Ophthalmic preparation for preventing and / or treating cataracts by eye drop administration

Abstract

An ophthalmic preparation for preventing and treating cataracts by instillation comprises an active substance for treating an ocular disease and a pharma- ceutically acceptable carrier or auxiliary material, the active substance for treating an ocular disease being an oxysterol including lanosterol or 25-hydroxycholesterol, the pharma-ceutically acceptable carrier or auxiliary material containing a surfactant, a thickener, a cosolvent and a solvent, the content of the oxysterol in the preparation is 0.01-5 mg / mL, the mass ratio of the surfactant, the thickener, the cosolvent and the oxysterol is (1-300):(1-100):(100-3000):1, and the remainder is the solvent. The preparation, after instillation, can selectively concentrate the active substance in the lens of an experimental animal, thereby accurately exerting a therapeutic effect on cataracts, and furthermore, since the active substance is not distributed in the aqueous humor or vitreous body, toxic side effects on other ocular tissues and the whole body can be avoided. This technology solves a technical problem that has long been desired to be solved in the field of ophthalmic drug delivery, and has extremely high clinical and social value.

Description

[Technical field]

[0001] The present invention is in the field of ophthalmic medications, and in particular relates to ophthalmic formulations for preventing and / or treating cataracts by eye drop administration. [Background technology]

[0002] Cataracts are caused by the gradual aging and clouding of the crystalline lens due to aging and other factors, which can affect vision by blocking the entry of light into the eye and can even lead to blindness. Cataracts are currently the most common blinding disease in the world, and according to a report by the World Health Organization (WHO), the rate of blindness due to cataracts is more than 40%, and the proportion of people over 50 years old who are blind due to cataracts is about 47.8%, accounting for 90% of blindness in developed countries. There are many cataract patients, and currently there is no reliable drug therapy to prevent or treat cataracts instead of surgery, and surgery is currently the only way to effectively treat cataracts (Jingjie Xu et al: Advances in pharmacotherapy of cataracts, Ann Transl Med 2020; 8(22): 1552).

[0003] In the course of investigating the development and progression of cataracts, researchers have found that oxysterols, including 25-hydroxycholesterol (25-HC, CAS#:2140-46-7) and lanosterol (LAN, CAS#:79-63-0), have the effect of reversing existing cataracts 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 α-crystallin partially restores transparency in cataract models, Science 350 (6261), 674-677, 2015).

[0004] The function of the crystalline lens is to transmit and focus light on the retina, and it is one of the refractive media of the eye. The crystallins in the crystalline lens contain three types of crystallins (α-, β-, and γ-crystallins) that are regularly arranged, and the alternating structure between crystallin molecules is the basis for determining the transparency and refractive index of the crystalline lens. The basis of the pathology of cataracts is abnormal folding and aggregation of crystallins, which changes the interactions between crystallins and reduces the fluidity and stability of crystallins, and the aggregated proteins cloud and make the crystalline lens opaque, preventing light from entering the eye, leading to the development of cataracts.

[0005] Lanosterol and 25-hydroxycholesterol can inhibit the polymerization of crystallins, improve the morphology of the lens, and restore transparency. Gestwicki et al. reported that oxysterols, including 25-hydroxycholesterol, can enhance the protective activity of α-crystallins and remove cataracts. Kang Zhang's team also reported that lanosterol can bind to β- and γ-crystallins, inhibit the polymerization of crystallins, and remove cataracts (Ling Zhao et. al., Nature 2015; Makley et. al., Science 2015).

[0006] LAN is an important intermediate in the cyclization reaction in the cholesterol biosynthesis pathway in the body, and is synthesized by lanosterol synthase (LSS). 25-hydroxycholesterol is also obtained by cholesterol synthesis by 25-hydroxycholesterol enzyme in the body. In pathological conditions, the concentration of these metabolic intermediates in the lens is not sufficient to effectively reverse cataracts, and active substances need to be supplemented. Oral administration is the most common method of supplementation, but since these active substances are mostly involved in biochemical processes throughout the body by being supplemented throughout the body, the intermediates that reach the lens are still insufficient to reach effective concentrations. In addition, the physiological activity of cholesterols is wide, so unpredictable pathophysiological reactions may occur when exogenous oxysterols are introduced by systemic administration. For example, lanosterol is a key intermediate in the endogenous biosynthesis of cholesterol, and 25-hydroxycholesterol is an oxidation product of cholesterol that is closely related to inflammation or infection, but it is also a liver X receptor (LXR) agonist, and ingestion of LXR agonists can cause hepatic lipogenesis and hypertriglyceridemia (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); Wu Tong, Du Hongjun, Research and progress of liver X receptors in ophthalmic diseases, New Advances in Ophthalmology, 39(9), 886-897, 2019).

[0007] Therefore, to prevent and treat cataracts using such active substances, it is optimal to deliver them locally to the eye.

[0008] However, the surface of the cornea at the front of the eye is covered with a tear film, and the cornea itself is composed of lipid, aqueous and mucin layers, so that in order for drug molecules to enter the anterior chamber and reach the lens through the pupil, they must penetrate the aqueous matrix layer of the cornea from the outside to the inside, and then pass through the lipid layer. In order for drugs to penetrate the natural anatomical, physiological and biochemical barriers of tissues to achieve the goal of preventing and treating cataracts, the technical challenges of conventional eye drops must be overcome (Thrimawithana, TR et al., Drug delivery to the lens for the management of cataracts, Advanced Drug Delivery Reviews(2018), 126, 185-194).

[0009] To date, ocular injections have been commonly used to deliver oxysterols to the lens across the corneal barrier. For example, researchers have injected nanoparticles prepared from lanosterol, polycaprolactone, lecithin, and phospholipid polyethylene glycol carboxyl (DSPE-PEG-COOH) into the vitreous cavity of cataract model animals (injected once every three days) and administered a lanosterol solution (15% cyclodextrin and 18% ethanol) at a concentration of 25 mM six times a week for six consecutive weeks, resulting in the disappearance of cataracts. However, when administered alone to experimental animals, it failed to treat cataracts (Ling Zhao et al., Nature, 2015; Kang Zhang and Shenyang Hou, US2017 / 0065617 A1). This shows that in the prior art, in order to overcome the ocular barrier, administration had to be carried out by an invasive method such as injection, and even if a lanosterol solution was administered directly into the eye, the drug could not smoothly pass through the barriers of the cornea and lens to enter the lens and exert its therapeutic effect.

[0010] However, active substances in the aqueous humor and / or vitreous body still cannot enter the lens and exert their effect unless they pass through the lens capsule. Many previous studies have shown that even if oxysterol active substances are directly injected into the vitreous body, they still have difficulty entering the lens and exerting their therapeutic effect due to the barrier effect of the lens capsule.

[0011] For example, when researchers injected a lanosterol thermogel prepared from poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) into the vitreous cavity of experimental rabbits (at a concentration of 400 mg / g), intravitreal LAN concentrations were maintained at >50 ng / 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). Nagai et al. injected lanosterol nanoparticles (2 mg / mL, once every two days) into the vitreous cavity of experimental rats for six consecutive weeks, but were unable to stop the progression of lens opacification (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).

[0012] In addition, in vitro experiments have further demonstrated that it is difficult for oxysterols to penetrate the lens barrier and enter the lens to exert their therapeutic effects. For example, Shanmugam et al. immersed the cataract nucleus obtained after cataract extraction in a lanosterol solution with a concentration of 25 mM for 6 days, but did not observe any significant improvement in the transparency of the cataract (Shanmugam et al., Effect of lanosterol on human cataract nucleus, Indian J Ophthalmol. 63(12): 888-8902015). In addition, Daszynski et al. immersed rat lenses in a 15 mM LAN liposome solution for 48 hours, or immersed lens fragments from cataract patients in lanosterol (0.20 mM lanosterol) or 25-hydroxycholesterol buffer (concentrations: 0.25 mM and 0.50 mM, 37°C, 72 hours), but in neither case was it possible to confirm that lanosterol or 25-hydroxycholesterol binds to crystallin to eliminate cataracts (Daszynski, et. al., Failure of oxysterols such as Lanosterol to Restore Lens Clarity from Cataracts, Scientific Reports (2019) 9: 8459, 14 pages).

[0013] It can be seen from this that even if the corneal barrier is penetrated by the invasive method of intravitreal injection, it can only increase the concentration of the drug in fundus tissues such as the retina, choroid and vitreous body, but still cannot effectively enter the crystalline lens for action. If intracrystalline injection is directly used, it may induce traumatic or secondary cataracts, and therefore cannot be practiced in clinical practice.

