CN113975373A

CN113975373A - Ophthalmic preparation

Info

Publication number: CN113975373A
Application number: CN202111370998.0A
Authority: CN
Inventors: 龚雁; 王正才; 包永波
Original assignee: Ningbo Eye Hospital
Current assignee: Ningbo Eye Hospital
Priority date: 2021-11-18
Filing date: 2021-11-18
Publication date: 2022-01-28

Abstract

The invention discloses an ophthalmic preparation, which comprises an antimicrobial peptide, wherein the antimicrobial peptide is akkerosine, and the akkerosine is used for inhibiting the growth of gram-negative bacteria, gram-positive bacteria and fungi. The ophthalmic preparation can be used as a medicament for treating bacterial infection and can also be used as a medicament for treating fungal infection.

Description

Ophthalmic preparation

Technical Field

The present invention relates to ophthalmic formulations, and more particularly to an ophthalmic formulation that inhibits the growth of bacteria and fungi.

Background

The eye preparation is a sterile preparation directly used for eyes to play a therapeutic role, the eye preparation containing substances such as beta-lactam or aminoglycoside can be used for inhibiting the growth of bacterial microorganisms, and the eye preparation containing substances such as triazole and polyene can be used for inhibiting the growth of fungi.

The antibacterial peptide is a polypeptide with broad-spectrum antibacterial property, and at present, the antibacterial peptide aciclovir peptide can be applied to cosmetics, for example, the invention patent with the patent authorization publication number of 'CN 110868886A', namely 'a biocompatible composition for rapidly synthesizing antibacterial cosmetic puff and a preparation method thereof' is that the aciclovir peptide is applied to the puff as an antibacterial agent. The Acksol peptide is a broad-spectrum antibacterial peptide and can be applied to other fields, in particular to medicines.

Disclosure of Invention

The present invention provides an antimicrobial ophthalmic preparation which is not capable of inhibiting both the growth of fungi and the growth of bacteria in the same preparation, and an object of the present invention is to provide an ophthalmic preparation which is capable of inhibiting both the growth of bacteria and fungi.

In order to achieve the above object, the present invention adopts the following technical solutions.

An ophthalmic formulation comprising an antimicrobial peptide which is an akkerosine peptide for inhibiting the growth of gram negative bacteria, gram positive bacteria and fungi.

Preferably, the aksol peptide comprises at least one of Is and Os.

Preferably, the Is amino acid sequence Is ILRWPPWWPWRRK, and the Os amino acid sequence Is RGGLCYCRGRFCVCVGR.

Preferably, the akkerosine is used for preventing bacteria in eyeballs from entering interstitial membranes after bacterial infection, reducing cell death, inhibiting vascular proliferation, inhibiting thickening of surface membrane structures of eyeballs and inhibiting interstitial proliferation of membranes.

Preferably, the akkerosine peptide is used to reduce the expression level of VEGF in the cornea and retina of the eye.

Preferably, the concentration of the akkerosine is between 100 μ M and 420 μ M.

Preferably, the gram-negative bacteria are escherichia coli and vibrio parahaemolyticus, the gram-positive bacteria are escherichia coli, and the fungus is candida albicans.

Preferably, the ophthalmic formulation is a liquid formulation or a semi-solid formulation.

Preferably, the liquid preparation is eye drops or eye lotion, and the semi-solid preparation is ophthalmic gel or ophthalmic cream.

Preferably, the eye drops comprise physiological saline for dissolving the akkerosine and pharmaceutically acceptable excipients.

Compared with the prior art, the ophthalmic preparation provided by the invention can be used for inhibiting the growth of gram-negative bacteria, gram-positive bacteria and fungi in ocular infection, is a broad-spectrum antibacterial preparation, and can inhibit the growth of bacteria and fungi in ocular infection.

Drawings

FIG. 1 is a schematic diagram of a culture medium of Staphylococcus aureus for the Acridtide antibacterial experiment in the ophthalmic preparation provided by the present invention;

FIG. 2 is a schematic diagram of an Escherichia coli culture medium for an experiment of antimicrobial Ackerosine in the ophthalmic preparation provided by the present invention;

FIG. 3 is a schematic diagram of the culture medium of Acridtide for antibacterial experiments and Parahemolytic vibrio in the ophthalmic preparation provided by the present invention;

FIG. 4 is a schematic diagram of the culture medium of Candida albicans for the Ackerosine antibacterial experiment in the ophthalmic preparation provided by the present invention;

