CN1552439A - Myocardium peptide and its use - Google Patents

Myocardium peptide and its use Download PDF

Info

Publication number
CN1552439A
CN1552439A CNA031413528A CN03141352A CN1552439A CN 1552439 A CN1552439 A CN 1552439A CN A031413528 A CNA031413528 A CN A031413528A CN 03141352 A CN03141352 A CN 03141352A CN 1552439 A CN1552439 A CN 1552439A
Authority
CN
China
Prior art keywords
copeptin
myocardium
ischemia
content
cardiac muscle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CNA031413528A
Other languages
Chinese (zh)
Other versions
CN1255183C (en
Inventor
孔祥平
万华印
李茹冰
杨联萍
曾平鲁
李恕
王日升
梁强
阮忠生
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian Zhen Ao Pharmaceutical Co Ltd
Original Assignee
Dalian Zhen Ao Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dalian Zhen Ao Pharmaceutical Co Ltd filed Critical Dalian Zhen Ao Pharmaceutical Co Ltd
Priority to CNB031413528A priority Critical patent/CN1255183C/en
Priority to EP04713508A priority patent/EP1661907B1/en
Priority to US10/567,286 priority patent/US7427663B2/en
Priority to AT04713508T priority patent/ATE545653T1/en
Priority to PCT/CN2004/000138 priority patent/WO2004108751A1/en
Priority to DK04713508.2T priority patent/DK1661907T3/en
Publication of CN1552439A publication Critical patent/CN1552439A/en
Application granted granted Critical
Publication of CN1255183C publication Critical patent/CN1255183C/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Landscapes

  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

A myocardial peptide, which is a polypeptide extracted from the heart of mammal other othan man and contains polypeptide, free amino acid, RNA and DNA, the method for testing its bioactivity, and its application in preparing the medicines for treating cardiovascular disease and reparing the damaged cardiac muscle are disclosed.

