Ophthalmic cyclosporine nano-micelle drug, preparation method therefor, and use thereof

Abstract

An ophthalmic cyclosporine nano-micelle drug, a preparation method therefor, and use thereof. The cyclosporine nano-micelle drug comprises: 0.1-10 parts by weight of a solubilizer for forming micelle particles, and 0.01-1 part by weight of cyclosporine loaded in the micelle particles. The solubilizer is a first solubilizer, or a combination of the first solubilizer and a second solubilizer.

Description

A cyclosporine nano-micelle drug for eye use and its preparation method and application Technical Field

[0001] The present application belongs to the field of biomedicine technology and relates to an ophthalmic cyclosporine nano-micelle drug and its preparation method and application. Background Art

[0002] Cyclosporine, also known as cyclosporine A, is a cyclic polypeptide composed of 11 amino acids and is a potent and selective immunosuppressant. In recent years, cyclosporine has been used in ophthalmology to treat dry eye (keratoconjunctivitis sicca), keratoconjunctivitis, and immune rejection after corneal transplantation. Its mechanism of action includes inhibiting T cell activation, thereby reducing the production of inflammatory cytokines, inhibiting cell apoptosis, increasing goblet cell density and mucin secretion, and increasing tear flow. Cyclosporine is a safe and effective long-term clinical medication with no systemic adverse reactions. It can significantly improve the symptoms of dry eye, keratoconjunctivitis, and related eye diseases.

[0003] Cyclosporine is a highly lipid-soluble drug, a white powder that dissolves readily in organic solvents such as acetonitrile and ethanol, while its water solubility in water is only 12 ng / mL (25°C). However, when administered as eye drops, the conjunctival sac volume per eye typically does not exceed 30 μL. Therefore, increasing the concentration of cyclosporine to increase the effective amount of drug acting on the eye and thereby enhance drug concentration in ocular tissues to achieve better results is a research direction that is urgently needed.

[0004] Currently, there are many cyclosporine formulations on the market, but most are oils or emulsions. These formulations often cause a strong burning sensation and irritation when applied topically to the eye. Therefore, there is an urgent need to develop new, safe, effective, and stable high-concentration cyclosporine ophthalmic products. Summary of the Invention

[0005] This application provides an ophthalmic cyclosporine nanomicelle drug, its preparation method, and application. This application utilizes nanomicelle technology to transform the poorly soluble drug cyclosporine into a high-concentration, stable ophthalmic solution formulation. This formulation contains no oil-phase components, exhibits minimal irritation, and features a simple and convenient preparation process, making it easy to scale up and apply.

[0006] In a first aspect, the present application provides an ophthalmic cyclosporine nano-micelle drug, which comprises, by weight: 0.1-10 parts of a solubilizer for forming micelle particles, and 0.01-1 parts of cyclosporine loaded in the micelle particles;

[0007] Wherein, the solubilizing agent is the first solubilizing agent, or a combination of the first solubilizing agent and the second solubilizing agent;

[0008] The second solubilizer is selected from any one or a combination of at least two of polyoxyethylene hydrogenated castor oil, polysorbate 80, polyoxyethylene lauryl ether, poloxamer 188, polyoxyl 40 stearate, and tyloxapol;

[0009] The first solubilizer is selected from polyethylene glycol 15-hydroxystearate and any one or a combination of at least two of the following structural compounds:

[0010] In the above structural compound, R is independently selected from C9-C26 alkyl; R1 is selected from C1-C5 alkylene; R2 is independently selected from H, methyl, ethyl; M+ is selected from Na + , K + or NH4 + ; The molecular weight of the polyethylene glycol chain segments is independently 500-20000.

[0011] The ophthalmic cyclosporine nanomicelle drug involved in this application uses the specific type of first solubilizer, or a combination of the first and second solubilizers, as described above. The nanomicelle technology solves the solubility problem of cyclosporine in water, allowing cyclosporine to be stably present in the aqueous solution at a higher concentration, which is conducive to the accumulation of higher concentrations of cyclosporine in ocular tissues, thereby facilitating the cyclosporine to take effect more quickly. Furthermore, the drug has low local irritation, which can improve patient comfort.

[0012] Cyclosporine cannot be successfully loaded into a stable micelle system using just any solubilizer. The second solubilizer used in this application is a commonly used solubilizer for ophthalmic drugs, is relatively inexpensive, and has good safety. However, a solution of the second solubilizer alone cannot directly dissolve cyclosporine. The present application has discovered that using the first solubilizer alone or combining the second solubilizer with the first solubilizer can directly dissolve cyclosporine. Stable nanomicelles with a high cyclosporine content can be prepared using a direct hydration method. This method, unlike thin film hydration methods, is simpler and more convenient, does not use any organic solvents, is safer and more environmentally friendly, and has a lower cost.