[0014] As described above, oxysterols can effectively prevent and treat cataracts in the lens, but currently there is no administration method that can be implemented in ophthalmic clinical practice to safely deliver drugs to the lens. Moreover, because the biological activity of oxysterols is broad, exogenous oxysterols must be avoided from entering the body, and the safety of these drugs cannot be ensured when administered by conventional systems such as oral, injection, and implant. The ideal administration method would be to deliver drugs to therapeutic concentrations in the lens by non-invasive eye drop administration, using a mechanism different from the delivery mechanism of conventional eye drop administration. Therefore, inventing an ophthalmic formulation that can safely and effectively deliver drugs to the lens by eye drop administration is a difficult problem that must be solved as soon as possible in the field of ophthalmic formulations.

[0015] However, the difficulty is not limited to the drug delivery part as mentioned above, but there is also a solubility problem when preparing a formulation with oxysterol as the active substance. Potential active substances such as oxysterols (e.g., lanosterol, etc.) belong to tetracyclic triterpenoids and are soluble in chloroform, ethanol, ether, n-propanol, isopropanol, N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), but have low solubility in water (Li and Forciniti, J. Chem. Eng. Data, 2020, 65, 2,. 436-445). Therefore, it is common to use organic solvents to increase solubility, but excessive use of organic solvents can irritate the eyes and pose a safety risk. In addition to organic solvents, researchers have used cyclodextrin or its derivatives as auxiliary materials (including cyclo-dextrins, CYD, α-cyclodextrin, β-cyclodextrin, and hydroxypropyl-β-cyclodextrin) and used inclusion technology to incorporate oxysterol molecules into the cyclodextrin cavity, then prepared the solution and used it for animal studies (Nguyen, CN108472303 A, 2016; JD Sciamanna, US2020 / 0360403A1). However, CYD inclusion compounds cannot penetrate biological membranes, and β-cyclodextrin forms a complex with cholesterol and is insoluble in the body, so it may accumulate in the kidneys and cause serious nephrotoxicity (RC Rowe, PJ Sheskey, PJ Weller, Handbook of Medicinal Supplements [M]. Beijing: Chemical Industry Press, 2005). Therefore, further development and application of CYD inclusion compounds are limited, and currently, only dog ​​products are sold on the Internet (trade name: Lanomax (registered trademark)).

[0016] Therefore, researching ophthalmic preparations that can solve the problem of drug solubility and penetrate the barriers of the cornea and lens, and realize the treatment of cataracts by eye drop administration, has very important clinical value and social significance. Summary of the Invention

[0017] An object of the present invention is to provide an ophthalmic preparation capable of delivering an active substance for treating an eye disease, such as an oxysterol substance, to the crystalline lens by eye drop administration and used for the treatment and prevention of cataracts.

[0018] The present invention provides an ophthalmic formulation for ophthalmic administration, the formulation comprising an active substance for treating an ophthalmic disease and a pharma- ceutically acceptable carrier or auxiliary material, the active agent for treating an eye disease is an oxysterol, including lanosterol or 25-hydroxycholesterol; The pharma- ceutically acceptable carrier or auxiliary material may include surfactants, thickeners, cosolvents and solvents; The content of oxysterol in the formulation is 0.01-5 mg / mL, the mass ratio of surfactant, thickener, cosolvent and oxysterol is (1-300):(1-100):(10-3000):1, and the remainder is solvent.

[0019] Furthermore, the content of oxysterol in the preparation is 0.01 to 2 mg / mL.

[0020] Furthermore, the content of oxysterol in the preparation is 0.05 to 0.5 mg / mL.

[0021] Further, the content of oxysterol in the formulation is 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, 1.5 mg / mL or 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL.

[0022] Further, the mass ratio of the surfactant, thickener, cosolvent and oxysterol is (6.7-250):(11-50):(100-2500):1, preferably (25-200):(11-48):(200-2500):1, and more preferably 25:12:(200-600):1.

[0023] Further, the surfactant is a non-ionic surfactant.

[0024] Further, the non-ionic surfactant is a polysorbate, a poloxamer, or an alkyl glucoside.

[0025] Furthermore, the thickening agent is a combination of at least two of the polymer compounds hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, povidone, carbomer, polyethylene glycol, poloxamer, polyvinyl alcohol, hydroxyethylcellulose, xanthan gum, hyaluronic acid or a salt thereof, alginic acid or a salt thereof, and carboxymethylcellulose or a salt thereof.

[0026] Furthermore, the thickener is a combination of two of the polymer compounds, and the weight ratio of the two polymer compounds is 1:(0.1 to 10), preferably 1:(0.6 to 5); Furthermore, the weight ratio of the two polymer compounds is 1:1.

[0027] Furthermore, the thickening agent is a combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose to povidone is 1:(1-2), preferably 1:(1-1.2). Or, the thickener is a combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone to hydroxypropyl methylcellulose is 1:(1-3), preferably 1:(1-1.5). Or, the thickener is a combination of povidone and carbomer, and the weight ratio of povidone to carbomer is 1:(0.5-2), preferably 1:1; Alternatively, the thickening agent is a combination of povidone and polyethylene glycol, and the weight ratio of povidone to polyethylene glycol is 1:(1 to 8), preferably 1:5.

[0028] Furthermore, the oxysterol is 25-hydroxycholesterol, and the thickener is a combination of at least two of the polymer compounds hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, povidone, carbomer, polyethylene glycol, poloxamer, polyvinyl alcohol, hydroxyethylcellulose, xanthan gum, hyaluronic acid or a salt thereof, alginic acid or a salt thereof, and carboxymethylcellulose or a salt thereof.

[0029] Furthermore, the oxysterol is 25-hydroxycholesterol, the thickener is a combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose to povidone is 1:(1-2), preferably 1:(1-1.2). Alternatively, the thickening agent is a combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone to hydroxypropyl methylcellulose is 1:(1 to 3), preferably 1:(1 to 1.5).

[0030] Furthermore, the oxysterol is lanosterol, and the thickener is a combination of at least two of the polymer compounds hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, povidone, carbomer, polyethylene glycol, poloxamer, polyvinyl alcohol, and hydroxyethylcellulose.

[0031] Furthermore, the oxysterol is lanosterol, the thickener is a combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose to povidone is 1:(1-2), preferably 1:(1-1.2). Or, the thickener is a combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone to hydroxypropyl methylcellulose is 1:(1-3), preferably 1:(1-1.5). Or, the thickener is a combination of povidone and carbomer, and the weight ratio of povidone to carbomer is 1:(0.5-2), preferably 1:1; Alternatively, the thickening agent is a combination of povidone and polyethylene glycol, and the weight ratio of povidone to polyethylene glycol is 1:(1 to 8), preferably 1:5.

[0032] Furthermore, the solvent in said pharma- ceutically acceptable carrier or auxiliary material is a polar solvent, preferably water.

[0033] Furthermore, the co-solvent in the pharma- ceutically acceptable carrier or auxiliary material is at least one selected from liquid polyethylene glycol, propylene glycol, glycerol, polyoxyethylene hydrogenated castor oil or castor oil polyoxyethylene ether, preferably liquid polyethylene glycol.

[0034] The formulation further comprises an active agent for treating an eye disease, a surfactant, a viscosity enhancer, and a co-solvent; The active substance for treating an eye disease is lanosterol, and the content thereof is 0.01 to 0.2 mg / mL; The surfactant is a polysorbate or a poloxamer, and the content thereof is 6.7 to 250 times that of lanosterol, the content of the thickening agent is 11 to 50 times that of lanosterol, the thickening agent is a combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose to povidone is 1: (1 to 1.2), or the thickening agent is a combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone to hydroxypropyl methylcellulose is 1: (1 to 1.5), or the thickening agent is a combination of povidone and carbomer, and the weight ratio of povidone to carbomer is 1:1, or the thickening agent is a combination of povidone and polyethylene glycol, and the weight ratio of polyethylene glycol to povidone is 1:5, The co-solvent is liquid polyethylene glycol, propylene glycol, glycerol, polyoxyethylene hydrogenated castor oil or castor oil polyoxyethylene ether, and its content is 100 to 2500 times that of lanosterol, and the solvent is water.

