CN103772498B

CN103772498B - Neuregulin and application thereof

Info

Publication number: CN103772498B
Application number: CN201310645946.9A
Authority: CN
Inventors: 周明东
Original assignee: ZESHENG SCIENCE AND TECHNOLOGY DEVELOPMENT Co Ltd SHANGHAI
Current assignee: Zensun Shanghai Science and Technology Ltd
Priority date: 2009-11-09
Filing date: 2009-11-09
Publication date: 2015-07-15
Anticipated expiration: 2029-11-09
Also published as: CN103772498A

Abstract

The invention provides a specific neuregulin polypeptide which can be applied to methods and compositions for preventing, treating or delaying various diseases or disorders.

Description

NRG and uses thereof

Technical field

The present invention relates to and have the neuregulin of high-affinity to ErbB2 acceptor and treat the purposes of various heart disease and disorder.

Background technology

Heart (ventricle) hypertrophy is a kind of important adaptability physiological response to the heart working pressure increased or demand.One of early stage cellular change occurred after loose stimulus effect is sarcostyle amplification (thickened chamber walls) of plastosome synthesis and the increase that is in proportion with individual cells, but cell quantity does not have (or few) to increase.

When ventricle is under pressure, initial reaction is the increase of sarcomere length.The increase of overall muscle mass subsequently.When load is too serious, myocardial contraction will weaken.In the slightest state, the reduction of this power development speed when weakening the reduction or isometric contraction that are presented as zero load myocardial contraction speed.When myocardial contraction weakens further, there is the decline more of zero load myocardium shortening speed, and decline with the development of isometric contraction muscle strength and contracted length.Now, circulation compensates and still can be provided by the increase of cardiac dilatation and myocardial mass, and it tends to maintain ventricle wall stress at normal level.When convergent force continues to decline, there will be obvious congestive heart failure, show as the decline of cardiac output or operational forces, and/or the rising of ventricular edv and diastolic pressure.

Transition from hypertrophy to heart failure is changed to feature with several cell tissue.Such as, normal loose cell has larger size, with strengthen and orderly contractile unit and stronger cell-ECM adhesion.Otherwise the cell of the hypertrophy of pathological state, its size is also comparatively large and have protein aggregation, shows disordering (sarcomere structure disturbance) and the poor cell-cell adherence (myofiber is disorderly) of contractile protein.Therefore, in the hypertrophy of pathological state, the unordered assembling of the increase of cell size and the gathering of contractile protein and sarcomere structure and the firmly interactional disappearance of cell-ECM are associated.

Nearly 500 ten thousand Americans suffer from heart failure, and annual newly-increased patient is more than 550,000.The drug main of Current therapeutic heart failure will concentrate on angiotensin-converting enzyme (ACE) inhibitor, and these vasodilators cause vasodilation, reduce blood pressure and reduce the workload of heart.Although the percentage ratio of mortality ratio declines have significant difference, mortality ratio actual after using ACE inhibitor declines and is on average only 3%-4%, and also has several potential side effect.

ACE inhibitor also with other drug coupling, such as purple foxglove, can systaltic strength be increased; And/or certain diuretic(s), get rid of more sodium and water in blood by causing kidney thus contribute to alleviating the workload of heart.But having at least one to study proves to use purple foxglove compared with placebo at II-III grade of Patients with Cardiac Failure, and what difference is its survival rate do not have.In addition, diuretic(s) can improve some symptom of heart failure, but is not suitable for for treatment separately.

Other selections of prevention or treatment heart failure also have corresponding limitation.Such as, heart transplantation is obviously more expensive than pharmacological agent and have more invasive, and further by the restriction with or without donor's heart.Using mechanism, such as biventricular cardiac pacemakers, is invasive and costly equally.Therefore, due to the deficiency of Current therapeutic means, need new remedy measures.

A kind of up-and-coming new treatment means comprises to patients with heart failure or has the patient of heart failure risk to use NRG (hereinafter referred to as " NRG ").NRGs, by GDF man group composition relevant in a structure, comprises NRG1, NRG2, NRG3 and NRG4 and isomer thereof.Such as, the isomer of the NRG1 identified, more than 15, can be divided into the large class of α and β two according to the difference in their basic Urogastron (EGF) similar districts.NRG-1 is mentioned in following documents and materials, as United States Patent (USP) 5, and 530,109,5,716,930,7,037,888; Lemke, Mol. Cell. Neurosci. 1996,7:247-262; Peles and Yarden, 1993, BioEssays 15:815-824,1993; Peles et al., 1992, Cell 69:205-216; Wen et al., 1992, Cell 69:559-572; Holmes et al., 1992, Science 256:1205-1210; Falls et al., 1993, Cell 72:801-815, Marchionni et al., 1993, Nature 362:312-8, complete being by reference herein incorporated of above content.NRG-2 mentions in following documents and materials, as Chang et al., 1997, Nature 387:509-512; Carraway et al., 1997, Nature 387:512-516; Higashiyama et al., 1997, J. Biochem. 122:675-680; Busfield et al., 1997, Mol. Cell. Biol. 17:4007-4014 and WO 97/09425, complete being by reference herein incorporated of above content.NRG-3 mentions in following documents and materials, as Hijazi et al., 1998, Int. J. Oncol. 13:1061-1067, and complete being by reference herein incorporated of its content.NRG-4 mentions in following documents and materials, as Harari et al., 1999, Oncogene. 18:2681-2689, and complete being by reference herein incorporated of its content.

