CN101966334A - Application of lecithin superoxide dismutase composite in preparation of medicines for treating and/or preventing cardiac ischemic injury - Google Patents
Application of lecithin superoxide dismutase composite in preparation of medicines for treating and/or preventing cardiac ischemic injury Download PDFInfo
- Publication number
- CN101966334A CN101966334A CN2010102995779A CN201010299577A CN101966334A CN 101966334 A CN101966334 A CN 101966334A CN 2010102995779 A CN2010102995779 A CN 2010102995779A CN 201010299577 A CN201010299577 A CN 201010299577A CN 101966334 A CN101966334 A CN 101966334A
- Authority
- CN
- China
- Prior art keywords
- superoxide dismutase
- lecithin
- sod
- lecithinized superoxide
- conjunction
- 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.)
- Pending
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to application of a lecithin superoxide dismutase composite in preparation of medicines for treating and/or preventing cardiac ischemic injury, wherein the lecithin superoxide dismutase composite is dipolymer combined with different molecule numbers of lecithin, and the concrete method comprises the following steps of: establishing a cardiac ischemic injury animal model; preparing a cardiac injury induced model for verifying a function of the cardiac ischemic injury animal model in cardiac protection treatment under the asepsis condition, spreading sacculus, blocking the sacculus for a long time and loosening the sacculus through injecting a contrast agent into the small pig coronary artery left anterior descending branch balloon implant so as to respectively establish a myocardial infarction model and a myocardial injury model through blood liquid infusion; and injecting the lecithin superoxide dismutase composite containing different molecule numbers of lecithin into the animal model so as to observe a new function of treating cardiac injury.
Description
Technical field
The present invention relates to the new purposes of Lecithinized superoxide dismutase (PC-SOD) compositions, particularly, the present invention relates to Lecithinized superoxide dismutase composition and treat and/or prevent application in the medicine of heart ischemia damage in preparation.
Background technology
The applicant's denomination of invention for disclose among the Chinese patent ZL200580034389.0 of " Lecithinized superoxide dismutase composition and preparation method thereof " PC-SOD monomer in the Lecithinized superoxide dismutase composition be comprise copper and/or zinc is coordinate, the sulfydryl of 111 cysteine by the amino more than 1 of the sulfurized superoxide dismutase from the people of ethoxy by following general formula (I):
The Lecithinized superoxide dismutase that the lecithin residue of expression replaces, this Lecithinized superoxide dismutase is a main component with m aforementioned amino is replaced (being that m represents bonded lecithin number on the superoxide dismutase composition monomer) by aforementioned lecithin residue Lecithinized superoxide dismutase (A), wherein, m is 1～4 integer, the meansigma methods of m is 1.5～2.4, this Lecithinized superoxide dismutase (A) is by the Lecithinized superoxide dismutase (a1) of the m=1 of 25～40 moles of %, the Lecithinized superoxide dismutase of the m=2 of 35～50 moles of % (a2), the Lecithinized superoxide dismutase (a4) of the m=4 of the Lecithinized superoxide dismutase of the m=3 of 10～20 moles of % (a3) and 5～15 moles of % forms, R in the general formula (I) is the alkyl of carbon number 8～30, and n is 2～10 integer.The Lecithinized superoxide dismutase composition of this patent disclosure is except that comprising above-mentioned Lecithinized superoxide dismutase (A), also comprise the Lecithinized superoxide dismutase (B) that the amino more than 5 from people's superoxide dismutase is replaced by the lecithin residue of above-mentioned general formula (I) expression, under normal conditions, the lecithin residue number that is present in the Lecithinized superoxide dismutase (B) is 5～8, and this patent is incorporated herein by reference in full at this.
Although myocardial preservation has developed for many years, in the middle of the clinical practice of the treatment of acute myocardial infarction and operation on heart, the method for myocardial preservation and effect await further developing.Find in the clinical practice that the outbreak of myocardial infarction is generally the obstruction of coronary artery or coronary arterial tree blood vessel, can cause the cardiac muscular tissue's degeneration and the necrosis in heart blood supply district usually.After of the obstruction removing of means such as operation with blood vessel, its histoorgan is carried out pouring into behind the ischemia again, tissue organ function is recovered, can increase the weight of the dysfunction and the structural damage of histoorgan on the contrary.Think that at present ischemia-reperfusion causes the basic mechanism that myocardial damage takes place to be that oxygen-derived free radicals produces in a large number, it causes calcium overload, endotheliocyte activation, complement system and renin-angiotensin system etc. and changes, the cytology basis that myocardial ischemia reperfusion injury takes place is an apoptosis of cardiac muscle, brings out arrhythmia, heart sudden death and the heart cardiovascular disease such as cardiac function due to pressing down goes down of pausing.Therefore myocardial preservation class medicine becomes the focus of vast researcher research and development.
At the damage that ischemia-reperfusion in the myocardial preservation treatment causes myocardial cell, on the one hand, still there is not the method for preventing and treating of general myocardial preservation at present, mainly use Na clinically
+/ H
+Exchanger inhibitor, Na
+/ Ca
2+Exchanger inhibitor, repolarization liquid, statins and platelet suppressant drug are treated, but its mechanism of action all belongs to the downstream of oxygen free radical injury, thereby are difficult to obtain ideal effect.On the other hand, though promptly there is researcher to propose to use superoxide dismutase treatment ischemical reperfusion injury in early days, but since during the superoxide dismutase intravenously administrable in the blood half-life extremely short and cellular affinity is poor, can't bring into play the effect of removing oxygen-derived free radicals in vivo.
