubated at room temperature in the dark. PBST was rinsed at room temperature. DAPI stained the nuclei. 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 ℃. Appropriate PBS was added to the wells to cover the cells and the cells were photographed under a fluorescent microscope.

4 statistical analysis

The distribution of the sarcomere in the cells was observed and classified into three classes according to the area occupied in the cardiomyocytes: 2/3, excellent (Well); 1/3-2/3, medium (Moderate); <1/3, difference (Poor)

All experimental data are expressed as ± SD, and single factor anova was performed using GraphPad Prism6 software, with p <0.05 indicating significant differences compared to Control group and p <0.01 indicating very significant differences compared to Control group.

5 analysis of the results of the experiment

The statistical results of the percentage of the area grade of the sarcomere in each group in the whole cells are shown in table 2:

table 2: percentage of different grades of sarcomere area grade in the whole cell

By analyzing the proportion of sarcomere to the total cell area, we can understand the status of primary cardiomyocytes. Compared with the Control group, the muscle segments of the Sham group are fewer, and after the NRG or NLF102 is added, the muscle segments are increased, which shows that the NRG or NLF102 has the function of promoting the reconstruction of the muscle segments. In addition, the action and effect of NLF102 are more significant than those of NRG.

Example 4 Elisa method to determine the half-life of intravenous NRG, NLF102 in rats

1 laboratory animal

Strain and source: wistar rat, supplied by Shanghai Sphall-Bikay laboratory animals Ltd

Sex and body weight: male, 210-250 g

2 course of experiment

2.1 sample preparation

2.1.1 test sample preparation

30ug/kg of recombinant human Neuregulin (NRG) or neuregulin long fragment NLF102 was injected into rats tail vein and orbital bleeds were performed at different time points post-dose, respectively. Standing at room temperature for 2h, and centrifuging to separate supernatant after blood coagulation. Rat sera containing neuregulin or neuregulin long fragment NLF102 were diluted 1:1 in dilution buffer for use. Here, NRG is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 1 and NLF102 is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 2.

2.1.2 preparation of standard: neulanglin and Neulanglin long fragment NLF102 standard samples were prepared from rat serum in the concentration ranges of 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml and 0ng/ml, and then diluted with dilution buffer at a ratio of 1: 1.

2.2 Experimental procedures

2.2.1 plate coating and sealing: NRG1-EGF domain antibody was diluted with coating buffer and 50uL was added to each reaction well in a 96-well plate, coated overnight at 4 degrees. 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 plate, draining the plate on absorbent paper, adding 50uL of corresponding standard sample or sample to be tested into each reaction well, 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-neuregulin antibody.

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

2.2.5 color development: washing the plate, adding the TMB substrate solution prepared temporarily into each reaction hole, reacting for 20 minutes at 37 ℃ in a dark place, and adding 50uL of 2M sulfuric acid to terminate the reaction.

2.2.6 detection: the absorbance of each reaction well at 450nM was measured and the neuregulin and protein content of the sample was calculated from the standard curve.

2.2.7 data were analyzed using GraphPad Prism 5.0 software.

3 results and discussion of the experiments

We injected 30ug/kg of recombinant human Neuregulin (NRG) and neuregulin long fragment NLF102 separately via the tail vein of rats and took orbital bleeds after administration separately and separated the sera, and then examined the neuregulin or NLF102 protein content in the sera at different time points, as shown in table 3, fig. 1:

table 3: half-life results statistics

Example 5 Long-term intravenous continuous administration of recombinant neuregulin polypeptide for testing the drug effect of anti-rat heart failure

1. Purpose of experiment

On a left coronary artery ligation heart failure rat model, recombinant human Neuregulin (NRG) and recombinant neuregulin NLF102 were administered by continuous intravenous drip using a medtronic insulin infusion pump, and the therapeutic effects of NRG and NLF102 on the rat heart failure model were compared. Here, NRG is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 1 and NLF102 is the neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 2.

