CN102233129A - Long-acting sustained release preparation for preventing or treating retinal damage, and preparation method thereof - Google Patents
Long-acting sustained release preparation for preventing or treating retinal damage, and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of pharmaceutical preparations, and relates to a long-acting sustained release preparation for preventing or treating retinal damage, and a preparation method thereof. The long-acting sustained release preparation takes erythropoietin (EPO) as an active component, takes dextran as a protective agent for the active component, and takes poly(lactic-co-glycolic acid), polylactic acid or polycaprolactone as a coating component to prepare sustained release microspheres, wherein the erythropoietin is coated by the poly(lactic-co-glycolic acid), polylactic acid or polycaprolactone. Proven by animal experiments, the sustained release microspheres of the long-acting sustained release preparation provided by the invention have the same protective actions on the ganglionic cells of damaged retinas by single vitreous chamber injection and repeated EPO protein vitreous chamber injection, and the sustained release microspheres are capable of avoiding a series of complications caused by many times of injection and overcoming the defects of repeated intraocular injection administration and gene therapy. The long-acting sustained release preparation provided by the invention adopts intraocular local administration, can reduce the dosage and treatment cost, and can not generate adverse effects on other organs or tissues in vivo.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, relate to a kind of long-acting slow-release preparation of retina injury and preparation method thereof that is used to prevent or treat.
Background technology
Bibliographical information, retina optic nerve disease kind is various, is one of important blinding factor, at present, Therapeutic Method still not yet in effect in clinical ophthalmology.Studies show that, after optic nerve sustains damage, the axon damage of retinal ganglial cells, cause the axoplasm transportation of aixs cylinder cribriform plate of sclera place to be obstructed, make the neurotrophic factor deficiency of supply nutrition, excitatory neurotransmitter discharges, oxidative stress and free radical produce in a large number, (Retinal Ganglion Cells, normal function RGCs) cause its degeneration death to have influenced retinal ganglial cells; The dead toxicity medium that is produced of retinal ganglial cells (RGCs) causes extracellular environment to change, and inspires adjacent healthy RGCs secondary degeneration, apoptosis, the minimizing of carrying out property of quantity.
Study verified multiple neurotrophic factor; as Brain Derived Neurotrophic Factor (Brain-derived NeurotrophicFactor; BDNF), ciliary neurotrophic factor (Ciliary Neurotrophic Factor; CNTF), neurogliocyte derived neurotrophic factor (Glial Cell Line-derived Neurotrophic Factor; GDNF), (Erythropoietin, EPO) grade has clear and definite protective effect to impaired retinal ganglial cells to erythropoietin.
But in the prior art, with the example that is applied as of EPO, the whole body administration needs dosage big, can cause angiogenesis increase and erythropoiesis and produces serious toxic and side effects, and be difficult to reach retina and bring into play its effective drug action; The vitreous chamber direct injection shows tangible neuroprotective in a short time, but because the half-life is short, it protects effect to weaken until disappearance very soon along with metabolism.In the clinical practice, for reaching permanently effective protective effect, need regularly intraocular injection repeatedly, this has just increased the complication that operation causes greatly, as hemorrhage, infection, concurrent cataract etc.Domestic literature has been reported local therapeutic scheme at vitreous body intracavitary administration EPO at this problem, wherein proposed to reach the method for treatment and prophylactic treatment retina injury at the solution of vitreous body intracavitary administration EPO, but after tested, EPO the half-life behind the vitreous body intracavitary administration still between 24-36 hour, show that its half-life do not obtain prolonging, so still can not substitute systemic administration.
Equally, have other neurotrophic factors of retinal neuronal cell protective effect, have identical shortcoming in retina optic nerve disease application facet and require study and capture.In order to overcome above-mentioned shortcoming; to reach long lasting neuroprotective; can avoid simultaneously a series of complication of causing because of multiple injection again, provide the long-acting slow-release preparation that to bring into play this class medicine of curative effect for a long time, lastingly to cause the concern of relevant research worker.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, a kind of long-acting slow-release preparation of retina injury and preparation method thereof that is used to prevent or treat is provided.
