CN101427997A - Eye drop with anti-cornea rebirth blood vessel function and preparation method thereof - Google Patents

Abstract

The invention relates to eye drops adopting arteannuinum succinate as the raw material for treating corneal neovascularization and a preparation method thereof. The arteannuinum succinate eye drops can be produced by adopting raw materials in the following parts by weight: 0.0096 to 0.0384 parts of arteannuinum succinate, 1.0032 to 12.028 parts of acid component in buffer solution, 0.106 to 18.0477 parts of alkaline component in buffer solution, 1.0 to 35.0 parts of thickener, 2.0 to 4.72 parts of isotonic reagent and 0.01 to 0.02 parts of antiseptics. The preparation method for the eye drops comprises the following steps: dissolving the arteannuinum succinate with NaHCO3, filtering and sterilizing for later use; dissolving appropriate amount of buffer solution, isotonic reagent and thickener into distilled water, sterilizing for 20 minutes with 100 to 120 DEG C high pressure to obtaining treatment fluid, and cooling the treatment fluid to room temperature for stand to by use; preparing the antiseptics with distilled water into high concentration storage solution, filtering, sterilizing and subpackaging for later use; in aseptic condition, adding the arteannuinum succinate into the treatment fluid according to the ratio of adding 16 to 66 Mul of arteannuinum succinate into 100 ml treatment fluid, uniformly blending, and at the same time, adding the antiseptics into the buffer solution according to the ratio of adding 50 to 100 Mul of antiseptics into 100 ml buffer solution, and subpackaging and producing eye drops in different specifications; preferably, the package per bottle is 0.5 ml.

Description

Has eye drop of anti-cornea rebirth blood vessel function and preparation method thereof

Technical field

The invention belongs to field of pharmaceutical preparations, relate to a kind of medicine for the treatment of the cornea new vessels, relating in particular to the artesunate is artesunate eye drop of raw material and preparation method thereof.

Background technology

The normal cornea tissue does not have blood vessel, and the cornea surrounding tissue ends at limbus of corneae and forms vasoganglion, and nutritional labeling diffuses into cornea thus.It is to keep its transparency and as one of key factor of important refractive media that cornea does not have blood vessel.In wearing of contact lens, ocular injury or chemical injury, infection, previously under disease such as history of operation, trichiasis and immunity oculopathy and the pathological state, blood capillary forms cornea rebirth blood vessel (CNV) by the limbus of corneae cornea of growing into.CNV has destroyed the normal microenvironment of cornea, causes the absolution of anterior ocular segment related immune to disappear, and becomes the high risk factor of corneal allograft rejection.In addition, the new vessels fragile structure, often because of angiorrbagia, ooze out with secondary fibrosis etc. and cause that the patient is blind, so CNV becomes one of the most common causes of blindness.In developed country, the ocular disease that the neovascularization cornea causes has accounted for the first place of blind reason; In China, the CNV that ocular injury (especially chemical burn) causes accounts for 10% of keratopathy, and severe visual is damaged to the patient and brought huge misery.

Along with people's corneal new vessels forms Study on Mechanism, on Drug therapy and operative treatment, obtained bigger progress, but therapeutic effect is not fully up to expectations, the hormonotherapy of inflammation-inhibiting for example, although can obviously suppress the development of cornea inflammation and new vessels, exist the healing of delay corneal epithelium, the self-regeneration of blocking-up cornea also can increase the weight of side effect such as corneal ulcer; The ARGON LASER PHOTOCOAGULATION IN therapy is used for the treatment of CNV recently, but the hot injury is involved the danger that surrounding tissue has further reduction vision; And the Biotherapeutics of application blood vessel hyperplasia inhibitive factor and blood vessel hyperplasia antagonist is evident in efficacy, but whole body administration side effect height, cost is higher, and most of method also is in the experimentation stage.Therefore need to seek more effective, safe and economical medicine and means.

Artemisinin-based drug mainly comes from Herba Artemisiae annuae, because of rapid-action, side effect is low, be difficult for producing drug resistance and can effectively treating chloroquine drug resistance plasmodium, and be used to the treatment of global subtertian malaria and cerebral malaria, artesunate (artesunate) is unique a kind of water-soluble arteannuin analog derivative, metabolism is that dihydroarteannuin plays a role in vivo, and antimalarial effect is remarkable, is mainly used in the treatment of cerebral malaria and various critical malaria.Research to artesunate in recent years is not limited only to treat malaria, finds that still it has antiinflammatory, antiviral, antitumor and immunoregulation effect and suppresses the outgrowth effect of tumor vessel.

