NANOCRYSTALLINE EYE DROP, PREPARATION METHOD AND USE THEREOF

Abstract

A nanocrystalline eye drop contains a double-soluble macromolecule, a single-soluble macromolecule, and a fat-soluble drug. The double-soluble macromolecule and the single-soluble macromolecule interact with each other to encapsulate the fat-soluble drug to form and stabilize a nanocrystalline. The drug can rapidly pass through a blood-ocular barrier into a vitreous body by means of special intercellular space infiltration and/or pinocytosis, and achieve an effective therapeutic effect by means of passive targeting and attachment.

Claims

1. A nanocrystalline eye drop, characterized by comprising a double-soluble macromolecule, a single-soluble macromolecule and a fat-soluble drug; the double-soluble macromolecule and the single-soluble macromolecule interact with each other to encapsulate the fat-soluble drug to form and stabilize a nanocrystalline.

2. The nanocrystalline eye drop according to claim 1, characterized in that the nanocrystalline eye drop is a solution or a suspension.

3. The nanocrystalline eye drop according to claim 1, characterized in that the fat-soluble drug comprises a targeting drug acting on vascular endothelial growth factor receptors and/or platelet-derived growth factor receptors.

4. The nanocrystalline eye drop according to claim 3, characterized in that the targeting drug comprises tyrosine kinase inhibitors, preferably, the tyrosine kinase inhibitors are selected from any one or more of the tinibs and their medical acceptable salts; more preferably, any one or more of axitinib, semaxanib, sorafenib, regorafenib, pazopanib, vandetanib, imatinib, nintedanib and sunitinib.

5. The nanocrystalline eye drop according to claim 1, characterized in that the double-soluble macromolecule is a macromolecular stabilizer containing both of hydrophilic group and lipophilic group, preferably a surface-active agent, more preferably any one or two of poloxamer, tweens, sodium dodecyl compounds, polyvinylpyrrolidone and polyethylene glycol compounds; preferably, the sodium dodecyl compound is sodium dodecyl sulfonate and/or sodium dodecyl sulfate; the polyethylene glycol compound is any one or more of PEG4000, PEG5000 or PEG6000.

6. The nanocrystalline eye drop according to claim 1, characterized in that the single-soluble macromolecule is a macromolecular suspending agent containing either hydrophilic group or lipophilic group, preferably any one or at least two of starch compounds, cellulose compounds or polycarboxylate compounds; more preferably, the cellulose compound is any one or at least two of chitosan, hyaluronic acid, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and sodium carboxymethyl cellulose; the starch compound comprises any one or at least two of sodium carboxymethyl starch, amylose and dextrin; the polycarboxylate compound is any one or at least two of PLA, PGA and PLGA.

7. The nanocrystalline eye drop according to claim 1, characterized in that the particle size of the nanocrystalline in the nanocrystalline eye drop is 200-1000 nm, preferably 300-800 nm; preferably, the mass ratio between the double-soluble macromolecule and the fat-soluble drug is 2-12:1 and preferably is 5-10:1; preferably, the mass ratio between the double-soluble macromolecule and the single-soluble macromolecule is 1-5:1 and preferably is 1-2:1.

8. The nanocrystalline eye drop according to claim 1, characterized in that the content of the fat-soluble drug in the nanocrystalline eye drop is 0.06-100 mg/mL.

9. A method for preparing the nanocrystalline eye drop according to claim 1, characterized by comprising the following steps: mixing the double-soluble macromolecule, the single-soluble macromolecule and the fat-soluble drug, and then reducing the particle size of the drug to form a nanocrystalline encapsulated stably.

10. The preparation method according to claim 9, characterized in that the nanocrystalline eye drop is prepared by mixing the double-soluble macromolecule and the single-soluble macromolecule to form a mixed solution; then, mixing the mixed solution and the fat-soluble drug to form an initial suspension; and then, grinding or homogenizing the initial suspension to form the nanocrystalline eye drop that stably encapsulates the fat-soluble drug.

11. The preparation method according to claim 9, characterized by comprising: mixing the double-soluble macromolecule, the single-soluble macromolecule and water to form the mixed solution and then mixing the mixed solution and the fat-soluble drug to form the initial suspension.

12. The preparation method according to claim 11, characterized in that a dosage of the double-soluble macromolecule is 4-1000 mg in every 100 mL of water in the mixed solution; and/or a dosage of the single-soluble macromolecule is 4-1000 mg in every 100 mL of water; preferably, the dosage of the double-soluble macromolecule is 10-300 mg in every 100 mL of water.

