CN116139083A - A kind of low concentration atropine nanoemulsion and its preparation method and application - Google Patents

Abstract

The invention provides a low-concentration atropine nanoemulsion and a preparation method and application thereof, and belongs to the technical field of medicines. The invention takes atropine as a main medicine, adopts oil and surfactant to successfully wrap the atropine in an oil phase, solves the problem of poor stability of aqueous solution of the atropine, realizes slow release of the atropine in eyes, improves the bioavailability of the medicine, reduces adverse reactions such as mydriasis, photophobia, dry eye and the like, has good biocompatibility, and solves the problem of irritation to eyes caused by overhigh local concentration of common eye drops. The nanoemulsion and the nanoemulsion with lower concentration have high bioavailability, have the effect of effectively delaying the development of myopia, and have high safety, small side effect and good application prospect.

Description

A low-concentration atropine nanoemulsion and its preparation method and application

Technical Field

The present invention relates to the field of medical technology, and in particular to a low-concentration atropine nanoemulsion and a preparation method and application thereof.

Background Art

Myopia is a phenomenon of refractive error. When the human eye is in a relaxed state, parallel light rays focus in front of the retina after passing through the eye's refractive system. This is called myopia. In terms of disease mechanism, it is known that it is mainly related to environmental factors (close work, outdoor activities, lighting, reading and writing habits, etc.) and genetic factors. According to the progression of the disease and pathological changes, it can be divided into two categories: simple myopia, which means that most patients have no pathological changes in the fundus, and the progression is slow. Vision can be corrected with appropriate lenses, and other visual function indicators are mostly normal; pathological myopia refers to pathological myopia. Pathological myopia mainly refers to the continuous increase in refractive power and continuous elongation of the eye axis in patients with high myopia with a refractive power ≥-6.00D, as well as a series of pathological changes in the posterior pole. Patients often have varying degrees of visual dysfunction.

At present, there are several main treatment methods for myopia. Simple myopia can be corrected mainly through frame glasses, corneal contact lenses and surgical correction; the treatment of related complications caused by pathological myopia can be achieved through laser photocoagulation, photodynamic therapy, anti-VEGF therapy and surgical treatment. The most widely used correction method is optical correction, which can change the refractive power of the eye's refractive surface by wearing frame glasses, contact lenses, etc. With the development of medical technology, refractive surgery has been continuously improved, and refractive surgery has also become a better choice for correcting myopia and obtaining clear and stable vision, such as laser minimally invasive femtosecond surgery; however, myopia generally deepens rapidly between the ages of 5 and 15. Most children and adolescents in this age group can only correct their vision by wearing glasses, and the anticholinergic drug low-concentration atropine has a certain control and delay effect on myopia in children and adolescents in clinical application.

At present, 0.01% atropine sulfate eye drops are widely used clinically to treat juvenile myopia. However, the delicate tissue barrier of the eye and the nasolacrimal duct clearance system result in a short retention time, poor targeting, and low bioavailability of traditional eye drops. Secondly, the ester bond in the atropine structure is easily hydrolyzed to produce biologically inactive tropic acid, which greatly reduces the efficacy of the drug and poses certain risks.

Summary of the invention

The object of the present invention is to provide a low-concentration atropine nanoemulsion and a preparation method and application thereof. The nanoemulsion of the present invention has the advantages of good drug stability, low irritation, high bioavailability and small side effects.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The invention provides a low-concentration atropine nanoemulsion. The preparation raw materials include 0.001-0.25 g atropine, 2-10 g oil, 0.2-10 g surfactant, 0.1-0.5 g co-surfactant, 0.05-3 g osmotic pressure regulator, buffer, 0.001-0.05 g metal ion chelator, pH regulator and water, based on a volume of 100 mL of water. The concentration of the buffer in the low-concentration atropine nanoemulsion is less than 100 mM.

The pH value of the low-concentration atropine nanoemulsion is 4.5 to 6.5;

In the low-concentration atropine nanoemulsion, the concentration of atropine is 0.001-0.25 wt %.

Preferably, based on a volume of 100 mL of water, the preparation raw materials include 0.001-0.1 g of atropine, 2-5 g of oil, 0.5-5 g of surfactant, 0.1-0.3 g of co-surfactant, 0.05-1.5 g of osmotic pressure regulator, buffer, 0.01-0.03 g of metal ion chelator, pH regulator and water; the concentration of the buffer in the low-concentration atropine nanoemulsion is <75 mM.

Preferably, the oil comprises one or more of medium chain oil, castor oil, soybean oil and mineral oil.

Preferably, the surfactant includes one or more of egg yolk lecithin, soybean lecithin, polyoxyethylene-40-hydrogenated castor oil, tyloxapol and polyethylene glycol-15-hydroxystearate.

Preferably, the co-surfactant includes one or more of poloxamer 188, polyethylene glycol 400 and propylene glycol.

Preferably, the osmotic pressure regulator includes one or more of glycerol, mannitol and sorbitol.

Preferably, the buffer comprises one of an acid-acid salt buffer system, a first salt-second salt buffer system and an amphoteric buffer.

Preferably, the metal ion chelator includes one or more of ethylenediaminetetraacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and penta(carboxymethyl)diethylenetriamine; or includes one or more of the salts or hydrates corresponding to ethylenediaminetetraacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and penta(carboxymethyl)diethylenetriamine.

The present invention provides a method for preparing the low-concentration atropine nanoemulsion described in the above technical solution, comprising the following steps:

dissolving a surfactant and atropine in oil to obtain an atropine oil solution;

dissolving a co-surfactant, an osmotic pressure regulator, a buffer system and a metal ion chelating agent in a portion of water to obtain an aqueous phase;

The atropine oil solution is added dropwise to the aqueous phase, and after shearing, the remaining amount of water is added to obtain a primary emulsion;

After the primary emulsion is subjected to high pressure homogenization, the pH value is adjusted to 4.5-6.5 using a pH adjuster to obtain a low concentration atropine nanoemulsion.

