CN101972470B - In-situ gel composition for eyes - Google Patents
In-situ gel composition for eyes Download PDFInfo
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Abstract
The invention relates to an in-situ gel composition for eyes, comprising the following materials: 50 mu g/ml-400 mu g/ml of nerve growth factor, 1 mg/ml-20 mg/ml of stabilizer, 3 mg/ml-10 mg/ml of ion sensitive gel and water; and the pH value of the in-situ gel composition is 4.5-7.5. In the invention, by using the composition and proportion and controlling the pH value of the in-situ gel composition to be within certain range, the in-situ gel preparation for the eyes containing nerve growth factors can be obtained. When being stored under the normal-temperature state, the preparation is liquid, and becomes a stable semisolid gel state after entering the eyes.
Description
Technical field
The present invention relates to a kind of ocular in-situ gel compositions.
Background technology
Advantages such as Laser in Situ Keratomileusis (LASIK) is light, safe owing to after-operation response, predictability is good are the main flow art formulas in the present cornea refractive surgery.But in the postoperative 6 months almost 50% case the xerophthalmia symptom appears, part patient has discomforts such as foreign body sensation, has influenced the satisfaction of patient to operation to some extent.Discover in appearance and the art of these symptom and signs that when cutting cornea flap, cutting angle membrane matrix, it is relevant to have damaged corneal nerve to some extent.The drug research of short corneal nerve injury repairing is present research focus.
Nerve growth factor (NGF) comes to light in the neurotrophic factor the earliest; Research is the most thorough at present; A kind of nerve growth regulatory factor with neuron nutrition and the dual biological function of short enation, it all has important regulation to the expression of growth, differentiation, growth, regeneration and the functional characteristic of maincenter and peripheral nerve unit.Nerve growth factor is to have the biological activity protein that promotes neuronal survival, differentiation and the effect of injured nerve Regeneration and Repair, is one of important biological material in the human body, and it plays an important role to promoting and keeping the human life function.It is to treat the ideal active drug of nerve injury at present, and its effect mainly is to improve the survival rate of injured nerve cell, promotes the injured nerve fiber along correct direction growth, becomes to making the nerve fiber of growth to be positioned target cell, forms functional connection.Use NGF both at home and abroad clinically more and more widely as various nerve injury reparations, comprise means traumatic, that physiological (old age) nerve injury is repaired, and be the unique effective biological preparation that is used for the nerve injury treatment at present both at home and abroad.
At present, the clinical NGF that uses is injection, uses inconvenience, needs housebroken professional person to participate in, and greatly limited its application clinically, and most of biological activity is degraded inactivation in conductive process and can not make NGF reach due optimum therapeuticing effect.
The eye that uses is clinically used the topical dosage form: eye drop, account for ophthalmic preparation more than 60%, and easy to use, cheap.But the holdup time is short, bioavailability is low.People's normal tear fluid capacity is about 7 μ l, if do not bat an eyelid, can hold the liquid about 30 μ l.General eye drop estimates to have approximately 70% medicinal liquid to overflow and cause damage from eye, if 90% medicinal liquid loss will be arranged nictation.The medicinal liquid major part of overflowing flows down along buccal, or gets into nasal cavity or digestive tract finally by systemic absorption through nasolacrimal duct.Hydrogel, prolong drug improve bioavailability in the holdup time of eye.But this gellike divided dose is inaccurate, and administration is inconvenient.Eye ointment, the long medicine holdup time, but inaccurate because of its dosage, be prone to cause and stick with paste that looking flakes with eyelid causes patient's poor compliance, and the preceding use of can only sleeping usually.Liposome has advantages such as the cornea of increasing permeability, slow release and reduction toxic reaction.But its drug loading is low, poor stability, and the big commercial production cost of asepticize is high, technical difficult.
Ocular in-situ gel: after the liquid condition administration, take place to change mutually at agents area immediately, form the ophthalmic preparation of semi-solid gel state.The FDA approved this series products: Timoptic-XE, PDR records.Also have two artificial tears's ocular in-situ gels that adopt poloxamer 407 preparations in Australia and Japan's listing.Compare with the topical dosage form with the tradition eye; Ocular in-situ gel has following characteristics: (1) is bioadhesive preferably; Closely contact with eye for a long time, overcome that conventional formulation can be washed away by tear very soon and the shortcoming that can't reach active drug concentration has improved bioavailability.(2) histocompatibility is good, and is easy to use, reduced administration frequency, and patient's compliance improves.(3) tridimensional network highly-hydrophilic, can with medicine be bound by wherein or its gap in, control drug release.(4) physicochemical property is special, because of it is runny liquid condition under vitro conditions, so be prone to fill, is convenient to suitability for industrialized production.(5) dosage is accurate, and long term administration also is difficult for causing general toxicity and untoward reaction.(6) bioadhesive preferably closely contacts with eye for a long time, overcome that conventional formulation can be washed away by tear very soon and the shortcoming that can't reach active drug concentration has improved bioavailability.