Granted publication number has been announced a kind of artesunate microsphere injection liquid in the Chinese patent literature of CN1208061C, use release polymer to grind artesunate, be prepared into microsphere injection liquid, be mainly used in the blood vessel hyperplasia that treatment cancer-related blood vessel hyperplasia and other disease cause, but this microsphere injection liquid does not fit into the treatment of eye table blood vessel hyperplasia.Reason is: on the one hand, often to use conveniently, the eye drop that welcome by the patient is main in the treatment of eye surface diseases, and this medicine is an injecting drug use, so use inconvenient aspect dosage form.In addition, be degraded to water and carbon dioxide after the slow release of polymer in the microsphere injection liquid, whether the carbon dioxide that discharges is influential to pH value and other physical signs of eye table, and whether influence impaired cornea and recover to await discussing, so the artesunate preparation in this patent documentation is not suitable for the treatment cornea rebirth blood vessel.

Summary of the invention

It is good to the objective of the invention is to disclose a kind of safety, the artesunate eye drop of treatment cornea rebirth blood vessel evident in efficacy.

Eye drop among the present invention with artesunate as active component, comprise in the preparation buffer, thickening agent, etc. ooze reagent and antiseptic.

Buffer can be phosphate buffer, borate buffer solution, citrate buffer, acetate buffer, aminoacid etc.Thickening agent can be hydroxypropyl emthylcellulose, carboxylic propyl methocel, hyaluronate sodium etc.Deng oozing reagent can be glucose, glycerol, sodium chloride, boric acid etc.Antiseptic can be thimerosal, benzalkonium bromide, p-Hydroxybenzoate etc.Preparation pH of the present invention transfers to 6.5-8.0 usually.

Artesunate eye drop of the present invention can be made by the raw material of following weight portion:

Artesunate 0.0096-0.0384 part;

Acid ingredient 1.0032-12.028 part in the buffer;

Alkaline components 0.106-18.0477 part in the buffer;

Thickening agent 1.0-35.0 part;

Deng oozing reagent 2.0-4.72 part;

Antiseptic 0.01-0.02 part.

Another object of the present invention provides the preparation method of above-mentioned eye drop:

1, get artesunate and dissolve with NaHCO3, stand-by after the filtration sterilization;

2, with the appropriate amount of buffer solution composition, etc. ooze reagent and thickening agent is dissolved in the distilled water, with 100-120 ℃ of autoclaving 20 minutes, working solution, it is standby to be cooled to room temperature;

3, antiseptic is configured to the high concentration storage liquid with distilled water, the filtration sterilization packing is stand-by;

4, under aseptic condition, artesunate solution is joined in the working solution according to the ratio that every 100ml adds 16 μ l～66 μ l, mix homogeneously, simultaneously antiseptic is joined in the buffer according to the ratio that every 100ml adds 50 μ l～100 μ l, the eye drop of different size is made in packing, preferably every bottle of packing 0.5ml.

Description of drawings

Fig. 1: alkali burn is rat cornea slit lamp microscope photo (* 20) after 11 days;

Fig. 2: the artesunate eye drop is to the assessment of alkali burn cornea therapeutic effect;

Fig. 3: artesunate is to the inhibitory action of HUVEC;

Fig. 4: the eye drop excipient to artesunate to the inhibiting influence of HUVEC;

Fig. 5: excipient is to the influence of HUVEC propagation;

Fig. 6: the former foster corneal epithelial cell (* 200) of being commissioned to train;

Fig. 7: artesunate is to the inhibitory action of corneal epithelial cells of rabbit.

Specific embodiment

Following experimental example and medicament prepare embodiment and only are used to purpose of the present invention is described, and can not limit the scope of the invention.

(1) experimental example---effect experiment

1, eye drop is to the therapeutical effect of the inductive rat corneal neovascularization of alkali burn

The rat corneal neovascularization Preparation of model is induced in alkali burn: select the volumetrical micropipettor rifle of 100 μ l-200 μ l head for use, level cuts the tip at distance afterbody 15mm place, and with blade with the incision skiving.Fill Cotton Gossypii in the rifle head, guarantee that head Cotton Gossypii degree of tightness is moderate, can fully contact the spherical surface of rat cornea, the paper adhesive plaster is sealed the mould afterbody, and this mold volumes is 220 μ l, and is standby behind the autoclave sterilization.Choose 15 of female SPF Sprague-Dawley (SD) rats of body weight 150g-230g, provide by Traditional Chinese Medicine University Of Guangzhou, raise the Animal Lab. in Zhongshan Ophthalmic Center, Sun Yat-sen University, eye surface diseases, modeling twice double ocellus Gernebcin eye drops every day first three day are got rid of in slit lamp examination.Then according to 45mg/kg body weight lumbar injection 3% pentobarbital sodium water solution.In self-control rat corneal alkali burn mould, splash into 220 μ l 1N NaOH.Mould is contacted rat cornea central authorities 35 seconds, use 30-60ml normal saline flushing conjunctival sac and cornea rapidly.As seen the middle canescence of circle is burnt speckle, diameter 4mm, eyes burn.Animal is divided into negative control at random, and (excipient adds suitable NaHCO ₃) group, 25 μ mol/L artesunate eye drop groups (be called for short artesunate group) and 0.1% dexamethasone positive controls (abbreviation Dexamethasone group), every day is respectively at 8:00am, 12:00pm, 4:00pm and 8:00pm dripping eyedrop, each every 20 μ l.Observed down rat eye in slit lamp on the 1st, 2,3,5,7,9,11 day after the alkali burn, observation index is inflammation index (comprises conjunctival congestion, corneal edema and ophthalmic ooze out or hemorrhage three), corneal epithelium, corneal opacity degree and new vessels area.And mark by following standard:

Conjunctival congestion: 0---does not have hyperemia

1---limbus of corneae vascular ring mild hyperaemia

The hyperemia of 2---limbus of corneae vascular ring moderate

The hyperemia of 3---limbus of corneae vascular ring severe, blood vessel is angry to be opened

Corneal edema: 0---does not have edema (corneal thickness is normal)

1---Mild edema (corneal thickness slightly increases)

2---intermediate edema (increase of corneal thickness moderate)

3---severe edema (increase of corneal thickness severe)

Ophthalmic oozes out or is hemorrhage: ooze out in the 0---anophthalmia or hemorrhage

The 1---ophthalmic oozes out or hemorrhage rat anterior chamber 1/4 volume that is less than

The 2---ophthalmic oozes out or hemorrhage rat anterior chamber 1/2 volume that is less than

The 3---ophthalmic oozes out or is hemorrhage greater than rat anterior chamber 1/2 volume, and the eyeball volume increases

Inflammation index is that conjunctival congestion, corneal edema and ophthalmic ooze out or the summation of hemorrhage three item ratings.

Corneal opacity degree: 0---corneal transparency

1---corneal nebula, iris is high-visible

2---corneal macula, iris are as seen

3---corneal leukoma, iris is invisible

Corneal epithelium fluorescein sodium dyeing assessment:

5 μ l1% fluorescein sodium are splashed in the rat conjunctival sac, and cotton swab is wiped unnecessary fluorescein sodium, and guarantees that it is uniformly distributed in anterior corneal surface.Slit lamp is observed with the cobalt blue lamp down.Cornea is divided into four parts, and each part is marked according to following standard, and final score is four part sums:

Fluorescent staining: 0---corneal epithelium dye-free

1---corneal epithelium colored spots is less than 30

2---corneal epithelium colored spots is less than the cornea half of the area

3--corneal epithelium colored spots the large tracts of land lamellar perhaps occurs greater than the cornea half of the area

Painted

After the alkali burn first day, the line of apsides in corneal epithelial defect district was measured in fluorescein sodium dyeing back, and calculates the defective region area.

The cornea rebirth blood vessel area measurement: measure cornea rebirth blood vessel length with micrometer under the slit lamp, and the shared hour number of blood vessel of record different length, calculate the new vessels area according to following formula:

A＝π×C×(R ²-(R-L) ²)/12

R is a SD rat corneal radii, and L is by being surveyed length of vessel, and C is a hour number

Experimental result:

With 15 SD rat random packet, 10 corneas of negative control group, 11 corneas of artesunate group, 8 corneas of Dexamethasone group, the treatment effectiveness evaluation index is the same, and the result is as follows:

(1) the artesunate eye drop to alkali burn after the influence of cornea rebirth blood vessel length and area.

(see Fig. 2-A) in the longest CNV length of each group of each point in time measurement behind the corneal alkali burn, calculate the CNV area and (see Fig. 2-B), artesunate group cornea C NV length and area are starkly lower than matched group (P＜0.05), referring to table 1 and table 2, first day artesunate group and Dexamethasone group be zero difference (P〉0.05) on CNV length and area, but two groups of obvious differences (P＜0.05) in the time point afterwards.Results suggest, the artesunate eye drop can suppress the growth of the inductive cornea rebirth blood vessel of alkali burn.

Hindered back 11 days, each is organized the slit lamp micro of cornea and sees Fig. 1, and the negative matched group of A, B are artesunate eye drop group, and C is a Dexamethasone group.

Table 1 artesunate eye drop is to the influence of rat corneal neovascularization length

Compare with negative control group: ^*P＜0.05, ^*P＜0.01

Table 2 artesunate eye drop is to the influence of rat corneal neovascularization area

Compare with negative control group: ^*P＜0.05, ^*P＜0.01

(2) antiinflammatory action of artesunate eye drop

The inflammation index of different time points artesunate eye drop group is starkly lower than negative control group, illustrates that the artesunate eye drop has the effect of remarkable inhibition cornea inflammatory reaction, the results are shown in Figure 2-C.

