CN1138785C - Application of growth factor neuregulin and its analogs - Google Patents

Abstract

The present invention relates to the application of growth factor neuregulin and the analogs of the growth factor neuregulin. The growth factor neuregulin and the analogs thereof are applied to promote the cell differentiation of myocardium, induce the reconstruction of sarcomere and cytoskeletal structures in the cells of the myocardium or the bonding among the cells, identify polypeptides or compounds capable of inhibiting the neuregulin to stimulate the cell differentiation of the myocardium and prepare medicine for treating cardiac diseases and cardiac failures.

Description

The new application of rhNRG-1BETA S1Q237 and analogue thereof

The present invention relates to influence application, especially rhNRG-1BETA S1Q237 and the application of analogue in improving heart patient myocardial function thereof of the polypeptide class of myocardial contraction unit's combination.

The population of heart failure affects 1.5%, the U.S. has 3,000,000 people to have a heart disease approximately, and is increasing with annual 400000 people's speed.Treatment to heart failure mainly is with hypertensin conversion enzyme (ACE) inhibitor and hydragog(ue) at present.ACE inhibitor was improved in heart failure the demonstration slowly in late period, only had the symptom that is less than 60% patient to be alleviated, and mortality ratio only reduces 15-20%.Heart transplantation is restricted owing to lacking donor's heart.Except digoxin, the prolonged application of other active drugs all can have side effects, and comprises arrhythmia and sudden death.

During heart development, myocardial cell's growth transfers hypertrophy to from propagation.The former is for increasing myocardial cell's quantity, and the latter then increases cell, synthetic DNA not, and cell is no longer division also.This conversion is accompanied by myocardial cell's ultimate differentiation during heart development.The conversion start from late embryo stage, end at the birth several weeks after.In the meantime, expression of gene specially refers to cell cycle and signal and is regrouped, and the phenotype of cell also is changed becomes cell-cell adhesion, and the contractile protein among the myocardial cell of ultimate differentiation is also made up more in an orderly manner.

After the stimulation of various pathology causes cardiac muscle impaired, or when myocardium work requirements increased, adult's cardiac hypertrophy was a kind of adaptive physiological response.Normal mastocyte body is big, have increase and to make up intact contractile unit bonding with strong iuntercellular, and contractile protein makes up unordered, intercellular bonding bad in the mastocyte of morbid state.

The align-err of myofiber and sarcostyle is myocardiac key character.The former is an iuntercellular bonded confusion, and the latter is the unordered combination of contractile protein in the cardiac muscle.They are subjected to the influence of special cells signal.Like this, some signals as somatomedin and hormone will change the bonding and sarcomeric structure of iuntercellular.Do not have these token stimulus, demonstrate cytoskeleton and the erroneous arrangement of sarcomere structure and separating of cell-cell interaction.Because rebuilding of myocardial cell's differentiation and heart cell, bonding and combination contractile protein is closely related, and these stimulate the factor of myocardial cell's differentiation may play conclusive effect to the assembling that strengthens sarcomere structure in the sophisticated myocardial cell.

The systematic research of myocardial cell's isolated model confirms that the stimulation on some physics, hormone, somatomedin and the pathology can excite independently phenotypic characteristic (Chien etc. (1991) FASEBJ.5:3037-3046 of some myocardial hypertrophies; Zhou etc., (1995) PNAS, USA, 92:7391-7395).At present, in these isolated models, there are three kinds of signal pipelines at least, relate to ras-, the downstream effect thing of rho and Gq albumen dependent form.

When the research to the signal pathway that excites myocardial cell's hypertrophic response makes substantial progress, but know seldom about the assembling mechanism of special stimulation myocardial cell's ultimate differentiation and the contractile protein that links with it relatively.The compound that can influence these processes may become the main novel treatment of a class that is used to handle various heart diseases.

Neuregulin (neuregulin) is the somatomedin of a kind of Urogastron (EGF) class.It can activate erbB acceptor in the EGF receptor family, and relates to other some biological respinses: as stimulating the secretion of mastocarcinoma cytodifferentiation and milk-protein; Induce neural lophocyte to be divided into the Schwann cell; Stimulate the synthetic of the interior acetylcholine receptor of skeletal muscle cell; And it is synthetic to promote that the myocardial cell survives with DNA.The viviperception proof neuregulin of making of the mice embryonic of neuregulin gene major defect is essential for heart and neurodevelopment.Yet information how to control cytodifferentiation and its downstream signal approach about neuregulin is limited.

In the heart of Fa Yuing, express in each comfortable endocardium lining of neuregulin and ErbB acceptor and the cardiac muscle in early days, because these two layers separately very wide, neuregulin must pass the space in two confluent monolayer cells, activates the ErbB acceptor again.Activating acceptor among these myocardial cells for promoting the muscle cell growth or moving to endocardium, is necessary thereby form the finger-type structure in two confluent monolayer cells.It is unclear that the differentiation whether neuregulin has stimulated the myocardial cell in the past.

The present invention is based on and find that neuregulin strengthens myocardial cell's differentiation, the combination of sarcomere and cytoskeleton and intercellular bonding.Neuregulin, the neuregulin polypeptide, the neuregulin derivative, or those have the compound of similar neuregulin effect, are abbreviated as NRG in the present invention.The inventor finds that now neuregulin and its cytosis may be applicable to detection, diagnosis and processing heart disease.

Therefore, the purpose of this invention is to provide the new application of neuregulin in external promotion myocardial cell differentiation.

Another object of the present invention provides that neuregulin sarcomere and cytoskeletal structure in inducing the myocardial cell are rebuild or the new application of iuntercellular in bonding.

A further object of the invention provides neuregulin can suppress the polypeptide of neuregulin stimulation myocardial cell differentiation or the new application in the compound in identification.

A further object of the invention provides the new application of neuregulin in preparation treatment heart trouble and heart failure drugs.

The present invention includes a kind of method of breaking up the external myocardial cell of impelling.This method comprises with the neuregulin of significant quantity handles the myocardial cell, thereby activates the map kinase approach and cause that the myocardial cell breaks up.

The present invention also comprise a kind of in external evoked myocardial cell sarcomere and cytoskeletal structure rebuild or iuntercellular adherent method.This method comprises with the neuregulin of significant quantity handles cell, thereby activates the map kinase approach, cause cyto-architectural rebuild or iuntercellular bonding.

Neuregulin can directly offer cell, also can produce by inducing those to relate to the genetic expression of neuregulin synthetic indirectly.Neuregulin can produce by self cell or by genetic expression in other cells.

