CN109966245A - A kind of brimonidine tartrate gellan gum type situ-gel eye drops and preparation method - Google Patents

A kind of brimonidine tartrate gellan gum type situ-gel eye drops and preparation method Download PDF

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CN109966245A
CN109966245A CN201910267066.XA CN201910267066A CN109966245A CN 109966245 A CN109966245 A CN 109966245A CN 201910267066 A CN201910267066 A CN 201910267066A CN 109966245 A CN109966245 A CN 109966245A
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gellan gum
eye drops
brimonidine tartrate
gel
situ
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王俏
汤湛
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Zhejiang Academy of Medical Sciences
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Zhejiang Academy of Medical Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics

Abstract

The invention discloses a kind of brimonidine tartrate gellan gum type situ-gel eye drops, including following raw material components in terms of 100mL: brimonidine tartrate 0.04-0.2g, gellan gum 0.4-0.55g, osmotic pressure regulator 2-5.5g, pH adjusting agent, 0.01-0.08g, thickener 0-0.2g, bacteriostatic agent 0-0.05g, adds water to 100mL.Brimonidine tartrate gellan gum type situ-gel eye drops of the present invention is by being made gellan gum type situ-gel eye drops for brimonidine tartrate drug, commercially available brimonidine tartrate eye drops in compared with the prior art, drug concentration in aqueous humor can be made to be maintained at higher level in a long time, highest drug concentration and accumulation medication amount greatly improve, increase bioavilability, drug frequency of usage can be reduced, a possibility that side effect occurs is reduced.

Description

A kind of brimonidine tartrate gellan gum type situ-gel eye drops and preparation method
Technical field
The present invention relates to eye drops technical fields, more particularly to a kind of brimonidine tartrate gellan gum type situ-gel Eye drops and preparation method.
Background technique
Glaucoma is one of irreversible most important reason of blindness, is common with characteristic optic atrophy and defect of visual field Feature, it is its main physical signs that pathologic intraocular pressure, which increases,.Epidemiological study the result shows that: the every raising 1mmHg of intraocular pressure suffers from glaucoma Probability is increased by 12%.The disease mainly causes since aqueous humor circulation is destroyed, and drug therapy mainly passes through inhibition room Moisture is secreted to reduce intraocular pressure, ensures that the optic nerve of patient and retina are not destroyed.Therefore, the generation of intraocular aqueous humor is reduced Facilitate to reduce intraocular pressure with the discharge for increasing aqueous humor, there is the drug of neuroprotection more to have for selection while reducing intraocular pressure Help the treatment of such disease.Foreign countries show about the compliance data of glaucoma medication, ordinary eye drops due to Leachability is big, rinses and nasolacrimal duct or spills into cheek, may result in side effect, such as allergic blepharitis and contact dermatitis, In addition fluid loss often leads to that therapeutic dose is not achieved, these factors lead to the compliance of patient for treatment's glaucoma eye drops Bad (being lower than 50%).The good tolerance of comfort level and drug of medication is the key that improve Treatment Compliance, Using the smallest drug dose, least times for spraying, to reach optimal drop intraocular pressure effect be current glaucoma medication Principle.
Brimonidine tartrate (Brimonidine Tartrate, BRT) is clinically to lead selectivity α to be applied at present 23 adrenergic receptor agonists have the production for reducing aqueous humor for reducing open-angle glaucoma or the intraocular pressure of intraocular hypertension patient Double action mechanism that is raw and increasing uveoscleral outflow, while there is function of protecting optic nerve.Nearly 3 years BRT eye drops Usage amount, which is dropped at home in the drug of intraocular pressure, is sure to occupy always the first two.Exactly because BRT significant effect, U.S. in terms of dropping intraocular pressure ALLERGAN company develops BRT eye drops (concentration 0.1-0.2%), and trade name Alphagan (Alphagan) exists The countries such as the U.S., China, Britain and Brazil get the Green Light and list.Its medicine frequency is usually 2-3 times/day, every time 1 drop.But Due to most of glaucoma patients use this kind of ordinary eye drops when because physiologically the reason of (as blink reflection, lacrimal secretion With nasolacrimal duct drainage etc.), it is easy to be eliminated rapidly, causes the residence time before cornea short, the absorption of drug is restricted, and eye is raw Object availability is poor, and dosage is also difficult to control, to be easy using excessive or frequently, and since nasolacrimal duct drainage is easy Into other nontarget areas, to generate side effect.Since the medicine is easier to have potential CNS inhibition by blood-brain barrier Effect, therefore children are disabled.The BRT eye drops of low concentration, although the adverse reaction effect of BRT can be reduced to a certain extent Fruit is still limited.Therefore, develop the novel form of the drug be it is necessary, to reduce BRT in the adverse reaction of ocular, reduce nose Mucous membrane is to the systemic Absorption of BRT, the effect of keeping drug to reduce intraocular pressure.
