CN102552249A - Application of edaravone - Google Patents
Application of edaravone Download PDFInfo
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- CN102552249A CN102552249A CN2012100022726A CN201210002272A CN102552249A CN 102552249 A CN102552249 A CN 102552249A CN 2012100022726 A CN2012100022726 A CN 2012100022726A CN 201210002272 A CN201210002272 A CN 201210002272A CN 102552249 A CN102552249 A CN 102552249A
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Abstract
The invention discloses application of edaravone, which is used for preparing a medicine preventing oxygen convulsion. The edaravone can effectively improve anti-oxidase level, remove oxygen radical, suppress nitric oxide synthase activity and reduce content of nitric oxide so as to effectively prevent oxygen convulsion, and is suitable for developing the medicine preventing oxygen convulsion.
Description
Technical field
The present invention relates to medical technical field, be specifically related to the purposes of Edaravone prevention oxygen convulsion.
Background technology
Oxygen convulsion has another name called convulsions type oxygen intoxication or acute oxygen poisoning, is after human body sucks high partial pressures oxygen (being higher than 200kPa) certain hour, a kind of disease of the similar epilepsy grand mal of clinical manifestation that the increase of neurocyte irritability causes.Oxygen convulsion mostly occurs in diving activity; Contraction of complete tetanus property and clonospasm can appear in the diver during outbreak; Loss of consciousness, and with urinary incontinence, the diver is often out of hand and cause diving accidents such as drowned or blow up; Have a strong impact on diver's safety, greatly limited use oxygen light diving apparatus and carried out military combat task, activity such as commercial diving operation and scientific investigation under water under water.In addition, clinical hyperbaric oxygen has been widely used in treating diseases such as decompression sickness, carbon monoxide poisoning, tissue necrosis and nerve injury, but also has the risk that oxygen convulsion takes place, particularly to some responsive persons.Therefore, effectively prevent oxygen convulsion, safety is used oxygen to greatest extent, and is all significant to military and civilian diving operation and clinical hyperbaric oxygentherapy.But the preventive measure that can take at present is quite passive, and the main pressure of restriction oxygen uptake and the time-histories of relying on prevented the oxygen convulsion generation, largely limit the application of high partial pressure of oxygen.
(3-methyl isophthalic acid-phenyl-2-pyrazolin-5-one MCI-186) is a kind of novel potent oxygen free radical scavenger and antioxidant to Edaravone, just is being widely used as a line first aid medicine of treatment apoplexy at present.So far do not see that the antioxidant Edaravone is used to prevent the report of oxygen convulsion.
Summary of the invention
The present invention will solve the technical problem that present shortage is effectively prevented the medicine of oxygen convulsion, and a kind of purposes of Edaravone is provided, and is used to prepare the medicine that prevents oxygen convulsion.
In order to solve the problems of the technologies described above, the present invention realizes through following technical scheme:
A kind of purposes of Edaravone is used to prepare the medicine that prevents oxygen convulsion.
The administering mode of said Edaravone comprises intramuscular injection or intravenous drip.
The dosage form of said Edaravone medicine comprises injection.
The present invention through Edaravone to the oxygen convulsion preclinical influence experiment proof that influences experiment and Edaravone to rat layer and Hippocampus mesophytization index of discharging first; Edaravone can significantly suppress the generation of oxygen convulsion; Explain that Edaravone can effectively prevent oxygen convulsion, be applicable to the prophylactic agent of exploitation oxygen convulsion.
Description of drawings
Below in conjunction with the accompanying drawing and the specific embodiment the present invention is done further detailed explanation.
