CN109966251A - The drug and preparation method and the drug effect method of inspection of inhibition choroidal neovascularization - Google Patents
The drug and preparation method and the drug effect method of inspection of inhibition choroidal neovascularization Download PDFInfo
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- CN109966251A CN109966251A CN201910288715.4A CN201910288715A CN109966251A CN 109966251 A CN109966251 A CN 109966251A CN 201910288715 A CN201910288715 A CN 201910288715A CN 109966251 A CN109966251 A CN 109966251A
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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
- A61K31/4965—Non-condensed pyrazines
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- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
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- 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
Abstract
The invention discloses the drug for inhibiting choroidal neovascularization and preparation methods and the drug effect method of inspection, belong to the novel delivery system technical field of Chinese medicine, include: phosphatidase 1 9-57%, cholesterol 5-25%, ligustrazine 2-13% and sucrose 23-60%, the process for preparing medicine is as follows: phosphatide and cholesterol being total to molten vacuum rotary steam, film is made；Dissolving films are incubated for obtain blank liposome solutions；Then it is incubated for obtain Ligustrazine Liposomes solution；Protective agent sucrose is added and is freeze-dried to obtain Ligustrazine Liposomes powder；The medicine effect method of inspection is as follows: adding water to redissolve, by miillpore filter to ophthalmic administration；The drug is evaluated Ocular irritation and monitored to the drug effect for swashing photogenic choroidal neovascularization.The present invention is at present clinically for treating the deficiencies of drug of AMD is poor there are expensive and patient compliance, it is intended to more safely and effectively therapeutic intervention is carried out to AMD, to achieve the purpose that the quality of life for improving AMD patient.
Description
Technical field
The present invention relates to a kind of drug and preparation methods and the drug effect method of inspection, new more particularly to a kind of inhibition choroid
The drug and preparation method and the drug effect method of inspection of angiogenic belong to the novel delivery system technical field of Chinese medicine.
Background technique
Choroidal neovascularization is one of major pathologic features of wet age related macular degeneration and important blinding
Factor.Age-related macular degeneration (AMD) is also known as senile macular degeneration (SMD), is a kind of progressive degeneration's disease, tires out
And eyes, the eyeground pathological changes of the non-infectious damage of macular area are shown as, are the main retinopathies for seriously threatening the elderly's visual function
One of become.
AMD is clinically divided into two kinds of atrophic type (also known as stemness) and exudative type (also known as moist).Wet age macula lutea
Denaturation, also known as Exudative Age is macular degeneration related or neovascular age-related macular degeneration, Zhan Suoyou AMD's
10-20%, but the AMD severe visual obstacle of 80-90% is caused by moist AMD, moist AMD macular area choroidal neovascularization
(CNV) cause bleeding the main reason for being visual loss.Currently, the clinical treatment of AMD is mainly anti-using intravitreal
VEGF Drug inhibition ocular angiogenesis is formed, photodynamic therapy, pupil thermotherapy etc..These method prevailing prices are expensive,
Complicated for operation and patient compliance is poor.
Therefore, a kind of effectively inexpensive and easy to use AMD therapeutic agent is found as the primary of current medical treatments AMD
Task.
Summary of the invention
The main object of the present invention is in order to for clinically there are expensive and sick for the drug for treating AMD at present
The deficiencies of people's poor compliance, provides the preparation side of a kind of drug for inhibiting choroidal neovascularization and its ophthalmic administration dosage form
Method and the drug effect method of inspection, it is intended to therapeutic intervention more safely and effectively be carried out to AMD, to reach the life for improving AMD patient
The purpose of bioplasm amount.
The purpose of the present invention can reach by using following technical solution:
It is a kind of for inhibiting the drug of choroidal neovascularization, include: that phosphatidase 1 9-57%, gallbladder are solid according to mass percent
Alcohol 5-25%, ligustrazine 2-13% and sucrose 23-60%.
Preferably, the phosphatide includes soybean lecithin, egg yolk lecithin, DPPC phosphatide and DSPC phosphatide.
Preferably, which includes: phosphatidase 3 8%, cholesterol 10%, ligustrazine 5%, sucrose according to mass percent
47%.