The present invention comprises identification can stimulate the polypeptide that the myocardial cell breaks up or the method for compound.This method comprises: when myocardial cell's induced differentiation thing neuregulin exists, with other polypeptide or compound contact cardiac muscle, and measure the development that the myocardial cell breaks up.The myocardial cell is exposed to neuregulin respectively, other polypeptide, or under the mixture of neuregulin and certain peptide species, measure the differentiation of cell.The differentiation assays of cell comprises that map kinase is at the analysis of Phosphorylation of different time, inhibition-p21 of cell cycle in the increase and decrease of DNA synthetic, the myocardial cell^CIP1The accumulation, cytoskeleton of phenotype combination, contractile unit of expression numerical value, contractile unit in the phenotype of Actin muscle fiber change and the mensuration of iuntercellular adherent phenotype.

Can stimulate in the method for polypeptide that the myocardial cell breaks up or compound in identification, first example is that cell is cultivated with the polypeptide or the compound of different concns, measures the various influences that the myocardial cell is broken up.

Second example is: whether the effect of discerning the differentiation of these polypeptide or compound inducing cell is better than the effect of the strong inductor-insulin-like growth factor (IGF-1) of myocardial cell's propagation.Cell is cultivated in the nutrient solution that adds IGF-1 or IGF-1 and other polypeptide or compound separately, measured these polypeptide or compound and suppress IGF-1 inducing DNA synthetic ability, and the assembling of mensuration sarcomere structure and intercellular bonding.

The 3rd example is: cell is put into to add phenylephrine (PE) separately or add test again cultivate with the nutrient solution of polypeptide or compound.The ability that these polypeptide or compound increase the myocardial cell's differentiation that is caused by PE is determined.The polypeptide that can stimulate the myocardial cell to break up can stimulate sarcomeric assembling, thereby cardiac function enhancing in many ways comprises single-minded acceptor such as the ErbB2, ErbB3 and the ErbB4 that activate neuregulin.

The present invention has also comprised a kind of identification and can suppress neuregulin and stimulate the polypeptide of myocardial cell's differentiation or the method for compound.This method is included in neuregulin and exists down, makes the polypeptide or the compound contact myocardial cell of test, and measures the restraining effect that they stimulate neuregulin.A kind of compound can be by blocking-up, compacting, and reverse or antagonism neuregulin stimulate the effect of myocardial cell's differentiation.Concrete steps are synthetic for the DNA that measures the myocardial cell.

The present invention has also comprised and has a kind ofly prevented that in Mammals adherent between the myocardial cell from separating and/or the methods of treatment of sarcomere erroneous arrangement.This method comprises the neuregulin of administration effective dose or compound.Methods of treatment is for directly handling the heart failure that is caused by bonding separation between the myocardial cell and/or sarcomere structural arrangement mistake.

The present invention has also comprised a kind of method that prevents or reduce the sick sickness rate of mammalian heart.This method comprises and prevents or reduce some polypeptide or the compound interfere with or compromise to neuregulin and receptor acting thereof that these influences produce in heart failure.

In another object lesson, handle or prevent the myocardial cell's that PE or IGF-1 transmit dysfunction with a kind of therapeutical agent that exactly likes the neuregulin effect.

In addition, the present invention has also comprised the method for the quality of a kind of definite curee's heart trouble or heart failure.This method comprises the neuregulin that can the myocardial cell that measure the curee or relevant cell express normal or enough levels or the ability of its ErbB acceptor.Do not have this ability person and indicate tool heart disease or quality in heart failure.

The present invention includes and use neuregulin, neuregulin polypeptide, neuregulin derivative or the compound with similar neuregulin effect prepare and are used to handle or treat heart trouble and medicine in heart failure.This medicine that makes by neuregulin also can select with certain compound that can suppress different loose ways of regeneration in conjunction with life-time service.Other selectable compositions comprise heart nutrition inhibitor (as the Ct-1 antagonist), ACE inhibitor such as captopril (captopril), human growth factor and the IGF-1 that is used for the congestive heart failure case, or other are used for the anti-hypertrophy of the case of other heart failure or cardiac disorders, myocardial nutrition, antiarrhythmic, or the Inotropic factor.

The present invention comprises that also producing recombinant human nerve regulates proteic method.

By cultivating the myocardial cell as model, the present inventor assessed the assembling of myocardial cell's differentiation and maturation and sarcomere and cytoskeletal structure or keep in the neuregulin signal.Regulate albumen (rhNRGB2) culturing embryo fetal rhythm myocyte with the recombinant human nerve of purifying, measure the influence of neuregulin pair cell signal.10^-8The map kinase that the M neuregulin produces at least 21 little durations activates lastingly, and 10^-10The rhNRGB2 of M only can produce of short duration activation.The inhibition p21 of Cdk^CIP1Be expressed in 10^-8M increases during concentration, 10^-10Do not increase during M.Increase NRG concentration, follow p21^CIP1The increase of expressing produces synthetic minimizing of DNA and causes ultimate differentiation.And when lower concentration, then observe the continuation of synthetic increase of DNA and cell proliferation.In addition, when rhNRGb2 with 10^-8M, or 10^-10When M concentration is mixed with IGF-1, do not see DNA synthetic negatively influencing, and blocked the myocardial cell that IGF-1 stimulated propagation significantly.For myocardial cell's differentiation of further assessment NRG stimulation, the sarcomere of cultured neonatal rat cardiac myocytes and cytoskeletal structure are measured with immunofluorescence dyeing (with the antibody of anti-actinine) with phalloidin dyeing.NRG has improved sarcomere and cytoskeletal structure and intercellular bonding significantly.Then there is not above-mentioned effect with IGF-1 or PE processing.When mixing with IGF-1 or PE with rhNRGB2, rhNRGB2 has improved cellularstructure.10^-8The rhNRGB2 of M concentration has shown sarcomere and the bonding improved maximum effect of iuntercellular.In addition, neuregulin is overlapping by the negative adjusting of the MHC-α of PE stimulus delivery.The function that NRG is pointed out in these discoveries is by two kinds of different approach: a kind of approach causes that the myocardial cell grows when lower concentration, and another kind of approach when high density by activating the transmission of map kinase approach enduringly, and cause that ultimate differentiation is with ripe.

The present invention can handle and prevent Mammals heart failure or myocardial cell's hypertrophy by the neuregulin that gives significant quantity.Preferable is to can be used for handling one to have suffered from the patient that maybe will suffer from heart failure.

The present invention can be used for also preventing that those patients are owing to using certain medicine to cause cardiac hypertrophy or congestive heart failure.In the method for invention, the neuregulin polypeptide can use this kind cause the medicine of heart disease before, simultaneously or use afterwards.

In the methods of treatment of invention, the neuregulin polypeptide can be for a long time or is injected patient's people vein acutely.

The present invention can combine with methods of treatment now and be used for processing in heart failure, combines as handling with ACE inhibitor.ACE inhibitor is the angiotensin converting enzyme inhibitor thing, and it can prevent that angiotensin i-converting is converted to Angiotensin II.ACE inhibitor can be benefited congested person in heart failure by reducing the opposing of whole body blood vessel and alleviating round-robin hyperemia.ACE inhibitor comprises that those trade marks are Accupril  (quinapril), Altace  (Ramipril), Capoten  (captopril), Lotensin  (benazepril), Monopril  (fosinopril), Prinivil  (lisinopril), Vasotec  (enalapril), and Zestril  (lisinopril).

