CN1498656A - Method and compsn. of nervous regulation protein for treating myocardial infarction - Google Patents
Method and compsn. of nervous regulation protein for treating myocardial infarction Download PDFInfo
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- CN1498656A CN1498656A CNA021451451A CN02145145A CN1498656A CN 1498656 A CN1498656 A CN 1498656A CN A021451451 A CNA021451451 A CN A021451451A CN 02145145 A CN02145145 A CN 02145145A CN 1498656 A CN1498656 A CN 1498656A
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Abstract
An application of the neuregulin, its functional fragment, the nucleic acid for coding said neuregulin or its functional, fragment, or the substances for increasing the output and function of said neuregulin in preventing, treating and relaxing the myocardial infarction is disclosed. Its components, reagent kit and composition are also disclosed.
Description
Technical field:
The present invention relates to be used to prevent, treat or delay the mammal especially composition and the method for human myocardium's infarction.More specifically say, the invention provides and a kind ofly prevent, treat or delay cardiac muscle of mammal infarction method, this method comprises needs or the mammal wishing prevention, treat or delay especially neuregulin or its function fragment of human administration effective dose or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function, thereby can prevent, treat or delay described myocardial infarction.The present invention neuregulin also is provided and improve neuregulin output and (or) material of function is used to prevent, treat or delays mammal especially compositions, test kit and the active drug composition of human myocardium's infarction.
Technical background:
Myocardial infarction (myocardial infarction) is a coronary occlusion, and blood flow interrupts, and makes the part cardiac muscle because of serious persistency ischemia local necrosis take place.Since myocardial infarction accompany simultaneously various complication as: the generation of arrhythmia, shock and congestive heart failure, myocardial infarction become the disease that has a strong impact on human health.When extensively (more than 40%) necrosis of cardiac muscle, cardiac output sharply descends and causes shock or heart failure.This disease is common in American-European countries, and its sickness rate is 3.5 ‰, and the U.S. has 100-150 ten thousand people that myocardial infarction takes place every year approximately.This disease is seldom seen in China, increase gradually in recent years in the past.Mainly be that to use the intravenous thrombolysis therapy be the reperfusion as treatment of representative to the treatment of acute myocardial infarction in recent years, this method to the mortality rate that reduces myocardial infarction, preserve cardiac function, improve prognosis and produced breakthrough progress.
Simultaneously, through the application of percutaneous transluminal coronary angioplasty (PTCA) and the implantation of intracoronary stent, to the acute myocardial infarction of some extremely serious type, cardiogenic shock or thromboembolism treatment have the patient of taboo person or thrombolytic failure, have increased lease of life.Auxiliary treatment when antiplatelet, anticoagulant are used for pouring into has again played positive effect.And the rational Application of ACEI, beta-blocker etc. has reduced the Myocardial Remodeling process behind the myocardial infarction, improves prognosis, has reduced mortality rate.Yet described treatment measure can limit and dwindle infarct size but the means of fundamentally treating at impaired myocardial cell are not arranged.
(Neuregulins is by NRG-1, NRG-2, NRG-3 NRGs) to neuregulin, and NRG-4 gene or nucleic acid (as cDNA) are encoded, and combines with ErbB2/ErbB4 or ErbB2/ErbB3, can activate the protein or the polypeptide of above-mentioned receptor.The adjustable multiple biological respinse of NRG and receptor ErbB signal transduction path: as stimulating the secretion of breast cancer cell differentiation and lactoprotein; Induce neural lophocyte to be divided into the Schwann cell; Stimulate the synthetic of the interior acetylcholinergic receptor of skeletal muscle cell; And it is synthetic to promote that myocardial cell survives with DNA.That at present, research is more deep is Neuregulin-1.Neuregulin-1 claims Neu Differentiation Factor (NDF) or Glial Growth Factor (GGF) again, be ErbB3 and ErbB4 part (Wen etc., Cell 69,559-572,1992; Holmes etc., Science 256,1205-1210; Pinkas-Kramarski etc., EMBO J.15,2452-2467,1996; Tzahar etc., Mol.Cell.Biol.16,5276-5287,1996).Because its extracellular fragment contains an EGF sample functional area (EGF-like domain), structurally belong to epidermal growth factor (EGF) the kinsfolk (Liu etc., Proc.Natl.Acad.Sci., 95:13024-13029.1998).It can be directly and the ErbBs receptors bind, activates the ErbBs receptor and also produce corresponding biologic activity.ErbBs is one group of receptor with tyrosine kinase activity that structure is close, has four members, ErbB1～4.ErbB1 is the EGF receptor, ErbB2～4th, the receptor of Neuregulin-1.In vivo, ErbB3 is expressed in the nervous tissue, and ErbB4 is expressed in neural and the cardiac muscular tissue, and ErbB2 then follows ErbB3 and ErbB4 coexpression.After part and ErbB3 or ErbB4 combination, ErbB3 or ErbB4 and ErbB2 form heterodimer, and cause intracellular signal transmission.The ErbB2/ErbB4 heterodimer in the activated cell signal growth, differentiation, the 26S Proteasome Structure and Function of myocardial cell had significant effects (Meyer etc., Nature 378:386-390,1995; Lee etc., Nature.378:394-398,1995; Gassmann etc., Nature 378:390-394,1995; Zhao etc., J Biol Chem.1998,273:10261-10269).
Applying gene location inactivation technology (Targeted Gene Knock-out) will cause the homozygote mutated embryonic growing death in early days in E10.5 days behind Neuregulin-1, ErbB2, the ErbB4 fixed point inactivation.Reason is that the cardiac structure of homozygote mutated embryonic does not have being differentiated to form of myocardium girder (trabeculae) fully, and ventricle becomes a cavity structure that enlarges, and causes heart early development imperfection.Simultaneously, embryo's neuroganglion is grown and is also had serious defective (Meyer etc., Nature 378:386-390,1995; Lee etc., Nature.378:394-398,1995; Gassmann etc., Nature 378:390-394,1995; Liu etc., Proc.Natl.Acad.Sci., 95:13024-13029.1998).
Early stage research to Neuregulin-1 and myocardial function mainly concentrates on cultivates myocardial cell.Thought at that time that Neuregulin-1 was similar to the influence of myocardial cell and the endotheliocyte factor-1 or Phenylephlin, be that it may divide with myocardial cell, survival, hypertrophy have certain contact (Baliga etc., Am JPhysiol 1999,277 (5 pt 2): H2026-H2037).WO00/37095 shows that Neuregulin-1 promotes the splitted effect of myocardial cell to only limit to the condition of culture at low concentration and serum-free, otherwise, Neuregulin-1 then shows the splitted inhibitory action of myocardial cell, confirms that NRG-1 promotes the myocardial cell differentiation.In addition, Douglas etc. studies confirm that the Neuregulin-1 amycin effectively suppress cultivating myocardial cell structural damage interaction energy (Douglas etc., Circulation 2002; 105:1551-1554).
