CN106963728A - A kind of preparation method and applications of docosahexaenoic acid subconjunctival injection liquid - Google Patents
A kind of preparation method and applications of docosahexaenoic acid subconjunctival injection liquid Download PDFInfo
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- CN106963728A CN106963728A CN201610021245.1A CN201610021245A CN106963728A CN 106963728 A CN106963728 A CN 106963728A CN 201610021245 A CN201610021245 A CN 201610021245A CN 106963728 A CN106963728 A CN 106963728A
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- docosahexaenoic acid
- subconjunctival injection
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- injection liquid
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- 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
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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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
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
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- 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
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- 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
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- Pharmacology & Pharmacy (AREA)
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- Ophthalmology & Optometry (AREA)
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Abstract
The invention discloses a kind of preparation method and applications of docosahexaenoic acid subconjunctival injection liquid, the docosahexaenoic acid stoste for taking concentration to be 250mg/ml is dissolved in ethanol, it is wholly absent with liquid nitrogen air-flow slow evaporation ethanol to liquid, add normal saline dilution docosahexaenoic acid stoste molten to 8~12 μ g/ml, produce.Concentration is used to treat and prevent ocular dry eyes disease for 8~12 μ g/ml docosahexaenoic acid subconjunctival injection liquid.The present invention is verified through zoopery, docosahexaenoic acid is administered by subconjunctival injection, it is capable of the activation of effective depression effect T cell (Th1 auxiliary cells and Th17 auxiliary cells), and then the immunization inflammatory reaction and promotion lacrimal tissue functional rehabilitation of Effective Regulation eye surface, significantly alleviate ocular keratoconjunctivitis sicca disease damage etc. and dry symptom, so as to reach the purpose of prevention and treatment xerophthalmia.
Description
Technical field
Receive field the present invention relates to medicine, the preparation method of specifically a kind of docosahexaenoic acid subconjunctival injection liquid and
It is applied.
Background technology
DHA (Docosahexaenoic acid, docosahexaenoic acid, scientific name：All-cis 1,7,10,13,16,19- bis- ten two
Carbon acid, is a kind of important long-chain polybasic unsaturated fatty acid (C22H32O2), belong to the serial polynary unsaturated lipids of ω -3
Fat acid (polyunsaturated fatty acids, PUFAs).Due to lacking the desaturase of Δ more than 9, people and other lactations
Animal itself can not synthesize DHA, it is necessary to be obtained by food, thus DHA is known as how unsaturated essential fatty acid again.
DHA is content highest essential fatty acid in brain and retina, and it accounts for the ratio difference of polyunsaturated fatty acid
For：Brain 40%, retina 60%, and 50% weight comes from DHA in neuronal cell film.DHA is for brain
Development, the improvement of the culture of notice and eyesight there is important influence, therefore, international aliphatic acid and lipid research association
It is recommended that pregnant woman and the daily DHA intakes of nursing women are 300mg.DHA nutritional intervention is grown up extremely to infantile health
Important, the pregnancy period rationally carries out DHA nutritional supplementations and is conducive to prenatal and postnatal care.In each histocyte of human body, in retina cell
DHA content highest, can effectively protect retina, significantly improve eyesight.Sufficient DHA can prevent retina thrombus
Formed, prevent lipid from oozing out, so as to effectively suppress retinal microvascular disease.Recent epidemiological studies investigation is aobvious
Show, DHA intake can effectively reduce the incidence of inflammation disease and autoimmune disease, thus infer, DHA has anti-
Scorching, immunosuppressive effect.
Xerophthalmia refers to that tear film stability declines caused by occurring exception due to tear matter, amount or dynamics, then triggers
The general name for a variety of diseases that ophthalmic uncomfortable and (or) eyeball surface infringement are characterized, also known as angle xerosis of conjunctiva.2007
International dry eyes special study is pointed out：Dry eyes are the eye surface diseases that a kind of many factors (tear and ocular) induce, and it can
To cause, eye is uncomfortable, tear film is unstable, vision disorder, and severe patient can cause eyeball surface to damage, while dry eyes companion
There is tear film permeability to increase and ocular immunization inflammatory reaction.According to American Women health research project (WHS), doctor's health
Research project (PHS) and other research projects statistics, in the U.S., there are about the old dry eyes of more than 4,910,000 50 years old altogether
Patient (3,230,000 women and 1,680,000 males), estimation is researched and analysed according to epidemiology statistics, there are about at present 5%-35% with
The xerophthalmia patients of upper different age group.
