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

Abstract

The present invention provides a low-concentration atropine nanoemulsion, its preparation method and application, belonging to the field of pharmaceutical technology. In the present invention, atropine is used as the main drug, and oil and surfactant are successfully used to encapsulate atropine in the oil phase, solving the problem of poor stability of atropine aqueous solution, realizing the slow release of atropine in the eye, improving the bioavailability of the drug, reducing adverse reactions such as mydriasis, photophobia, and dry eye, having good biocompatibility, and overcoming the problem of irritation to the eye caused by too high local concentration of ordinary eye drops. The nanoemulsion and lower-concentration nanoemulsion of the present invention have high bioavailability, have the effect of effectively delaying the development of myopia, have high safety, small side effects, and good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a low-concentration atropine nanoemulsion, a preparation method thereof, and an application thereof. Background Art

[0002] Myopia is a refractive error phenomenon. When the human eye is in a relaxed accommodation state, parallel light rays are focused in front of the retina after passing through the eye's refractive system, which is called myopia. In terms of disease mechanism, it is mainly known to be related to environmental factors (such as near work, outdoor activities, lighting, reading and writing habits, etc.) and genetic factors. According to the course of the disease and pathological changes, it can be divided into two categories: simple myopia, that is, most patients have no pathological changes in the fundus, the progression is slow, and the vision can be corrected with appropriate lenses, and most other visual function indicators are normal; pathological myopia mainly refers to patients with high myopia with a refractive degree ≥ -6.00D, whose refractive degree continues to increase and the eye axis continues to extend, and a series of pathological changes occur in the posterior pole. Patients often have varying degrees of impairment in visual function.

[0003] At present, the main treatment methods for myopia are as follows. The correction of simple myopia can mainly be achieved through frame glasses, contact lenses, and surgical correction; the treatment of related complications caused by pathological myopia can be through laser photocoagulation treatment, photodynamic therapy, anti-VEGF treatment, and surgical treatment. The most widely used correction method is optical correction, and the refractive power of the eye refractive surface can be changed 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 full 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 low-concentration atropine, an anticholinergic drug, has a certain control and delaying effect on the myopia of children and adolescents in clinical applications.

[0004] At present, 0.01% atropine sulfate eye drops are widely used clinically to treat adolescent myopia. However, due to the delicate tissue barrier of the eye and the nasolacrimal duct clearance system, the traditional eye drops have a short retention time, poor targeting, and low bioavailability; secondly, the ester bond in the atropine structure is easily hydrolyzed to produce tropic acid that does not have biological activity, greatly reducing the drug efficacy and posing certain potential risks. Summary of the Invention

[0005] The purpose of the present invention is to provide a low-concentration atropine nanoemulsion, a preparation method thereof, and an application thereof. The nanoemulsion of the present invention has the advantages of good drug stability, small irritation, high bioavailability, and small side effects.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a low-concentration atropine nanoemulsion. Based on 100 mL of the volume of water, the raw materials for preparation include 0.001 - 0.25 g of atropine, 2 - 10 g of oil, 0.2 - 10 g of surfactant, 0.1 - 0.5 g of co-surfactant, 0.05 - 3 g of osmotic pressure regulator, buffer, 0.001 - 0.05 g of metal ion chelator, pH regulator and water; the concentration of the buffer in the low-concentration atropine nanoemulsion < 100 mM;

[0008] The pH value of the low-concentration atropine nanoemulsion is 4.5 - 6.5;

[0009] In the low-concentration atropine nanoemulsion, the concentration of atropine is 0.001 - 0.25 wt%.

[0010] Preferably, based on 100 mL of the volume of water, the raw materials for preparation 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 < 75 mM.

[0011] Preferably, the oil includes one or more of medium-chain oil, castor oil, soybean oil and mineral oil.

[0012] Preferably, the surfactant includes one or more of egg yolk lecithin, soybean phospholipid, polyoxyethylene-40-hydrogenated castor oil, tyloxapol and polyethylene glycol-15-hydroxystearate.

[0013] Preferably, the co-surfactant includes one or more of poloxamer 188, polyethylene glycol 400 and propylene glycol.

[0014] Preferably, the osmotic pressure regulator includes one or more of glycerol, mannitol and sorbitol.

[0015] Preferably, the buffer includes one of an acid-acid salt buffer system, a first salt-second salt buffer system and an amphoteric buffer.

[0016] 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 several 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.

[0017] The present invention provides a method for preparing the low-concentration atropine nanoemulsion described in the above technical solution, comprising the following steps:

[0018] Dissolve a surfactant and atropine in oil to obtain an atropine oil solution;

[0019] Dissolve a co-surfactant, an osmotic pressure regulator, a buffer system and a metal ion chelator in a part of water to obtain an aqueous phase;

[0020] Drop the atropine oil solution into the aqueous phase, perform shearing and then add the remaining water to obtain a primary emulsion;

[0021] After subjecting the primary emulsion to high-pressure homogenization treatment, adjust the pH value to 4.5-6.5 using a pH regulator to obtain a low-concentration atropine nanoemulsion.

[0022] 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 ophthalmic drugs.

[0023] 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 encapsulated in the oil phase, which can reduce the direct contact between atropine and the external aqueous phase, thereby reducing the degradation of atropine in water; atropine is relatively stable in the pH range of 4-5, and the degradation degree becomes deeper when the acidity or alkalinity is too strong. The external aqueous phase of the present invention uses a pH regulator to buffer the pH of the nanoemulsion, avoiding the sharp decrease in the pH of the nanoemulsion after atropine degrades into tropic acid, and further accelerating the degradation of atropine; metal ions can promote the degradation of drugs. Adding a metal ion chelator can chelate the metal ions introduced by the instruments 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.

[0024] 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 chelator and water). The atropine release process is to first diffuse 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, which slows down the diffusion rate of internal atropine from the oil phase to the external aqueous phase. Therefore, slow release of atropine in the eye can be achieved.