Particularly, the present invention is used to prevent or treat the long-term sustained-release administration of long-acting slow-release preparation employing erythropoietin sustained-release micro-spheres method of retina injury, prior art can be solved and the difficult problem that this class disease of retina injury needs for a long time frequent drug administration by injection can't be overcome for a long time, make the scheme of more acceptant treatment of retina injury patient and control, alleviate patient's misery.。
It is active component that long-acting slow-release preparation of the present invention adopts erythropoietin (EPO), and glucosan is the active component protective agent, and the polylactic acid-glycolic guanidine-acetic acid gathers (PLGA), polylactic acid (PLA) or polycaprolactone (PCL) and makes sustained-release micro-spheres for the parcel composition; Its particle diameter of described sustained-release micro-spheres is: 0.10-500 μ m, its percentage by weight of each component is that the percentage by weight of polylactic acid-glycolic guanidine-acetic acid, polylactic acid or polycaprolactone is 50%-99%, erythropoietin is 0.5%-20%, and glucosan is 0.5%-30%.
Each component of above-mentioned sustained-release micro-spheres prepares the erythropoietin polyoses grain by the low temperature induction phase disengagement method, the method of reuse oil-in-water/oily Bao Gu (S/O/W) or oil bag oil/oily Bao Gu (S/O/O) prepares erythropoietin sustained-release micro-spheres (EPO-PLGA), described polylactic acid-glycolic guanidine-acetic acid, polylactic acid or polycaprolactone parcel erythropoietin; Prepared erythropoietin sustained-release micro-spheres is a long-acting slow-release preparation, can be used for prevention or treatment retina injury.
Among the present invention, described retina injury comprises: retina cell damage or retinal tissue damage.Wherein the retina cell damage comprises: retinal pigment epithelium, retinal neuronal cell, retinal endothelial cell or pericyte's damage.
Among the present invention, described retinal tissue damage comprises degeneration of macula.
Among the present invention, described retinal neuronal cell comprises group down: cone cell, rod cell, bipolar cell, Muller cell, horizontal cell, amacrine cell, ganglionic cell.
Among the present invention, described cell injury mainly is an apoptosis.
Long-acting slow-release preparation erythropoietin sustained-release micro-spheres of the present invention wraps solid method (S/O/O) preparation by following oil-in-water/solid method (S/O/W) of oil bag or oil bag oil/oil:
1. the preparation of erythropoietin dextran microparticles
A) percentage by weight that will account for the erythropoietin microsphere in advance be the erythropoietin of 0.5%-20% to be mixed with concentration expressed in percentage by weight be 0.01%-10% solution, the percentage by weight 0.5-30% that glucosan accounts for the erythropoietin microsphere is mixed with the solution of the 1%-30% of concentration expressed in percentage by weight; The preparation concentration expressed in percentage by weight is the polyglycol solution of 1%-30%;
B) above-mentioned erythropoietin solution, dextran solution, PEG solution are mixed and mixing according to certain volume ratio;
C) and then with above-mentioned steps b) mixture of prepared mix homogeneously is refrigerating chamber pre-freeze 8-32 hour, lyophilizing then,
D) with above-mentioned steps c) powder after the lyophilizing utilizes organic solvent dissolution Polyethylene Glycol (PEG), centrifugally remove the organic solution that supernatant is PEG, triplicate, volatilization is done and is collected then, and the reuse organic solvent is removed the dextran microparticles that poly-ethanol (PEG) promptly gets erythropoietin;
2. the preparation of erythropoietin sustained-release micro-spheres
A) after 1. the microgranule of erythropoietin joins polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA) or polycaprolactone (PCL) organic solution that concentration expressed in percentage by weight is 5-25% with step, form suspension, promptly stir in the oil phase (O) or whirlpool etc. makes it homodisperse and forms uniform suspension;
B) completing steps a) being formed the erythropoietin microsphere suspension, to be added to sodium chloride solution and the weight percent concentration that weight percent concentration is 0%-10% be 1%-10% surfactant emulsifying 1-5min, and the sodium chloride solution of then it being transferred to weight percent concentration and be 1%-10% solidified 1-4 hour; Wash the microsphere of collecting with water three to five times again and remove surfactant and sodium chloride;
C) with completing steps b) the sample lyophilizing remove moisture and promptly obtain exsiccant its particle diameter of erythropoietin sustained-release micro-spheres and be: 0.10-500 μ m.
