CN1311867C - Nerve growth factor (NGF) liposome - Google Patents
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Abstract
The present invention discloses a novel medicament form of a nerve growth factor (NGF) liposome, which belongs to the field of the novel medicament form of a protein polypeptide drug in pharmacology. The structure of the present invention discloses in an attached figure 1. Phospholipid is dispersed in water to obtain a single layer or multi-layer microcapsules, and a water-solubility medicine NGF is entrapped in a water phase in each liposome microcapsule. The NGF liposome mainly comprises materials, such as phospholipid, cholesterol, distearoyl phosphatidyl ethanolamine-polyethyleneglycol (2000) (DSPE-PEG-2000) and NGF. A reverse evaporation method is utilized to obtain the NGF liposome of which the encapsulation efficiency is more than 34% after the materials are preferably selected by a prescription. That the in vivo stability of the NGF liposome is higher than that of NGF is discovered, and the half-life in vivo is prolonged by about 4 times; that the brain tissue drug concentration of the NGF liposome is higher than that of NGF after nasal administration is discovered (attached figure 2). Moreover, the cognitive ability and the memory ability of a rat with senile dementia can be obviously improved. The optimal preparation for the nasal administration of the NGF liposome is an NGF liposome gel spraying agent.
Description
This invention of technical field has proposed the feasibility with the preparation technology of the theory of liposome entrapment nerve growth factor (NGF) and NGF Liposomal formulation and nasal-cavity administration treatment central nervous system disease, belongs to protein and peptide drugs technical field in the pharmacy.
Background technology nerve growth factor (nerve growth factor, NGF) be one of most important bioactive molecule of nerve system of human body, having the dual function of short neurite-outgrowth and trophic nerve concurrently, is the unique energy found so far has repair to central nervous system and peripheral nervous system lesions bioactive substance [1].Calendar year 2001 injection NGF obtains country's one kind biological product New Drug Certificate that national medical Surveillance Authority issues in China, and this is that the NGF preparation of listing is produced in first approval in the world.The NGF that uses at present is the intramuscular injection freeze-dried powder preparation, mainly is 2.5s NGF in the preparation, adds 5% mannitol and 0.1% human blood protein makes protective agent.Its indication is the n-hexane toxic peripheral neuropathy, is mainly used in the nerve injury that treatment causes owing to wound, optic neuropathy, [2] such as polyneuritis, herpes zoster, facial paralysis.In addition, NGF also has excellent protection and repair to the nervus centralis cholinergic neuron, but because NGF is difficult for passing through blood brain barrier, treats central nervous system disease at present and also only limit to the direct administration of the ventricles of the brain.Once there are some researches show, give Alzheimer people ventricles of the brain direct injection NGF, can obviously alleviate patient's cognitive dysmnesia [3], yet this administering mode easily causes damage and infects, brought inconvenience to clinical practice, limited its application the central nervous system.Rising along with China's aged tendency of population ratio, the incidence probability of degeneration neuropathy is in rising trend, consumer groups might further enlarge, and market capacity also will increase, and it is imperative therefore to develop the NGF central nervous system uses in brain appropriate formulation and administering mode.
The anatomical research proof exists olfactory mucosa epithelium path and olfactory neural pathway between nasal cavity and cranial cavity, for the administration of macromolecular drug via intranasal application provides approach [4] directly into brain.Experimental results show that those are positioned at the central nervous system, and the curative effect medicine relevant with brain function is such as being used for parkinson, the medicine of Alzheimer or pain, the extremely low medicine of concentration in conventional route of administration hypencephalon especially, the per nasal mucosal delivery is gone into the brain route of administration and is had very big advantage.Frey[5] etc. studied the NGF nasal-cavity administration the earliest, the result shows
125The NGF of I labelling promptly occurs in olfactory bulb at 20min behind the nasal-cavity administration, and also there is distribution at other positions in the brain, but measure less.Analyze its reason, may easily be degraded, and easily removed and caused the drug level reduction by the nose cilium owing to it by proteolytic enzyme and aminopeptidase that nasal cavity exists.Be head it off, can adopt suitable enzyme inhibitor, thickening agent and with pharmaceutical carrier (as liposome, liposome is that phospholipid disperses to get in water, and it structurally is the single or multiple lift vesicle.And liposome has biocompatibility and biodegradability, is present excellent drug carrier [6]) the bag method of carrying increases the stability of NGF, and then increase its cerebral tissue medicine intake, realize curative effect preferably.
