CN116509796A - Pharmaceutical composition containing aromatic ketone and preparation method and application thereof - Google Patents

Abstract

The invention discloses a pharmaceutical composition containing aromatic ketone, a preparation method and application thereof. The pharmaceutical composition comprises: aromatic ketones and pharmaceutically acceptable polymers; wherein the aromatic ketone is vanillyl ethanone or pharmaceutically acceptable salt thereof, and the polymer is povidone and/or copovidone. The pharmaceutical composition is safe and effective, has no stimulation, has good curative effect on treating and/or preventing xerophthalmia and xerophthalmia, remarkably improves tear secretion and cornea sensitivity, and effectively inhibits the generation of cornea neovascularization; in addition, the expression of HMGB1, RAGE, TLR2, TLR4 and cytokines can be inhibited, the corneal inflammation is reduced or eliminated, and a new solution is provided for the treatment and/or prevention of xerophthalmia, keratitis and other Guan Yanbu diseases.

Description

Pharmaceutical composition containing aromatic ketone and preparation method and application thereof

Technical Field

The invention belongs to the field of ophthalmic medicines, and in particular relates to a pharmaceutical composition containing aromatic ketone, a preparation method and application thereof.

Background

Dry eye is a tear dyscrasia eye disease which is caused by various factors and mainly causes dry eyes, and is often accompanied by the symptoms of reduced corneal sensitivity, photophobia, blurred vision and the like, and brings great inconvenience to the life and work of patients. Moreover, with the development of society and the advancement of technology, dry eye patients are increasing; epidemiological investigation shows that the prevalence of dry eye is about 7.8% -33.78%, and the prevention and treatment work is becoming more and more important.

The cornea is an important barrier of the eye against outside microbial bacteria. Because the cornea is directly exposed to the outside, the cornea is easily damaged by a plurality of factors such as machinery, infection, chemicals, burns and the like. The health of cornea affects the health state of eyeball and can repair damaged cornea effectively to prevent the disease from going deep.

Vanillyl ethanone (Apocynin, apoo for short), chemical name: the 4 '-hydroxy-3' -methoxyacetophenone has weak vanilla taste, can be used in the perfume industry, is also an important organic synthesis raw material, can be used for synthesizing the mental medicines iloperidone, novel antimalarials and the like, and is commonly used as a cardiotonic, diuretic and the like in medicine, see CN 110903178A.

Povidone, a polymer consisting essentially of 1-vinyl-2-pyrrolidone groups, has different degrees of polymerization resulting in different molecular weights, which can be characterized by the viscosity of an aqueous povidone solution relative to water, and is expressed by a K value, which is generally between 10 and 120, which is widely used in pharmaceutical formulations, mainly in solid formulations, and also as an adhesive, suspending agent, coating material, etc. (see handbook of pharmaceutical excipients (original fourth edition), [ english ] r.c. ro, [ american ] p.j. Scherns, [ english ] p.j. Weller code Zheng Jun democratic, chemical industry publication 2005).

The copovidone is a copolymer of vinyl pyrrolidone and vinyl acetate, has white to milky powder appearance, keeps good water solubility and cohesiveness of the povidone, has wider dissolution performance and stronger surface activity, and is carried in United states pharmacopoeia, european pharmacopoeia, japanese pharmaceutical excipients handbook and the like.

Until now, there have been no reports of a related literature on the combination of vanillyl ethanone or a salt thereof with povidone, or the combination of vanillyl ethanone or a salt thereof with povidone for the prevention and/or treatment of dry eye, keratitis (or for the reduction of the expression level of HMGB1, RAGE, IL-6, NF- κ B, TNF- α, etc.).

Disclosure of Invention

In view of the problems and/or disadvantages of the prior art, it is an object of the present invention to provide a pharmaceutical composition comprising an aromatic ketone (vanillyl ethanone or a salt thereof), and a method for preparing the same and use thereof. The pharmaceutical composition containing aromatic ketone (vanilloid or vanilloid salt) is safe, effective, non-irritating and good in stability, and provides a new potential choice for treating and/or preventing eye diseases such as xerophthalmia and keratitis.

The present invention provides a pharmaceutical composition comprising: aromatic ketones and pharmaceutically acceptable polymers; wherein the aromatic ketone is vanillyl ethanone or pharmaceutically acceptable salt thereof, and the polymer is povidone and/or copovidone.

In a preferred embodiment of the invention (pharmaceutical composition) the weight ratio of aromatic ketone to polymer is 1 (2-50), e.g. 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:16, 1:18, 1:20, 1:22, 1:25, 1:30, 1:35 or 1:45; preferably, the weight ratio of the aromatic ketone to the polymer is 1 (9-25); more preferably, the weight ratio of aromatic ketone to polymer is 1 (12-22), such as 1:15 or 1:18.

In a preferred embodiment of the invention (pharmaceutical composition) the encapsulation efficiency of the aromatic ketone is at least 80%, for example 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%; preferably, the encapsulation efficiency of the aromatic ketone is not less than 90% or not less than 95%.

In a preferred embodiment of the present invention (pharmaceutical composition), the pharmaceutical composition satisfies one or more of the following conditions i to iii:

i. the aromatic ketone is vanillyl ethanone;

ii. The polymer is copovidone;

iii, the K value of the polymer is 25-35; preferably, the K value of the polymer is 25.2-30.8;

preferably, the pharmaceutical composition satisfies the conditions i to iii simultaneously.