Description

Cardiac muscle Copeptin and uses thereof
Technical field
The present invention relates to a kind of myocardium Copeptin and uses thereof, specifically, the present invention relates to a kind of myocardium Copeptin that from the health mammal heart except that human, extracts and uses thereof, belong to the biochemical technology field.
Background technology
" myocardial preservation " is the focus of the inside and outside section of heart research in recent years always.Data shows recently, and many variations take place in the myocardial cell of hypoxic-ischemic, comprises intracellular calcium overload, free-radical generating, and membrane damage, (adenosine triphosphate, ATP) level descends ATP, oxygen exhaustion etc.
Becoming better and approaching perfection day by day and popularizing of cardiac operation for numerous patients have removed misery, improved people's quality of life.Along with people to improving constantly that myocardial preservation requires, the basis is also more and more deep with clinical research.The myocardial preservation of clinical department of cardiac surgery comprises: reach the postoperative myocardial preservation before the art, in the art, but the emphasis of myocardial preservation still concentrates on the ischemical reperfusion injury aspect of prevention cardiac muscle in the extracorporeal circulation process.To this, basic and clinical scientist has carried out deep multi-faceted research, mainly comprise: (1) heart perfusion: mainly concentrate on dabbling mode, for example: along irritate, contrary irritates, irritate simultaneously, be interrupted or continue irritate, whether add and use hemocyte filter etc.(2) temperature of infusion liquid: mainly contain room temperature, low temperature etc.(3) component of infusion liquid: as add with oxygen free radical scavenger (superoxide dismutase, reduced glutathion, aprotinin, puerarin etc.).More than these measures all improved the pathological change of myocardial ischemia-reperfusion injury to a certain extent.Abroad also have phosphokinase (Neoton, Italy Ou Hui pharmaceutical factory) places infusion liquid or take trimetazidine (vasorel, France's Les Laboratoires servier) makes cardiac muscle under pathological conditions, improve metabolism, set forth some from aspects such as cell, subcellular structure, oxygen-derived free radicals, energy metabolism, calcium ion (Ca2+) over loadings and discovered.But these researchs or emphasis is placed on the makeup energy aspect isolatedly; or the defense mechanism disengaging of a pre-antisitic defect and body self is come; thereby caused the clinical effectiveness of myocardial preservation unsatisfactory, therefore, research and development more efficient drug protection cardiac muscle is very necessary.
In order to intervene myocardial ischemia and protection cardiac muscle, studied many medicines over nearly 20 years, as beta-blocker, calcium antagonist, converting enzyme inhibitor, various oxygen free radical scavengers etc., but it is clinical still uncertainly to myocardial protection effect.The medicine of existing treatment myocardial ischemia does not also have a kind ofly can definitely reduce myocardial infarction, to resisting myocardial ischemia.The late nineteen eighties, people observe drosophila larvae through the high heat treatment of short-term, its salivary gland cell multifibres dyes body " bulk " mode and changes, showing that this regional gene is transcribed is activated, and claim that this is heat shock effect (heatshock reaction, HSR, Anna Rev Biochem 1986,55:1151).Find later on laboratory animal in succession through heat-shock pretreatment, can be obviously to the myocardial damage due to the anti-ischemia/reperfusion.Marry was called " ischemic preconditioning " (ischemia precondition with this phenomenon in 1986, IP), discover that further this phenomenon and heat shock induction myocardial cell synthesize one group of new protein-heat shock protein (heat shock proteins, HSPs) or claim stress protein (stress protein, SP) relevant.These research reports in succession show that cardiac muscle self has powerful endogenous protection mechanism; Find from the prokaryote to the eukaryote again later on, from plant, animal to the people, no matter cultured cell or whole machine body, heat shock all has the synthetic expression of HSPs when handling, and show following characteristics: (1) except that heat shock can induce HSP synthetic, many factors are still arranged, all can induce HSP synthetic as ischemia, anoxia, ethanol, heavy metallic salt, myocardium pressure load, medicine and most of morbid state, and " cross tolerance phenomenon " can occur; (2) on the HSP structure high conservative is arranged, as fruit bat and yeast HSP 70There is 72% amino acid sequence identical, human HSP 70Gene and fruit bat HSP 70Gene has 73% homology, HSP 9078% homology is arranged.These are structural similar, guaranteed the homogeny on the function (Burdon:Biochem, J.1986,240:313); (3) HSP has the Best Times scope to myocardial protective effect, claims " window(s) of opportunity " (window ofopportunity) again, exceeds certain time limit just to lose its protective effect (Perdriget:Curr Surg 1989,23); (4) HSP is present in whole biosphere, is present in the various cells of higher mammal body.HSP sInduce and not only strengthen myocardial function and recover, and also strengthen the recovery of myocardium endothelial function, prolong the cardiac arrest time.Above-mentioned discovery is applied to the donor protection of heart transplantation, the treatment of ischemic myocardium, the preparation of extracorporeal circulation cardioplegic solution etc.; can break through the conventional medicament constraint; from strengthening the anti-damage of inductor inner cell potential itself, provide a kind of new way with milestone significance.
People such as Pennica (1995) clone cardiotrophin-1 (CT-1) gene in succession in myocardial cell, and express in E scherichia coli.Studies show that CT-1 is a kind of cytokine with immunomodulating and induced growth effect, its natural being present in the myocardial cell, can making myocardial cell to anti-hypoxia, pyritous infringement, and have the effect that suppresses apoptosis of cardiac muscle.Deep discovers, CT-1 is in cardiac muscle cells and in vivo can induce HSP to express, and this inducing action is and a kind of gp of being called 130The cell surface polypeptide relevant, it activates gp 130After, make HSP by NF-IL-6/NF-IL--6 β and tyrosine kinase pathway 70, HSP 90The expression that reaches the HSP small-molecule substance is strengthened, thereby strengthens myocardial cell tolerance anoxia and pyritous ability.Research shows that also CT-1 can promote the synthetic of myocardial cell structural protein, and the major axis that increases cell makes contraction stronger.The research of the Myotrophin of gene recombinaton is another problem of myocardial cell stimulating factor.Parames (1997) proves that Myotrophin promotes that the myocardial cell growth is relevant with protein kinase-c.Myotrophin and Cardiotrophin may be functional similarities in the myocardial cell, but the different cytokine of active cell growth pathway has all shown protecting myocardial cell, promote the function of growth.
Existing all kinds of cardiovascular disease therapies medicine, remove converting enzyme inhibitor and have the generation of retardance somatomedin, Profilin matter is synthetic, alleviates outside the effect of myocardial hypertrophy (Hypertrophy), and other medicines all do not have the direct effect of regulating the cardiac muscle growth, breaking up, repair.In recent years, abroad begun to pay attention to using pharmaceutical methods to induce the protective capability of cardiac muscle self, promoted the research of myocardial cell regeneration etc., the research of Cardiotrophin and Myotrophin as transducible gene.On the other hand, by the various pass through mechanism of extracellular signal triggering, the propagation or the reconstruct of regulation and control cardiac muscle, vascular cell.But all these researchs all are in animal experiment or preclinical study stage.
Above-mentioned research clearly proves: under the imperfect as yet situation of extracorporeal circulation methods of myocardial protection; design a kind of harmless to body; before again can Rhizoma Atractylodis Macrocephalae, in the art and the medicine of postoperative protection cardiac muscle; to exploring the control of myocardial ischemia and reperfusion injury; provide new thinking and approach, with significant.
ZL94102798 discloses a kind of CMGSP and preparation method thereof, choose the heart of healthy young mammals, adopt that mechanical system is smashed to pieces ,-20 ℃ of dark freezing-dissolve post-heating 60-100 ℃, the centrifugal 3000rpm in ℃ dark freezing-dissolve back again-20, hold back post-degerming-packing-lyophilizing-packing through negative pressure, obtain molecular weight less than 20000 daltonian polypeptide class active substances.
ZL94102799 discloses a kind of CMGSP (GMGSP) with the synthetic and protein synthesis of the DNA that stimulates former generation cardiac muscle cells, is that preparation is extracted from the heart of healthy young mammals, and is stable in the pH2-9 scope; The heating 95-100 ℃ 10 minutes, 60-70 ℃ of 30 minutes following biological activity do not change; At the multiple protein hydrolytic enzyme, loss of bioactivity under 37 ℃ of 2 hours conditions; Under 22 ℃ of-30 ℃ of conditions of aqueous solution, can form polymer but biological activity changes not obvious; Adding under the 3%-8% mannitol lyophilizing air-proof condition, room temperature storage 1.5 years was stored 2 years for 4 ℃, stored 3 years for-20 ℃, and biological activity does not change; HPLC the analysis showed that: described GMGSP is made up of four components, and relative peak of each component and retention time are respectively: 10.4% (2.88 minutes), 6.4% (3.93 minutes), 36.3% (5.09 minutes), 7.3% (7.41 minutes), the equal biologically active of each component; Analyze the two band molecular weight that show through SDS-PAGE and be respectively 8500Da, 10800Da, HPLC analyzes number-average molecular weight 9800Da, weight average molecular weight 10500Da, 2 components all have biological activity.
But above-mentioned patented technology has just been carried out separation, purification and simple active testing roughly to this biologically active peptide, and its concrete composition, purposes and effect are not described in detail.
Summary of the invention
The object of the present invention is to provide a kind of myocardium Copeptin, the main active ingredient of described myocardium Copeptin is a polypeptide, can directly act on myocardial cell, promotes the reparation that cardiac muscle damages under multiple impairment factor, for alleviating induced myocardial injury in the operation on heart, promote the reparation of damage that a new approach is provided.
Another goal of the invention of the present invention provides the purposes of this cardiac muscle Copeptin aspect preparation treatment cardiovascular disease medicine.
The present invention's goal of the invention again provides the purposes of this cardiac muscle Copeptin aspect preparation treatment myocardial ischemia-reperfusion injury medicine.
To achieve these goals, the technical solution used in the present invention is: a kind of myocardium Copeptin is never to comprise the polypeptide that extracts in people's the health mammal heart, its content of peptides is 75%～90%, free amino acid is 6%～15%, rna content is less than 2%, DNA (deoxyribonucleic acid) content is less than 7.5%, and weight average molecular weight is less than 10000 dalton.
Wherein saidly do not comprise that people's healthy mammal comprises pig, cattle, sheep, rabbit, horse etc., preferred young mammals comprises piglets, milk cattle, milk goat, newborn rabbit, newborn horse etc., most preferably piglets.
The weight average molecular weight of described myocardium Copeptin is less than 10000 dalton, can be 1000～10000 dalton, and preferred weight average molecular weight is 2000～8 000 dalton, and most preferred weight average molecular weight is 2000～5000 dalton.
Described myocardium Copeptin biological activity in pH3～8 scopes is stable, and to the E.C. 3.4.21.64 sensitivity, 85 ℃ of 10min biological activitys do not change, and is stable under freezing or lyophilisation condition.
Described myocardium Copeptin isoelectric focusing electrophoresis shows 2-6 bar colored zone, and preferred isoelectric focusing electrophoresis shows 2 bands, and wherein pI 10.92 is with painted dark person.
Described myocardium Copeptin has a stable maximum absorption band at ultra-violet absorption spectrum 190-210nm place.Preferably one maximum absorption band person is arranged at ultra-violet absorption spectrum 200 ± 2nm place.
Adopt sulfosalicylic acid method to identify in the described myocardium Copeptin of demonstration and do not contain protein.
Myocardium Copeptin vigor of the present invention is at least 2.2.
Also can comprise excipient in the myocardium Copeptin of the present invention, its weight ratio consists of:
Cardiac muscle Copeptin 15～20
Excipient 100～375,
Be preferably 18～20: 200～375.
Excipient can be mannitol, trehalose, lactose, sucrose or other lyophilizing adjuvant, is preferably mannitol.
In order to remove thermal source, also can comprise active carbon in the myocardium Copeptin of the present invention, its content is about 0.1%.
Myocardium Copeptin of the present invention is analyzed through FPLC, the cardiac muscle Copeptin mainly contains 5 component peaks, the percentage area adds up to 90%～95% relatively, through the activity inspection, 5 component peaks all can promote former myocyte and the anoxia oxygen supply myocardial cell succinate dehydrogenase vigor again that nourishes heart of being commissioned to train, and wherein P1 peak activity is stronger.
Myocardium Copeptin of the present invention can be made by the following method: the health mammal ventricular muscles that will not comprise the people is cleaned, chopping, add sterile purified water homogenate, homogenate is freezing repeatedly, thaw 3～4 times, be heated to 65～95 ℃ of filter cleaners, obtain coarse filtration liquid with the filtration of sheet frame filter, the reuse hollow fiber column ultrafilter, obtain fine straining liquid, use the ultrafilter membrane ultrafiltration, molecular cut off is less than the myocardium peptide cellulose solution of 10000Da, reverse osmosis concentration obtains finished product through quality examination, filtration sterilization, fill, lyophilization at last.
The addition of wherein said sterile purified water is 0.5～4 times of mammiferous ventricular muscles; The rotating speed of described homogenate is 1000～5000rpm/min.
Described freezing for being lower than under-5 ℃ the temperature freezing 24～72 hours, preferably-20 ℃～-30 ℃ freezing 36～48 hours down; Described mode of heating is for adopting water proof heating or direct heating, and temperature is 70～90 ℃, and the time is preferably adopted the water proof heating for being no more than 2 hours, and temperature is 75 ℃～80 ℃, and the time is for being no more than 1 hour.
