An ophthalmic preparation for preventing and / or treating cataract by eye drop administration

Abstract

The application provides an eye preparation for preventing and treating cataract by eye drop, which is composed of an active substance for treating eye diseases and a pharmaceutically acceptable carrier or excipient; the active substance for treating eye diseases is oxidized cholestanol, including lanosterol or 25-hydroxycholestanol; the pharmaceutically acceptable carrier or excipient contains a surfactant, a tackifier, a cosolvent and a solvent; the content of the oxidized cholestanol in the preparation is 0.01-5 mg / mL; the mass ratio of the surfactant, the tackifier, the cosolvent and the oxidized cholestanol is (1-300):(1-100):(100-3000):1, and the rest is the solvent. After the eye drop of the preparation, the active substance can be selectively enriched in the lens of experimental animals, so that the effect of treating cataract can be accurately exerted, and the active substance is not distributed in the aqueous humor and the vitreous body, thereby avoiding the toxic and side effects on other eye tissues and the whole body. The application solves the technical problem which people in the field of ophthalmic drug delivery have been eager to solve but have failed to obtain success, and has a very high clinical application value and social value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of ophthalmic drugs, and particularly relates to an ophthalmic preparation for preventing and / or treating cataract by eye drop administration. BACKGROUND

[0002] Cataract is a kind of disease that the lens gradually ages and becomes cloudy due to age and other reasons, which blocks the light from entering the eye and affects vision or even causes blindness. Cataract is the most common cause of blindness in the world. According to the report of the World Health Organization (WHO), the rate of cataract-induced blindness is more than 40%, and the proportion of people over 50 years old who are blind due to cataract is about 47.8%. In developed countries, cataract accounts for 90% of the rate of blindness. There are a large number of cataract patients, and there is currently no reliable drug treatment method to replace surgical prevention or treatment of cataract. Surgical treatment is the only effective method for treating cataract (Jingjie Xu et al: Advances in pharmacotherapy of cataracts, Ann Transl Med 2020; 8(22): 1552).

[0003] In the process of exploring the occurrence and development of cataract, researchers found that oxysterols including 25-hydroxycholesterol (25-HC, CAS#: 2140-46-7) and lanosterol (LAN, CAS#: 79-63-0) have the effect of reversing the already occurring cataract 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 lens is one of the refractive media of the eye, which transmits and focuses light onto the retina. The lens proteins include three types of crystallins, α-, β-, and γ-crystallins, which are regularly arranged, and the intermolecular structure between the crystallins is the basis for determining the transparency and refractive index of the lens. The pathological basis of cataract is the abnormal folding and aggregation of crystallins, which changes the interaction between crystallins, reduces the fluidity and stability of crystallins, and the aggregated proteins produce lens turbidity and opacity, so that light cannot enter the eye, resulting in cataract.

[0005] Lanosterol and 25-hydroxycholesterol can prevent the aggregation of crystallins, improve the lens morphology and restore transparency. Gestwicki et al. reported that oxidized cholesterols, including 25-hydroxycholesterol, can enhance the protective activity of α-crystallin and can clear cataracts. The team of Kang Zhang reported that lanosterol binds to β- and γ-crystallins, preventing the aggregation of crystallins, and can clear cataracts (Ling Zhao et. al., Nature 2015; Makley et. al., Science 2015).

[0006] LAN is a key cyclization reaction intermediate in the in vivo biosynthesis pathway of cholesterols, synthesized by Lanosterol synthase (LSS); 25-hydroxycholesterol is synthesized from cholesterols by 25-hydroxycholesterol synthase in vivo. In pathological conditions, the concentration of these in vivo metabolic intermediates in the lens is not sufficient to reach levels effective in reversing cataracts, and it is necessary to supplement the active substances. Oral supplementation is the most common method, but these active substances are supplemented systemically, and most participate in systemic biochemical processes, and the intermediates that reach the lens are still not sufficient to reach effective concentrations. In addition, since cholesterols have a wide range of physiological activities, introducing exogenous oxidized cholesterols in a systemic manner can produce unpredictable pathophysiological reactions. For example, lanosterol is a key intermediate in the biosynthesis of cholesterols in vivo, and 25-hydroxycholesterol is an oxidized product of cholesterols, which is closely related to inflammation or infection, and is also a liver X receptor (LXR) agonist, and the intake of LXR agonists can lead to liver fat synthesis 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 Progress of Liver X Receptor in Ophthalmic Diseases, Ophthalmic New Progress, 39(9), 886-897, 2019).

