WO2024027451A1 - 一种环孢素乳剂及其制备方法 - Google Patents

一种环孢素乳剂及其制备方法 Download PDF

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WO2024027451A1
WO2024027451A1 PCT/CN2023/105857 CN2023105857W WO2024027451A1 WO 2024027451 A1 WO2024027451 A1 WO 2024027451A1 CN 2023105857 W CN2023105857 W CN 2023105857W WO 2024027451 A1 WO2024027451 A1 WO 2024027451A1
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oil
water
cyclosporine
emulsion
osmotic pressure
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PCT/CN2023/105857
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English (en)
French (fr)
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张树荣
严贤龙
薛琦
牛璐
王振恒
熊璎珞
戴佩旻
王韫哲
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珠海亿胜生物制药有限公司
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Publication of WO2024027451A1 publication Critical patent/WO2024027451A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention relates to the field of preparations, and in particular to a cyclosporine emulsion and a preparation method thereof.
  • Cyclosporine is a calcineurin inhibitor and an effective immunosuppressant. It has less toxic effects than other immunosuppressants. It can reversibly and selectively change the function of T lymphocytes and block the lymphokine gene. Transcription, interfering with the transmission of original information, inhibiting the release of interleukin-2 (IL-2), interferon and other immune factors, and exerting anti-inflammatory effects. After ocular administration, cyclosporine can alleviate the symptoms of dry eye (keratoconjunctivitis sicca) by inducing the inactivation of calcineurin and preventing the release of pro-inflammatory cytokines such as IL-2.
  • Cyclosporine is almost insoluble in water and has good fat solubility.
  • Commercially available eye drops are generally dissolved in vegetable oil to form a solution or emulsification technology is used to prepare an oil-in-water emulsion.
  • Oil solutions used for topical administration to the eyes are highly irritating and suffer from poor patient compliance.
  • products already on the market and They are all sub-microemulsions with an average particle size of more than 100nm. They are opaque and milky white in color and will blur the patient's vision after being dropped into the eyes.
  • the cationic surfactant sitalonium chloride is used to enhance the retention time of the drug on the ocular surface, but the use of cationic surfactants increases the occurrence of adverse reactions such as eye pain, eye irritation, and eye congestion.
  • Nanoemulsion is a translucent to almost transparent dispersion system with a particle size of less than 100 nm formed by self-assembly of oil, water, interface stabilizers, etc. Nanoemulsions are used for ocular drug delivery. Their nano-sized particles can increase the permeability of drugs in ocular tissue and improve drug efficacy. Higher clarity can improve patient compliance, and have high development value.
  • oil-in-water ophthalmic emulsions need to solve two major technical problems: 1) emulsion particle stability and encapsulation stability issues.
  • the ionic strength of physiological tears or artificial tear substitutes will affect the stability of the interfacial film of the emulsion, thereby causing emulsion particles to Aggregation and drug leakage affect the safety and effectiveness of the preparation; 2)
  • the pH value of the preparation gradually decreases, affecting the quality of the preparation.
  • Oil-in-water emulsions usually use sodium hydroxide or hydrochloric acid to adjust the pH value, but during storage, the pH value will gradually decrease as the oil is hydrolyzed into free fatty acids.
  • buffer salts to form a buffer system can improve the stability of the pH value, but an increase in ionic strength will affect the stability of the interfacial film, leading to aggregation of milk particles and drug leakage.
  • the field of oil-in-water ophthalmic emulsions urgently needs to provide a A combination of stabilizers that can produce synergistic stabilizing effects together form an interface composite film.
  • the composite film can remain stable in a high ionic strength environment (such as a buffer salt solution), thereby achieving the pH value, particle size and encapsulation rate of the preparation. The average is stable in the long run.
  • the object of the present invention is to provide a cyclosporine emulsion with controllable particle size (for example, the average particle size is below 50 nm) and a high encapsulation rate (more than 90%).
  • Another object of the present invention is to provide a cyclosporine emulsion with long-term stable pH value, particle size and encapsulation efficiency.
  • the present invention provides an oil-in-water cyclosporine emulsion comprising an interface stabilizer comprising a combination of 15-hydroxystearate polyethylene glycol ester and a phospholipid.
  • the invention also provides a method for preparing an oil-in-water cyclosporine emulsion, the emulsion comprising an interface stabilizer, an osmotic pressure regulator and a buffer salt, the interface stabilizer comprising 15-hydroxystearic acid polyethylene glycol A combination of esters and phospholipids, the method comprising
  • the cyclosporine emulsion has improved interfacial film stability, which can effectively inhibit the migration of cyclosporine into the aqueous medium and inhibit the aggregation of emulsion particles, thereby achieving long-term stability in terms of pH value, particle size and encapsulation rate.
  • Significant improvement Due to the penetration promotion and higher clarity of nano-sized particles, the emulsion has higher efficacy and better patient compliance.
  • the present invention relates to an oil-in-water cyclosporine emulsion, preferably a nanoemulsion, comprising an interfacial stabilizer comprising a combination of polyethylene glycol 15-hydroxystearate and a phospholipid.