Or,
1) preparation erythropoietin dextran microparticles
With above-mentioned step 1.;
2) preparation erythropoietin sustained-release micro-spheres
D) after 1. the microgranule of erythropoietin joins polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA) or polycaprolactone (PCL) organic solution that concentration expressed in percentage by weight is 5-25% with step, form suspension, promptly stir in the oil phase (O) or whirlpool etc. makes it homodisperse and forms uniform suspension;
E) completing steps a) is formed the erythropoietin microsphere suspension and be added to Oleum Gossypii semen, mineral oil emulsifying 1-5min, it is transferred in petroleum ether or the ether solidified 1-4 hour then; Then the microsphere of collecting is removed residual organic solvent three to five times with the ether washing;
F) with completing steps b) sample volatilize and remove organic solvent and obtain exsiccant erythropoietin sustained-release micro-spheres, its particle diameter is: 0.10-500 μ m.
In the method for the invention, the weight percent concentration of described erythropoietin is: 0.01%-20%,
The weight percent concentration of described glucosan is: 1%-30%, the molecular weight of glucosan are 10000-5000000 dalton;
The molecular weight of described PEG is: 2, and 000-300,000 dalton, the weight percent concentration in aqueous solution is 1%-40%;
The dextran microparticles of described erythropoietin, its particle grain size size be at 0.01 μ m-5 μ m,
Described oil phase (O) is: the organic solution of polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL) or their any mixture;
Organic solvent in the described organic solution is: dichloromethane, ethyl acetate, acetonitrile, heptane, chloroform or acetone organic solution among the present invention, are good with the organic solution of dichloromethane, ethyl acetate, acetonitrile or their combination in any;
The organic solution concentration of described polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL) or their any mixture is: the weight percent concentration of polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL) or their any mixture is 5%-30%;
Described polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA), their molecular weight of polycaprolactone (PCL) are 5000-500000 dalton;
Described surfactant is: polyvinyl alcohol (PVA), Polyethylene Glycol (PEG), polyvinylpyrrolidone (PVP) or poloxamer (poloxmer);
Described polyvinyl alcohol (PVA), Polyethylene Glycol (PEG), polyvinylpyrrolidone (PVP) or their molecular weight of poloxamer (poloxmer) are respectively: 10000-1000000 dalton, 4000-300000 dalton, 20000-400000 dalton or 10000-400000 dalton;
The weight percent concentration of described surface active agent polyvinyl alcohol (PVA) is 0.5%-10% or the solution that contains 0.5%-10% sodium chloride, the weight percent concentration of Polyethylene Glycol (PEG) is 0.5%-20% or the saline solution that contains 0.5%-10% sodium chloride, polyvinylpyrrolidone (PVP) weight percent concentration is 0.5%-20% or to contain weight percent concentration be saline solution such as 0.5%-10% sodium chloride, poloxamer (poloxmer) weight percent concentration is 0.5%-20% or contains saline solution such as 0.5%-10% sodium chloride.
It is mixed that described promoting erythrocyte sustained-release micro-spheres and 0.01M PBS press constant weight percentage ratio, and its ratio is 1: 0.5-1: 20.
Erythropoietin sustained-release micro-spheres long-acting slow-release preparation of the present invention can adopt topical in eye vitreous body, subcutaneous injection or lumbar injection approach; all can produce good protective effect to impaired retina; sustained-release micro-spheres can reduce administration number of times, the complication of avoiding multi-pass operation to cause.
The present invention studies the long-effective protection effect of the impaired retinal ganglion cell of contusion of optic nerve adult SD rats by erythropoietin sustained-release micro-spheres vitreous chamber is injected; the result shows; do not treat with contusion of optic nerve only and to compare; this erythropoietin sustained-release micro-spheres can obviously improve the survival rate of impaired retinal ganglial cells; and this sustained-release micro-spheres single vitreous chamber injection can reach erythropoietin albumen and repeat repeatedly the vitreous chamber injection with the retinal ganglial cells protective effect of imitating; and significantly alleviate the complication that causes because of multiple injection; experiment confirm, this erythropoietin sustained-release micro-spheres has significant long lasting protective effect to impaired retinal ganglial cells.
Description of drawings
The sem photograph of Fig. 1 erythropoietin dextran microparticles.
The sem photograph of Fig. 2 erythropoietin sustained-release micro-spheres.
After Fig. 3 contusion of optic nerve 5 days, TUNEL detected the RGCs apoptosis situation of respectively organizing, and visible normal retina section (A, B, C) ganglion cell layer of retina does not detect the RGCs of apoptosis, each experimental group (not treatment group: D-F; EPO-PLGA group: G-I; EPO group: J-L; PLGA group: M-O; PBS group: P-R) the positive RGCs of all visible TUNEL, maximum with PLGA group, PBS group TUNEL positive cell, apoptosis is the most obvious.EPO group and EPO-PLGA organize rarely seen a small amount of TUNEL positive cell.(figure A, D, G, J, M, P are that TUNEL detects picture; Figure B, E, H, K, N, Q are DAPI mark transfect cell nuclear; Figure C, F, I, L, O, R are that TUNEL and DAPI merge.)