[0007] Therefore, ocular local delivery is the best way for this class of active substances to prevent and treat cataracts.

[0008] However, the corneal surface of the anterior segment of the eye is covered by a tear film, and the cornea itself is composed of a lipid layer, a water-like layer, and a mucin layer, from the outside to the inside, the drug molecules have to penetrate into the aqueous stroma layer of the cornea, and then penetrate through the lipid layer, to enter the anterior chamber, and then reach the lens through the pupil. To achieve the purpose of preventing and treating cataracts by overcoming 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 (2018), 126, 185-194).

[0009] So far, to overcome the corneal barrier and deliver oxidized cholesterols to the lens, ocular injection is usually used. For example, researchers injected nanoparticles prepared from lanosterol, polycaprolactone, lecithin and phospholipid polyethylene glycol carboxyl (DSPE-PEG-COOH) into the vitreous cavity of animal cataract models (once every 3 days), combined with eye drops, using a 25mM lanosterol solution (15% cyclodextrin and 18% ethanol) with a concentration of 25mM, 6 times a week, for 6 weeks, the cataract subsided; however, the experimental animals were given eye drops alone, and the cataract could not be treated (Ling Zhao et al., Nature, 2015; Kang Zhang and Shenyang Hou, US2017 / 0065617A1). It shows that in the prior art, to overcome the eye barrier, invasive administration such as injection must be used, and direct eye drops with lanosterol solution cannot make the drug pass through the cornea and lens barrier to enter the lens to play a therapeutic role.

[0010] However, the active substance in the aqueous humor or / and vitreous body still has to pass through the lens capsule membrane to enter the lens to play a role. Many existing studies have found that even if the oxidized cholesterols active substance is directly injected into the vitreous body, it is still difficult to enter the lens to play a curative effect because of the barrier effect of the lens capsule membrane.

[0011] For example, researchers injected experimental rabbits with lanosterol thermogel (Thermogel) prepared with poly(lactide-co-glycolide)-polyethylene glycol-poly(lactide-co-glycolide) (PLGA-PEG-PLGA) into the vitreous cavity (concentration 400 mg / g), and the intravitreal LAN concentration could be 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 experimental rats with lanosterol nanoparticle injection into the vitreous cavity (concentration 2 mg / mL, once every 2 days), for 6 weeks, which could not block the progress 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 further confirmed that oxidized cholesterol is difficult to break through the barrier of the lens to play a therapeutic role in the lens, for example, Shanmugam et al. soaked the cataract nucleus obtained after cataract surgery in a 25 mM lanosterol solution for 6 days, and the cataract clarity did not improve significantly (Shanmugam et al., Effect of lanosterol on human cataract nucleus, Indian J Ophthalmol. 63(12): 888-890, 2015); Daszynski et al. soaked rat lenses in 15 mM LAN liposome solution for 48 hours, or cataract patient lens fragments in lanosterol (0.20 mM lanosterol) or 25-hydroxycholesterol buffer (concentration: 0.25 mM and 0.50 mM, 37°C, 72 hours), and no lanosterol or 25-hydroxycholesterol was seen to bind to crystallins to solve the cataract problem (Daszynski, et. al., Failure of oxysterols such as Lanosterol to Restore Lens Clarity from Cataracts, Scientific Reports (2019) 9:8459, 14 pages).

[0013] Therefore, even if the invasive vitreous injection method is used to penetrate the corneal barrier, only a higher concentration of the drug can be obtained in the retinal, choroidal and vitreous fundus tissues, but it still cannot effectively enter the lens to play a role. If direct lens injection is used, it may induce traumatic or secondary cataracts, which cannot be implemented in clinical practice.