(3) the artesunate eye drop does not hinder the healing of corneal epithelial cell

Since the 5th day, the scoring of artesunate eye drop corneal epithelial transparency was starkly lower than negative control group, referring to Fig. 2-D.24 hours corneal epitheliums have begun self-regeneration after the alkali burn, and the treatment that damages the reparation of the fast transferring of early stage corneal epithelium and normal healing corneal and alkali burn is significant.We go up damaged area to alkali burn after 24 hours measurement shows that the damaged area of negative control group is 6.820 ± 1.647mm ², the artesunate group is 4.733 ± 0.917mm ², Dexamethasone group is 5.322 ± 1.141mm ²The artesunate group is compared difference significance (P＜0.05) with negative control group, the results are shown in Figure 2-F, illustrate that the artesunate eye drop is to the migration that damages early stage corneal epithelium and the without hindrance effect of healing, in the scoring of each time point corneal epithelial in later stage, find that also the artesunate eye drop does not influence the healing of corneal epithelium, the results are shown in Figure 2-E.

2, the artesunate eye drop external to the effect of human umbilical vein endothelial cell inhibition of proliferation

(1) human umbilical vein endothelial cell former is commissioned to train foster

Under aseptic condition, behind healthy parturient childbirth, get neonatal umbilical cord immediately, extrude the blood in the cord vessels, be immersed in and contain two anti-(penicillin 100u/ml, among the aseptic PBS of pre-cooling streptomycin 100 μ l/ml), with containing two anti-quiet several of PBS lavation umbilicus, in umbilical vein, pour into 0.25% trypsin 8ml～10ml behind the remaining bloodstain of Ex-all, and in 37 ℃ of water-baths or incubator, digest 20min.The trypsin that will contain endotheliocyte injects 50ml taper centrifuge tube, adds calf serum 2ml cessation reaction and contains two anti-PBS irrigating solutions flushing tube chambers with 30ml, collects the effluent centrifuge tube of packing in the lump.The centrifugal 10min of 1000r/min abandons supernatant, adds complete culture solution, fully is mixed and made into cell suspension, is transferred in the culture bottle of 2% gelatin bed board 5%CO ₂, 37 ℃ of static cultivation culture fluid compositions are that M199 contains 20%FBS and 30 μ g/ml ECGS (upstate company), change a culture fluid, and whether express the VIII factor with identification of cell purity with immunofluorescence chemical detection cell in 3 days.Getting 2-6 uses for research for cell.

(2) the artesunate eye drop is to the influence of Human umbilical vein endothelial cells propagation

With endotheliocyte with 4 * 10 ⁴Individual/hole is inoculated in 12 orifice plates, in 37 ℃, 5%CO ₂Condition under cultivate, treat cell attachment after, be replaced by culture fluid that contains 2%FBS and the artesunate that adds variable concentrations, set up the PBS negative control group, 3 every group multiple holes, drug effect is after 48 hours, every hole adds the MTT of 200 μ l5mg/ml, 37 ℃, 5%CO ₂Condition under continued to hatch 2-4 hour, abandon supernatant, every hole adds 2mlDMSO dissolving, treats that granule fully dissolves the back and measures the absorbance of every hole at the 570nm place, obtains cell survival rate with the absorbance/matched group absorbance of processed group.

(3) artesunate eye drop excipient is to the influence of artesunate anti-vascular endothelial cell proliferation function

For whether the excipient that detects selected eye drop influences the performance of artesunate pharmacological action, select for use mtt assay to observe artesunate--the NaHCO of artesunate eye drop and same medicine concentration ₃Solution is to the difference of human umbilical vein endothelial cell propagation influence.Method is: with endotheliocyte with 2 * 10 ⁴Individual/hole is inoculated in 24 orifice plates, in 37 ℃, 5%CO ₂Condition under cultivate, treat cell attachment after, be replaced by the culture fluid that contains 2%FBS and grouping, set up PBS negative control group, 25 μ mol/L artesunate--NaHCO ₃The various eye drop groups of solution group and identical artesunate concentration, 4 every group multiple holes, drug effect is after 48 hours, and every hole adds the MTT of 100 μ l 5mg/ml, 37 ℃, 5%CO ₂Condition under continued to hatch 2-4 hour, abandon supernatant, every hole adds 1mlDMSO dissolving, treats that granule fully dissolves the back and measures the absorbance of every hole at the 570nm place, obtains cell survival rate with the absorbance/matched group absorbance of processed group.

(4) the eye drop excipient is to the influence of human umbilical vein endothelial cell propagation

For detecting in the artesunate eye drop, except artesunate, whether other compositions are arranged to the influential effect of the propagation of human umbilical vein endothelial cell, the eye drop excipient that other compositions that still adopt MTT to observe eye drop are combined into is to the influence of vascular endothelial cell, method is the same, still select 24 orifice plates for use, set up the PBS matched group, with the corresponding various excipient of above artesunate eye drop, every group 4 multiple holes, behind the drug effect 48 hours, every hole adds the MTT of 100 μ l 5mg/ml, 37 ℃, 5%CO ₂Condition under continued to hatch 2-4 hour, abandon supernatant, every hole adds 1mlDMSO dissolving, treats that granule fully dissolves the back and measures the absorbance of every hole at the 570nm place, obtains cell survival rate with the absorbance/matched group absorbance of processed group.