[0025] The results of the examples show that through the screening of excipients such as the concentrations of surfactants and stabilizers, and by controlling the preparation conditions (such as shear rate and time, homogenization pressure and number of times, etc.), the nanoemulsion prepared by the present invention has a nano-scale particle size and appropriate hydrophilic-lipophilic properties, making it easier to penetrate the epithelial cell layer of the corneal barrier (constituted by 3 - 6 layers of lipid-rich and tightly connected epithelial cells, a hydrophilic drug barrier) and the stromal layer (composed of water, collagen, proteoglycans, and corneal stromal cells, accounting for 90% of the corneal thickness, a lipophilic drug barrier). 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 residence time in the conjunctival sac, greatly improve the bioavailability in the eye, and achieve the same myopia retardation effect as the commercially available preparation, atropine sulfate eye drops (the concentration of atropine in atropine sulfate eye drops is 0.01 wt%), which is beneficial to reducing adverse reactions such as mydriasis, photophobia, and blurred near vision caused by excessive local concentration. Moreover, the oil used in the nanoemulsion can delay the evaporation of tears, and the phospholipids used can supplement the corresponding lipid components, avoiding dry eye symptoms caused by long-term use of atropine sulfate eye drops. The atropine nanoemulsion and the nanoemulsion with a lower concentration described in the present invention have high bioavailability, have the effect of effectively delaying the development of myopia, have high safety, few side effects, and good application prospects.

[0026] The low-concentration atropine nanoemulsion provided by the present invention has a high encapsulation efficiency (above 80%), small and uniform particle size, and excellent stability. After accelerating and long-term tests at 40°C and 25°C for 6 months, the appearance and particle size distribution have no significant changes compared with those on day 0. The content of the main drug atropine is between 90% and 110% of the labeled amount, meeting the pharmacopoeia standards, and the content of the related substance, tropic acid, does not exceed 0.2%, meeting the requirements of the Chinese Pharmacopoeia for the impurity limit of the raw material drug. Description of the Drawings

[0027] Figure 1 Transmission electron microscope (A) and atomic force microscope image (B) of the nanoemulsion prepared in Example 3;

[0028] Figure 2 Results of the influencing factor test for Experimental Group 1 (examining phosphate concentration) (A. content; B. encapsulation efficiency; C. particle size; D. PDI);

[0029] Figure 3 Results of the influencing factor test for Experimental Group 2 (examining phosphate type) (A. content; B. encapsulation efficiency; C. particle size; D. PDI);

[0030] Figure 4Figure showing the test results of influencing factors for Experimental Group 3 (investigating the concentration of EDTA-2Na) (A. content; B. encapsulation efficiency; C. particle size; D. PDI);

[0031] Figure 5 Figure showing the test results of influencing factors for Experimental Group 4 (investigating the final emulsion pH) (A. content; B. encapsulation efficiency; C. particle size; D. PDI);

[0032] Figure 6 Figure showing the in vitro release curve of the atropine nanoemulsion and atropine sulfate eye drops prepared in Example 3;

[0033] Figure 7 Figure showing the evaluation results of the irritation test of the atropine nanoemulsion prepared in Example 3 (A. Observation of the rabbit eye morphology under a slit lamp; B. Irritation scoring table; C. Results of eye tissue sections after multiple administrations);

[0034] Figure 8 Figure showing the results of the pre-corneal retention investigation in rabbits (A. Group of Example 3; B. Group of atropine sulfate eye drops);

[0035] Figure 9 Figure showing the results of the corneal permeability investigation in rabbits (A. Group of Example 3; B. Group of atropine sulfate eye drops; C. Fluorescence semi-quantification diagram);

[0036] Figure 10 Figure showing the change results of the tear film break-up time in guinea pigs before and after administration of each group. Detailed implementation manners

[0037] The present invention provides a low-concentration atropine nanoemulsion. Based on 100 mL of the volume of water, the preparation raw materials include 0.001 - 0.25 g of atropine, 2 - 10 g of oil, 0.2 - 10 g of surfactant, 0.1 - 0.5 g of co-surfactant, 0.05 - 3 g of osmotic pressure regulator, buffer, 0.001 - 0.05 g of metal ion chelator, pH regulator and water; the concentration of the buffer in the low-concentration atropine nanoemulsion < 100 mM;

[0038] The pH value of the low-concentration atropine nanoemulsion is 4.5 - 6.5;

[0039] In the low-concentration atropine nanoemulsion, the concentration of atropine is 0.001 - 0.25 wt%.

[0040] In the present invention, unless otherwise specified, the required preparation raw materials are all commercially available products well-known to those skilled in the art.

[0041] The water used in the low-concentration atropine nanoemulsion of the present invention is preferably sterile injection water.

[0042] Based on 100 mL of the volume of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.001 - 0.25 g of atropine, preferably 0.01 - 0.1 g. In the low-concentration atropine nanoemulsion of the present invention, the concentration of atropine is 0.001 - 0.25 wt%, more preferably 0.005 - 0.01 wt%.

[0043] Based on 100 mL of the volume of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 2 - 10 g of oil, preferably 2 - 5 g. In the present invention, the oil preferably includes one or more of medium-chain oil, castor oil, soybean oil, and mineral oil. When there are two or more of the above oils, the present invention has no special limitation on the ratio of different types of oils, and it can be adjusted according to actual needs. The medium-chain oil in the present invention is preferably medium-chain triglyceride.

[0044] Based on 100 mL of the volume of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.2 - 10 g of surfactant, preferably 0.5 - 5 g, more preferably 3 - 4 g. In the present invention, the surfactant preferably includes one or more of egg yolk lecithin, soybean phospholipid, 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 there are two or more of the above surfactants, the present invention has no special limitation on the ratio of different types of surfactants, and it can be adjusted according to actual needs. The present invention uses phospholipids similar to cell membrane components as surfactants, realizing the slow release of atropine in the eye, improving the bioavailability of the drug, reducing adverse reactions such as mydriasis, photophobia, and dry eye, and having good biocompatibility.

[0045] Based on 100 mL of the volume of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.1 - 0.5 g of co-surfactant, preferably 0.1 - 0.3 g. In the present invention, the co-surfactant preferably includes one or more of poloxamer 188, polyethylene glycol 400, and propylene glycol. When there are two or more of the above co-surfactants, the present invention has no special limitation on the ratio of different types of co-surfactants, and it can be adjusted according to actual needs.

