Traditional Chinese medicine microemulsion eye drops as well as preparation method and application thereof

Abstract

The invention discloses traditional Chinese medicine microemulsion eye drops as well as a preparation method and application thereof. The traditional Chinese medicine microemulsion eye drops comprise the following components in percentage by weight: 0.001%-2.0% of baicalein, 0.001%-2.0% of borneol, 0.1%-20% of oil for injection, 0.1%-10% of a surfactant, 0.1%-10% of phospholipid, 0.1%-5% of an osmotic pressure regulator and the balance of water. The oil-in-water emulsion containing the borneol and the baicalein is prepared by combined application of the baicalein and the borneol, so that a mutual synergistic effect is achieved, the opportunity that the medicine enters cornea and eye inflammation parts is increased, the functions that the medicine rapidly enters eye tissues and the curative effect is enhanced are realized, meanwhile, the residence time in eyes is obviously prolonged, and the curative effect is improved. In addition, the cornea permeability of the medicine can be increased, and the treatment of AMD patients in a non-invasive administration mode and the treatment of various types of eyestrain are realized.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to a traditional Chinese medicine microemulsion eye drop, a preparation method and an application thereof. Background Art

[0002] Age-related macular degeneration (AMD) is a neuroretinal degenerative disease and the main cause of blindness in people over 50 years old. In 2020, there were approximately 196 million AMD patients worldwide, and it is predicted that this number will increase to 288 million in 2040. With the acceleration of aging in my country, the number of people with visual impairment caused by AMD will increase rapidly. Clinically, AMD is divided into two categories: dry (atrophic) and wet (neovascular), of which dry AMD accounts for about 90% of the total number. Most AMD patients start with dry AMD and progress to wet AMD later. Therefore, dry AMD is also considered to be a risk factor or starting stage for wet AMD. In recent years, due to the application of anti-vascular endothelial growth factor drugs, the treatment of wet AMD has made significant progress, but there is no specific treatment for dry AMD. Therefore, AMD is still the main cause of blindness in my country, and the development of new AMD therapeutic drugs is crucial for clinical treatment.

[0003] Baicalein, also known as baicalein, is one of the main active ingredients in the dried root of Scutellaria baicalensis, a perennial herbaceous plant of the genus Scutellaria in the Lamiaceae family. It belongs to the flavonoids. Baicalein is a yellow needle-shaped crystal, soluble in acetone, alcohol, ethyl acetate, etc., and almost insoluble in water. Domestic and foreign studies have shown that it has anti-inflammatory, anti-oxidative stress, antibacterial and antiviral effects. It has shown broad prospects in inhibiting ocular inflammatory reactions, resisting oxidative stress damage, and preventing and treating eye diseases such as keratitis and retinopathy. However, the properties of baicalein being poorly soluble in water and having poor bioavailability make it difficult for its ordinary preparations to exert biological activity through traditional ocular administration.

[0004] At the same time, the baicalein-related preparations currently on the market in China include baicalein aluminum capsules, compound scutellaria tablets, Shuanghuanglian granules / soft capsules, Qingkailing tablets / soft capsules, and other traditional Chinese medicine formula preparations such as scutellaria baicalensis, gegenqinlian capsules, etc. for gastrointestinal administration, Shuanghuanglian oral liquid, Shuanghuanglian mixture, Qingkailing oral liquid, etc. for oral liquid, and bear gall bladder scutellaria eye drops for eyes. It is not difficult to find that the current preparations containing baicalein are mostly traditional Chinese medicine compound preparations. Due to the presence of many unknown ingredients in compound preparations, the pharmacological effects of the preparations are not obvious, and they are prone to cause various unexpected adverse reactions.

[0005] The eyeball is a highly enclosed organ, which makes it difficult to deliver drugs into the eye, especially to the diseased tissue of the fundus. At present, macromolecular drugs for fundus diseases are generally injected, and macromolecular drugs need to be delivered to the posterior segment of the eye through invasive intravitreal injection to achieve their purpose. Long-term and frequent injections, on the one hand, bring psychological pressure to patients and doctors, and on the other hand, they also cause trauma to the eyes, thus affecting the treatment effect.

[0006] The drug delivery logic of small molecule eye drops is different from invasive injection. For ophthalmic diseases, eye drops are easy to be accepted by patients because of their advantages such as convenient administration, non-invasiveness, and low price. Therefore, the administration of eye drops into the conjunctival sac is still the most commonly used method of administration for the treatment of ocular diseases. However, the ocular bioavailability of traditional eye drops is extremely low (less than 5%).

[0007] Therefore, in order to increase the ocular absorption of the drug, prolong the residence time in the eye, improve the bioavailability, and obtain better local therapeutic effects, it is urgently necessary to provide a new formulation of baicalin components that increases the corneal permeability and bioavailability of baicalin, reduces the frequency of drug use and eye irritation. Summary of the invention

[0008] The purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide a Chinese medicine microemulsion eye drops with high corneal permeability and bioavailability, low eye irritation and improved efficacy, as well as a preparation method and application thereof.

[0009] To achieve the above object, the technical solution of the present invention is as follows:

[0010] A Chinese medicine microemulsion eye drop comprises the following components in percentage by weight: 0.001% to 2.0% of baicalein, 0.001% to 2.0% of borneol, 0.1% to 20% of injection oil, 0.1% to 10% of surfactant, 0.1% to 10% of phospholipid, 0.1% to 5% of osmotic pressure regulator, and the balance is water.