13. An application of the nanocrystalline eye drop according to claim 1 in preparation of a drug used for treating fundus oculi diseases or/and ocular surface diseases.

14. The application according to claim 13, the fundus oculi diseases comprising diseases related to fundus neovascularization and the ocular surface diseases comprising diseases related to ocular surface neovascularization; preferably, the diseases related to fundus neovascularization comprise any one or more of age-related macular degeneration, retinal vein occlusion macular edema, central retinal vein occlusion, diabetic retinopathy, diabetic macular edema, or impaired vision, neovascular glaucoma and eye tumors caused by choroidal neovascularization secondary to pathological myopia; preferably, the diseases related to ocular surface neovascularization comprise any one or more of viral keratitis, corneal neovascularization caused by physical and/or chemical trauma, corneal transplantation, corneal neovascularization, ocular surface neovascularization and pterygium, corneal neovascularization complicated by pterygium, corneal neovascularization due to corneal transplantation rejection, and deficiency of corneal stem cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the technical solution in the examples of the present invention or in the prior art, a brief introduction of the accompanying drawings that are required to describe the example is given below.

[0063] FIG. 1 shows an SEM phenogram of a drug of a nanocrystalline eye drop in Example 1.

[0064] FIG. 2 shows an SEM phenogram of a drug of a nanocrystalline eye drop in Example 2.

[0065] FIG. 3 shows an SEM phenogram of a drug of a nanocrystalline eye drop in Example 3.

[0066] FIG. 4 shows an SEM phenogram of a drug of a nanocrystalline eye drop in Example 4.

[0067] FIG. 5 shows a fluorescence contrast image of eyes of animals in a pharmacodynamic test in which a CNV model is built for laser-induced mice eyes.

DETAILED DESCRIPTION OF THE PREFERRED EXAMPLES

[0068] To make the objectives, technical solutions, and advantages of the examples of the present invention clearer, the technical solution in the examples of the present invention will be described clearly and completely below. Unless otherwise specified in the examples, the examples were carried out according to conventional conditions or the conditions recommended by manufacturers. Reagents or instruments used, without specific manufacturers, are conventional products purchased from the market.

[0069] Further detailed descriptions to the characteristics and performances of the present invention are made in combination with the examples.

Example 1

[0070] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0071] The nanocrystalline eye drop of this example includes a double-soluble macromolecule, a single-soluble macromolecule and a fat-soluble drug. The double-soluble macromolecule and the single-soluble macromolecule interact with each other to encapsulate the fat-soluble drug.

[0072] The double-soluble macromolecule is poloxamer 188; the single-soluble macromolecule is HPC μF; and the fat-soluble drug is axitinib as a targeting drug, with the effects on both a vascular endothelial growth factor receptor and a platelet-derived growth factor receptor.

[0073] The mass ratio of poloxamer 188 to HPC μF is 5:1, and that of poloxamer 188 to axitinib is 10:1.

[0074] The preparation method of the nanocrystalline eye drop according to this example includes the following steps of:

dispersing 0.5 g poloxamer 188 and 0.1 g HPC μF into 50 mL of purified water, and preparing a mixed solution through heating and stirring;
dispersing 50 mg axitinib into the mixed solution to prepare an initial suspension with a concentration of 1 mg/mL;
transferring the initial suspension into a planetary ball mill for rapid grinding for 2 h at 350 rpm under temperature of 0° C. A grinding container is a 100 mL sealing cup made of zirconia; a grinding bead is a spherical bead made of zirconia, with a particle size of approximately 0.3-0.4 mm. A finished product prepared through decompression filtration via a filter membrane is an axitinib drug nanosuspension.

Examples 2-9

[0075] A nanocrystalline eye drop and a preparation method thereof according to the present invention are provided in the Examples 2-9.

[0076] When the nanocrystalline eye drops provided in the Examples 2-9 are compared with that provided in the Example 1, a double-soluble macromolecule, a single-soluble macromolecule and a fat-soluble drug have the same type. The prepared nanocrystalline eye drops have the same structure, with a difference in specific compounds used, or/and the mass ratio of the compounds.

[0077] The preparation methods of the nanocrystalline eye drops provided in the Examples 2-9 are basically the same as that in the Example 1, with a difference in operating conditions.

Example 2

[0078] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0079] In the nanocrystalline eye drop, the double-soluble macromolecule is poloxamer 188; the single-soluble macromolecule is HPC μF; the fat-soluble drug is axitinib; the mass ratio of the double-soluble macromolecule to the single-soluble macromolecule is 5:1; and that of poloxamer 188 to axitinib is 5:1.

