A preparation method of griseofulvin eye drops

Abstract

The invention discloses a preparation method of griseofulvin eye drops, and belongs to the technical field of pharmaceutical preparations. Hemoglobin is dissolved in water as an aqueous phase, and griseofulvin is dissolved in an organic solvent as an organic phase; the organic phase is added to the aqueous phase, and the organic solvent is removed after stirring; dopamine hydrochloride is then added, and then a Tris buffer solution is added under stirring conditions and continued to be stirred; finally, a cell-penetrating peptide is added and stirred to react to obtain griseofulvin eye drops. The cell-penetrating peptide is used to penetrate the eye barrier and is combined with the nanostructure formed by hemoglobin, griseofulvin and dopamine. The nanopreparation can carry drugs to the posterior segment of the eye by local eye drops.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparation preparation, and particularly relates to a method for preparing griseofulvin eye drops. Background Art

[0002] The eyeball can be divided into two parts according to its anatomical structure: the anterior segment of the eye (cornea, conjunctiva, anterior chamber, etc.) and the posterior segment of the eye (vitreous body, retina, choroid, etc.). Many ophthalmic diseases that cause visual impairment occur in tissues such as the retina and choroid in the posterior segment of the eye, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and choroidal neovascularization (CNV). These diseases are characterized by abnormal angiogenesis (neovascularization) in the posterior segment of the eye (retina or choroid), leading to vascular leakage, which in turn leads to hemorrhage, edema, and retinal detachment, and directly causes vision loss. Neovascularization is driven by many factors, the most prominent of which is vascular endothelial growth factor.

[0003] At present, the most commonly used method for drug treatment of CNV and other posterior ocular tissue diseases is to use intravitreal injection of anti-VEGF (vascular endothelial growth factor) preparations (such as ranibizumab, bevacizumab, etc.), because intravitreal injection can deliver higher concentrations of drugs directly to the posterior ocular tissue. However, the half-life of general drugs in the vitreous is short, and repeated injections are required to achieve the therapeutic purpose. This traumatic administration method increases the risk of lens damage, vitreous hemorrhage, retinal detachment, and endophthalmitis. In addition, a considerable number of patients do not respond to anti-VEGF treatment, and there is currently a lack of drugs that replace VEGF as a target. Systemic administration (intravenous or oral) is an easily accepted method of administration, but due to the influence of the blood-retinal barrier (BRB) and systemic distribution, the concentration of general drugs entering the posterior ocular tissue from the circulatory system is limited, requiring large doses and frequent administration, which is bound to cause systemic toxic and side effects. Topical administration is a convenient, non-invasive way of drug delivery. However, due to the barrier effects of the cornea, conjunctiva and tear film, as well as nasolacrimal duct loss, the intraocular bioavailability of traditional ophthalmic preparations is very low, and the concentration transported to the posterior segment of the eye is even lower. This method of drug delivery is currently mainly used to treat anterior segment tissue diseases. The development of new target drug preparations with non-invasive drug delivery methods is of great significance for the treatment of neovascularization in the posterior segment of the eye.

[0004] Recent studies have found that ferrochelatase is the terminal enzyme in heme biosynthesis and is essential for angiogenesis in vitro and in vivo. Griseofulvin can act on ferrochelatase and inhibit choroidal neovascularization to a certain extent, but griseofulvin is poorly soluble, which limits its application in the eye. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing griseofulvin eye drops, which can carry drugs to the posterior segment of the eye by local eye drops and can be used for non-invasive treatment of neovascularization in the posterior segment of the eye including age-related macular degeneration.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing griseofulvin eye drops comprises the following steps: dissolving 1-200 parts by weight of hemoglobin in water as an aqueous phase, and dissolving 0.1-5 parts by weight of griseofulvin in an organic solvent as an organic phase; adding the organic phase to the aqueous phase, stirring, and removing the organic solvent; then adding 1-200 parts by weight of dopamine hydrochloride, and then adding a Tris buffer solution with a pH value of 8.0-9.0 under stirring conditions and continuing to stir; finally adding 1-100 parts by weight of a cell-penetrating peptide, and stirring for reaction to obtain the griseofulvin eye drops;

[0008] The volume ratio of the aqueous phase to the organic phase is 1:(0.2-8); the volume ratio of the aqueous phase to the Tris buffer solution is 1:(1-100); and the hemoglobin concentration in the aqueous phase is 5-50 mg / mL.

