CN101232893A - Pharmaceutical composition for the treatment of nerve damage comprising blood plasma or serum - Google Patents
Pharmaceutical composition for the treatment of nerve damage comprising blood plasma or serum Download PDFInfo
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- CN101232893A CN101232893A CNA2006800280383A CN200680028038A CN101232893A CN 101232893 A CN101232893 A CN 101232893A CN A2006800280383 A CNA2006800280383 A CN A2006800280383A CN 200680028038 A CN200680028038 A CN 200680028038A CN 101232893 A CN101232893 A CN 101232893A
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Abstract
The present invention relates to a pharmaceutical composition for the treatment of nerve damage, and more particularly to a pharmaceutical composition for the treatment of nerve damage, which contains hlood plasma or serum as an active ingredient. The inventive composition regenerates nerve cells after spinal nerve damage and provides complete structural continuity in the spinal nerve lesion sites. Thus, the composition is useful for the treatment of nerve damage.
Description
Technical field
The present invention relates to be used for the treatment of the ingredient of nerve injury, preferably comprise the ingredient that is used for the treatment of nerve injury as active component of the blood plasma or the serum of medicine effective dose.
Background technology
Nervous system is divided into the peripheral nervous system central nervous system that unifies, and peripheral nervous system comprises except the brain that belongs to the central nervous system and all nerves the spinal cord.Nervous system also can be divided into the somatic nervous system autonomic nervous system of unifying, and wherein somatic nervous system comprises cranial nerve and spinal nerves, and autonomic nervous system comprises sympathetic nerve and parasympathetic nervous.Single neurocyte comprises cyton, dendron and aixs cylinder, wherein dendron is used for transmission and gets excited to cyton, and aixs cylinder derives cyton with neural impulse.At the end of aixs cylinder, several synapses are terminal to link to each other, and is connected to other neurocyte or target cell.Aixs cylinder comprises the myelin that is surrounded with schwann cell, and Lang Feijie.
The typical treatment of the neurological disorder relevant with nerve cell death or damage is by promoting that neurite promotes neuranagenesis to outgrowth, perhaps suppressing nerve cell death.The neuranagenesis promoter, known up to now, comprise nerve growth factor (NGF; Levi-Montalcini R. and Hamburger V.J.Exp.Zool.123:233,1953), Brain Derived Neurotrophic Factor (BDNF; Leibrock etc., Nature, 341:149,1989), neurotrophic factor-3 (NT-3; Maisonpierre etc., Science, 247:1446,1990), ciliary neurotrophic factor (CNTF; Lin etc., Science, 246:1023,1990), insulin and insulin like growth factor (Aizenman etc., Brain Res.406:32,1987; Bothwell, J.Neurosci.Res.8:225,1982; Recio-Pinto etc., J.Neurosci.6:1211,1986; Near etc., PNAS, 89:11716,1992; Shemer etc., J.Biol.Chem.262:7693,1987; Anderson etc., Acta Physiol.Scand.132:167,1988; Kanje etc., Brain Res.475:254,1988; Sjoberg and Kanje, Brain Res.485:102,1989; .Growth Factors such as Nachemson, 3:309,1990; Re-cio-Pento etc., J.Neurosci.Res.19:312,1988; Matteson etc., J.CellBiol.102:1949,1986; Edbladh etc., Brain Res.641:76,1994), activin (Schubert etc., Nature, 344:868,1990), alizarinopurpurin (Berman etc., Cell, 51:135,1987), fibroblast growth factor (D.Gospodarowicz etc., Cell Differ.19:1,1986; Baird, A. and Bohlen, P. fibroblast growth factor .In: peptide growth factor and receptor 1. thereof (eds.Sporn, M.B. and Roberts, A.B.) 369-418.Spring-Verlag, Berlin, Heidelberg, 1990; And Walter, M.A. etc., Lymphokine Cytokine Res.12:135,1993), estrogen (Toran-Allerand etc., J.Steroid Biochem.Mol.Biol.56:169,1996; McEwen, B.S. etc., Brain Res.Dev.Brain.Res.87:91,1995), platelet-derived somatomedin (PDGF; United States Patent (USP) 6,506,727), the fibrin degradation factor (as, tissue plasminogen's activator), azelon inhibitor (defibrinogenating agents) (for example, Agkistrodon halys Ahylysantinfarctase, urokinase, streptokinase or anticonvulsant; U.S. Patent Publication No. 2003/0219431 A1) or the like.The material of known inhibition cell death comprises thrombomodulin isoreagent (U.S. Patent Publication 2004/0002446 A1).
