WO2023030430A1 - 一种滴眼给药预防和/或治疗白内障的眼用制剂 - Google Patents

一种滴眼给药预防和/或治疗白内障的眼用制剂 Download PDF

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WO2023030430A1
WO2023030430A1 PCT/CN2022/116432 CN2022116432W WO2023030430A1 WO 2023030430 A1 WO2023030430 A1 WO 2023030430A1 CN 2022116432 W CN2022116432 W CN 2022116432W WO 2023030430 A1 WO2023030430 A1 WO 2023030430A1
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content
preparation
povidone
lens
lanosterol
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董庆
张舒
薛陆兵
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成都瑞沐生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • 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
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • 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
    • 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
    • A61K9/08—Solutions
    • 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
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts

Definitions

  • the invention belongs to the field of ophthalmic drugs, in particular to an ophthalmic preparation for preventing and/or treating cataract by eye drop administration.
  • Cataracts are the gradual aging and cloudiness of the lens due to aging and other reasons, blocking light from entering the eye, affecting vision and even blindness. Cataract is currently the most common blinding eye disease in the world. According to the report of the World Health Organization (WHO), the blinding rate of cataract accounts for more than 40%. The blinding rate of people over 50 years old is about 47.8%. In developed countries, it accounts for 90% of the blinding rate. %. There are many cataract patients, and there is currently no reliable drug therapy to replace surgery to prevent or treat cataracts. Surgical treatment is currently the only confirmed and effective method for the treatment of cataracts (Jingjie Xu et al: Advances in pharmacotherapy of cataracts, Ann Transl Med 2020; 8( 22): 1552).
  • oxidized cholesterol molecules include 25-hydroxycholesterol (25-hydroxycholesterol, 25-HC, CAS#: 2140-46-7) and lanosterol (Lanosterol , LAN, CAS#: 79-63-0) has the effect of reversing the cataract that has occurred and restoring the transparency of the lens (Ling Zhao et.al., Lanosterol reverses protein aggregation in cataracts, Nature 523,607–611, 2015; Makley et.al ., Pharmacological chaperone for ⁇ -crystallin partially restores transparency in cataract models, Science 350(6261), 674-677, 2015).
  • the function of the lens is to transmit and gather light to the retina, which is one of the refractive media of the eye.
  • the crystallins in the lens include ⁇ -, ⁇ - and ⁇ -three kinds of crystallins ( ⁇ -, ⁇ -, ⁇ -crystallins), which are regularly arranged.
  • the interactive structure between crystallin molecules determines the transparency and refraction of the lens. basis of the rate.
  • the pathological basis of cataract is the abnormal folding and aggregation of crystal proteins, which changes the interaction between crystal proteins and reduces the fluidity and stability of crystal proteins. The aggregated proteins make the lens cloudy and opaque, and light cannot enter the eyes, resulting in cataracts.
  • Lanosterol and 25-hydroxycholesterol can prevent the aggregation of crystal proteins, improve the shape of the lens and restore transparency.
  • Gestwicki et al. reported that oxidized cholesterol, including 25-hydroxycholesterol, can enhance the protective activity of ⁇ -crystallin and clear cataracts.
  • Kang Zhang's team reported that lanosterol binds to ⁇ - and ⁇ -crystallins, prevents the polymerization of crystallins, and can clear cataracts (Ling Zhao et.al., Nature 2015; Makley et.al., Science 2015).
  • LAN is a key cyclization reaction intermediate in the cholesterol biosynthesis pathway in vivo, which is catalyzed by lanosterol synthase (LSS); 25-hydroxycholesterol is synthesized by 25-hydroxycholesterol enzyme in vivo.
  • LDS lanosterol synthase
  • 25-hydroxycholesterol is synthesized by 25-hydroxycholesterol enzyme in vivo.
  • Oral supplementation is the most common method, but these active substances are supplemented through the whole body, and most of them participate in the biochemical process of the whole body, and the intermediates that reach the lens are still not enough to reach the effective concentration.
  • lanosterol is a key intermediate in the biosynthesis of cholesterol in the body.
  • 25-Hydroxycholesterol is an oxidation product of cholesterol, which is closely related to inflammation or infection. It is also a liver X receptor (LXR) agonist.
  • topical delivery in the eye is the best way to prevent and treat cataracts with this type of active substance.
  • the surface of the cornea in the anterior segment of the eyeball is covered by a tear film, and the cornea itself is composed of a lipid layer, a watery layer, and a mucin layer. It is only possible to enter the anterior chamber and reach the lens through the pupil. In order to achieve the purpose of preventing and treating cataracts through the natural barriers of tissue anatomy, physiology and biochemistry, it is necessary to overcome the technical difficulties of existing eye drops (Thrimawithana, T.R. et al., Drug delivery to the lens for the management of cataracts, Advanced Drug Delivery Reviews (2016), 126, 185-194).
  • researchers injected nanoparticles prepared from lanosterol, polycaprolactone, lecithin, and phospholipid polyethylene glycol carboxyl (DSPE-PEG-COOH) into the vitreous cavity of an animal cataract model (1 injection every 3 days) , combined with eye drops, using a concentration of 25mM lanosterol solution (15% cyclodextrin and 18% ethanol), 6 times a week, after 6 weeks, the cataract disappeared; Cannot treat cataract (Ling Zhao et al., Nature, 2015; Kang Zhang and Shenyang Hou, US2017/0065617 A1).
  • lanosterol thermal gel prepared from polylactide-lactide copolymer-polyethylene glycol copolymer-polylactide-lactide copolymer (PLGA-PEG-PLGA)
  • Intravitreal injection of experimental rabbits concentration 400mg/g
  • intravitreal LAN concentration can be maintained at > 50ng/mL for 3 weeks
  • Lei Lv et al. Quantitation of lanosterol in the vitreous humor of rabbits after ocular administration of lanosterol/thermogel formulation by ultra high performance liquid chromatography-tandem mass spectrometry with the electrospray ionization mode, J Chromatogr.A, 2017; 1519:83-90).
  • the drug can only obtain a high concentration in the retina, choroid and vitreous fundus tissues, but still cannot effectively enter the lens to play a role. If the lens is injected directly, it may induce traumatic or post-cataract, which cannot be implemented in clinical practice.
  • oxidized cholesterol can effectively prevent and treat cataracts in the lens, but there is currently no ophthalmic clinically feasible drug delivery method to safely deliver it to the lens. And because they have a wide range of physiological activities, it is necessary to prevent exogenous oxidized cholesterol from entering the human body, and the existing systemic administration such as oral administration, injection, and implantation cannot guarantee the safety of such drugs.
  • the use of non-invasive eye drop administration is different from the delivery mechanism of traditional eye drop administration, and it is an ideal delivery method to make the drug reach a therapeutic concentration in the lens. Therefore, it is an urgent problem to be solved in the field of ophthalmic preparations to invent ophthalmic preparations that can safely and effectively deliver drugs to the lens through eye drops.
  • oxidized cholesterol such as lanosterol
  • DMF N,N-dimethylformamide
  • DMSO Dimethyl sulfoxide
  • the object of the present invention is to provide an ophthalmic preparation capable of delivering active substances for treating eye diseases, such as oxidized cholesterol substances, to the lens through eye drops for treating and preventing cataracts.
  • the invention provides an ophthalmic preparation for eye drop administration, which is composed of an active substance for treating eye diseases and a pharmaceutically acceptable carrier or auxiliary material;
  • the active substance for treating eye diseases is oxidized cholesterol, which includes lanosterol or 25-hydroxycholesterol;
  • the pharmaceutically acceptable carrier or adjuvant contains the following components: surfactant, tackifier, co-solvent and solvent;
  • the content of oxidized cholesterol in the preparation is 0.01-5mg/mL; the mass ratio of surfactant, tackifier, cosolvent and oxidized cholesterol is (1-300):(1-100):(10- 3000): 1, the balance is solvent.
  • the content of oxidized cholesterol in the above preparation is 0.01-2 mg/mL.
  • the content of oxidized cholesterol in the above preparation is 0.05-0.5 mg/mL.
  • the content of oxidized cholesterol in the above preparation is: 0.01mg/mL, 0.05mg/mL, 0.1mg/mL, 0.15mg/mL, 0.2mg/mL, 0.25mg/mL, 0.3mg/mL, 0.35 mg/mL, 0.4mg/mL, 0.45mg/mL, 0.5mg/mL, 0.55mg/mL, 0.6mg/mL, 0.65mg/mL, 0.7mg/mL, 0.75mg/mL, 0.8mg/mL, 0.85 mg/mL, 0.9mg/mL, 0.95mg/mL, 1mg/mL, 1.5mg/mL or 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL or 5mg/mL.
  • the mass ratio of the above-mentioned surfactant, tackifier, co-solvent and oxidized cholesterol is (6.7-250):(11-50):(100-2500):1, preferably (25-200) :(11-48):(200-2500):1, more preferably 25:12:(200-600):1.
  • the above-mentioned surfactant is a nonionic surfactant.
  • nonionic surfactant is polysorbate, poloxamer or alkyl glucoside.
  • the above-mentioned thickener is a combination of at least two of the following polymer compounds: hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, povidone, carbomer, polyethylene glycol alcohol, poloxamer, polyvinyl alcohol, hydroxyethylcellulose, xanthan gum, hyaluronic acid or its salt, alginic acid or its salt, carboxymethylcellulose or its salt.
  • the above-mentioned tackifier is a combination of two of the polymer compounds, and the weight ratio of the two polymer compounds is 1:(0.1-10), preferably 1:(0.6-5);
  • the weight ratio of the above two polymer compounds is 1:1.
  • the above-mentioned thickening agent is the combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose and povidone is 1:(1 ⁇ 2), preferably 1:(1 ⁇ 1.2 );
  • Or described tackifier is the combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone and hydroxypropyl methylcellulose is 1:(1 ⁇ 3), is preferably 1:(1 ⁇ 1.5);
  • the tackifier is a combination of povidone and carbomer, the weight ratio of povidone and carbomer is 1:(0.5 ⁇ 2), preferably 1:1;
  • the thickening agent is a combination of povidone and polyethylene glycol, and the weight ratio of polyethylene glycol and povidone is 1:(1-8), preferably 1:5.
  • the above-mentioned oxidized cholesterol is 25-hydroxycholesterol
  • the thickener is a combination of at least two of the following macromolecular compounds: hydroxypropyl methylcellulose, hydroxypropyl cellulose, methyl Cellulose, povidone, carbomer, polyethylene glycol, poloxamer, polyvinyl alcohol, hydroxyethyl cellulose, xanthan gum, hyaluronic acid or its salt, alginic acid or its salt, carboxymethyl cellulose or its salts.
  • the above-mentioned oxidized cholesterol is 25-hydroxycholesterol
  • the thickener is a combination of povidone and hydroxypropyl cellulose
  • the weight ratio of hydroxypropyl cellulose and povidone is 1 :(1 ⁇ 2), preferably 1:(1 ⁇ 1.2);
  • Or described tackifier is the combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone and hydroxypropyl methylcellulose is 1:(1 ⁇ 3), is preferably 1:(1 ⁇ 1.5).
  • the above-mentioned oxidized cholesterol is lanosterol
  • the thickener is a combination of at least two of the following macromolecular compounds: hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, Povidone, Carbomer, Macrogol, Poloxamer, Polyvinyl Alcohol, Hydroxyethylcellulose.
  • the above-mentioned oxidized cholesterol is lanosterol
  • the thickener is a combination of povidone and hydroxypropyl cellulose
  • the weight ratio of hydroxypropyl cellulose and povidone is 1:(1 ⁇ 2 ), preferably 1:(1 ⁇ 1.2);
  • Or described tackifier is the combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone and hydroxypropyl methylcellulose is 1:(1 ⁇ 3), is preferably 1:(1 ⁇ 1.5);
  • the tackifier is a combination of povidone and carbomer, the weight ratio of povidone and carbomer is 1:(0.5 ⁇ 2), preferably 1:1;
  • the thickening agent is a combination of povidone and polyethylene glycol, and the weight ratio of polyethylene glycol and povidone is 1:(1-8), preferably 1:5.
  • the solvent in the above-mentioned pharmaceutically acceptable carrier or excipient is a polar solvent, preferably water.
  • the co-solvent in the above-mentioned pharmaceutically acceptable carrier or excipient is selected from at least one of liquid polyethylene glycol, propylene glycol, glycerol, polyoxyethylene hydrogenated castor oil or castor oil polyoxyethylene ether, preferably It is liquid polyethylene glycol.