Fig. 1 is oxygen convulsion pathogeny of the present invention and the Edaravone mechanism of action sketch map to oxygen convulsion;
Fig. 2 is that the embodiment of the invention 1 Edaravone is to preclinical experimental result and the electroencephalogram exemplary plot of influencing of oxygen convulsion FED;
Fig. 3 be in the embodiment of the invention 2 Edaravone to the experimental result picture that influences of MDA in cortex and the hippocampal tissue;
Fig. 4 be in the embodiment of the invention 2 Edaravone to H in cortex and the hippocampal tissue
2O
2The experimental result picture that influences with NO;
Fig. 5 be in the embodiment of the invention 2 Edaravone to the experimental result picture that influences of antioxidase in cortex and the hippocampal tissue (SOD, GPx, CAT);
Fig. 6 be in the embodiment of the invention 2 Edaravone to the experimental result picture that influences of NOS in cortex and the hippocampal tissue.
The specific embodiment
In order to seek the medicine of effective prevention oxygen convulsion, the present invention finds the new purposes of Edaravone prevention oxygen convulsion finally through repeatedly experiment repeatedly.Face the pathogeny of oxygen convulsion and the mechanism of action of Edaravone down and briefly set forth (as shown in Figure 1).
The concrete mechanism of oxygen convulsion is not clear as yet, but clear and definite oxygen-derived free radicals (ROS) is its main paathogenic factor.The basic pathogenesis of oxygen convulsion can be summarized as follows: under the hyperbaric oxygen (HBO) that continues exposes, and the partial pressure of oxygen (pO in the cerebral tissue
2) increase.The partial pressure of oxygen that increases causes that on the one hand ROS increases; The oxidation resistance that surpasses superoxide dismutase (SOD), glutathion peroxidase (GPx) and catalase antioxidases such as (CAT) in the cerebral tissue; Neurocyte suffers oxidative damage, and N-methyl-D-aspartate (NMDA) receptor is activated, and excitatory transmitter increases; Inhibitory transmitter reduces, thereby causes oxygen convulsion; On the other hand, after the partial pressure of oxygen in the cerebral tissue increased, nitric oxide synthetase (NOS) activity increased, and L-arginine (L-arginine) content increases, thereby causes nitric oxide (NO) content to increase.Under the effect of NO, the cerebrovascular function imbalance, vasodilation, cerebral blood flow increases, and the oxygen of brain supplies to increase, and further increases the partial pressure of oxygen in the brain, causes oxygen convulsion.
The present invention confirms through following embodiment; Edaravone (Edv) is active through removing ROS, improve the antioxidase level and suppressing NOS; Can effectively suppress the cerebral tissue lipid peroxidation injury that hyperbaric oxygen causes; Alleviate the oxidative damage of neurocyte, prolong oxygen convulsion incubation period, thus the effect of performance prevention oxygen convulsion.
Following embodiment of the present invention gives a certain amount of Edaravone in advance in whole animal; Carry out the oxygen convulsion modeling again; Observe the effect of Edaravone prevention oxygen convulsion, further study the influence of Edaravone again the lipid peroxidation in rat layer and the hippocampal tissue, ROS, NO, antioxidase and NOS level.
1) animal is prepared: healthy male SD rat, to purchase in the U.S. than triumphant company, and the The 2nd Army Medical College animal center provides, and raises to body weight 250 ± 10g to be used for experiment.
2) oxygen convulsion Preparation of model: after animal put into hyperbaric oxygen chamber; At first use the air velocity washing of tanks 5min of pure oxygen with 1L/s; Monitor oxygen concentration simultaneously, treat oxygen concentration＞99% after, at the uniform velocity be forced into timing behind the 6ATA with the speed of 1ATA/min; It is stable to keep-up pressure, and continues ventilation (0.3L/s) in pressurization and the voltage stabilizing process.In the process-exposed, bilge shop one deck sodica calx is to absorb the CO that animal is breathed out
2, temperature remains on 22～26 ℃ in the cabin.
3) animal divides into groups
12 of SD rats are divided into normal saline matched group and Edaravone 3mg/kg body weight group at random.Be used for experiment after the body weight standard up to standard.