[0013] The specific point values ​​that can be selected from the above 0.1-10 parts include but are not limited to 0.1 part, 0.3 part, 0.5 part, 0.8 part, 1 part, 1.2 part, 1.5 part, 1.8 part, 2 parts, 2.5 parts, 2.7 parts, 3 parts, 3.2 parts, 3.5 parts, 4 parts, 4.3 parts, 4.5 parts, 4.8 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, etc.; the specific point values ​​that can be selected from the above 0.01-1 parts include but are not limited to 0.01 part, 0.03 part, 0.05 part, 0.08 part, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, 1 part, etc.

[0014] The C9-C26 alkyl group refers to a C9 alkyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, a C18 alkyl group, a C19 alkyl group, a C20 alkyl group, a C21 alkyl group, a C22 alkyl group, a C23 alkyl group, a C24 alkyl group, a C25 alkyl group, and a C26 alkyl group. The C1-C5 alkylene group refers to a C1 alkylene group, a C2 alkylene group, a C3 alkylene group, a C4 alkylene group, and a C5 alkylene group.

[0015] The molecular weight of the polyethylene glycol chain segments can be independently selected to be 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 10000, 13000, 15000, 20000, etc. Other specific values ​​within this numerical range can be selected and will not be described in detail here.

[0016] In one embodiment, the first solubilizer is selected from any one or a combination of at least two of polyethylene glycol 15-hydroxystearate, TPGS (vitamin E polyethylene glycol succinate), mPEG-DSPE (methoxy-polyethylene glycol-phosphatidylethanolamine), mPEG-DPPE (methoxy-polyethylene glycol-dipalmitoylphosphatidylethanolamine), ALC-0159 (methoxy polyethylene glycol ditetradecyl acetamide), mPEG-DMG (1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol), and mPEG-DSG (methoxy polyethylene glycol disuccinimidyl glutarate); in the above structural compounds, the molecular weight of the polyethylene glycol segments is independently 500-20,000.

[0017] The molecular weight of the polyethylene glycol segments in the above compounds is independently 500-20,000, for example, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 10,000, 13,000, 15,000, 20,000, etc. Other specific values ​​within this numerical range can be selected and will not be described in detail here.

[0018] In one embodiment, the ratio of the mass of the solubilizer to the mass of cyclosporine is not less than 3:1, for example, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, etc. Other specific values ​​within this numerical range can be selected and will not be repeated here.

[0019] In one embodiment, when the solubilizer is a combination of a first solubilizer and a second solubilizer, the mass ratio of the second solubilizer to the first solubilizer is not higher than 10:1, for example, 9.5:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.1:1, etc. Other specific point values ​​within this numerical range can be selected, and they will not be repeated here.

[0020] In one embodiment, the particle size of the ophthalmic cyclosporine nanomicelle drug is 5-50 nm, for example, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, etc. Other specific values ​​within this numerical range can be selected and will not be described in detail here.

[0021] In a second aspect, the present application provides a method for preparing the ophthalmic cyclosporine nanomicelle drug according to the first aspect, the preparation method comprising:

[0022] Mixing cyclosporine with a solubilizer and water to dissolve the cyclosporine to obtain the ophthalmic cyclosporine nanomicelle drug (the drug is in the form of an aqueous solution);

[0023] Alternatively, cyclosporine is mixed with a solubilizing agent and water, the cyclosporine is dissolved, and then freeze-dried to obtain the ophthalmic cyclosporine nanomicelle drug (the drug is in the form of freeze-dried powder).

[0024] The ophthalmic cyclosporine nanomicelles described in this application can be prepared using a direct hydration method, which allows cyclosporine to completely dissolve within 4 hours. Unlike most methods for preparing polymer micelles, such as thin film hydration, this method is simple and convenient, does not use any organic solvents, and is conducive to industrial scale-up production and is safe and environmentally friendly. Furthermore, the resulting ophthalmic cyclosporine nanomicelle drug system is very stable and has a long shelf life, facilitating storage and transportation.

[0025] In one embodiment, the preparation method comprises: mixing cyclosporine with an aqueous solution of a solubilizer to dissolve the cyclosporine to obtain the ophthalmic cyclosporine nanomicelle drug (the drug is in the form of an aqueous solution);

[0026] Alternatively, cyclosporine is mixed with an aqueous solution of a solubilizer, the cyclosporine is dissolved, and then freeze-dried to obtain the ophthalmic cyclosporine nanomicelle drug (the drug is in the form of freeze-dried powder).

[0027] In one embodiment, the dissolution is carried out at 0-70°C, for example, 2°C, 4°C, 10°C, 25°C, 30°C, 35°C, 40°C, 50°C, 60°C, 70°C, etc. Other specific values ​​within this numerical range can be selected and will not be described in detail here. The above preparation method has no special requirements for temperature, and the product can be prepared at 0-70°C.

[0028] In one embodiment, the dissolving is performed by any one or at least two of the following methods:

[0029] (1) Stirring; (2) Oscillation; (3) Ultrasonic treatment.

[0030] In one embodiment, the final concentration of cyclosporine in the system after dissolution is 0.2-10 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, etc.