Respectively organized the comparison of RGCs apoptosis rate after Fig. 4 contusion of optic nerve in 5 days,
*Expression is compared with not treatment group, P＜0.001.
Respectively organize RGCs density after Fig. 5 contusion of optic nerve in 4W, 8 weeks relatively,
*Represent that each experimental group compares P＜0.001 with not treatment group.
The specific embodiment
Below embodiments of the invention are elaborated: following examples have provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
1. the dextran microparticles for preparing erythropoietin
A) percentage by weight that will account for the erythropoietin microsphere in advance is respectively 0.5%, 10% or 20% erythropoietin and is mixed with concentration expressed in percentage by weight and is respectively 0.01%, 10% or 20% solution, and the percentage by weight that respectively glucosan (molecular weight be 10000,2500000 or 5000000 daltonian) is accounted for the erythropoietin microsphere is respectively 0.5%, 15% or 30%, and to be mixed with concentration expressed in percentage by weight be 1%, 15% or 30% solution; Respectively Polyethylene Glycol (molecular weight is 2000,150000 or 300000) is mixed with concentration expressed in percentage by weight and is respectively 1%, 20% or 40% solution.
B) above-mentioned erythropoietin solution is pressed erythropoietin solution: dextran solution: PEG solution is according to volume ratio=50: 1: 200,1: 1: 8 or 2: 3: 12 mixed and mixings of volume ratio;
C) then with above-mentioned steps b) mixed evenly, in refrigerating chamber pre-freeze 8-32 hour, lyophilizing then,
D) with above-mentioned steps c) powder after the lyophilizing utilizes organic solvent dissolution Polyethylene Glycol (PEG), centrifugally remove the organic solution that supernatant is PEG, triplicate, volatilization is done and is collected then, the reuse organic solvent is removed the dextran microparticles that poly-ethanol (PEG) promptly obtains erythropoietin, and the part by weight of erythropoietin in the microgranule and glucosan was respectively 1: 1,10: 15 or 20: 30;
2. prepare the erythropoietin sustained-release micro-spheres
A) microgranule of the erythropoietin that the 1. corresponding ratio of step is prepared is got 1mg respectively, 2.5mg or 3mg to join concentration expressed in percentage by weight be 30% 330mg polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA), or the dichloromethane solution of polycaprolactone (PCL) (the erythropoietin sustained-release micro-spheres of preparation is contained 0.5% erythropoietin and 0.5% glucosan), concentration expressed in percentage by weight is 15% 50mg polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA), or the dichloromethane solution of polycaprolactone (PCL) (the erythropoietin sustained-release micro-spheres of preparation is contained 10% erythropoietin and 15% glucosan), or the polylactic acid-glycolic guanidine-acetic acid (PLGA) of 5% 140mg, or the dichloromethane solution of polycaprolactone (PCL) (the erythropoietin sustained-release micro-spheres of preparation is contained 20% erythropoietin and 30% glucosan), form suspension.Be to stir in the oil phase (O) or whirlpool etc. makes it homodisperse and forms uniform suspension;
B) the erythropoietin microsphere suspension that completing steps a) is formed be added to respectively weight percent concentration be 0.5% sodium chloride solution and weight percent concentration be 1%PVA, PVP or PEG surfactant 4mL in emulsifying 1-5min, then it being transferred to weight percent concentration is to solidify 1-4 hour among 10% the sodium chloride solution 1000mL; Wash the microsphere of collecting with water 3-5 time then and remove surfactant and sodium chloride;
C) with completing steps b) the sample lyophilizing remove moisture and obtain exsiccant erythropoietin sustained-release micro-spheres.
The about 1-2 μ of the Scanning Electron Microscope photos reveal size m of the erythropoietin glucose microgranule of preparation, (as shown in Figure 1); The about 40-100 μ of the Scanning Electron Microscope photos reveal size m (as shown in Figure 2) of the promoting erythrocyte sustained-release micro-spheres of preparation.
The erythropoietin sustained-release micro-spheres of above-mentioned preparation and 0.01M PBS according to 1: 0.5,1: 1,1: 0.5 or 1: 2 mixing, are used for the vitreous administration of adult SD rats eyes.