The present invention provides the use of the low-concentration atropine nanoemulsion described in the above technical solution or the low-concentration atropine nanoemulsion prepared by the preparation method described in the above technical solution in the preparation of ocular drugs.

The atropine nanoemulsion provided by the present invention is an oil-in-water emulsion, that is, the outside is water and the inside is oil. Atropine is wrapped in the oil phase, which can reduce the direct contact between atropine and the external water phase, thereby reducing the degradation of atropine in water; atropine is relatively stable in the pH range of 4 to 5, and the degree of degradation is deepened when the acidity or alkalinity is too strong. The external water phase of the present invention uses a pH regulator to buffer the pH of the nanoemulsion to avoid the pH of the nanoemulsion from being sharply reduced after atropine is degraded into tropic acid, further accelerating the degradation of atropine; metal ions can promote the degradation of drugs, and the addition of metal ion chelators can chelate the metal ions introduced by the instrument during the production process, thereby reducing degradation and improving stability. Therefore, the present invention can solve the problem of poor stability of atropine aqueous solution.

The nanoemulsion provided by the present invention is composed of an oil phase, a surfactant and an aqueous phase (including a co-surfactant, an osmotic pressure regulator, a buffer system, a metal ion chelating agent and water), and the atropine release process is first diffused from the internal oil phase to the external aqueous phase, and then from the external aqueous phase to the eye tissues such as tears and cornea. The present invention encapsulates atropine in the oil phase, and the oil phase has a certain viscosity, so that the diffusion speed of the internal atropine from the oil phase to the external aqueous phase is slowed down, thereby realizing the slow release of atropine in the eye.

The results of the examples show that the nanoemulsion prepared by the present invention has a nanometer-level particle size and suitable hydrophilic and lipophilic properties by screening auxiliary materials such as surfactant and stabilizer concentrations and controlling the preparation conditions (such as shear rate and time, homogenization pressure and number of times, etc.), making it easier to penetrate the epithelial cell layer (composed of 3 to 6 layers of lipid-rich and tightly connected epithelial cells, a hydrophilic drug barrier) and the matrix layer (composed of water, collagen, proteoglycans and corneal stromal cells, accounting for 90% of the corneal thickness, a lipophilic drug barrier) of the corneal barrier, and the large specific surface area promotes the absorption of atropine in the cornea. In addition, the nanoemulsion can interact with the lipid components in the tear film, prolong its retention time in the conjunctival sac, greatly improve the intraocular bioavailability, and achieve a lower concentration (the concentration of atropine in the nanoemulsion is 0.005wt%). The atropine nanoemulsion has a comparable effect of delaying myopia with the commercially available preparation of atropine sulfate eye drops (the concentration of atropine in atropine sulfate eye drops is 0.01wt%), which is conducive to reducing adverse reactions such as mydriasis, photophobia, and near vision blur caused by excessive local concentration. Furthermore, the oil used in the nanoemulsion can delay the evaporation of tears, and the phospholipids used can supplement the corresponding lipid components to avoid dry eye symptoms caused by long-term use of atropine sulfate eye drops. The atropine nanoemulsion of the present invention and the lower concentration nanoemulsion have high bioavailability, have the effect of effectively delaying the development of myopia, are highly safe, have small side effects, and have good application prospects.

The low-concentration atropine nanoemulsion provided by the present invention has a high encapsulation rate (more than 80%), a small and uniform particle size, and excellent stability. After the nanoemulsion is placed at 40°C and 25°C for accelerated and long-term tests for 6 months, the appearance and particle size distribution are not significantly changed compared with 0 days, the content of the main drug atropine is between 90% and 110% of the labeled amount, which meets the pharmacopoeia standard, and the content of the related substance--tropic acid does not exceed 0.2%, which meets the requirements of the Chinese Pharmacopoeia for the impurity limit of raw materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a transmission electron microscope (A) and an atomic force microscope (B) image of the nanoemulsion prepared in Example 3;

Figure 2 is a graph showing the test results of influencing factors of experimental group 1 (investigating phosphate concentration) (A. content; B. encapsulation efficiency; C. particle size; D. PDI);

Figure 3 is a graph showing the test results of influencing factors of experimental group 2 (investigating phosphate types) (A. content; B. encapsulation efficiency; C. particle size; D. PDI);

Figure 4 is a graph showing the test results of influencing factors of experimental group 3 (investigating EDTA-2Na concentration) (A. content; B. encapsulation efficiency; C. particle size; D. PDI);

Figure 5 is a graph showing the test results of influencing factors of experimental group 4 (investigation of final milk pH) (A. content; B. encapsulation efficiency; C. particle size; D. PDI);

FIG6 is an in vitro release curve of the atropine nanoemulsion and atropine sulfate eye drops prepared in Example 3;

7 is a diagram showing the evaluation results of the irritation test of the atropine nanoemulsion prepared in Example 3 (A. morphological observation of rabbit eyes under slit lamp; B. irritation score sheet; C. eye tissue section results after multiple administrations);

FIG8 is a graph showing the results of the investigation of anterior corneal retention in rabbit eyes (A. Example 3 group; B. atropine sulfate eye drops group);

FIG9 is a graph showing the results of the investigation of corneal permeability of rabbits (A. Example 3 group; B. atropine sulfate eye drops group; C fluorescence semi-quantitative graph);

FIG10 is a graph showing the change in tear film breakup time of guinea pigs in each group before and after administration.

DETAILED DESCRIPTION

The invention provides a low-concentration atropine nanoemulsion. The preparation raw materials include 0.001-0.25 g atropine, 2-10 g oil, 0.2-10 g surfactant, 0.1-0.5 g co-surfactant, 0.05-3 g osmotic pressure regulator, buffer, 0.001-0.05 g metal ion chelator, pH regulator and water, based on a volume of 100 mL of water. The concentration of the buffer in the low-concentration atropine nanoemulsion is less than 100 mM.

