Nano-micelle ophthalmic preparation as well as preparation method and application thereof

Abstract

The invention relates to a nano-micelle ophthalmic preparation as well as a preparation method and application thereof, the nano-micelle ophthalmic preparation is prepared by taking cyclosporine, a solubilizer and an aqueous medium as raw materials through a film hydration method, and the solubilizer is polyoxyethylene hydrogenated castor oil. According to the invention, an ophthalmic preparation product with a specific formula and a specific preparation process is creatively designed, the used solubilizer only contains a single component polyoxyethylene hydrogenated castor oil, and other solubilizers, organic solvents or oily solvents do not need to be added into the formula; the cyclosporine can stably exist in an aqueous solution at a relatively high concentration, so that the cyclosporine can be easily accumulated at a relatively high concentration in eye tissues. And meanwhile, the use safety of the product is greatly improved, and the manufacturing cost is reduced. According to the preparation technology, polyoxyethylene hydrogenated castor oil and cyclosporin can form a long-term stable nano-micelle system in an aqueous solvent, the micelle particle size is smaller than 50 nm, and the system is clear and transparent.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a nano-micelle eye preparation and a preparation method and application thereof. Background Art

[0002] Cyclosporin A (CsA), also known as cyclosporin A or cyclosporin, is a cyclic polypeptide compound composed of 11 amino acids. It is a powerful immunosuppressant. Cyclosporin can be used to treat eye diseases such as dry eye. Systemic administration of cyclosporin can cause serious toxic side effects such as kidney damage, central nervous system damage, and liver damage. Therefore, the application of systemic cyclosporin is greatly limited. These toxic side effects can be avoided by local administration methods such as eye drops.

[0003] Cyclosporine is a highly lipid-soluble drug in the form of white powder. It is easily soluble in organic solvents such as acetonitrile and ethanol, and its water solubility in water is only 12ng / mL (25°C). However, when administered in the form of eye drops, the conjunctival sac volume of each eye usually does not exceed 30μL. Therefore, increasing the concentration of cyclosporine to increase the effective amount of drug acting on the eye and increasing the drug concentration in the eye tissue to achieve better effects are urgent research directions.

[0004] In order to improve the water solubility of cyclosporine, many solubilizers are used to prepare cyclosporine ophthalmic preparations. However, most of them add oil phase or other organic solvents such as ethanol to the formula. Oil or organic solvents are usually used for local use in the eye, which usually has a strong burning sensation and irritation. There are also some formulas that use solubilizers with certain toxicity or high cost, which limits their widespread use. There are also some formulas that have high solubility of cyclosporine, but poor long-term stability. After being placed at room temperature or high and low temperatures for a long time, cyclosporine will undergo crystal changes, resulting in cyclosporine crystals or precipitation. Therefore, it is urgent to develop new high-concentration cyclosporine eye drops that are safe and effective, have good long-term stability, and have low preparation costs. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a nanomicelle ophthalmic preparation and a preparation method and application thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a nanomicelle ophthalmic preparation, wherein the nanomicelle ophthalmic preparation is prepared by a thin film hydration method using cyclosporine, a solubilizer and an aqueous vehicle as raw materials, and the solubilizer is polyoxyethylene hydrogenated castor oil.

[0008] Based on the drawbacks of poor safety, poor long-term stability, high preparation cost or low cyclosporin concentration of existing cyclosporin ophthalmic preparations, the present invention creatively designs an ophthalmic preparation product with a specific formula and a specific preparation process, wherein the solubilizer used is only a single component of polyoxyethylene hydrogenated castor oil, and no other solubilizers are needed, and no organic solvents such as ethanol, methanol, etc., are needed to be added to the formula, and no oily solvents are needed to be added, so that cyclosporin can be stably present in the aqueous solution at a higher concentration, which is conducive to the accumulation of a higher concentration of cyclosporin in the eye tissue, thereby facilitating the cyclosporin to take effect faster. At the same time, this greatly improves the safety of the product and reduces the manufacturing cost.

[0009] The nano-micelle ophthalmic preparation of the present invention uses a thin film hydration method for preparation, and polyoxyethylene hydrogenated castor oil can form a long-term stable nano-micelle system with cyclosporine in an aqueous solvent, the micelle particle size is less than 50nm, and the system is clear and transparent. If other preparation processes such as aqueous solution method are used, the long-term stability of the product is poor, and obvious precipitation will occur after long-term storage.

