Cyclosporine emulsion and method for producing the same

Abstract

The present invention relates to a cyclosporine emulsion and a method for producing the same. In particular, the oil-in-water cyclosporine emulsion contains an interfacial stabilizer, and the interfacial stabilizer includes a combination of polyethylene glycol 15-hydroxystearate and phospholipids. The pH value, particle size, and encapsulation rate of the emulsion are stable over a long period of time.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals, and particularly to a cyclosporine emulsion and a method for producing the same.

Background Art

[0002] Cyclosporine is an effective immunosuppressant as a calcineurin inhibitor, has a lower toxic effect compared to other immunosuppressants, and selectively changes the function of T lymphocytes reversibly, thereby blocking the transcription of lymphokine genes, interfering with the transmission of original information, suppressing the release of interleukin-2 (IL-2), interferon and other immune factors, and can play an anti-inflammatory role. After topical administration of cyclosporine to the eye, it induces the inactivation of calcineurin phosphatase and blocks the release of pro-inflammatory cytokines such as IL-2, thereby alleviating the symptoms of dry eye (xerophthalmia).

[0003] Cyclosporine is almost insoluble in water but has good lipophilicity. Commercially available eye drops are generally prepared as a solution by dissolving in vegetable oil or as an oil-in-water emulsion using an emulsification technique. Oily solution agents are highly irritating when used for topical administration to the eye, and patient compliance is poor. In the field of oil-in-water ophthalmic emulsions, Restasis (registered trademark) and Ikervis (registered trademark), both of which are already on the market, are both submicron emulsions with an average particle size of 100 nm or more, and their properties are opaque milky white, which will blur the patient's vision after eye drops. Furthermore, Restasis (registered trademark) has a large particle size and no components are added to promote drug retention and penetration in the eye, so its bioavailability is low. Ikervis (registered trademark) uses cetalkonium chloride, a cationic surfactant, to extend the retention time of the drug on the eye surface, but the use of the cationic surfactant increases the occurrence of side effects such as eye pain, eye irritation, and eye congestion.

[0004] Nanoemulsion is a translucent to almost transparent dispersion system with a particle size of 100 nm or less formed by self-assembly of oil, water, surfactant, etc. Nanoemulsion is applied to ocular administration. Its nano-sized particle size can increase the permeability of drugs into ocular tissues and enhance the drug efficacy. Higher transparency can improve patient compliance and has very high development value.

[0005] Currently, oil-in-water ophthalmic emulsions need to solve two major technical problems: 1) the problems of the stability of emulsion particles and the stability of encapsulation. For example, the ionic strength of physiological tears or artificial tear substitutes affects the stability of the interfacial film of the emulsion, which further leads to the aggregation of emulsion particles and the leakage of drugs, affecting the safety and efficacy of the preparation. 2) In the case of long-term stability, the pH value of the preparation gradually decreases, affecting the quality of the preparation. The pH value of oil-in-water emulsions is usually adjusted using sodium hydroxide or hydrochloric acid. However, as the oil and fat are hydrolyzed into free fatty acids during the storage process, the pH value gradually decreases. When a buffer system is formed using buffer salts, the stability of the pH value can be enhanced, but the increase in ionic strength affects the stability of the interfacial film, which further leads to the aggregation of emulsion particles and the leakage of drugs. In view of this, in the field of oil-in-water ophthalmic emulsions, there is an urgent need to provide a combination of stabilizers that can produce a synergistic stabilizing effect, jointly maintain stability in a high ionic strength environment (such as a buffer salt solution), and further achieve long-term stability of the pH value, particle size, encapsulation rate, etc. of the preparation.

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a cyclosporine emulsion having a controllable particle size (for example, an average particle size of 50 nm or less) and a high encapsulation rate (90% or more).

[0007] Another object of the present invention is to provide a cyclosporine emulsion with long-term stability of pH value, particle size, and encapsulation rate.