The member of NRG and EFG receptor family combines, and this receptor family comprises EGFR, ErbB2, ErbB3 and ErbB4, and wherein each acceptor plays an important role in various kinds of cell function, comprises Growth of Cells, differentiation and survival.They are all protein tyrosine kinase receptors, are made up of an extracellular ligand binding domains, membrane spaning domain and cytoplasmic tyrosine kinase structural domain.After NRG is bonded to the extracellular domain of ErbB3 or ErbB4, can change thus cause between ErbB3, ErbB4 and ErbB2 and form heterodimer by induced conformational, or ErbB4 self forms homodimer, will cause the phosphorylation of C-terminal domains in recipient cell like this.Then the intracellular domain of phosphorylation other signal protein in cell is combined, activate corresponding downstream AKT or ERK signal transduction path, and induce a series of cell response, such as stimulate or antiproliferative effect, cytodifferentiation, apoptosis, cell migration or cell adhesion.In these acceptors, mainly ErbB2 and ErbB4 expresses at heart.

There are some researches show the similar district of the EGF of NRG1, about 50 to 64 amino acid, be enough to combine and activate these acceptors.Research in the past shows that NRG-1 β (NRG-1 β) can with high-affinity directly in conjunction with ErbB3 and ErbB4.Orphan receptor ErbB2 can form heterodimer with ErbB3 or ErbB4 and its avidity is higher than ErbB3 or ErbB4 homodimer.The formation of neurodevelopmental result of study prompting sympathetic nervous system needs complete NRG-1 β, ErbB2 and ErbB3 signal transducting system.Target causes embryonic death due to heart development defect after destroying NRG-1 β or ErbB2 or ErbB4.Nearest research has also highlighted NRG-1 β, ErbB2 and ErbB4 and has had vital role at cardiovascular development and the adult normal heart function aspects of maintenance.Research shows that NRG-1 β can strengthen the weave construction of the sarcomere of adult cardiomyocytes.The similar district of NRG-1 β EGF that short-term uses a kind of restructuring can significantly improve or prevent the deterioration of the myocardial function of three kinds of different animal heart failure models.The more important thing is, the survival of NRG-1 β energy significant prolongation heart failure animal.These effects make NRG-1 β be expected to become a kind of broad-spectrum curing thing or lead compound for the treatment of the heart failure that multiple common disorder causes.But, still need a kind of more effective method applying NRG, can be used for clinically preventing, treating or postpone heart failure and/or cardiac hypertrophy.

Summary of the invention

summary of the invention

First aspect of the present invention there is provided NRG polypeptide ErbB acceptor being had to high-affinity.In certain embodiments, NRG polypeptide comprises the EGF functional domain of neuregulin.In certain embodiments, NRG polypeptide comprises the EGF functional domain of rhNRG-1beta S177-Q237 β 2 isomer.In certain embodiments, the NRG polypeptide of high-affinity is had to comprise the aminoacid sequence of SEQ ID NO:5 to ErbB acceptor.

NRG polypeptide of the present invention has stronger avidity than wild type full-length neuregulin to ErbB acceptor.Except being connected with ErbB acceptor, NRG polypeptide of the present invention also possesses the biological function of one or more natural neuregulin.

The preparation of NRG polypeptide can according to any correlation technique well know in the art.The typical technology preparing NRG polypeptide is provided at this.In certain embodiments, NRG polypeptide can be restructuring.In certain embodiments, NRG polypeptide is synthesis, such as, by liquid phase or Solid phase peptide synthesis.

Another aspect of the present invention there is provided NRG polypeptide relevant nucleic acid, carrier and host cell.This nucleic acid or its complementary sequences encode NRG polypeptide or fragment wherein.Nucleic acid can be DNA or RNA of double-strand or strand, can be inserted in suitable carrier so that the expression of propagation and NRG polypeptide.Improved carrier is transferred in suitable host cell, as expressed the host cell of restructuring NRG polypeptide.

Another aspect of the present invention there is provided NRG polypeptide in treatment and the application of non-therapeutic aspects.Particularly NRG polypeptide is in prevention, the application treating or postpone various heart disease and disorder.Correspondingly, the invention provides the pharmaceutical preparation comprising NRG polypeptide and related methods for the treatment of.

Another aspect of the present invention there is provided the method for the treatment of Mammals heart failure.In certain embodiments, the method comprises in NRG polypeptide injection mammalian body.

Another aspect of the present invention there is provided the method for ErbB receptor phosphorylation in inducing cell.In certain embodiments, the method comprises with NRG polypeptide exposing cell.

Another Fang Ming of the present invention there is provided and induces and maintain the activation of the AKT signal path in heart cell.In certain embodiments, the method comprises with NRG polypeptide contact heart cell.

Another Fang Ming of the present invention there is provided and induces and maintain the activation of the ERK signal path in heart cell.In certain embodiments, the method comprises with NRG polypeptide contact heart cell.

picture brief description

Fig. 1 shows NRG55, NRG57 and NRG59 of different concns to the impact of myocardial cell AKT phosphorylation.GADPH in contrast.

detailed Description Of The Invention

a. lexical or textual analysis

Except as otherwise definition, it is identical that all technology used herein and scientific and technical terminology and general technical staff of the technical field of the invention understand implication.Mentioned herein to all patents, application, disclosed application and complete all being by reference herein incorporated of other publications.If the definition that this part proposes contrary with the definition proposed in the patent be incorporated herein by reference, application, disclosed application and other publications or inconsistent time, then with this part propose definition be as the criterion.

Unless specifically stated otherwise, being meant to " at least one " or " one or more than one " this " " used.