Therefore for solving NGF stability and open up its range of application in vivo the central nervous system, the present invention adopts liposome entrapment NGF, and has developed NGF liposome preparation technology and to NGF liposome nasal cavity administrated preparation---and gel spray is write out a prescription preferably; Than NGF arranged the half-life in the longer body after animal experiment study is found NGF liposome vascular drug delivery, distribution and pharmacodynamics in rat cerebral tissue behind the nasal-cavity administration all are better than free NGF.Illustrate that the NGF liposome has more wide application prospect than NGF clinically.By literature search, the research report that does not have the NGF liposome at present.
Summary of the invention
1. one of innovative point of this invention is to propose with liposome entrapment nerve growth factor (NGF).
Nerve growth factor (NGF) is a protein and peptide drugs, the biological activity height, and consumption is few, and the half-life weak point is degraded by enzyme easily in vivo.Adopt liposome entrapment NGF can avoid NGF to be degraded, improve its stability, prolong the half-life in its body by the body endoenzyme.NGF is that phospholipid disperses to get in water with the liposome that is characterized as of liposome entrapment, and it structurally is the single or multiple lift microcapsule.Water soluble drug NGF bag is loaded in aqueous phase (seeing accompanying drawing 1) in the liposome.Liposome is different with the micelle that is made of surfactant, and it when diluted, can not break because of concentration reduces in body fluid, has the bioactive ability of NGF that the better protect bag carries.Phospholipid is biomembranous constituent, has biological degradability and biocompatibility, so liposome is excellent drug carrier of NGF.
2. two of the innovative point of this invention preparation technologies that are the NGF liposome.
The material of preparation liposome mainly is phospholipid and cholesterol.Phospholipid can adopt phospholipid of natural soybean (main component is a phosphatidylcholine), Ovum Gallus domesticus Flavus lecithin and distearoyl phosphatidylcholine, palmitoylphosphatidyl choline (DPPC), Phosphatidylserine, phosphatidic acid, hydrolecithin etc.Here preferred crude soya bean lecithin (purity＞95%).
The method for preparing the NGF liposome has reverse evaporation, multi-emulsion method, and freeze-thaw method, calcium is induced fusion method, injection method etc.Preferred reverse evaporation.Soybean lecithin SPC, cholesterol CHol and DSPE-Polyethylene Glycol (2000) are (DSPE-PEG-2000), after the suitable oils dissolving, add an amount of NGF, the vortex vibration ultrasonic 1～2min in back makes it form uniform w/o type emulsion, 25～30 ℃ of following reduction vaporizations continue rotary evaporation 10～20min after form the gel state material.Replenish a certain amount of normal saline then and make its aquation, be dispersed into uniform liposome turbid liquor.The NGF liposome of preparation is characterized as transparency liquid, has blue opalescence, and mean diameter is less than 100nm.
Add DSPE-PEG-2000 in the NGF liposome preparation technology preparation and help the dispersion of liposome at aqueous phase, rotary evaporation generally is not higher than 37 ℃, and ultrasonic time is no more than 2min, helps protecting the NGF biological activity.
Utilize freezing supercentrifugal process further to improve the NGF liposome encapsulation.NGF liposome centrifugal 10～30min of 10000～40000rpm under-10～4 ℃ with preparation removes the part supernatant, can improve the NGF liposome encapsulation.
Above-mentioned NGF liposome preparation technology is not appeared in the newspapers.In liposome preparation technology, the adding of DSPE-PEG-2000 and improve the protein and peptide drugs envelop rate with freezing high speed centrifugation and belong to method innovation.
3. three of the innovative point of this invention be to find that the NGF liposome has better body internal stability than NGF.
NGF is a protein and peptide drugs, and the half-life weak point is degraded by enzyme easily in vivo.In vivo half-life is about 9 minutes behind the NGF intravenously administrable, carries NGF after the administration of rat femoral vein with the long circulating liposomes bag, and the half-life extends to 35 minutes in the body, has improved 4 times approximately, has embodied its superiority in clinical practice.