In recent years, the disadvantage lower for the bioavilability for solving traditional ophthalmically acceptable liquid preparation, domestic and foreign scholars grind Bioavilability of a series of new drug delivery system come drug when improving local administration in eye, such as nano suspension, soft are studied carefully Cream, intraocular inserting agent and gel etc..Compared to traditional solution, these dosage forms are able to extend drug in the reservation of eye Between.But since it will cause eye-blurred (such as ointment), or since patient tolerability poor (such as intraocular inserting agent) influences, and It is not widely accepted.And ocular in-situ gel is a kind of macromolecule polymer solution, is administered with solution state, and in administration portion The advantages of position mutually becomes the drug delivery system of gel state, which has both solution and gel, and the dosage form is due to before phase change Viscosity is small, therefore dosage is accurate；Gel viscosity is big after phase transformation, colorless and transparent, can extend drug in the delay of eye Between, the problems such as reducing administration frequency, improve drug bioavailability, patient tolerability also can be improved, it has also become research hotspot.This Kind can be caused in the solution-gel phase transformation that eyeball surface occurs by 3 kinds of factors: temperature, pH and ion.1. responsive to temperature type has Poloxamer, cellulose derivative, xylan etc., wherein the most commonly used is poloxamers.2. pH is sensitive, there is shell poly- using more Sugar and carbomer.3. ionic is sensitive, such as gellan gum, alginates, beta-carrageenin.Compared with temperature-sensitive situ-gel, from Polymer concentration needed for sub- sensitive in-situ gel is lower；Compared with the situ-gel of pH sensitivity, ion-activited in situ gel can be incited somebody to action Prescription pH is adjusted to eye optimal pH, therefore to Ocular irritation minimum.Its ionic situ-gel is especially with gellan gum (gellan Gum) optimal for the development prospect of matrix.The gellan gum of low concentration forms anion polysaccharide in aqueous solution, and ionic strength increases Afterwards, gelling agent is become from solution, the ratio that cation increases gel-forming in tear is consequently increased.It is solidifying as eye in-situ Gel matrix, the prominent feature of gellan gum have (1) dosage low: general 0.6% hereinafter, small to Eye irritation；(2) it convenient for administration, is formed Gel is fast: the gellan gum aqueous solution of low concentration is a kind of solution of low-viscosity, shows extraordinary mobility, clicks and enters and intraocularly touch To after micro tear, in the presence of water, the cation in tear lures that gellan gum forms chain bridge from random coil and is transformed into Double helix is then assembled by double helix and forms combined area to form gel.(3) comfort enhances, and be evenly distributed: it is artificial Shear thinning behavior is shown in tear, i.e., stirring or eyelid frequently blink and viscosity can be made to decline, and static, viscosity can increase again Gel is presented, is conducive to ophthalmic administration and the distribution in eyeball surface.
Medicinal gellan gum (Gellan Gum) is initially the one kind developed by U.S.'s CP Kelco company the 1980s Microorganism edible glue is a kind of linear anion heteroglycan, by glucose, glucuronic acid, rhamnose with 2: 1: 1 molar ratio Quaternary repetitive unit is connected into, main polymer chain, relative molecular mass about 0.5 × 10 are formed6, structural formula is shown below:
Its research for having been used for a variety of drugs, such as timolol maleate, Ciprofloxacin Hydrochloride, Indomethacin, methanesulfonic acid Pefloxacin and gatifloxacin etc..Though there are the listing of timolol maleate situ-gel, brimonidine tartrate in foreign countries at present Compared with timolol maleate, the mechanism of action is different, and brimonidine tartrate is α 2- 3 adrenergic receptor agonists, In animal and human body use fluorometry studies have shown that, with dual mechanism of action: both reducing aqueous humor Generation, and increase uveal scleral outflow.Timolol maleate is non-selective β-adrenergic receptor retarding agent, drop Intraocular pressure exact mechanism is unclear, and intraocular pressure marks and the research of aqueous humor fluorophotometric prompts this product reducing iop and reduces aqueous humor life At correlation.In terms of the two side effect, brimonidine tartrate is to cardiovascular and lung function influence very little, and maleic acid thiophene Lip river You are then obvious.There is document to show that brimonidine tartrate also compares timolol maleate to retinal nerve fibre layer damage It is few, and the visual field progress of difference unlikely occurs.
Summary of the invention
The present invention is poor for brimonidine tartrate correlation eye drops eye bioavilability in the prior art, is easy to make It with excessive or frequently, and is easy to enter other nontarget areas by nasolacrimal duct drainage, the problems such as generation side effect, provide A kind of better brimonidine tartrate gellan gum type situ-gel eye drops of using effect.
