CN104984326A - Recombinant human basic fibroblast growth factor gel without bacteriostatic agent and preparation method thereof - Google Patents
Recombinant human basic fibroblast growth factor gel without bacteriostatic agent and preparation method thereof Download PDFInfo
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- CN104984326A CN104984326A CN201510413146.3A CN201510413146A CN104984326A CN 104984326 A CN104984326 A CN 104984326A CN 201510413146 A CN201510413146 A CN 201510413146A CN 104984326 A CN104984326 A CN 104984326A
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Abstract
The invention provides a recombinant human basic fibroblast growth factor gel without a bacteriostatic agent. The recombinant human basic fibroblast growth factor gel comprises the following components: 1*10<6> to 9*10<6> IU of recombinant human basic fibroblast growth factors, 0.01 to 5.0 g of albumen stabilizers, 0.1 to 20.0 g of thickeners, 0.1 to 10.0 g of pH regulators, 0.1 to 10.0 g of buffering agents and 0.1 to 50.0 g of humectants, and water for injection is added to a constant volume of 1000 ml. The invention further provides a preparation method of the recombinant human basic fibroblast growth factor gel without the bacteriostatic agent. The recombinant human basic fibroblast growth factor gel without the bacteriostatic agent is disposable, so as to prevent eyes from being stimulated by the bacteriostatic agent and avoid microbial contamination which possibly occurs in the processes of use and storage.
Description
Technical field
The invention belongs to pharmaceutical field, be specifically related to recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent and preparation method thereof.
Background technology
Basic fibroblast growth factor (bFGF) is a kind of endogeneous activity factor closely-related with the agglutination of tissue injury, has significant facilitation to tissue injury healing, repairing of neural injury and revascularization.The single chain polypeptide that bFGF is made up of 146 aminoacid, molecular weight is 17KD.Clinical practice shows, rh-bFGF has significant Promotive union effect to human body burn, wound, operation wound etc., and without local and whole body toxic and side effects.BFGF not only can accelerated wound healing, and can reduce the formation of cicatrix, in the regeneration promoting the tissue injurys such as burn, wound and decubital ulcer and reparation, illustrate outstanding and original effect.
The recombined human alkaline fibroblast growth factor gelling agent gone on the market at present is mainly external-use gel and does not can be applicable to eye.Its preparation mostly is many metering packings, repeatedly uses.Behind preparation Kaifeng, be easy to using and be subject to microbial contamination in preservation process, and then produce various potential safety hazard.In order to prevent microorganism secondary pollution problem, in multi-dose formulation, all with the addition of various antibacterial (preservtive).Although antibacterial is preventing there is positive effect in microbial contamination, it also can cause a lot of untoward reaction.It is reported that the existence of antibacterial can produce zest to the superficial cell of eye.In eye drop, antibacterial directly can affect the composition of tear, change the microenvironment of eyeball surface, making close-connected epithelial cell structural damage originally, there is exfoliation of corneal epithelium, defect, epithelial erosion in severe patient, and then generation corneal ulcer, even corneal solution, perforation, blind etc.In recent years, abuse by antibacterial the problem caused more and more to receive publicity.In addition, for being used for the treatment of wound, the ophthalmic preparation of the direct and Wound contact such as operation such as hinder, burns, scalds and fester at the wound surface damage, the infringement of antibacterial to wound surface also be can not ignore.
Summary of the invention
Technical problem to be solved by this invention is the recombined human alkaline fibroblast growth factor gelling agent of the not bacteriostatic agent providing a kind of not bacteriostatic agent.
The invention provides a kind of recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent, it is characterized in that, comprise following component: recombination human basic fibroblast growth factor 1 × 10
6~ 9 × 10
6iU, protein stabiliser 0.01 ~ 5.0g, thickening agent 0.1 ~ 20.0g, pH adjusting agent 0.1 ~ 10.0g, buffer agent 0.1 ~ 10.0g, wetting agent 0.1 ~ 50.0g, inject and be settled to 1000ml with water.
Further, described pH adjusting agent is one or more mixing in triethanolamine, sodium hydroxide, sodium bicarbonate, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, Borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid.