Further, a kind of preparation method of the drug as above-mentioned for inhibiting choroidal neovascularization, it is characterised in that:
Include the following steps:
Phosphatide and cholesterol are codissolved in methylene chloride by step (1), and film is made in vacuum rotary steam；
Ammonium sulfate is added in step (2), and dissolving films are incubated for obtain blank liposome solutions；
Blank liposomes liquid solution is put into row dialysis by step (3)；
Step (4) mixes the blank liposomes liquid solution dialysed with ligustrazine medical fluid, be incubated for Ligustrazine Liposomes are molten
Liquid；
Step (5) dialyses Ligustrazine Liposomes solution, and protective agent sucrose is added, is freeze-dried to obtain ligustrazine rouge
Plastid powder.
Preferably, the method that film is made in the step (1) is to depressurize rotary evaporation under the conditions of 30-65 DEG C of temperature
0.5-3h forms film.
Preferably, it includes Probe Ultrasonic Searching method and high-pressure homogeneous for reducing the method for partial size after step (2) dissolving films
Method, the method for the Probe Ultrasonic Searching are ice bath probe sonication 1-5min.
Preferably, the step (2) and the middle temperature being incubated for of step (4) are water-bath, and temperature is 30-60 DEG C.
Preferably, the dialysis medium in step (5) dialysis procedure is pure water, dialysis time 2-6h, the step
Suddenly the dialysis medium in (3) dialysis procedure is physiological saline, 5% glucose or 10% sucrose solution.
Preferably, the dialysis process in the step (3) and step (5) is Bag filter method, supercentrifugal process or Portugal
Polysaccharide gel column method.
Further, a kind of drug effect method of inspection of the above-mentioned drug for inhibiting choroidal neovascularization, including it is as follows
Step:
Step (6) after adding the lipidosome freeze-dried powder water to redissolve, is given by eye of the miillpore filter to experimental animal
Medicine；
The drug being evaluated Ocular irritation and monitored after step (7), administration, photogenic experiment is swashed to 532nm
The drug effect of animal choroidal neovascularization；
The process evaluated after the administration to Ocular irritation is as follows: preparing healthy several of experimental rabbit, Suo Youdong
Object selects one eye to drip Ligustrazine Liposomes solution, and another eye drips physiological saline as a control group, then observes by the naked eye
Carry out Driaze scoring, pathological section, enzyme-linked immunosorbent assay cornea and aqueous humor inflammatory factor IL-1 β and TNF-α content
To evaluate the safety of the ophthalmically acceptable liposome of ligustrazine；
The drug effect process that described monitoring drug swashs photogenic experimental animal choroidal neovascularization to 532nm is as follows:
Step (7.1), grouping: preparing SPF grades male SD experimental mouse several, is randomly divided into 4 groups, 4 groups of classification point
It Wei not saline control group, ligustrazine bulk pharmaceutical chemicals group, Ligustrazine Liposomes group and Normal group；
Step (7.2) prepares model: carrying out examination of eyes to all experimental mouses before experiment, it is ensured that eyes prosthomere and eye
Bottom is showed no exception；After giving adaptable fed at least 1 week, using Normal group as blank control, other three groups of experimental mouses
Carry out the modeling of 532nm laser；
Step (7.3) judges modeling success or not: modeling after two weeks, passes through Optical coherence tomography and pathological section
Whether judgment models succeed, and model success is indicated if seeing the capillary for having new life；
The expression of step (7.4), test experience mouse train of thought embrane-associated protein: after modeling success, eye drop administration at least two weeks,
Then optical coherence tomography and pathological section, Immunofluorescence test experimental mouse choroid are carried out to experimental mouse eyes
The expression of CD31 albumen, the expression of immunohistochemistry test experience mouse choroid vegf protein；
Step (7.5), contrast and experiment and analysis experimental data.
