CN102871969A - Application of erythropoietin microspheres to preparation of drugs for treating motor complications in Parkinson's disease - Google Patents

Abstract

The invention discloses application of erythropoietin microspheres to preparation of drugs for treating motor complications in the Parkinson's disease. The erythropoietin microspheres are obtained by means of preparing one of naturally extracted erythropoietin, genetic recombination expression erythropoietin, polyethylene glycol modified erythropoietin, glycosylation modified erythropoietin or human serum fused erythropoietin with polysaccharides into particles, and preparing the particles with sustained-release materials into the microspheres. The application has the advantages that a novel medicinal use of the erythropoietin microspheres is cultivated, a novel application field is developed, treating and preventing the motor complications in the Parkinson's disease by the erythropoietin microspheres have the advantages of long-acting sustained release, no toxic and side effects, fine compliance, patient pain relieving, low cost and the like and are easily accepted by patients, and the erythropoietin microspheres are simple in preparation process and environment-friendly and have excellent application prospects in treating and preventing the motor complications in the Parkinson's disease.

Description

The erythropoietin microsphere is controlled application in the parkinson disease motor complication medicine in preparation

Technical field

The present invention relates to the new purposes of erythropoietin microsphere, specifically, being the erythropoietin microsphere controls purposes in the parkinson disease motor complication medicine in preparation.

Background technology

Parkinson disease (Parkinson ' s disease, PD) be the common nervous system degeneration illness of middle-aged and elderly people, mainly reach the nigrostriatum path dopamine mediator that causes thus with black substance dense area degeneration of dopaminergic neurons disappearance and reduce relevant.Through clinical practice for many years, it is generally acknowledged, levodopa is still and the most effectively treats the parkinson disease medicine.But after the levodopa prolonged application, symptom fluctuation, unusual fluctuation disease (1evodopa-induced dyskinesia can appear in most of patient, LID) and mental symptom, it is the PD motor complication, laboratory finds that high concentration dopamine (dopamine, DA) and levodopa can because autoxidation produces free radical, can cause the neurocyte degeneration necrosis in addition, therefore, the common prevention of associating other drug and treatment PD and PD motor complication are very urgent.

Research in recent years shows, the generation of PD motor complication is activated in close relations with direct path and the signal transduction pathways such as downstream cAMP deopendent protein kinase (PKA), ERK thereof of expressing the D1 receptor, the phosphorylated protein that its downstream signal transducin dopamine and adenosine cyclophosphate are regulated-32(dopamine and cAMP-regulated phosphoprotein of Mr 32000, the change of albumen Thr75 site Expression of phosphorylated DARPP-32) may participate in the morbidity of unusual fluctuation disease (LID).

Aspect the prevention of PD motor complication, mainly adopt COMT inhibitor, amantadine, DR agonist, L-dopa methyl ester or ethyl ester etc. at present.Such as Chinese patent literature CN 200880108979.7, date of publication on August 18th, 2010, a kind of " being used for improving the medicament of Parkinsonian motor complication or mental symptom " disclosed, described medicament has the effect of 5-hydroxy tryptamine 1A acceptor portion agonist, simultaneously dopamine D 3 receptor is had the agonist effect, have for the motor complication of supervening when the repetitively administered levodopa and improve and the effect of delayed onset.But the side effect that Western medicine prevention and treatment produce is larger, easily forms drug resistance, unsuitable long-term taking.

Erythropoietin (Erythropoictin, EPO) is a kind of glycoprotein hormones that participates in regulating the differentiation of erythrocyte precursor, propagation, apoptosis inhibit, can increase erythrocytic number.More and more studies show that in recent years; EPO is all bringing into play very extensive and complicated effect in neurodevelopment, neuroprotective and adult neuron reparation, existence, regenerative process, and thinks that its neuroprotective is a kind of direct effect that it promotes the erythropoiesis function that do not rely on.In addition, EPO and receptor thereof (Erythropoictin receptor, EPO-R) all have expression at Rodents, mammals and human neuron and glial cell.Have been reported about the experimentation of erythropoietin to the protective effect of PD model and mechanism thereof, but be used for treating and prevent the application of PD motor complication to yet there are no report about the microsphere that erythropoietin and polysaccharide, slow-release material prepare.