4. five of the innovative point of this invention be to find that NGF liposome nasal-cavity administration has better advantage at more free NGF aspect the treatment central nervous system disease.
Because NGF is difficult to by blood brain barrier, has limited its application aspect the central nervous system for a long time.Can increase the distribution of medicine behind the NGF nasal-cavity administration, but effect is not the best at cerebral tissue.Behind liposome entrapment NGF, nasal-cavity administration can obviously increase the drug level of NGF in rat cerebral tissue.After giving the treatment of Alzheimer rat, find that NGF liposome nasal-cavity administration can obviously improve the cognitive memory ability of rat than NGF, the function that has excellent protection and repair impaired cholinergic neuron.NGF liposome nasal-cavity administration is expected to treat central nervous system disease.
5. four of the innovative point of this invention be to propose NGF liposome nasal-cavity administration gel spray prescription.
NGF liposome nasal cavity administrated preparation the best is a gel spray.Prescription has added enzyme inhibitor on the basis of NGF liposome, antibacterial, thickening agent and frozen-dried supporting agent etc.This nasal cavity administrated preparation is made into lyophilized powder earlier, face with preceding disperse with distilled water after, can use.Because NGF is the highly active protein polypeptide, makes lyophilized powder and helps keeping its biological activity.Gel spray is made simple, easy to use, add the clearance rate that gel-type vehicle can reduce the nose cilium in the prescription, prolong drug is in the holdup time of nasal cavity, make medicine can keep drug level for a long time at nasal cavity, making spray can increase the dispersion absorption area of medicine at nasal cavity as far as possible, makes the medicine assimilation effect best.
The specific embodiment
Embodiment 1 NGF liposome preparation technology
Get an amount of soybean lecithin SPC by optimizing prescription, cholesterol CHol and DSPE-Polyethylene Glycol (2000) are (DSPE-PEG-2000), after the suitable oils dissolving, add an amount of NGF, the vortex vibration ultrasonic 1min in back makes it form uniform w/o type emulsion, 28 ℃ of following reduction vaporizations continue rotary evaporation 10min after form the gel state material.Replenish a certain amount of normal saline then and make its aquation, be dispersed into uniform liposome turbid liquor.The NGF liposome of preparation at-10 ℃ of centrifugal 20min of following 20000rpm, is removed the part supernatant, obtain envelop rate 34% above NGF liposome.The NGF liposome of preparation is a transparency liquid, has blue opalescence, and mean diameter is 64.03nm.
It optimizes prescription:
The ratio of phospholipid and cholesterol is 1: 1～1: 2 (weight ratio),
Phospholipid and DSPE-PEG-2000 ratio are 100: 8～100: 1 (weight ratio),
NGF and fat material ratio are 1: 50～1: 100 (weight ratio)
Embodiment 2 NGF liposome intravenously administrable body internal stabilities
One, experimental technique: get 36 of SD male rats, be divided into 2 groups, be respectively NGF and NGF liposome group.After the anesthesia of 15% urethane, the femoral vein administration, dosage is every 100g body weight 6uCi (20ug)
125I-NGF (NGF liposome group dosage by the bag NGF amount of carrying).Respectively at administration 5,10,15,30,60, get blood 1.0ml from the carotid artery intubate behind the 120min.Blood sample 5000rpm is centrifugal, and 5min obtains serum, gets the capable SDS-PAGE gel electrophoresis of 20ul serum about 4 hours, cuts gel strips at NGF molecular weight place with γ calculating instrument counting 1min, according to counting with
125The standard curve of I-NGF relation with contents calculates serum Chinese medicine content, according to C～t relation, with 3P-87 computed in software pharmacokinetic parameter.
Two, experimental result:
The pharmacokinetic parameters of table 1 NGF and NGF liposome intravenously administrable
| K e/min -1×10 3 | t/min | |
| NGF NGF-SSL | 25.5±3.1 7.89±2.84 | 9 35 |
Three, conclusion: NGF is a protein and peptide drugs, degraded by enzyme easily in vivo, half-life is short, in vivo half-life is about 9 minutes behind the NGF intravenously administrable, carry NGF after the administration of rat femoral vein with the long circulating liposomes bag, the half-life extends to 35 minutes in the body, has improved 4 times approximately, has embodied its superiority in clinical practice.