  • the above preparation includes the following components;
  • Active substance for treating eye diseases lanosterol, the content of which is 0.01-0.2 mg/mL;
  • Surfactant polysorbate or poloxamer, the content of which is 6.7 to 250 times that of lanosterol;
  • Viscosifier its content is 11 ⁇ 50 times of lanosterol, and described viscosifier is the combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose and povidone is 1:(1 ⁇ 1.2); Or, described tackifier is the combination of povidone and hydroxypropyl methylcellulose; The weight ratio of povidone and hydroxypropyl methylcellulose is 1:(1 ⁇ 1.5); Or , the tackifier is a combination of povidone and carbomer, and the weight ratio of povidone and carbomer is 1:1; or the tackifier is a combination of povidone and polyethylene glycol, The weight ratio of polyethylene glycol and povidone is 1:5;
  • Co-solvent liquid polyethylene glycol, propylene glycol, glycerin, polyoxyethylene hydrogenated castor oil or castor oil polyoxyethylene ether, the content of which is 100-2500 times that of lanosterol; the solvent is water.
  • the above preparation includes the following components;
  • Active substance for the treatment of eye diseases 25-hydroxycholesterol, the content of which is 0.1 mg/mL;
  • Viscosifier its content is 12 times of 25-hydroxycholesterol, and described viscosifier is the combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose and povidone is 1:1 Or, the thickening agent is the combination of povidone and hydroxypropyl methylcellulose; The weight ratio of povidone and hydroxypropyl methylcellulose is 1:1;
  • Co-solvent liquid polyethylene glycol or glycerol, the content of which is 100-1750 times that of 25-hydroxycholesterol; the solvent is water.
  • the pharmaceutically acceptable carrier or adjuvant of the above preparation also contains any one or more of an osmotic pressure regulator, a pH regulator, and a preservative;
  • the osmotic pressure regulator is any one or more of glucose, sodium chloride, potassium chloride, mannitol, sorbitol, sodium citrate, potassium citrate and glycerol;
  • the pH regulator is hydrochloric acid, sodium hydroxide, acetic acid or its salt, citric acid or its salt, fumaric acid, succinic acid, sorbic acid, phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, boric acid, borax, tartaric acid any one or more of its salts;
  • Described preservative is sorbic acid, chlorobutanol, sodium chlorite, sodium perborate, quaternary ammonium salts (comprising benzalkonium chloride, benzalkonium bromide, polyquaternium-1, cetyl bromide Alkyltrimethylammonium), paraben esters (including methylparaben, ethylparaben, propylparaben), any one or more of phenylmercuric nitrate; preferably, the quaternary ammonium salts include Benzalkonium chloride, benzalkonium bromide, polyquaternium-1 and/or cetyltrimethylammonium bromide, the paraben esters include methylparaben, ethylparaben and/or propylparaben ester.
  • quaternary ammonium salts comprising benzalkonium chloride, benzalkonium bromide, polyquaternium-1, cetyl bromide Al
  • the above-mentioned ophthalmic preparation contains a nanosome structure, and the nanosome is formed by self-assembly of the carrier or adjuvant components of the ophthalmic preparation; the nanosome contains active substances for treating eye diseases.
  • the above-mentioned nano bodies are spherical and have a particle size of 5-900 nm, preferably 5-50 nm and/or 200-700 nm.
  • the present invention also provides a method for preparing the above preparation, comprising the following steps:
  • step (1) Add active substances for treating eye diseases to the mixed solution obtained in step (1), add or not add a cosolvent, disperse and mix to obtain an initial suspension;
  • step (3) Stir and disperse and/or homogeneously disperse the primary suspension obtained in step (2) to obtain the product.
  • the dispersion in step (2) is selected from at least one of mechanical stirring dispersion, magnetic stirring dispersion, vortex shaking dispersion, shear dispersion, homogeneous dispersion, grinding dispersion, and ultrasonic dispersion.
  • the present invention also provides the use of the above preparation in the preparation of medicines for preventing and treating human or animal lens diseases.
  • the above-mentioned medicine is a medicine for preventing and treating cataract, preferably a medicine for reducing lens protein aggregation and lens turbidity.
  • the above-mentioned medicine is a medicine preparation for ocular administration, preferably a medicine for topical administration to the eye.
  • Oxidized cholesterols include 25-hydroxycholesterol (25-HC) and lanosterol (LAN), which can interact with crystal proteins in the lens, thereby preventing the polymerization of crystal proteins, improving the shape of the lens and restoring transparency. It can reverse the cataract that has occurred and restore the transparency of the lens, so as to achieve the purpose of treating and preventing cataract.
  • Oxidized cholesterol administered systemically through oral administration, intramuscular injection, and intravenous injection participates in the biochemical process of the whole body, and cannot reach an effective therapeutic concentration in the lens; LAN and 25-HC injected into the vitreous cannot enter the lens through the capsule; lens injection Traumatic or post-cataract can result.
  • Eye drop administration is a safe and convenient way of administration, but to treat fundus diseases, it is necessary to penetrate the eye barrier and deliver the drug safely and effectively to the fundus lesions.
  • the present invention reduces the concentration of the preparation as much as possible to minimize the risk under the premise of achieving the therapeutic effect. Due to mechanical actions such as blinking, only about 10% of the eye drops remain on the ocular surface, most of them are discharged from the lacrimal duct with tears, and a small part enters the nasal cavity through the nasolacrimal duct, and even absorbs into the blood circulation through the nasal cavity. The lower the concentration of the preparation, the smaller the risk to the tissues and organs, and the smaller the systemic side effects.
  • the concentration of lanosterol (not higher than 4.68mM) in the preparation of the present invention is lower than the concentration claimed by other patents, which can reduce toxic and side effects as much as possible.
  • the lens after instillation The concentration of LAN can reach 2.3-2.5 times of the original content, and the transmittance can reach 44-52%.
  • Another feature of the preparation of the present invention is the formulation advantage.
  • the treatment of cataract is a long-term medication, and it is particularly important to avoid active adjuvants in the formulation.
  • a low-concentration formulation with anti-infection activity may cause drug resistance of the ocular surface and nasolacrimal duct flora, and even Induce infection.
  • the concentration of the present invention is low; on the other hand, the preparation does not need to use preservatives, and long-term use has no potential complications.
  • oxidized cholesterol has poor water solubility
  • solvents such as ethanol and DMSO are commonly used to prepare oxidized cholesterol injections or eye drops.
  • Alcohol solvents are irritating to the eyes and their dosage must be controlled.
  • DMSO as a co-solvent may increase the solubility of the active substance, but it may cause more active substances to enter the systemic circulation. For example, injection of 0.5% LAN solution prepared with 15% DMSO, 20% ethanol or polysorbate 80 solution into rat vitreous not only causes vitreous opacity but also risks promoting lens opacity.
  • Crystalline proteins are highly ordered, tightly arranged and highly transparent, and injection will cause damage to their structure, resulting in traumatic cataracts. It is very difficult to deliver LAN to the lens by injection (Enguin, CN108472303A, 2016; Zheng Qinyuan, Yidan Gapal, 201610720166.X; Noriaki Nagai, et al: The Intravitreal Injection of Lanosterol Nanoparticles Rescues Lens Structure Collapse at an Early Stage in Shumiya Cataract Rats, Int. J. Mol. Sci. 2020, 21, 1048).
  • the inventor invented the use of non-ionic surfactants as solubilizers, or/and tackifiers, or/and joint use of cosolvents, while applying technologies such as physical stirring and mixing, high-speed shear dispersion and high-pressure homogenization It can be processed by means of processing, and if necessary, it can be heated and ultrasonicated to prepare a solution of oxidized cholesterol LAN or 25-HC with water as the main medium, and has good stability.
  • the prepared aqueous solution containing the oxidized cholesterol is detected by a laser particle size detector, and its main particle size distribution is 5-50 nm and/or 200-700 nm, and spherical particles can be observed under an electron microscope.
  • the water-soluble solvent prepared by the invention is compatible with tears; then the oxidized cholesterol enters the lens through the cornea and lens capsule.
  • the eye drops with water as the main medium of the oxidized cholesterol prepared by the present invention after eye drops to the experimental animals, detect that their concentration in the lens obviously increases, and in Aqueous humor and vitreous oxidized cholesterol content is little (rat test) or not detected (rabbit test).
  • the present inventor observed in the cataract model test of subcutaneous injection of sodium selenite solution in rats that the eye drops prepared by the present invention can delay the occurrence and development of cataract in experimental animals.
  • Senior dogs (15 years old) suffering from cataract use the LAN eye drops prepared by the present invention, and after 20 days of eye drops, it can be observed that the turbidity of the dog lens obviously subsides.
  • the present invention proves through experiments that the ophthalmic preparation of the present invention can efficiently and precisely deliver the active substance into the lens of the experimental animal after being instilled in the eye. Cataract prevention effect.
  • the oxidized cholesterol eye drops prepared by the present invention have the advantages of safety and effectiveness, especially the unexpected discovery that after eye drops of experimental animals, oxidized cholesterol was enriched in the lens; (New Zealand rabbit drops In the eye absorption test, LAN is enriched in the lens), while the concentration in the aqueous humor and vitreous is very low, indicating that the eye drops of the present invention have high target tissue selectivity.
  • the eye drops of the present invention can not only selectively and efficiently deliver the oxidized cholesterol active substances to the lens through eye drops, but also supplement the cataract caused by the lack of oxidized cholesterol; Unpredictable pathophysiological reactions in vivo.
  • Oxidized cholesterol in the lens can prevent the polymerization of crystal proteins and remove cataracts; however, oxidized cholesterol can produce cardiovascular system complications in the body, which is harmful to the body. So far, there is no technology in the pharmaceutical field to deliver drugs, including oxidized cholesterol, into the lens in a non-invasive way.
  • the new low-concentration eye drop of the present invention realizes the directional delivery of the drug into the lens, and its most obvious clinical advantage is that the oxidized cholesterol is delivered to the lens through the eye drop, and the content of the active substance in the invented cataract eye drop is achieved. Low, the bioavailability is very high after eye drops administration, effectively increasing the concentration of oxidized cholesterol in the lens and treating cataract. Because of the low concentration and high local bioavailability of the eye, it greatly reduces the possibility that the drug may enter the body through the conjunctiva and nasal cavity and produce toxic side effects.
  • the active substances for the treatment of eye diseases referred to in the present invention are: the active substances for the treatment of human or animal eye diseases, which exist or do not exist in the body.
  • the nano-body referred to in the present invention is: a nano-scale spherical aggregate formed by self-assembly of components of a drug carrier or auxiliary material in a solvent.
  • the solvent referred to in the present invention is: a liquid capable of dissolving components of a pharmaceutical carrier or auxiliary material.
  • Surfactants referred to in the present invention are: substances that can significantly reduce the surface tension of liquids; nonionic surfactants referred to in the present invention refer to surfactants that do not dissociate in water.
  • the eye drop administration of the present invention is: a method of administration by dripping a medicinal solution into the eye, which belongs to the route of mucosal administration.
  • liquid polyethylene glycol (liquid PEG) of the present invention is: polyethylene glycol that is liquid at normal temperature and pressure, preferably polyethylene glycol with a weight average molecular weight of no more than 1000.
  • Fig. 1 is the transmission electron micrograph of the sample that embodiment 3 makes.
  • Fig. 2 is the transmission electron micrograph of the sample prepared in Example 3 after being stained with a staining agent.
  • Fig. 3 is a photo showing the cloudiness of the lens before and after the ophthalmic preparation of the present invention is instilled in cataract dogs.
  • Fig. 4 is the particle size distribution diagram of the sample (0 day) prepared in Example 5.
  • Fig. 5 is the particle size distribution figure of the sample (room temperature 15 days) that embodiment 5 makes.
  • the reagents or instruments used in the present invention can be purchased from commercially available products. If no specific conditions are specified, use them according to conventional conditions or conditions suggested by the manufacturer.
  • Dispersing machine T25 easy clean digital, IKA company (Germany);
  • API 4000 triple quadrupole mass spectrometer (Applied Biosystems, USA);
  • the property detection method of preparation of the present invention is as follows
  • the freezing point depression of a solution is measured to determine its osmolarity.
  • Operation Clean the probe of the STY-1A osmometer: take three portions of 100 ⁇ L distilled water into 3 sample tubes, and after the instrument is preheated, screw the sample tube containing 100 ⁇ L distilled water onto the instrument probe, select to clean 3 times, and click " Wash", repeat three times.
  • Detection After filling in the sample information in the instrument information form, click "Test”; pipette 100 ⁇ L of sample into the sample tube with a pipette gun, gently screw on the instrument, and click "Start” for detection. The detection was repeated three times, and the average value of the three detection results was taken as the detection result.
  • the osmotic pressure has not reached isotonicity, use the above-mentioned osmotic pressure regulator to make it reach or approach isotonicity.
  • FE20 acidity meter is calibrated with pH buffer solution (pH is 4.00, 6.86 and 9.18 respectively), rinses the electrode with pure water, absorbs excess water with cellulose-free paper, immerses in the liquid sample to be tested and press the read button to start measurement, The data obtained after the reading is stable is the pH value of the sample.
  • pH adjusting agent is NaOH and HCl, phosphoric acid and phosphoric acid salt (as sodium dihydrogen phosphate, hydrogen phosphate disodium), citric acid and citrate (as sodium citrate), boric acid and borax; when actually carrying out animal experiments, detect the pH value of gained liquid as not reaching ophthalmic preparation requirement, regulate with above-mentioned pH adjuster.