The present invention filters with the sheet frame filter and obtains coarse filtration liquid, with obtaining the fine straining liquid of molecular weight less than 12kDa behind the hollow fiber column ultrafilter; The ultrafiltration of reuse ultrafilter membrane is dammed and is obtained the fine straining liquid of molecular weight less than 10kDa; Wherein said sheet frame filter belongs to the bio-pharmaceuticals conventional equipment, selects for use less than 10 μ middling speed filter paper, preferably is less than or equal to 5 μ middling speed filter paper, is the sheet frame filter of XAS03-172/8 such as the model of Guangzhou medicine instrument institute production; It is F60 that hollow fiber column can be selected model for use, can filter the liquid of molecular weight less than 12kDa, such as the hollow fiber column of Switzerland Zinpro Corp.; The ultrafilter membrane specification is 1～10kDa, such as the product of Millipore company.The reverse osmosis concentration post is the product of Millipore company.
Filtration sterilization of the present invention and fill are known in those skilled in the art.
Freeze drying equipment commonly used is adopted in lyophilization of the present invention: freezer dryer; Detailed process is: made the interior shelf temperature of hothouse reach-15 ℃～-20 ℃ in 5～40 minutes, reach-18 ℃～-20 ℃ in preferred 20～30 minutes, through 20～40 minutes, make products temperature reach-25～-35 ℃ again, reach-30～-35 ℃ in preferred 25～35 minutes.Keep temperature in the condenser being dropped to-40～-50 ℃ in 1～3 hour, evacuation again, when vacuum reaches 90～100KPa, be communicated with hothouse and condenser, stop the refrigeration of drying baker, when being 10～15Pa, the vacuum of drying baker begins to heat up, programming rate is 2～5 ℃/min, is warming up to 5～15 ℃, is incubated 3～6 hours, preferably be warmed up to 8～12 ℃, continue 4～5 hours with 3～4 ℃/min speed.Continuation is warming up to 15～25 ℃ with 8～16 ℃/min speed, continues 3～8 hours, preferably is warmed up to 18～22 ℃ with 10～12 ℃/min speed, continues 4～6 hours.Continuation is warming up to 30～35 ℃ with 7～15 ℃/min speed, continues 1～4 hour, preferably is warmed up to 33～35 ℃ with 9～12 ℃/min speed, continues 1.5～2 hours.Continuation is warming up to 50～60 ℃ with 4～8 ℃/min speed, continues 1～3 hour, preferably is warmed up to 54～58 ℃ with 5～7 ℃/min speed, continues 1.5～2 hours.Enter temperature-fall period, make temperature reduce to 40-50 ℃ in 10～30min, continue 8-15 hour, make temperature reduce to 45～48 ℃ in preferred 15～20min, continue 9～12 hours, obtain the qualified myocardium Copeptin dried frozen aquatic products of outward appearance, take out goods and seal.
In the preparation process of myocardium Copeptin of the present invention, also can add the adjuvant of freeze-dried product commonly used in the gained cardiac muscle peptide cellulose solution, such as being mannitol, trehalose, lactose, sucrose or other lyophilizing adjuvant.Easily form lattice after adding adjuvant, play the carriage effect, stablize the character of these goods.
Adopt preparation method of the present invention, filter type through the filtration of sheet frame filter, hollow fiber column ultrafilter and ultrafilter membrane ultrafiltration, can obtain required for the present invention after the reverse osmosis concentration less than 10000 dalton cardiac muscle Copeptin, compare with ZL in the background technology 94102798, working time is short, and the treating capacity of product is many, obtains the concentration height of product, active big, be difficult for producing pyrogen.
The invention provides the purposes of described myocardium Copeptin aspect preparation treatment cardiovascular drugs.
The present invention also provides the purposes of described myocardium Copeptin aspect preparation treatment myocardial ischemia and reperfusion injury medicine.
Myocardium Copeptin of the present invention and patent ZL94102798 and the disclosed CMGSP of ZL94102799 (GMGSP) are relatively, has obviously high external biological activity, myocardium Copeptin of the present invention active unit be CMGSP 3-5 doubly, the drug effect correction data shows in the body, and it has obvious favourable influence to myocardium creatine phosphokinase release due to myocardial ischemia-reperfusion injury and lactic acid dehydrogenase activity and free fatty and mda content.
Myocardium Copeptin of the present invention is for can directly act on myocardial cell; the reparation (as ischemia, drug intoxication etc.) of promotion injury of myocardium under multiple impairment factor; promote protein synthesis, reduce oxygen free radical injury, reduce calcium overload, induce endogenous protection, improve the medicine of myocardial metabolism function; for alleviating induced myocardial injury in the operation on heart, promote the reparation of damage that a new approach is provided.
The main pharmacodynamics result of study of cardiac muscle Copeptin is as follows:
1. myocardium Copeptin can obviously alleviate the myocardial ultrastructure damage due to the perfusion of myocardial ischemia-again, make its near or recover normal (Fig. 6-12 black-and-white photograph, table 13).
2. the visceral pericardium electrocardiogram shows that the ST section that myocardium Copeptin can obviously resist due to the cat myocardial ischemia raises, and reduces myocardial ischemia scope (table 14-15).
3. myocardium Copeptin can obviously lower increase (the table 16-21) of myocardium creatine phosphokinase release and lactic acid dehydrogenase activity and free fatty and mda content due to myocardial ischemia-reperfusion injury.
4. myocardium Copeptin can reduce myocardial oxygen consumption (table 22).
5. myocardium Copeptin 5,10mg/kg can obviously reduce the Electrocardiographic ST of myocardial infarction Cor Sus domestica adventitia, reduce NST, and dwindle myocardial infarct size, dead due to quivering in arrhythmia, the chamber that the acute myocardial ischemia pig is occurred have certain therapeutical effect, and blood pressure, heart rate are not had obvious influence (table 23, Figure 13).
Description of drawings
Fig. 1 cardiac muscle Copeptin HPLC molecular weight collection of illustrative plates
Fig. 2 cardiac muscle Copeptin FPLC separation and purification collection of illustrative plates
Fig. 3 cardiac muscle Copeptin isoelectric point determination collection of illustrative plates
Fig. 4 cardiac muscle Copeptin UV scanning collection of illustrative plates
Fig. 5 cardiac muscle Copeptin HPLC differentiates collection of illustrative plates
Fig. 6 cardiac muscle Copeptin is to the normal control group of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury
Fig. 7 cardiac muscle Copeptin is to the normal saline matched group of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury
Fig. 8 cardiac muscle Copeptin to the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury damage matched group after, add water 50ml and bromine liquid number droplet again, shake up, boiled 15 minutes, drive away excessive bromine.Be cooled to room temperature, thin up filters to 1000ml, and filtrate is stock solution, stores in the brown bottle, puts in the refrigerator and preserves.Face the time spent, get stock solution and use water as doubly amount dilution, promptly.
Specification Curve of Increasing
The preparation precision of reference substance solution takes by weighing dry bovine serum albumin(BSA) reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides) 100mg, puts in the 100ml measuring bottle, and thin up is to scale, shake up, precision is measured 10.0ml, puts in the 100ml measuring bottle, thin up shakes up and get final product to scale.
The preparation precision of calibration curve is measured reference substance solution 0.0,0.2,0.4,0.6,0.8 and 1.0ml, put respectively in the tool plug test tube, and each Guan Jiashui makes into 1.0ml, and every pipe adds alkaline copper solution 5.0ml, shakes up, and room temperature was placed 10 minutes. Add fast successively again phenol reagent 0.5ml, shake up immediately, put in the water-bath (35 ℃) and be incubated 30 minutes. Take out, be cooled to room temperature, make blank with reference substance solution 0.0ml pipe, measure trap at 660nm wavelength place according to AAS (two appendix IV of Chinese Pharmacopoeia nineteen ninety-five version A). Take trap as ordinate, protein concentration is abscissa, the drawing standard curve.
Determination method is got this product, is dissolved in water respectively or dilutes and quantitatively be transferred in the 50ml measuring bottle, adds water to scale, shakes up. The accurate 10.0ml that draws puts in the 50ml measuring bottle, adds water to scale, shakes up. The accurate 1.0ml that draws puts in the tool plug test tube, plays the mensuration trap from " adding the alkaline copper test solution " under the sighting target directrix curve preparation, checks in respective concentration at calibration curve, calculates, and get final product.
Results and analysis
Measured result sees Table 4.
The every 1ml of measured result myocardium peptide solution contains polypeptide in the 2.6-2.8mg scope; Every bottle of injection myocardium peptide contains 9.2-9.9mg. For guaranteeing solution and preparation content constant, we stipulate that myocardium peptide solution content of peptides should be greater than every milliliter of 2.5mg, and every bottle of content of peptides of injection myocardium peptide is at 9.0-11.0mg.
The measurement result of three kinds of content assaying methods of table 4. relatively
Assay method
Biuret method forint-phenol law nitriding (polypeptide organic nitrogen)
Solution (mg/ml)
960419        1.65        2.7          1.58
960422        1.85        2.8          1.74
960423        2.00        2.6          1.46
Preparation (mg/ bottle)
960501        3.26        9.9          3.61
960502        3.63        9.2          3.92
960503        3.73        9.2          3.72
Annotate:*Biuret method is measured by automatic clinical chemistry analyzer
Three kinds of assay methods are inconsistent as can be seen from Table 4, and the result is quite different. The reaction principle of considering forint-phenol law is the phenolic group reaction of aromatic amino acid, and specificity is better, and is easy and simple to handle. Select specific reference substance and the equal drawing standard curve of each mensuration can overcome non-linear relation, we select forint-phenol law to measure the content of peptides of myocardium peptide solution and injection myocardium peptide.
(3) proportion of composing analysis
These goods mainly are comprised of polypeptide, measure respectively its organic nitrogen content, deduct free amino acid organic nitrogen content by total organic nitrogen content and are polypeptide organic nitrogen content.
Reagent and method the same (seeing nitriding).
Stable in pH3-8 scope heart carnosine element biologically active as can be seen from Table 1.
Analyze through FPLC, myocardium peptide mainly contains 5 component peaks, and the percentage area adds up to 90%-95% relatively. Active check that 5 component peaks all can promote Primary cultured myocardial cells and cardiac myocyte under hypoxia-reoxygenation Succinate Dehydrogenase Activity (table 2), wherein P1 peak activity strong (seeing Fig. 2). The myocardium peptide content of peptides accounts for 75-90%, and free amino acid accounts for 6-15%, and trace dna and trace element are still arranged. Isoelectric focusing electrophoresis shows 2 colored zones, and wherein pI 10.92 is with painted dark (seeing Fig. 3).
The impact (mtt assay) of each Components On Myocytes enzyme activity of table 2 myocardium peptide (n=8, x ± s)
The OD value (x ± s)
The 5 μ g/ml t values of dividing into groups
Normal group 0.344 ± 0.014**       9.93
Injury by Adriamycin group 0.272 ± 0.015
P1              0.318±0.004 **       6.344
P2              0.295±0.012 **       3.39
P3              0.309±0.012 **       5.45
P4              0.317±0.017 **       5.61
P5              0.303±0.014 **       4.27
Myocardium peptide group 0.298 ± 0.005**       3.47
Annotate: compare with the adriamycin group,**P＜0.01
5 component peaks all can promote the former myocyte's succinate dehydrogenase vigor that nourishes heart of being commissioned to train as can be seen from Table 2.
1. the discriminating of polypeptide
The carnosine cellulose content of coring is the myocardium peptide cellulose solution 1ml of 2.5mg/ml, adds water 2ml dissolving, adds biuret reagent 2ml[biuret reagent preparation method: get copper sulfate (CuSO 45H 2O) 0.75g and sodium potassium tartrate tetrahydrate (NaKC 4H 4O 64H 2O) 3g adds the about 250ml dissolving of water, under agitation add 10% sodium hydroxide test solution 150ml, and thin up is stored in the plastic bottle to 500ml.], mixing shows blue purple, is and contains polypeptide; Through 6 batches of inspections, myocardium Copeptin of the present invention all shows blue purple, promptly contains polypeptide.
2. assay
(1) semimicro Kjeldahl
Cardiac muscle peptide cellulose solution, lot number 960419,960422,960423; Injection cardiac muscle Copeptin, 960501,960502,960503, face the time spent water and be dissolved to desired concn.
Reagent of sulfuric acid: chemical pure, proportion 1.84; Digestive pharmaceutical: copper sulfate (CuSO 4.5H2O) 1 part with 10 parts of common porphyrize mixings of potassium sulfate (K2SO4); 12.5mol/L sodium hydroxide solution; 2% boric acid absorption liquid; 10% sodium tungstate; 0.33mmol/L sulphuric acid; Mixed indicator: 5 parts of 0.2% (W/V) bromocresol green alcoholic solutions mix for 2 parts with 0.1% (W/V) C.I. 13020. alcoholic solution and are made into; 0.01mol/L hydrochloric acid.
Computing formula:
Figure A0314135200091
Algoscopy is measured according to N2 method (seeing two appendix VII of Chinese Pharmacopoeia nineteen ninety-five version D, second method).
Inorganic nitrogen: precision is measured test sample 5ml, adds water 3ml, adds 10% sodium tungstate 1ml, and 0.33mmol/L sulphuric acid 1ml shakes up, and leaves standstill 30 minutes, filters.Precision is measured filtrate 5ml and sodium hydroxide test solution 5ml, adds in the alembic, shines under the total nitrogen item with the method determination of distillation.
Total nitrogen: get one bottle of injection cardiac muscle Copeptin, accurately add water 4ml dissolving, precision is measured 2.0ml; Precision is measured myocardium peptide cellulose solution 2.0ml, measures according to the determination of total nitrogen content method respectively.
Organic nitrogen amount=total nitrogen-inorganic nitrogen amount
Annotate: (1) because test sample is easy to generate foam in still-process, brings sodium hydroxide into condensing tube when measuring inorganic nitrogen, flow into and collect in the liquid, make measurement result higher, should before distillation, add 10% sodium tungstate and 0.33mmol/L sulphuric acid and remove Organic substance, get filtrate and measure inorganic nitrogen.
(2) test sample is Main Ingredients and Appearance with the peptide material, influences measurement result owing to bringing inorganic matter generation inorganic nitrogen in process of production into.This law is measured inorganic nitrogen after measuring total nitrogen, deduct the inorganic nitrogen amount as the organic nitrogen amount with total nitrogen.
Result and analysis
The nitrogen content (seeing Table 3) of different lot number cardiac muscle peptide cellulose solutions and preparation.
The different lot number test sample of table 3. nitrogen determination result
Cardiac muscle peptide cellulose solution (the injection cardiac muscle Copeptin (mg nitrogen/bottle) of mg nitrogen/ml)
Lot number 960419??960422??960423??960501??960502??960503
Total nitrogen 1.788???1.926???1.628???3.816???4.250???4.100
Organic nitrogen 1.589???1.743???1.460???3.612???3.919???3.722
Inorganic nitrogen 0.199???0.183???0.168???0.204???0.331???0.378
Table 3 shows that myocardium peptide cellulose solution organic nitrogen content is between 1.46-1.74mg/ml, and injection cardiac muscle Copeptin organic nitrogen content is in 3.61-3.92mg/ bottle scope.Average content is respectively 1.60mg nitrogen/ml and 3.75mg nitrogen/bottle.
(2) forint--phenol method (Folin-phenol)