Means for Solving the Problems

[0008] The present invention provides an oil-in-water type cyclosporine emulsion containing an interfacial stabilizer, wherein the interfacial stabilizer includes a combination of polyethylene glycol 15-hydroxystearate and phospholipids.

[0009] The present invention further provides a method for producing an oil-in-water type cyclosporine emulsion, which includes an interfacial stabilizer containing a combination of polyethylene glycol 15-hydroxystearate and phospholipids, an osmotic pressure regulator, and a buffer salt, and the method comprises: 1) uniformly mixing an oil and an interfacial stabilizer, adding cyclosporine thereto and dissolving it to form an oil phase; 2) dissolving a part of the osmotic pressure regulator in water, and stirring and mixing the oil phase and the water containing the osmotic pressure regulator to form a primary emulsion; 3) dissolving the remaining buffer salt and osmotic pressure regulator in water to form a solution, and adding the primary emulsion to the solution and uniformly mixing them. The present invention provides a method for producing an oil-in-water type cyclosporine emulsion.

Advantages of the Invention

[0010] The above cyclosporine emulsion has improved interfacial film stability, can effectively suppress the transfer of cyclosporine to an aqueous medium, and can suppress the aggregation of emulsion particles. Therefore, the long-term stability is greatly improved in terms of pH value, particle size, and encapsulation rate. Due to the permeability promoting property caused by the nano-sized particle size and higher transparency, this emulsion has higher drug efficacy and better patient compliance.

Embodiments for Carrying out the Invention

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0012] For all numerical ranges mentioned in this disclosure, it should be understood that all specific numerical values within that range, and sub-ranges limited by any two numerical values within that range, are disclosed. For example, in the case of 1 to 20, specific numerical values such as 1, 2, 3, 3.5, 4.5, 10, 12, 15, 20, etc., and sub-ranges such as 1 to 5, 2 to 6, 3.5 to 7.5, 15 to 20, etc., are disclosed.

[0013] The present invention relates to an oil-in-water type cyclosporine emulsion containing an interfacial stabilizer, preferably a nanoemulsion, and the interfacial stabilizer includes a combination of polyethylene glycol ester of 15-hydroxystearic acid and phospholipids.

[0014] In one embodiment, the oil-in-water type cyclosporine emulsion further contains a buffer salt.

[0015] In one embodiment, the phospholipids are selected from soybean phospholipids, egg yolk phospholipids, or any combination thereof, preferably selected from soybean phospholipid S100, egg yolk phospholipid E80, or any combination thereof.

[0016] In one embodiment, the mass ratio of cyclosporine to polyethylene glycol ester of 15-hydroxystearic acid is 1:30 to 1:45.

[0017] In one embodiment, the mass ratio of phospholipids to polyethylene glycol ester of 15-hydroxystearic acid is 1:14 to 1:31.

[0018] In one embodiment, the content of polyethylene glycol 15-hydroxystearate is 2.25 to 13.5 g / 100 ml.

[0019] In one embodiment, the buffer salt is a phosphate buffer salt, and preferably, the buffer salt concentration is 0.01 mol / L to 0.05 mol / L in terms of phosphate group.

[0020] In one embodiment, the oil-in-water type cyclosporine emulsion further contains an osmotic pressure regulator, and preferably, the osmotic pressure regulator is selected from sodium chloride, sorbitol, or any combination thereof, and preferably, the content of the osmotic pressure regulator is 0.2 to 1 g / 100 ml.

[0021] In one embodiment, the average particle size of the particles of the emulsion is 50 nm or less, preferably 10 to 40 nm, more preferably 20 to 30 nm, preferably, the particle size of 90% of the particles of the emulsion is 90 nm or less, preferably, the pH value of the emulsion is 6.5 to 8.0, preferably 6.8 to 7.6, more preferably 7.0 to 7.4, preferably, the encapsulation rate of the emulsion is 90% or more, preferably 95% or more, preferably, the emulsion is a transparent milky liquid, preferably, the emulsion is an eye drop, and preferably, the oil is selected from medium-chain triglycerides, soybean oil, castor oil, olive oil, or fish oil.