  • Embodiment 1 the preparation of ophthalmic preparation of the present invention
  • the materials and ratios used are shown in Table 1.
  • the preparation process first weigh polysorbate 80, PVP K12, HPMC and liquid PEG (PEG400) and put them into a 100mL polypropylene centrifuge tube, add an appropriate amount of water for injection and stir for 30 minutes, and then put them into Add 6.0mg of lanosterol, add water for injection to 60ml, continue to stir and mix for 10 minutes to obtain a mixed solution, use a disperser to disperse the mixed solution at a speed of 12000-15000 per minute for 3 minutes, wait for the foam to disappear after stopping the machine, and transfer the dispersed solution to a high pressure homogenizer.
  • Detection instrument Agilent 1100 high performance liquid chromatograph
  • Particle size test results (main particle size and its distribution ratio): particle size 67.7nm (46.2%) and 75.8nm (38.9%), PdI (Polydispersity Index, distribution coefficient): 0.276;
  • the product was placed in the dark at 40°C for 30 days, and the appearance did not change significantly.
  • Animal eye drops absorption test results Three rats (6 eyes) were given 20 ⁇ L/eye eye drops for both eyes. After 1.5 hours, the animals were euthanized and the vitreous body and lens were quickly taken out to detect the content of LAN. Test results: lanosterol content in the lens: 5.16 ⁇ 1.90 ( ⁇ g/mL); in the vitreous: 0.045 ⁇ 0.091 ( ⁇ g/mL).
  • Embodiment 2 the preparation of ophthalmic preparation of the present invention
  • Particle size test results particle size 441.8nm (100.0%), PdI: 0.190;
  • Detection instrument Agilent 1100 high performance liquid chromatograph
  • the product was placed in the dark at 40°C for 15 days, and the appearance did not change significantly.
  • Embodiment 3 the preparation of ophthalmic preparation of the present invention
  • Particle size test results particle size 416.3nm (100.0%), PdI: 0.214; HPLC content: 0.078mg/mL.
  • the product was placed in the dark at 2-8°C for 30 days, and the appearance did not change significantly.
  • Animal lens eye drops absorption test results 2 New Zealand rabbits (4 eyes) were given eye drops, 50 ⁇ L/eye, 1.5 hours later, the animals were euthanized, and the aqueous humor, vitreous body and lens were quickly taken, and the content of LAN was detected; Test results: no lanosterol was detected in the aqueous humor and vitreous body (below the detection limit, LCQ ⁇ 0.002 ⁇ g/mL); the lanosterol content in the lens was 2.72 ⁇ 0.16( ⁇ g/mL); New Zealand rabbits, 2 eyes, no administration) detected the lanosterol content in the lens to be 0.76 ⁇ 0.01 ( ⁇ g/mL).
  • Embodiment 4 the preparation of ophthalmic preparation of the present invention
  • Particle size test results particle size 531.6nm (100.0%), PdI: 0.165; HPLC content: 0.077mg/mL.
  • the product was placed at room temperature in the dark for 15 days, and the appearance did not change significantly.
  • the particle size test result 935.1nm (88.2%), PdI: 0.340, HPLC content test result: 0.074mg/mL.
  • Embodiment 5 the preparation of ophthalmic preparation of the present invention
  • Particle size test results particle size: 401.3nm (98.8%); PdI: 0.221; HPLC content: 0.063mg/mL.
  • the product was placed at room temperature in the dark for 15 days, and the appearance did not change significantly.
  • the particle size test result 613.2nm (100.0%), PdI: 0.350, HPLC content test result: 0.064mg/mL.
  • Embodiment 6 the preparation of ophthalmic preparation of the present invention
  • the particle size is 10.7nm (99.1%); PdI: 0.224; HPLC content: 0.070mg/mL.
  • the product was placed at room temperature in the dark for 15 days, and the appearance did not change significantly.
  • the particle size test result 11.2nm (91.7%), PdI: 0.246, and the HPLC content test result: 0.069mg/mL.
  • Embodiment 7 the preparation of ophthalmic preparation of the present invention
  • the particle size is 472.5nm (99.4%); PdI: 0.205; HPLC content: 0.072mg/mL.
  • the product was placed in the dark at 2-8°C for 15 days, and the appearance did not change significantly.
  • the particle size test result 513.2nm (97.1%), PdI: 0.235, HPLC content test result: 0.070mg/mL.
  • Embodiment 8 the preparation of ophthalmic preparation of the present invention
  • Particle size detection results particle size 508.5nm (97.7%); PdI: 0.245; HPLC content: 0.080mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the appearance did not change significantly.
  • the particle size test result 423.5nm (90.1%), PdI: 0.223, and the HPLC content test result: 0.078mg/mL.
  • Animal lens absorption test results three rats (6 eyes) were given eye drops, 20 ⁇ L/eye, 1.5 hours later, the animals were euthanized and the lenses were taken out quickly, and the content of lanosterol was detected; the test results: wool in the lens The sterol content was 5.68 ⁇ 1.60 ( ⁇ g/mL).
  • Embodiment 9 the preparation of ophthalmic preparation of the present invention
  • Particle size detection results particle size 572.2nm (85.3%); PdI: 0.529; HPLC content: 0.085mg/mL.
  • the product was placed in the dark at 2-8°C for 15 days, and the appearance did not change significantly.
  • Animal lens absorption test results Three rats (6 eyes) were given eye drops of 20 ⁇ L/eye. After 1.5 hours, the animals were euthanized and the lenses were quickly taken out to detect the content of lanosterol in them; test results: lanosterol in the lens The content was 4.01 ⁇ 1.65 ( ⁇ g/mL).
  • Embodiment 10 the preparation of ophthalmic preparation of the present invention
  • the particle size is 12.8nm (99.0%); PdI: 0.175; HPLC content: 0.073mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the appearance did not change significantly.
  • Embodiment 11 the preparation of ophthalmic preparation of the present invention
  • Particle size detection results particle size 455.6nm (99.7%); PdI: 0.249; HPLC content: 0.051mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the appearance did not change significantly.
  • Embodiment 12 the preparation of ophthalmic preparation of the present invention
  • the particle size is 420.0nm (100.0%); PdI: 0.205; HPLC content: 0.037mg/mL.
  • the product was placed in the dark at 40°C for 30 days, and the appearance did not change significantly.
  • Embodiment 13 the preparation of ophthalmic preparation of the present invention
  • the particle size is 474.3nm (98.7%); PdI: 0.229; HPLC content: 0.089mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the appearance did not change significantly.
  • Embodiment 14 the preparation of ophthalmic preparation of the present invention
  • the particle size is 136.6nm (86.3%); PdI: 0.542; HPLC content: 0.009mg/mL.
  • the product was placed in the dark at 2-8°C for 15 days, and the appearance did not change significantly.
  • the particle size test result 214.2nm (98.6%), PdI: 0.232, and the HPLC content test result: 0.008mg/mL.
  • Embodiment 15 preparation of ophthalmic preparation of the present invention
  • the particle size is 397.2nm (90.1%); PdI: 0.649; HPLC content: 0.015mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the appearance did not change significantly.
  • Embodiment 16 the preparation of ophthalmic preparation of the present invention
  • the particle size is 13.2nm (98.3%); PdI: 0.235; HPLC content: 0.071mg/mL.
  • the product was placed in the dark at 2-8°C for 15 days, and the appearance did not change significantly.
  • the particle size test result 13.8nm (93.6%), PdI: 0.316, and the HPLC content test result: 0.069mg/mL.
  • Embodiment 17 the preparation of ophthalmic preparation of the present invention
  • Particle size test results particle size 527.5nm (99.4%); PdI: 0.240; HPLC content: 0.058mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the appearance did not change significantly.
  • the particle size is 16.86nm (69.0%); PdI: 1.000; HPLC content: 0.051mg/mL.
  • Particle size test results particle size 369.8nm (82.8%); PdI: 0.554, HPLC content of the supernatant of the example: 0.025 mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the flocculent suspension was precipitated.
  • the particle size test result 462.1nm (80.2%), PdI: 0.979, and the HPLC content test result of the supernatant of the example: 0.027mg/mL.
  • Particle size detection results particle size 313.7nm (74.9%); PdI: 0.587, HPLC content of the supernatant of the example: 0.029mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the flocculent suspended matter precipitated.
  • the particle size test result 161.4nm (100.0%), PdI: 0.231, the HPLC test result of the supernatant: 0.023mg/mL, and the active substance content was low.
  • the materials and ratios used are shown in Table 1.
  • the preparation process and content detection are the same as in Example 1, and the obtained solution has flocculent suspended matter. After the product was placed at room temperature in the dark for 15 days, the flocculent suspended matter aggregated and precipitated.
  • Particle size test results particle size 292.2nm (72.6%); PdI: 1.000, HPLC content: 0.029mg/mL.
  • the product was placed in the dark at 40°C for 15 days, and the appearance did not change significantly.
  • Animal lens absorption test results 2 rats (4 eyes) were given eye drops, 20 ⁇ L/eye, 1.5 hours later, the animals were euthanized and the lens was quickly taken out, and the content of LAN was detected; test results: lanosterol in the lens The content is 2.00 ⁇ 0.41 ( ⁇ g/mL), which is almost at the same level as that of the blank control eye, indicating that the sample of this comparative example cannot effectively enter the animal lens.
  • HPMC hydroxypropyl methylcellulose
  • PVP povidone
  • HPC hydroxypropyl cellulose
  • PEG polyethylene glycol with an average molecular weight ⁇ 5000Da
  • CMC-Na Sodium carboxymethyl cellulose
  • PEG-60 hydrogenated castor oil polyoxyethylene hydrogenated castor oil.
  • the eye drop system of the present invention contains at least one surfactant, two thickeners and an appropriate amount of solubilizers, the prepared product is stable, and the absorption rate of the animal lens is high. ;
  • the eye drop system of the present invention contains a surfactant, no or only a thickening agent; or when there is no surfactant, the stability of the prepared product is relatively poor (see Comparative Examples 2, 3 and 4).
  • the formulated product will have a great impact on the absorption of the animal lens, which will cause the active substance to fail to effectively enter the lens (comparative example 5).
  • Animal lens samples are homogenized (physiological saline is added to the lens at a ratio of 1:4, homogenized), take 50 ⁇ L, add methanol 175 ⁇ L, vortex mix for 2 minutes, and centrifuge at 12000 rpm for 10 minutes at 4 °C, and take the supernatant for LC-MS/MS analyze.
  • LC-MS/MS test conditions are as follows, LC-20AD high performance liquid chromatography system (SHIMADZU)-API4000 triple quadrupole mass spectrometer (Applied Biosystems company), equipped with Fortis Pace C18 5UM 2.1X30mm chromatographic column, column temperature 40 ° C, flow The phase is methanol:water (95:5), the flow rate is 0.4 mL/min, and the injection volume is 10 ⁇ L; the mass spectrometer uses an atmospheric pressure chemical ionization source (Atomsperic Pressure Chemical Ionization, APCI). The mass spectrometry conditions are shown in the table below:
  • the ophthalmic preparation of the present invention can effectively transport lanosterol through the lens barrier and accumulate in the lens through eye drops, and hardly remain in the vitreous.
  • test preparation Example 15
  • eyedrops After euthanasia at each time point, the lens and vitreous were collected immediately to detect the drug content in the lens and vitreous. Since the lens of the animal almost does not contain 25-HC, no control eyes were set.
  • C is the concentration of the active substance in the lens after eye drop administration
  • C0 is the concentration ( ⁇ g/mL) of the active substance in the lens of the eye without eye drop administration.
  • the preparation of the present invention can achieve a very high lens absorption rate on the basis of a low active substance concentration, and the effective absorption rate of the rabbit lens is even as high as 100%. It shows that the utilization rate of the preparation of the present invention is very high, and the active substance for treating cataract is delivered directionally into the lens, effectively increasing the concentration of oxidized cholesterol in the lens, treating cataract, and avoiding systemic absorption and toxic side effects.
  • Example 3 Poodles (age 15 years old, male, body weight 4.0 kg), keep the dogs in an indoor home environment, regularly and quantitatively provide dog food and drinking water, and treat them completely as pets during the experiment.
  • the preparation of Example 3 was instilled once a day, about 30 ⁇ L each time. After 20 days of eye drop administration, the range of autonomous activities of the dog was significantly increased; comparison of photographs showed that the partial regression of cataract after 20 days was observed (Fig. 3).
  • the experimental animals were anesthetized by inhalation of isoflurane and topically anesthetized by lidocaine to observe the turbidity of the intraocular lens of the rats, record the time of cataract formation, and score according to the following criteria:
  • Grade 6 Manifested as a mature cataract involving the entire lens.
  • Grade 5 Presents with nuclear opacities that do not involve the lens cortex.
  • Grade 4 It manifests as partial nuclear opacity.
  • Grade 3 Shows diffuse nuclear opacity with some cortical scatter.
  • Grade 2 Presents with mild nuclear opacities, diffused by swollen fibers or posterior subscapular opacities 2 to 3 days after selenite injection.