Result of study shows, the Neuregulin-1/ErbB signaling system not only in early days fetal development period heart and nervous system are played an important role, and substantial connection is arranged with the pathological changes of myocardial cell.Up-to-date result of study shows that Neuregulin-1 has important function to the cardiac function of adult.Antineoplastic agent Herceptin (anti-ErbB Humanized monoclonal antibodies) finds to have the cardiac toxicity side effect in clinical practice, especially unite can make up to 28% cancer patient when using with amycin and suffer from heart failure; And the adult cardiomyocytes cell of the damage of biopsy is carried out In vitro culture, PRELIMINARY RESULTS finds that Neuregulin-1 can recover the function of myocardial cell; Show that this signaling system has important effect to the physiological function of human adult heart.In addition, in aforementioned Neuregulin-1 gene inactivation experiment, because embryo's early stage death is difficult to observe the effect of Neuregulin-1 in becoming systemic heart.Use Cre-Loxp enzyme action reconstruction experiment technology, a kind of experiment mice has been cultivated in KF Lee and Kenneth R.Chien cooperation, the ErbB2 acceptor gene of this mice is by conditionality ground inactivation (Conditional Gene Knockout), and this gene mutation betides mice develop than the later stage and be limited in the myocardial cell.In carrying the mouse cardiac muscle of this mutant gene, the Neuregulin-1/ErbB signaling system is blocked, and mice suffers from DCM (dilated cardiomyopathy) as a result.This experimental result further specifies the Neuregulin-1/ErbBs signal to adult heart physiological function significant (Crone etc., Nat Med 8 (5): 459-465,2002).
The present invention is by the nucleic acid with effective dose neuregulin or its function fragment or encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function treatment myocardial infarction, repair impaired myocardial cell, fundamentally improve myocardial function, improve the biological effect of cardiac muscle, thereby this class patient cardiac function is improved, prolong life cycle, mortality rate reduces, and quality of life improves.
Summary of the invention:
The invention provides a kind of prevention, treat or delay cardiac muscle of mammal infarction method, this method comprises needs or the mammal wishing prevention, treat or delay especially neuregulin or its function fragment of human administration effective dose or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function, thereby prevention, treat or delay myocardial infarction.
The present invention also provides prevention, treated or has delayed the especially compositions of human myocardium's infarction of mammal, said composition comprises neuregulin or its function fragment of effective dose or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) medicine of the material of function and the prevention of effective dose or treatment myocardial infarction.
The present invention also provides prevention, treated or has delayed the especially effective ingredient of human myocardium's infarction of mammal, this effective ingredient comprises neuregulin or its function fragment of effective dose or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function, thereby prevention, treat or delay myocardial infarction.
The present invention also provides and has been used for prevention, treats and delays the myocardial infarction test kit, and this test kit comprises described effective ingredient and the operation instruction thereof that places container.
Description of drawings:
Fig. 1. the technology path of the structure design of people's neuregulin engineered strain.The gene mapping of people's neuregulin has about 13 exons structures in chromosome 8P12.Neuregulin β 2 fragments that make up are made up of 61 aminoacid, and theoretical molecular is 7055D, and SDS-PAGE electrophoresis apparent molecular weight is 6500-7000D, isoelectric point, IP is about 6.5, the sugar based site contains three disulfide bond, is fit to express with the escherichia coli system.Select for use PET22b as expression plasmid, transformed into escherichia coli BL21 behind the importing people neuregulin gene obtains the high expressed recombinant human nerve through screening and regulates proteic engineered strain.
Fig. 2. the amplification of neuregulin gene.From 5 months total RNA of embryo and brain tissue extraction of people and mRNA, reverse transcription becomes cDNA, as template, carries out RT-PCR and obtains genes of interest.Get pcr amplification product through 1.5% sepharose electrophoresis, visible special 183bp size amplified fragments conforms to the design fragment length.Swimming lane 1 is that the amplification size is the purpose fragment of 183bp, and swimming lane 2,3 is DNA Markers.
Fig. 3 .PET22b plasmid physical map
Fig. 4. recombinant human nerve is regulated the structure of protein expressing plasmid.With the calcium chloride sedimentation method with the gene clone of people's neuregulin to the PET22b expression plasmid, be built into recombinant human nerve and regulate protein expressing plasmid (PET22b-people's neuregulin), this albumen efficiently expresses under the driving of T7 promoter.Expressing gene N end inserts from the NdeI site, and the C end has terminator to be right after aminoacid place in the end, and gained albumen does not form fusion rotein with any aminoacid.Cut out the correct fragment of about 183bp through enzyme action.Transformant is extracted double-stranded DNA and is carried out nucleotide sequence mensuration after enzyme action is identified.
Fig. 5. efficient expression engineering is identified.The engineering bacteria clone (BL21-PET22b-people's neuregulin) that PCR and enzyme action identify learns from else's experience, the a series of single colony inoculations of random choose are in the 2mlLB-Amp fluid medium, put 37 ℃, 250rpm overnight incubation, be inoculated in by a certain percentage then in the 20ml LB-Amp culture fluid, 37 ℃ are cultured to OD
600Reach 1.0 o'clock adding IPTG and induce 4h, collect thalline.Collect inclusion body behind the broken bacterium, 15%SDS-PAGE electrophoresis, thin slice scan analysis, Western-blotting identify, obtain a strain through repeated screening and stablize the engineering bacteria of high-expression target proteins neuregulin (BL21-PET22b-people's neuregulin).The destination protein that this bacterial strain is expressed accounts for about 10% of bacterial protein.Behind the broken bacterium of high-pressure homogenization, destination protein exists with the inclusion body form.Engineering bacteria is after IPTG induces 4h, and thalline is through the SDS-PAGE electrophoresis, and inclusion body accounts for about 20% of bacterial protein; Reorganization neuregulin specific activity＞5 * 10 behind the purification
3EU/mg shows that the neuregulin engineered strain successfully constructs.On SDS-PAGE, the Western Blot that when the screening engineering bacteria, carries out, the basis of Determination of biological activity, carry out people's neuregulin amino acid composition analysis, N-terminal sequence analysis, the result shows that expressed recombinant human nerve adjusting Argine Monohydrochloride sequence conforms to design.
Mode carries out an invention
For clear open invention, be divided into following trifle and describe in detail.
A. definition
Unless other definition is arranged, technology and scientific terminology have the same implication that the common skill personnel with the technical field of the invention generally understand as used herein for all.Here all patents of reference, application, disclosed application and other publications all are incorporated herein by reference.If the definition that this section is set forth with this with reference in patent, application, disclosed application and other publications definition of setting forth when opposite or inconsistent, the definition that this section is set forth is better than the definition of other lists of references.
In the meaning of this used " " is " at least one " or " one or more than one ".
Be meant with ErbB2/ErbB4 or ErbB2/ErbB3 in the meaning of this used " neuregulin " or " neuregulin " or " NRG " to combine, can activate the protein or the polypeptide of above-mentioned receptor.It can activate above-mentioned receptor, and regulates and control multiple biological respinse: as stimulating the secretion of mastocarcinoma cell differentiation and lactoprotein; Induce neural lophocyte to be divided into the Schwann cell; Stimulate the synthetic of the interior acetylcholinergic receptor of skeletal muscle cell; And it is synthetic to promote that myocardial cell survives with DNA.Common neuregulin can be by for example NRG-1, NRG-2, NRG-3, NRG-4 gene or nucleic acid (as cDNA) encoded protein matter or polypeptide.Because the indivedual amino acid whose change in the non-functional area of polypeptide does not have influence (see to its function, e.g., .Molecular Biology of the Gene such as Watson, 4th Edition, 1987, TheBejacmin/Cummings Pub.co., p.224), here, NRG also comprises reservation NRG conserved amino acid sequence, but to the constant NRG isomer of the therapeutical effect of myocardial infarction.