[0035] Alternatively, the formulation comprises an active substance for treating an eye disease, a surfactant, a viscosity enhancer, and a co-solvent as ingredients, The active substance for treating eye diseases is 25-hydroxycholesterol, and its content is 0.1 mg / mL; The surfactant is a polysorbate, and its content is 25 to 250 times that of 25-hydroxycholesterol; The content of the thickening agent is 12 times that of 25-hydroxycholesterol, and the thickening agent is a combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose to povidone is 1:1, or the thickening agent is a combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone to hydroxypropyl methylcellulose is 1:1; The co-solvent is liquid polyethylene glycol or glycerol, the content of which is 100 to 1750 times that of 25-hydroxycholesterol, and the solvent is water.

[0036] In addition, the pharma- ceutically acceptable carrier or auxiliary material of the formulation may further comprise any one or more of an osmolality adjusting agent, a pH adjusting agent, and a preservative; the osmolality adjusting agent is any one or more of glucose, sodium chloride, potassium chloride, mannitol, sorbitol, sodium citrate, potassium citrate, and glycerin; The pH adjuster is any one or more of hydrochloric acid, sodium hydroxide, acetic acid or a salt thereof, citric acid or a salt thereof, fumaric acid, succinic acid, sorbic acid, phosphoric acid, disodium dihydrogen phosphate, disodium hydrogen phosphate, boric acid, borax, and tartaric acid or a salt thereof; The preservative is any one or more of sorbic acid, chlorobutanol, sodium chlorite, sodium perborate, quaternary ammonium salts (including benzalkonium chloride, benzalkonium bromide, polyquaternium-1, cetyltrimethylammonium bromide), hydroxybenzene esters (including methylparaben, ethylparaben, propylparaben), phenylmercuric nitrate, preferably the quaternary ammonium salts include benzalkonium chloride, benzalkonium bromide, polyquaternium-1 and / or cetyltrimethylammonium bromide, and the hydroxybenzene esters include methylparaben, ethylparaben and / or propylparaben.

[0037] Further, the ophthalmic formulation comprises a nanoparticle structure formed by self-assembly of components of a carrier or auxiliary material of the ophthalmic formulation, the nanoparticle comprising an active substance for treating an ophthalmic disease.

[0038] Furthermore, the nanoparticles are spherical and have a particle size of 5 to 900 nm, preferably 5 to 50 nm and / or 200 to 700 nm.

[0039] The present invention also provides a method for preparing the above formulation, the method comprising the steps of: (1) adding a surfactant and a thickener to a solvent and mixing them to obtain a mixed solution; (2) adding an active substance for treating an eye disease to the mixture obtained in step (1) and dispersing and mixing with or without the addition of a co-solvent to obtain an initial suspension; and step (3) dispersing the initial suspension obtained in step (2) by stirring and / or homogenizing.

[0040] Furthermore, the dispersion in step (2) is at least one selected from dispersion by mechanical stirring, dispersion by magnetic stirring, dispersion by vortex shaking, dispersion by shearing, dispersion by homogenization, dispersion by pulverization, and dispersion by ultrasonic waves.

[0041] The present invention further provides a use of the formulation in the preparation of a medicament for preventing or treating a lens disease in a human or animal.

[0042] Furthermore, the drug is a drug for preventing and treating cataracts, and is preferably a drug for reducing lens protein aggregation and reducing lens opacity.

[0043] Furthermore, the drug is a drug formulation for ocular administration, preferably a drug for local administration to the eye.

[0044] Oxysterols include 25-hydroxycholesterol (25-HC) and lanosterol (LAN), which can interact with lens proteins in the lens, thereby inhibiting the polymerization of crystallin, improving the morphology of the lens and restoring its transparency, thus having the effect of reversing cataracts that have already developed and restoring the transparency of the lens, thus achieving the purpose of treating and preventing cataracts. Oxysterols administered systemically by oral, intramuscular, intravenous, etc., cannot reach an effective therapeutic concentration in the lens because they are involved in the biochemical processes of the whole body. In addition, LAN and 25-HC injected into the vitreous body cannot penetrate the lens capsule and enter the lens. In addition, lens injections cause traumatic or secondary cataracts.

[0045] Although eye drops are a safe and convenient administration method, in order to treat ocular fundus diseases, it is necessary to allow the drug to pass through the ocular barrier and deliver the drug to the site of the ocular fundus lesion safely and effectively.

[0046] The present invention is based on the principle of optimally designing the therapeutic effect vs. risk ratio, and minimizes the risk while achieving the therapeutic effect. Only about 10% of the eye drops remain on the ocular surface due to mechanical actions such as blinking, and most of them are discharged from the lacrimal canaliculus together with tears, and a small portion enters the nasal cavity through the nasolacrimal duct, and is further absorbed through the nasal cavity and enters the blood circulation. The lower the concentration of the formulation, the smaller the risk to tissues and organs, and the fewer toxic side effects throughout the body. The concentration of lanosterol in the formulation of the present invention (not exceeding 4.68 mM) is lower than the concentration claimed by other patents, and can reduce toxic side effects as much as possible. At the same time, when 20 μL of this formulation is dropped into the conjunctival sac at a concentration of 0.1 μg / μL, about 0.2 μg actually remains on the ocular surface, and the volume of the rat lens is 0.03 cm. 3 Based on this calculation (diameter = 3.87 mm), the LAN concentration in the lens after instillation reached 2.3 to 2.5 times the original content, and the transmittance reached 44 to 52%. In the case of rabbits, 50 uL of the formulation of the present invention was dropped at a concentration of 0.11 μg / μL, and assuming that 10% remained on the ocular surface, the lens of the rabbit (diameter = 7.9 mm, volume = 0.258 cm) 3), the amount of lanosterol entering the lens reached 3.6 times the original concentration, and the transmittance reached nearly 100% (AB 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, (copyright) Springer Science+Business Media, LLC 2013, Ophthalmic Drugs and Formulations supervised by Junshun Gaku, China Light Industry Press, 2010, P6). As can be seen, although the concentration of oxysterol used in the present invention is low, the utilization rate is very high, which not only reduces the potential risk of systemic toxic side effects, but also ensures the dosage and efficacy.

[0047] Another feature of the formulation of the present invention is the advantage of the formulation.Because the treatment of cataract is a long-term medication, it is particularly important to avoid active auxiliary materials in the formulation, for example, the formulation with low concentration of anti-infective activity may cause drug resistance of ocular surface and nasolacrimal flora, and even induce infection.In one aspect, the present invention has low concentration, and in another aspect, the formulation does not require the use of preservatives, and there is no risk of complications in long-term use.

[0048] Oxysterols have low water solubility, and it has been reported in the literature that solvents such as ethanol and DMSO are commonly used to prepare injections or eye drops of oxysterols. The amount of alcohol-based solvents used must be controlled because they are irritating to the eye. When DMSO is used as a co-solvent, the solubility of the active substance can be increased, but there is a risk that a larger amount of the active substance will enter the systemic circulation. For example, when a 0.5% LAN solution prepared with 15% DMSO and 20% ethanol or polysorbate 80 solution is injected into the vitreous of rats, it not only causes vitreous opacification, but also has the risk of promoting lens opacification.

[0049] Lens proteins are highly ordered and densely arranged, and have high transparency, so injection may damage the structure and cause traumatic cataract. It is difficult to deliver LAN to the lens using an injection route (Nguyen, CN108472303 A, 2016; Cheng Qinyuan, Jiapaer Zeyidan, 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).

[0050] As a result of many experiments, the inventors of the present invention have found that the present invention uses a nonionic surfactant as a solubilizer and / or a thickener and / or a co-solvent in combination, and processes the solution by applying technical means such as mixing by physical stirring, dispersion by high-speed shearing, and high-pressure homogenization, and if necessary, heat or ultrasonic treatment, to prepare a solution of oxysterol LAN or 25-HC mainly in water, which has very good stability. The prepared aqueous solution containing oxysterol was measured by a laser particle sizer, and the main particle size distribution was in the range of 5-50 nm and / or 200-700 nm, and spherical particles were observed by observation under an electron microscope. The water-soluble solvent prepared by the present invention is mixed with the tear fluid, and then the oxysterol passes through the cornea and the lens capsule to enter the lens. In a lens absorption test using eye drops administered to live animals, a water-based eye drop containing oxysterols prepared according to the present invention was administered to experimental animals, and a significant increase in the concentration of these oxysterols was detected in the lens, but the content of oxysterols in the aqueous humor and vitreous body was low (rat experiments) or not detected at all (rabbit experiments).