  • Grade 1 is the initial sign of nuclear opacity.
  • the ophthalmic preparation of the present invention is administered by eye drops, and the active substance can be effectively enriched in the lens, and has the functions of preventing cataract, delaying the development of cataract and improving cataract.
  • the present invention provides an ophthalmic preparation for preventing and treating cataract by eye drops.
  • the ophthalmic preparation of the present invention has excellent stability. Effect. Moreover, no active substances are detected in the aqueous humor and vitreous around the lens, avoiding systemic toxic and side effects, and solving the technical problems that people have been eager to solve in the field of ophthalmic drug delivery but have not been successful, and have extremely high clinical application value.

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Abstract

一种滴眼给药防治白内障的眼用制剂，由治疗眼病的活性物质和药学上可接受的载体或辅料组成；治疗眼病的活性物质为氧化型胆甾醇，包括羊毛甾醇或25-羟基胆甾醇；所述药学上可接受的载体或辅料含有：表面活性剂、增粘剂、助溶剂和溶剂；所述制剂中氧化型胆甾醇含量为0.01～5mg/mL；表面活性剂、增粘剂、助溶剂和氧化型胆甾醇的质量比为(1～300):(1～100):(100～3000):1，余量为溶剂。所述制剂在滴眼给药后，可以使活性物质选择性富集在实验动物的晶状体，精准发挥治疗白内障的作用，并且活性物质不分布在房水、玻璃体中，避免对其它眼组织和对全身的毒副作用。解决了眼科用药递送领域人们一直渴望解决的技术难题，具有极高的临床应用价值和社会价值。

Description

一种滴眼给药预防和/或治疗白内障的眼用制剂 技术领域
本发明属于眼用药物领域，具体涉及一种滴眼给药预防和/或治疗白内障的眼用制剂。
背景技术
白内障是因为年龄增长等原因导致晶状体逐渐老化浑浊，阻挡光线进入眼内从而影响视力甚至失明。白内障为目前世界上最常见致盲眼病，根据世界卫生组织(WHO)报告白内障致盲率占40％以上，50岁以上人群因白内障致盲比例约47.8％，在发达国家占致盲率的90％。白内障患者众多，目前尚无可靠的药物治疗方法替代手术预防或治疗白内障，手术治疗是目前唯一确定有效的治疗白内障的方法(Jingjie Xu et al：Advances in pharmacotherapy of cataracts，Ann Transl Med 2020；8(22):1552)。
在探索白内障发生发展的过程中，研究人员发现氧化型胆甾醇类分子(oxysterols)包括25-羟基胆甾醇(25-hydroxycholesterol，25-HC，CAS#：2140-46-7)和羊毛甾醇(Lanosterol，LAN，CAS#：79-63-0)具有逆转已经发生的白内障，恢复晶状体透明的作用(Ling Zhao et.al.,Lanosterol reverses protein aggregation in cataracts,Nature 523,607–611，2015；Makley et.al.,Pharmacological chaperone forα-crystallin partially restores transparency in cataract models,Science 350(6261),674-677，2015)。
晶状体的功能是传送和聚集光线到视网膜，是眼的屈光介质之一。晶状体内的晶体蛋白包括α-、β-和γ-三种晶体蛋白(α-、β-、γ-crystallins)，它们有规律地排列，晶体蛋白分子之间交互式结构是决定晶状体透明度和折射率的基础。白内障的病理基础是晶体蛋白异常折叠、聚集，改变了晶体蛋白之间的相互作用，降低晶体蛋白的流动性和稳定性，聚集的蛋白产生晶状体浑浊、不透明，光线不能进入眼睛，产生白内障。
羊毛甾醇和25-羟基胆甾醇能阻止晶体蛋白的聚合，可改善晶状体形态和恢复透明。Gestwicki等报道，氧化型胆甾醇，包括25-羟基胆甾醇等能增强α-晶体蛋白的保护活性，可以清除白内障。Kang Zhang团队报道羊毛甾醇与β-和γ-晶体蛋白结合，阻止晶体蛋白的聚合，可以清除白内障(Ling Zhao et.al.,Nature 2015；Makley et.al.,Science 2015)。
LAN是体内胆甾醇生物合成通路的关键环合反应中间体，由羊毛甾醇酶(Lanosterol synthase，LSS)催化合成；25-羟基胆甾醇是由体内25-羟基胆甾醇酶催化胆甾醇合成得到。在病理状态下，这些体内代谢中间体在晶状体的浓度不足以达到有效逆转白内障的水平，需要补充活性物质。口服等补充方式是最常见的方法，但这些活性物质通过全身补充，大多参与全身的生物化学过程，抵达晶状体的中间体仍然不足以达到有效浓度。此外，由于胆甾 醇类有广泛的生理活性，以全身给药的方式引入外源性的氧化胆甾醇可能产生难以预料的病理生理反应。例如羊毛甾醇是体内生物合成胆甾醇的关键中间体，25-羟基胆甾醇是胆甾醇的氧化产物，与炎症或感染有密切关系，也是肝X受体(LXR)激动剂，摄入LXR激动剂可能导致肝脂肪合成和高甘油三酯血症(Willinger，et al.,Oxysterols in intestinal immunity and inflammation,Journal of Internal Medicine,2019,285；367–380；Donovan Duc,et al.,Oxysterols in Autoimmunity,Int.J.Mol.Sci.2019,20,4522,1-16；Cystger,et al.Nat.Rev.Immunol.14(11),731-743(2014)；吴桐，杜红俊，肝脏X受体在眼科疾病中的研究进展，眼科新进展，39(9)，886-897，2019)。
因此，在眼部局部递送是这一类活性物质预防治疗白内障的最佳途径。
然而，眼球前段的角膜表面有泪膜覆盖，角膜本身由脂质层、水样层和粘蛋白层组成，由外向内，药物分子要渗透进入角膜的水性基质层、继而又要穿透通过脂质层，才有可能进入前房，经瞳孔到达晶状体。要实现药物透过组织解剖学、生理学和生物化学方面的天然屏障，到达防治白内障的目的，就必须克服现有滴眼剂的技术难题(Thrimawithana,T.R.et al.,Drug delivery to the lens for the management of cataracts，Advanced Drug Delivery Reviews(2018),126,185-194)。
目前为止，为克服角膜屏障，将氧化型胆甾醇类递送到晶状体，通常采用眼球注射。例如：研究人员将由羊毛甾醇、聚已内酯、卵磷脂和磷脂聚乙二醇羧基(DSPE-PEG-COOH)制备的纳米微粒注射到动物白内障模型的玻璃体腔中(每3天注射1次)，联合滴眼给药，使用浓度为25mM羊毛甾醇溶液(15％环糊精和18％乙醇)，每周6次，持续6周后，白内障消退；然而单独给实验动物滴眼给药，却不能治疗白内障(Ling Zhao et al.,Nature,2015；Kang Zhang and Shenyang Hou,US2017/0065617 A1)。说明现有技术中，要克服眼屏障必须采用注射等侵入性方式给药，而直接以羊毛甾醇溶液滴眼给药，则无法使得药物顺利通过角膜、晶状体的屏障进入晶状体中发挥治疗作用。
然而，在房水或/和玻璃体里的活性物质仍然必须穿过晶状体囊膜才能进入晶状体发挥作用。现有的诸多研究发现，即使将氧化胆甾醇活性物质直接注射到玻璃体中，因为晶状体囊膜的屏障作用，其依然难以进入晶状体中发挥疗效。
例如，研究人员将用聚已交酯丙交酯共聚物-聚乙二醇共聚物-聚已交酯丙交酯共聚物(PLGA-PEG-PLGA)制备的羊毛甾醇热凝胶剂(Thermogel)给实验兔玻璃体腔注射(浓度400mg/g)，玻璃体内LAN浓度可维持在>50ng/mL达3周(Lei Lv et al.,Quantitation of lanosterol in the vitreous humor of rabbits after ocular administration of lanosterol/thermogel formulation by ultra high performance liquid chromatography-tandem mass spectrometry with the electrospray ionization mode,J Chromatogr.A，2017；1519:83-90)。Nagai等人给实验大鼠玻璃体腔注射羊毛甾醇纳米微粒注射液(浓度为2mg/mL，每2天1次)，持续6周，不能阻断晶状体浑浊化的进程(Noriaki Nagai,et al: The Intravitreal Injection of Lanosterol Nanoparticles Rescues Lens Structure Collapse at an Early Stage in Shumiya Cataract Rats,Int.J.Mol.Sci.2020,21,1048)。
此外，还有体外实验进一步证实氧化性胆甾醇难以突破晶状体的屏障进入晶状体中发挥治疗作用，例如Shanmugam等将白内障摘除术后取得的白内障核浸泡在浓度为25mM的羊毛甾醇溶液里6天，白内障澄明度无明显改善(Shanmugam et al.,Effect of lanosterol on human cataract nucleus,Indian J Ophthalmol.63(12):888-890,2015)；Daszynski等将大鼠晶状体浸泡在15mM LAN脂质体溶液浸泡48小时，或白内障病人晶状体碎片浸泡在羊毛甾醇(0.20mM lanosterol)或25-羟基胆甾醇缓冲液中(浓度：0.25mM和0.50mM，37℃，72小时)，都没有看到羊毛甾醇或25-羟基胆甾醇能结合晶体蛋白解决白内障问题(Daszynski,et.al.,Failure of oxysterols such as Lanosterol to Restore Lens Clarity from Cataracts，Scientific Reports(2019)9:8459,14 pages)。
由此可见，即使采用侵入性的玻璃体注射方式透过角膜屏障，也仅可使药物在视网膜、脉络膜和玻璃体等眼底组织获得较高浓度，却仍然无法有效进入晶状体发挥作用。如果直接采用晶状体注射，则可能诱导外伤性或后发性白内障，无法在临床中实施。
综上所述，氧化型胆甾醇在晶状体里能有效地防治白内障，但现在没有眼科临床可实施的给药方法将其安全递送到晶状体。且由于它们有广泛的生理活性，必须避免外源性的氧化型胆甾醇进入人体，现有的口服、注射、植入等系统给药不能保证此类药物的安全性。采用无创性滴眼给药，有别于传统滴眼给药的递送机理，使药物在晶状体达到治疗浓度是理想的给药方式。因此，发明能通过滴眼给药的方式将药物安全有效地递送到晶状体的眼用制剂，是眼科制剂领域迫切需要解决的难题。