[0022] The present invention further provides a method for producing an oil-in-water type cyclosporine emulsion comprising an interfacial stabilizer containing a combination of polyethylene glycol 15-hydroxystearate and phospholipids, an osmotic pressure regulator, and a buffer salt, comprising: 1) uniformly mixing an oil and an interfacial stabilizer, adding cyclosporine thereto and dissolving it to form an oil phase; 2) dissolving a part of the osmotic pressure regulator in water, and stirring and mixing the oil phase and the water containing the osmotic pressure regulator to form a primary emulsion; 3) Dissolve the remainder of the buffer salt and the osmotic pressure regulator in water to form a solution, and add the primary emulsion to the solution and mix uniformly, to provide a method for producing an oil-in-water type cyclosporine emulsion.

[0023] As the buffer salt, a phosphate buffer salt is preferred.

[0024] The technical features in the various embodiments mentioned above can be combined with each other without contradiction to form another technical solution.

[0025] Examples Example 1 The cyclosporine nanoemulsion is produced using polyethylene glycol 15-hydroxystearate and soy lecithin S100 as stabilizers, disodium hydrogen phosphate and sodium dihydrogen phosphate as pH regulators, and sodium chloride as an osmotic pressure regulator, and the formulation composition is as follows.

Table 1

[0026] Manufacturing method: 1) Add polyethylene glycol 15-hydroxystearate, soy lecithin S100 and oil to Container 1, heat to 60°C to 80°C, stir until uniformly mixed, add cyclosporine, stir and dissolve to form an oil phase. 2) Add about 9% of the total amount of water for injection to Container 2, add an appropriate amount of the osmotic pressure regulator while stirring, and continue stirring until dissolved to form the primary emulsion aqueous phase. 3) Add the primary emulsion aqueous phase to the oil phase while stirring, gradually raise the temperature to 60°C to 80°C while stirring, and continue stirring for 5 to 15 minutes to form a primary emulsion. 4) Add about 50% of the total amount of water for injection to Container 3, add the pH regulator and the remaining osmotic pressure regulator while stirring, and continue stirring until dissolved to obtain a diluted aqueous phase. 5) Add the primary emulsion to the diluted aqueous phase while stirring, rinse container 2 several times with an appropriate amount of water for injection, transfer the rinsing water to container 3, and continue stirring for 5 - 10 minutes. 6) Add water for injection up to the total formulation volume and stir uniformly.

[0027] Example 2 Cyclosporine nanoemulsion is prepared using polyethylene glycol 15 - hydroxystearate and egg yolk phospholipid E80 as stabilizers, disodium hydrogen phosphate and sodium dihydrogen phosphate as pH adjusters, and sodium chloride as an osmotic pressure adjuster. The formulation composition is as follows.

Table 2

[0028] Manufacturing method: 1) Add polyethylene glycol 15 - hydroxystearate, egg yolk phospholipid E80, and oil to container 1, heat to 60°C - 80°C, stir until uniformly mixed, add cyclosporine, and stir to dissolve to form an oil phase. 2) Add about 9% of the total formulation volume of water for injection to container 2, add an appropriate amount of osmotic pressure adjuster while stirring, and continue stirring until dissolved to form a primary emulsion aqueous phase. 3) Add the primary emulsion aqueous phase to the oil phase while stirring, gradually raise the temperature to 60°C - 80°C while stirring, and continue stirring for 5 - 15 minutes to form a primary emulsion. 4) Add about 50% of the total formulation volume of water for injection to container 3, add the pH adjuster and the remaining osmotic pressure adjuster while stirring, and continue stirring until dissolved to obtain a diluted aqueous phase. 5) Add the primary emulsion to the diluted aqueous phase while stirring, rinse container 2 several times with an appropriate amount of water for injection, transfer the rinsing water to container 3, and continue stirring for 5 - 10 minutes. 6) Add water for injection up to the total formulation volume and stir uniformly.