The present invention can combine with processing heart disease such as hypertensive pharmacological agent.For example, the neuregulin polypeptide can be used with endothelial factor acceptor opposing agent, as the antibody of endothelial factor acceptor, and peptide class and small molecules opposing agent; β-adrenoceptor opposing agent such as carvedilol; α₁-adrenoceptor opposing agent; Antioxidant; The compound of multiple effect (as β-encapsulant/α-encapsulant/antioxidant); Carvedilol-compounds or the compound combination of the multiple function of tool in carvedilol; And somatomedin etc.

The neuregulin analogue combines usefulness separately or with other loose depressor approach promotor or the molecule of resisting known loose ways of regeneration, can be as the medicine of handling the Mammals heart failure.

Describe in further detail with reference to following accompanying drawing below.

Fig. 1 represents that neuregulin reduces DNA synthetic situation in the embryonic myocardium cell under different concns.The myocardial cell of purifying cultivates in 96 orifice plates of collagen coating.Add carrier (X) in contrast, rhNRGB2 (▲), or IGF-1 (◆) are in different concentration.Cell after cultivating 24 hours with [³H] the thymidine act of union ([³H] thymidine incorporation) mensuration DNA.The mean value of data data from 5 culture dish adds the standard error gained.

Fig. 2 represents that neuregulin stimulates the lasting activation that causes map kinase in the myocardial cell.(a) cell is cultivated with serum-free medium, adds carrier (-), or adds 10^-8M, or 10^-10The rhNRGb2 (+) of M, the time is as shown in showing.Collect processed cell and do the Western blotting with special P hospo-Mark antibody.Western blotting with anti-erbB 2 antibody comes stdn.(b) band intensity is quantitative with densometer, two time curves: map kinase is to the reaction of 10-8M part (▲) and 10-10M part (zero).

Fig. 3 represents that the neuregulin stimulation has increased p21 in the myocardial cell^CIP1Expression.(a) serum (5%FBS) exist or non-existent condition under with 10^-8M, or 10^-10The rhNRGb2 of M stimulated the cultivation myocardial cell 24 hours.Cell is done the analysis of Western blotting after with the cracking of electrophoresis sample loading buffer.P21^CIP1Use anti-p21^CIP1Monoclonal antibody measuring.The protein content of application of sample expresses stdn by measuring erbB2.(b) in the density analysis result of the first row band, white or horizontal stripe rod are the p21 without ligand stimulation^CIP1Express, grey or vertical stripe rod are through the lower concentration ligand stimulation, and reaching black or twill rod is to stimulate through high density.

Fig. 4 represents that neuregulin suppresses IGF-1 and stimulates the DNA among the myocardial cell synthetic.With optimum concn (10^-9M) IGF-1 is 10^-8M, or 10^-10There is or does not exist the cell that stimulates down in the 96 hole culture dish in the rhNRGb2 of M.Use after 24 hours [³H] DNA is synthetic for thymidine act of union mensuration.Rod among the figure has shown that the mean value that 5 holes are measured adds the standard error, and the result is from least three times repeated experiments.

Fig. 5 represents Actin muscle fiber combinations among the enhanced myocardial cell of neuregulin institute.Neonatal cardiac myocytes is with adding or not adding 10^-8The serum-free medium of M rhNRGb2 is fixed with phalloidin after cultivating, dyeing.At the Ultraluminescence microscopically, the myocardium myo filamentous actin manifests red fiber at cell, takes pictures with 40 times of mirrors.

Fig. 6 represents that proteic assembling of neuregulin institute enhanced sarcomere and combination and iuntercellular are bonding.Neonatal cardiac myocytes adds 10 with serum-free medium (a)^-10The rhNRGb2 of M (b) adds 10^-8The rhNRGb2 of M (c) cultivated 48 hours.Antibody with anti-α-actinine carries out immunity-fluorescent dye then.At the Ultraluminescence microscopically, the heart actinine in the sarcomere manifests green fiber.Take pictures with 40 times of mirrors.

Fig. 7 represent neuregulin improved sarcomere structure and iuntercellular in the myocardial cell that IGF-1 stimulates bonding.The myocardial cell adds 10 with serum-free medium (a)^-9The IGF-1 of M, (b) (or) add 10^-9The IGF-1 of M and 10^-8The rhNRGb2 of M cultivated 48 hours.Then cell fixation is also carried out immunity-fluorescent dye with the antibody of anti-α-actinine.At the Ultraluminescence microscopically, the heart actinine in the sarcomere manifests green fiber.Take pictures with 40 times of mirrors.

Fig. 8 represent neuregulin improved sarcomere structure and iuntercellular in the myocardial cell that PE stimulates bonding.The myocardial cell is added 10 with serum-free medium (a)^-4The PE of M (b) (or) add 10^-4The PE of M and 10^-8The rhNRGb2 of M cultivated 48 hours.Then cell fixation is also carried out immunity-fluorescent dye with the antibody of anti-α-actinine.At the Ultraluminescence microscopically, the heart actinine in the sarcomere manifests green fiber.Take pictures with 40 times of mirrors.Two kinds of cell phenotypes stimulate observed separately by PE and are labeled as A and B.

Fig. 9 represents that neuregulin induces the expression of the myoglobulin heavy chain αYi Gouti (MHC-α) of PE-compacting.The myocardial cell cultivates with serum-free medium and adds PE respectively, NRG, or PE and NRG.Relevant MHC-α and the expression level of MHC-β are measured with the PT-PCR in low cycle (20 cycles).PCR output is represented this two expression levels that gene is relative.

Utilize the stripped system of Cardiomyocyte Differentiation, with two fixed by α-adrenaline promoter and The hormone that IGF-1 produces and the stimulation of growth factor are made comparisons, and neuregulin stimulates the function of differentiation reaction Obtain confirming. The present invention has confirmed that the neuregulin differentiation pathway is present among the cardiac muscle cell, and neural the accent The joint polypeptide can activate these approach. Because cardiac muscle cell's differentiation comprises the combination of sarcomeres structure and reaches Therefore the process that iuntercellular is bonding the invention provides one and processes and prevent sarcomeres knot among the cardiac muscle cell The process useful of the bonding wrong combination of the combination of structure and iuntercellular, the method also can strengthen myocardiac heart merit Can, identification activates polypeptide or the compound of Cardiomyocyte Differentiation approach.