In a preferred embodiment of the invention (pharmaceutical composition), the pharmaceutical composition is made by a process comprising the steps of: dispersing or dissolving the aromatic ketone and the pharmaceutically acceptable polymer together in an organic solvent, uniformly mixing, and removing the organic solvent by rotary evaporation at 35-45 ℃ to obtain the aromatic ketone;

preferably, the organic solvent is an alcohol solvent or a halogenated hydrocarbon solvent, and/or the dosage of the organic solvent corresponding to each milligram of the aromatic ketone is 0.5-20 mL;

more preferably, the organic solvent is methanol or ethanol, and/or the amount of the organic solvent corresponding to each milligram of the aromatic ketone is 0.5-5 mL.

In a preferred embodiment of the present invention (pharmaceutical composition), the pharmaceutical composition is a solid or liquid formulation; and/or, in the pharmaceutical composition, the aromatic ketone is in a therapeutically effective amount.

According to common knowledge in the art or actual needs, the pharmaceutical composition of the invention can also comprise pharmaceutically acceptable auxiliary materials; the pharmaceutically acceptable excipients may be conventional excipients for pharmaceutical formulations in the art (e.g., eye drops), or may be conventional choices.

In a preferred embodiment of the present invention (pharmaceutical composition), the pharmaceutical composition is a liquid formulation, the solvent of which is selected from pharmaceutically acceptable water, PBS buffer or sodium carboxymethyl cellulose aqueous solution;

preferably, when the content of aromatic ketone in the liquid preparation is 5mg/mL, the liquid preparation satisfies one or more of the following conditions (1) to (3):

(1) the average micelle diameter of the liquid preparation is 1 to 80nm, for example 5nm, 8nm, 10nm, 15nm, 20nm, 25nm, 30nm, 40nm, 50nm, 60nm or 70nm; preferably 10 to 20nm;

(2) the polydispersity of the liquid preparation is less than or equal to 0.5; preferably 0.4 or less, for example 0.1 to 0.4;

(3) the Zeta potential of the liquid preparation is-20 to 0mV, for example-18 mV, -15mV, -12mV, -10mV, -8mV, -5mV or-2 mV; preferably-15 to-5 mV;

more preferably, the liquid formulation satisfies the conditions (1) to (3) at the same time when the aromatic ketone content in the liquid formulation is 5 mg/mL.

In a preferred embodiment of the invention (pharmaceutical composition), the pharmaceutical composition is an ophthalmic formulation;

preferably, the ophthalmic formulation satisfies at least one of the following uses a-e:

a. the ophthalmic preparation is used for treating and/or preventing xerophthalmia;

b. the ophthalmic preparation is used for inhibiting cornea neovascularization;

c. the ophthalmic preparation is used for inhibiting the expression of HMGB 1;

d. the ophthalmic formulation is an ophthalmic formulation for inhibiting expression of RAGE, TLR2 or TLR 4;

e. the ophthalmic preparation is an ophthalmic preparation for inhibiting the expression of cytokines IL-6, NF-kappa B, TNF-alpha or VEGF.

The invention also provides a preparation method of the medicinal composition, which comprises the following steps: dispersing or dissolving aromatic ketone and pharmaceutically acceptable polymer in organic solvent, mixing, and rotary evaporating at 35-45 deg.c to eliminate organic solvent to obtain solid product; and then optionally comprises: dissolving or dispersing the obtained solid product in the solvent of the liquid preparation, filtering and sterilizing to obtain the liquid preparation.

Preferably, the organic solvent is an alcohol solvent or a halogenated hydrocarbon solvent, and/or the dosage of the organic solvent corresponding to each milligram of aromatic ketone is 0.5-20 mL.

More preferably, the organic solvent is methanol or ethanol, and/or the dosage of the organic solvent corresponding to each milligram of aromatic ketone is 0.5-5 mL.

The invention also provides application of the medicinal composition in preparing an ophthalmic preparation.

Preferably, the ophthalmic formulation meets at least one of the above uses a-e.

The invention has the positive progress effects that:

the test result shows that the pharmaceutical composition containing aromatic ketone (vanilloid or the salt thereof) is safe, effective and non-irritating, has good curative effect on treating and/or preventing xerophthalmia, remarkably improves tear secretion and cornea sensitivity, and effectively inhibits the generation of cornea neovascularization; in addition, the expression of HMGB1, RAGE, TLR2, TLR4 and cytokines (IL-6, NF-kappa B, TNF-alpha, VEGF and the like) can be inhibited, the corneal inflammation is reduced or eliminated, and a new solution is provided for the treatment and/or prevention of symptoms Guan Yanbu such as xerophthalmia, keratitis and the like.

Drawings

FIG. 1 is an external view of APO-VA64 eye drops in example 1 of the present invention and a control thereof.

Fig. 2 shows the encapsulation efficiency of APO and VA64 in different mass ratios.

FIG. 3 shows the change in encapsulation efficiency of APO-VA64 eye drops stored for 12 weeks.

FIG. 4 shows the change in micelle size of APO-VA64 eye drops stored for 12 weeks.

FIG. 5 shows the change in polydispersity index (PDI) of APO-VA64 eye drops stored for 12 weeks.

FIG. 6 shows the change in Zeta potential of APO-VA64 eye drops stored for 12 weeks.

FIG. 7 shows antioxidant activity at various concentrations (ABTS method).

FIG. 8 shows antioxidant activity (ABTS method) at various times.

FIG. 9 shows antioxidant activity at various concentrations (FRAP method).

FIG. 10 shows antioxidant activity (FRAP) at various times.

FIG. 11 is an in vitro release profile of APO-VA64 eye drops.

FIG. 12 shows vascular congestion in the allantoic-trypan blue staining experiments of chick embryos from different dosing groups.

Fig. 13 shows trypan blue uptake for different dosing groups.

Fig. 14 is a graph showing the eye irritation observation of rabbits in different administration groups.

Fig. 15 is a diagram of histopathological observations of rabbit eyes from different dosing groups.

