CN101987084B - Chitosan-collagen sustained-release eye drop and preparation method - Google Patents
Chitosan-collagen sustained-release eye drop and preparation method Download PDFInfo
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Abstract
The invention belongs to the field of pharmaceutics and particularly relates to a chitosan-collagen levofloxacin hydrochloride sustained-release eye drop and a preparation method thereof. The eye drop comprises the following components: 0.1 to 0.5 grams of levofloxacin hydrochloride, 0.6 to 3.0 grams of chitosan, 0.13 to 0.7 grams of collagen and 3 to 17 micrograms of bacteriostat. The preparation method comprises: adding a pH regulator to regulate the pH value to 6.0 to 7.0; adding an osmotic pressure regulator to regulate the osmotic pressure to 270 to 280mOsmol/kg; and diluting with aqueous solution of ethylic acid to 100 milliliters. In the invention, the levofloxacin hydrochloride sustained-release eye drop is prepared by using chitosan and collagen a thickener and synergist, the formula of the preparation is qualified, the preparation process is simple, and the preparation has the advantages of desirable treatment effect, high bioavailability, long treatment action, fewer toxic and side effects, and the like, avoids stimulating eyes and arousing allergy and is the first choice in clinic and daily use.
Description
Technical field
The invention belongs to pharmaceutical field, particularly a kind of hydrochloric acid ofloxacin slow-release eye drop that contains chitosan and collagen and preparation method thereof.
Background technology
Common eye drops is prone to behind eye drip by the tear dilution, and discharges from lacrimal passage very soon, thereby its bioavailability is low, and action time is of short duration, needs frequent drug administration repeatedly.In order to prolong the drug treating time of eye drop, reduce the medication number of times, reduce toxicity, improve curative effect, begun the research of spacetabs type ophthalmic preparation both at home and abroad.Eye use slow releasing preparation, splashes into behind the ophthalmic and can form the thin film of one deck at eyeball surface, can be as common eye drops most of the loss, thereby make medicine keep the treatment time of growing relatively at eye, the curative effect reinforcement; Slow releasing preparation has certain viscosity, and flowability is little, the transparency is good, do not influence sight line, can effectively overcome ophthalmic and be difficult for being detained medicine, drips medicine shortcoming often, does not have the greasy feeling of Eye ointments again, has so both guaranteed therapeutic effect, has made things convenient for the patient again.
Common commercially available levofloxacin hydrochloride eye drop mainly contains the Lang Yue that Beijing sharp auspicious medicine (group) limited company produces, central thickening agent and the slow-release material of not adding, and every day 3-5 time course of treatment, 1-2 drips at every turn.The Helen that Zhengda Furuida Pharmaceutical Co., Ltd., Shandong produces mainly adds hyaluronate sodium as a kind of stickiness excipient, and the retarding action time, be every day 3-5 time the course of treatment, and 1-2 drips at every turn.What have bibliographical information now mainly is to increase stickiness excipient such as carbomer and hyaluronate sodium, also is the one-side drug treating time that delays.
Bibliographical information chitosan and ofloxacin preparation gel eyedrop is arranged, but all be preliminary formulating, not molding as yet.In order to strengthen the anti-infectious function of levofloxacin hydrochloride eye drop at eye; Promote the healing of eye wound; Overcome the defective that it be prone to run off, needs frequent drug administration, the present invention prepares out a kind of levofloxacin hydrochloride sustained-release eye drop with chitosan and collagen protein as thickening agent and synergist and levofloxacin hydrochloride first.
Summary of the invention
In order to overcome the defective of prior art, one of the object of the invention is to propose the levofloxacin hydrochloride eye drop that contains chitosan and collagen protein that a kind of bioavailability is high, promote the eye wound healing.
Another object of the present invention is to provide preparation to contain the method for the levofloxacin hydrochloride sustained-release eye drop of chitosan and collagen protein.
The present invention proposes a kind of chitosan and collagen sustained-release eye drop of containing; Comprise following each component: levofloxacin hydrochloride 0.1-0.5g, chitosan 0.6-3.0g, collagen protein 0.13-0.7g; Antibacterial 3-17mg; Add pH regulator agent adjust pH to 6.0-7.0, add osmotic pressure regulator and transfer osmotic pressure to 270-280mOsmol/kg, the reuse aqueous acetic acid is diluted to 100ml.
Preferably; Each component of this eye drop is: levofloxacin hydrochloride 0.3-0.5g, chitosan 1.8-3.0g, collagen protein 0.4-0.7g; Antibacterial 10-17mg; Add pH regulator agent adjust pH to 6.2-6.5, add osmotic pressure regulator and transfer osmotic pressure to 280mOsmol/kg, the reuse aqueous acetic acid is diluted to 100ml.
The method for preparing of sustained-release eye drop according to the invention is characterized in that:
A) get chitosan 0.6-3.0g and add in the aqueous acetic acid fully swelling, stir solution A, get collagen protein 0.13-0.7g and add in the aqueous acetic acid fully swelling; Be stirred to fully dissolve solution B, solution A slowly be added in the solution B stir, add levofloxacin hydrochloride 0.1-0.5g; Stir; In mixed liquor, add antibacterial 3-17mg, the pH value that adds pH regulator agent accent mixed liquor is to 6.0-7.0, and the osmotic pressure that adds osmotic pressure regulator accent mixed liquor again is 270-280mOsmol/kg; Add aqueous acetic acid at last and be diluted to 100ml, fully mixing;
B) with mixed liquor warp 0.2 μ microporous filter membrane rustless steel filter pressurization aseptic filtration, filtrate is carried out high temperature sterilize, aseptic subpackaged, promptly get the levofloxacin hydrochloride sustained-release eye drop.
The time of said high temperature sterilize is 20-40 minute.
Said antibacterial is selected from a kind of in Nepal gold compound ester sodium, benzalkonium bromide, the sorbic acid.