[0011] The present invention also discloses a method for preparing the above-mentioned traditional Chinese medicine eye drops, comprising the following steps:

[0012] (1) mixing injection oil, phospholipids, baicalein and borneol to obtain an oil phase;

[0013] (2) mixing an osmotic pressure regulator, a surfactant, and water to obtain an aqueous phase;

[0014] (3) adding the oil phase to the water phase and mixing them at room temperature to obtain colostrum;

[0015] (4) After the colostrum is mixed with water, high pressure homogenization and filtration sterilization are performed in sequence to obtain the Chinese medicine microemulsion eye drops.

[0016] The present invention also discloses a use of the above-mentioned Chinese medicine microemulsion eye drops, or the Chinese medicine microemulsion eye drops prepared by the above-mentioned preparation method in preparing medicines for treating eye diseases or preparing medicines for relieving eye fatigue.

[0017] Implementing the embodiments of the present invention will have the following beneficial effects:

[0018] (1) The Chinese medicine microemulsion eye drops provided by the present invention are prepared by using injectable oil, phospholipids, baicalin, and borneol as the oil phase and an aqueous phase (osmotic pressure regulator, surfactant, and water) to prepare an oil-in-water emulsion containing borneol and baicalin. By appropriately combining borneol and baicalin in a certain ratio and controlling the preparation process conditions, a synergistic effect is achieved, thereby increasing the chance of the drug entering the cornea and the site of ocular inflammation, achieving the function of the drug quickly entering the ocular tissue and enhancing the therapeutic effect, and significantly prolonging the retention time in the eye. In addition, the corneal permeability of the drug can be increased, and the intraocular or posterior segment diseases can be treated by eye drops, thereby making up for the shortcomings of existing eye drops.

[0019] (2) The present invention loads borneol and baicalein into microemulsion together, and utilizes the "guiding meridians upward" effect of borneol to promote drug penetration through the cornea and increase the distribution of drugs in ocular tissues. From the perspective of baicalein's anti-oxidative stress and anti-inflammatory effects, the present invention achieves the effects of clearing active oxygen in the posterior ocular tissues and reducing inflammation.

[0020] In summary, the present invention combines baicalein with borneol to allow baicalein to enter the posterior segment of the eye, and at the same time screens the ratio of the two, which can not only reduce the irritation of borneol to the eyes, but also effectively solve the problem that baicalein is poorly soluble in water, improve the bioavailability of the active ingredients of the drug, and achieve excellent synergistic effects, thereby achieving non-invasive treatment of intraocular or posterior segment diseases, especially non-invasive drug administration for patients with early and mid-stage AMD and treatment of various types of eye fatigue. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the appearance of the preparations of Example 1 and Example 2.

[0022] Figure 2 Schematic diagram of the particle size measurement results of Example 1.

[0023] Figure 3 Schematic diagram of in vitro release of Example 1 and Example 2.

[0024] Figure 4 The irritation observation diagrams of Example 1, Example 2 and normal saline are shown.

[0025] Figure 5 The irritating pathological tissue sections of Example 1, Example 2 and normal saline are shown.

[0026] Figure 6 This is the drug-time curve diagram of rabbit eye tissue distribution in Example 1 and Example 2.

[0027] Figure 7 It is the area under the drug-time curve of rabbit eye tissue distribution in Example 1 and Example 2.

[0028] Figure 8 The pharmacopathological tissue sections of Example 1, Example 2, commercially available preparations, normal group and model group mice are shown.

[0029] Fig. 9 The diagram shows the cell apoptosis of mice in Example 1, Example 2, commercial preparation, normal group and model group.

[0030] Fig.10 It is a quantitative graph of cell apoptosis in mice of Example 1, Example 2, commercial preparation, normal group and model group.

[0031] Fig.11 The ROS staining images of mice in Example 1, Example 2, commercial preparation, normal group and model group are shown.

[0032] Fig.12 It is a quantitative graph of the fluorescence intensity of ROS staining of mice in Example 1, Example 2, commercial preparation, normal group and model group.

[0033] Fig.13 This is a graph showing the results of detecting the inflammatory factor IL-6 in mice in Example 1, Example 2, commercially available preparations, the normal group, and the modeling group.

[0034] Fig.14 This is a graph showing the results of detecting the inflammatory factor MCP-1 in mice in Example 1, Example 2, commercially available preparations, the normal group, and the modeling group.

[0035] Fig.15 This is a graph showing the results of detecting the inflammatory factor TNF-α in mice in Example 1, Example 2, commercially available preparations, the normal group, and the modeling group. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0037] The invention discloses a Chinese medicine microemulsion eye drop, which comprises the following components in percentage by weight: 0.001% to 2.0% of baicalein, 0.001% to 2.0% of borneol, 0.1% to 20% of injection oil, 0.1% to 10% of surfactant, 0.1% to 10% of phospholipid, 0.1% to 5% of osmotic pressure regulator, and the balance is water.

[0038] Specifically, the Chinese medicine microemulsion eye drops disclosed in the present invention produce a synergistic effect after reasonable compatibility, wherein:

[0039] The Chinese medicine Borneol is a crystal obtained by extracting the resin and volatile oil processed products of borneol. It was first recorded in "Ming Yi Bie Lu" and is often used as a "adjuvant drug" and "channel-guiding drug". It is also recorded in many ancient prescriptions of ophthalmology in my country. The channel-guiding effect of Borneol is reflected in changing the direction or site of action of other drugs, or making their effects focus or concentrate on specific directions and sites. For example, in preparations such as Compound Danshen Dripping Pills, Mayinglong Hemorrhoid Suppositories, and Suxiao Jiuxin Pills, Borneol is an adjuvant drug, which plays a role in channel-guiding and permeation-promoting in the prescription. In addition, the inventors found that the compatibility of Borneol with baicalin can enhance the improvement of baicalin on endothelial dysfunction after cerebral ischemia, indicating the channel-guiding and permeation-promoting effect of Borneol. At the same time, the inventors also found that its function of "leading drugs upward" can act on the "blood-ocular barrier". For example, borneol can promote the entry of drugs such as quercetin, guarenzepine, and puerarin into the eyes. This may be related to the fact that borneol makes the movement of the phospholipid bilayer of the corneal epithelial cell membrane more orderly, increases corneal permeability, and provides a method for ocular drug delivery.