[0046] Based on 100 mL of the volume of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.05 to 3 g of an osmotic pressure regulator, preferably 0.05 to 1.5 g, and more preferably 1 g. In the present invention, the osmotic pressure regulator preferably includes one or more of glycerol, mannitol, and sorbitol. When two or more of the above-mentioned osmotic pressure regulators are used, the present invention has no special limitation on the ratio of different types of osmotic pressure regulators, and it can be adjusted according to actual needs.

[0047] Based on 100 mL of the volume 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.

[0048] Based on 100 mL of the volume of water, the raw materials for preparing the low-concentration atropine nanoemulsion provided by the present invention include 0.001 to 0.05 g of a metal ion chelator, preferably 0.01 to 0.03 g, and more preferably 0.01 g. 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 several of the salts or hydrates corresponding to the above-mentioned 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 two or more of the above-mentioned metal ion chelators are used, the present invention has no special limitation on the ratio of different types of metal ion chelators, and it can be adjusted according to actual needs.

[0049] Based on 100 mL of the volume 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 this 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, and it can reach the required pH value.

[0050] The components of the low-concentration atropine nanoemulsion described in the present invention are preferably made into single-dose packaging preparations or multi-dose packaging preparations. The multi-dose packaging preparations preferably further include the addition of preservatives, and the preservatives preferably include one or several of cetylpyridinium chloride, benzalkonium chloride, benzalkonium bromide, methylparaben, ethylparaben, sorbic acid, chlorobutanol, and thimerosal. When two or more of the above preservatives are used, the present invention has no special limitation on the ratio of different types of preservatives, and it can be adjusted according to actual needs. The present invention has no special limitation on the addition amount of the preservatives, and it can be adjusted according to actual multi-dose requirements.

[0051] The present invention provides a preparation method of the low-concentration atropine nanoemulsion described in the above technical solution, including the following steps:

[0052] Dissolve the surfactant and atropine in oil to obtain an atropine oil solution;

[0053] Dissolve the co-surfactant, osmotic pressure regulator, buffer, and metal ion chelator in part of the water to obtain an aqueous phase;

[0054] Drop the atropine oil solution into the aqueous phase, perform shearing, and then add the remaining water to obtain a primary emulsion;

[0055] After subjecting the primary emulsion to high-pressure homogenization treatment, use a pH regulator to adjust the pH value to 4.5 - 6.5 to obtain a low-concentration atropine nanoemulsion.

[0056] In the present invention, the temperature of the oil is preferably 55 - 70 °C, more preferably 60 °C; both the part of the water and the remaining water are preferably sterile injection water; the temperature of the part of the water is preferably 55 - 70 °C, more preferably 65 °C; the present invention has no special limitation on the temperature of the remaining water, and room temperature is acceptable; the volume ratio of the part of the water to the remaining water is preferably 4:1; the temperature of the aqueous phase is preferably 60 - 75 °C.

[0057] In the present invention, the shearing speed is preferably 8000 - 15000 rpm, more preferably 10000 rpm, and the time is preferably 10 min.

[0058] 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 - 12 times, more preferably 10 times, and 1 cycle is recorded when the homogenizer pump reaches 30; the pressure for each high-pressure homogenization treatment is independently preferably 700 - 1000 bar, more preferably 850 bar, and the time is independently preferably 10 min.

[0059] After adjusting the pH value to 4.5 - 6.5, the present invention preferably sterilizes the obtained product by passing it through a 0.22 μm filter membrane to obtain a low-concentration atropine nanoemulsion. The present invention has no special limitation on the sterilization by passing through a 0.22 μm filter membrane, and it can be carried out according to the processes well-known in the art.

[0060] The present invention preferably dilutes the final emulsion of the prepared low-concentration atropine nanoemulsion by a certain multiple according to the actual production conditions and requirements, and then further removes the free drug in the aqueous phase by ultrafiltration to improve the encapsulation efficiency of the nanoemulsion.

[0061] The present invention provides the application 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 ophthalmic drugs. The present invention has no special limitation on the application method, and it can be applied according to the methods well-known in the art.

[0062] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0063] In Examples 1 - 14, the concentration of the buffer system in the atropine nanoemulsion is 50 mM; the medium-chain oil is medium-chain triglycerides for injection (abbreviated as MCT). The manufacturer is Zhonghang (Tieling) Pharmaceutical Co., Ltd.

[0064] Example 1

[0065] Atropine 0.01 g; medium-chain oil 2.00 g; egg yolk lecithin S100 3.00 g; poloxamer 188 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust the pH to 5.00 with hydrochloric acid; injection water 100 mL.

[0066] Preparation method: Accurately weigh the prescription amount of egg yolk lecithin S100 and atropine, dissolve them in medium-chain triglyceride at 60 °C, and stir to obtain an atropine oil solution; dissolve the prescription amount of poloxamer 188, glycerol, sodium dihydrogen phosphate anhydrous, disodium hydrogen phosphate anhydrous, and EDTA-2Na in 80 mL 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 10000 rpm for 10 min, then add the remaining 20 mL 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 850 bar, adjust the pH value of the final emulsion to 5.00 with hydrochloric acid, and sterilize by passing through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0067] Example 2

[0068] Atropine 0.01 g; medium-chain triglyceride 2.00 g; egg yolk lecithin PC-98T 3.00 g; poloxamer 188 0.30 g; glycerol 1.00 g; disodium hydrogen phosphate anhydrous 0.60 g; sodium dihydrogen phosphate anhydrous 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; water for injection 100 mL.

[0069] Preparation method: Accurately weigh the prescription amount of egg yolk lecithin PC-98T and atropine, dissolve them in medium-chain triglyceride at 60 °C, and stir to obtain an atropine oil solution; dissolve the prescription amount of poloxamer 188, glycerol, sodium dihydrogen phosphate anhydrous, disodium hydrogen phosphate anhydrous, and EDTA-2Na in 80 mL 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 10000 rpm for 10 min, then add the remaining 20 mL 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 850 bar, adjust the pH value of the final emulsion to 5.00 with hydrochloric acid, and sterilize by passing through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0070] Example 3

[0071] Atropine 0.01 g; medium-chain triglyceride 2.00 g; egg yolk lecithin E80 3.00 g; poloxamer 188 0.30 g; glycerol 1.00 g; disodium hydrogen phosphate anhydrous 0.60 g; sodium dihydrogen phosphate anhydrous 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; water for injection 100 mL.