As everyone knows, the nervus centralis of brain and spinal cord does not have regeneration capacity.Owing to this reason, do not have method can treat for example wound of spinal cord injury, perhaps for example Alzheimer's disease and Parkinson's disease such neurodegenerative diseases.
Even there is the neurocyte of some research report spinal cord to show regeneration capacity, these researchs are incomplete, because regenerative response is of short duration and aixs cylinder death.The reason of this jejune reaction does not find as yet.
Have restriction when using aforesaid known neuranagenesis to promote that material is treated nerve injury, several limited Therapeutic Method are used for the treatment of the damage of spinal nerves.Up to date, for secondary injury being reduced to minimum degree, commitment in spinal nerve injury, the Drug therapy that is used for spinal nerves comprises steroid methyl meticortelone (the steroidmethylprednisolone) (Hall that gives heavy dose to vein, E.D.Adv.Neurol.59:241,1993; Bracken, M.B.J.Neurosurg.93:175,2000; Bracken, M.B.Cochrane Database Syst.Rev.2,2000; Koszdin, etc., Anesthesiology, 92:156,2000).Yet showing, nearest analytical data should forbid this Drug therapy (Hurlbert, R.J.J.Neurosurg.93:1,2000; Pointillart etc., Spinal Cord, 38:71,2000; Lankhorst etc., Brain Res.859:334,2000), and other medicines still are in experimental period.
In recent years, obtained marked improvement (Ramer, M.S. etc., J.Neurosci.21:2651,2001) in the understanding of restriction spinal nerves regeneration factor with based on the spinal nerves success of this understanding development is regenerated aspect tactful.Based on these research, think that Nogo is a kind of (Chen, M.S. etc., Nature, 403:434,2000) in the regenerated inhibitive factor of nervus centralis.Yet because the inhibitory action of enemy Nogo causes aixs cylinder that regeneration is partly only arranged, and other regeneration inhibitive factor can exist.Can illustrate the regeneration inhibitive factor by MAG (glycoprotein that myelin is relevant), inose albumen, ganglioside, ephrin, the neurite-outgrowth guiding factor (netrin), axon guidance factor (semaphorin) or the like.Although these factors are carried out important function really in the successful regeneration of spinal nerves, in spinal nerves regeneration, also should consider other demand.These demands comprise the propagation support that maximizes aixs cylinder, prominent function, the guiding expansion regrowth of optimization live axle in the part damage, adjust suitable persistence and minimize adverse effect, comprise initial trauma, and inflammation and wound generate.Based on this consideration, people such as Priestley advise that the regeneration of the spinal nerves cell that damages can be by the Fn Fiberonectin (Priestley, J.V. etc., the Journalof Physiology-Paris 96 that stimulate and neutrophilic granulocyte is intertwined, 123-133,2002).
Yet there is the problem of the local effectiveness of some part of neurocyte in the nervous cell regenerating process in the neuranagenesis by above-mentioned somatomedin, and owing to spent a large amount of funds in the production of somatomedin and purification process, this is uneconomic.
Therefore the present invention makes huge effort for the growth of the more effective neuranagenesis factor, found that after the mouse model with spinal nerve injury is handled by mammiferous blood plasma or serum, this moment nervous cell regenerating, and have the regenerating nerve injury site and have the structure persistence complete, thereby finish the present invention with other site.
Summary of the invention
The object of the present invention is to provide a kind of ingredient for the treatment of nerve injury, it comprises that the blood plasma of medicine effective dose or serum are as active component.
In order to achieve the above object, the invention provides a kind of ingredient for the treatment of nerve injury, it comprises the blood plasma of medicine effective dose or serum as active component.
This composition exists with any form in the group that is preferably selected from paste, solution, suspension and gel composition in topical according to the present invention.Simultaneously, the content of described blood plasma or serum is preferably the 0.1-99.9wt% of this composition weight.
In the present invention, nerve injury preferably refers to peripheral nerve injury or spinal nerve injury.Nerve injury refers to that preferably its nerve is cut off.This damage, the nerve of its cut-out, the neurite lengths of preferably having cut off is greater than the damage of 10mm.
In the present invention, peripheral nerve injury preferably is selected from the nervous function disorder of a kind of disease association in the group of being made up of diabetic neuropathy, acromegaly, hypothyroidism, AIDS, leprosy, Lyme disease, systemic lupus erythematosus (sle), rheumatoid arthritis, siogren's syndrome, periarteritis nodosa, Wei Genashi granulomatosis, cranial arteritis and sarcoidosis.