But also there is not a kind of ocular in-situ gel that includes nerve growth factor at present.
Summary of the invention
Technical problem to be solved by this invention is, a kind of ocular in-situ gel that includes nerve growth factor is provided.
Why up to now the ocular in-situ gel of nerve growth factor does not still appear including on the market; Be because also do not find a kind of suitable proportioning; Make the ocular in-situ gel that includes nerve growth factor before getting into eye, be liquid, and after getting into eyes, become stable semi-solid gel state.
The inventor has successfully developed this ocular in-situ gel that includes nerve growth factor.
In order to solve the problems of the technologies described above, the present invention provides following technical scheme:
A kind of ocular in-situ gel compositions comprises:
Nerve growth factor 50 μ g/ml～400 μ g/ml;
Stabilizing agent 1mg/ml～20mg/ml;
Ion-sensitive type gel 3mg/ml～10mg/ml;
Water;
The pH value of this situ-gel compositions is 4.5～7.5.
Preferably, said stabilizing agent is an aminoacid.
Preferably, said stabilizing agent is the mixture of aminoacid and non-ionic surface active agent.
Preferably, said stabilizing agent is aminoacid and acetate.
Preferably, said aminoacid is alanine, glycine and arginic mixture.
Preferably, also comprise the pH value buffer agent.
Preferably, said pH value buffer agent is phosphate, acetate, citric acid and salt, carbonate.
Preferably, the ion-sensitive type gel is selected from gellan gum.
Preferably, this situ-gel compositions splashes into ophthalmic and forms gel in the external liquid condition that is.
Preferably, also comprise antiseptic.
Preferably, said antiseptic is selected from benzyl alcohol, phenol, metacresol, methyl butex and propylparaben, trichlorine tert-butylamine alcohol, benzalkonium chloride, benzalkonium bromide, hibitane.
Preferably, the amount of said antiseptic is 0mg/ml～100mg/ml.
Preferably, also comprise osmotic pressure regulator.
Preferably, said osmotic pressure regulator is selected from mannitol, sorbitol, sodium chloride, sodium citrate.
Preferably; Also comprise medical hydrophilic high molecular material, contain in the following material one or more in the said medical hydrophilic high molecular material: methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl emthylcellulose, polyvinylpyrrolidone, poloxamer, polyvinyl alcohol, hyaluronate sodium, chitosan, sodium glycerophosphate.
In the present invention, through using above-mentioned composition and proportioning, and the pH value of control situ-gel compositions can obtain including the ocular in-situ gel preparation of nerve growth factor within certain scope.When said preparation was preserved under the room temperature state, said preparation was liquid, after getting into eyes, just becomes stable semi-solid gel state.
The NGF ocular in-situ gel of Isodose is compared with common NGF eye drop, and the NGF ocular in-situ gel is administered once and can reaches the common NGF eye drop identical effectiveness that is administered three times.
The specific embodiment
For those skilled in the art can understand technical scheme of the present invention better, will make further elaboration to technical scheme of the present invention below.
The 1st technical scheme provided by the invention is that a kind of ocular in-situ gel compositions comprises:
Nerve growth factor 50 μ g/ml～400 μ g/ml;
Stabilizing agent 1mg/ml～20mg/ml;
Ion-sensitive type gel 3mg/ml～10mg/ml;
Water;
The pH value of this situ-gel compositions is 4.5～7.5.
Wherein, μ g/ml is a mcg/ml.
In above-mentioned technical scheme, use the performance of the ocular in-situ gel compositions of ion-sensitive type gel, be better than the performance of the ocular in-situ gel compositions of serviceability temperature responsive type gel.The responsive to temperature type gel, before use, might be because of the rising gelation of ambient temperature in transportation and storing process.And the ion-sensitive type gel does not receive the influence of ambient temperature, and has reduced the consumption of gel-type vehicle.Thereby good superiority is being arranged aspect fill, transportation, the storage.