2. Experimental Material

2.1 Experimental animals

2.1.1 line, source: wistar rat, supplied by Shanghai Sphall-Bikay laboratory animals Ltd

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

2.2 reagent drugs

2.2.1 excipients: the Shanghai Zesheng science and technology development company, the dosage form is: freeze-dried powder, specification: 2 mgAlb/bottle

2.2.2 NLF102 solution: the Shanghai Zesheng science and technology development company, the dosage form is: solution, specification: 0.485mg/ml

2.2.3 recombinant human Neuregulin (NRG) final product: the Shanghai Zesheng science and technology development company, the dosage form is: freeze-dried powder, specification: 250 ug/piece

2.2.4 Isoflurane: rewarded life science ltd, specifications: 100 ml/bottle

3. Experimental equipment 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: meidunli, type: MMT-712EWS, MMT-722NAS/L

4. Experimental methods

4.1 establishment of Heart failure model caused by coronary artery ligation in rat

Rats were anesthetized with isoflurane at 4% concentration by gas anesthesia, fixed lying on their back after anesthesia, and sterilized with 75% alcohol after chest unhairing. After the left anterior skin of the chest is incised, the chest muscles are separated bluntly, 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 separated bluntly by hemostatic forceps, the heart is extruded from the chest by matching with the two hands for extrusion, the heart is fully exposed, the pulmonary inflation and heartbeat conditions are observed, the left auricle and the pulmonary artery cone are fully exposed, and the anterior descending branch of the left coronary artery is ligated by 6-0 operation suture between the left auricle and the pulmonary artery cone. And then forcefully squeezing the chest to exhaust air, suturing the muscles and the skin of the chest, feeding the rat in a cage after an operation, and closely observing the condition of the rat, if the occurrence of acute arrhythmia is found, massaging the heart for 3-5 minutes in an emergency.

4.2 Experimental groups and dosing

4.2.1 Experimental grouping situations

The heart function of the rats is detected by using a B ultrasonic machine Vivid E95 respectively 2 weeks, 3 weeks and 4 weeks after operation, and the rats with the EF value ranging from 28.6 to 42.4 percent are selected for the next experiment after the 4-week cardiac ultrasonography. The rats are randomly divided into 3 groups according to the heart super-junction fruits, an excipient group, an NRG 6 mu g/kg group and an NLF 1029.6 mu g/kg group, wherein the EF value of the excipient group and the EF value of the NRG group are both 37.3%, the EF value of the NLF102 group is 37.4%, 12 rats are arranged in each group, an insulin pump is applied every day for 8 hours of caudal vein administration, the administration is continuously carried out for 10 days, the NRG administration dose is 0.75 mu g/kg/h, the administration volume is 5ml/kg, the administration concentration is 1.2 mu g/ml, the NLF102 administration dose is 1.2 mu g/kg/h, the administration volume is 5ml/kg, the administration concentration is 1.92 mu g/ml, and the heart super-junction detection is carried out again on the 1 day after the administration is finished. In the sham operation group, 13 coronary arteries were not ligated by threading, and no drug administration was performed.

4.2.2 dispensing methods

1) Excipient: adding 1ml of normal saline into each bottle of 2 mgAlb/bottle to prepare mother liquor, adding 49.76ml of normal saline into 0.24ml of mother liquor to dilute into 9.6 mu g/ml Alb solution

2) NRG: adding 1ml of normal saline into each 250 mu g NRG bottle to prepare a mother solution, adding 49.76ml of normal saline into 0.24ml of the mother solution to dilute the mother solution into 1.2 mu g/ml NRG solution

3) NLF 102: 0.485mg/ml, 0.20ml of the solution is added with 49.80ml of normal saline to be diluted into NLF102 solution of 1.92 mu g/ml