Advantageous effects of the invention:
(1), the drug provided by the invention for inhibiting choroidal neovascularization, according to the generation machine of choroidal neovascularization
System, based on promoting blood circulation effect of chuanxiong piperazine and pharmacological action, and since ligustrazine ordinary preparation such as injection and tablet wait until
Effective dose up to eyeground is few, by the biocompatibility of matrix material, constructs in a kind of modern nanotechnology of fusion and tradition
Medical theoretical new and effective Chinese medicine eye liposome delivery systems help to extend pharmaceutical release time, improve its targeting
Property and bioavilability.
(2), compared with prior art, Chinese medicine thinks that AMD belongs to the drug provided by the invention for inhibiting choroidal neovascularization
In ophthalmology blood trouble, control that promoting blood circulation and removing obstruction in channels with peaceful blood and the promoting flow of qi and blood circulationization is tired etc. according to course of disease difference；Chuanxiong medicinal effects are main
Including two aspect of promoting the circulation of qi and promoting blood circulation, it is one of effective component of Rhizoma Chuanxiong that ligustrazine, which is the alkaloid extracted in Rhizoma Chuanxiong, is had identical
Effect is clinically mainly used for treating the multisystem diseases such as ischemic angiocardiopathy and cerebrovascular, based on Chinese medicine to the understanding of AMD, by river
Drug of the rhizome of chuanxiong piperazine as treatment AMD.
(3), the drug provided by the invention for inhibiting choroidal neovascularization, is prepared into liposome eye drops for ligustrazine,
Improving convenient administration mode, that is, intravitreal drug leads to the problem of patient compliance difference, and liposome is with fabulous
Biocompatibility, high osmosis, the eye residence time that drug can be extended, the advantages that improving the bioavilability of drug.
Detailed description of the invention
Fig. 1 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The transmission electron microscope picture of the Ligustrazine Liposomes freeze-dried powder of preferred embodiment；
Fig. 2 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The grain size distribution of the Ligustrazine Liposomes freeze-dried powder of preferred embodiment；
Fig. 3 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The DSC of the Ligustrazine Liposomes of preferred embodiment schemes；
Fig. 4 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The XRD diagram of the Ligustrazine Liposomes of preferred embodiment；
Fig. 5 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The preparation group cornea and physiological saline group cornea comparative diagram of the staining pathologic section of preferred embodiment；
Fig. 6 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The preparation group and physiological saline group comparative diagram of the Inflammatory Factors Contents of preferred embodiment；
Fig. 7 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
OCT figure behind the modeling of 532nm laser 2 weeks of preferred embodiment；
Fig. 8 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
Pathological section behind the modeling of 532nm laser 2 weeks of preferred embodiment；
Fig. 9 is the one of the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The comparative diagram of OCT figure before the administration of preferred embodiment and after administration 14 days；
Figure 10 is the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The comparative diagram of four groups of pathological sections behind administration 2 weeks of one preferred embodiment；
Figure 11 is the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The comparative diagram of four groups of CD31 immunofluorescences of one preferred embodiment；
Figure 12 is the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The schematic diagram of the CD31 immunofluorescence average optical density value of one preferred embodiment；
Figure 13 is the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The comparative diagram of four groups of VEGF immunohistochemistry figures of one preferred embodiment；
Figure 14 is the drug according to the invention for inhibiting choroidal neovascularization and preparation method and the drug effect method of inspection
The schematic diagram of four groups of VEGF immunohistochemistry average optical density values of one preferred embodiment.
Specific embodiment
To make the more clear and clear technical solution of the present invention of those skilled in the art, below with reference to examples and drawings
The present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1
The drug and preparation method provided in this embodiment for inhibiting choroidal neovascularization, comprising: soybean lecithin
120mg, cholesterol 30mg, ligustrazine 15mg and sucrose 150mg prepare Rhizoma Chuanxiong using the ammonium sulphate gradient in active loading method
Piperazine liposome weighs soybean lecithin 120mg, cholesterol 30mg is dissolved in 6ml methylene chloride, 45 DEG C of decompression rotary evaporation 1h shapes
At film, it is added the ammonium sulfate 8ml of 0.2mol/L, ultrasonic dissolution, Probe Ultrasonic Searching 2min is so that partial size point under condition of ice bath
Cloth is uniform, and 45 DEG C of water-baths are incubated for 20min, blank liposome is put into the bag filter that molecular weight is 3500, and dialysis medium is 100
The physiological saline of times volume, for 24 hours, timing replacement dialysis medium takes out blank liposomes liquid solution, 15mg ligustrazine is dissolved in for dialysis
It in PBS liquid, is mixed with blank liposome, is incubated for 20min in 45 DEG C of water-baths up to Ligustrazine Liposomes.