Further feature among the present invention and embodiment are explained by following detailed description and accompanying drawing.
Description of drawings
Fig. 1 shows the result of the BBB detection with injured nerve cells mouse model.
Fig. 2 shows the result of the grid walking detection with injured nerve cells mouse model.
Fig. 3 is for showing mouse model (A: normal group; B: matched group; And C: the photo of footprint analysis result processed group).
Fig. 4 shows the result of the electric physiological detection of the sensory latency with injured nerve cells mouse model.
Fig. 5 shows the histology pictures of the injury site of contrast Mus group and normal mice group.
The specific embodiment
The term " spinal nerve injury " that is used for herein is meant all damages that take place when external force acts on spinal cord.Spinal nerve injury comprises primary injury and the secondary injury in the Pathophysiology term.The primary injury of spinal nerves is meant the disruptive tissue injury of health that is caused by bump.The primary injury of human body by mechanical external force as clash into, oppress, draw and tear, incision or the like causes.According to modal mechanism, primary injury be because and continuously compressing one bump that works cause, and mainly break owing to compression fracture, fracture dislocation, gunshot wound and marrow intercalated disc.
The secondary injury of spinal nerves is to be caused by the biochemical medium that discharges from the tissue of primary injury.The biochemical substances that is produced by secondary injury causes the Pathophysiology signal process and damaged tissue little by little, thereby causes cell death.Other biochemical substances that the primary injury tissue discharges continues the vicious cycle of disorganization.The Pathophysiology pathological changes of this secondary comprises various biochemical variation of the energy metabolism that can cause cell membrane damage, blood vessel injury, inflammation, electrolyte disturbance, change and oxidative stress.
The degree of nerve injury can be assessed by several different modes.These methods comprise Frankel categorizing system, ASIA (American Spinal Injury Association) categorizing system, barthel index (the Wells of Yale's categorizing system, exercise index scale, improvement, J.D. and Nicosia, S.J.Spinal CordMed.18:33,1994) or the like.The patient can be divided into complete or incomplete spinal cord injury.Spinal cord injury can refer to after traumatic injury muscular strength within 24 hours and muscle sense completely loses or anal sphincter shrinks, the situation of the sensory deprivation of rumpbone and bulbocavernous reflex forfeiture completely.Incomplete spinal cord injury is meant and remains with componental movement and sensory function at least below injury site.Incomplete spinal cord injury can be divided into anterior cornual syndrome, posterior cord syndrome, central cord syndrome, lateral funiculus syndrome, radicular syndrome or the like.
The animal model that forms traumatic spinal cord injury is advantageously used in sets up pathophysiological mechanism and assessment curative effect (Anderson, T.E. and Strokes, B.T.J.Neurotrauma, 9:S135,1992; Blight, A.R.Central Nervous System Trauma, 2:299,1985; Blight, A.R. etc., McGraw Hill: New York, 1367-1379,1966).These animal models comprise weight decline model (Kuhn, P.L. and Wrathall, J.R.J.Neurotrauma, 15:125,1998), crush injury model (Bunge, R.P. etc., Advances in Neurology.FJ Seil (ed), Raven Press: New York, 75-89,1993; Bunge, R.P. etc., neuranagenesis, reorganization and reparation .FJ Seil (ed), Leppincott-Raven Publishers:Philadelphia, 305-315,1997), dampen model (Stokes, B.T. etc., J Neurotrauma, 9:187,1992) or the like.Dampen model and be suitable for imitating acute not perforated traumatic injury most.The damage that forms is by tissue examination assessment (for example, optics or electron microscope technique, dyeing and spike; Gruner, J.A.J.Neurotrauma, 9:123,1992); Electricity physiology result's measurement (for example, evoked potential; J.Neurotrauma such as Metz, 17:1,2000) or the behavior assessment (for example, go out locomotor activity or on the inclined-plane degree of stability of position; Basso etc., J.Neurotrauma, 13:343,1996).
On the other hand, the present invention relates to treat the ingredient of the cut peripheral nerve injury of aixs cylinder, especially the neurite lengths of Qie Duaning is greater than several millimeters (for example, greater than 10 millimeters).Neuronic reproducing characteristic has the ability of limited recovery nervous pathway function in the peripheral nervous system.New aixs cylinder is extended arbitrarily, and is connected with unsuitable target through the wrong direction of being everlasting, and causes dysfunction thus.For example, if the motor nerve damages, the aixs cylinder of regrowth is brought into wrong muscle, causes paralysis like this.In addition, (for example, greater than 10 millimeters) place suitable neuranagenesis can not occur, is because neurite is failed when they need growth or axon growth at the opposite way round greater than several millimeters in the neurite lengths of having cut off.
