CN103203009A - New application of partial metabolite of pentapeptide in preparation of myocardial ischemia resistant product - Google Patents
New application of partial metabolite of pentapeptide in preparation of myocardial ischemia resistant product Download PDFInfo
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Abstract
The invention relates to the technical field of biomedicine, and in particular relates to a new application of partial metabolite of pentapeptide in preparation of a myocardial ischemia resistant product. The amino acid sequence of the small peptide is shown as Seq No.2, No.3, No.4, No.5, No.6 and No.7. Through pharmacological experiments of acute rat myocardial ischemia and damage models, the small peptide and the metabolite thereof can remarkably reduce the range of rat myocardial damage and myocardial infarction caused by myocardial ischemia, reduce the content of lactic dehydrogenase and creatine kinase in myocardial ischemia rat serum and have the function of preventing and treating the myocardial ischemia, so the small peptide and the metabolite thereof can be used for preparing a medicament for preventing and treating the myocardial ischemia.
Description
Technical field
The present invention relates to technical fields such as medicine, food, reagent, specifically relate to the new purposes of part metabolite of a kind of little peptide of biological medicine technology aspect, more particularly relate to a kind of pentapeptide and be the part cylinder metabolism-ure of mononuclear cell migration inhibitory factor for the preparation of the new purposes of the product that resists myocardial ischemia, specifically a kind ofly again relate to 6 application of little peptide in preparation resists myocardial ischemia product.
Background technology
Raising along with people's living standard, cardiovascular disease has become the principal disease of harm humans health, Epidemiological study in recent years shows, population of China cardiovascular disease incidence rate and mortality rate all are lasting ascendant trend, and age of onset reduces gradually, become a kind of serious threat human health, influenced people's commonly encountered diseases of quality of life, frequently-occurring disease, especially the acute myocardial ischemia serious harm mankind's is healthy.
The research overview of myocardial ischemia
(1) general introduction
Myocardial ischemia refers to that the hemoperfusion of heart reduces, and causes the oxygen supply of heart to reduce, and energy metabolism of myocardial is undesired, can not support a kind of pathological state of heart operate as normal; Be that a certain position of heart blood is for serious or not enough fully, cause this position cardiac muscle oxygen and nutrient substance is under-supply or interrupt, metabolite is piled up the pathological state cause, can cause myocardial necrosis (infarction) when serious, how by coronary spasm, arteria coronaria is serious or total blockage causes.
The coronary stenosis that coronary atherosclerosis causes or obturation are to cause main, the modal cause of disease of myocardial ischemia, and then cause myocardial ischemia-anoxemia, the heart disease that causes thus i.e. big homely " coronary heart disease " said, so coronary heart disease is myocardial ischemia " arch-criminal ".
The health of myocardial ischemia serious harm middle-aged and elderly people, in recent years along with growth in the living standard, coronary atherosclerosis presents the trend of rejuvenation, and the performance of myocardial ischemia also appears in some youngsters of 20～30 years old.
(2) classification of diseases
1, latent coronary heart disease
Latent coronary heart disease refers to not have clinical symptoms, but the coronary heart disease of myocardial ischemia objective evidence (change of electrocardiogram typical case ischemic ST, myocardial blood flow perfusion minimizing etc.) is arranged.Discover among acute sudden the dead of about 25%～50%, not have the angina pectoris history of attack before death in a large number in recent years, but in nearly 90% the postmortem, find that these have serious coronary atherosclerosis pathological changes per capita.Even in the patient that acute myocardial infarction takes place, also still have 30% patient not have symptom.Silent ischemia can occur in the dissimilar angina pectoriss.
Patient's transference cure but still have myocardial ischemia to exist after treating, this is the important indicator of prognosis mala.This type patient also may transfer myocardial infarction suddenly to, also may develop into myocardial fibrosis gradually and cardiac enlargement occur, and heart failure or arrhythmia take place, and individual patient also may be died suddenly.Therefore, the silent myocardial ischemia should cause enough attention of people, in time finds, and gives early Clinics and Practices.
2, angina pectoris
It is coronary insufficiency, its characteristics of clinical syndrome that rapid, the temporary transient ischemia of cardiac muscle and anoxia cause are paroxysmal shirtfront squeezing sample pain perception, can emit to left upper extremity, often betide the labour, catch cold, excited, when being satiated with food etc., continue several minutes, disappearances such as rest or sublingual administration nitroglycerin.
3, myocardial infarction type
Be on the basis of coronary artery pathological changes, coronary blood take place for sharply reducing or interrupting, make seriously and the enduringly part cardiac muscle acute necrosis due to the acute ischemia of corresponding cardiac muscle.Myocardial infarction often is due to secondary thrombus forms on the basis of coronary atherosclerosis pathological changes.
4, ischemic cardiomyopathy
Be to cause myocardial cell minimizing, necrosis, myocardial fibrosis, MS to form owing to atherosclerotic lesion makes the oxygen supply of cardiac muscle and aerobic imbalance.It is stiff that its characteristics are that heart becomes, and enlarges gradually, and arrhythmia and heart failure take place.
5, sudden death type
Be spontaneous generation, unexpected sudden death.Sudden death coronary heart disease was sent out well season in midwinter, how little age is, be because on atherosclerotic basis, coronary vasospasm or thromboembolism take place, cause myocardium acute ischemia, cause local electric physiologic derangement, cause (Chen Zaijia, Gao Runlin chief editor due to the temporary transient severe arrhythmia (quivering as the chamber) etc.Coronary heart disease.Beijing: People's Health Publisher, 2002).
(3) pathogenic factor
The common reason of myocardial ischemia is coronary atherosclerosis, secondly also has inflammation (rheumatic, syphilis, mucocutaneous lymphnode syndrome and thromboangitis obliterans etc.), spasm, thromboembolism, connective tissue disease, wound and congenital malformation etc. multiple.Epidemiological study finds, the significant risk factor relevant with atherosclerosis is that hyperlipemia, hypertension, diabetes, smoking, obesity, homocysteine increase, physical exertion is few, advanced age and male etc.
Its pathogenesis thinks mainly that at present myocardial ischemia causes that apoptosis, calcium ion overload, a large amount of oxygen-derived free radicals produce and the lipid peroxidation thing strengthens, the activation of the cytokine of the intervention of inflammatory factor and participation cell signalling etc.
(4) clinical manifestation
Clinical angina pectoris, myocardial infarction, arrhythmia and the heart failure of showing as.
Myocardial ischemia is commonly encountered diseases and the frequently-occurring disease of middle-aged and elderly people, is in the people of this age level, in daily life, if following situation will in time be sought medical advice, as early as possible indagation.
1, occur behind the breastbone when fatigue or psychentonia or the precordial fullness pain, or tighten sample pain, and shoulder, left upper arm radiation left, continue 3～5 minutes, the back spontaneous remission person that has a rest, the time with profuse sweating.
2, occur during physical exertion uncomfortable in chest, cardiopalmus, breathe hard spontaneous remission person during rest.
3, the throat pain relevant with motion and burn feeling, constriction, toothache etc. appear.
4, heavy meal, cold, chest pain, person uncomfortable in chest appear after drinking.
5, sleep medicated pillow night when low, feel chest distress, need high pillow clinostatism side to feel comfortable person; Sleep soundly or daytime unexpected chest pain, cardiopalmus, dyspnea during flat crouching, need sit up immediately or stand can alleviation person.
6, sexual life or firmly occur nervous, uncomfortable in chest, out of breath during defecation or chest pain is uncomfortable.
7, Tu Fa bradycardia, blood pressure reduce or the person of fainting.
(5) diagnosis is differentiated
1, the relevant risk factor inspection of coronary heart disease
For finding myocardial ischemia as early as possible, the health check-up of should regularly being correlated with of the people more than 40 years old is understood the risk factor that has or not coronary heart disease relevant, as inspections such as blood fat, blood pressure, blood glucose, cervical region Vascular Ultrasonography, cardiac ultrasonic, electrocardiograms.
2, be subjected to stress test inspection
If belong to the high-risk group of myocardial ischemia, whether the inquiry of will sending for a doctor possesses typical coronary heart disease symptom, determines whether to need to accept stress test, arranges CT as treadmill electrocardiogram, adenosine load cardiac ultrasonic kinetocardiogram and arteria coronaria more.