FIG. 16 is a graph of corneal fluorescence intensity contrast (flap-cut tiling) for APO-VA 64.

FIG. 17 is a graph (vertical cross section) showing comparison of fluorescence intensity of APO-VA64 cornea.

Fig. 18 shows corneal haze score results for different dosing groups.

Fig. 19 is a graph of sodium corneal fluorescein staining from a slit lamp observation of mice from different dosing groups.

FIG. 20 shows the rate of corneal epithelium loss for mice from different dosing groups based on sodium fluorescein staining statistics.

FIG. 21 is a graph showing red staining of the cornea of mice from different dosing groups observed with a slit lamp.

FIG. 22 shows the rate of corneal epithelium loss in mice from different dosing groups based on tiger red staining statistics.

Figure 23 is corneal sensitivity for different dosing groups.

Fig. 24 shows the amount of lacrimal secretion of the different administration groups (day 6).

Fig. 25 shows the amount of lacrimal secretion (day 7) in the different administration groups.

Fig. 26 shows the amount of lacrimal secretion of the different administration groups (day 9).

Fig. 27 shows the amount of lacrimal secretion of different administration groups (day 11).

Fig. 28 shows the amount of lacrimal secretion of different administration groups (day 13).

Fig. 29 is a slit lamp examination image of corneal neovascularization in different dosing groups.

Fig. 30 is a corneal neovascularization profile for various dosing groups.

Fig. 31 is a histopathological observation of the different administration groups.

Fig. 32 is an SEM image of the different dosing groups.

Fig. 33 shows HMGB1 expression levels in corneal tissue from different dosing groups.

FIG. 34 shows IL-6 expression levels in corneal tissue from different administration groups.

FIG. 35 shows NF- κB expression levels in corneal tissue of different dosing groups.

FIG. 36 shows TNF-. Alpha.expression levels in corneal tissue from different administration groups.

FIG. 37 shows the results of PCR analysis of HMGB1 in corneal tissue from different dosing groups.

FIG. 38 shows the results of PCR analysis of IL-6 in corneal tissue from different administration groups.

FIG. 39 shows the results of PCR analysis of NF- κB in corneal tissue of different dosing groups.

FIG. 40 shows the results of PCR analysis of RAGE in corneal tissue from different dosing groups.

FIG. 41 shows the results of PCR analysis of TLR2 in corneal tissue from different dosing groups.

FIG. 42 shows the results of PCR analysis of TLR4 in corneal tissue from different dosing groups.

FIG. 43 shows the results of PCR analysis of TNF-. Alpha.in corneal tissue from different administration groups.

FIG. 44 shows the results of PCR analysis of VEGF in corneal tissue from different dosing groups.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

In the present invention, the specific conditions are not noted, and the reagents or instruments used are conventional products commercially available, which are not noted to the manufacturer, or the conditions suggested by the manufacturer.

For example, the number of the cells to be processed,

vanillyl ethanone (chemical name: 4 '-hydroxy-3' -methoxyacetophenone, english name: apocynin, abbreviated APO): shanghai Aba Ding Shenghua technologies Co., ltd;

copovidone VA64 (linear copolymer of N-vinylpyrrolidone (NVP) and Vinyl Acetate (VA), abbreviated as VA 64): basf corporation (Shanghai);

PBS buffer (ph=7.2 to 7.4): qingdao cloud mountain biotechnology limited;

coumarin 6 (Cou-6): sigma-Aldrich company;

new Zealand white rabbits: qingdao Kangda biological Co., ltd (Qingdao China);

male BALB/c mice (8 weeks old): experimental animals of Jinan Pengyue were bred to the company Limited.

In the invention, the statistical comparison between two groups adopts t test, the statistical comparison between multiple groups adopts spss11.5 software (spinc, chicago) to conduct variance analysis, and P <0.05 indicates significance.

In connection with the definition of terms used in the present invention, unless otherwise indicated, the initial definition provided by the terms herein applies to the term throughout; to the extent that terms are not specifically defined herein, they should be given meanings that would be able to be given to those skilled in the art in light of the disclosure and/or the context.

Example 1 preparation of APO-VA64 nanomicelle eye drops

(1) 25mg of vanillyl ethanone and 450mg of copovidone VA64 (the mass ratio of vanillyl ethanone to copovidone VA64 is 1:18) are dissolved or dispersed in ethanol (for example, 20 mL) together, and are uniformly mixed, and then the ethanol is completely removed by rotary evaporation under the conditions of 40 ℃ and reduced pressure, so as to obtain a solid product;

(2) Dissolving the solid product obtained in the step (1) in 5mL PBS buffer solution (pH=7.2-7.4), and filtering and sterilizing with a 0.22 μm filter membrane to obtain APO-VA64 eye drops, wherein the appearance is shown in figure 1; APO-VA64 (APO content 5 mg/mL), APO (solvent PBS buffer, APO concentration 5 mg/mL), VA64 (solvent PBS buffer, VA64 concentration 90 mg/mL), water as control.

Examples 2 to 5 preparation of APO-VA64 nanomicelle eye drops

In examples 2 to 5, the amount of vanillyl ethanone (25 mg) was kept unchanged, and the amount of copovidone VA64 was adjusted so that the mass ratio of vanillyl ethanone to copovidone VA64 was 1:9, 1:12, 1:15 and 1:21, respectively, and the APO-VA64 eye drops were prepared under the same conditions as in example 1.

Effect example 1

1. Solubility of

The results of fig. 1 show that: APO cannot be completely dissolved in PBS buffer and precipitated at the bottom of the vial; and VA64 and APO-VA64 can be dissolved in PBS buffer solution and are colorless transparent liquid, and no difference is observed by naked eyes compared with water.