[0051] The inventors of the present invention observed that the eye drops prepared according to the present invention can delay the onset and progression of cataracts in experimental animals in a rat cataract model test in which sodium selenite solution was subcutaneously injected.When the LAN eye drops prepared according to the present invention were used on an elderly dog ​​(15 years old) suffering from cataracts, it was observed that the opacity of the dog's lens had obviously disappeared 20 days after administration.

[0052] Tests have demonstrated that the ophthalmic preparation of the present invention, after instillation, can deliver active substances highly efficiently and accurately into the lenses of experimental animals, and can act as a chaperone for lens proteins, inhibiting the polymerization of crystallin, removing cataracts, and exerting the effects of treating and preventing cataracts.

[0053] It was unexpectedly found in animal experiments that the oxysterol eye drops prepared according to the present invention have the advantages of being safe and effective, in particular, it was unexpectedly discovered that after instillation into the eyes of experimental animals, oxysterols were concentrated in the lens (LAN was concentrated in the lens in an eye drop absorption test in New Zealand rabbits), while the concentrations in the aqueous humor and vitreous were very low, indicating that the eye drops of the present invention have high target tissue selectivity.

[0054] The eye drops of the present invention can selectively and highly efficiently deliver oxysterol active substances to the lens by instillation, thereby providing supplementation in cataracts caused by oxysterol deficiency and avoiding the occurrence of unpredictable pathophysiological reactions in the body caused by cholesterols that have a wide range of physiological activities.

[0055] Oxysterols in the lens can inhibit the polymerization of crystallin and remove cataracts, but oxysterols can cause cardiovascular complications in the body and are harmful to the body. So far, there is no technology in the pharmaceutical field that delivers drugs containing oxysterols to the lens in a non-invasive drug delivery manner. The new low-concentration eye drops of the present invention realize the targeted delivery of drugs into the lens, and its most obvious clinical advantage is that oxysterols are delivered to the lens by eye drops, and the active substance content of the invented cataract eye drops is kept low and the bioavailability after eye drops is very high, which can effectively increase the concentration of oxysterols in the lens to treat cataracts. The low concentration and high local bioavailability in the eye greatly reduce the possibility of drugs entering the body through the conjunctiva, nasal cavity, etc. and causing toxic side effects.

[0056] The active substances for treating eye diseases referred to in the present invention are in vivo or non-in vivo active substances for treating eye diseases in humans or animals.

[0057] The nanoparticles referred to in the present invention are nano-level spherical aggregates formed by self-organization of components of drug carriers or auxiliary materials in a solvent.

[0058] A solvent as referred to in the present invention is a liquid capable of dissolving the components of the drug carrier or auxiliary material.

[0059] The surfactant referred to in this invention is a substance that can significantly reduce the surface tension of a liquid. The nonionic surfactant referred to in this invention is a surfactant that does not dissociate in water.

[0060] The eye drop administration described in the present invention is an administration method in which a drug solution is dropped into the eye, and belongs to the mucosal administration route.

[0061] The liquid polyethylene glycol (liquid PEG) according to the present invention is a polyethylene glycol that is liquid at room temperature and pressure, and preferably has a weight-average molecular weight of 1,000 or less.

[0062] Naturally, based on the above content of the present invention, various other forms of modifications, substitutions or changes can be made in light of general technical knowledge and conventional means in this field, on the premise that they do not deviate from the above basic technical idea of ​​the present invention.

[0063] The above content of the present invention will be described in more detail below by way of specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention. Any technology realized based on the above content of the present invention belongs to the scope of the present invention. [Brief description of the drawings]

[0064] [Figure 1] FIG. 1 is a transmission electron microscope image of the sample obtained in Example 3. [Diagram 2] FIG. 2 is a transmission electron microscope image of the sample obtained in Example 3 after staining with a staining agent. [Diagram 3] FIG. 3 is a set of photographs showing the opacification of the lens of a dog with cataract before and after instillation of the ophthalmic preparation of the present invention. [Figure 4] FIG. 4 is a particle size distribution diagram of the sample obtained in Example 5 (0 days). [Diagram 5] FIG. 5 is a particle size distribution diagram of the sample obtained in Example 5 (at room temperature for 15 days). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] The reagents or equipment used in the present invention can be obtained by purchasing commercial products, and unless specific conditions are specified, they are used according to conventional conditions or conditions recommended by the manufacturer. Lanosterol (CAS: 79-63-0), content: 99.9% (HPLC, Chengdu Puteid Biotechnology Co., Ltd., Chengdu). 25-Hydroxycholesterol (CAS: 2140-46-7, content ≧98%, Shanghai McKinlin Biochemical Technology Co., Ltd., Shanghai).

[0066] Some of the equipment and facilities are as follows:

[0067] ES225SM-DR(E) Electronic Analytical Balance, Preciasa (Switzerland) DF-101S heat collecting type constant temperature heating magnetic stirrer, Yingyu High Technology Equipment Factory, Gunyi City (Henan, China) WH-2 Micro Vortex Mixer, Shanghai Luxi Analytical Instrument Factory Co., Ltd. (Shanghai, China).

[0068] Disperser: T25 Easy Clean Digital, IKA (Germany) KQ-500 type ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd. (Kunshan, China) AH-NANO Plus high pressure homogenizer, Antao Sina Rice Technology (Suzhou) Co., Ltd. (China) Mettler Toledo FE20 pH meter, Mettler Toledo (Switzerland) NS-90 nanoparticle size analyzer, Zhuhai Oumei Equipment Co., Ltd. (Zhuhai, China) Agilent 1100 HPLC high performance liquid chromatography, Agilent Technologies (USA) API 4000 triple quadrupole mass spectrometer (Applied Biosystems, USA) STY-1A osmotic pressure meter, Tianjin Tianda Tianfa Science and Technology Co., Ltd. (Tianjin, China) Zetasizer Nano ZS, nano particle size and zeta potential analyzer, Malvern, UK.

[0069] The properties of the formulation of the present invention were measured as follows.

[0070] Particle size measurement method 1 mL of the sample prepared in the Examples or Comparative Examples was transferred to a sample cell, the measurement temperature was set to 40° C., the sample cell was set in the NS-90 nanoparticle size analyzer, and the measurement was started. Each sample was measured three times, and the average of the three measurement results was taken and shown as the measurement results, particle size (light intensity distribution, and %), and polydispersity index (PdI, Polydispersity Index) of the sample.

[0071] Osmolality Measurement Method The freezing point depression of the solution was measured, and the osmolarity was measured. Operation: Cleaning the probe of the STY-1A osmometer: Put 100 μL of distilled water into three sample tubes, preheat the instrument, and then screw the sample tube containing 100 μL of distilled water onto the instrument probe, select "clean" for three times, click "clean", and repeat three times. Measurement: Enter sample information in the instrument information table and click "test". Transfer 100 μL of sample to the sample tube with a pipette gun, lightly screw it onto the instrument, and click "start" for measurement. The measurement was repeated three times, and the average value of the three measurement results was used as the measurement result. When actually conducting animal experiments, if the osmotic pressure did not reach isotonicity, the above osmotic regulator was used to adjust it to isotonicity or close to isotonicity.

[0072] How to measure pH value The FE20 pH meter was calibrated with each pH buffer solution (pH 4.00, 6.86, and 9.18, respectively), the electrode was washed with pure water, excess water was absorbed with fiberless paper, and the electrode was immersed in the liquid sample to be measured, and the measurement was started by pressing the read button. The data obtained after the reading stabilized was taken as the pH value of the sample.

[0073] The solutions obtained in the examples were measured, and if the pH was <5 or >9, it was necessary to adjust the pH to 6-8 with an acid or alkali. Common pH adjusters are NaOH and HCl, phosphoric acid and phosphates (e.g., sodium dihydrogen phosphate, disodium hydrogen phosphate), citric acid and citrates (e.g., sodium citrate), boric acid and borax. When actually conducting animal experiments, if the pH value of the liquid obtained by measurement did not meet the requirements for an ophthalmic preparation, it was adjusted with the pH adjusters mentioned above.