The effect of PH regulator is the acid-base value regulating eye drop, makes it equal with the acid-base value of tear or close, to reduce the zest of eye drop, and makes drug substance stable, improves drug effect.
Further, described thickening agent is one or more mixing in dextrin and derivant, sodium alginate and derivant thereof, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl emthylcellulose, hydroxypropyl cellulose, methylcellulose, carboxymethyl cellulose, arabic gum, tragcanth, guar gum, carbopol, phytohemagglutinin, hyaluronic acid, xanthan gum, chitosan, tyloxapol.
Further, described wetting agent is glycerol, one or more mixing of propylene glycol.
The effect of wetting agent is the lubrication improving gel.
Further, described protein stabiliser is one or more mixing in mannitol, sodium lauryl sulphate, glycosides propylhomoserin, tween, dextran, hyaluronic acid, chondroitin sulfate, heparin.
The effect of protein stabiliser is the stability in order to increase eye drop principal agent, improves drug effect.
Further, described buffer agent be citric acid receive/citric acid buffering to, phosphate-buffered to one or more mixing to, glycine-triethylamine buffering centering of, tris-hydrochloride buffer.
Further, the recombined human alkaline fibroblast growth factor gelling agent of described not bacteriostatic agent adopts the independent packaging of not bacteriostatic agent, and the volume scope of the container of the independent packaging of described not bacteriostatic agent is that 0.1 ~ 0.5ml/ props up.
The present invention also provides the preparation method of the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent, comprises the following steps:
S1: take thickening agent 0.1 ~ 20.0g, pH adjusting agent 0.1 ~ 10.0g are dissolved in appropriate water for injection, sterilizing 30min;
S2: take recombination human basic fibroblast growth factor 1 × 10
6~ 9 × 10
6iU, protein stabiliser 0.01 ~ 5.0g, buffer agent 0.1 ~ 10.0g and wetting agent 0.1 ~ 50g are dissolved in appropriate water for injection;
S3: the medicinal liquid of step S2 gained is mixed with step S1 gained medicinal liquid after 0.01 ~ 10.00 μm of filtering with microporous membrane 5 times, and is settled to 1000ml with water for injection, stir;
S4: fill step S3 gained medicinal liquid is in the packing container of not bacteriostatic agent, and the volume scope of container is that 0.1 ~ 0.5ml props up.
The recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent provided by the invention is without antibacterial, disposable, can avoid the stimulation that the existence of antibacterial produces eye, and the microbial contamination using and may occur in storage process.
Detailed description of the invention
Embodiment 1
The embodiment of the present invention provides a kind of recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent, comprises following component: recombination human basic fibroblast growth factor 1 × 10
6iU, mannitol 0.01g, polyvinyl alcohol 0.1g, sodium hydroxide 0.1g, phosphate-buffered, to 0.1g, glycerol 0.1g, inject and are settled to 1000ml with water.
The embodiment of the present invention also provides a kind of preparation method of recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent, comprises the following steps:
S1: weighing polyvinyl alcohol 0.1g, sodium hydroxide 0.1g is dissolved in water for injection, 121 DEG C of pressure sterilizing 30min;
S2: take recombination human basic fibroblast growth factor 1 × 10
6iU, mannitol 0.01g, phosphate-buffered 0.1g, glycerol 0.1g are dissolved in water for injection;
S3: the medicinal liquid of step S2 gained is mixed with step S1 gained medicinal liquid after 0.01 ~ 10.00 μm of filtering with microporous membrane 5 times, and is settled to 1000ml with water for injection, stir;
S4: detecting step S3 gained medicinal liquid, after qualified, under hundred grades of environment, liquid drug is in the packing container of not bacteriostatic agent, and the volume scope of container is that 0.1 ~ 0.5ml/ props up.
Embodiment 2
The embodiment of the present invention provides a kind of recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent, comprises following component: recombination human basic fibroblast growth factor 9 × 10
6iU, sodium lauryl sulphate 5.0g, carboxymethyl cellulose 20.0g, sodium carbonate 10.0g, citric acid receive/citric acid buffering to 10.0g, propylene glycol 50g, inject and be settled to 1000ml with water.