3, coronarography inspection
Evaluation by noninvasive test needing to determine whether further adopting the goldstandard----of diagnosis of coronary heart disease, and-coronarography is made a definite diagnosis, with degree and the scope of clear and definite arteria coronaria pathological changes.
Expert's viewpoint: be judged as the patient of typical coronary heart disease symptom by having the cardiologist of enriching clinical experience, can consider directly to carry out the coronary angiography inspection, implant frame treatment in case of necessity, and there is no need to do earlier the cardiovascular diseases branch of many row CT(Chinese Medical Association of arteria coronaria, China cardiovascular diseases magazine editorial board, Chinese circulation magazine editorial board.Diagnosis of Acute Myocardial Infarction and treatment guide.China cardiovascular diseases magazine, 2001,29(2): 270).
(6) disease treatment
1, Therapeutic Principle
Because myocardial ischemia has the danger that myocardial infarction and sudden death take place, when therefore finding myocardial ischemia, reach early treatment.The atherosclerotic generation of active prevention as taking place, is answered active treatment, prevents and treats pathological development and strives for reverse.Complication person has been taken place, should in time treat, control worsens, and prolongs patient's life-span.
Treatment mainly contains Drug therapy, surgical operation, intervention, stem cell transplantation and gene therapy etc. to modern medicine to primary disease.
2, Drug therapy
Drug therapy to coronary heart disease mainly is divided into nitrate esters, beta-blocker, calcium ion channel blocker, anticoagulant medicine and antiplatelet aggregation class, Statins and blood vessel converting enzyme inhibitor (abbreviation: ACEI) etc. at present.But these medicines also all exist comparatively significant side effects.Side effect such as drug dependence and various untoward reaction can appear during long-term a large amount of administration.
Can be summarized as the ABCDENT scheme:
A. antiplatelet drug (as aspirin, clopidogrel) is prevented and treated thrombosis, prevention coronary artery and cerebral artery thrombosis thromboembolism;
B. receptor blocking agent (as metoprolol or its slow releasing tablet), decreased heart rate reduces myocardium De Mao oxygen, the prevention sudden death;
C. calcium ion antagonist;
D. statins (as atorvastatin, Rosuvastatin etc.) reduces the cholesterol in the blood plasma, stablizes artery plaque, prevents the speckle formation thrombosis that comes off;
E.RAS system blocking agent (as benazepril and valsartan etc.) prevention remodeling ventricle improves cardiac function etc.;
N. nitric acid lipid drug (as the different Pyrusussuriensis fat of single nitric acid): coronary artery dilator increases blood supply of cardiac muscle;
T. thrombolytic drug dissolves the thrombosis of acute formation, is used for acute myocardial infarction.
3, interventional therapy
Be that percutaneous coronary intervention (pci) (is called for short: PCI): stenting.
Percutaneous coronary intervention is the PCI art, is the milestone event on the coronary heart disease treatment history over nearly 30 years, this be a kind of without surgery open breast, invasive methods coronarius is opened in the sense of patient's no pain.Have the advantages that wound is little, recovery is fast, the hospital stays is short, widely the patient accepts.From early stage simple balloon dilatation, to the naked metal rack epoch, develop into the coating stent of medicine epoch till now, become the important means for the treatment of primary disease.So for paresthesia epilepsy is typical or unstable, Drug therapy is undesirable, the patient of noninvasive test prompting myocardial ischemia advises the inspection of row coronary angiography, capable stent endoprosthesis in case of necessity.
4, operative treatment
The surgery coronary artery bypass: complicated multivessel disease, unprotect left side trunk pathological changes person can select (cardiovascular diseases branch of Chinese Medical Association, Chinese cardiovascular diseases's magazine editorial board, Chinese circulation magazine editorial board.Diagnosis of Acute Myocardial Infarction and treatment guide.China cardiovascular diseases magazine, 2001,29(2): 270, and Li Yan, Guo Zhaojiang, Jia Guoliang.Restenosis research behind the systematic scientific method and the percutaneous coronary intervention.2002 23 05 phases of volume of medical science and philosophy).
(7) disease prevention
1, prevention method
The patient of the patient's of A, myocardial ischemia diet myocardial ischemia grasps several principles at diet, notes less salt low fat light diet, eats vegetables such as Radix Ipomoeae, Fructus Lycopersici esculenti, Radix Dauci Sativae more, and these all are the food that can improve the patient body resistivity.Drink a little green tea, contain a spot of theophylline in the Folium Camelliae sinensis, certain diuresis is arranged, patient's curing myocardial ischemia is had certain help, also have vitamin C in the Folium Camelliae sinensis, can play a well arteriosclerotic effect of control, but unsuitable overrich.Adhere to eating a little Auricularia every day, can help to reduce blood viscosity, improve myocardial ischemia.
To note not great rejoicing great sorrow on B, the myocardial ischemia patient emotion, keep sufficient sleep.Form good living habit, the timing defecation can not be overworked.
C, should modest movement, promote the foundation of myocardium collateral circulation.
D, the state of an illness are as the treatment of will in time going to a doctor that changes.
2, prophylactic agent
The ABCDE of coronary heart disease primary prevention: the primary prevention scheme of coronary heart disease comprises can be summarized as ABCDE.
A. aspirin;
B. beta-blocker;
C. calcium ion antagonist;
D. Statins lipid regulating agent;
E. (Chen Zaijia, Gao Runlin edits angiotensin converting enzyme inhibitor.Coronary heart disease.Beijing: People's Health Publisher, 2002).
(8) Epidemiological study
Ischemic heart desease is common cardiovascular disease, and sickness rate is than higher clinically, and myocardial ischemia mostly occurred after 40 years old, and average prevalence is about 6.49%.
Along with the raising of living standards of the people, myocardial ischemia is the trend that rises year by year in the prevalence of China at present.Become second largest lethal factor after malignant tumor at the mortality rate of Chinese cardiovascular disease.According to the WHO statistics, in China, in per 100000 people, just there were 54 people to die from ischemic heart desease in 2002, mortality rate accounts for 23.4% of all cardiovascular disease, occupies first.
The Rui Yaocheng of Second Military Medical University, PLA professor seminar discovers that mononuclear cell migration inhibitory factor (being called for short: pentapeptide MLIF or MLIF) has the effect of control ischemic cerebrovascular and myocardial ischemia, has applied for national inventing patent and authorized (patent No. is respectively ZL200810200610.0 and ZL200910055203.X).MLIF is the pentapeptide of finding from the Entamoeba histolytica of aseptic culture (Met-Gln-Cys-Asn-Ser) at first.
Therefore; working out a kind of effective Therapeutic Method, to stop potential pathogenic process be very necessary; in conjunction with existing chemical compound or medicine; it is still imperative therefrom excavate to seek the little product that resists myocardial ischemia of novel, definite ingredients, determined curative effect, untoward reaction; medicine especially for aspects such as prevention, diagnosis, detection, protection and treatment myocardial ischemia has very important significance, and can produce remarkable social benefit and economic benefit.
But, by literature search etc., up to the present, still find no the part metabolite of this pentapeptide for the preparation of the report of aspects such as the product that resists myocardial ischemia.
Summary of the invention
This finds that the technical problem of required solution is the new purposes that discloses a kind of part metabolite of little peptide, namely the part metabolite of this little peptide has the new role that resists myocardial ischemia, can be used in the preparation product that resists myocardial ischemia, to overcome the above-mentioned defective that prior art exists.
That is to say; the present invention is directed to the deficiencies in the prior art; research and theory study by experiment; purpose is the new purposes that a kind of part metabolite of little peptide will be provided; be intended to the new purposes of the part metabolite of clear and definite little peptide; namely the part metabolite of this little peptide is as resisting myocardial ischemia the new purposes of product, for prevention, diagnosis, detection, protection, treatment and the research that resists myocardial ischemia provides more effective, product more targetedly.
Described little peptide for the preparation of the product that resists myocardial ischemia refers to the little peptide for the preparation of the product that resists myocardial ischemia, comprise that some metabolite comprises that tetrapeptide QS(is called for short in the body of pentapeptide mononuclear cell migration inhibitory factor MLI: QS), tetrapeptide MN(is called for short: MN), tripeptides CS(is called for short: CS), tripeptides MC(is called for short: MC), dipeptides MQ(is called for short: MQ), dipeptides NS(is called for short: NS), add up to 6 kinds pentapeptide and metabolite (QS, MN, CS, MC, MQ, NS) thereof.