Summary of the invention

The objective of the invention is for deficiency of the prior art, provide a kind of erythropoietin microsphere to control application in the parkinson disease motor complication medicine in preparation.

For achieving the above object, the technical scheme taked of the present invention is:

The erythropoietin microsphere is controlled application in the parkinson disease motor complication medicine in preparation, described erythropoietin microsphere is: a kind of in the erythropoietin that naturally extracted erythropoietin, dna recombinant expression erythropoietin, polyethyleneglycol modified erythropoietin, glycosylation modified erythropoietin or human serum merge is prepared into the erythropoietin polyoses grain with polysaccharide, and described erythropoietin polyoses grain is prepared into the erythropoietin microsphere with slow-release material again.

Described erythropoietin polyoses grain refers to that erythropoietin and polysaccharide prepare by lyophilization or spray drying in polyglycol solution, described polysaccharide is a kind of in glucosan or the sodium alginate.

Described slow-release material is a kind of in polylactic acid (PLA), PLGA (PLGA), polycaprolactone (PCL), polylactic acid-polyglycol (PLA-PEG) or the PLGA-Polyethylene Glycol (PLGA-PEG).

Described erythropoietin microsphere is that the method by the method for oil-in-water-oily Bao Gu (S/O/W) or oil-in-water-oil bag oil-oily Bao Gu (S/O/O/W) prepares.

Described erythropoietin microsphere is by subcutaneous injection or intracranial injection.

The injected dose of described erythropoietin microsphere is 100,000 units～5,000,000 units/kg.

The frequency of injection of described erythropoietin microsphere be 7 days～60 days once.

The invention has the advantages that:

1, the present invention has excavated the new medical application of the special microsphere of erythropoietin, has opened up a new application;

2, this erythropoietin microsphere as Drug therapy and prevention PD motor complication have long-acting slow-release, have no side effect, the advantage such as compliance is good, alleviate patient's misery, price is low, be easy to be accepted by the patient;

3, the erythropoietin microspheres is simple, and is environmentally friendly, in treatment and prevention PD motor complication good application prospect arranged.

Description of drawings

Accompanying drawing 1 is cerebrospinal fluid of rats EPO content results.

Accompanying drawing 2 is that SABC detects EPO result in the rat brain.

Accompanying drawing 3 is that SABC detects EPO-R result in the rat brain.

Accompanying drawing 4 is that SABC detects rat substantia nigra TH cell result.

Accompanying drawing 5 is substantia nigra dopaminergic neuron count results in the rat brain.

Accompanying drawing 6 is testing results of rat substantia nigra TH.

Accompanying drawing 7 is testing results of rat striatum TH.

The specific embodiment

Below in conjunction with accompanying drawing the specific embodiment provided by the invention is elaborated.

The preparation of embodiment 1 erythropoietin microsphere

One, preparation erythropoietin polyoses grain

1. prepare respectively 10%(w/w with ultra-pure water) Polyethylene Glycol (PEG) aqueous solution and 10%(w/w) glucan aqueous solution.Accurately taking by weighing the naturally extracted erythropoietin of 800mg is dissolved in the 7.2ml ultra-pure water stand-by.

2. under 0 ℃～4 ℃ conditions, be the ratio mixing of 1:1:5,1:1:10,1:1:20 or 1:1:40, the then abundant mixing of vortex concussion 30s～60s according to volume ratio with above-mentioned glucan aqueous solution, erythropoietin aqueous solution and PEG aqueous solution.

3. the mixed solution freeze overnight that erythropoietin, PEG and glucosan is formed, then vacuum lyophilization.

4. with step 3. the sample of gained remove PEG continuous phase three times with washed with dichloromethane, to carry an erythropoietin polyoses grain.

The particle diameter of the erythropoietin polyoses grain that obtains is 0.3～5 μ m, the smooth rounding of these vitreous body granules, and particle size distribution is even, and the structure of erythropoietin is protected preferably, has avoided inactivation in the dosage form preparation process.