  • the oil-in-water cyclosporine emulsion further comprises a buffer salt.
  • the phospholipid is selected from soybean phospholipid, egg yolk phospholipid or any combination thereof, preferably, is selected from soybean phospholipid S100, egg yolk phospholipid E80 or any combination thereof.
  • the mass ratio of cyclosporine to polyethylene glycol 15-hydroxystearate is 1:30 to 1:45.
  • the mass ratio of phospholipid to polyethylene glycol 15-hydroxystearate is 1:14 to 1:31.
  • the content of polyethylene glycol 15-hydroxystearate is 2.25-13.5g/100ml.
  • the buffer salt is a phosphate buffer salt.
  • the buffer salt concentration is 0.01 mol/L to 0.05 mol/L based on phosphate radicals.
  • the oil-in-water cyclosporine emulsion further comprises an osmotic pressure regulator; preferably, the osmotic pressure regulator is selected from sodium chloride, sorbitol, or any combination thereof; preferably, the osmotic pressure regulator Content is 0.2 ⁇ 1g/100ml.
  • the average particle diameter of the particles of the emulsion does not exceed 50 nm, preferably 10 to 40 nm, more preferably 20 to 30 nm; preferably, 90% of the particles of the emulsion have a particle diameter of no more than 90 nm; 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 not less than 90%, preferably not less than 95%; preferably, the emulsion is Transparent milky liquid; preferably, the emulsion is eye drops; preferably, the oil is selected from medium chain triglycerides, soybean oil, castor oil, olive oil or fish oil.
  • the invention also provides a method for preparing an oil-in-water cyclosporine emulsion, the emulsion comprising an interface stabilizer, an osmotic pressure regulator and a buffer salt, the interface stabilizer comprising 15-hydroxystearic acid polyethylene glycol A combination of esters and phospholipids, the method comprising
  • the buffer salt is preferably phosphate buffer salt.
  • cyclosporine nanoparticles using 15-hydroxystearate polyethylene glycol ester and soybean lecithin S100 as stabilizers, disodium hydrogen phosphate and sodium dihydrogen phosphate as pH regulators, sodium chloride and sorbitol as osmotic pressure regulators Emulsion, the prescription composition is as follows:
  • the particle size is measured according to the third method of Part Four General Chapter 0982 of the 2020 edition of the "Chinese Pharmacopoeia”; the detection method of the encapsulation efficiency is as follows:
  • cyclosporine reference substance Take an appropriate amount of cyclosporine reference substance, add 500 ⁇ l of methanol to dissolve it, and then quantitatively dilute it with 50% methanol solution to prepare a solution containing approximately 0.1 mg of cyclosporine reference substance per 1 ml.
  • Samples were prepared according to the prescription process of Example 1, and the properties, pH value, particle size and encapsulation rate of the cyclosporine nanoemulsion were examined. The results are shown in Table 1. Samples were prepared according to the prescription 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 examined. The results are shown in Table 2.
  • Samples were prepared according to the prescription process of Example 1, Example 2, and Example 3, and the stability of the samples at 40°C high temperature and 5000 ⁇ 500 lx light was investigated. The results are shown in Tables 3 and 4. Samples were prepared according to the prescription process of Example 1, and the intermediate condition test at 30°C and the long-term test at 15-20°C were conducted. The results are shown in Tables 5 and 6.
  • the pH value, particle size and encapsulation rate of cyclosporine nanoemulsion have good stability, indicating that the use of buffer salt can improve the pH value stability of the emulsion.
  • the composition of 15-hydroxystearate polyethylene glycol ester and phospholipid The interface composite membrane can withstand high ionic strength and ensure the stability of the emulsion particles and encapsulation during the storage process of the nanoemulsion.
  • Low-dose test product 0.05% cyclosporine nanoemulsion, prepared according to the prescription process of Example 5.
  • a medium-dose test product 0.1% cyclosporine nanoemulsion, prepared according to the prescription process of Example 1.
  • High-dose test product 0.3% cyclosporine nanoemulsion, prepared according to the prescription process of Example 4.
  • Blank auxiliary material prepared according to the prescription process of Example 1, except that it does not contain cyclosporine.
  • Test animals C57BL/6JShjh mice, weighing 15.4-20.4g, female.
  • Test method 150 healthy female C57BL/6JShjh mice were selected on D-1. 21 mice with no abnormalities in both eyes were selected without modeling and 125 mice received a low-humidity environment combined with subcutaneous injection of scopolamine ( 2 times/day, 0.75 mg/mL, 0.3 mL/animal/time) to induce modeling, and the day of the first modeling was recorded as D1.
  • D5 selected 15 unmodeled animals with similar corneal sodium fluorescein staining scores and tear secretion volumes to be divided into the non-modelling group, and selected 90 modeled animals with similar corneal sodium fluorescein staining scores and tear secretion volumes, taking both modeling into account.
  • Tear secretion volume Baoding mice, use clean toothless forceps to hold a truncated commercial phenol red cotton thread, fix the cotton thread in the middle of the conjunctival sac of the lower eyelid of the mouse for 30 seconds, and measure the wet length of the cotton thread.