FIG. 5 Is a schematic representation of colony formation of Ackerosine group IS preservative challenge for 28 days in ophthalmic formulations provided by the present invention;

figure 6 is a schematic representation of osgroup colony formation challenging for 28-day preservation of akkerosine in an ophthalmic formulation provided by the present invention;

FIG. 7 is a statistical representation of the colony counts of Staphylococcus aureus-infected PBS and ocular secretion cultures;

FIG. 8 is a statistical representation of the colony counts of E.coli infected PBS and ocular secretion cultures;

FIG. 9 is a micrograph of tear Giemsa staining results;

FIG. 10 is a graphical representation of slit lamp shots in a rat staphylococcus aureus infection;

FIG. 11 is a graph showing slit lamp imaging results in E.coli infection in rats;

FIG. 12 is a micrograph of the HE staining of the cornea of the eye;

FIG. 13 is a graph showing immunohistochemical results on the surface and bottom of the eyeball;

FIG. 14 is a graph showing the relative expression levels of corneal VEGF;

FIG. 15 is a graph showing the relative expression levels of retinal VEGF.

Detailed Description

In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

The invention provides an ophthalmic preparation, which comprises akkersol peptide, wherein the akkersol peptide Is antibacterial peptide and comprises at least one of Is and Os. Wherein the amino acid sequence of the Is ILRWPPWWPWRRK, the molecular weight Is 1.78kDa, and the molar mass Is 1779.1 g/mol; the Os amino acid sequence is RGGLCYCRGRFCVCVGR, the molecular weight is 1.90kDa, and the molar mass is 1900.3 g/mol.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

According to the American Cosmetic Association (CTFA) standards, initial mold and bacteria inoculations were controlled at 10000CFU/mL and 1000000CFU/mL, respectively, requiring a 90% reduction in mold, a 99.9% reduction in bacteria at day 7, and a continuing reduction in bacteria count over 28 days.

All experimental data in the examples were obtained after three measurements, and the correlation analysis and statistics were performed by Graphpad prism 8.0. Comparisons between groups were analyzed using the t-test. Represents P <0.05, # represents P <0.05, and the differences are statistically significant.

First embodiment

Antimicrobial experiments of akkerosine in ophthalmic formulations.

The experimental steps are as follows: the experiment was performed in a Phosphate Buffer Saline (PBS) liquid medium system. PBS (phosphate buffer solution)The process is as follows: weighing 8g NaCl, 0.2g KCl and 1.42g Na₂HPO₄，0.27g KH₂PO₄Placing the mixture into a 1L beaker, adding about 800mL of deionized water into the beaker, fully stirring and dissolving, dropwise adding concentrated hydrochloric acid to adjust the pH value to 7.4, and then adding deionized water to a constant volume of 1L. Sterilizing at high temperature and high pressure, and storing at room temperature.

The inoculation amount of staphylococcus aureus, escherichia coli and vibrio parahaemolyticus is 1000000 CFU/mL; the inoculation amount of Candida albicans was 10000 CFU/mL.

Preparing the following mother liquor: 2000000CFU/mL Staphylococcus aureus, 2000000CFU/mL Escherichia coli, 3000000CFU/mL Vibrio parahaemolyticus, and 50000CFU/mL Candida albicans. Is and Os were solubilized in sterile PBS to final concentrations of 260. mu.M and 420. mu.M, respectively.

The mother liquor, PBS buffer, Is dissolved in PBS and Os dissolved in PBS were added to 10mL centrifuge tubes in the amounts shown in the following table, and the group to which neither Is nor Os was added, i.e., PBS group was used as the control group. Mixing, storing in 37 deg.C incubator, taking 100 μ L each on days 2, 4, and 7, spreading on LB solid culture medium, and counting colony number after culturing.

The results of the experiments with reference to fig. 1, 2, 3 and 4 show that: according to the antibacterial experiment of the aksotide, the control groups of staphylococcus aureus, escherichia coli, vibrio parahaemolyticus and candida albicans grow full of colonies, the experiment group added with the antibacterial peptide does not grow obvious colonies of staphylococcus aureus, vibrio parahaemolyticus and candida albicans, the group Is does not form an escherichia coli colony on the seventh day, and the group Os forms a plurality of obvious escherichia coli colonies on the seventh day.

Second embodiment

Challenge experiment for 28 days of preservation.