Two, preparation erythropoietin microsphere

1. take by weighing 1g polyvinyl alcohol (PVA) and 5g NaCl, add ultra-pure water 94g, then heat, stir, to the PVA complete swelling, stopped heating continues slowly to stir, until solution be clear and without bubble after, stop to stir, be put in 4 ℃ of refrigerators stand-by.PVA concentration is 1%(w/w in this solution), NaCl concentration is 5%(w/w).

2. accurately take by weighing slow-release material PLGA (PLGA) 200mg, the 800mg that adds methylene chloride, vortex oscillation, PLGA is dissolved in the dichloromethane, gets the dichloromethane solution of PLGA, concentration is 20%(w/w), matching while using is in case the dichloromethane volatilization.

3. take by weighing erythropoietin polyoses grain 10mg, be dispersed in the dichloromethane solution of the PLGA of 0.5g step in 2., magnetic agitation 15min, rotating speed 1800rpm, fully stir so that the erythropoietin polyoses grain is dispersed in the dichloromethane solution of PLGA, the gained colostrum is scattered in contains 1%(w/w) PVA and 5%(w/w) in the aqueous solution of sodium chloride, rotating speed homogenate with 2200rpm becomes emulsion (S/O/W), in 1min, transfer to 4 ℃ 10%(w/w) solidify in the aqueous solution of sodium chloride, stir the protein microsphere of collecting ageing after 3～4 hours, with ultra-pure water washing three times, spend the night ice phase pre-freeze, again vacuum lyophilization makes particle diameter at the microsphere of 50 μ m～120 μ m.

Need to prove, erythropoietin can also be a kind of in the erythropoietin that merges of dna recombinant expression erythropoietin, polyethyleneglycol modified erythropoietin, glycosylation modified erythropoietin or human serum.

Described erythropoietin polyoses grain can also be that erythropoietin and polysaccharide prepare by spray drying in polyglycol solution, and described polysaccharide can also be sodium alginate.

Described slow-release material can also be a kind of in polylactic acid (PLA), polycaprolactone (PCL), polylactic acid-polyglycol (PLA-PEG) or the PLGA-Polyethylene Glycol (PLGA-PEG).

Described erythropoietin microsphere can also adopt oil-in-water-method of oil bag oil-oily Bao Gu (S/O/O/W) to prepare and (see for details: Yuan W, Wu F, Guo M, Jin T. Development of protein delivery microsphere system by a novel S/O/O/W multi-emulsion. Eur J Pharm Sci, 2009,36 (2-3): 212-218. Yuan Wei grace, Wu Fei, Ge Meiyan, Jin Tuo. with the method preparation conveying protein microsphere system of a kind of oil-in-water of novelty-oil bag oil-oily Bao Gu, Europe medicament Scientific Magazine, 2009,36(2-3): 212-218).The erythropoietin microsphere of preparation can be used for subcutaneous injection or intracranial injection, plays the effect of slow release and treatment PD motor complication.

The preclinical test of the microspheres in treating PD motor complication of embodiment 2 naturally extracted erythropoietin preparations

One, experimental technique

1. unusual fluctuation disease (levodopa-induced dyskinesia, LID) model preparation

After the pentobarbital anesthesia of rats by intraperitoneal injection 3%, strict flat cranium position is fixed on the rat brain stereotactic apparatus, iodophor disinfection skin of head after the cropping, cut scalp along median line, 15% hydrogen peroxide burns periosteum, expose skull suture and bregma, determine the bregma coordinate, show " rat brain stereotaxic atlas " according to bag new people etc., determine right side medial forebrain bundle (medial forebrain bundle, MFB) injection site: 1. 3.7mm behind the bregma, sagittal suture right side 1.7mm, 8.0mm under the skull, front tooth line 2.4mm; 2. 4.4mm behind the bregma, sagittal suture right side 1.2mm, 8.0mm under the skull, front tooth line 2.4mm.By above-mentioned definite injection site boring, microsyringe extraction 6-OHDA 6 μ l(with 10 μ l specifications are dissolved in 0.2% vitamin C normal saline, concentration 4 μ g/ μ l), every some injection 3 μ l, injection speed is 1 μ l/min approximately, the slowly withdraw of the needle behind let the acupuncture needle remain at a certain point the 5min, move back 4mm let the acupuncture needle remain at a certain point again 5min, until withdraw from fully, the skin suture otch is put cage and is fed.After 3 weeks, rats by intraperitoneal injection apomorphine (0.5mg/kg) is induced rotation, average speed〉7 times/min is successful PD rat model.After utilizing 6-OHDA to prepare the PD rat model, lumbar injection LDME/benserazide (50 mg/kg LDME and 25 mg/kg benserazides are dissolved in and contain in the 0.2% ascorbic sterilization normal saline) 4 weeks, preparation LID rat model.