Said pH regulator agent is selected from a kind of in sodium hydroxide, potassium hydroxide, the ammonia.
Said osmotic pressure regulator is selected from a kind of in sodium acetate, potassium acetate, the ammonium acetate.
The weight percent concentration of said aqueous acetic acid is 0.2-0.5%, is 0.4% for good with concentration.
The main component levofloxacin hydrochloride of eye drop of the present invention (purchasing the weighing apparatus Yu Pharmaceutical Co of Pu Kang group in Nanyang, lot number 20070302) is mainly used in treatment conjunctivitis and keratitis; Chitosan (is purchased the biological company limited of aokang in Shandong; Lot number 20070214) in eye drop of the present invention as synergist and thickening agent; Be the natural polysaccharide carapace rope that extracts in the Crustacean take off the acetyl product; Contain multiple hydrophilic functional group in chitosan and the derivative molecular thereof, have good hydrophilicity and bioadhesive; Collagen protein (purchase Yi Erkang biological engineering development centre, lot number in Beijing: 20070123) in eye drop of the present invention as synergist and tackifier.
Further specify the slow release drug action that the present invention contains the levofloxacin hydrochloride sustained-release eye drop of chitosan and collagen protein through preparation technology, pharmacokinetics test, pharmacodynamics test, safety evaluatio below.
One materials and methods
1 instrument and reagent
Day island proper Tianjin LC-10A high performance liquid chromatograph (LC-10Atvp high pressure pump, SPD-10Mavp UV-detector, SCL-10Avp chromatographic work station); Electronic analytical balance (METTLER AE240); Levofloxacin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute; Lot number 130455-200604), levofloxacin hydrochloride slow release eye drop (self-control, lot number 080310,080311,080312), chitosan, collagen, Nepal's gold compound ester sodium, benzalkonium bromide, sorbic acid, glacial acetic acid, sodium acetate, potassium acetate, ammonium acetate, sodium hydroxide, potassium hydroxide, ammonia are medicinal specification; Acetonitrile and purified water are chromatographically pure.
Sample sets: the levofloxacin hydrochloride self-control eye drop (lot number: 20080601) that adopts prescription of the present invention and method for preparing to process;
Matched group: 0.3% levofloxacin hydrochloride eye drop (Beijing auspicious pharmacy of profit group; Trade name: Lang Yue; Lot number 080501), 0.3% levofloxacin hydrochloride eye drop (Shandong Bausch & Lomb Freda Pharmaceutical Co., Ltd., trade name: Helen; Lot number 080901011) levofloxacin reference substance, metronidazole reference substance (Chinese pharmaceutical biological product is checked institute)
25 of rabbit (male, body weight 5.0 ± 0.2kg, Beijing dimension tonneau China animal center)
Two experimental sections
The preparation of 1 levofloxacin hydrochloride sustained-release eye drop and process route
1.1 confirmed the concentration and the inventory of chitosan and collagen through orthogonal experiment
According to the preliminary experiment situation; The relevant investigation factor of confirming slow-release material is chitosan concentration (A), collagen protein kind (B), collagen concentration (C); Each factor has arranged 3 levels to carry out preferably, and experimental factor and level arrangement see the following form 1, and the preparation scoring is that index comprehensive is considered with color sample, clarity, stretchability respectively; Each item index satisfaction person is 10 minutes, between other media and 0～10.Result: A
2＞A
1＞A
3, B
2＞B
1＞B
3, C
3＞C
2＞C
1, factor primary and secondary relation is A → B → C, optimal case is A
2B
2C
3, promptly the concentration of chitosan is 1.8%, the collagen protein kind is 1 collagen type (M about 2000, albumen 95%), collagen concentration 0.4%.
Table 1 factor level design table
1.2 confirmed the concentration and the ingredient proportion of chitosan through preliminary pharmacokinetic data
This laboratory is processed slow-release material with chitosan and collagen; And it is prepared the levofloxacin hydrochloride eye drop as medicine carrying substrate; Behind the lagophthalmos single-dose; Pharmacokinetic parameters presentation of results levofloxacin hydrochloride all meets the first order kinetics characteristics in three kinds of preparations, relative bioavailability result shows that this preparation has significantly improved the bright happy bioavailability of commercially available general formulation.
Under fixedly chitosan concentration and ratio, select the collagen aqueous acetic acid of variable concentrations to prepare the slow release eye drop.Medicine shows that for experimental result bioavailability increases with the increase of collagen concentration, but receives the restriction of dissolubility, so the concentration of this experimental selection collagen is 0.4%.
Under fixing glue original content and ratio, select the chitosan aqueous acetic acid of variable concentrations to prepare the slow release eye drop.Medicine shows that for experimental result bioavailability increases with the increase of chitosan concentration, but receives the restriction of viscosity, so the concentration of this experimental selection chitosan collagen is 1.8%.
1.3 confirmed best relevant parameter such as pH value and osmotic pressure through the addition of additives.
The selection of pH value:
According to pharmacopeia regulation, the pH value of ophthalmic preparation should be between 6～7, and the pH value of the aqueous solution of levofloxacin hydrochloride is about 5.0, and collagen protein and chitosan are about in the aqueous acetic acid about 5.0 at pH value and are swelling to dissolving.Experiment shows: the slow release eye drop of configuration is about in 6.3 the environment adularescent material at pH value and separates out.In order to reduce the zest of preparation to eye, consider that simultaneously levofloxacin hydrochloride and slow-release material are the most stable in PH neutrality, therefore transfer pH value to 6.20 with a kind of in sodium hydroxide, potassium hydroxide or the ammonia as the pH regulator agent.