  • a summary of tear secretion is shown in Table 7.
  • Corneal sodium fluorescein staining test After instilling fluorescein sodium solution (1.5 ⁇ L, 0.5%) into the upper conjunctival sac of the animal, starting from staining for about 1.5 minutes, use 1.25 mL of sterile saline to flush the animal's conjunctival sac every about 10 seconds. , 3 times in a row, and after each rinse, use tissue paper to dry the physiological saline around the animal's eyes. After staining for about 5 minutes, use a slit lamp (+ cobalt blue filter) to observe the ocular surface, take pictures, and score.
  • Scoring standard The cornea of each eye is divided into 5 areas (1-central area, 2-superior, 3-temporal, 4-nasal, 5-inferior).
  • the staining score of each area is up to 8 points, of which 0 points means corresponding
  • a score of 1 means that the area of dotted coloring is 1% to 25% of the area of the corresponding area.
  • a score of 2 means that the area of dotted coloring is 26% to 50% of the area of the corresponding area.
  • a score of 3 means that the area of dotted coloring is 26% to 50% of the area of the corresponding area. 51% to 75% of the area area.
  • a score of 4 means that the area of dotted coloring is 76% to 100% of the area of the corresponding area.
  • the area of the coloring area occupies the corresponding area, that is, 1 % ⁇ 25%, 26% ⁇ 50%, 51% ⁇ 75%, 76% ⁇ 100%, respectively, will be given 1, 2, 3, and 4 extra points.
  • the maximum total score for each eye is 40 points.
  • the total corneal fluorescein sodium staining score was calculated for each eye.
  • the summary table of corneal fluorescein sodium staining scores is shown in Table 8.
  • N represents the number of eyes of the animal.
  • a p ⁇ 0.05
  • b p ⁇ 0.05
  • c p ⁇ 0.05
  • d p ⁇ 0.05
  • e p ⁇ 0.05
  • f p ⁇ 0.05
  • N represents the number of eyes of the animal.
  • a p ⁇ 0.05
  • b p ⁇ 0.05
  • c p ⁇ 0.05
  • d p ⁇ 0.05
  • e p ⁇ 0.05
  • f p ⁇ 0.05
  • a low-humidity environment combined with subcutaneous injection of scopolamine can successfully induce a decrease in tear secretion in mice.