The effort of repairing peripheral nerve injury by surgical method causes different effects.In some cases, the stitching step that is used to the concentricity of the teleneuron that obtains to have cut off stimulates the formation of scar tissue, and it is considered to suppress axonal regeneration.Even form the position of reducing at scar tissue, successful neuranagenesis still is limited to the nerve injury less than 10mm.In addition, when damage or neuropathy affect the nerves cell itself cyton or cause under the situation of extensive degeneration of far-end aixs cylinder, the repair ability of peripheral neurons significantly is suppressed.Composition of the present invention makes nervous cell regenerating, and the complete structural continuity in nerve injury site is provided, and can treat neurite lengths that mammal cut off thus greater than 10 millimeters peripheral nerve injury.
On the other hand, the present invention relates to be used for the treatment of peripheral nervous pathological changes that disease causes and with the ingredient of the state of disease association.The peripheral nervous disorder that disease causes can be a diabetic neuropathy.Diabetic neuropathy do not cause by the peripheral nervous disorder clinically, but the typical disease that takes place owing to diabetic symptom.These neuropathy comprise the symptom that the autonomic nerve system of peripheral nervous system is unified and taken place in the somatic nervous system.Diabetic neuropathy is that infringement is in nerve under the skin, causes a symptom in following at least: the paralysis of finger, hands, toe and foot and tingling; Myasthenia of limbs; The P﹠B of extremity.The peripheral nervous pathological changes that disease causes can be and acromegaly, hypothyroidism, AIDS, leprosy, Lyme disease, systemic lupus erythematosus (sle), rheumatoid arthritis, siogren's syndrome, periarteritis nodosa, Wei Genashi granulomatosis, neuropathy that cranial arteritis is relevant with sarcoidosis.
Composition of the present invention makes nervous cell regenerating, and the structural continuity completely of nerve injury is provided, and can treat peripheral nervous pathological changes and relevant situation with above-mentioned disease association thus.
The blood plasma that is used as active component in the present invention is meant typical humoral substance in the mammalian, in other words, the straw-colored liquid substance of from isolated cells and cell debris, obtaining, and this material and its composition are by the well-known (Westerman of the document of this area, P. plasma protein, VII-1 to VII-13,2002; Wendy, Y.C. etc., the plasma protein guide, the full content of blood WARF (Foundation for Blood Research)-these documents will be quoted into the application as a reference at this).
The serum that composition in the present invention is used as active component typically refers to the part of the Fibrinogen removed, thrombin etc. from blood plasma.
Blood plasma or serum that composition in the present invention is used as active component comprise blood plasma or the serum of separating from various mammals, comprising: the mankind, non-human primates, for example, domestic animal such as sheep, goat, pig, horse, Canis familiaris L. and cattle, primates, Rodents or the like.
Blood plasma in the present invention or serum are to use conventional method, can easily separate obtaining from blood as centrifugalize, sedimentation or Filtration.Centrifugal separation is precipitation blood cell realization in blood plasma under appropriate condition.For example, about 1, the 400rpm centrifugal blood can be enough to precipitation in 10 minutes and comprise platelet and erythrocyte and leukocytic all cells fragment.The supernatant that comprises blood plasma can easily be separated from the cell of preparation.
This Filtration can be realized by filtering blood in the filter that is suitable for washed corpuscles from blood plasma.This filter preferably can make the easily filterable microporous membrane of protein.
Except the fresh liquid blood plasma preparation and liquid preparation that from the blood that extracts, obtain by centrifuging or sedimentation, before blood plasma or serum use, be known with multi-form store method, for example, fresh food frozen goods, cryoprecipitate preparation, lyophilized goods or concentrated product.The above-mentioned in the present invention blood plasma or the form of ownership of serum all can use.
FFP prepares by centrifugal blood, after extracting blood within 6 hours, and about 1, centrifugal 15 minutes washed corpuscleses of 400rpm and blood plasma, and approximately-40 ℃ freezing to-18 ℃.FFP is melted in about 30 ℃～37 ℃ warm water afterwards to be used.