The selection of osmotic pressure:
The osmotic pressure of eye drop must keep basically identical with 0.9% sodium chloride injection, and osmotic pressure and ion concentration are directly proportional.Experiment shows: the concentration of dilute acetic acid solution that is used for dissolving collagen and chitosan is about 0.4%, and the reuse sodium hydroxide is regulated pH value to 6.25, this moment solution osmotic pressure about 230mOsmol/kg.Final add a kind of in sodium acetate, potassium acetate or the ammonium acetate and transfer osmotic pressure to 280mOsmol/kg,, measure isosmotic solution through cryoscopic method by the sample of prescription configuration as osmotic pressure regulator.
The selection of antibacterial:
These article are multiple-unit container liquid ophthalmic preparation, and patient repeatedly uses very easy infection, need to add antibacterial.Nepal gold compound ester sodium, benzalkonium bromide and sorbic acid are a kind of of many uses, safe in utilization, food preservative freshness retaining agents that consumption is few, good water solubility not only, and the pH value scope of application is also very wide, and antimicrobial spectrum is expanded.These article are selected a kind of as antibacterial in Nepal's gold compound ester sodium, benzalkonium bromide or the sorbic acid, and dosage is selected 0.01% of its common dose.
The quality control of 2 levofloxacin hydrochloride sustained-release eye drops of the present invention
2.1 set up the serial of methods of the content of high effective liquid chromatography for measuring levofloxacin hydrochloride
2.1.1 chromatographic condition is established chromatographic column: DIKMA-DiamonsiL-C
18(250mm * 4.6mm, 5 μ m) chromatographic column; Mobile phase: water (containing 0.05M citric acid and 0.1M ammonium acetate)-acetonitrile=80: 20; Flow velocity: 1.0ml/mim; Detect wavelength: 294nm; Column temperature: 30 ℃; Sample size: 10 μ l.
2.1.2 sample, reference substance, blank article are handled
The preparation precision of reference substance stock solution takes by weighing the about 10mg of levofloxacin reference substance and puts in the 100ml volumetric flask, adds the 0.1mol/L dissolve with hydrochloric acid solution and quantitatively is diluted to scale, shakes up, and promptly gets.
The accurate about 1ml of levofloxacin hydrochloride slow release eye drop that draws of the preparation of need testing solution puts into the 100ml measuring bottle, adds the mobile phase dissolving and quantitatively is diluted to scale, shakes up, and filters.Precision is measured subsequent filtrate 5ml, places the 50ml measuring bottle, adds the mobile phase dissolving and quantitatively is diluted to scale, and precision is measured 10 μ l and injected high performance liquid chromatograph.
The accurate about 1ml of blank substrate that draws of the preparation of blank solution puts into the 100ml measuring bottle, adds the mobile phase dissolving and quantitatively is diluted to scale, shakes up, and filters.Precision is measured subsequent filtrate 5ml, places the 50ml measuring bottle, adds the mobile phase dissolving and quantitatively is diluted to scale, and precision is measured 10 μ l and injected high performance liquid chromatograph.
2.1.3 accurate reference substance stock solution 1.0,2.0,4.0,6.0,8.0 and the 10.0ml of drawing of the drafting of standard curve; Put respectively in the brown measuring bottle of 100ml; Add mobile phase and be diluted to scale, shake up, be made into the solution that concentration is 1.0,2.0,4.0,6.0,8.0 and 10.0 μ g/ml.Accurate respectively each 10 μ l that draw inject hplc determination, the record chromatogram, and returning with the peak area integrated value is vertical coordinate, reference substance concentration is abscissa, carries out linear regression.The regression equation of asking is: A=37189C-2820.8, r=0.9998.The result shows: levofloxacin is good in 1.0 μ g/ml-10.0 μ g/ml concentration range internal linear relation.Chromatogram is seen Fig. 1.
2.1.4 it is an amount of that recovery test takes by weighing levofloxacin reference substance each three minutes by recipe quantity 80%, 100%, 120% precision, adds in the blank substrate of recipe quantity ratio mix homogeneously respectively.The about 1ml of accurate respectively absorption puts into the 100ml measuring bottle, adds the mobile phase dissolving and quantitatively is diluted to scale, shakes up, and filters.Precision is measured subsequent filtrate 5ml, places the 50ml measuring bottle, adds the mobile phase dissolving and quantitatively is diluted to scale, and precision is measured 10 μ l injection high performance liquid chromatograph respectively.Average recovery rate is 100.8%, and RSD is 1.1%, and the result sees table 2.
The determination of recovery rates result of table 2 levofloxacin
2.1.5 it is that the standard solution of 4.0 μ g/ml repeats sample introduction 6 times continuously that the levofloxacin concentration of linear use will " 2.1.3 " be done in precision test down; The RSD that calculates the gained peak area is 0.35%; The relative standard deviation that shows continuous 6 its peak areas of sample introduction of same sample liquid is all less than 1.0%, and precision is good.
2.1.6 sample size mensuration is got three lot sample article and carried out assay, sample is by " 2.1.2 " method processing down, and the mensuration result sees table 3.
Table 33 lot sample article assay results
2.2 levofloxacin hydrochloride stability of formulation test
2.2.1 influence factor's test
Exposure experiments to light: the eye drop for preparing is divided in some eyedrops bottles; Put under (4500 ± 500) Lx strong illumination, in 0,5 and 10d sampling investigate indexs such as pH value, content, results sample each item index and comparison in 0 day: color has intensification slightly; PH value reduces; Content reduces, and points out instability under these article illumination condition, answers lucifuge to store.
Hot test: the eye drop for preparing is divided in some eyedrops bottles; Put 60 ℃ and 40 ℃; In 0,5 and 10d sampling investigate indexs such as pH value, content, 60 ℃ of samples of high temperature with 0 day relatively: color has intensification slightly, the pH value reduction; Content reduces, and indicates these article unstable under 60 ℃ of conditions of high temperature.40 ℃ of samples of high temperature and comparison in 0 day: each item index does not have significant change, indication sample steady quality under 40 ℃ of conditions.