  • Low, medium, and high doses of the test article and commercially available control substances were administered for 5 days in a row, which significantly reduced the tear secretion in model mice.
  • the administration was extended to 10 days, the low, medium and high doses of the test article and the commercially available reference substance still showed a certain improvement effect, and the effect of the test article was more obvious from the mean value.
  • Low-humidity environment combined with subcutaneous injection of scopolamine can successfully induce an increase in the corneal fluorescein sodium staining score of mice.
  • Low, medium, and high doses of the test article and commercially available control substances can significantly reduce the corneal fluorescein sodium staining score in mice after continuous administration for 5 days.
  • Sodium staining score, administration extended to 10 days can still reduce the fluorescein sodium staining score of mouse corneas.
  • the effects of medium and high doses of the test article are better than those of the commercially available control substances, and the high dose of the test article can restore the corneal sodium fluorescein staining score of model mice to close to normal levels.
  • the cyclosporine ophthalmic emulsion of the present invention can improve the efficacy.

Abstract

一种水包油环孢素乳剂及其制备方法，包含界面稳定剂，界面稳定剂包含15-羟基硬脂酸聚乙二醇酯和磷脂的组合。水包油环孢素乳剂的pH值、粒径和包封率长期稳定。

Description

一种环孢素乳剂及其制备方法 技术领域
本发明涉及制剂领域，尤其是涉及一种环孢素乳剂及其制备方法。
背景技术
环孢素是一种钙调神经磷酸酶抑制剂和有效的免疫抑制剂，相较其他免疫抑制剂毒性作用更小，可通过可逆的选择性地改变T淋巴细胞的功能，阻止淋巴激活素基因的转录，干扰原信息的传递，抑制白细胞介素-2(IL-2)、干扰素和其他免疫因子的释放，发挥抗炎作用。环孢素眼部给药后，可通过诱导钙神经蛋白磷酸酶失活，阻止IL-2等原炎细胞因子的释放，从而缓解干眼(角结膜干燥症)的症状。
环孢素在水中几乎不溶，其脂溶性良好，市售滴眼剂一般将其溶解于植物油中制成溶液剂或采用乳化技术制备成水包油乳剂。油溶液剂用于眼局部给药刺激性强，患者顺应性差。水包油眼用乳剂领域，已上市的和均为平均粒径100nm以上的亚微乳，性状呈不透明的乳白色，滴眼后会模糊患者视力。此外，因粒径较大且未添加促进药物在眼内停留或促渗透的组分，生物利用度较低；使用了阳离子表面活性剂西他氯铵加强药物在眼表的保留时间，但阳离子表面活性剂的使用增加了眼痛、眼刺激、眼充血等不良反应的发生。
纳米乳剂是由油、水、界面稳定剂等自组装形成的粒径100nm以下的半透明至几乎透明的分散体系。纳米乳剂应用于眼部给药，其纳米尺寸的粒径可增加药物在眼组织的渗透性而提高药效，更高的澄明度可提高患者顺应性，具有很高的开发价值。
目前水包油眼用乳剂需要解决两大技术难题：1)乳粒稳定性和包封稳定性问题，例如生理泪液或人工替代泪液的离子强度会影响乳剂的界面膜稳定性，进而导致乳粒聚集和药物渗漏，影响制剂的安全性和有效性；2)长期稳定性时，制剂pH值逐渐下降，影响制剂的质量。水包油乳剂通常使用氢氧化钠或盐酸调节pH值，但在储存过程中，随着油脂水解成游离脂肪酸，pH值会逐渐下降。使用缓冲盐组成缓冲体系可以提高pH值的稳定性，但离子强度增大会影响界面膜稳定性，进而导致乳粒聚集和药物渗漏。鉴于此，水包油眼用乳剂领域，亟需提供一 种能够产生协同稳定作用的稳定剂组合，共同构成界面复合膜，复合膜可在较高的离子强度环境下(如缓冲盐溶液中)保持稳定，进而实现制剂pH值、粒径和包封率等均长期稳定。