[0031] In one embodiment, when the solubilizing agent is a combination of a first solubilizing agent and a second solubilizing agent, the final concentration of the first solubilizing agent in the mixed system is 2-30 mg / mL, such as 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 25 mg / mL, 28 mg / mL, 30 mg / mL, etc.; the final concentration of the second solubilizing agent in the mixed system is 0.01-20 mg / mL, such as 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, etc. Other specific point values ​​within the above numerical range can be selected and will not be repeated here.

[0032] In a third aspect, the present application provides use of the ophthalmic cyclosporine nanomicelle drug according to the first aspect in the preparation of an ophthalmic cyclosporine preparation.

[0033] In a fourth aspect, the present application provides cyclosporine eye drops, comprising the ophthalmic cyclosporine nanomicelle drug according to the first aspect; the final concentration of cyclosporine in the eye drops is 0.2-10 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, etc.

[0034] In one embodiment, the components of the cyclosporine eye drops further include pharmaceutical excipients, which include any one or a combination of at least two of an osmotic pressure regulator, a pH regulator, a thickener, a preservative, and a solvent.

[0035] In a fifth aspect, the present application provides a method for preparing the cyclosporine eye drops according to the fourth aspect, the method comprising: mixing and dissolving the ophthalmic cyclosporine nanomicelle drug and optional pharmaceutical excipients in a solvent, filtering the solution to remove insoluble particles, and obtaining the cyclosporine eye drops.

[0036] Compared with the prior art, this application has the following beneficial effects:

[0037] This application addresses the solubility issue of cyclosporine in water through an innovative carrier system. This allows for the preparation of high-concentration cyclosporine micellar solutions, which facilitate higher drug concentration accumulation in ocular tissues, thereby promoting faster drug onset. The system also exhibits low local irritation, improving patient compliance and tolerance. The micellar solution exhibits excellent stability, facilitating storage and transportation. Furthermore, this ophthalmic nanodrug delivery system is produced using a direct hydration method. Unlike thin-film hydration methods used to prepare most polymer micelles, this method does not use any organic solvents, making it suitable for industrial scale-up, safe, environmentally friendly, and cost-effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG1 is a graph showing the change in cyclosporine concentration in the cornea over time after use of the product of the present application and the control product.

[0039] FIG2 is a graph showing the change in cyclosporine concentration in the bulbar conjunctiva over time after use of the product of the present application and the control product.

[0040] FIG3 is a graph showing the change in cyclosporine concentration in the upper and lower eyelids over time after use of the product of the present application and the control product.

[0041] FIG4 is a graph showing the change in cyclosporine concentration in the sclera over time after use of the product of the present application and the control product. DETAILED DESCRIPTION

[0042] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0043] The cyclosporine mentioned in the following content was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; polyethylene glycol 15-hydroxystearate was purchased from BASF; vitamin E polyethylene glycol succinate (abbreviated as TPGS, molecular weight 1513, of which the molecular weight of the PEG segment is approximately 1000) was purchased from Shanghai Lianlu Industrial Co., Ltd.; mPEG-2000-DSPE was purchased from Jiangsu Southeast Nano Co., Ltd.; mPEG5000-DPPE was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; ALC-0159 was purchased from Meiluo Technology Co., Ltd.; mPEG2000-DMG was purchased from MedChemExpress; mPEG2000-DSG was purchased from MedChemExpress; and hydropropyl methylcellulose (labeled viscosity 4000) was purchased from Tai'an Ruitai Cellulose Co., Ltd.

[0044] The polyoxyethylene hydrogenated castor oil mentioned below was purchased from BASF; polysorbate 80 was purchased from Jiangxi Yipsheng Pharmaceutical Co., Ltd.; polyoxyethylene lauryl ether was purchased from Sigma-Aldrich; and polyoxyl 40 stearate was purchased from Beijing Puxitang Biotechnology Co., Ltd.

[0045] The instruments used in the following content include: laser particle size analyzer (Malvern, zetasizer ZSP); high performance liquid chromatograph (Shimadzu LC-20-AT); high performance liquid chromatography mass spectrometry (Shimadzu LCMS8050); other reagents or instruments not specified are conventional products that can be purchased commercially.

[0046] Cyclosporine content was determined using the external standard method. Samples were centrifuged and the supernatant was collected before testing. High-performance liquid chromatography (HPLC) testing conditions were: an Agilent Zorbax C18 column, 5 μm, 4.6×250 mm, flow rate 1.0 mL / min, column temperature 70°C, detection wavelength 210 nm, injection volume 20 μL. The mobile phase was a ratio of water: acetonitrile: tert-butyl methyl ether: 85% phosphoric acid solution (1040:860:100:2, by volume), with isocratic elution.

[0047] Example 1

[0048] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0049] Place 8.6 mL of pure water in a flask and stir continuously at 25°C. Add 430 mg of TPGS and allow it to dissolve gradually. Take 3 mL of this solution and add 12 mL of pure water to obtain a 1% TPGS solution. Add 10 mg of cyclosporine to 5 mL of this solution, raise the solution temperature to 60°C, and continue stirring until the cyclosporine dissolves.