[0080] A process nanocrystalline eye drop is prepared under conditions of grinding temperature of 0° C., a rotation speed of 350 rpm and grinding time of 2 h.

Example 3

[0081] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0082] In the nanocrystalline eye drop, the double-soluble macromolecule is Tween 80; the single-soluble macromolecule is HPC μF; the targeting drug is axitinib; the mass ratio of the double-soluble macromolecule to the single-soluble macromolecule is 5:1; and that of Tween 80 to axitinib is 10:1.

[0083] A process nanocrystalline eye drop is prepared under conditions of grinding temperature of 3° C., a rotation speed of 350 rpm and grinding time of 1.5 h.

Example 4

[0084] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0085] In the nanocrystalline eye drop, the double-soluble macromolecule is Tween 80; the single-soluble macromolecule is HPMC E5; the targeting drug is axitinib; the mass ratio of the double-soluble macromolecule to the single-soluble macromolecule is 5:1; and that of Tween 80 to axitinib is 10:1. A process nanocrystalline eye drop is prepared under conditions of grinding temperature of 5° C., a rotation speed of 350 rpm and grinding time of 3 h.

Example 5

[0086] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0087] In the nanocrystalline eye drop, the double-soluble macromolecule is a mixture of PEG4000, PEG5000 and sodium dodecyl sulfonate; the single-soluble macromolecule is a sodium carboxymethyl starch; the targeting drug is regorafenib; the mass ratio of the double-soluble macromolecule to the single-soluble macromolecule is 3:1; and that of the double-soluble macromolecule to regorafenib is 12:1.

[0088] A process nanocrystalline eye drop is prepared under conditions of grinding temperature of 2° C., a rotation speed of 350 rpm and grinding time of 2.5 h.

Example 6

[0089] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0090] In the nanocrystalline eye drop, the double-soluble macromolecule is poloxamer 188; the single-soluble macromolecule is PLGA; the targeting drug is vandetanib; the mass ratio of the double-soluble macromolecule to the single-soluble macromolecule is 1:1; and that of poloxamer 188 to vandetanib is 5:1.

[0091] A process nanocrystalline eye drop is prepared under conditions of grinding temperature of 0° C., a rotation speed of 500 rpm and grinding time of 3 h.

Example 7

[0092] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0093] In the nanocrystalline eye drop, the double-soluble macromolecule is a mixture of PEG6000 and Tween 80; the single-soluble macromolecule is a sodium carboxymethyl starch; the targeting drug is regorafenib; the mass ratio of the double-soluble macromolecule to the single-soluble macromolecule is 3:1; and that of the double-soluble macromolecule to regorafenib is 12:1.

[0094] A process nanocrystalline eye drop is prepared under conditions of grinding temperature of 2° C., a rotation speed of 350 rpm and grinding time of 2.5 h.

Example 8

[0095] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0096] In the nanocrystalline eye drop, the double-soluble macromolecule is sodium dodecyl sulfate; the single-soluble macromolecule is chitosan; the targeting drug is sorafenib; the mass ratio of the double-soluble macromolecule to the single-soluble macromolecule is 4:1; and that of sodium dodecyl sulfate to sorafenib is 6:1.

[0097] A process nanocrystalline eye drop is prepared under conditions of grinding temperature of 3° C., a rotation speed of 450 rpm and grinding time of 1.5 h.

Example 9

[0098] This example discloses a nanocrystalline eye drop and a preparation method thereof according to the present invention.

[0099] In the nanocrystalline eye drop, the double-soluble macromolecule is Tween 80; the single-soluble macromolecule is hyaluronic acid; the targeting drug is sunitinib; the mass ratio of the double-soluble macromolecule to the single-soluble macromolecule is 2.5:1; and that of Tween 80 to sunitinib is 8:1. A process nanocrystalline eye drop is prepared under conditions of grinding temperature of 3° C., a rotation speed of 480 rpm and grinding time of 2 h.

Characterization

[0100] The nanocrystalline eye drops prepared in the Examples 1˜4 are subject to SEM detection, with detection results as shown in FIGS. 1-4.

[0101] FIG. 1 is an SEM diagram of the Example 1. Referring to FIG. 1, nanocrystallines in the nanocrystalline eye drop of the Example 1 are flake-like, and a part of the nanocrystallines has an adhesion phenomenon, with a particle size between 100 nm and 800 nm.

[0102] FIG. 2 is an SEM diagram of the Example 2. Referring to FIG. 2, nanocrystallines in the nanocrystalline eye drop of the Example 2 are flake-like, and a part of the nanocrystallines adhesion phenomenon is not obvious, with a particle size between 100 nm and 2 μm and wider distribution in the particle size.