In Chinese patent ZL200580034389.0, mention the effect of PC-SOD compositions aspect treatment ulcerative colitis, interstitial pneumonia.Yet, still do not have the report that the PC-SOD compositions that contains different lecithin (PC) molecular number is used at present in the heart ischemia injury in treating.
Summary of the invention
The inventor is by discovering, Lecithinized superoxide dismutase composition carries out structural modification by using lecithin to superoxide dismutase (SOD) albumen, can strengthen the affinity of itself and cell membrane, enables to enter in the cell to play a role.The PC-SOD that contains different lecithin molecules numbers by combination; be about to have the PC-SOD of prolong half-life effect and have and manage active PC-SOD than Johnson ﹠ Johnson and make up the compositions that makes in accordance with the appropriate ratio; can be when improving the PC-SOD cellular affinity; have stronger pharmaceutically active, thereby treating myocardial ischemia damage is had substantive protective effect.
The purpose of this invention is to provide Lecithinized superoxide dismutase composition and treat and/or prevent application in the medicine of heart ischemia damage in preparation, wherein, described Lecithinized superoxide dismutase composition is to be combined with the dimer that different molecular is counted lecithin.
Wherein, described heart ischemia damages the myocardial cell injury that too much causes for free radical.
Wherein, described Lecithinized superoxide dismutase composition is the dimer that is combined with 2-8 molecule lecithin.
Wherein, in the described Lecithinized superoxide dismutase composition, Lecithinized superoxide dismutase in conjunction with 2 molecule lecithin accounts for 6.25-16 mole %, Lecithinized superoxide dismutase in conjunction with 3 molecule lecithin accounts for 18-40 mole %, Lecithinized superoxide dismutase in conjunction with 4 molecule lecithin accounts for 12.25-25 mole %, Lecithinized superoxide dismutase in conjunction with 5 molecule lecithin accounts for 8-20 mole %, Lecithinized superoxide dismutase in conjunction with 6 molecule lecithin accounts for 1-4 mole %, Lecithinized superoxide dismutase in conjunction with 7 molecule lecithin accounts for 1-6 mole %, accounts for 0.25-2.25 mole % in conjunction with the Lecithinized superoxide dismutase of 8 molecule lecithin.
Wherein, effective dose every day of described Lecithinized superoxide dismutase composition is 10-160mg/60kg.
The present invention injects contrast agent and makes balloon expandable, shutoff a period of time by Chinese miniature pig left anterior descending coronary artery (LAD) is implanted sacculus, unclamps sacculus then and makes blood reperfusion make up myocardial infarction and myocardial damage model; The Lecithinized superoxide dismutase composition for preparing is injected in the described animal model, treats and/or prevents application in the medicine of heart ischemia damage in preparation by every treatment evaluation of indexes being judged said composition.
Compared with prior art, the present invention has following beneficial effect at least:
1, the medicine that contains the Lecithinized superoxide dismutase composition composition can pour into beginning moment seizure oxygen-derived free radicals again; thereby effectively alleviate reperfusion injury; it shows as the infarct size that significantly reduces ischemic myocardium; effectively reduce ARR generation; and can improve ventricular systole; help to reduce the generation of heart failure behind the PCI; therefore, to have the myocardial preservation ability be that Lecithinized superoxide dismutase composition has been expanded new clinical practice field to Lecithinized superoxide dismutase composition.
2, the present invention is that experimental animal, balloon occluder technology make up animal model with Chinese miniature pig, because the heart of Chinese miniature pig is the most similar to the structure of human heart, therefore overcome when adopting the animal model that cardiac structures such as Mus or dog and human body differ greatly in the conventional art, the test data existence is than the problem of big-difference; The experimental data that obtains by test method of the present invention is more near the truthful data in the anthropometric dummy, thereby the new purposes of treatment of said composition can be described more accurately.
Though 3, the superoxide dismutase that exists at present is effective to various diseases associated with inflammation, decompose rapidly after the administration or discharge through kidney, in clinical lesion, there is not therapeutic effect.And will giving SOD by PCization, PCization SOD has cellular affinity, increased molecular particle size simultaneously, in inflammation, accumulated in damaged cells film place easily, can also suppress discharge through kidney, therefore can remove organized enzyme significantly at the illness position, obtain effective clinical effectiveness.
4, Lecithinized superoxide dismutase composition of the present invention has better therapeutic effect than the Lecithinized superoxide dismutase composition in conjunction with the lecithin (PC) of a certain number (for example 4 molecules) merely: compare with the SOD of unmodified, reduced the enzymatic activity of SOD after the PCization, though PCization is effective to disease, survival rate does not replenish the reduction of enzymatic activity in its cellular affinity or the high blood.Enzymatic activity high is low more more in conjunction with number for PC, but cellular affinity is high more, and the present invention has obtained the compositions of enzymatic activity the best by this two aspect of balance.The present invention makes up according to certain ratio in conjunction with the PC-SOD that number replaces in conjunction with PC-SOD and the high PC that number replaces by hanging down PC, the low PC-SOD that replaces can give play to powerful enzymatic activity in compositions, demonstrate high-efficiency, the high PC-SOD that replaces can increase the affinity of compositions and cell membrane, make drug molecule accumulate in lesions position for a long time, half-life of prolong drug significantly, improved blood drug level, after blood drug level improves, patient's dosage every day can reduce greatly, thereby saved cost for clinical practice, improved patient's compliance.
5, Lecithinized superoxide dismutase composition long half time of the present invention, enzymatic activity intensity height can make the drug level that arrives heart significantly improve, thereby can give play to very strong pharmacologically active in operation on heart.