Be meant with ErbB2/ErbB4 or ErbB2/ErbB3 in this used " EGF spline structure territory " or " epidermal growth factor-like domain " or " EGF-like domain " and combine, has polypeptide structure with EGF receptors bind zone same structure, common " EGF spline structure territory " can be by NRG-1, NRG-2, NRG-3, NRG-4 gene or nucleic acid (as cDNA) coding.About EGF spline structure territory referring to WO 00/64400, Holmes etc., Science, 256:1205-1210 (1992); U.S.PatentNos.5,530,109 and5,716,930; 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.Biochern., 122:675-680 (1997); And WO 97/09425..
Be meant the function fragment or the analog of neuregulin or encode prevention, treatment or the retarding action of this segmental nucleic acid reservation at this used " function fragment or analog " myocardial infarction.Usually, these function fragments or analog keep 50% prevention at least, treat or delay the effect of myocardial infarction, better under the situation, these function fragments or analog keep 60%, 70%, 80% at least, 90%, 95%, 99% or 100% prevents, treats or delay the effect of myocardial infarction.
Be meant the material that can improve NRG gene transcription, translation effect this used " improving the material of above-mentioned neuregulin output ", or improve modification behind the NRG gene translation material and (or) improve the material of NRG precursor communication, or improve the material of NRG half-life.
Be meant the material that can improve the neuregulin prevention, treat or delay the effect of myocardial infarction this used " improving the material of above-mentioned neuregulin function ", or the material of raising neuregulin part sensitivity in the pathway of cell on that prevents, treats or delay myocardial infarction, or the material of reduction neuregulin antagonist action, but do not comprise neuregulin and code nucleic acid thereof.
Be meant the material that two or more combination of chemicals forms in this used " compositions ".
Refer to two kinds of oncogenes at this used " erb ", erb A and erb B are with erythroblastosis virus relevant (a kind of acute convertibility retrovirus).
Being meant at this used " effective dose that is used for the treatment of the chemical compound of specified disease " is enough to improve, or reduces the consumption with the symptom of this disease association in some way.Such consumption can be used as single dose or according to its effective taking dose of prescription.This consumption possibility cure diseases, but typical situation is to take in order to improve disease symptoms.For reaching the doing well,improving of expectation, may need to take continuously.
In the meaning of this used " treatment " is the method that the symptom of any state, discomfort or disease is improved or have useful variation.Treatment comprises that also wherein any Drug therapy of compositions is used.
At this, be meant any alleviating by taking the symptom " improvement " that specific Drug therapy compositions reaches specific discomfort, persistent or of short duration no matter be permanent or temporary transient, need only this alleviate can give the credit to or with take compositions relation arranged.
Be meant the production method of using the recombinant nucleic acid method in this used " recombination method production ".This method is with the well-known coded proteinic molecular biology method of expression of nucleic acid with cloning.
This used " complementary " when being meant two nucleic acid molecules, the meaning is that two nucleotide sequences can be hybridized, and preferably is lower than 25%, is more preferably to be lower than 15%, even to be more preferably be to be lower than 5%, most preferably is not have the mistake pairing at relative nucleotide place.Preferably under stringent condition, these two molecular hybridizations.
In the wrong pairing of this used decision percentile " stringency of hybridization " following regulation:
High stringency: 0.1 * SSPE, 0.1%SDS, 65 ℃;
Medium stringency: 0.2 * SSPE, 0.1%SDS, 50 ℃; (also referring to appropriate stringency)
Low stringency: 1.0 * SSPE, 0.1%SDS, 50 ℃;
Be interpreted as using buffer, salt and the temperature of equity, can obtain suitable stringency.
Be meant at this used " carrier (or plasmid) " and be used for discontinuous element that the foreign DNA transfered cell is expressed or duplicated.Select and use this kind carrier in this technical staff's technical scope, to be widely known by the people.Expression vector comprise can expressible dna carrier, its DNA effectively with regulating and controlling sequence, for example promoter region links together.Regulating and controlling sequence can influence the segmental expression of this segment DNA.So expression vector is meant that recombinant DNA or RNA constitute thing, for example plasmid, phage, recombinant virus or other carriers, in a single day they import in the appropriate host cell, causes the DNA that clones to express.Suitable expression vector is fully aware of to those skilled in the art, and comprises the carrier that can duplicate and keep free state or be integrated into the carrier of host cell gene group in eukaryotic cell and/or prokaryotic cell.
Be meant transcription DNA or the RNA fragment of controlling with its DNA that effectively is connected or RNA at this used " promoter region or promoter element ".Promoter region comprises is enough to allow the particular sequence of RNA polymerase identification, combination and transcription initiation.This part of promoter region is meant promoter.And promoter region also comprises regulates RNA polymerase identification, combination and the active sequence of transcription initiation.These sequences can be the cis acting factors or trans acting factor responded.According to the character of regulating, promoter can be composing type or adjustment type.Consider that being used for procaryotic typical promoter comprises phage t7 and T3 promoter and similar promoter.
Be meant nucleotide effect sequence and regulate sequence in that this is used " effectively connect and effectively in conjunction with ", for example promoter, enhancer, transcribe and translation termination site and other signal sequences and DNA between functional relationship.For example, make single-minded identification, combination and transcribe the RNA polymerase of this segment DNA can transcribing from initial this segment DNA of promoter for the effective physics and functional relationship that is meant between DNA and promoter that be connected of DNA and promoter, this relation.For ease of optimization expression and/or in vitro transcription, be necessary to remove, increase or change clone's 5 ' not translator units, no matter so that eliminate unnecessary, potential inappropriate substituting translation initiation codon or other be transcribe or translation skill on disturb or reduce the sequence of expressing.Additive method is right after 5 of start codon ' insertion with the ribosome binding site consensus sequence in addition, can strengthen expression.(reference example, Kozak, journal of biological chemistry (J.Biol.Chem., 266:19867-19870 (1991).The hope of this correction (or needs) can be decided by experience.
" myocardial infarction " is coronary occlusion, and blood flow interrupts, and makes the part cardiac muscle because of serious persistency ischemia local necrosis take place.
B. prevent, treat or delay the method for cardiac muscle of mammal infarction
The invention provides a kind of prevention, treat or delay cardiac muscle of mammal infarction method, this method comprises needs or the mammal wishing prevention, treat or delay especially neuregulin or its function fragment of human administration effective dose or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function, thereby prevention, treat or delay myocardial infarction.
Method of the present invention can be used for for example mice, rat, rabbit, cat, Canis familiaris L., pig, milch cow, cattle, sheep, goat, horse, monkey and the prevention of other inhuman primate of any mammal, treats or delay the cardiac muscle of mammal infarction.Preferably, method of the present invention is used for human prevention, treats or delays the cardiac muscle of mammal infarction.