Term used herein is as follows:

" neuregulin or neuregulin analog " be some can activate ErbB2/ErbB4 or The molecule of the different bigeminy protein tyrosine kinase of ErbB2/ErbB3 is such as neuregulin isomers, nerve modulation EGF zone in the albumen, neuroregulation protein mutant and any nerve modulation egg that can activate above-mentioned acceptor The gene outcome of white class. " neuregulin " used among the present invention is following polypeptide, this polypeptid system people god Through regulating a fragment ofalbumen β 2 isomers, wherein comprised receptor binding domain EGF-class district.

The order of cDNA is:

AGC CAT CTT GTA AAA TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGA GGG GAG TGC TTC ATG GTG AAA GAC CTT TCA AAC CCC TCG AGA TAC TTG TGC AAG TGC CCA AAT GAG TTT ACT GGT GAT CGC TGC CAA AAC TAC GTA ATG GCC AGC TTC TAC AAG GCG GAG GAG CTG TAC CAG

Amino acid sequence according to above-mentioned cDNA sequential encoding is:

SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYV MASFYKAEELYQ

" Cardiomyocyte Differentiation " is a kind of state, it is characterized by the synthetic reduction of DNA greater than 10%, the synthetic inhibition that is stimulated by other factors of DNA is greater than 10%, the sarcomeres structure that fully makes up and intercellular bonding, to the lasting activation of map kinase, with p21^CIP1Expressing increases.

" enhancing of orderly sarcomeres or sarcomeres structure " is a kind of state, and it is thin to it is characterized by cardiac muscle Contractile protein shows in line row by immunity-fluorescent staining of α-actinine among the born of the same parents. The moving egg of α-accessorius in the cell The white in line photography that is listed in available microexamination in the chart and links to each other is distinguished.

" mistake combination or the arrangement of sarcomeres or sarcomeres structure " is opposite with above-mentioned definition meaning.

" combination of cytoskeletal structure or the combination of enhancing " is a kind of state, it is characterized by by to the cardiac muscle cell The shown straight actin fiber of phalloidine dyeing. Actin fiber straight in the cell is at chart In available microexamination and the photography that links to each other distinguish.

" unordered combination or the arrangement of cytoskeletal structure " is opposite with above-mentioned definition meaning.

" the lasting activation of map kinase " refers to that the phosphorylation state P42/44 of map kinase is maintained until in cell Few 24 hours.

“p21^CIP1Express to increase " refer to p21^CIP1Expressing increases by 50% and keep at least 24 little at least in cell The time.

" processing of heart disease " comprises the method that all are suitable, such as intravenous injection neuregulin polypeptide, base Because of methods for the treatment of, remove to process the heart with gene insertion heart or other cells of coding neuregulin or derivative Dirty disease. For example, transport the neuregulin gene with adenovirus or the virus of being combined with adenovirus as carrier Enter heart or other cells, transfected cell can be expressed justacrine neuregulin polypeptide then, thereby Activate the ErbB (acceptor) in the cardiac muscle cell.

Term " processing " is used for this with similar word and is often referred to pharmacology and/or the physiological effect that a plan obtains. Should Effect can be prevention: completely or partially prevent a kind of disease or symptom; It also can be treatment: complete or section Branch ground is treated a kind of disease and/or is provided this disease opposite effect. " processing " covered mammal at this The processing of disease is especially to the processing of human diseases. And comprise:

Prevent that this disease from not yet being diagnosed at those but show effect among the pretreated curee.

Control this kind disease. As control disease progression or alleviate this disease, make disease degeneration etc.

The present invention directly processes those diseases with heart disease and correlated condition such as dangerous development in heart failure The people.

" heart failure " refers to that cardiac function is undesired, heart can not pump with the speed that is fit to enough blood with Satisfy the tissue of metabolism. Scope in heart failure comprises congested heart failure, miocardial infarction, and tachycardia is (fast The speed cardiac arrhythmia), family's hypertrophic cardiomyopathy, ischaemic heart disease, the heart disease of spontaneous expansion, and cardiac muscle Scorching. Ischaemic, hyperemia, rheumatic, and any factor can both cause heart failure in the spontaneous form. Chronic myocardial hypertrophy is a kind of remarkable morbid state of the precursor of congestive heart failure and heart stopping collecting.

" processing " relates to the mensuration for the treatment of processing and preventing or preventing. The disorderly patient who is inclined to of disorder or tool of having had needs Process. Loose can being caused by any reason that retinoic acid is replied comprises geneogenous, viral, spontaneous , the reason of heart or muscle nutrition, or the infringement such as the miocardial infarction that cause because of ischaemic or ischaemic are drawn Rise. Processing is used to stop or slows down loose process, handles with great care the dirty ischaemic miocardial infarction that causes damage After. These medicaments should be used immediately, can prevent or reduce hypertrophy.

In the following example, adopted following these reagent and antibody:

IGF (Boehringer); Collagenase (Worthington); Pancreatin (Gibco BRL); MEK1 (MAPKK) inhibitor (PD98059) (New England); [methyl-3H] thymidine (Amersham); Anti--erbB2 monoclonal antibody (Novocastra); Mono-clonal IgG2b; P21^CIP1(F-5) (Santa Cruz); Anti--Tyrosine O-phosphate horseradish peroxidase (HRPO) link coupled monoclonal antibody, RC20 (Transduction Laboratories); Anti--α-sacromeric actin monoclonal antibody (clone 5c5), the anti-rabbit Ig of HRPO-link coupled and anti--mouse Ig (Sigma); PhosphoPlus p44/42MAP kinases (Thr202/Tyr204) antibody box (New England); Anti--FLAG M1 affinity gel and anti--FLAG M2 monoclonal antibody (Eastman Kodak).

The expression and purification ofembodiment 1 reorganization NRGb2

A. the encode amplification of people NRGb2 cDNA

From the human breast cancer cell strain MDAMB-231 of energy high expression level NGR, separate whole RNA with Rneasy Mini test kit.Synthetic first chain DNA, the cDNA segment of NRG is by pcr amplification, forward primer (GATCAAGCTTAGCCATCTTGTAAAATGTGCG) comprise the homing sequence and a Hind III site (adding the person of rolling off the production line) of four kinds of Nucleotide, the primer (5-GATC reverse with NRGb2AAGCTTCTACTGGTACAGCTCCTCCGC-3), contain a homing sequence, a Hind III site (adding the person of rolling off the production line) is with terminator codon in the frame (black matrix with add the person of rolling off the production line).

To the encode EGF district (amino-acid residue 177-237) of people NRGb2 isomer (rhNRGb2) inserts pFLAG1 expression vector (IBI).RhNRGb2 links to each other with the N-end of 1FLAG-polypeptide at it.In the kytoplasm of bacillus coliDH 5 alpha, express.Affinitive layer purification with the monoclonal antibody of anti-FLAGM1.RhNRGb2 purity behind the purifying surpasses 90%.