Cardiac muscle peptide cellulose solution, lot number 960419,960422,960423; Injection is Copeptin diligently, lot number 960501,960502,960503; Face the time spent water and be dissolved into suitable concentration.Reference substance: bovine serum albumin (Nat'l Pharmaceutical ﹠ Biological Products Control Institute) lot number 9607.
Instrument 7221 type spectrophotometers, Shanghai.
The reagent preparation:
4% sodium carbonate liquor is got sodium carbonate (Na2CO310H2O) 4g and is dissolved in water and is diluted to 100ml, shakes up.
0.2mol/L sodium hydroxide solution is got sodium hydroxide (NaOH) 0.8g, is dissolved in water and dilutes 100ml, shakes up.
1% copper-bath is got copper sulfate (CuSo45H2O) 1g and is dissolved in water and is diluted to 100ml, shakes up, promptly.
2% Soluble tartar. solution is got Soluble tartar. (K2C4H4O61/2H2O) 2g, is dissolved in water and is diluted to 100ml, shakes up.
The alkaline copper test solution faces with before getting test solution 1 and 2 each 25ml, test solution 3 and 4 each 0.5ml, mixing.
The phenol test solution is got sodium tungstate (Na2WO42H2O) 100g, sodium molybdate (Na 2MoO 4.2H 2O) 25g puts in the 1500ml flask, adds water 700ml, 85% phosphoric acid 50ml, hydrochloric acid 100ml, on connect little the boiling of return duct (with the rubber closure of cork or tinfoil parcel) and refluxed 10 hours, take off condensing tube, add lithium sulfate (Li 2SO 4) 150g, complete molten after, add water 50ml and bromine liquid number droplet again, shake up, boiled 15 minutes, drive away excessive bromine.Be cooled to room temperature, thin up filters to 1000ml, and filtrate is stock solution, stores in the brown bottle, puts in the refrigerator and preserves.Face the time spent, get stock solution and use water as doubly amount dilution, promptly.
Standard curve is drawn
The preparation precision of reference substance solution takes by weighing exsiccant bovine serum albumin reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides) 100mg, puts in the 100ml measuring bottle, and thin up is to scale, shake up, precision is measured 10.0ml, puts in the 100ml measuring bottle, thin up shakes up promptly to scale.
The preparation precision of standard curve is measured reference substance solution 0.0,0.2,0.4,0.6,0.8 and 1.0ml, put respectively in the tool plug test tube, and each Guan Jiashui makes into 1.0ml, and every pipe adds alkaline copper solution 5.0ml, shakes up, and room temperature was placed 10 minutes.Add phenol reagent 0.5ml more successively fast, shake up immediately, put in the water-bath (35 ℃) and be incubated 30 minutes.Take out, be cooled to room temperature, make blank, measure trap at 660nm wavelength place according to spectrophotography (two appendix IV of Chinese Pharmacopoeia nineteen ninety-five version A) with reference substance solution 0.0ml pipe.With the trap is ordinate, and protein concentration is an abscissa, the drawing standard curve.
Algoscopy is got this product, is dissolved in water respectively or dilutes and quantitatively be transferred in the 50ml measuring bottle, adds water to scale, shakes up.The accurate 10.0ml that draws puts in the 50ml measuring bottle, adds water to scale, shakes up.The accurate 1.0ml that draws puts in the tool plug test tube, plays the mensuration trap from " adding the alkaline copper test solution " under the sighting target directrix curve preparation, checks in respective concentration on standard curve, calculates, promptly.
Result and analysis
Measured result sees Table 4.
The every 1ml of measured result cardiac muscle peptide cellulose solution contains polypeptide in the 2.6-2.8mg scope; Injection cardiac muscle Copeptin contains 9.2-9.9mg for every bottle.Constant for guaranteeing solution and formulation content, we stipulate that myocardium peptide cellulose solution content of peptides should inject every bottle of content of peptides of myocardium Copeptin at 9.0-11.0mg greater than every milliliter of 2.5mg.
The measurement result of three kinds of content assaying methods of table 4. relatively
Assay method
Biuret method forint-phenol law nitriding (polypeptide organic nitrogen)
Solution (mg/ml)
960419????????1.65????????2.7??????????1.58
960422????????1.85????????2.8??????????1.74
960423????????2.00????????2.6??????????1.46
Preparation (mg/ bottle)
960501????????3.26????????9.9??????????3.61
960502????????3.63????????9.2??????????3.92
960503????????3.73????????9.2??????????3.72
Annotate: *Biuret method is measured by automatic clinical chemistry analyzer
Three kinds of assay methods are inconsistent as can be seen from Table 4, and the result is quite different.The reaction principle of considering forint-phenol law is the phenolic group reaction of aromatic amino acid, and specificity is better, and is easy and simple to handle.Select for use specific reference substance and the equal drawing standard curve of each mensuration can overcome non-linear relation, we select for use forint-phenol law to measure the content of peptides of myocardium peptide cellulose solution and injection cardiac muscle Copeptin.
(3) proportion of composing analysis
These goods mainly are made up of polypeptide, measure its organic nitrogen content respectively, deduct free amino acid organic nitrogen content by total organic nitrogen content and are polypeptide organic nitrogen content.
Reagent and method the same (seeing nitriding).
The injection of 3 lot numbers cardiac muscle Copeptin free amino acid nitrogen content sees Table 5 as a result.
Table 5 injection cardiac muscle Copeptin free amino acid nitrogen content
Injection cardiac muscle Copeptin
Lot number ??960501????960502????960503
Free amino acid total nitrogen g/L ??0.257?????0.285?????0.28
Injection cardiac muscle Copeptin organic nitrogen content and free amino acid nitrogen content be (seeing Table 6) relatively
Table 6 total nitrogen and free amino acid nitrogen content are relatively
Injection cardiac muscle Copeptin
Lot number ??960501????960502????960503
Polypeptide organic nitrogen g/L ??3.612?????3.919?????3.722
Free Amino Nitrogen g/L ??0.257?????0.285?????0.286
The free amino acid nitrogen content accounts for total organic nitrogen amount percentage ratio (%) ??6.643?????6.823?????7.135
From table 5,6 as can be seen, and injection cardiac muscle Copeptin free amino acid nitrogen content only accounts for the 6.643%-7.135% of test sample nitrogen content, shows that the test sample polypeptide accounts for the overwhelming majority of total nitrogen.In view of this product polypeptide is the Main Ingredients and Appearance of biologic activity and the stable and controllable of considering production technology, we should reach 75%-90% at regulation injections cardiac muscle Copeptin polypeptide ratio.
3. UV scanning analysis
Day island proper Tianjin 2201 type ultraviolet spectrophotometers are measured according to spectrophotography (two appendix IV of Chinese Pharmacopoeia nineteen ninety-five version A).
The result shows that myocardium peptide cellulose solution has maximum absorption band at the 199.8-201.2nm place, and injection cardiac muscle Copeptin shows maximum absorption band (Fig. 4) at the 200.4-201.8nm place.Show that 3 batches of solution are consistent with 3 batches of preparation UV scanning collection of illustrative plates, the test sample main component is a polypeptide, and myocardium Copeptin reparation technology is stable.
The UV scanning absorbing wavelength of the myocardium Copeptin of table 7
Lot number absorbing wavelength (nm)
Solution (mg/ml)
960422????????????????????????????200.4
960423????????????????????????????199.8
960419????????????????????????????201.2
Preparation (mg/ bottle)
960501????????????????????????????200.4
960502????????????????????????????201.8
960503????????????????????????????201.6
4. proteinic discriminating
Get the myocardium Copeptin 2ml of the present invention's cardiac muscle peptide cellulose content 2.5mg/ml, add 20% sulfosalicylic acid solution 1ml, do not produce muddiness, three batches of solution of myocardium Copeptin and the demonstration of preparation measurement result all do not contain protein.Check protein with sulfosalicylic acid method, both can monitor that the shown blue purple of also provable biuret method is polypeptide rather than other material simultaneously to the protein that may sneak into.
5. molecular weight and peptide chromatogram are checked
HPLC method determining molecular weight
The HP1050 chromatograph of liquid
Chromatographic condition: mobile phase: sodium sulfate (0.1mol/L)-sodium dihydrogen phosphate (0.05mol/L)-sodium azide (0.05%), transfer pH to 6.8 with NaOH; Flow velocity: 0.35ml/min; Post: TOSOH TSK G2000sw 7.5mm * 300mm; Column temperature: 25 ℃; Detect wavelength: 280nm; Sample size: 10 μ l.
Get cytochrome C (MW=12400) respectively, aprotinin (MW=6700) and vitamin B 12(MW=1355) in right amount, make the reference substance solution of suitable concentration respectively with mobile phase.Get 1 bottle of test sample injection cardiac muscle Copeptin, content of peptides 10mg makes the need testing solution that every 1ml contains 5mg with mobile phase.Get reference substance solution and need testing solution respectively, press the chromatographic condition injecting chromatograph, measure its retention time respectively.Compare the regression equation of product with method of least square, correlation coefficient must not be less than 0.99.Press following formula drawing standard curve and calculate the test sample molecular weight.
IgMW＝A+BtR
IgMW＝6.8405-0.1219tR?γ＝-0.9990
MW is a molecular weight in the formula, and A is a constant, and B is a slope, tR be retention time (minute).
The results are shown in Table 5,6.
The retention time of the relative percentage area of table 8 injection cardiac muscle Copeptin chromatographic peak
Lot number Chromatographic peak retention time (min)
????P1??????P2??????P3??????P4??????P5
??960501 ????30.5????31.8????32.8????35.0????38.7
??960502 ????30.5????31.2????32.8????35.0????38.7
??960503 ????31.2????????????32.7????35.0????38.7
??X ????30.7????31.5????32.7????35.0????38.7
The peak molecular weight of table 9 injection cardiac muscle Copeptin
Lot number Molecular weight (dalton)
????P1??????P2??????P3??????P4??????P5
??960501 ????6023????4588????3665????2233????969
??960502 ????6027????5261????3688????2234????971
??960503 ????5165????3709????????????2236????875
????X ????5736????4519????3676????2234????971
X＝6.7444-0.09696×Y＝-0.9937
The range of molecular weight distributions of injection cardiac muscle Copeptin is at 922-6027Da, and the maximum molecular weight scope shows that at 5214-6027Da (Fig. 1) this test sample is a micromolecule polypeptide, and molecular weight is difficult for producing anaphylaxis less than 10000 dalton.
6. nucleic acid
Injection cardiac muscle Copeptin, every bottle contains polypeptide 10mg.Adding distil water 4ml dissolving adds the extracting of equal-volume phenol once again.Get supernatant and measure dna content.With resetting and adding the 10mol/L ammonium acetate of long-pending no water-cooled ethanol of amphiploid and 1/20 volume on this, put-80 ℃, 30min, 12000rpm * 20min abandons supernatant, and precipitation is dissolved in the 4ml distilled water.This sample is used to measure rna content.
(1) rna content is measured
The preparation of standard curve:
Get 6 in test tube, according to the form below adds reagent
Addition ml
Admixture 0 ?1 ?2 ?3 ?4 ?5
The RNA titer 0 ?0.2 ?0.4 ?0.6 ?0.8 ?1.0
Distilled water 1.0 ?0.8 ?0.6 ?0.4 ?0.2 ?0
Orcinol reagent 3.0 ?3.0 ?3.0 ?3.0 ?3.0 ?3.0
With each pipe mixing, put and heat 20min in the burning water, take out, cold water is cooled to room temperature,, with " 0 " number pipe zeroing, measures and respectively manages absorbance under the 670nm wavelength with spectrophotometer.With the rna content is abscissa, and absorbance is a vertical coordinate, the drawing standard curve.
The sample rna content is measured:
Get 4 in test tube, be marked with " blank pipe " and " measuring pipe " respectively.In blank pipe, add distilled water 1.0ml, in measuring pipe, add RNA sample solution 1.0ml, every then pipe adds orcinol reagent 3.0ml, and mixing is put 20min in the boiling water, take out, psychrolusia is cooled to room temperature,, returns to zero with the blank pipe under the 670nm wavelength with spectrophotometer, working sample pipe absorbance is found rna content and is averaged on standard curve.
(2) dna content is measured
The preparation of standard curve:
Get 6 in test tube, according to the form below adds reagent
Addition ml
Admixture 0 ?1 ?2 ?3 ?4 ?5
The RNA titer 0 ?0.2 ?0.4 ?0.6 ?0.8 ?1.0
DH 2O 1.0 ?0.8 ?0.6 ?0.4 ?0.2 ?0
Diphenylamines reagent 3.0 ?3.0 ?3.0 ?3.0 ?3.0 ?3.0
With each pipe mixing, put in 60 ℃ of water-baths and heat 60min, take out, cold water is cooled to room temperature, uses spectrophotometer, under the 595nm wavelength, with " 0 " number pipe zeroing, measures and respectively manages absorbance.With the dna content is abscissa, is vertical coordinate with the absorbance, the drawing standard curve.
The sample DNA assay:
Get 3 in test tube, be marked with " blank pipe " and " measuring pipe " (3) respectively, in blank pipe, add distilled water 1.0ml, in measuring pipe, add DNA sample solution 1.0ml, every then pipe adds diphenylamines reagent 3.0ml, mixing is put heating 60min taking-up in 60 ℃ of water-baths, puts psychrolusia and is cooled to room temperature.Under the 595nm wavelength, return to zero working sample pipe absorbance with the blank pipe with spectrophotometer.On standard curve, find dna content, average.
The result shows: injection cardiac muscle Copeptin contains RNA for every bottle and is no more than 200 μ g (2%); DNA is no more than 750 μ g (7.5%).
7. vigor
Be subjected to the reagent thing: injection cardiac muscle Copeptin lot number is 960501,960502,960503,960101, and content of peptides is the 10mg/ bottle.
Adopt former generation myocardial cell culture method.
Experimental result: (the results are shown in Table 10).
The myocardium Copeptin vitality test of table 10 t value (n=6)
Lot number t value
960501?????????????????5.8
960502?????????????????3.2
960503?????????????????7.9
960101?????????????????7.8
8.HPLC method is differentiated injection cardiac muscle Copeptin
Instrument: HP1100, Module liquid chromatograph No DE 70300954
Chromatographic condition: mobile phase: methanol: water=10: 90
Post: ymc-park ODS-A A-302 150mm * 4.6mm I.D S-5 μ m 120A No041543847 (W)
Column temperature: 26 ℃.Detect wavelength: 254nm.Flow velocity: 0.8ml/min.Sample size: 10 μ l.
Algoscopy: get test sample, every bottle adds mobile phase 10ml, after treating to dissolve fully, is for experiment.
The test sample lot number is respectively 960101,960501,960502,960503,961101,961103,971201,980301.
The result shows: 10 batches of test samples mainly show 4-5 main peak, and relative percentage peak area is greater than 85%, each main peak retention time very close (seeing Fig. 7, table 11,12).
Each main peak retention time of table 11
Retention time (min)
Main peak 101 501 502 503 1,101 1,102 1,103 201 301
P1??????1.922????1.901????1.920????1.915????1.925????1.924????1.949????1.954????1.990
P2??????2.506????2.504????2.500????2.502????2.643????2.639????2.640????2.638????2.638
P3??????2.654????2.651????2.646????2.645
P4??????3.156????3.144????3.134????3.128????3.124????3.114????3.117????3.115????3.115
P5??????4.240????4.203????4.181????4.159????4.148????4.128????4.133????4.129????4.107
The relative percentage peak area of each main peak of table 12
Percentage area %
Main peak 101 501 502 503 1,101 1,102 1,103 201 301
P1??????18.3????12.3????13.4????15.7????13.4????13.5????11.4????14.4????16.1
P2??????10.6????11.7????9.2?????12.4????5.2?????5.2?????5.1?????5.8?????4.7
P3??????10.4????13.3????11.1????8.8