发明内容
本发明的目的是提供一种粒径可控(例如，平均粒径在50nm以下)和包封率高(90％以上)的环孢素乳剂。
本发明的另一个目的是提供一种pH值、粒径和包封率长期稳定的环孢素乳剂。
本发明提供了一种水包油环孢素乳剂，包含界面稳定剂，所述界面稳定剂包含15-羟基硬脂酸聚乙二醇酯和磷脂的组合。
本发明还提供了一种制备水包油环孢素乳剂的方法，所述乳剂包含界面稳定剂、渗透压调节剂和缓冲盐，所述界面稳定剂包含15-羟基硬脂酸聚乙二醇酯和磷脂的组合，所述方法包括
1)将油和界面稳定剂混合均匀，向其中加入环孢素并溶解，形成油相；
2)将渗透压调节剂的一部分溶于水，将油相和含有渗透压调节剂的水搅拌混合，形成初乳；以及
3)将缓冲盐和余量的渗透压调节剂溶于水形成溶液，将初乳加入至所述溶液并混匀。
所述环孢素乳剂具有提高的界面膜稳定性，可有效抑制环孢素向水介质中迁移，并抑制乳粒的聚集，从而使得pH值、粒径和包封率方面的长期稳定性得到显著改善。因纳米尺寸粒径的促渗透性和更高的澄明度，使得该乳剂具有更高的药效和更好的患者顺应性。
具体实施方式
除非另外定义，本文所用的所有技术和科学术语具有本领域技术人员通常理解的相同含义。在冲突的情况下，以包括定义在内的本文件为准。下面描述优选的方法和材料，但是与本文所述那些类似或等同的方法和材料可用于实施或测试本发明。本文公开的材料、方法和实例仅是说明性的，而非旨在限制。
对于本公开内容涉及的所有数值范围，应理解为公开了该范围内所有具体数值，以及该范围内任意两个数值限定的子范围。例如，对于1-20，应理解为公 开了1、2、3、3.5、4.5、10、12、15、20等具体数值，以及1-5，2-6，3.5-7.5，15-20等子范围。
本发明涉及一种水包油环孢素乳剂，优选纳米乳剂，包含界面稳定剂，所述界面稳定剂包含15-羟基硬脂酸聚乙二醇酯和磷脂的组合。
在一个实施方案中，所述水包油环孢素乳剂还包含缓冲盐。
在一个实施方案中，所述磷脂选自大豆磷脂、蛋黄磷脂或其任何组合，优选地，选自大豆磷脂S100、蛋黄磷脂E80或其任何组合。
在一个实施方案中，环孢素与15-羟基硬脂酸聚乙二醇酯的质量比为1：30～1：45。
在一个实施方案中，磷脂与15-羟基硬脂酸聚乙二醇酯的质量比为1：14～1：31。
在一个实施方案中，15-羟基硬脂酸聚乙二醇酯的含量为2.25～13.5g/100ml。
在一个实施方案中，所述缓冲盐为磷酸缓冲盐，优选地，以磷酸根计，缓冲盐浓度为0.01mol/L～0.05mol/L。
在一个实施方案中，所述水包油环孢素乳剂还包含渗透压调节剂；优选地，渗透压调节剂选自氯化钠、山梨醇或其任何组合；优选地，渗透压调节剂的含量为0.2～1g/100ml。
在一个实施方案中，所述乳剂的粒子的平均粒径不超过50nm，优选10～40nm，更优选20～30nm；优选地，所述乳剂的90％的粒子的粒径不超过90nm；优选地，乳剂的pH值为6.5～8.0，优选6.8～7.6，更优选7.0～7.4；优选地，乳剂的包封率为不低于90％，优选不低于95％；优选地，所述乳剂为透明的乳状液体；优选地，所述乳剂为滴眼剂；优选地，所述油选自中链甘油三酸酯、大豆油、蓖麻油、橄榄油或鱼油。
本发明还提供了一种制备水包油环孢素乳剂的方法，所述乳剂包含界面稳定剂、渗透压调节剂和缓冲盐，所述界面稳定剂包含15-羟基硬脂酸聚乙二醇酯和磷脂的组合，所述方法包括
1)将油和界面稳定剂混合均匀，向其中加入环孢素并溶解，形成油相；
2)将渗透压调节剂的一部分溶于水，将油相和含有渗透压调节剂的水搅拌混合，形成初乳；以及
3)将缓冲盐和余量的渗透压调节剂溶于水形成溶液，将初乳加入至所述溶液 并混匀。
所述缓冲盐优选磷酸缓冲盐。
以上提及的各个实施方案中的技术特征在不产生矛盾的前提下可以相互组合成另外的技术方案。
实施例
实施例1
以15-羟基硬脂酸聚乙二醇酯和大豆磷脂S100为稳定剂，磷酸氢二钠和磷酸二氢钠为pH调节剂，氯化钠为渗透压调节剂制备环孢素纳米乳剂，处方组成如下：
制备方法：
1)将15-羟基硬脂酸聚乙二醇酯、大豆磷脂S100和油加入容器1中，加热至60℃～80℃，搅拌混合均匀；加入环孢素搅拌溶解，成油相；
2)向容器2中加入约配制总量9％的注射用水，搅拌下加入适量渗透压调节剂，继续搅拌至溶解，成初乳水相；
3)搅拌下，将初乳水相加入到油相中，在搅拌下逐渐升温至60℃～80℃，继续搅拌5～15min，成初乳；
4)向容器3中加入约配制总量50％的注射用水，搅拌下加入pH调节剂和剩余的渗透压调节剂，继续搅拌至溶解，得稀释水相；
5)搅拌下，将初乳加入到稀释水相中，用适量注射用水分次冲洗容器2并将冲洗水转移至容器3中，继续搅拌5～10min；
6)加注射用水至配制总量，搅拌均匀。
实施例2
以15-羟基硬脂酸聚乙二醇酯和蛋黄磷脂E80为稳定剂，磷酸氢二钠和磷酸二氢钠为pH调节剂，氯化钠为渗透压调节剂制备环孢素纳米乳剂，处方组成如下：
制备方法：
1)将15-羟基硬脂酸聚乙二醇酯、蛋黄磷脂E80和油加入容器1中，加热至60℃～80℃，搅拌混合均匀；加入环孢素搅拌溶解，成油相；