The optimization prescription of embodiment 3 NGF liposome nasal-cavity administration gel sprays
NGF liposome nasal-cavity administration gel spray prescription:
| The range of choice | Preferably | |
| NGF liposomase inhibitor antibacterial thickening agent proppant | The bacitracin benzalkonium bromide, the EDTA sodium carboxymethyl cellulose, hypromellose, Polyethylene Glycol, polyvinylpyrrolidone K30, carbopol 1974NF mannitol, sorbitol, sucrose, bovine serum albumin, the human blood protein | Press embodiment 1 described preparation technology's preparation, contain NGF 100ug bacitracin 2mg 0.01% benzalkonium bromide 1% Hydroxypropyl methylcellulose, 5% sweet mellow wine and 0.1% human blood protein |
In order to protect the NGF biological activity, by the NGF liposome nasal gel spray of above-mentioned optimization prescription preparation, packing 2ml (containing 100ugNGF) is lyophilizing 30 hours under the 0.22mbar in temperature for-40 ℃ of pressure in 5ml spray special-purpose bottle.Disperse with the 2ml sterilized water before using.
This gel spray is made simple, and is easy to use, adds the clearance rate that gel-type vehicle can reduce the nose cilium in the prescription, makes medicine be difficult for running off from nasal cavity, and the droplet of ejection is thinner, is evenly distributed at nasal cavity, makes the medicine assimilation effect best.
Embodiment 4 NGF liposome (NGF-SSL) nasal-cavity administrations treatment central nervous system aspect disease
1.NGF the tissue distribution of liposome nasal-cavity administration
One, experimental technique:
Get 54 of SD male rats, be divided into 2 groups, be followed successively by NGF, NGF liposome (NGF-SSL).Separate its one-sided carotid artery and femoral vein with 15% urethane anesthesia back, the ligation of carotid artery proximal part, distal end is done the carotid artery intubate.Do tracheal intubation simultaneously to keep breathing, cut an osculum on the esophagus that exposes, a polyethylene tube is inserted straight-through nasal cavity rear portion, the gelatin of injection debita spissitudo is made adhesive nose palate passage is shut, in case medicinal liquid enters the oral cavity loss from nasal cavity after the administration.
The nasal-cavity administration position is 0.4cm in the nostril, and dosage is every 100g body weight 6uCi (20ug)
125I-NGF.The administration of bilateral nostril respectively at administration 30,60, is got blood 1.0ml from intubate behind the 120min, uses peristaltic pump with 0.5mlmin then
-1Flow velocity by carotid artery intubate perfusion normal saline 5min, cut off rat contralateral seventh cervical vein then, with 1.0mlmin
-1Flow velocity continue perfusion 10min, cut off opposing carotid at last, continue perfusion 15min. and eliminate medicine residual in the brain blood capillary to the influence that cerebral tissue distributes with this, accurately estimate medicine and cross over the ability that blood brain barrier enters cerebral tissue.
The cerebral tissue each several part is made 30% cerebral tissue suspension after with the ultrasonic homogenate of 0.5%TritonX-100, gets supernatant [7] behind the centrifugal 10min of 10000rpm.Accurately get the part supernatant, 35 ℃ of vacuum dryings, an amount of dissolved in distilled water of reuse concentrates 10～20 times.Get supernatant behind the centrifugal 10min of 10000rpm once more.Liver, spleen, lung, nephridial tissue are made 10% homogenate after with 0.5%TritonX-100 homogenate, directly the centrifuging and taking supernatant.The capable SDS-PAGE electrophoresis of supernatant after electrophoresis finishes, cuts the gel strips of NGF molecular weight place's spike, calculates drug level according to standard curve.
Two, experimental result:
1. in the distribution results of rat cerebral tissue's each several part:
30min behind NGF and NGF liposome (NGF-SSL) nasal-cavity administration, behind 60min and the 120min at the cerebral tissue olfactory bulb, striatum, Hippocampus, cerebellum, the drug level of brain stem and prefrontal cortex is seen accompanying drawing 2.As can be seen from the figure, NGF liposome (NGF-SSL) group at the drug level of abundant striatum of olfactory bulb and cholinergic neuron and hippocampus all apparently higher than NGF administration group, this is because the NGF bag is loaded in the interior aqueous phase of long circulating liposomes, liposome has reduced the interaction of NGF and nasal cavity endoenzyme, the corresponding intake of NGF at cerebral tissue that increased.