Ligustrazine Liposomes solution is fitted into processed bag filter, is dialysis medium with the pure water of 100 times of volumes
Dialyse 4h, to remove non-encapsulated ligustrazine free drug, the Ligustrazine Liposomes solution in bag filter is collected, in ligustrazine rouge
150mg freeze drying protectant sucrose is added in plastid solution, vacuum freeze drying obtains Ligustrazine Liposomes freeze-dried powder for 24 hours.
Embodiment 2
Unlike the first embodiment, the drug and preparation method provided in this embodiment for inhibiting choroidal neovascularization, packet
It includes: soybean lecithin 120mg, cholesterol 30mg, ligustrazine 20mg and sucrose 150mg, using the ammonium sulfate in active loading method
Gradient method prepares Ligustrazine Liposomes, weighs soybean lecithin 120mg, cholesterol 30mg is dissolved in 6ml methylene chloride, and 45 DEG C subtract
It presses rotary evaporation 1h to form film, is added the ammonium sulfate 8ml of 0.2mol/L, ultrasonic dissolution, Probe Ultrasonic Searching under condition of ice bath
For 2min so that particle diameter distribution is uniform, 37 DEG C of water-baths are incubated for 20min, and blank liposome is put into the bag filter that molecular weight is 3500
In, dialysis medium is the physiological saline of 100 times of volumes, and for 24 hours, timing replacement dialysis medium takes out blank liposomes liquid solution for dialysis,
20mg ligustrazine is dissolved in PBS liquid, is mixed with blank liposome, is incubated for 20min in 45 DEG C of water-baths up to Ligustrazine Liposomes.
Ligustrazine Liposomes solution is fitted into processed bag filter, is dialysis medium with the pure water of 100 times of volumes
Dialyse 4h, to remove non-encapsulated ligustrazine free drug, the Ligustrazine Liposomes solution in bag filter is collected, in ligustrazine rouge
150mg freeze drying protectant is added in plastid solution, vacuum freeze drying obtains Ligustrazine Liposomes freeze-dried powder for 24 hours.
Embodiment 3
Unlike embodiment 1 and embodiment 2, the drug and system provided in this embodiment for inhibiting choroidal neovascularization
Preparation Method, comprising: natural phospholipid 120mg, cholesterol 60mg, ligustrazine 18mg and sucrose 180mg, using in active loading method
Ammonium sulphate gradient prepares Ligustrazine Liposomes, weighs natural phospholipid 120mg, cholesterol 60mg is dissolved in 6ml methylene chloride, and 45
DEG C decompression rotary evaporation 1h forms film, is added the ammonium sulfate 8ml of 0.3mol/L, ultrasonic dissolution, pops one's head under condition of ice bath
For ultrasonic 2min so that particle diameter distribution is uniform, 37 DEG C of water-baths are incubated for 20min, and blank liposome is put into the dialysis that molecular weight is 3500
In bag, dialysis medium is the physiological saline of 100 times of volumes, and for 24 hours, it is molten to take out blank liposome for timing replacement dialysis medium for dialysis
18mg ligustrazine is dissolved in PBS liquid, mixes with blank liposome by liquid, is incubated for 20min in 37 DEG C of water-baths up to ligustrazine lipid
Body.
Ligustrazine Liposomes solution is fitted into processed bag filter, is dialysis medium with the pure water of 100 times of volumes
Dialyse 4h, to remove non-encapsulated ligustrazine free drug, the Ligustrazine Liposomes solution in bag filter is collected, in ligustrazine rouge
180mg freeze drying protectant is added in plastid solution, vacuum freeze drying obtains Ligustrazine Liposomes freeze-dried powder for 24 hours.