Some metabolite comprises that tetrapeptide QS, tetrapeptide MN, tripeptides CS, tripeptides MC, dipeptides MQ, dipeptides NS, dipeptides QC(are called for short in described pentapeptide MLIF and the body thereof: QC), add up to 8 kinds pentapeptide and metabolite (MLIF, QS, MN, CS, MC, MQ, NS, QC) thereof to be known compound, its aminoacid sequence is as follows respectively:
SEQID?NO：1：Met-Gln-Cys-Asn-Ser；
SEQ?ID?NO：2：Gln-Cys-Asn-Ser；
SEQ?ID?NO：3：Met-Gln-Cys-Asn；
SEQ?ID?NO：4：Cys-Asn-Ser；
SEQ?ID?NO：5：Met-Gln-Cys；
SEQ?ID?NO：6：Met-Gln；
SEQ?ID?NO：7：Asn-Ser；
SEQ?ID?NO：8：Gln-Cys；
That is: 1. the mononuclear cell migration inhibitory factor (be called for short: aminoacid sequence MLIF) is shown in SEQ ID NO:1;
2. the aminoacid sequence of tetrapeptide QS is shown in SEQ ID NO:2;
3. the aminoacid sequence of tetrapeptide MN is shown in SEQ ID NO:3;
4. the aminoacid sequence of tripeptides CS is shown in SEQ ID NO:4;
5. the aminoacid sequence of tripeptides MC is shown in SEQ ID NO:5;
6. the aminoacid sequence of dipeptides MQ is shown in SEQ ID NO:6;
7. the aminoacid sequence of dipeptides NS is shown in SEQ ID NO:7;
8. the aminoacid sequence of dipeptides QC is shown in SEQ ID NO:8;
More than 8 kinds of pentapeptides and metabolite thereof finished syntheticly according to existing routine techniques by Guangdong 8+1 Medicine Co.,Ltd, purity reaches more than 98%, has realized commercially producing synthetic.
The described product that resists myocardial ischemia refers in the technical fields such as medicine, food or reagent; the product that is used for prevention, diagnosis, detection, protection, treatment and research myocardial ischemia disease and directly related disease thereof; be to comprise in medicine, reagent, food or the health product etc. one or more; preferred agents and health product, further preferred agents.
Described myocardial ischemia disease and directly related disease thereof refer to comprise one or more in latent coronary heart disease, angina pectoris, myocardial infarction type, ischemic cardiomyopathy or the sudden death type disease etc., and the present invention has prevention and therapeutic effect preferably to the early stage symptom of above-mentioned myocardial ischemia disease.
One, technical conceive
Independent development research and development original new drug is a present urgent task of China, the new combination of discovery novel drugs, existing medicine or new purposes etc. all are effectively quick approach, particularly finding the shortcut that the new purposes etc. of existing medicine or its compositions takes effect especially fast, also is the advantage place of Chinese original new drug development.
The inventor (is called for short: further further investigation discovery on research basis MLIF) at the mononuclear cell migration inhibitory factor, by cause the pharmacological testing of myocardial infarction and ischemia model through the rat coronary artery ligation, the part cylinder metabolism-ure of this pentapeptide can significantly reduce rat myocardial cell damage and myocardial infarct size due to the myocardial ischemia, reduce rats with myocardial ischemia serum lactate dehydrogenase (SLD) and creatine kinase, show that it has the effect of control myocardial ischemia, therefore can be used for preparation control myocardial ischemia drug; But, do not find that QC prevents and treats the activity of myocardial ischemia.
Ischemic heart desease is common cardiovascular disease, has become second largest lethal factor after malignant tumor at the mortality rate of Chinese cardiovascular disease, and the mortality rate of ischemic heart desease accounts for first of all cardiovascular disease.Myocardial ischemia disease has become important death cause and important social and the family burden of Chinese person in middle and old age's population; develop the product that resists myocardial ischemia; especially the product that prevents, diagnoses, protects and treat aspects such as myocardial ischemia disease particularly medicine has remarkable social benefit, economic benefit; economic pressures and the monitoring burden of family can be obviously alleviated, also patient's life quality can be obviously improved.
Generally speaking, the inventor is by carrying out the screening of system to little peptide and metabolite thereof, and deduction is in the drug effect of aspects such as resisting myocardial ischemia, one of its main path by this little peptide and metabolite thereof particularly its metabolite bring into play, result of study also proves and has confirmed that the part metabolite of this little peptide has significant function of resisting myocardial ischemia.According to this idea and thinking, the inventor passes through experimentation and analysis and theory study repeatedly, successfully obtains result of study and the application product of expecting.
Two, for the preparation of the research of the little peptide of the product that resists myocardial ischemia
Originally discover that 6 little peptides in the little peptide MLIF cylinder metabolism-ure have the pharmacological action of control myocardial ischemia.
The invention provides 6 little peptides, the i.e. part interior metabolism product of this pentapeptide (QS, MN, CS, MC, MQ, NS), its aminoacid sequence are respectively SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, the SEQ ID NO:7 application in preparation control myocardial ischemia drug.
Cause the pharmacological testing of myocardial infarction and ischemia model through the rat coronary artery ligation, the result shows, 6 little Toplink significantly reduce rat myocardial cell damage and myocardial infarct size due to the myocardial ischemia, reduce rats with myocardial ischemia serum lactate dehydrogenase (SLD) and creatine kinase, show that it has the effect of control myocardial ischemia, therefore can be used for preparation control myocardial ischemia drug; But, do not find that QC prevents and treats the activity of myocardial ischemia.Compare with MLIF, these six little peptides have synthetic relatively easy, lower-cost advantage.
Therefore, the inventor further conscientiously observe little peptide and metabolite thereof namely this pentapeptide and metabolite thereof to the influence of control myocardial ischemia, deeply inquire into the machine-processed place of little peptide and metabolite thereof control myocardial ischemia, anatomize the effect aspect the control myocardial ischemia of little peptide and metabolite thereof, in the hope of providing new thinking, approach and experimental data to the pathogenetic research of myocardial ischemia disease patient.
Three, for the preparation of activity and the effect of the little peptide of the product that resists myocardial ischemia
The present invention has carried out many-sided test to pentapeptide and the metabolite thereof for the preparation of the product that resists myocardial ischemia in the activity of the aspects such as prevention, diagnosis, protection, treatment and research of myocardial ischemia disease or its directly related disease etc.
For the preparation of the test at aspects such as prevention, diagnosis, protection and treatment myocardial ischemia diseases of this pentapeptide of the product that resists myocardial ischemia and metabolite thereof
(1) protective effect that Acute Myocardial Ischemia in Rats is damaged
1, laboratory animal: rat.
2, experiment grouping: with the experimental rat random packet.
3, experimental procedure: behind the anesthetized rat, its dorsal position is fixed.Cut thoracic wall and cut off the 4th and the 5th rib along left border of sternum at the 5th intercostal place, breast left side, cut off pericardium, expose heart, under the ramus descendens anterior arteriae coronariae sinistrae clump, wear stitching thread, the ligation stitching thread causes myocardial ischemia, and heart is put back to the thoracic cavity, discharges intrathoracic residual gas with hands, close the thoracic cavity, the whole breast of opening is finished fast to closing the breast operating process.Sham operated rats rat ramus descendens anterior arteriae coronariae sinistrae clump is threading only, not ligation arteria coronaria.The little peptide of each little peptide group rat difference tail vein injection low dosage and high dose behind the coronary ligation, sham operated rats and model group injection are with the normal saline of volume.Rat is put to death in anesthesia behind the ischemia.Get blood, centrifuging and taking serum is measured creatine kinase and (is called for short: CK) and lactic acid dehydrogenase (abbreviation: LDH).It is dirty to core, under the coronary ligation line, be parallel to coronary sulcus, with the ventricular muscles crosscut, place nitro blue tetrazolium (to be called for short: N-BT) dye liquor cardiac muscle, take out jolting dyeing back, normal myocardium dyes and is skipper, and the infarct cardiac muscle is then not painted, separates infarct under anatomical lens, weigh respectively, account for the percentage ratio (%) of myocardium weight as the index of weighing infarction size with infarct cardiac muscle weight.