The preparation method of the recombined human alkaline fibroblast growth factor gelling agent of the not bacteriostatic agent that the present embodiment provides is identical with embodiment 1.
Embodiment 3
The embodiment of the present invention provides a kind of recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent, comprises following component: recombination human basic fibroblast growth factor 6 × 10
6iU, hyaluronic acid 1g, sodium alginate 3g, sodium hydrogen phosphate 2.5g, phosphate-buffered, to 1.5g, glycerol 30g, inject and are settled to 1000ml with water.
The preparation method of the recombined human alkaline fibroblast growth factor gelling agent of the not bacteriostatic agent that the present embodiment provides is identical with embodiment 1.
Embodiment 4
The embodiment of the present invention provides a kind of recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent, comprises following component: recombination human basic fibroblast growth factor 4 × 10
6iU, chondroitin sulfate 1.5g, hydroxypropyl emthylcellulose 4.5g, hydrochloric acid 1.5g, phosphate-buffered, to 4.5g, glycerol 15g, inject and are settled to 1000ml with water.
The preparation method of the recombined human alkaline fibroblast growth factor gelling agent of the not bacteriostatic agent that the present embodiment provides is identical with embodiment 1.
Experiment one
Stability experiment
BFGF biologic activity uses the method recorded in Chinese Pharmacopoeia 2010 editions the 3rd annex XG, and adopt BalB/c 3T3 cell, mtt assay measures.Stability experiment result shows, and this product at room temperature about can deposit 3 months without obvious loss of activity, can meet normal instructions for use.Under 4 ~ 8 DEG C of conditions of storage, place and still keep original activity after 2 years.Outside place's activity, in stability test process, formulation aesthetics, pH value, viscosity, aseptic, active every test all do not find significant change.In sum, under 4 ~ 8 DEG C of storage conditions, invention formulation has good stability.
Experiment two
Skin allergy is tested
The recombined human alkaline fibroblast growth factor gelling agent of the not bacteriostatic agent adopting embodiment 3 obtained, be applied to the normal and tested district of damaged skin of Cavia porcellus with every gram containing 20 μ g, 40 μ g, 80 μ g, 100 μ g (being equivalent to 1 times of people's quantity, 2 times, 4 times, 5 times) respectively, area is 2cm
2result shows, a drug and repeatedly medication, matched group and administration group comparative observation, the equal < of skin wound repair intensity 0.5 point, illustrate not bacteriostatic agent recombined human alkaline fibroblast growth factor gelling agent a drug and repeatedly medication to Cavia porcellus the complete and equal nonirritant of damaged skin.
Experiment three
Extracorporeal releasing test
Method with reference to DiBase MD etc. is carried out, and release in vitro meets zero order kinetics pattern.
Clinical practice situation of the present invention is as follows:
The recombined human alkaline fibroblast growth factor gelling agent of the not bacteriostatic agent that clinical practice the present embodiment 3 is obtained, directly spread upon on the damage wound surface because of wound (as operation wound, burn, scald, ulcer), can promote that wound tissue heals, shorten healing time, curative effect is better, and to no skin irritation, easy to use, be easy to clean convenient operation, be applicable to clinical.
To tire evaluation: clinical application effect by doctor by subjective symptoms and finding Comprehensive Assessment of having a medical check-up, be divided into recovery from illness, effective, effectively, invalid four classes, recovery from illness, effective, effectively add up to effectively, the recombined human alkaline fibroblast growth factor gelling agent treatment group of bacteriostatic agent does not compare with matched group (zinc sulfadiazine), result shows: the not recombined human alkaline fibroblast growth factor gelling agent clinical verification of bacteriostatic agent, treatment burn, scald and ulcer, effective percentage is respectively more than 95%, prove that the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent is feasible, more reasonable.And matched group effective percentage is more than 74%.
