CN112741805A

CN112741805A - Antifungal eye drops and preparation method thereof

Info

Publication number: CN112741805A
Application number: CN202110168153.7A
Authority: CN
Inventors: 李景果; 石刘奇; 栗占荣; 张俊杰; 祝磊
Original assignee: Henan Provincial Peoples Hospital
Current assignee: Henan Provincial Peoples Hospital
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2021-05-04
Anticipated expiration: 2041-02-07
Also published as: CN112741805B

Abstract

The invention belongs to the technical field of polymer chemistry, biomedical materials and pharmacy, and discloses antifungal eye drops and a preparation method thereof. The eye drops are prepared from the following raw materials in percentage by g/mL: 0.1-1% of eye drop medicine, 0.5-10% of high-molecular graft copolymer, a pH regulator, an osmotic pressure regulator and the balance of water for injection; wherein the macromolecular graft polymer is chitosan-g- (cyclodextrin +3, 4-dihydroxyphenyl propionic acid). The invention obviously reduces the high drug concentration of the clinical common eye drops and greatly reduces the side effect caused by the drug; the eye drops of the invention prolong the detention time of the carrier and the drug on the ocular surface, thereby shortening the administration frequency and the administration times of patients; the eye drops have excellent antibacterial effect in vitro and show excellent curative effect of clinical first-line medication (voriconazole) in vivo.

Description

Antifungal eye drops and preparation method thereof

Technical Field

The invention belongs to the technical field of polymer chemistry, biomedical materials and pharmacy, and particularly relates to antifungal eye drops and a preparation method thereof.

Background

Fungal keratitis (Fungal keratitis) is a corneal infectious ophthalmic disease caused by fungi, has a high blinding rate, and the incidence rate is currently increasing year by year in developing countries. The main cause of fungal keratitis is post-traumatic infection. Failure to cure fungal keratitis rapidly and effectively can progress to corneal perforation and even endophthalmitis, which can occur at rates as high as 50%. Therefore, it is crucial to take effective measures after a definite diagnosis to prevent further deterioration. However, there are fewer effective commercial antifungal agents, and the two major barriers in the eye (the tear film barrier and the corneal barrier) are urgently seeking more effective ophthalmic formulations to cure fungal keratitis.

In recent years, biomaterials have been continuously applied in ocular drug delivery to overcome intraocular barriers to increase drug concentration at focal sites. Although some formulations have been used clinically, such as creams, gels, contact lenses, etc., eye drops remain the most common and widespread means for treating diseases associated with the anterior segment of the eye. Patient compliance during topical administration is a critical factor in the treatment of the entire disease. Frequent administration of high doses of eye drops for achieving a pharmacological effect often leads to severe side effects and ocular discomfort, such as temporary blurred vision and chronic injury, and further leads to poor patient compliance. The existing antifungal eye drops commonly used in clinic have the disadvantages of higher required drug concentration, short retention time, higher administration frequency and administration frequency during treatment, and greatly reduced patient compliance.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide the antifungal eye drops which can prolong the retention time of the eye drops, reduce the administration frequency and the administration times and increase the compliance of patients and the preparation method thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

an antifungal eye drop, the percentage is counted by g/mL, the eye drop is prepared by the following raw materials: 0.1-1% of eye drop medicine, 0.5-10% of high-molecular graft copolymer, a pH regulator, an osmotic pressure regulator and the balance of water for injection; wherein the macromolecular graft polymer is chitosan-g- (cyclodextrin +3, 4-dihydroxyphenyl propionic acid), the pH regulator regulates the pH of the eye drops to 4.3-6.5, and the osmotic pressure regulator regulates the osmotic pressure of the eye drops to 330 +/-3.2 mOsm/kg.

Preferably, the eye drop drug is an antifungal drug or an immunosuppressant.

Preferably, the antifungal drug is econazole.

Preferably, in the high-molecular graft copolymer, the grafting rate of the cyclodextrin is 20-80%, and the grafting rate of the 3, 4-dihydroxyphenyl propionic acid is 1-15%.

Preferably, the cyclodextrin is alpha-cyclodextrin, beta-cyclodextrin or gamma-cyclodextrin.

The preparation method of the antifungal eye drops comprises the following steps: firstly, weighing a high-molecular graft polymer, adding injection water with dissolving equivalent, and stirring until the high-molecular graft polymer is completely dissolved; then, weighing the eye drop medicine, adding the eye drop medicine into the solution, and stirring until the eye drop medicine is completely dissolved; and finally, adding water for injection to a constant volume, adding a pH regulator and an osmotic pressure regulator to regulate the pH and the osmotic pressure, and sterilizing to obtain the required eye drops.