[0040] Therefore, based on these insights, the inventors loaded borneol and baicalein into microemulsions together, and used the two together to allow baicalein to enter the posterior segment of the eye. At the same time, the ratio of the two was screened, which not only reduced the irritation of borneol to the eye, but also effectively solved the problem of baicalein being poorly soluble in water, ultimately achieving non-invasive treatment of posterior segment eye diseases.

[0041] Further, in order to increase the ocular absorption of the drug, prolong the retention time in the eye, improve the bioavailability, and obtain better local therapeutic effects, the droplets in the Chinese medicine microemulsion eye drops provided by the present invention are nanometer-sized and uniform droplets, which dissolve the drug and absorb it into the body, which can reduce the degradation in and out of the body, and the solubility is improved relative to the aqueous solution, while increasing the retention of the eye, it will not increase the foreign body sensation in the eye, and can improve the bioavailability. At the same time, the present invention uses phospholipids to replace a part of the surfactant, increase the paracellular transport, and promote drug absorption.

[0042] By further optimizing and limiting the weight ratio relationship between the components in the Chinese medicine microemulsion eye drops of the present invention, the dosage and efficacy of each component can be specifically adjusted and controlled, thereby achieving a better synergistic compatibility effect between the components.

[0043] In a specific embodiment, the Chinese medicine microemulsion eye drops include the following components in weight percentage: 0.01% to 1.0% baicalein, 0.01% to 1.0% borneol, 2% to 15% injection oil, 0.1% to 10% surfactant, 0.2% to 1.5% phospholipid, 0.5% to 3% osmotic pressure regulator, and the balance is water.

[0044] In a specific embodiment, the mass ratio of baicalein to borneol is 10:1-1:1. Preferably, the mass ratio is 5:1-1:1. More preferably, the mass ratio is 5:3.

[0045] In a specific embodiment, the mass ratio of baicalein to oil for injection is 1:20-1:200; preferably, the mass ratio is 1:50-1:200.

[0046] In a specific embodiment, the mass ratio of the injection oil to the phospholipid is 15:1-1:3.

[0047] In a specific embodiment, the injection oil includes one or more of MCT, LCT and structured triglycerides. Preferably, the injection oil includes one or more of MCT and LCT.

[0048] In a specific embodiment, the osmotic pressure regulator includes one or more of glycerol, sucrose, trehalose, xylitol and sodium chloride.

[0049] In a specific embodiment, the surfactant includes one or more of Tween-80, Solutol HS-15 and poloxamer.

[0050] In a specific embodiment, the surfactant includes one or more of 0.3% to 1.2% Tween-80, 0.2% to 0.9% Solutol HS-15, and 0.5% to 1.6% Poloxamer 188.

[0051] In a specific embodiment, the phospholipids include one or both of glycerophospholipids and sphingomyelin.

[0052] In a specific embodiment, the phospholipids include one or more of natural phospholipids, semi-synthetic phospholipids and fully synthetic phospholipids.

[0053] In a specific embodiment, the natural phospholipids include one or more of egg yolk phospholipids, soybean phospholipids, EPG, ESM, phosphatidylinositol and myocardial phospholipids.

[0054] In one embodiment, the semi-synthetic phospholipids include hydrogenated soybean lecithin (HSPC).

[0055] In a specific embodiment, the fully synthetic phospholipids include one or more of phosphatidylcholine, phosphatidylglycerol, cardiolipin and distearoylphosphatidylethanolamine (DSPE).

[0056] In a specific embodiment, the phosphatidylcholine includes one or more of DOPC, DSPC, DPPC, DMPC and DLPC.

[0057] In a specific embodiment, the phosphatidylglycerol includes one or more of DOPG, DSPG, DPPG, DMPG, and DLPG.

[0058] In a specific embodiment, the phospholipids preferably include one or more of 0.3% to 0.7% egg yolk phospholipids E80, 0.6% to 1.2% soybean phospholipids PC70, and 0.6% to 1.2% soybean phospholipids PC50.

[0059] In a specific embodiment, the Chinese medicine microemulsion eye drops further include 0.001% to 1% by weight of an antioxidant to improve the stability of the system.

[0060] In a specific embodiment, the antioxidant includes one or more of sodium bisulfite, sodium sulfite, sodium thiosulfate, ascorbic acid, citric acid and disodium edetate.

[0061] In a specific embodiment, the average particle size of the droplets in the Chinese medicine microemulsion eye drops is less than 200 nm.

[0062] In a specific embodiment, the pH value of the Chinese medicine microemulsion eye drops is 5-9.

[0063] The present invention also discloses a method for preparing the above-mentioned traditional Chinese medicine eye drops, comprising the following steps:

[0064] (1) Mix injection oil, phospholipids, baicalein and borneol to obtain an oil phase.

[0065] (2) Mixing an osmotic pressure regulator, a surfactant and water to obtain an aqueous phase.

[0066] (3) At room temperature, the oil phase is added to the water phase and mixed to obtain colostrum.

[0067] (4) After the colostrum is mixed with water, it is subjected to high-pressure homogenization and filtration sterilization in sequence to obtain a Chinese medicine microemulsion eye drop.

[0068] In a specific embodiment, step (1) specifically comprises: dissolving baicalein and borneol in ethanol, adding injection oil and phospholipids to the obtained mixed solution, stirring at room temperature for 10 minutes until a clear and transparent liquid is obtained, and obtaining an oil phase.