[0072] Preparation method: Accurately weigh the prescribed amounts of egg yolk lecithin E80 and atropine, dissolve them in medium-chain triglyceride at 60 °C, and stir to obtain an atropine oil solution; dissolve the prescribed amounts of poloxamer 188, glycerol, sodium dihydrogen phosphate anhydrous, disodium hydrogen phosphate anhydrous, and EDTA-2Na in 80 mL of sterile injection water 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 10000 rpm for 10 min, then add the remaining 20 mL of injection water to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize 10 times at a homogenization pressure of 850 bar, adjust the pH value of the final emulsion to 5.00 with hydrochloric acid, and sterilize through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0073] Example 4

[0074] Atropine 0.01 g; Medium-chain triglyceride 2.00 g; Egg yolk lecithin E80 4.00 g; Poloxamer 188 0.30 g; Glycerol 1.00 g; Disodium hydrogen phosphate anhydrous 0.60 g; Sodium dihydrogen phosphate anhydrous 0.03 g; EDTA-2Na 0.01 g; Adjust pH to 5.00 with hydrochloric acid; Injection water 100 mL.

[0075] Preparation method: Accurately weigh the prescribed amounts of egg yolk lecithin E80 and atropine, dissolve them in medium-chain triglyceride at 60 °C, and stir to obtain an atropine oil solution; dissolve the prescribed amounts of poloxamer 188, glycerol, sodium dihydrogen phosphate anhydrous, disodium hydrogen phosphate anhydrous, and EDTA-2Na in 80 mL of sterile injection water 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 10000 rpm for 10 min, then add the remaining 20 mL of injection water to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize 10 times at a homogenization pressure of 850 bar, adjust the pH value of the final emulsion to 5.00 with hydrochloric acid, and sterilize through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0076] Example 5

[0077] Atropine 0.01 g; Medium-chain triglyceride 2.00 g; Egg yolk lecithin E80 5.00 g; Poloxamer 188 0.30 g; Glycerol 1.00 g; Disodium hydrogen phosphate anhydrous 0.60 g; Sodium dihydrogen phosphate anhydrous 0.03 g; EDTA-2Na 0.01 g; Adjust pH to 5.00 with hydrochloric acid; Injection water 100 mL.

[0078] Preparation method: Accurately weigh the prescribed amounts of egg yolk lecithin E80 and atropine and dissolve them in medium-chain triglyceride at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amounts of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL 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 10,000 rpm for 10 min, then add the remaining 20 mL 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 850 bar, adjust the pH of the final emulsion to 5.00 with hydrochloric acid, and sterilize by passing through a 0.22-μm filter membrane to obtain the nanoemulsion.

[0079] Example 6

[0080] Atropine 0.01 g; medium-chain triglyceride 2.00 g; egg yolk lecithin E80 1.50 g; polyethylene glycol-15-hydroxystearate 1.50 g; poloxamer 188 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust the pH to 5.00 with hydrochloric acid; water for injection 100 mL.

[0081] Preparation method: Accurately weigh the prescribed amounts of egg yolk lecithin E80, polyethylene glycol-15-hydroxystearate, and atropine and dissolve them in castor oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amounts of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL 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 10,000 rpm for 10 min, then add the remaining 20 mL 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 850 bar, adjust the pH of the final emulsion to 5.00 with hydrochloric acid solution, and sterilize by passing through a 0.22-μm filter membrane to obtain the nanoemulsion.

[0082] Example 7

[0083] Atropine 0.01 g; medium-chain triglyceride 2.00 g; egg yolk lecithin E80 1.50 g; polyoxyl 40 hydrogenated castor oil 1.50 g; poloxamer 188 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust the pH to 5.00 with hydrochloric acid; water for injection 100 mL.

[0084] Preparation method: Accurately weigh the prescribed amounts of egg yolk lecithin E80, polyoxyl 40 hydrogenated castor oil, and atropine, and dissolve them in medium-chain triglyceride at 60 °C. Stir to obtain an atropine oil solution. Dissolve the prescribed amounts of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL 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 10,000 rpm for 10 min, then add the remaining 20 mL 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 850 bar, adjust the pH of the final emulsion to 5.00 with hydrochloric acid, and sterilize by passing through a 0.22-μm filter membrane to obtain the nanoemulsion.

[0085] Example 8

[0086] Atropine 0.01 g; medium-chain triglyceride 5.00 g; egg yolk lecithin E80 3.00 g; poloxamer 188 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; water for injection 100 mL.

[0087] Preparation method: Accurately weigh the prescribed amounts of egg yolk lecithin E80 and atropine, and dissolve them in medium-chain triglyceride at 60 °C. Stir to obtain an atropine oil solution. Dissolve the prescribed amounts of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL 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 10,000 rpm for 10 min, then add the remaining 20 mL 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 850 bar, adjust the pH of the final emulsion to 5.00 with hydrochloric acid, and sterilize by passing through a 0.22-μm filter membrane to obtain the nanoemulsion.

[0088] Example 9

[0089] Atropine 0.01 g; medium-chain triglyceride 10.00 g; egg yolk lecithin E80 3.00 g; poloxamer 188 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; water for injection 100 mL.

[0090] Preparation method: Accurately weigh the prescribed amounts of egg yolk lecithin E80 and atropine and dissolve them in medium-chain triglyceride at 60 °C, and stir to obtain an atropine oil solution; dissolve the prescribed amounts of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL 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 10,000 rpm for 10 min, then add the remaining 20 mL 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 850 bar, adjust the pH value of the final emulsion to 5.00 with hydrochloric acid, and sterilize through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0091] Example 10

[0092] Atropine 0.01 g; castor oil 2.00 g; egg yolk lecithin E80 3.00 g; poloxamer 188 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; water for injection 100 mL.