2)向容器2中加入约配制总量9％的注射用水，搅拌下加入适量渗透压调节剂，继续搅拌至溶解，成初乳水相；
3)搅拌下，将初乳水相加入到油相中，在搅拌下逐渐升温至60℃～80℃，继续搅拌5～15min，成初乳；
4)向容器3中加入约配制总量50％的注射用水，搅拌下加入pH调节剂和剩余的渗透压调节剂，继续搅拌至溶解，得稀释水相；
5)搅拌下，将初乳加入到稀释水相中，用适量注射用水分次冲洗容器2并将冲洗水转移至容器3中，继续搅拌5～10min；
6)加注射用水至配制总量，搅拌均匀。
实施例3
以15-羟基硬脂酸聚乙二醇酯和大豆磷脂S100为稳定剂，磷酸氢二钠和磷酸二氢钠为pH调节剂，氯化钠和山梨醇为渗透压调节剂制备环孢素纳米乳剂，处方组成如下：
制备方法：
1)将15-羟基硬脂酸聚乙二醇酯、大豆磷脂S100和油加入容器1中，加热至60℃～80℃，搅拌混合均匀；加入环孢素搅拌溶解，成油相；
2)向容器2中加入约配制总量9％的注射用水，搅拌下加入适量渗透压调节剂，继续搅拌至溶解，成初乳水相；
3)搅拌下，将初乳水相加入到油相中，在搅拌下逐渐升温至60℃～80℃，继续搅拌5～15min，成初乳；
4)向容器3中加入约配制总量50％的注射用水，搅拌下pH调节剂和剩余的渗透压调节剂，继续搅拌至溶解，得稀释水相；
5)搅拌下，将初乳加入到稀释水相中，用适量注射用水分次冲洗容器2并将冲洗水转移至容器3中，继续搅拌5～10min；
6)加注射用水至配制总量，搅拌均匀。
实施例4
以15-羟基硬脂酸聚乙二醇酯和大豆磷脂S100为稳定剂，磷酸氢二钠和磷酸二氢钠为pH调节剂，氯化钠为渗透压调节剂制备环孢素纳米乳剂，处方组成如下：
制备方法：
1)将15-羟基硬脂酸聚乙二醇酯、大豆磷脂S100和油加入容器1中，加热至60℃～80℃，搅拌混合均匀；加入环孢素搅拌溶解，成油相；
2)向容器2中加入约配制总量27％的注射用水，搅拌下加入适量渗透压调节剂，继续搅拌至溶解，成初乳水相；
3)搅拌下，将初乳水相加入到油相中，在搅拌下逐渐升温至60℃～80℃，继续搅拌5～15min，成初乳；
4)向容器3中加入约配制总量30％的注射用水，搅拌下加入pH调节剂和剩余的渗透压调节剂，继续搅拌至溶解，得稀释水相；
5)搅拌下，将初乳加入到稀释水相中，用适量注射用水分次冲洗容器2并将冲洗水转移至容器3中，继续搅拌5～10min；
6)加注射用水至配制总量，搅拌均匀。
实施例5
以15-羟基硬脂酸聚乙二醇酯和大豆磷脂S100为稳定剂，磷酸氢二钠和磷酸二氢钠为pH调节剂，氯化钠为渗透压调节剂制备环孢素纳米乳剂，处方组成如下：
制备方法：
1)将15-羟基硬脂酸聚乙二醇酯、大豆磷脂S100和油加入容器1中，加热至60℃～80℃，搅拌混合均匀；加入环孢素搅拌溶解，成油相；
2)向容器2中加入约配制总量4.5％的注射用水，搅拌下加入适量渗透压调节剂，继续搅拌至溶解，成初乳水相；
3)搅拌下，将初乳水相加入到油相中，在搅拌下逐渐升温至60℃～80℃，继续搅拌5～15min，成初乳；
4)向容器3中加入约配制总量25％的注射用水，搅拌下加入pH调节剂和剩余的渗透压调节剂，继续搅拌至溶解，得稀释水相；
5)搅拌下，将初乳加入到稀释水相中，用适量注射用水分次冲洗容器2并将冲洗水转移至容器3中，继续搅拌5～10min；
6)加注射用水至配制总量，搅拌均匀。
实施例6
以15-羟基硬脂酸聚乙二醇酯和大豆磷脂S100为稳定剂，磷酸氢二钠和磷酸二氢钠为pH调节剂，氯化钠为渗透压调节剂制备环孢素纳米乳剂，处方组成如下：

制备方法：
1)将15-羟基硬脂酸聚乙二醇酯、大豆磷脂S100和油加入容器1中，加热至60℃～80℃，搅拌混合均匀；加入环孢素搅拌溶解，成油相；
2)向容器2中加入约配制总量9％的注射用水，搅拌下加入适量渗透压调节剂，继续搅拌至溶解，成初乳水相；
3)搅拌下，将初乳水相加入到油相中，在搅拌下逐渐升温至60℃～80℃，继续搅拌5～15min，成初乳；
4)向容器3中加入约配制总量50％的注射用水，搅拌下加入pH调节剂和剩余的渗透压调节剂，继续搅拌至溶解，得稀释水相；
5)搅拌下，将初乳加入到稀释水相中，用适量注射用水分次冲洗容器2并将冲洗水转移至容器3中，继续搅拌5～10min；
6)加注射用水至配制总量，搅拌均匀。
实施例7
以15-羟基硬脂酸聚乙二醇酯和大豆磷脂S100为稳定剂，磷酸氢二钠和磷酸二氢钠为pH调节剂，氯化钠为渗透压调节剂制备环孢素纳米乳剂，处方组成如下：

制备方法：
1)将15-羟基硬脂酸聚乙二醇酯、大豆磷脂S100和油加入容器1中，加热至60℃～80℃，搅拌混合均匀；加入环孢素搅拌溶解，成油相；
2)向容器2中加入约配制总量9％的注射用水，搅拌下加入适量渗透压调节剂，继续搅拌至溶解，成初乳水相；
3)搅拌下，将初乳水相加入到油相中，在搅拌下逐渐升温至60℃～80℃，继续搅拌5～15min，成初乳；
4)向容器3中加入约配制总量50％的注射用水，搅拌下加入pH调节剂和剩余的渗透压调节剂，继续搅拌至溶解，得稀释水相；
5)搅拌下，将初乳加入到稀释水相中，用适量注射用水分次冲洗容器2并将冲洗水转移至容器3中，继续搅拌5～10min；
6)加注射用水至配制总量，搅拌均匀。