[0069] In a specific embodiment, step (2) specifically comprises: stirring the osmotic pressure regulator, the surfactant and about 10% of the prescription amount of purified water in a water bath at 40° C. to 60° C. to obtain an aqueous phase.

[0070] In a specific embodiment, in step (3), mixing comprises: adding the oil phase to the water phase and shearing at 15000 rpm to 20000 rpm for 5 min to 7 min.

[0071] In a specific embodiment, in step (4), the pressure of high-pressure homogenization is 800 bar to 1000 bar; and the number of high-pressure homogenization is 15 to 25 times.

[0072] The present invention also discloses a use of the above-mentioned Chinese medicine microemulsion eye drops, or the Chinese medicine microemulsion eye drops prepared by the above-mentioned preparation method in preparing medicines for treating eye diseases or preparing medicines for relieving eye fatigue.

[0073] In one embodiment, the drug for treating an eye disease is a drug for treating age-related macular degeneration.

[0074] In one embodiment, the drug for relieving eye fatigue is a drug for preventing dry age-related macular degeneration.

[0075] In a specific embodiment, the drug for treating eye diseases or relieving eye fatigue is a drug that relieves cellular oxidative damage or inhibits cellular inflammatory response.

[0076] In one embodiment, the drug can be diluted to a certain concentration to form a hypotonic or isotonic form and then administered topically to the eye.

[0077] The following are specific embodiments

[0078] The reagents in the following examples are all conventional products that can be purchased from the market. If no specific conditions are specified in the examples, conventional conditions are used.

[0079] Embodiment 1:

[0080] A Chinese medicine microemulsion eye drop, comprising the following components in percentage by weight: 0.05% baicalein; 0.03% borneol; 5% MCT vegetable oil; 0.6% egg yolk lecithin E80; 0.9% Tween-80 surfactant; 2.2% glycerol as an osmotic pressure regulator; 0.01% sodium sulfite as an antioxidant;

[0081] The preparation method comprises the following steps:

[0082] (1) MCT and egg yolk lecithin E80 were taken, and baicalein and borneol completely dissolved in 10 mL of ethanol were added, and the mixture was stirred and dissolved at room temperature until a clear and transparent liquid was obtained; the ethanol was removed by vacuum rotary evaporation at 45° C. to obtain a uniform oil phase.

[0083] (2) Take glycerol for injection, add Tween-80, add about 80% purified water, and stir at 60°C until dissolved to prepare the aqueous phase.

[0084] (3) The organic phase was added dropwise to the aqueous phase under shearing conditions of 3000 rpm, the speed was adjusted to 16000 rpm, and shearing was performed for 5 min to obtain colostrum. The colostrum was transferred to a measuring cylinder and the volume was fixed to 100 mL. The colostrum was transferred to a high-pressure homogenizer and homogenized at a pressure of 800 bar. After passing through 0.22 μm, baicalin microemulsion was obtained.

[0085] Embodiment 2:

[0086] A Chinese medicine microemulsion eye drop, comprising the following components in percentage by weight: 0.05% baicalein; 0.03% borneol; 10% MCT vegetable oil; 0.6% egg yolk lecithin E80; 0.9% Tween-80 surfactant; 2.2% glycerol osmotic pressure regulator; 0.01% sodium sulfite antioxidant;

[0087] The preparation method of this embodiment is the same as that of embodiment 1.

[0088] Embodiment 3:

[0089] A Chinese medicine microemulsion eye drop, comprising the following components in percentage by weight: 0.05% baicalein; 0.03% borneol; 5% MCT vegetable oil; 0.6% soybean lecithin S100; 0.9% Tween-80 surfactant; 2.2% glycerol osmotic pressure regulator; 0.01% sodium sulfite antioxidant;

[0090] The preparation method of this embodiment is the same as that of embodiment 1.

[0091] Embodiment 4:

[0092] A Chinese medicine microemulsion eye drop, comprising the following components in percentage by weight: 0.05% baicalein; 0.03% borneol; 5% MCT vegetable oil; 0.6% soybean lecithin PC50; 0.9% Tween-80 surfactant; 2.2% glycerol osmotic pressure regulator; 0.01% sodium sulfite antioxidant;

[0093] The preparation method of this embodiment is the same as that of embodiment 1.

[0094] Embodiment 5:

[0095] A Chinese medicine microemulsion eye drop, comprising the following components in percentage by weight: 0.05% baicalein; 0.03% borneol; 5% MCT vegetable oil; 0.6% egg yolk lecithin E80; 0.9% HS-15 surfactant; 2.2% glycerol as an osmotic pressure regulator; 0.01% sodium sulfite as an antioxidant;

[0096] The preparation method of this embodiment is the same as that of embodiment 1.

[0097] Embodiment 6:

[0098] A Chinese medicine microemulsion eye drop, comprising the following components in percentage by weight: 0.05% baicalein; 0.03% borneol; 5% MCT vegetable oil; 0.6% egg yolk lecithin E80; 1.8% Tween-80 surfactant; 2.2% glycerol as an osmotic pressure regulator; 0.01% sodium sulfite as an antioxidant;

[0099] The preparation method of this embodiment is the same as that of embodiment 1.

[0100] Embodiment 7:

[0101] A Chinese medicine microemulsion eye drop, comprising the following components in percentage by weight: 0.05% baicalein; 0.01% borneol; 5% MCT vegetable oil; 0.6% egg yolk lecithin E80; 0.9% Tween-80 surfactant; 2.2% glycerol osmotic pressure regulator; 0.01% sodium sulfite antioxidant;

[0102] The preparation method of this embodiment is the same as that of embodiment 1.