[0093] Preparation method: Accurately weigh the prescribed amounts of egg yolk lecithin E80 and atropine and dissolve them in castor oil at 60 °C, and stir to obtain an atropine oil solution; dissolve the prescribed amounts of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL 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 10,000 rpm for 10 min, then add the remaining 20 mL 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 850 bar, adjust the pH value of the final emulsion to 5.00 with hydrochloric acid, and sterilize through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0094] Example 11

[0095] Atropine 0.01 g; soybean oil 2.00 g; egg yolk lecithin E80 3.00 g; poloxamer 188 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; water for injection 100 mL.

[0096] Preparation method: Weigh accurately the prescribed amounts of egg yolk lecithin E80 and atropine and dissolve them in soybean oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amounts of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL of sterile injection water 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 10,000 rpm for 10 min, then add the remaining 20 mL of injection water to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize 10 times at a homogenization pressure of 850 bar, adjust the pH of the final emulsion to 5.00 with hydrochloric acid, and sterilize by passing through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0097] Example 12

[0098] Atropine 0.01 g; medium-chain oil 2.00 g; egg yolk lecithin E80 3.00 g; polyethylene glycol 400 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; injection water 100 mL.

[0099] Preparation method: Weigh accurately the prescribed amounts of egg yolk lecithin E80 and atropine and dissolve them in soybean oil at 60°C, and stir to obtain an atropine oil solution; dissolve the prescribed amounts of polyethylene glycol 400, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL of sterile injection water 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 10,000 rpm for 10 min, then add the remaining 20 mL of injection water to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize 10 times at a homogenization pressure of 850 bar, adjust the pH of the final emulsion to 5.00 with hydrochloric acid, and sterilize by passing through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0100] Example 13

[0101] Atropine 0.01 g; medium-chain oil 2.00 g; egg yolk lecithin E80 3.00 g; propylene glycol 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.6 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; injection water 100 mL.

[0102] Preparation method: Weigh accurately the prescribed amounts 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 amounts of propylene glycol, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL of sterile injection water 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 10000 rpm for 10 min, then add the remaining 20 mL of injection water to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize 10 times at a homogenization pressure of 850 bar, adjust the pH value of the final emulsion to 5.00 with hydrochloric acid, and sterilize by passing through a 0.22 μm filter membrane to obtain the nanoemulsion.

[0103] Example 14

[0104] Atropine 0.01 g; medium-chain oil 5.00 g; egg yolk lecithin E80 3.00 g; poloxamer 188 0.30 g; glycerol 1.00 g; anhydrous disodium hydrogen phosphate 0.60 g; anhydrous sodium dihydrogen phosphate 0.03 g; EDTA-2Na 0.01 g; adjust pH to 5.00 with hydrochloric acid; injection water 100 mL.

[0105] Preparation method: Weigh accurately the prescribed amounts 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 amounts of poloxamer 188, glycerol, anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, and EDTA-2Na in 80 mL of sterile injection water 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 10000 rpm for 10 min, then add the remaining 20 mL of injection water to obtain a primary emulsion; transfer the primary emulsion to a high-pressure homogenizer, homogenize 10 times at a homogenization pressure of 850 bar, adjust the pH value of the final emulsion to 5.00 with hydrochloric acid; dilute the final emulsion by 10 times, use a peristaltic pump / ultrafiltration membrane package system, adjust the peristaltic speed to 8 rpm, chromatograph 2 times, and sterilize by passing through a 0.22 μm filter membrane to obtain the nanoemulsion after removing the free form.

[0106] Characterization and testing

[0107] Use a Malvern laser particle size analyzer and a high-performance liquid chromatograph to test the particle size, PDI, and encapsulation efficiency of the nanoemulsions prepared in Examples 1 to 14. The results are shown in Table 1.

[0108] The formula for calculating the encapsulation efficiency is:

[0109]

[0110] where 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, Vwater is the volume of the external aqueous phase.

[0111] Table 1 Prescription process of atropine ophthalmic nanoemulsion with low concentration

[0112]

[0113] Note: PDI refers to the Polydispersity Index, which represents the degree of uniformity of particle size distribution. Generally, it is required to be less than 0.3.

[0114] In Table 1, according to Examples 1 to 7, it can be seen that the surfactant egg yolk lecithin E80 as the main surfactant, with a dosage of 3 g, can improve the encapsulation efficiency; 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 efficiency; according to Examples 1, 12 to 13, it can be seen that poloxamer 188 as the co-surfactant can improve the encapsulation efficiency.

[0115] Example 14 is the encapsulation efficiency of the preparation after removing the free drug in the external aqueous phase. The results show that the encapsulation efficiency of the nanoemulsion can be significantly improved by removing the free drug through the ultrafiltration method.

[0116] Figure 1 are the transmission electron microscope (A) and atomic force microscope images (B) of the nanoemulsion prepared in Example 3; from Figure 1 it can be seen that the prepared nanoemulsion is a uniformly distributed round sphere, and it also shows that the interface layer of the nanoemulsion has strong rigidity and can still maintain a complete morphology under dry conditions.

[0117] Test examples

[0118] 1) Influence factor test --- High-temperature accelerated test

[0119] Experimental group 1: Based on Example 3, the stability of atropine ophthalmic nanoemulsion at 40 °C for 10 days was investigated when the concentrations of phosphate buffer were 20, 50, and 100 mM 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 atropine ophthalmic nanoemulsion were used as indicators to screen the concentration of the buffer. The results are as Figure 2 shown. Too high or too low buffer salt concentration will reduce the drug content of the emulsion. It is speculated that the reason is that the salt concentration affects the hydration layer on the surface of the emulsion, affecting the stability of the emulsion and promoting the diffusion of the drug to the external aqueous phase; too low buffer salt concentration weakens the buffering effect, and as atropine degrades to tropic acid, the pH decreases, further promoting the hydrolysis of atropine.

[0120] Table 2 Concentrations of buffer agents under different buffer systems

[0121]

[0122] Experimental group 2: Based on Example 3, when the buffers were boric acid / borax and citric acid / sodium citrate, the stability of atropine ophthalmic nanoemulsion stored at 40 °C for 10 days was investigated. Samples were taken on days 0, 5, and 10 respectively, and the drug content, related substance content, appearance, and particle size distribution of atropine ophthalmic nanoemulsion were used as indicators to screen the types of buffers. The results are as Figure 3 shown. The stabilizing abilities of buffer salts were in the order of phosphate, borate, and citrate. It is speculated that the possible reason is that citrate can form an ion pair with atropine, resulting in 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 aqueous phase and accelerates the hydrolysis of atropine.