Experimental result:

(1) the artesunate eye drop can effectively suppress the propagation of vascular endothelial cell

The artesunate eye drop can significantly suppress vascular endothelial cell proliferation, the OD of PBS matched group, 6.25 μ mol/L artesunate eye drop groups, 12.5 μ mol/L artesunate eye drop groups, 25 μ mol/L artesunate eye drop groups, 50 μ mol/L artesunate eye drop groups, 100 μ mol/L artesunate eye drop groups ₅₇₀Be respectively: 0.688 ± 0.158,0.511 ± 0.082,0.369 ± 0.053,0.199 ± 0.074,0.076 ± 0.156,0.056 ± 0.046 result shows the propagation of artesunate eye drop dose-dependent inhibition HUVEC.The result is referring to Fig. 3, n=3

(2) each composition of artesunate eye drop and excipient are to the influence of the anti-HUVEC proliferation function of artesunate

With each composition group in the eye drop and, make representative solutions, experimentize, experiment shows, thickening agent in the artesunate eye drop (hydroxypropyl emthylcellulose and carboxylic propyl methocel), osmotic pressure regulator (sodium chloride) and antiseptic (benzalkonium bromide and thimerosal) the anti-angiogenic proliferative effect to artesunate in selected concentration does not influence, n=4, the PBS group, 25 μ mol/L artesunate solution groups, and the artesunate eye drop of 25 μ mol/L is respectively organized a1, b1, c1, d1, e1, f1, g1, a2, b2, c2, d2, e2, f2 and g2, wherein a1, b1, c1, d1, e1, f1, g1, a2, b2, c2, d2, e2, f2 and g2 represent the eye drop OD of different prescriptions respectively ₅₇₀Be respectively 0.691 ± 0.129,0.379 ± 0.060,0.361 ± 0.074,0.374 ± 0.107,0.281 ± 0.078,0.274 ± 0.114,0.356 ± 0.032,0.358 ± 0.058,0.273 ± 0.1133,0.335 ± 0.0469,0.370 ± 0.077,0.354 ± 0.0641,0.354 ± 0.050,0.331 ± 0.0358,0.371 ± 0.053,0.417 ± 0.093, the relation of medicine and cell survival rate (%) is referring to Fig. 4 (n=4).

(3) the excipient composition in the eye drop is to the influence of the propagation of HUVEC

The OD of PBS group, a1, b1, c1, d1, e1, f1, g1, a2, b2, c2, d2, e2, f2 and g2 group (each excipient of a1, b1, c1, d1, e1, f1, g1, a2, b2, c2, d2, e2, f2 and g2) for prescription eye drop together ₅₇₀Be respectively 0.566 ± 0.0382,0.635 ± 0.055,0.626 ± 0.039,0.615 ± 0.008,0.578 ± 0.054,0.641 ± 0.079,0.61475 ± 0.063,0.518 ± 0.084,0.624 ± 0.101,0.582 ± 0.029,0.491 ± 0.0312,0.524 ± 0.065,0.604 ± 0.113,0.587 ± 0.0725,0.542 ± 0.037, experiment is found, there be not of the propagation not influence of the eye drop excipient of artesunate to HUVEC, the result is referring to accompanying drawing 5, n=4.3, eye drop is at external toxic action to corneal epithelial cells of rabbit

(1) the former foster corneal epithelial cells of rabbit of being commissioned to train

Under aseptic condition, get cornea, remove limbus of corneae after, clean cornea with the PBS solution that contains mycillin.In a clean culture dish, drip 3～4 of the 1:1 mixed liquors of 0.25% trypsin and 0.2%EDTA, above-mentioned rabbit corneal epithelial surface is placed on the mixture slaking liquid down, put in 37 ℃ of incubators and digest 25～30min, add 1～2 hyclone and stop digestion.The flip angle diaphragm washes epithelial surface gently with DMEM (or R1640) culture fluid, and the centrifugal 5～6min of flushing liquor (1000r/min) abandons supernatant, adds the epithelial cell culture fluid in every cornea 4mL ratio, the piping and druming mixing.The blood cell counting plate counting makes 150 * 10 ⁶～200 * 10 ⁶/ L cell suspension is inoculated in epithelial cell in 12 or 6 well culture plates of spreading gelatin (2%) with every hole 2mL, puts 37 ℃, 5%CO ₂Cultivate in the incubator, culture fluid was changed once in 2～3 days.To digest back or indigested cornea and be placed on that endothelium faces down in the aseptic plate, be cut into several piece, put in culture dish or 6 orifice plates, epithelial surface is downward, add a small amount of culture fluid, make around the firm submergence cornea tissue of liquid and 37 ℃, 5%CO are put in the surface of not submergence cornea tissue piece ₂Cultivate in the incubator, add the part culture fluid behind the 24h, culture fluid was changed once in 2～3 days.The culture fluid composition is that RMPI1640 contains 20%FBS and 10-20ng/mlEGF, after cytokeratin 3/12 SABC is identified, get the 1st generation cell carry out subsequent experimental.