4, result: ischemia 5h behind the rat coronary ligation, model group ischemic region myocardial infarction.With model group relatively, 6 little peptides (QS, MN, CS, MC, MQ, NS) all can alleviate ischemic region myocardial infarction weight, reduce the percentage ratio of infarct/whole-heartedly, and difference have significance meaning (P＜0.05, P＜0.01).With model group relatively, little peptide QC does not significantly dwindle the percentage ratio of ischemic region myocardial infarction weight and infarct/whole-heartedly.Illustrate that 6 little peptides (QS, MN, CS, MC, MQ, NS) have protective effect to treating myocardial ischemia damage.
Cause myocardial ischemia behind the rat coronary ligation 5h, LDH and CK are released into blood in the myocardial cell injury, cell, cause LDH and the rising of CK content in the serum; Model group rat blood serum LDH and CK and sham operated rats comparing difference highly significant (P＜0.01).Compare with model group, 6 little peptides (QS, MN, CS, MC, MQ, NS) all can reduce CK and LDH content in the serum, and difference has significance meaning (P＜0.05, P＜0.01).Compare with model group, little peptide QC does not significantly reduce CK and LDH content in the serum.Illustrate that 6 little peptides (QS, MN, CS, MC, MQ, NS) have protective effect to treating myocardial ischemia damage.
Above-mentioned experimental result shows that QS, MN, CS, MC, these 6 little Toplink of MQ, NS reduce coronary ligation rat myocardium block area, reduce serum CK and LDH, show that it has protective effect to treating myocardial ischemia damage.And little peptide QC does not have the ischemic myocardial protection effect.Therefore, these 6 little peptides (QS, MN, CS, MC, MQ, NS) can be used for preparation control myocardial ischemia drug.
The little peptide of addressing for the preparation of the product that resists myocardial ischemia of the present invention can be applied to the patient who needs treatment with the form of compositions, said compositions comprise the treatment effective dose for the preparation of the little peptide of the product that resists myocardial ischemia and pharmaceutically acceptable carrier.
Above specifically experiment showed, proves that all the present invention is a kind of safe, efficient, stable, inexpensive control myocardial ischemia disease and medicine or the health product of directly related disease thereof for the preparation of little peptide and the pharmaceutical composition of the product that resists myocardial ischemia.
In sum, the present invention has carried out theory study and experimentation to the part metabolite of little peptide, finds that the part metabolite level of this pentapeptide has very important effect for prevention, diagnosis, detection, protection, treatment and research myocardial ischemia disease.Therefore, the product that can resist myocardial ischemia at the part metabolite preparation of this pentapeptide, preferred agents and food, further preferred part metabolite with this pentapeptide of the present invention is the medicine that raw material prepares.
Four, for the preparation of the purposes of the part metabolite of the little peptide of the product that resists myocardial ischemia
1, general introduction
The purpose of this invention is to provide a kind of product for prevention, diagnosis, detection, protection, treatment and research myocardial ischemia disease and directly related disease thereof; comprise in medicine, reagent, the food etc. one or more; preferred agents and health product, further preferred agents.
Completed acute toxicity testing proves, the mouse stomach administration surpasses 3.2g/kg to the maximum tolerated dose of this active site, shows that this single component is safe and reliable, solved biological medicine complicated component, active constituent content is low and has contained the problem of toxic component.Therefore, can be used for the preparation product that resists myocardial ischemia for the preparation of the little peptide of the product that resists myocardial ischemia and compositions thereof, is medicine and the health product that feedstock production forms with the present invention for the preparation of the little peptide of the product that resists myocardial ischemia preferably, further preferred agents.
2, for the preparation of the little peptide of the product that resists myocardial ischemia and using method and the requirement of compositions thereof
The present invention can unite use separately or with other active component for the preparation of the little peptide of the product that resists myocardial ischemia; comprise for the preparation of the product that is used for prevention, diagnosis, detection, protection, treatment and research myocardial ischemia disease and directly related disease thereof; comprise medicine, reagent, food etc., especially medicine.
Aspect concrete use, the little peptide for the preparation of the product that resists myocardial ischemia of the present invention can use separately, can also use with other many chemical substances.These chemical substances biologically active or have the function for the treatment of disease whether no matter, comprise miscellaneous function as collaborative amplification, antagonism or alleviate for the preparation of the side effect of the little peptide of the product that resists myocardial ischemia etc. that these chemical substances are to comprise in pharmaceutically acceptable carrier, food, natural product, chemical synthetic drug or the human medication etc. one or more; Preferably include in pharmaceutically acceptable carrier or the food etc. one or more; Further preferred pharmaceutically acceptable carrier.
" pharmaceutically acceptable carrier " used herein comprises one or more in any He all physiology suitable solvent, disperse medium, afterbirth, antibacterial and antifungal, isotonic agent or the absorption delay agent etc.The example of pharmaceutically acceptable carrier comprises one or more water, saline, phosphate-buffered saline, glucose, glycerol or ethanol etc. and in the compositions one or more thereof.In many cases, in said composition, preferably include isotonic agent, for example, sugar, such as in the polyhydric alcohol of mannitol, sorbitol, sorbitol or the sodium chloride etc. one or more.Pharmaceutically acceptable carrier can also comprise a spot of auxiliary substance, one or more in wetting agent or emulsifying agent, antiseptic or the buffer etc. for example, and they have strengthened this effect duration or effectiveness for the preparation of the little peptide of the product that resists myocardial ischemia.
From concrete classification, said pharmaceutically acceptable carrier refers to the pharmaceutical carrier of medicine and pharmacology field routine, comprises excipient, as in starch or the water etc. one or more; Lubricant is as in glycerol or the magnesium stearate etc. one or more; Disintegrating agent is as microcrystalline Cellulose etc.; Filler is as in starch or the lactose etc. one or more; Bonding agent is as in pregelatinized Starch, dextrin, cellulose derivative, alginate, gelatin or the polyvinylpyrrolidone etc. one or more; Osmotic pressure regulator is as in glucose, sucrose, sorbitol or the mannitol etc. one or more; Diluent is as water etc.; Disintegrating agent is as in agar, calcium carbonate or the sodium bicarbonate etc. one or more; Absorption enhancer is as quaternary ammonium compound etc.; Surfactant is as hexadecanol etc.; Absorption carrier is as in Kaolin or the soap clay etc. one or more; Lubricant is as in Pulvis Talci, calcium stearate, magnesium stearate or the Polyethylene Glycol etc. one or more; In addition, can also in compositions, add other adjuvant, as in flavouring agent or the sweeting agent etc. one or more.
For example, with the little peptide dissolving of active component for the preparation of the product that resists myocardial ischemia, suspendible or (for example be emulsifiable in the suitable aqueous solvent, distilled water, in normal saline or the Green's solution etc. one or more) or in the oil-based solvent (for example, vegetable oil is olive oil for example, Oleum sesami, Oleum Gossypii semen, in Semen Maydis oil or the propylene glycol etc. one or more) in, can make ejection preparation, wherein (for example can contain dispersant in the solvent, polyoxyethylene sorbitan monoleate, polyoxyethylene hardened castor oil 60, Polyethylene Glycol, benzyl alcohol, in chlorobutanol or the phenol etc. one or more), osmotic pressure regulator (for example, sodium chloride, glycerol, the D9-mannose, in D-sorbitol or the glucose etc. one or more).In this case, if necessary, can add additive, for example solubilizing agent (for example, one or more in sodium salicylate or the sodium acetate etc.), stabilizing agent (for example, human serum albumin etc.), analgesic (for example, benzyl alcohol etc.) etc.
Of the present invention and the little peptide for the preparation of the product that resists myocardial ischemia can also unite use with the form of compositions, particularly with other chemical substance such as medicine animal especially mammal is comprised that people or other animals treat compositions for use or similar compositions.Described mammal, comprise in people, mice, rat, sheep, monkey, cattle, pig, horse, rabbit, dog, chimpanzee, baboon, Adeps seu carnis Rhiopithecus roxellanae, macaque or the Rhesus Macacus etc. one or more, in preferred people, mice, rat, monkey, pig, rabbit or the dog etc. one or more, one or more in further preferred people, rat or the monkey etc.For example, can with the present invention for the preparation of the little peptide of the product that resists myocardial ischemia add be suitable for to curee's Pharmaceutical composition in.Usually, this Pharmaceutical composition comprises the present invention for the preparation of little peptide and the pharmaceutically acceptable carrier of the product that resists myocardial ischemia.