[0123] Experimental group 3: Based on Example 3, when the concentrations of metal ion chelating agents were 0%, 0.01%, 0.03%, and 0.05% respectively, the stability of atropine ophthalmic nanoemulsion stored at 40 °C for 10 days was investigated. Samples were taken on days 0, 5, and 10 respectively, and the drug content, related substance content, appearance, and particle size distribution of atropine ophthalmic nanoemulsion were used as indicators to screen the concentrations of metal ion chelating agents. The results are as Figure 4 shown. Compared with day 0, the degradation rate of atropine in the formulation without chelating agent was significantly higher than that of the formulations containing chelating agent. Secondly, adding 0.01% chelating agent could achieve an obvious effect of delaying the hydrolysis of atropine.

[0124] Experimental group 4: Based on Example 3, when the final emulsion pH values were 4.50, 5.00, 5.50, 6.00, and 7.40 respectively, the stability of atropine ophthalmic nanoemulsion stored at 40 °C for 10 days was investigated. Samples were taken on days 0, 5, and 10 respectively, and the drug content, related substance content, appearance, and particle size distribution of atropine ophthalmic nanoemulsion were used as indicators to screen the influence of pH on the formulation stability. The experimental results are as Figure 5 shown. Compared with day 0, there were no obvious changes in the entrapment efficiency, particle size, and PDI of each group of emulsions after 10 days of acceleration, but the content was greatly affected by pH, and the emulsion with pH 5.00 was the most stable.

[0125] Control group: Under the same above conditions, an equal-concentration atropine sulfate solution control group was set up, and samples were taken at the same time points to investigate the drug content and related substance content of the control group.

[0126] The results showed that at day 10, the content of atropine sulfate in the control group was less than 90%, which did not meet the pharmacopoeia requirements; meanwhile, the related substance content was greater than 0.2% (detected by high-performance liquid chromatography), and the atropine sulfate aqueous solution had hydrolyzed and deteriorated.

[0127] 2) Long-term accelerated stability test

[0128] Experimental group: The atropine nanoemulsion prepared in Example 3 above was placed in a clean weighing bottle that had been equilibrated in a desiccator for 24 h, and placed at 40 °C and 25 °C. Samples were taken at the 1st, 2nd, 3rd, and 6th months respectively. The drug content, related substance content, appearance, and particle size distribution of the atropine nanoemulsion were tested.

[0129] Control group: Under the same conditions as above, a control group of atropine sulfate solution with the same concentration was set up, and samples were taken at the same time points to investigate the drug content and related substance content of the control group.

[0130] The results are shown in Tables 3 - 6.

[0131] Table 3 Accelerated test results of the atropine nanoemulsion prepared in Example 3 Batch number: 20220329 - 01 / 02 Specification: 10 mL: 1 mg Accelerated conditions: 40 ± 2 °C, RH 75% ± 5%

[0132]

[0133]

[0134] Table 4 Accelerated test results of commercially available atropine sulfate eye drops

[0135] Batch number: 210502 Specification: 0.4 mL: 0.04 mg Accelerated conditions: 40 ± 2 °C, RH 75% ± 5%

[0136]

[0137] Table 5 Long - term test results of the 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%

[0138]

[0139] Table 6 Long - term test results of commercially available atropine sulfate eye drops Batch number: 210502 Specification: 0.4 mL: 0.04 mg Long - term conditions: 25 ± 2 °C, RH 25% ± 5%

[0140]

[0141]

[0142] As can be seen from 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, meeting the pharmacopoeia standards; the appearance was uniform without stratification, and the particle size distribution met the requirements, showing no significant changes compared with the data at day 0; the content of the related substance tropic acid was less than 0.2% (detected by high performance liquid chromatography), lower than that in the control group, and meeting the requirements of the Chinese Pharmacopoeia for the impurity limits of the active pharmaceutical ingredient.

[0143] In the control group: at the 6th month, the content of atropine sulfate in the control group decreased significantly, 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.

[0144] 3) In vitro release study

[0145] 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 accurately pipetted into the supply cell, 7 mL of the release medium, artificial tears with pH 7.4, was measured and added to the receiving cell. A semi-permeable membrane with a molecular weight cut-off of 1000 Da was placed between the supply cell and the receiving cell; the temperature was set at 35 °C and the rotation speed was 300 rpm. 2 mL of the sample was pipetted at 15, 30, 45, 60, 90, 120, 150, and 180 min respectively, and an equal volume of fresh artificial tears at the same temperature was added as a supplement. After filtering the sample through a 0.22 μm filter membrane, the drug concentration was determined by high performance liquid chromatography, and the cumulative release Q was calculated according to the following formula:

[0146]

[0147] where C n represents the concentration of the drug in the receiving cell at the sampling time point t, μg / mL; C i is the concentration of the drug in the receiving cell at the previous time point before the sampling time t, μg / mL; V0 represents the volume of the receiving cell, which is 7 mL, and V r represents the volume of the received liquid sampled (2 mL), and W is the total initial amount of the drug, μg. With the sampling time point as the abscissa and the cumulative release percentage (%) as the ordinate, a fitting graph was made, and the results are as Figure 6 shown.

[0148] From Figure 6It can be seen that 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%. This indicates that the release rate of atropine in the external aqueous phase is similar to that of the eye drops. After 15 minutes, the release rate of the nanoemulsion significantly slows down, reflecting that the drug has been successfully encapsulated in the internal oil phase or inserted into the interfacial film, enabling the drug to be slowly released. The release amount within 3 hours is nearly 70%. The release of atropine is relatively fast and is completely released within 3 hours, indicating that the nanoemulsion can achieve a longer therapeutic effect of the drug.