B. the recombinate expression of NRGb2

The PCR product of coding NRGb2 is with Hind III degraded and be cloned into PFLAG.1 expression vector (International Biotechnologies).After being transformed into E.coli DH5 α, the bacterium colony of measuring the cDNA clone who contains the NRGb2 coding is stored in the glycerine.The pFLAG.1 prokaryotic expression carrier is designed to produce a soluble recombinant protein, this albumen is expressed in the kytoplasm of transform bacteria, wherein mixed the boot sequence (ompA) of 8 amino-acid residue pFLAG.1 and 21 amino-acid residues, boot sequence will fuse the albumen transposition to the kytoplasm space, and the ompA order is cut in the transposition process.The fused albumen of reorganization can-FLAG monoclonal antibody anti-with M1 affinity chromatography come purifying.

The E.coli that transforms reaches 0.8 37 ℃ of shaking culture until the OD600 value, and sec.-propyl-1-sulphur-β-D pyrans (type) galactoside that adds 500 μ M was then hatched 2 hours.The expression level of each E.coli cellular constituent of decision is divided into 3 parts, centrifugal collecting cell with sec.-propyl-1-sulphur-β-D pyrans (type) galactoside inductive 100ml nutrient solution in initial experiment.A cell precipitation is suspended in the SDS-PAGE sample loading buffer again, isolates whole cellular constituents.By adding 5ml extraction buffer A[50mM Tris-HCl (pH8.0), 5mM EDTA, 0.25mg/ml N,O-Diacetylmuramidase, 50 μ g/ml NaN₃], add 0.5ml extraction buffer B[1.5MNaCl again, 0.1M CaCl₂, 0.1M MgCl₂, 0.02 μ g/ml DNase I, 0.2mM NaVO₃, 0.2mM phenylmethylsulfonyl fluoride, 0.2mM leupeptin, 0.2mM aprotinin], second part of precipitation is used to whole cell precipitations are classified into solvable and insoluble composition.The suspension that produces is used centrifugal 1 hour of 18000xg, tells dissolving and insoluble composition.Insoluble composition suspends again with the SDS-PAGE sample loading buffer, and solvable composition uses the sample loading buffer equal-volume of 2X to mix.At last, the 3rd part of cell precipitation is resuspended in the OS damping fluid [0.5M sucrose, 0.03M Tris-HCl (pH8.0) and 1mM EDTA] of 8ml and uses 3500xg centrifugal 10 minutes at 10 ℃.Cell suspends to discharge cytoplasmic protein again with the 5ml frozen water at once.Then 10 ℃ with 3500xg centrifugal 10 minutes, collect supernatant liquor, 10 μ l supernatant liquors are mixed with SDS-PAGE sample loading buffer with volume, after electrophoresis and the transfer, albumen is with resisting-FLAGM2 monoclonal antibody and enhanced chemoluminescence method (ECL, Amersham) detection.The M2 detection of antibodies contains the FLAG albumen of ompA-excision and non-excision form.

C. the purifying of recombinant protein

NRGb2 at the kytoplasm space expression fuses protein bound resisting-the monoclonal affinity chromatography separation and purification of FLAG M1 with only excising FLAG with ompA-.This antibody only combines with the FLAG polypeptide when calcium ion exists; Therefore, the kytoplasm cellular component is with containing 2mM CaCl₂TBS (150mM NaCl and 50mMTris-HCl, pH7.5) and cross post.Be combined in albumen on the post with containing the TBS wash-out of 2mM EDTA, then with M2 anti--antibody of FLAG does the Western blotting and examines the blue SDS-PAGE glue of Ma Shi staining analysis purity.

The elementary cultivation ofembodiment 2 mouse cardiac muscle cells

Mice embryonic (E11.5-12.5) is used to prepare primary myocardial cell.Heart tissue is separated as previously mentioned from the embryo under aseptic condition.(PBS pH7.4) gives a baby a bath on the third day after its birth time with the 5ml phosphoric acid buffer.Tissue was degraded 15 minutes at 37 ℃ with 0.05% trypsinase/0.57mM EDTA.Abandon isolated cells, the tissue that stays is with containing the PBS of 0.41mg/ml collagenase and 0.6mg/ml pancreatin 37 ℃ of concussions (100rpm) 15-30 minute.Two steps were handled institute's isolated cells mix with the cold calf serum (FBS) of equal volume, stop DeR, at room temperature 1500rpm is centrifugal 5 minutes.Remove supernatant liquor, precipitation adds 10%FBS with the improved Eagle substratum of Dulbecco (DMEM) (Gibco BRL), the 10mM glutamine, and 100 units/ml penicillin and Streptomycin sulphate suspend again.By being coated in culture dish in advance 3-5 time (1 hour/time), the myocardial cell is concentrated.Again with cell in the culture dish in pretreated 6 holes of gelatin or 96 holes, final densities is respectively 2.5 * 10⁵Cells/well or 2 * 10³Cells/well was cultivated 20 hours.Before 37 ℃ of processing of serum-free medium (DMEM), removing serum with NRG or other somatomedins.

Use this method, the present inventor can obtain the myocardial cell's (data do not provide) greater than 90% routinely.With monoclonal anti α-sarcomere actin antibody (clone 5c5) (Sigma) pair cell carry out immunity-fluorescent dye.Examine under a microscope evaluation.

Embodiment 3 myocardial cells' immunocytochemistry

After cardiac muscle cells is washed with PBS, add 0.1%Triton X-100 with 4% Paraformaldehyde 96 and fix 30 minutes in room temperature.The fixed cell was at room temperature hatched 45 minutes with anti--a-actinine (actinine) antibody (SIGMA) then with the PBS blocking-up that contains 5% skimmed milk 30 minutes.After washing with PBS, (Silenus Laboratory, Australia) link coupled antibody (SIGMA) was at room temperature hatched 30 minutes with anti--mouse IgG and FITC.After washing with PBS, with cell with containing 1% Ursol D (1mg/ml, glycerine Sigma), dress lid, sealing.With the Ultraluminescence microscopy of 40 times of mirrors and take pictures.For phalloidin dyeing, cell is at room temperature used 4% formaldehyde fixed 1 hour.After washing secondary with PBS, cell in the dark dyes hour with phalloidin.(1mg/ml, glycerine system sample Sigma) was at the Ultraluminescence test under microscope with containing 1% Ursol D with cell after cell was washed five times with PBS.

P185^NeuPhosphorylation with map kinase

The myocardial cell of serum-free culture handles different time deexcitation erbB acceptor and map kinase with rhNRGb2 in 6 orifice plates.Make cell separate damping fluid (50mM Hepes, pH7.5,150mMNaCl, 10% glycerine, 1% (v/v) Triton X-100,1.5mM MgCl in the 200ml cold cracking immediately₂, 1mM ethylene glycol triethylammonium tetrakis (EGTA), 10mM trisodium phosphate, 10mM NaF, 2mM sodium orthovanadate, 1mM phenylmethylsulfonyl fluoride (PMSF), 10mg/ml aprotinin and 10mg/ml leupeptin) and middle cracking.The lysate that produces is used HRPO-link coupled monoclonal antibody RC20 (1: 2,000) surveys the erbB acceptor, with the map kinase of single-minded p44/42 map kinase antibody (1: 1000) the detection phosphorylation of phosphoric acid, with chemoluminescence (ECL) the immunoblotting detection system of strengthening (Amersham).Anti-erbB acceptor is used to stdn application of sample albumen.The band that fluorescently-labeled albumen produces on film comes quantitatively with densometer.