For the preparation of the compositions of the little peptide of the product that resists myocardial ischemia particularly pharmaceutical composition various forms can be arranged, comprise in the dosage forms such as liquid, semisolid and solid for example one or more; Wherein said pharmaceutical composition comprises that the little peptide for the preparation of the product that resists myocardial ischemia for the treatment of effective dose is active component, and one or more pharmaceutically acceptable carriers.
Pharmaceutical composition for the preparation of the little peptide of the product that resists myocardial ischemia can adopt conventional production method well known in the art to make various dosage forms, and active component is mixed with one or more carriers, is made into required dosage form then.For example, little peptide for the preparation of the product that resists myocardial ischemia of the present invention can further be made dosage forms such as granule, capsule, tablet, pill by adding suitable adjuvant, and described adjuvant can be selected from one or more in starch, dextrin, lactose, silicon dioxide, calcium hydrogen phosphate, cyclodextrin, microcrystalline Cellulose, sodium carboxymethyl cellulose, magnesium stearate, the Pulvis Talci etc.
Described dosage form comprises one or more in tablet, capsule, granule, suspensoid, Emulsion, solution, syrup or the injection etc., takes one or more route of administration in oral or the injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more) etc. to carry out prevention, diagnosis, detection, protection, treatment or the scientific research of myocardial ischemia disease and directly related disease thereof.
It is that 0.5%～99% active component is for the preparation of the little peptide of the product that resists myocardial ischemia that pharmaceutical composition preferably contains weight ratio, further preferably contain weight ratio and be 60%～99% active component for the preparation of the little peptide of the product that resists myocardial ischemia, most preferably contain weight ratio and be 95%～99% active component for the preparation of the little peptide of the product that resists myocardial ischemia.
For the preparation of the pharmaceutical composition of the little peptide of the product that resists myocardial ischemia generally must be aseptic and produce under the condition of storage stable.Said composition can be mixed with solution, microemulsion, dispersion liquid, liposome or other is suitable for the ordered structure of high drug level.By with aequum should be for the preparation of the little peptide of the product that resists myocardial ischemia with a kind of of required mentioned component or combine and add that also then carrying out aseptic filtration prepares aseptic parenteral solution in the appropriate solvent.Generally speaking, add in the aseptic solvent that contains basic disperse medium and required above-mentioned other composition and prepare dispersion liquid by being somebody's turn to do little peptide for the preparation of the product that resists myocardial ischemia.Under the situation for the preparation of the sterile powder of aseptic parenteral solution, the preparation method of recommendation is vacuum drying and lyophilized preparation.For example, by passing through to keep required granular size such as the coating of lecithin, under the situation of dispersion liquid and by using surfactant, can keeping the adequate liquidity of solution.By comprising that in said composition the medicament (for example Monostearate or gelatin) that postpones to absorb can reach the prolongation absorption of injectable composition.
When being used for the patient, the little peptide dosage for the preparation of the product that resists myocardial ischemia of the present invention is 10～220mg/kgd, and this dosage or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.
The present invention can comprise that for the preparation of little peptide and the Pharmaceutical composition thereof of the product that resists myocardial ischemia the present invention of " treatment effective dose " or " prevention effective dose " is for the preparation of the little peptide of the product that resists myocardial ischemia." treatment effective dose " refers at the dosage of necessity and effectively reaches the amount of required therapeutic effect under the time.Can be according to such as the patient's condition, age, sex and the body weight of individuality and should cause at this individuality that the factors such as ability of required reaction changed for the preparation of the little peptide of the product that resists myocardial ischemia for the preparation of the treatment effective dose of the little peptide of the product that resists myocardial ischemia.The treatment effective dose also refers to that this surpasses the amount of its any toxicity or harmful effect for the preparation of the useful therapeutic effect of the little peptide of the product that resists myocardial ischemia." prevention effective dose " refers to effectively reach the amount of required preventive effect under necessary dosage and time.Because preventive dose is used for the ill preceding or early stage curee of disease, the prevention effective dose is usually less than the treatment effective dose.The present invention is 10～220mg/kg for the preparation of the treatment of the little peptide of the product that resists myocardial ischemia or the typical non-limiting scope of prevention effective dose, more preferably 20～100mg/kg.Should note, dose value will change according to the disease type of desiring to alleviate and seriousness, when that is to say for the patient, little peptide dosage or consumption for the preparation of the product that resists myocardial ischemia of the present invention decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.In addition; should understand; for any specific curee; should along with the time according to individual need and give with or supervision give with the people's of described compositions professional judgement and adjust the given dose system; and the dosage range that this paper sets only be illustrative, scope or the practice of the compositions of can't requirement for restriction protecting.
That is to say, need be according to object, route of administration, institute's disease of treat and the situation etc. for the treatment of, variation the present invention for the preparation of the little peptide of the product that resists myocardial ischemia at every turn and/or dosage or the consumption of every day.For example, give mammal through vein, adult (as body weight 60kg) especially, the single dose of described little peptide for the preparation of the product that resists myocardial ischemia is about 20～180mg, preferred about 100mg, preferred administration every day 1～3 time.Can adjust dosage unit, so that best required reaction (for example, treatment or prevention are replied) to be provided.For example, can single heavy dose of administration can give several divided doses or reduce or increase dosage in proportion according to the urgency for the treatment of situation in a period of time.The non-intestinal compositions that preparation is easy to the unified dosage unit form of administration and dosage is especially favourable.Dosage unit form used herein refers to be suitable for the physical separation unit of dosage unit of the mammalian subject of desire treatment; The calculating that each unit contains scheduled volume is used for together producing the active matter of required therapeutic effect for the preparation of the little peptide of the product that resists myocardial ischemia with required pharmaceutical carrier.The specification of dosage unit form of the present invention, determine and depend on that directly following (a) should be for the preparation of the specific characteristic of the little peptide of the product that resists myocardial ischemia and the particular treatment of desiring to reach or preventive effect and (b) mixing this individual sensitivity interior in restriction for the preparation of in the technology of the little peptide of the product that resists myocardial ischemia that be used for the treatment of by following.
3, for the preparation of the little peptide of the product that resists myocardial ischemia and pharmaceutical dosage form and the route of administration of compositions thereof
Of the present invention for the preparation of the product that resists myocardial ischemia little peptide and the product of being used for prevention, diagnosis, detecting of preparation of compositions, protection, treatment and research myocardial ischemia disease and directly related disease thereof, wherein the product according to the requirement preparation of beverage, food technology field can be used in prevention, protection and treats myocardial ischemia disease and directly related disease thereof; Can be used in patient's treatment or health care according to the product of the requirement of medical technical field preparation, can either be directly used in the medicine of preparation treatment or health care separately, also can mix with many chemical substances or make up, directly or indirectly for the preparation of the medicine for the treatment of or keeping healthy.Chemical substance described here is above described identical with this section.
In the present invention, required material comprises raw material of the present invention, above-mentioned matching used chemical substance etc., all should adopt the material of food stage or pharmaceutical grade according to practical situation and needs.
Little peptide and compositions thereof for the preparation of the product that resists myocardial ischemia of the present invention can be with the whole bag of tricks administration known in the art, although route of administration/administering mode of recommending in many therapeutic use is spray or oral administration.But the technical staff will appreciate that route of administration/administering mode changes with required result.In some concrete enforcement, this reactive compound can avoid the quick carrier that discharges together preparation example such as empty release formulation with this chemical compound of protection, comprises that graft transmission system, transdermal paste one or more in transmission system or the microcapsule transmission system etc.In addition, can also use biodegradable, biocompatible polymer, for example one or more in ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or the polylactic acid etc.Prepare the equal patent applied for of many methods of this preparation or generally known to those skilled in the art (referring to for example Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson edits, Marcel Dekker, Inc., New York, 1978).
Little peptide and compositions thereof for the preparation of the product that resists myocardial ischemia of the present invention usually by one or more modes in oral or the parenteral etc., are applied to the patient who needs this treatment.