然而，除上述的药物递送环节存在的困难外，要制备以氧化型胆甾醇为活性物质的制剂还存在溶解性问题。氧化型胆甾醇等潜在活性物质(例如羊毛甾醇)属于四环三萜类化合物，溶于氯仿、乙醇、乙醚、正丙醇、异丙醇、N,N-二甲基甲酰胺(DMF)和二甲亚砜(DMSO)等，但在水中溶解度低(Li and Forciniti,J.Chem.Eng.Data，2020,65,2,436–445)，因此，目前常用有机溶剂来提高溶解度，但过量使用有机溶剂对眼睛有刺激作用，存在潜在的安全隐患。除有机溶剂外，还有研究人员采用环糊精或其衍生物作为辅料(cyclo-dextrins,CYD，包括α-环糊精、β-环糊精、羟丙基-β-环糊精)，利用包合技术使氧化胆甾醇类分子包入环糊精空穴，配制成溶液用于动物研究(恩固因，CN108472303 A，2016；J.D.Sciamanna,US2020/0360403A1)。但是CYD包合物不能透过生物膜，并且β-环糊精与胆甾醇形成复合物在体内不溶解，在肾脏蓄积，还会导致严重肾毒性(R.C.罗，P.J.舍斯基，P.J.韦勒，《药用辅料手册》[M].北京：化学工业出版社，2005)，会限制其进一步开发应用，目前仅有犬用产品在互联网销售(商品名：
Figure PCTCN2022116432-appb-000001
)。
因此，研究一种能够克服药物溶解性问题以及突破角膜、晶状体屏障的 眼用制剂，实现滴眼给药治疗白内障，将具有非常重要的临床价值和社会意义。
发明内容
本发明的目的在于提供一种能够通过滴眼给药方式，将治疗眼病的活性物质，如氧化型胆甾醇类物质递送到晶状体，用于治疗、预防白内障的眼用制剂。
本发明提供了一种滴眼给药的眼用制剂，它由治疗眼病的活性物质和药学上可接受的载体或辅料组成；
所述治疗眼病的活性物质为氧化型胆甾醇，所述氧化型胆甾醇包括羊毛甾醇或25-羟基胆甾醇；
所述药学上可接受的载体或辅料含有如下成分：表面活性剂、增粘剂、助溶剂和溶剂；
所述制剂中氧化型胆甾醇含量为0.01～5mg/mL；表面活性剂、增粘剂、助溶剂和氧化型胆甾醇的质量比为(1～300):(1～100):(10～3000):1，余量为溶剂。
进一步地，上述制剂中氧化型胆甾醇含量为0.01～2mg/mL。
进一步地，上述制剂中氧化型胆甾醇含量为0.05～0.5mg/mL。
更进一步地，上述制剂中氧化型胆甾醇含量为：0.01mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.25mg/mL、0.3mg/mL、0.35mg/mL、0.4mg/mL、0.45mg/mL、0.5mg/mL、0.55mg/mL、0.6mg/mL、0.65mg/mL、0.7mg/mL、0.75mg/mL、0.8mg/mL、0.85mg/mL、0.9mg/mL、0.95mg/mL、1mg/mL、1.5mg/mL或2mg/mL、2.5mg/mL、3mg/mL、3.5mg/mL、4mg/mL、4.5mg/mL或5mg/mL。
进一步地，上述表面活性剂、增粘剂、助溶剂和氧化型胆甾醇的质量比为(6.7～250):(11～50):(100～2500):1，优选为(25～200):(11～48):(200～2500):1，更优选为25:12:(200～600):1。
进一步地，上述表面活性剂为非离子表面活性剂。
更进一步地，上述非离子表面活性剂为聚山梨酯、泊洛沙姆或烷基葡萄糖苷。
进一步地，上述增粘剂为如下高分子化合物中的至少两种的组合：羟丙基甲基纤维素、羟丙基纤维素、甲基纤维素、聚维酮、卡波姆、聚乙二醇、泊洛沙姆、聚乙烯醇、羟乙基纤维素、黄原胶、透明质酸或其盐、海藻酸或其盐、羧甲基纤维素或其盐。
更进一步地，上述增粘剂为所述高分子化合物中的两种的组合，两种高分子化合物的重量比为1:(0.1～10)，优选为1:(0.6～5)；
更进一步地，上述两种高分子化合物的重量比为1:1。
更进一步地，上述增粘剂为聚维酮和羟丙基纤维素的组合，羟丙基纤维素和聚维酮的重量比为1:(1～2)，优选为1:(1～1.2)；
或所述增粘剂为聚维酮和羟丙基甲基纤维素的组合，聚维酮和羟丙基甲基纤维素的重量比为1:(1～3)，优选为1:(1～1.5)；
或所述增粘剂为聚维酮和卡波姆的组合，聚维酮和卡波姆的重量比为1:(0.5～2)，优选为1:1；
或所述增粘剂为聚维酮和聚乙二醇的组合，聚乙二醇和聚维酮的重量比为1:(1～8)，优选为1:5。
更进一步地，上述氧化型胆甾醇为25-羟基胆甾醇，所述增粘剂为如下高分子化合物中的至少两种的组合：羟丙基甲基纤维素、羟丙基纤维素、甲基纤维素、聚维酮、卡波姆、聚乙二醇、泊洛沙姆、聚乙烯醇、羟乙基纤维素、黄原胶、透明质酸或其盐、海藻酸或其盐、羧甲基纤维素或其盐。
更进一步地，上述所述氧化型胆甾醇为25-羟基胆甾醇，所述增粘剂为聚维酮和羟丙基纤维素的组合，羟丙基纤维素和聚维酮的重量比为1:(1～2)，优选为1:(1～1.2)；
或所述增粘剂为聚维酮和羟丙基甲基纤维素的组合，聚维酮和羟丙基甲基纤维素的重量比为1:(1～3)，优选为1:(1～1.5)。
更进一步地，上述氧化型胆甾醇为羊毛甾醇，所述增粘剂为如下高分子化合物中的至少两种的组合：羟丙基甲基纤维素、羟丙基纤维素、甲基纤维素、聚维酮、卡波姆、聚乙二醇、泊洛沙姆、聚乙烯醇、羟乙基纤维素。
更进一步地，上述氧化型胆甾醇为羊毛甾醇，所述增粘剂为聚维酮和羟丙基纤维素的组合，羟丙基纤维素和聚维酮的重量比为1:(1～2)，优选为1:(1～1.2)；
或所述增粘剂为聚维酮和羟丙基甲基纤维素的组合，聚维酮和羟丙基甲基纤维素的重量比为1:(1～3)，优选为1:(1～1.5)；
或所述增粘剂为聚维酮和卡波姆的组合，聚维酮和卡波姆的重量比为1:(0.5～2)，优选为1:1；
或所述增粘剂为聚维酮和聚乙二醇的组合，聚乙二醇和聚维酮的重量比为1:(1～8)，优选为1:5。
进一步地，上述药学上可接受的载体或辅料中的溶剂为极性溶剂，优选为水。
进一步地，上述药学上可接受的载体或辅料中的助溶剂选自液态聚乙二醇、丙二醇、丙三醇、聚氧乙烯氢化蓖麻油或蓖麻油聚氧乙烯醚中的至少一种，优选为液态聚乙二醇。
进一步地，上述制剂包括如下组分；
治疗眼病的活性物质：羊毛甾醇，其含量为0.01～0.2mg/mL；
表面活性剂：聚山梨酯或泊洛沙姆，其含量为羊毛甾醇的6.7～250倍；
增粘剂：其含量为羊毛甾醇的11～50倍，所述增粘剂为聚维酮和羟丙基纤维素的组合，羟丙基纤维素和聚维酮的重量比为1:(1～1.2)；或，所述增粘剂为聚维酮和羟丙基甲基纤维素的组合；聚维酮和羟丙基甲基纤维素的重量比为1:(1～1.5)；或，所述增粘剂为聚维酮和卡波姆的组合，聚维酮和卡波姆的重量比为1:1；或所述增粘剂为聚维酮和聚乙二醇的组合，聚乙二醇和聚维酮的重量比为1:5；
助溶剂：液态聚乙二醇、丙二醇、丙三醇、聚氧乙烯氢化蓖麻油或蓖麻油聚氧乙烯醚，其含量为羊毛甾醇的100～2500倍；溶剂为水。
或，上述制剂包括如下组分；
治疗眼病的活性物质：25-羟基胆甾醇，其含量为0.1mg/mL；
表面活性剂：聚山梨酯，其含量为25-羟基胆甾醇的25～250倍；
增粘剂：其含量为25-羟基胆甾醇的12倍，所述增粘剂为聚维酮和羟丙基纤维素的组合，羟丙基纤维素和聚维酮的重量比为1:1；或，所述增粘剂为聚维酮和羟丙基甲基纤维素的组合；聚维酮和羟丙基甲基纤维素的重量比为1:1；
助溶剂：液态聚乙二醇或丙三醇，其含量为25-羟基胆甾醇的100～1750倍；溶剂为水。
进一步地，上述制剂的药学上可接受的载体或辅料还含有渗透压调节剂、pH调节剂、防腐剂中的任意一种或多种；
所述渗透压调节剂为葡萄糖、氯化钠、氯化钾、甘露醇、山梨醇、枸橼酸钠、枸橼酸钾和甘油中的任意一种或多种；
所述pH调节剂为盐酸、氢氧化钠、醋酸或其盐、柠檬酸或其盐、富马酸、琥珀酸、山梨酸、磷酸、磷酸二氢钠、磷酸氢二钠、硼酸、硼砂、酒石酸或其盐中的任意一种或多种；
所述防腐剂为山梨酸、三氯叔丁醇、亚氯酸钠、过硼酸钠、季铵盐类(包括苯扎氯铵、苯扎溴铵、聚季銨盐-1、溴化十六烷基三甲铵)、羟苯酯类(包括羟苯甲酯、羟苯乙酯、羟苯丙酯)、硝酸苯汞中的任意一种或多种；优选地，所述季铵盐类包括苯扎氯铵、苯扎溴铵、聚季铵盐-1和/或溴化十六烷基三甲铵，所述羟苯酯类包括羟苯甲酯、羟苯乙酯和/或羟苯丙酯。
进一步地，上述眼用制剂含有纳米小体的结构，所述纳米小体由眼用制剂的载体或辅料的成分自组装形成；所述纳米小体中包含有治疗眼病的活性物质。
更进一步地，上述纳米小体为球形，其粒径为5～900nm，优选为5～50nm和/或200～700nm。
本发明还提供了制备上述的制剂的方法，包括以下步骤：
(1)向溶剂中加入表面活性剂、增粘剂，混合得到混合液；
(2)向步骤(1)得到的混合液中加入治疗眼病的活性物质，加入或不加助溶剂，分散混合得到初悬液；
(3)将步骤(2)得到的初悬液进行搅拌分散和/或均质分散，即得。
进一步地，步骤(2)中所述分散选自机械搅拌分散、磁力搅拌分散、涡旋振摇分散、剪切分散、均质分散、研磨分散、超声分散中的至少一种。
本发明还提供了上述的制剂在制备防治人或动物晶状体疾病的药物中的用途。
进一步地，上述药物是防治白内障的药物，优选为减少晶状体蛋白聚集、减少晶状体浑浊的药物。
进一步地，上述药物是眼部给药的药物制剂，优选为眼局部给药的药物。
氧化型胆甾醇类包括25-羟基胆甾醇(25-HC)和羊毛甾醇(LAN)，在晶状体里能和晶状蛋白相互作用，从而阻止晶体蛋白的聚合，改善晶状体形态和恢复透明的作用，对已经发生的白内障有逆转且恢复晶状体透明的作用，达到治疗、预防白内障的目的。通过口服、肌肉注射、静脉注射等全身给药的氧化型胆甾醇参加全身的生化过程，无法在晶状体达到有效治疗浓度；注射到玻璃体的LAN、25-HC无法透过囊膜进入晶状体；晶状体注射会导致外伤性或后发性白内障。
滴眼给药途径是安全、方便的给药方式，但要治疗眼底疾病需要将药物穿透眼屏障，安全、有效地递送到眼底病灶部位。
本发明按照治疗效果/风险比的优化设计原则，在达到治疗效果的前提下，尽量降低制剂的浓度以使风险最小化。滴眼液由于眨眼等机械性动作，只有约10％的滴眼液保留在眼表，大部分都随泪液从泪小管排出，小部分通过鼻泪管进入鼻腔，甚至经鼻腔吸收进入血循环。制剂浓度越低组织器官承受的风险越小，产生的全身毒副作用越小。本发明的制剂中羊毛甾醇的浓度(不高于4.68mM)低于其他专利声称的浓度，可以尽可能降低毒副作用。与此同时，本制剂0.1μg/μL的浓度滴入20μL于睑结膜囊，实际有约0.2μg停留在眼表，按照大鼠晶状体体积0.03cm 3计算(直径＝3.87mm)，滴眼后晶状体的LAN浓度可以达到原有含量的2.3-2.5倍，透过率达到44-52％。以兔为例，滴50uL含0.11μg/μL浓度的本发明制剂，以停留在眼表10％计进入兔晶状体(直径＝7.9mm，体积＝0.258cm 3)，其进入晶状体的羊毛甾醇的量可以达到原有浓度的3.6倍，透过率几乎达到100％(A.B.Weir and M.Collins(eds.),Assessing Ocular Toxicology in Laboratory Animals,1 Molecular and Integrative Toxicology,DOI 10.1007/978-1-62703-164-6_1,
Figure PCTCN2022116432-appb-000002
Springer Science+Business Media,LLC 2013；凌沛学主编《眼科药物与制剂学》，中国轻工出版社，2010，P6)。可见，本发明尽管所用的氧化型胆甾醇浓度低，但利用率非常高，不但能够降低潜在的全身毒副作用风险，而且能够充分保证给药剂量和药效。
本发明制剂的另一特点是配方优势，治疗白内障是长期用药，配方中避免有活性的辅料特别重要，如低浓度具有抗感染活性的配方可能造成眼表和鼻泪管菌群耐药，甚至诱发感染。本发明一方面浓度低，另一方面，制剂无需使用防腐剂，长期使用没有潜在的并发症。
文献报道，氧化型胆甾醇水溶性差，为配制氧化型胆甾醇类注射液或滴眼液，常用乙醇和DMSO等溶剂。醇类溶剂对眼有刺激作用，必须控制其用量。用DMSO作助溶剂，虽然可增加活性物质溶解度，但可能使更多量的活性物质进入体循环。例如，用15％DMSO、20％乙醇或加聚山梨酯80溶液配制的0.5％LAN溶液注射到大鼠玻璃体，不仅有导致玻璃体浑浊而且还有促进晶状体混浊的危险。
晶状蛋白的高度有序紧密排列和高度透明，注射会造成其结构受损，导致外伤性白内障。用注射途径递送LAN到晶状体是很困难的(恩固因，CN108472303A，2016；郑钦元，责祎旦·加帕尔，201610720166.X；Noriaki Nagai,et al:The Intravitreal Injection of Lanosterol Nanoparticles Rescues Lens Structure Collapse at an Early Stage in Shumiya Cataract Rats,Int.J.Mol.Sci.2020,21,1048)。