In the present invention, through using above-mentioned composition and proportioning, and the pH value of control situ-gel compositions can obtain including the ocular in-situ gel preparation of nerve growth factor within certain scope.When said preparation was preserved at room temperature, said preparation was liquid, after getting into eyes, just becomes stable semi-solid gel state.For ion-sensitive type gel wherein, for example: be selected from gellan gum.
Wherein, the English of said nerve growth factor is nerve growth factor, also abbreviates NGF in the present invention as.NGF is that the target tissue cell of being arranged by effector neuron synthesizes and excretory a kind of nerve growth regulatory factor with neuron nutrition and the dual biological function of short enation.Can keep sensation, sympathetic neuron survival, promote the reparation of injured nerve fiber, lymphocyte, mononuclear cell and neutrophilic granulocyte propagation, differentiation, wound healing etc.
Present nerve growth factor mainly is the Mus source and from people's Placenta Hominis, extracts, i.e. people source, and the NGF that has only the Mus source of listing at present, the people source do not take official written reply.The external NGF kind that does not have listing, the domestic producer that produces official written reply that takes has: Livzon Pharmaceutical Factory, Livzon Group, product specification: 30 μ g (biological activity is not less than 15000AU)/bottle, injection; Xiamen Beidazhilu Biological Engineering Co., Ltd, specification: 18 μ g (>=9000AU)/, injection, 6000AU (12 μ g)/bottle, lyophilized formulations, 2000AU (4 μ g)/prop up lyophilized formulations; Wuhan Haite Bio-pharmaceutical Co., Ltd, specification: 20 μ g (>=9000AU)/, injectable powder.
The 2nd technical scheme provided by the invention is the improvement to the 1st technical scheme, and improvements are that said stabilizing agent is an aminoacid.Do not adopt the human serum albumin in the present invention, because the compatibility between human serum albumin and other compositions is bad.
The 3rd technical scheme provided by the invention is the improvement to the 1st technical scheme, and improvements are that said stabilizing agent is the mixture of aminoacid and non-ionic surface active agent.
The 4th technical scheme provided by the invention is the improvement to the 1st technical scheme, and improvements are that said stabilizing agent is aminoacid and acetate.
The 5th technical scheme provided by the invention is the improvement to the 2nd～4 technical scheme, and improvements are that said aminoacid is alanine, glycine and arginic mixture.When said aminoacid is alanine, glycine and arginic mixture, then be more prone to obtain the excellent ocular in-situ gel preparation of the compatibility.
The 6th technical scheme provided by the invention is the improvement to the 1st～5 technical scheme, and improvements are, also comprise the pH value buffer agent.The use of pH value buffer agent; Can be so that the pH value of ocular in-situ gel compositions of the present invention be comparatively stable; That is to say; When introducing the substance of acid or alkalescence, the pH value of ocular in-situ gel combination of the present invention changes little, and this formulation selection for the ocular in-situ gel preparation provides great degree of freedom.In former technology; In the ocular in-situ gel compositions, there is delicate balance between each component; In case this balance is broken, will be difficult to so recover once more, this just makes the preparation of ocular in-situ gel preparation and research under point-device condition, to carry out.
The 7th technical scheme provided by the invention is the improvement to the 6th technical scheme, and improvements are that said pH value buffer agent is phosphate, acetate, citric acid and salt, carbonate.Though the kind of pH value buffer agent does not have special restriction, as long as can be used in field of medicaments, the above-mentioned pH value buffer agent of preferred use.
Said ion-sensitive type gel is selected from gellan gum, with Ca in the tear
2+, Na
+, K
+Form stable hydrogen bond Deng complexing of metal ion, and be gel state.Synthermal responsive type gel phase ratio can not make things convenient for fill, transportation and storage because of the rising gelation of ambient temperature.In lyophilized powder redissolution process; The present invention utilizes the gellan gum gel of gellan gum preparation to show good superiority, the gel phase ratio that macromolecular materials such as same poloxamer, hypromellose form, and the gellan gum gel has greatly shortened the redissolution time; And redissolve effective, stable performance.
The 8th technical scheme provided by the invention is the improvement to the 1st～7 technical scheme, and improvements are that the ion-sensitive type gel is selected from gellan gum (Geuam gum).