[0014] In summary, oxidized cholesterol can effectively prevent and treat cataracts in the lens, but there is no ophthalmic clinical drug delivery method to safely deliver it to the lens. And because they have a wide range of physiological activities, exogenous oxidized cholesterol must be avoided from entering the human body, and existing oral, injection, implantation and other systemic drug delivery cannot guarantee the safety of such drugs. Non-invasive eye drops are different from the delivery mechanism of traditional eye drops, and the drug reaches the therapeutic concentration in the lens, which is an ideal drug delivery method. Therefore, the invention can safely and effectively deliver drugs to the lens by eye drop administration, and the ophthalmic preparation is an urgent problem to be solved in the field of ophthalmic preparations.

[0015] However, in addition to the above-mentioned difficulties in drug delivery, there are also solubility problems in preparing preparations with oxidized cholestanol as the active substance. Potential active substances such as oxysterols (e.g., lanosterol) belong to tetracyclic triterpenoids, which are soluble in chloroform, ethanol, diethyl 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, currently, organic solvents are often used to improve solubility, but excessive use of organic solvents can irritate the eyes and pose potential safety hazards. In addition to organic solvents, some researchers have used cyclodextrins or their derivatives as excipients (cyclo-dextrins, CYD, including α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin) to prepare solutions for animal studies by using inclusion technology to package oxysterol molecules into cyclodextrin cavities (Engein, CN108472303 A, 2016; J. D. Sciamanna, US2020 / 0360403A1). However, CYD inclusions cannot penetrate biological membranes, and β-cyclodextrin complexes with cholestanol are not soluble in the body and accumulate in the kidneys, causing severe kidney toxicity (R.C. Luo, P.J. Sheski, P.J. Wheeler, Handbook of Pharmaceutical Excipients [M]. Beijing: Chemical Industry Press, 2005), which limits its further development and application. Currently, only dog products are sold on the Internet (trade name: ).

[0016] Therefore, it is of great clinical value and social significance to develop an eye preparation that can overcome the solubility problem of the drug and break through the corneal and lens barriers, allowing eye drops to deliver the active substance for treating cataracts. SUMMARY

[0017] The present application aims to provide an eye preparation that can deliver active substances for treating eye diseases, such as oxysterols, to the lens through eye drops, for treating and preventing cataracts.

[0018] The present application provides an eye preparation for eye drops, which is composed of an active substance for treating eye diseases and a pharmaceutically acceptable carrier or excipient;

[0019] The active substance for treating eye diseases is oxysterol, which includes lanosterol or 25-hydroxycholestanol.

[0020] The pharmaceutically acceptable carrier or excipient contains the following components: a surfactant, a tackifier, a cosolvent, and a solvent.

[0021] The content of the oxidized cholesterols in the preparation is 0.01-5 mg / mL; the mass ratio of the surfactant, the viscosity increasing agent, the co-solvent and the oxidized cholesterols is (1-300):(1-100):(10-3000):1, and the rest is solvent.

[0022] Further, the content of the oxidized cholesterols in the above preparation is 0.01-2 mg / mL.

[0023] Further, the content of the oxidized cholesterols in the above preparation is 0.05-0.5 mg / mL.

[0024] Further, the content of the oxidized cholesterols in the above preparation 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.

[0025] Further, the mass ratio of the surfactant, the viscosity increasing agent, the co-solvent and the oxidized cholesterols in the above preparation 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.

[0026] Further, the surfactant in the above preparation is a non-ionic surfactant.

[0027] Further, the non-ionic surfactant in the above preparation is polysorbate, poloxamer or alkyl glucoside.

[0028] Further, the viscosity increasing agent in the above preparation is a combination of at least two of the following high molecular compounds: hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, 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 salt.

[0029] Further, the viscosity increasing agent in the above preparation is a combination of two of the above high molecular compounds, and the weight ratio of the two high molecular compounds is 1:(0.1-10), preferably 1:(0.6-5).

[0030] Further, the weight ratio of the above two high molecular compounds is 1:1.