本发明人经过大量试验，发明采用非离子型表面活性剂作用增溶剂，或/和增黏剂、或/和助溶剂联合使用，同时应用物理搅拌混合、高速剪切分散和高压均质等技术手段进行加工处理，必要时可加热、超声，配制得到以水为主要介质的氧化胆甾醇LAN或25-HC的溶液，并且具有很好的稳定性。所制备的含有所述氧化胆甾醇的水溶液经激光粒度检测仪检测，其主要粒径分布在5～50nm和/或200～700nm，在电子显微镜下可观察到球状微粒。本发明制备的水溶性溶剂与泪液相溶；继而氧化胆甾醇穿过角膜和晶状体囊膜进入晶状体。在活体动物滴眼给药晶状体吸收试验中，用本发明制备的氧化胆甾醇的以水为主要介质的滴眼液，给实验动物滴眼后，检测到它们在晶状体里浓度明显增加，而在房水和玻璃体中则氧化胆甾醇含量很少(大鼠试验)或没有检测到(兔试验)。
本发明人在大鼠皮下注射亚硒酸钠溶液的白内障模型试验中观察到，本发明制备的滴眼液可延缓实验动物白内障的发生和发展。患有白内障的高龄犬(15岁龄)使用本发明配制的LAN滴眼液，滴眼20天后可观察到犬晶状体的浑浊明显消退。
本发明通过试验证明，本发明的眼用制剂在滴眼后，活性物质可高效、精准递送到实验动物晶状体内，作为晶状蛋白的伴侣，可阻止晶体蛋白的聚合，清除白内障，发挥治疗、预防白内障的效果。
在动物试验中，意外地发现本发明制备的氧化型胆甾醇滴眼剂具有安全、有效的优点，特别是意外发现，实验动物滴眼后，氧化型胆甾醇富集在晶状体；(新西兰兔滴眼吸收试验LAN富集在晶状体)，而在房水和玻璃体里的浓度很低，说明本发明的滴眼剂具有高度靶组织选择性。
本发明滴眼剂既能将氧化型胆甾醇类活性物质通过滴眼有选择性的高效递送到晶状体，补充了氧化型胆甾醇缺乏导致的白内障；又可避免具有广泛生理活性的胆甾醇类在体内产生难以预料的病理生理反应。
晶状体内的氧化胆甾醇能阻止晶体蛋白的聚合，清除白内障；但是，氧化胆甾醇在体内可产生心血管系统并发症，对身体有害。迄今为止，在制药 领域尚无以无创伤的药物递送方式把药物，包括氧化胆甾醇输送到晶状体里的技术。本发明的新型低浓度滴眼剂实现了把药物定向递送到晶状体里，其最明显的临床优势是把氧化胆甾醇经滴眼递送到晶状体，做到了所发明的白内障滴眼剂里活性物质含量低，滴眼给药后生物利用度非常高，有效地增加了晶状体里氧化胆甾醇的浓度，治疗白内障。因为浓度低、眼局部生物利用度高，极大地减少了药物可能经过结膜和鼻腔等进入身体并生产毒副作用。
本发明所指治疗眼病的活性物质是：治疗人或动物的眼病的、存在或不存在于机体内的活性物质。
本发明所指的纳米小体是：药物载体或辅料的成分在溶剂中自组装形成的纳米级的球形聚集体。
本发明所指的溶剂是：能溶解药物载体或辅料的成分的液体。
本发明所指表面活性剂是：能够显著降低液体表面张力的物质；本发明所指非离子型表面活性剂是指在水中不解离的表面活性剂。
本发明所述滴眼给药是：将药液滴入眼内的一种给药方法，属于粘膜给药途径。
本发明所述的液态聚乙二醇(液态PEG)是：在常温常压下为液态的聚乙二醇，优选为重均分子量不超过1000的聚乙二醇。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为实施例3制得的样品的透射电镜图。
图2为实施例3制得的样品加染色剂染色后的透射电镜图。
图3为白内障犬使用本发明眼用制剂滴眼前后的晶状体浑浊情况示照片。
图4为实施例5制得的样品(0天)的粒径大小分布图。
图5为实施例5制得的样品(室温15天)的粒径大小分布图。
具体实施方式
本发明所用试剂或仪器可以通过市售产品购买获得，未注明具体条件的，按照常规条件或制造商建议的条件使用。羊毛甾醇(CAS：79-63-0)，含量：99.9％(HPLC，成都普菲德生物技术有限公司，成都)；25-羟基胆甾醇(Hydroxycholesterol，CAS:2140-46-7，含量≥98％，上海麦克林生化科技有限公司，上海)。
部分仪器设备如下：
ES225SM-DR(E)电子分析天平，Precisa公司(瑞士)；
DF-101S集热式恒温加热磁力搅拌器，巩义市英峪高科仪器厂(河南，中国)；
WH-2微型涡旋混合仪，上海沪西分析仪器厂有限公司(上海，中国)；
分散机：T25 easy clean digital，IKA公司(德国)；
KQ-500型超声清洗仪，昆山市超声波仪器有限公司(昆山，中国)；
AH-NANO Plus高压均质机，安拓思纳米技术(苏州)有限公司(中国)；
Mettler Toledo FE20pH meter，梅特勒-托利多公司(瑞士)；
NS-90纳米粒度分析仪，珠海欧美克仪器有限公司(珠海，中国)；
安捷伦1100HPLC高效液相色谱仪，安捷伦科技有限公司(美国)；
API 4000三重四极杆质谱仪(美国Applied Biosystems公司)；
STY-1A渗透压测定仪，天津市天大天发科技有限公司(天津，中国)；
Zetasizer Nano ZS，纳米粒度及Zeta电位分析仪，马尔文公司(英国)。
本发明制剂的性质检测方法如下
粒径检测方法：
将1mL实施例或对比例制备得到的样品转移至样品池中，检测温度设置为40℃，将样品池放入NS-90纳米粒度分析仪，开始检测。每个样品重复检测3次，取3次检测结果的平均值为该样品检测结果为粒度(以光强分布，及占比％)和多分散指数(PdI，Polydispersity Index)表示。
渗透压检测方法：
测量溶液的冰点下降来测定其渗透压摩尔浓度。操作：清洗STY-1A渗透压测定仪探头：取三份100μL蒸馏水至3只样品管中，待仪器预热后，将装有100μL蒸馏水的样品管旋上仪器探头，选择清洗3次，点击“清洗”，重复三次。检测：在仪器信息表中填入样品信息后，点击“测试”；用移液枪移取100μL样品至样品管中，轻轻旋上仪器，点击“启动”检测。重复检测三次，取3次检测结果的平均值为检测结果。在实际进行动物实验时，渗透压如未达到等渗，用上述所述渗透压调节剂，使其达到或接近等渗。
pH值检测方法：
FE20型酸度计分别用pH缓冲溶液(pH分别为4.00、6.86和9.18)校准，电极用纯净水冲洗后，用无纤维纸吸去多余水份，浸入待检测液体样品中按读数键开始测量，在读数稳定后所得数据，即为样品pH值。
检测实施例得到的溶液若pH<5，或>9，则需要用酸或碱调节至pH6～8，常用的pH调节剂为NaOH和HCl，磷酸和磷酸盐(如磷酸二氢钠、磷酸氢二钠)，柠檬酸和柠檬酸盐(如柠檬酸钠)，硼酸和硼砂；实际进行动物实验时，检测所得液体的pH值如未达到眼用制剂要求，用上述所述pH调节剂调节。
实施例1、本发明眼用制剂的制备
所用物料及比例如表1所示，制备过程：首先分别称取聚山梨酯80、PVP K12、HPMC和液态PEG(PEG400)投入到100mL聚丙稀离心管，加入适量注射用水搅拌30分钟，然后投入6.0mg羊毛甾醇，加注射用水至60ml，继续搅拌混合10min后得混合液，混合液用分散机在转速每分钟12000～15000转分散3分钟，停机后待泡沫消失，将分散液转移至高压均质机，控制温度5±5℃，在压力400Bar左右均质2分钟，然后提高压力至1200～1400Bar均质20分钟，减压至500Bar均质2分钟后排出，泡沫自然消失后得到澄明均质液。检测pH值和滲透压，加柠檬酸钠(0.10g)和氯化钠(0.4g)，用0.1N HCl或0.1N NaOH调节至pH7.0，滲透压：302mOsmol/kg。溶液经滤膜减压过滤，得产品为溶液。
HPLC产品浓度检测：
检测仪器:安捷伦1100高效液相色谱仪；
色谱条件:Agilent ZORBAX Eclipse Plus C18,4.6x100mm 3.5μm色谱柱；流速1.0mL/min，检测波长205nm，流动相:MeOH(60％)-乙腈(40％)等度洗脱。样品用流动相5倍稀释后取10μL注入液相色谱仪。HPLC含量检测结果：0.098mg/mL。
粒径检测结果(主要粒径及其分布占比)：粒径67.7nm(46.2％)和75.8nm(38.9％)，PdI(Polydispersity Index，分布系数)：0.276；
Zeta电位检测平均值：-5.03±4.33mV(25℃)。
产品在40℃避光放置30天，外观无明显变化，粒径检测结果：40.68nm(88.8％)，PdI：0.249，HPLC含量检测结果：0.093mg/mL。
动物滴眼吸收试验结果：取三只大鼠(6只眼)，双眼滴眼给药20μL/眼，1.5小时后，动物安乐死并迅速取玻璃体和晶状体，并检测LAN的含量。检测结果：羊毛甾醇含量，晶状体里：5.16±1.90(μg/mL)；玻璃体里：0.045±0.091(μg/mL)。
实施例2、本发明眼用制剂的制备
所用物料及比例如表1所示，制备过程同实施例1，得到溶液；
粒径检测结果：粒径441.8nm(100.0％)，PdI：0.190；
HPLC含量检测：
检测仪器:安捷伦1100高效液相色谱仪；
色谱条件:Agilent ZORBAX Eclipse Plus C18,4.6x100mm 3.5μm色谱柱；流速0.8mL/min，检测波长：205nm，流动相:MeOH(85％)-0.1％H 3PO 4(15％)等度洗脱。样品用流动相5倍稀释后取10μL注入液相色谱仪。HPLC含量检测结果：0.095mg/mL。
产品在40℃避光放置15天，外观无明显变化，粒径检测结果：413.2nm(94.5％)，PdI：0.214，HPLC含量检测结果：0.090mg/mL。
实施例3、本发明眼用制剂的制备
所用物料及比例如表1所示，制备过程与含量检测同实施例1，得到溶液；
粒径检测结果：粒径416.3nm(100.0％)，PdI：0.214；HPLC含量：0.078mg/mL。
产品在2-8℃避光放置30天，外观无明显变化，粒径检测结果：478.5nm(98.2％)，PdI：0.245，HPLC含量检测结果：0.077mg/mL。
动物晶状体滴眼吸收实验结果：取新西兰兔2只(4只眼)滴眼给药，50μL/眼，1.5小时后，动物安乐死并迅速取房水、玻璃体和晶状体，并检测其中LAN的含量；检测结果：房水和玻璃体里未检测到羊毛甾醇(低于检测限，LCQ≤0.002μg/mL)；晶状体里羊毛甾醇含量为2.72±0.16(μg/mL)；空白本底对照动物(1只新西兰兔，2只眼，不给药)检测到晶状体里羊毛甾醇含量为0.76±0.01(μg/mL)。
实施例4、本发明眼用制剂的制备
所用物料及比例如表1所示，制备过程与含量检测同实施例1，得到溶液；
粒径检测结果：粒径531.6nm(100.0％)，PdI：0.165；HPLC含量：0.077mg/mL。
产品室温避光放置15天，外观无明显变化，粒径检测结果：935.1nm(88.2％)，PdI：0.340，HPLC含量检测结果：0.074mg/mL。
实施例5、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径：401.3nm(98.8％)；PdI：0.221；HPLC含量：0.063mg/mL。
产品室温避光放置15天，外观无明显变化，粒径检测结果：613.2nm(100.0％)，PdI：0.350，HPLC含量检测结果：0.064mg/mL。
动物滴眼晶状体吸收试验结果：取三只大鼠(6只眼)滴眼给药，20μL/眼，1.5小时后，动物安乐死并迅速取晶状体，并检测其中羊毛甾醇的含量；检测结果：晶状体里羊毛甾醇含量为2.89±0.60(μg/mL)；
实施例6、本发明眼用制剂的制备
所用物料及比例如表1所示，制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径10.7nm(99.1％)；PdI：0.224；HPLC含量： 0.070mg/mL。
产品室温避光放置15天，外观无明显变化，粒径检测结果：11.2nm(91.7％)，PdI：0.246，HPLC含量检测结果：0.069mg/mL。
动物晶状体吸收试验结果：取三只大鼠(6只眼)滴眼给药20μL/眼，1.5小时后，动物安乐死并迅速取晶状体，并检测其中羊毛甾醇的含量；检测结果：晶状体里羊毛甾醇含量为4.83±2.15(μg/mL)。
实施例7、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径472.5nm(99.4％)；PdI：0.205；HPLC含量：0.072mg/mL。