Any suitable neuregulin, or its function fragment, the nucleic acid of neuregulin or its function fragment of perhaps encoding, or improve above-mentioned neuregulin output and (or) material of function can both be used for this method.In the present invention, neuregulin by with the ErbB2-ErbB4 receptors bind, thereby the effect that produces anti-myocardial infarction, wherein neuregulin can be selected from neuregulin 1, neuregulin 2, neuregulin 3 or neuregulin 4.Neuregulin 1 can be neuregulin α 2 or neuregulin β 2.Neuregulin β 2 fragments wherein include the aminoacid sequence of SEQID:1.In other example, United States Patent(USP) Nos. 6,252,051,6,121,415 (NRG3), 6,087,323,6,033,906, US2002002276 and WO01/81540 (NRG-4), WO01/64877 (NRG1), WO01/64876 (NRG1AG1), WO01/58948 (neuregulin-beta), WO01/26607, WO00/70322, WO00/64400, WO99/18976, WO98/02540, WO96/30403, WO96/15812, and GenBank Accession Nos. such as BC017568, BC007675, AF142632, AF194439, AF194438, HS2NRG12, HS2NRG08, HS2NRG07 AF083067, AF076618, AF045656, AF045655, AF045654, MAU96612, the disclosed neuregulin of AF010130, or its function fragment, perhaps the encode nucleic acid of neuregulin or its function fragment is used for this method.Preferably, the open neuregulin of GenBank Accession No.NT_007995 (gi:18570363) is used for this method.The conservative nucleotide sequence of existence is replaced but is not influenced its active any neuregulin, or its function fragment can be used for this method.
In the present invention, the myocardial infarction that causes of any reason all can obtain prevention, treats or delay.For example, the myocardial infarction that causes of coronary artery thrombosis, coronary vasospasm and deficiency myocardial blood supply all can obtain prevention, treats or delay.
In the present invention, the clinical symptoms of myocardial infarction and complication can obtain prevention, treat or delay.For example, arrhythmia, ventricular arrhythmia, atrioventricular block, arrhythmia, supraventricular tachy-arrhythmia, shock, heart failure can obtain prevention, treat or delay slowly.
In the present invention, wherein neuregulin can dwindle the area of myocardial infarction, impels the myocardial infarction blood vessel hyperplasia, thereby produces the effect of anti-myocardial infarction.
In the present invention, any medicine that can prevent, treat or delay myocardial infarction and clinical symptoms and complication thereof can be used for this method.For example the aspirin of the long-term oral low dose of myocardial infarction patient or dipyridamole are considered to the effect of prevention myocardial infarction recurrence to antiplatelet gathering and adhesion.The medicine that for example prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be thrombolytic such as urokinase, streptokinase, tissue-type plasminogen activator (t-PA), Single-chain Urokinase-type Plasminogen Activator (SCUPA), former streptokinase complex of anisyl chemical fibre lyase (APSAC) etc.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be treating irregular heart pulse medicine such as lignocaine, mexiletine, procainamide, bretylium tosylate, disopyramide, tocainide, atropine or quinidine adrenocortical hormone, isoproterenol or ephedrine etc.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be a vasoconstrictor: dopamine, dobutamine, metaradrine (aramine), norepinephrine etc.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be that vasodilator can be selected nitroglycerin, sodium nitroprusside, phentolamine, isosorbide dinitrate, nifedipine or scopolamine etc. for use.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be beta-blocker such as atenolol, metoprolol, Propranolol, sotalol or plug Luo Er etc.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be ACEI such as captopril, ramipril, fosinopril, lisinopril, enalapril or quinapril etc.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be calcium ion antagonist such as nifedipine, verapamil, sieve's Ah Horizon or diltiazem etc.Promote the myocardial metabolism medicine: vitamin C, coenzyme A, sodium inosinate, cytochrome C, vitamin B6 etc.And medicine such as Folium Digitalis Purpureae class, amiodarone, quinidine, antazoline.Hyaluronidase, morphine or Pethidine and diuretic glucocorticoid such as methyl meticortelone.Anticoagulant such as heparin, warfarin or dicoumarol or phenindione can be used for this method.
C. prevent, treat or delay effective ingredient, compositions and the test kit of myocardial infarction
The invention provides a kind of active drug composition that is directly used in prevention, treats or delays the cardiac muscle of mammal infarction, this active drug composition comprises neuregulin or its function fragment of effective dose or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function.
Any suitable neuregulin, or its function fragment, the nucleic acid of perhaps encode neuregulin or its function fragment can both be used for this compositions.In the present invention, neuregulin by with the ErbB2-ErbB4 receptors bind, thereby the effect that produces anti-myocardial infarction, wherein neuregulin can be selected from neuregulin 1, neuregulin 2, neuregulin 3, neuregulin 4.Neuregulin 1 is neuregulin α 2 or neuregulin β 2.Neuregulin β 2 fragments wherein include the aminoacid sequence of SEQ ID:1.
In the present invention, wherein neuregulin can dwindle the area of myocardial infarction, impels the myocardial infarction blood vessel hyperplasia, thereby produces the effect of anti-myocardial infarction.The nucleic acid of neuregulin or its function fragment or encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function, dosage can be from 25 μ g to 2500 μ g.
The invention provides a kind of compositions that is directly used in prevention, treats or delay the cardiac muscle of mammal infarction, said composition comprises neuregulin or its function fragment of effective dose or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) medicine of the material of function and the prevention of effective dose, treatment myocardial infarction.
Anyly suitable can prevent, treat or delay myocardial infarction and clinical symptoms and complication medicine thereof and can be used for said composition.For example the aspirin of the long-term oral low dose of myocardial infarction patient or dipyridamole are considered to the effect of prevention myocardial infarction recurrence to antiplatelet gathering and adhesion.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be thrombolytic such as urokinase, streptokinase, tissue-type plasminogen activator (t-PA), Single-chain Urokinase-type Plasminogen Activator (SCUPA) or the former streptokinase complex of anisyl chemical fibre lyase (APSAC) etc.Treating irregular heart pulse medicine such as lignocaine, mexiletine, procainamide, bretylium tosylate, disopyramide, tocainide, atropine or quinidine adrenocortical hormone, isoproterenol or ephedrine etc.Vasoconstrictor: dopamine, dobutamine, metaradrine (aramine) or norepinephrine etc.Vasodilator can be selected nitroglycerin, sodium nitroprusside, phentolamine, isosorbide dinitrate, nifedipine or scopolamine etc. for use.Beta-blocker such as atenolol, metoprolol, Propranolol, sotalol or plug Luo Er etc.ACEI such as captopril, ramipril, fosinopril, lisinopril, enalapril or quinapril etc.Calcium ion antagonist such as nifedipine, verapamil, sieve's Ah Horizon or diltiazem etc.Promote the myocardial metabolism medicine: vitamin C, coenzyme A, sodium inosinate, cytochrome C, vitamin B6 etc.And medicine such as Folium Digitalis Purpureae class, amiodarone, quinidine, antazoline.Hyaluronidase, morphine or Pethidine and diuretic glucocorticoid such as methyl meticortelone.Anticoagulant such as heparin, warfarin or dicoumarol or phenindione can be used for said composition.
The present invention also provides and has been used for prevention, treats and delays the myocardial infarction test kit, and this test kit comprises described effective ingredient and the operation instruction thereof that places container.