[0103] Embodiment 8:

[0104] A Chinese medicine microemulsion eye drop, comprising the following components in percentage by weight: 0.05% baicalein; 0.05% borneol; 5% MCT vegetable oil; 0.6% egg yolk lecithin E80; 0.9% Tween-80 surfactant; 2.2% glycerol osmotic pressure regulator; 0.01% sodium sulfite antioxidant;

[0105] The preparation method of this embodiment is the same as that of embodiment 1.

[0106] Comparative Example 1:

[0107] The invention discloses a baicalein microemulsion eye drop, which comprises the following components in percentage by weight: 0.05% baicalein; 5% MCT as vegetable oil; 0.6% egg yolk lecithin E80; 0.9% Tween-80 as surfactant; 2.2% glycerol as osmotic pressure regulator; and 0.01% sodium sulfite as antioxidant.

[0108] The preparation method of this embodiment is the same as that of embodiment 1.

[0109] Comparative Example 2:

[0110] The only difference between this comparative example and Example 1 is that the preparation method of the oil phase is different.

[0111] Specifically, MCT and egg yolk lecithin E80 are taken, baicalein and borneol are added, and the mixture is stirred at room temperature for 30 minutes; the aqueous phase preparation method is as follows: glycerol for injection is taken, Tween-80, sodium sulfite and about 80% purified water are added, and the mixture is stirred at 60°C until dissolved to obtain the aqueous phase; colostrum is added dropwise to the aqueous phase under a shearing condition of 3000 rpm, the rotation speed is adjusted to 16000 rpm, and the mixture is sheared for 5 minutes to obtain colostrum; final milk is prepared by adjusting the volume of the colostrum to 100 mL, transferring the colostrum to a high-pressure homogenizer, and homogenizing the mixture under a pressure of 800 bar to obtain final milk; and baicalein microemulsion eye drops containing the traditional Chinese medicine borneol are obtained by filling the mixture.

[0112] Commercially available preparations:

[0113] The main active ingredients of esculetin eye drops are digitalis glycosides and esculetin glycosides. They can improve the microcirculation of the retinal macula, enhance the delivery of retinal nutrients, restore damaged nerve cells and remove metabolites. The two ingredients work together to prevent the retina from being damaged or atrophied. Since there is no effective drug for dry age-related macular degeneration, it is used as a commercial preparation for intervention.

[0114] Application Example 1: Detection of average particle size and particle size distribution (PDI) of baicalein microemulsion eye drops of Examples 1-8 and Comparative Examples 1-2

[0115] Table 1: Average particle size of baicalein microemulsion eye drops

[0116]

[0117] Application Example 2: Stability of Baicalin Microemulsion Eye Drops from Examples 1-8 and Comparative Examples 1-2

[0118] The eye drops prepared in Examples 1-8 and Comparative Examples 1-2 were placed in an environment at a temperature of 60°C ± 2°C and a relative humidity of 75 ± 5% for 10 days, the appearance of the samples was observed, and the average particle size of the emulsion was detected. The test results are shown in Table 2 below:

[0119] Table 2: Stability test results

[0120]

[0121] Application Example 3: In vitro release study

[0122] The preparations of Example 1 and Comparative Example 1 were examined. The release medium used in this experiment was artificial tears containing 0.5% SDS and 0.2% ascorbic acid as the release medium.

[0123] The in vitro release characteristics of baicalein microemulsion were determined by transdermal diffusion instrument.

[0124] The specific experimental steps are as follows: fix the semipermeable membrane between the supply pool and the receiving pool, place a magnetic stirrer in the receiving pool, place 0.5mL of baicalin microemulsion eye drops in the supply pool, add 7.5mL to the receiving pool, place the diffusion pool in the transdermal diffusion instrument, set the temperature to 34°C, and the speed to 300rpm. Take samples at 0.5, 1, 2, 3, 4, 6, 8, and 12h, take out the release medium in the receiving pool, and add 7.5mL of fresh release medium. Determine the baicalin content in the release medium by HPLC, calculate and evaluate the in vitro release of baicalin, and conduct three parallel experiments for each sample.

[0125] Cumulative release (Q n ) and cumulative release percentage (F%) are obtained by formula (1) and formula (2) respectively:

[0126]

[0127]

[0128] Where Q n is the cumulative release at each time point; C0 is the measured drug concentration at the 0th time point; V0 is the sampling volume at the 0th time point; n is the number of samplings; C i is the measured drug concentration at the i-th time point; V i is the sampling volume at the i-th time point; F% is the cumulative release percentage at each time point; C 总 is the total drug concentration.

[0129] The sampling time was fitted with the cumulative release percentage (F%) of the drug to obtain the in vitro release curves of the baicalein solution and the baicalein microemulsion eye drops containing the traditional Chinese medicine borneol. Figure 3 .

[0130] According to the cumulative release amount and cumulative release percentage at each time point, the in vitro drug release curve was drawn, and the in vitro drug release curve was fitted by zero-order release, first-order release, Higuchi equation and Ritger-Peppas equation. The in vitro release results of baicalein microemulsion eye drops containing Chinese medicine borneol are shown in Table 1.

[0131] Table 3 In vitro release fitting results of baicalein microemulsion eye drops containing Chinese medicine borneol

[0132]

[0133] The release data of Example 1 and Comparative Example 1 were fitted with zero-order, first-order and Higuchi diffusion equations and regression analysis was performed, and the correlation coefficients of the regression equations were compared. The in vitro release fitting results showed that the in vitro release of both was more consistent with the first-order equation.