In the above-described embodiments, soybean lecithin can be changed into the phospholipid materials such as egg yolk lecithin, DPPC, DSPC, it is molten
Probe Ultrasonic Searching method can be changed into high-pressure homogeneous method by the method for degrading film, dialyse blank liposome when can be by medium of dialysing
Physiological saline changes 5% glucose or 10% sucrose solution into, removes in Ligustrazine Liposomes solution processes of dialysing non-encapsulated
The Bag filter method that free ligustrazine drug uses can change supercentrifugal process or sephadex column method into.
As Figure 1-Figure 4, the interpretation of result of comprehensive above-described embodiment, as a result as follows:
In the above-described embodiments, as depicted in figs. 1 and 2, pass through the form of transmission electron microscope observing Ligustrazine Liposomes, discovery
Liposome is at the spherical shape of rule, and surface is smooth, and partial size is smaller, narrow distribution.
Using the encapsulation rate (EE%) of dialysis measurement Ligustrazine Liposomes solution, result 56.2%；
Drug quality/dispensing gross mass × 100% in EE%=liposome
The situation of change of EE (%), PDI and nanometer particle size (nm) is as follows: in above-described embodiment
Table 1: the situation of change of EE (%), the PDI and nanometer particle size (nm) of each embodiment and comparative example
As shown in Table 1, compared with other embodiments, the encapsulation rate with higher of embodiment 1 and lesser partial size, distribution
It is narrow.
In the above-described embodiments, as shown in figure 3, the curve 3 of Ligustrazine Liposomes freeze-dried powder 1 and physical mixture have it is bright
Aobvious difference, physical mixture are the simple superposition of ligustrazine and blank liposome freeze-dried powder, Rhizoma Chuanxiong occur at 90.7 DEG C
The characteristic peak of piperazine is the peak of blank liposome at 290.6 DEG C, and Ligustrazine Liposomes appearance at 300.7 DEG C, ligustrazine are special
Sign peak has disappeared, and Ligustrazine Liposomes peak is slightly different with blank liposome, shows that ligustrazine is encapsulated in liposome.
In the above-described embodiments, as shown in figure 4, physical mixture 3 occurs the spy of ligustrazine bulk pharmaceutical chemicals 1 at 10 ° or so
Peak is levied, illustrates that physical mixture is the simple superposition of blank liposome and drug, Ligustrazine Liposomes 2 and physical mixture 3
It broadens and gets higher compared to the diffraction maximum between 10 ° -20 °, illustrate that drug encapsulation property after liposome changes, it was demonstrated that ligustrazine
Liposome preparation success.This result is consistent with the result of DSC.
Further, the obtained drug for inhibiting choroidal neovascularization in the above-described embodiments, needs to be used for eye to it
Portion's irritation makes evaluation, after adding a small amount of water to redissolve Ligustrazine Liposomes freeze-dried powder obtained in above-described embodiment first,
0.22 μm of miillpore filter is crossed after lipidosome freeze-dried powder is redissolved and is used for Ocular irritation evaluation, and concrete operations are as follows, health
New Zealand White Rabbit 6, all animal selection right eyes drip Ligustrazine Liposomes solution, and left eye drips physiological saline as a control group, often
, lagophthalmos is made after eye drip to be closed 10s days administration for 3 times, every time 50 μ L, successive administration 7 days.Progress Driaze is observed by the naked eye to comment
Point, pathological section, enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, elisa) measurement cornea and
The modes such as aqueous humor inflammatory factor IL-1 β and TNF-α content evaluate the safety of the ophthalmically acceptable liposome of ligustrazine.
It 7 days after eye drip, observes by the naked eye, for Ligustrazine Liposomes group compared with physiological saline group, eye has no red and swollen existing
As cornea clarification, conjunctiva has no congested.According to 2 irritation Draize standards of grading of table, Ligustrazine Liposomes group total score is 2 points,
The result shows that Ligustrazine Liposomes multiple dosing is non-stimulated to lagophthalmos.