The pH value of the low-concentration atropine nanoemulsion is 4.5 to 6.5;

In the low-concentration atropine nanoemulsion, the concentration of atropine is 0.001-0.25 wt %.

In the present invention, unless otherwise specified, the required raw materials for preparation are all commercially available products well known to those skilled in the art.

The water used in the low-concentration atropine nanoemulsion of the present invention is preferably sterile water for injection.

Based on the volume of 100 mL of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.001 to 0.25 g of atropine, preferably 0.01 to 0.1 g. In the low-concentration atropine nanoemulsion of the present invention, the concentration of atropine is 0.001 to 0.25 wt%, more preferably 0.005 to 0.01 wt%.

Based on the volume of 100mL of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 2 to 10g of oil, preferably 2 to 5g. In the present invention, the oil preferably includes one or more of medium-chain oil, castor oil, soybean oil and mineral oil. When the oil is two or more of the above, the present invention has no special restrictions on the ratio of different types of oils, and can be adjusted according to actual needs. The medium-chain oil of the present invention is preferably medium-chain triglyceride.

Based on the volume of 100mL of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.2 to 10g of surfactant, preferably 0.5 to 5g, and more preferably 3 to 4g. In the present invention, the surfactant preferably includes one or more of egg yolk lecithin, soybean lecithin, polyoxyethylene-40-hydrogenated castor oil, tyloxapol and polyethylene glycol-15-hydroxystearate; the egg yolk lecithin is preferably egg yolk lecithin S100, egg yolk lecithin PC-98T or egg yolk lecithin E80. When the surfactant is two or more of the above, the present invention has no special restrictions on the ratio of different types of surfactants, and can be adjusted according to actual needs. The present invention uses phospholipids similar to cell membrane components as surfactants, realizes the slow release of atropine in the eye, improves the bioavailability of the drug, reduces adverse reactions such as mydriasis, photophobia, dry eyes, and has good biocompatibility.

Based on the volume of 100 mL of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.1 to 0.5 g of a cosurfactant, preferably 0.1 to 0.3 g. In the present invention, the cosurfactant preferably includes one or more of poloxamer 188, polyethylene glycol 400 and propylene glycol. When the cosurfactant is two or more of the above, the present invention has no special limitation on the ratio of different types of cosurfactants, and can be adjusted according to actual needs.

Based on the volume of 100mL of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.05 to 3g of an osmotic pressure regulator, preferably 0.05 to 1.5g, and more preferably 1g. In the present invention, the osmotic pressure regulator preferably includes one or more of glycerol, mannitol, and sorbitol. When the osmotic pressure regulator is two or more of the above, the present invention has no special limitation on the ratio of different types of osmotic pressure regulators, and can be adjusted according to actual needs.

Based on the volume of 100 mL of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include a buffer; the concentration of the buffer in the low-concentration atropine nanoemulsion is <100 mM, more preferably <75 mM, and further preferably 50 mM. In the present invention, the buffer preferably includes one of an acid-acid salt buffer system, a first salt-second salt buffer system, and an amphoteric buffer. In the present invention, the acid-acid salt buffer system preferably includes citric acid/sodium citrate, acetic acid/sodium acetate or boric acid/sodium borate; the first salt-second salt buffer system preferably includes sodium dihydrogen phosphate/disodium hydrogen phosphate or sodium dihydrogen phosphate/sodium citrate; the amphoteric buffer preferably includes HEPES, MOPS, PIPES or MES.

Based on the volume of 100mL of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.001 to 0.05g of metal ion chelator, preferably 0.01 to 0.03g, and more preferably 0.01g. In the present invention, the metal ion chelator preferably includes one or more of ethylenediaminetetraacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and penta(carboxymethyl)diethylenetriamine; or includes one or more of the salts or hydrates corresponding to the ethylenediaminetetraacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and penta(carboxymethyl)diethylenetriamine; the present invention has no special limitation on the salts or hydrates, and the corresponding salts or hydrates well known in the art can be used. When the metal ion chelator is two or more of the above, the present invention has no special limitation on the ratio of different types of metal ion chelators, and can be adjusted according to actual needs.

Based on the volume of 100 mL of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include a pH regulator, and the pH regulator makes the pH value of the low-concentration atropine nanoemulsion 4.5 to 6.5, more preferably 5.0. The present invention controls the pH value to ensure the stability of the low-concentration atropine nanoemulsion during storage and use and the safety of the eye drops to the eyes. In the present invention, the pH regulator is preferably hydrochloric acid or sodium hydroxide solution; the present invention has no special limitation on the concentration of the hydrochloric acid or sodium hydroxide solution, as long as the desired pH value can be reached.

The components of the low-concentration atropine nanoemulsion of the present invention are preferably made into a single-dose packaged preparation or a multi-dose packaged preparation, and the multi-dose packaged preparation preferably also includes the addition of a preservative, and the preservative preferably includes one or more of cetaxel chloride, benzalkonium chloride, benzalkonium bromide, methylparaben, ethylparaben, sorbic acid, chlorobutanol and thimerosal. When the preservative is two or more of the above, the present invention has no special restrictions on the ratio of different types of preservatives, and can be adjusted according to actual needs. The present invention has no special restrictions on the amount of the preservative added, and can be adjusted according to actual multi-dose needs.

The present invention provides a method for preparing the low-concentration atropine nanoemulsion described in the above technical solution, comprising the following steps:

dissolving a surfactant and atropine in oil to obtain an atropine oil solution;

dissolving a co-surfactant, an osmotic pressure regulator, a buffer and a metal ion chelating agent in part of the water to obtain an aqueous phase;

The atropine oil solution is added dropwise to the aqueous phase, and after shearing, the remaining amount of water is added to obtain a primary emulsion;

After the primary emulsion is subjected to high pressure homogenization, the pH value is adjusted to 4.5-6.5 using a pH adjuster to obtain a low concentration atropine nanoemulsion.