[0074] Example 1 Preparation of Ophthalmic Formulations of the Invention The materials and their proportions used are shown in Table 1, and the manufacturing process is as follows. First, polysorbate 80, PVP K12, HPMC, and liquid PEG (PEG400) were weighed and placed in a 100mL polypropylene centrifuge tube, and an appropriate amount of water for injection was added and stirred for 30 minutes. Then, 6.0mg of lanosterol was added, and water for injection was added until the volume reached 60mL. After stirring and mixing for 10 minutes, a mixture was obtained, and the mixture was dispersed for 3 minutes at a rotation speed of 12000-15000 rpm using a dispersing machine. After the machine was stopped and the bubbles disappeared, the dispersion was transferred to a high-pressure homogenizer, the temperature was controlled at 5±5℃, and the mixture was homogenized for 2 minutes at a pressure of about 400 Bar, then the pressure was increased to 1200-1400 Bar and homogenized for 20 minutes, then the pressure was reduced to 500 Bar and homogenized for 2 minutes, and then the mixture was discharged. After the bubbles disappeared naturally, a transparent homogenized liquid was obtained. The pH value and osmolality were measured, sodium citrate (0.10 g) and sodium chloride (0.4 g) were added, and the pH was adjusted to 7.0 with 0.1 N HCl or 0.1 N NaOH, and the osmolality was 302 mOsmol / kg. The solution was filtered under reduced pressure through a filter membrane to obtain the product as a solution.

[0075] HPLC product concentration measurement: Measurement equipment: Agilent 1100 high performance liquid chromatography.

[0076] Chromatography conditions: Agilent ZORBAX Eclipse Plus C18, 4.6×100mm 3.5μm column: flow rate 1.0mL / min, measurement wavelength 205nm, mobile phase: MeOH (60%)-acetonitrile (40%) isocratic elution. After diluting the sample 5 times with the mobile phase, 10μL was taken and injected into the liquid chromatograph. HPLC content measurement result: 0.098mg / mL.

[0077] Particle size measurement results (main particle size and its distribution ratio): particle size 67.7 nm (46.2%) and 75.8 nm (38.9%), PdI (Polydispersity Index): 0.276.

[0078] Average zeta potential measurement: -5.03 ± 4.33 mV (25°C).

[0079] The product was stored at 40°C for 30 days away from light with no significant change in appearance. Particle size: 40.68 nm (88.8%), PDI: 0.249, HPLC content: 0.093 mg / mL.

[0080] Results of animal eye drop absorption test: Three rats (6 eyes) were administered 20μL per eye in both eyes, and 1.5 hours later, the rats were euthanized and the vitreous and lens were quickly removed to measure the LAN content. Results: Lanosterol content in the lens: 5.16±1.90(μg / mL), in the vitreous: 0.045±0.091(μg / mL).

[0081] Example 2 Preparation of Ophthalmic Formulations of the Invention The materials and proportions used are shown in Table 1, and the preparation process was the same as in Example 1 to obtain a solution.

[0082] Particle size measurement result: Particle size 441.8nm (100.0%), PdI: 0.190.

[0083] HPLC content measurement: Measurement equipment: Agilent 1100 high performance liquid chromatography.

[0084] Chromatography conditions: Agilent ZORBAX Eclipse Plus C18, 4.6 × 100 mm 3.5 μm column: flow rate 0.8 mL / min, measurement wavelength 205 nm, mobile phase: MeOH (85%)-0.1% H 3 PO 4 (15%) isocratic elution. After diluting the sample 5 times with the mobile phase, 10μL was taken and injected into the liquid chromatograph. HPLC content measurement result: 0.095mg / mL.

[0085] The product was stored at 40°C for 15 days away from light with no significant change in appearance. Particle size: 413.2 nm (94.5%), PdI: 0.214, HPLC content: 0.090 mg / mL.

[0086] Example 3 Preparation of Ophthalmic Formulations of the Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0087] Particle size measurement results: Particle size 416.3nm (100.0%), PdI: 0.214, HPLC content: 0.078mg / mL.

[0088] The product was stored at 2-8°C for 30 days away from light, and no significant changes in appearance were observed. Particle size: 478.5 nm (98.2%), PdI: 0.245, HPLC content: 0.077 mg / mL.

[0089] Results of the absorption test by instillation into the lens of animals: Two New Zealand rabbits (4 eyes) were prepared, 50 μL per eye was instilled, and 1.5 hours later, the rabbits were euthanized and the aqueous humor, vitreous body, and lens were quickly removed and the LAN content was measured. Measurement results: No lanosterol was detected in the aqueous humor and vitreous body (below the detection limit, LCQ≦0.002 μg / mL). The lanosterol content in the lens was 2.72 ± 0.16 (μg / mL). In the blank background control animal (1 New Zealand rabbit, 2 eyes, no drug administration), the lanosterol content in the lens was 0.76 ± 0.01 (μg / mL).

[0090] Example 4 Preparation of Ophthalmic Formulations of the Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0091] Particle size measurement result: Particle size 531.6 nm (100.0%), PdI: 0.165. HPLC content: 0.077mg / mL.

[0092] The product was left at room temperature for 15 days away from light with no significant change in appearance. Particle size: 935.1 nm (88.2%), PdI: 0.340, HPLC content: 0.074 mg / mL.

[0093] Example 5 Preparation of Ophthalmic Formulations of the Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0094] Particle size measurement result: Particle size 401.3nm (98.8%), PDI: 0.221. HPLC content: 0.063mg / mL.

[0095] The product was left at room temperature for 15 days away from light with no significant change in appearance. Particle size: 613.2 nm (100.0%), PdI: 0.350, HPLC content: 0.064 mg / mL.

[0096] Results of the animal lens absorption test using eye drops: Three rats (6 eyes) were prepared, 20μL was administered per eye, and 1.5 hours later, the rats were euthanized and the lenses were quickly removed and the lanosterol content was measured. Measurement results: The lanosterol content in the lenses was 2.89±0.60(μg / mL).

[0097] Example 6 Preparation of Ophthalmic Formulations of the Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0098] Particle size measurement result: Particle size 10.7nm (99.1%), PDI: 0.224. HPLC content: 0.070mg / mL.

[0099] The product was left at room temperature for 15 days away from light with no significant change in appearance: particle size: 11.2 nm (91.7%), PdI: 0.246, HPLC content: 0.069 mg / mL.

[0100] Results of the animal lens absorption test: Three rats (6 eyes) were prepared, 20μL was instilled per eye, and 1.5 hours later, the rats were euthanized and the lenses were quickly removed and the lanosterol content was measured. Measurement results: The lanosterol content in the lenses was 4.83±2.15(μg / mL).

[0101] Example 7 Preparation of Ophthalmic Formulations of the Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0102] Particle size measurement result: Particle size 472.5nm (99.4%), PDI: 0.205. HPLC content: 0.072mg / mL.

[0103] The product was stored at 2-8°C for 15 days away from light, with no significant change in appearance. Particle size: 513.2 nm (97.1%), PdI: 0.235, HPLC content: 0.070 mg / mL.

[0104] Example 8 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0105] Particle size measurement result: Particle size 508.5nm (97.7%), PDI: 0.245. HPLC content: 0.080mg / mL.

[0106] The product was stored at 40°C for 15 days away from light with no significant change in appearance. Particle size: 423.5 nm (90.1%), PdI: 0.223, HPLC content: 0.078 mg / mL.

[0107] Results of animal lens absorption test: Three rats (6 eyes) were prepared, 20μL was instilled per eye, 1.5 hours later the rats were euthanized and the lenses were quickly removed and the lanosterol content was measured. Measurement results: The lanosterol content in the lenses was 5.68±1.60(μg / mL).

[0108] Example 9 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0109] Particle size measurement result: Particle size 572.2nm (85.3%), PDI: 0.529. HPLC content: 0.085mg / mL.

[0110] The product was stored at 2-8°C for 15 days away from light, with no significant change in appearance. Particle size: 583.8 nm (80.2%), PdI: 0.545, HPLC content: 0.083 mg / mL.

[0111] Results of the animal lens absorption test: Three rats (6 eyes) were prepared, 20μL was instilled per eye, and 1.5 hours later, the rats were euthanized and the lenses were quickly removed and the lanosterol content was measured. Measurement results: The lanosterol content in the lenses was 4.01±1.65(μg / mL).

[0112] Example 10 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 2, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0113] Particle size measurement result: Particle size 12.8nm (99.0%), PDI: 0.175. HPLC content: 0.073mg / mL.

[0114] The product was stored at 40°C for 15 days away from light with no significant change in appearance. Particle size: 13.2 nm (91.5%), PdI: 0.222, HPLC content: 0.072 mg / mL.

[0115] Example 11 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1. The preparation process was the same as in Example 1, and the content measurement was the same as in Example 2 to obtain a solution.

[0116] Particle size measurement result: Particle size 455.6nm (99.7%), PdI: 0.249. HPLC content: 0.051mg / mL.

[0117] The product was stored at 40°C for 15 days away from light with no significant change in appearance. Particle size: 216.8 nm (95.6%), PdI: 0.351, HPLC content: 0.049 mg / mL.