The content of the invention
It is an object of the invention to provide a kind of docosahexaenoic acid subconjunctival injection for the treatment of and prevention for scheroma
The preparation method and applications of liquid, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides following technical scheme：
A kind of preparation method of docosahexaenoic acid subconjunctival injection liquid, takes the docosahexaenoic acid stoste that concentration is 250mg/ml
It is dissolved in ethanol, is wholly absent with liquid nitrogen air-flow slow evaporation ethanol to liquid, adds normal saline dilution docosahexaenoic acid
Stoste is molten to 8~12 μ g/ml, produces.
A kind of application of described docosahexaenoic acid subconjunctival injection liquid, by 22 carbon that concentration is 8~12 μ g/ml
Acid subconjunctival injection liquid is used to treat and prevent ocular dry eyes disease, and per injection amount is 4~6 μ L.
Compared with prior art, the beneficial effects of the invention are as follows：It is of the invention to be verified through zoopery, docosahexaenoic acid
Be administered by subconjunctival injection, can effectively depression effect T cell (Th1 auxiliary cells and Th17 auxiliary cells) swash
It is living, and then the immunization inflammatory reaction and promotion lacrimal tissue functional rehabilitation of Effective Regulation eye surface, significantly alleviate ocular angle conjunctiva
Inflammation damnification etc. dries symptom, so as to reach the purpose of prevention and treatment xerophthalmia.
Brief description of the drawings
Fig. 1 is the structural formula figure of docosahexaenoic acid.
Fig. 2 is the effect exemplary view after docosahexaenoic acid subconjunctival injection liquid is injected.
Fig. 3 is mouse cornea fluorescein sodium dyeing observation figure.
Fig. 4 is that mouse cornea fluorescein sodium dyes score value and cornea Tear secretion compares figure.
Fig. 5 is the PAS stain control figures of conjunctival tissue.
Fig. 6 is goblet cell counting compares figure in conjunctival tissue.
Fig. 7 is that the positive rate that effect TH1 auxiliary cells are expressed compares figure.
Fig. 8 is the design sketch for the positive rate that effect TH1 auxiliary cells are expressed.
Fig. 9 is that the positive rate that effect TH17 auxiliary cells are expressed compares figure.
Figure 10 is the positive rate design sketch that effect TH17 auxiliary cells are expressed.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Ground is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based on this
Embodiment in invention, the every other implementation that those of ordinary skill in the art are obtained under the premise of creative work is not made
Example, belongs to the scope of protection of the invention.
Embodiment 1
See Fig. 2, effect exemplary view after DHA parenteral solution subconjunctival injections.20 health C57BL/6J black female mices (18~
20g) it is divided into experimental group (10) and control group (10), using the mixed liquor of ketalar and xylazine by mouse
After anesthesia, eyeball bulbar conjunctiva is fully exposed, using No. 3 (30G) syringe needles with horizontal direction and eyeball into about 15 ° of angles, by pin
Head is pierced under the bulbar conjunctiva away from mouse cornea edge about 1~2mm temporos side, gently provokes bulbar conjunctiva, Hamilton is used afterwards
The milli mm of 701RN TLC syringes (10 μ L, syringe needle 26s G) inserting needle about 2, slowly by 5 μ L (experimental groups：DHA
Parenteral solution；Control group：Normal saline solution) parenteral solution injection canthus, visible bulbar conjunctiva swells into transparent fish bubble sample at this.
A small amount of artificial tears for being free of preservative is dripped in injection after finishing, mouse is put back in cage.Subconjunctival injection is respectively in dry eyes modeling
Carry out within first 24 hours, (7 days) 192 hours after (3 days), modeling in 72 hours after modeling.