CPP obtains by separating white precipitate (cryoprotein) (comprise the multiple factor: for example VIII:C, Fibrinogen, XIII and Fn Fiberonectin), its be when the FFP of a unit about 4 ℃ melt after, and approximately-40 ℃ to-18 ℃ the time, produce when freezing again.For its use, cryoprecipitate preparation is to melt or melt (for using then at about 4 ℃ fast) by placing it in to spend the night in the refrigerator (1 ℃～6 ℃) in water-bath.
Spendable concentrated blood plasma is isolating blood plasma from blood, with isolating blood plasma with concentrate after concentrating agents mixes, concentrating agents is dextranomer, SEPHDEX, dextramine for example, polyacrylamide, Bio Gel P, silicon gel, zeolite, glucosan glue (DEBRISAN), commissure agarose, starch and alginate separate concentrating agents then in spissated blood plasma.
In one embodiment of the invention, can use blood plasma or the serum of from blood bank (Blood Bank), buying.Liquid preparation (for example, the Gibco of the powder of for example from blood bank, buying, Invitrogen company (InvitrogenCorporation)
TMLittle chicken serum, Gibco
TMLowlenthal serum, Gibco
TMLittle sheep blood serum, Gibco
TMPorcine blood serum, Gibco
TMRabbit anteserum) or the serum product of GeminiBio-Products (USA) (for example, can use little chicken serum (Cat.#100-161), dog serum (Cat.#100-160), donor donkey serum (Cat.#100-151), donor lowlenthal serum (Cat.#100-109), donor Mus serum (Cat.#100-155), cat serum (Cat.100-153), guinea pig serum (Cat.#100-130), monkey serum (Cat.#100-154), mice serum (Cat.#100-113), porcine blood serum (Cat.#100-115), rabbit anteserum (Cat.#100-116), rat blood serum (Cat.#100-150) or sheep serum (Cat.#100-117).Determine not react from testing result from these preparations that prepare in the mammiferous blood plasma unit that comprises the people, be negative with the antibody response of HIV-1 and HIV-2 with the antibody of hbs antigen (HBsAg) and hepatitis C (HCV).All groups that are used to prepare the blood plasma of these goods all do not have pathogen through standard tests.
Blood plasma or the serum of use except that described goods is preferably the deactivation envelope virus, and for example HIV, hepatitis B virus and the hepatitis C virus (HCV) in blood plasma or the serum is to reduce the potential risk that infectious agent is propagated.Universal method in the method for deactivation blood plasma comprises that pasteurization, dry heat treatment, steam treatment, organic solvent/detergent mixture (for example handle, three (n-butyl)/phosphate/polysorbate80s), low pH (pH4), cohn fractionation, chromatography, nanofiltration method.Nearest ultraviolet radiation, gamma-radiation radiation, iodinate develop.Use blood plasma to suit through gamma-radiation radiation, methylene blue are handled and the lasting circulation deactivation of steam treatment is present in the virus in the blood plasma.
After the blood plasma that uses in the present invention or serum carried out powdered by heating, lyophilization or other suitable dry technology, blood plasma or serum can use.For example blood plasma or serum can use at lyophilization below-40 ℃ a couple of days (as about 7 days) Cheng Fenhou.
For treating nerve injury effectively, blood plasma that uses in the ingredient of the present invention or serum should act on target site easily.For this purpose, according to the present invention the composition of suitable use be as acceptable auxiliary in active component and one or more pharmacy or the preparation of carrier-bound goods form with blood plasma or serum.This employed " acceptable in the pharmacy " term be meant can with blended carrier of active component or adjuvant and harmless to object.In the present invention, blood plasma or serum are that 0.1-99.9wt% with the ingredient gross weight exists.Optionally blood plasma or serum can not combine with adjuvant, carrier or diluent and use separately.
But according to blood plasma of the present invention or serum topical.The component that is suitable for topical comprises semi-solid phase, semi-fluid bulk phase or liquid phase component, for example paste, gel, solution, emulsion or suspension.The preferred route of administration of composition of invention can be intramuscular, subcutaneous or endermic injection or infusion.The optional form that is able to lyophilized powder of its component, its with ooze through physiological body fluid etc. and use after buffered medium is rebuild with suitable pH.These components can use known equipment of pharmaceutical field or method to mix different compositions, and dissolving or rub mixture preparation [Remington ' s PharmaceuticalScience, 18th Edition, 1990, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87:Blaug, Seymour)].Preferred active component can become paste by formulation.