P21^CIP1Proteic expression

With sample loading buffer (50mM Tris-Cl, pH6.8,100mM DTT, 2%SDS, 0.1% tetrabromophenol sulfonphthalein and 10% glycerine) cracking, and use p21 with the rhNRGb2 of different concns separately or add mek inhibitor PD98059 and stimulate, with the cell of serum-free culture 24 hours^CIP1Antibody (1: 1,000) is done immunoblotting assay.With the anti-mouse Ig of HRPO-coupling (1: 3,000), p21^CIP1Used ECL immunoblotting systems measurement.The protein content of application of sample comes stdn by measuring the erbB2 acceptor.The immunity spot comes quantitatively with densometer.

The thymidine act of union

Behind the serum-free 24 hours, cell was cultivated 12 hours at the DMEM of serum-free with rhNRGb2 or IGF-I, add then [methyl-³H] thymidine (0.5m Ci/ hole) is washed secondary with 5% Tricholroacetic Acid of ice, and the PBS ofice washes 5 times.With 100ml 1%SDS dissolved cell, liquid dodges the numeration of numeration meter.All data add the standard table of error with mean number and reach.

DNA among the elementary mice embryonic myocardial cell (E11.5-12.5) synthesizes is stimulating the back evaluated through rhNRGb2, and the growth of research cell is to the reaction of NRG.In Fig. 1,10^-10The rhNRGb2 of M concentration makes about 2 times of the synthetic increase of DNA.Yet when ligand concentration increased, DNA is synthetic to be reduced significantly.Opposite with the reaction to rhNRGb2, recombinant human IGF-1 only causes cell proliferative response, and this reaction is irrelevant with the somatomedin concentration of using.It is not because the E.coli albumen in the rhNRGb2 reagent is caused that the NRG of high density suppresses the DNA synthetic, because these albumen do not show the negative interaction to myocardial cell's propagation from after purifying the bacterium of FLAG carrier transfection.

The activation of erbB receptor family has excited the chain reaction of an interaction of molecules, finally causes the stimulation of map kinase.As preceding report, the lasting pair cell differentiation of map kinase activatory is conclusive (Traverse etc. (1994) Current Biology 4:694-701).With 10^-8M or 10^-10After the rhNRGb2 of M handles, by studying map kinase activatory time course with discerning the single-minded phosphoric acid-map kinase antibody of p42/p44 map kinase that is activated.As shown in Figure 2, when high consumption rhNRGb2, the activation energy of p42/p44 map kinase at least 21 hours lastingly, and when lower concentration, the activation of map kinase is of short duration and fall back to basal level in less than 3 hours.

Because the of short duration activation and the p21 of map kinase^CIP1Transient expression directly relevant, and p21^CIP1Accumulation cause the cell cycle to rest on the G1 phase, whether the inventor proposes that the lasting activation of map kinase causes a high-level p21^CIP1The problem of expressing.Show p21 among Fig. 3^CIP1The increase of expressing only is observed with the rhNRGb2 of high density the time.This is to p21^CIP1The effect of expressing is independent of cell culture condition, because with serum-free and when containing the serum nutrient solution, all observe similar effect.10^-8M rhNRGb2 increases p21^CIP1Expression can be lastingly at least 24 hours.Like this in the myocardial cell that NRG stimulates, p21^CIP1Lastingly the DNA synthetic being suppressed of expressing is conclusive.

Synthetic by the DNA that measures the myocardial cell behind mixing rhNRGb2 and the IGF-I, the myocardial cell who estimates the NRG transmission breaks up whether resist the propagation that is stimulated by other somatomedins.This problem is extremely important, because in vivo, the myocardial cell is exposed under multiple polypeptides hormone and the somatomedin.In this experiment, the optimum concn (10 of IGF-1^-9M) with 10^-8M, or 10^-10The NRG of M is in conjunction with usefulness.As shown in Figure 4, with single different with NRG, the NRG of lower concentration produces very little negative effect to DNA is synthetic, and the NRG of high density has blocked the reaction that the myocardial cell stimulates IGF-1 significantly.Back one result supports above data: special intracellular approach is the NRG activated by high density.

By with the immunity-fluorescent dye of the anti-sarcomere of specificity or fribrillin α-actinine antibody and phalloidin dyeing, the phenotype of assessment cultured neonatal rat cardiac myocytes to the Actin muscle fiber.

Cell in serum-free medium demonstrates the unordered combination of cytoskeleton (Fig. 5), and adds the Actin muscle fiber (Fig. 5) that the NRG cultured cells has orderly combination.Fig. 6 shows, in the cell with the immunity-fluorescent dye of anti-α-actinine antibody, compares with the cell that does not add NRG, and NRG has improved sarcomeric assembling significantly.The concentration of NRG has also been played the part of a key player: the NRG (10 of high density^-8M) more organized sarcomere (Fig. 6) have been produced.Intercellular bonding also by NRG, special stimulation when high density strengthens.The cellularstructure of being handled by NRG also compares with IGF-1 or PE stimulated cells.IGF-1 pair cell sarcomere and the intercellular bonding tangible effect (Fig. 7) that do not have, however the stimulation of PE has produced two types cell: type A cell is similar to the NRG stimulated cells, and tool fully breaks up and very organized sarcomere structure; Type B cell has a big cell surface to amass and relative relatively poor sarcomere structure and intercellular bonding (Fig. 8).Importantly be, when cell by NRG (10^-8When M) stimulating simultaneously with IGF-1, compare with the IGF-1 stimulated cells with independent, they have shown improved sarcomere structure (Fig. 7).As NRG (10^-8When M) being used from irritation cell with PE one, only type A cell is observed (Fig. 8).Myocardial cell's differentiation is accompanied by the increase of MHC-alpha expression and the minimizing that MHC-β expresses with maturation.PE has suppressed the expression (Fig. 9) of MHC-α as the loose factor of a brute force.Yet NRG has the positive-effect that the MHC-alpha expression is confirmed separately, and can offset the negative effect to the MHC-alpha expression (Fig. 9) that PE-transmits.

The evidence that provides shows that the concentration of NRG is an important factor of the of short duration or persistent state of activation of decision map kinase.The lasting state of activation of map kinase has produced Cdk inhibitor p21 in the embryonic myocardium cell^CIP1The increase of expression level, and follow the DNA synthetic to reduce.This finds to provide tangible proof for the final result of ligand concentration gradient decision cytodifferentiation and fetal development, and further provides signal pathway in the cell how to distinguish molecule observation based on the strength of signal of ligand concentration.