[0029] Example 3 Cyclosporine nanoemulsion is prepared using polyethylene glycol 15 - hydroxystearate and soy lecithin S100 as stabilizers, disodium hydrogen phosphate and sodium dihydrogen phosphate as pH adjusters, and sodium chloride and sorbitol as osmotic pressure regulators, and the formulation composition is as follows.

Table 3

[0030] Manufacturing method: 1) Add polyethylene glycol 15 - hydroxystearate, soy lecithin S100 and oil to Container 1, heat to 60°C - 80°C, stir until uniformly mixed, add cyclosporine, and stir to dissolve to form an oil phase. 2) Add about 9% of the total formulated amount of water for injection to Container 2, add an appropriate amount of osmotic pressure regulator while stirring, and continue stirring until dissolved to form the primary emulsion aqueous phase. 3) Add the primary emulsion aqueous phase to the oil phase while stirring, gradually raise the temperature to 60°C - 80°C while stirring, and continue stirring for 5 - 15 minutes to form a primary emulsion. 4) Add about 50% of the total formulated amount of water for injection to Container 3, add the pH adjuster and the remaining osmotic pressure regulator while stirring, and continue stirring until dissolved to obtain a diluted aqueous phase. 5) Add the primary emulsion to the diluted aqueous phase while stirring, rinse Container 2 several times with an appropriate amount of water for injection, transfer the rinsing water to Container 3, and continue stirring for 5 - 10 minutes. 6) Add water for injection to make up the total formulated amount and stir uniformly.

[0031] Example 4 Cyclosporine nanoemulsion is prepared using polyethylene glycol 15 - hydroxystearate and soy lecithin S100 as stabilizers, disodium hydrogen phosphate and sodium dihydrogen phosphate as pH adjusters, and sodium chloride as an osmotic pressure regulator, and the formulation composition is as follows.

Table 4

[0032] Manufacturing method: 1) Add polyethylene glycol ester of 15 - hydroxystearic acid, soybean phospholipid S100 and oil to Container 1, heat to 60°C - 80°C, stir until uniformly mixed, add cyclosporine, and stir until dissolved to form an oil phase. 2) Add about 27% of the total formulated amount of water for injection to Container 2, add an appropriate amount of osmotic pressure regulator while stirring, and continue stirring until dissolved to form a primary emulsion aqueous phase. 3) Add the primary emulsion aqueous phase to the oil phase while stirring, gradually raise the temperature to 60°C - 80°C while stirring, and continue stirring for 5 - 15 minutes to form a primary emulsion. 4) Add about 30% of the total formulated amount of water for injection to Container 3, add a pH adjuster and the remaining osmotic pressure regulator while stirring, and continue stirring until dissolved to obtain a diluted aqueous phase. 5) Add the primary emulsion to the diluted aqueous phase while stirring, rinse Container 2 several times with an appropriate amount of water for injection, transfer the rinsing water to Container 3, and continue stirring for 5 - 10 minutes. 6) Add water for injection up to the total formulated amount and stir uniformly.

[0033] Example 5 The cyclosporine nanoemulsion is prepared using polyethylene glycol ester of 15 - hydroxystearic acid and soybean phospholipid S100 as stabilizers, disodium hydrogen phosphate and sodium dihydrogen phosphate as pH adjusters, and sodium chloride as an osmotic pressure regulator, and the formulation composition is as follows.