The 9th technical scheme provided by the invention is the improvement to the 1st～8 technical scheme, and improvements are that said composition also comprises antiseptic.For medical composition, keep the characteristic of medicine to help the storage of medical composition, so preferably also comprise antiseptic.
The 10th technical scheme provided by the invention is the improvement to the 9th technical scheme; Improvements are that said antiseptic is selected from benzyl alcohol, phenol, metacresol, methyl butex and propylparaben, trichlorine tert-butylamine alcohol, benzalkonium chloride, benzalkonium bromide, hibitane.The introducing of employed antiseptic does not have special restriction among the present invention, only otherwise influencing technical problem to be solved by this invention gets final product.Putting before this, preferably using the antiseptic of the above-mentioned type.
The 11st technical scheme provided by the invention is the improvement to the 9th and 10 technical scheme, and improvements are that the amount of said antiseptic is 0mg/ml～100mg/ml.As long as the amount of antiseptic can play the effect of antiseptic,, more preferably below the 50mg/ml, also can adopt below the 25mg/ml so be preferably below the 100mg/ml.Under the prerequisite that can play the antiseptic effect, add antiseptic as far as possible less.
The 12nd technical scheme provided by the invention is the improvement to the 1st～11 technical scheme, and improvements are, also comprise osmotic pressure regulator.
The 13rd technical scheme provided by the invention is the improvement to the 12nd technical scheme, and improvements are that said osmotic pressure regulator is selected from mannitol, sorbitol, sodium chloride, sodium citrate.
The 14th technical scheme provided by the invention is the improvement to the 1st～13 technical scheme; Improvements are; Also comprise medical hydrophilic high molecular material, contain at least a in the following material in the said medical hydrophilic high molecular material: methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl emthylcellulose, polyvinylpyrrolidone, poloxamer, polyvinyl alcohol, hyaluronate sodium, chitosan, sodium glycerophosphate.
More than technical scheme provided by the present invention is illustrated, below in conjunction with providing concrete embodiment, but protection scope of the present invention is not limited to these embodiment.
Embodiment 1
The proportioning of present embodiment is as shown in the table:
| NGF | 200μg/ml |
| Alanine | 3.3mg/ml |
| Glycine | 3.3mg/ml |
| Arginine | 3.3mg/ml |
| Gellan gum | 5mg/ml |
| Benzyl alcohol | 9mg/ml |
| Water for injection | In right amount |
Method for preparing: it is an amount of to get water for injection, and dispersive while stirring adding gellan gum stirs down in 90 ℃, gellan gum is dissolved fully form transparent solution, is cooled to room temperature.NGF, alanine, glycine, arginine, benzyl alcohol are dissolved in earlier in an amount of water for injection, after the aseptic filtration, add in the above-mentioned gellan gum solution for preparing, and water for injection is supplemented to capacity, stirs, and is aseptic subpackaged.
Embodiment 2
The proportioning of present embodiment is as shown in the table:
| NGF | 200μg/ml |
| Alanine | 3.3mg/ml |
| Glycine | 3.3mg/ml |
| Arginine | 3.3mg/ml |
| Gellan gum | 4mg/ml |
| Benzyl alcohol | 9mg/ml |
| Acetate buffer | Transfer to pH5.5 |
| Mannitol | 50mg/ml |
| Water for injection | In right amount |
Method for preparing: it is an amount of to get water for injection, and dispersive while stirring adding gellan gum, benzyl alcohol stir down in 90 ℃, gellan gum is dissolved fully form transparent solution, is cooled to room temperature.NGF, alanine, glycine, arginine, mannitol are dissolved in earlier in the pH5..5 acetate buffer, after the aseptic filtration, adopt the lyophilizing of frozen drying method.Before facing usefulness, gellan gum solution adds in the NGF lyophilizing, the dissolving mixing.
Above-mentioned gellan gum is the Microbial exopolysaccharides of U.S. kelco company exploitation.It is to belong to (Pseudomonaseloden) under neutrallty condition by the single brain bacillus of vacation waterweed; With the glucose is carbon source; Ammonium nitrate is in the culture medium that nitrogenous source and some inorganic salts were made into, and the extracellular polysaccharide colloid that produces through aerobic fermentation is a kind of novel bright gel of full impregnated.Ca in gellan gum and the tear
2+, Na
+, K
+Form stable hydrogen bond Deng complexing of metal ion, and be gel state.