Description of drawings
Fig. 1 is the experimental implementation flow chart of embodiment 1;
Fig. 2 be among the embodiment 1 the PC-SOD compositions to the influence of Chinese miniature pig myocardial ischemia reperfusion injury; Wherein, Fig. 2 A represents to pour into the region area there was no significant difference again in each total left ventricle area of organizing; Fig. 2 B represent to pour into again gave in preceding 30 minutes PC-SOD (0.3,1,3mg/kg, n=7), (n=8) compares with model control group, can significantly reduce no resurgent area, * * p＜0.01, * * * p＜0.005;
Fig. 3 is the influence that the PC-SOD compositions is pressed changing value (Δ LVEDP) among the embodiment 1 to Chinese miniature pig left ventricular end diastolic; Balloon occluder causes Chinese miniature pig myocardial ischemia and caused Δ LVEDP significantly to raise (compare with sham operated rats * * * p＜0.005) in 120 minutes; Pour into again gave in preceding 3 minutes PC-SOD (1mg/kg, n=7), when pouring into 180 minutes again, the rising of Δ LVEDP significantly is suppressed (##p＜0.01, with PC-SOD 0mg/kg administration group relatively);
Fig. 4 be among the embodiment 2 the PC-SOD compositions to the influence of Chinese miniature pig myocardial ischemia reperfusion injury; Wherein, Fig. 4 A represents to pour into the region area there was no significant difference again in each total left ventricle area of organizing; Fig. 4 B represented to pour into preceding 30 minutes again or poured into and gave PC-SOD (0.3mg/kg, n=7～8) in preceding 3 minutes, and (n=8) compares with model control group, all can significantly reduce no resurgent area, * * p＜0.01, * * * p＜0.005;
Fig. 5 is the influence that the PC-SOD compositions is pressed changing value (Δ LVEDP) among the embodiment 2 to Chinese miniature pig left ventricular end diastolic; Balloon occluder causes Chinese miniature pig myocardial ischemia and caused Δ LVEDP significantly to raise (compare with sham operated rats * * * p＜0.005) in 120 minutes; Pour into again gave in preceding 3 minutes PC-SOD (0.3mg/kg, n=7), when pouring into 180 minutes again, the rising of Δ LVEDP significantly is suppressed (##p＜0.01, with PC-SOD 0mg/kg administration group relatively); And
Fig. 6 is a curve when containing different molecular and counting the medicine of PC-SOD compositions intravenous administration of PC.
The specific embodiment
Below will make further detailed description to the present invention, but this should be interpreted as that scope of the present invention only limits to following examples by embodiment.Allly all belong to scope of the present invention based on the technology that content of the present invention realized.Obviously, according to content of the present invention,, under the prerequisite that does not break away from basic fundamental thought of the present invention, can also make modification, replacement or the change of other various ways according to the ordinary skill knowledge and the customary means of this area.
1. experiment material
Laboratory animal: 35 of Chinese miniature pigs, culture the center available from the emerging laboratory animal of Beijing section, male and female are regardless of, body weight 20～30kg;
The experiment medicine:
Test sample: the Lecithinized superoxide dismutase composition lyophilized formulations, its preparation method is as follows:
In the 10L flask, add injection distilled water 4500mL, add boric acid 13g, NaOH 1.8g and KCl 68g dissolving more successively.Then, adding SOD aqueous solution (50g, SOD concentration 56.8mg/mL), when stirring with 210rpm, in chromatography room is 8 ℃ isopropyl alcohol 1000mL with 16.5mL/ minute speed dropping temperature, modulates SOD solution.
7g lecithin derivant is dissolved in isopropyl alcohol 500mL, removes insoluble matter with the hydrophobicity filter then.In filtrate, add isopropyl alcohol, make total capacity reach 4000mL, make the lecithin derivative solution.Solution temperature is 8 ℃.Then in aforementioned SOD solution, drips this lecithin derivative solution 2000mL with 33mL/ minute speed, will in 210rpm stirred 4 hours and must solution as reactant liquor.
Employing is dissolved with the 50mmol/L borate buffer of NaCl and 1: 1 eluting solvent of methanol, and the SOD dissolving of unmodified is separated out.Then, adopt to be dissolved with the 50mmol/mL borate buffer of NaCl and the eluting solvent of methanol, the part that dissolving is washed out is as PC-SOD compositions part.Behind the ultrafiltration and concentration, utilize to separate and use HPLC, isolate with the PC-SOD compositions in the corresponding part in each peak of PC-SOD, gained is partly carried out MALDI TOF-MS to be analyzed, measure molecular weight and the segment of PC-SOD, Lecithinized superoxide dismutase in conjunction with 2 molecule lecithin in the PC-SOD dimer that makes accounts for 13 moles of %, Lecithinized superoxide dismutase in conjunction with 3 molecule lecithin accounts for 40 moles of %, Lecithinized superoxide dismutase in conjunction with 4 molecule lecithin accounts for 25 moles of %, Lecithinized superoxide dismutase in conjunction with 5 molecule lecithin accounts for 20 moles of %, accounts for 2 moles of % in conjunction with the Lecithinized superoxide dismutase of 8 molecule lecithin.
With the above-mentioned PC-SOD dimer compositions that makes and commonly used lyophilizing adjuvant mutually mixed every lyophilized formulations that contains 40mg self-control Lecithinized superoxide dismutase dimer compositions and refined sucrose, be test sample;
Other drug:
Pentobarbital sodium: Shanghai Ke Feng chemical reagent company limited
Diazepam: Anhui Hua Yuan medicine limited company
Ultravist: Bayer HealthCare Co
Sodium chloride injection: the two crane pharmaceutical Co. Ltds in Beijing
Heparin sodium: Sichuan Bei Ao Biology Pharmacy Co., Ltd
Thioflavin: ADVANCE SCIENTIFIC ﹠amp; The CHEMICAL company limited
Evan ' s indigo plant: Europe, Shanghai Veda instrument Science and Technology Ltd.