2 eye irritation experimental grade criterion of table
As shown in Figure 5, left figure 1 is staining pathologic section preparation group cornea figure, and right figure 2 is physiological saline group cornea figure, disease
Reason slice is the results show that Ligustrazine Liposomes group and two groups of corneal epithelial cells of physiological saline group are continuous, hypothallus fiber alignment
Rule is showed no inflammatory cell.Corneal epithelium, prezone film layer, corneal stroma, posterior limiting lamina and endothelial cell structural integrity,
Cell arrangement is neat, and each confluent monolayer cells boundary is clear.Show that Ligustrazine Liposomes do not have irritation to cornea.
It will be appreciated from fig. 6 that 1 being Inflammatory Factors Contents preparation group picture in figure, 2 be physiological saline group in figure, with physiological saline group
Compare, the inflammatory factor IL-1 β and TNF-α content of Ligustrazine Liposomes group cornea and aqueous humor there are no significant difference (P >
0.05), show that Ligustrazine Liposomes group not will cause inflammatory reaction to lagophthalmos, thus can illustrate the safety of Ligustrazine Liposomes
Preferably.
In conclusion Ligustrazine Liposomes are smaller for ophthalmic applications irritation, safety is preferable.
After adding a small amount of water to redissolve Ligustrazine Liposomes freeze-dried powder produced above, after lipidosome freeze-dried powder is redissolved
0.22 μm of miillpore filter is crossed for ophthalmic administration.Investigate its medicine that photogenic choroidal neovascularization in rat is swashed to 532nm
Effect.Concrete operations are as follows:
SPF grades male SD rat 24, it is randomly divided into 4 groups, every group 6, respectively saline control group, ligustrazine is former
Expect medicine group, Ligustrazine Liposomes group, Normal group.The process of model preparation are as follows: all rats carry out eye inspection before testing
It looks into, eyes prosthomere and eyeground are showed no exception.After giving adaptable fed 1 week, rat is randomly divided into 4 groups, wherein one group of work
For blank control, other mouse carry out the modeling of 532nm laser.First with 10% chloraldurate (3.5ml/kg) in being infused in rat abdominal cavity
Anesthesia is penetrated, expands pupil with compound tropicamide eye drops drop eyes, using 532nm laser through slit-lamp microscope in full retina
It is solidifying around the equidistant light that carried out between 2 retinal vessels of optic disk under mirror, 50 μm of spot diameter, laser energy 320mW, the time
0.12s has bubble generation or hyporrhea after light of being subject to is solidifying.
Whether modeling after two weeks, is succeeded by Optical coherence tomography (OCT) and pathological section judgment models, such as Fig. 7
It is thickened for layer of retina,neuroepithelial shown in OCT, RPE composite layer is discontinuous, partial thickening, reflection enhancement, and boundary is unclear；Fig. 8
For pathological section tela chorioidea's structure disturbance as the result is shown, it is dispersed in inflammatory cell infiltration and fibroblast proliferation, seeing has new life
Capillary then indicate model success, continuous eye drop administration two weeks after model success, 3 times/day, 1 50 μ L.Two weeks
Afterwards, optical coherence tomography is carried out to rat eye, then puts to death rat, won eyeball and fixed in 4% paraformaldehyde, into
Row pathological section, the expression of Immunofluorescence test rat choroid CD31 albumen, immunohistochemistry detect rat choroid VEGF egg
White expression.
OCT result such as Fig. 9 shows that after two weeks with modeling, the focal area of Ligustrazine Liposomes group rat eye obviously has
It is reduced.The result of slice such as Figure 10 is shown: being respectively as follows: normal group of 1- from 1 group of-four group；2- physiological saline group；3- ligustrazine medicine
Object group；4- Ligustrazine Liposomes group, Ligustrazine Liposomes group totally six samples, in addition to a choroid of right eye cellular layer arrangement is disorderly
Disorderly, being dispersed in right inflammatory cell infiltration while seeing has newborn capillary.Remaining sample tissue cells marshalling, there are no bright
Aobvious histopathological lesions change.And six samples of modeling group are normal in addition to a left eye institutional framework, have no apparent tissue
Pathological lesions change.Remaining sample standard deviation Jian You tela chorioidea structure disturbance, is dispersed in inflammatory cell infiltration and fibrocyte increases
Raw, seeing has newborn capillary.