The ocular in-situ gel preparation that above embodiment 1～2 is prepared be liquid when under room temperature (0 ℃～30 ℃) condition, preserving, and after the entering eyes, just becomes stable semi-solid gel state.
To continue some character of research ocular in-situ gel compositions of the present invention below
Test Example 1
NGF and medical macromolecular materials compatibility test
With NGF stock solution (30 μ g) and medical macromolecular materials solution proportional mixing, adopt chicken dorsal ganglion method to detect the biological activity of NGF.
1.1 instrument:
Super-clean bench: model DL-CJ-2N (Harbin Donglian Electronic & Technology Development Co., Ltd.); High-pressure sterilizing pot: model YXQG02 (Xinhua Medical Apparatus Co., Ltd. Shandong); CO2 gas incubator: model MCO-15AC (SANYO GS), inverted microscope: model C KX41 (Olympus).
1.2 reagent and test solution: calf serum (U.S. invitrogen Life Technologies, Inc.), the high sugared culture fluid (U.S. invitrogen Life Technologies, Inc.) of DMEM, Mus tail collagen (Sigma company), normal saline (Shangdong Hualu Pharmaceutical Co., Ltd.).
DMEM is a kind of culture medium that contains each seed amino acid and glucose.
1.3 operating procedure:
1.3.1 get the glass cell bottle behind the autoclaving, after frozen Mus tail collagen is melted, get 50ul (microlitre) approximately by each cell bottle, at the bottom of evenly coating bottle with the elbow Glass rod in 1/3 place, remove bottle cap, placed the super-clean bench natural drying two days.
1.3.2 add the DMEM culture fluid 2ml of 10% calf serum in each cell bottle, capped, soaked overnight.
1.3.3 during experiment, in plate, pour the normal saline of sterilization into, under anatomical lens, get the dorsal root ganglion of instar chicken embryo on the 7th～9, be inoculated in the culture bottle that scribbles Mus tail collagen 3～5 neuroganglions of every bottle graft kind.Be incubated 2 hours down in 37 ℃.
1.3.4NGF dilution of sample:
Sample thief adds the DMEM culture fluid, dilutes 5000 times as testing sample.
1.3.5NGF dilution with reference to article (labelled amount is that 1000AU/ props up):
Get 1 and add serum-free DMEM culture fluid 1.0ml and be dissolved as tested mother solution, get tested mother solution 0.5ml and add DMEM culture fluid 9.5ml and be made into A liquid, get A liquid 0.3ml and add DMEM culture fluid 4.7ml and be made into reference to article and contrast liquid.
1.3.6 make 3 times of doubling dilutions with the NGF sample after the above-mentioned preparatory dilution with reference to article with serum-free DMEM culture fluid, sample is made 6 to 7 dilution factors (should be diluted to and negative findings occur), adds in the culture bottle, establishes blank simultaneously.In 5%CO2, cultivated 18～24 hours in 37 ℃ of CO2 gas incubators.
1.3.7 observed result under inverted microscope, according to the neuroganglion enation in various degree, each do respectively classification record (with #, ++ ++, +++, ++ ,+,-represent).
1.3.8 judge:
It is negative that neuroganglion does not have enation, representes with "-"; Neuroganglion enation but more sparse is represented with "+"; Obviously growth is represented with " ++ "; Growth is better with " +++" expression; It is best with " ++ ++ " expression to grow; Excessive inhibition occurring representes with " # ".Usually, from the dilution factor that negative findings occurs begin to get the back several the 3rd and the 4th liang of dilution factor the best conduct of growth judge endpoint calculation tire (tire=AU/ml).If these two dilution factors look good equally, then get both meansigma methodss.