The solid products of APO, APO-VA64 were tested for their solubility in water, PBS and artificial tears (example 1: mass ratio of APO to VA64 1: 18), respectively, and the results are shown in table 1.

Table 1 solubility comparison data

2. Micelle size, polydispersity index, and Zeta potential

The measurement was performed by Dynamic Light Scattering (DLS) method, and the result showed that: the APO-VA64 eye drops obtained in example 1 (mass ratio of APO to VA64 is 1:18, APO content is 5 mg/ml) had a micelle size of 14.12+ -1.24 nm, a polydispersity index (PDI: polydispersity index) of 0.163+ -0.089 and zeta potential of- (8.86+ -0.98) mV.

3. Encapsulation efficiency

The measurement is carried out by adopting high performance liquid chromatography: the prepared APO-VA64 nano micelle eye drops are filtered through a 0.22 mu m filter membrane, and unencapsulated APO is separated by filtration. The solution of APO-VA64 before and after filtration is diluted with a suitable solvent (e.g., methanol) to disrupt the micelles. And measuring the APO concentration by adopting a high performance liquid chromatography. Encapsulation efficiency, which is the ratio of the concentration of APO detected after filtration to the concentration of APO detected before filtration.

High Performance Liquid Chromatography (HPLC):

reversed phase chromatographic column: agilent ZORBAX SB-C18 (250 mm. Times.4.60 mm,5 μm); mobile phase: methanol-water (55:45, v/v); G1314A uv detector: the detection wavelength is 274nm; column temperature 25 ℃; the flow rate is 1.0mL/min; G1367A sample injector. The retention time of APO was 4.2min.

The detection result is shown in fig. 2, when the mass ratio of APO to VA64 is 1:9, the encapsulation efficiency is 88.28±1.63%, and when the mass ratio of APO to VA64 is 1:18 or higher, the encapsulation efficiency is >99%, which is close to 100%.

4. Storage stability

The APO-VA64 eye drops prepared in example 1 were packaged, sealed, and then stored at 4 ℃ and 25 ℃ in a dark place, respectively. Every two weeks, the encapsulation efficiency, micelle size, polydispersity index (PDI) and Zeta potential were measured to evaluate the storage stability of APO-VA64 eye drops, and the measurement results are shown in fig. 3, 4, 5 and 6, respectively.

The results show that the encapsulation efficiency can be maintained above 90% after the polymer is stored for 12 weeks at the temperature of 4 ℃ and 25 ℃, the micelle size is in the range of 10-20 nm, the polydispersity index (PDI) is in the range of 0.1-0.5, the Zeta potential is in the range of-15 to-5 mV, and the polymer has good stability.

Effect example 2

In vitro antioxidant Activity

The antioxidant activity of APO-VA64 (example 1: APO to VA64 mass ratio of 1:18) was measured by 2,2' -diamine bis (3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS) and Fe (III) reducing power (FRAP), APO concentration in APO-VA64 was 6.25, 12.5, 25, 50 and 100. Mu.g/mL, respectively, APO concentration in PBS buffer was 0.11, 0.23, 0.45, 0.90 and 1.80mg/mL, respectively, and the results were shown in FIGS. 7 to 10.

Wherein, the ABTS method detects the antioxidant activity of APO-VA64 nano micelle eye drops under different concentrations as shown in figure 7; the ABTS method is used for detecting the antioxidant activity of APO-VA64 nano micelle eye drops at different times as shown in figure 8; the FRAP method is used for detecting the antioxidant activity of the APO-VA64 nano micelle eye drops under different concentrations as shown in figure 9; the FRAP method is used for detecting the antioxidant activity of APO-VA64 nano micelle eye drops at different times as shown in figure 10.

From the above results, it can be seen that APO has better antioxidant activity than VA64, and APO-VA64 exhibits stronger antioxidant activity than APO.

Effect example 3

In vitro Release study

Test solution:

APO-VA64 nano micelle eye drops (example 1: the mass ratio of APO to VA64 is 1:18, and the APO content is 5 mg/mL);

APO (PBS buffer as solvent, APO content 5 mg/mL).

1mL of the test solution was placed in dialysis bags (molecular weight cut-off [ MWCO ] =3500 Da), and then the dialysis bags were placed in simulated tears (pH=7.4, 37 ℃) and incubated in a shaker at 100rpm and 37.+ -. 1 ℃, 1mL of the sample was collected at different time points (1 mL of simulated tears were simultaneously fed in) and diluted with methanol, and then analyzed by HPLC, resulting in an in vitro release profile as shown in FIG. 11.

The results in fig. 11 show that APO-VA64 is able to release APO faster than APO (P <0.05, there is a significant difference), and that the cumulative release rate of APO is only 30.50±0.29% for the 6h test results, whereas the cumulative release rate of APO-VA64 is as high as 92.94±0.38%.

Effect example 4

1. Irritation evaluation: chick embryo allantoic membrane (HET-CAM) -trypan blue staining experiment

Chick embryo allantoic membrane-trypan blue staining (CAM-TBS) method was used to evaluate the irritation of APO-VA64 eye drops. Experimental chick embryos were randomly dosed into groups of 5 animals each:

(1) 0.9% NaCl: physiological saline;

(2) 0.01% bac: benzalkonium chloride aqueous solution;

(3) 1% SDS: an aqueous solution of sodium dodecyl sulfate;

(4) 0.1M NaOH: an aqueous sodium hydroxide solution;

(5) APO: the solvent is PBS buffer solution, and the APO content is 5mg/mL;

(6) VA64: the solvent is PBS buffer solution, and the VA64 content is 90mg/mL;

(7) APO & VA64 (physical mix): the solvent is PBS buffer solution, and each milliliter contains 5mg of APO and 90mg of VA64;

(8) 1mg/mL APO-VA64: the mass ratio of APO to VA64 is 1:18, and the APO content is 1mg/mL;

(9) 5mg/mL APO-VA64: the mass ratio of APO to VA64 was 1:18 and the APO content was 5mg/mL (example 1).