Humid test: the eye drop for preparing is divided in some eyedrops bottles; Put and (put dry bottom) in the close drying device of relative humidity 20% ± 2% under 25 ℃ with the potassium acetate saturated solution; In 0,5 and 10d sampling investigate indexs such as pH value, content; With 0 day relatively: each item index does not have significant change, indication sample steady quality under 20% ± 2% and 25 ℃ of condition.
2.2.2 accelerated test
Get 3 batches of eye drop and place (40 ± 2) ℃; Relative humidity 20% ± 2% condition held six months; Indexs such as pH value, content are investigated in sampling 0,1,2,3,6 the end of month,, with 0 day relatively: appearance character does not have significant change, the pH value kept stable; The sign content of content is lower slightly, but all in acceptability limit.
The bioavailability of 3 levofloxacin hydrochloride sustained-release eye drops
Materials and methods
1) material
1.1 instrument
The high performance liquid chromatogram appearance is Tianjin, island LC-2020AHT model, and chromatographic work station is Shimadzu CLASS-VP, electronic balance model AE240 (Tianjin, island)
1.2 medicine and reagent
Adopt the levofloxacin hydrochloride self-control eye drop that prescription of the present invention and method for preparing process (lot number: 20080601), levofloxacin reference substance (Chinese pharmaceutical biological product check institute), metronidazole reference substance (mark in doing; Tianjin medicine inspecting institute), 0.3% levofloxacin hydrochloride eye drop (Beijing auspicious pharmacy of profit group, trade name: Lang Yue; Lot number 080501); 0.3% levofloxacin hydrochloride eye drop (Shandong Bausch & Lomb Freda Pharmaceutical Co., Ltd., trade name: Helen, lot number 080901011)
1.3 animal
25 of rabbit (male, body weight 5.0 ± 0.2kg, Beijing dimension tonneau China animal center)
1.4 chromatographic condition
Chromatographic column: Diamonsil C
18(5 μ m, 200 * 4.6mm), mobile phase: the acetonitrile-water phase (contains 1molL
-1Citric acid, 0.1molL
-1Ammoniom-Acetate)=and 20-80, flow velocity: 1mLmin, wavelength: 294nm, column temperature: 30 ℃, sample size: 10 μ L.
2) method
2.1 the preparation of standard curve
Title 5mg levofloxacin is used 0.1molL
-1HCl dissolving standardize solution gets concentration 0.5mgmL in the 10mL measuring bottle
-1, claim that again mark gets concentration 1mgmL with the dissolve with methanol standardize solution in the 10mL measuring bottle in the 10mg
-1, all be stored in 4 ℃ of refrigerators.Use 0.1molL
-1HCl dilutes levofloxacin to 0.25,0.50,1.0,2.5,5.0,10,20 μ gmL
-1, interior mark is diluted to 100 μ gmL with methanol
-1Get blank rabbit aqueous humor 90 μ L, add the levofloxacin solution mixing of the above-mentioned a series of concentration of 10 μ L, promptly get standard curve,, analyze by 2.2 " aqueous humor sample pre-treatments " operation.
2.2 aqueous humor sample pre-treatments
Get 100 μ L aqueous humor samples, add 100 μ gmL
-1Interior mark 10 μ L, mixing adds 200 μ L phosphate buffer (pH=7.4) mixings, adds chloroform extraction 2 times, each 800 μ L, combining extraction liquid, N
2Dry up, redissolve to 100 μ L, sample introduction with methanol.
2.3 medication
25 rabbits are divided into 5 groups at random, every group 5 10, adopt own control, cross-over experiment design, 1 week of cleaning phase at interval; Splash into 50 μ L self-control eye drop with pipettor respectively at fornix under each rabbit eyes, Lang Yue, the Helen is after administration 0.17,0.5; 0.75,1,1.5,2,3; 4,6,8h gets the preceding about 100 μ L of aqueous humor of lagophthalmos, and sample preservation is in-20 ℃, and is to be measured.
2.4 data analysis
Use 3p97, Microsoft Excel 2003 and Origin 6.0 carry out date processing and mapping, calculate elimination rate constant k and half-life t
1/2, TG-AUC AUC
0-tTry to achieve AUC with the trapezoidal area method
0-∞=AUC
0-t+ C
n/ λ, calculate relative bioavailability Fr with formula:
With paired t check the self-control eye drop is carried out the relative bioavailability statistical analysis with bright happy, Helen.
The result
3) analytical method conclusive evidence
3.1 specificity
Get blank aqueous humor sample,, analyze by 2.3 " aqueous humor sample pre-treatments " operation.Levofloxacin hydrochloride and interior target retention time are respectively 5.4min and 4.3min, and through sample drawing after more blank aqueous humor figure and the administration, showing does not have endogenous interference, sees Fig. 2.
3.2 standard curve
Concentration with determinand in the aqueous humor sample is abscissa, and determinand is a vertical coordinate with the ratio of interior target peak area, with weighted least-squares method (W=1/C
2) carrying out regressing calculation, the linear regression equation of gained is standard curve.Obtaining typical regression equation is y=0.0323+2.944x (r=0.9996), and the range of linearity is 0.025～2.0 μ gmL
-1, mark bent lower limit 0.025 μ gmL
-1
3.3 precision and accuracy
(concentration is respectively 0.05,0.5 and 2.0 μ gmL to repeat 3 different basic, normal, high three concentration of analyzing batch
-1) quality-control sample evaluation accuracy (R.E. representes with relative error) and precision (R.S.D. representes with relative standard deviation); Relative error R.E. is the average of measured value and the percent error value between the theoretical value; Relative standard deviation R.S.D. be standard deviation divided by meansigma methods, each concentration is carried out 6 sample analyses, adopt two factor factorials method of analysis of variance computational analysis method batch in and betweenrun precision; The result sees table 4, shows that precision and accuracy are up to specification.