(2) artesunate is to the toxic action of corneal epithelial cells of rabbit

Whether have inhibitory action, and then observe the toxic and side effects of artesunate at external corneal epithelial cell with mtt assay if detecting artesunate corneal epithelial cell proliferation.The corneal epithelial cells of rabbit of getting for the 1st generation is with 2 * 10 ⁴The density in individual/hole is inoculated in 24 orifice plates, in 37 ℃, 5%CO ₂Condition under cultivate, after treating cell attachment and stretching out normal morphology, be replaced by the culture fluid that contains 3%FBS and be divided into PBS negative control group, NaHCO ₃Matched group, 50 μ mol/L, 100 μ mol/L, 200 μ mol/L, 400 μ mol/L artesunate groups, 4 every group multiple holes, drug effect is after 48 hours, and every hole adds the MTT of 100 μ l 5mg/ml, 37 ℃, 5%CO ₂Condition under continued to hatch 2-4 hour, abandon supernatant, every hole adds 1mlDMSO dissolving, treats that granule fully dissolves the back and measures the absorbance of every hole at the 570nm place, obtains cell survival rate with the absorbance/matched group absorbance of processed group.

Experimental result:

Former be commissioned to train foster the 1st generation corneal epithelial cells of rabbit, the cellular immunization chemical identification cell purity of keratin 3/12 in the cell, find all express keratin 3/12 of nearly all cell, the results are shown in Figure 6, A be the 1st generation corneal epithelial cells of rabbit the phase contrast microscope photo, B is the PBS group, and C is 3/12 group of a keratin.

Because of the artesunate eye drop can significantly suppress HUVEC propagation at 50 μ mol/L places, thus 50 μ mol/L chosen as the least concentration group, simultaneously for getting rid of solvent NaHCO ₃The effect of corneal epithelial cell is set up with the suitable NaHCO of artesunate maximum concentration group 400 μ mol/L content ₃As negative control 2.PBS group and NaHCO ₃Group OD ₅₇₀Be respectively 0.601 ± 0.054 and 0.589 ± 0.102, the OD570 of 50 μ mol/L, 100 μ mol/L, 200 μ mol/L and 400 μ mol/L artesunate groups is respectively 0.545 ± 0.099,0.4525 ± 0.065,0.426 ± 0.112 and 0.3735 ± 0.128, result proof has slight inhibitory action to corneal epithelial cells of rabbit during up to 200 μ mol/L when artesunate concentration, explanation is external, artesunate in 100 μ mol/L and the following concentration of 100 μ mol/L to the corneal epithelial cells of rabbit free of toxic effects.

Three, eye irritant test

(1) single-dose eye irritant test

4 of new zealand rabbits (providing by Guangdong Province's Experimental Animal Center) are provided,, male and female hold concurrently half, body weight 2.5-3.0kg, every rabbit left eye splashes into the 0.1ml excipient, and right eye splashes into 0.1ml50 μ mol/L artesunate eye drop.Eyelid about 10s then gently sleeps.Eye was checked in 1,24,48,72 hour, the 4th day, the 5th day and the 7th day that after administration observe cornea, iris, conjunctiva irritant reaction degree and local response situation, observation index and standards of grading see Table 4.

(2) multiple dosing eye irritant test

4 of new zealand rabbits (providing by Guangdong Province's Experimental Animal Center) are provided,, male and female hold concurrently half, body weight 2.5-3.0kg, every rabbit left eye splashes into the 0.1ml excipient, and right eye splashes into 0.1ml50 μ mol/L artesunate eye drop.Eyelid about 10s then gently sleeps.Be administered six times every day, and one hour once.1,24,48,72 hour, the 4th day, the 5th day and the 7th day be to checking eye before administration every day and after the last administration, check and evaluation methodology the same.

Experimental result:

Show by single medication and repeatedly medication result, but this medication does not have tangible irritant reaction.Repeatedly slight conjunctival congestion appearred after the administration in 48 hours in 1/4 example the last time after the medication behind 1 hour, administration, recovered normal in 72 hours after the last administration, do not have during the administration and change, according to the eye irritation evaluation criterion, it is 0.5 minute that the multiple dosing eye stimulates comprehensive mean scores best result, belongs to nonirritant.The result shows that artesunate eye drop continuous use does not have obvious irritation.