After the sample solution was contacted with chick embryo allantoic membrane (CAM) for 5min, the vascular comparison of the CAM is shown in FIG. 12, and the trypan blue uptake is shown in FIG. 13 (X: represents P <0.05 compared to 0.9% NaCl #; represents P <0.05 compared to 0.01% BAC; $: represents P <0.05 compared to 1% SDS; $: represents P <0.05 compared to 0.1M NaOH).

Besides obvious vascular rupture bleeding conditions of blood vessels of chick embryo allantois of 1% SDS and 0.1M NaOH (both positive control groups), other groups such as APO, VA64, APO & VA64, APO-VA64 and the like and normal saline (negative control) have no rupture and bleeding phenomena when blood vessels of chick embryo allantois observed by naked eyes, and the trypan blue absorption capacity of the chick embryo allantois basically equivalent to that of the normal saline (negative control), so that the chick embryo allantois has no obvious irritation to the chick embryo allantois.

2. Eye irritation test in rabbits

New Zealand white rabbits were used as experimental animals, and were randomly administered in the following groups, each group having 3 animals:

(1) PBS buffer: a negative control;

(2) 0.5% sds: sodium dodecyl sulfate aqueous solution, positive control;

(3) APO: the solvent is PBS buffer solution, the APO content is 5mg/ml, and 0.5% sodium carboxymethylcellulose is added (plays a role of suspending);

(4) VA64: the solvent is PBS buffer solution, and the VA64 content is 90mg/ml;

(5) APO & VA64 (physical mix): the solvent is PBS buffer solution, each milliliter contains 5mg of APO and 90mg of VA64, and 0.5 percent of sodium carboxymethyl cellulose is added;

(6) APO-VA64: the mass ratio of APO to VA64 was 1:18 and the APO content was 5mg/mL (example 1).

The right eye of each group of white rabbits was subjected to eye drop (the left eye was used as a self-control), 50. Mu.L each time, once every 30min, and 13 times in total. After 24 hours from the last eye drop, eye tissues such as cornea, conjunctiva and the like of the rabbit eye are observed and recorded by a slit lamp. After the rabbit was sacrificed, the eyeball was fixed in a 10% formaldehyde solution, embedded, sectioned, stained with hematoxylin-eosin (HE) or with snow-wav Periodate (PAS) for histopathological observation.

The results are shown in fig. 14 and 15; wherein, the diagrams of the irritation observation of rabbit eyes of different administration groups are shown in fig. 14; histopathological observations of rabbit eyes from different dosing groups are shown in figure 15.

The results showed that 0.5% sds (positive control) exhibited significant irritating symptoms: edema, congestion, secretions, and the like; in other groups like APO, VA64, APO & VA64, APO-VA64, and the like were similar to cornea, conjunctiva, and other ocular tissues of PBS (negative control) group, no obvious adverse symptoms and histopathological changes were observed, further indicating that: APO-VA64 eye drops are not significantly irritating to the eyes and are safe for application to the eyes.

Effect example 5

Mouse cornea absorption contrast test

APO-VA64 and APO were labeled with coumarin 6 (Cou-6), respectively (coumarin 6 concentration 50 μg/mL), and mice were randomly divided into two groups (9 per group): one group of eye drops is used for dropping coumarin 6-marked APO-VA64 (the mass ratio of APO to VA64 is 1:18 in example 1, and the APO content is 5 mg/mL), and the other group of eye drops is used for dropping coumarin 6-marked APO (the solvent is PBS buffer solution, and the APO content is 5 mg/mL) suspension; 5 μl/eye at a time, once every 10 minutes, 4 times, and 30, 60 and 90 minutes after the end of the last administration, mice (3 each) were sacrificed, the cornea was isolated and rinsed with physiological saline; mouse cornea was spread with a cut flap or a vertical cross section on a slide glass, and the penetration of the drug into the cornea was observed using a fluorescence microscope (olympus BX53F2, tokyo, japan).

The results are shown in fig. 16 and 17; wherein, the contrast graph (cut-flap tiling) of the corneal fluorescence intensity of APO-VA64 is shown in FIG. 16, and the contrast graph (vertical cross section) of the corneal fluorescence intensity of APO-VA64 is shown in FIG. 17. The results show that at the same time point (30 minutes), coumarin 6-labeled APO-VA64 exhibited much stronger fluorescence intensity than coumarin 6-labeled APO, indicating that APO in APO-VA64 was significantly better absorbed through the cornea than APO alone. Further, the fluorescence intensity gradually becomes weaker with the increase of time.

Effect example 6

1. Establishing benzalkonium chloride (BAC) induced ocular surface injury (xerophthalmia) model

Molding was performed using male BALB/c mice (about 20 g), and 5 μl of benzalkonium chloride in PBS (BAC concentration 0.45%) was administered to the right eye 2 times daily (9 a.m. and 9 a.m.) for 6 consecutive days to induce ocular inflammation, and subsequent dosing experiments were performed after successful molding on day 7.