Embodiment 2
See the comparison of the experimental group of mouse ocular dry eyes assessment and control group after Fig. 3-4, subconjunctival injection DHA parenteral solutions.Will experiment
Group (10) is with control group (10) mouse after conjunctival injection is carried out 24 hours, and concentration is 0.5mg/mL's
Scopolamine hydrobromide injection thigh both sides are alternately subcutaneously injected (1ml/ times, 3 times/day), and by mouse exposed to holding
In continuous air-flow (humidity ＜ 30%, daily at least 18h), mouse dry eye model induction is carried out with this.Continue after 10 days, it is right
Each group mouse carries out the dyeing of corneal epithelium fluorescein sodium and Schimer's test.
The plain sodium (10mg/ml) of 5 μ L liquid fluorescents is instilled in Fig. 3, the dyeing of corneal epithelium fluorescein sodium, mouse conjunctival sac
After 60s, corneal epithelium fluorescein sodium coloring case is observed under microscope cobalt blue light and is scored, appraisal result is shown in Fig. 4 (A).
Fig. 4 (A), is equally divided into 4 quadrants by cornea and scores respectively, by four quadrants of cornea and nose, temporo side bulbar conjunctiva
All score values are added and produce last score value.Standards of grading：Non-coloring：0 point；Point-like is coloured, 1 point；Slice colouring, 2
Point.The score value in each region is added and obtains last score value (total score：12 points).As a result confirm：DHA parenteral solution groups angle
The dyeing of film epithelium fluorescein sodium is substantially less than control group, and DHA parenteral solutions significantly alleviate corneal epithelial wound.
Fig. 4 (B), Schimer's test gently pulls open mouse palpebra inferior, clamps phenol red cotton thread with tweezers, is placed in small rathole
In outer canthus subordinate's conjunctival sac, stop after 15s and take out.Measure the RED sector length soaked by tear, every eye retest 3
It is secondary to average (each test interval 30min), mouse cornea epithelium should be avoided contact with or scratched in experiment.As a result confirm：
DHA parenteral solution group lacrimal secretion is significantly higher than control group, and DHA parenteral solutions are obviously promoted mouse lacrimal secretion.
Embodiment 3
After seeing that Fig. 5-7, measurement are terminated, cervical vertebra Tuo Baifa puts to death mouse, rounds an eyeball, and frozen section carries out PAS dyeing, light
Microscopic observation.And take the fresh cervical lymph node of mouse to be digested, separate parallel flow cytometry, detect dry eyes mouse neck
The change of lymph node Th1 auxiliary cells and Th17 auxiliary cell quantity.
Fig. 5 and Fig. 6, the PAS dyeing of conjunctival tissue and goblet cell are counted：By the whole eye including upper palpebra inferior
Ball is put into tissue freezing section's OCT embedding mediums, is put into rapidly in -80 DEG C of refrigerators and is frozen at least 2 hours, and frost is cut after taking-up
Piece is the sample of 5 μ m-thicks, is uniformly fitly laid on slide section with fine, soft fur pen, is carried out using 4% paraformaldehyde
After fixation, PAS dyeing is carried out with reference to reagent operation explanation, i.e., with being entered after 0.5% strong oxidizer periodate oxidation with Schiff's reagent
The processing of one step, dyes wind rose person with kytoplasm and confirms as Conjunctival Goblet Cells, count and average respectively.As a result confirm：
DHA parenteral solution group Conjunctival Goblet Cells density is significantly higher than control group, and it is close that DHA parenteral solutions significantly improve Conjunctival Goblet Cells
Degree.