产品在2-8℃避光放置15天，外观无明显变化，粒径检测结果：513.2nm(97.1％)，PdI：0.235，HPLC含量检测结果：0.070mg/mL。
实施例8、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径508.5nm(97.7％)；PdI：0.245；HPLC含量：0.080mg/mL。
产品40℃避光放置15天，外观无明显变化，粒径检测结果：423.5nm(90.1％)，PdI：0.223，HPLC含量检测结果：0.078mg/mL。
动物晶状体吸收试验结果：取三只大鼠(6只眼)滴眼给药，20μL/眼，1.5小时后，动物安乐死并迅速取晶状体，并检测其中羊毛甾醇的含量；检测结果：晶状体里羊毛甾醇含量为5.68±1.60(μg/mL)。
实施例9、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径572.2nm(85.3％)；PdI：0.529；HPLC含量：0.085mg/mL。
产品在2-8℃避光放置15天，外观无明显变化，粒径检测结果：583.8nm(80.2％)，PdI：0.545，HPLC含量检测结果：0.083mg/mL。
动物晶状体吸收试验结果：取三只大鼠(6只眼)滴眼给药20μL/眼，1.5小时后，动物安乐死并迅速取晶状体，并检测其中羊毛甾醇的含量；检测结果：晶状体里羊毛甾醇含量为4.01±1.65(μg/mL)。
实施例10、本发明眼用制剂的制备
所用物料及比例如表2所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径12.8nm(99.0％)；PdI：0.175；HPLC含量：0.073mg/mL。
产品40℃避光放置15天，外观无明显变化，粒径检测结果：13.2nm(91.5％)，PdI：0.222，HPLC含量检测结果：0.072mg/mL。
实施例11、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程同实施例1，含量检测同实施例2，得到溶液；
粒径检测结果，粒径455.6nm(99.7％)；PdI：0.249；HPLC含量：0.051mg/mL。
产品40℃避光放置15天，外观无明显变化，粒径检测结果：216.8nm(95.6％)，PdI：0.351，HPLC含量检测结果：0.049mg/mL。
实施例12、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径420.0nm(100.0％)；PdI：0.205；HPLC含量：0.037mg/mL。
产品40℃避光放置30天，外观无明显变化，粒径检测结果：452.0nm(94.0％)，PdI：0.256，HPLC含量检测结果：0.033mg/mL。
实施例13、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程同实施例1，含量检测同实施例2，得到溶液；
粒径检测结果，粒径474.3nm(98.7％)；PdI：0.229；HPLC含量：0.089mg/mL。
产品40℃避光放置15天，外观无明显变化，粒径检测结果：425.2nm(88.6％)，PdI：0.245，HPLC含量检测结果：0.087mg/mL。
实施例14、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径136.6nm(86.3％)；PdI：0.542；HPLC含量：0.009mg/mL。
产品2-8℃避光放置15天，外观无明显变化，粒径检测结果：214.2nm(98.6％)，PdI：0.232，HPLC含量检测结果：0.008mg/mL。
实施例15、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程同实施例1，含量检测同实施例2，得到溶液；
粒径检测结果，粒径397.2nm(90.1％)；PdI：0.649；HPLC含量：0.015mg/mL。
产品40℃避光放置15天，外观无明显变化，粒径检测结果：345.6nm(85.6％)，PdI：0.431，HPLC含量检测结果：0.014mg/mL。
实施例16、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径13.2nm(98.3％)；PdI：0.235；HPLC含量：0.071mg/mL。
产品在2-8℃避光放置15天，外观无明显变化，粒径检测结果：13.8nm(93.6％)，PdI：0.316，HPLC含量检测结果：0.069mg/mL。
实施例17、本发明眼用制剂的制备
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径527.5nm(99.4％)；PdI：0.240；HPLC含量：0.058mg/mL。
产品40℃避光放置15天，外观无明显变化，粒径检测结果：161.3nm(100.0％)，PdI：0.505，HPLC含量检测结果：0.052mg/mL。
对比例1
所用物料及比例如表1所示,制备过程与含量检测同实施例1，得到溶液；
粒径检测结果，粒径16.86nm(69.0％)；PdI：1.000；HPLC含量：0.051mg/mL。
室温放置静置过夜产生沉淀，此制剂稳定性较差。
对比例2
所用物料及比例如表1所示，制备过程与含量检测同实施例1，得到含有絮状悬浮物溶液；
粒径检测结果：粒径369.8nm(82.8％)；PdI：0.554，实施例上清液HPLC含量：0.025mg/mL。
产品40℃避光放置15天，絮状悬浮物产生沉淀，粒径检测结果：462.1nm(80.2％)，PdI：0.979，实施例上清液HPLC含量检测结果：0.027mg/mL。
对比例3
所用物料及比例如表1所示，制备过程与含量检测同实施例1，得到含有絮状悬浮物溶液；
粒径检测结果：粒径313.7nm(74.9％)；PdI：0.587，实施例上清液HPLC含量：0.029mg/mL。
产品40℃避光放置15天，絮状悬浮物产生沉淀，粒径检测结果：161.4nm(100.0％)，PdI：0.231，上清液HPLC含量检测结果：0.023mg/mL，活性物质含量低。
对比例4
所用物料及比例如表1所示，制备过程与含量检测同实施例1，得到溶液有絮状悬浮物。产品室温避光放置15天后，絮状悬浮物聚集产生沉淀。
对比例5
所用物料及比例如表1所示，制备过程与含量检测同实施例1，得到溶液；
粒径检测结果：粒径292.2nm(72.6％)；PdI：1.000，HPLC含量：0.029mg/mL。
产品40℃避光放置15天，外观无明显变化，粒径检测结果：188.3nm(100.0％)，PdI：0.217，HPLC含量检测结果：0.026mg/mL，活性物质含量降低。
动物晶状体吸收试验结果：取2只大鼠(4只眼)滴眼给药，20μL/眼，1.5小时后，动物安乐死并迅速取晶状体，并检测其中LAN的含量；检测结果：晶状体里羊毛甾醇含量为2.00±0.41(μg/mL)，与空白对照眼相比几乎处于同一水平，说明本对比例的样品不能有效进入动物晶状体。
表1实施例和对比例用量信息
Figure PCTCN2022116432-appb-000003
Figure PCTCN2022116432-appb-000004
注释：1、HPMC：羟丙基甲基纤维素；2、PVP：聚维酮；3、HPC：羟丙基纤维素；4、PEG：聚乙二醇平均分子量≤5000Da；5、CMC-Na：羧甲基纤维素钠盐；6、PEG-60氢化蓖麻油：聚氧乙烯氢化蓖麻油。
从实施例和对比例的结果可以看出：本发明的滴眼液体系中含有至少1种表面活性剂、2种增黏剂和适量的助溶剂，所制备的产品稳定，动物晶状体吸收率高；
当本发明的滴眼液体系中含有1种表面活性剂，没有或仅含1种增黏剂；或没有表面活性剂时，所制备的产品稳定性均比较差(见对比例2、3和4)。
如果在制剂配方中添加离子型高分子，所配制产品在短时间放置后，会产生沉淀，稳定性不好(见对比例1)；
如果表面活性剂用量超出本发明的范围，所配制的产品对动物晶状体吸收有很大影响，会导致活性物质无法有效进入晶状体(对比例5)。
以下通过实验例证明本发明的有益效果。
实验例1、SD大鼠眼吸收试验(LAN)
健康成年SD大鼠6只，SPF级，180～220g，均为雄性，呼吸麻醉后对每只大鼠双眼分别滴眼给予受试制剂(实施例1)20μL(浓度0.1mg/mL)，滴眼后1.5小时动物安乐死后，立即采集晶状体及玻璃体，保存于-80℃待测，用LC/MS/MS方法检测晶状体和玻璃体药品内LAN的含量。
动物样品处理如下：
动物玻璃体样品匀浆后，取10μL，加入40μL70％甲醇，超声2min，涡旋1min后，加入甲醇175μL，涡旋混合2min，在4℃转速12000rpm离心10min，取上清液用于液相色谱-串联质谱(LC-MS/MS)分析。
动物晶状体样品匀浆(生理盐水按1:4加入晶状体，匀浆)后，取50μL，加入甲醇175μL，涡旋混合2min，4℃转速12000rpm离心10min，取上清液用于LC-MS/MS分析。
LC-MS/MS测试条件如下，LC-20AD高效液相色谱系统(SHIMADZU)-API4000三重四极杆质谱仪(Applied Biosystems公司)，配置Fortis Pace C18 5UM 2.1X30mm色谱柱，柱温40℃，流动相为甲醇：水(95:5)，流速为0.4mL/min，进样体积为10μL；质谱仪选用大气压化学电离源(Atomsperic Pressure Chemical Ionization,APCI)。质谱条件如下表所示：
Figure PCTCN2022116432-appb-000005
Figure PCTCN2022116432-appb-000006
检测结果如表2所示：
表2大鼠滴眼后晶状体和玻璃体内羊毛甾醇含量(Mean±SD)
Figure PCTCN2022116432-appb-000007
大鼠本底值检测，取2只SD大鼠(4只眼)，动物安乐死并迅速取晶状体，按同样方法处理样品并检测LAN的含量。检测结果：羊毛甾醇含量：2.23±0.86(μg/mL)。可见，本发明的眼用制剂，通过滴眼给药的方式可以有效输送羊毛甾醇透过晶状体屏障，并在晶状体富集。
上述结果说明，本发明的眼用制剂，通过滴眼给药的方式可以有效输送羊毛甾醇透过晶状体屏障，并在晶状体富集，几乎不会留于玻璃体中。
实验例2、新西兰兔眼吸收试验(LAN)
健康成年新西兰兔，SPF级，2-2.5kg，全雄性，共7只，选取6只兔子进行受试物(实施例3)滴眼给药，均为单次给药50μL，剩余1只兔子双眼均不给药，做为本底对照；给药后在1.5小时安乐处死动物，采集房水、晶状体及玻璃体，并检测房水、晶状体及玻璃体内药物含量，样品处理及LC/MS/MS检测方法同大鼠眼吸收试验，检测结果列入表3：
表3兔滴眼后晶状体、房水和玻璃体内羊毛甾醇含量(Mean±RSD)
Figure PCTCN2022116432-appb-000008
Figure PCTCN2022116432-appb-000009
注：BLOQ:低于检测限(LOQ＝0.001μg/mL)，未检测到。
上述结果进一步证实了本发明的眼用制剂，通过滴眼给药的方式，可以有效输送羊毛甾醇透过晶状体屏障，在晶状体富集，并且不在玻璃体、房水中富集。
实验例3、SD大鼠眼吸收试验(25-HC)
健康成年SD大鼠6只，SPF级，180～220g，均为雄性，呼吸麻醉后对每只大鼠双眼分别滴眼给予受试制剂(实施例15)20μL(浓度0.1mg/mL)，每个时间点安乐死后，立即采集晶状体及玻璃体，检测晶状体及玻璃体内药物含量。由于动物晶状体内本就几乎不含25-HC，故不设置对照眼。
具体而言，大鼠呼吸麻醉后，各组动物双眼各滴制剂受试物(实施例15)20μL(浓度0.1mg/mL)，给药后1.5h，用二氧化碳安乐死3只动物，迅速采集双眼晶状体，保存于-80℃待测。样品处理加等体积0.10mM的乙酸银甲醇溶液，其它处理及测试条件同实验例2。
Figure PCTCN2022116432-appb-000010
检测结果如表4所示：
表4大鼠滴眼后晶状体25-羟基胆甾醇(25-HC)的含量
Figure PCTCN2022116432-appb-000011
在房水和玻璃体样品中未检测到25-羟基胆甾醇(低于检测限，LCQ＝0.001μg/mL)。上述结果证实了本发明的眼用制剂，通过滴眼给药的方式，可以有效输送25-羟基胆甾醇透过晶状体屏障，在晶状体富集。