Be used for when oral, can be made into conventional solid preparation such as in tablet, powder, granule or the capsule etc. one or more.When implementing, the present invention can be together oral with for example inert diluent or assimilable edible carrier for the preparation of the little peptide of the product that resists myocardial ischemia.Should (with its composition altogether, if desired) can also be wrapped in hard or soft shell gelatin capsules, be pressed into tablet or directly add in curee's the meals for the preparation of little peptide of the product that resists myocardial ischemia.About oral therapeutic administration, described little peptide for the preparation of the product that resists myocardial ischemia can be added with excipient and use with one or more forms in edible tablet, buccal tablet agent, lozenge, capsule, suspension, syrup or wafer etc.
For to give the present invention the little peptide for the preparation of the product that resists myocardial ischemia outside the parenterai administration, may need with preventing that the material of its inactivation from together giving for the preparation of the little peptide coating of the product that resists myocardial ischemia or with this little peptide for the preparation of the product that resists myocardial ischemia this.The reactive compound that replenishes can also be added in the said composition.In the specific implementation, the present invention is prepared altogether for the preparation of the little peptide of the product that resists myocardial ischemia and one or more other medicines that can be used for the treatment of disease and/or give altogether.Thisly unite use, can utilize this medicine that gives than low dosage primely, therefore avoid possible toxicity or the complication relevant with various monotherapies.
Make in liquid preparation such as water preparation, oil-suspending agent or other liquid preparation one or more, as in syrup, tincture or the elixir etc. one or more; When being used for parenteral, can be made in solution, water preparation or the oiliness suspending agent etc. of injection one or more.
Little peptide of the present invention pharmaceutics routinely is prepared into injection, injectable powder, tablet, capsule or other non-injecting pathway preparation.
In the above-described type of service, one or more in other preparations that preferred form is tablet, coated tablet, capsule or other non-injecting pathways etc., further one or more in preferred tablet or the capsule etc., preferred tablet especially.
In sum; the product that the present invention can be used for preventing, diagnose, detect, protect, treating and study myocardial ischemia disease and directly related disease thereof for the preparation of little peptide and the compositions thereof of the product that resists myocardial ischemia; preferred agents and health product, further preferred agents.
Five, technology speciality
The present invention is through the pharmacology test of myocardial ischemia disease, and the result shows that the little Toplink of part cylinder metabolism-ure of this pentapeptide molecule significantly reduces.Compare with similar protein drug, such little peptide effect is strong; And because molecular weight is little, be easy to by blood brain barrier, have the saturating sexual clorminance of significant nicergoline.
The part metabolite of little peptide provided by the invention has good prospects for application in preparation control myocardial ischemia disease; provide a kind of new medicine and application forms to originate for preventing, diagnose, detect, protect, treat and study myocardial ischemia disease and directly related disease thereof; thereby to existing resist myocardial ischemia product systems particularly medicine carried out improvement, improved; thereby expanded the new medical usage of little peptide and metabolite thereof, also provide a kind of new pharmaceutical intervention means for the control myocardial ischemia.
Little peptide and metabolite pharmacological action thereof for the preparation of the product that resists myocardial ischemia of the present invention is stronger, and the effect of prevention, diagnosis, detection, protection, treatment and research myocardial ischemia disease and directly related disease thereof is obvious; Safety and low toxicity have overcome the side effect that existing common drug causes; The quality of the pharmaceutical preparations for preparing is stable, and the source is abundant, and technology is simple, and the yield height is more suitable for large-scale production and commercial application of industry such as medicine, reagent and industry, has a good application prospect; Can be used for preparing the product of prevention, diagnosis, detection, protection, treatment and research myocardial ischemia disease and directly related disease thereof; little peptide of the present invention and metabolite thereof pharmaceutics routinely are prepared into other preparations of injection, tablet, capsule or non-injecting pathway; can play a role to greatest extent; and the scope of application is wide especially; therefore apply easily, can have a tremendous social and economic benefits in the short period of time.
In a word, the present invention studies the myocardial ischemia disease targetedly, has found a kind of new product that resists myocardial ischemia, active adaption modern medical service and the job demand of scientific research field and the needs of human nature service, made beyond thought achievement.The present invention is for the safe raw material that prevents, diagnoses, detects, protects, treats and study aspects such as myocardial ischemia disease and directly related disease thereof, has important value to developing Chinese existing medicine, improvement and the existing medical level of raising.
The specific embodiment
The present invention has studied existing disease and the means of prevention thereof of resisting myocardial ischemia, and the application of the part metabolite of little peptide as a kind of product that resists myocardial ischemia newly is provided, and is convenient to the safe handling of industries such as medical treatment, reagent.
The present invention finally need be prepared into the product that resists myocardial ischemia and use, and will enumerate embodiment below and further specify.If any problem, can contact directly 13386272938 or 15900897066 with the inventor.Provide several prescriptions and using method and some experimental study contents by aforementioned summary of the invention in above-mentioned some experimental datas that provide and the following example, but the research contents that list in the place that should be appreciated that the present invention is not limited to this, should also be appreciated that term as used herein only is used for describing certain embodiments, and be not limitation of the invention.
The present invention has studied the new pharmacological action of existing little peptide for the preparation of the product that resists myocardial ischemia and new purposes; a kind of raw material that can be used in preparation prevention, diagnosis, detection, protection, treatment and products such as research myocardial ischemia disease and directly related disease thereof is provided, has been convenient to the safe handling in medical industry and fields such as relevant industries such as food, beverage.
(1) for the preparation of the little peptide of the product that resists myocardial ischemia in the test aspect prevention, diagnosis, protection and the treatment myocardial ischemia disease
In order to set forth activity of the present invention and effect better, be described in detail below by example, the little peptide sample for the preparation of the product that resists myocardial ischemia wherein comes from Chinese market.
Below just the present invention the protective effect of Acute Myocardial Ischemia in Rats damage specifically is described below:
1, laboratory animal: SD rat, 200～220 of body weight.
2, experiment grouping: with the experimental rat random packet: sham operated rats, model group, low dose group, high dose group;
Facing the time spent now is mixed with injection with normal saline, the tail vein injection administration.
3, experimental procedure: etherization SD rat, dorsal position is fixed rapidly.Cut thoracic wall and cut off the 4th and the 5th rib along left border of sternum at the 5th intercostal place, breast left side, cut off pericardium, expose heart, under the ramus descendens anterior arteriae coronariae sinistrae clump, wear 1 stitching thread, the ligation stitching thread causes myocardial ischemia, and heart is put back to the thoracic cavity, discharges intrathoracic residual gas with hands as far as possible, close the thoracic cavity rapidly, the whole breast of opening is finished in 30 seconds to closing the breast operating process.Sham operated rats rat ramus descendens anterior arteriae coronariae sinistrae clump is threading only, not ligation arteria coronaria.15 minutes each little peptide group rats little peptide of tail vein injection low dosage and high dose respectively behind the coronary ligation, sham operated rats and model group injection are with the normal saline of volume.Rat is put to death in anesthesia behind the ischemia 5h.Get blood, centrifuging and taking serum is measured creatine kinase and lactic acid dehydrogenase.It is dirty to core, under the coronary ligation line, be parallel to coronary sulcus, wait heavy back that the ventricular muscles crosscut is become 5,5 cardiac muscles are placed the nitro blue tetrazolium dye liquor, take out behind the jolting dyeing 15min, normal myocardium dyes and is skipper, and the infarct cardiac muscle is then not painted, separates infarct under anatomical lens, weigh respectively, account for the percentage ratio (%) of myocardium weight as the index of weighing infarction size with infarct cardiac muscle weight.
4, data and statistical analysis: experimental data with
Expression is adopted variance analysis with SPSS10.0 software, carries out the significance test of difference.
The above equal proof of experiment and other experiments of the present invention, the present invention for the preparation of the little peptide of the product that resists myocardial ischemia with and pharmaceutical composition be a kind of safe, efficient, stable, inexpensive control myocardial ischemia disease and medicine or the health product of directly related disease thereof.
(2) for the preparation of the little peptide of the product that resists myocardial ischemia in resist myocardial ischemia dosage form aspect the product of preparation
Little peptide for the preparation of the product that resists myocardial ischemia can adopt method well known in the art to make various dosage forms, as tablet, capsule, granule, suspensoid, Emulsion, solution, syrup, injection etc., take oral or route of administration such as injection (comprising intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection), mucosa dialysis are carried out the control of myocardial ischemia disease.