Formalin solution: contain and the chemical industry company limited Tianjin
2. experimental technique
2.1 the structure of Chinese miniature pig myocardial damage model
Lumbar injection pentobarbital sodium, diazepam are with Animal Anesthesia, and tracheal intubation is connected to respirator, connect cardiac monitoring.The pars inguinalis otch separates a burst arteriovenous, and 6F sheath pipe is inserted in puncture, under C type arm X-ray machine perspective, utilize cardiografin radiography location, seal wire is inserted left anterior descending coronary artery (LAD), sacculus is put to left anterior descending branch far-end 1/3～1/2 place (second diagonal angle props up following).
Inject contrast agent and make balloon expandable, shutoff LAD unclamped sacculus and makes perfusion again after 2 hours, continued to observe 3 hours.
2.2 grouping and administration
According to random table, animal is divided into totally 5 groups of sham operated rats, model group and basic, normal, high dosed administration groups, every group of 7～8 animals.
Sham operated rats (Sham) is withdrawn from the sacculus placement after 2 hours, continue to observe 3 hours again.
Model control group (control), balloon occluder LAD unclamped sacculus and makes perfusion again after 2 hours, continued to observe 3 hours.
Basic, normal, high dosed administration group is balloon occluder 1.5 hours, promptly pours into before 30 minutes respectively with 0.3,1 again, the dosage intravenous injection Lecithinized superoxide dismutase composition solution of 3mg/kg (with the Lecithinized superoxide dismutase composition note).
2.3 infarct size evaluation
After pouring into 3 hours again, put to death animal and take out heart, inject the thioflavin (thioflavin-S) of 1ml/kg 4% in the left coronary artery, make perfusion area painted again, it is not painted not have the stream district again; The ligation anterior descending branch once more at the balloon occluder place, Evan ' the s indigo plant of reinjecting makes to show blue outside the ligation district, and it is blue that the ligation district does not show.Specimen after the dyeing places formalin fixing.
After the animal hearts specimen is fixed more than 48 hours in formalin, be divided into 5～6 myocardium short-axis slice along long axis of heart, it is blue that the non-ligation of result district cardiac muscle is, and the ligation district does not have the stream district again and do not develop the color (being kermesinus) under ultraviolet light, stream place displaing yellow is arranged again.The use digital camera is taken pictures, and adopts computer area measurement left ventricle locular wall myocardial area, ligation district (not having blue) myocardial area (promptly pouring into myocardial area again) and does not have resurgent district (not developing the color under the fluorescence) area; Calculate LA (perfusion cardiac muscle again) scope according to pouring into myocardial area/left ventricle locular wall myocardial area again, according to no resurgent district area/pour into myocardial area again to calculate myocardium ANR (no resurgent scope).
2.4 left ventricular end diastolic presses (LVEDP) to estimate
Each is organized behind the preceding 5min of balloon occluder (OCC) (baseline), OCC 120min, when pouring into 60min, 120min, 180min again, pigtail catheter is sent into left ventricle, and working pressure sensing suit adopts direct tremie method to detect left ventricular end diastolic and presses.
2.5 arrhythmia evaluation
A situation arises for the animal arrhythmia in the record experimentation, and quiver in the chamber, the chamber is pounced on and counted the severe arrhythmia, and chamber speed, conduction block, premature beat or premature ventricular beat and multi-joint rule etc. are counted slight arrhythmia.Respectively organize the situation that arrhythmia takes place.
2.6 ultrasonic cardiography inspection
Putting Ultrasound Instrument pops one's head in body surface, get left-ventricular short-axis papillary muscles horizontal section, before AMI 120min behind 5min, the AMI, pour into behind 60min, 120min, the 180min 5 different time points again and adopt real-time radiography and intermittently trigger technology such as imaging and carry out ultrasonic examination, inject the 1mL acoustic contrast through the right lateral thigh vein with injection system at the uniform velocity, continue the record image that 30s disappears to myocardial visualization before contrast-medium injection.The experimental implementation flow process of sham operated rats, model group and each administration group as shown in Figure 1.
3. date processing and analysis
Adopt the SPSS statistical software that experimental data is carried out one factor analysis of variance (OnewayANOVA).
4. experimental result
4.1 infarct size evaluation
The result shows, all do not observe no resurgent zone in the animal hearts specimen section of sham operated rats, model group and each administration group all can be observed no resurgent zone, and the region area of the perfusion again ratio of each group does not have significant difference (Fig. 2 A), the balloon occluder position that proves each animal does not have significant difference, and the equal significance of no resurgent region area of three dosage groups is lower than model group (Fig. 2 B).
4.2 left ventricular end diastolic presses (LVEDP) to estimate
During deal with data, be to eliminate the individual variation between animal, with every animal shutoff 120min and the measured value when pouring into 180min again deduct the baseline value (before the balloon occluder) of this animal, obtain difference (Δ LVEDP) after, comparison between organizing.
By the result as can be known and sham operated rats relatively, model group and each treatment group balloon occluder is left front a 120min falls and after, Δ LVEDP significantly raises, and each treatment group and model group do not have significant difference (one factor analysis of variance).The proof balloon occluder has produced appreciable impact to the left chamber diastolic function of animal.