实验例8：环孢素纳米乳剂质量特性考察
本申请中，粒径按照《中国药典》2020年版四部通则0982第三法测定；包封率的检测方法如下：
取本品200μl加入到葡聚糖凝胶G-25微型柱的顶端，2000rpm/min离心1min，以水为洗脱液进行洗脱，向微型柱顶端加水200μl，2000rpm/min离心2min，重复洗脱4次，收集洗脱液于5ml量瓶中，加甲醇破乳并稀释至刻度，摇匀，即得。取环孢素对照品适量，加500μl的甲醇溶解后，再用50％的甲醇溶液定量稀释制成每1ml中约含环孢素对照品0.1mg溶液。用十八烷基硅烷键合硅胶为填充剂；以乙腈-水(70:30)为流动相；流速为每分钟1.0ml；不锈钢管和柱温为80℃；检测波长为210nm。分别取上述溶液，注入液相色谱仪，记录色谱图，按外标法以峰面积计算，即得。
按实施例1处方工艺制备样品，考察环孢素纳米乳剂的性状、pH值、粒径和包封率，结果见表1。按实施例2、实施例3、实施例4、实施例5、实施例6和实施例7处方工艺制备样品，考察环孢素纳米乳剂的性状、pH值和粒径，结果见表2。
表1实施例1环孢素纳米乳剂质量特性
表2实施例2～6环孢素纳米乳剂质量特性
结论：环孢素纳米乳剂的性状、pH值、粒径和包封率符合限度要求。
实验例9：环孢素纳米乳剂稳定性试验
按实施例1、实施例2、实施例3处方工艺制备样品，进行40℃高温和5000±500lx光照稳定性的考察，结果见表3-表4。按实施例1处方工艺制备样品，进行30℃中间条件试验及15～20℃长期试验的考察，结果见表5-表6。
表3实施例1～3环孢素纳米乳剂高温稳定性试验结果
表4实施例1、实施例3环孢素纳米乳剂光照稳定性试验结果
表5实施例1环孢素纳米乳剂中间条件稳定性试验结果
表6实施例1环孢素纳米乳剂长期稳定性试验结果
结论：环孢素纳米乳剂的pH值、粒径和包封率的稳定性良好，说明缓冲盐的使用可以提高乳剂的pH值稳定性，15-羟基硬脂酸聚乙二醇酯和磷脂组成的界面复合膜可以耐受较高的离子强度，保证纳米乳剂储存过程中的乳粒和包封的稳定性。
实验例10：环孢素纳米乳剂对干燥应激所致小鼠干眼的药效学试验
1、受试药物：
低剂量的供试品：0.05％环孢素纳米乳剂，按实施例5处方工艺制备。
中剂量的供试品：0.1％环孢素纳米乳剂，按实施例1处方工艺制备。
高剂量的供试品：0.3％环孢素纳米乳剂，按实施例4处方工艺制备。
市售对照品：规格0.1％。
空白辅料：按实施例1处方工艺制备，除了不含环孢素。
2、受试动物：C57BL/6JShjh小鼠，体重15.4-20.4g，雌性。
3、试验方法：150只健康雌性C57BL/6JShjh小鼠，于D-1选择双眼眼部无异常的21只小鼠不进行造模和125只小鼠连续5天接受低湿环境联合皮下注射东莨菪碱(2次/天，0.75mg/mL，0.3mL/只/次)诱导造模，首次造模当天记为D1。D5选择角膜荧光素钠染色评分、泪液分泌量相近的15只未造模动物分入未造模组，选择角膜荧光素钠染色评分、泪液分泌量相近的90只造模动物，同时兼顾造模前的基础分值，依双眼角膜荧光素钠染色评分均值随机分入2～7组(15只/组)，依次为模型对照组、空白辅料组、市售对照组、供试品低、中、高剂量组。D6～D15，2～7组动物继续接受造模(方法同前)，第1～2组不给药，第3～7组分别给予空白辅料、Ikervis、0.05％、0.1％和0.3％环孢素纳米乳剂，双眼滴眼给药(3次/天，间隔约3小时，3μL/眼)。D-1、D5、D10、D15对各组动物进行泪液分泌量检查及角膜荧光素钠染色试验。
4、评价方法：
泪液分泌量：保定小鼠，用洁净的无齿镊夹持截短后的市售酚红棉线，将棉线固定于小鼠下眼睑结膜囊中部30s，测量棉线染湿长度。泪液分泌量总结表见表7。
角膜荧光素钠染色试验：将荧光素钠溶液(1.5μL，0.5％)滴入动物上结膜囊后，自染色约1.5min开始，每隔约10s使用1.25mL的无菌生理盐水冲洗动物结膜囊，连续3次，每次冲洗完毕用面纸吸干动物眼周生理盐水。染色约5min时使用裂隙灯(+钴蓝滤光片)观察眼表，拍照，评分。评分标准：每眼角膜分为5个区域(1-中央区、2-上方、3-颞侧、4-鼻侧、5-下方)，每个区域染色评分最高8分，其中0分表示对应区域未见着色，1分表示点状着色面积为对应区域面积的1％～25％，2分表示点状着色面积为对应区域面积的26％～50％，3分表示点状着色面积为对应区域面积的51％～75％，4分表示点状着色面积为对应区域面积的76％～100％，若着色区致密和/或可见明显融合，依着色区占对应区域的面积大小，即1％～25％、26％～50％、51％～75％、76％～100％，分别给予1、2、3、4分的加分。每眼总分最高40分。计算每眼角膜荧光素钠染色总分。角膜荧光素钠染色评分总结表见表8。
表7泪液分泌量总结表(mm)
注：N表示动物眼数。和未造模组比较，a表示p≤0.05；和模型对照组比较，b表示p≤0.05；和空白辅料组比较，c表示p≤0.05；和市售对照组比较，d表示p≤0.05；和供试品低剂量组比较，e表示p≤0.05；和供试品中剂量组比较，f表示p≤0.05。
表8角膜荧光素钠染色评分总结表