The composition of invention can comprise at least a being selected from the group that following additional complex forms: viscosity is adjusted agent, for example Cera Flava, three Glyceryl Behenates (glyceryl tribenhenate) and glyceryl three myristic acid glycerol salt (glyceryl trimyristate); Buffer, for example potassium metaphosphate (potassium metaphosphate), dipotassium hydrogen phosphate (potassium phosphate), single alkali sodium acetate (monobasic sodium acetate) and anhydrous citric acid sodium (anhydrous sodiumcitrate); Surface activity energy agent such as alkali metal fatty acid, dimethyl two hydrocarbon ammonium halides (dimethyldialkyl ammonium halide), alkyl pyridine halogenide (alkyl pyridinium halide), alkylsulfonate (alkyl sulfonate), fat oxidation amine (fatty amine oxide) and 2-quaternary ammonium alkyl imidazoline (2-alkyl imidazoline quaternary ammonium); Thickening agent is as paraffin, silicon oxide, cetostearyl alcohol (cetostearyl alcohol) and Cera Flava; Diluent, for example mannitol, sorbitol, starch, kaolin and sucrose; Antiseptic, for example p-Hydroxybenzoate (paraben); Basifier (alkalifying agents), for example ammonium carbonate, diethanolamine, ethanolamine, potassium hydroxide, sodium carbonate, sodium bicarbonate and sodium hydroxide; Acidulant, for example hydrochloric acid, nitric acid, acetic acid, aminoacid and citric acid; Antioxidant, for example ascorbic acid, sodium ascorbate, BHA, BHT, thioglycerol and sodium sulfoxylate formaldehyde; Emulsifying or suspending agent, for example PVP, gel, natural sugar, arabic gum, guar gum, agar, Bentonite, sodium carboxymethyl cellulose and Polyethylene Glycol; Stabilizing agent; And stain, for example ferrum oxide, titanium dioxide and aluminum color lake (aluminum lake).
The dosage of blood plasma or serum should consider that the degree of patient's health status and nerve injury suitably must determines according to the present invention.For the adult, the dosage that is used to damage is each about 0.0001～5mg/cm
2
Embodiment
Hereinafter, the present invention will describe in further detail in conjunction with the embodiments.Yet be to be understood that these embodiment are used for the purpose of interpretation, and be not as restriction for the scope of the invention.
Embodiment 1: contain the preparation of the paste of blood plasma
People source blood constituent (Korea S central authorities Blood Center, FFP) through HIV, HCV and HBV check melts at 30 ℃, mix with normal saline solution in the ratio of 10g/5cc then, thereby preparation contains the paste of blood plasma.
Embodiment 2: the preparation with rat model of spinal cord injury
The female Mus of use in detection (SD rat (Sprague-Dawley), about two months are big; Body weight 200-220g; Obtain Univ Yonsei Seoul, Korea S from medical zoopery portion).These rats remain on illumination/12 hour dark circulation in 12 hours, and allow freely to obtain food and water, and these are to assert that according to the management of laboratory animal evaluation mechanism animal of association's formulation is according to supporting and using committee's guide to raise.For BBB detects and grid walking detection (grid walk test), began animal is carried out basic ambulation training in preceding 14 days at surgical operation.At surgical operation preceding 3 days, animal is carried out the basic evaluation of behavior and movable function, selected 25 rats to be used for detecting like this.
The Mus of choosing carries out holonarcosis with the mixture of the xylazine of the ketamine of 25mg/ml and 1.3mg/ml by 2kg/ml, according to Schucht, P. wait people's method (ExperimentalNeurology, 176:143,2002) accept L2 veutro laminectomy (L2 VentralLaminectom).For prevention infection, after the surgical operation neutralization, press the 5mg/100g body weight/day and give animal muscle injection of antibiotics cefalexin.Open the second lumbar vertebra of each rat, use micro rongeur at aperture (1mm of the outside of the arc vertebra in the left side of rat puncture
2).The blade of blade holder is inserted in the hole,, form the traumatic injury of spinal column abdominal part like this via cerebral dura mater arc vertebra outside cutting to the right side.After surgical operation, rat is placed on its body temperature of protection on the warm sawdust, massages the following part of its abdominal part every day 3-4 time, continuous 7 days, to discharge the inclusions of bladder, recovers fully until its autonomous bladder control.