Embodiment 2
1. the dextran microparticles for preparing erythropoietin
With the step of embodiment 1 1..
A) microgranule of the erythropoietin that the 1. corresponding ratio of step is prepared is got 1mg respectively, 2.5mg or 3mg to join concentration expressed in percentage by weight be 30% 330mg polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA), or the dichloromethane solution of polycaprolactone (PCL) (the erythropoietin sustained-release micro-spheres of preparation is contained 0.5% erythropoietin and 0.5% glucosan), concentration expressed in percentage by weight is 15% 50mg polylactic acid-glycolic guanidine-acetic acid (PLGA), polylactic acid (PLA), or the dichloromethane solution of polycaprolactone (PCL) (the erythropoietin sustained-release micro-spheres of preparation is contained 10% erythropoietin and 15% glucosan), or the polylactic acid-glycolic guanidine-acetic acid (PLGA) of 5% 140mg, or the dichloromethane solution of polycaprolactone (PCL) (the erythropoietin sustained-release micro-spheres of preparation is contained 20% erythropoietin and 30% glucosan), form suspension.Be to stir in the oil phase (O) or whirlpool etc. makes it homodisperse and forms uniform suspension;
B) completing steps a) is formed the erythropoietin microsphere suspension and be added to emulsifying 1-5min among Oleum Gossypii semen, silicone oil or the mineral oil 4mL that the weight percent concentration weight percent concentration is 1%PVP, poloxamer or PEG respectively, it is transferred in the petroleum ether of 200mL or the ether solidified 1-4 hour then; Then the microsphere of collecting is removed residual organic solvent three to five times with the ether washing;
C) with completing steps b) sample volatilize and remove organic solvent and obtain exsiccant erythropoietin sustained-release micro-spheres.
The about 1-2 μ of the Scanning Electron Microscope photos reveal size m (as shown in Figure 1) of the erythropoietin glucose microgranule of preparation, the about 40-100 μ of the Scanning Electron Microscope photos reveal size m (as shown in Figure 2) of the promoting erythrocyte sustained-release micro-spheres of preparation.
The vitreous administration that the erythropoietin sustained-release micro-spheres of above-mentioned preparation and 0.01M PBS is used for the adult SD rats eyes according to 1: 0.5,1: 1,1: 0.5 or 1: 2 mixing.
Embodiment 3
Apoptosis original position apoptosis detection method (TUNEL detection) is observed the protection effect of long-acting slow-release preparation EPO sustained-release micro-spheres of the present invention to impaired retinal ganglial cells (RGCs)
1. laboratory animal and grouping: adult male SD rats (about 200g), adaptability is fed after 7 days and is checked eyes, and it is clear to look into refractive media, circle such as isocoria, the light reflex sensitivity, the optical fundus is no abnormal.With the left eye is the contusion of optic nerve eye, and right eye is the normal control eye.Difference according to contusion of optic nerve rear vitreous body chamber drug administration by injection is divided into following five groups: not treatment group (vitreous chamber is not injected), EPO group (vitreous chamber injection EPO), EPO-PLGA group (vitreous chamber injection EPO-PLGA microsphere), PBS group (vitreous chamber injection 0.01M PBS), PLGA group (vitreous chamber injection PLGA microsphere).
2. animal model is set up and administration: behind chlore-ammonia ketone (80mg/kg) and xylazine (12mg/kg) the lumbar injection general anesthesia, the all local cleaning-sterilizings of SD rat left eye go in ring and cut off outer canthus portion bulbar conjunctiva, open Tenon ' s capsule, passivity is separated Suspensory ligament, the tractive eyeball exposes optic nerve forward, uses microhemostat, the power of holding is 70g, about 1mm place's clamping optic nerve caused optic nerve injury in 60 seconds behind ball, the stitch ball conjunctiva, but enlightening sieve eye ointment is coated with eye.The Marcus-gun pupil appearred on the 2nd day in postoperative, and eyeball does not have obviously outstanding, and the optical fundus does not have the bleeder and is the model success.Right eye is not performed the operation as normal control.Prepared microsphere is suspended from 0.01M PBS, behind the optic nerve injury, not treatment group will not be injected by vitreous chamber, EPO organizes vitreous body intracavitary administration EPO10IU (5 μ L) at once, the EPO-PLGA group gives vitreous body intracavitary administration EPO-PLGA sustained-release micro-spheres 0.2mg (5 μ L at once, contain EPO 20IU), the PBS group gives vitreous body intracavitary administration 0.01M PBS (5 μ L) at once, and the PLGA group gives the vitreous body intracavitary administration blank PLGA microsphere 0.2mg (5 μ L) at once.Postoperative reached postoperative at that time and observed down every 1 day operating microscope, intraocular hemorrhage, concurrent cataract, intraocular infection etc. did not occur, included experiment in.