The present invention also provides the test kit that carries out therapeutic scheme of the present invention.This test kit comprise place one or more containers no matter separately or with other medicament unite the neuregulin of the treatment effective dose of use, or its function fragment, the nucleic acid of perhaps encode neuregulin or its function fragment or improve above-mentioned neuregulin output and (or) material of function.Preferred medicament forms is and physiological saline solution that the acceptable sterile liquid is united use in glucose solution or buffer soln or other pharmacy.Compositions also can lyophilizing or drying; In this case, test kit further can optionally comprise the pharmaceutically acceptable solution that is stored in the container, and preferred sterile solution forms injection solution to reassemble into complex.Typical pharmaceutically acceptable solution is normal saline and glucose solution.
In another embodiment, test kit of the present invention further comprises the entry needle of one piece of preferred aseptic packaging that is used for injectable composition or the ethanol liner of syringe and/or packing.Being suitable for doctor or patient uses the indicative explanation of this compositions optionally to be included in above the test kit.
D. neuregulin using method, dosage and route of administration
Neuregulin, or its function fragment, perhaps encode neuregulin or its function fragment nucleic acid or improve above-mentioned neuregulin output and (or) material of function uses prescription, dosage and route of administration, during especially as pharmaceutical composition, can be determined according to the method known to the present technique field.(referring to, for example, Lei Mingdun: the pharmaceutics science with put into practice (Remington:The Science andPractice of Pharmacy), Alfonso R.Gennaro (editor), Mack publishing house, in April, 1997; Therapeutic peptide and albumen: prescription, processing and transmission system (Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems), Banga, 1999; With the development (Pharmaceutical Formulation Development of Peptides andProteins) of peptide and albumen pharmaceutical formulation, Hovgaard and Frkjr (editor), Taylor﹠amp; Francis company, 2000; The medical application of liposome (Medical Applications of Liposomes), Lasic and Papahadjopoulos (editor), Elsevier Science, 1998; Gene therapy study course (Textbook of Gene Therapy), Jain, Hogrefe﹠amp; Huber publishing house, 1998; Adenovirus: the basic biology of gene therapy (Adenoviruses:Basic Biology to Gene Therapy), 15 volumes, Seth, LandesBioscience, 1999; The drug design of bio-pharmaceuticals and development (Biopharnaceutical Drug Designand Development), Wu-Pong and Rojanasakul (editor), Humana publishing house, 1999; Blood vessel on the therapeutics takes place: to clinical (Therapeutic Angiogenesis:FromBasic Science to the Clinic), 28 roll up Dole etc. (editor), Springer-Verlag New York, 1999 from basic science).Neuregulin, or its function fragment, the nucleic acid of perhaps encode neuregulin or its function fragment, can be mixed be used for oral, rectally, local application, the inhalation medication, oral cavity medicine (for example Sublingual), injecting drug use is (for example, subcutaneous, intramuscular, Intradermal, venous), percutaneous dosing or other route of administration that is fit to.Under any given situation, only route of administration will depend on character and the order of severity and the used specific neuregulin of the situation of will treating, or its function fragment, the nucleic acid of perhaps encode neuregulin or its function fragment or improve above-mentioned neuregulin output and (or) Substance Properties of function.
Neuregulin, or its function fragment, the nucleic acid of perhaps encode neuregulin or its function fragment or improve above-mentioned neuregulin output and (or) material of function can use separately.Preferred mode is, neuregulin, or its function fragment, the nucleic acid of perhaps encode neuregulin or its function fragment or improve above-mentioned neuregulin output and (or) material of function, use jointly with acceptable carrier on the Drug therapy or excipient.Any suitable Drug therapy acceptable carrier or excipient can be used for this method.(referring to, for example, Lei Mingdun: the pharmaceutics science with put into practice (Remington:The Science and Practice of Pharmacy), Alfonso R.Gennaro (editor), Mack publishing house, in April, 1997).
This method can be used separately.Perhaps, this method can be united use with other anti-myocardial infarction method.This method also can be united the aspirin that uses the long-term oral low dose of myocardial infarction patient for example or dipyridamole to antiplatelet gathering and adhesion with other anti-myocardial infarction medicament, is considered to the effect of prevention myocardial infarction recurrence.The medicine that for example prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be thrombolytic such as urokinase, streptokinase, tissue-type plasminogen activator (t-PA), Single-chain Urokinase-type Plasminogen Activator (SCUPA), former streptokinase complex of anisyl chemical fibre lyase (APSAC) etc.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be treating irregular heart pulse medicine such as lignocaine, mexiletine, procainamide, bretylium tosylate, disopyramide, tocainide, atropine or quinidine adrenocortical hormone, isoproterenol or ephedrine etc.Vasoconstrictor: dopamine, dobutamine, metaradrine (aramine), norepinephrine etc.Vasodilator can be selected nitroglycerin, sodium nitroprusside, phentolamine, isosorbide dinitrate, nifedipine or scopolamine etc. for use.Beta-blocker such as atenolol, metoprolol, Propranolol, sotalol or plug Luo Er etc.The medicine that prevents, treats or delay myocardial infarction and clinical symptoms and complication thereof can be ACEI such as captopril, ramipril, fosinopril, lisinopril, enalapril or quinapril etc.Calcium ion antagonist such as nifedipine, verapamil, sieve's Ah Horizon or diltiazem etc.Promote the myocardial metabolism medicine: vitamin C, coenzyme A, sodium inosinate, cytochrome C, vitamin B6 etc.And medicine such as Folium Digitalis Purpureae class, amiodarone, quinidine, antazoline.Hyaluronidase, morphine or Pethidine and diuretic glucocorticoid such as methyl meticortelone.Other anti-myocardial infarction medicament such as anticoagulant such as heparin, warfarin or dicoumarol or phenindione etc.Other anti-myocardial infarction treatment or medicament can be used in neuregulin, or its function fragment, perhaps the encode nucleic acid of neuregulin, or its function fragment or improve above-mentioned neuregulin output and (or) before the use of the material of function, among or afterwards.For embodiment, neuregulin, or its function fragment, the nucleic acid of the neuregulin of perhaps encoding, or its function fragment can use jointly with anti-myocardial infarction medicament.Any viral vector that is suitable for gene therapy can be united in the use at this and used.For example, adenovirus vector (U.S. Patent No. 5,869,305), monkey disease poisonous carrier (U.S. Patent No. 5,962,274), condition replication form human immune deficiency type viral vector (U.S. Patent No. 5,888,767), retrovirus retrovirus, simian virus-40, herpes simplex virus amplicon vector and vaccinia virus vector can use.And these genes can change the non-virus carrier system over to, liposome for example, and in liposome, lipid protects DNA or other biological substance not oxidated in coacervation process.
According to the present invention, no matter separately or with other medicament, carrier or excipient to unite the neuregulin of use, or its function fragment, perhaps the encode nucleic acid of neuregulin or its function fragment, or improve above-mentioned neuregulin output and (or) material of function can system be used for any route of administration, such as injection in the spongy body, subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, oral or local application.This method can be used the ejection preparation with the antiseptic that contains interpolation of unit dosage form, ampoule injection or multi-dose container.This prescription can adopt the suspension in oil or the water quality excipient, solution or emulsion form and can comprise prescription reagent, for example suspending agent, stabilizing agent and/or dispersant.Active component also can be a powdery, so as before use with the carrier, aseptic water or other solvent that does not contain thermal source that are fit to.Local application of the present invention can adopt foam, gelinite, frost, ointment, transdermal ointment or ointment.