[0009] Preferably, the griseofulvin eye drops are nanoparticles with a particle size of less than 200 nm formed by griseofulvin, hemoglobin and dopamine; and the cell-penetrating peptide is combined with the dopamine in the nanoparticles.

[0010] Preferably, the amino acid sequence of the cell-penetrating peptide is RKKRRQRRRC.

[0011] Preferably, the organic solvent is acetone.

[0012] Compared with the prior art, the present invention has the following technical advantages.

[0013] (1) The present invention utilizes cell-penetrating peptides to penetrate the ocular barrier and combines them into nanostructures formed by hemoglobin, griseofulvin and dopamine to prepare eye drops. The griseofulvin eye drops prepared by the present invention have a significant inhibitory effect on angiogenesis and are more conducive to the treatment of posterior segment diseases of the eye by eye drop administration.

[0014] (2) The particle size of the eye drops prepared according to the method of the present invention is uniform and stable. Experiments have shown that the particle size of the eye drops conforms to the normal distribution and the particle size is less than 200 nm.

[0015] (3) The eye drops prepared according to the method of the present invention have a high drug encapsulation rate. Experiments have shown that the encapsulation rate exceeds 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Fluorescence distribution diagram of eye slices after eye drops of the preparation of Example 4 of the present invention;

[0017] Figure 2 : Representative images of the inhibition of angiogenesis by the preparation of Example 5 of the present invention (A is the blank control group, B is the Example 5 group, scale bar = 200 μm);

[0018] Figure 3 : Particle size distribution diagram of the preparation of Example 1 of the present invention;

[0019] Figure 4 : Transmission electron micrograph of the preparation of Example 2 of the present invention (scale bar = 100 μm). DETAILED DESCRIPTION

[0020] The present invention is further described below by way of examples. It should be correctly understood that the examples are only used to illustrate the present invention, rather than to limit the present invention, and simple modifications made on the premise of the method of the present invention are within the scope of protection claimed by the present invention. Example 1

[0021] (1) 0.1 mg of griseofulvin was dissolved in 2 mL of acetone as the organic phase, and 50 mg of hemoglobin was dissolved in 10 mL of deionized water as the aqueous phase;

[0022] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and stirring was continued for 0.2 hours. The organic solvent was removed by rotary evaporation, and then 1 mg of dopamine hydrochloride was added under stirring conditions;

[0023] (3) Add 10 ml of Tris buffer solution (pH = 8.5) under stirring and continue stirring for 1 hour;

[0024] (4) Add 1 mg of cell-penetrating peptide TAT under stirring, and continue stirring for 1 hour to obtain griseofulvin eye drops. Example 2

[0025] (1) 5 mg of griseofulvin was dissolved in 10 mL of acetone as the organic phase, and 200 mg of hemoglobin was dissolved in 10 mL of deionized water as the aqueous phase;

[0026] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and the stirring was continued for 10 hours. The organic solvent was removed by rotary evaporation, and then 200 mg of dopamine hydrochloride was added under stirring conditions;

[0027] (3) Add 100 ml of Tris buffer solution (pH = 8.5) under stirring and continue stirring for 48 hours;

[0028] (4) Add 20 mg of cell-penetrating peptide TAT under stirring conditions, and continue stirring for 48 hours to obtain griseofulvin eye drops. Example 3

[0029] (1) 0.1 mg of griseofulvin was dissolved in 8 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0030] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and the stirring was continued for 5 hours. The organic solvent was removed by rotary evaporation, and then 20 mg of dopamine hydrochloride was added under stirring conditions;

[0031] (3) Add 100 ml of Tris buffer solution (pH = 8.5) under stirring and continue stirring for 10 hours;

[0032] (4) Add 10 mg of cell-penetrating peptide TAT under stirring conditions, and continue stirring for 10 hours to obtain griseofulvin eye drops. Example 4

[0033] (1) 0.1 mg of griseofulvin and 1 mg of coumarin-6 were dissolved in 8 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0034] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and the stirring was continued for 5 hours. The organic solvent was removed by rotary evaporation, and then 20 mg of dopamine hydrochloride was added under stirring conditions;

[0035] (3) Add 10 ml of Tris buffer solution (pH = 8.5) under stirring and continue stirring for 10 hours;

[0036] (4) Add 10 mg of cell-penetrating peptide TAT under stirring conditions, and continue stirring for 10 hours to obtain griseofulvin eye drops. Example 5