In the present invention, the temperature of the oil is preferably 55-70°C, more preferably 60°C; the partial water and the remaining water are preferably sterile water for injection; the temperature of the partial water is preferably 55-70°C, more preferably 65°C; the present invention has no special limitation on the temperature of the remaining water, room temperature is sufficient; the volume ratio of the partial water to the remaining water is preferably 4:1; the temperature of the water phase is preferably 60-75°C.

In the present invention, the shearing speed is preferably 8000-15000 rpm, more preferably 10000 rpm, and the shearing time is preferably 10 min.

In the present invention, the high-pressure homogenization treatment is preferably carried out by a homogenizer; the number of cycles of the high-pressure homogenization treatment is preferably 5 to 12 times, more preferably 10 times, and 30 times of the homogenizer pump is recorded as 1 cycle; the pressure of each high-pressure homogenization treatment is independently preferably 700 to 1000 bar, more preferably 850 bar, and the time is independently preferably 10 minutes.

After adjusting the pH value to 4.5-6.5, the present invention preferably sterilizes the obtained product through a 0.22 μm filter membrane to obtain a low-concentration atropine nanoemulsion. The present invention has no special limitation on the sterilization through a 0.22 μm filter membrane, and can be carried out according to a process well known in the art.

The present invention preferably dilutes the final emulsion of the prepared low-concentration atropine nanoemulsion by a certain multiple according to actual production conditions and requirements, and then uses ultrafiltration to further remove free drugs in the aqueous phase to improve the encapsulation rate of the nanoemulsion.

The present invention provides the use of the low-concentration atropine nanoemulsion described in the above technical solution or the low-concentration atropine nanoemulsion prepared by the preparation method described in the above technical solution in the preparation of ocular drugs. The present invention has no special limitation on the method of application, and the application can be carried out according to methods well known in the art.

The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

In Examples 1 to 14, the concentration of the buffer system in the atropine nanoemulsion is 50 mM; the medium-chain oil is medium-chain triglycerides (MCT) for injection, and the manufacturer is AVIC (Tieling) Pharmaceutical Co., Ltd.

Example 1

Atropine 0.01g; medium chain oil 2.00g; egg yolk lecithin S1003.00g; poloxamer 1880.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin S100 and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, use hydrochloric acid to adjust the final emulsion pH to 5.00, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 2

Atropine 0.01g; medium chain oil 2.00g; egg yolk lecithin PC-98T 3.00g; poloxamer 1880.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin PC-98T and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 3

Atropine 0.01g; medium chain oil 2.00g; egg yolk lecithin E80 3.00g; poloxamer 188 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 4

Atropine 0.01g; medium chain oil 2.00g; egg yolk lecithin E804.00g; poloxamer 1880.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 5

Atropine 0.01g; medium chain oil 2.00g; egg yolk lecithin E80 5.00g; poloxamer 1880.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 6

Atropine 0.01g; medium-chain oil 2.00g; egg yolk lecithin E80 1.50 g; polyethylene glycol-15-hydroxystearate 1.50g; poloxamer 188 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust the pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80, polyethylene glycol-15-hydroxystearate and atropine, dissolve them in castor oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenizing pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid solution, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 7

Atropine 0.01g; medium-chain oil 2.00g; egg yolk lecithin E80 1.50g; polyoxyethylene-40-hydrogenated castor oil 1.50g; poloxamer 188 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust the pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80, polyoxyethylene-40-hydrogenated castor oil and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 8

Atropine 0.01g; medium chain oil 5.00g; egg yolk lecithin E80 3.00g; poloxamer 188 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 9

Atropine 0.01g; medium chain oil 10.00g; egg yolk lecithin E80 3.00g; poloxamer 188 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 10

Atropine 0.01g; castor oil 2.00g; egg yolk lecithin E80 3.00g; poloxamer 188 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust the pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in castor oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Embodiment 11

Atropine 0.01g; soybean oil 2.00g; egg yolk lecithin E80 3.00g; poloxamer 188 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust the pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in soybean oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 12

Atropine 0.01g; medium chain oil 2.00g; egg yolk lecithin E80 3.00 g; polyethylene glycol 4000.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust the pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in soybean oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of polyethylene glycol 400, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Example 13

Atropine 0.01g; medium chain oil 2.00g; egg yolk lecithin E80 3.00 g; propylene glycol 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.6g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in soybean oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of propylene glycol, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize it 10 times at a homogenization pressure of 850bar, adjust the final emulsion pH to 5.00 with hydrochloric acid, and sterilize it through a 0.22μm filter membrane to obtain a nanoemulsion.

Embodiment 14

Atropine 0.01g; medium chain oil 5.00g; egg yolk lecithin E80 3.00g; poloxamer 188 0.30g; glycerol 1.00g; anhydrous disodium hydrogen phosphate 0.60g; anhydrous sodium dihydrogen phosphate 0.03g; EDTA-2Na 0.01g; hydrochloric acid to adjust pH to 5.00; water for injection 100mL.

Preparation method: accurately weigh the prescribed amount of egg yolk lecithin E80 and atropine, dissolve them in medium-chain oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amount of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous sodium hydrogen phosphate, and EDTA-2Na in 80mL of sterile water for injection at 65°C, and stir to obtain an aqueous phase; drop the atropine oil solution into the aqueous phase at 60°C, shear at a shear rate of 10000rpm for 10min, and then add the remaining 20mL of water for injection to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize 10 times at a homogenization pressure of 850bar, and adjust the pH value of the final emulsion to 5.00 with hydrochloric acid; take the final emulsion and dilute it to 10 times, use a peristaltic pump/ultrafiltration membrane bag system, adjust the peristaltic speed to 8rpm, chromatograph twice, and sterilize through a 0.22μm filter membrane to obtain a nanoemulsion after free removal.