P4??????37.4????45.4????47.8????38.6????43.8????43.4????47.3????46.8????42.2
P5??????13.2????8.8?????9.6?????15.9????22.8????22.6????22.9????18.5????23.6
4 ℃ of test samples of storing 3 years to 11 months are analyzed at aforementioned chromatographic condition, the retention time of the test sample of 10 lot numbers is very approaching, and the ratio (the relative percentage area addition at 3 peaks is greater than 66%) of selecting main peak 1,4,5 relative retention time is as index of discrimination.Main peak 1,4,5 relative retention time ratios should be 1: 1.61: 2.14 (± 0.1) as calculated.
Embodiment 2
The present invention has carried out the test of main pharmacodynamics to described myocardium Copeptin, has observed myocardium Copeptin to the influence of myocardium morphological indexes, physical signs and biochemical indicator and to the influence of myocardial oxygen consumption on whole and isolated myocardium ischemia and Ischemia-reperfusion Injury model.Result of study is as follows:
1, myocardium Copeptin is to the influence of the damage of the myocardial ultrastructure due to the Ischemia-reperfusion Injury
The list of references method, behind the ligation rat coronary artery LAD 5min, sublingual vein is injected myocardium Copeptin or contrast medicine, unclamps ligature behind the myocardial ischemia 10min, irritates 30min again, writes down the II ECG that leads simultaneously.Irritate to finish the back again from abdominal aortic blood, take out heart, after aorta perfusion is clean towards Xian, with 6% glutaraldehyde 0.1M sodium cacodylate buffer liquid perfusion and fixing 2h, get left front wall ischemic myocardium again, be cut into 1mm with normal saline 3Fritter immerse in the 4% glutaraldehyde 0.1M sodium cacodylate buffer liquid fixing, in order to making electron microscope specimen.After osmic acid was fixing, serial acetone dehydration was cut into slices after 618 epoxy resin embeddings and the polymerization, and every animal cuts 4 embedded blocks.20 of every treated animal random picture, the egative film multiplying power is 12000, observes ultrastructural change, presses the pathological changes kind and the order of severity classification definite value of mitochondrion, cardiac muscle fiber and the damage of other composition.Experiment is divided into 7 groups, is respectively sham-operation (P-O) group; Ischemia-reperfusion Injury (I-R) group; Ischemia-filling group again+normal saline or general Luo Naier (I-R+N.S, I-R+Pro) group; Ischemia-reperfusion Injury+3 dosage groups of myocardium Copeptin (I-R+MTP).
The myocardium Copeptin of table 13 is to the histological influence of Ischemia-reperfusion Injury rat heart muscle Electronic Speculum sxemiquantitative (n=20, x ± s)
Group dosage pathological changes value ± SD
(mg/Kg)
Sham-operation--0.36 ± 0.46
Ischemia-reperfusion Injury--1.97 ± 1.4 △ △ △
Ischemia-reperfusion Injury+normal saline--2.68 ± 1.3 *
Ischemia-reperfusion Injury+myocardium Copeptin 1.0 1.85 ± 1.6 *
5.0???????????0.73±0.96 ***
10.0??????????0.33±0.42 ***
Ischemia-reperfusion Injury+propranolol 2.0 0.71 ± 0.84 * *
Annotate: compare △ △ △ P＜0.01 with Sham-operated control group; Compare with the Ischemia-reperfusion Injury group, *P＞0.05, * *P＜0.01
Can find that by test myocardium Copeptin can obviously alleviate the myocardial ultrastructure damage due to the perfusion of myocardial ischemia-again, make its near or recover normal (seeing Fig. 6-12).
2. myocardium Copeptin is to the influence of myocardial ischemia
The list of references method also corrects, cat opens cuts off LAD place pericardium after breast exposes heart, cause acute myocardial ischemia 10min with the plastic bushing compression method, unclamp 30min, the cloth sheet that will have 5 groups of (3 every group) electrodes is sewn in ischemic myocardium pericardium place, write down every group of I, II, the III epicardial electrogram of leading, go femoral arteriography record arteriotony simultaneously.Get ischemia 1 ', 4 ', 7 ' and irritate 1 again ', 5 ', 10 ', 20 ' and continue blocking-up 1 ', 5 ', 10 ', 15 ', 20 ', 30 ', 40 ', 50 ', 60 ' be some writing time.Raise or the millivolt number that descends represent ST section change with the ST section.Every cat blocking-up 5 times, the 5min vein is given different medicines before the 4th blocking-up, continues blocking-up LAD the 5th time, and in continuing blocking-up back 20min, 30min, 40min vein give the myocardium Copeptin of various dose, and gains in depth of comprehension are satisfied with to continue administration behind the blocking-up 30min.∑ △ ST and ∑ NST when statistics is respectively organized the 3rd, 4,5 blocking-up respectively.Experiment is divided into ischemia-reperfusion group (I-R); Ischemia-reperfusion+normal saline group (I-R+N.S); Ischemia-reperfusion+myocardium Copeptin 2.0,5.0, (I-R+MTP 2.0,5.0,10.0mg/kg) for the 10.0mg/kg group; And totally 6 groups of ischemia-reperfusions+propranolol group 2.0mg/kg (I-R+Pro).
The myocardium Copeptin preventive administration of table 14 is to the influence of cat epicardial electrogram ischemic stage ∑ △ ST and ∑ NST (x ± s)
Group dosage n ∑ △ ST ∑ NST
(mg/Kg) (mV) (individual)
The Ischemia-reperfusion Injury group--10 109 ± 32 28.9 ± 5.2
Ischemia-reperfusion Injury+normal saline group--10 115 ± 24 *31.1 ± 5.1 *
Ischemia-reperfusion Injury+myocardium Copeptin group 2.0 6 70.8 ± 16 * *19.5 ± 4.8 * *
5.0???????6?????37.8±12 ***???9.33±3.9 ***
Annotate: compare with the Ischemia-reperfusion Injury group, *P＞0.05; * *P＜0.01
The administration of the myocardium Copeptin therapeutic of table 15. is to the influence of cat epicardial electrogram ∑ △ ST and ∑ NST (x ± s)
Group dosage n ∑ △ ST ∑ NST
(mg/Kg) (mV) (individual)
Ischemia group--12 40.5 ± 10 11.1 ± 2.1
Ischemia+normal saline group--12 40.0 ± 12 *11.2 ± 1.5 *
Ischemia+myocardium Copeptin group 2.0 6 29.9 ± 2.9 * *8.67 ± 2.2 *
5.0???????6?????25.6±5.7 ***???7.33±1.5 ***
10.0??????6?????19.7±4.0 ***???6.17±1.2 ***
Ischemia+propranolol group 2.0 6 22.8 ± 6.4 * *6.17 ± 1.5 * *
Annotate: compare with the Ischemia-reperfusion Injury group, *P＞0.05, *P＜0.05, * *P＜0.01
Show that by the visceral pericardium electrocardiogram ST section that myocardium Copeptin can obviously resist due to the cat myocardial ischemia raises, and reduces the myocardial ischemia scope.
3. myocardium Copeptin is to the influence of myocardium creatine phosphokinase release due to myocardial ischemia-reperfusion injury and lactic acid dehydrogenase activity and free fatty and mda content
(1) the list of references method is made myocardial ischemia-animal model of reperfusion injury, sublingual vein injection MTP or verapamil (verapamil, Ver) back 5min ligation rat coronary artery left anterior descending branch 10min, pour into 30min again, with the ECG that leads that physiograph is observed continuously and record II leads more, to irritate again and get left painstaking effort 2ml after finishing, heart is got left ventricular apex portion cardiac muscle behind aorta perfusion, 4 ℃ of preservations detect in the 48h.Experiment grouping: experiment be divided into Sham-operated control group (pseudo-operation, P-O); The Ischemia-reperfusion Injury group (ischemia-reperfution, I-R); Ischemia-reperfusion Injury+normal saline group (I-R+N.S); 7 groups of Ischemia-reperfusion Injury+myocardium Copeptin 0.5,2.0,10.0mg/Kg group (I-R+MTP) and Ischemia-reperfusion Injury+verapamil 1.0mg/Kg (I-R+Ver) etc., every group of 8-10 animal.
The myocardium Copeptin preventive administration of table 16. is to Ischemia-reperfusion Injury rat heart muscle and the active influence of plasma C PK (x ± s)
Group dosage n cardiac muscle CPK plasma C PK
(mg/Kg)????????(u/100mg?pro)????(u/100ml)
Sham-operation 10 980 ± 63 164 ± 64
Ischemia-reperfusion Injury 10 522 ± 65 △ △ △ 374 ± 54 △ △ △
Ischemia-reperfusion Injury+normal saline 8 501 ± 59 *337 ± 48 *
Ischemia-reperfusion Injury+myocardium Copeptin 0.5 8 732 ± 98 * *210 ± 50 * *
2.0?????8?????904±95 ***??????157±31 ***
10.0????8?????976±95 ***??????134±24 ***
Ischemia-reperfusion Injury+verapamil 1.0 8 886 ± 115 * *192 ± 60 * *
Ischemia pours into+CMGSP 5.0 8 890 ± 97 again and again * *199 ± 35 * *
Annotate: compare △ △ △ P＜0.01 with Sham-operated control group; Compare with the Ischemia-reperfusion Injury group, *P＞0.05, * *P＜0.01
The myocardium Copeptin preventive administration of table 17. is to Ischemia-reperfusion Injury rat heart muscle and the active influence of blood plasma LDH (x ± s)
Group dosage n cardiac muscle LDH blood plasma LDH
(mg/kg)???????(u/mg?pro)??????(u/ml)
Sham-operation--10 76.7 ± 19 40.9 ± 9.5
Ischemia-reperfusion Injury--10 110 ± 27 △ △ △, 120 ± 20 △ △ △
Ischemia-reperfusion Injury+normal saline--8 112 ± 19 *116.2 ± 12 *
Ischemia-reperfusion Injury+myocardium Copeptin 0.5 8 97.1 ± 12 *93.9 ± 17 * *
2.0?????8?????76.3±22 ***?????59.7±12 ***
10.0????8?????64.8±17 ***?????52.6±13 ***
Ischemia-reperfusion Injury+verapamil 1.0 8 75.1 ± 23 * *46.7 ± 8.8 * *
Ischemia-reperfusion Injury+CMGSP 5.0 8 83.0 ± 17 * *60.9 ± 15 * *
Annotate: compare with Sham-operated control group, compare with the Ischemia-reperfusion Injury group △ △ △ P＜0.01, *P＞0.05, * *P＜0.01
The myocardium Copeptin preventive administration of table 18. is to the influence of Ischemia-reperfusion Injury rat heart muscle and contents of mda (x ± s)
Group dosage n cardiac muscle MDA blood plasma MDA
(mg/kg)???????(nmol/100mg?pro)????(nmol/ml)
Sham-operation 10 68.3 ± 8.4 22.3 ± 1.8
Ischemia-reperfusion Injury 10 135 ± 10 △ △ △ 63.6 ± 11 △ △ △
Ischemia-reperfusion Injury+normal saline 8 127 ± 15 *58.4 ± 11 *
Ischemia-reperfusion Injury+myocardium Copeptin 0.5 8 73.1 ± 13 * *38.1 ± 6.2 * *
2.0?????8??????60.5±10.4 ***?????27.7±5.5 ***
10.0????8??????49.8±9.4 ***??????25.5±5.1 ***
Ischemia-reperfusion Injury+verapamil 1.0 8 66.6 ± 19.8 * *24.9 ± 6.6 * *
Ischemia-reperfusion Injury+CMGSP 5.0 8 75.2 ± 9.7 * *39.2 ± 5.3 * *
Annotate: compare △ △ △ P＜0.01 with Sham-operated control group; Compare with the Ischemia-reperfusion Injury group, *P＞0.05, * *P＜0.01
The myocardium Copeptin of table 19 is to the influence of Ischemia-reperfusion Injury rat blood serum FFA content (n=8, x ± s)
Group dose F FA
(mg/Kg)????????(μmol/100ml)
Sham-operation--60.6 ± 7.8
Ischemia-reperfusion Injury--129 ± 26 △ △ △
Ischemia-reperfusion Injury+normal saline--121 ± 10 *
Ischemia-reperfusion Injury+myocardium Copeptin 1.0 85.4 ± 5.0 * *
5.0????????????77.7±7.1 ***
10.0???????????71.4±11 ***
Ischemia-reperfusion Injury+propranolol 2.0 77.1 ± 6.4 * *
Ischemia-reperfusion Injury+CMGSP 5.0 89.2 ± 6.7 * *
Annotate: compare △ △ △ P＜0.01 with Sham-operated control group; Compare with the Ischemia-reperfusion Injury group, *P＞0.05, * *P＜0.01
Myocardium Copeptin of the present invention and patent ZL94102798 and the disclosed CMGSP of ZL94102799 (GMGSP) are relatively, has obviously high external biological activity, myocardium Copeptin of the present invention active unit be CMGSP 3-5 doubly, the drug effect correction data shows in the body, and it has obvious favourable influence (seeing Table 16-19) to myocardium creatine phosphokinase release due to myocardial ischemia-reperfusion injury and lactic acid dehydrogenase activity and free fatty and mda content.
(2) the list of references method is made isolated rat Langendorffs heart anoxia-reoxygenation injury animal model, with high Ca2+, the K-H liquid that low K+ and continuing charges into mist carries out the Langendorffs perfusion, and two platinum filaments are colluded respectively in the apex of the heart and left room root, recording ecg.LAD ligation 10min unclamps 15min, the administration group before ligation 5min to unclamp the back 5min all to contain the K-H liquid perfusion of respective concentration medicine.8min and the effluent that unclamps back 2min are measured relevant index before collecting ligation respectively, after the ligation.Perfusion is got left chamber front and back walls cardiac muscle after finishing, and 4 ℃ of preservations detect CPK, LDH and MDA in the 48h.The experiment be divided into Sham-operated control group (pseudo-operation, P-O); Anoxia-reoxygenation group (anoxia-reoxygenation, A-R); (final concentration, A-R+MTP) group and anoxia-reoxygenation+verapamil 1.0 μ g/Kg (A-R+Ver) etc. are 6 groups, every group of 10 animals for anoxia-reoxygenation+myocardium Copeptin 10,50,100 μ g/ml.
The active influence of arteria coronaria effluent CPK during to the perfusion of isolated rat myocardial ischemia-again of the myocardium Copeptin of table 20. (n=10, x ± s)
Arteria coronaria effluent CPK (U/L)
Group dosage
Ischemic stage, irritate the phase again before (μ g/ml) ischemia
Sham-operation--15.3 ± 1.5 16.5 ± 1.8 17.1 ± 2.0
Lack blood-reperfusion--16.3 ± 2.3 △, 24.8 ± 2.7 △ △ △, 35.4 ± 4.3 △ △ △
Lack blood-reperfusion
+ myocardium Copeptin 10 16.1 ± 2.6 *20.7 ± 1.7 * *22.7 ± 2.3 * *
50?????15.6±1.7 *???17.9±2.7 ***???19.0±2.3 ***
100????15.5±2.7 *???15.3±2.1 ***???16.5±2.4 ***
Lack blood-reperfusion
+ verapamil 1 16.3 ± 2.0 *16.2 ± 2.8 * *16.0 ± 1.8 * *
Annotate: compare △ P＞0.05, △ △ △ P＜0.01 with Sham-operated control group; With ischemia-hemoperfusion group compares again,
*P＞0.05， ***P＜0.01
The myocardium Copeptin of table 21. is to isolated rat myocardial ischemia-active influence of perfusion arteria coronaria effluent LDH again (n=10, x ± s)
Arteria coronaria effluent LDH (U/L)
Group dosage
Ischemic stage, irritate the phase again before (μ g/ml) ischemia
Sham-operation--11.8 ± 0.79 12.6 ± 1.1 11.8 ± 0.69
Lack blood-reperfusion--11.7 ± 0.83 △, 17.3 ± 1.9 △ △ △, 24.7 ± 1.7 △ △ △
Lack blood-reperfusion
+ myocardium Copeptin 10 12.0 ± 0.58 *13.4 ± 1.1 * *15.3 ± 1.4 * *
50??????????11.8±0.53 *????12.9±1.1 ***??????13.4±0.76 ***
100?????????11.2±0.55 *????12.2±0.79 ***?????12.9±0.93 ***
Lack blood-reperfusion
+ verapamil 1 11.4 ± 0.78 *13.0 ± 0.62 * *14.3 ± 0.95 * *
Annotate: compare △ P＞0.05, △ △ △ P＜0.01 with Sham-operated control group; With ischemia-hemoperfusion group compares again,
*P＞0.05， ***P＜0.01
Show that by test myocardium Copeptin can obviously lower increasing of myocardium creatine phosphokinase release and lactic acid dehydrogenase activity and free fatty and mda content due to myocardial ischemia-reperfusion injury.
4. myocardium Copeptin is to the influence of myocardial oxygen consumption
The anesthesia of Canis familiaris L. pentobarbital sodium, tracheal intubation, the artificial respiration leads physiograph monitoring electrocardiogram and aortic pressure with the RM-86 type more.Breast is opened in the left side, exposes heart, from apex of the heart intubate to left ventricle, heart left side constant pressure and rate of pressure change (± dp/dt max).For understanding coronary circulation and myocardium O 2Metabolic variation, the LCA of separation Canis familiaris L. is surveyed coronary flow with electromagnetic flowmeter, calculates coronary resistance.Extremely crown from the external jugular vein intubate of Canis familiaris L., extract arterial blood and coronary sinus blood simultaneously, measure blood O with blood gas analyzer (ABL-3 type, Denmark) 2Content, calculating myocardium O 2Uptake ratio and cardiac muscle consumption O 2Amount.The arterial blood ph CO that keeps Canis familiaris L. in the experimentation 2With O 2Divide and be pressed in normal range.MTP dosage is 2,5,10mg/kg, two spacing of doses 30min.Continuous record parameters after the administration is until returning to control value substantially.After the administration 2,5,10,30min extracting arterial blood and coronary sinus blood are surveyed vim and vigour content, calculating myocardium O 2Uptake ratio and cardiac muscle consumption O 2Amount is observed MTP to myocardium O 2Metabolic influence.