Firstly, mother liquor of 2000000CFU/mL of staphylococcus aureus, escherichia coli, vibrio parahaemolyticus and candida albicans Is prepared, Is and Os are dissolved by sterile PBS, and the final concentrations are 260 mu M and 420 mu M respectively. The stock solution was added to a 10mL centrifuge tube according to the following table, and the group to which neither Is nor Os was added, i.e., the PBS group was used as a control group. After mixing, the mixture was stored in a 37 ℃ incubator, 25. mu.L of each LB solid medium was applied to 12-well plates on

days

7, 14, 21, and 28, and the number of colonies was counted after the culture.

Referring to fig. 5 and 6, the experimental results show that: in the 28-day preservative challenge, through a coating experiment, staphylococcus aureus, escherichia coli, vibrio parahaemolyticus and candida albicans all form obvious colonies in a culture dish within 28 days in a control group; in the group Is, within 28 days, staphylococcus aureus, escherichia coli, vibrio parahaemolyticus and candida albicans do not form obvious colonies in a culture dish; in the Os group, no obvious colonies were formed in the culture dish by Staphylococcus aureus, Vibrio parahaemolyticus and Candida albicans within 28 days.

In 28-day experiments, the sterilization rate of Is on staphylococcus aureus, escherichia coli, vibrio parahaemolyticus and candida albicans reaches 100%. The sterilization rate of Os on staphylococcus aureus, vibrio parahaemolyticus and candida albicans reaches 100%.

Third embodiment

60 clean-grade rats were taken and all rats were subjected to slit lamp microscopy on eyes to ensure that the eyes of the rats were free of defects and were randomly divided into six groups, namely a1, a2, B1, B2, C1 and C2, and 10 rats were used in each group. The A1 and A2 groups are control groups, the B1 and B2 groups are antibiotic treatment groups, and the C1 and C2 groups are Acxol peptide treatment groups.

All the rats in 6 groups were anesthetized by intraperitoneal injection of 2% phenobarbital sodium (the dose of the phenobarbital sodium is 35mg/kg), the corneal epithelium of the left eye of the rat was scraped by a 26-gauge needle to form a superficial wound without damaging the stroma layer, and the right eye of the rat was used as a control eye without molding treatment. The groups A1, B1 and C1 were infected with Staphylococcus aureus suspension drops at equal concentrations on the corneal surface, and the groups A2, B2 and C2 were infected with Escherichia coli suspension drops at equal concentrations on the corneal surface. The groups a1 and a2 were used as model control groups, and saline was applied as eye drops. Group B1: all rats were given equal volume of cefazolin eye drops for treatment 6h after infection, 1 dose every 2h for 3 consecutive days. Group B2: all rats were given an equal volume of tobramycin eye drops for treatment 6h post-infection, 1 dose every 2h for 3 consecutive days. Group C1, C2: all rats were treated with 100 μ L of eyedrops of 100 μ M solution of akkerosine Is dissolved in physiological saline 6h after infection, 1 time every 2h for 3 consecutive days. At the end of the experiment on day 3, the rats were deeply anesthetized with 2% phenobarbital sodium and then sacrificed by cervical dislocation. Collecting blood through the main vein of abdominal cavity, centrifuging, collecting serum, and storing at-80 deg.C. And (3) taking the left eye on the experimental side and the right eye on the control side for tissue fixation, preparing paraffin sections, quickly freezing part of corneal tissues by liquid nitrogen, storing at-80 ℃, and detecting immunoblotting, qPCR and the like.

To evaluate the bacteriostatic effect of akkerosine in the rat eyeball, a viable count assay was performed. Groups of rats were anesthetized on day 3. After injecting 10. mu.L of sterile PBS onto the surface of the eye on the experimental side of the rat, the mixture of PBS and eye secretion (10. mu.L total) was aspirated out with a tip. 10 μ L of the mixture was dropped into LB solid medium for plating, incubated at 37 ℃ overnight, and photographed. The number of plate colony formations was counted by IPP 6.0.

Results of viable count assay referring to fig. 7 and 8, the results show that the number of colonies formed by the groups C1 and C2 of the aksol peptide treatment is minimal.

To evaluate the bacteriostatic effect of akkerosine in the rat eyeball, a Giemsa staining experiment of tears was also performed. The tears of the rats 3 days after infection were collected from the ocular surface, smears were made by dropping the tears onto a glass slide, and after naturally drying, 100. mu.L of Giemsa staining solution was dropped and stained for 30 seconds. 200 μ L of PBS was added dropwise to the surface of the Giemsa stain and rinsed with tap water after 5 minutes. The results were photographed by an optical microscope (DM500, Leica).