" EGF sample functional domain " used herein or " EGF-like domain " refer to by neuregulin coded by said gene can in conjunction with and activate the polypeptide fragment of ErbB2, ErbB3, ErbB4 or its allos or homodimer, and with the EGF receptorbinding region that describes in following reference, there is structural similarity: WO 00/64400; Holmes etc., Science, 256:1205-1210 (1992); United States Patent (USP) 5,530,109 and 5,716,930; Hijazi etc., Int. J. Oncol., 13:1061-1067 (1998); Chang etc., Nature, 387:509-512 (1997); Carraway etc., Nature, 387:512-516 (1997); Higashiyama etc., J. Biochem., 122:675-680 (1997); And WO 97/09425, complete being by reference herein incorporated of above content.In certain embodiments, EGF sample functional domain combines and activates ErbB2/ ErbB4 or ErbB2/ ErbB3 heterodimer.In certain embodiments, EGF sample functional domain comprises the receptor binding domain amino acid of NRG-1.In certain embodiments, EGF sample functional domain refers to the 177-226 position of NRG-1,177-237 position or 177-240 amino acids.In certain embodiments, EGF sample functional domain comprises the receptor binding domain amino acid of NRG-2.In certain embodiments, EGF sample functional domain comprises the receptor binding domain amino acid of NRG-3.In certain embodiments, EGF sample functional domain comprises the receptor binding domain amino acid of NRG-4.In certain embodiments, EGF sample functional domain comprises United States Patent (USP) 5,834, the aminoacid sequence described in 229: Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro (SEQ ID NO:11).

" effective dose " of the activeconstituents for the treatment of specified disease used herein is the dosage being enough to improve or reduce in some way this disease related symptom.This dose perhaps can cure diseases, but is used to the symptom improving disease in typical situation.

" activeconstituents " used herein is used to diagnosis, cures, alleviates, treats or prevent people or other animals to suffer from the disease, or strengthens any material of health or Mental health.

" improvement " of particular disorder symptom used herein refer to by use specific activeconstituents and permanent or temporarily, continue or mitigation symptoms instantaneously, and this alleviate can owing to or relevant with using this reagent.

Any mode that the direction that " treatment " or " process " used herein refers to can make discomfort, the symptom of disorder or disease is improved or become better changes.Its effect can be preventative, and such as preventing a certain disease or its symptom from occurring wholly or in part, also can be curative, such as partially or completely cures the disadvantageous effect that a certain disease and/or this disease cause.Treatment also comprises any pharmaceutical use of composition described herein.

" carrier (or plasmid) " used herein refers to for allogeneic dna sequence DNA being introduced cell with the dispersed components thereof of expressing or copy wherein.Select and use these carriers very familiar for the skilled person.Expression vector comprises the carrier of the DNA of energy Regulation and expression sequence connection, and regulating and controlling sequence such as promoter region can affect the expression of these DNA fragmentations.Therefore, expression vector refers to DNA or the RNA assembly of recombinating, such as plasmid, phage, recombinant virus or other carriers, just causes the DNA of clone to express when being introduced into suitable host cell.Suitable expression vector is that those skilled in the art extensively knows, comprises those carriers copied at eukaryotic cell and/or prokaryotic cell prokaryocyte and those keep free or those are incorporated into the carrier of host cell gene group.

The state that " Cardiomyocyte Differentiation " used herein refers to following situation is feature, DNA synthesizes minimizing more than 10%, the DNA synthesis that suppresses other factors to stimulate is greater than 10%, the combination of orderly sarcomere and cell-ECM glue connection, the continuous activation of map kinase and p21 ^cip1enhanced expressing.Further discussion is see WO00/37095, and its content is intactly herein incorporated by reference.

" ejection fraction (ejection fraction) " or " EF " used herein refers to the ratio of the blood that heartbeat pumps from the left ventricle be full of.Can define with following formula: (left ventricular end-diastolic volume-left ventricular end-systolic volume)/left ventricular end-diastolic volume.

" contraction fraction (fractional shortening) " or " FS " used herein refers to the ratio of left ventricular contraction state and the change of diastole state diameter.Can define with following formula: (left ventricular end diastolic dimension-left ventricular end-systolic dimension)/left ventricular end diastolic dimension.

" heart failure " used herein refers to that heart can not with the core function abnormality of the speed pump blood of the organization need of metabolism.Heart failure comprises various disease states as congestive heart failure, myocardial infarction, rapidity arrhythmia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, myocarditis etc.Heart failure can be caused by many factors, includes, but are not limited to: ischemic, inborn, rheumatism an or former form.Chronic cardiac hypertrophy is a kind of significantly morbid state, is the omen of congestive heart failure and cardiac arrest.

" myocardial infarction " used herein refers to that the patchiness of the part cardiac muscle that the serious and lasting ischemic that blocking of coronary artery or blood flow ceases cause causes is downright bad.

Used herein " arrangement that is orderly, that strengthen of sarcomere or sarcomere structure " refers to the state being feature with the proper alignment of contractile protein of being shown by α in myocardial cell-actinine immunofluorescence dyeing.In cell, the proper alignment of α-actinine can be identified by microscope and connected camera installation thereof." disorder or irregular of sarcomere or sarcomere structure " used herein is the situation contrary with " orderly, the arrangement that strengthens of sarcomere or sarcomere structure ".

Used herein " arrangement that is orderly or that strengthen of cytoskeletal structure " refers to the state being feature with actin fiber ordered arrangement through Phalloidine (phalloidin) dyeing display in myocardial cell.In cell, the proper alignment of actin fiber can be identified, as the illustration in picture of the present invention by microscope and connected camera installation thereof." disorder or irregular of cytoskeletal structure " used herein refers to the situation contrary with " cytoskeletal structure orderly, or the arrangement strengthened ".

The implication of " albumen " and " polypeptide " or " peptide " used herein is identical, unless separately clearly stated in literary composition.

" continuous activation of map kinase " used herein is map kinase in phalangeal cell---the phosphorylation state of p42/44 maintains at least 21 hours.Have further discussion inside WO00/37095, its content is incorporated herein by reference.