2. in the distribution results of its hetero-organization of rat:
30min behind NGF and the NGF liposome nasal-cavity administration, behind 60min and the 120min liver, spleen, lung, the drug level of kidney is seen accompanying drawing 3.Relatively the NGF nasal-cavity administration distributes (accompanying drawing 2 and 3) as can be seen at the medicament contg of cerebral tissue and its hetero-organization, drug main will be distributed in cerebral tissue behind the nasal-cavity administration, and the amount that is distributed in its hetero-organization after Nasal Mucosa Absorption is gone into blood is less, illustrating that nasal-cavity administration has brain targeting, is a kind of good approach that medicine is gone into brain.
Three, conclusion:
NGF is better than NGF with the liposome entrapment nasal-cavity administration in the distribution of cerebral tissue, and especially the drug level at striatum relevant with degenerative disease and hippocampus can improve 2～3 times approximately, is a more potential nasal-cavity administration brain targeting preparation.
2.NGF the pharmacodynamic study of liposome (NGF-SSL) nasal-cavity administration
One, experimental technique:
1.NGF and NGF liposome (NGF-SSL) pharmacodynamic study step:
1) gets 50 of SD male rats, raise two days later, with evenly grouping of water maze laboratory screening.
2) rat is divided into 4 groups, is respectively normal saline intravenous injection (NS-iv), NGF intravenous injection (NGF-iv), NGF nasal-cavity administration (NGF-na), NGF-SSL nasal-cavity administration (NGF-SSL-na) group.Dosage is NGF:10ug/100gbw, administration once a day, successive administration 7 days.
3) make the Alzheimer model: SD male rat lumbar injection 10% chloral hydrate anesthesia (0.4ml/100g body weight), head is fixed on the rat brain stereotaxic instrument, fontanel was a basic point in the past, according to rat brain stereotaxic atlas [8], determine the Meynert nuclear location, coordinate is brain median line side 2.0mm, before the bregma-1.4mm, 7.0mm under the cerebral dura mater.After electricity consumption is drilled in the vertical sphenotresia in coordinate place, be inserted into target spot gently along boring, slowly inject the amino-(3-hydroxy-5-isoxazolyl)acetic acid. (IBO) of 5ug/ml with the 10ul microsyringe, each 2ul of both sides, let the acupuncture needle remain at a certain point injection back 1min is with the bonding wound skin of seccotine.
4) after the modeling two days, the animal activity that affranchises substantially continued administration 7 days, once a day.
5) behavioristics's measuring: adopt following water maze laboratory and diving tower experimental technique.
6) two animals of every group selection are cooked AchE dyeing, and method is as follows.
2. pharmacodynamic index:
2.1 water maze laboratory: water maze swimming case is made 25 ± 1 ℃ of water temperatures by vinyon.Training in first day: at first rat is placed 10s on safety board and conform, respectively rat is put into the second, the three successively then, the 4th cecum, to far away, distance gradually extends by near.When rat swims over to the platform place, and after climbing up platform, carry out next cecum training.Training finishes second day same time and carries out test of memory: at first rat is placed 10s on safety board, rat is placed in the water of safety board vicinity then, make it climb up platform automatically 1 time.During experiment rat is placed the 5th cecum, start stopwatch simultaneously, time and errors number that record is reached home.The setting experimental period is 180s, surpass 180s can not the person of swimming out of by 180s.Repetitive memory experiment in the 3rd day.
2.2 diving tower experiment: this device is separated into 2 for passive avoidance trained reflex case with the black plastic dividing plate, and bottom screen (spacing 0.5cm) can be switched on.Training in first day: rat is put into the diving tower instrument, and the 60s that conforms passes to the 36V alternating current then immediately, jumps after rat is shocked by electricity and scurries.Training time is 180s.Memory achievement measure: behind the 24h rat put into the same mensuration case once 60s that conforms, then rat is placed on the safety board, energising timing 180s, the time that the record rat jumps off safety board for the first time is incubation period, and the errors number of after this jumping off platform.Be 180s writing time.48 hours repetitive memory achievement measures.