The myocardium Copeptin of the quiet notes of table 22. is to the influence (changing value % after the administration) of Cor Canitis flesh oxygen consumption and myocardium coefficient of oxygen utilization
Time myocardial oxygen consumption cardiac muscle coefficient of oxygen utilization
(min)?????(MVO 2)????????????????(O 2?ext)
Cardiac muscle Copeptin 2mg/kg (N=8)
2?????????-23.0±26??????????????-4.00±13
5?????????-21.0±13 ***??????????0±8.0
10????????-18.0±14??????????????-2.00±6.0
20????????-9.00±12??????????????-2.00±12
Cardiac muscle Copeptin 5mg/kg (N=7)
2?????????-36.0±24 **???????????-5.00±13
5?????????-26.0±21 **???????????6.00±8.0
10????????-19.0±15 **???????????6.00±6.0 *
20????????-8.00±10??????????????-14.0±35
Cardiac muscle Copeptin 10mg/kg (N=6)
5?????????-22.0±25??????????????9.00±5 ***
10????????-21.0±14 **???????????2.00±5.0
20????????-8.00±4.0 *???????????3.00±4.0
30????????-10.0±7.0 *???????????-6.00±15
Propranolol 2mg/kg (N=6)
2?????????-31.0±13 *????????????-3.00±1.0
5?????????-30.0±13 ***??????????3.00±7.0
10????????-33.0±10 ***??????????3.00±8.0
30????????-32.0±14 ***??????????3.00±9.0
With before the administration relatively: *P＞0.05, *P＜0.05, * *P＜0.01
5. myocardium Copeptin is to the influence of myocardial infarction
Body weight 20.9 ± 4.0kg adult healthy miniature pig, male, ear vein is injected 3% pentobarbital sodium 30mg/kg anesthesia.Tracheal intubation connects SC-3 type electric pulmotor pedestrian worker positive pressure respiration.Left side III intercostal is opened breast, exposes heart, separates anterior descending coronary (about 1/3 position of centroid point), wears 0 under it #Silk thread is in order to ligation; Myocardial surface is placed the fixed epicardial lead of multiple spot of 20 points under ligature, myocardial electrical signals is recorded in RM-6300 type eight through ZYS1-I type numerical control visceral pericardium scanner, AB-601G bioelectric amplifier and leads on the RTA-1200 type heat battle array monitor of physiograph, normal voltage 1mV=1mm, the visceral pericardium electrocardiogram of 20 points of self-timing mapping changes.Femoral arteriography connects the AP-641G blood pressure amplifier through TP-400T type pressure transducer and measures mean arterial pressure (MBP) to ventral aorta; The subcutaneous insertion needle electrode of extremity through AC-601G ecg amplifier measurement standard II lead electrocardiogram (ECGII), and with ECG electric signal input AT-601G cardiotachometer, is measured heart rate (HR).Femoral venous catheter is used for administration and fluid infusion.
Experiment divides 4 groups, 25 of shared animals, the solvent control group is when venoclysis mannitol 120mg/kg, have 5 to survive 5 because of the chamber death of quivering, all the other respectively organize 5 every group, through femoral vein difference infusion cardiac muscle Copeptin 5,10mg/kg, positive drug verapamil 0.25mg/kg, the administration volume is 2ml/kg, and infusion velocity is 2ml/min.Operation finishes and treats to trace the visceral pericardium electrocardiogram after every index is stablized, ligation anterior descending branch subsequently, record visceral pericardium electrocardiogram contrasts before as administration behind the 5min, intravenous infusion administration then, after the record administration 5,10,15,20,25,30,45,60,90,120, the visceral pericardium ECG ST section lift-off value of ECGII, MBP, HR and 20 points of 180min, and try to achieve summation (ST), with this index as the measurement degree of myocardial ischemia.The point that the rising of visceral pericardium ECG ST section is surpassed 2mV is decided to be the ischemia point, calculates total ischemia and counts (NST), with this index as the myocardial ischemia scope.3h sacrificed by exsanguination animal after the administration, immediately take out heart, cut ventricle, clean remained blood, the following ventricle in ligation position is pressed the thick crown section of 5mm, 1%TTC lucifuge dyeing 30min under the room temperature, again 5 double-edged ischemic regions of cardiac muscle and non-ischemic region are traced on transparent film, clip white infarct district film is weighed, and is heavy divided by 10 center of area chamber sarcoglia sheets, calculates the percentage ratio that infarct accounts for ventricular weight under the ligature.
Experiment grouping, dosage and administering mode
Group infusion of drug dosage infusion velocity
(mg/kg)?????(ml/min)
Solvent control group mannitol 120 2
Tried thing low dose group cardiac muscle Copeptin+mannitol 5+,120 2
Tried object height dosage group cardiac muscle Copeptin+mannitol 10+,120 2
Positive drug matched group verapamil 0.25 2
Table 23 venoclysis cardiac muscle Copeptin is to the influence of pig myocardium infarction size
Drug dose number of animals infarction size
(mg/kg)????????(n)???????????(％)
Solvent control-5 19.4 ± 3.02
Cardiac muscle Copeptin 55 11.8 ± 3.13 *
Cardiac muscle Copeptin 10 5 10.2 ± 3.2 *
Verapamil 0.25 5 12.5 ± 3.4 *
Annotate: compare with the solvent control group: *P＜0.05, *P＜0.01.
Evidence, cardiac muscle Copeptin 5,10mg/kg can obviously reduce the Electrocardiographic ST of myocardial infarction Cor Sus domestica adventitia, reduce NST, and dwindling myocardial infarct size, dead due to being quivered in arrhythmia, the chamber of the appearance of acute myocardial ischemia pig have certain therapeutical effect, and blood pressure, heart rate are not had obvious influence.
Embodiment 3
Getting 1 kilogram of certified milk pig ventricular muscles cleans, chopping, add 1 kilogram of sterile purified water with the homogenate of 3000rpm/min rotating speed, homogenate is freezing 24 hours at-20 ℃, after thawing 3 times repeatedly, water proof is heated to 75 ℃, with model is sheet frame filter (available from the Guangzhou medicine instrument institute) filter cleaner of XAS03-172/8, select 10u middling speed filter paper for use, obtain coarse filtration liquid, (specification is F60 to coarse filtration liquid with hollow fiber column, available from Switzerland Zinpro Corp.) ultrafiltration, obtaining molecular weight is 12Kd fine straining liquid, with ultrafilter membrane (specification is 10Kd, Millipore company) ultrafiltration, molecular cut off is the myocardium peptide cellulose solution 150ml of 9500Da, with the reverse osmosis concentration post concentrated product of Millipore company.
The solution process quality examination of described myocardium Copeptin is to meeting quality standard, filtration sterilization then, fill, again through lyophilization, device therefor is a freezer dryer, makes that shelf temperature reaches-20 ℃ in the hothouse in 20 minutes, through 30 minutes, make products temperature reach-35 ℃ again; Keep temperature in the condenser being dropped to-50 ℃ in 2 hours, evacuation again, when vacuum reaches 100KPa, be communicated with hothouse and condenser, stop the refrigeration of drying baker, when the vacuum of drying baker begins to heat up during for 15Pa, programming rate is 3 ℃/min, is warming up to 15 ℃, is incubated 3 hours, continuation is warming up to 22 ℃ with 10 ℃/min speed, continues 5 hours; Continuation is warming up to 35 ℃ with 10 ℃/min speed, continues 2 hours; Continuation is warming up to 50 ℃ with 5 ℃/min speed, continues 1 hour.Enter temperature-fall period, make temperature reduce to 40 ℃ in the 20min, continue 10 hours, promptly obtain the qualified myocardium Copeptin dried frozen aquatic products of outward appearance, take out goods and seal.
Described myocardium Copeptin by analysis, content of peptides is 85%, free amino acid is 8%, rna content 1%, DNA (deoxyribonucleic acid) content 6%, molecular weight 9500 dalton.
Embodiment 4
Getting 1 kilogram of certified milk cattle ventricular muscles cleans, chopping, add 1 kilogram of sterile purified water with the homogenate of 5000rpm/min rotating speed, homogenate is freezing 48 hours at-30 ℃, after thawing 4 times repeatedly, water proof is heated to 90 ℃, with model is sheet frame filter (available from the Guangzhou medicine instrument institute) filter cleaner of XAS03-172/8, select 8u middling speed filter paper for use, obtain coarse filtration liquid, (specification is F60 to coarse filtration liquid with hollow fiber column, available from Switzerland Zinpro Corp.) ultrafiltration, obtaining molecular weight is 12Kd fine straining liquid, with ultrafilter membrane (specification is 5Kd, Millipore company) ultrafiltration, molecular cut off is the myocardium peptide cellulose solution 150ml of 5000Da, with the reverse osmosis concentration post concentrated product of Millipore company.
The solution process quality examination of described myocardium Copeptin is to meeting quality standard, filtration sterilization then, fill, again through lyophilization, device therefor is a freezer dryer, makes that shelf temperature reaches-18 ℃ in the hothouse in 40 minutes, through 20 minutes, make products temperature reach-25 ℃ again; Keep temperature in the condenser being dropped to-40 ℃ in 1 hour, evacuation again, when vacuum reaches 95KPa, be communicated with hothouse and condenser, stop the refrigeration of drying baker, when the vacuum of drying baker begins to heat up during for 12Pa, programming rate is 2 ℃/min, is warming up to 10 ℃, is incubated 5 hours, continuation is warming up to 25 ℃ with 16 ℃/min speed, continues 3 hours; Continuation is warming up to 30 ℃ with 15 ℃/min speed, continues 1 hour; Continuation is warming up to 60 ℃ with 8 ℃/min speed, continues 2 hours.Enter temperature-fall period, make temperature reduce to 46 ℃ in the 30min, continue 8 hours, promptly obtain the qualified myocardium Copeptin dried frozen aquatic products of outward appearance, take out goods and seal.
Described myocardium Copeptin by analysis, content of peptides is 78%, free amino acid is 15%, rna content 2%, DNA (deoxyribonucleic acid) content 5%, molecular weight 5000 dalton.
Embodiment 5
Getting 1 kilogram of certified milk rabbit ventricular myocyte cleans, chopping, add 1 kilogram of sterile purified water with the homogenate of 1000rpm/min rotating speed, homogenate is freezing 72 hours at-10 ℃, after thawing 3 times repeatedly, water proof is heated to 85 ℃, with model is sheet frame filter (available from the Guangzhou medicine instrument institute) filter cleaner of XAS03-172/8, select 5u middling speed filter paper for use, obtain coarse filtration liquid, (specification is F60 to coarse filtration liquid with hollow fiber column, available from Switzerland Zinpro Corp.) ultrafiltration, obtaining molecular weight is 11Kd fine straining liquid, with ultrafilter membrane (specification is 3Kd, Millipore company) ultrafiltration, molecular cut off is the myocardium peptide cellulose solution 150ml of 2000Da, with the reverse osmosis concentration post concentrated product of Millipore company.
The solution process quality examination of described myocardium Copeptin is to meeting quality standard, filtration sterilization then, fill, again through lyophilization, device therefor is a freezer dryer, makes that shelf temperature reaches-15 ℃ in the hothouse in 10 minutes, through 25 minutes, make products temperature reach-30 ℃ again; Keep temperature in the condenser being dropped to-45 ℃ in 2.5 hours, evacuation again, when vacuum reaches 90KPa, be communicated with hothouse and condenser, stop the refrigeration of drying baker, when the vacuum of drying baker begins to heat up during for 10Pa, programming rate is 5 ℃/min, is warming up to 5 ℃, is incubated 6 hours, continuation is warming up to 15 ℃ with 8 ℃/min speed, continues 8 hours; Continuation is warming up to 32 ℃ with 7 ℃/min speed, continues 4 hours; Continuation is warming up to 55 ℃ with 4 ℃/min speed, continues 3 hours.Enter temperature-fall period, make temperature reduce to 50 ℃ in the 10min, continue 15 hours, promptly obtain the qualified myocardium Copeptin dried frozen aquatic products of outward appearance, take out goods and seal.
Described myocardium Copeptin by analysis, content of peptides is 90%, free amino acid is 6%, rna content 1%, DNA (deoxyribonucleic acid) content 3%, molecular weight 2000 dalton.
Embodiment 6
With embodiment 3, different is that molecular cut off is the myocardium peptide cellulose solution of 4000Da, through quality examination to meeting quality standard, filtration sterilization then, fill, according to following prescription:
Cardiac muscle Copeptin 20mg
Mannitol 375mg
Active carbon 0.005mg
Water for injection adds to 5ml
Bottling places freezer dryer, by making that shelf temperature reaches in the hothouse in 30 minutes-20 ℃, through 40 minutes, make products temperature reach-35 ℃ again, keep temperature in the condenser being dropped to-50 ℃ in 3 hours, evacuation again, when vacuum reaches 95Kpa, be communicated with hothouse and condenser, stop the refrigeration of drying baker, when the vacuum of drying baker begins to heat up during for 15Pa, programming rate is 3 ℃/min, is warming up to 10 ℃, is incubated 4 hours.Continuation is warmed up to 20 ℃ with 12 ℃/min speed, continues 5.5 hours.Continuation is warmed up to 30 ℃ with 12 ℃/min speed, continues 1.5 hours.Continuation is warmed up to 60 ℃ with 6 ℃/min speed, continues 2 hours.Enter temperature-fall period, make temperature reduce to 48 ℃ in the 20min, continue 9 hours.Obtain the qualified myocardium Copeptin dried frozen aquatic products of outward appearance, take out goods and seal.
Lyophilization obtains myocardium peptide cellulose content 2.0mg/ml finished product.Described myocardium Copeptin by analysis, content of peptides is 80%, free amino acid is 12%, rna content 2%, DNA (deoxyribonucleic acid) content 6%, molecular weight 4000 dalton.
Embodiment 7
With embodiment 3, different is the ventricular muscles that raw material adopts the certified milk horse, molecular cut off is the myocardium peptide cellulose solution of 8000Da, gained cardiac muscle Copeptin by analysis, content of peptides is 84.5%, and free amino acid is 6%, rna content 2%, DNA (deoxyribonucleic acid) content 7.5%, molecular weight 8000 dalton.
Embodiment 8
With embodiment 3, different is also to contain trehalose in the myocardium peptide cellulose solution, and the set of dispense ratio is: myocardium Copeptin 15mg/ml, trehalose 200mg/ml.
Embodiment 9
With embodiment 3, different is the ventricular muscles that raw material adopts health pig, also contains lactose in the myocardium peptide cellulose solution, and the set of dispense ratio is: myocardium Copeptin 18mg/ml, lactose 250mg/ml.
Embodiment 10
With embodiment 6, different is that molecular cut off is the myocardium peptide cellulose solution of 1000Da, and wherein myocardium Copeptin solution component is:
Cardiac muscle Copeptin 16mg
Sucrose 300mg
Active carbon 0.005mg
Water for injection adds to 5ml
Gained cardiac muscle Copeptin by analysis, content of peptides is 82%, free amino acid is 12%, rna content 2%, DNA (deoxyribonucleic acid) content 4%, molecular weight 1000 dalton.
Embodiment 11
The test of cardiac muscle Copeptin preliminarily stabilised
1. the result of injection cardiac muscle Copeptin influence factor test, accelerated test shows, is removing the rapid flavescence of appearance luster under outer package, humidity＞75% and the temperature＞37 ℃ condition.Moisture content increases, and vigor reduces.
2. room temperature keeps sample and investigates the result show that except that the outward appearance color and luster became little yellow, other project did not have change in the time of 480～540 days, stored under 4 ℃ of conditions, and each investigates the project no change.Show that injection cardiac muscle Copeptin stored under humidity 45%～90% and room temperature condition 150 days at least, appearance character, content and vigor do not have change, under 4 ℃ of conditions, can store at least 480 days.