Experiment four
Eye irritation is tested
Experiment purpose
Observe the recombined human alkaline fibroblast growth factor gelling agent single of the obtained not bacteriostatic agent of the present embodiment 3 and irritant reaction that repeatedly eye produces after eye drip, for data for clinical drug use provides foundation.
Laboratory animal
Japan large ear rabbit: provided by Chengdu, Chengdu military area command hospital general Animal House
Credit number: SCXK (army) 2002-016
Experimental agents
The recombined human alkaline fibroblast growth factor gelling agent of the not bacteriostatic agent that the present embodiment 3 is obtained
Sodium fluorescein: Mingxing Pharmaceuticals Co., Ltd., Guangzhou City produces
Experimental technique and result
Single-dose irritant test
Preliminary election animal: get the healthy large ear rabbit of Japan 10 without hindering more than 2Kg, male and female dual-purpose, single only raising, hangs cage card labelling.Test in first 24 hours and the eyes of every animal are checked, inspection method: (1) perusal: cornea is without muddiness, and conjunctiva is without hyperemia, edema and secretions, and pupil is circular, and both sides etc. are large, good to luminous reflectance.(2) slit lamp examination: corneal transparency, without nebula, class's nebula; Iris texture is clear, without congested, edema.(3) fluorescent staining: get Sodium fluorescein, faces the used time with normal saline dilution 5 times, and every rabbit is clean with normal saline flushing after dripping, and observing cornea end tinter is that cornea is normal.
Be used for testing through the Healthy Rabbits of above-mentioned inspection without eye part disease, have the animal of eye irritation symptom, corneal defect and conjunctival damage can not be used for experiment.
Get satisfactory animal 6 with after picric acid labelling for experiment, every rabbit left eye drips the recombined human alkaline fibroblast growth factor gelling agent 0.1ml of not bacteriostatic agent, right eye drips normal saline 0.1ml and compares, compressing nasolacrimal duct when dripping medicine, and makes eye passive closed 10 seconds.Within after administration 1,2,4,24,48,72 hour, check every lagophthalmos, inspection method, with inspection method during preliminary election animal, is scored by the irritant reaction situation of table one eye.If eye has no any irritation after 72 hours, then no longer observe; If have irritation after 72 hours, then extend and observe the time limit to 21 day, to observe the reversible case of irritant reaction.
Each inspection, all should record eye reactor score value (grade form is in table 1).Except observing listed conjunctiva, cornea and injury of iris, other are viewed also should record and report.
Table 1 Eye irritation reaction score criteria
Evaluation of result:
Scoring system: the irritant reaction score value of the cornea of each of each animal, iris and conjunctiva is added to obtain total mark observing time, by the integration summation of a group divided by number of animals, obtains last score value.Its stimulation degree is judged by table 2.
Table 2 eye irritation evaluation criterion
| Score value | Evaluate |
| 0-3 | Nonirritant |
| 4-8 | Slight zest |
| 9-12 | Moderate zest |
| 13-16 | Intensity zest |
Result of the test: the recombined human alkaline fibroblast growth factor gelling agent single-dose of bacteriostatic agent does not occur irritative response to lagophthalmos, and score value is 0, refers to table 3.
Multiple dosing irritant test
Preliminary election animal: get the healthy Japan large ear rabbit 10 without hindering more than 2kg, male and female dual-purpose, single only raising, hangs cage card labelling.Test in first 24 hours and check by the eyes of inspection method to every animal during single-dose stimulation test preliminary election animal, be used for testing through the Healthy Rabbits of above-mentioned inspection without eye part disease, have the animal of eye irritation symptom, corneal defect and conjunctival damage can not be used for experiment.
Get satisfactory animal 6 with after picric acid labelling for experiment, every rabbit left eye drips the recombined human alkaline fibroblast growth factor gelling agent 0.1ml of not bacteriostatic agent, right eye drips normal saline 0.1ml and compares, compressing nasolacrimal duct when dripping medicine, and makes eye passive closed 10 seconds.Successive administration 4 weeks, administration every day 3 times, within 1,2,4,24,48,72 hour, check every lagophthalmos before administration every day and after last administration, inspection method, with inspection method during animal preliminary election, is scored by the irritant reaction situation of table one eye.If eye has no any irritation after 72 hours, then no longer observe; If have irritation after 72 hours, then extend and observe the time limit to 21 day, to observe the reversible case of irritant reaction.