Table 5

[0034] Manufacturing method: 1) Add polyethylene glycol 15 - hydroxystearate, soy lecithin S100 and oil to Container 1, heat to 60°C to 80°C, stir until uniformly mixed, add cyclosporine, and stir to dissolve to form an oil phase. 2) Add about 4.5% of the total formulation volume of water for injection to Container 2, add an appropriate amount of osmotic pressure regulator while stirring, and continue stirring until dissolved to form the aqueous phase of the primary emulsion. 3) Add the aqueous phase of the primary emulsion to the oil phase while stirring, gradually raise the temperature to 60°C to 80°C while stirring, and continue stirring for 5 to 15 minutes to form the primary emulsion. 4) Add about 25% of the total formulation volume of water for injection to Container 3, add the pH regulator and the remaining osmotic pressure regulator while stirring, and continue stirring until dissolved to obtain a diluted aqueous phase. 5) Add the primary emulsion to the diluted aqueous phase while stirring, rinse Container 2 several times with an appropriate amount of water for injection, transfer the rinsing water to Container 3, and continue stirring for 5 to 10 minutes. 6) Add water for injection up to the total formulation volume and stir uniformly.

[0035] Example 6 The cyclosporine nanoemulsion is manufactured using polyethylene glycol 15 - hydroxystearate and soy lecithin S100 as stabilizers, disodium hydrogen phosphate and sodium dihydrogen phosphate as pH regulators, and sodium chloride as an osmotic pressure regulator, and the formulation composition is as follows.

Table 6

[0036] Manufacturing method: 1) Add polyethylene glycol 15 - hydroxystearate, soy lecithin S100 and oil to Container 1, heat to 60°C to 80°C, stir until uniformly mixed, add cyclosporine, and stir to dissolve to form an oil phase. 2) Add about 9% of the total formulation volume of water for injection to Container 2, add an appropriate amount of osmotic pressure regulator while stirring, and continue stirring until dissolved to form the primary emulsion aqueous phase. 3) Add the primary emulsion aqueous phase to the oil phase while stirring, gradually raise the temperature to 60°C to 80°C while stirring, and continue stirring for 5 to 15 minutes to form the primary emulsion. 4) Add about 50% of the total formulation volume of water for injection to Container 3, add the pH regulator and the remaining osmotic pressure regulator while stirring, and continue stirring until dissolved to obtain the diluted aqueous phase. 5) Add the primary emulsion to the diluted aqueous phase while stirring, rinse Container 2 several times with an appropriate amount of water for injection, transfer the rinsing water to Container 3, and continue stirring for 5 to 10 minutes. 6) Add water for injection up to the total formulation volume and stir uniformly.

[0037] Example 7 Cyclosporine nanoemulsion is manufactured using polyethylene glycol 15-hydroxystearate and soybean phospholipid S100 as stabilizers, disodium hydrogen phosphate and sodium dihydrogen phosphate as pH regulators, and sodium chloride as an osmotic pressure regulator, and the formulation composition is as follows.

Table 7

[0038] Manufacturing method: 1) Add polyethylene glycol 15-hydroxystearate, soybean phospholipid S100, and oil to Container 1, heat to 60°C to 80°C, stir until uniformly mixed, add cyclosporine, and stir until dissolved to form the oil phase. 2) Add about 9% of the total formulation volume of water for injection to Container 2, add an appropriate amount of osmotic pressure regulator while stirring, and continue stirring until dissolved to form the primary emulsion aqueous phase. 3) Add the primary emulsion aqueous phase to the oil phase while stirring, gradually raise the temperature to 60°C to 80°C while stirring, and continue stirring for 5 to 15 minutes to form the primary emulsion. 4) Add about 50% of the total amount of water for injection to Container 3, add the pH adjuster and the remaining osmotic pressure adjuster while stirring, and continue stirring until dissolved to obtain a diluted aqueous phase. 5) Add the primary emulsion to the diluted aqueous phase while stirring, rinse Container 2 several times with an appropriate amount of water for injection, transfer the rinsing water to Container 3, and continue stirring for 5 to 10 minutes. 6) Add water for injection up to the total amount of formulation and stir uniformly.