[0134] Application example 4: In vitro safety study

[0135] Example 1, Example 8, Example 9 and Comparative Example 1 were used to conduct a red blood cell hemolysis test. As an alternative to the eye irritation test (Draize test), the red blood cell hemolysis test uses the amount of hemoglobin released after the red blood cells are damaged as an indicator to evaluate the damage effect of the red blood cell membrane. It has the advantages of high sensitivity and simple operation, high reliability, and is more sensitive than the eye irritation test, and can be used to predict eye irritation.

[0136] The specific experimental steps are:

[0137] (1) Use a disposable syringe to draw about 10 mL of blood from the dorsal middle ear artery of a New Zealand white rabbit. Immediately stir with a sterilized glass rod to remove fibrinogen from the blood. Add physiological saline and mix well. Centrifuge at 2000 rpm for 10 min at 4°C. Aspirate the supernatant. Continue to add 0.9% physiological saline to wash twice under the same conditions, each time for 5 min until the supernatant no longer appears red. Measure the red blood cells and prepare a 2% red blood cell suspension with 0.9% physiological saline. Keep the operation at 4°C.

[0138] (2) Mix the red blood cell suspension with Example 1 in a test tube at a ratio of 1:1, incubate at 32°C for 30 min, terminate the reaction on ice, centrifuge at 800 rpm for 10 min at 4°C, take the supernatant, and repeat the above operation with 0.9% saline as a negative control and distilled water as a complete hemolysis control. Take 200 μL of the supernatant to a 96-well ELISA plate, measure the absorbance at 540 nm and 575 nm with an ELISA reader, and calculate the hemolysis percentage (HR).

[0139]

[0140] Example 8, Example 9 and Comparative Example 1 were used to repeat the above experimental steps instead of Example 1. The hemolysis percentage results are shown in Table 4.

[0141] Table 4: Hemolysis rate test results

[0142]

[0143] The experimental results show that when the concentration of borneol is 0.5mg / mL, the hemolysis rate is close to 100%, but some studies have shown that 1mg / mL borneol eye drops are almost non-irritating. This may be because the Draize experiment is based on whole animal experiments and has many influencing factors. Considering that the hemolysis experiment is more sensitive, the concentration of borneol used is controlled below 0.5mg / mL.

[0144] Application Example 5: In vivo safety study

[0145] The safety of the preparations of Example 1 and Comparative Example 1 was investigated. The safety of the baicalein microemulsion eye drops preparation containing borneol was investigated by using the Draize eye irritation test and pathological examination.

[0146] The specific experimental steps are:

[0147] (1) Draize eye irritation test: 50 μL of the baicalin microemulsion eye drops containing Chinese medicine borneol prepared in Example 1 was dripped into the right eye of each rabbit, and physiological saline was dripped into the left eye for comparison. After administration, the rabbit's eyes were passively closed for 8-10 seconds. The drug was administered 4 times a day, with an interval of 4 hours each time, and the administration was continued for 7 days. The eye condition before each administration and the reaction of the cornea, iris, and conjunctiva at 4 hours, 12 hours, and 24 hours after the last administration were observed. At the same time, 2% sodium fluorescein was dripped on the corneal surface after the last administration of the day, and the rabbit was manually blinked to make the sodium fluorescein evenly dispersed on the rabbit's eye surface. The eye damage was detected with cobalt blue light of a slit lamp. If there is damage on the ocular surface, green fluorescence will appear on the cornea under the irradiation of cobalt blue light. If there is no damage, green fluorescence will not be observed in the eye.

[0148] (2) Pathological examination: After the completion of the eye irritation study, all rabbits were killed by intravenous injection of air. Then, the rabbit eyeballs were immediately removed and placed in eyeball fixative. For histopathological analysis, each eye tissue was paraffin sectioned and stained with hematoxylin-eosin (H&E). The cornea, sclera, iris and conjunctiva were observed under an optical microscope for inflammatory cell infiltration, hemorrhage, necrosis, etc.

[0149] (3) In the Draize eye irritation test, the baicalein microemulsion eye drops containing borneol, a traditional Chinese medicine, were non-irritating to the cornea, iris, and conjunctiva. Figure 4 , Figure 5 As shown in the results, after 7 days of continuous administration, there was no significant difference between the baicalin microemulsion eye drops preparation group containing Chinese medicine borneol and the control group, and there was no obvious inflammatory reaction, indicating that the preparation had low eye irritation. The results further confirmed the safety of the baicalin microemulsion eye drops preparation containing Chinese medicine borneol.

[0150] Comparative Example 1 was used to replace Example 1 to repeat the above experimental steps and observe the irritation results. The results showed that Comparative Example 1 had no effect on the corneal structure and integrity.

[0151] Application example 6: Investigation of tissue distribution in rabbit eyes

[0152] The preparations of Example 1 and Comparative Example 1 were used to investigate the distribution of baicalin in rabbit eyes. The HPLC-MS / MS method was used to investigate the content of baicalein in various tissues.

[0153] The specific implementation steps are:

[0154] (1) 0.1 mL of the preparation prepared in Example 1 was dripped into the conjunctival sac of a rabbit, and the rabbit's eyelids were forced to close for 15 seconds. The eyeballs were removed 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration. The surface of the eyeball was repeatedly rinsed with physiological saline solution, the eyeball was cut open, the cornea, sclera, vitreous body, and lens were separated, and the eyeballs were dried with filter paper and set aside.

[0155] (2) Accurately weigh each separated tissue, add 5 mL of acetonitrile, homogenize, centrifuge at 4000 rpm for 10 min, take the supernatant, blow dry under a nitrogen flow, dissolve the residue in 500 μL of methanol, vortex for 5 min, centrifuge at 12000 rpm (4°C) for 10 min, and take 10 μL of the supernatant for injection.

[0156] (3) Make a drug-time curve graph and calculate the area under the curve.