[0103] FIG. 3 is an SEM diagram of the Example 3. Referring to FIG. 3, nanocrystallines in the nanocrystalline eye drop of the Example 3 are minor block-shaped particles, with a particle size approximately between 100 nm and 600 nm.

[0104] FIG. 4 is an SEM diagram of the Example 4. Referring to FIG. 4, nanocrystallines in the nanocrystalline eye drop of the Example 4 are minor block-shaped particles, with a particle size approximately between 300 nm and 800 nm.

[0105] Comparative example 1: the nanocrystalline eye drop prepared according to the preparation method of the Example 1 differs in that the double-soluble macromolecule used is a substance only having a hydrophilic group, namely sodium stearate; and the nanocrystalline eye drop prepared by the double-soluble macromolecule may be a gel status, instead of a nanocrystalline structure.

[0106] Comparative example 2: the nanocrystalline eye drop prepared according to the preparation method of the Example 1 differs in that the double-soluble macromolecule used is a substance only having a fat-soluble group, namely glyceryl tristearate; and the nanocrystalline eye drop prepared by the double-soluble macromolecule may be a microspherulitic structure, instead of a nanocrystalline structure.

[0107] Comparative example 3: the nanocrystalline eye drop prepared according to the preparation method of the Example 1 differs in that the single-soluble macromolecule used is a substance only having a hydrophilic substrate and an oleophylic substrate, namely lecithin; and the nanocrystalline eye drop prepared by the single-soluble macromolecule may be a gel status, a microspherulitic structure or other structures, instead of a nanocrystalline structure.

[0108] Comparative example 4: the nanocrystalline eye drops are prepared according to the preparation method provided in the Example 1, except that the axitinib is ground first, and the grinding conditions are the same as those in the Example 1, and then the ground axitinib is mixed with a mixed solution, but nanocrystallines cannot be obtained by this method.

Stability Determination

[0109] The nanocrystalline eye drops of Example 1 to 4 and comparative examples 1 to 4 are placed at 25±5° C. and a relative humidity of 60±10% for 30 days, and then D90, D50 and D10 of the nanocrystalline eye drops are detected, and detection results are shown in a table 1.

[0110] Among them, the apparatus used for detection is Microtrac S3500, the detection conditions are: a wet method, dispersion medium water, a flow rate 60%, ultrasonic power 30 w, and ultrasonic time 120 S; a detection process: setting experiment parameters according to the above experiment conditions; filling a wet sampler with water, starting internal circulation, and starting zero adjustment at the same time; and after the zero adjustment of an instrument is passed, adding prepared nanocrystalline suspension dropwise until the concentration reaches a concentration range specified by the instrument, starting internal ultrasound, and testing PSD results after the ultrasound.

TABLE-US-00001 TABLE 1 Detection Results Time 0 day 30 days Particle size distribution D90(μm) D50(μm) D10(μm) D90(μm) D50(μm) D10(μm) Example 1 1.312 0.522 0.244 1.51 0.544 0.245 Example 2 1.367 0.477 0.226 1.356 0.472 0.229 Example 3 1.746 0.878 0.446 2.02 0.865 0.421 Example 4 1.887 0.932 0.437 2.815 0.974 0.412

[0111] It can be seen from the table 1 that the nanocrystalline eye drops provided by the examples of the present invention have good stability, and drugs are not easy to aggregate. However, since the substances prepared in the comparative examples 1 to 4 are not nanocrystallines, during a storage process, the drugs aggregate quickly and the stability is poor.

[0112] The nanocrystalline eye drops of the Example 1 to 4 are placed at 25±5° C. and the relative humidity of 60±10% for 60 days, and then the content of the nanocrystalline eye drops are analyzed. The nanocrystalline eye drops are filtered with a 0.45 μm membrane, and a filtrate is used as a test solution; API is dissolved by adding methanol to prepare a reference solution with API content of 0.1 mg/ml. The content is determined by an external standard method. Specific detection conditions are shown in Table 2, and specific detection results are shown in Table 3.

TABLE-US-00002 TABLE 2 Analyses Conditions Chromatographic conditions Agilent 1100 high performance Apparatus liquid chromatograph system Chromatographic Agilent Eclipse XDB-C18 4.6 × 150 mm, 5 μm column Detection 260 nm wavelength Mobile phase 20 mM phosphate buffer(pH = 3.0) − acetonitrile (40:60, v/v) Flow rate 1.0 ml/min Column temperature 35° C. External standard 0.1 API mg/ml (methanol) Injection volume 10 μl Retention time 1.94 min

TABLE-US-00003 TABLE 3 Detection Results API concentration Relative Sample No. mg/mL Peak area percentage % Example 1 0.1 4684 99.34 Example 2 4627 98.14 Example 3 4640 98.41 Example 4 4629 98.18 Raw material 4687 99.41 External standard 4715 100 substance

[0113] It can be seen from the table 3 that the nanocrystalline eye drops provided by the examples of the present invention have good stability, and the effective content of the drugs can be guaranteed.