[0050] Example 2

[0051] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0052] Place 14 mL of pure water in a flask and continue stirring at 25°C. Add 200 mg of mPEG-2000-DSPE and allow it to gradually dissolve. Add 20 mg of cyclosporine to this solution, raise the temperature to 60°C, and continue stirring until the cyclosporine dissolves.

[0053] Example 3

[0054] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0055] Place 64 mL of pure water in a flask and stir continuously at 25°C. Add 894 mg of mPEG-2000-DSPE and allow it to gradually dissolve. Add 28 mg of cyclosporine to 13.7 mL of this solution, raise the temperature to 60°C, and continue stirring until the cyclosporine dissolves.

[0056] Example 4

[0057] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0058] Place 3.6 mL of pure water in a flask and continue stirring at 25°C. Add 50 mg of mPEG-5000-DPPE and allow it to gradually dissolve. Add 5 mg of cyclosporine and raise the solution temperature to 60°C. Continue stirring until the cyclosporine dissolves.

[0059] Example 5

[0060] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0061] Place 3.6 mL of pure water in a flask and continue stirring at 25°C. Add 50 mg of ALC-0159 and allow it to gradually dissolve. Add 5 mg of cyclosporine, raise the solution temperature to 60°C, and continue stirring until the cyclosporine dissolves.

[0062] Example 6

[0063] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0064] Place 3.6 mL of pure water in a flask and continue stirring at 25°C. Add 50 mg of mPEG 2000-DSPE and allow it to gradually dissolve. Add 10 mg of cyclosporine, raise the solution temperature to 60°C, and continue stirring until the cyclosporine dissolves.

[0065] Example 7

[0066] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0067] Place 3.6 mL of pure water in a flask and continue stirring at 25°C. Add 50 mg of mPEG 2000-DMG and allow it to gradually dissolve. Add 5 mg of cyclosporine, raise the solution temperature to 60°C, and continue stirring until the cyclosporine dissolves.

[0068] Example 8

[0069] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0070] Place 3.6 mL of pure water in a flask and continue stirring at 25°C. Add 50 mg of mPEG 2000-DSG and allow it to gradually dissolve. Add 5 mg of cyclosporine, raise the solution temperature to 60°C, and continue stirring until the cyclosporine dissolves.

[0071] Example 9

[0072] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0073] (1) Accurately weigh polyoxyethylene hydrogenated castor oil, add pure water to dissolve, and prepare a 20 mg / mL solution; accurately weigh MPEG-2000-DSPE, add pure water to dissolve, and prepare a 20 mg / mL solution.

[0074] (2) Take 2 mL of MPEG-2000-DSPE solution, 3 mL of polyoxyethylene hydrogenated castor oil solution and 2 mL of purified water and mix them evenly. Add 10 mg of cyclosporine and continue stirring at 60°C until the cyclosporine is dissolved.

[0075] Example 10

[0076] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0077] (1) Accurately weigh polyoxyethylene hydrogenated castor oil, add pure water to dissolve, and prepare a 20 mg / mL solution; accurately weigh MPEG-2000-DSPE, add pure water to dissolve, and prepare a 40 mg / mL solution.

[0078] (2) Take 2 mL of MPEG-2000-DSPE solution and mix it evenly with 5 mL of polyoxyethylene hydrogenated castor oil solution, add 20 mg of cyclosporine, and continue stirring at 25°C until the cyclosporine is dissolved.

[0079] Example 11

[0080] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0081] (1) Accurately weigh polysorbate 80, dissolve it in pure water, and prepare a 20 mg / mL solution; accurately weigh MPEG-2000-DSPE, dissolve it in pure water, and prepare a 20 mg / mL solution.

[0082] (2) Take 2 mL of MPEG-2000-DSPE solution, 3 mL of polysorbate 80 solution and 2 mL of purified water and mix them evenly. Add 10 mg of cyclosporine and continue stirring at 4°C until the cyclosporine is dissolved.

[0083] Example 12

[0084] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0085] (1) Accurately weigh polyoxyethylene hydrogenated castor oil and dissolve it in pure water to prepare a 20 mg / mL solution. Accurately weigh MPEG-2000-DMG and dissolve it in pure water to prepare a 20 mg / mL solution.

[0086] (2) Take 2.25 mL of MPEG-2000-DMG solution, 2.75 mL of polyoxyethylene hydrogenated castor oil solution and 2 mL of purified water and mix them evenly. Add 10 mg of cyclosporine and continue stirring at 40°C until the cyclosporine is dissolved.

[0087] Example 13

[0088] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0089] (1) Accurately weigh polyoxyethylene lauryl ether (Brij 35), dissolve it in pure water to prepare a 20 mg / mL solution; accurately weigh MPEG-2000-DSG, dissolve it in pure water to prepare a 20 mg / mL solution.

[0090] (2) Take 2.5 mL of MPEG-2000-DSG solution, 2.5 mL of lauryl alcohol polyoxyethylene ether solution and 2 mL of purified water and mix them evenly. Add 10 mg of cyclosporine and continue stirring at 25°C until the cyclosporine is dissolved.