3.4 extraction recovery
Getting blank aqueous humor sample 90 μ L adds 10 μ L levofloxacin hydrochlorides and makes into concentration 0.05,0.5,2.0 μ gmL
-1, other gets 0.1molL
-1HCl 90 μ L add 10 μ L levofloxacin hydrochlorides and make into concentration 0.05,0.5,2.0 μ gmL
-1, parallel 6 samples of each concentration.Calculating is levofloxacin hydrochloride peak area percentage ratio under two conditions, and the result sees table 4, and the result shows that the response rate under the variable concentrations is stable.
Accuracy, precision, the extraction recovery of table 4 levofloxacin hydrochloride in rabbit aqueous humor
3.5 medicine is for parameter and relative bioavailability
Preparation levofloxacin hydrochloride eye drop receives test preparation C, and (chitosan concentration is 1.8%; Collagen concentration is 0.4%), (chitosan concentration is 1.8% to levofloxacin hydrochloride eye drop D; Do not add collagen) and levofloxacin hydrochloride eye drop E (collagen concentration is 0.4%, does not add chitosan).After giving the levofloxacin hydrochloride eye drop and receiving test preparation C and two kinds of reference preparations (levofloxacin hydrochloride eye drop D and E), parameter A UC
0-t, AUC
0-∞, k, t
1/2The result see table 5.With AUC
0-tCalculate, draw the levofloxacin hydrochloride eye drop and receive test preparation C that the relative bioavailability of D and E is respectively 119.2%, 182.5%, levofloxacin hydrochloride mean concentration-time graph is seen Fig. 3 in the aqueous humor of 5 rabbits.
The medicine of table 5 self-control eye drop C, D, E is for parameter A UC
0-t, AUC
0-∞, k, t
1/2(n=5)
After giving the levofloxacin hydrochloride eye drop and receiving test preparation and two kinds of reference preparations (Lang Yue and Helen), parameter A UC
0-t, AUC
0-∞, k, t
1/2The result see table 6.With AUC
0-tCalculate, draw the self-control eye drop Lang Yue and Helen's relative bioavailability is respectively 213.2%, 110.4%, levofloxacin hydrochloride mean concentration-time graph is seen Fig. 4 in the aqueous humor of 5 rabbits.
Table 6 self-control eye drop, Lang Yue, Helen's medicine is for parameter A UC
0-t, AUC
0-∞, k, t
1/2(n=5)
Conclusion:
This experiment is with homemade levofloxacin hydrochloride slow release eye drop; Behind the lagophthalmos single-dose; Pharmacokinetic parameters presentation of results levofloxacin hydrochloride all meets the first order kinetics characteristics in three kinds of preparations; Relative bioavailability result shows that adding collagen artifact availability in the prescription increases; And collagen protein can also promote reparation and the cell growth of the damage of corneal epithelial cell, so on the basis of levofloxacin hydrochloride chitosan eye drop, add collagen again, is the innovation on the early stage working foundation.
This experiment is with homemade levofloxacin hydrochloride slow release eye drop; Behind the lagophthalmos single-dose; Pharmacokinetic parameters presentation of results levofloxacin hydrochloride all meets the first order kinetics characteristics in three kinds of preparations; Relative bioavailability result shows that this preparation has significantly improved bright happy bioavailability (p=0.002), does not have significant difference (p=0.959) with Helen's bioavailability.
The safety testing of 4 levofloxacin hydrochloride sustained-release eye drops of the present invention
4.1 eye irritant test
Get 6 of healthy White Rabbits, male and female half and half, body weight (2.5 ± 0.3) kg, the levofloxacin hydrochloride eye drop is dripped in a glance, and another eye drips normal saline.Continuous seven days, every day three times.24h after last instils, 48h and 72h observe the animal eyes.Family's rabbit cornea does not have untoward reaction such as hyperemia, edema, ulcer and muddiness, iris, and conjunctiva and secretions are all normal, compare no significant difference with normal saline, this eye drop anophthalmia IR.
4.2 skin irritation test
Get 25 of healthy albino guinea-pigs, body weight (300-400) g is divided into test specimen group (through the levofloxacin hydrochloride eye drop of sterilization), negative control (normal saline) and positive control (0.4%2-sulfydryl benzene cake thiazole).Percutaneous test comprises that Intradermal induces, and the part is induced and excited, and removes to stick 24h behind the thing, 48h, and skin is show speckle, edema phenomenon not, and the test specimen treated animal is kept the score and surpassed the negative control treated animal and do not keep the score, and this eye drop does not have sensitization.
4.3 acute toxicity test
4.3.1 experimental animal:
6 of Kunming kind white mice, male and female half and half, body weight 17-20g.
Animal origin: Beijing Vital River Experimental Animals Technology Co., Ltd.
Credit number: SCXK (capital) 2007-0001
4.3.2 test method: select the test method of limiting the quantity of for use.
Adopt tail vein constant speed injection slow release eye drop, dosage is 5000mg/kg.After injection, observed to 14 days at once and record animal general state, toxicity performance and dead animal number, and the record body weight change, see table 6.
4.3.3 result of the test:
The weight of animals log before and after table 6 test
Injection back animal the observation period in order, movable, appetite is normal, breathes steadily toxic reactions such as no convulsions, paralysis and death.The weight of animals day by day increases.
4.3.4 evaluation of result:
Levofloxacin hydrochloride self-control eye drop LD
50Greater than 5000mg/kg.No acute toxic reaction under this dosage.