The microsphere injection liquid pretreatment

Naturally extracted erythropoietin prepares the method for microsphere and sees embodiment 1.

Carboxymethyl cellulose (CMC) solvent formula: 0.5%CMC, 5% mannitol, 0.1% tween 80 is dissolved in water.

The EPO microsphere injection liquid: 123mg embodiment 1 prepared EPO microsphere is dissolved in the 7.5mlCMC solvent, obtains containing the suspension of EPO and CMC, i.e. EPO microsphere injection liquid, wherein, the final concentration of EPO is 10000 U/ml.

Model grouping and behavioristics measure

At random the LID rat model is divided into: 1. LID organizes (the LID rat is no longer cooked further processing), 2. EPO microsphere 100,000 units/kg organizes (microsphere that LID rat skin lower injection or intracranial injection dosage are the naturally extracted EPO preparation of 100,000 active units/kg rat), 3. EPO microsphere 1,000,000 units/kg organizes (microsphere that LID rat skin lower injection or intracranial injection dosage are the naturally extracted EPO preparation of 1,000,000 active units/kg rat), 4. EPO microsphere 5,000,000 units/kg organizes (microsphere that LID rat skin lower injection or intracranial injection dosage are the naturally extracted EPO preparation of 5,000,000 active units/kg rat).Other is provided with sham operated rats: normal SD rats, do not do any processing.Each is organized rat and induces rotation through the lumbar injection apomorphine, records rotating cycle in its 30 minutes, calculates the average rotating cycle of respectively organizing the rat per minute.

Cerebrospinal fluid of rats extracts

Rat is at injection EPO microsphere injection liquid after 1 week, give the pentobarbital anesthesia of lumbar injection 3% after, strict flat cranium position is fixed on the rat brain stereotactic apparatus, iodophor disinfection skin of head after the cropping, dissecting instrument is transformed by the 18G syringe needle, and needle point 1.5mm makes an angle of 90 degrees into pliers.The 30G pin connects the 1ml syringe and is used for collecting cerebrospinal fluid.Transform medicated pillow blunt separation top layer muscle, expose atlanto-occipital membrane, thrust atlanto-occipital membrane with 30 degree angles from the tail end of otch with the 30G syringe needle that connects 1ml immediately, enter approximately 1mm, the extraction cerebrospinal fluid.Cerebrospinal fluid is injected the EP pipe of 0.5ml ,-80 ℃ of lower preservations are stand-by.

Immunohistochemical staining

The 12h of rat after finishing injection, randomly draw 4 rat etherizations for every group, pour into successively normal saline 100ml and the 4% paraformaldehyde 300ml of pre-cooling through heart, broken end is got brain, fixing 8h after in the identical fixative, piece, gradient alcohol dehydration, dimethylbenzene are transparent through repairing, use paraffin embedding behind the waxdip.The row paraffin section, slice thickness is 5 μ m.Get the crown aspect of striatum and adopt streptavidin peroxidase to link method (SP method) row immunohistochemical staining, step is as follows: paraffin section de-waxing is to water; 3% hydrogen peroxide room temperature lucifuge is hatched 5min, to eliminate the endogenous catalase activity; The 1nmol/l EDTA-Tris-HCl microwave heating of pH7.7 is repaired; 1%BSA-PTS(0.5%Triton-X100) room temperature sealing 20min; Overnight incubation in 4 ℃ of wet boxes of TH acceptor monoclonal antibody (1:400,1%BSA dilution); The biotin labeling two that drips dilution is anti-, hatches 20min for 37 ℃; Drip the horseradish peroxidase-labeled strepto-avidin of dilution, hatch 20min for 37 ℃; Benzidine (DAB) chromogenic reagent, tap water flushing, afterwards dehydration, transparent, mounting.All use the abundant rinsing of 0.01mol/l PBS between each step, PBS replaces primary antibodie as negative control.SABC each observation area of cutting into slices is got 5 not overlapped views at random, observe in high power lens (10 * 40) is lower, adopt OLYMPUS-IX50 to become phase system to take, Image-Pro Plus 5.1 imgae processing softwares carry out semi-quantitative analysis, calculate TH receptor positive cell index (IOD)=Positive area * correction optical density value (measurement zone optical density-background indensity).