When being used for the patient, dosage determines according to the situation of patient's age, body weight, route of administration and symptom.
The present invention prepares injectable powder and generally adopts conventional freeze-drying, with water as solvent, the steps include: to take in preparation resist myocardial ischemia little peptide or its metabolite of product, add excipient, be dissolved in water, add active carbon, filtration sterilization, plug is partly rolled in fill, lyophilization, tamponade are rolled lid and are got final product.Used excipient is selected from one or more in mannitol, gelatin hydrolysate, glucose, lactose, the dextran etc.Every bottle contains little peptide or its metabolite 10～100mg.
The present invention prepares injectable powder also can adopt spray drying method, with water as solvent, the steps include: to take in preparation resist myocardial ischemia little peptide or its metabolite of product, add or do not add excipient (excipient is the same), be dissolved in water, add active carbon, filtration sterilization, spray drying, aseptic subpackaged, tamponade is rolled lid and is got final product.Every bottle contains little peptide or its metabolite 10～100mg.
When the present invention prepares small-volume injection, preparation gets final product as solvent with water for injection, also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzoic acid, dimethyl acetylamide, pH regulator agent, surfactant, cyclodextrin, antioxidant, complexing of metal ion agent, the antibacterial.Injection can be mixed with solution, microemulsion, emulsion, liposome, microsphere, microcapsule or other is suitable for the ordered structure of high drug level, wherein can comprise the medicament that postpones absorption, for example Monostearate, gelatin, ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or polylactic acid etc. absorb with the prolongation that reaches injectable composition.Every contains little peptide or its metabolite 10～100mg.
The present invention prepares glucose infusion liquid or sodium chloride transfusion, with water for injection as solvent, adding the preparation of an amount of glucose or sodium chloride gets final product, also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzoic acid, dimethyl acetylamide, pH regulator agent, surfactant, antioxidant, cyclodextrin, complexing of metal ion agent, the antibacterial.Every bottle contains little peptide or its metabolite 10～100mg.
The present invention prepares oral formulations such as tablet, capsule, granule, oral liquid, and adjuvant can be lactose, starch, dextrin, stearate etc., technology preparation routinely.Can comprise the high molecular polymer carrier, as hydroxypropyl methylcellulose or polyoxyethylene etc., discharge with the prolongation that reaches Orally administered composition.
As biological technology products, the present invention also can adopt conventional biotechnology well known in the art to prepare little peptide or its metabolite expression vector is direct or indirect as resisting myocardial ischemia product, preferably with injection by intravenous injection or testis local injection mode, enter in the body; In addition, can also be by other multiple method, chemical compound and routes of administration that can directly or indirectly help to improve this little peptide or its metabolite function, all can be as the especially embodiment of medicine of product that resists myocardial ischemia.
But the preferred pharmaceutics routinely of little peptide of the present invention and metabolite thereof is prepared into other preparations of injection, tablet, capsule or non-injecting pathway.
All are ripe methods as for the preparation method for the preparation of the little peptide of the product that resists myocardial ischemia, delivered at public publications such as multiple periodicals and magazines and patent documentations, just repeat no more in the present invention.
In the present invention, the example of the above-described specific embodiment and the following stated all is in order to set forth the present invention better, is not to limit scope of invention.
Mode below by embodiment further is described in detail the present invention.
Used animal, instrument and equipment, reagent and preparation thereof etc. all are from above-mentioned description or meet relevant conventional requirement in the experiment of following examples.
The preparation of embodiment 1, little peptide
1. the aminoacid sequence of tetrapeptide (QS) is shown in SEQ ID NO:2: Gln-Cys-Asn-Ser.
2. the aminoacid sequence of tetrapeptide (MN) is shown in SEQ ID NO:3: Met-Gln-Cys-Asn.
3. the aminoacid sequence of tripeptides (CS) is shown in SEQ ID NO:4: Cys-Asn-Ser.
4. the aminoacid sequence of tripeptides (MC) is shown in SEQ ID NO:5: Met-Gln-Cys.
5. the aminoacid sequence of dipeptides (MQ) is shown in SEQ ID NO:6: Met-Gln
6. the aminoacid sequence of dipeptides (NS) is shown in SEQ ID NO:7: Asn-Ser.
7. the aminoacid sequence of dipeptides (QC) is shown in SEQ ID NO:8: Gln-Cys.
More than little peptide synthetic by Guangdong 8+1 Medicine Co.,Ltd, purity is more than 98%.
Embodiment 2, to the protective effect of Acute Myocardial Ischemia in Rats damage
Table 1, little peptide to the influence of coronary ligation rat myocardium block (n=10,
)
| Group | Weigh mg whole-heartedly | Infarct weight (mg) | Infarct/(%) whole-heartedly |
| Sham operated rats | 687.2±56.9 | 0±0 | 0±0 |
| Model group | 692.6±53.4 | 198.3±41.6 | 28.5±4.9 |
| The MQ low dose group | 685.4±41.6 | 137.9±35.1 ** | 20.2±5.5 ** |
| The MQ high dose group | 702.8±32.4 | 132.8±30.5 ** | 18.7±3.8 ** |
| ? | ? | ? | ? |
| The MN low dose group | 680.2±50.0 | 144.6±33.7 ** | 21.3±4.6 ** |
| The MN high dose group | 700.7±66.9 | 135.7±47.1 ** | 19.5±7.0 ** |
| ? | ? | ? | ? |
| The MC low dose group | 691.3±54.5 | 143.8±29.5 ** | 20.9±4.2 ** |
| The MC high dose group | 715.6±65.6 | 140.6±30.4 ** | 19.9±5.2 ** |
| ? | ? | ? | ? |
| The QS low dose group | 715.7±37.1 | 143.0±43.2 ** | 19.9±5.5 ** |
| The QS high dose group | 728.0±55.5 | 133.3±18.7 ** | 18.3±2.4 ** |
| ? | ? | ? | ? |
| The CS low dose group | 702.3±74.2 | 165.0±22.1 * | 23.9±4.6 * |
| The CS high dose group | 686.7±43.2 | 131.7±18.8 ** | 19.2±2.8 ** |
| ? | ? | ? | ? |
| The NS low dose group | 680.3±31.2 | 166.5±36.7 ** | 24.4±4.9 * |
| The NS high dose group | 680.2±500.8 | 144.5±33.7 * | 21.3±4.6 * |
| ? | ? | ? | ? |
| The QC low dose group | 711.1±70.3 | 193.8±23.1 | 27.4±3.6 |
| The QC high dose group | 700.0±50.0 | 176.9±30.8 | 25.2±4.7 |
Compare with model group,
*P＜0.05;
*P＜0.01.
1, laboratory animal: the SD rat, male, the cleaning level, 200～220 of body weight are available from the 2012-0002 of Shanghai Slac Experimental Animal Co., Ltd. (ticket number SCXK(Shanghai)).
2, experiment grouping: experimental rat is divided into 10 groups at random: sham operated rats, model group, low dose group are 0.5mg/kg, high dose group is 2mg/kg.
Facing the time spent now is mixed with injection with normal saline, tail vein injection administration, administration volume 10ml/kg.
3, experimental procedure: etherization SD rat, dorsal position is fixed rapidly.Cut off the 4th and the 5th rib at breast left side the 5th intercostal place incision thoracic wall and along left border of sternum 2mm place, cut off pericardium, expose heart, under the ramus descendens anterior arteriae coronariae sinistrae clump, wear 1 No. 0/3 stitching thread, the ligation stitching thread causes myocardial ischemia, and heart is put back to the thoracic cavity, discharges intrathoracic residual gas with hands as far as possible, close the thoracic cavity rapidly, the whole breast of opening is finished in 30 seconds to closing the breast operating process.Sham operated rats rat ramus descendens anterior arteriae coronariae sinistrae clump is threading only, not ligation arteria coronaria.15 minutes each little peptide group rats little peptide of tail vein injection low dosage and high dose respectively behind the coronary ligation, sham operated rats and model group injection are with the normal saline of volume.Rat is put to death in anesthesia behind the ischemia 5h.Get blood, centrifuging and taking serum is measured creatine kinase and lactic acid dehydrogenase (Shanghai Foxing Changzheng medical science Co., Ltd's product: determination of lactate dehydrogenase test kit, lot number: P1201031; Creatine kinase is measured test kit, lot number: 01207021.Beijing Fu Litai medical science and technology company limited is produced semi-automatic analyser, model: SCREEN MASTER3000).It is dirty to core, under the coronary ligation line, be parallel to coronary sulcus, wait heavy back that the ventricular muscles crosscut is become 5,5 cardiac muscles are placed nitro blue tetrazolium (N-BT) dye liquor, take out behind the jolting dyeing 15min, normal myocardium dyes and is skipper, and the infarct cardiac muscle is then not painted, separates infarct under anatomical lens, weigh respectively, account for the percentage ratio (%) of myocardium weight as the index of weighing infarction size with infarct cardiac muscle weight.