" collaborative ", the result for the treatment of of the improvement that " synergistic effect " or similar terms are used to be described through one or more treatment reagent of associating and one or more retinoid compounds at this and obtain.Although inside some field synergistic effect mean be greater than add and effect (such as, 1+1=3), at medical field, one add and (1+1=2) or be less than add and (1+1=1.6) effect also can be collaborative.Such as, if the expansion of the ventricular muscle cell hypertrophy that can suppress 50% used separately by one of two kinds of medicines, two kinds of drug combinations just can not be look to can to stop the development of ventricular muscle cell hypertrophy completely.In many cases, due to unacceptable side effect, two kinds of medicines can not be used together.In other cases, mutual antagonism between medicine, the development slowing down ventricular muscle cell hypertrophy when conbined usage is less than 50%.Therefore, if the development that two kinds of drug combinations slow down ventricular muscle cell hypertrophy is greater than 50% and does not increase unacceptable side effect, just synergistic effect is obtained.

" cardiac hypertrophy " used herein refers to the situation that feature is following: the increase of Ventricular Myocytes size, the increase of cell size is enough to cause making clinical diagnosis to patient or being enough to assert that cell becomes large (such as, twice larger than non-mastocyte or more).It may along with the activation with embryonic gene expression of gathering of contractile protein in Cardiomyocytes.

The method being used for detecting ventricular muscle cell hypertrophy has in vitro method and intracorporal method two kinds.The method of vitro detection ventricular muscle cell hypertrophy comprises those methods described in WO00/37095, the increase that the increase of such as cell size and atrial natriuretic peptide (ANP) are expressed.The change of cell size is used to a kind of points-scoring system measure loose degree.Useful inverted phase contrast microscope is observed these and is changed, and loose degree is weighed with the scoring yardstick of artificial 7-0, and 7 points represent completely loose cell, and 3 points represent the cell do not stimulated.State representated by 3 points and 7 points can respectively see Fig. 2 A and B in (1982) Circulation Res. 51:787-801 such as Simpson.Loose scoring and cell surface amass (μm ²) relation be found linearly (relation conefficient=0.99).In the hypertrophy of phyenlephrinium induction, the hypertrophy scoring not exposing (normally) cell is 3, and it is 581 μm that cell surface amasss ²; And the hypertrophy scoring of mastocyte is 7 completely, surface-area is 1811 μm ², or be about 200% of normal value.Loose scoring is the surface-area of the cell of 4 is 771 μm ², or more about than non-exposed cell 30%; Loose scoring is the surface-area of the cell of 5 is 1109 μm ², or more about than non-exposed cell 90%; Loose scoring is the surface-area of the cell of 6 is 1366 μm ², or more about than non-exposed cell 135%.There is ventricular muscle cell hypertrophy and mainly comprise cells show size increase (loose scoring 3.5) going out about 15% or more.Can reflect that the difference of maximum hypertrophic response ability induced by loose inductor by above-mentioned analytical procedure scoring.Such as, the maximum value that the cell size that endothelin (endothelin) is induced increases is approximately loose scoring 5 points.

" cardiac hypertrophy suppression " used herein refers to that characterizing one of loose parameter has reduction relative in loose situation, or stops one of loose parameter of sign to increase relative to normal circumstances.Such as, the suppression of ventricular muscle cell hypertrophy shows relative to reducing in loose situation by measuring cell size.The suppression of ventricular muscle cell hypertrophy refers to and will reduce 10% or more relative to size cell size viewed under loose situation.Under preferred condition, loose suppression means that the minimizing of cell size is 50% or more.Loose analysis meter point-score when reference phyenlephrinium is inductor, these minimizings are equivalent to loose score about 6.5 or less, 5.0-5.5 and 4.0-5.0 respectively.When using different inductors, show suppression by the score value measuring the maximum cell size (or loose scoring) when existing relative to inductor.

Prevent ventricular muscle cell hypertrophy from being by stoping cell size to be determined relative to normal cell increase under being enough to induce loose inducer concentrations.Such as, cell size increases less than 200% relative to non-inducing cell when the inductor of maximal stimulation concentration exists to prevent hypertrophy from referring to.Under preferred condition, cell size is less than 135% relative to non-inducing cell increase to prevent hypertrophy from meaning; Under most preferred condition, cell size is less than 90% relative to non-inducing cell increase to prevent hypertrophy from meaning.Relative to score analytical method loose when being inductor with phyenlephrinium, when the phyenlephrinium of maximal stimulation concentration exists, loose hypertrophy is prevented to be respectively about 6.0-6.5,5.0-5.5 and 4.0-5.0.

Loose in vivoassay comprises to be measured cardio-vascular parameters such as blood pressure, heart rate, body circulation resistance, convergent force, heartbeat strength, concentric hypertrophy or distensibility hypertrophy, left ventricular systolic pressure, left ventricle mean pressure, ventricular end diastolic pressure, cardiac output, often to fight index (stroke index), Histological parameter and ventricle size and chamber wall thickness.Be used for the development and suppression that measure body ventricular myocyte hypertrophy animal model comprises pressure overload mouse model, function mouse model, transgene mouse model and Rat of Myocardial Infarction model lose in right ventricle.Be used for the existence of evaluating patient ventricular muscle cell hypertrophy, development and suppression medical procedures be widely known by the people, comprise and measure diastole and shrinkage parameters, assessment ventricular weight and pulmonary vein flow.

Loose can comprise factor congenital virus, inborn, cardiotrophin, muscle nutrition from any factor responded to vitamin A acid, or as the result of ischemic or ischemia injury such as myocardial infarction.Typically, carry out treating to stop or slow down loose process, especially after heart and injury, after such as ischemic occurs.Preferably, be treatment myocardial infarction, reagent gives immediately after myocardial infarction, to prevent or to alleviate hypertrophy.

" activity unit " or " 1U " used herein refers to the consumption of the standard prod that can cause 50% maximum reaction.In other words, in order to measure the activity unit of a certain active ingredient, EC50 must be measured.Such as, if the EC50 of a certain product is 0.067 μ g/ml, then this amount is exactly 1 unit.Furthermore, if employ 1 this product of μ g, just 14.93U(1/0.067 is the use of).EC50 can be measured by any method known in the art, comprise the method that contriver in the following examples uses.The mensuration of activity unit is important for the quality control of genetic engineering product and Clinical practice medicine, and the product of different pharmaceuticals and/or different batches can be made like this to come quantitatively with same standard.