Characterization and testing

The particle size, PDI and encapsulation efficiency of the nanoemulsions prepared in Examples 1 to 14 were tested using a Malvern laser particle size analyzer and a high performance liquid chromatograph. The results are shown in Table 1.

The encapsulation efficiency calculation formula is:

Wherein, C _total is the total drug concentration of the nanoemulsion, C _water is the free drug concentration in the external aqueous phase, V _total is the total volume of the preparation, and V _water is the volume of the external aqueous phase.

Table 1 Prescription process of low concentration atropine ophthalmic nanoemulsion

Note: PDI refers to the polydispersity index, which indicates the uniformity of particle size distribution and is generally required to be less than 0.3.

In Table 1, according to Examples 1 to 7, the surfactant egg yolk lecithin E80 as the main surfactant can improve the encapsulation rate at an amount of 3 g; according to Examples 8 to 11, it can be seen that 1 g of medium-chain oil as the oil phase can improve the encapsulation rate; according to Examples 1, 12 to 13, it can be seen that poloxamer 188 as a co-surfactant can improve the encapsulation rate.

Example 14 shows the encapsulation efficiency of the preparation after removing the free drug in the aqueous phase. The results show that the encapsulation efficiency of the nanoemulsion can be significantly improved after removing the free drug by ultrafiltration.

FIG. 1 is a transmission electron microscope (A) and an atomic force microscope (B) of the nanoemulsion prepared in Example 3; FIG. 1 shows that the prepared nanoemulsion is a uniformly distributed round sphere, and also shows that the nanoemulsion interface layer has strong rigidity and can still maintain a complete morphology under dry conditions.

Test Case

1) Influencing factor test---high temperature accelerated test

Experimental group 1: Based on Example 3, the stability of atropine ophthalmic nanoemulsion placed at 40°C for 10 days was investigated when the concentration of phosphate buffer was 20, 50, and 100 mM, respectively. Samples were taken on the 0th, 5th, and 10th days, and the concentration of the buffer was screened based on the drug content, related substance content, appearance shape, and particle size distribution of the atropine ophthalmic nanoemulsion. The results are shown in Figure 2. Too high or too low a buffer salt concentration will reduce the drug content of the emulsion. The reason is speculated that the salt concentration affects the hydration layer on the surface of the emulsion, affects the stability of the emulsion, and promotes the diffusion of the drug to the external aqueous phase; too low a buffer salt concentration weakens the buffering effect, and as atropine is degraded into a tropic acid solution, the pH decreases, further promoting the hydrolysis of atropine.

Table 2 Buffer concentrations in different buffer systems

Experimental Group 2: Based on Example 3, the stability of atropine ophthalmic nanoemulsion placed at 40°C for 10 days was investigated when the buffer was boric acid/borax and citric acid/sodium citrate. Samples were taken on the 0th, 5th and 10th days, and the types of buffers were screened based on the drug content, related substance content, appearance shape and particle size distribution of the atropine ophthalmic nanoemulsion. The results are shown in Figure 3. The stability of the buffer salts is phosphate, borate and citrate in turn. It is speculated that the possible reason is that citrate can form an ion pair with atropine, so that there is a large concentration difference between the inner and outer phases of the emulsion, which promotes the diffusion of atropine from the oil phase to the outer water phase, and accelerates the hydrolysis of atropine.

Experimental Group 3: Based on Example 3, the stability of atropine ophthalmic nanoemulsion placed at 40°C for 10 days was investigated when the concentration of the metal ion chelator was 0%, 0.01%, 0.03%, and 0.05%, respectively. Samples were taken on the 0th, 5th, and 10th days, and the concentration of the metal ion chelator was screened based on the drug content, related substance content, appearance shape, and particle size distribution of the atropine ophthalmic nanoemulsion. The results are shown in Figure 4. Compared with the 0th day, the atropine degradation rate of the preparation without the addition of the chelator was significantly higher than that of the preparation containing the chelator. Secondly, the addition of 0.01% chelator can achieve the effect of significantly delaying the hydrolysis of atropine.

Experimental Group 4: Based on Example 3, the stability of the atropine ophthalmic nanoemulsion placed at 40°C for 10 days was investigated when the final emulsion pH was 4.50, 5.00, 5.50, 6.00, and 7.40, respectively. Samples were taken on the 0th, 5th, and 10th days, and the drug content, related substance content, appearance shape, and particle size distribution of the atropine ophthalmic nanoemulsion were used as indicators to screen the effect of pH on the stability of the preparation. The experimental results are shown in Figure 5. Compared with the 0th day, the encapsulation efficiency, particle size, and PDI of each group of emulsions accelerated for 10 days did not change significantly, but the content was greatly affected by pH, and the pH 5.00 emulsion was the most stable.

Control group: Under the same conditions as above, a control group of atropine sulfate solution of equal concentration was set up, and samples were taken at the same time points to examine the drug content and the content of related substances in the control group.

The results showed that on the 10th day, the content of atropine sulfate in the control group was less than 90%, which did not meet the requirements of the pharmacopoeia; at the same time, the content of related substances was greater than 0.2% (detected by high performance liquid chromatography), and the atropine sulfate aqueous solution had undergone hydrolysis and deterioration.

2) Long-term accelerated stability test

Experimental group: The atropine nanoemulsion prepared in Example 3 was placed in a clean weighing bottle that had been equilibrated in a desiccator for 24 hours, placed at 40°C and 25°C, and samples were taken at 1, 2, 3, and 6 months, respectively. The drug content, related substance content, appearance, shape, and particle size distribution of the atropine nanoemulsion were tested.