In Vivo Animal Experiment

Example 10

Pharmacodynamic Experiment

[0114] Inhibition experiments to rabbit ocular surface alkali burnt corneal neovascularization (CNV) by using the drugs prepared in the Examples 1 and 4 are applied.

[0115] Ten New Zealand male rabbits, 2.0 to 2.5 kg, 3 to 4-month-old are divided into 1 normal control group (one animal and two eyes); 3 experiment groups (a model group, an Example 1 group and an Example 4 group), each group has 3 animals and 6 eyes, their corneas were burnt with 1 mol/L NaOH solution, CNV observed significantly. On the first day (Day 2) after the modeling, the administration the drugs (concentration: 0.1 mg/ml) started, 30 μl/eye/time, 3 times/day; On the tenth day (Day 11) after the administration, to observe the length and the numbers of corneal neovascularization (NV) distribution by clock directions on the rabbit eyes are and to calculate the corneal neovascularization area. The numbers are corrected based on acquired images using Photoshop CS, and the corneal neovascularization area is processed by Image Pro Plus; An area formula: S=C/12×3.1416×[R2−(R−L)2], the C represents the numbers by clock direction points when a corneal edge grows with the NV to non-NV in the picture, the R represents the length from an edge where a cornea contacts with a sclera to the center of the cornea in the picture, and the L represents the length from the root of new neovascularization of the edge where the cornea contacts with the sclera to the tail end of the NV in the cornea in the picture, and the longest neovascularization is acquired in each clock direction.

Test results:

TABLE-US-00004 Left eye Right eye New New vascular New vascular New numbers by vascular numbers by vascular Animal clock area clock area No. Group direction (S-mm.sup.2) direction (S-mm.sup.2) 1 Model 9 81.57 8 66.35 2 group 4 30.65 9 71.24 3 12 138.52 12 113.39 4 Example 1 0 0.00 0 0.00 5 group 4 11.18 0 0.00 6 0 0.00 3 14.98 7 Example 4 6 34.80 7 25.12 8 group 0 0.00 0 0.00 9 0 0.00 4 15.34 10 Normal 0 0.00 0 0.00 control group

[0116] The experiment results show that administrated the drugs prepared in the Example 1 and 4 have very lower number and area of neovascularization than those of the model group, indicating that these two test drugs have a significant inhibition effect on the neovascularization.

Example 11

Animal Vitreous Body Absorption Experiment

[0117] The nanocrystalline eye drops prepared in Example 1-4 and the eye drops of the comparative example 1-4 are used to carry out animal vitreous body absorption experiments.

[0118] Sixty-six healthy male adult SD rats are selected, two for each group, a total of 33 groups. One group (4 eyes) is a blank control group, 40 μl of normal saline is dropped, and samples are taken 10 minutes after a sample liquid is added. The remaining 32 groups are testing groups, every 4 groups as one series, there is total of 8 series; to each series animals the nanocrystalline eye drops which are prepared in Example 1 to 4 and the comparative examples 1 to 4, are instilled 20 μl for each eye, respectively. The sampling time for each group in each series is set at different time point as 30 minutes, 60 minutes, 120 minutes and 240 minutes after instillation.

[0119] The specific sampling is to collect vitreous bodies of both eyes quickly after an animal is sacrificed by breaking a neck and store the vitreous bodies at −80° C. Thereafter, the vitreous body sample is homogenized, dilution, according to a standard sample preprocessing process with methanol or acetonitrile to obtain a liquid sample for liquid chromatography mass spectrometry analysis (LC/MS/MS) to determine the target compound concentration. LC/MS conditions: referring to SHIMADUZ No. C126. Sample analysis and process: an LC/MS/MS method is used to determine the drug concentration in the vitreous bodies, specific detection conditions are shown in table 4, and specific detection results are shown in table 5. However, no drug is detected in the vitreous body samples of comparative examples 1 to 4.