Protein immunoblot

After rat was injected 12h the last time, etherization was randomly drawed 4 rat sacrificed by decapitation for every group.Open rapidly cranium and get brain, separate fast the bilateral striatum on ice, every 10mg tissue adds 100 μ l protein lysate RIPA(and contains 50mM pH7.4 Tris, 150mM NaCl, 1%Triton X-100,1% sodium deoxycholate, 0.1% SDS and sodium orthovanadate, sodium fluoride, EDTA, leupeptin etc.) and 1 μ l protease inhibitor PMSF, ultrasonic abundant homogenate, 30 min of cracking on ice, then place 4 ℃ of centrifuges in the centrifugal 30min of 14000rpm, draw supernatant to another EP pipe, extract total protein, measure behind the protein concentration in-80 ℃ of preservations.

The protein electrophoresis solution preparation.1 * electrophoretic buffer (Running Buffer): 14.4g/L glycine, 3g/L Tris base, 1g/L dodecyl sodium sulfate.Transferring film buffer (Transter Buffer): 19.375 g Tris base, 0.75 g glycine, the 200ml methanol plus water is to 1000ml.1 * sample-loading buffer (Sample Buffer): 62.5mM Tris-HCl (pH6.8,25 ℃), 2% w/v dodecyl sodium sulfate, 10% glycerol, 50mM dithiothreitol, DTT, 0.01%w/v bromophenol blue or phenol red.10 * TBS (Tris-buffered saline): sealing buffer (Blocking Buffer): 1 * TBS, 0.1% tween 20 and 5% w/v defatted milk powder.First antibody diluent: 1 * TBS, 0.1% tween 20 and 5% sealer.Wash?Buffer?TBS/T：1×TBS，0.1%?Tween-20。

Join glue with the special-purpose stick mixing tank of Bio-Rad company, the sample that adds degeneration with 15 μ l microsyringes or 20 μ l EPPENDORF liquid-transfering guns whenever adds one in well, and water washes needle tubing.Elder generation electrophoresis 30min, again voltage stabilizing 150V electrophoresis 60min under 100V.The albumen transferring film shifts 90min under the electric current of 250mA, gets film after the transferring film.After the transferring film with washing film 5min under the 25ml TBS room temperature; Sealing buffer room temperature lower seal with 25ml is closed 1h; Wash Buffer with 15ml washes 3 times, each 5min; By the anti-solution of rabbit (titre is 1:1000) of respective concentration dilution first antibody TH antibody (titre is 1:400) or anti-β-actin, in the primary antibodie dilution buffer liquid of 10ml, 4 ℃ of jogs are hatched film and are spent the night; Wash Buffer with 15ml washes 3 times, each 5min; Add the second antibody (IgG that the HRP of anti-rabbit links to each other; 1:1000) in the sealing buffer of 10ml under the room temperature jog hatch film 1h; Wash Buffer with 15ml washes 3 times, each 5min.The ECL colour developing mixed liquor of configuration 10ml is added on the pvdf membrane, puts in the room temperature and reacts, and removes unnecessary liquid, calculates the product of each sample destination protein band OD value and area value with the Imagelab image analysis software, thereby it is quantitative to carry out protein band density.The immunoblotting protein band of each sample draws a density ratio with after β-actin compares, and carries out statistical analysis again.

Statistical analysis

Adopt SPSS13.0 software to carry out statistical procedures.Data represent with mean ± standard error, use the t of group check and one factor analysis of variance and add up, take P＜0.05 as significance level.