[0010] Preferably, the degree of polymerization of polyoxyethylene in the polyoxyethylene hydrogenated castor oil is 30-80, such as polyoxyethylene 35 hydrogenated castor oil, polyoxyethylene 40 hydrogenated castor oil, polyoxyethylene 60 hydrogenated castor oil, and the like.

[0011] Preferably, the aqueous vehicle comprises water or phosphate buffered saline.

[0012] Preferably, the mass ratio of the solubilizer to cyclosporine is not less than 5:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 15:1, etc. More preferably, the mass ratio is not less than 9:1.

[0013] The amount of solubilizer used in the ophthalmic preparation product of the present invention relative to cyclosporin is relatively small, and as long as the mass ratio of solubilizer to cyclosporin is not less than 5:1, cyclosporin can be stably present in the aqueous solution at a relatively high concentration. When the mass ratio is not less than 9:1, the long-term stability is better.

[0014] Preferably, the mass ratio of the aqueous vehicle to cyclosporine is not less than 333:1, such as 350:1, 400:1, 450:1, 500:1, 600:1, 700:1, 800:1, 1000:1, 1500:1, etc. More preferably, the mass ratio is not less than 800:1.

[0015] The amount of aqueous medium used in the ophthalmic preparation product of the present invention relative to cyclosporine can reach a lower level, that is, cyclosporine can be stably present in the aqueous solution at a higher concentration. When the mass ratio is not less than 800:1, the long-term stability is better.

[0016] Preferably, the particle size of the drug-loaded micelles prepared by the thin film hydration method is less than 50 nm.

[0017] The micelle particles in the nano-micelle ophthalmic preparation of the present invention have small and uniform particle sizes, good tissue permeability and high bioavailability.

[0018] In a second aspect, the present invention provides a method for preparing the nanomicelle ophthalmic preparation according to the first aspect, the preparation method comprising:

[0019] The organic solution containing polyoxyethylene hydrogenated castor oil and cyclosporine is subjected to solvent removal treatment to form a film, and then mixed with an aqueous vehicle for hydration treatment to form a clear and transparent system, namely the nano-micelle ophthalmic preparation.

[0020] The preparation method of the nano-micelle ophthalmic preparation of the present invention has simple process and is easy to operate, and is very suitable for industrial large-scale production.

[0021] Preferably, the solvent includes any one of ethanol, methanol, acetonitrile, acetone, chloroform or dichloromethane, or a combination of at least two thereof.

[0022] Preferably, the solvent removal treatment method includes rotary evaporation or reduced pressure distillation.

[0023] Preferably, the hydration treatment comprises oscillation treatment or ultrasonic treatment.

[0024] The preparation dosage form formed after the above hydration treatment is a solution, and it can also be further freeze-dried to obtain a powder for storage.

[0025] In a third aspect, the present invention provides use of the nanomicelle ophthalmic preparation according to the first aspect in the preparation of cyclosporine eye drops.

[0026] In a fourth aspect, the present invention provides a cyclosporine eye drop, wherein the raw materials for preparing the cyclosporine eye drop include the nanomicelle ophthalmic preparation according to the first aspect and a pharmaceutically acceptable excipient.

[0027] Preferably, the auxiliary material comprises any one of an osmotic pressure regulator, a pH regulator, a thickener or a preservative, or a combination of at least two thereof.

[0028] Preferably, the osmotic pressure regulator comprises any one of sodium chloride, mannitol, sorbitol, glucose, glycerol, polyethylene glycol or propylene glycol, or a combination of at least two thereof.

[0029] Preferably, the pH adjuster comprises any one or a combination of at least two of phosphate buffer, boric acid, sodium borate, tromethamine, tromethamine hydrochloride buffer, sodium hydroxide, hydrochloric acid, citric acid or sodium citrate.

[0030] Preferably, the thickener comprises any one of hyaluronic acid or its salt, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, polyvinyl pyrrolidone, chitosan, carbomer, glycerol or polyoxyethylene, or a combination of at least two thereof.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Based on the drawbacks of poor safety, poor long-term stability, high preparation cost or low cyclosporin concentration of existing cyclosporin ophthalmic preparations, the present invention creatively designs an ophthalmic preparation product with a specific formula and a specific preparation process, wherein the solubilizer used is only a single component of polyoxyethylene hydrogenated castor oil, and no other solubilizers are needed, and no organic solvents such as ethanol, methanol, etc., are needed to be added to the formula, and no oily solvents are needed to be added, so that cyclosporin can be stably present in the aqueous solution at a higher concentration, which is conducive to the accumulation of a higher concentration of cyclosporin in the eye tissue, thereby facilitating the cyclosporin to take effect faster. At the same time, this greatly improves the safety of the product and reduces the manufacturing cost.