[0037] (1) 1 mg of griseofulvin was dissolved in 8 mL of acetone as the organic phase, and 50 mg of hemoglobin was dissolved in 2 mL of deionized water as the aqueous phase;

[0038] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and the stirring was continued for 2 hours. The organic solvent was removed by rotary evaporation, and then 20 mg of dopamine hydrochloride was added under stirring conditions;

[0039] (3) Add 10 ml of Tris buffer solution (pH = 8.5) under stirring and continue stirring for 5 hours;

[0040] (4) Add 10 mg of cell-penetrating peptide TAT under stirring conditions, and continue stirring for 5 hours to obtain griseofulvin eye drops.

[0041] The amino acid sequences of the TAT peptides in Examples 1-5 of the present invention are all RKKRRQRRRC.

[0042] Comparative Example 1

[0043] (1) 1 mg of griseofulvin was dissolved in 8 mL of tetrahydrofuran as the organic phase, and 50 mg of hemoglobin was dissolved in 2 mL of deionized water as the aqueous phase;

[0044] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and the stirring was continued for 2 hours. The organic solvent was removed by rotary evaporation, and then 20 mg of dopamine hydrochloride was added under stirring conditions;

[0045] (3) Add 10 ml of Tris buffer solution (pH = 8.5) under stirring and continue stirring for 5 hours;

[0046] (4) Add 10 mg of cell-penetrating peptide TAT under stirring and continue stirring for 5 hours.

[0047] Comparative Example 2

[0048] (1) 1 mg of griseofulvin was dissolved in 8 mL of acetone as the organic phase, and 50 mg of human serum albumin was dissolved in 2 mL of deionized water as the aqueous phase;

[0049] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and the stirring was continued for 2 hours. Then, 0.2% glutaraldehyde was added for cross-linking for 2 hours, and the organic solvent was removed by rotary evaporation. Then, 20 mg of dopamine hydrochloride was added under stirring conditions;

[0050] (3) Add 10 ml of Tris buffer solution (pH = 8.5) and continue stirring for 5 hours;

[0051] (4) Add 10 mg of cell-penetrating peptide TAT under stirring and continue stirring for 5 hours.

[0052] Comparative Example 3

[0053] (1) 8 mL of acetone as the organic phase, 2 mg of griseofulvin, 20 mg of hemoglobin, and 50 mg of dopamine hydrochloride dissolved in 10 mL of Tris buffer solution (pH = 8.5) as the aqueous phase;

[0054] (2) Add the organic phase dropwise to the aqueous phase under stirring, continue stirring for 4 h, and remove the organic solvent by rotary evaporation;

[0055] (3) Add 10 mg of cell-penetrating peptide TAT under stirring and continue stirring for 5 hours.

[0056] Comparative Example 4

[0057] (1) 1 mg of icariin was dissolved in 8 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0058] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and the stirring was continued for 5 hours. The organic solvent was removed by rotary evaporation, and then 20 mg of dopamine hydrochloride was added under stirring conditions;

[0059] (3) Add 100 ml of Tris buffer solution (pH = 8.5) under stirring and continue stirring for 10 hours;

[0060] (4) Add 10 mg of cell-penetrating peptide TAT under stirring and continue stirring for 10 hours.

[0061] Comparative Example 5

[0062] (1) 1 mg of griseofulvin was dissolved in 8 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0063] (2) The organic phase was added dropwise to the aqueous phase under stirring conditions, and the stirring was continued for 5 hours. 0.2% glutaraldehyde cross-linking agent was added and stirred for 2 hours. The organic solvent was removed by rotary evaporation, and then 20 mg of dopamine hydrochloride was added under stirring conditions;

[0064] (3) Add 100 ml of Tris buffer solution (pH = 8.5) under stirring and continue stirring for 10 hours;

[0065] (4) Add 10 mg of cell-penetrating peptide TAT under stirring and continue stirring for 10 hours.

[0066] Comparative Example 6

[0067] (1) Weigh 1 mg of griseofulvin and dissolve it in 5 ml of DMSO (dimethyl sulfoxide). Weigh 30 mg of dopamine hydrochloride and add 15 ml of Tris buffer solution (pH = 8.5). Mix the two and stir to react for 12 hours.