In some example, the unit of NRG is determined, as WO03/099300 and Sadick by the activity measuring NRG by Kinase Receptor Activation enzyme-linked immunosorbent assay (KIRA-ELISA) et al., 1996, Analytical Biochemistry, describes in detail in 235:207-14, and its content is herein incorporated by reference and intactly.In brief, the method measures ErbB2 activation and the phosphorylation of the adherent breast cancer cell line MCF-7 of NRG induction.Carry out dissolving film albumen with Triton X-100 lysate, acceptor is wherein coated on catching with the ErbB2 specific antibody (as H4) of ErbB3 or ErbB4 no cross reaction in ELISA hole.The phosphorylation degree of acceptor is measured by anti phosphotyrosine antibody ELISA.

b. NRG

NRG polypeptide provided by the invention can in conjunction with ErbB acceptor.In certain embodiments, NRG polypeptide is strengthened ErbB receptor affinity.In certain embodiments, NRG polypeptide contains neuregulin EGF functional area.In certain embodiments, NRG polypeptide contains the EGF functional area of rhNRG-1beta S177-Q237 β 2 isomer.In certain embodiments, NRG polypeptide contain SEQ ID NO:3,5,7, the aminoacid sequence of 9.

In certain embodiments, NRG polypeptide contains the aminoacid sequence of SEQ ID NO:3.In more excellent embodiment, NRG polypeptide is the aminoacid sequence of SEQ ID NO:3.

In certain embodiments, NRG polypeptide contains the aminoacid sequence of SEQ ID NO:5.In more excellent embodiment, NRG polypeptide is the aminoacid sequence of SEQ ID NO:5.

In certain embodiments, NRG polypeptide contains the aminoacid sequence of SEQ ID NO:7.In more excellent embodiment, NRG polypeptide is the aminoacid sequence of SEQ ID NO:7.

NRG polypeptide of the present invention has stronger avidity than wild type full-length neuregulin to ErbB acceptor.Except being connected with ErbB acceptor, NRG polypeptide also possesses the biological function of one or more natural neuregulin.

the preparation of NRG polypeptide

The preparation of NRG polypeptide can according to any obvious correlation technique.The typical technology preparing NRG polypeptide can be see, and as United States Patent (USP) 7,226,907 and 5,367,060, WO94/026298 and WO03/099300, its content is intactly herein incorporated by reference.

The preparation of NRG polypeptide of the present invention can according to any correlation technique well know in the art.In certain embodiments, NRG polypeptide is synthesis, such as, by liquid phase or Solid phase peptide synthesis, see Merrifield, and 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, its content is intactly herein incorporated by reference.

In the embodiment optimized, NRG polypeptide can obtain from natural resource, synthesis or commercial sources.In certain embodiments.In certain embodiments, NRG polypeptide can be obtained by purifying after recombinant expression protein.

NRG polypeptide can be purified by any technology known in the art, as high performance liquid chromatography, and ion exchange chromatography, gel electrophoresis, affinity chromatography etc.Those skilled in the art know the purification condition of concrete NRG polypeptide.

the use of NRG polypeptide

NRG polypeptide can judge to use according to those skilled in the art.The example of this respect comprises the method for following file description: United States Patent (USP) 7,226,907 and 5,367,060, WO94/026298 and WO03/099300, its content is intactly herein incorporated by reference.

NRG polypeptide to treatment a series of disease and disorder effective.Typical disease and disorder comprise heart disease as heart failure, viral myocarditis, expansion (hyperemia) type myocardosis (DCM), cardiac toxic or myocardial infarction.

In certain embodiments, the invention provides a kind of method, treat heart failure by the NRG polypeptide using effective dose.

NRG polypeptide may be used in the form of a pharmaceutical preparation.

The method of application of NRG polypeptide is judged by person skilled in the art, including, but not limited to oral, intravenous injection, gastric infusion, rectal administration, abdomen (film) intracavitary administration or intracerebroventricular administration.

In a preferred embodiment, the composition for administration is pharmaceutical preparation.Pharmaceutical preparation can be containing prevention or one or more preventions of therapeutic dose or the medicament (such as, containing NRG polypeptide and other prevention or the complex body of healing potion) for the treatment of, and pharmaceutically acceptable carrier or vehicle.In certain embodiment and herein, " pharmaceutically acceptable " refer to passed through by national correlation department or be can be used for animal particularly on the person in US-built pharmacy or other by what the pharmacopedics extensively approved was recorded." carrier " refers to thinner, adjuvant (as Freund's complete adjuvant and Freund's incomplete adjuvant), vehicle or other carrier helping therapeutical agent to use.Pharmaceutical carrier can be aseptic liquid, Ru Shui and oil, and oil comprises the oil of oil, animal oil, vegetables oil or synthesis, as peanut oil, soybean oil, mineral oil, sesame wet goods and so on.Water by the carrier of the best during intravenous (IV) drug preparation.When preparing injection type mixture, salt solution, glucose and glycerol liquids can be adopted.Description is had in " Lei Mingdun pharmacopedics " (Remington ' s Pharmaceutical Sciences) that the example of suitable pharmaceutical carriers is write at E.W. Martin.

Typical pharmaceutical preparation and formulation contain one or more vehicle.The those of skill in the art of suitable vehicle pharmaceutical field are known, include but not limited to starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, mica, sodium-chlor, skimmed milk powder, propylene, ethylene glycol, water, alcohol etc. and so on.Whether certain vehicle is applicable to being incorporated into pharmaceutical preparation or formulation depends on the known several factors in this field, includes but not limited to, formulation is applied to the special active ingredient in the mode of patient and formulation.If needed, preparation or single formulation can containing micro-wetting agent, emulsifying agent or pH buffer reagents.