May be used for Drug therapy acceptable composition of the present invention and method and be included in, but be not limited only to United States Patent(USP) Nos. 5,736,154; 6,197,801 B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374 B1; With 5,686, in 102.
The big young pathbreaker of treatment or prevention Chinese medicine therapeutic dose changes with the order of severity and the route of administration of the situation of treatment.Dosage and dose frequency also will change according to patient self age, body weight, disease condition and reaction.
How and when, the doctor in charge should know because drug toxicity or disadvantageous effect stop, interrupt maybe low dosage being adjusted in treatment.On the contrary, the doctor how and when should know because clinical effect deficiency (having got rid of the toxicity seondary effect) is adjusted to high level with treatment.
Any suitable route of administration can be used.Dosage form comprises tablet, lozenge, cachet, dispersion, suspension, solution, capsule, ointment and similar form.(reference, Lei Mingdunshi Drug therapy science)
In actual applications, no matter separately or with other medicament to unite the neuregulin of use, or its function fragment, perhaps encode neuregulin or its function fragment nucleic acid or improve above-mentioned neuregulin output and (or) material of function, can treat hybrid technology according to conventional medicament and form tight interpolation mixture as active component and Drug therapy carrier or excipient such as beta-schardinger dextrin-and 2-hydroxyl-propyl group-beta-schardinger dextrin-.Carrier is according to the expectation occupation mode, and part or injecting drug use are taked multiple formulation forms.When preparation is used for the compositions of injection, for example intravenous injection or intravenous infusion, identical pharmacy medium can use, and water, ethylene glycol, oil, buffer, sugar, antiseptic, liposome and other have the medium known to the people of present technique field technical ability.The example of injectable composition includes, but not limited to glucose, normal physiological saline solution or other solution of 5%w/v.The nucleic acid of neuregulin or its function fragment or encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function, dosage can be from 25 μ g to 2500 μ g.Treat the neuregulin of administration, or its function fragment, the nucleic acid of perhaps encode neuregulin or its function fragment or improve above-mentioned neuregulin output and (or) accumulated dose of the material of function can contain volume at a bottle and use from 1 milliliter to 2000 milliliters intravenous fluid.According to used accumulated dose, the diluent liquid volume will change to some extent.
The amplification of embodiment 1. neuregulin genes
Neuregulin gene mapping has about 13 exons structures in chromosome 8P12.The neuregulin that makes up is made up of 61 aminoacid, and theoretical molecular is 7055D, and SDS-PAGE electrophoresis apparent molecular weight is 6500-7000D, and isoelectric point, IP is about 6.5, and the sugar based site contains three disulfide bond, is fit to express with the escherichia coli system.Select for use PET22b as expression plasmid, transformed into escherichia coli BL21 behind the importing people neuregulin gene obtains the high expressed recombinant human nerve through screening and regulates proteic engineered strain.
The amplification of neuregulin gene.From 5 months total RNA of embryo and brain tissue extraction of people and mRNA, reverse transcription becomes cDNA, as template, carries out RT-PCR and obtains genes of interest.Get pcr amplification product through 1.5% sepharose electrophoresis, visible special 183bp size amplified fragments conforms to the design fragment length.
With the calcium chloride sedimentation method with the gene clone of people's neuregulin to the PET22b expression plasmid, be built into recombinant human nerve and regulate protein expressing plasmid (PET22b-people's neuregulin), this albumen efficiently expresses under the driving of T7 promoter.Expressing gene N end inserts from the NdeI site, and the C end has terminator to be right after aminoacid place in the end, and gained albumen does not form fusion rotein with any aminoacid.Cut out the correct fragment of about 183bp through enzyme action.Transformant is extracted double-stranded DNA and is carried out nucleotide sequence mensuration after enzyme action is identified.The result confirms that the people's neuregulin sequence on the expression vector is entirely true.
The order of neuregulin cDNA is (SEQ ID NO:2):
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 (SEQ ID NO:1):
SHLVKCAEKEKIFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ
The engineering bacteria clone (BL21-PET22b-people's neuregulin) that PCR and enzyme action identify learns from else's experience, the a series of single colony inoculations of random choose are in 2ml LB-Amp fluid medium, put 37 ℃, 250rpm overnight incubation, be inoculated in by a certain percentage then in the 20ml LB-Amp culture fluid, 37 ℃ are cultured to OD
600Reach 1.0 o'clock adding IPTG and induce 4h, collect thalline.Collect inclusion body behind the broken bacterium, 15%SDS-PAGE electrophoresis, thin slice scan analysis, Western-blotting identify, obtain a strain through repeated screening and stablize the engineering bacteria of high-expression target proteins neuregulin (BL21-PET22b-people's neuregulin).The destination protein that this bacterial strain is expressed accounts for about 10% of bacterial protein.Behind the broken bacterium of high-pressure homogenization, destination protein exists with the inclusion body form.Engineering bacteria is after IPTG induces 4h, and thalline is through the SDS-PAGE electrophoresis, and inclusion body accounts for about 20% of bacterial protein; Reorganization neuregulin specific activity＞5 * 10 behind the purification
3EU/mg shows that the neuregulin engineered strain successfully constructs.On SDS-PAGE, the Western Blot that when the screening engineering bacteria, carries out, the basis of Determination of biological activity, carry out people's neuregulin amino acid composition analysis, N-terminal sequence analysis, the result shows that expressed recombinant human nerve adjusting Argine Monohydrochloride sequence conforms to design.
Embodiment 3.NRG-1 is to the treatment experiment of myocardial infarction animal
Experiment material and method
Animal: 300-350g, male, SD rat (providing by Shanghai Medical Univ's Experimental Animal Center) are provided for use.
Model production method: according to Johns and Olson (Johns, T.N.P., and B.J.Olson.Experimental myocardial infarction.I.A method of coronary occlusion in smallanimals.Ann.Surg.140:675-682,1954.) ligation rat left coronary artery, the heart is done in ligation after one week super, selects EF (ejection fraction) decline 70% left and right sides rat to be successful myocardial infarction model.
Grouping and processing: be divided into 4 groups, 10 every group.Sham operated rats: undergo surgery but not ligation rat left coronary artery, do not carry out any processing; Negative control group: give Rat of Myocardial Infarction afterbody injection PBS; Positive controls: irritate stomach verapamil 8mg/kg (nine good fortune pharmaceutical factories, Shanghai), every day 2 times to Rat of Myocardial Infarction; Experimental group: give Rat of Myocardial Infarction afterbody injection neuregulin 90 μ g/kg/d, successive administration 11 days.All rats are in the super observation of preceding 1 day of processing, processing the 5th day and the 11st right overhead.Put to death at the 11st day, carry out pathological analysis.
Experimental result:
1.NRG-1 effect to Rat of Myocardial Infarction LVEDD and LVESD
As shown in table 1, compare with the PBS group, NRG-1 can reduce the effect of Rat of Myocardial Infarction left ventricular end diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD).The 5th day, the 11st day NRG-1 of administration can reduce the effect (P＜0.05) of Rat of Myocardial Infarction left ventricular end diastolic dimension (LVEDD).NRG-1 can reduce Rat of Myocardial Infarction left ventricular end-systolic dimension (LVESD), and the NRG-1 group compares the 5th day P＜0.05th of administration, administration the 11st day, P＜0.001 with the PBS group.