[0033] The nano-micelle ophthalmic preparation of the present invention uses a thin film hydration method for preparation, and polyoxyethylene hydrogenated castor oil can form a long-term stable nano-micelle system with cyclosporine in an aqueous solvent, the micelle particle size is less than 50nm, and the system is clear and transparent. If other preparation processes such as aqueous solution method are used, the long-term stability of the product is poor, and obvious precipitation will occur after long-term storage. DETAILED DESCRIPTION

[0034] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0035] The cyclosporine involved in the following content was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; polyoxyethylene hydrogenated castor oil was purchased from BASF.

[0036] The instruments used in the following contents include: laser particle size analyzer (Malvern, zetasizer ZSP); high performance liquid chromatograph (Shimadzu LC-20-AT).

[0037] The cyclosporine content was measured by the external standard method. The sample was centrifuged to obtain the supernatant before detection. The conditions for high performance liquid chromatography detection were: 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 water: acetonitrile: tert-butyl methyl ether: phosphoric acid solution (85%) = 520: 430: 50: 1 (volume ratio), isocratic elution.

[0038] Example 1

[0039] This embodiment provides a nano-micelle ophthalmic preparation, and the preparation method thereof is as follows:

[0040] 9 mg of cyclosporine and 45 mg of polyoxyethylene hydrogenated castor oil were placed in a 100 mL round-bottom flask, 10 mL of anhydrous ethanol was added, and after sufficient dissolution, the anhydrous ethanol was removed by a rotary evaporator to obtain a uniformly dispersed film, and then 10 mL of purified water was added and hydrated to dissolve the film to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0041] Example 2

[0042] This embodiment provides a nano-micelle ophthalmic preparation, and the preparation method thereof is as follows:

[0043] 9 mg of cyclosporine and 200 mg of polyoxyethylene hydrogenated castor oil were placed in a 100 mL round-bottom flask, 10 mL of dichloromethane was added, and after sufficient dissolution, the dichloromethane was removed by a rotary evaporator to obtain a uniformly dispersed film, and then 5 mL of purified water was added and hydrated to dissolve the film to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0044] Example 3

[0045] This embodiment provides a nano-micelle ophthalmic preparation, and the preparation method thereof is as follows:

[0046] 9 mg of cyclosporine and 250 mg of polyoxyethylene hydrogenated castor oil were placed in a 100 mL round-bottom flask, 10 mL of methanol was added, and after sufficient dissolution, the methanol was removed by a rotary evaporator to obtain a uniformly dispersed film, and then 3 mL of water was added and hydrated to dissolve the film to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0047] Example 4

[0048] This embodiment provides a nano-micelle ophthalmic preparation, and the preparation method thereof is as follows:

[0049] 9 mg of cyclosporine and 120 mg of polyoxyethylene hydrogenated castor oil were placed in a 100 mL round-bottom flask, 10 mL of methanol was added, and after sufficient dissolution, the methanol was removed by a rotary evaporator to obtain a uniformly dispersed film, and then 10 mL of water was added and hydrated to dissolve the film to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0050] Comparative Example 1

[0051] This comparative example provides a nano-micelle ophthalmic preparation, and its preparation method is as follows:

[0052] 120 mg of polyoxyethylene hydrogenated castor oil was placed in a glass bottle, heated at 40°C until the polyoxyethylene hydrogenated castor oil was completely melted, 9 mg of cyclosporine was added, and the mixture was fully stirred at 40°C until the cyclosporine was evenly dispersed. Then, 10 mL of purified water was added, and the mixture was fully stirred at 25°C until the system became clear and transparent to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0053] Comparative Example 2

[0054] This comparative example provides a nano-micelle ophthalmic preparation, and its preparation method is as follows:

[0055] 120 mg of polyoxyethylene hydrogenated castor oil was placed in a glass bottle, heated at 55° C. until the polyoxyethylene hydrogenated castor oil was completely melted, 9 mg of cyclosporine was added, and the mixture was stirred at 55° C. for 5 minutes. The mixture was cooled to 35° C., 10 mL of purified water was added, and the mixture was fully stirred at 25° C. until the system became clear and transparent to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0056] Comparative Example 3