A kind of brimonidine tartrate gellan gum type situ-gel eye drops, including following raw material components in terms of 100mL:
Preferably, the osmotic pressure regulator is at least one of mannitol, glycerol, glucose, 1,2-PD. It is furthermore preferred that the osmotic pressure regulator is mannitol.
Preferably, the pH adjusting agent is tromethamine.About pH adjusting agent, general report is with buffer salt body System, for example, commercially available brimonidine tartrate eye drops using sodium citrate and citric acid as buffer solution system, and it is of the invention Because ocular in-situ gel is made using gellan gum, the situ-gel of ionic encounters sodium, potassium, magnesium, calcium plasma can be by liquid Body is transformed into gel, so to avoid using, the present invention is preferred using tromethamine etc. as pH adjusting agent.
Preferably, the thickener be methylcellulose, carboxymethyl cellulose, in hydroxypropyl methyl cellulose at least It is a kind of.Thickener can not also add.
Preferably, the bacteriostatic agent is benzalkonium chloride, in benzalkonium bromide, p-hydroxybenzoate (methylparaben) It is at least one.Bacteriostatic agent is referred to as preservative, avoids bacteria breed for playing after eye drops packaging is opened, makes to drip The rotten effect of ocular fluid.Since some patients may be sensitive to preservative, cause ophthalmic uncomfortable, so anti-corrosion can also be removed Agent ingredient is changed to the packaging of odd-numbered day dosage, use as early as possible behind Kaifeng.
Preferably, the pH value of the brimonidine tartrate gellan gum type situ-gel eye drops is 6.0-7.0, infiltration Pressure is 260-310mOsmol/kg.
The pH of normal tear fluid is 7.4 or so, and has certain buffer capacity, and eye drops is diluted after instilling eye by tear, The buffer function of tear can neutralize ophthalmic solution, so can mitigate apparent sense of discomfort.But eye drops excessively meta-acid when, solidifiable The protein of eye mucous membrane；Excessively meta-alkalescence can then make the epithelial cell of a mucous membrane harden or expand.Therefore, eye drops pH is excessively high Or it is too low all can be irritant to eye.As pH it is improper caused by irritation, the secretion of tear can be increased, drug is caused to flow rapidly Mistake or even corneal damage.The pH of eye drops generally can be in 5.5~7.8 range of pH.There is document to think brimonidine tartrate More more stable than at 7.0 or more 6.5 or so, the pH of commercially available eye drops is 6.5, when considering that situ-gel does not add tromethamine PH be about 4, between adjustable pH to 6.0~7.0.
The present invention also provides the preparation methods of the brimonidine tartrate gellan gum type situ-gel eye drops, including Following steps:
(1) part water, stirring swelling are added in gellan gum；
(2) after dissolving remaining raw material with remainder water, in the gellan gum after being added to step (1) swelling, stirring is equal It is even to get the brimonidine tartrate gellan gum type situ-gel eye drops.
Preferably, the water that step (1) is added is the 60~70% of total Water, temperature is 90 ± 5 DEG C, when stirring is swollen Between be 15~20min.
Preferably, step (2) filtration sterilization after mixing evenly.Filtration sterilization can use 0.1 μm~0.22 μm aperture Filter membrane be filtered degerming.
Since the water that gellan gum meets higher temperature could leach faster, so first that gellan gum use is higher in the preparation Temperature be prepared into highly concentrated solution, while at this time due to do not contain main ingredient object brimonidine tartrate, high-temperature is to drug Influence it is relatively small；Then main ingredient is mixed with the dissolution of other ingredients, is added in gellan gum solution.It finally needs to be kept for 40 DEG C The above filtration sterilization, when because more than temperature thus, the viscosity of eye-drops solution is comparatively smaller very than 25 DEG C of room temperatures It is more that (it is smaller that gellan gum solution temperature gets over high viscosity, and such as in 10rpm revolving speed, 35 DEG C of viscosity is 6cP or so, 25 DEG C of viscosity For 125cP), 40 DEG C or more of viscosity can be in 3cP hereinafter, viscosity is small can be smoothly through filtering with microporous membrane.
Brimonidine tartrate gellan gum type situ-gel eye drops of the present invention is by by brimonidine tartrate drug system At gellan gum type situ-gel eye drops, compared with the prior art in commercially available brimonidine tartrate eye drops, can be longer Drug concentration in aqueous humor is set to be maintained at higher level in time, highest drug concentration and accumulation medication amount greatly improve, increase Bioavilability, it is possible to reduce drug frequency of usage reduces a possibility that side effect occurs.
Detailed description of the invention
Fig. 1 is different ionic strength in embodiment 1 to the influence testing result figure of 4 viscosity of prescription.
Fig. 2 is the variation detection knot of different prescription viscosity when being 40: 28 with the volume proportion of artificial tears in embodiment 1 Fruit figure.
Fig. 3 be in embodiment 1 prescription 3 when being 40: 28 with artificial tears' volume proportion different shear rate to viscosity shadow Ring testing result figure.