4.4 the test of pesticide effectiveness
4.4.1 reagent
Sample sets: the levofloxacin hydrochloride self-control eye drop (lot number: 081201) that adopts prescription of the present invention and method for preparing to process;
Matched group: 0.3% levofloxacin hydrochloride eye drop (Beijing auspicious pharmacy of profit group, trade name: Lang Yue, lot number: 080501);
Matched group: 0.3% levofloxacin hydrochloride eye drop (Shandong Bausch & Lomb Freda Pharmaceutical Co., Ltd., trade name: Helen, lot number: 080901011);
Matched group: 0.9% sodium chloride injection (normal saline, Otsuka Pharmaceutical (China) Co., Ltd., lot number: 8C93B).
4.4.2 laboratory animal: 2 of healthy new zealand white rabbits, body weight 2.5-2.8kg, eyes are no abnormal.
Animal origin: Beijing Vital River Experimental Animals Technology Co., Ltd..
Licence numbering: SCXK (capital) 2007-0001
4.4.3 test strain: staphylococcus aureus [CMCC (B) 26003]
Source: Nat'l Pharmaceutical & Biological Products Control Institute.
4.4.4 test method:
The foundation of (1) damage type bacterial conjunctivitis model:
The preparation of infectious bacteria suspension: the staphylococcus aureus liquid that will cultivate 2 days with 0.9% sodium chloride injection is diluted to 5 * 109/ml (adopting the standard opacity tube to confirm).
Conjunctival damage and conjunctiva infect: draw 4mm * 4mm "+" with aseptic No. 6 syringe needles in the rabbit upper tarsal conjunctiva, be advisable just to scratch, the pulling eye eyelid becomes little cup-shaped gently, splashes into 2 infectious bacteria suspensions to the conjunctiva scar carefully, restores eyelid gently.
The treatment of (2) damage type bacterial conjunctivitis
Sample sets (levofloxacin hydrochloride self-control eye drop) and 3 matched groups (normal saline, bright happy eye drop and Helen's eye drop) are established in test; To infecting conjunctivitis animal continuous use 7 days; Every day at the upper and lower noon each 1 time; Each 0.1ml observes eye and sheds tears and secretions situation, conjunctival congestion and edematous condition.
4.4.5 result of the test:
(1) behind the infection of staphylococcus aureus 24h, lagophthalmos is aubergine, and with obvious edema and make eye be semi-closure and shape, a large amount of secretions soak eyelid, eyelashes and region near the eyes.
(2) therapeutic effect of bacterial conjunctivitis is seen table 7
The ocular injury catalog of keeping the score is respectively organized in table 7 test
The result shows that levofloxacin hydrochloride self-control eye drop therapeutic effect is better than matched group, particularly obviously is superior to the contrast of Lang Yue and normal saline.
Three brief summaries
Levofloxacin hydrochloride is a third generation fluoroquinolone antibacterial agent; Have good water solubility, active strong, wide spectrum, toxic and side effects is low and characteristics such as clinical efficacy height; And has a good permeability; Infection has good curative effect to ophthalmology such as bacterial conjunctivitis and keratitis, has potential applicability in clinical practice widely.Find through experimental study; The present invention has not only increased the viscosity of medicinal liquid through the adding of chitosan; Prolonged the medicine holdup time within the eye, increased the absorption of medicine, and chitosan is a kind of natural product; It is wide, cheap to originate, so be that a kind of novel eye that the value utilized is arranged very much is with thickening agent and synergist.Collagen protein is a kind of naturally occurring very biological substance of usury usefulness value that has; Show through experimental study of the present invention: collagen protein not only can promote the reparation and the cell growth of the damage of corneal epithelial cell; But also can be used as the carrier of ophthalmic administration; Medicine is discharged in conjunctival sac gradually, in the treatment of ophthalmic diseases, use quite extensive.
In order to strengthen the anti-infectious function of levofloxacin hydrochloride eye drop at eye, overcome the defective that it is prone to run off, need frequent drug administration, promote the healing of eye wound simultaneously.The present invention is thickening agent and synergist with chitosan and collagen protein, has prepared the levofloxacin hydrochloride sustained-release eye drop, and the said preparation prescription is qualified, and preparation technology is easy.Adopt the content of high effective liquid chromatography for measuring levofloxacin hydrochloride, and set up the method for its quality control, all in acceptability limit, preparation stability is good for content and parameters in the stability test.
This experiment is with homemade levofloxacin hydrochloride slow release eye drop; Behind the lagophthalmos single-dose; Pharmacokinetic parameters presentation of results levofloxacin hydrochloride all meets the first order kinetics characteristics in three kinds of preparations; Relative bioavailability result shows that adding collagen artifact availability in the prescription increases; And collagen protein can also promote reparation and the cell growth of the damage of corneal epithelial cell, so on the basis of levofloxacin hydrochloride chitosan eye drop, add collagen again, is the innovation on the early stage working foundation.Homemade levofloxacin hydrochloride eye drop and commercially available bright pleasing with the Helen are compared relative bioavailability; The result shows: this preparation is compared bioavailability and significantly improves (p=0.002) with bright please, and compares bioavailability with the Helen and does not have significant difference (p=0.959).
In sum; The beneficial effect that contains the levofloxacin hydrochloride sustained-release eye drop of chitosan and collagen protein of the present invention is: the present invention has that curative effect is good, bioavailability is high; Prolong effects such as curative effect, toxic and side effects are little; Experiment shows sustained-release eye drop of the present invention to the non-stimulated no sensitization of eyes, and effect all is superior to existing product.
Description of drawings
Fig. 1 is the HPLC chromatogram of levofloxacin reference substance (A), sample (B) and blank substrate (C);
Fig. 2 is levofloxacin hydrochloride and an interior target chromatogram in the aqueous humor, and wherein Fig. 2-a is blank aqueous humor chromatogram; Fig. 2-b is that blank aqueous humor adds levofloxacin hydrochloride and interior target chromatogram; Fig. 2-c is levofloxacin hydrochloride and an interior target chromatogram (mark in the 1-, 2-levofloxacin hydrochloride) in the aqueous humor after the administration;
Fig. 3 is a levofloxacin hydrochloride logarithmic mean concentration-time curve (n=5) in the rabbit aqueous humor;
Fig. 4 is a levofloxacin hydrochloride logarithmic mean concentration-time curve (n=5) in the rabbit aqueous humor.