[0091] Example 14

[0092] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0093] (1) Accurately weigh polysorbate 80, dissolve it in pure water, and prepare a 20 mg / mL solution; accurately weigh polyethylene glycol 15-hydroxystearate, dissolve it in pure water, and prepare a 20 mg / mL solution.

[0094] (2) Take 3 mL of polysorbate 80 solution and 5 mL of polyethylene glycol 15-hydroxystearate solution and mix them evenly. Add 10 mg of cyclosporine and continue stirring at 60°C until the cyclosporine is dissolved.

[0095] Example 15

[0096] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0097] (1) Accurately weigh polyoxyethylene hydrogenated castor oil, add pure water to dissolve it, and prepare a 20 mg / mL solution; accurately weigh MPEG-5000-DPPE, add pure water to dissolve it, and prepare a 20 mg / mL solution.

[0098] (2) Take 3 mL of MPEG-5000-DPPE solution and mix it evenly with 5 mL of polyoxyethylene hydrogenated castor oil solution, add 10 mg of cyclosporine, and continue stirring at 60°C until the cyclosporine is dissolved.

[0099] Example 16

[0100] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0101] (1) Accurately weigh poloxamer 188, dissolve it in pure water, and prepare a 20 mg / mL solution; accurately weigh polyethylene glycol 15-hydroxystearate, dissolve it in pure water, and prepare a 20 mg / mL solution.

[0102] (2) Accurately weigh MPEG-2000-DSPE and dissolve it in pure water to prepare a 20 mg / mL solution; take 2.5 mL of MPEG-2000-DSPE solution, 2 mL of polyethylene glycol 15-hydroxystearate, and 2.5 mL of poloxamer 188 solution and mix them evenly; add 15 mg of cyclosporine and continue shaking at 25°C until the cyclosporine is dissolved.

[0103] Example 17

[0104] This embodiment provides an ophthalmic cyclosporine nano-micelle drug, the preparation method of which is as follows:

[0105] (1) Accurately weigh polyoxyl 40 stearate, add pure water to dissolve, and prepare a 20 mg / mL solution; accurately weigh ALC-0159, add pure water to dissolve, and prepare a 20 mg / mL solution.

[0106] (2) Take 1 mL of ALC-0159 solution and 6 mL of polyoxyl 40 stearate solution, mix them evenly, add 5 mg of cyclosporine, and continue stirring at 25°C until the cyclosporine is dissolved.

[0107] Example 18

[0108] This example provides an ophthalmic cyclosporine nanomicelle drug. The preparation method differs from that of Example 1 only in step (2). Other conditions are consistent with those of Example 1. 2 mL of MPEG-2000-DSPE solution, 3 mL of polyoxyethylene hydrogenated castor oil solution, and 2 mL of purified water are mixed uniformly. 30 mg of cyclosporine is added, and stirring is continued at 60°C. The results show that after 10 hours, the solution remains turbid, with a large number of undissolved drug particles. The solution is filtered through a 0.22 μm membrane, and the cyclosporine concentration is determined by HPLC, which is 1.721 mg / mL.

[0109] Example 19

[0110] This example provides an ophthalmic cyclosporine nanomicelle drug. The preparation method differs from that of Example 1 only in step (2). Other conditions are consistent with those of Example 1. 0.4 mL of MPEG-2000-DSPE solution, 4.6 mL of polyoxyethylene hydrogenated castor oil solution, and 2 mL of purified water are mixed uniformly. 10 mg of cyclosporine is added, and the mixture is stirred continuously at 60°C. The results show that some drug particles remain undissolved in the solution. The solution is filtered through a 0.22 μm membrane, and the cyclosporine concentration is determined by HPLC, which is 0.3914 mg / mL.

[0111] Comparative Example 1

[0112] The preparation method of this comparative example differs from that of Example 1 only in that TPGS is replaced with an equal mass of polyoxyethylene castor oil 40 (purchased from BASF), while other process operations remain unchanged. The results show that cyclosporine cannot be dissolved after addition, resulting in drug precipitation and failure to form micelles.

[0113] Comparative Example 2

[0114] The preparation method of this comparative example differs from that of Example 1 only in that TPGS is replaced with an equal mass of poloxamer 407, and other process operations remain unchanged. The results show that after 10 hours, the solution is still turbid, cyclosporine fails to dissolve, and nanomicelles cannot be formed.

[0115] Comparative Example 3

[0116] The preparation method of this comparative example differs from that of Example 1 only in that TPGS is replaced with an equal mass of poloxamer 188, and other process operations remain unchanged. The results show that after 10 hours, the solution is still turbid, cyclosporine fails to dissolve, and nanomicelles cannot be formed.

[0117] Comparative Example 4

[0118] The preparation method of this comparative example differs from that of Example 1 only in that TPGS is replaced with an equal mass of mPEG2000-PLGA (Mn 4500) (purchased from Sigma Aldrich). The results show that the auxiliary material cannot be completely dissolved in water at 25°C.

[0119] Comparative Example 5

[0120] The preparation method of this comparative example differs from that of Example 1 only in that TPGS is replaced with an equal amount of mPEG750-PLA (Mn 1000) (purchased from Sigma Aldrich). The results show that the excipient cannot be completely dissolved in water at 25°C.