2.3 the histochemistry of acetylcholinesterase [9]:
1. tissue is fixing: the SD rat is got cerebral tissue behind the left ventricle perfusion.Cerebral tissue fixing 4h in 4% paraformaldehyde solution, put 30% sucrose solution (0.1mol/LPBS, pH7.4) 4 ℃ are saturated to and sink to the bottom, and do freezing crown section, thickness is 20um.
2. AchE dyeing: section is embathed twice with 0.1mol/L sodium-acetate buffer (pH6.0).Room temperature is at reactant liquor (32.5ml 0.1mol/L sodium-acetate buffer (pH6.0), 2ml 0.1mol/L trisodium citrate, 5ml 0.03mol/LCuSO4,9.5ml H
2O, 1ml 0.005mol/L K3FeCN6,25mg acetyl cholinesterase iodide) middle reaction 30min, magnetic stirrer solution.Section adds 1% (NH successively then
4)
2SO
4Handle 1min, 0.1%AgNO
3Solution reaction 1min.Gradient dehydration in the ethanol, transparent processing in the dimethylbenzene, neutral gum mounting.
3. integral optical density is measured: with the integral optical density of AchE in the image analysis system measurement unit area, quantitative parameter is every 5949412.00um
2The area that interior AchE positive cell covers and the product of optical density.Measure surcingle shell nuclear CPU and hippocampal CA, 3 sections of same area are got in every group of experiment, do statistical analysis with the meansigma methods of AchE positive cell integral optical density.All results do the t check by SPSS 8.0 statistical packages and handle.
Two, experimental result:
1. rat behavior is learned result of study
SD rat nasal-cavity administration carries out the experiment of water maze and diving tower after 2 weeks, experimental result sees Table 2.
Table 2 NGF and NGF liposome behind intravenously administrable and nasal-cavity administration to the influence (n=4～10) of the ability of learning and memory of rat
| Normal saline (iv) | NGF(iV) | NGF(na) | NGF-SSL(na) | ||
| The water maze diving tower | Swimming time (S) errors number incubation period (S) errors number | 152.17± 43.06 22.00±7.35 33.67±12.93 3.50±1.38 | 121.75±50.89 16.05±5.72 90.80±64.56 2.40±1.34 | 30.17± 14.08 **△ 4.50± 3.21 **△ 106.17± 61.11 * 1.00±0.89 * | 11.05± 1.87 **△△ 0.80± 0.45 **△△ 156.17± 37.15 **△ 0.33±0.75 **△ |
NS (iv) is a matched group:
*P＜0.05,
*P＜0.01;
NGF (iv) is a matched group: Δ P＜0.05, Δ Δ P＜0.01
Each is organized rat and shows through water maze laboratory and diving tower experimental result: with normal saline tail intravenously administrable (natural saline, NS is (iv)) organize and compare with NGF intravenously administrable group (NGF is (iv)), NGF and NGF-SSL nose administration (NGF (na), NGF-SSL (na)) rat memory ability obviously improves.Especially the memory achievement of NGF-SSL (na) group has significance increases.This shows that NGF-SSL nose administration group has good superiority aspect the raising animal memory ability.
2.AchE Histochemical studies result
Each treated animal brain tissue slice and carry out the AchE histochemical stain after, the cholinergic neuron number is more, presents rufous, feminine gender is a Lycoperdon polymorphum Vitt.Relatively the coloration result of each administration group as can be seen; the dyeing of NGF-SSL (na) group is rufous; and NGF (na) is slightly shallow, and NGF (iv) (iv) organizes gray with NS, illustrates that NGF-SSL (na) group has good protection and repair for the rat cholinergic neuron.(seeing accompanying drawing 4)
Select striatum CPU district to carry out the measurement of integral optical density, the results are shown in accompanying drawing 5.The result shows that NGF-SSL (na) group all has the highest integral optical density, is decremented to NGF (na) successively, NGF (iv), the normal saline group.
Three, conclusion:
The administration of NGF via intranasal application is better than intravenously administrable for the therapeutic effect of Alzheimer, and nasal-cavity administration is an effective way of treatment central nervous system disease; Can protect NGF to avoid the nasal cavity endoenzyme with liposome entrapment NGF degrades, further improves the therapeutic effect of NGF to Alzheimer.NGF liposome nasal-cavity administration is the effective ways of a comparatively ideal treatment central nervous system disease.