实验例4、本发明部分制剂的动物吸收利用率
滴眼剂滴眼后，只有约10％药物进入内眼，其余大部分经过结膜和鼻腔进入身体体系(凌沛学主编《眼科药物与制剂学》，中国轻工出版社，2010，P6)，因此，按照如下标准计算本发明制剂的利用率：
对于以大鼠或兔为实验动物的实施例而言，总吸收率为：A％＝[(C-C 0)V 晶状体/V 给药]*100％，有效吸收率为A％/10％。
其中，C是滴眼给药后晶状体中活性物质的浓度，C 0是未滴眼给药的眼晶状体中活性物质的浓度(μg/mL)。大鼠晶状体：直径＝3.87mm，V 晶状体＝0.03cm 3；
兔眼晶状体：直径＝7.9mm，V 晶状体＝0.258cm 3；
实施例1、3、8的LAN浓度为0.1mg/mL，即0.1μg/μL；滴眼剂给药量：大鼠：V 给药＝20uL*0.1μg/μL＝2μg；兔：V 给药＝50μL*0.1μg/μL＝5μg。
因此，计算得到实施例1、3、8的制剂总吸收率和有效吸收率结果如表5所示：
表5本发明部分制剂的动物吸收率
Figure PCTCN2022116432-appb-000012
可见，本发明制剂在活性物质浓度较低的基础上，还能够取得非常高的晶状体吸收率，兔晶状体有效吸收率甚至高达100％。说明本发明制剂的利用率非常高，把治疗白内障的活性物质定向递送到晶状体里，有效地增加了晶状体里氧化胆甾醇的浓度，治疗白内障，又避免了全身吸收和避免了毒副作用。
实验例5、白内障犬给药观察试验
贵宾犬(年龄15岁，雄性，体重4.0公斤)，将犬保持在室内居家环境，定时定量投放犬食和饮水，在实验期间完全作为宠物对待。每日用实施例3的制剂滴眼1次，每次一滴约30μL。滴眼给药20天后，犬的自主活动范围明显增大；拍照比较可观察到在20天后白内障的部分消退(图3)。
实验例6、大鼠药效试验
1.试验方法A
选取9日龄新生SD大鼠每日观察睁眼情况，当出现轻微睁眼时(约13日龄)，皮下注射亚硒酸钠溶液(Na 2SeO 3，20μmol/kg)，注射体积为2ml/kg，隔一天再注射一次同样剂量的亚硒酸钠溶液。首次注射亚硒酸钠溶液之后，动物随机分为二组，对照组和给药组，其中对照组每只鼠的双眼作对照，用生理盐水(NS)滴眼，给药组的双眼用受试药物(实施例3)滴眼。给药方案如表6：
表6给药方案
Figure PCTCN2022116432-appb-000013
2.白内障观察与等级评分
试验动物吸入异氟烷麻醉，利多卡因表面麻醉后观察大鼠眼内晶状体浑浊度，记录白内障形成时间，并按以下标准评分：
6级：表现为成熟白内障，累及整个晶状体。
5级：表现为不涉及晶状体皮质的核混浊。
4级：表现为部分核混浊。
3级：表现为弥漫性核混浊，伴有一些皮质散射。
2级：表现为轻微核混浊，在注射亚硒酸盐2到3天后，肿胀的纤维或后肩胛下混浊产生散射。
1级：是核混浊的初始迹象。
3.试验观察结果如表7
表7试验观察结果
Figure PCTCN2022116432-appb-000014
试验结果，在开始滴眼的第2天，模型对照组(滴生理盐水)动物的18只眼睛全部产生4级白内障(100％)；而给药组(滴受试药)有10/16只眼睛(62.5％)产生4级白内障；第6天，模型组66.7％的受试眼发生5级白内障，试验组受试眼发生5级白内障的比例为50％。
4、试验方法B
选取9日龄新生SD大鼠每日观察睁眼情况，当出现轻微睁眼时(约13日龄)，皮下注射亚硒酸钠溶液(Na 2SeO 3，20μmol/kg)，注射体积为2ml/kg。动物随机分为模型组和给药组，模型组动物双眼各给予10μL生理盐水(NS)滴眼，3次/天；给药组动物双眼各给予10μL受试药物(实施例3)滴眼，3次/天，给药方案参考表6。试验观察结果如表8所示：
表8试验观察结果
Figure PCTCN2022116432-appb-000015
给试验大鼠滴眼给药第15天(D15)，模型组大鼠眼白内障3级的发生率为68.75％，有1/16只眼发生白内障4级；给药组大鼠眼白内障3级的发生率为28.57％，无眼发生白内障4级。
上述结果证实，本发明眼用制剂通过滴眼的方式给药，活性物质能够有效在晶状体里富集，具有预防白内障、延缓白内障发展进程和改善白内障的作用。
综上，本发明提供了一种滴眼给药防治白内障的眼用制剂，本发明眼用制剂稳定性优异，在滴眼给药后，活性物质富集在试验动物晶状体，发挥治疗、防治白内障的效果。而且，晶状体周围的房水和玻璃体没有检测到活性物质，避免了系统毒副作用，解决了眼科用药递送领域人们一直渴望解决但始终未能获得成功的技术难题，在具有极高的临床应用价值。

Claims (25)

  1. 一种滴眼给药的眼用制剂，其特征在于，它由治疗眼病的活性物质和药学上可接受的载体或辅料组成；
    所述治疗眼病的活性物质为氧化型胆甾醇，所述氧化型胆甾醇包括羊毛甾醇或25-羟基胆甾醇；
    所述药学上可接受的载体或辅料含有如下成分：表面活性剂、增粘剂、助溶剂和溶剂；
    所述制剂中氧化型胆甾醇含量为0.01～5mg/mL；表面活性剂、增粘剂、助溶剂和氧化型胆甾醇的质量比为(1～300):(1～100):(100～3000):1，余量为溶剂。
  2. 根据权利要求1所述的制剂，其特征在于，所述制剂中氧化型胆甾醇含量为0.01～2mg/mL。
  3. 根据权利要求2所述的制剂，其特征在于，所述氧化型胆甾醇含量为0.05～0.5mg/mL，或0.01～0.2mg/mL。
  4. 根据权利要求1所述的制剂，其特征在于，所述制剂中氧化型胆甾醇含量为：0.01mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.25mg/mL、0.3mg/mL、0.35mg/mL、0.4mg/mL、0.45mg/mL、0.5mg/mL、0.55mg/mL、0.6mg/mL、0.65mg/mL、0.7mg/mL、0.75mg/mL、0.8mg/mL、0.85mg/mL、0.9mg/mL、0.95mg/mL、1mg/mL、1.5mg/mL或2mg/mL、2.5mg/mL、3mg/mL、3.5mg/mL、4mg/mL、4.5mg/mL或5mg/mL。
  5. 根据权利要求1所述的眼用制剂，其特征在于，所述表面活性剂、增粘剂、助溶剂和氧化型胆甾醇的质量比为(6.7～250):(11～50):(100～2500):1，优选为(25～200):(11～48):(200～2500):1，更优选为25:12:(200～600):1。
  6. 根据权利要求根据权利要求1～5任一项所述的制剂，其特征在于，所述表面活性剂为非离子表面活性剂。
  7. 根据权利要求6所述的制剂，其特征在于，所述非离子表面活性剂为聚山梨酯、泊洛沙姆或烷基葡萄糖苷。
  8. 根据权利要求1～5任一项所述的制剂，其特征在于，所述增粘剂为如下高分子化合物中的至少两种的组合：羟丙基甲基纤维素、羟丙基纤维素、甲基纤维素、聚维酮、卡波姆、聚乙二醇、泊洛沙姆、聚乙烯醇、羟乙基纤维素、黄原胶、透明质酸或其盐、海藻酸或其盐、羧甲基纤维素或其盐。
  9. 根据权利要求8所述的制剂，其特征在于，所述氧化型胆甾醇为25-羟基胆甾醇，所述增粘剂为如下高分子化合物中的至少两种的组合：羟丙基甲基纤维素、羟丙基纤维素、甲基纤维素、聚维酮、卡波姆、聚乙二醇、泊洛沙姆、聚乙烯醇、羟乙基纤维素、黄原胶、透明质酸或其盐、海藻酸或其盐、羧甲基纤维素或其盐。
  10. 根据权利要求8所述的制剂，其特征在于，所述氧化型胆甾醇为羊毛甾醇，所述增粘剂为如下高分子化合物中的至少两种的组合：羟丙基甲基 纤维素、羟丙基纤维素、甲基纤维素、聚维酮、卡波姆、聚乙二醇、泊洛沙姆、聚乙烯醇、羟乙基纤维素。
  11. 根据权利要求8～10任一项所述的制剂，其特征在于，所述增粘剂为所述高分子化合物中的两种的组合，两种高分子化合物的重量比为1:(0.1～10)，优选为1:(0.6～5)，更优选为1:1。
  12. 根据权利要求1～5任一项所述的制剂，其特征在于：所述药学上可接受的载体或辅料中的溶剂为极性溶剂，优选为水。
  13. 根据权利要求1～5任一项所述的制剂，其特征在于，所述药学上可接受的载体或辅料中的助溶剂选自液态聚乙二醇、丙二醇、丙三醇、聚氧乙烯蓖麻油、聚氧乙烯氢化蓖麻油、蓖麻油聚氧乙烯醚中的至少一种。
  14. 根据权利要求1～13任一项所述的制剂，其特征在于，它包括如下组分；
    治疗眼病的活性物质：羊毛甾醇，其含量为0.01～0.2mg/mL；
    表面活性剂：聚山梨酯或泊洛沙姆，其含量为羊毛甾醇的6.7～250倍；
    增粘剂：其含量为羊毛甾醇的11～50倍，所述增粘剂为聚维酮和羟丙基纤维素的组合，羟丙基纤维素和聚维酮的重量比为1:(1～1.2)；或，所述增粘剂为聚维酮和羟丙基甲基纤维素的组合；聚维酮和羟丙基甲基纤维素的重量比为1:(1～1.5)；或，所述增粘剂为聚维酮和卡波姆的组合，聚维酮和卡波姆的重量比为1:1；或所述增粘剂为聚维酮和聚乙二醇的组合，聚乙二醇和聚维酮的重量比为1:5；
    助溶剂：液态聚乙二醇、丙二醇、丙三醇、聚氧乙烯氢化蓖麻油或蓖麻油聚氧乙烯醚，其含量为羊毛甾醇的100～2500倍；溶剂为水。
  15. 如权利要求14所述的制剂，其特征在于，所述表面活性剂含量为羊毛甾醇的25～200倍；所述增粘剂的含量为羊毛甾醇的11～48倍；所述助溶剂的含量为羊毛甾醇的200～2500倍。
  16. 根据权利要求1～13任一项所述的制剂，其特征在于，它包括如下组分；
    治疗眼病的活性物质：25-羟基胆甾醇，其含量为0.1mg/mL；
    表面活性剂：聚山梨酯，其含量为25-羟基胆甾醇的25～250倍；
    增粘剂：其含量为25-羟基胆甾醇的12倍，所述增粘剂为聚维酮和羟丙基纤维素的组合，羟丙基纤维素和聚维酮的重量比为1:1；或，所述增粘剂为聚维酮和羟丙基甲基纤维素的组合；聚维酮和羟丙基甲基纤维素的重量比为1:1；
    助溶剂：液态聚乙二醇或丙三醇，其含量为25-羟基胆甾醇的100～1750倍；溶剂为水。
  17. 如权利要求16所述的制剂，其特征在于，所述表面活性剂含量为25-羟基胆甾醇的25倍；所述增粘剂的含量为羊毛甾醇的12倍；所述助溶剂的含量为羊毛甾醇的300倍。
  18. 根据权利要求1～17任一项所述的制剂，其特征在于，所述制剂中药学上可接受的载体或辅料还含有渗透压调节剂、pH调节剂、防腐剂中的任意一种或多种；
    所述渗透压调节剂为葡萄糖、氯化钠、氯化钾、甘露醇、山梨醇、枸橼酸钠、枸橼酸钾和甘油中的任意一种或多种；
    所述pH调节剂为盐酸、氢氧化钠、醋酸或其盐、柠檬酸或其盐、富马酸、琥珀酸、山梨酸、磷酸、磷酸二氢钠、磷酸氢二钠、硼酸、硼砂、酒石酸或其盐中的任意一种或多种；
    所述防腐剂为山梨酸、三氯叔丁醇、亚氯酸钠、过硼酸钠、季铵盐类(包括苯扎氯铵、苯扎溴铵、聚季銨盐-1、溴化十六烷基三甲铵)、羟苯酯类(包括羟苯甲酯、羟苯乙酯、羟苯丙酯)、硝酸苯汞中的任意一种或多种；优选地，所述季铵盐类包括苯扎氯铵、苯扎溴铵、聚季铵盐-1和/或溴化十六烷基三甲铵，所述羟苯酯类包括羟苯甲酯、羟苯乙酯和/或羟苯丙酯。
  19. 根据权利要求1～18任一项所述的制剂，其特征在于，所述眼用制剂含有纳米小体的结构，所述纳米小体由眼用制剂的载体或辅料的成分自组装形成；所述纳米小体中包含有治疗眼病的活性物质。
  20. 根据权利要求19所述的制剂，其特征在于，所述纳米小体为球形，其粒径为5～900nm，优选为5～50nm和/或200～700nm。
  21. 一种制备权利要求1～20任一项所述的制剂的方法，其特征在于，包括以下步骤：
    (1)向溶剂中加入表面活性剂、增粘剂，混合得到混合液；
    (2)向步骤(1)得到的混合液中加入治疗眼病的活性物质，加入或不加助溶剂，分散混合得到初悬液；
    (3)将步骤(2)得到的初悬液进行搅拌分散和/或均质分散，即得。
  22. 根据权利要求21所述的方法，其特征在于，步骤(2)中所述分散选自机械搅拌分散、磁力搅拌分散、涡旋振摇分散、剪切分散、均质分散、研磨分散、超声分散中的至少一种。
  23. 权利要求1～20任意一项所述的制剂在制备防治人或动物晶状体疾病的药物中的用途。
  24. 根据权利要求23所述的用途，其特征在于，所述药物是防治白内障的药物，优选为减少晶状体蛋白聚集、减少晶状体浑浊的药物。
  25. 根据权利要求23所述的用途，其特征在于，所述药物是眼部给药的药物制剂，优选为眼局部给药的药物。
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