After pouring into 180min again, the Δ LVEDP of model group has downward trend, but compare before the perfusion again and do not have significant difference, the Δ LVEDP value of middle dosage (1mg/kg) administration group significantly is lower than model group, proves that Lecithinized superoxide dismutase composition can significantly improve the recovery situation of left ventricular diastolic function between flush phase again with the 1mg/kg administration.Compare with model group, though the Δ LVEDP of low dose group and high dose group has reduction trend, because standard deviation is bigger, thus there is not significant difference, shown in Fig. 3 and table 1.
Table 1 Δ LVEDP (measured value-baseline value)
1. experiment material
Laboratory animal: small-sized 28 of China, culture the center available from the emerging laboratory animal of Beijing section, male and female are regardless of, body weight 20～30kg;
The experiment medicine:
Test sample: the Lecithinized superoxide dismutase composition lyophilized formulations, its preparation method is as follows:
In the 20L flask, add injection distilled water 6000mL, add boric acid 15g, NaOH 2.3g and KCl 76g dissolving more successively.Then, adding SOD aqueous solution (65g, SOD concentration 60.3mg/mL), when stirring with 210rpm, in chromatography room is 8 ℃ isopropyl alcohol 1000mL with 16.5mL/ minute speed dropping temperature, modulates SOD solution.
12g lecithin derivant is dissolved among the isopropyl alcohol 500mL, removes insoluble matter with the hydrophobicity filter then.In filtrate, add isopropyl alcohol, make total capacity reach 6000mL, make the lecithin derivative solution.Solution temperature is 8 ℃.Then in aforementioned SOD solution, drips this lecithin derivative solution 3000mL with 33mL/ minute speed, will in 210rpm stirred 4 hours and must solution as reactant liquor.
Employing is dissolved with the 50mmol/L borate buffer of NaCl and 1: 1 eluting solvent of methanol, and the SOD dissolving of unmodified is separated out.Then, adopt to be dissolved with the 50mmol/mL borate buffer of NaCl and the eluting solvent of methanol, the part that dissolving is washed out is as PC-SOD compositions part.Behind the ultrafiltration and concentration, utilize to separate and use HPLC, isolate with the PC-SOD compositions in the corresponding part in each peak of PC-SOD, gained is partly carried out MALDI TOF-MS to be analyzed, measure molecular weight and the segment of PC-SOD, Lecithinized superoxide dismutase in conjunction with 2 molecule lecithin in the PC-SOD dimer that makes accounts for 16 moles of %, Lecithinized superoxide dismutase in conjunction with 3 molecule lecithin accounts for 35 moles of %, Lecithinized superoxide dismutase in conjunction with 4 molecule lecithin accounts for 24 moles of %, Lecithinized superoxide dismutase in conjunction with 5 molecule lecithin accounts for 15 moles of %, Lecithinized superoxide dismutase in conjunction with 6 molecule lecithin accounts for 4 moles of %, accounts for 6 moles of % in conjunction with the Lecithinized superoxide dismutase of 7 molecule lecithin.
With the above-mentioned PC-SOD dimer compositions that makes and commonly used lyophilizing adjuvant mutually mixed every lyophilized formulations that contains 40mg self-control Lecithinized superoxide dismutase dimer compositions and refined sucrose, be test sample;
Other drug:
Pentobarbital sodium: Shanghai Ke Feng chemical reagent company limited
Diazepam: Anhui Hua Yuan medicine limited company
Ultravist: Bayer HealthCare Co
Sodium chloride injection: the two crane pharmaceutical Co. Ltds in Beijing
Heparin sodium: Sichuan Bei Ao Biology Pharmacy Co., Ltd
Thioflavin: ADVANCE SCIENTIFIC ﹠amp; The CHEMICAL company limited
Evan ' s indigo plant: Europe, Shanghai Veda instrument Science and Technology Ltd.
Formalin solution: contain and chemical industry company limited pentobarbital sodium Tianjin:
2. experimental technique
2.1 Chinese miniature pig myocardial ischemia-reperfusion model
With embodiment 1.
2.2 grouping and administration
According to random table, with animal be divided into sham operated rats, model group, again pour into preceding 30 minutes administration groups, pour into preceding 3 minutes administration groups again, totally 4 groups, every group of 7～8 animals.
Sham operated rats (Sham) is withdrawn from the sacculus placement after 2 hours, continue to observe 3 hours again.
Model control group (control), balloon occluder LAD unclamped sacculus and makes perfusion again after 2 hours, continued to observe 3 hours.
Pour into preceding 30 minutes administration groups again,, promptly pour into preceding 30 minutes dosage intravenous injection Lecithinized superoxide dismutase composition solution again with 0.3mg/kg (with the Lecithinized superoxide dismutase composition note) balloon occluder 1.5 hours.
Pour into preceding 3 minutes administration groups again, pouring into 3 minutes dosage intravenous injection Lecithinized superoxide dismutase composition solution again with 0.3mg/kg (with the Lecithinized superoxide dismutase composition note).
2.3 infarct size evaluation
With embodiment 1.
2.4 left ventricular end diastolic presses (LVEDP) to estimate
With embodiment 1.
2.5 arrhythmia evaluation
With embodiment 1.
2.6 ultrasonic cardiography inspection
With embodiment 1.
3. date processing and analysis
With embodiment 1.
4. experimental result
4.1 infarct size evaluation
The result shows, all do not observe no resurgent zone in the animal hearts specimen section of sham operated rats, model group and administration group all can be observed no resurgent zone, and the region area of the perfusion again ratio of administration group does not have significant difference (Fig. 4 A), the balloon occluder position that proves each animal does not have significant difference, the equal significance of no resurgent region area of administration group is lower than model group (Fig. 4 B), model group, pour into preceding 30 minutes administration groups again, the I/R ratio that pours into preceding 3 minutes administration groups again is respectively 39.47 ± 5.89%, 28.94 ± 7.37% and 18.33 ± 8.19%.