Pharmaceutical preparation contains vehicle well known in the art or the vehicle of publication on such as American Pharmacopeia (USP) SP (XXI)/NF (XVI).In general, lactose free preparation contains active ingredient, tackiness agent/weighting agent, and the pharmaceutically compatible and acceptable lubricant of dosage.Typical lactose free formulation contains active ingredient, Microcrystalline Cellulose, pre-gelatinized starch and Magnesium Stearate.

Pharmaceutical preparation of the present invention and formulation contain the compound that one or more reduce active ingredient rate of decomposition.This compound is called " stablizer " herein, and it includes but are not limited to antioxidant as xitix, pH damping fluid or salt buffer.

Pharmaceutical preparation and single formulation can adopt following form: solution, suspension, emulsion, tablet, capsule, powder, sustained release form etc. and so on.Oral preparation contains the N.F,USP MANNITOL of carrier as pharmaceutical grade, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate etc. of standard.What this medicament and formulation contained the purifying of prevention or therapeutic dose plays prevention or the medicament of therapeutic action, in order to can better must be administered to patient can and a certain amount of carrier mix with figuration.Medicine type should be applicable to the mode of administration.In the embodiment optimized, pharmaceutical preparation or single formulation should be aseptic and use in an appropriate form, preferably to liking animal, preferred to liking Mammals, most preferred to liking people.

The form of the pharmaceutical preparation containing NRG should be able to adapt to its administering mode.Administering mode is interior including, but not limited to injection (such as vein, muscle, subcutaneous or intradermal injection), oral, oral cavity (as sublingual), suctions, nose, by skin, locally, by mucous membrane, tumour, in synovial membrane and rectal administration.In certain particular, the formulation of preparation can with reference to certain conventional procedure, be such as used for preparing with in vein or subcutaneous or intramuscular injection, oral, nose or topical modes be administered to the conventional procedure of the pharmaceutical preparation of people.In certain embodiments, the form of pharmaceutical preparation meets the usual manner of subcutaneous administrations.Typically, the preparation of intravenous administration mode is sterile isotonic solution.If needed, said preparation also can containing solubilizing agent and local anesthetic as lignocaine to alleviate the pain of injection site.

Formulation is including, but not limited to following form: tablet, caplet, capsule are as soft elastic gelatin capsule, cachet, tablet, lozenge, dispersion agent, suppository, ointment, poultice (poultice), paste, powder, dressing, emulsion, plaster, solution, patche, gaseous solvents (as nasal spray or sucker), colloid; Liquid dosage form is applicable to patient that is oral or mucosa delivery, and liquid dosage form comprises suspension (as moisture or non-aqueous suspension, oil-in-water emulsion or water-in-oil emulsion), solution and panacea; Liquid dosage form is applicable to the patient of injecting drug use; Sterile solid (as crystal or noncrystal) becomes the liquid dosage form of applicable injecting drug use patient by rebuild.

Different according to purposes, the preparation of NRG polypeptide, shape and formulation classification are also by difference.For example, the formulation for acute treatment disorder may contain more NRG polypeptide than the formulation of same disease long-term treatment.Similarly, there is the formulation of result for the treatment of also different for various cancers.Similarly, the amount of active ingredient that contains of injection type is fewer than the amount of the oral dosage form for the treatment of same disease or disorder.It is different that those skilled in the art know other special form comprised in above-mentioned preparation way and the present invention.Refer to " Lei Mingdun pharmacopedics ", the 18th edition, Mack press, Easton, Pennsylvania (1990).

The administration of NRG polypeptide can be carried out, including, but not limited to these with any approach according to the judgement of those skilled in the art: oral, intravenous injection, gastric infusion, duodenal administration, Intraperitoneal medication or intracerebroventricular administration.

c. dosage and route of administration

The difference of the route of administration along with disease or the character of uncomfortable situation and the difference of severity and active ingredient changes by the consumption of the NRG in the present invention.Specific factor along with each patient also changes by administration frequency and dosage, depend on specific treatment (such as, therapeutic or prophylactic formulation), disorder, the severity of disease or discomfort, route of administration and age, body weight, response situation, also have the past medication history of patient.The dose-response curve that can obtain from external or animal model test macro is to infer effective dose.

The using dosage imitated of NRG comprises the NRG (e.g., about 1 microgram per kilogram of body weight is to about 500 milligrams of pers kilogram of body weight, about 100 microgram pers kilogram of body weight to about 5 milligrams of pers kilogram of body weight or about 1 microgram per kilogram of body weight extremely about 50 microgram pers kilogram of body weight) of administration object per kilogram of body weight how many milligrams or microgram.As given the dosage of patient, be that the weight of patient's per kilogram of body weight bioactive peptide used is 0.001mg/kg-15mg/kg in typical case.Suitable consumption also has: 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.

The using dosage imitated of NRG also comprise administration object per kilogram of body weight how many units (U) or unit vol NRG (as, about 1U per kilogram of body weight is to about 5,000U per kilogram of body weight, about 10U per kilogram of body weight are to about 1,000U per kilogram of body weight or about 100U per kilogram of body weight extremely about 500U per kilogram of body weight).As given the dosage of patient, be that the unit of patient's per kilogram of body weight bioactive peptide used is 10U/kg-1,000U/kg in typical case.Suitable consumption also has: 1U/kg-10,000U/kg, 1U/kg-5,000U/kg, 10U/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.