After the surgical operation of nerve injury 3 days, carry out BBB and detect the open walking ability that (Basso, D.M.Beattie, M.S. and Bresnahan, J.C.J.Neurotrauma, 12:1,1995) are used to assess rat.Rat is put into the transparent Yurisangja with a coarse surface, observes 4-5 minute.Animal comprises that according to a plurality of standards the position of joint motion, load-bearing, front and back football association tonality and afterbody carries out classification, marks with 0-21, and wherein 0 is that metapedes does not have the ability that moves, and 21 be that animal has normal locomotor activity.
After the surgical operation of spinal nerve injury 3 days, carry out the grid walking detect (Z ' Graggen etc., J.Neurosci.18; 4744,1998) to assess the motor capacity of rat.In the grid walking detects, allow rat on 1 meter long parallel metallic rod, to walk, keep regular paces circulation ability with assessment.Also allow rat walking downwards on the bar that has a down dip, with the control ability of assessment back leg.Step number by the number error is implemented assessment.10 mistakes the step show that rat can not regular walking, can not control extremity.0-1 mistake step shows not damage of rat, has normal behavioral competence.
In 25 animals, BBB detects with grid walking detection and shows that 20 animals have similar mark, and its average mark is approximately 11 fens in BBB detects, be approximately 6 fens in the grid walking detects.Determine mark according to double-blind method by two observers.Remain 5 animals and show different significantly behaviors, depart from average mark significantly, therefore from laboratory animal, get rid of.
Embodiment 3: the mouse model of vertebra damage contains the processing of plasma fraction
Select 20 animals in embodiment 2,10 animals (processed group) are handled in the following manner.The spinal nerve injury of the second lumbar vertebra bone of 10 animals partly is used in the paste that contains blood plasma of preparation among the embodiment 1.Sew up the back musculature then, skin is sewed up with surgical clamp.Simultaneously, remaining 10 animals (matched group) replace paste to handle with normal saline solution, sew up with above-mentioned same mode then.
Embodiment 4:BBB detects and the grid walking detects
In embodiment 3,10 animals (processed group) are handled at its spinal nerve injury position with the paste that 100 μ l (2mg/ μ l) contain blood plasma.In embodiment 3,10 animals (matched group) are handled its nerve injury position with normal saline solution, detect beginning in 3 days week enforcement at interval after the surgical operation of spinal nerve injury according to method BBB detection of describing among the embodiment 2 and grid walking.Fig. 1 shows the result that BBB detects, and Fig. 2 shows the result that the grid walking detects.As seeing from testing result, processed group and matched group reached the peak value of recovery in 35 days behind surgical operation.In addition, as two samples graceful-see that processed group detects (p＜0.05) and grid walking at BBB and detects that there are significant difference in recovery and matched group in (p＜0.05) in Whitney U (Mann-Whitney U) statistical analysis.
Embodiment 5: the footprint analysis
After the surgical operation of spinal nerve injury 35 days, ink was caught in the vola of processed group, matched group and normal mice, walking on blank sheet of paper then.According to a plurality of standards, comprise step-length (distance between the vola of two continuous paces), support base (distance between left vola and the right vola), the anglec of rotation (by the angle of cut between the line of the angle initialization of vola and toe) (Kunkel-Bagden, E. etc., Exp Neurol.119,153,1993) carry out the footprint analysis of each animal.Fig. 3 shows the footprint analysis photo of normal group, matched group, processed group.As visible in Fig. 3, display process group footprint is similar to normal group.
Embodiment 6: electric Physiological Analysis
Behind the surgical operation of spinal nerve injury 35 days, matched group and processed group Mus exposed the sciatic nerve of its sensorimotor cortex and its back leg with the urethanes anesthesia of the dosage of press the every kg body weight of 1.5g/.Insert the 0.5mm concentric needle electrode in the place ahead (rostrocaudal) of about 1-2mm in pro-cranium left side and preceding cranium approximately 1mm place with the right side sensorimotor cortex 1mm degree of depth, the electric pulse that re-uses 3mA finds and transmits the sciatic position of optimum signal to back leg.Off-line is in optimal stimulus position analysis response strength and sensory nerve waiting time.Fig. 4 shows the preclinical result of sensory nerve by two independent sample t-detection.As result displayed among Fig. 4 as seen, the sciatic nerve average latency of processed group significantly is lower than the average latency of matched group (p＜0.05).The paste that this explanation contains blood plasma makes nervous cell regenerating, and aixs cylinder connects by regenerated neurocyte, thereby realizes normal function.