Chitosan-g- (cyclodextrin +3, 4-dihydroxyphenylpropionic acid) (CS-g- (CD + HCA) for short) can be prepared according to the prior art and comprises the following steps:

s1 Synthesis of CD-COOH (monocarboxylated cyclodextrin): sodium chloroacetate (ClCH) was added₂COONa) in alkaline conditions, then adding hydrochloric acid (HCl) to adjust the pH value for acidification;

s2, synthesis of CS-g-CD: grafting monocarboxylated cyclodextrin onto chitosan main chain through amidation reaction and controlling the reaction material ratio to synthesize CS-g-CD with different grafting rate;

s3 Synthesis of CS-g- (CD + HCA): grafting 3, 4-dihydroxyphenyl propionic acid (HCA) to a chitosan main chain through amidation reaction by controlling the charge ratio of the reaction, and synthesizing CS-g- (CD + HCA) with different grafting ratios;

the detailed steps are as follows:

s1, synthesizing CD-COOH: weighing CD and NaOH, dissolving with water for injection, and adding ClCH in corresponding amount₂COONa, and performing oil bath reaction for 5 hours at the temperature of 50 ℃; then regulating the pH value of the system to 6-7 by hydrochloric acid, dropwise adding the system into an excessive organic solvent, filtering and collecting precipitates, and drying to obtain CD-COOH;

s2, synthesis of CS-g-CD: weighing CD-COOH and NHS, dissolving with water for injection, replacing air, carrying out water bath at 4 ℃ for 2h, adding EDC, and carrying out water bath at 4 ℃ for reaction for 2 h; n is a radical of₂Adding chitosan in the atmosphere, and reacting for 24 hours at room temperature; centrifuging, filtering, dialyzing, and lyophilizing to obtain CS-g-CD;

s3 Synthesis of CS-g- (CD + HCA): weighing CS-g-CD and NHS, dissolving with water for injection, replacing air, carrying out water bath at 4 ℃ for 2h, adding EDC, and carrying out water bath at 4 ℃ for reaction for 2 h; n is a radical of₂Adding HCA under the atmosphere, and reacting for 24h at room temperature; centrifuging, filtering, dialyzing, and lyophilizing to obtain CS-g- (CD + HCA).

The invention has the beneficial effects that: the invention obviously reduces the high drug concentration of the clinical common eye drops and greatly reduces the side effect caused by the drug; the eye drops of the invention prolong the detention time of the carrier and the drug on the ocular surface, thereby shortening the administration frequency and the administration times of patients; the antibacterial result shows that: the eye drops have excellent antibacterial effect in vitro and show excellent curative effect of clinical first-line medication (voriconazole) in vivo.

Drawings

FIG. 1: of CS, CS-g-CD and CS-g- (CD + HCA)¹H NMR spectrum.

FIG. 2: ex vivo corneal permeability experiments.

FIG. 3: results of pharmacokinetic experiments in cornea (a) and aqueous humor (b).

FIG. 4: results of in vitro antifungal experiments.

FIG. 5: results of in vivo antifungal experiments.

Detailed Description

In the following description of specific embodiments, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein and is therefore not limited to the specific embodiments disclosed below.

EXAMPLE 1 Synthesis of a Polymer graft copolymer

A high molecular graft copolymer having the formula: CS-g- (CD + HCA).

The synthetic route is as follows:

the synthesis steps are as follows:

s1, synthesizing CD-COOH: firstly, 9.73 g of alpha-CD and 7.2 g of NaOH are put into a flask and dissolved by 30 mL of injection water; 1.165 g of ClCH was added₂COONa, and carrying out oil bath reaction for 5 hours at 50 ℃; adjusting pH of the solution to 6-7 with hydrochloric acid, adding the product dropwise into excessive acetone for precipitation, freezing at 4 deg.C, vacuum filtering, collecting precipitate, and vacuum drying for 24 hr to obtain white powder: monocarboxylated cyclodextrins (abbreviated: CD-COOH);

s2, synthesis of CS-g-CD: placing CD-COOH (3.093 g, 3 mmol) and NHS (0.379 g, 3.3 mmol) in a flask equipped with a magnetic stirring bar, dissolving with 180 mL of water for injection, displacing 3 times the air, placing the flask in a water bath at 4 ℃ for reaction for 2h, adding EDC (0.63 g, 3.3 mmol), reacting at 4 ℃ for 2 h; under magnetic stirring, N₂Adding 0.587 g of Chitosan (CS) under protection, and reacting at room temperature for 24 h; dialyzing with water for injection for three days, and freezing to obtain CS-g-CD (abbreviated as CC);