The specific embodiment
Below in conjunction with accompanying drawing embodiments of the invention are done further explain.
Embodiment 1
Prescription:
Levofloxacin hydrochloride 0.5g
Chitosan 0.6g
Collagen protein 0.4g
Nepal gold compound ester sodium 3mg
PH value is 6.25
Osmotic pressure 280mOsmol/kg
Add aqueous acetic acid to 100ml
Its preparation method:
A) getting chitosan 0.6g, to be sprinkled into 54ml, concentration be abundant swelling in 0.4% the aqueous acetic acid, stir solution A, getting collagen protein 0.4g, to be sprinkled into 36ml, concentration be abundant swelling in 0.4% the aqueous acetic acid; Stir 2 hours to fully dissolve solution B, solution A slowly be added in the solution B stir, add levofloxacin hydrochloride 0.5g; Stir; In mixed liquor, add Nepal gold compound ester sodium 3mg, add the pH value to 6.25 that sodium hydroxide solution is transferred mixed liquor, it is 280mOsmol/kg that the adding sodium acetate is transferred the osmotic pressure of mixed liquor; Add aqueous acetic acid at last and be diluted to 100ml, fully mixing;
B) with mixed liquor warp 0.2 μ microporous filter membrane rustless steel filter pressurization aseptic filtration, filtrate was carried out high temperature sterilize 30 minutes, aseptic subpackaged, promptly get the levofloxacin hydrochloride sustained-release eye drop.
Embodiment 2
Prescription:
Levofloxacin hydrochloride 0.3g
Chitosan 1.8g
Collagen protein 0.4g
Nepal gold compound ester sodium 10mg
PH value is 6.2
Osmotic pressure 280mOsmol/kg
Add aqueous acetic acid to 100ml
Its preparation method:
A) getting chitosan 1.8g, to be sprinkled into 59ml, concentration be abundant swelling in 0.4% the aqueous acetic acid, stir solution A, getting collagen protein 0.4g, to be sprinkled into 36ml, concentration be abundant swelling in 0.4% the aqueous acetic acid; Stir 2 hours to fully dissolve solution B, solution A slowly be added in the solution B stir, add levofloxacin hydrochloride 0.3g; Stir; In mixed liquor, add Nepal gold compound ester sodium 10mg, add the pH value to 6.2 that sodium hydroxide solution is transferred mixed liquor, the osmotic pressure that adds sodium acetate accent mixed liquor again is 280mOsmol/kg; Add aqueous acetic acid at last and be diluted to 100ml, fully mixing;
B) with mixed liquor warp 0.2 μ microporous filter membrane rustless steel filter pressurization aseptic filtration, filtrate was carried out high temperature sterilize 30 minutes, aseptic subpackaged, promptly get the levofloxacin hydrochloride sustained-release eye drop.
Embodiment 3
Prescription:
Levofloxacin hydrochloride 0.5g
Chitosan 3.0g
Collagen protein 0.13g
Benzalkonium bromide 17mg
PH value is 6.5
Osmotic pressure 280mOsmol/kg
Add aqueous acetic acid to 100ml
Its preparation method:
A) getting chitosan 3.0g, to be sprinkled into 60ml, concentration be abundant swelling in 0.5% the aqueous acetic acid, stir solution A, getting collagen protein 0.13g, to be sprinkled into 30ml, concentration be abundant swelling in 0.5% the aqueous acetic acid; Stir 2 hours to fully dissolve solution B, solution A slowly be added in the solution B stir, add levofloxacin hydrochloride 0.5g; Stir; In mixed liquor, add benzalkonium bromide 17mg, add the pH value to 6.0 that potassium hydroxide solution is transferred mixed liquor, the osmotic pressure that adds potassium acetate accent mixed liquor again is 270mOsmol/kg; Add aqueous acetic acid at last and be diluted to 100ml, fully mixing;
B) with mixed liquor warp 0.2 μ microporous filter membrane rustless steel filter pressurization aseptic filtration, filtrate was carried out high temperature sterilize 20 minutes, aseptic subpackaged, promptly get the levofloxacin hydrochloride sustained-release eye drop.
Embodiment 4
Prescription:
Levofloxacin hydrochloride 0.1g
Chitosan 1.8g
Collagen protein 0.7g
Sorbic acid 10mg
PH value is 6.0
Osmotic pressure 280mOsmol/kg
Add aqueous acetic acid to 100ml
Its preparation method:
A) getting chitosan 1.8g, to be sprinkled into 55ml, concentration be abundant swelling in 0.2% the aqueous acetic acid, stir solution A, getting collagen protein 0.7g, to be sprinkled into 35ml, concentration be abundant swelling in 0.2% the aqueous acetic acid; Stir 2 hours to fully dissolve solution B, solution A slowly be added in the solution B stir, add levofloxacin hydrochloride 0.1g; Stir; In mixed liquor, add sorbic acid 10mg, add the pH value to 7.0 that ammonia is transferred mixed liquor, the osmotic pressure that adds ammonium acetate accent mixed liquor again is 270mOsmol/kg; Add aqueous acetic acid at last and be diluted to 100ml, fully mixing;
B) with mixed liquor warp 0.2 μ microporous filter membrane rustless steel filter pressurization aseptic filtration, filtrate was carried out high temperature sterilize 40 minutes, aseptic subpackaged, promptly get the levofloxacin hydrochloride sustained-release eye drop.