Control group: Under the same conditions as above, a control group of atropine sulfate solution of equal concentration was set up, and samples were taken at the same time points to examine the drug content and the content of related substances in the control group.

The results are shown in Tables 3 to 6.

Table 3 Accelerated test results of atropine nanoemulsion prepared in Example 3 Batch number: 20220329-01/02 Specification: 10 mL: 1 mg Acceleration conditions: 40 ± 2 ° C, RH 75% ± 5%

Table 4 Results of accelerated test of commercially available atropine sulfate eye drops

Batch number: 210502 Specification: 0.4mL: 0.04mg Acceleration conditions: 40±2℃, RH75％±5％

Table 5 Long-term test results of atropine nanoemulsion prepared in Example 3 Batch number: 20220329-01/02 Specification: 10 mL: 1 mg Long-term conditions: 25 ± 2 ° C, RH 25% ± 5%

Table 6 Long-term test results of commercially available atropine sulfate eye drops Batch number: 210502 Specification: 0.4mL: 0.04mg Long-term conditions: 25±2℃, RH25%±5%

As shown in Tables 3 to 6, in the experimental group: the content of the main drug atropine was detected to be between 90% and 110% of the labeled amount, which met the pharmacopoeia standards; the appearance was uniform and not stratified, and the particle size distribution met the requirements, with no significant changes compared with the 0-day data; the content of the related substance - tropic acid was less than 0.2% (detected by HPLC), which was lower than that of the control group and met the requirements of the Chinese Pharmacopoeia for the impurity limits of raw materials.

Control group: At the 6th month, the content of atropine sulfate in the control group was significantly reduced, the content of related substances was greater than 0.2% (detected by HPLC), and the aqueous solution of atropine sulfate had been hydrolyzed and deteriorated.

3) In vitro release study

The Franz vertical diffusion cell method was used to investigate the in vitro release of commercially available atropine sulfate eye drops and the atropine nanoemulsion prepared in Example 3. 0.5 mL of 0.01% commercially available atropine sulfate eye drops and 0.01 wt% atropine nanoemulsion prepared in Example 3 (n=5) were precisely pipetted and placed in the supply pool, 7 mL of release medium pH 7.4 artificial tears were measured and added to the receiving pool, and a semipermeable membrane with a molecular weight cutoff of 1000 Da was placed between the supply pool and the receiving pool; the temperature was set to 35°C, the speed was 300 rpm, and 2 mL of sample was pipetted at 15, 30, 45, 60, 90, 120, 150, and 180 min, respectively, and an equal volume of isothermal fresh artificial tears was added at the same time. After the sample was filtered with a 0.22 μm filter membrane, the drug concentration was determined by high performance liquid chromatography, and the cumulative release rate Q was calculated according to the following formula:

Wherein, _Cn represents the concentration of the drug in the receiving pool at the sampling time point t, μg/mL; _Ci represents the concentration of the drug in the receiving pool at the previous time point of sampling time t, μg/mL; _V0 represents the volume of the receiving pool, i.e. 7mL, _Vr represents the volume of the sampled receiving solution (2mL), and W represents the initial total amount of drug, μg. With the sampling time point as the abscissa and the cumulative release percentage (%) as the ordinate, a fitting diagram was drawn, and the results are shown in Figure 6.

As shown in Figure 6, within 15 minutes, the release rate of the nanoemulsion prepared in Example 3 is not much different from that of the commercially available eye drops, and the cumulative release amount is about 18%, indicating that the release rate of atropine free in the external aqueous phase is similar to that of the eye drops. After 15 minutes, the release rate of the nanoemulsion slows down significantly, reflecting that the drug is successfully encapsulated in the inner oil phase or inserted into the interface membrane, so that the drug can be slowly released. The release amount is nearly 70% within 3 hours, and the release of atropine is faster, and it has been completely released within 3 hours, indicating that the nanoemulsion can achieve a longer therapeutic effect of the drug.

4) Safety evaluation of rabbit ocular administration

Three healthy Japanese white rabbits with no eye diseases and weighing 2-2.5 kg were taken. The rabbit eye conditions were observed and recorded using a slit lamp before administration. 40 μL of normal saline was instilled into the left eye as a blank control, and 40 μL of atropine nanoemulsion prepared in Example 3 was instilled into the conjunctival sac of the right eye. After application, the eyelids were gently closed for about 10 seconds. The drug was administered once a day for 7 consecutive days. The rabbit eyes were observed before and after the last administration of the drug every day, and the scores were recorded in combination with the "Eye Irritation Reaction Scoring Standard" of the State Food and Drug Administration in 2014. The results are shown in Figure 7, which is a diagram of the irritation test evaluation results of the atropine nanoemulsion prepared in Example 3 (A. Rabbit eye morphology observation under slit lamp; B. Irritation score table; C. Eye tissue section results after multiple administrations); As shown in Figure 7, there was no obvious irritation in the atropine nanoemulsion eye drops group, and there was a small amount of secretion on the eyelids during the period, but it was completely within the normal range. The irritation scores of the nanoemulsion group were all between 0 and 3, indicating that the atropine nanoemulsion eye drops were non-irritating after administration. In addition, the morphology of the rabbit eyes before administration, after a single administration eye irritation test, and after multiple administration eye irritation tests were recorded using a slit lamp. The results showed that there was no significant change in the morphology of the rabbit eyes after multiple administrations compared with the saline group, indicating that the atropine nanoemulsion eye drops were highly safe and had good biocompatibility. Compared with the saline group, no histological inflammation and toxicological changes were observed in the anterior segment of the eye (cornea, conjunctiva, iris) and the posterior segment of the eye (retina), indicating that the atropine nanoemulsion provided by the present invention is a safe ocular delivery system suitable for direct application in the conjunctival sac.