Results of tear staining in rats referring to fig. 9, the results showed that the number of normal eyeball epithelial cells was reduced in the a1 staphylococcus aureus model group, and the cell nuclei were lost and the cell morphology was condensed. B1 after the cefazolin eye drops are treated, the number of normal epithelial cells is increased, and a small amount of cells invade into the interior of the cells. After the C1 akkukotide group is treated, the number of normal epithelial cells of cells is increased, no obvious bacterial invasion phenomenon is found in the cells, but partial epithelial cells are heterotypic. A2 in Escherichia coli model group, epithelial cell shrinkage, and cell nucleus heterotypic. The number of epithelial cells in the B2 tobramycin-treated group was restored, but significant bacterial invasion was observed in the cells. The number of normal cells in group C2 was smaller than that in group B2, but the invasion of the cells into the remaining cells was not observed.

In order to evaluate the protective effect of the akkerosine in preventing and treating bacterial infection after eye injury, eyeball slit lamp examination, corneal sodium fluorescein staining and eyeball tissue HE staining experiments on rats are carried out.

Examining an eyeball slit lamp: the rat eyes were slit-lamp microscopically examined on days 0 and 3, respectively, using a slit-lamp.

Corneal fluorescein sodium staining: after 3 days of infection, the rats were anesthetized, the eyes of all the rats were stained with corneal fluorescein sodium, 5 μ L of 1% fluorescein sodium (solarbio) was dropped on the canthus portion of the eyes to stain for 3 minutes, and the eyeball of the rat was photographed by a cobalt blue lamp. The results were calculated by Image-Pro Plus 6.0(IPP 6.0).

The eyeball slit lamp examination and the identification standard of the corneal fluorescein sodium staining are as follows:

(1) conjunctival hyperemia: grade 0 is no obvious hyperemia; grade 1 is mild hyperemia of blood vessels, but less than 2 quadrants, without bulbar conjunctival edema; grade 2 is marked by blood vessel filling and tortuosity which is more than 2 quadrants and concomitant bulbar conjunctival edema; grade 3 is the complete conjunctival vascular hyperemia tortuosity with bulbar conjunctival edema and ciliary hyperemia.

(2) Area of corneal ulcer: after 2% corneal fluorescein sodium staining, a picture is collected by a slit lamp microscope under cobalt blue light with 16 times magnification, and the area of the ulcer area is measured by ImageJ software.

(3) Corneal neovascularization: grade 0, no new blood vessels; grade 1 neovascularization is less than 1 quadrant; grade 2 neovasculature occupies 1 or 2 quadrants; grade 3 neovasculature occupies 2 or 3 quadrants; grade 4 neovasculature occupies 3 or 4 quadrants and even covers the entire corneal surface.

HE staining: placing fresh eyeball tissue in 10% neutral formaldehyde solution, fixing for 24 hr, and dehydrating with alcohol. After paraffin embedding, 5 μm paraffin sections were prepared. The sections were paraffin-dissolved in an oven at 65 ℃ for 20 minutes. Soaking in xylene for 20 min, performing gradient alcohol rehydration, performing hematoxylin staining for 3 min, washing with tap water for 1 min, and differentiating with 1% alcohol hydrochloric acid for 3 s. 0.1% NaHCO₃After reverse blue, eosin stain for 5 seconds. Dehydrating with 95% alcohol, oven drying, and sealing with neutral gum after xylene is transparent. The results were photographed by an optical microscope.

Referring to fig. 10 and 11, the results of slit lamp microscopy and sodium corneal fluorescein staining experiments show that, 3 days after the rats in groups A1 and A2 are respectively infected with staphylococcus aureus and escherichia coli, the eyeball shows turbidity and the blood vessel grows. Under a cobalt blue lamp, a large area of bacterial invasion damage (green fluorescent part on the eyeball surface) can be seen. After the Acksol peptides of the C1 and C2 groups are treated, the staining area of fluorescein sodium is reduced, and the reduction effect of the staining area of fluorescein sodium in Escherichia coli infection of the Acksol peptides is stronger.

The results of HE staining pathological detection are shown in FIG. 12, and the results show that the membrane structure shown by rat eyeballs is obviously thickened after the staphylococcus aureus and escherichia coli are infected, especially the staphylococcus aureus model group. The A1 Staphylococcus aureus model group showed massive cell death and increased mesentery on the surface of the eyeball, consistent with the turbid eyeball characteristics under the slit lamp. The cornea structure of the eyes of group C1 was significantly thinner than that of group A1, with a small number of dead cells in the mesentery. The cell structure on the surface of the eyeball of the C2 group is relatively complete, and no obvious bacterial invasion is found in the mesentery.