Embodiment 5
Prescription:
Levofloxacin hydrochloride 0.3g
Chitosan 0.9g
Collagen protein 0.6g
Nepal gold compound ester sodium 8mg (the another kind of antibacterial of replaceable one-tenth)
PH value is 6.2
Osmotic pressure 280mOsmol/kg
Add aqueous acetic acid to 100ml
Its preparation method:
A) getting chitosan 0.9g, to be sprinkled into 50ml, concentration be abundant swelling in 0.2% the aqueous acetic acid, stir solution A, getting collagen protein 0.6g, to be sprinkled into 40ml, concentration be abundant swelling in 0.2% the aqueous acetic acid; Stir 2 hours to fully dissolve solution B, solution A slowly be added in the solution B stir, add levofloxacin hydrochloride 0.3g; Stir; In mixed liquor, add Nepal gold compound ester sodium 8mg, add the pH value to 6.5 that potassium hydroxide solution is transferred mixed liquor, the osmotic pressure that adds potassium acetate accent mixed liquor again is 280mOsmol/kg; Add aqueous acetic acid at last and be diluted to 100ml, fully mixing;
B) with mixed liquor warp 0.2 μ microporous filter membrane rustless steel filter pressurization aseptic filtration, filtrate was carried out high temperature sterilize 35 minutes, aseptic subpackaged, promptly get the levofloxacin hydrochloride sustained-release eye drop.
Claims (8)
1. one kind contains chitosan and collagen sustained-release eye drop, it is characterized in that: this eye drop comprises following each component:
Levofloxacin hydrochloride 0.1-0.5g, chitosan 0.6-3.0g, collagen protein 0.13-0.7g; Antibacterial 3-17mg; Add pH regulator agent adjust pH to 6.0-7.0, add osmotic pressure regulator and transfer osmotic pressure to 270-280mOsmol/kg, the reuse aqueous acetic acid is diluted to 100ml.
2. sustained-release eye drop as claimed in claim 1 is characterized in that: this eye drop comprises following each component:
Levofloxacin hydrochloride 0.3-0.5g, chitosan 1.8-3.0g, collagen protein 0.4-0.7g; Antibacterial 10-17mg; Add pH regulator agent adjust pH to 6.2-6.5, add osmotic pressure regulator and transfer osmotic pressure to 280mOsmol/kg, the reuse aqueous acetic acid is diluted to 100ml.
3. according to claim 1 or claim 2 sustained-release eye drop is characterized in that: said antibacterial is selected from a kind of in Nepal's gold compound ester sodium, benzalkonium bromide, the sorbic acid; Said pH regulator agent is selected from a kind of in sodium hydroxide, potassium hydroxide, the ammonia; Said osmotic pressure regulator is selected from a kind of in sodium acetate, potassium acetate, the ammonium acetate.
4. according to claim 1 or claim 2 sustained-release eye drop, it is characterized in that: the weight percent concentration of said aqueous acetic acid is 0.2-0.5%.
5. the method for preparing of a sustained-release eye drop as claimed in claim 1 is characterized in that:
A) get chitosan 0.6-3.0g and add in the aqueous acetic acid fully swelling, stir solution A, get collagen protein 0.13-0.7g and add in the aqueous acetic acid fully swelling; Be stirred to fully dissolve solution B, solution A slowly be added in the solution B stir, add levofloxacin hydrochloride 0.1-0.5g; Stir; In mixed liquor, add antibacterial 3-17mg, the pH value that adds pH regulator agent accent mixed liquor is to 6.0-7.0, and the osmotic pressure that adds osmotic pressure regulator accent mixed liquor again is 270-280mOsmol/kg; Add aqueous acetic acid at last and be diluted to 100ml, fully mixing;
B) with mixed liquor warp 0.2 μ microporous filter membrane rustless steel filter pressurization aseptic filtration, filtrate is carried out high temperature sterilize, aseptic subpackaged, promptly get the levofloxacin hydrochloride sustained-release eye drop.
6. the method for preparing of sustained-release eye drop as claimed in claim 5 is characterized in that: said antibacterial is selected from a kind of in Nepal's gold compound ester sodium, benzalkonium bromide, the sorbic acid; Said pH regulator agent is selected from a kind of in sodium hydroxide, potassium hydroxide, the ammonia; Said osmotic pressure regulator is selected from a kind of in sodium acetate, potassium acetate, the ammonium acetate.
7. sustained-release eye drop as claimed in claim 5 is characterized in that: the weight percent concentration of said aqueous acetic acid is 0.2-0.5%.
8. sustained-release eye drop as claimed in claim 5 is characterized in that: the time of said high temperature sterilize is 20-40 minute.
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| CN2009101625235A CN101987084B (en) | 2009-07-31 | 2009-07-31 | Chitosan-collagen sustained-release eye drop and preparation method |
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| CN111848830A (en) * | 2019-04-30 | 2020-10-30 | 苏州大学 | Application of fluorine-containing compound modified chitosan as drug carrier and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1403158A (en) * | 2002-08-23 | 2003-03-19 | 中国科学院兰州化学物理研究所 | Eye drops containing chitosan derivative |
| CN101461777A (en) * | 2009-01-06 | 2009-06-24 | 河北科技大学 | Disposable levofloxacin hydrochloride eye drops without bacteriostatic agent and preparation method thereof |
| CN101461779A (en) * | 2009-01-06 | 2009-06-24 | 河北科技大学 | Disposable ofloxacin eye drops without bacteriostatic agent and preparation method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1403158A (en) * | 2002-08-23 | 2003-03-19 | 中国科学院兰州化学物理研究所 | Eye drops containing chitosan derivative |
| CN101461777A (en) * | 2009-01-06 | 2009-06-24 | 河北科技大学 | Disposable levofloxacin hydrochloride eye drops without bacteriostatic agent and preparation method thereof |
| CN101461779A (en) * | 2009-01-06 | 2009-06-24 | 河北科技大学 | Disposable ofloxacin eye drops without bacteriostatic agent and preparation method thereof |
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