Fig. 4 is the cumulative in vitro release rate-of brimonidine tartrate situ-gel and commercially available eye drops made from embodiment 2 Time plot.
Fig. 5 is the body of brimonidine tartrate situ-gel made from embodiment 3 and commercially available eye drops in the in vitro sclera of rabbit Outer accumulation permeability-time plot.
Fig. 6 be embodiment 3 brimonidine tartrate situ-gel with commercially available eye drops in the external of rabbit isolated cornea Accumulate permeability-time plot.
Fig. 7 is brimonidine tartrate situ-gel made from embodiment 4 and commercially available eye drops drug in rabbit aqueous humor Concentration time curve figure.
Fig. 8 is the rabbit varieties of intraocular pressure of brimonidine tartrate situ-gel and commercially available eye drops made from embodiment 2 and 7 Figure.
Specific embodiment
Embodiment 1
According to the form below 1 prepares different prescriptions, can screen the amount of different gellan gums and hydroxypropyl methyl cellulose (HPMC).
The gelling ability of 1 different auxiliary material of table proportion.
Note :+: gelling, but shake fugitive；++: gelling rapidly is shaken not fugitive；+++: gelling, shaking are less susceptible to rapidly It dissipates.
Preparation method: it takes gellan gum to be added in appropriate 90 DEG C or so purified waters, the knot of weight percent content 0.6% is made Gellan gum solution, stirring swelling are complete.Then dilution can obtain the gellan gum of 0.3,0.4,0.45,0.5% various concentration respectively；It takes 0.2g HPMC is dissolved in 25mL pure water, is taken 2.5mL that 0.6% gellan gum of 7.5mL is added, that is, is formed prescription 5；1.25mL is taken to add 0.6% gellan gum water supplement of 7.5mL forms prescription 4, remaining prescription is prepared by similar approach to 10mL.
Gelling ability measurement: take 2mL artificial tears (34 DEG C, sodium-chloride water solution 6.7gL-1, sodium bicarbonate aqueous solution 2.0g·L-1, CALCIUM CHLORIDE DIHYDRATE aqueous solution 0.8gL-1) be placed in tool plug test tube, 34 DEG C of water-bath heat preservations are in situ by 100 μ L Gel is added in test tube, observes and records the time of gel-forming and dissolution.
In addition, matching (ion with tear using the viscosity that Brookfield DV-2T viscosimeter can measure different prescriptions Intensity) and revolving speed (shear rate) situation of change.Tear can constantly be secreted by eye after administration, therefore by situ-gel and not Artificial tears in proportion are uniformly mixed, and are added in specimen cup, balance and start to measure it after 5min temperature is 34 DEG C, revolving speed is Under 10RPM, the case where viscosity changes over time, each sample measures 4 times respectively.By taking prescription 4 as an example, viscosity is with ionic strength The case where variation, sees Fig. 1, and before artificial tears are not added, the viscosity of situ-gel is 7cP or so, and the artificial of different ratio is added After tear, viscosity can play very big variation with the variation of ionic strength.This is increasing and increase reduction with tear amount Process, embody the characteristic of ion situ-gel.Prescription with the volume proportion of artificial tears be 40: 28 when overall viscosity most Height, each prescription at this moment the case where visible Fig. 2.Influence of the shear rate to prescription viscosity is also very high, right by taking prescription 3 as an example Viscosity change when prescription and the volume proportion of artificial tears are 40: 28 is shown in Fig. 3, and shear rate is higher, viscosity show it is smaller, this The characteristic of shear thinning can be shown in artificial tears by demonstrating gellan gum, i.e., stirring or eyelid frequently blink and can make under viscosity Drop, static, viscosity can increase presentation gel again, be conducive to ophthalmic administration and the distribution in eyeball surface.
The proportioning test result of these types of gellan gum and HPMC can be used as the foundation of selection gellan gum weight percent content, Gellan gum is mostly important, preferably can be 0.4-0.5%.