5) Fluorescence retention and corneal permeability studies

Retention study: Japanese big-eared rabbits were placed in a fixed box with their heads fixed. The lower eyelids were lifted and the conjunctival sac was pulled into a ring. The left eye was used as the test eye, and 40 μL of atropine nanoemulsion eye drops containing sodium fluorescein were instilled with a pipette; the right eye was used as the control eye, and 40 μL of atropine sulfate eye drops containing sodium fluorescein were instilled. After administration, the eyes were closed for 10 seconds, and the eyes were kept closed at fixed time intervals of 0 seconds, 3 minutes, 5 minutes, 10 minutes, 15 minutes, and 25 minutes (Group A); (0 seconds, 3 minutes, 5 minutes, 8 minutes, 10 minutes In group B, the fluorescence elimination of the eye after administration was observed under cobalt blue light under the slit lamp, and the results are shown in Figure 8. As shown in Figure 8, after administration to the rabbit eye, the fluorescence of the atropine nanoemulsion basically disappeared at 18 minutes, while the fluorescence of the atropine sulfate eye drops was almost not observed at 15 minutes after administration, and the fluorescence disappeared. Compared with the situation of the 0-minute preparation in the eye, it can be seen that the area of the nanoemulsion spread in the eye is slightly larger than that of the eye drops group. In summary, the ocular retention ability of the atropine nanoemulsion is slightly stronger than that of the atropine sulfate eye drops.

Corneal permeability study

Using luminescent DiI fluorescent dye (red) as a fluorescent marker, as a substitute for atropine, DiI-water-soluble eye drops (prepared with the excipients of atropine sulfate eye drops (excluding the active ingredient of atropine sulfate) and DiI fluorescent dye) and DiI-nanoemulsion eye drops (prepared with the excipients of atropine nanoemulsion prescription (excluding atropine) and DiI fluorescent dye in Example 3) were prepared, and the drug distribution behavior was observed using a fluorescence microscope. The specific operation is as follows: 2 Japanese big-eared rabbits were taken, and DiI-nanoemulsion eye drops were taken to the right eye and DiI-water-soluble eye drops to the left eye, respectively, and the administration was performed every 5 minutes, for a total of 3 times. The timing started from the last administration, and the rabbits were killed by injecting air into the ear vein at 30 minutes, 60 minutes, and 120 minutes, respectively, and the rabbits were quickly and completely eyeballed, fixed with eyeball fixative, and the sclera, cornea, and retina were frozen and sectioned, and the distribution of the preparation in the eye was observed by fluorescence microscopy after DAPI staining, and the fluorescence distribution was semi-quantitatively analyzed using software Image J. The results are shown in Figure 9, which is a graph of the results of the investigation of corneal permeability of rabbit eyes (A. Example 3 group; B. Atropine sulfate eye drops group; C fluorescence semi-quantitative graph). The cornea is mainly composed of the epithelium, endothelium, and stroma. As shown in Figure 9, the red fluorescence in the DiI nanoemulsion eye drops is more distributed in the corneal epithelium and stroma, and the fluorescence intensity increases with time, while the DiI water-soluble eye drops increase first with time, reaching a peak at 60min, and then decreasing, indicating that it is eliminated faster in the eye. It shows that the atropine nanoemulsion provided by the present invention can prolong the retention time of the drug in the cornea and increase the corneal permeability of the drug.

6) Pharmacodynamic studies:

Experimental grouping: Before enrollment, the eye diseases of guinea pigs in each group were screened to exclude common eye diseases and systemic diseases such as cataracts, congenital myopia, and corneal diseases. After screening for eye health and refraction (refraction of 2.0-7.0D, binocular anisometropia <2.0D, binocular pupil diameter difference <0.1mm), the guinea pigs were enrolled. Independent sample t test, the differences between the two comparisons were not statistically significant (all p>0.05), and then the guinea pigs were enrolled. They were randomly divided into a blank control group, a myopia model group, a 0.01wt% atropine nanoemulsion eye drops group (atropine nanoemulsion of Example 3), a 0.005wt% atropine nanoemulsion eye drops group (prepared with 0.01wt% atropine nanoemulsion) and atropine sulfate eye drops group, 5 in each group.

Construction of the form-deprivation guinea pig myopia model: A No. 6 translucent non-toxic latex balloon produced by Suzhou Gaofei Import and Export Trading Co., Ltd. was used as a special headgear to cover the left eye (OD) of the guinea pigs in the experimental group, and the right eye (OS) was used as the control eye. After the experiment began, the blank control group did not receive any treatment, and the other four groups of guinea pigs all covered their left eyes with latex balloons. Cut the latex balloon into a shape that fits the guinea pig's head and fix it with medical tape to ensure that the left eye is covered and the right eye is exposed. Check the coverage of the guinea pig's left eye at irregular intervals every day to confirm whether the eye mask is displaced, falls off, or blocks the contralateral eye, and whether it can completely cover the experimental eye without causing pressure on the eyeball or affecting its blinking.

Dosage regimen: 14 days after the modeling was successful, 0.005wt%, 0.01wt% atropine nanoemulsion eye drops (ATRNE) and atropine sulfate eye drops were dripped into the conjunctival sac of guinea pigs, one drop per day, about 40μL, containing 4μg of drug, for 21 days and 42 days, and the modeling state was restored after each administration, for a total of 56 days.

Measurement of biological parameters: The guinea pigs in each group were tested with YZ24 strip retinoscope and ophthalmic AB ultrasound diagnostic instrument before the experiment, 14 days after modeling, 21 days and 42 days after administration, and the refractive error (RE), anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD) and axial length (AL) were recorded.