Inspection method, scoring system and evaluation of result method are with single-dose irritant test.
Result of the test: administration 4 weeks, check lagophthalmos before administration every day, all do not occur irritative response, score value is 0.Within after last administration 1,2,4,24,48,72 hour, check, there is not irritative response in result, score value is 0 yet.Refer to table 4.Result of the test shows that the recombined human alkaline fibroblast growth factor gelling agent multiple dosing of not bacteriostatic agent is to lagophthalmos nonirritant.
The recombined human alkaline fibroblast growth factor gelling agent of table 4 not bacteriostatic agent is marked to lagophthalmos zest
Conclusion: not bacteriostatic agent the medication of recombined human alkaline fibroblast growth factor gelling agent single and repeatedly after medication to the equal nonirritant reaction of lagophthalmos.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for those skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent, is characterized in that, comprise following component: recombination human basic fibroblast growth factor 1 × 10
6~ 9 × 10
6iU, protein stabiliser 0.01 ~ 5.0g, thickening agent 0.1 ~ 20.0g, pH adjusting agent 0.1 ~ 10.0g, buffer agent 0.1 ~ 10.0g, wetting agent 0.1 ~ 50.0g, inject and be settled to 1000ml with water.
2. described in the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent as claimed in claim 1, pH adjusting agent is triethanolamine, sodium hydroxide, sodium bicarbonate, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, Borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, sulphuric acid, nitric acid, one or more mixing in phosphoric acid.
3. the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent as claimed in claim 1, it is characterized in that, described thickening agent is one or more mixing in dextrin and derivant, sodium alginate and derivant thereof, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl emthylcellulose, hydroxypropyl cellulose, methylcellulose, carboxymethyl cellulose, arabic gum, tragcanth, guar gum, carbopol, phytohemagglutinin, hyaluronic acid, xanthan gum, chitosan, tyloxapol.
4. the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent as claimed in claim 1, is characterized in that, described wetting agent is one or more mixing in glycerol, propylene glycol.
5. the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent as claimed in claim 1, it is characterized in that, described protein stabiliser is one or more mixing in mannitol, sodium lauryl sulphate, glycosides propylhomoserin, tween, dextran, hyaluronic acid, chondroitin sulfate, heparin.
6. the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent as claimed in claim 1, it is characterized in that, described buffer agent be citric acid receive/citric acid buffering to, phosphate-buffered to one or more mixing to, glycine-triethylamine buffering centering of, tris-hydrochloride buffer.
7. the recombined human alkaline fibroblast growth factor gelling agent of not bacteriostatic agent as claimed in claim 1, it is characterized in that, the recombined human alkaline fibroblast growth factor gelling agent of described not bacteriostatic agent adopts the independent packaging of not bacteriostatic agent, and the volume scope of the container of the independent packaging of described not bacteriostatic agent is that 0.1 ~ 0.5ml/ props up.
8. the preparation method of the not recombined human alkaline fibroblast growth factor gelling agent of bacteriostatic agent, is characterized in that, comprise the following steps:
S1: take thickening agent 0.1 ~ 20.0g, pH adjusting agent 0.1 ~ 10.0g is dissolved in water for injection, sterilizing 30min;
S2: take recombination human basic fibroblast growth factor 1 × 10
6~ 9 × 10
6iU, protein stabiliser 0.01 ~ 5.0g, buffer agent 0.1 ~ 10.0g and wetting agent 0.1 ~ 50g are dissolved in water for injection;
S3: the medicinal liquid of step S2 gained is mixed with step S1 gained medicinal liquid after 0.22 ~ 0.45 μm of filtering with microporous membrane 5 times, and is settled to 1000ml with water for injection, stir;
S4: fill step S3 gained medicinal liquid is in the packing container of not bacteriostatic agent, and the volume scope of container is that 0.1 ~ 0.5ml/ props up.
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