Fig. 7-10, effect Th1 (Fig. 7 and Fig. 8), the positive rate ratio of Th17 (Fig. 9 and Figure 10) auxiliary cell expression
Compared with：The mouse cervical lymph node of separating experiment group and control group, is prepared into cell suspending liquid, is determined with direct fluorescent marker method,
Using different sulphur hydracid fluorescein (FITC) and phycoerythrin (PE) fluorescein.Every part of 3 pipes of sample point mark CD4- respectively
FITC, IFN γ-PE or IL17-PE and negative control pipe, (U.S. BD is public for each pipe plus the corresponding μ L of monoclonal antibody 10
Department) and dye solution, 30min is reacted, 1000 leave heart 5min, abandon supernatant, PBS liquid is washed 2~3 times, plus
The μ L of PBS 400, are detected after mixing using flow cytometer (LSR II, U.S. company BD).As a result confirm：
The positive rate of DHA parenteral solution group effects Th1, Th17 auxiliary cell expression is substantially less than control group, and DHA parenteral solutions significantly press down
The activation of effector T cell processed.
In summary, result above shows, docosahexaenoic acid (DHA) subconjunctival injection liquid is by reducing effect T
The activation of cell and then the immunization inflammatory reaction of Effective Regulation eye surface, are entered by promoting lacrimal secretion, improving goblet cell density
And promote lacrimal tissue functional rehabilitation, have in advance so that corneal epithelial wound triggered to ocular angle conjunctiva xerophthalmia etc. dries symptom
The anti-effect with treatment.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, and without departing substantially from this hair
In the case of bright spirit or essential attributes, the present invention can be realized in other specific forms.Which point therefore, no matter come from
See, embodiment all should be regarded as exemplary, and be it is nonrestrictive, the scope of the present invention by appended claims without
It is that described above is limited, it is intended that all changes fallen in the implication and scope of the equivalency of claim are included at this
In invention.Any reference in claim should not be considered as to the claim involved by limitation.
Claims (2)
1. a kind of preparation method of docosahexaenoic acid subconjunctival injection liquid, it is characterised in that it is the 20 of 250mg/ml to take concentration
Two carbon acid stostes are dissolved in ethanol, are wholly absent with liquid nitrogen air-flow slow evaporation ethanol to liquid, add normal saline dilution
Docosahexaenoic acid stoste is molten to 8~12 μ g/mL, produces.
2. a kind of application of docosahexaenoic acid subconjunctival injection liquid as claimed in claim 1, it is characterised in that be by concentration
8~12 μ g/mL docosahexaenoic acid subconjunctival injection liquid is used to treat and prevent ocular dry eyes disease, per injection amount
For 4~6 μ L.
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| CN201610021245.1A CN106963728A (en) | 2016-01-13 | 2016-01-13 | A kind of preparation method and applications of docosahexaenoic acid subconjunctival injection liquid |
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| CN201610021245.1A CN106963728A (en) | 2016-01-13 | 2016-01-13 | A kind of preparation method and applications of docosahexaenoic acid subconjunctival injection liquid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108096181A (en) * | 2018-02-05 | 2018-06-01 | 西安医学院 | DHA, EPA are in the application for the oral drugs for preparing treatment xerophthalmia |
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| CN1850062A (en) * | 2006-02-24 | 2006-10-25 | 无锡杰西医药科技有限公司 | Fatty acid eye nano preparations for dry eye disease |
| CN101043884A (en) * | 2004-07-01 | 2007-09-26 | 谢彭斯眼科研究公司 | Compositions and methods for treating eye disorders and conditions |
| CN101991535A (en) * | 2010-11-16 | 2011-03-30 | 王京南 | Docosahexaenoic acid (DHA) ester fat emulsion intravenous injection and manufacturing method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101043884A (en) * | 2004-07-01 | 2007-09-26 | 谢彭斯眼科研究公司 | Compositions and methods for treating eye disorders and conditions |
| CN1850062A (en) * | 2006-02-24 | 2006-10-25 | 无锡杰西医药科技有限公司 | Fatty acid eye nano preparations for dry eye disease |
| CN101991535A (en) * | 2010-11-16 | 2011-03-30 | 王京南 | Docosahexaenoic acid (DHA) ester fat emulsion intravenous injection and manufacturing method thereof |
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Cited By (1)
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| CN108096181A (en) * | 2018-02-05 | 2018-06-01 | 西安医学院 | DHA, EPA are in the application for the oral drugs for preparing treatment xerophthalmia |
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