3. retina section original position apoptosis detects: postoperative 5 days is with normal saline 200～250mL, 4% paraformaldehyde 200mL takes out eyeball after the heart perfusion is fixing, after eyeball is in 4% paraformaldehyde, fix 2 hours, successively in 20% sucrose, 30% sucrose soaked overnight, cut off cornea along limbus of corneae, remove crystal and vitreous body.Fill up eyeball with the OCT embedding medium, make continuous frozen section, thickness 10 μ m along the eyeball sagittal plane.The original position apoptosis detects and adopts original position apoptosis detection method (TUNEL staining).The frozen section room temperature is dried, with 4% paraformaldehyde 15-20 ℃ fixedly behind the 20min, 0.01M PBS washes 30min, and reuse is freshly prepared to contain 0.1%Triton X-100, the slow liquid of 0.1% sodium citrate soaks 2min for 4 ℃ towards liquid, 0.01M PBS washes 2min, washes 2 times.Behind the dry specimen periphery microscope slide, drip the TUNEL mixed reaction solution, in 37 ℃ of following 60min, 0.01M PBS washes 2min, washes 3 times.Add DAPI reactant liquor (1 μ g/mL), in 37 ℃ of following 10min, last 0.01M PBS flushing 2min washes 3 times.Dry slide, the glycerol mounting.Fluorescence microscope.
4. result: normal SD rats retina section TUNEL detects visible ganglion cell layer of retina negative (shown in Fig. 3 A-C), does not see apoptotic cell.After the contusion of optic nerve 5 days, each experimental group retinal ganglion cells apoptosis peaked, and all visible TUNEL positive cell of each group is wherein maximum with not treatment group, PBS group, PLGA group TUNEL positive cell, apoptosis the most obvious (as Fig. 3 D-F, M-O is shown in the P-R); EPO group and EPO-PLGA group are (as Fig. 3 G-I, shown in the J-L) ganglion cell layer of retina also has the TUNEL positive cell, but obviously be less than not treatment group, PBS group, PLGA group, tool significant difference (p＜0.001) (as shown in Figure 4), and no difference of science of statistics (p＞0.05) between EPO group and the EPO-PLGA group.
The result shows that EPO-PLGA sustained-release micro-spheres of the present invention has remarkable protective effect to impaired RGCs, and protection effect and epo protein are with imitating.
The contrary mark of DiI detects the long-effective protection effect of long-acting slow-release preparation EPO sustained-release micro-spheres of the present invention to impaired retinal ganglial cells (RGCs)
Step 1. with embodiment 3 in 1. identical;
2. animal model is set up and administration: behind chlore-ammonia ketone (80mg/kg) and xylazine (12mg/kg) the lumbar injection general anesthesia, the all local cleaning-sterilizings of SD rat left eye go in ring and cut off outer canthus portion bulbar conjunctiva, open Tenon ' s capsule, passivity is separated Suspensory ligament, the tractive eyeball exposes optic nerve forward, uses microhemostat, the power of holding is 70g, about 1mm place's clamping optic nerve caused optic nerve injury in 60 seconds behind ball, the stitch ball conjunctiva, but enlightening sieve eye ointment is coated with eye.The Marcus-gun pupil appearred on the 2nd day in postoperative, and eyeball does not have obviously outstanding, and the optical fundus does not have the bleeder and is the model success.Right eye is not performed the operation as normal control.Prepared microsphere is suspended from 0.01M PBS, behind the optic nerve injury, not treatment group will not be injected by vitreous chamber, EPO group respectively at once with modeling after 4 all vitreous body intracavitary administration EPO10IU (5 μ L), the EPO-PLGA group gives vitreous body intracavitary administration EPO-PLGA sustained-release micro-spheres 0.2mg (5 μ L at once, contain EPO 20IU), the PBS group is respectively at giving vitreous body intracavitary administration 0.01M PBS (5 μ L) 4 weeks at once with after the modeling, and the PLGA group gives the vitreous body intracavitary administration blank PLGA microsphere 0.2mg (5 μ L) at once.Postoperative reached postoperative at that time and observed down every 1 day operating microscope, intraocular hemorrhage, concurrent cataract, intraocular infection etc. did not occur, included experiment in.