Embodiment 2
Preparation method: it takes gellan gum to be added in appropriate 90 DEG C or so purified waters, the knot of weight percent content 0.6% is made Gellan gum solution, stirring swelling are complete；It separately takes the hydroxypropyl methyl cellulose of recipe quantity 15mL purified water to dissolve, tartaric acid is added Brimonidine, glycerol, tromethamine, p-hydroxybenzoate, stirring and dissolving, above-mentioned weight percent content, which is added, is 0.6% gellan gum solution 70mL, then plus purified water to 100mL, 0.22 μm of filtering with microporous membrane dispenses to obtain the final product, tartaric acid bromine Mo Niding weight percent content is 0.1%, gellan gum final weight percent concentration 0.42%.PH value is 6.5, and osmotic pressure is 260mOsmol/kg。
Extracorporeal releasing experiment: precision pipettes 1.0mL brimonidine tartrate ocular fluid (1mg/mL) respectively, with the present embodiment 2 Dialysis sack is tightened in the bag filter that molecule interception is 3500 and is placed on cillin bottle by homemade gellan gum situ-gel In.The artificial tearful eyes of 25mL are added to be placed in constant-temperature table as dispersive medium, and by cillin bottle, temperature controls the concussion at 34 DEG C Frequency 100rpm.At regular intervals (15min, 30min, 45min, 60min, 75min, 90min, 120min, 150min, 180min), 1mL is sampled, and adds isothermal artificial tearful eyes in equal volume in time.Sample is after being centrifuged, using Syrups by HPLC Medicament contg.Chromatographic column: C18Column (Diamonsil, 4.6mm × 150mm, 5 μm, Dikma company), mobile phase: acetonitrile: Chinese holly Rafter acid buffer (0.017mol/L, triethylamine tune pH=3) volume ratio is 0.08: 0.92；Flow velocity: 1.0mL/min；Detect wave It is long: 248nm；Column temperature: 30 DEG C；Sample volume: 20 μ L.
As a result see Fig. 4, in Fig. 4, prescription 1 is the product that embodiment 2 is prepared；It compares as the tartaric acid bromine with concentration not Buddhist nun determines ordinary eye drops and (dilutes one times by the commercially available brimonidine tartrate eye drops that concentration is 2mg/mL to obtain.Commercially available winestone Sour every milliliter of Brimonidine eye drops (2mg/mL) contains: brimonidine tartrate 2mg, benzalkonium chloride 0.05mg, polyvinyl alcohol 14mg, sodium chloride 7.0mg, sodium citrate 4.5mg, citric acid 0.5mg and purified water.), test number of repetition n=4.
By the testing result of Fig. 4 it is found that compared with brimonidine tartrate ordinary eye drops, prepared by the embodiment of the present invention 2 Gellan gum group discharge no notable difference.
Embodiment 3
Preparation method: taking recipe quantity gellan gum to be added in 90 DEG C or so the purified waters of 700mL, and stirring swelling is complete；Separately take Brimonidine tartrate, mannitol, tromethamine, benzalkonium chloride are set in bottle, and about 150mL purified water is taken to be stirred to dissolve, It is added in above-mentioned gellan gum solution, takes about 100mL pond to wash bottle and be added again.Mixed solution adds purified water to 1000mL, and 40 DEG C or more 0.22 μm of filtering with microporous membrane, dispense to obtain the final product.Brimonidine tartrate weight percent content is 0.2%, gellan gum Final weight percent concentration 0.48%.PH value is 6.74, osmotic pressure 289mOsmol/kg.
The in vitro sclera of rabbit (or cornea) penetration study: after new zealand rabbit pneumatic needl is put to death, eyeball is taken immediately, it is extra to remove Tissue, carefully isolates sclera (or cornea).Fresh in vitro sclera (or cornea) is fixed on self-control arc port Franz diffusion cell Supply pool and reception tank between, make epithelial layer towards supply pool (or cornea on the outside of towards supply pool), be added in acceptance pool The STF solution about 3mL of Fresh catches up with most bubble, and temperature is simultaneously controlled in 34 DEG C by magnetic agitation, then respectively in supply pool The mixed solution of 80 μ L BRT-ISG+14 μ LSTF or mixing for commercially available+14 μ L STF of brimonidine tartrate eye drops of 80 μ L is added Close liquid.Start timing after preparation is added, take out 100 μ L samples from reception tank at regular intervals, while supplementing isometric etc. Warm STF solution, HPLC method measure drug concentration.Figure is done to accumulate infiltration capacity Q and time t.
As a result as shown in Fig. 5 and Fig. 6, compared with existing commercially available brimonidine tartrate eye drops, the embodiment of the present invention 3 The in vitro sclera of rabbit (Fig. 5) and cornea (Fig. 6) of the gellan gum group of preparation permeate on the whole without notable difference.
Embodiment 4
Preparation method: taking recipe quantity gellan gum to be added in 90 DEG C or so the purified waters of 600-700mL, and stirring swelling is complete； It separately takes tromethamine, mannitol, brimonidine tartrate to be placed in small beaker and about 50 DEG C of 100mL or so purified water stirrings is added Dissolution, is added in above-mentioned gellan gum solution, takes to swing for about 100mL moisture 2-3 times and washes small beaker and be added again.Mixed solution adds purifying Water to 1000mL, 50-60 DEG C of 0.22 μm of filtering with microporous membrane dispenses to obtain the final product.Brimonidine tartrate degree is 0.2%, the final mass percent concentration of gellan gum is 0.45%.PH value is 7.0, osmotic pressure 293mOsmol/kg.