The above model mice were randomly grouped (28 to 30 per group) and given the following drug treatments:

(1) PBS buffer;

(2) HA (sodium hyaluronate): the solvent is PBS buffer solution, and the concentration is 0.1%;

(3) APO: the solvent is PBS buffer solution, the APO content is 5mg/ml, and 0.5% sodium carboxymethylcellulose is added (plays a role of suspending);

(4) VA64: the solvent is PBS buffer solution, and the VA64 content is 90mg/ml;

(5) APO & VA64 (physical mix): the solvent is PBS buffer solution, each milliliter contains 5mg of APO and 90mg of VA64, and 0.5 percent of sodium carboxymethyl cellulose is added;

(6) Low dose APO-VA64: the mass ratio of APO to VA64 is 1:18, and the APO content is 1mg/mL;

(7) High dose APO-VA64: the mass ratio of APO to VA64 was 1:18 and the APO content was 5mg/mL (example 1).

Eye drop administration is carried out 3 times a day (9 am, 3 pm and 9 pm) from 7 days of molding, 5 mu L/eye each time, and 7 days (i.e. 7 th to 13 th days) are continued; during the dosing period, each group of mice continued to be given 5. Mu.L of benzalkonium chloride in PBS (BAC concentration 0.60%, once: 6. Mu.m.A). Healthy normal mice were used as controls (28 to 30 mice).

2. Corneal haze score

Each group of mice (n=10) was scored for corneal haze.

The results of corneal haze scores for the different dosing groups are shown in FIG. 18 (X: P <0.05 compared to PBS #; P <0.05 compared to HA #; $ P <0.05 compared to VA64 $ P <0.05 compared to APO; @ P <0.05 compared to APO & VA 64).

The results show that after the eye is locally instilled with benzalkonium chloride (BAC) for 6 days, typical symptoms of ocular surface damage, impaired corneal transparency and corneal haze score of about 10 minutes can be observed, the corneal transparency is improved and haze score is greatly reduced after the eye is treated by the APO-VA64 eye drop for 7 days (day 13), and compared with APO, VA64, APO & VA64 groups and the like, the significant difference (P < 0.05) exists.

3. Eye surface slit lamp observation

Sodium fluorescein staining: after staining with 0.25% sodium fluorescein solution, the staining of the sodium fluorescein in the cornea of mice of different groups was recorded by photographing with slit lamp, and the corneal epithelial area defect rate (n=10) was counted and calculated.

The results are shown in figures 19 and 20 (x: showing P <0.05 compared to PBS;, # denotes P <0.05 compared to HA, & gt denotes P <0.05 compared to VA64, & gt denotes P <0.05 compared to APO, @ denotes P <0.05 compared to APO & VA 64. The images of corneal fluorescein sodium staining of mice from different dosing groups were observed with a slit lamp as shown in fig. 19; the rate of corneal epithelial defects in mice from different dosing groups according to the fluorescein sodium staining statistics is shown in figure 20.

The results show that after 6 days of local instillation of benzalkonium chloride (BAC) in eyes, the area defect rate of the corneal epithelium is as high as more than 90 percent (sodium fluorescein staining), the corneal epithelium is obviously repaired after 5 days of treatment (11 th day) by the administration of APO-VA64 eye drops, and the defect rate is lower than 15 percent (sodium fluorescein staining), and has obvious difference (P < 0.05) compared with APO, VA64, APO & VA64 and the like.

Dyeing tiger red: after staining with 0.25% tiger red solution, observing with slit lamp, photographing to record the staining condition of the tiger red on the cornea of mice in different groups, and counting and calculating the area defect rate of the corneal epithelium (n=10).

The results are shown in fig. 21 and 22 (x: showing P <0.05 compared to PBS;, # denotes P <0.05 compared to HA, & gt denotes P <0.05 compared to VA64, & gt denotes P <0.05 compared to APO, @ denotes P <0.05 compared to APO & VA 64. The slit lamp is used for observing the red staining images of the cornea of the mice of different administration groups as shown in figure 21; the rate of corneal epithelial defects in mice from different dosing groups based on tiger red staining statistics is shown in figure 22.

The results show that after the eye is locally instilled with benzalkonium chloride (BAC) for 6 days, the area defect rate of the corneal epithelium is as high as more than 90 percent (tiger red staining), the corneal epithelium is obviously repaired after the eye is treated by the APO-VA64 eye drop for 7 days (13 th day), and the defect rate is remained below 15 percent (tiger red staining), and has obvious difference (P < 0.05) compared with APO, VA64, APO & VA64 and the like.

4. Cornea sensitivity detection

Detection using a Cochet-Bonnet perceptron (france): the eyelashes of the eyelids of the mice are cleaned, the eyeballs are exposed, the contact pins are used for touching the cornea, the length of the contact pins of the instrument is initially adjusted to be 6.0cm at the maximum scale, then the length of the contact pins of the instrument is sequentially decreased by 0.5 until frequent blink reflection occurs to the mice, and the scale of the contact pins at the moment is recorded to be the measured value of the cornea sensitivity of the mice (n=10).

Corneal sensitivity results for the different dosing groups are shown in figure 23 (x: shows P <0.05 compared to normal healthy mice;, # denotes P <0.05 compared to PBS, & gt denotes P <0.05 compared to HA, & gt denotes P <0.05 compared to VA64, @ denotes P <0.05 compared to APO,% & gt denotes P <0.05 compared to APO & VA 64.

The results showed that after 6 days of local instillation of benzalkonium chloride (BAC) in the eyes, the corneal nerve was damaged, the corneal sensitivity was reduced to 1.9.+ -. 0.6cm, and the corneal sensitivity was restored to 5.7.+ -. 0.3cm after 7 days of treatment with APO-VA64 eye drops (day 13), which was comparable to normal levels in healthy mice with significant differences (P < 0.05) in APO, VA64, APO & VA64, etc.