注：N表示动物眼数。和未造模组比较，a表示p≤0.05；和模型对照组比较，b表示p≤0.05；和空白辅料组比较，
c表示p≤0.05；和市售对照组比较，d表示p≤0.05；和供试品低剂量组比较，e表示p≤0.05；和供试品中剂量组比较，f表示p≤0.05。
5、实验结果
低湿环境联合东莨菪碱皮下注射能成功诱导小鼠泪液分泌量的减少，低、中、高剂量的供试品和市售对照品连续给药5天对模型小鼠的泪液分泌量的降低均有显著的改善作用，给药延长至10天时低、中、高剂量的供试品和市售对照品仍可见一定的改善作用且从均值上看供试品的效果更明显。
低湿环境联合东莨菪碱皮下注射能成功诱导小鼠角膜荧光素钠染色评分的升高，低、中、高剂量的供试品和市售对照品连续给药5天均可显著降低小鼠角膜荧光素钠染色评分，给药延长至10天时仍可降低小鼠角膜荧光素钠染色评分， 其中中、高剂量的供试品效果更优于市售对照品，且高剂量的供试品可使模型小鼠的角膜荧光素钠染色评分恢复接近至正常水平。
结论：本发明的环孢素眼用乳剂可以提高药效。
虽然本发明某些特征已经在本文中阐释和描述，但本领域技术人员将想到许多修改、替代、变更和等同。因此，应理解的是，所附权利要求书意在涵盖落入本发明真实精神范围之内的所有此类修改和变更。

Claims (10)

  1. 一种水包油环孢素乳剂，优选纳米乳剂，包含界面稳定剂，所述界面稳定剂包含15-羟基硬脂酸聚乙二醇酯和磷脂的组合。
  2. 根据权利要求1所述的水包油环孢素乳剂，还包含缓冲盐。
  3. 根据权利要求1所述的水包油环孢素乳剂，其中所述磷脂选自大豆磷脂、蛋黄磷脂或其任何组合，优选地，选自大豆磷脂S100、蛋黄磷脂E80或其任何组合。
  4. 根据权利要求1所述的水包油环孢素乳剂，其中环孢素与15-羟基硬脂酸聚乙二醇酯的质量比为1：30～1：45。
  5. 根据权利要求1所述的水包油环孢素乳剂，其中磷脂与15-羟基硬脂酸聚乙二醇酯的质量比为1：14～1：31。
  6. 根据权利要求1所述的水包油环孢素乳剂，其中15-羟基硬脂酸聚乙二醇酯的含量为2.25～13.5g/100ml。
  7. 根据权利要求2所述的水包油环孢素乳剂，其中缓冲盐为磷酸缓冲盐，优选地，以磷酸根计，缓冲盐浓度为0.01mol/L～0.05mol/L。
  8. 根据权利要求1所述的水包油环孢素乳剂，还包含渗透压调节剂；优选地，渗透压调节剂选自氯化钠、山梨醇或其任何组合；优选地，渗透压调节剂的含量为0.2～1g/100ml。
  9. 根据权利要求1所述的水包油环孢素乳剂，所述乳剂的粒子的平均粒径不超过50nm，优选10～40nm，更优选20～30nm；优选地，所述乳剂的90％的粒子的粒径不超过90nm；优选地，乳剂的pH值为6.5～8.0，优选6.8～7.6，更优选7.0～7.4；优选地，乳剂的包封率为不低于90％，优选不低于95％；优选地，所述乳剂为透明的乳状液体；优选地，所述乳剂为滴眼剂；优选地，所述油选自中链甘油三酸酯、大豆油、蓖麻油、橄榄油或鱼油。
  10. 一种制备水包油环孢素乳剂的方法，所述乳剂包含界面稳定剂、渗透压调节剂和缓冲盐，所述界面稳定剂包含15-羟基硬脂酸聚乙二醇酯和磷脂的组合，所述方法包括
    1)将油和界面稳定剂混合均匀，向其中加入环孢素并溶解，形成油相；
    2)将渗透压调节剂的一部分溶于水，将油相和含有渗透压调节剂的水搅拌混合，形成初乳；以及
    3)将缓冲盐和余量的渗透压调节剂溶于水形成溶液，将初乳加入至所述溶液并混匀。
PCT/CN2023/105857 2022-08-03 2023-07-05 一种环孢素乳剂及其制备方法 WO2024027451A1 (zh)

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CN102525887A (zh) * 2012-01-16 2012-07-04 无锡信仁堂药物技术有限公司 含有环孢素的眼用乳剂凝胶及其制备方法
CN106692052A (zh) * 2015-11-13 2017-05-24 天津金耀集团有限公司 一种环孢素眼用乳剂组合物
CN106821987A (zh) * 2017-03-16 2017-06-13 四川大学 一种载含酚羟基难溶性药物的脂质体及制备方法和应用
CN111249442A (zh) * 2019-10-09 2020-06-09 沈阳药科大学 一种脂质纳米囊滴眼剂及其制备方法
CN114796111A (zh) * 2021-01-28 2022-07-29 北京德立福瑞医药科技有限公司 一种含有难溶性药物的浓缩液以及由其制备的乳剂

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CN102525887A (zh) * 2012-01-16 2012-07-04 无锡信仁堂药物技术有限公司 含有环孢素的眼用乳剂凝胶及其制备方法
CN106692052A (zh) * 2015-11-13 2017-05-24 天津金耀集团有限公司 一种环孢素眼用乳剂组合物
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