The expression of Immunofluorescence test rat choroid CD31 albumen, as shown in Figure 11 and 12,1-4 is respectively 1- in Figure 11
Physiological saline group, 2- ligustrazine medicine group, 3- Ligustrazine Liposomes group, normal group of 4-, 1-4 is 1- physiological saline respectively in Figure 12
Group, 2- ligustrazine medicine group, 3- Ligustrazine Liposomes group, normal group of 4-, after administration 14 days, physiological saline group CD31 green fluorescence
Positive expression is obvious, and the CD31 positive expression of Ligustrazine Liposomes group and ligustrazine medicine group is lower than physiological saline group.Image J
Known to software analysis: the CD31 albumen of Ligustrazine Liposomes group and ligustrazine medicine group coloring average optical density value MOD is below
Physiological saline group, but Ligustrazine Liposomes group CD31 albumen coloring average optical density value significantly lower than physiological saline group (P <
0.05)。
Immunohistochemistry detects the expression of rat choroid vegf protein, and as shown in Figure 13 and 14,1-4 is 1- respectively in Figure 13
Physiological saline group, 2- ligustrazine medicine group, 3- Ligustrazine Liposomes group, normal group of 4-, 1-4 is 1- physiological saline respectively in Figure 14
Group, 2- ligustrazine medicine group, 3- Ligustrazine Liposomes group, normal group of 4-, after administration 14 days, physiological saline group VEGF brown color sun
Property expression it is obvious, be higher than Ligustrazine Liposomes group and ligustrazine medicine group.Known to the analysis of 6.0 software of Image Pro Plus: river
The vegf protein of rhizome of chuanxiong piperazine liposome group and ligustrazine medicine group coloring average optical density value MOD is below physiological saline group, but river
The vegf protein coloring average optical density value of rhizome of chuanxiong piperazine liposome group is significantly lower than physiological saline group (P < 0.05).
To sum up, the drug provided by the above embodiment for inhibiting choroidal neovascularization and preparation method and drug effect inspection party
Method, according to the generting machanism of choroidal neovascularization, based on promoting blood circulation effect of chuanxiong piperazine and pharmacological action, and due to Rhizoma Chuanxiong
Effective dose that piperazine ordinary preparation such as injection and tablet etc. reach eyeground is few, then by the biocompatibility of matrix material,
A kind of new and effective Chinese medicine eye liposome delivery systems for merging modern nanotechnology and traditional Chinese medicine theory are constructed, are had
Help extend pharmaceutical release time, improves its targeting and bioavilability.
The above, further embodiment only of the present invention, but scope of protection of the present invention is not limited thereto, and it is any
Within the scope of the present disclosure, according to the technique and scheme of the present invention and its design adds those familiar with the art
With equivalent substitution or change, protection scope of the present invention is belonged to.
Claims (10)
1. inhibiting the drug of choroidal neovascularization, it is characterised in that: according to mass percent include: that phosphatidase 1 9-57%, gallbladder are solid
Alcohol 5-25%, ligustrazine 2-13% and sucrose 23-60%.
2. the drug according to claim 1 for inhibiting choroidal neovascularization, it is characterised in that: the phosphatide includes soybean
Lecithin, egg yolk lecithin, DPPC phosphatide and DSPC phosphatide.
3. the drug according to claim 1 for inhibiting choroidal neovascularization, it is characterised in that: according to mass percent packet
It includes: phosphatidase 3 8%, cholesterol 10%, ligustrazine 5% and sucrose 47%.
4. a kind of preparation method of the drug as described in claim 1 for inhibiting choroidal neovascularization, it is characterised in that: including
Following steps:
Phosphatide and cholesterol are codissolved in methylene chloride by step (1), and film is made in vacuum rotary steam；
Ammonium sulfate is added in step (2), and dissolving films are incubated for obtain blank liposome solutions；
Blank liposomes liquid solution is put into row dialysis by step (3)；
Step (4) mixes the blank liposomes liquid solution dialysed with ligustrazine medical fluid, is incubated for obtain Ligustrazine Liposomes solution；
Step (5) dialyses Ligustrazine Liposomes solution, and protective agent sucrose is added, is freeze-dried to obtain Ligustrazine Liposomes
Powder.