Table 1.NRG-1 is to the effect of Rat of Myocardial Infarction LVEDD and LVESD
LVEDD(mm) LVESD(mm)
Day?0 Day?5 Day?11 Day?0 Day?5 Day?11
Sham operated rats 0.570 ± 0.037 0.553 ± 0.050 0.465 ± 0.046 0.258 ± 0.051 0.236 ± 0.029 0.156 ± 0.039
Verapamil 0.712 ± 0.084 0.739 ± 0.119 0.727 ± 0.131 0.557 ± 0.101 0.531 ± 0.127 0.523 ± 0.170
PBS group 0.705 ± 0.117 0.792 ± 0.083 0.847 ± 0.101 0.558 ± 0.119 0.594 ± 0.110 0.677 ± 0.105
NRG-1 0.730±0.108 0.718±0.068
*?0.727±0.092
*?0.575±0.119 0.463±0.096
*?0.460±0.142
*
2.NRG-1 effect to Rat of Myocardial Infarction left ventricle EF and Fs
As shown in table 2, compare with the PBS group, NRG-1 can improve the effect (P＜0.05) of rat left ventricular ejection fraction (EF) (P＜0.001) and left ventricle shortening mark (Fs).As seen, NRG-1 can obviously improve the effect of rat cardiac function.
Table 2.NRG-1 is to the effect of Rat of Myocardial Infarction left ventricle EF and Fs
EF Fs
D0 D5 D11 D0 D5 D11
Sham operated rats 89.320 ± 4.908 91.080 ± 2.708 95.040 ± 3.133 54.980 ± 6.467 57.380 ± 4.378 65.640 ± 7.501
Verapamil 49.492 ± 12.132 61.527 ± 11.473 60.786 ± 16.693 22.146 ± 6.629 28.933 ± 7.726 29.757 ± 11.213
PBS 48.275±10.307 51.050±9.049 42.650±6.355 21.333±5.585 25.450±7.628 18.310±3.185
NRG-1 49.269±11.440 72.973±11.586
*?75.720±10.780
*21.723±6.429 35.785±10.779
*?41.280±10.980
*
3.NRG-1 effect to Rat of Myocardial Infarction left ventricle IVS and promotion blood capillary proliferation
As shown in table 3, compare with the PBS group, NRG-1 has the zone thin blood vessel hyperplasia effect of internal hair (P＜0.05) of the fibrosis of promotion to Rat of Myocardial Infarction interventricular septal thickness (IVS) and to Rat of Myocardial Infarction.
Table 3.NRG-1 is to the effect of Rat of Myocardial Infarction left ventricle IVS and promotion blood capillary proliferation
The IVS capillary vessel number (individual/mm2)
D0 D5 D11 D11
Sham operated rats 0.173 ± 0.006 0.178 ± 0.008 0.187 ± 0.012 0.000 ± 0.000
Verapamil 0.169 ± 0.017 0.164 ± 0.013 0.162 ± 0.028 7.775 ± 2.309
PBS 0.162±0.014 0.162±0.009 0.146±0.016 6.490±2.173
NRG-1 0.164±0.018 0.161±0.020 0.182±0.041
* 10.123±2.995
*
SEQUENCE?LISTING
＜110〉Zesheng Science and Technology Development Co Ltd, Shanghai
＜120〉neuregulin is used for the method and composition of myocardial infarction treatment
<130>
<140>
<141>2002-11-08
<150>
<151>
<160>2
<170>FastSEQ?for?Windows?Version?4.0
<210>1
<211>183
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>1
agccatcttg?taaaatgtgc?ggagaaggag?aaaactttct?gtgtgaatgg?aggggagtgc 60
ttcatggtga?aagacctttc?aaacccctcg?agatacttgt?gcaagtgccc?aaatgagttt 120
actggtgatc?gctgccaaaa?ctacgtaatg?gcgagcttct?acaaggcgga?ggagctgtac 180
cag
<210>2
<211>61
<212>PRT
＜213〉homo sapiens (Homo sapiens
<400>2
Ser?His?Leu?Val?Lys?Cys?Ala?Glu?Lys?Glu?Lys?Thr?Phe?Cys?Val
1 5 10 15
Asn?Gly?Gly?Glu?Cys?Phe?Met?Val?Lys?Asp?Leu?Ser?Asn?Pro?Ser
20 25 30
Arg?Tyr?Leu?Cys?Lys?Cys?Pro?Asn?Glu?Phe?Thr?Gly?Asp?Arg?Cys
35 40 45
Gln?Asn?Tyr?Val?Met?Ala?Ser?Phe?Tyr?Lys?Ala?Glu?Glu?Leu?Tyr?Gln
50 55 60
Claims (33)
1. compositions, said composition comprises neuregulin or its function fragment of effective dose or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) medicine of the material of function and the prevention of effective dose or treatment myocardial infarction forms.
2. compositions according to claim 1, wherein neuregulin by with the ErbB2-ErbB4 receptors bind, thereby the effect that produces anti-myocardial infarction.
3. compositions according to claim 1, wherein neuregulin impels the myocardial infarction blood vessel hyperplasia, thereby produces the effect of anti-myocardial infarction.
4. compositions according to claim 1, wherein neuregulin is selected from neuregulin 1, neuregulin 2, neuregulin 3 or neuregulin 4.
5. compositions according to claim 4, wherein neuregulin 1 is neuregulin α 2 or neuregulin β 2.
6. according to claim 1 described compositions, wherein the neuregulin fragment is neuregulin β 2 fragments, contains the aminoacid sequence of SEQ ID:1.
7. according to claim 1 described compositions, wherein the medicine of prevention or treatment myocardial infarction comprises ACEI, beta-blocker, calcium ion antagonist, aspirin, atropine, nitroglycerin, scopolamine and thrombolytic class medicine.
8. according to claim 7 described compositionss, it is wherein arbitrary that ACEI is selected from captopril, ramipril, fosinopril, lisinopril, enalapril, quinapril, cilazapril (cilazapril) or perindopril (perindopril).
9. according to claim 7 described compositionss, it is wherein arbitrary that beta-blocker is selected from atenolol, metoprolol, Propranolol, sotalol or plug Luo Er.
10. according to claim 7 described compositionss, it is wherein arbitrary that calcium ion antagonist is selected from nifedipine, verapamil, sieve's Ah Horizon or diltiazem .
11. according to claim 7 described compositionss, it is wherein arbitrary that thrombolytic class medicine is selected from urokinase, streptokinase, tissue-type plasminogen activator (t-PA), Single-chain Urokinase-type Plasminogen Activator (SCUPA) or the former streptokinase complex of anisyl chemical fibre lyase (APSAC).
12. compositions according to claim 1 comprises that the pharmacology can accept carrier or excipient.
13. a test kit, this test kit comprise described compositions and the operation instruction thereof that is used to prevent, treat and delay myocardial infarction of the claim 1 that places container.