4) experimental procedure:
(1) brain electrode is implanted: rats by intraperitoneal injection pentobarbital (50mg/kg), treat the rat holonarcosis after, be fixed on the brain solid positioner.Make its head top maintenance level,, cut off head skin, separate subcutaneous tissue, peel off, wipe off periosteum, expose skull with 75% cotton ball soaked in alcohol sterilization skin.Appropriate location on the selected respectively skull uses cranial drill to get out the aperture of the degree of depth as 0.5mm: cortical electrode, 4mm behind the bregma, the other 2mm that opens in a center line left side; Reference electrode, 4mm behind the bregma, the other 2mm that opens in the center line right side.The short end of electrode after the sterilization is implanted aperture, electrode is fixed in skull, skin suture with the zinc phosphate adhesive that mixes up.The equal sterile working of overall process.Postoperative continue to be raised rat 3 days, and temperature 24-26 ℃, humidity 40-60% is freely intake and ingested.
(2) administration: administration group dosage by 3mg/kg before hyperbaric oxygen exposure 30min gives Edaravone, lumbar injection.The normal saline matched group gives 0.9% normal saline of equivalent, lumbar injection.
(3) oxygen convulsion is measured incubation period: with big rat holder the rat trunk is fixed, placed separately in the compression chamber, connect the electrode of surveying electroencephalogram, wherein the ground electrode lead is connected to rat forehead fur.Open physiograph (POWERLAB/8SP, ADInstrument company, Australia), operation eeg recording software.With pure oxygen washing of tanks (1L/s); Treat in the oxygen analyser display module that oxygen concentration reaches 99% when above; Beginning at the uniform velocity is forced into 6ATA with the speed of 1ATA/min, and it is stable to keep-up pressure, the opening entry brain; Continue to be exposed to and occur discharge (FED) and convulsions grand mal first on the electroencephalogram, the time of record FED appearance.Temperature maintenance is at 22～26 ℃ in the cabin.After expose finishing, at the uniform velocity be decompressed to normal pressure, deliver from vault with the speed of 1ATA/min.
The result is as shown in Figure 2, Edaravone significant prolongation FED incubation period, shows that Edaravone can effectively prevent the outbreak of oxygen convulsion.
1) animal is prepared and divides into groups: healthy male SD rat, to purchase in the U.S. than triumphant company, and the The 2nd Army Medical College animal center provides, and raises to body weight 250 ± 10g to be used for experiment.Animal is assigned randomly in three groups, is respectively normal control group, normal saline matched group and Edaravone group.The normal control group is left intact, and the laggard horizontal high voltage oxygen of the normal saline of normal saline group lumbar injection and Edaravone equivalent exposes, and the laggard horizontal high voltage oxygen of Edaravone group lumbar injection Edaravone (3mg/kg) exposes.
Hyperbaric oxygen exposure: normal saline group and the laggard cabin of Edaravone treated animal administration 30min, washing of tanks, pressurization and voltage stabilizing process are with " embodiment 1 ".Open-assembly time is 20min.After expose finishing, at the uniform velocity be decompressed to normal pressure, deliver from vault with the speed of 1ATA/min.
2) draw materials: behind the rat deliver from vault, the excessive pentobarbital of lumbar injection (100mg/kg) makes rat euthanasia, and broken end takes out cortex and hippocampal tissue rapidly, puts into-80 ℃ of refrigerators behind the liquid nitrogen freezing and preserves.