4.2 left ventricular end diastolic presses (LVEDP) to estimate
Data processing method is with embodiment 1.
By the result as can be known and sham operated rats relatively, model group and administration group balloon occluder is left front a 120min falls and after, Δ LVEDP significantly raises, and each treatment group and model group do not have significant difference (one factor analysis of variance).The proof balloon occluder has produced appreciable impact to the left chamber diastolic function of animal.
After pouring into 180min again, the Δ LVEDP of model group has downward trend, but compare before the perfusion again and do not have significant difference, the Δ LVEDP value of middle dosage (1mg/kg) administration group significantly is lower than model group, proves that Lecithinized superoxide dismutase composition can significantly improve the recovery situation of left ventricular diastolic function between flush phase again with the 1mg/kg administration.Compare with model group, though the Δ LVEDP of low dose group and high dose group has reduction trend, because standard deviation is bigger, thus there is not significant difference, shown in Fig. 5 and table 2.
Table 2 Δ LVEDP (measured value-baseline value)
According to embodiment 1 and embodiment 2 as can be known, Lecithinized superoxide dismutase composition pours into administration in preceding 3～30 minutes time range again behind myocardial ischemia, all can effectively reduce myocardial infarction area, and reduce the left ventricular end diastolic that pours into again after 180 minutes and press, prove that Lecithinized superoxide dismutase composition can alleviate the damage that myocardial ischemia-reperfusion causes.Though on data, the effect of improving of pouring into administration in preceding 3 minutes again is better than pouring into administration in preceding 30 minutes again, pours into administration in preceding 30 minutes again and also can obtain certain effect of improving.
Proof thus, Lecithinized superoxide dismutase composition can be effective to treatment and myocardial ischemia reperfusion injury diseases associated, for example is used to improve the prognosis situation etc. that coronary artery leads to art (thrombolytic, PTCA or Coronary Artery Bypass) again.
Simultaneously, think that Lecithinized superoxide dismutase composition also can be used for the prognosis of clinical improvements PTCA in the mode of blood vessel stent drug coating.
The pharmacodynamics of PC-SOD compositions that contains identical PC molecular number for the pharmacodynamics specific activity of verifying the PC-SOD compositions that contains different PC molecular number is active better, the pharmacokinetics test that the present invention measures blood drug level by the ELISA method compares, and curve as shown in Figure 6 during medicine.
1. experiment material
Laboratory animal: laboratory animal is 60 of SPF level Sprague-Dawley (SD) rats, available from Beijing Vital River Experimental Animals Technology Co., Ltd., and male and female half and half, body weight 175～205g;
The experiment medicine:
Test sample:
The Lecithinized superoxide dismutase composition lyophilized formulations, its preparation method is as follows:
In the 20L flask, add injection distilled water 6000mL, add boric acid 15g, NaOH 2.3g and KCl 76g dissolving more successively.Then, adding SOD aqueous solution (65g, SOD concentration 60.3mg/mL), when stirring with 210rpm, in chromatography room is 8 ℃ isopropyl alcohol 1000mL with 16.5mL/ minute speed dropping temperature, modulates SOD solution.
Lecithin derivant 10g is dissolved among the isopropyl alcohol 500mL, removes insoluble matter with the hydrophobicity filter then.In filtrate, add isopropyl alcohol, make total capacity reach 6000mL, make the lecithin derivative solution.Solution temperature is 8 ℃.Then in aforementioned SOD solution, drips this lecithin derivative solution 3000mL with 33mL/ minute speed, will in 210rpm stirred 4 hours and must solution as reactant liquor.
Employing is dissolved with the 50mmol/L borate buffer of NaCl and 1: 1 eluting solvent of methanol, and the SOD dissolving of unmodified is separated out.Then, adopt to be dissolved with the 50mmol/mL borate buffer of NaCl and the eluting solvent of methanol, the part that dissolving is washed out is as PC-SOD compositions part.Behind the ultrafiltration and concentration, utilize to separate and use HPLC, isolate with the PC-SOD compositions in the corresponding part in each peak of PC-SOD, gained is partly carried out MALDI TOF-MS to be analyzed, measure molecular weight and the segment of PC-SOD, Lecithinized superoxide dismutase in conjunction with 2 molecule lecithin in the PC-SOD dimer that makes accounts for 12 moles of %, Lecithinized superoxide dismutase in conjunction with 3 molecule lecithin accounts for 36 moles of %, Lecithinized superoxide dismutase in conjunction with 4 molecule lecithin accounts for 22 moles of %, Lecithinized superoxide dismutase in conjunction with 5 molecule lecithin accounts for 20 moles of %, Lecithinized superoxide dismutase in conjunction with 6 molecule lecithin accounts for 2 moles of %, Lecithinized superoxide dismutase in conjunction with 7 molecule lecithin accounts for 6 moles of %, and the Lecithinized superoxide dismutase that contains 8 molecule lecithin accounts for 2 moles of %.
With the above-mentioned PC-SOD dimer compositions that makes and commonly used lyophilizing adjuvant mutually mixed every lyophilized formulations that contains 40mg self-control Lecithinized superoxide dismutase dimer compositions and refined sucrose, be test sample;
The PC-SOD compositions, PC-SOD compositions that contains 4 molecule PC and the PC-SOD compositions that contains 8 molecule PC that contain 2 molecule PC, these PC-SOD compositionss that contain single molecular number are self-control, particularly, be that the above-mentioned PC-SOD compositions that contains 2-8 molecule PC for preparing is made behind reversed phase chromatography method purification.