Table 3 an irritant reaction score value standard

The eye irritant reaction	Score value
		Cornea
Do not have muddy	0
		Be dispersed in or the diffusivity muddiness, iris is high-visible	1
Translucent areas is easily differentiated, and iris is smudgy	2
		The canescence translucent areas occurs, the iris details is unclear, and the pupil size reluctantly as seen	3
Cornea is opaque, and iris is beyond recognition	4
		Iris
Normally
		0
Gauffer is obviously deepened, congested, swelling, mild hyperaemia around the cornea, and pupil still responds to light			1
	The visible necrosis of hemorrhage/naked eyes/to light reactionless (or wherein a kind of)	2
Conjunctiva
	Congested (referring to palpebral conjunctiva and bulbar conjunctiva)
Blood vessel is normal			0

The congestion of blood vessel is cerise	1
		The congestion of blood vessel is peony, and blood vessel is difficult for differentiating	2
Diffusivity hyperemia is aubergine	3
		Edema
No edema	0
		Slight edema (containing eyelid)	1
Obviously edema is accompanied the part ectropion of lid	2
		Edema is to the nearly semi-closed of eyelid	3
Edema to eyelid surpasses semi-closed	4
		Secretions
No secretions	0
		A small amount of secretions	1
Secretions makes eyelid and eyelashes are moist or adhesion	2
		Secretions makes the moist or adhesion in whole eye district	3
Maximum total mark	16

The eye irritation evaluation criterion

Score value	Estimate
		0-3.9	Nonirritant
4-8.9	Slight zest
		9-12.9	The moderate zest
13-16	Strong and stimulating

(2) preparation embodiment

Below the preparation embodiment with borate buffer solution as the buffer composition, sodium chloride as isotonic agent, carboxylic propyl methocel or hydroxypropyl emthylcellulose as thickening agent, thimerosal or benzalkonium bromide as antiseptic, as mentioned above, this preparation embodiment only is used to illustrate the present invention, can not be used to limit scope of invention.

Artesunate 0.0096-0.0384 part;

Boric acid (H ₃BO ₃) 9.30-12.028 part;

Borax (Na ₂B ₄O ₇10H ₂O) 0.573-4.775 part;

Hydroxypropyl emthylcellulose (or carboxylic propyl methocel) 1.5-3.0 part;

Sodium chloride 2.2-2.4 part;

Benzalkonium bromide (or thimerosal) 0.01-0.02 part.

Artesunate is mixed with the artesunate that concentration is 60mg/ml-NaHCO3 solution, stand-by after the filtration sterilization.It is 2% that thimerosal is mixed with the solution concentration that concentration is with distilled water, is sub-packed in the vial stand-by after the filtration sterilization.It is 2% that benzalkonium bromide is mixed with the solution concentration that concentration is with distilled water, is sub-packed in the vial stand-by after the filtration sterilization.Get boric acid 12.4g be dissolved in make boric acid solution in the 1000ml distilled water, molten Borax 19.1g makes borax soln in the 1000ml distilled water standby.

1, wooden invention eye drop, in the 100ml eye drop, get sodium chloride 0.22g, carboxylic propyl methocel 0.15g is dissolved in boric acid-borate buffer solution of 100ml, wherein boric acid solution 97ml, borax soln 3ml, high pressure steam sterilization 20 minutes, cooling back aseptic condition adds artesunate solution 16 μ l, benzalkonium bromide solution 50 μ l, pH6.7 down.

2, eye drop of the present invention, in the 100ml eye drop, getting sodium chloride 0.22g, hydroxypropyl emthylcellulose 0.15g is dissolved in boric acid-borate buffer solution of 100ml, wherein boric acid solution 97ml, borax soln 3ml, high pressure steam sterilization 20 minutes, cooling back aseptic condition adds artesunate solution 33 μ l, thimerosal solution 100 μ l, pH6.7 down.

3, eye drop of the present invention, in the 100ml eye drop, getting sodium chloride 0.22g, hydroxypropyl emthylcellulose 0.15g is dissolved in boric acid-borate buffer solution of 100ml, wherein boric acid solution 90ml, borax soln 10ml, high pressure steam sterilization 20 minutes, the cooling back adds artesunate solution 66 μ l, benzalkonium bromide solution 100 μ l, pH7.36.

4, eye drop of the present invention, in the 100ml eye drop, get sodium chloride 0.22g, carboxylic propyl methocel 0.15g is dissolved in boric acid-borate buffer solution of 100ml, wherein boric acid solution 90ml, borax soln 10ml, high pressure steam sterilization 20 minutes, the cooling back adds artesunate solution 16 μ l, thimerosal solution 50 μ l, pH7.36.