[0039] Experimental Example 8: Investigation of the Quality Characteristics of Cyclosporine Nanoemulsion In this application, the particle size is measured according to Method 3 of General Rule 0982, Section IV of the Chinese Pharmacopoeia (2020 Edition), and the detection method for the encapsulation rate is as follows.

[0040] Take 200 μl of this product and add it to the tip of a Sephadex G-25 microcolumn, centrifuge at 2000 rpm for 1 minute, elute with water as the eluent, add 200 μl of water to the tip of the microcolumn, centrifuge at 2000 rpm for 2 minutes, repeat the elution 4 times, collect the eluent in a 5-ml volumetric flask, add methanol to break the emulsion, dilute to the mark, and shake well. Take an appropriate amount of the cyclosporine reference substance (standard substance), add 500 μl of methanol to dissolve it, and then quantitatively dilute it with a 50% methanol solution to prepare a solution containing about 0.1 mg of cyclosporine reference substance per 1 ml. Use octadecylsilane-bonded silica gel as the filler, acetonitrile-water (70:30) as the mobile phase, the flow rate is 1.0 ml / min, the temperature of the stainless steel column and the column is 80 °C, and the detection wavelength is 210 nm. Take the above solutions respectively, inject them into a liquid chromatography device, record the chromatogram, and calculate as the peak area according to the external standard method.

[0041] Samples were prepared according to the formulation and process of Example 1, and the properties, pH value, particle size, and encapsulation rate of the cyclosporine nanoemulsion were investigated, and the results are shown in Table 1. Samples were prepared according to the formulation and process of Example 2, Example 3, Example 4, Example 5, Example 6, and Example 7, and the properties, pH value, and particle size of the cyclosporine nanoemulsion were investigated, and the results are shown in Table 2.

Table 8

Table 9

[0042] Conclusion: The properties, pH value, particle size, and encapsulation rate of the cyclosporine nanoemulsion meet the limiting requirements.

[0043] Experimental Example 9: Stability Test of Cyclosporine Nanoemulsion Samples were prepared according to the formulation and process of Example 1, Example 2, and Example 3, and the stability against high temperature of 40 °C and light irradiation of 5000 ± 500 lx was investigated, and the results are shown in Table 3 and Table 4. Samples were prepared according to the formulation and process of Example 1, and the intermediate condition test at 30 °C and the long-term test at 15 - 20 °C were investigated, and the results are shown in Table 5 and Table 6.

Table 10

Table 11

Table 12

Table 13

[0044] Conclusion:The pH value, particle size, and encapsulation rate stability of the cyclosporine nanoemulsion are good. The use of buffer salts can improve the pH value stability of the emulsion. The interfacial composite film composed of polyethylene glycol 15-hydroxystearate and phospholipids can withstand a relatively high ionic strength, which means that the stability of emulsion particles and encapsulation during the storage process of the nanoemulsion is ensured.

[0045] Experimental Example 10: Pharmacodynamic Test of Cyclosporine Nanoemulsion on Dry Eye in Mice Induced by Dry Stress 1. Test drug: Low-dose test sample: 0.05% cyclosporine nanoemulsion prepared according to the formulation and process of Example 5. Medium-dose test sample: 0.1% cyclosporine nanoemulsion prepared according to the formulation and process of Example 1. High-dose test sample: 0.3% cyclosporine nanoemulsion prepared according to the formulation and process of Example 4. Commercially available control: Ikervis (registered trademark), specification 0.1%. Blank adjuvant material: Prepared according to the formulation and process of Example 1, except that it does not contain cyclosporine.

[0046] 2. Test animals: C57BL / 6JShjh mice, body weight 15.4 - 20.4 g, female.