3) mensuration of malonaldehyde (MDA) in the cerebral tissue: get suitably frozen cortex and Hippocampus, carry out homogenate with Western and IP cell pyrolysis liquid (green the skies company, Jiangsu), the ratio that tissue weight accounts for lysate is 10%.The homogenate protein concentration of back that finish with BCA determination of protein concentration test kit (green the skies company, Jiangsu) working sample.Be ready to MDA detection kit (green skies company; Jiangsu) after; Suitably solution is as blank in centrifuge tube, to add 0.1ml homogenate, lysate or PBS etc., and adding 0.1ml variable concentrations standard substance are used for the production standard curve, add the 0.1ml sample and are used for measuring; Add 0.2ml MDA testing liquid subsequently.Behind the mixing, 100 ℃ or boiling water bath heating 15min.Water-bath is cooled to room temperature, the centrifugal 10min of 1000g room temperature.Get 200 μ l supernatants and join in 96 orifice plates, measure absorbance with ELIASA at 532nm subsequently.After calculating the MDA content in the sample solution, represent the MDA content in the sample through the protein content of Unit Weight.The result shows that Edaravone can significantly reduce the horizontal (see figure 3) of MDA in the cerebral tissue, explains that Edaravone can suppress the lipid oxidation damage that hyperbaric oxygen causes.
4) H in the cerebral tissue
2O
2Mensuration with NO: 1. H
2O
2Mensuration: bark fetching layer and Hippocampus, the ratio that adds 200 microlitre Western and IP cell pyrolysis liquid (green the skies company, Jiangsu) according to every 10mg tissue is carried out homogenate.4 ℃ of centrifugal 5min of about 12000g get supernatant.Melt the hydrogen peroxide detectable on ice or on the ice-water bath earlier, in detecting hole or detector tube, add 50 μ l sample or standard substance, in each hole, add 100 μ l hydrogen peroxide detectable then.Vibrate gently or beat mixing, room temperature is placed 30min.Measure A560 then immediately, calculate the concentration of hydrogen peroxide in the sample at last according to standard curve.2. the mensuration of NO: with tissue sample homogenate, the centrifugal 5min of 12000g gets supernatant.With homogenate 10mmol/L KNO
2Be diluted to 2,5,10,20,50 μ mol/L.Be ready to NO and measure test kit (green the skies company, Jiangsu), add standard substance, sample and detectable to specifications successively, measure A540 after room temperature (25 ℃) is hatched 10min.Calculate the concentration of NO in the sample according to the standard substance opisometer.The result shows that Edaravone can significantly reduce cerebral tissue H
2O
2With the horizontal (see figure 4) of NO, explain that Edaravone can suppress the lipid oxidation damage that hyperbaric oxygen causes.
5) mensuration of superoxide dismutase (SOD), glutathion peroxidase (GPx) and three kinds of antioxidases of catalase (CAT) in the cerebral tissue: the 1. detection of SOD: animal obtains cortex and Hippocampus after removing blood with normal saline (containing the 0.16mg/ml heparin sodium) perfusion.Get Western and the IP cell pyrolysis liquid that an amount of tissue adds the green skies (green the skies company, Jiangsu) and in ice bath, carry out homogenate (tissue concentration is 10%), 4 ℃ of centrifuging and taking supernatants.Be ready to SOD detection kit (green the skies company, Jiangsu), in 96 orifice plates, add sample and various blanks hole, add behind the enzyme working solution fully mixing.Hatch 20min for 37 ℃, measure absorbance at 450nm then, calculate the SOD enzyme activity.2. the detection of GPx: sample is prepared to detect with SOD.After being ready to test kit, in centrifuge tube, adding successively and detect buffer, testing sample and GPx testing liquid, mixing.After adding 4 microlitre 15mmol/L peroxide reagent solutions, with the suitable mixing of agitator.Measure A340 down at 25 ℃, whenever measured once METHOD FOR CONTINUOUS DETERMINATION 3min at a distance from 30 seconds.Calculate the GPx enzyme activity.3. the mensuration of CAT: bark fetching layer and Hippocampus with Western and IP cell pyrolysis liquid (green the skies company, Jiangsu) cracking tissue (tissue concentration is 10%), add isopyknic catalase again and detect the buffer dilute sample.Be ready to CAT detection kit (green the skies company, Jiangsu), first bioassay standard curve.Reference reagent box description is got 10 μ l samples to the 1.5ml plastic centrifuge tube, and adding catalase detection buffer to volume is 40 microlitres, mixing.Add 10 microlitre 250mmol/L hydrogenperoxide steam generators again, with the rapid mixing of pipettor.25 ℃ of reaction 3min.Finish the back and add 450 μ l catalase reaction stop buffers, put upside down mixing with cessation reaction.In another plastic centrifuge tube, add 40 μ l catalases immediately and detect buffer, add 10 μ l again and stopped the also above-mentioned reaction system of mixing, mixing is therefrom got 10 μ l and is joined in the hole in 96 orifice plates.Add 200 μ l colour developing working solution.Measure A520 after hatching 20min for 25 ℃, it is active to calculate CAT.The result shows, compares with matched group, and Edaravone can significantly improve the horizontal (see figure 5) of SOD in the cerebral tissue, GPx, three kinds of antioxidases of CAT, alleviates the oxidative damage of neurocyte.