[0157] (4) The baicalein microemulsion eye drops preparation of comparative example 1 was substituted for the baicalein microemulsion eye drops preparation containing borneol of the present example to investigate tissue distribution.

[0158] The tissue distribution results of different preparations in rabbit eyes over time are as follows Figure 6 The areas under the concentration-time curves of the two groups of preparations in different tissues are as follows: Figure 7 The drug-time curve results showed that the drug concentration of Example 1 in each tissue was significantly increased, and the differences between the groups were significant. Among them, the AUC of the cornea, conjunctiva, sclera, iris and vitreous 0-24 The AUC values ​​of aqueous humor, lens and retina were 2.35, 2.80, 4.17, 2.9 and 5.65 times those of comparative example 1. 0-24 The increase was more than 10 times, and the difference was statistically significant (P < 0.05). The experimental results showed that the addition of borneol can effectively increase the concentration of baicalein in the eye.

[0159] Application Example 7: Pharmacodynamics Study

[0160] The pharmacodynamics of Example 1, Comparative Example 1 and commercially available preparations were investigated. The pharmacodynamics of the preparations were investigated by comparing the pathological tissue sections, retinal cell apoptosis, retinal reactive oxygen levels and related inflammatory factor levels with those of the normal group and the modeling group.

[0161] The specific implementation steps are:

[0162] (1) 40 male C57BL / 6 mice were used. After one week of adaptive culture, 32 mice were selected to establish a dry AMD model (NaIO3 was injected into the tail vein to establish a retinal damage model). After modeling, they were randomly divided into 4 groups, 8 mice in each group, namely the modeling group, Example 1, Comparative Example 1 and the commercial preparation group. The mice in the normal group were injected with physiological saline.

[0163] (2) Drug administration began one day after modeling. Eyeballs of mice were removed 14 and 28 days after administration, and paraffin sections of various eye tissues were cut and stained with hematoxylin-eosin (H&E) for histopathological analysis. DAPI and TUNEL staining were performed to analyze retinal cell apoptosis. Frozen sections were embedded and stained with DHE fluorescent probe and DAPI to detect retinal reactive oxygen levels. The levels of inflammatory factors in the tissues were measured after eyeball homogenization to observe the therapeutic effect.

[0164] (3) Histopathological section results are shown in Figure 8 . The results showed that after 14 days of administration, the cells in each layer of the retina of the normal group of mice had good morphology, complete structure, neat arrangement of cells and uniform density. The outer plexiform layer (OPL) of the modeling group mice had collapsed, the outer nuclear layer (ONL, yellow arrow) was disordered, thinned, the cell density decreased and wavy changes, and the photoreceptor (PR) layer was observed to be disordered and thinned, and the entire retina was significantly thinned, the RPE structure was atrophied and pigmented (blue arrow). As time went on, the damage was aggravated on the 28th day. In the comparative example 1 group, there was thinning of each layer of the retina, reduced cell density and wavy changes, but there were still significant differences with the modeling group. Comparative example 1 had similar conditions to the modeling group, and phenomena such as reduced cell density could be observed, but there were still significant differences, indicating that baicalin could play a role in protecting the retina; compared with the comparative example 1 group, the damage of Example 1 and the commercially available preparation was relatively mild, and the RPE layer had plaques and reduced cell numbers rather than disappearance, and the degree of disorder and cell reduction of ONL and PR were significantly lighter, and the cells were relatively neatly arranged. These results show that the preparation of Example 1 protects the structure and morphology of the retina. After 28 days of treatment, the retinal thickness of Example 1 was greater than that of the commercially available preparation and Comparative Example 1, indicating that the preparation containing baicalein and borneol can improve its therapeutic effect.

[0165] TUNEL retinal apoptosis results are shown in Fig. 9. After 14 and 28 days of treatment, the TUNEL kit was used to detect cell apoptosis in the retina of the model mice. When cells are apoptotic, they will be marked in red, while normal cells will be marked in blue. The apoptosis in the tissue can be determined by detecting the proportion of TUNEL-stained cells. There were no apoptotic cells in the retinal sections of the normal group of mice, and there was no obvious red fluorescence. However, TUNEL+ (apoptotic) cells were detected in the INL and ONL of the other groups to varying degrees. In order to quantify the amount of cell apoptosis, the apoptotic cells and total cells in each group were counted using ImageJ software, and the apoptotic rate was calculated (see Fig.10 ). There were statistical differences between the normal group and each group (p<0.5); the number of labeled cells in the modeling group was the largest, indicating that sodium iodate would cause apoptosis in the retinal cells of mice, and the number of apoptotic cells increased over time; the apoptosis rates of the retinal parts of the mice in Example 1, the commercially available preparation and Comparative Example 1 were all lower than those in the modeling group, and the number of apoptotic cells in Example 1 and the commercially available preparation was significantly lower than that in Comparative Example 1 and the modeling group, and the difference was statistically significant (p<0.01), indicating that the preparation in Example 1 could successfully enter the fundus and inhibit the apoptosis induced by sodium iodate, which was consistent with the results of histopathological sections.

[0166] The results of retinal reactive oxygen species levels are shown in Fig.11 The fluorescence intensity was quantified as shown in Fig.12 . The results showed that the level of reactive oxygen in the modeling group was significantly higher than that in other groups. It should be pointed out that ROS was also detected in the normal control group, because the body will also produce a certain amount of ROS under healthy conditions. The experimental results showed that the fluorescence intensity of Example 1, the commercially available preparation and Comparative Example 1 were significantly lower than that of the modeling group, proving that baicalein is similar to the commercially available preparation and can quench the reactive oxygen in the eye and can remove ROS in the retina; at the same time, the fluorescence intensity of Example 1 is lower than that of Comparative Example 1, proving that borneol can enhance the anti-ROS effect of the preparation; there is no significant difference between Example 1 and the commercially available preparation, but the relative deviation of Example 1 is smaller and the precision is better. The ROS results are consistent with the results of HE staining and TUNEL.