Two, result

1. various dose EPO microsphere is on the impact of rat model number of revolutions

After each organizes rat injection apomorphine, its average rotating cycle the results are shown in Table 1, in the rats in sham-operated group 30 minutes average rotation number be 0 ± 2 circle/minute, in the LID group rat 30 minutes average rotation number be 19 ± 2 circles/minute, compare P＜0.001(*) with sham operated rats; 100,000 active units/kg EPO microsphere group rotation number be 15 ± 2 circles/minute, compare without significant difference with LID group; 1,000,000 active units/kg EPO microsphere group rotation number be 4 ± 1 circles/minute, compare P＜0.001(#) with LID group; 5,000,000 active units/kg EPO microsphere group rotation number be 2 ± 1 circles/minute, compare P＜0.001(#) with LID group.The result shows that the microsphere of naturally extracted EPO preparation can reduce the not autonomous number of revolutions of LID rat, and wherein, high dose EPO microsphere has fairly obvious therapeutic effect.

Table 1 various dose EPO microsphere is on the impact of rat model number of revolutions

2. cerebrospinal fluid EPO testing result

Extract cerebrospinal fluid of rats after 1 week of injection EPO microsphere, Western blot detects EPO content, the results are shown in accompanying drawing 1.As can be seen from Figure, the cerebrospinal fluid of rats EPO content of EPO microsphere group is compared obvious increase with sham operated rats, P＜0.001 (*); The optical density value of EPO microsphere group is higher than sham operated rats far away.Wherein EPO microsphere dosage is 1,000,000 active units/kg rat.

The SABC testing result

(1) EPO and EPO-R testing result

EPO detects negatively in the rats in sham-operated group brain, and (EPO detects obviously positively injection EPO microsphere in the rat brain of 1,000,000 active units/kg), illustrates to have injected behind the EPO microsphere can detect more EPO positive neuron in the rat brain.See also Fig. 2, A: rats in sham-operated group brain EPO dyeing, without positive; B:EPO microsphere group (1,000,000 active units/kg), obviously positive; Scale: 200 μ m, P＜0.001 (* *).

Similar, behind the injection EPO microsphere in the rat brain EPO-R on the neuron express and phenomenon occurs increasing, particularly injected the EPO microsphere of 1,000,000 active units/kg after, the EPO-R great expression in the rat brain on the neuron.See also Fig. 3, A: rats in sham-operated group brain EPO-R dyeing, without positive; B:EPO microsphere group (1,000,000 active units/kg) rat brain EPO-R dyeing, obviously positive; Scale: 200 μ m, P＜0.001 (* *).

(2) testing result of substantia nigra dopaminergic neuron

Compare with normal SD rats; the damage side black substance TH positive cell of LID model group obviously reduces; and as seen the LID rat that gives EPO microsphere protection damages the TH positive cell of side and obviously increases than the LID model group; as seen the EPO microsphere has protective effect to substantia nigra dopaminergic neuron, and wherein EPO microsphere dosage is 1,000,000 active units/kg rat.See also Fig. 4, A: the rats in sham-operated group black substance, damage side; B: rats in sham-operated group black substance, right side; C:LID rat model black substance is damaged side; D:LID rat model black substance, strong side; E:EPO microsphere group rat substantia nigra is damaged side; F:EPO microsphere group rat substantia nigra, strong side; Scale: 200 μ m.

(3) substantia nigra dopaminergic neuron count results

To black substance TH positive cell counting, and self do not damage side and compare and draw TH positive neuron percentage ratio.Statistical result sees also Fig. 5, show among the figure that LID group rat positive cell is 19 ± 3%, with sham operated rats than P＜0.01(*), EPO microsphere group is 86 ± 6%, organize than P＜0.01(**), wherein EPO microsphere dosage is 1,000,000 active units/kg rat with LID.

Testing result

Compare with sham operated rats, the TH of the black substance damage side of LID group and EPO microsphere group (1,000,000 active units/kg rat) all obviously reduces, but the TH content of EPO microsphere group is compared many with the LID group, P＜0.001(**), testing result sees also Fig. 6, L: damage side, U: do not damage side, compare with sham operated rats, the TH content of LID group obviously reduces, P＜0.001(*), compare with the LID group, the TH content of EPO microsphere group increases, P＜0.001(**).But in striatum, the TH of EPO microsphere group damage side obviously increases, and prompting EPO microsphere has protective effect to striatal dopamine neuron; wherein EPO microsphere dosage is 1,000,000 active units/kg rat; testing result sees also Fig. 7, L: damage side, U: do not damage side; compare with sham operated rats; the TH content of LID group obviously reduces, P＜0.001(*), compare with the LID group; the TH content of EPO microsphere group significantly increases, P＜0.001(**).