5. the preparation method of the drug according to claim 4 for inhibiting choroidal neovascularization, it is characterised in that: the step
Suddenly the method that film is made in (1) is to depressurize rotary evaporation 0.5-3h under the conditions of 30-65 DEG C of temperature to form film.
6. the preparation method of the drug according to claim 4 for inhibiting choroidal neovascularization, it is characterised in that: the step
Suddenly reducing the method for partial size after (2) dissolving films includes Probe Ultrasonic Searching method and high-pressure homogeneous method, the side of the Probe Ultrasonic Searching
Method is ice bath probe sonication 1-5min.
7. the preparation method of the drug according to claim 4 for inhibiting choroidal neovascularization, it is characterised in that: the step
Suddenly the temperature in (2) and step (4) is water-bath, and temperature is 30-60 DEG C.
8. the preparation method of the drug according to claim 4 for inhibiting choroidal neovascularization, it is characterised in that: the step
Suddenly the dialysis medium in (5) dialysis procedure is pure water, dialysis time 2-6h, the dialysis in step (3) dialysis procedure
Medium is physiological saline, 5% glucose or 10% sucrose solution.
9. the preparation method of the drug according to claim 4 for inhibiting choroidal neovascularization, it is characterised in that: the step
Suddenly the dialysis process in (3) and step (5) is Bag filter method, supercentrifugal process or sephadex column method.
10. a kind of drug effect method of inspection of the drug as described in claim 1 for inhibiting choroidal neovascularization, feature exist
In: include the following steps:
Step (6), the lipidosome freeze-dried powder is added water redissolve after, by miillpore filter to the ophthalmic administration of experimental animal；
The drug being evaluated Ocular irritation and monitored after step (7), administration, photogenic experimental animal is swashed to 532nm
The drug effect of choroidal neovascularization；
The process evaluated after the administration to Ocular irritation is as follows: preparing healthy several of experimental rabbit, all animals choosings
One eye drop Ligustrazine Liposomes solution is selected, another eye drips physiological saline as a control group, and successive administration at least 5 days, then
It observes by the naked eye and carries out Driaze scoring, pathological section, enzyme-linked immunosorbent assay cornea and aqueous humor inflammatory factor IL-1 β
The safety of the ophthalmically acceptable liposome of ligustrazine is evaluated with TNF-α content, if eye has no red and swollen phenomenon, cornea clarification, conjunctiva is not
See hyperemia, then shows that Ligustrazine Liposomes multiple dosing is non-stimulated to experiment lagophthalmos；
The drug effect process that described monitoring drug swashs photogenic experimental animal choroidal neovascularization to 532nm is as follows:
Step (7.1), grouping: prepare SPF grades male SD experimental mouse several, be randomly divided into 4 groups, 4 groups of classification are respectively
Saline control group, ligustrazine bulk pharmaceutical chemicals group, Ligustrazine Liposomes group and Normal group；
Step (7.2) prepares model: carrying out examination of eyes to all experimental mouses before experiment, it is ensured that eyes prosthomere and eyeground are equal
No abnormality seen；After giving adaptable fed at least 1 week, using D group Normal group as blank control, other three groups of experimental mouses into
Row 532nm laser modeling；
Step (7.3) judges modeling success or not: modeling after two weeks, is judged by Optical coherence tomography and pathological section
Whether model succeeds, and model success is indicated if seeing the capillary for having new life；
The expression of step (7.4), test experience mouse train of thought embrane-associated protein: after modeling success, eye drop administration at least two weeks, then
Optical coherence tomography and pathological section, Immunofluorescence test experimental mouse choroid CD31 albumen are carried out to experimental mouse eyes
Expression, the expression of immunohistochemistry test experience mouse choroid vegf protein；
Step (7.5), contrast and experiment and analysis experimental data.
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