[0068] Verification Example

[0069] 1. The griseofulvin eye drops obtained in Example 4 were filtered through a 0.45 μm filter membrane and then administered to SD rats by eye drops. The rats were killed 0.5 h later, the entire eyeballs were removed, fixed with 4% paraformaldehyde and dehydrated, frozen sections of the eyeballs were made, and the distribution of fluorescence in the eyes was observed.

[0070] Figure 1 The results show that the nanoparticles labeled with the fluorescent probe coumarin-6 can penetrate into the posterior segment of the rat eye, providing a basis for griseofulvin to exert its therapeutic effect.

[0071] 2. Starve human umbilical vein epithelial cells (HUVEC cells): replace the complete culture medium with DMEM / F12 culture medium containing 0.2% fetal bovine serum (FBS) and culture for 24 hours. Place the matrix gel (Matrix-Gel™ matrix gel) (standard type) on ice and melt at 4°C, place the 96-well plate on ice, add 50µl matrix gel to each well of the 96-well plate, and then incubate at 37°C for 45 minutes to solidify the matrix gel. Digest the HUVEC cells, resuspend them in DMEM / F12 containing 10% FBS and count them. Add 100µl cell suspension (containing 30,000 cells) to each well of the 96-well plate, add the DMEM / F12 culture medium of Example 5 to one group, and add the same amount of DMEM / F12 to the blank control group. After incubation for 4 hours, observe the angiogenesis under a microscope.

[0072] The results are as follows Figure 2 As shown, the vascular nodes of the embodiment of the present invention are significantly lower than those of the blank control group, which proves that the griseofulvin eye drops prepared by the present invention have an inhibitory effect on angiogenesis.

[0073] 3. The products obtained in Examples 1-5 and Comparative Examples 1-6 were filtered using a 0.45 μm microporous filter membrane. The particle size and polydispersity index of the obtained products were monitored using a laser particle size analyzer. The results are shown in Table 1.

[0074] Table 1. Particle size and polydispersity index (PDI) of various examples

[0075]

[0076] Figure 3 Table 1 shows that the particle size of the eye drops prepared in the embodiment of the present invention conforms to the normal distribution, the particle size is less than 200 nm, and the particle size is uniform.

[0077] 4. The product obtained in Example 1 was filtered using a 0.45 μm microporous filter membrane, the resulting liquid was negatively stained with phosphotungstic acid, and the morphology was observed using a transmission electron microscope.

[0078] like Figure 4 As shown, the eye drops prepared in the embodiment of the present invention are approximately spherical in shape and have a relatively uniform particle size.

[0079] 5. The products obtained in Examples 1-5 and Comparative Examples 1, 2, 3, and 5 were filtered through a 0.45 micron microporous membrane, 200 microliters of the obtained liquid was diluted 5 times with DMSO to break the emulsion, and the drug content was detected by high performance liquid chromatography. The liquid phase conditions were as follows: chromatographic column: Diamonsil Plus C18, 250×4.6mm, 5μm; mobile phase acetonitrile: water = 55:45; flow rate 1mL / min; column temperature: 37°C; injection volume: 20μL; detection wavelength: 296nm. The drug encapsulation efficiency was calculated, and the results are shown in Table 2.

[0080] Table 2. Encapsulation efficiency of various examples

[0081]

[0082] As shown in Table 2, griseofulvin is easily encapsulated in the preparation prepared by the present invention, and the encapsulation rate is relatively high, exceeding 90%.

Claims

1. A method for preparing griseofulvin eye drops, characterized in that: Dissolve 1-200 parts by weight of hemoglobin in water as an aqueous phase, and dissolve 0.1-5 parts by weight of griseofulvin in an organic solvent, acetone, as an organic phase; add the organic phase to the aqueous phase, stir, and then remove the organic solvent; then add 1-200 parts by weight of dopamine hydrochloride, and then add a Tris buffer solution with a pH of 8.0-9.0 under stirring and continue stirring; finally, add 1-100 parts by weight of a cell-penetrating peptide, and stir to react to obtain griseofulvin eye drops; The volume ratio of the aqueous phase to the organic phase is 1:(0.2-8); the volume ratio of the aqueous phase to the Tris buffer solution is 1:(1-100); the hemoglobin concentration in the aqueous phase is 5-50 mg / mL; The griseofulvin eye drops are nanoparticles with a particle size of less than 200 nm formed by griseofulvin, hemoglobin and dopamine; the cell-penetrating peptide is combined with the dopamine in the nanoparticles; The amino acid sequence of the cell-penetrating peptide is RKKRRQRRRC.

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