[0121] Comparative Example 6

[0122] The preparation method of this comparative example differs from that of Example 9 only in that the MPEG-2000-DSPE solution in step (2) is replaced by a polyoxyethylene hydrogenated castor oil solution:

[0123] (1) Accurately weigh polyoxyethylene hydrogenated castor oil and dissolve it in pure water to prepare a 20 mg / mL solution.

[0124] (2) 5 mL of polyoxyethylene hydrogenated castor oil solution was mixed evenly with 2 mL of purified water, 10 mg of cyclosporine was added, and stirring was continued at 60°C. The results showed that after 10 hours, the solution was still turbid, cyclosporine was not dissolved, and nanomicelles could not be formed.

[0125] Comparative Example 7

[0126] The preparation method of this comparative example differs from that of Example 11 only in that the MPEG-2000-DSPE solution in step (2) is replaced with a polysorbate 80 solution:

[0127] (1) Accurately weigh polysorbate 80 and dissolve it in pure water to prepare a 20 mg / mL solution.

[0128] (2) 5 mL of polysorbate 80 solution was mixed evenly with 2 mL of purified water, and 10 mg of cyclosporine was added. The mixture was stirred continuously at 4°C. The results showed that after 10 hours, the solution was still turbid, and cyclosporine was not dissolved and could not form nanomicelles.

[0129] Comparative Example 8

[0130] The preparation method of this comparative example differs from that of Example 13 only in that the MPEG-2000-DSG solution in step (2) is replaced with a lauryl alcohol polyoxyethylene ether solution:

[0131] (1) Accurately weigh polyoxyethylene lauryl ether (Brij 35) and dissolve it in pure water to prepare a 20 mg / mL solution.

[0132] (2) 5 mL of lauryl alcohol polyoxyethylene ether solution was mixed evenly with 2 mL of purified water, 10 mg of cyclosporine was added, and stirring was continued at 25°C. The results showed that after 10 hours, the solution was still turbid, cyclosporine was not dissolved, and nanomicelles could not be formed.

[0133] Test Example 1

[0134] In this test example, the products obtained in Examples 1-17 were characterized by particle size using a laser particle size analyzer. The results are shown in Table 1.

[0135] Table 1

[0136] As can be seen from the data in Table 1, the ophthalmic cyclosporine nanomicelle drug involved in the present application can be prepared by using a direct hydration method, and the system is colorless, clear and transparent, with small and uniform particle size.

[0137] Application Example 1

[0138] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0139] Dissolve 530 mg of disodium hydrogen phosphate (dihydrate), 470 mg of sodium dihydrogen phosphate (anhydrous), and 50 mg of sodium chloride in 10 mL of pure water, and set aside 1 mL. Dissolve 300 mg of the thickener, hypromellose, in 50 mL of pure water, and set aside 1 mL. Mix 7 mL of the product from Example 2 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22 μm filter to remove insoluble particles.

[0140] Application Example 2

[0141] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0142] Dissolve 795 mg of disodium hydrogen phosphate (dihydrate), 705 mg of sodium dihydrogen phosphate (anhydrous), and 75 mg of sodium chloride in 15 mL of pure water, taking 2.8 mL for later use. Dissolve 173 mg of the thickener, hypromellose, in 20 mL of pure water, taking 3.3 mL for later use. Mix 13.7 mL of the product from Example 3 with the two solutions, and add pure water to a final volume of 27.7 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0143] Application Example 3

[0144] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0145] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and take 0.5 mL for later use. Dissolve 100 mg of the thickener, hypromellose, in 15 mL of pure water, and take 0.8 mL for later use. Mix 3.6 mL of the product from Example 4 with the two solutions, and add pure water to a final volume of 5 mL. Filter the solution through a 0.22 μm filter to remove insoluble particles.

[0146] Application Example 4

[0147] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0148] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and take 0.5 mL for later use. Dissolve 100 mg of the thickener, hypromellose, in 15 mL of pure water, and take 0.8 mL for later use. Mix 3.6 mL of the product from Example 5 with the two solutions, and add pure water to a final volume of 5 mL. Filter the solution through a 0.22 μm filter to remove insoluble particles.

[0149] Application Example 5

[0150] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0151] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and take 0.5 mL for later use. Dissolve 100 mg of the thickener, hypromellose, in 15 mL of pure water, and take 0.8 mL for later use. Mix 3.6 mL of the product from Example 6 with the two solutions, and add pure water to a final volume of 5 mL. Filter the solution through a 0.22 μm filter to remove insoluble particles.

[0152] Application Example 6

[0153] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0154] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and set aside 1 mL. Dissolve 30 mg of thickener PVP-K90 in 1 mL of pure water. Mix the product from Example 9 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0155] Application Example 7

[0156] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0157] Dissolve 530 mg of disodium hydrogen phosphate (dihydrate), 470 mg of sodium dihydrogen phosphate (anhydrous), and 50 mg of sodium chloride in 10 mL of pure water, and set aside 1 mL. Dissolve 10 mg of the thickener, hypromellose, in 10 mL of pure water, and set aside 1 mL. Mix the product from Example 10 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22 μm filter to remove insoluble particles.