TABLE-US-00005 TABLE 4 Analytic Conditions Chromatographic Conditions SHIMADZU LC-20AD high performance Apparatus liquid chromatograph system Chromatographic INERTSIL ODS-3.5 μm 4.6 × 50 mm column Mobile phase Methanol: 5 mM ammonium acetate (containing 0.1% of formic acid) water (9:1, Wv) Flow rate 0.5 ml/min Column temperature 30° C. Internal standard XPS2497 (25 ng/ml) Injection volume 5 μl Retention time 1.47 min

TABLE-US-00006 TABLE 5 Analytic Results Single Detection concentration administration (ng/mL) Sample No. Time (h) 0.5 1.0 2.0 4.0 Example 1 20 μL/eye 11.8 440 38.5 0 Example 2 26.1 3.5 5.2 0 Example 3 92.8 81.7 7.0 0 Example 4 0 771 278 43.3 Comparative 0 0 0 0 Example 1 Comparative 0 0 0 0 example 2 Comparative 0 0 0 0 example 3 Comparative 0 0 0 0 Example 4 Control group 0 0 0 0

[0120] It can be seen from the table 5 that the nanocrystalline eye drops prepared in the examples of the present invention have good absorption, and the drugs can quickly pass through a blood-ocular barrier to enter the vitreous bodies, however, the eye drops prepared after changing the formulation or operation of the examples of the present invention cannot pass through the blood-ocular barrier to enter the vitreous bodies.

Example 12

Animal Pharmacodynamic Experiment

[0121] The pharmacodynamic study of a laser-induced mouse choroidal neovascularization (CNV) model

1) Sample Preparation

[0122] High-dose group: a sample with the drug concentration of 1 mg/ml prepared based on the conditions in the Example 13 (table 8 experiment conditions and result—No. 1);
Medium-dose group: 4 times dilution of the high-dose group;
Low-dose group: 4 times dilution of the medium-dose group.

2) Experiment Animal Preparation

[0123] Forty C57Bl/6c mice, 6-8 weeks old, 18-25 g, half male and half female, without abnormality in both eyes are chosen for laser-induced modeling.

[0124] Among them, the laser modeling refers to laser induction on fundi in both eyes of the mice to construct a CNV model, and the number of laser burns per eye is 3; laser parameters are wavelength 532 nm, power 120 mW, a light spot diameter 100 μm, and exposure time 100 ms.

[0125] The mice successfully modeled by laser photocoagulation are randomly divided into the following 4 groups:

TABLE-US-00007 TABLE 6 Experiment Conditions Administration Group concentration The number of animals No. Group (mg/ml) Female Male 1 Vehicle control group 0 4 4 2 Low-dose group 0.0625 4 4 3 Medium-dose group 0.25 4 4 4 High-dose group 1.0 4 4

3) Dosing Frequency and Cycle

[0126] Eye drop instillation begins on the seventh day after modeling, 4 times/day, 5 μL/eye/time, for 14 consecutive days. Normal saline was administrated to the vehicle control group in the same manner.

[0127] Fundus photography (FP) is used to observe the retinal morphology of the fundi, and fundus angiography (FFA) is used to observe the leakage of the choroidal neovascularization.

4) Results

[0128]

TABLE-US-00008 TABLE 7 Experiment Results Improvement of light spot leakage of the mouse CNV model by testing samples Time The average score of light spot leakage Two weeks after the Subject Before the administration administration Vehicle control group 2.67 2.52 Low-dose group 2.63 2.06 Medium-dose group 2.61 2.13 High-dose group 2.61 1.83 Note: the average score of light spot leakage = [(0-level light spot number × 0) + (1-level light spot number × 1) + (2-level light spot number × 2) + (3-level light spot number × 3)] ÷ 4 total light spots (that is, the number of effective light spots).

[0129] The fluorescein pictures of the animal eyes before and after the administration the nanocrystalline eye drops are shown in FIG. 5.

[0130] The experiment results show that, compared with the vehicle control group, the three testing groups of the present invention can reduce the eye spot light leakage of experimental animals, indicating that the nanocrystalline eye drops of the present invention can effectively reach the bottom of the eye and play a therapeutic role. It shows that the nanocrystalline eye drop according to the present invention can effectively reach the fundus and play a therapeutic role.

Example 13

Relationship Between the Drug Formulation and Animal Vitreous Body Absorption

[0131] In this example, different nanocrystalline eye drops are prepared and used for animal study to investigate the relationship between different nanocrystalline eye drops and the absorption of animal vitreous body.