4.3 Observation index

4.3.1 cardiac function testing

After the rats were anesthetized with 4% isoflurane by a gas anesthetic machine, the left side was fixed on an operation plate in a horizontal position. The rat head was fixed in the breathing mask of a gas anesthesia machine and maintained with isoflurane at a concentration of 2%. The breast is unhaired, sterilized by 75% alcohol, smeared with a coupling agent, and detected by a rat heart ultrasonic probe. Selecting a B-mode, placing a cardiotachograph probe on the left side of the sternum, pointing the probe at 2-3 o' clock, cutting the heart vertically to the long axis direction of the heart by the sound beam, adjusting the probe to the level of two papillary muscles, obtaining the short axis section of the left ventricle of the papillary muscle level, collecting a section of dynamic image of the papillary muscle plane of the left ventricle, and storing. Select "M-mode" to keep the probe to the left of the papillary muscleAdjusting an M-shaped sampling line to pass through the weakest point of the front wall pulse, adjusting the focal length, collecting an M-shaped curve (the left ventricular cavity and the front and rear walls of the left ventricle 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) D³The left ventricular end-diastolic and end-systolic volumes, EDV, ESV, were calculated, and the Ejection Fraction (EF) value was calculated, EF ═ EDV-ESV/EDV × 100%.

4.4 data processing

For all experimental data

Represents, one-way analysis of variance, P, using GraphPadprism6 software<0.05 indicated a significant difference between groups, P<0.01 indicated a very significant difference between groups.

5. Results of the experiment

5.1 Heart super fruits

The LVEDd and LVEDs of the model control group before the grouping administration are respectively 0.956 + -0.076 cm and 0.806 + -0.065 cm, and the LVEDd and LVEDs are respectively 0.991 + -0.075 cm and 0.851 + -0.068 cm after the administration; LVEDd and LVEDs of NRG administration group are respectively 0.952 + -0.086 cm and 0.802 + -0.076 cm before administration, and 0.969 + -0.075 cm and 0.810 + -0.078 cm after administration; LVEDd and LVEDs of the NLF102 administration group are respectively 0.930 +/-0.059 cm and 0.783 +/-0.054 cm before administration, and 0.936 +/-0.060 cm and 0.781 +/-0.075 cm after administration; the EF value of the model control group before the grouping administration is 37.3 +/-3.2 percent, and the EF value after the administration is 33.9 +/-4.9 percent; the EF value of the NRG administration group before administration is 37.3 +/-3.8 percent, and the EF value after administration is 38.9 +/-4.8 percent; the EF value of the NLF102 administration group before administration is 37.4 +/-2.9%, and the EF value after administration is 39.2 +/-6.6%. After administration, the EF value of heart failure rats in NRG and 1NLF102 treatment groups is obviously increased, and compared with that in a model control group, the EF value of the heart failure rats in the two groups is obviously different (p < 0.05). 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 treatment effect on heart failure rats, and the treatment effect of the NLF102 is superior to that of NRG.

TABLE 4NRG, NLF-102 Pump Tail veinLong-time administration drug effect experiment heart super-junction fruit for treating rat heart failure

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

The scope of the 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 and 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.

Claims (10)

1. The neuregulin polypeptide contains an amino acid sequence shown in SEQ ID NO. 2.
2. The neuregulin polypeptide of claim 1, which has the amino acid sequence of SEQ ID NO. 2.
3. Use of neuregulin polypeptide for the manufacture of a medicament for preventing, treating or delaying heart failure in a mammal.
4. The use of claim 3, wherein the neuregulin polypeptide comprises the amino acid sequence of SEQ ID NO 2
5. A pharmaceutical preparation comprises neuregulin polypeptide and a pharmaceutically acceptable carrier, excipient or diluent, wherein the neuregulin polypeptide comprises an amino acid sequence shown in SEQ ID NO. 2.
6. The pharmaceutical preparation of claim 5, wherein the neuregulin polypeptide has an amino acid sequence of SEQ ID NO. 2.
7. A method for treating heart failure, comprising administering to an individual in need thereof an effective amount of neuregulin polypeptide comprising the amino acid sequence of SEQ ID NO. 2.
8. The method of claim 7, wherein the neuregulin polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 2.
9. The method of claim 7, wherein the individual is a human.
10. The method of claim 7, wherein the neuregulin polypeptide is administered intravenously.

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