[0149] 4) Safety evaluation of drug administration to rabbit eyes

[0150] Three healthy Japanese white rabbits with no eye diseases and a body weight of 2 - 2.5 kg were selected. Before drug administration, the eyes of the rabbits were observed and recorded using a slit lamp. 40 μL of normal saline was instilled into the left eye as a blank control, and 40 μL of the atropine nanoemulsion prepared in Example 3 was instilled into the conjunctival sac of the right eye. After instillation, the eyelids were gently closed for about 10 s. The drug was administered once a day for 7 consecutive days. The eyes of the rabbits were observed before each drug administration and after the last drug administration. The scores were recorded according to the "Eye Irritation Response Score Standard" of the National Medical Products Administration in 2014. The results are as Figure 7 shown, Figure 7 Figure showing the evaluation results of the irritation test of the atropine nanoemulsion prepared in Example 3 (A. Observation of the rabbit eye morphology under a slit lamp; B. Irritation score table; C. Results of eye tissue sections after multiple drug administrations); It can be Figure 7 seen that there was no obvious irritation in the atropine nanoemulsion eye drop group. There was a small amount of secretion on the eyelids during the period, but it was completely within the normal range. The irritation scores in the nanoemulsion group were all between 0 and 3, indicating that there was no irritation after administration of the atropine nanoemulsion eye drops. In addition, the morphology of the rabbit eyes before drug administration, after a single-dose eye irritation test, and after multiple-dose eye irritation tests was photographed using a slit lamp. The results showed that there was no significant change in the morphology of the rabbit eyes after multiple drug administrations compared with the normal saline group, indicating that the atropine nanoemulsion eye drops have high safety and good biocompatibility. Compared with the normal saline group, no histological inflammation and toxicological changes were observed in the anterior segment (cornea, conjunctiva, iris) and posterior segment (retina) of the eye; it shows that the atropine nanoemulsion provided by the present invention is a safe ocular delivery system and is suitable for direct application to the conjunctival sac.

[0151] 5) Study on fluorescence retention and corneal permeability

[0152] Retention study: Place Japanese white rabbits in a fixed box, fix their heads, lift the lower eyelids, pull the conjunctival sac into a ring, use the left eye as the test eye, and drop 40 μL of atropine nanoemulsion eye drops containing sodium fluorescein into it with a pipette; use the right eye as the control eye and drop 40 μL of atropine sulfate eye drops containing sodium fluorescein. After administration, close the eyes for 10 s, and observe the elimination of fluorescence in the eyes under cobalt blue light at fixed time intervals of 0 s, 3 min, 5 min, 10 min, 15 min, 25 min (Group A); (0 s, 3 min, 5 min, 8 min, 10 min, 15 min (Group B). The results are as follows Figure 8 shown. It can be seen from Figure 8 the figure that after eye drops administration to rabbits, the fluorescence of atropine nanoemulsion basically disappeared at 18 min, while almost no fluorescence could be observed in the atropine sulfate eye drops at 15 min after administration. Comparing the situation of the 0 min preparation in the eye, it can be seen that the spreading area of the nanoemulsion in the eye is slightly larger than that of the eye drops group. In summary, the eye retention ability of atropine nanoemulsion is slightly stronger than that of atropine sulfate eye drops.

[0153] Corneal permeability study

[0154] Use the luminescent DiI fluorescent dye (showing red) as a fluorescent marker and as an atropine substitute. Prepare DiI-water-soluble eye drops (prepared from the excipients of atropine sulfate eye drops (excluding the active ingredient of atropine sulfate) and DiI fluorescent dye) and DiI-nanoemulsion eye drops (prepared from the excipients of the atropine nanoemulsion in Example 3 (excluding atropine) and DiI fluorescent dye) respectively, and observe the drug distribution behavior with a fluorescence microscope. The specific operation is as follows: Take 2 Japanese white rabbits, instill DiI-nanoemulsion eye drops into the right eye and DiI-water-soluble eye drops into the left eye respectively, administer the drugs every 5 min, and administer the drugs 3 times in total. Start timing from the last administration. At 30 min, 60 min, and 120 min, inject air into the marginal ear vein to sacrifice the rabbits quickly and remove the eyeballs intact, fix them with an eyeball fixative, make tissue cryosections of the sclera, cornea, and retina, observe the distribution of the preparation in the eye under a fluorescence microscope after DAPI staining, and perform semi-quantitative analysis of the fluorescence distribution using the software Image J. The results are as follows Figure 9 shown, Figure 9 which is the result diagram of the corneal permeability investigation of rabbits (A. Group of Example 3; B. Atropine sulfate eye drops group; C. Fluorescence semi-quantitative diagram). The cornea is mainly composed of the epithelium, endothelium, and stroma. It can be seen from Figure 9It can be seen that more red fluorescence in the DiI nanoemulsion eye drops is distributed in the corneal epithelium and stromal layer, and with the increase of time, the fluorescence intensity increases. While the DiI water-soluble eye drops first increase with the prolongation of time, reach the peak at 60 min, and then show a decreasing trend, indicating that it is eliminated faster in the eye. This shows that the atropine nanoemulsion provided by the present invention can prolong the residence time of the drug in the cornea and increase the corneal permeability of the drug.

[0155] 6) Pharmacodynamic study:

[0156] Experimental grouping: Before enrollment, the ocular diseases of guinea pigs in each group were screened to exclude common ocular diseases and systemic diseases such as cataracts, congenital myopia, and corneal diseases. After screening for eye health and refractive power (refractive power of 2.0 - 7.0 D, binocular anisometropia < 2.0 D, binocular pupil diameter difference < 0.1 mm), they were enrolled. After independent sample t-tests showed no significant differences in pairwise comparisons (all p > 0.05), they were enrolled. They were randomly divided into a blank control group, a myopia model group, a 0.01 wt% atropine nanoemulsion eye drop group (the atropine nanoemulsion of Example 3), a 0.005 wt% atropine nanoemulsion eye drop group (prepared with the 0.01 wt% atropine nanoemulsion), and a tropicamide eye drop group, with 5 in each group.

[0157] Construction of form-deprivation myopia model in guinea pigs: A No. 6 semi-transparent 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 experimental guinea pigs, and the right eye (OS) was used as the self-control eye. After the experiment started, the blank control group was not treated with anything, and the left eyes of the other four groups of guinea pigs were covered with latex balloons. The latex balloon was cut into a shape that fit the guinea pig's head and fixed with medical tape to ensure that the left eye was covered and the right eye was exposed. The covering condition of the left eye of the guinea pigs was checked irregularly every day to confirm whether the eye mask had shifted, fallen off, blocked the contralateral eye, and could completely cover the experimental eye without pressing on the eyeball and not affecting its blinking.