Determination of tear film breakup time: The tear film breakup time (BUT) of each group of guinea pigs was measured before administration and on the 21st and 42nd days of administration. In a light-proof environment with normal temperature and appropriate humidity, 0.1wt% fluorescein solution was dropped on the ocular surface, and the cornea was observed with a handheld slit lamp using cobalt blue light, and a stopwatch was used to time the cornea. The time when the first black spot appeared on the corneal surface after the last blink was recorded. Each eye was measured 3 times in parallel, and the results were recorded to calculate the average value.

The results are shown in Tables 7 to 16 and Figure 10.

Table 7 Refraction (X±S, D) of experimental eyes (OD) in each group on the 0th and 14th day of modeling and the 21st and 42nd day of treatment

Table 8 Refractive power (X±S, D) of normal eyes (OS) in each group on the 0th and 14th day of modeling and the 21st and 42nd day of treatment

Table 9 Axial length of each group of experimental eyes (OD) on the 0th day, 14th day and after treatment (X±S, mm)

Table 10 Axial length of normal eyes (OS) in each group on day 0, day 14 and after treatment (X±S, mm)

Table 11 Depth of vitreous cavity of experimental eyes (OD) in each group on day 0, day 14 and after treatment (X±S, mm)

Table 12 Depth of vitreous cavity of normal eyes (OS) in each group on day 0, day 14 and after treatment (X±S, mm)

Table 13 Anterior chamber depth (X±S, mm) of each group of experimental eyes (OD) on the 0th day, 14th day and after treatment

Table 14 Anterior chamber depth (X±S, mm) of normal eyes (OS) in each group on day 0, day 14 and after treatment

Table 15 Lens thickness (X±S, mm) of each group of experimental eyes (OD) on the 0th day, 14th day and after treatment

Table 16 Lens thickness of normal eyes (OS) in each group on day 0, day 14 and after treatment (X±S, mm)

As shown in Tables 7 to 16 and Figure 10, low-concentration atropine ophthalmic nanoemulsion (0.005wt%) and atropine sulfate eye drops have a certain therapeutic effect on form deprivation guinea pig myopia, slowing down the progression of guinea pig myopia by about 1D, while delaying the growth of the eye axis. Relative to atropine sulfate eye drops (0.01wt%), the myopia effect of the 0.01wt% nanoemulsion eye drops provided by the present invention is better, indicating that the atropine nanoemulsion provided by the present invention can increase the relative bioavailability of atropine. In addition, the tear film breakup time can be increased, providing a new idea for alleviating the dry eye symptoms caused by long-term use of atropine sulfate eye drops.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

1. a low concentration atropine nanoemulsion, is characterized in that, with the volume 100mL of water as benchmark, preparation raw material comprises atropine 0.001～0.25g, oil 2～10g, surfactant 0.2～10g, cosurfactant 0.1～0.5 g, osmotic pressure regulator 0.05～3g, buffering agent, metal ion chelating agent 0.001～0.05g, pH regulator and water; The concentration of described buffering agent in low concentration atropine nanoemulsion＜100mM; The pH value of the low concentration atropine nanoemulsion is 4.5～6.5; In the low-concentration atropine nanoemulsion, the concentration of atropine is 0.001-0.25 wt%. 2. the low-concentration atropine nanoemulsion according to claim 1, is characterized in that, with the volume 100mL of water as benchmark, preparation raw material comprises atropine 0.001～0.1g, oil 2～5g, tensio-active agent 0.5～5g, auxiliary surface Active agent 0.1-0.3g, osmotic pressure regulator 0.05-1.5g, buffering agent, metal ion chelating agent 0.01-0.03g, pH regulator and water; the concentration of the buffering agent in the low-concentration atropine nanoemulsion is less than 75mM. 3. low concentration atropine nanoemulsion according to claim 1, is characterized in that, described oil comprises one or more in medium chain oil, castor oil, soybean oil and mineral oil. 4. low concentration atropine nanoemulsion according to claim 1, is characterized in that, described surfactant comprises egg yolk lecithin, soybean lecithin, polyoxyethylene-40-hydrogenated castor oil, tyloxapol and polyethylene glycol One or more of alcohol-15-hydroxystearate. 5. low concentration atropine nanoemulsion according to claim 1, is characterized in that, described cosurfactant comprises one or more in poloxamer 188, polyethylene glycol 400 and propylene glycol. 6. low concentration atropine nanoemulsion according to claim 1, is characterized in that, described osmotic pressure regulator comprises one or more in glycerol, mannitol and sorbitol. 7. low concentration atropine nanoemulsion according to claim 1, is characterized in that, described buffering agent comprises the one in acid-salt buffer system, first salt-second salt buffer system and amphoteric buffering agent. 8. low-concentration atropine nanoemulsion according to claim 1, is characterized in that, described metal ion chelating agent comprises ethylenediaminetetraacetic acid, ethylene glycol-two (beta-amino ethyl ether)-N, N, N ' , one or more of N'-tetraacetic acid and penta(carboxymethyl)diethylenetriamine; or include the ethylenediaminetetraacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N , one or more of the corresponding salts or hydrates of N,N',N'-tetraacetic acid and penta(carboxymethyl)diethylenetriamine. 9. the preparation method of the described low concentration atropine nanoemulsion of any one of claim 1～8, is characterized in that, comprises the following steps: Dissolving surfactant and atropine in oil to obtain atropine oil solution; Dissolving the co-surfactant, osmotic pressure regulator, buffer and metal ion chelating agent in part of the water to obtain an aqueous phase; Adding the atropine oil solution dropwise to the water phase, adding the remainder of water after shearing, to obtain the primary emulsion; After the primary emulsion is subjected to high-pressure homogeneous treatment, a pH regulator is used to adjust the pH value to 4.5-6.5 to obtain a low-concentration atropine nanoemulsion. 10. The application of the low-concentration atropine nanoemulsion described in any one of claims 1 to 8 or the low-concentration atropine nanoemulsion prepared by the preparation method described in claim 9 in the preparation of eye medicine.

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