14. a prevention, treat or delay the method for cardiac muscle of mammal infarction, this method comprises neuregulin or its function fragment of needs or the administration effective dose wishing this prevention, treat or delay or the nucleic acid of encode this albumen or function fragment, or improve above-mentioned neuregulin output and (or) material of function, thereby can prevent, treat or delay described myocardial infarction.
15. method according to claim 14, wherein neuregulin by with the ErbB2-ErbB4 receptors bind, thereby the effect that produces anti-myocardial infarction.
16. method according to claim 14, wherein neuregulin impels the myocardial infarction blood vessel hyperplasia, thereby produces the effect of anti-myocardial infarction.
17. method according to claim 14, wherein neuregulin is selected from neuregulin 1, neuregulin 2, neuregulin 3 or neuregulin 4.
18. method according to claim 17, wherein neuregulin 1 is neuregulin α 2 or neuregulin β 2.
19. according to claim 14 described methods, wherein the neuregulin fragment is neuregulin β 2 fragments, contains the aminoacid sequence of SEQ ID:1.
20. method according to claim 14, the nucleic acid of neuregulin or its function fragment or encode this albumen or function fragment wherein, or improve above-mentioned neuregulin output and (or) material of function, can antagonism myocardial infarction LVEDD and the raising of LVESD.
21. method according to claim 14, the nucleic acid of neuregulin or its function fragment or encode this albumen or function fragment wherein, or improve above-mentioned neuregulin output and (or) material of function, left ventricular ejection fraction reduction effect that can the antagonism myocardial infarction.
22. method according to claim 14, wherein mammal is human.
23. method according to claim 14, the clinical symptoms of myocardial infarction are selected from, and left ventricle enlarges, ventricular systolic function descends or filling pressure raises wherein arbitrary.
24. method according to claim 14, the nucleic acid of neuregulin or its function fragment or encode this albumen or function fragment wherein, or improve above-mentioned neuregulin output and (or) material of function can accept carrier with the pharmacology or excipient uses.
25. method according to claim 14 is comprising using neuregulin or its function fragment.
26. method according to claim 14 is comprising the nucleic acid that uses coding neuregulin or its function fragment.
27. method according to claim 14 wherein also comprises the medicine that uses prevention or treatment myocardial infarction.
28. according to claim 27 described methods, wherein the medicine of prevention or treatment myocardial infarction comprises ACEI, beta-blocker, calcium ion antagonist, aspirin, atropine, nitroglycerin, scopolamine and thrombolytic class medicine.
29. according to claim 29 described methods, it is wherein arbitrary that ACEI is selected from captopril, ramipril, fosinopril, lisinopril, enalapril, quinapril, cilazapril (cilazapril) or perindopril (perindopril).
30. according to claim 29 described methods, it is wherein arbitrary that beta-blocker is selected from atenolol, metoprolol, Propranolol, sotalol or plug Luo Er.
31. according to claim 29 described methods, it is wherein arbitrary that calcium ion antagonist is selected from nifedipine, verapamil, sieve's Ah Horizon or diltiazem .
32. according to claim 29 described methods, it is wherein arbitrary that thrombolytic class medicine is selected from urokinase, streptokinase, tissue-type plasminogen activator (t-PA), Single-chain Urokinase-type Plasminogen Activator (SCUPA) or the former streptokinase complex of anisyl chemical fibre lyase (APSAC).
33. method according to claim 14, the nucleic acid of neuregulin or its function fragment or encode this albumen or function fragment wherein, or improve above-mentioned neuregulin output and (or) material of function uses in vivo.
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|---|---|---|---|
| CNA021451451A CN1498656A (en) | 2002-11-08 | 2002-11-08 | Method and compsn. of nervous regulation protein for treating myocardial infarction |
| US12/879,023 US8785387B2 (en) | 2002-05-24 | 2010-09-10 | Neuregulin based methods and compositions for treating cardiovascular disease |
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| CNA021451451A CN1498656A (en) | 2002-11-08 | 2002-11-08 | Method and compsn. of nervous regulation protein for treating myocardial infarction |
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| CNA021451451A Withdrawn CN1498656A (en) | 2002-05-24 | 2002-11-08 | Method and compsn. of nervous regulation protein for treating myocardial infarction |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007076701A1 (en) * | 2005-12-30 | 2007-07-12 | Zensun (Shanghai) Science & Technology Limited | Extended release of neuregulin for improved cardiac function |
| EP1963360A1 (en) * | 2005-12-02 | 2008-09-03 | Zensun (Shanghai) Science and Technology Limited | Neuregulin variants and methods of screening and using thereof |
| US7612164B2 (en) | 1998-12-21 | 2009-11-03 | Zensun (Shanghai) Science and Technologies Ltd. | Cardiac muscle function and manipulation |
| EP2528616A1 (en) * | 2010-01-29 | 2012-12-05 | Zensun (Shanghai) Science and Technology Limited | Neuregulin based compositions and uses thereof for preventing, treating or delaying the myocardial ischemia-reperfusion injury |
| CN103432681A (en) * | 2007-05-25 | 2013-12-11 | 上海泽生科技开发有限公司 | Medicine preparation and device comprising NRG (neuregulin) |
| US8785387B2 (en) | 2002-05-24 | 2014-07-22 | Zensun (Shanghai) Science & Technology Limited | Neuregulin based methods and compositions for treating cardiovascular disease |
| CN104784679A (en) * | 2007-05-25 | 2015-07-22 | 上海泽生科技开发有限公司 | Safe dose of neuregulin applied to people |
| CN105497876A (en) * | 2014-09-24 | 2016-04-20 | 上海泽生科技开发股份有限公司 | Method and composition for using neuregulins in preventing, treating or delaying cardiac ventricular arrhythmias |
| CN108064164A (en) * | 2014-10-17 | 2018-05-22 | 上海泽生科技开发股份有限公司 | Neuregulin is used to prevent, treat or postpone the method and composition of the heart failure of ejection fraction reservation |
| US10702585B2 (en) | 2014-01-03 | 2020-07-07 | Zensun (Shanghai) Science & Technology, Co., Ltd. | Formula of neuregulin preparation |
| CN111407881A (en) * | 2019-01-07 | 2020-07-14 | 上海泽生科技开发股份有限公司 | Methods and compositions for neuregulin to prevent, treat or delay myocardial damage |
| CN111840517A (en) * | 2019-04-28 | 2020-10-30 | 上海泽生科技开发股份有限公司 | Method for long-acting prevention, treatment or delay of heart injury by using neuregulin |
| CN113677699A (en) * | 2019-04-01 | 2021-11-19 | 伊莱利利公司 | Neuregulin-4 compounds and methods of use |
| US11179323B2 (en) | 2013-05-22 | 2021-11-23 | Zensun (Shanghai) Science & Technology, Co., Ltd. | Extended release of neuregulin for treating heart failure |
| US11253573B2 (en) | 2011-10-10 | 2022-02-22 | Zensun (Shanghai) Science & Technology, Co., Ltd. | Compositions and methods for treating heart failure |
-
2002
- 2002-11-08 CN CNA021451451A patent/CN1498656A/en not_active Withdrawn
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| CN105497876A (en) * | 2014-09-24 | 2016-04-20 | 上海泽生科技开发股份有限公司 | Method and composition for using neuregulins in preventing, treating or delaying cardiac ventricular arrhythmias |
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