The preclinical test of the microspheres in treating PD motor complication of embodiment 3 dna recombinant expression erythropoietin preparation

The preparation method of dna recombinant expression erythropoietin microsphere is seen embodiment 1, and experimental technique replaces naturally extracted EPO to prepare microsphere with embodiment 2 with dna recombinant expression EPO, and then subcutaneous or intracranial injection detects corresponding index to the LID rat.The microsphere of dna recombinant expression EPO preparation sees Table 2 to the impact of rat model number of revolutions.

Table 2 various dose dna recombinant expression EPO microsphere is on the impact of rat model number of revolutions

As can be seen from the table, the microsphere of dna recombinant expression EPO preparation can reduce the not autonomous number of revolutions of LID rat, and wherein, high dose EPO microsphere has fairly obvious therapeutic effect.

Each group rat is carried out cerebrospinal fluid EPO detection, SABC detection and Western blot to be detected; Fig. 1 to Fig. 7 result among testing result and the embodiment 2 is basically identical; behind injection dna recombinant expression EPO microsphere; EPO-R in the rat brain on the neuron expresses and phenomenon occurs increasing; after particularly having injected high dose dna recombinant expression EPO microsphere; EPO-R great expression in the rat brain on the neuron, visible dna recombinant expression EPO microsphere has protective effect to black substance and striatal dopaminergic neuron.Prove absolutely that the microsphere of dna recombinant expression EPO preparation has the effect for the treatment of parkinson disease motor complication.

The preclinical test of the microspheres in treating PD motor complication that embodiment 4 polyethyleneglycol modified erythropoietin prepare

The preparation method of polyethyleneglycol modified erythropoietin microsphere is seen embodiment 1, and experimental technique replaces naturally extracted EPO to prepare microsphere with embodiment 2 with polyethyleneglycol modified EPO, and then subcutaneous or intracranial injection detects corresponding index to the LID rat.The microsphere of polyethyleneglycol modified EPO preparation sees Table 3 to the impact of rat model number of revolutions.

The polyethyleneglycol modified EPO microsphere of table 3 various dose is on the impact of rat model number of revolutions

As can be seen from the table, the microsphere of polyethyleneglycol modified EPO preparation can reduce the not autonomous number of revolutions of LID rat, and wherein, high dose EPO microsphere has fairly obvious therapeutic effect.

Each group rat is carried out cerebrospinal fluid EPO detection, SABC detection and Western blot to be detected; Fig. 1 to Fig. 7 result among testing result and the embodiment 2 is basically identical; behind the polyethyleneglycol modified EPO microsphere of injection; EPO-R in the rat brain on the neuron expresses and phenomenon occurs increasing; after particularly having injected the polyethyleneglycol modified EPO microsphere of high dose; EPO-R great expression in the rat brain on the neuron, visible polyethyleneglycol modified EPO microsphere has protective effect to black substance and striatal dopaminergic neuron.The microsphere that proves absolutely polyethyleneglycol modified EPO preparation has the effect for the treatment of parkinson disease motor complication.

The preclinical test of the microspheres in treating PD motor complication that embodiment 5 glycosylation modified erythropoietin prepare

The preparation method of glycosylation modified erythropoietin microsphere is seen embodiment 1, and experimental technique replaces naturally extracted EPO to prepare microsphere with embodiment 2 with glycosylation modified EPO, and then subcutaneous or intracranial injection detects corresponding index to the LID rat.The microsphere of glycosylation modified EPO preparation sees Table 4 to the impact of rat model number of revolutions.

The glycosylation modified EPO microsphere of table 4 various dose is on the impact of rat model number of revolutions

As can be seen from the table, the microsphere of glycosylation modified EPO preparation can reduce the not autonomous number of revolutions of LID rat, and wherein, high dose EPO microsphere has fairly obvious therapeutic effect.