Generally speaking, for various diseases described herein, the amount ranges of NRG every day of recommending in method of the present invention is: every day is about 0.001mg to 1000mg.Under particular case, the scope of the medication total amount of every day can be: 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.To patient arrange treatment time, start use low dosage, such as every day about 0.1 μ g-1 μ g, if necessary can increase to μ g-1 every day about 20,000 μ g, can single dose use also can gradation use, depend on the W-response of patient.In some cases, the dosage of active ingredient used is necessary to exceed the scope said in this place, and this is apparent for the person of ordinary skill of the art.In addition, it should be noted that clinician or treating physician it will be appreciated that response situation according to individual patient thus how and when to interrupt, adjust or stopped treatment.In some particular cases, the usage quantity of NRG is about 1U/ days-10,000U/ days.In some specific cases, the usage quantity of NRG is about 1U/ days-5,000U/ days.In some specific cases, the usage quantity of NRG is about 10U/ days-2,000U/ days.In some specific cases, the usage quantity of NRG is about 10U/ days-1,000U/ day.In some specific cases, the usage quantity of NRG is about 100U/ days-200U/ skies.

NRG can also carry out administration by dosage schedule or " treatment cycle ".In treatment cycle, the dosage of every day is listed in detail above.Treatment cycle can continue 2 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.

In some particular cases, in treatment cycle, every day all uses NRG.In some specific cases, in a treatment cycle, the SM time of NRG is 3,4,5,6,7,8,9,10,11 or 12 days.In some particular cases, give NRG at the first day of a treatment cycle, then do not give NRG one day for the treatment of cycle remainder or a couple of days.In some specific cases, in a treatment cycle, give NRG every day and continue 3,5,7 or 10 days, time then not administration remaining in this cycle.

Embodiment

embodiment 1 NRG chemiluminescent polypeptide synthesizes

NRG polypeptide NRG53 (SEQ ID NO:3), NRG polypeptide NRG55 (SEQ ID NO:4), NRG polypeptide NRG57 (SEQ ID NO:5), NRG polypeptide NRG59 (SEQ ID NO:6), NRG polypeptide NRG61 (SEQ ID NO:1) are synthesized by gill biochemical corp, Shanghai.

NRG61 is containing, for example lower aminoacid 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. people NRG-1 aminoacid sequence 177-237.

The nucleotide sequence that above-mentioned aminoacid sequence is corresponding is: agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga ggagctgtac cag (SEQ ID NO:2).

The aminoacid sequence of NRG polypeptide EGF53 is as follows: 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 (SEQ ID NO:3).

The nucleotide sequence that above-mentioned aminoacid sequence is corresponding is: agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt actggtgatc gctgccaaaa ctacgtaatg gcgagcttc (SEQ ID NO:4).

The aminoacid sequence of NRG polypeptide NRG55 is as follows: 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:5).

The nucleotide sequence that above-mentioned aminoacid sequence is corresponding is: agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaag (SEQ ID NO:6).

The aminoacid sequence of NRG polypeptide NRG57 is as follows: 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 (SEQ ID NO:7).

The nucleotide sequence that above-mentioned aminoacid sequence is corresponding is: agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga g (SEQ ID NO:8).

The aminoacid sequence of NRG polypeptide NRG59 is as follows: 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 (SEQ ID NO:9).

The nucleotide sequence that above-mentioned aminoacid sequence is corresponding is: agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga ggagctg (SEQ ID NO:10).

Aforementioned polypeptides is dissolved in respectively Buffer 1 (0.1M Na ₂hPO ₄, 0.1M citric acid, 6M urea, 1mM EDTA-Na ₂, 5mM DTT, pH8.0) in, concentration is 1mg/ml, and lysate places room temperature 1 hour, by 1ml lysate and 9ml Buffer 2(0.01M Na ₂hPO ₄, 0.01M citric acid, 6M urea, 1mM EDTA-Na ₂, 0.5mM GSSG, 0.5mM GSH, pH8.0) and mixing, polypeptide final concentration is 0.1mg/ml.Again by mixed solution stirring at room temperature 90 minutes, place 16 hours for 4 DEG C afterwards.Also collect with sephadex G25 separating sample and be dissolved in Buffer 3(20mM Na ₂hPO ₄, 20mM NaH ₂pO ₄, pH6.0) in.

the combination of the NRG polypeptide of embodiment 2 acceptor and chemosynthesis

Collect MCF-7 cell, counting, centrifugal and be resuspended in DMEM(10% serum, 9 μ g/ml Regular Insulin) in, cell density is 5 × 10 ⁴/ ml.Spread 96 orifice plates, every hole adds 100 μ l suspension, and 37 DEG C are spent the night.Within second day, wash three times cells with PBS, change liquid serum-free DMEM and cultivate 24 hours.

Liquid (50mM Na is buffered with bag ₂cO ₃-NaHCO ₃, pH9.6) and dilute ErbB2 antibody H4(ErbB2 monoclonal antibody, pool is raw) to 6 μ g/ml, be added in 96 orifice plates, every hole 50 μ l.4 DEG C of reactions are spent the night and are made antibody and harden to close.

Suck the DMEM nutrient solution in MCF-7 cell, NRG, NRG53, NRG55, NRG57, NRG59, NRG61 DMEM is added in hand-hole after serial dilution, every hole 100 μ l.Blank only adds DMEM.Hatch 20 minutes for 37 DEG C, wash one time with PBS damping fluid, then add 100 μ l/ hole lysis buffer (50mM Hepes, pH8.0,150mM NaCl, 2mM sodium orthovanadate, 0.01% thimerosal, 1% Triton X-100 and 1 proteinase inhibitor mixes partially/25ml), 4 DEG C of cracking 30 minutes, light balance on a plank makes cell come off from plate afterwards, centrifugal 15,000rpm 15 minutes.

The plate of antibody bag quilt rinses 5 times by washing lotion (10mM PBS, pH7.4,0.05% Tween 20), and every hole adds the washing lotion of 200 μ l 5% skimmed milks, hatches 2 hours for 37 DEG C, then washes 3 times by washing lotion.