Table 2, little peptide to the influence of coronary ligation rat blood serum LDH and CK (n=10,
IU/L)
| Group | CK | LDH |
| Sham operated rats | 2373.1±421.5 | 1381.8±268.0 |
| Model group | 4406.7±1027.0 ** | 2631.0±537.4 ** |
| The MQ low dose group | 3117.8±624.2 ** | 2054.2±328.3 * |
| The MQ high dose group | 2995.6±852.9 ** | 1813.3±474.1 ** |
| ? | ? | ? |
| The MN low dose group | 3367.0±989.9 * | 2184.2±399.4 * |
| The MN high dose group | 3044.3±417.8 ** | 2088.7±285.6 * |
| ? | ? | ? |
| The MC low dose group | 3344.3±812.1 * | 2103.6±479.1 * |
| The MC high dose group | 3155.0±662.3 ** | 2011.4±270.8 * |
| ? | ? | ? |
| The QS low dose group | 3036.2±884.7 ** | 2063.5±643.5 * |
| The QS high dose group | 2887.7±803.9 ** | 1899.7±434.5 ** |
| ? | ? | ? |
| The CS low dose group | 3562.4±523.9 * | 2199.6±409.3 * |
| The CS high dose group | 3126.0±882.8 ** | 2010.5±665.7 * |
| ? | ? | ? |
| The NS low dose group | 3524.2±717.7 * | 2174.0±242.1 * |
| The NS high dose group | 3236.3±847.5 ** | 2107.3±253.1 * |
| ? | ? | ? |
| The QC low dose group | 4220.0±772.0 | 2527.7±271.5 |
| The QC high dose group | 4369.4±570.7 | 2480.8±419.8 |
Compare with model group,
*P＜0.05;
*P＜0.01.
4, data and statistical analysis: experimental data with
Expression is adopted variance analysis with SPSS10.0 software, carries out the significance test of difference.
5, result:
By shown in the table 1, ischemia 5h behind the rat coronary ligation, model group ischemic region myocardial infarction, myocardial infarction district weight is 198.3 ± 41.6mg, the percentage ratio of infarct/whole-heartedly is 28.5 ± 4.9%.With model group relatively, 6 little peptides (QS, MN, CS, MC, MQ, NS) all can alleviate ischemic region myocardial infarction weight, reduce the percentage ratio of infarct/whole-heartedly, and difference have significance meaning (P＜0.05, P＜0.01).With model group relatively, little peptide QC does not significantly dwindle the percentage ratio of ischemic region myocardial infarction weight and infarct/whole-heartedly.Illustrate that 6 little peptides (QS, MN, CS, MC, MQ, NS) have protective effect to treating myocardial ischemia damage.Detailed results sees Table 1.
By table 2 as seen, cause myocardial ischemia behind the rat coronary ligation 5h, LDH and CK are released into blood in the myocardial cell injury, cell, cause LDH and the rising of CK content in the serum; Model group rat blood serum LDH and CK are respectively 2631.0 ± 537.4IU/L and 4406.7 ± 1027.0IU/L, with sham operated rats comparing difference highly significant (P＜0.01).Compare with model group, 6 little peptides (QS, MN, CS, MC, MQ, NS) all can reduce CK and LDH content in the serum, and difference has significance meaning (P＜0.05, P＜0.01).Compare with model group, little peptide QC does not significantly reduce CK and LDH content in the serum.Illustrate that 6 little peptides (QS, MN, CS, MC, MQ, NS) have protective effect to treating myocardial ischemia damage.Detailed results sees Table 2.
Above-mentioned experimental result shows that QS, MN, CS, MC, these 6 little peptides (0.5mg/kg and 2mg/kg) of MQ, NS can reduce coronary ligation rat myocardium block area, reduces serum CK and LDH, shows that it has protective effect to treating myocardial ischemia damage.And little peptide QC does not have the ischemic myocardial protection effect.Therefore, 6 little peptide QS, MN, CS, MC, MQ, NS can be used for preparation control myocardial ischemia drug.
The above the present invention that specifically experiment showed, is a kind of safe, efficient, stable, inexpensive control myocardial ischemia disease and medicine or the health product of directly related disease thereof for the preparation of little peptide and the pharmaceutical composition of the product that resists myocardial ischemia.
Claims (6)
1. the purposes of part metabolite in preparation resists myocardial ischemia product of a pentapeptide MLIF, the part metabolite of described pentapeptide MLIF are that the aminoacid sequence of tetrapeptide QS, tetrapeptide MN, tripeptides CS, tripeptides MC, dipeptides MQ, dipeptides NS is shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.
2. the purposes of compositions in preparation resists myocardial ischemia product of the part metabolite of a pentapeptide MLIF, the part metabolite of described pentapeptide MLIF are that the aminoacid sequence of tetrapeptide QS, tetrapeptide MN, tripeptides CS, tripeptides MC, dipeptides MQ, dipeptides NS is shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.
3. purposes according to claim 1 and 2; it is characterized in that; the described product that resists myocardial ischemia refers in medicine, food or the reagent technical field; be used for prevention, diagnosis, detect, the product of protection, treatment and research myocardial ischemia disease and directly related disease thereof, be to comprise in medicine, reagent, food or the health product one or more.
4. purposes according to claim 3 is characterized in that, described myocardial ischemia disease and directly related disease thereof refer to comprise one or more in latent coronary heart disease, angina pectoris, myocardial infarction type, ischemic cardiomyopathy or the sudden death type disease.
5. purposes according to claim 4 is characterized in that, described myocardial ischemia disease and directly related disease thereof refer to the early stage of latent coronary heart disease, angina pectoris, myocardial infarction type, ischemic cardiomyopathy or sudden death type disease.
6. purposes according to claim 1 and 2, it is characterized in that, the described product that resists myocardial ischemia be with the part metabolite of pentapeptide MLIF routinely pharmaceutics be prepared into injection, injectable powder, tablet, capsule or other non-injecting pathway preparation and use.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103599523A (en) * | 2013-11-25 | 2014-02-26 | 中国人民解放军第二军医大学 | Application of pentapeptide metabolite in preparation of anti-inflammatory medicine |
| CN103611149A (en) * | 2013-11-25 | 2014-03-05 | 中国人民解放军第二军医大学 | Pentapeptide metabolite and application of pentapeptide metabolite in preparation of medicament for preventing and treating ischemic cerebrovascular diseases |
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| CN101361961B (en) * | 2008-09-27 | 2011-12-21 | 中国人民解放军第二军医大学 | Use of polypeptides micro-molecule in preparing medicine for preventing and treating ischemic cerebrovascular disease |
| 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 |
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| CN101361961B (en) * | 2008-09-27 | 2011-12-21 | 中国人民解放军第二军医大学 | Use of polypeptides micro-molecule in preparing medicine for preventing and treating ischemic cerebrovascular disease |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103599523A (en) * | 2013-11-25 | 2014-02-26 | 中国人民解放军第二军医大学 | Application of pentapeptide metabolite in preparation of anti-inflammatory medicine |
| CN103611149A (en) * | 2013-11-25 | 2014-03-05 | 中国人民解放军第二军医大学 | Pentapeptide metabolite and application of pentapeptide metabolite in preparation of medicament for preventing and treating ischemic cerebrovascular diseases |
| CN103611149B (en) * | 2013-11-25 | 2015-05-27 | 中国人民解放军第二军医大学 | Pentapeptide metabolite and application of pentapeptide metabolite in preparation of medicament for preventing and treating ischemic cerebrovascular diseases |
| CN103599523B (en) * | 2013-11-25 | 2016-08-17 | 中国人民解放军第二军医大学 | Pentapeptide metabolite application in preparing anti-inflammatory drug |
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