2. test method: get 60 rats, be divided into 3 groups at random, every group 20, male and female half and half, intravenous contain respectively 2-8 molecule PC the PC-SOD compositions, contain 2 molecule PC PC-SOD, contain 4 molecule PC PC-SOD, contain the PC-SOD of 8 molecule PC, dosage is 10mg/kg, take a blood sample in 0min, 5min, 15min, 30min, 1hr, 2hr, 4hr, 6hr, 8hr, 24hr, 48hr, 72hr, 96hr, 120hr after the administration, according to the drug level in the ELISA method mensuration serum.
The ELISA algoscopy: preparation 10mM phosphate buffer (pH7.0), buffer I (sample diluting liquid), * 10PBS solution, buffer II (enzymic-labelled antibody diluent), cleanout fluid (0.05% polysorbas20/PBS), confining liquid, chromophoric solution, reaction stop solution (2M H
2SO
4).
With buffer solution I with 100 times of rat blood serum diluted samples.If when also needing further to dilute, 100 times of diluents (buffer solution I) of normal machin serum can be used as diluent.To culture plate (immune culture plate I, 439454, add insolubilized antibody (15 μ g/mL) the 100 μ L of PBS dilution in each aperture Nunc), 4 ℃ of hatchings about 16 hours down.The hatching back adds 200 μ L cleanout fluid in aperture, clean 3 times.Add 200 μ L confining liquids then in each aperture, hatching is 1 hour under the room temperature.Culture plate is cleaned 3 times in the hatching back, adds standard solution, QC sample or sample solution 100 μ L, and hatching is 2 hours under the room temperature.Culture plate is cleaned 3 times in the hatching back, adds enzymic-labelled antibody (2.5mg/mL) the 100 μ L with buffer solution II dilution in each aperture, and hatching is 2 hours under the room temperature.Culture plate is cleaned 3 times in the hatching back, adds chromophoric solution 100 μ L, and (dark place) hatching added reaction again and stops solution 50 μ L after 15 minutes under the room temperature, measured absorbance at the 492nm place with microplate reader.The result as shown in Figure 6.
As shown in Figure 6, the PC-SOD that contains low molecular number PC has higher activity, but eliminate very fast, the PC-SOD that contains high score subnumber PC is because the content height of PC, improved cell affine prolonged the half-life, but the connection of PC can make the activity of SOD reduce greatly, it is very low to reach peak blood drug level, do not reach therapeutic effect, PC-SOD compositions of the present invention makes up according to specific ratio by the PC-SOD that will contain suitable PC number, half-life of prolong drug greatly when having high pharmaceutically active makes remaining on the valid density of blood drug level long period, can play long lasting therapeutic effect to myocardial damage.
Claims (5)
1. Lecithinized superoxide dismutase composition treats and/or prevents application in the medicine of heart ischemia damage in preparation, and wherein, described Lecithinized superoxide dismutase composition is to be combined with the dimer that different molecular is counted lecithin.
2. application according to claim 1, the myocardial cell injury that wherein said heart ischemia damage too much causes for free radical.
3. application according to claim 1 and 2, wherein, described Lecithinized superoxide dismutase composition is the dimer that is combined with 2-8 molecule lecithin.
4. according to each described application in the claim 1 to 3, wherein, in the described Lecithinized superoxide dismutase composition, Lecithinized superoxide dismutase in conjunction with 2 molecule lecithin accounts for 6.25-16 mole %, Lecithinized superoxide dismutase in conjunction with 3 molecule lecithin accounts for 18-40 mole %, Lecithinized superoxide dismutase in conjunction with 4 molecule lecithin accounts for 12.25-25 mole %, Lecithinized superoxide dismutase in conjunction with 5 molecule lecithin accounts for 8-20 mole %, Lecithinized superoxide dismutase in conjunction with 6 molecule lecithin accounts for 1-4 mole %, Lecithinized superoxide dismutase in conjunction with 7 molecule lecithin accounts for 1-6 mole %, accounts for 0.25-2.25 mole % in conjunction with the Lecithinized superoxide dismutase of 8 molecule lecithin.