[0167] The results of related inflammatory factor level detection are shown in Fig.13 , Fig.14 , Fig.15. Compared with the normal group, the levels of IL-6, MCP-1 and TNF-α inflammatory factors in the eyeballs of the modeling group mice were significantly increased (P<0.0001). Compared with the modeling group, after the mice were treated with different eye drop preparations, the levels of the three inflammatory factors were reduced to varying degrees, and the differences were significant. After eye drops in Example 1, the levels of the three inflammatory factors were significantly reduced (P<0.001). After eye drops in Comparative Example 1, the levels of inflammatory factors IL-6 and TNF-α were not significantly reduced, and there were significant differences compared with Example 1 (P<0.05). The results show that the eye drops in Example 1 can effectively reduce the levels of IL-6, MCP-1 and TNF-α inflammatory factors in the eye, showing good anti-inflammatory effects. In addition, the commercially available preparations did not significantly reduce the levels of inflammatory factors IL-6 and TNF-α, similar to Comparative Example 1, and both were significantly different from Example 1 (P<0.05). It is consistent with the above-mentioned efficacy experimental results.

[0168] In summary, the baicalin microemulsion eye drops containing the traditional Chinese medicine borneol prepared by the present invention can increase the corneal permeability and bioavailability of baicalin, improve the therapeutic effect, and reduce the frequency of drug use. It is expected to provide new ideas for the treatment and prevention of AMD as a potential therapeutic drug.

[0169] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Chinese medicine microemulsion eye drops, characterized in that: The composition includes the following weight percentages: 0.001% to 2.0% baicalein, 0.001% to 2.0% borneol, 0.1% to 20% injection oil, 0.1% to 10% surfactant, 0.1% to 10% phospholipid, 0.1% to 5% osmotic pressure regulator, and the balance is water.

2. The Chinese medicine microemulsion eye drops according to claim 1, characterized in that: The Chinese medicine microemulsion eye drops include the following components in percentage by weight: 0.01% to 1.0% baicalein, 0.01% to 1.0% borneol, 2% to 15% injection oil, 0.1% to 10% surfactant, 0.2% to 1.5% phospholipid, 0.5% to 3% osmotic pressure regulator, and the balance is water.

3. The Chinese medicine microemulsion eye drops according to claim 1 or 2, characterized in that: The mass ratio of the baicalein to the borneol is 10:1-1:1; The mass ratio of the baicalein to the injection oil is 1:20-1:200; The mass ratio of the injection oil to the phospholipid is 15:1-1:3; The injection oil includes one or more of MCT, LCT and structured triglycerides; The osmotic pressure regulator includes one or more of glycerol, sucrose, trehalose, xylitol and sodium chloride; The surfactant includes one or more of Tween-80, Solutol HS-15 and poloxamer; The phospholipids include one or both of glycerophospholipids and sphingomyelin; The phospholipids include one or more of natural phospholipids, semi-synthetic phospholipids and fully synthetic phospholipids; The natural phospholipids include one or more of egg yolk phospholipids, soybean phospholipids, EPG, ESM, phosphatidylinositol and myocardial phospholipids; The semi-synthetic lecithin includes hydrogenated soybean lecithin; The fully synthetic phospholipids include one or more of phosphatidylcholine, phosphatidylglycerol, myocardial phospholipids and distearoylphosphatidylethanolamine.

4. The important microemulsion eye drops according to claim 1, characterized in that: The Chinese medicine microemulsion eye drops also include an antioxidant in an amount of 0.001% to 1% by weight; The antioxidant includes one or more of sodium bisulfite, sodium sulfite, sodium thiosulfate, ascorbic acid, citric acid and disodium ethylenediaminetetraacetate.

5. The Chinese medicine microemulsion eye drops according to claim 1, characterized in that: The average particle size of the droplets in the Chinese medicine microemulsion eye drops is less than 200 nm; The pH value of the traditional Chinese medicine microemulsion eye drops is 5-9.

6. A method for preparing the Chinese medicine eye drops according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) mixing injection oil, phospholipids, baicalein and borneol to obtain an oil phase; (2) mixing an osmotic pressure regulator, a surfactant, and water to obtain an aqueous phase; (3) adding the oil phase to the water phase and mixing them at room temperature to obtain colostrum; (4) After the colostrum is mixed with water, high pressure homogenization and filtration sterilization are performed in sequence to obtain the Chinese medicine microemulsion eye drops.

7. The preparation method according to claim 6, characterized in that: In step (1), the mixing temperature is 20°C to 25°C; In step (2), the mixing temperature is 40°C to 60°C; In step (3), the mixing includes: adding the oil phase to the water phase and shearing at 15000 rpm to 20000 rpm for 5 min to 7 min; In step (4), the pressure of the high-pressure homogenization is 800 bar to 1000 bar; the number of times of the high-pressure homogenization is 15 to 25 times.

8. Use of the Chinese medicine microemulsion eye drops as claimed in any one of claims 1 to 5, or the Chinese medicine microemulsion eye drops prepared by the preparation method as claimed in any one of claims 6 to 7 in preparing drugs for treating eye diseases or preparing drugs for relieving eye fatigue.

9. The use according to claim 8, characterized in that: The drug for treating eye diseases is a drug for treating age-related macular degeneration; The drug for relieving eye fatigue is a drug for preventing dry age-related macular degeneration.

10. The use according to claim 8, characterized in that: The medicine for treating eye diseases or relieving eye fatigue is a medicine for alleviating cell oxidative damage or inhibiting cell inflammatory response.

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