s3 Synthesis of CS-g- (CD + HCA): CS-g-CD (4.59 g) and NHS (133.8 mg, 1.162 mmol) were placed in a flask equipped with a magnetic stir bar, dissolved with 300 mL of water for injection, 3 times air was replaced, the flask was placed in a water bath at 4 ℃ for reaction for 2h, EDC (171.4 mg, 0.894 mmol) was added, and reaction was carried out at 4 ℃ for 2 h; under magnetic stirring, N₂HCA (147.9 mg, 0) was added with protection.813 mmol), reacting at room temperature for 24 h; dialyzed against water for injection for three days, and frozen to obtain CS-g- (CD + HCA) (abbreviated as CCH).

Of CS, CC and CCH in S1-S3¹The H NMR spectrum is shown in FIG. 1. In the spectrum of CC, the peak of 3.3-4.0 ppm is the sum of the characteristic peak of chitosan repeating unit (3.6-4.0 ppm) and the characteristic peak of cyclodextrin (3.3-3.8 ppm); 4.9-5.0 ppm of peaks ascribed to methylene hydrogens (-OCH) on carboxylated CDs₂-) according to the formula (I); peaks at 1.8-2.0 ppm attributable to the methyl group (-CH) of chitosan₃) (ii) a Peaks at 2.7-2.9 ppm were assigned to the characteristic peaks (-CH) of the residues on the chitosan backbone₂OH). In the spectrum of CCH, peaks of 6.5-6.8 ppm are assigned to characteristic peaks on the benzene ring. These results are consistent with literature reports, demonstrating the successful synthesis of the target polymer; calculating the substitution degree of the CD to be 50 percent of the chitosan main chain according to the area integral ratio of the peak (CD) at the position of 4.9-5.0 ppm and the peak (CS) at the position of 2.7-2.9 ppm; the degree of substitution of HCA was calculated to be 14% of the chitosan main chain from the area integral ratio of the peak (benzene ring) at the 6.5-6.8 ppm position to the peak (CS) at the 2.7-2.9 ppm position.

EXAMPLE 2 preparation of 0.1wt% CCH/ECZ eye drops

Firstly, weighing 15 g of CCH copolymer in example 1, adding 600 mL of water for injection, and stirring until the CCH copolymer is completely dissolved; then, 1 g of Econazole (ECZ) is added into the solution and stirred until the Econazole (ECZ) is completely dissolved to obtain a transparent solution; then, adding water for injection to a constant volume of 1L, adjusting pH to 4.5 with NaOH solution and osmotic pressure to 330 mOsm/kg with sodium chloride solution, and sterilizing at high temperature and high pressure (121 ℃, 0.1 MPa) to obtain 0.1wt% CCH/ECZ eye drops. The whole operation process is carried out at room temperature.

COMPARATIVE EXAMPLE 1- -0.1wt% CC/ECZ eye drops preparation

Firstly, weighing 15 g of CC copolymer of example 1, adding 600 mL of water for injection, and stirring until the CC copolymer is completely dissolved; then, 1 g of Econazole (ECZ) is added into the solution and stirred until the Econazole (ECZ) is completely dissolved to obtain a transparent solution; then, adding water for injection to a constant volume of 1L, adjusting pH to 4.5 with NaOH solution and osmotic pressure to 320 mOsm/kg with sodium chloride solution, and sterilizing at high temperature and high pressure (121 deg.C, 0.1 MPa) to obtain 0.1wt% CC/ECZ eye drop. The whole operation process is carried out at room temperature.

COMPARATIVE EXAMPLE 2 preparation of 0.1wt% ECZ suspension

Weighing 20 mg of Econazole (ECZ) drug substance to dissolve in 2 mL of a mixed solvent of methanol and acetonitrile (1: 1, v/v); the mixture was added dropwise to 20 mL of PBS (pH 7.4) with ultrasonic stirring for 30 min, the organic solvent was removed by vacuum distillation through a rotary evaporator, and then the mixture was washed and concentrated 3 times repeatedly by an ultrafiltration centrifugal filtration apparatus (molecular weight cut-off: 100,000 Da) to remove free econazole, and further filtered to remove large econazole aggregates, to obtain a 0.1wt% ECZ suspension.