Enchylema after cracking is added by every hole 90 μ l and wraps by plate accordingly, hatch 1 hour for 37 DEG C, wash 5 times by washing lotion afterwards, the horseradish peroxidase (HRP) adding 100 μ l suitable concns joins phosphotyrosine antibody (Santa Cruz biotechnology), hatches 1 hour for 37 DEG C.Washing lotion washes 5 times, adds fresh ready HRP substrate solution (50mM citric acid, 100mM Na ₂hPO ₄, pH5.0,0.2mg/ml tetramethyl benzidine (TMB), 0.003%H ₂o ₂), hatch 10 minutes altogether for 37 DEG C.Last every hole adds 50 μ l 2M H ₂sO ₄destroy HRP active in termination reaction.In microplate reader (BIORAD Model 550) upper measurement 450nm every hole OD value, EC50 is the concentration of the NRG polypeptide reaching maximum light absorption value half.EC50 value is lower, and the avidity of acceptor and NRG polypeptide is higher.

The EC50 value of NRG (total length wild-type neuregulin β 2 peptide section), NRG53, NRG55, NRG57, NRG59 and NRG61 is as shown in table 1.

The EC50 value of table 1 NRG, NRG53, NRG55, NRG57, NRG59 and NRG61

Sample	EC50(μg/ml)
		NRG	0.2772
NRG53	0.814
		NRG55	0.0492
NRG57	0.9783
		NRG59	0.4605
NRG61	1.439

As shown in table 1, the EC50 value of NRG55 is far below the EC50 value of NRG

in embodiment 3 myocardial cell, NRG polypeptide is on the impact of AKT phosphorylation

In order to study the polypeptide that synthesizes in myocardial cell to the impact of ErbB2 and ErbB4 signal path, take the heart cell of neonate rat left ventricle.

In the substratum having serum, cultivate heart cell, cultivate two days, when cell covers with about 80%, change substratum into serum free medium.Cultivate after 24 hours, each hole adds separately NRG polypeptide or the neuregulin of the synthesis of different amount, reacts 20 minutes.Suck substratum afterwards, add gel-loading buffer with lysing cell, collect sample afterwards and loading, start gel electrophoresis and do western blot analysis.

What Fig. 1 showed is that the NRG polypeptide of different concns is on the impact of AKT phosphorylation.As shown in Figure 1, NRG57 and NRG59 almost can reach the equivalent efficacy of NRG, and NRG55 has stronger phosphorylation than NRG.Result display NRG55 may be the more effective polypeptide for the treatment of heart disease.

Scope of the present invention is not limited to the description of embodiment.Can carry out many modifications and variations to the present invention, and not deviate from its spirit and scope, this is to it will be readily apparent to those skilled in the art that.Specific embodiments described here is provided by means of only embodiment, and the present invention only limited by whole categories of claims and equivalence with it.

<110> Zesheng Science and Technology Development Co Ltd, Shanghai

<120> NRG and uses thereof

<130> ZS-024-228

<150> US 61/118,566

<151> 2008-11-28

<160> 11

<210> 1

<211> 61

<212> PRT

The <213> mankind

<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> 183

<212> DNA

The <213> mankind

<400> 2

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc 60

ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt 120

actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga ggagctgtac 180

cag 183

<210> 3

<211> 53

<212> PRT

The <213> mankind

<400> 3

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

50

<210> 4

<211> 159

<212> DNA

The <213> mankind

<400> 4

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc 60

ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt 120

actggtgatc gctgccaaaa ctacgtaatg gcgagcttc 159

<210> 5

<211> 55

<212> PRT

The <213> mankind

<400> 5

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

50 55

<210> 6

<211> 165

<212> DNA

The <213> mankind

<400> 6

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc 60

ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt 120

actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaag 165

<210> 7

<211> 57

<212> PRT

The <213> mankind

<400> 7

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

50 55

<210> 8

<211> 171

<212> DNA

The <213> mankind

<400> 8

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc 60

ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt 120

actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga g 171

<210> 9

<211> 59

<212> PRT

The <213> mankind

<400> 9

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

50 55

<210> 10

<211> 177

<212> DNA

The <213> mankind

<400> 10

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc 60

ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagttt 120

actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga ggagctg 177

<210> 11

<211> 23

<212> PRT

The <213> mankind

<400> 11

Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met

1 5 10 15

Val Lys Asp Leu Ser Asn Pro

20

Claims

1. a NRG polypeptide, its aminoacid sequence is the amino acid of in SEQ ID NO:1 front 57 and 59.

2. NRG polypeptide according to claim 1, its aminoacid sequence is SEQ ID NO:7.

3. NRG polypeptide according to claim 1, its aminoacid sequence is SEQ ID NO:9.

4. a pharmaceutical preparation, it contains NRG polypeptide and pharmaceutically acceptable carrier, vehicle or thinner, and the aminoacid sequence of described NRG polypeptide is the amino acid of in SEQ ID NO:1 front 57 and 59.

5. pharmaceutical preparation according to claim 4, the aminoacid sequence of described NRG polypeptide is SEQ ID NO:7.

6. pharmaceutical preparation according to claim 4, the aminoacid sequence of described NRG polypeptide is SEQ ID NO:9.

7. a NRG polypeptide is for the preparation of the purposes in treatment heart failure drugs, comprise the NRG polypeptide using effective dose to the individuality of needs treatment, the aminoacid sequence of described NRG polypeptide is the front amino acid of 53,57 and 59 in SEQ ID NO:1.

8. purposes according to claim 7, the aminoacid sequence of wherein said NRG polypeptide is SEQ ID NO:3.

9. purposes according to claim 7, the aminoacid sequence of wherein said NRG polypeptide is SEQ ID NO:7.

10. purposes according to claim 7, the aminoacid sequence of wherein said NRG polypeptide is SEQ ID NO:9.

11. purposes according to claim 7, wherein said individuality is people.

12. purposes according to claim 7, wherein NRG polypeptide is by intravenously administrable.