Rabbit eyes irritation test: take quarantine qualified, new zealand rabbit 4 health, half male and half female.Using androgynous left and right sides Own control, every rabbit left eye give test sample, and right eye, which is given, to be compareed.The head Right deviation for making animal, allow tested left eye obliquely upward, The palpebra inferior for gently mentioning side eyes, by test sample, (the ophthalmically acceptable gellan gum of the brimonidine tartrate that embodiment 4 is prepared is solidifying in situ Jelly) 1 drop (about 50 μ L) directly drip in the eyelid 10s that in conjunctival sac, gently sleeps.0.9%NaCl is added dropwise with same procedure in right side eyes Aqueous solution is as control.Successive administration 14 days.1,2,4,24,48 and after preceding and last time is administered are administered for the first time in daily Eye irritation reaction inspection being carried out with ophthalmoscope and is scored within 72 hours, eye irritation is experiments have shown that nonirritant.
Pharmacokinetic experiments in rabbit aqueous humor:
Take weight in the healthy new zealand white rabbit of 2.5-3.0kg as experimental animal.20% crow of rabbit auricular vein injection is drawn Smooth (dosage 1g/kg) anesthesia, in due course after-teeming are penetrated, and guarantee that animal is constantly in narcosis in entirely examination experimentation.It is inserting Before entering probe, 1% Tropicamide first is instilled in eye and amplifies pupil.Eyelid is strutted with eye speculum, assists visiting by 27G injection needle Needle is inserted into aqueous humor, and injection needle is inserted into from an end margin of cornea across cornea, the other end that anterior chamber reaches cornea is then crossed Edge, then probe sample collecting terminal is carefully inserted into the beveled end of injection needle, then injection needle is slowly extracted out so that probe On dialysis membrane be located at the centre of anterior chamber, close threading hole with adhesive of medical.PH7.4 woods lattice are perfused into probe for micro syringe pump Family name's buffer salt solution balances 1h, is thus capable of sufficiently recovering anti-aqueous humor in intraocular pressure and camera oculi anterior.
Commercially available every milliliter of brimonidine tartrate eye drops (2mg/mL) contain: brimonidine tartrate 2mg, benzalkonium chloride 0.05mg, polyvinyl alcohol 14mg, sodium chloride 7.0mg, sodium citrate 4.5mg, citric acid 0.5mg and purified water.
Images of left and right eyes gives situ-gel and commercially available brimonidine tartrate eye drops (2mg/mL) 50 μ L respectively, is being administered Afterwards 30,60,90,120,150,180,210,240,270,300min collect aqueous humor microdialysis sample, with efficient liquid phase-string Join mass spectroscopy drug concentration.
Liquid matter system: Agilent liquid matter system (Anjelen Sci. & Tech. Inc, the U.S.), module composition: Agilent1290 liquid phase systems, 6460 triple quadrupole mass spectrometer of Agilent, MassHunter WorkstationSoftware B07 software.
LC/MS/MS condition:
Liquid-phase condition: it uses chromatographic column ZORBAX SB-C18 (3.0 × 150mm, 3.5 μm)；Mobile phase: A:0.1% formic acid Water；B: acetonitrile, isocratic elution program: 0min~2.5min (a sample needle time): 50%B；Flow velocity: 0.4mL/min；Sample introduction Amount: 5 μ L；40 DEG C of column temperature.
Mass Spectrometry Conditions: atmospheric pressure electrospray ion source (ESI): positive ion mode, multiple reaction Salbutamol Selected Ion Monitoring (MRM)；
Source parameters:
Dry temperature degree (Gas Temp): 300 DEG C
Dry gas stream speed；(Gas Flow)5L/min
Atomization gas pressure: (Nebulizer) 45psi
Sheath temperature degree: 250 DEG C of (Sheath Gas Temp)
Sheath gas: (Sheath Gas Flow) 11L/min
Capillary voltage: (Capillary) 3500V
Spray nozzle voltage: (Nozzle Voltage) 500V；
Mass spectral analysis parameter is shown in Table 2.
The MRM mass spectral analysis parameter of 2 compound of table.
The drug concentration measured is compared and analyzed, is handled with DAS2.0 pharmacokinetics software.Drug concentration-in aqueous humor Time graph result is shown in Fig. 7, and pharmacokinetic parameters are shown in Table 3 (n=10).The results show that commercially available stone acid Brimonidine eye drops aqueous humor For interior concentration always in relatively low level, area under the concentration-time curve (AUC) is 412.6 ± 204.3mg/Lmin, highest Concentration (Cmax) is 3.1 ± 1.4mg/L, and situ-gel prepared by the embodiment of the present invention 4 can make aqueous humor medicine in a long time Object concentration is held in higher drug concentration level, and AUC is 1397.1 ± 444.6mg/Lmin, and Cmax is 8.2 ± 2.6mg/ L.The AUC of situ-gel is 3.38 times of commercially available eye drops, and Cmax is 2.67 times of commercially available eye drops, and the two has extremely significant property poor Different (p < 0.01).