And (3) performance testing:

1. experiment on permeability of cornea in vitro

Four normal cornea disease-free rabbits were randomly divided into two groups: group A (0.1 wt% CCH/ECZ eye drops prepared in example 2) and group B (0.1 wt% ECZ suspension prepared in control example 2). Animals were sacrificed by anesthesia with 10% (w/v, g/mL) chloral hydrate and the cornea gently sheared and immersed in physiological saline to ensure that the area of the sheared cornea was greater than 0.785 cm². The iris is peeled off by a pair of tweezers in normal saline, then the iris is transferred to sterile normal saline for cleaning (the whole process is injected: the operation in the normal saline is ensured as much as possible, and the dehydration of the cornea is prevented), and the iris-Bi-Side diffusion pool is further arranged on a Side-Bi-Side diffusion pool which is stable in advance in water bath at 37 ℃.5 mL of artificial tears are added into the donor pool and the receptor pool respectively immediately, the rotating speed of the magnetic stirrer is adjusted to 700 rpm, and the balance is stabilized for 30 min. 1 mL of the drug was added to the donor cell, 200. mu.L was taken from the receptor cell at different time intervals, and 200. mu.L of artificial tear was added. And adding 200 mu L of methanol into the taken sample for dilution, centrifuging, taking supernatant, drying, concentrating by 2 times, and analyzing the content of the econazole by using a high performance liquid testing methodology. Calculating to obtain the drug permeability, wherein different time intervals are abscissa, plotting, and calculating the apparent permeability coefficient of the drug:

Papp = (dQ/dt)/AC₀

dQ/dt: a relatively stable slope in the curve during drug permeation; a: exposed area of cornea (0.785 cm)²）； C₀: for supplying toInitial drug concentration in the body pool.

The results of the in vitro permeability experiments are shown in FIG. 2, and the cumulative permeation amount and the apparent permeability coefficient of the drug are shown in Table 1. The ECZ suspension group had little penetration of econazole within 4 h; the CCH/ECZ eye drops group has higher econazole permeation amount, and the drug permeation amount is increased sequentially along with the time. At 4h, the apparent permeability coefficient of the CCH/ECZ group drug is 14 times that of the suspension group. The reasons for this may be: the cation-rich CCH opens up the tight junctions of the corneal epithelium, which in turn increases the drug permeability of econazole.

2. Pharmacokinetic Studies

Normal japanese big ear white rabbits (2-3 kg, cornea disease-free) were used for ocular pharmacokinetic experiments, randomly divided into 3 groups: experimental group (0.1 wt% CCH/ECZ eye drops prepared in example 2), control group 1 (0.1 wt% CC/ECZ eye drops prepared in control example 1) and control group 2 (0.1 wt% ECZ suspension prepared in control example 2). After a single 50 μ L dose, tears were collected with 6 mm filter paper sheets at various time points, animals were anesthetized and sacrificed, and aqueous humor and cornea were collected. The content of the econazole in different parts is analyzed by using a high performance liquid testing methodology, and the data is processed by using software DAS2.0 to obtain pharmacokinetic parameters Tmax, Cmax, AUC and the like. The results are shown in FIG. 3 and the details are shown in Table 2.

As can be seen from Table 2: in cornea, the CCH/ECZ eye drops improve the drug concentration by 1.22 times (vs CC/ECZ) and 73.9 times (vs ECZ Susp), AUC_0→4hIncreases by 1.96 (vs CC/ECZ) and 48.9 times (v)sECZ Susp); in aqueous humor, the medicine concentration of CCH/ECZ eye drops is improved by 1.6 times and AUC (oral administration time) compared with CC/ECZ eye drops_0→4hThe improvement is 9.0 times, and no econazole drug is detected in the ECZ Susp group.

In addition, as can be seen from fig. 3: at the same time point, after the eye drops are administrated, the concentration of the medicine is obviously improved no matter in cornea or aqueous humor; area under the graph line (AUC)_0→4h) The content of the compound in the eye drops is higher than that in a control group, which shows that the bioavailability of the compound in the eye drops is greatly improved by a strategy of prolonging the retention time of the compound on the ocular surface.

3. In vitro antifungal experiments

In vitro antifungal experiments were performed to test the minimum bactericidal concentration using the most common fungal keratitis strains of Fusarium solani as the study subjects, 0.1wt% CCH/ECZ eye drops prepared in example 2 as the experimental group, and commercial antifungal eye drops voriconazole eye drops (0.1 wt%) and natamycin eye drops (0.1 wt%) as the positive control group. The specific process is as follows: isolating Fusarium solani from patients with fungal keratitis, incubating to RPMI-1640 agar culture dish, culturing at 28 deg.C in fungal incubator, subculturing for 7 d, picking out white colony, and diluting to 10%⁶CFU/mL fungus liquid for later use. Preparing 0.5, 1, 2 mug/mL liquid medicine (100 muL) by adopting a microdilution method, adding fungus liquid medicine (100 muL) to cultivate for 1 d, and diluting for a plurality of times (10 times)³-10⁵Double), 100. mu.L were plated on RPMI 1640 agarose plates and colonies were formed and recorded after 48 h incubation at 28 ℃.