List of references:
[1] Cai Weidong, Fang Huang, " progress of the short neuranagenesis of nerve growth factor ", Chinese orthopedics magazine, 2003,11 (22): 1564-1566.
[2]http://www.bioway-pku.com/page/ngf9.htm
[3]Eriksdotter JM，Nordberg A，Amberla Ket al.“Intracerebroventricular infusion of nerve growthfactor in three patients with Alzheimer's disease.”Dement Geriatr cogn Disord，1998，9(5)：246-257.
[4]Lisbeth IBS，“Transport of drugs from the nasal cavity to the central nervous system”，Eur JPharm Sci.2000，11：1-18.
[5]Frey WH，Liu J，Chen X et al，“Delivery of125I-NGF to the brain via the olfactory route”，DrugDelivery，1997，4(2)：87-92.
[6] flat its can be edited, " modern medicinal agents ", Chinese Medicine science and technology publishing house, Beijing, 1998, p589.
[7]Wihong P，William AB，Abba JK，“Permeability of the blood-brain barrier to neurotrophins”，Brain Res.，1998，788：87-94.
[8]George paxinos，Charles Watson，“The rat Brain in stereotaxic coordinates”，Compact thirdedition，Academic Press，Printed in USA，fig 24.
[9]Hedreen JC，Bacon SJ，Price DL，“A modified Histochemical Technique to VisualizeAcetylcholinesterase-containing Axons”，J. Histochemistry and Cytochemistry，1985，33(2)：134-140.
Claims (8)
1, a kind of nerve growth factor preparation that is used for nasal-cavity administration, it is characterized in that said preparation contains liposome microcapsule and the nerve growth factor that is wrapped in wherein, also contains enzyme inhibitor, thickening agent, antiseptic and proppant, described antiseptic is selected from benzalkonium bromide, EDTA: described thickening agent is selected from hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose, Polyethylene Glycol, polyvinylpyrrolidone, carbopol; Described proppant is selected from mannitol and human blood protein; The weight ratio of described nerve growth factor and liposome microcapsule is 1: 50～1: 200, and described liposome microcapsule contains phospholipid, cholesterol, and DSPE-Polyethylene Glycol complex.
2, preparation according to claim 1, wherein said Polyethylene Glycol are Macrogol 2000.
3, preparation according to claim 1, wherein said enzyme inhibitor are bacitracin, and described antiseptic is a benzalkonium bromide, and described thickening agent is a hydroxypropyl emthylcellulose.
4, preparation according to claim 1, said preparation are gel, spray, solution, suspensoid or injectable powder.
5, preparation according to claim 4, said preparation are gel spray.
6, preparation according to claim 1, wherein said phospholipid is selected from soybean lecithin, Ovum Gallus domesticus Flavus lecithin, distearoyl phosphatidylcholine, palmitoylphosphatidyl choline, Phosphatidylserine, phosphatidic acid, hydrolecithin.
7, preparation according to claim 6, wherein said phospholipid are soybean lecithin.
8, the preparation method of the described preparation of claim 7, it is characterized in that this method is a reverse evaporation, concrete steps are as follows: with soybean lecithin, after cholesterol and DSPE-Polyethylene Glycol complex dissolves with suitable oils, add an amount of nerve growth factor, the vortex vibration ultrasonic 1～2min in back makes it form uniform w/o type emulsion, 25～30 ℃ of following reduction vaporizations are after form the gel state material, continue rotary evaporation 10～20min, replenish a certain amount of normal saline then and make its aquation, be dispersed into uniform liposome turbid liquor, freezing then high speed centrifugation promptly.
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Effective date of registration: 20081121 Address after: Department of physical medicine, School of pharmacy, Peking University Health Science Center, Xueyuan Road 38, Beijing, Haidian District, 100083 Co-patentee after: Xie Ying Patentee after: Hou Xin Pu Co-patentee after: Wang Junbo Address before: Department of physical medicine, School of pharmacy, Peking University Health Science Center, Xueyuan Road 38, Beijing, Haidian District, 100083 Co-patentee before: Xie Ying Patentee before: Hou Xin Pu Co-patentee before: Wang Junbo |