Claims (16)

1. myocardium Copeptin, it is characterized in that it being never to comprise the polypeptide that extracts in people's the health mammal heart, its content of peptides is 75%～90%, free amino acid is 6%～15%, rna content is less than 2%, DNA (deoxyribonucleic acid) content is less than 7.5%, and weight average molecular weight is less than 10000 dalton.
2. myocardium Copeptin according to claim 1 is characterized in that describedly not comprising that people's healthy mammal comprises pig, cattle, sheep, rabbit, horse.
3. myocardium Copeptin according to claim 2 is characterized in that the preferred young mammals of described mammal, comprises piglets, milk cattle, milk goat, newborn rabbit, newborn horse, most preferably is piglets.
4. myocardium Copeptin according to claim 1, the molecular weight that it is characterized in that described myocardium Copeptin is 1000～10000 dalton.
5. myocardium Copeptin according to claim 1 is characterized in that the molecular weight of described myocardium Copeptin is preferably 2000～8000 dalton, and most preferred weight average molecular weight is 2000～5000 dalton.
6. according to any one described myocardium Copeptin of claim 1～5, it is characterized in that described myocardium Copeptin biological activity in pH3～8 scopes is stable, to the E.C. 3.4.21.64 sensitivity, 85 ℃ of 10min biological activitys do not change, and are stable under freezing or lyophilisation condition.
7. according to any one described myocardium Copeptin of claim 1～5, it is characterized in that described myocardium Copeptin isoelectric focusing electrophoresis shows 2-6 bar colored zone.
8. myocardium Copeptin according to claim 7 is characterized in that the preferred isoelectric focusing electrophoresis of described myocardium Copeptin shows 2 bands, and wherein pI 10.92 is with painted dark person.
9. according to any one described myocardium Copeptin of claim 1～5, it is characterized in that described myocardium Copeptin has a stable maximum absorption band at ultra-violet absorption spectrum 190-210nm place.
10. myocardium Copeptin according to claim 8 is characterized in that described myocardium Copeptin preferably has a maximum absorption band person at ultra-violet absorption spectrum 200 ± 2nm place.
11., it is characterized in that described myocardium Copeptin vigor is at least 2.2 according to any one described myocardium Copeptin of claim 1～5.
12. according to any one described myocardium Copeptin of claim 1～5, it is characterized in that also can comprising excipient in the described myocardium Copeptin, its weight ratio is: myocardium Copeptin: excipient=15～20: 100～375, preferably 18～20: 200～375, excipient can be mannitol, trehalose, lactose, sucrose or other lyophilizing adjuvant, is preferably mannitol.
13. myocardium Copeptin according to claim 12 is characterized in that also comprising active carbon, its content is about 0.1%.
14., it is characterized in that described myocardium Copeptin analyzes through FPLC according to any one described myocardium Copeptin of claim 1～5, mainly contain 5 component peaks, the percentage area adds up to 90%～95% relatively.
15. the purposes of the described myocardium Copeptin of claim 1 aspect preparation treatment cardiovascular disease medicine.
16. the purposes of the described myocardium Copeptin of claim 1 aspect preparation treatment myocardial ischemia and reperfusion injury medicine.
CNB031413528A 2003-06-04 2003-06-04 Myocardium peptide and its use Expired - Lifetime CN1255183C (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CNB031413528A CN1255183C (en) 2003-06-04 2003-06-04 Myocardium peptide and its use
EP04713508A EP1661907B1 (en) 2003-06-04 2004-02-23 Method of preparing cardio myopeptidin
US10/567,286 US7427663B2 (en) 2003-06-04 2004-02-23 Cardio myopeptidin, the production and the use thereof
AT04713508T ATE545653T1 (en) 2003-06-04 2004-02-23 METHOD FOR PRODUCING CARDIO MYOPEPTIDINE
PCT/CN2004/000138 WO2004108751A1 (en) 2003-06-04 2004-02-23 A cardio myopeptidin, the production and the use thereof
DK04713508.2T DK1661907T3 (en) 2003-06-04 2004-02-23 Process for the preparation of cardiomyopeptidine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB031413528A CN1255183C (en) 2003-06-04 2003-06-04 Myocardium peptide and its use