3 brimonidine tartrate situ-gel of table and pharmacokinetic parameters (n=10) of the commercially available eye drops in lagophthalmos.
Pharmacokinetic parameters Unit Commercially available eye drops Situ-gel eye drops
AUC(0-t) mg·min·L-1 412.6±204.3** 1397.1±444.6
MRT(0-t) min 110.3±18.2** 140.1±17.5
t1/2 min 65.0±19.2* 75.6±17.7
Tmax min 60.0±21.2** 93.0±29.0
Cmax mg·L-1 3.1±1.4** 8.2±2.6
* the commercially available original position VS P > 0.05；
The commercially available original position VS * P < 0.01.
Embodiment 5
Preparation method: it takes recipe quantity gellan gum to be added in about 700mL purified water, is warming up to 90-95 DEG C, stirs to clarify； It separately takes methylcellulose to add 100mL purifying water-swellable completely, adds 1,2-PD, tromethamine, tartaric acid bromine not Buddhist nun It is fixed, it stirs evenly, is added in above-mentioned gellan gum solution.It is swung and is washed in medicinal liquid bottle addition mixed liquor in two times with purified water 100mL again. Mixed liquor adds purified water to 1000mL, is kept for 50-60 DEG C of temperature, 0.22 μm of filtering with microporous membrane dispenses to obtain the final product, and tartaric acid bromine is not It is 0.1% that Buddhist nun, which determines degree, and the final mass percent concentration of gellan gum is 0.4%.PH value is 6.0, and osmotic pressure is 286mOsmol/kg。
Embodiment 6
Preparation method: it takes recipe quantity gellan gum to be added in about 60mL purified water, is warming up to 90 DEG C or so, stirring shakes up；Separately Other auxiliary materials and brimonidine tartrate are taken, adds about 50 DEG C of 20mL or so purified waters, is stirred to dissolve, it is molten that above-mentioned gellan gum is added In liquid.It is swung and is washed in medicinal liquid bottle addition mixed liquor in two times with a small amount of purified water again.Mixed liquor adds purified water to 100mL, 40 DEG C with To obtain the final product, brimonidine tartrate degree is 0.2% to 0.22 μm of miillpore filter of upper mistake, the final mass percentage of gellan gum Concentration is 0.55%.PH value is 6.5, osmotic pressure 291mOsmol/kg.
Rabbit eyes irritation test: take quarantine qualified, new zealand rabbit 4 health, half male and half female.Using androgynous left and right sides Own control, every rabbit left eye give test sample, and right eye, which is given, to be compareed.The head Right deviation for making animal, allow tested left eye obliquely upward, The palpebra inferior for gently mentioning side eyes, by test sample, (the ophthalmically acceptable gellan gum of the brimonidine tartrate that embodiment 6 is prepared is solidifying in situ Jelly) 1 drop (about 50 μ L) directly drip in the eyelid 10s that in conjunctival sac, gently sleeps.Commercially available tartaric acid is added dropwise with same procedure in right side eyes Brimonidine eye drops is as control.Successive administration 14 days.In it is daily be administered for the first time before and last time be administered after 1,2, 4, Eye irritation reaction inspection is carried out with ophthalmoscope and is scored within 24,48 and 72 hours.Eye irritation is experiments have shown that two medicines are non-stimulated Property.
Embodiment 7
Preparation method: it takes recipe quantity gellan gum to be added in about 60mL purified water, is warming up to 95 DEG C, shakes up；Separately take carboxymethyl Cellulose adds water 20mL swelling completely, and tromethamine, glycerol, brimonidine tartrate is added, stirs evenly, above-mentioned knot is added In gellan gum solution.It is added in mixed liquor with a small amount of purifying pond wash liquid bottle again.Mixed liquor adds purified water to 100mL, crosses 0.22 μ M miillpore filter, dispenses to obtain the final product.Brimonidine tartrate degree is 0.04%, and the final mass percentage of gellan gum is dense Degree is 0.4%.PH value is 6.0, osmotic pressure 310mOsmol/kg.
Embodiment 8
Drop intraocular pressure experiment: new zealand rabbit, male and female have concurrently, make ocular hypertension model, and auricular vein injecting anesthetic is used 1mL syringe cornea edge, which punctures, extracts aqueous humor 0.1mL, reinjects 0.3% Carbomer solution 0.1mL, Tonopen intraocular pressure The daily variation surveyed intraocular pressure and observe lagophthalmos of meter, measurement 3 times, take its average value every time.It is greater than 22mmHg with intraocular pressure and continues l Show modeling success in week.Successful 8 rabbits of modeling (16 eyes) are taken, are randomly divided into 4 groups, every group of 4 eyes are respectively dropped into 0.2% The brimonidine tartrate of commercially available brimonidine tartrate eye drops, 0.1% and 0.04% situ-gel (embodiment 2 and 7) each 50 μ L, drop 1 time daily, physiological saline eye drops is as blank control.Intraocular pressure is measured, measurement 3 times, take its average value every time.Intraocular pressure becomes Fig. 8 is shown in change.0.1% and 0.04% brimonidine tartrate situ-gel and commercially available brimonidine tartrate eye drip as seen from the figure It is suitable that intraocular pressure effect drops in liquid.