[0047] 3. Test method:150 healthy female C57BL / 6JShjh mice were selected. On D-1, 21 mice without eye abnormalities in both eyes were selected and not modeled. 125 mice were continuously exposed to a low-humidity environment for 5 days and scopolamine was subcutaneously injected (twice a day, 0.75 mg / mL, 0.3 mL / mouse / time) to induce modeling. The first day of modeling was designated as D1. On D5, 15 non-modeled animals with similar corneal fluorescein sodium staining scores and tear secretion amounts were selected and classified into the non-modeled group. 90 modeled animals with similar corneal fluorescein sodium staining scores and tear secretion amounts were selected. At the same time, considering the baseline scores before modeling, they were randomly divided into 2 - 7 groups (15 animals / group) according to the average value of the corneal fluorescein sodium staining scores of both eyes, which were in order the model control group, the blank excipient group, the commercial control group, and the low, medium, and high dose groups of the test sample. From D6 to D15, the animals in groups 2 - 7 continued to be modeled (the method was the same as before), groups 1 - 2 were not administered, and groups 3 - 7 were given blank excipient, Ikervis, 0.05%, 0.1%, and 0.3% cyclosporine nanoemulsion respectively, and instilled into both eyes (three times a day, at approximately 3-hour intervals, 3 μL per eye). On D-1, D5, D10, and D15, tear secretion amount examinations and corneal fluorescein sodium staining tests were performed on the animals in each group.

[0048] 4. Evaluation method: Tear secretion amount: The mice were restrained, and a commercially available shortened phenol red cotton thread was clamped using clean non-toothed forceps. The cotton thread was fixed in the middle of the lower conjunctival sac of the mouse for 30 seconds, and the stained wet length of the cotton thread was measured. The summary table of tear secretion amount is shown in Table 7.

[0049] Corneal fluorescein sodium staining test: After dropping a fluorescein sodium solution (1.5 μL, 0.5%) into the superior conjunctival sac of the animal, the conjunctival sac of the animal was rinsed three times continuously with 1.25 mL of sterile physiological saline every about 10 seconds starting from about 1.5 minutes of staining. After each rinsing was completed, the physiological saline around the animal's eyes was absorbed with tissue paper. After staining for about 5 minutes, the ocular surface was observed using a slit lamp (+ cobalt blue filter), photographs were taken, and scores were given. Scoring criteria: Each cornea of the eye was divided into five regions (1 - central region, 2 - upper region, 3 - temporal region, 4 - nasal region, 5 - lower region). The staining score for each region was up to 8 points, among which 0 points indicated that the corresponding region was not colored, 1 point indicated that the dot-like colored area was 1% - 25% of the area of the corresponding region, 2 points indicated that the dot-like colored area was 26% - 50% of the area of the corresponding region, 3 points indicated that the dot-like colored area was 51% - 75% of the area of the corresponding region, and 4 points indicated that the dot-like colored area was 76% - 100% of the area of the corresponding region. If the colored area was dense and / or clearly fused, 1, 2, 3, and 4 points were respectively given according to the size of the area occupied by the colored area in the corresponding region, that is, 1% - 25%, 26% - 50%, 51% - 75%, 76% - 100%. The total score for each eye was up to 40 points. The total score of corneal fluorescein sodium staining was calculated for each eye. The summary table of corneal fluorescein sodium staining scores is shown in Table 8.

Table 14

Table 15

[0050] 5. Experimental results Subcutaneous injection of scopolamine in combination with a low-humidity environment can successfully induce a decrease in the tear secretion volume of mice. When the low-dose, medium-dose, and high-dose test samples and the commercial control product are administered continuously for 5 days, all of them have a significant improvement effect on the decrease in the tear secretion volume of model mice. When the administration period is extended to 10 days, the low-dose, medium-dose, and high-dose test samples and the commercial control product still show a certain improvement effect, and from the average value, the effect of the test sample is more obvious.

[0051] Subcutaneous injection of scopolamine in combination with a low-humidity environment can successfully induce an increase in the corneal fluorescein sodium staining score of mice. When the low-dose, medium-dose, and high-dose test samples and the commercial control product are administered continuously for 5 days, all of them can significantly reduce the corneal fluorescein sodium staining score of mice. Even when the administration period is extended to 10 days, the corneal fluorescein sodium staining score of mice can still be reduced. Among them, the effects of the medium-dose and high-dose test samples are superior to those of the commercial control product, and moreover, the high-dose test sample can almost restore the corneal fluorescein sodium staining score of model mice to the normal level.