3. the superior colliculus labelling survival retinal ganglial cells that drives in the wrong direction: before the SD rat conventional treatment.With electric drill (the about 1.1mm of bit diameter) in the boring of the corresponding position of bilateral superior colliculus, in the other 1.2mm that opens of posterior, 2.0mm forward, inserting needle is injected DiI 5 μ L to degree of depth 3.2mm place with the microsyringe of 10 μ L, the stitching scalp, suture is coated with antibiotic.
4. retina shop sheet ganglion cell counts: 4 weeks of postoperative, 8 weeks are used normal saline 200～250mL respectively, 4% paraformaldehyde 200mL takes out the left eye eyeball after the heart perfusion is fixing, after eyeball is in 4% paraformaldehyde, fix 2 hours, in 0.01M PBS, remove anterior ocular segment and vitreous body, separate retina, retina be tiled on the microscope slide of gelatinization, retinal periphery do 4 places perpendicular to the otch of optic disc so that the retina tiling, buffering glycerol mounting, fluorescence microscope is observed down.Four quadrants of every retina are taken 12 200 times fluorescence photo altogether apart from optic disc 1/6,3/6,5/6 radius, and the RGCs of labelling in each visual field is counted, and ask the meansigma methods of RGCs density (individual/mm
2).
5. result: retina shop sheet RGCs counting shows, normal SD rats RGCs density is 2387.69 ± 164.87 (individual/mm
2); In 4 weeks of postoperative, not treatment group, EPO group, EPO-PLGA group, PBS group and PLGA group RGCs density are (individual/mm
2) be respectively 748.30 ± 58.81,1418.52 ± 154.91,1296.68 ± 157.55,804.43 ± 86.44 and 821.72 ± 52.13; In 8 weeks of postoperative, not treatment group, EPO group, EPO-PLGA group, PBS group and PLGA group RGCs density are (individual/mm
2) be respectively 532.74 ± 35.14,1083.66 ± 57.82,913.98 ± 37.72,548.01 ± 47.00 and 561.96 ± 34.23; EPO group and more not treatment group of EPO-PLGA group have tangible retinal ganglial cells (RGCs) protective effect, and statistical significance is (P＜0.001) significantly, and no significant difference (P＞0.05) between EPO group and EPO-PLGA group.(as shown in Figure 5).
Experimental result show single vitreous chamber injection EPO-PLGA sustained-release micro-spheres to the protection effect of impaired RGCs with repeat regular vitreous chamber injection epo protein with imitating; the result shows that also single injection can significantly reduce because of the complication of duplicate injection to causing; can reduce dosage and treatment cost, can not produce harmful effect other organ or tissue in the body.The result confirms that EPO-PLGA sustained-release micro-spheres of the present invention has long lasting retinal ganglial cells protective effect.
Claims (10)
- One kind the prevention or the treatment retina injury long-acting slow-release preparation, it is characterized in that, with the erythropoietin is active component, and glucosan is the active component protective agent, and the polylactic acid-glycolic guanidine-acetic acid is poly-, polylactic acid or polycaprolactone are made sustained-release micro-spheres for the parcel composition; Described polylactic acid-glycolic guanidine-acetic acid, polylactic acid or polycaprolactone parcel erythropoietin;The percentage by weight of the described sustained-release micro-spheres of each ingredients constitute is: polylactic acid-glycolic guanidine-acetic acid, polylactic acid or polycaprolactone are 50%-99%, and erythropoietin is 0.5%-20%, and glucosan is 0.5%-30%.
- 2. by the long-acting slow-release preparation of described prevention of claim 1 or treatment retina injury, it is characterized in that its particle diameter of described sustained-release micro-spheres is: 0.10-500 μ m.