Embodiment 7: histologic analysis
Behind the surgical operation of spinal nerve injury 35 days, matched group Mus and processed group Mus are implemented euthanasia through injecting excessive anesthetis.The second lumbar vertebra at spinal nerve injury position and coupled thoracic vertebra, third lumbar vertebra and fourth lumbar vertebra are removed, the formalin buffer of residue tissue immersion 10% 48 hours, and embedding in paraffin.Organize the sagittal plane that is cut into 2-5 μ m thickness in the vertical, the preparation tissue segments is placed on the microscope slide continuously.Microscope slide dyes with h and E.Fig. 5 A to 5C is the enlarged photograph (5 times, 10 times and 20 x magnifications) of matched group tissue, and Fig. 5 D to 5F is the enlarged photograph (5 times, 10 times and 20 x magnifications) of processed group tissue.Shown in Fig. 5 D to 5F, the injury site of processed group shows complete widely structural continuity, thereby the medicine that shows invention makes nervous cell regenerating and has essence curative effect to spinal nerve injury.On the other hand, shown in Fig. 5 A to 5C, the injury site of matched group is rotten.
Industrial applicibility
Aforesaid, the invention provides the ingredient of the treatment of nerve injury, it comprises blood plasma or serum that effective dose is gone up in treatment.Ingredient comprises according to blood plasma of the present invention or serum having the curative effect that makes injured nerve cell regeneration and healing, thereby neurocyte has normal function.
Even specific embodiments of the invention are described in detail, those skilled in the art can understand this description and only put down in writing preferred embodiment, and are not used in the restriction of explaining scope of the present invention.
Claims (7)
1. ingredient for the treatment of nerve injury, it comprises the blood plasma or the serum as active component of medicine effective dose.
2. ingredient according to claim 1 is characterized in that, exists with any form in the group that is selected from paste, solution, suspension and gel composition at topical.
3. ingredient according to claim 1 is characterized in that the content of described blood plasma or serum is based on the 0.1-99.9wt% of composition weight.
4. ingredient according to claim 1 is characterized in that, nerve injury is peripheral nerve injury or spinal nerve injury.
5. ingredient according to claim 1 is characterized in that, nerve injury is meant neural cut damage.
6. ingredient according to claim 5 is characterized in that, nerve injury be meant in this damage that nerve is cut off and cut neurite lengths greater than 10mm.
7. ingredient according to claim 1, it is characterized in that, peripheral nerve injury be with diabetic neuropathy, acromegaly, hypothyroidism, AIDS, leprosy, Lyme disease, systemic lupus erythematosus (sle), rheumatoid arthritis, siogren's syndrome, periarteritis nodosa, Wei Genashi granulomatosis, cranial arteritis, sarcoidosis in a kind of nervous function disorder of disease association.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020050073654 | 2005-08-11 | ||
| KR1020050073653 | 2005-08-11 | ||
| KR20050073654 | 2005-08-11 |
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| CN101232893A true CN101232893A (en) | 2008-07-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800280383A Pending CN101232893A (en) | 2005-08-11 | 2006-08-08 | Pharmaceutical composition for the treatment of nerve damage comprising blood plasma or serum |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106267348A (en) * | 2016-08-26 | 2017-01-04 | 于海龙 | Repair spinal cord or the albumin glue complex of spinal nerve injury and preparation, using method |
| CN106581062A (en) * | 2016-12-29 | 2017-04-26 | 田野 | Mixture for improving memory and preparation method and application thereof |
| CN106581063A (en) * | 2016-12-29 | 2017-04-26 | 田野 | Blood plasma isolate for enhancing memory, and preparation method and application thereof |
-
2006
- 2006-08-08 CN CNA2006800280383A patent/CN101232893A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106267348A (en) * | 2016-08-26 | 2017-01-04 | 于海龙 | Repair spinal cord or the albumin glue complex of spinal nerve injury and preparation, using method |
| CN106581062A (en) * | 2016-12-29 | 2017-04-26 | 田野 | Mixture for improving memory and preparation method and application thereof |
| CN106581063A (en) * | 2016-12-29 | 2017-04-26 | 田野 | Blood plasma isolate for enhancing memory, and preparation method and application thereof |
| CN106581063B (en) * | 2016-12-29 | 2018-11-06 | 北京豪思生物科技有限公司 | It is a kind of to be used to enhance blood plasma isolate of memory and its preparation method and application |
| CN106581062B (en) * | 2016-12-29 | 2020-09-18 | 江苏豪思睦可生物科技有限公司 | Mixture for improving memory and preparation method and application thereof |
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Open date: 20080730 |