[0057] This comparative example provides a nano-micelle ophthalmic preparation, and its preparation method is as follows:

[0058] 120 mg of polyoxyethylene hydrogenated castor oil was placed in a glass bottle, heated at 55° C. until the polyoxyethylene hydrogenated castor oil was completely melted, 9 mg of cyclosporine was added, and the mixture was stirred at 55° C. for 5 minutes. The mixture was cooled to 35° C., 5 mg of octylphenol polyoxyethylene ether-40 and 10 mL of purified water were added, and the mixture was fully stirred at 25° C. until the system became clear and transparent to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0059] Comparative Example 4

[0060] This comparative example provides a nano-micelle ophthalmic preparation, and its preparation method is as follows:

[0061] 120 mg of polyoxyethylene hydrogenated castor oil was placed in a glass bottle, 10 mL of pure water was added, and the mixture was heated at 40° C. until the polyoxyethylene hydrogenated castor oil was completely dissolved in the water. Then, 9 mg of cyclosporine was added, and the mixture was stirred sufficiently at 40° C. until the cyclosporine was evenly dispersed and the system became clear and transparent to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0062] Comparative Example 5

[0063] This comparative example provides a nano-micelle ophthalmic preparation, and its preparation method is as follows:

[0064] 120 mg of polyoxyethylene hydrogenated castor oil was placed in a glass bottle, 10 mL of pure water was added, and the mixture was heated at 40° C. until the polyoxyethylene hydrogenated castor oil was completely dissolved in the water. Then, 9 mg of cyclosporine was added, and the mixture was stirred at 25° C. until the cyclosporine was evenly dispersed and the system became clear and transparent to obtain a micellar solution. The micellar solution was filtered through a 0.22 μm microporous filter membrane to remove insoluble particles to obtain a nanomicelle ophthalmic preparation.

[0065] Test Example 1

[0066] The particle size of the products obtained in Examples 1-4 and Comparative Examples 1-5 was characterized by a laser particle size analyzer. The results are shown in Table 1.

[0067] Table 1

[0068] sample Particle size (nm) PDI Example 1 28.10 0.239 Example 2 32.87 0.183 Example 3 32.55 0.231 Example 4 30.23 0.205 Comparative Example 1 30.37 0.105 Comparative Example 2 33.61 0.106 Comparative Example 3 32.43 0.225 Comparative Example 4 32.11 0.195 Comparative Example 5 28.19 0.183

[0069] It can be seen from the data in Table 1 that the micelle particle size in the nano-micelle ophthalmic preparation system of the present invention is small and uniform.

[0070] Test Example 2

[0071] The products obtained in Examples 1-4 and Comparative Examples 1-5 were placed at 35° C. for 30 days, and their appearances were recorded. The cyclosporine content (mg / mL) was determined by high performance liquid chromatography. The results are shown in Table 2.

[0072] Table 2

[0073]

[0074] From the data in Table 2, it can be seen that the nano-micelle ophthalmic preparation system of the present invention has good stability. After being placed at 35° C. for 30 days, the cyclosporine content has basically no significant change, while the cyclosporine content of the product of the comparative example is significantly reduced, and the stability is poor.

[0075] Test Example 3

[0076] The products prepared in Examples 1-4 were placed at 35° C. for 12 months, and the cyclosporine content (mg / mL) was measured by HPLC at 0 month, 1 month, 2 months, 3 months, 6 months, and 12 months, respectively. The results are shown in Table 3.

[0077] Table 3

[0078] sample 0 months 1 month 2 months 3 months 6 months 12 months Example 1 0.883 0.865 0.459 / / / Example 2 1.754 1.733 0.715 / / / Example 3 2.903 2.809 0.613 / / / Example 4 0.885 0.871 0.863 0.863 0.921 0.914

[0079] From the data in Table 3, it can be seen that the nano-micelle ophthalmic preparation system of Example 4 of the present invention has the best long-term stability. After being placed at 35° C. for 12 months, the cyclosporine content has basically no significant change.

[0080] Application Example 1

[0081] This application example provides a cyclosporine eye drops, the preparation method of which is as follows:

[0082] Disodium hydrogen phosphate (dihydrate) 48 mg, sodium dihydrogen phosphate (anhydrous) 52 mg, and sodium chloride 48 mg were dissolved in 1 mL of pure water; 30 mg of thickener PVP-K90 was dissolved in 1 mL of pure water; the product of Example 4 was mixed with the above two solutions, and purified water was added to reach a final volume of 10 mL. The solution was filtered through a 0.22 micron filter to remove insoluble particles to obtain cyclosporine eye drops.