5. according to each described application in the claim 1 to 4, wherein, effective dose every day of described Lecithinized superoxide dismutase composition is 10-160mg/60kg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102995779A CN101966334A (en) | 2010-09-29 | 2010-09-29 | Application of lecithin superoxide dismutase composite in preparation of medicines for treating and/or preventing cardiac ischemic injury |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102995779A CN101966334A (en) | 2010-09-29 | 2010-09-29 | Application of lecithin superoxide dismutase composite in preparation of medicines for treating and/or preventing cardiac ischemic injury |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101966334A true CN101966334A (en) | 2011-02-09 |
Family
ID=43545597
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102995779A Pending CN101966334A (en) | 2010-09-29 | 2010-09-29 | Application of lecithin superoxide dismutase composite in preparation of medicines for treating and/or preventing cardiac ischemic injury |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101966334A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106983574A (en) * | 2017-05-02 | 2017-07-28 | 南京医科大学第附属医院 | A kind of small balloon-system of zoopery heart infarction model and preparation method |
| CN107137700A (en) * | 2017-04-27 | 2017-09-08 | 张国瑜 | A kind of composition based on stem cell source excretion body and its application in the medicine for preparing treatment myocardial infarction |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0654681A (en) * | 1991-06-07 | 1994-03-01 | L T T Kenkyusho:Kk | Lecithinized superoxide dismutase and medicine containing the same as active ingredient |
| JPH09117279A (en) * | 1995-10-25 | 1997-05-06 | Samu Kenkyusho:Kk | Lecithinized superoxide dismutase and medicine comprising the same as active ingredient |
| JP2002080395A (en) * | 2000-08-31 | 2002-03-19 | Ltt Institute Co Ltd | Agent for treatment of myocardial lesion |
| CN101035891A (en) * | 2004-10-12 | 2007-09-12 | 日本株式会社Ltt生物医药 | Lecithinized superoxide dismutase composition and process for producing the same |
-
2010
- 2010-09-29 CN CN2010102995779A patent/CN101966334A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0654681A (en) * | 1991-06-07 | 1994-03-01 | L T T Kenkyusho:Kk | Lecithinized superoxide dismutase and medicine containing the same as active ingredient |
| JPH09117279A (en) * | 1995-10-25 | 1997-05-06 | Samu Kenkyusho:Kk | Lecithinized superoxide dismutase and medicine comprising the same as active ingredient |
| JP2002080395A (en) * | 2000-08-31 | 2002-03-19 | Ltt Institute Co Ltd | Agent for treatment of myocardial lesion |
| CN101035891A (en) * | 2004-10-12 | 2007-09-12 | 日本株式会社Ltt生物医药 | Lecithinized superoxide dismutase composition and process for producing the same |
Non-Patent Citations (2)
| Title |
|---|
| GERTJAN J.M.等: "Lecithinized copper,zinc-superoxide dismutase as a protector against doxorubicin-induced cardiotoxicity in mice", 《TOXICOLOGY AND APPLIED PHARMACOLOGY》 * |
| GERTJAN J.M.等: "Lecithinized copper,zinc-superoxide dismutase as a protector against doxorubicin-induced cardiotoxicity in mice", 《TOXICOLOGY AND APPLIED PHARMACOLOGY》, vol. 194, no. 2, 15 January 2004 (2004-01-15), pages 180 - 188 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107137700A (en) * | 2017-04-27 | 2017-09-08 | 张国瑜 | A kind of composition based on stem cell source excretion body and its application in the medicine for preparing treatment myocardial infarction |
| CN106983574A (en) * | 2017-05-02 | 2017-07-28 | 南京医科大学第附属医院 | A kind of small balloon-system of zoopery heart infarction model and preparation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115427427B (en) | Prostate specific membrane antigen targeting compound and preparation method and application thereof | |
| US12054458B2 (en) | 18F-tagged inhibitors of prostate specific membrane antigen (PSMA) and their use as imaging agents for prostate cancer | |
| CN112043838B (en) | ACE2 receptor targeted nuclide polypeptide probe, and preparation method and application thereof | |
| EA037778B1 (en) | Labeled inhibitors of prostate specific membrane antigen (psma), their use as imaging agents and pharmaceutical agents for the treatment of prostate cancer | |
| DK172391B1 (en) | High Molecular Compound for Use as a Non-Radioactive Carrier, Radiolabeled High Molecular Compound, Method for Preparation thereof, and Method for Improved Labeling of an Asialoglycoprotein Acceptor Conducting Compound | |
| CN103936815A (en) | Dual-functional oleanolic acid derivatives as well as preparation method and application thereof | |
| CN102898507A (en) | Thrombolysis oligopeptide-imidazolidine binary conjugate, preparation method and uses thereof | |
| CN101966334A (en) | Application of lecithin superoxide dismutase composite in preparation of medicines for treating and/or preventing cardiac ischemic injury | |
| US12128111B2 (en) | Glyco-metal-organic frameworks-based hepatic targeted therapeutic drug and preparation method thereof | |
| CN104045823B (en) | A kind of Enoxolone derivative and its preparation method and application | |
| CN103547270A (en) | A pharmaceutical composition for inhibiting recurrence, aggravation and metastasis of hepatocarcinoma | |
| CN101066982B (en) | Amino acid-glucose derivative 99m TC compound and its prepn process | |
| CN117186087A (en) | Carbonic anhydrase IX targeted radioactive diagnosis and treatment medicine and preparation method thereof | |
| US20230277699A1 (en) | Tumor stroma imaging agent and preparation method thereof | |
| CN101851313B (en) | Technetium-99m-marked poly N-vinyl benzyl-D-lactose amide composition and preparation method | |
| CN107586321B (en) | Preparation method of F-18 labeled modified Dimer-San A probe | |
| AU2013226944B2 (en) | Inhibitory agent for body cavity fluid accumulation | |
| CN110812487A (en) | Preparation method and application of mitochondrial gene therapy vector MI-linker-U-binding | |
| CN107674098A (en) | One kind is containing aryl boric acid99mTc complexs and its medicine box formula and application | |
| TW200824697A (en) | Use of ATP in controlled regional reperfusion as treatment during acute myocardial infarction | |
| CN118496299A (en) | PSMA targeted radioactive diagnosis and treatment integrated medicine and synthesis and application thereof | |
| CN104887656B (en) | Application of the L-dopa methyl ester hydrochloride in the medicine for preparing preventing and treating rheumatoid arthritis | |
| KR20010040676A (en) | Bibapcitide-based pharmaceutical compositions for imaging and treating thrombi | |
| CN101906137B (en) | Conjugate of Arg-Gly-Asp chain and two fatty alcohol chains, synthesis and medical application thereof | |
| CN115581690A (en) | Application of magnolol or aromatic ring amino substituted derivative of honokiol in preparation of anti-myocardial ischemia drugs and pharmaceutical composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110209 |