Stability test: light blue polyester of high density eye-drop liquid bottle is distributed by preparation prepared by 3 prescription of embodiment and is sealed (every 5mL) is used as sample afterwards, does primary contributions factorial experiments and accelerated test,
1. influence factor is tested
(1) hot test: sample is respectively placed in 40 DEG C, places 10d in 60 DEG C of insulating box.And it is taken in the 0th, 5,10d Sample investigates preparation indices.
(2) strong illumination is tested: sample being set in the light incubator, is put under conditions of Yu Zhaodu 4500lx ± 500lx 10d is set, is sampled in 5d and 10d, investigates the indices of sample.
2. accelerated test: sample is placed in the constant incubator under the conditions of 40 ± 2 DEG C of temperature, relative humidity are 25 ± 5% In 6 months, sampled respectively at 1,2,3,6 month, investigate the indices of sample.
Test result:
Influence factor test: (1) 4 hot test: be the results are shown in Table.
Stability (n=3) under 4 hot conditions of table
The results show that with initial phase ratio, after being placed 10 days under the conditions of 40 DEG C and 60 DEG C of high temperature respectively, appearance, pH value and Content is substantially unchanged, and osmotic pressure is substantially also unchanged at 40 DEG C, and osmotic pressure value is slightly increased under 60 DEG C of high temperature, but still In human eye adaptation range.Show that said preparation is stablized under the conditions of 40 DEG C of high temperature, but 60 DEG C of hot conditions have certain shadow to it It rings.
(2) intense light irradiation is tested: the results are shown in Table 5.
Stability (n=3) under 5 illumination condition of table
The results show that with initial phase ratio, after being placed 10 days under the conditions of 4500lx ± 500lx, appearance, pH value and content base This is unchanged, and impurity slightly increases.
Accelerated test: 6 be the results are shown in Table.
6 accelerated test result (n=3) of table
The results show that accelerating after placing 6 months with initial phase ratio, appearance, pH value and content, related substance etc. slightly become Change, but still in the normal range.It is tested in conjunction with influence factor, shows that preparation should be protected from light shady place preservation.Generally speaking, it puts for a long time It sets more stable.

Claims (10)

1. a kind of brimonidine tartrate gellan gum type situ-gel eye drops, which is characterized in that including following original in terms of 100mL Expect component:
2. brimonidine tartrate gellan gum type situ-gel eye drops as described in claim 1, which is characterized in that the infiltration Pressure regulator is at least one of mannitol, glycerol, glucose, 1,2- propylene glycol thoroughly.
3. brimonidine tartrate gellan gum type situ-gel eye drops as claimed in claim 2, which is characterized in that the infiltration Pressure regulator is mannitol thoroughly.
4. brimonidine tartrate gellan gum type situ-gel eye drops as described in claim 1, which is characterized in that the pH Regulator is tromethamine.
5. brimonidine tartrate gellan gum type situ-gel eye drops as described in claim 1, which is characterized in that the increasing Thick dose is at least one of methylcellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose.
6. brimonidine tartrate gellan gum type situ-gel eye drops as described in claim 1, which is characterized in that the suppression Microbial inoculum is at least one of benzalkonium chloride, benzalkonium bromide, p-hydroxybenzoate.
7. brimonidine tartrate gellan gum type situ-gel eye drops as described in claim 1, which is characterized in that the wine The pH value of stone acid Brimonidine gellan gum type situ-gel eye drops is 6.0-7.0, osmotic pressure 260-310mOsmol/kg.
8. the preparation method of brimonidine tartrate gellan gum type situ-gel eye drops as described in claim 1~7 is any, It is characterized in that, comprising the following steps:
(1) part water, stirring swelling are added in gellan gum；
(2) it after dissolving remaining raw material with remainder water, in the gellan gum after being added to step (1) swelling, stirs evenly, i.e., Obtain the brimonidine tartrate gellan gum type situ-gel eye drops.
9. preparation method as claimed in claim 8, which is characterized in that step (1) be added water be total Water 65~ 70%, temperature is 90 ± 5 DEG C, and stirring swelling time is 15~20min.
10. preparation method as claimed in claim 8, which is characterized in that step (2) filtration sterilization after mixing evenly.
CN201910267066.XA 2019-04-03 2019-04-03 A kind of brimonidine tartrate gellan gum type situ-gel eye drops and preparation method Pending CN109966245A (en)

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Application publication date: 20190705