[0158] Application Example 8

[0159] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0160] Dissolve 795 mg of disodium hydrogen phosphate (dihydrate), 705 mg of sodium dihydrogen phosphate (anhydrous), and 75 mg of sodium chloride in 15 mL of pure water, and set aside 1 mL. Dissolve 100 mg of sodium hyaluronate (thickener) in 15 mL of pure water, and set aside 1 mL. Mix the product from Example 11 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0161] Application Example 9

[0162] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0163] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and set aside 1 mL. Dissolve 200 mg of the thickener, hypromellose, in 15 mL of pure water, and set aside 1 mL. Mix the product from Example 12 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0164] Application Example 10

[0165] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0166] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and set aside 1 mL. Dissolve 30 mg of thickener PVP-K90 in 1 mL of pure water. Mix the product from Example 13 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0167] Application Example 11

[0168] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0169] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and set aside 1 mL. Dissolve 30 mg of thickener PVP-K90 in 1 mL of pure water. Mix the product from Example 14 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0170] Application Example 12

[0171] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0172] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and set aside 1 mL. Dissolve 200 mg of the thickener, hypromellose, in 15 mL of pure water, and set aside 1 mL. Mix the product from Example 15 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0173] Application Example 13

[0174] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0175] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and set aside 1 mL. Dissolve 30 mg of thickener PVP-K90 in 1 mL of pure water. Mix the product from Example 16 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0176] Application Example 14

[0177] This application example provides a cyclosporine eye drop, the preparation method of which is as follows:

[0178] Dissolve 96 mg of disodium hydrogen phosphate (dihydrate), 105 mg of sodium dihydrogen phosphate (anhydrous), and 95 mg of sodium chloride in 2 mL of pure water, and set aside 1 mL. Dissolve 30 mg of thickener PVP-K90 in 1 mL of pure water. Mix the product from Example 17 with the two solutions, and add pure water to a final volume of 10 mL. Filter the solution through a 0.22-μm filter to remove insoluble particles.

[0179] Test Example 2

[0180] In this test example, the products obtained in Example 1 and Application Examples 1-14 were placed at 35° C. for 30 days, and the cyclosporine content was measured before and after placement using a high performance liquid chromatograph. The results are shown in Table 2.

[0181] Table 2

[0182] As can be seen from the data in Table 2, the cyclosporine nano-micelle drug and cyclosporine eye drops for ophthalmology involved in the present application do not show significant changes in cyclosporine content after being placed at 35° C. for 30 days, indicating good stability.

[0183] Test Example 3

[0184] This test case is used to conduct eye irritation tests on the products prepared using Examples 1-14:

[0185] Twenty-eight healthy rabbits were randomly divided into 14 groups of two rabbits each. Each rabbit received 50 μL of the test product in the left eye and 50 μL of normal saline in the right eye, with the eyelids gently closed for 10 seconds. Administration was continued four times daily for seven consecutive days. Local reactions in the cornea, iris, and conjunctiva were observed before dosing and 1, 2, 4, 24, 48, and 72 hours after the last dose, and a total score was calculated. The irritation score for normal saline was 0. See Table 3 for scoring criteria and methods.

[0186] Table 3

[0187] The result judgment criteria are shown in Table 4 below:

[0188] Table 4

[0189] The eye irritation test results of each group showed that the total scores of Application Examples 1-14 groups were all 0 points.

[0190] It can be seen from this that the ophthalmic cyclosporine nanomicelle drug and cyclosporine eye drops involved in this application have no obvious irritating effect on the rabbit eyes and are safe and mild.

[0191] Test Example 4

[0192] This test case performs a pharmacokinetic test on the product prepared in Example 2:

[0193] A total of 60 healthy rabbits were randomly divided into 20 groups, with 3 rabbits in each group. 10 groups of experimental rabbits were given 35 μL of the product prepared in Example 2 in both eyes, and the other 10 groups of experimental rabbits were given a control product (0.05% cyclosporine eye drops II produced by Shenyang Xingqi Eye Medicine Co., Ltd.). ) 35 μL. Experimental rabbits in groups 1-10 were sacrificed at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours after administration, and the upper and lower eyelids and conjunctiva were removed. Immediately thereafter, the rabbits underwent enucleation, and the eyeballs were dissected, with the cornea and sclera separated. After the tissues were ground and homogenized, the cyclosporine content in the tissues was analyzed using high-performance liquid chromatography-mass spectrometry. The pharmacokinetic parameters C of cyclosporine in the cornea, conjunctiva, upper and lower eyelids, and sclera were as follows: max and AUC (0-t) See Table 5. The changes in cyclosporine drug concentrations in the cornea, conjunctiva, upper and lower eyelids, and sclera over time are shown in Figures 1 to 4, where the X-axis represents time (h) and the Y-axis represents the cyclosporine drug concentration in the tissue (ng / g).