5. Tear secretion test

The change in the tear secretion of the mice was measured by phenol red cotton lines (n=10). Grabbing the mice, placing the cotton thread on the lower eyelid conjunctiva of the mice at a position which is far from the outer corner of the eye by using an ophthalmic tweezer and is one third of the outer corner of the eye for 15 seconds, measuring the length of a wet dyeing part of the phenol red cotton thread by using a millimeter ruler and recording, wherein the test results of the 6 th day, the 7 th day, the 9 th day, the 11 th day and the 13 th day are shown in fig. 24, fig. 25, fig. 26, fig. 27 and fig. 28 respectively (the test results of the P <0.05 compared with a normal healthy mouse are shown in the specification;, # denotes P <0.05 compared to PBS, & gt denotes P <0.05 compared to HA, & gt denotes P <0.05 compared to VA64, @ denotes P <0.05 compared to APO,% & gt denotes P <0.05 compared to APO & VA 64.

The results showed that after 6 days of ocular instillation of benzalkonium chloride (BAC), the amount of tear secretion was significantly reduced and the administration of APO-VA64 eye drops was treated for 7 days (day 13), the amount of tear secretion had recovered to near or equivalent to normal levels, with significant differences (P < 0.05) compared to APO, VA64, APO & VA64, etc.

6. Cornea neovascularization investigation

The neovascularization of the mouse cornea was observed with a slit lamp, and the neovascularization distribution was calculated.

The results are shown in fig. 29 and fig. 30 (x: showing P <0.05 compared to PBS;, # denotes P <0.05 compared to HA, & gt denotes P <0.05 compared to VA64, & gt denotes P <0.05 compared to APO, @ denotes P <0.05 compared to APO & VA 64. Wherein, the slit lamp examination images of the corneal neovascularization of the different administration groups are shown in figure 29; corneal neovascularization profiles for the different dosing groups are shown in figure 30.

The results show that after 6 days of local instillation of benzalkonium chloride (BAC) in the eyes, severe corneal Neovascularization (NV) was observed, and the neovascularization reached the center of the cornea, the percentage of the neovascularization was about 50%, and the percentage of the neovascularization was significantly reduced by the administration of APO-VA64 eye drops for 7 days (day 13), effectively inhibiting neovascularization, with significant differences (P < 0.05) compared with APO, VA64, APO & VA64, etc.

7. Histopathological examination

On day 14, histopathological observations were made on the mice eyeballs after staining with hematoxylin-eosin (HE) or with snow-fu Periodate (PAS).

Histopathological observations of the different dosing groups are shown in fig. 31. The results show that the cornea of the PBS group has serious edema and inflammation characteristics, the epithelial defect, a large number of blood vessels and matrix arrangement disorder, and the whole cornea is seriously infiltrated by inflammatory cells; conjunctival tissue is much thicker than normal healthy conjunctival tissue, and the entire conjunctiva also has a large amount of inflammatory cell infiltration; the number of goblet cells decreased. In contrast, the cornea and conjunctiva of APO-VA64 group were administered without abnormal symptoms, which were substantially equivalent to normal healthy tissues.

8. Corneal epithelium and corneal endothelial examination

On day 14, the corneal epithelium and corneal endothelium were observed with a Scanning Electron Microscope (SEM) for each group of mice, healthy normal mice and mice on day 6 of molding as controls.

SEM images of the different dosing groups are shown in figure 32. The results show that administration of APO-VA64 group significantly reduced benzalkonium chloride (BAC) induced corneal damage, which tended to restore the normal corneal epithelium and endothelium.

9. ELISA (enzyme-Linked immunosorbent assay)

On day 14, four corneas were collected as one sample, three samples were used for each group, and HMGB1, IL-6, NF- κ B, TNF- α were detected using a mouse ELISA kit (Shanghai enzyme-Linked Biotechnology Co., shanghai, china).

The results are shown in FIGS. 33-36 (x: representing P <0.05 compared to normal healthy mice #; representing P <0.05 compared to PBS; $ representing P <0.05 compared to HA $ representing P <0.05 compared to VA64; @ representing P <0.05 compared to APO;:%: representing P <0.05 compared to APO & VA 64); wherein, the HMGB1 expression level of the cornea tissue of different administration groups is shown in figure 33; IL-6 levels in corneal tissue from different dosing groups are shown in FIG. 34; NF- κb levels of corneal tissue from different dosing groups are shown in fig. 35; TNF- α levels for corneal tissue from different dosing groups are shown in FIG. 36. The results show that the APO-VA64 can effectively inhibit the expression of HMGB1, IL-6 and NF-kappa B, TNF-alpha, so that the HMGB is in a normal level, and compared with the APO, VA64, APO & VA64 and the like, the APO-VA64 has significant difference (P is less than 0.05).

10. Quantitative PCR analysis

On day 14, three corneas were pooled into one sample, and three samples were used for each group to determine the mRNA levels of inflammatory cytokines. Total RNA was extracted from the cornea using the Nucleospin RNA kit (BD Biosciences, canada). Primers for inflammatory cytokines (including HMGB1, IL-6, NF-. Kappa. B, RAGE, TLR2, TLR4, TNF-. Alpha., VEGF) were synthesized using the first strand cDNA Synthesis kit (PrimeScript 1st Strand cDNA Synthesis Kit, daidan TaKaRa, china). Real-time PCR was performed using TaqMan reagents and Applied Biosystems 7500 real-time PCR system (canada Applied Biosystems). The circulation conditions are as follows: 95℃for 10 seconds and then 45 two cycles (95℃for 15 seconds, 60℃for 1 minute). The data was analyzed using sequence detection system software (Applied Biosystems) with GAPDH as an internal control. The mRNA levels of each target gene were normalized, first to GAPDH, and then to normal healthy control mice.