Publications (2)

Publication Number Publication Date
CN1552439A true CN1552439A (en) 2004-12-08
CN1255183C CN1255183C (en) 2006-05-10

Family

ID=34323986

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB031413528A Expired - Lifetime CN1255183C (en) 2003-06-04 2003-06-04 Myocardium peptide and its use

Country Status (1)

Country Link
CN (1) CN1255183C (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010006476A1 (en) * 2008-07-15 2010-01-21 大连珍奥药业有限公司 Myocardial peptide, preparation method and uses thereof
CN101012455B (en) * 2005-11-14 2011-05-18 大连珍奥药业有限公司 Deactivation method of biochemistry substance, preparing method of cardiomyopeptidin and use of cardiomyopeptidin
CN101265292B (en) * 2007-03-16 2011-09-14 大连珍奥药业有限公司 Polypeptides substances, preparing method and use thereof
CN101612384B (en) * 2009-07-23 2012-06-20 中国人民解放军第二军医大学 Application of polypeptide small molecule MLIF in preparing medicine for preventing and treating myocardial ischemia
CN106546694A (en) * 2016-11-01 2017-03-29 青海盐湖工业股份有限公司 A kind of method for determining organic amine content
CN106668069A (en) * 2015-11-04 2017-05-17 张志强 Bile extract as well as extracting method and application thereof
CN108478781A (en) * 2018-05-08 2018-09-04 大连理工大学 The lyophilized technique of injection cardiac muscle peptide
CN114982962A (en) * 2022-05-31 2022-09-02 中科康盛(河北)生物科技有限公司 Formula for improving anoxia endurance function of heart and raw material preparation method
CN115684608A (en) * 2022-11-03 2023-02-03 大连珍奥药业股份有限公司 Metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide and application thereof

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101012455B (en) * 2005-11-14 2011-05-18 大连珍奥药业有限公司 Deactivation method of biochemistry substance, preparing method of cardiomyopeptidin and use of cardiomyopeptidin
CN101265292B (en) * 2007-03-16 2011-09-14 大连珍奥药业有限公司 Polypeptides substances, preparing method and use thereof
WO2010006476A1 (en) * 2008-07-15 2010-01-21 大连珍奥药业有限公司 Myocardial peptide, preparation method and uses thereof
CN101612384B (en) * 2009-07-23 2012-06-20 中国人民解放军第二军医大学 Application of polypeptide small molecule MLIF in preparing medicine for preventing and treating myocardial ischemia
CN106668069A (en) * 2015-11-04 2017-05-17 张志强 Bile extract as well as extracting method and application thereof
CN106546694A (en) * 2016-11-01 2017-03-29 青海盐湖工业股份有限公司 A kind of method for determining organic amine content
CN108478781A (en) * 2018-05-08 2018-09-04 大连理工大学 The lyophilized technique of injection cardiac muscle peptide
CN114982962A (en) * 2022-05-31 2022-09-02 中科康盛(河北)生物科技有限公司 Formula for improving anoxia endurance function of heart and raw material preparation method
CN115684608A (en) * 2022-11-03 2023-02-03 大连珍奥药业股份有限公司 Metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide and application thereof
CN115684608B (en) * 2022-11-03 2024-01-09 大连珍奥药业股份有限公司 Metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide and application thereof

Also Published As

Publication number Publication date
CN1255183C (en) 2006-05-10

Similar Documents

Publication Publication Date Title
CN1774257A (en) Method for preparing a centella asiatica extracts rich in madecassoside and in terminoloside
CN1925845A (en) Powdered compositions of sensitive active materials in an at least partially amorphous state
CN1560228A (en) Method of whole manual culture for chinese caterpillar fungus
CN1520314A (en) Drugs contg. chymase inhibitor and ace inhibitors as active ingredients
CN1552439A (en) Myocardium peptide and its use
CN1931236A (en) Medicine composition of red sage and rhodiola root
CN1616014A (en) Chinese medicine composition for treating diabetes and its preparing method
CN1923241A (en) Medicine composition containing epimedium extract, uncaria extract, and gastrodine, and its preparation and use
CN1256347C (en) Fusion protein of kininogen D5 and tumor necrosis factor related apoptosis-inducing ligand(D5-TRAIL), its preparation and use thereof
CN1694719A (en) Casein derived peptides and uses thereof in therapy
CN1535711A (en) Medicine composition for curing burn, scald and mucocutaneous ulcer
CN1552733A (en) Preparation of myocardium peptide
CN1709498A (en) Ginseng-astragalus blood-sugar lowering soft capsule, and its preparing and detecting method
CN1557352A (en) Novel usage of chicory aqueous extract
CN1879891A (en) Method for expressing human insulin for reducing blood sugar by transgenic lucid ganoderma
CN1183150C (en) Prepn process, medicine prepn and new medicinal use of sophoricosids
CN1557347A (en) Medicine for treating cardiac and cerebral vascular diseases and its preparation
CN1912113A (en) Protein and nuclein of antrodia camphoratu antioxidase and preparation method and use thereof
CN1615994A (en) Method for preparing Chinese medicine composition for treating ischemic brain apoplexy
CN1089092C (en) Diterpene-kind compound anticancer drug capable of promoting differentiation and depressing proliferation and preparation method and use thereof
CN1193773C (en) Capsule agent of chanhua mycelia and preparing method thereof
CN1919202A (en) Pharmaceutical composition of pharmacy acceptable salt of carthamus tinctorius yellow colour and ferulaic acid
CN1586604A (en) Zedoary injection preparation and its preparing method
CN1292002C (en) Arginine deacylase fusion protein, method for preparation and application
CN101041690A (en) Recombinant dog hookworm coagulate peptide resistant 5 mutant, its encoding gene, preparation and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
ASS Succession or assignment of patent right

Owner name: ZHEN'AO GROUP CORP., LTD.

Free format text: FORMER OWNER: ZHENAO PHARMACEUTICAL CO., LTD., DALIAN CITY

Effective date: 20080718

C41 Transfer of patent application or patent right or utility model
TR01 Transfer of patent right

Effective date of registration: 20080718

Address after: Double D port, Dalian, Liaoning Province, No. 88, No. 1, life

Patentee after: ZHEN-AO GROUP Co.,Ltd.

Address before: Double D port, Dalian, Liaoning Province, No. 88, No. 1, life

Patentee before: DALIAN ZHEN-AO PHARMACEUTICAL Co.,Ltd.

TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20190613

Address after: 116000 Jin 7 Road, Advanced Equipment Manufacturing Park, Dalian Economic and Technological Development Zone, Liaoning Province

Patentee after: Dalian Zhenao Pharmaceutical Co.,Ltd.

Address before: 116620, Liaoning, Dalian double D port, life all the way No. 88

Patentee before: ZHEN-AO GROUP Co.,Ltd.

CX01 Expiry of patent term

Granted publication date: 20060510

CX01 Expiry of patent term