1.4 qualified limit standard: >=15000AU/ props up
1.5 computing formula:
1.6 outcome record:
Reference article outcome record: indicate active:
1000AU/ props up
| Liquid numbering to be measured | 1 | 2 | 3 | 4 | 5 | 6 |
| Indicate active (AU/ml) | 3 | 1 | 1/3 | 1/9 | 1/27 | 1/81 |
| Preparatory extension rate | 1000/3 | 1000 | 3000 | 9000 | 27000 | 81000 |
| With reference to the article growth | # | ++++ | +++ | ++ | + | - |
Negative control:
-
1.7 the result judges:
| Sample | The result judges |
| NGF+ poloxamer 407+ poloxamer 188 | The compatibility is good |
| NGF+ human albumin+poloxamer 407+ poloxamer 188 | White precipitate occurs, the compatibility is poor |
| NGF+ alanine+glycine+arginine+poloxamer 407+ poloxamer 188 | The compatibility is good |
| The NGF+ carbomer | The compatibility is good |
| The NGF+ gellan gum | The compatibility is good |
| The NGF+ chitosan | The compatibility is good |
| The NGF+ sodium alginate | The compatibility is good |
| The NGF+ hyaluronate sodium | The compatibility is good |
| The NGF+ hydroxypropyl emthylcellulose | The compatibility is good |
Test Example 2
The adding of stabilizing agent influences NGF ocular in-situ gel stability test
Traditional NGF preparation adopts the human albumin as protective agent more; But there is very big limitation in end user's blood albumin, and is prone to and consistency problem appears in macromolecular material, therefore; We use aminoacid, acetate as stabilizing agent, and investigate the influence of stabilizing agent to the NGF ocular in-situ gel.
With the NGF ocular in-situ gel place that humidity is 92%, temperature is incubator preserve separately 0 day, 5 days, 15 days, 20 days, January, February of 40 ℃, adopts the chick embryonic dorsal root ganglion method to detect the biological activity of NGF then.
The prescription that uses in an embodiment is as shown in the table:
| Prescription 1 | Prescription 2 | |
| NGF | 50μg/ml | 50μg/ml |
| Alanine | 3.3mg/ml | |
| Glycine | 3.3mg/ml | |
| Arginine | 3.3mg/ml | |
| Gellan gum | 5mg/ml | 5mg/ml |
| Water for injection | In right amount | In right amount |
The determination of activity result is as shown in the table:
| 0 day | 5 days | 15 days | 20 days | January | February | |
| Prescription 1 | ++++ | ++ | - | - | - | - |
| Prescription 2 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ |
The result shows that the gel stability that does not contain stabilizing agent is very poor, and the amino acid whose adding of stabilizing agent can make the stability of NGF situ-gel significantly improve.
Test Example 3
The length of eye holdup time is to weigh an important indicator of eye exterior-applied formulation, and the fluorescein method is adopted in this experiment, and the eye that has carried out NGF ocular in-situ gel (embodiment 1) and eye drop respectively is detained experiment, and the two is contrasted.
The green fluorescent protein that in NGF ocular in-situ gel (embodiment 1) and eye drop, adds trace respectively.With 6 fixing heads of healthy rabbits; Mention palpebra inferior; Pull into little cup-shaped to conjunctival sac, in rabbit conjunctiva of left eye capsule, splash into green fluorescent protein NGF ocular in-situ gel 40 μ l, in the conjunctival sac of these 6 rabbit right eyes, splash into 40 μ l green fluorescent protein NGF eye drops as contrast simultaneously.After the administration; Made rabbit each passive closed 10 seconds; Rabbit is placed on observation under blue light 470nm (nanometer) wavelength (green fluorescent protein has green fluorescence under the irradiation of 450～490nm blue light); The fluorescence of conjunctiva and cornea green fluorescent protein disappears the time of taking off after the record administration, and disappearing with growing most of two position fluorescence, to take off timing definition be the eye holdup time.
Following table is the eye holdup time of NGF ocular in-situ gel (embodiment 1) and eye drop to measure the result
Conclusion: compare the eye holdup time phenomenal growth of NGF ocular in-situ gel with traditional eye drop.
Test Example 4
Rabbit eye irritation experiment: eyes are the most responsive organs, and the substrate adjuvant in this research is macromolecular compound, so need the zest of investigation preparation to lagophthalmos.
Adopt animal consubstantiality left and right sides self matching type:
The single-dose irritant experiment: 1 of NGF ocular in-situ gel (embodiment 1) is splashed in the rabbit conjunctiva of right eye capsule, and left eye splashes into normal saline and makes blank.After the administration, the passive closure of lagophthalmos eyelid about 10 seconds, 6,24,48,72 hours, 7 days eye local response situation after the record administration.
The multiple dosing irritant experiment: NGF ocular in-situ gel (embodiment 1) is directly splashed into the rabbit right eye with clinical consumption, and left eye splashes into normal saline and makes blank.After the administration, the passive closure of rabbit eyes eyelid about 10 seconds, every day 2 times, continuous 2 weeks.Observe eye situation before administration every day (observe once before administration every day, totally twice, get the best result in twice), and 12h after the last administration (hour), the situation of each rabbit corneal, iris and conjunctiva when 24h, 48h, 72h.