[0118] Example 12 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0119] Particle size measurement results: Particle size 420.0nm (100.0%), PdI: 0.205. HPLC content: 0.037mg / mL.

[0120] The product was stored at 40°C for 30 days away from light, with no significant change in appearance. Particle size: 452.0 nm (94.0%), PdI: 0.256, HPLC content: 0.033 mg / mL.

[0121] Example 13 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1. The preparation process was the same as in Example 1, and the content measurement was the same as in Example 2 to obtain a solution.

[0122] Particle size measurement result: Particle size 474.3nm (98.7%), PdI: 0.229. HPLC content: 0.089mg / mL.

[0123] The product was stored at 40°C for 15 days away from light with no significant change in appearance. Particle size: 425.2 nm (88.6%), PdI: 0.245, HPLC content: 0.087 mg / mL.

[0124] Example 14 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0125] Particle size measurement result: Particle size 136.6nm (86.3%), PdI: 0.542. HPLC content: 0.009mg / mL.

[0126] The product was stored at 2-8°C for 15 days away from light, with no significant change in appearance. Particle size: 214.2 nm (98.6%), PdI: 0.232, HPLC content: 0.008 mg / mL.

[0127] Example 15 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1. The preparation process was the same as in Example 1, and the content measurement was the same as in Example 2 to obtain a solution.

[0128] Particle size measurement results: Particle size 397.2 nm (90.1%), PdI: 0.649. HPLC content: 0.015mg / mL.

[0129] The product was stored at 40°C for 15 days away from light with no significant change in appearance. Particle size: 345.6 nm (85.6%), PdI: 0.431, HPLC content: 0.014 mg / mL.

[0130] Example 16 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0131] Particle size measurement result: Particle size 13.2 nm (98.3%), PdI: 0.235. HPLC content: 0.071mg / mL.

[0132] The product was stored at 2-8°C for 15 days away from light, with no significant change in appearance. Particle size: 13.8 nm (93.6%), PdI: 0.316, HPLC content: 0.069 mg / mL.

[0133] Example 17 Preparation of Ophthalmic Formulations of the Present Invention The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0134] Particle size measurement result: Particle size 527.5nm (99.4%), PdI: 0.240. HPLC content: 0.058mg / mL.

[0135] The product was stored at 40°C for 15 days away from light, with no significant change in appearance. Particle size: 161.3 nm (100.0%), PdI: 0.505, HPLC content: 0.052 mg / mL.

[0136] Comparative Example 1 The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0137] Particle size measurement result: Particle size 16.86nm (69.0%), PdI: 1.000. HPLC content: 0.051mg / mL.

[0138] After standing overnight at room temperature, a precipitate formed. This formulation has poor stability.

[0139] Comparative Example 2 The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution containing phlox-like suspended matter.

[0140] Particle size measurement results: particle size 369.8 nm (82.8%), PdI: 0.554, HPLC content of the supernatant of the example: 0.025 mg / mL.

[0141] The product was left out of the light for 15 days at 40°C, whereupon a precipitation occurred with a phlox-like suspended solid: particle size 462.1 nm (80.2%), PdI 0.979, HPLC content of the supernatant of the example: 0.027 mg / mL.

[0142] Comparative Example 3 The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution containing phlox-like suspended matter.

[0143] Particle size measurement results: particle size 313.7 nm (74.9%), PdI: 0.587, HPLC content of the supernatant of the example: 0.029 mg / mL.

[0144] The product was left out of the light for 15 days at 40°C, whereupon a precipitation occurred with a floxed suspended solid. Particle size: 161.4 nm (100.0%), PdI: 0.231, HPLC content of the supernatant: 0.023 mg / mL, low content of active substance.

[0145] Comparative Example 4 The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution containing phlox-like suspended matter. The product was left at room temperature for 15 days away from light, and the phlox-like suspended matter aggregated and precipitated.

[0146] Comparative Example 5 The materials and proportions used are shown in Table 1, and the preparation process and content measurement were the same as in Example 1 to obtain a solution.

[0147] Particle size measurement results: particle size 292.2nm (72.6%), PdI: 1.000, HPLC content: 0.029mg / mL.

[0148] The product was stored at 40°C for 15 days away from light, with no significant change in appearance. Particle size: 188.3 nm (100.0%), PdI: 0.217, HPLC content: 0.026 mg / mL, and the content of active substance was reduced.

[0149] Results of animal lens absorption test: Two rats (4 eyes) were prepared, and 20 μL was instilled per eye. After 1.5 hours, the rats were euthanized and the lenses were quickly removed and the LAN content was measured. Measurement results: The lanosterol content in the lens was 2.00 ± 0.41 (μg / mL). This was almost the same level as the blank control eye, indicating that the sample of this comparative example could not effectively enter the animal lens.

[0150] Table 1. Information on the amounts used in the examples and comparative examples TIFF2024533923000001.tif252170TIFF2024533923000002.tif66170Notes: 1.HPMC: Hydroxypropyl methylcellulose. 2.PVP: Povidone. 3.HPC: Hydroxypropyl cellulose. 4.PEG: Polyethylene glycol average molecular weight ≦5000Da. 5.CMC-Na: Carboxymethylcellulose sodium salt. 6.PEG-60 hydrogenated castor oil: Polyoxyethylene hydrogenated castor oil.

[0151] From the results of the Examples and Comparative Examples, it can be seen that the ophthalmic solution system of the present invention contains at least one surfactant, two thickeners and an appropriate amount of co-solvent, and the prepared product is stable and has a high absorption rate by the lens of an animal.

[0152] When the ophthalmic solution system of the present invention contains one surfactant and none or only one thickening agent, or does not contain a surfactant, all of the products prepared have low stability (see Comparative Examples 2, 3 and 4).

[0153] When an ionic polymer was added to the formulation, the prepared product precipitated upon standing for a short period of time, and had poor stability (see Comparative Example 1).

[0154] If the amount of surfactant used is outside the range of the present invention, the prepared product may have a significant effect on the absorption by the animal's lens, and the active substance may not be able to effectively enter the lens (Comparative Example 5).

[0155] The beneficial effects of the present invention will now be demonstrated by experimental examples.

[0156] Experimental Example 1. Ocular absorption test in SD rats (LAN) Six healthy adult SD rats (SPF grade, 180-220g, all male) were given respiratory anesthesia, and then 20μL (concentration 0.1mg / mL) of the test formulation (Example 1) was instilled into both eyes of each rat. 1.5 hours after instillation, the rats were euthanized, and the lenses and vitreous bodies were immediately collected and stored at -80℃ until measurement. The LAN content in the lenses and vitreous bodies was measured by LC / MS / MS.

[0157] Animal samples were treated as follows.

[0158] After homogenizing the vitreous samples from the animals, 10 μL was taken, 40 μL of 70% methanol was added, and the samples were sonicated for 2 min and vortexed for 1 min. Then, 175 μL of methanol was added, vortexed for 2 min, and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was used for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis.

[0159] After homogenizing the animal lens samples (1:4 saline solution was added to the lens and homogenized), 50 μL was taken, 175 μL of methanol was added, vortex mixed for 2 minutes, centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was taken for LC-MS / MS analysis.

[0160] The measurement conditions for LC-MS / MS were as follows: LC-20AD high-performance liquid chromatography system (SHIMADZU)-API4000 triple quadrupole mass spectrometer (Applied Biosystems), Fortis Pace C18 5UM 2.1X30mm column, column temperature 40℃, mobile phase methanol:water (95:5), flow rate 0.4mL / min, injection volume 10μL, and atmospheric pressure chemical ionization (APCI) mass spectrometer. The mass analysis conditions are shown in the table below.

[0161] TIFF2024533923000003.tif103170The measurement results are shown in Table 2.

[0162] Table 2. Lanosterol content in the lens and vitreous of rats after instillation (Mean ± SD) TIFF2024533923000004.tif27170

[0163] The background value of the rats was measured, and two SD rats (four eyes) were taken and euthanized, and the lenses were quickly removed and sampled in the same manner to measure the LAN content. Measurement results: lanosterol content: 2.23±0.86 (μg / mL). As can be seen from the above, the ophthalmic preparation of the present invention can effectively transport lanosterol through the lens barrier and concentrate it in the lens by eye drop administration.

[0164] The above results indicate that the ophthalmic formulation of the present invention, when administered by eye drop administration, can effectively transport lanosterol to penetrate the lens barrier and concentrate it in the lens, with almost no residue remaining in the vitreous.