[0194] Table 5

[0195] As shown in Table 5 and Figures 1-4, the cyclosporine nano-micelle drug and cyclosporine eye drops involved in this application significantly increased the cyclosporine concentration and exposure (AUC (0-t) ), indicating that it may produce better results in actual clinical use.

[0196] The applicant declares that while the above-mentioned embodiments are used to illustrate the technical solutions of this application, this application is not limited to the above-mentioned embodiments, which does not mean that this application must rely on the above-mentioned embodiments in order to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent replacements for the raw materials of the products of this application, the addition of auxiliary ingredients, and the selection of specific methods, etc., fall within the scope of protection and application of this application.

[0197] The preferred embodiments of the present application are described in detail above. However, the present application is not limited to the specific details of the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the scope of protection of the present application.

[0198] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner unless there is any contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

Claims

1. A cyclosporine nano-micelle drug for eye use, comprising, by weight: 0.1-10 parts of a solubilizing agent for forming micelle particles, and 0.01-1 parts of cyclosporine loaded in the micelle particles; Wherein, the solubilizing agent is the first solubilizing agent, or a combination of the first solubilizing agent and the second solubilizing agent; The second solubilizer is selected from any one or a combination of at least two of polyoxyethylene hydrogenated castor oil, polysorbate 80, polyoxyethylene lauryl ether, poloxamer 188, polyoxyl 40 stearate, and tyloxapol; The first solubilizing agent is selected from polyethylene glycol 15-hydroxystearate and any one or a combination of at least two of the following structural compounds: Wherein, R is independently selected from C9-C26 alkyl; R1 is selected from C1-C5 alkylene; R2 is independently selected from H, methyl, ethyl; M+ is selected from Na + , K + or NH4 + ; The molecular weight of the polyethylene glycol chain segments is independently 500-20000.

2. The ophthalmic cyclosporine nano-micelle drug according to claim 1, wherein: The first solubilizer is selected from any one of polyethylene glycol 15-hydroxystearate, TPGS, mPEG-DSPE, mPEG-DPPE, ALC-0159, mPEG-DMG, and mPEG-DSG, or a combination of at least two thereof; in the above structural compounds, the molecular weights of the polyethylene glycol segments are independently 500-20,000.

3. The ophthalmic cyclosporine nano-micelle drug according to claim 1, wherein: The ratio of the mass of the solubilizing agent to the mass of cyclosporine is not less than 3:

1.

4. The ophthalmic cyclosporine nano-micelle drug according to claim 1, wherein: When the solubilizer is a combination of a first solubilizer and a second solubilizer, the mass ratio of the second solubilizer to the first solubilizer is no higher than 10:

1.

5. The ophthalmic cyclosporine nano-micelle drug according to claim 1, wherein: The particle size of the ophthalmic cyclosporine nano-micelle drug is 5-50 nm.

6. A method for preparing the ophthalmic cyclosporine nano-micelle drug according to any one of claims 1 to 5, comprising: Mixing cyclosporine with a solubilizing agent and water to dissolve the cyclosporine, thereby obtaining the ophthalmic cyclosporine nano-micelle drug; Alternatively, cyclosporine is mixed with a solubilizing agent and water, the cyclosporine is dissolved, and then freeze-dried to obtain the ophthalmic cyclosporine nano-micelle drug.

7. The method for preparing the ophthalmic cyclosporine nano-micelle drug according to claim 6, wherein: The dissolving is carried out at 0-70°C; The dissolution is carried out by any one or at least two of the following methods: (1) Stirring; (2) Oscillation; (3) Ultrasonic treatment.

8. The method for preparing the ophthalmic cyclosporine nano-micelle drug according to claim 6, wherein: The final concentration of the cyclosporine after dissolution in the system is 0.2-10 mg / mL.

9. The method for preparing the ophthalmic cyclosporine nano-micelle drug according to claim 6, wherein: When the solubilizer is a combination of a first solubilizer and a second solubilizer, the final concentration of the first solubilizer in the mixed system is 2-30 mg / mL; the final concentration of the second solubilizer in the mixed system is 0.01-20 mg / mL.

10. Use of the ophthalmic cyclosporine nano-micelle drug according to any one of claims 1 to 5 in the preparation of an ophthalmic cyclosporine preparation.

11. A cyclosporine eye drop, the components of which include the ophthalmic cyclosporine nano-micelle drug according to any one of claims 1 to 5; wherein: The final concentration of cyclosporine in the eye drops is 0.2-10 mg / mL.

12. The cyclosporine eye drops according to claim 11, wherein The components of the cyclosporine eye drops also include pharmaceutical excipients, which include any one of an osmotic pressure regulator, a pH regulator, a thickener, a preservative, and a solvent, or a combination of at least two of them.

13. A method for preparing the cyclosporine eye drops according to claim 11 or 12, comprising: The ophthalmic cyclosporine nano-micelle drug and optional pharmaceutical excipients are mixed and dissolved in a solvent, and the solution is filtered to remove insoluble particles to obtain the cyclosporine eye drops.

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