The results are shown in FIGS. 37-44 (X: P < 0.05; #: P < 0.05; &: P < 0.05; $: P < 0.05; @: P < 0.05; and @: P < 0.05; respectively, in comparison with normal healthy mice; and:%: P < 0.05; in comparison with APO & VA64; respectively). Wherein, the PCR analysis results of HMGB1 in cornea tissues of different administration groups are shown in FIG. 37; the results of PCR analysis of IL-6 in corneal tissue from different dosing groups are shown in FIG. 38; the results of PCR analysis of NF- κB in corneal tissue from different dosing groups are shown in FIG. 39; the results of PCR analysis of RAGE in corneal tissue from different dosing groups are shown in FIG. 40; the results of PCR analysis of TLR2 in corneal tissue from different dosing groups are shown in figure 41; the results of PCR analysis of TLR4 in corneal tissue from different dosing groups are shown in figure 42; the results of PCR analysis of TNF-. Alpha.in corneal tissue from different dosing groups are shown in FIG. 43; the results of PCR analysis of VEGF in corneal tissue from different dosing groups are shown in FIG. 44; the results show that APO-VA64 can effectively inhibit mRNA transcription level of HMGB1, IL-6, RAGE, TLR2, TLR4, TNF-alpha and VEGF.

The invention is, of course, capable of other numerous embodiments and of being practiced in accordance with the invention and carried out by those skilled in the art without departing from the spirit and spirit of the invention, and it is intended that all such modifications and/or variations be regarded as being within the scope of the appended claims.

Claims (10)

1. A pharmaceutical composition comprising: aromatic ketones and pharmaceutically acceptable polymers; wherein the aromatic ketone is vanillyl ethanone or pharmaceutically acceptable salt thereof, and the polymer is povidone and/or copovidone.

2. The pharmaceutical composition of claim 1, wherein the weight ratio of said aromatic ketone to said polymer is 1 (2-50); preferably, the weight ratio of the aromatic ketone to the polymer is 1 (9-25); more preferably, the weight ratio of the aromatic ketone to the polymer is 1 (12-22).

3. The pharmaceutical composition of claim 1, wherein the aromatic ketone has an encapsulation efficiency of at least 80%; preferably, the encapsulation efficiency of the aromatic ketone is not less than 90% or not less than 95%.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition satisfies one or more of the following conditions i to iii:

i. the aromatic ketone is vanillyl ethanone;

ii. The polymer is copovidone;

iii, the K value of the polymer is 25-35; preferably, the K value of the polymer is 25.2-30.8;

preferably, the pharmaceutical composition satisfies the conditions i to iii simultaneously.

5. A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is prepared by a process comprising the steps of: dispersing or dissolving the aromatic ketone and the pharmaceutically acceptable polymer together in an organic solvent, uniformly mixing, and removing the organic solvent by rotary evaporation at 35-45 ℃ to obtain the aromatic ketone;

preferably, the organic solvent is an alcohol solvent or a halogenated hydrocarbon solvent, and/or the dosage of the organic solvent corresponding to each milligram of the aromatic ketone is 0.5-20 mL;

more preferably, the organic solvent is methanol or ethanol, and/or the amount of the organic solvent corresponding to each milligram of the aromatic ketone is 0.5-5 mL.

6. A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is a solid or liquid formulation; and/or, in the pharmaceutical composition, the aromatic ketone is in a therapeutically effective amount.

7. A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is a liquid formulation, and the solvent in the liquid formulation is selected from pharmaceutically acceptable water, PBS buffer or sodium carboxymethyl cellulose aqueous solution;

preferably, when the content of aromatic ketone in the liquid preparation is 5mg/mL, the liquid preparation satisfies one or more of the following conditions (1) to (3):

(1) the average diameter of the micelle of the liquid preparation is 1-80 nm; preferably 10 to 20nm;

(2) the polydispersity of the liquid preparation is less than or equal to 0.5; preferably less than or equal to 0.4;

(3) the Zeta potential of the liquid preparation is-20 mV to 0mV; preferably-15 to-5 mV;

more preferably, the liquid formulation satisfies the conditions (1) to (3) at the same time when the aromatic ketone content in the liquid formulation is 5 mg/mL.

8. A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is an ophthalmic formulation;

preferably, the ophthalmic formulation satisfies at least one of the following uses a-e:

a. the ophthalmic preparation is used for treating and/or preventing xerophthalmia;

b. the ophthalmic preparation is used for inhibiting cornea neovascularization;

c. the ophthalmic preparation is used for inhibiting the expression of HMGB 1;

d. the ophthalmic formulation is an ophthalmic formulation for inhibiting expression of RAGE, TLR2 or TLR 4;

e. the ophthalmic preparation is an ophthalmic preparation for inhibiting the expression of cytokines IL-6, NF-kappa B, TNF-alpha or VEGF.

9. A method of preparing a pharmaceutical composition according to any one of claims 1 to 8, comprising the steps of: dispersing or dissolving the aromatic ketone and the pharmaceutically acceptable polymer together in an organic solvent, uniformly mixing, and removing the organic solvent by rotary evaporation at 35-45 ℃ to obtain a solid product; and then optionally comprises: dissolving or dispersing the obtained solid product in a solvent of a liquid preparation, filtering and sterilizing to obtain the liquid preparation;

preferably, the organic solvent is an alcohol solvent or a halogenated hydrocarbon solvent, and/or the dosage of the organic solvent corresponding to each milligram of the aromatic ketone is 0.5-20 mL;

more preferably, the organic solvent is methanol or ethanol, and/or the amount of the organic solvent is 0.5-5 mL per milligram of the aromatic ketone.

10. Use of a pharmaceutical composition according to any one of claims 1 to 8 for the preparation of an ophthalmic formulation.