- 3. press the preparation method of the long-acting slow-release preparation of described prevention of claim 1 or treatment retina injury, it is characterized in that, adopt oil-in-water/solid method (S/O/W) of oil bag or the solid method (S/O/O) of oily bag oil/oil bag, comprise that step is as follows:1. prepare the erythropoietin dextran microparticlesA) erythropoietin of 0.5%-20% being mixed with concentration expressed in percentage by weight is 0.01%-10% solution, and the glucosan of 0.5-30% is mixed with the solution of concentration expressed in percentage by weight 1%-30%; The preparation concentration expressed in percentage by weight is the polyglycol solution of 1%-30%;B) above-mentioned erythropoietin solution, dextran solution or polyglycol solution are mixed and mixing according to volume ratio;C) with above-mentioned steps b) mixture of the mix homogeneously of preparation is refrigerating chamber pre-freeze 8-32 hour, lyophilizing then,D) with above-mentioned steps c) powder organic solvent dissolution Polyethylene Glycol after the lyophilizing, the centrifugal supernatant of removing, triplicate, volatilization is done and is collected, the reuse organic solvent is removed Polyethylene Glycol, the dextran microparticles of erythropoietin;2. prepare the erythropoietin sustained-release micro-spheresA) dextran microparticles of the erythropoietin that 1. step is made adding concentration expressed in percentage by weight is polylactic acid-glycolic guanidine-acetic acid, polylactic acid or the polycaprolactone organic solution of 5-25%, forms suspension;B) suspension of step a) being added weight percent concentration is that sodium chloride solution and the weight percent concentration of 0%-10% is 1%-10% surfactant emulsifying 1-5min, and the sodium chloride solution of then it being transferred to weight percent concentration and be 1%-10% solidified 1-4 hour; Wash the microsphere of collecting with water three to five times and remove surfactant and sodium chloride;C) moisture is removed in the sample lyophilizing of step b) and made the erythropoietin sustained-release micro-spheres; Or,1) preparation erythropoietin dextran microparticlesWith above-mentioned step 1.;2) preparation erythropoietin sustained-release micro-spheresD) after the dextran microparticles of the erythropoietin that 1. step is made adding concentration expressed in percentage by weight is polylactic acid-glycolic guanidine-acetic acid, polylactic acid or polycaprolactone (PCL) organic solution of 5-25%, form suspension;E) suspension that step d) is formed is added to Oleum Gossypii semen, mineral oil emulsifying 1-5min, then it is transferred in petroleum ether or the ether to solidify 1-4 hour; Then the microsphere of collecting is removed residual organic solvent three to five times with the ether washing;F) sample of step e) volatilized remove organic solvent, exsiccant sustained-release micro-spheres.
- 4. by the described preparation method of claim 3, it is characterized in that the molecular weight of described glucosan is 10000-5000000 dalton.
- 5. by the described preparation method of claim 3, it is characterized in that the molecular weight of described Polyethylene Glycol is: 2,000-300,000 dalton.
- 6. by the described preparation method of claim 3, it is characterized in that the dextran microparticles particle diameter of described erythropoietin is 0.01 μ m-5 μ m.
- 7. by the described preparation method of claim 3, it is characterized in that the molecular weight of described polylactic acid-glycolic guanidine-acetic acid, polylactic acid or polycaprolactone is 5000-500000 dalton.
- 8. by the described preparation method of claim 3, it is characterized in that, described surfactant is: polyvinyl alcohol, Polyethylene Glycol, polyvinylpyrrolidone or poloxamer, molecular weight is respectively: 10000-1000000 dalton, 4000-300000 dalton, 20000-400000 dalton or 10000-400000 dalton.
- 9. by the long-acting slow-release preparation of described prevention of claim 1 or treatment retina injury, it is characterized in that described retina injury is selected from: retina cell damage or retinal tissue damage.
- 10. the long-acting slow-release preparation of claim 1 is preparing the purposes for the treatment of in retina cell damage or the retinal tissue damage medicine, and wherein, erythropoietin sustained-release micro-spheres and 0.01M PBS are by 1 in the described medicine: 0.5-1: 20 percentage by weights are mixed.
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| CN111712228B (en) * | 2017-09-15 | 2024-05-07 | 奥叙拉尔有限公司 | Ophthalmic pharmaceutical composition |
| RU2705723C1 (en) * | 2018-06-26 | 2019-11-11 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Южно-Уральский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО ЮУГМУ Минздрава России) | Rectal suppositories with erythropoietin, having reparative and antioxidant activity |
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| CN108938600B (en) * | 2018-07-11 | 2021-10-08 | 南京锐利施生物技术有限公司 | Microspheres for intravitreal injection and double protection of antibody drugs and preparation method thereof |
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| CN114870023A (en) * | 2022-05-12 | 2022-08-09 | 成都市第三人民医院 | Slow-release optic nerve protection drug nano synthetic material and preparation method and application thereof |
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