Following table is an eye irritation test grade form
| The investigation factor | The eye irritation reaction | Score value |
| Conjunctiva | Blood vessel is clear, color and luster is pale red | 0 |
Following table is the zest evaluation criterion
| The stimulation degree | Integration |
| Nonirritant | The 0-3 branch |
| Slight zest | The 4-8 branch |
| The moderate zest | The 9-12 branch |
| The severe zest | The 13-16 branch |
Following table is that single-dose is to rabbit eye irritation experimental result
Following table is multiple dosing irritant experiment result
Rabbit eye irritation experiment conclusion:
1. single-dose: conjunctiva and iris do not have hyperemia, redness and swelling, and cornea does not have phenomenons such as muddiness.The zest scoring is zero.
2. multiple dosing: lagophthalmos has slight conjunctival congestion phenomenon, counts 1 fen, and 1 to 2 day congested phenomenon disappears after the drug withdrawal, and eye IR average integral all is no more than 1 fen.
NGF ocular in-situ gel (embodiment 1) single and multiple dosing are non-stimulated to lagophthalmos
Test Example 5
The NGF ocular in-situ gel is to the zooscopy of LASIK post-operative cornea neuranagenesis effect
1. test method
Choose the Belgian rabbit of medical fitness and implement the bilateral lasik surgery, right eye is a self-controlled group, and left eye is a test group, gives normal saline and variable concentrations NGF ocular in-situ gel respectively.The successful rabbit of operation is divided into 5 groups at random, 6 every group, totally 24.Every rabbit left eye of group I, group II, group III, group IV, group V treats for respectively variable concentrations NGF ocular in-situ gel 0,50,100,200,400 μ g/ml, and right eye is given normal saline.Every group from postoperative administration in first day, 20 μ l/ time, and every day 2 times, administration time is 6 months.Before art, postoperative 1d, 14d, 1m, 3m, 6m measure operation eye and the neural quantity of contrast cornea.
2. result of the test
Each group test rabbit corneal nerve fiber quantity of LASIK postoperative is seen table 7.
Following table is represented: each time point corneal nerve fiber number of LASIK postoperative
| Time | NS | NGF50μg/ml | NGF?100μg/ml | NGF200μg/ml | NGF400μg/ml |
| 0d | 42.60±2.38 | 41.75±2.14 | 42.61±1.27 | 41.03±1.52 | 42.54±1.39 |
| 1d | 2.60±0.38 | 2.30±0.39 | 2.42±0.27 | 2.31±0.33 | 2.39±0.35 |
| 14d | 3.90±0.77 | 4.35±0.51 | 4.87±0.66 * | 5.21±0.54 * | 5.82±0.71 * |
| 1m | 6.83±0.75 | 7.01±0.58 | 7.34±0.69 * | 8.89±0.63 ** | 9.22±0.51 ** |
| 3m | 13.52±1.97 | 16.87±1.95 * | 26.06±2.14 ** | 30.04±2.26 ** | 33.72±2.40 ** |
| 6m | 26.47±2.47 | 30.22±2.68 * | 41.14±3.17 ** | 44.97±2.18 ** | 45.39±2.52 ** |
Compare with the NS group,
*P＜0.05;
*P＜0.01
Each test dose group of conclusion: NGF all has the effect that promotes that LASIK post-operative cornea nerve fiber quantity increases, and is dose-effect relationship, and each dose groups all has significant difference than matched group.