[0132] The ball-milling method according to this example comprises the following steps:

1) respectively weighing, placing, and stirring double-soluble macromolecules and single-soluble macromolecule in a container containing 50 mL of purified water; heating in hot water bath (50-70° C.), continuing stirring till full dissolution;
2) weighing and placing a drug into the solution prepared in Step 1, starting a shearing machine to shear for 3-5 min at about 10000 rpm and get a preliminary suspension;
3) transferring the preliminary suspension prepared in Step 2 into a ball mill, where the ball-milling container is 100 ml sealing cup, and the grinding ball is ZrOZ beads with 0.3-0.4 mm (or 0.1-0.2 mm) in diameter; grinding for 2 h at 0° C.-10° C. and 350 rpm; filtering the obtained material by Buchner funnel and filter membrane under diminished pressure, and collecting the filtrate to obtain the nanocrystalline eye drop; recovering grinding ball.

[0133] The high-pressure homogenization method according to this comprises the following steps:

A. respectively weighing and placing double-soluble macromolecule and single-soluble macromolecule in a container containing 50 mL of purified water; stirring and heating (50-70° C. water bath) till full dissolution;
B. weighing and placing a drug into the solution prepared in Step A, starting a shearing machine to shear for 3-5 min at 10000-15000 rpm to prepare a preliminary suspension;
C. transferring the preliminary suspension prepared in Step B into a high-pressure homogenization machine, and controlling temperature to 5-10° C.; setting the pressure to not more than 1500 bar, and recycling for 15-20 times; finally recycling once at homogenization pressure of about 100-200 bar, discharging the solution, and performing membrane filtration to obtain homogeneous liquid.

[0134] The test method for drug content in rat vitreous body according to this example comprises the following steps:

selecting and grouping healthy adult SD rats with 4-6 eyes for each group; adding the prepared eye drop at 20 μl per eye dropwise, respectively; sacrificed the animals at set time point (1 h) to collect the vitreous body of both eyes and store the vitreous body at −80° C.; after homogenating, fully mixing the vitreous body with methyl alcohol or acetonitrile, filtration, obtaining filtrate as an analytical sample; determining the concentration of the target compound by a liquid chromatography-mass spectrometry (LC/MS), and calculating the API content in the test sample according to the API standard curve obtained under same analysis condition, as shown in Table 8.

TABLE-US-00009 TABLE 8 Experiment conditions and results Rat vitreous concentration S/N Test Conditions Test method Properties (ng/ml) 1 Targeting drug, axitinib 50 mg; High pressure Suspension 92.8 Double-soluble macromolecule, Tween 80 homogenization 0.5 g; Single-soluble macromolecule, HPC method EF 0.5 g 2 Targeting drug, axitinib 50 mg; High pressure Suspension 20.3 Double-soluble macromolecule, Tween 80 homogenization 0.5 g; Single-soluble macromolecule, HPC method HF 0.5 g 3 Targeting drug, axitinib 50 mg; Ball-milling Suspension 67.8 Double-soluble macromolecule, poloxamer method 0.5 g; Single-soluble macromolecule, HPC HF 0.3 g 4 Targeting drug, axitinib 50 mg; Ball-milling Suspension 22.7 Double-soluble macromolecule, Tween 80 method 0.5 g; Single-soluble macromolecule, HPMC E5 0.3 g 5 Targeting drug, axitinib 50 mg; Ball-milling Suspension 14.4 Double-soluble macromolecule, Tween 80 method 0.3 g; Single-soluble macromolecule, HPMC E5 0.3 g 6 Targeting drug, axitinib 5 mg; High pressure Suspension 3.6 Double-soluble macromolecule, Tween 80 homogenization 0.5 g; Single-soluble macromolecule, method HPMC E5 0.3 g 7 Targeting drug, axitinib 5 mg; High pressure Suspension 42.3 Double-soluble macromolecule, Tween 80 homogenization 0.5 g; Single-soluble macromolecules, HPC method EF 0.3 g. 8 Targeting drug, axitinib 12.5 mg; Ball-milling Suspension 80.8 Double-soluble macromolecule, Tween 80 method 0.3 g; Single-soluble macromolecules, HPC EF 0.3 g. 9 Targeting drug, axitinib 50 mg; Ball-milling Suspension 74.0 Double-soluble macromolecule, Tween 80 method 0.5 g; Single-soluble macromolecule, HPC EF 0.5 g 10 Targeting drug, axitinib 12.5 mg; Ball-milling Suspension 70.9 Double-soluble macromolecule, Tween 80 method 31 mg; Single-soluble macromolecule: HPC EF 31 mg. 11 Targeting drug, axitinib 0.05 g; High pressure Suspension 32.5 Double-soluble macromolecule, poloxamer homogenization 0.5 g; Single-soluble macromolecule, method HPMC E5 0.3 g 12 Targeting drug, Axitinib 5 mg; High pressure Solution 1.7 Double-soluble macromolecule, Tween 80 homogenization 2 mg; Single-soluble macromolecules, method HPMC E5 2 mg. 13 Targeting drug, axitinib 10 mg; High pressure Solution 6.3 Double-soluble macromolecule, Tween 80 homogenization 2 mg; Single-soluble macromolecules, method HPMC E5 2 mg. 14 Targeting drug, axitinib 50 mg; High pressure Suspension 4.3 Double-soluble macromolecule, Tween 80 homogenization 25 mg; Single-soluble macromolecule: method HPMC E5 25 mg, CMC-Na 25 mg. 15 Targeting drug, axitinib 5 mg; High pressure Suspension 38.5 Double-soluble macromolecule, povidone homogenization K30 150 mg; Single-soluble method macromolecule, HPC HF 20 mg. 16 Targeting drug, axitinib 5 mg; High pressure Suspension 46.3 Double-soluble macromolecule, povidone homogenization K30 100 mg; Single-soluble method macromolecule, HPC EF 67 mg. 17 Targeting drug, sunitinib 10 mg; High pressure Solution 120.5 Double-soluble macromolecule, povidone homogenization K30 100 mg; Single-soluble method macromolecule, HPC HF 20 mg. 18 Targeting drug, sunitinib 10 mg; High pressure Solution 219.4 Double-soluble macromolecule, povidone homogenization K30 100 mg; Single-soluble method macromolecule, HPC EF 67 mg. Note: Water (50 ml) is used as the medium of the experiments in the table.