6) mensuration of nitric oxide synthetase (NOS) in the cerebral tissue: animal obtains cortex and Hippocampus after removing blood with normal saline (containing the 0.16mg/ml heparin sodium) perfusion.Getting 0.1g organizes the normal saline 0.9ml that adds 9 times to be prepared into 10% tissue homogenate, the centrifugal 10min of 3000rpm.Get supernatant 50 μ l and survey the NOS vigor.(company is built up in Nanjing to be ready to NOS mensuration test kit; Nanjing), blank is managed, TNOS measures and manages, iNOS (induced NOS) blank is managed and iNOS mensuration pipe to establish TNOS (NOS always), in mensuration is managed, adds distilled water, supernatant and all ingredients; Mixing; Measure the absorbance of each pipe at the 530nm place, calculate TNOS and iNOS value, cNOS (structural type NOS) value is the poor of TNOS value and iNOS value.The result shows that Edaravone can significantly reduce the TNOS and the horizontal (see figure 6) of cNOS of cerebral tissue, alleviates the oxidative damage of neurocyte and vascular endothelial cell.
The above embodiment has only expressed embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with accompanying claims.
Claims (3)
1. the purposes of an Edaravone is characterized in that, is used to prepare the medicine that prevents oxygen convulsion.
2. purposes according to claim 1 is characterized in that the administering mode of said Edaravone comprises intramuscular injection or intravenous drip.
3. purposes according to claim 1 is characterized in that the dosage form of said medicine comprises injection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106668056A (en) * | 2016-11-08 | 2017-05-17 | 中国人民解放军第二军医大学 | Application of associated adenosine and nNOS inhibitor in preparation of medicine or food for preventing oxygen poisoning of central nervous system |
| CN110090225A (en) * | 2019-04-19 | 2019-08-06 | 济南康和医药科技有限公司 | A kind of Edaravone sodium chloride injection and preparation method thereof |
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| CN1440749A (en) * | 2003-03-24 | 2003-09-10 | 南昌弘益科技有限公司 | Edaravone injection for treating acute cerebral thrombus and its prepn |
| CN101966182A (en) * | 2010-09-25 | 2011-02-09 | 西安力邦制药有限公司 | Compound brain protection preparation and preparation method thereof |
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- 2012-01-05 CN CN2012100022726A patent/CN102552249A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1440749A (en) * | 2003-03-24 | 2003-09-10 | 南昌弘益科技有限公司 | Edaravone injection for treating acute cerebral thrombus and its prepn |
| CN101966182A (en) * | 2010-09-25 | 2011-02-09 | 西安力邦制药有限公司 | Compound brain protection preparation and preparation method thereof |
Non-Patent Citations (3)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106668056A (en) * | 2016-11-08 | 2017-05-17 | 中国人民解放军第二军医大学 | Application of associated adenosine and nNOS inhibitor in preparation of medicine or food for preventing oxygen poisoning of central nervous system |
| CN110090225A (en) * | 2019-04-19 | 2019-08-06 | 济南康和医药科技有限公司 | A kind of Edaravone sodium chloride injection and preparation method thereof |
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Application publication date: 20120711 |