Test Example 6
NGF ocular in-situ gel (embodiment 1) is used for the neural repairing and treating of LASIK post-operative cornea lobe relatively with the NGF eye drop
Adopt the randomized, double-blind prospective control study; The between twenty and fifty myope of 30 examples (60) row LASIK is divided into NGF ocular in-situ gel group 15 examples (30) and NGF eye drop group 15 examples (30) at random; 2 groups of patients LASIK postoperative of being expert at is accepted NGF ocular in-situ gel or the local eye dripping of NGF eye drop respectively, all patient's medications 10 days, NGF ocular in-situ gel group every day 1 time; NGF eye drop group every day 3 times, check art respectively before, 6 months neural density of cornea of postoperative.Postoperative 10 days, 30 routine patients 1～2 nerve fiber of subcutaneous stringer except that 16 corneas are visible, and the complementary angle film is not seen upward subcutaneous plexus nervorum.NGF ocular in-situ gel group and NGF eye drop group no significant difference.In 6 months cutting region of postoperative under the corneal epithelium shallow hypothallus nerve fiber do not reach level before the art yet.2 groups of newborn nerve fiber quantity of cornea flap central area all obviously increase, and NGF ocular in-situ gel group is superior to NGF eye drop group.All do not find adverse events such as cornea rebirth blood vessel, conjunctival congestion, epithelial cell peel off, hypothallus edema, endothelium minimizing among 2 groups of patients.The result shows that NGF has facilitation in the reparation of LASIK postoperative corneal nerve fiber, and NGF ocular in-situ gel and NGF eye drop are all safe and effective, and the medication process is not found systemic adverse reactions, and local response is slight.But it is excessive that the NGF ocular in-situ gel is difficult for, and can reduce the eye drip number of times, and administration number of times only is 1/3 of an eye drop, and the NGF ocular in-situ gel is compared foreign body sensation decline significantly with the NGF eye drop.Situ-gel is superior to eye drop.
Claims (10)
1. ocular in-situ gel compositions comprises:
Nerve growth factor 50 μ g/ml～400 μ g/ml;
Stabilizing agent 1mg/ml～20mg/ml;
Ion-sensitive type gel 3mg/ml～10mg/ml;
Water;
The pH value of this situ-gel compositions is 4.5～7.5;
Wherein, said stabilizing agent is an aminoacid;
Said ion-sensitive type gel is selected from gellan gum (Geuam gum).
2. ocular in-situ gel compositions according to claim 1 is characterized in that, said aminoacid is alanine, glycine and arginic mixture.
3. ocular in-situ gel compositions according to claim 1 is characterized in that, also comprises the pH value buffer agent.
4. ocular in-situ gel compositions according to claim 3 is characterized in that, said pH value buffer agent is phosphate, acetate, citric acid and salt, carbonate.
5. ocular in-situ gel compositions according to claim 1 is characterized in that, also comprises antiseptic.
6. ocular in-situ gel compositions according to claim 5 is characterized in that, said antiseptic is selected from benzyl alcohol, phenol, metacresol, methyl butex and propylparaben, trichlorine tert-butylamine alcohol, benzalkonium chloride, benzalkonium bromide, hibitane.
7. according to claim 5 or 6 described ocular in-situ gel compositionss, it is characterized in that the amount of said antiseptic is 0mg/ml～100mg/ml.
8. ocular in-situ gel compositions according to claim 1 is characterized in that, also comprises osmotic pressure regulator.
9. ocular in-situ gel compositions according to claim 8 is characterized in that said osmotic pressure regulator is selected from mannitol, sorbitol, sodium chloride, sodium citrate.
10. ocular in-situ gel compositions according to claim 1; It is characterized in that; Also comprise medical hydrophilic high molecular material, contain at least a in the following material in the said medical hydrophilic high molecular material: methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl emthylcellulose, polyvinylpyrrolidone, poloxamer, polyvinyl alcohol, hyaluronate sodium, chitosan, sodium glycerophosphate.
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| CN103432632B (en) * | 2013-09-16 | 2015-11-25 | 姚静 | A kind of thermosensitive in situ gel compositions and preparation method |
| FR3024363B1 (en) * | 2014-08-01 | 2018-08-10 | Synolyne Pharma | THERMOGELIFIABLE COMPOSITION |
| EP3287140B1 (en) * | 2015-04-21 | 2021-06-02 | Staidson (Beijing) Biopharmaceuticals Co., Ltd. | Nerve growth factor composition and powder injection |
| DK3287139T3 (en) * | 2015-04-21 | 2021-09-13 | Beijing Staidson Medical Tech Co Ltd | Nerve growth factor composition and injection powder |
| ES2893952T3 (en) * | 2015-04-21 | 2022-02-10 | Staidson Beijing Biopharmaceuticals Co Ltd | Composition of nerve growth factor and powder for injection |
| CN110947036B (en) * | 2019-12-25 | 2022-03-15 | 广州聚明生物科技有限公司 | Lacrimal passage suppository and preparation method thereof |
| CN111317814B (en) * | 2020-04-23 | 2020-12-18 | 中国中医科学院眼科医院 | Borneol and neurotrophic factor combined composition and application |
| CN115068395B (en) * | 2022-06-29 | 2023-06-09 | 贵州中医药大学 | Liquid eye mask and preparation method thereof |
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