In order to detect the expression level of Vascular Endothelial Growth Factor (VEGF) on the surface and bottom of the eyeball, immunohistochemistry is adopted to detect the VEGF in the eye.

Paraffin sections were removed and rehydrated, and then placed in 0.01M citric acid buffer (pH 6.0) for antigen retrieval in an autoclave at 121 ℃ for 20 minutes. And naturally cooling to room temperature after antigen retrieval. Dropwise adding 3% H₂O₂After 15 minutes of inactivation of endogenous peroxidase (3% hydrogen peroxide), antigen blocking was performed for 30 minutes using 1% Bovine Serum Albumin (BSA) dissolved in PBS. Rabbit anti-rat VEGF (diluted 1: 50, dissolved in 1% BSA) was added dropwise and incubated overnight at 4 ℃. The next day, after the primary antibody was decanted, the membrane was washed 3 times with membrane washing buffer (Tris Buffered Saline Tween, TBST) prepared as follows: putting 8.8g of NaCl and 20mL of 1mol/L Tris-HCl buffer solution into a 1L beaker, adding 800mL of deionized water into the 1L beaker, stirring to dissolve, adding 0.5mL of Tween 20(Tween 20), fully mixing, adding deionized water to a constant volume of 1L, and storing at 4 ℃. The goat anti-rabbit-IgG secondary antibody conjugated with horseradish peroxidase (diluted 1: 100) is added dropwise, incubated for 60 minutes at room temperature, and washed 3 times by washing TBST. And (5) developing with the DAB developing solution. After hematoxylin counterstaining, alcohol is dehydrated in a gradient way. And (5) sealing the neutral gum after the xylene is transparent. The results were observed by light microscopy and analyzed using IPP 6.0.

Immunohistochemical results referring to fig. 13, 14 and 15 in combination, the results suggested that treatment with akkerosine significantly reduced the expression levels of surface and basal VEGF in the eye, with the differences being statistically significant (P <0.05) in both the akkerosine treated group compared to the control group.

The acremotide contained in the ophthalmic preparation provided by the invention is a broad-spectrum antibacterial substance and can inhibit the growth of gram-negative bacteria, gram-positive bacteria and fungi, so that the ophthalmic preparation provided by the invention can be used for inhibiting the growth of the gram-negative bacteria, the gram-positive bacteria and the fungi in ocular infection and protecting eyeballs.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent flow transformations made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. An ophthalmic preparation comprising an antimicrobial peptide which is an akkerosine peptide for inhibiting the growth of gram-negative bacteria, gram-positive bacteria and fungi.

2. The ophthalmic formulation of claim 1, wherein the akkerosine peptide comprises at least one of Is and Os.

3. The ophthalmic formulation of claim 2, wherein the Is amino acid sequence Is ILRWPWWPWRRK and the Os amino acid sequence Is RGGLCYCRGRFCVCVGR.

4. The ophthalmic formulation according to any of the claims 3, wherein the Ackerosine is used for preventing bacteria in the eyeball from entering the mesenchyme after bacterial infection, reducing cell death, inhibiting vascular proliferation, inhibiting thickening of the surface membrane structure of the eyeball, inhibiting the mesenchyme increase.

5. The ophthalmic formulation according to claim 3, characterized in that the Ackerosine is used to reduce the VEGF expression level in the cornea and retina of the eye.

6. The ophthalmic formulation according to claim 3, characterized in that the concentration of said Acxotide is between 100 and 420 μ M.

7. The ophthalmic formulation according to any of the claims 3, characterized in that the gram-negative bacteria are Escherichia coli and Vibrio parahaemolyticus, the gram-positive bacteria are Escherichia coli, and the fungus is Candida albicans.

8. The ophthalmic formulation according to claim 3, characterized in that it is a liquid formulation or a semisolid formulation.

9. The ophthalmic formulation according to claim 8, wherein the liquid formulation is an eye drop or an eye lotion and the semi-solid formulation is an ophthalmic gel or an ophthalmic cream.

10. The ophthalmic formulation according to any of claims 1 to 9, characterized in that the eye drops comprise physiological saline dissolving the akkerosine and a pharmaceutical excipient.