[0165] Experimental Example 2. New Zealand Rabbit Ocular Absorption Test (LAN) Six of the seven healthy adult New Zealand rabbits (SPF grade, 2-2.5 kg, all male) were selected and the test product (Example 3) was administered in a single dose of 50 μL each, while the remaining rabbit was not administered to either eye and served as a background control. The rabbits were euthanized 1.5 hours after administration, and the aqueous humor, lens and vitreous were collected, and the drug content in the aqueous humor, lens and vitreous was measured. The sample treatment and LC / MS / MS measurement methods were the same as those used in the rat ocular absorption test, and the measurement results are shown in Table 3.

[0166] Table 3. Lanosterol content in the lens, aqueous humor and vitreous of rabbits after instillation (Mean ± RSD) TIFF2024533923000005.tif38170Note: BLOQ: below limit of detection (LOQ=0.001μg / mL), not detected.

[0167] The above results further demonstrate that the ophthalmic formulation of the present invention, when administered by eye drop, can effectively transport lanosterol across the lens barrier and concentrate it in the lens, but not in the vitreous or aqueous humor.

[0168] Experimental Example 3. Ocular absorption test in SD rats (25-HC) Six healthy adult SD rats (SPF grade, 180-220g, all male) were given respiratory anesthesia, and then 20μL (concentration 0.1mg / mL) of the test formulation (Example 15) was instilled into both eyes of each rat. After euthanasia at each timing, the lens and vitreous were immediately collected and the drug content in the lens and vitreous was measured. Since the lens of animals originally contains almost no 25-HC, no control eye was prepared.

[0169] Specifically, after administering respiratory anesthesia to the rats, 20 μL (concentration 0.1 mg / mL) of the test formulation (Example 15) was dripped into both eyes of each group of animals, and 1.5 h after administration, three animals were euthanized with carbon dioxide, and the lenses of both eyes were promptly collected and stored at -80°C until measurement. For sample treatment, an equal volume of 0.10 mM silver acetate methanol solution was added, and other treatments and test conditions were the same as in Experimental Example 2.

[0170] TIFF2024533923000006.tif21170The measurement results are shown in Table 4.

[0171] Table 4. 25-hydroxycholesterol (25-HC) content in the lens of rats after instillation TIFF2024533923000007.tif23170

[0172] 25-hydroxycholesterol was not detected in the aqueous humor and vitreous samples (below detection limit, LCQ=0.001 μg / mL). The above results demonstrate that the ophthalmic formulation of the present invention can effectively transport 25-hydroxycholesterol through the lens barrier and concentrate it in the lens by eye drop administration.

[0173] Experimental Example 4. Absorption and utilization rate of some formulations of the present invention in animals After instillation of eye drops, only about 10% of the drug enters the inside of the eye, and the majority of the remaining enters the body via the conjunctiva and nasal cavity (Ophthalmic Drugs and Formulations, edited by Ling Pei Xue, China Light Industry Press, 2010, p. 6). Therefore, the utilization rate of the formulation of the present invention was calculated according to the following criteria:

[0174] In the examples using rats or rabbits as experimental animals, the total absorption rate was calculated as A% = [(CC 0 )V 水晶体 / V 投与 ]*100%, and the effective absorption rate was A% / 10%.

[0175] where C is the concentration of the active substance in the lens after instillation, and C 0 is the concentration of active substance in the lens of an untreated eye (μg / mL). Rat lens: diameter = 3.87 mm, V 水晶体 =0.03cm 3 .

[0176] Rabbit eye lens: diameter = 7.9mm, V 水晶体 =0.258cm 3 .

[0177] The LAN concentration in Examples 1, 3, and 8 was 0.1 mg / mL, i.e., 0.1 μg / μL. Eye drop dosage: Rat: V 投与 =20uL*0.1μg / μL=2μg. Rabbit: V 投与 =50μL*0.1μg / μL=5μg.

[0178] The total absorption rate and effective absorption rate calculated for the preparations of Examples 1, 3, and 8 are shown in Table 5.

[0179] Table 5. Absorption rate of some formulations of the present invention in animals TIFF2024533923000008.tif79170

[0180] As can be seen from the above, in addition to the low concentration of active substance, the preparation of the present invention can also achieve a very high lens absorption rate, and the effective absorption rate of rabbit lens can reach 100%, which indicates that the preparation of the present invention has a very high utilization rate, and can deliver the active substance for treating cataracts to the lens, thereby effectively increasing the concentration of oxysterols in the lens to treat cataracts, and also avoids systemic absorption and toxic side effects.

[0181] Experimental Example 5: Observational study of administration to dogs with cataracts A toy poodle (15 years old, male, weighing 4.0 kg) was used, and the dog was placed in a home environment indoors, given food and water at regular times and in fixed quantities, and treated as a pet during the experiment. The formulation of Example 3 was instilled into the eyes once a day, about 30 μL each time. 20 days after administration of the eye drops, the range of spontaneous movements of the dog was significantly expanded. By comparing the photographs, partial disappearance of the cataract was observed after 20 days (Figure 3).

[0182] Experimental Example 6: Efficacy test on rats 1. Test Method A Nine-day-old newborn SD rats were selected and observed daily for eye opening. When the eyes were slightly opened (approximately 13 days of age), sodium selenite solution (Na 2 SeO 3 , 20 μmol / kg) was injected subcutaneously at an injection volume of 2 ml / kg, and the same dose of sodium selenite solution was injected once every other day. After the first injection of sodium selenite solution, the animals were randomly divided into two groups, a control group and a treatment group, in which both eyes of each mouse were instilled with saline (NS) as a control, and the treatment group was instilled with the test drug (Example 3) in both eyes. The dosing schedule is shown in Table 6.

[0183] Table 6. Dosing regimen TIFF2024533923000009.tif39170

[0184] 2. Cataract observation and classification The test animals were anesthetized by inhaling isoflurane and then topically anesthetized with lidocaine. The degree of opacity of the lens in the rat's eye was then observed, and the time of cataract formation was recorded and classified according to the following criteria. Level 6: Manifests as a mature cataract affecting the entire lens. Level 5: Manifested as nuclear opacity without affecting the lens cortex. Level 4: Manifests as partial nuclear opacity. Level 3: appeared as diffuse nuclear opacity with cortical scattering. Level 2: Appears as minor nuclear opacity, and 2-3 days after selenite injection, the opacity becomes scattered due to swollen fibers or retroscapular. Level 1: Early signs of nuclear opacification.

[0185] 3. The test observation results are shown in Table 7.

[0186] Table 7. Test observation results TIFF2024533923000010.tif66170

[0187] The results showed that on the second day after the start of instillation, all 18 eyes (100%) of the animals in the model control group (saline instillation) developed level 4 cataracts, whereas 10 / 16 eyes (62.5%) of the treated group (test drug instillation) developed level 4 cataracts. On the sixth day, 66.7% of the test eyes in the model group developed level 5 cataracts, whereas the proportion of test eyes in the test group that developed level 5 cataracts was 50%.

[0188] 4. Test Method B Nine-day-old newborn SD rats were selected and observed daily for eye opening. When the eyes were slightly opened (approximately 13 days of age), sodium selenite solution (Na 2 SeO 3, 20 μmol / kg) was subcutaneously injected at an injection volume of 2 ml / kg. The animals were randomly divided into a model group and a treatment group, and 10 μL of saline (NS) was instilled into each of the two eyes of the animals in the model group three times a day, and 10 μL of the test drug (Example 3) was instilled into each of the two eyes of the animals in the treatment group three times a day. See Table 6 for the dosing schedule. The test results are shown in Table 8.

[0189] Table 8. Test Observation Results TIFF2024533923000011.tif44170

[0190] On the 15th day (D15) after the administration of eye drops to the test rats, the incidence of level 3 cataract in the eyes of the model group rats was 68.75%, and 1 / 16 eyes developed level 4 cataract. The incidence of level 3 cataract in the eyes of the treated group rats was 28.57%, and no eyes developed level 4 cataract.

[0191] From the above results, it was confirmed that the ophthalmic preparation of the present invention can be administered by eye drop, and can effectively concentrate the active substance in the lens, thereby preventing, delaying the progression of, and even improving cataracts.

[0192] As described above, the present invention provides an ophthalmic preparation for preventing and treating cataracts by instillation, and the ophthalmic preparation of the present invention has excellent stability, and after instillation, the active substance is concentrated in the lens of the test animal, and exerts the effect of treating and preventing cataracts. Furthermore, the active substance is not detected in the aqueous humor and vitreous body around the lens, avoiding toxic side effects of the system and solving the technical difficulties that have long been desired but have not been achieved in the field of ophthalmic drug delivery, and has great value in clinical application.