Each group rat is carried out cerebrospinal fluid EPO detection, SABC detection and Western blot to be detected; Fig. 1 to Fig. 7 result among testing result and the embodiment 2 is basically identical; behind the glycosylation modified EPO microsphere of injection; EPO-R in the rat brain on the neuron expresses and phenomenon occurs increasing; after particularly having injected the glycosylation modified EPO microsphere of high dose; EPO-R great expression in the rat brain on the neuron, visible glycosylation modified EPO microsphere has protective effect to black substance and striatal dopaminergic neuron.The microsphere that proves absolutely glycosylation modified EPO preparation has the effect for the treatment of parkinson disease motor complication.

The preclinical test of the microspheres in treating PD motor complication of the erythropoietin preparation that embodiment 6 human serums merge

The preparation method of the erythropoietin microsphere that human serum merges is seen embodiment 1, and experimental technique is with embodiment 2, and the EPO that merges with human serum replaces naturally extracted EPO to prepare microsphere, and then subcutaneous or intracranial injection detects corresponding index to the LID rat.The microsphere of the EPO preparation that human serum merges sees Table 5 to the impact of rat model number of revolutions.

The EPO microsphere that table 5 various dose human serum merges is on the impact of rat model number of revolutions

As can be seen from the table, the microsphere of the EPO preparation that human serum merges can reduce the not autonomous number of revolutions of LID rat, and wherein, high dose EPO microsphere has fairly obvious therapeutic effect.

Each group rat is carried out cerebrospinal fluid EPO detection, SABC detection and Western blot to be detected; Fig. 1 to Fig. 7 result among testing result and the embodiment 2 is basically identical; behind the EPO microsphere that the injection human serum merges; EPO-R in the rat brain on the neuron expresses and phenomenon occurs increasing; after particularly having injected the EPO microsphere of high dose human serum fusion; EPO-R great expression in the rat brain on the neuron, the EPO microsphere that visible human serum merges has protective effect to black substance and striatal dopaminergic neuron.The microsphere that proves absolutely the EPO preparation that human serum merges has the effect for the treatment of parkinson disease motor complication.

The application of erythropoietin microsphere of the present invention in preparation treatment parkinson disease motor complication medicine; the EPO microsphere can reduce the not autonomous number of revolutions of LID rat; EPO-R behind the injection EPO microsphere on the interior neuron of rat brain expresses and phenomenon occurs increasing; the EPO microsphere has protective effect to black substance and striatal dopaminergic neuron, can be used as effective ancillary drug of PD motor complication treatment.

The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.

Claims (7)

1. the erythropoietin microsphere is controlled application in the parkinson disease motor complication medicine in preparation, it is characterized in that, described erythropoietin microsphere is: a kind of in the erythropoietin that naturally extracted erythropoietin, dna recombinant expression erythropoietin, polyethyleneglycol modified erythropoietin, glycosylation modified erythropoietin or human serum merge is prepared into the erythropoietin polyoses grain with polysaccharide, and described erythropoietin polyoses grain is prepared into the erythropoietin microsphere with slow-release material again.

2. application according to claim 1, it is characterized in that, described erythropoietin polyoses grain refers to that erythropoietin and polysaccharide prepare by lyophilization or spray drying in polyglycol solution, described polysaccharide is a kind of in glucosan or the sodium alginate.

3. application according to claim 1 is characterized in that, described slow-release material is a kind of in polylactic acid, PLGA, polycaprolactone, polylactic acid-polyglycol or the PLGA-Polyethylene Glycol.

4. application according to claim 1 is characterized in that, described erythropoietin microsphere is that the method by the method for oil-in-water-oily Bao Gu or oil-in-water-oil bag oil-oily Bao Gu prepares.

5. application according to claim 1 is characterized in that, described erythropoietin microsphere is by subcutaneous injection or intracranial injection.

6. application according to claim 1 is characterized in that, the injected dose of described erythropoietin microsphere is 100,000 units～5,000,000 units/kg.

7. application according to claim 1 is characterized in that, the frequency of injection of described erythropoietin microsphere be 7 days～60 days once.

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