The importance of ligand concentration in cytodifferentiation is guessed based on following observation:

1) gradient of the pattern of fetal development and part is got in touch.

When 2) exsomatizing, the differentiation of ligand concentration pair cell is crucial.

3) the too much expression of acceptor has changed the final result of their response ligand stimulations in the cell.

Consider these observations, the map kinase that NRG concentration relies in the embryonic myocardium cell activates has set up one to further describing the model of the continuous cell signal of erbB acceptor to the reaction mechanism of ligand concentration change.

NRG is that the notion of myocardial cell's differentiation factor is by the p21 among the NRG inducing embryo myocardial cell^CIP1The discovery of expressing is supported.Because p21^CIP1Be defined as the inhibition of Cdk fully, and Cdk can promote the G1 in the cell cycle to enter the S phase, so p21^CIP1The increase of expressing is crucial to initial ultimate differentiation.(Dias etc. (1994) Semin.Diagn Pathol.11:3-14) p21 when the ultimate differentiation of in vivo myocardial cell that this notion was also found in the past (Parker etc. (1995) Science 267:1024-1027) takes place with the skeletal muscle cytodifferentiation^CIP1Expressing increase supports.In one process of back, p21^CIP1Express to increase and finally to cause withdrawing from the cell cycle and begin differentiation.Because p21^CIP1The generation of expressing increase is early than other Cycle Regulation, and it can be used as the early sign of skeletal muscle differentiation.As confirming here, in the myocardial cell that NRG stimulates, p21^CIP1Expression is accompanied by the DNA synthetic and reduces, the p21 that hint NRG-stimulates^CIP1The physiology role who expresses.In addition, by the ERK kinase inhibitor to map kinase and p21^CIP1The p21 that stimulates of inhibition evaluation NRG-^CIP1Expression is direct result of map kinase activated.

Persistent map kinase activation is to induce p21 in the cardiac muscle cells^CIP1Constitutive expression is necessary, otherwise the activation of of short duration map kinase causes of short duration p21^CIP1Express.The latter is not enough to regulate the activity of Cdk, because p21^CIP1To degrade soon and constitutive expression to the blocking-up cyclin/Cdk mixture be essential.In the PC12 cell, persistent map kinase approach is limited to reply the distinctive signal from the NGF acceptor.Persistent map kinase activates and makes PC12 cytodifferentiation neuroblast.Yet this approach is not replied the strength of signal of NRG based on concentration in the myocardial cell intermediate energy region.

Further support NRG is that the evidence of a differentiation factor is when myocardium precursor cell is divided into the myocardial cell, and NRG stimulates the assembling of sarcomere and cytoskeletal structure.Observation in the past also point out the high cell of differentiation degree have more orderly sarcomere (Rumynatsev, P.P. (1977) International ReviewCytology 51, p.187-273).Compare with PE or IGF-1 stimulated cells, the NRG stimulated cells has the best sarcomere of combination.The more important thing is that when NGF mixed with PE or IGF-1, NRG had greatly improved sarcomere, point out that NRG stimulates the sarcomere assembling to preponderate when other cell signals exist.The negative adjusting to the MHC-a expression that the overlapping PE-of NRG passes on, this shows that NRG relates to the adult of keeping contractile protein.Pointed out that as former research NRG, ErbB2 and ErbB4 express in the heart of growing up, NRG should play an important role in keeping myocardial cell's differentiation state.

With two very important features are arranged in the myocardiac heart failure patient body: they are sarcostyle and sarcomeric erroneous arrangement.The former is that the iuntercellular adherent is lax, and the latter is the lax of sarcomere combination.The state of these pathology is present in widely from the heart failure of hyperemia to the myocardosis that enlarges, and seriously influences heart function.Do not handle the way that iuntercellular is bonding and sarcomere assemble now.NRG has been identified in assembling and the process of keeping the bonding and sarcomere structure of iuntercellular and has played a role.NRG stimulates the result of myocardial cell's differentiation and assembling sarcomere structure to point out that the myocardial cell breaks up to rebuild with its cellularstructure to interrelate.This conclusion with myocardial cell differentiation total observation during heart development is conformed to: the myocardial cell of differentiation always has the sarcomere of abundant combination.

Put it briefly, NRG is that myocardial cell's differentiation factor is supported by following evidence:

1) NRG stimulates persistent map kinase to activate;

2) NRG increases p21^CIP1Express;

3) DNA of NRG inhibition IGF-1 reception and registration is synthetic; And

4) NRG stimulates the myocardial cell to assemble sarcomere and cytoskeletal structure;

5) NRG stimulates the expression of adult form mhc gene.

Preparation and application that embodiment 4 treatments are filled a prescription with promotor

By neuregulin or its analogue and selectable physiology acceptable carrier, excipient or the stablizer that mixing has required purity, can make the promotor prescription of treatment cardiac disorders, and can freeze-drying or the form of the aqueous solution preserve.(Remington’s?Pharmaceutical?Sciences，16^th?edition，Oslo，A.，Ed.，1980)。Dosage that acceptable carrier, excipient or stablizer are used and concentration are nontoxic to the recipient, and contain: damping fluid, and as phosphoric acid, citric acid and other organic acid damping fluid; Antioxidant comprises vitamins C; Lower molecular weight (being less than 10 amino-acid residues) peptide; Protein, as serum albumin, gelatin, or immunoglobulin (Ig); Hydrophilic polymer such as polyvinylpyrrolidone; Amino acid, as glycine, glutamine, l-asparagine, arginine or Methionin; Monose, disaccharide and other steamed bun stuffed with sugars are drawn together glucose, seminose, or dextrin; Chelating reagent is as EDTA; Ethanol sugar is as mannitol or Sorbitol Powder; Become salt ion, as sodium; Nonionogenic tenside, as Tween, Pluronics or polyvinyl alcohol (PEG).Antagonist also is fit to connect with a kind of non-albumen polymer (as polyoxyethylene glycol, polypropylene glycol, or polyalkylene class (polyalkylenes)).The consumption of carrier can account for the 1-99% of total amount, and the best is the 90-99% of weight.

It is aseptic that the neuregulin analogue is used in vivo.The neuregulin analogue generally will be preserved with freeze-drying or solution form.Before freeze-drying and reconstruct, analogue should pass through aseptic filtering membrane.

The neuregulin analogue composition of treatment usefulness generally is placed in the container at aseptic population place, an intravenous solution bag or the bottle of the stopper that is penetrated by entry needle is arranged.Only using in long-term mode with a kind of the carrying out in the following approach of neuregulin analogue: injection or perfusion be by intravenously, intraperitoneal, and in the brain, intramuscular, intraocular, intra-arterial, or in the damaging part, oral or with lastingly-release system is following to be annotated.The neuregulin analogue can be by inculcating successive administration, if or clearance rate enough low, take interimly to concentrate injection, or give blood flow or lymph.Better administering mode be heart as direct target, thereby minimum is reduced in the side effect of neuregulin analogue.