Claims

1. An ophthalmic preparation for ophthalmic administration, comprising an active substance for treating an eye disease and a pharmaceutically acceptable carrier or auxiliary material; the active agent for treating an eye disease is an oxysterol, including lanosterol or 25-hydroxycholesterol; The pharmaceutically acceptable carrier or auxiliary material comprises surfactants, thickeners, cosolvents and solvents, The formulation contains 0.01-5 mg / mL of oxysterol, and the weight ratio of surfactant, thickener, cosolvent, and oxysterol is (1-300):(1-100):(100-3000):1, with the remainder being solvent. A formulation characterized by:

2. 2. The formulation of claim 1, wherein the oxysterol content in the formulation is 0.01 to 2 mg / mL.

3. 3. The formulation according to claim 2, wherein the content of the oxysterol is 0.05-0.5 mg / mL or 0.01-0.2 mg / mL.

4. 2. The formulation of claim 1, wherein the oxysterol content in the formulation is 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, or 5 mg / mL.

5. 2. The ophthalmic preparation of claim 1, wherein the weight ratio of the surfactant, thickener, cosolvent and oxysterol is (6.7-250):(11-50):(100-2500):1, preferably (25-200):(11-48):(200-2500):1, and more preferably 25:12:(200-600):

1.

6. 2. The formulation of claim 1, wherein the surfactant is a non-ionic surfactant.

7. 7. The formulation according to claim 6, wherein the non-ionic surfactant is a polysorbate, a poloxamer or an alkylglucoside.

8. 2. The formulation according to claim 1, wherein the thickener is a combination of at least two of the polymer compounds hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, povidone, carbomer, polyethylene glycol, poloxamer, polyvinyl alcohol, hydroxyethylcellulose, xanthan gum, hyaluronic acid or a salt thereof, alginic acid or a salt thereof, and carboxymethylcellulose or a salt thereof.

9. 9. The formulation according to claim 8, wherein the oxysterol is 25-hydroxycholesterol, and the thickener is a combination of at least two of the polymer compounds hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, povidone, carbomer, polyethylene glycol, poloxamer, polyvinyl alcohol, hydroxyethylcellulose, xanthan gum, hyaluronic acid or a salt thereof, alginic acid or a salt thereof, and carboxymethylcellulose or a salt thereof.

10. 9. The formulation of claim 8, wherein the oxysterol is lanosterol, and the thickener is a combination of at least two of the polymer compounds hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, povidone, carbomer, polyethylene glycol, poloxamer, polyvinyl alcohol, and hydroxyethylcellulose.

11. The formulation according to claim 8, characterized in that the thickener 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), and more preferably 1:

1.

12. 2. The formulation according to claim 1, characterized in that the solvent in the pharmaceutically acceptable carrier or auxiliary material is a polar solvent, preferably water.

13. The formulation according to claim 1, characterized in that the co-solvent in the pharmaceutically acceptable carrier or auxiliary material is at least one selected from liquid polyethylene glycol, propylene glycol, glycerol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, and castor oil polyoxyethylene ether.

14. The composition comprises an active substance for treating an eye disease, a surfactant, a thickener, and a co-solvent, the active substance for treating eye diseases is lanosterol, and its content is 0.01 to 0.2 mg / mL; the surfactant is polysorbate or poloxamer, and its content is 6.7 to 250 times that of lanosterol; the content of the thickener is 11 to 50 times that of lanosterol, the thickener is a combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose to povidone is 1: (1 to 1.2), or the thickener is a combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone to hydroxypropyl methylcellulose is 1: (1 to 1.5), or the thickener is a combination of povidone and carbomer, and the weight ratio of povidone to carbomer is 1:1, or the thickener is a combination of povidone and polyethylene glycol, and the weight ratio of polyethylene glycol to povidone is 1:5, The co-solvent is liquid polyethylene glycol, propylene glycol, glycerol, polyoxyethylene hydrogenated castor oil or castor oil polyoxyethylene ether, and its content is 100 to 2500 times that of lanosterol, and the solvent is water. The formulation according to claim 1, characterized in that

15. The formulation according to claim 14, characterized in that the content of the surfactant is 25 to 200 times that of lanosterol, the content of the thickener is 11 to 48 times that of lanosterol, and the content of the co-solvent is 200 to 2500 times that of lanosterol.

16. The composition comprises an active substance for treating an eye disease, a surfactant, a thickener, and a co-solvent, the active substance for treating eye diseases is 25-hydroxycholesterol, and its content is 0.1 mg / mL; the surfactant is a polysorbate, and its content is 25 to 250 times that of 25-hydroxycholesterol; the content of the thickener is 12 times that of 25-hydroxycholesterol, the thickener is a combination of povidone and hydroxypropyl cellulose, and the weight ratio of hydroxypropyl cellulose to povidone is 1:1, or the thickener is a combination of povidone and hydroxypropyl methylcellulose, and the weight ratio of povidone to hydroxypropyl methylcellulose is 1:1; The co-solvent is liquid polyethylene glycol or glycerol, the content of which is 100 to 1750 times that of 25-hydroxycholesterol, and the solvent is water. The formulation according to claim 1, characterized in that

17. 17. The formulation of claim 16, wherein the surfactant content is 25 times that of 25-hydroxycholesterol, the viscosity increasing agent content is 12 times that of 25-hydroxycholesterol, and the co-solvent content is 300 times that of 25-hydroxycholesterol.

18. The pharmaceutically acceptable carrier or auxiliary material in the formulation further comprises any one or more of an osmolality adjusting agent, a pH adjusting agent, and a preservative; the osmolality adjusting agent is any one or more of glucose, sodium chloride, potassium chloride, mannitol, sorbitol, sodium citrate, potassium citrate, and glycerin; the pH adjuster is any one or more of hydrochloric acid, sodium hydroxide, acetic acid or a salt thereof, citric acid or a salt thereof, fumaric acid, succinic acid, sorbic acid, phosphoric acid, disodium dihydrogen phosphate, disodium hydrogen phosphate, boric acid, borax, and tartaric acid or a salt thereof; The preservative is any one or more of sorbic acid, chlorobutanol, sodium chlorite, sodium perborate, quaternary ammonium salts (including benzalkonium chloride, benzalkonium bromide, polyquaternium-1, and cetyltrimethylammonium bromide), hydroxybenzene esters (including methylparaben, ethylparaben, and propylparaben), and phenylmercuric nitrate, and preferably, the quaternary ammonium salts include benzalkonium chloride, benzalkonium bromide, polyquaternium-1, and / or cetyltrimethylammonium bromide, and the hydroxybenzene esters include methylparaben, ethylparaben, and / or propylparaben. The formulation according to claim 1, characterized in that

19. The formulation of claim 1, wherein the ophthalmic formulation comprises a nanoparticle structure formed by self-assembly of components of a carrier or auxiliary material of the ophthalmic formulation, the nanoparticles comprising an active substance for treating an ophthalmic disease.

20. 20. The formulation according to claim 19, characterized in that the nanoparticles are spherical and have a particle size of 5 to 900 nm, preferably 5 to 50 nm and / or 200 to 700 nm.

21. A method for preparing a formulation according to any one of claims 1 to 20, comprising the steps of: Step (1) of adding a surfactant and a thickener to a solvent and mixing them to obtain a mixed solution; Step (2) adding an active substance for treating an ophthalmic disease to the mixture obtained in step (1), and dispersing and mixing with or without a co-solvent to obtain an initial suspension; and step (3) of dispersing the initial suspension obtained in step (2) by stirring and / or homogenizing. A method characterized by:

22. 22. The method according to claim 21, wherein the dispersion in step (2) is at least one selected from dispersion by mechanical stirring, dispersion by magnetic stirring, dispersion by vortex shaking, dispersion by shearing, dispersion by homogenization, dispersion by pulverization, and dispersion by ultrasonic waves.

23. Use of the formulation according to any one of claims 1 to 20 in the preparation of a medicament for preventing or treating a lens disease in humans or animals.

24. The use according to claim 23, characterized in that the drug is a drug for preventing and treating cataracts, preferably a drug for reducing lens protein aggregation and reducing lens opacity.

25. 24. The use according to claim 23, characterized in that the drug is a drug formulation for ocular administration, preferably a drug for topical administration to the eye.

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