The experimental results are shown in figure 4, Nat represents the natamycin eye drop positive Control group, Vor represents the voriconazole eye drop positive Control group, CCH/ECZ represents the experimental group, and Control represents that the addition amount of the untreated liquid medicine is 0. The results show that: compared with a control group, when the concentration of the eye drops is 1 mug/mL, no obvious colony grows; the positive control groups (voriconazole eye drops and natamycin eye drops) all had fungal colonies present. Compared with a positive control group, the eye drop of the invention shows excellent antifungal performance in vitro.

4. In vivo antifungal assay

A C57BL/6 mouse is taken as a research object, Fusarium solani is inoculated to the cornea of the mouse by a cross-hatch method, and a mouse model of fungal keratitis is established to research the pharmacodynamics of the eye drops. The 0.1wt% CCH/ECZ eye drops of the invention example 2 are used as an experimental group, the commercial antifungal eye drops voriconazole eye drops (0.1 wt%) are used as a positive control group, and the physiological saline is used as a blank control group. The specific process is as follows: c57 mice were anesthetized with 10% chloral hydrate (4 mL/kg). Under the operating microscope, the sterilized trephine was marked on the cornea of the right eye of the mouse, which was left untreated. Then, a cross-scratch was made in the marked circular area with a 45-degree knife, and then hyphae were introduced into the cross-scratch area using a sterilized bamboo stick. After 12 h, 5 μ L of the drug was instilled to the eyes of the mice, and the drug was administered once every 12 h for 7 d. The pathological changes of the mouse cornea were observed and recorded at 1, 3, 5 and 7 d, respectively, using a slit lamp.

The pathological changes of the cornea are shown in FIG. 5, wherein Control represents a blank Control group, Vor represents a positive Control group, and CCH/ECZ represents an experimental group. As can be seen from fig. 5: the blank group perforated at 3 d and progressively worsened severely; while the positive control and experimental groups had intact corneal epithelium. Compared with a positive control group, after the eye drops disclosed by the invention act, the cornea is transparent, and the clinical symptoms without edema are obviously relieved; at time 5 d, the treatment effect was significantly better than that of the positive control group. The results show that: the eye drops have excellent in-vivo antibacterial performance, shorten the course of disease, and can effectively reduce the administration frequency and the administration times (the existing clinical administration standard of voriconazole eye drops (0.1 wt%) is once for 2h and 8 times for one day, while the eye drops can be administered once for 12 h and 2 times for one day), thereby greatly increasing the compliance of patients.

Claims

1. An antifungal eye drop, which is characterized in that: the eye drops are prepared from the following raw materials in percentage by g/mL: 0.1-1% of eye drop medicine, 0.5-10% of high-molecular graft copolymer, a pH regulator, an osmotic pressure regulator and the balance of water for injection; wherein the macromolecular graft polymer is chitosan-g- (cyclodextrin +3, 4-dihydroxyphenyl propionic acid), the pH regulator regulates the pH of the eye drops to 4.3-6.5, and the osmotic pressure regulator regulates the osmotic pressure of the eye drops to 330 +/-3.2 mOsm/kg.

2. The antifungal eye drop of claim 1 wherein: the eye drop medicine is antifungal medicine or immunosuppressant.

3. An antifungal eye drop as claimed in claim 2, wherein: the antifungal drug is econazole.

4. The antifungal eye drop of claim 1 wherein: in the high-molecular graft copolymer, the grafting rate of cyclodextrin is 20-80%, and the grafting rate of 3, 4-dihydroxyphenyl propionic acid is 1-15%.

5. The antifungal eye drop of claim 1 wherein: the cyclodextrin is alpha-cyclodextrin, beta-cyclodextrin or gamma-cyclodextrin.

6. A process for the preparation of antifungal eye drops as claimed in any of claims 1 to 5, characterized in that: firstly, weighing a high-molecular graft polymer, adding injection water with dissolving equivalent, and stirring until the high-molecular graft polymer is completely dissolved; then, weighing the eye drop medicine, adding the eye drop medicine into the solution, and stirring until the eye drop medicine is completely dissolved; and finally, adding water for injection to a constant volume, adding a pH regulator and an osmotic pressure regulator to regulate the pH and the osmotic pressure, and sterilizing to obtain the required eye drops.