[0135] The result finally obtained by a great number of the experiment researches shows:

1) the double-soluble macromolecule and the single-soluble macromolecule have different chemical groups, polymerization patterns and polymerization degrees, which causes that they are very different in physicochemical property, lipid-water partition and stabilization for and biocompatibility with the targeting drug, etc. For example, compared with the eye drop prepared by using the hydroxypropyl methyl cellulose (HPMC E5), the eye drop prepared by using the hydroxypropyl cellulose (HPC μF or HF) as the single-soluble macromolecule and the Tween as the double-soluble macromolecule in the case of other conditions unchanged, is obviously better absorbed by the vitreous body of animals. The eye drop prepared by using the poloxamer+HPC HF is 1 time higher than that prepared by using poloxamer+HPMC E5 with respect to the absorbent concentration inside the vitreous body of animals;
2) the mass ratio between the double-soluble macromolecule and the targeting drug affects the absorption of the nanocrystalline eye drop inside the vitreous body of animals;
3) the final concentration of the double-soluble macromolecule or/and the single-soluble macromolecule in the eye drop will affect the absorption of the targeting drug; when the concentration of the double-soluble macromolecule is lower than 0.6 mg/ml, it is obvious to affect the drug absorption inside the vitreous body of animals;
4) such conditions as type, mass ratio and preparation technology of the targeting drug, the double-soluble macromolecule or/and the single-soluble macromolecule for preparing the nanocrystalline eye drop are different, which will result in different absorption of the prepared eye drop inside the vitreous body.

[0136] In conclusion, in the present invention, it is available to wrap the fat-soluble drug through interaction between the double-soluble macromolecule and the single-soluble macromolecule and further to form the nanocrystalline eye drop. Due to the hydrophily of the double-soluble macromolecule, the nanocrystalline eye drop is affiliative to the aqueous phase on the ocular surface. As the nanocrystalline eye drop is affiliative to lipid phase after contact with the ocular surface, it is helpful for the nanocrystalline eye drop to permeate the focus on fundus vitreous body. The nanocrystalline particle of drug is smaller, which is also good for penetratively enter the posterior segment.

[0137] Selecting small molecular tinib kinase inhibitors, the fat-soluble drug is as the preferred way in the present invention, since the small molecular tinib kinase inhibitor is easier to permeate into tissue than the macromolecular biological medicines. The pharmacodynamic study shows that the nanocrystalline eye drop of the present invention can ensure the effect of treating the neovascularization diseases of eye by using the targeting drug acting on VEGFR and/or PDGFR.

[0138] Through strict control for the types, mass ratio of the double-soluble macromolecule, the single-soluble macromolecule and the fat-soluble drug, and preparation technology, the present invention has the advantage that the prepared eye drop has stable property, is uneasy to accumulate or settle, and can fast go through the blood-ocular barrier to enter the fundus.

[0139] The above examples are part of examples of the present invention, but not all examples. The protection scope of the present invention is not limited by the detailed description of the examples according to the present invention, and these examples are only selected to describe the present invention. Based on the examples of the present invention, all other examples obtained by those skilled in the art without creative work shall fall within the protection scope of the present invention.