Lastingly-suitable example of delivery formulations comprises the semi-transparent matrix of proteic hydrophobic polymer, as film or minigel.Lastingly-example of release matrix comprises polyester, hydrogel such as polyacrylic acid 2-hydroxyethyl-methyl esters resin ((1981) J.Biomed.Mater.Res.15:167-277 such as Langer and Langer (1982) Chem.Tech.12:98-105) or polyvinyl alcohol, polylactide (US Patent No 3,773,919, EP58,481), the multipolymer of L-L-glutamic acid and γ-ethyl-L-glutaminate (Sidman etc. (1983) Biopolymers22:547-556), the vinyl-vinyl acetate copolymer of non-degraded (Langer etc. (1981)The same), degradable poly lactic coglycolic acid such as Lupron Depot^TM(the injectable centrosome of forming by poly lactic coglycolic acid and leuprolideacetate), and poly--D-(-)-3-hydroxybutyric acid (EP 133,988).

The neuregulin analogue also can wrap in the minigel of preparation, for example by condensation technique or by interfacial polymerization technology (Walocel MT 20.000PV or gelatin-minigel and poly--[methyl-methacrylate resin] minigel, at the colloidal transfer system (for example, liposome, albumin centrosome, microemulsion, small-particle and Caplet) in, or in big latex.These technology are published inRemington ' s Pharmaceutical SciencesIn (the same).

Some polymkeric substance (as ethane-acetic acid ethyenyl ester and lactic-co-glycolic acid) can discharge molecule and surpass 100 days, and some hydrogel then discharges molecule in a short time.Molecule in being enclosed in capsule is long-time to be kept in vivo, but as their variabilities or the cohesion as a result that are exposed in 37 ℃ of humidity, causes loss of biological activity.Can design stable mechanism if adopt suitable additive and development specific polymers matrix components etc.

Lastingly-composition of the neuregulin analogue that discharges also comprises the analogue of lipid parcel.The preparation method who contains the liposome of neuregulin analogue is: DE 3,218, and 121; Epstein etc. (1985) Proc.Natl.Acad.Sci.USA 82:3688-3692; Hwang etc. (1980) Proc.Natl.Acad.Sci.USA77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent No. 4,485,045 and 4,544,545; With EP 102,324.Common liposome volume very little (about 200-800) is single-layer type, and lipid composition contains the cholesterol greater than 30% mole number, the ratio of cholesterol regulating and obtain best result of treatment.One suitable lasting-object lesson of release formulation is at EP647, in 449.

It is fixed that the NRG effective dose that is used for the treatment of will be come according to treatment target, route of administration and patient's situation.The doctor generally needs titration dosage and regulates route of administration as required to obtain best result of treatment.

NRG can select to combine with other factors to use and handle congested heart failure, and these factors comprise ACE inhibitor, CT-1 inhibitor, human growth factor and/or IGF-1.If be applied, the effective level of these factors will be determined by the doctor.The using and regulate with method known to those skilled in the art and determine of dosage obtaining congested optimum therapeuticing effect in heart failure, and considered the use of hydragog(ue) and some diseases (as ypotension and kidney damage) etc. ideally.In addition, dosage also depends on the factor such as used drug type and patient's particular case to be treated.In general, if medicine does not add promotor, consumption will be identical; But should provide the effective dose of treatment according to factors such as the kind of the negative interaction, treatment situation, patient's type, promotor and the medicine that exist and total amounts.

ACE inhibitor for example, the enalapril of test usefulness is 5mg, if patient can bear, rises to 10-20mg every day, once a day.As another example, patient initially tries the captopril of oral 6.25mg, increases dosage then step by step, and is when patient can bear to every day that secondary or three total amounts are 25mg, titratable to 50mg secondary every day or three times.Whether the level of bearing by measuring blood pressure drops or following hypotensive symptom to estimate.If any above-mentioned indication, dosage rises to 100mg secondary every day or three times.Captopril combines with hydrochlorothiazide as effective composition and is applied, and it has the shell of the slowly-releasing of a protection captopril to arrive colon up to captopril as the pH stable centers.The captopril of producing has tablet or capsule.About the discussion of captopril and other ACE inhibitor dosage, use, indication and contraindication be at Physicians Desk Reference, Medical Economics Data Production Co., Montvale, NJ.2314-2320 can find in (1994).

In the example of the injectable therapeutic component of a neuregulin, prescription comprises the salt solution of 1% neuregulin and 99%, and wherein neuregulin is a polypeptide.In the example of the injectable therapeutic component of another neuregulin, prescription comprises 5% neuregulin polypeptide, and 1% ACE inhibitor captopril is with 94% salt solution.

Claims (11)

1. neuregulin or its function fragment that contains EGF spline structure territory are in the purposes that is used for preparing treatment, prevent or postponing described Mammals heart failure drugs.

2. purposes according to claim 1, the function fragment that wherein said neuregulin or its contain EGF spline structure territory is by combining with acceptor ErbB2-ErbB3 or acceptor ErbB2-ErbB4, activate the map kinase approach, thereby by activating this approach treatment, prevent or delay the Mammals heart failure.

3. purposes according to claim 1, thus wherein said neuregulin or its function fragment that contains EGF spline structure territory be by activate the map kinase approach induce connect between sarcomere, cytoskeletal structure or myocardial cell among the myocardial cell rebuild treatment, prevent or postpone described mammiferous heart failure.

4. purposes according to claim 1, thus wherein said neuregulin or its function fragment that contains EGF spline structure territory are by stimulating myocardial cell's differentiation treatment, prevent or postpone described mammiferous heart failure.

5, purposes according to claim 1, wherein said EGF spline structure territory fragment contains aminoacid sequence: SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKA EELYQ.

6. purposes according to claim 1, wherein said Mammals is the people.

7. purposes according to claim 6, wherein said people suffers from or suspects and suffer from heart failure.

8. purposes according to claim 7, wherein said heart failure is a kind of morbid state, is selected from congestive heart failure, myocardial infarction, quick arrhythmia, family's hypertrophic cardiomyopathy, ischemic heart disease, spontaneous dilated cardiomyopathy and myocarditis.

9. purposes according to claim 7, the existence form of wherein said heart failure have ischemic, congenital, rheumatic or spontaneous in heart failure.

10. purposes according to claim 7, wherein said heart failure be by in the described mammalian body between the myocardial cell adherent separate and/or the sarcomere construction error is arranged and caused.

11. purposes according to claim 1, wherein said neuregulin or its contain, and the function fragment in EGF spline structure territory can be accepted carrier with pharmacology or the vehicle preparation is used.

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