[0083] Test Case

[0084] Conduct eye irritation test on the product prepared according to Example 1:

[0085] Take 5 healthy rabbits, drip 50μL of the test product into the left eye of each rabbit, and 50μL of normal saline into the right eye, and gently close the eyelids for 10 seconds. Administer the drug 4 times a day for 7 consecutive days (cyclosporine eye drops). Observe the local reactions of the cornea, iris, and conjunctiva of the rabbits before administration and 1, 2, 4, 24, 48, and 72 hours after the last administration every day, and calculate the total score (average score). The irritation score of normal saline is 0 points. Test scoring criteria and methods: See the table below.

[0086]

[0087]

[0088] The result judgment criteria are shown in the following table:

[0089] Thrill Level Total score Criteria 1 0-3 points Non-irritating 2 4-8 points Mild irritation 3 9-12 points Moderate irritation 4 13-16 points Intensity of stimulation

[0090] The total score of the eye irritation test result is 0, indicating that the nano-micelle ophthalmic preparation and cyclosporine eye drops of the present invention have no obvious irritation to the eyes of rabbits and are safe and mild.

[0091] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0092] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0093] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A nanomicelle ophthalmic preparation, characterized in that: The nano-micelle ophthalmic preparation is prepared by a thin film hydration method using cyclosporine, a solubilizer and an aqueous vehicle as raw materials, and the solubilizer is polyoxyethylene hydrogenated castor oil.

2. The nanomicelle ophthalmic preparation according to claim 1, characterized in that: The polymerization degree of polyoxyethylene in the polyoxyethylene hydrogenated castor oil is 30-80; Preferably, the aqueous vehicle comprises water or phosphate buffered saline.

3. The nanomicelle ophthalmic preparation according to claim 1 or 2, characterized in that: The mass ratio of the solubilizing agent to cyclosporine is not less than 5:1; Preferably, the mass ratio of the aqueous vehicle to cyclosporine is not less than 333:

1.

4. The nanomicelle ophthalmic preparation according to any one of claims 1 to 3, characterized in that: The particle size of the drug-loaded micelles prepared by the thin film hydration method is less than 50 nm.

5. The method for preparing the nanomicelle ophthalmic preparation according to any one of claims 1 to 4, characterized in that: The preparation method comprises: The organic solution containing polyoxyethylene hydrogenated castor oil and cyclosporine is subjected to solvent removal treatment to form a film, and then mixed with an aqueous vehicle for hydration treatment to form a clear and transparent system, namely the nano-micelle ophthalmic preparation.

6. The method for preparing the nanomicelle ophthalmic preparation according to claim 5, characterized in that: The solvent includes any one of ethanol, methanol, acetonitrile, acetone, chloroform or dichloromethane, or a combination of at least two thereof; Preferably, the solvent removal treatment method includes rotary evaporation or reduced pressure distillation; Preferably, the hydration treatment comprises oscillation treatment or ultrasonic treatment.

7. Use of the nanomicelle ophthalmic preparation according to any one of claims 1 to 4 in the preparation of cyclosporine eye drops.

8. A cyclosporine eye drop, characterized in that: The raw materials for preparing the cyclosporine eye drops include the nanomicelle ophthalmic preparation according to any one of claims 1 to 4 and pharmaceutically acceptable excipients.

9. The cyclosporine eye drops according to claim 8, characterized in that The auxiliary materials include any one of an osmotic pressure regulator, a pH regulator, a thickener or a preservative, or a combination of at least two of them.

10. The cyclosporine eye drops according to claim 9, characterized in that The osmotic pressure regulator includes any one or a combination of at least two of sodium chloride, mannitol, sorbitol, glucose, glycerol, polyethylene glycol or propylene glycol; Preferably, the pH adjuster comprises any one or a combination of at least two of phosphate buffer, boric acid, sodium borate, tromethamine, tromethamine hydrochloride buffer, sodium hydroxide, hydrochloric acid, citric acid or sodium citrate; Preferably, the thickener comprises any one of hyaluronic acid or its salt, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, polyvinyl pyrrolidone, chitosan, carbomer, glycerol or polyoxyethylene, or a combination of at least two thereof.

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