[0052] Conclusion: The cyclosporine ophthalmic emulsion of the present invention can improve the drug efficacy.

[0053] Although some features of the present invention have been described and explained in this specification, those skilled in the art will doubtless conceive of many modifications, alternatives, variations, and equivalents. Accordingly, the appended claims are to be understood as intended to cover all such modifications and variations that fall within the true spirit of the present invention.

Claims

**Claim 1** An oil-in-water type cyclosporine emulsion containing an interfacial stabilizer, preferably a nanoemulsion, wherein the interfacial stabilizer comprises a combination of polyethylene glycol 15-hydroxystearate and a phospholipid. An oil-in-water type cyclosporine emulsion. **Claim 2** The oil-in-water type cyclosporine emulsion according to claim 1, further comprising a buffer salt. **Claim 3** The phospholipid is selected from soybean phospholipid, egg yolk phospholipid, or any combination thereof, preferably selected from soybean phospholipid S100, egg yolk phospholipid E80, or any combination thereof. The oil-in-water type cyclosporine emulsion according to claim 1. **Claim 4** The mass ratio of cyclosporine to polyethylene glycol 15-hydroxystearate is 1:30 to 1:

45. The oil-in-water type cyclosporine emulsion according to claim 1. **Claim 5** The mass ratio of phospholipid to polyethylene glycol 15-hydroxystearate is 1:14 to 1:

31. The oil-in-water type cyclosporine emulsion according to claim 1. **Claim 6** The content of polyethylene glycol 15-hydroxystearate is 2.25 to 13.5 g / 100 ml. The oil-in-water type cyclosporine emulsion according to claim 1. **Claim 7** The buffer salt is a phosphate buffer salt, preferably the buffer salt concentration is 0.01 mol / L to 0.05 mol / L in terms of phosphate group. The oil-in-water type cyclosporine emulsion according to claim 2. **Claim 8** Further containing an osmotic pressure regulator, preferably the osmotic pressure regulator is selected from sodium chloride, sorbitol, or any combination thereof, preferably the content of the osmotic pressure regulator is 0.2 to 1 g / 100 ml. The oil-in-water type cyclosporine emulsion according to claim 1. **Claim 9** The average particle size of the emulsion particles is 50 nm or less, preferably 10 to 40 nm, more preferably 20 to 30 nm. Preferably, the particle size of 90% of the emulsion particles is 90 nm or less. Preferably, the pH value of the emulsion is 6.5 to 8.0, preferably 6.8 to 7.6, more preferably 7.0 to 7.

4. Preferably, the encapsulation rate of the emulsion is 90% or more, preferably 95% or more. Preferably, the emulsion is a transparent milky liquid. Preferably, the emulsion is an eye drop. Preferably, the oil is selected from medium-chain triglycerides, soybean oil, castor oil, olive oil or fish oil. The oil-in-water type cyclosporine emulsion according to claim 1.

10. A method for producing an oil-in-water type cyclosporine emulsion, comprising an interfacial stabilizer containing a combination of polyethylene glycol 15-hydroxystearate and phospholipids, an osmotic pressure regulator, and a buffer salt, 1) uniformly mixing an oil and an interfacial stabilizer, adding cyclosporine thereto and dissolving it to form an oil phase; 2) dissolving a part of the osmotic pressure regulator in water, and stirring and mixing the oil phase and the water containing the osmotic pressure regulator to form a primary emulsion; 3) dissolving the buffer salt and the remainder of the osmotic pressure regulator in water to form a solution, and adding the primary emulsion to the solution and uniformly mixing them. A method for producing an oil-in-water type cyclosporine emulsion.

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