[0158] Drug administration plan: Starting from 14 days after modeling (i.e., after successful modeling), 0.005 wt%, 0.01 wt% atropine nanoemulsion eye drops (ATRNE), and tropicamide eye drops were instilled into the conjunctival sac of the guinea pigs, one drop per day, about 40 μL, with a drug content of 4 μg. They were continuously administered for 21 days and 42 days, and the modeling state was restored after each administration, for a total of 56 days.

[0159] Measurement of biological parameters: Before the experiment, 14 days after modeling, on the 21st and 42nd days after administration, the guinea pigs in each group were refracted using a YZ24 streak retinoscope and an ophthalmic A / B ultrasonic diagnostic instrument, and the refractive error (RE), anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL) were recorded.

[0160] Measurement of tear film break-up time: The tear film break-up time (BUT) of the guinea pigs in each group was measured before administration and on the 21st and 42nd days after administration. In an environment with normal temperature, appropriate humidity, and light avoidance, a 0.1 wt% fluorescein solution was dropped onto the ocular surface. The slit lamp was held and the cornea was observed using cobalt blue light. At the same time, a stopwatch was used to time, and the time when the first black spot appeared on the corneal surface after the last blink was recorded. Each eye was measured in parallel 3 times, and the average value was calculated from the recorded results.

[0161] The results are shown in Tables 7 - 16 and Figure 10 。

[0162] Table 7 Refractive error (X±S, D) of the experimental eyes (OD) in each group on the 0th, 14th day of modeling, and the 21st and 42nd days of treatment

[0163]

[0164] Table 8 Refractive error (X±S, D) of the normal eyes (OS) in each group on the 0th, 14th day of modeling, and the 21st and 42nd days of treatment

[0165]

[0166] Table 9 Axial length (X±S, mm) of the experimental eyes (OD) in each group on the 0th, 14th day of modeling, and after treatment

[0167]

[0168]

[0169] Table 10 Axial length (X±S, mm) of the normal eyes (OS) in each group on the 0th, 14th day of modeling, and after treatment

[0170]

[0171] Table 11 Vitreous chamber depth (X±S, mm) of the experimental eyes (OD) in each group on the 0th, 14th day of modeling, and after treatment

[0172]

[0173]

[0174] Table 12 Vitreous cavity depth of normal eyes (OS) in each group on the 0th day, 14th day of model establishment and after treatment (X±S, mm)

[0175]

[0176] Table 13 Anterior chamber depth of experimental eyes (OD) in each group on the 0th day, 14th day of model establishment and after treatment (X±S, mm)

[0177]

[0178] Table 14 Anterior chamber depth of normal eyes (OS) in each group on the 0th day, 14th day of model establishment and after treatment (X±S, mm)

[0179]

[0180] Table 15 Lens thickness of experimental eyes (OD) in each group on the 0th day, 14th day of model establishment and after treatment (X±S, mm)

[0181]

[0182] Table 16 Lens thickness of normal eyes (OS) in each group on the 0th day, 14th day of model establishment and after treatment (X±S, mm)

[0183]

[0184]

[0185] From Tables 7 - 16, Figure 10 it can be seen that both the low - concentration atropine ophthalmic nanoemulsion (0.005 wt%) and atropine sulfate eye drops have a certain therapeutic effect on form - deprivation myopia in guinea pigs, slowing down the myopia progression of guinea pigs by about 1 D and simultaneously delaying the axial length growth of the eyes. Compared with atropine sulfate eye drops (0.01 wt%), the 0.01 wt% nanoemulsion eye drops provided by the present invention have a better myopia - treating effect, indicating that the atropine nanoemulsion provided by the present invention can increase the relative bioavailability of atropine. In addition, it can increase the tear film break - up time, providing a new idea for alleviating the dry eye symptoms caused by long - term use of atropine sulfate eye drops.

[0186] The above - mentioned are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can still be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A low-concentration atropine nanoemulsion, characterized in that, Based on 100 mL of the volume of water, the raw materials are 0.001 - 0.25 g of atropine, 2 - 5 g of oil, 0.5 - 5 g of surfactant, 0.1 - 0.3 g of co-surfactant, 0.05 - 3 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 < 75 mM; The pH value of the low-concentration atropine nanoemulsion is 4.5 - 6.5; The surfactant is egg yolk lecithin or soybean phospholipid; the oil is medium-chain oil; the co-surfactant is one of poloxamer 188, polyethylene glycol 400 and propylene glycol; the pH regulator is hydrochloric acid or sodium hydroxide solution; the buffer is boric acid / sodium borate or sodium dihydrogen phosphate / disodium hydrogen phosphate; the metal ion chelator is one or more of ethylenediaminetetraacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and penta(carboxymethyl)diethylenetriamine or their corresponding salts.

2. The low-concentration atropine nanoemulsion according to claim 1, wherein The osmotic pressure regulator includes one or more of glycerol, mannitol and sorbitol.

3. The preparation method of the low-concentration atropine nanoemulsion according to any one of claims 1 or 2, comprising the following steps: Dissolve the surfactant and atropine in the oil to obtain an atropine oil solution; Dissolve the co-surfactant, osmotic pressure regulator, buffer and metal ion chelator in part of the water to obtain an aqueous phase; Drop the atropine oil solution into the aqueous phase, perform shearing and then add the remaining water to obtain a primary emulsion; Perform high-pressure homogenization treatment on the primary emulsion, and use a pH regulator to adjust the pH value to 4.5 - 6.5 to obtain a low-concentration atropine nanoemulsion; The high-pressure homogenization treatment is carried out by a homogenizer; the number of cycles of the high-pressure homogenization treatment is 5 - 12 times, and 1 cycle is recorded when the homogenizer pump is at 30; the pressure of each high-pressure homogenization treatment is 700 - 1000 bar.

4. The application of the low-concentration atropine nanoemulsion according to any one of claims 1 - 2 in the preparation of ophthalmic drugs.

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