5, eye drop of the present invention, in the 100ml eye drop, getting sodium chloride 0.23g, hydroxypropyl emthylcellulose 0.15g is dissolved in boric acid-borate buffer solution of 100ml, wherein boric acid solution 85ml, borax soln 15ml, high pressure steam sterilization 20 minutes, the cooling back adds artesunate solution 33 μ l, benzalkonium bromide solution solution 100 μ l, pH7.6.

6, eye drop of the present invention, in the 100ml eye drop, get sodium chloride 0.23g, carboxylic propyl methocel 0.15g is dissolved in boric acid-borate buffer solution of 100ml, wherein boric acid solution 75ml, borax soln 25ml, high pressure steam sterilization 20 minutes, the cooling back adds artesunate solution 66 μ l, thimerosal solution 100 μ l, pH7.94.

Claims (10)

1, a kind of eye drop with anti-cornea rebirth blood vessel function, it with artesunate as active component, and comprise buffer, thickening agent, etc. ooze reagent and antiseptic, each parts by weight of raw materials is as follows:

Artesunate 0.0096-0.0384 part;

Acid ingredient 1.0032-12.028 part in the buffer;

Alkaline components 0.106-18.0477 part in the buffer; (or being expressed as buffer composition 0.106-18.0477 part)

Thickening agent 1.0-35.0 part;

Deng oozing reagent 2.0-4.72 part;

Antiseptic 0.01-0.02 part.

2,, it is characterized in that it is made by the raw material of following weight portion according to the described eye drop of claim 1:

Artesunate 0.0096-0.0384 part;

Boric acid (H ₃BO ₃) 9.30-12.028 part;

Borax (Na ₂B ₄O ₇10H ₂O) 0.573-4.775 part;

Hydroxypropyl emthylcellulose 1.5-3.0 part;

Sodium chloride 2.2-2.4 part;

Benzalkonium bromide 0.01-0.02 part.

3,, it is characterized in that it is made by the raw material of following weight portion according to the described eye drop of claim 1:

Artesunate 0.0096-0.0384 part;

Sodium dihydrogen phosphate (NaH ₂PO ₄2H ₂O) 1.0032-8.0256 part;

Sodium hydrogen phosphate (Na ₂HPO ₄12H ₂O) 4.0106-18.0477 part;

Carboxylic propyl methocel 1.5-3.0 part;

Sodium chloride 4.22-4.75 part;

Thimerosal 0.01-0.02 part.

4,, it is characterized in that it is made by the raw material of following weight portion according to claim 1 or 2 described eye drops:

0.0096 part of artesunate;

Boric acid (H ₃BO ₃) 12.028 parts;

Borax (Na ₂B ₄O ₇10H ₂O) 0.573 part;

1.5 parts of hydroxypropyl emthylcelluloses;

2.2 parts in sodium chloride;

0.01 part of benzalkonium bromide.

5,, it is characterized in that it is made by the raw material of following weight portion according to claim 1 or 3 described eye drops:

0.0192 part of artesunate;

Sodium dihydrogen phosphate (NaH ₂PO ₄2H ₂O) 1.0032 parts;

Sodium hydrogen phosphate (Na ₂HPO ₄12H ₂O) 18.0477 parts;

2.0 parts of carboxylic propyl methocels;

4.25 parts in sodium chloride;

0.02 part of thimerosal.

6,, it is characterized in that its pH is 6.5-8.0 according to the described eye drop of claim 1.

7,, it is characterized in that buffer can be phosphate buffer, borate buffer solution, citrate buffer, acetate buffer, aminoacid according to the described eye drop of claim 1; Thickening agent can be hydroxypropyl emthylcellulose, carboxylic propyl methocel, hyaluronate sodium; Deng oozing reagent can be glucose, glycerol, sodium chloride, boric acid; Antiseptic can be thimerosal, benzalkonium bromide, p-Hydroxybenzoate.

8, a kind of preparation method with eye drop of anti-cornea rebirth blood vessel function comprises:

A. get artesunate NaHCO ₃Dissolving becomes artesunate-NaHCO ₃Solution, stand-by after the filtration sterilization;

B. with the appropriate amount of buffer solution composition, etc. ooze reagent and thickening agent is dissolved in the distilled water, with 100-120 ℃ of autoclaving 20 minutes, working solution, it is standby to be cooled to room temperature;

C. antiseptic is configured to the high concentration storage liquid with distilled water, the filtration sterilization packing is stand-by;

D. under aseptic condition, artesunate solution is joined in the working solution according to the ratio that every 100ml adds 16 μ l～66 μ l, mix homogeneously, simultaneously antiseptic is joined in the buffer according to the ratio that every 100ml adds 50 μ l～100 μ l, the eye drop of different size is made in packing, preferably every bottle of packing 0.5ml.

9, described according to Claim 8 eye drop is characterized in that artesunate-NaHCO ₃The concentration of solution is 60mg/ml.

10, described according to Claim 8 eye drop, the concentration that it is characterized in that the antiseptic storage liquid is 2%.

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