[0031] Further, the above tackifier 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);

[0032] or the tackifier is a combination of povidone and hydroxypropyl methyl cellulose, the weight ratio of povidone and hydroxypropyl methyl cellulose is 1:(1-3), preferably 1:(1-1.5);

[0033] or the tackifier is a combination of povidone and carbomer, the weight ratio of povidone and carbomer is 1:(0.5-2), preferably 1:1;

[0034] or the tackifier is a combination of povidone and polyethylene glycol, the weight ratio of polyethylene glycol and povidone is 1:(1-8), preferably 1:5.

[0035] Further, the above oxidized cholesterols are 25-hydroxycholesterol, and the tackifier is a combination of at least two of the following high molecular compounds: hydroxypropyl methyl cellulose, 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 salt.

[0036] Further, the above oxidized cholesterols are 25-hydroxycholesterol, and the tackifier 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);

[0037] or the tackifier is a combination of povidone and hydroxypropyl methyl cellulose, the weight ratio of povidone and hydroxypropyl methyl cellulose is 1:(1-3), preferably 1:(1-1.5).

[0038] Further, the above oxidized cholesterols are lanosterol, and the tackifier is a combination of at least two of the following high molecular compounds: hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose, povidone, carbomer, polyethylene glycol, poloxamer, polyvinyl alcohol, hydroxyethyl cellulose.

[0039] Further, the above oxidized cholesterols are lanosterol, and the tackifier 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);

[0040] or the viscosity increasing agent is a combination of povidone and hydroxypropyl methylcellulose, the weight ratio of povidone to hydroxypropyl methylcellulose being 1: (1-3), preferably 1: (1-1.5);

[0041] or the viscosity increasing agent is a combination of povidone and carbomer, the weight ratio of povidone to carbomer being 1: (0.5-2), preferably 1:1;

[0042] or the viscosity increasing agent is a combination of povidone and polyethylene glycol, the weight ratio of polyethylene glycol to povidone being 1:5.

[0043] Further, the solvent in the above pharmaceutically acceptable carrier or adjuvant is a polar solvent, preferably water.

[0044] Further, the co-solvent in the above pharmaceutically acceptable carrier or adjuvant is selected from at least one of liquid polyethylene glycol, propylene glycol, glycerol, polyoxyethylene hydrogenated castor oil or castor oil polyoxyethylene ether, preferably liquid polyethylene glycol.

[0045] Further, the above preparation comprises the following components:

[0046] active substance for treating eye diseases: lanosterol, the content of which is 0.01-0.2 mg / mL;

[0047] surfactant: polysorbate or poloxamer, the content of which is 6.7-250 times that of lanosterol;

[0048] viscosity increasing agent: the content of which is 11-50 times that of lanosterol, the viscosity increasing agent is a combination of hydroxypropyl cellulose and povidone, the weight ratio of hydroxypropyl cellulose to povidone being 1: (1-1.2); or, the viscosity increasing agent is a combination of povidone and hydroxypropyl methylcellulose, the weight ratio of povidone to hydroxypropyl methylcellulose being 1: (1-1.5); or, the viscosity increasing agent is a combination of povidone and carbomer, the weight ratio of povidone to carbomer being 1:1; or the viscosity increasing agent is a combination of povidone and polyethylene glycol, the weight ratio of polyethylene glycol to povidone being 1:5;

[0049] co-solvent: liquid polyethylene glycol, propylene glycol, glycerol, polyoxyethylene hydrogenated castor oil or castor oil polyoxyethylene ether, the content of which is 100-2500 times that of lanosterol; the solvent is water.

[0050] or, the above preparation comprises the following components:

[0051] active substance for treating eye diseases: 25-hydroxycholesterol, the content of which is 0.1 mg / mL;

[0052] surfactant: polysorbate, the content of which is 25-250 times that of 25-hydroxycholesterol;

[0053] a viscosity-increasing agent, which is a combination of povidone and hydroxypropyl cellulose, the weight ratio of hydroxypropyl cellulose to povidone being 1:1, or a combination of povidone and hydroxypropyl methyl cellulose, the weight ratio of povidone to hydroxypropyl methyl cellulose being 1:1, the content of the viscosity-increasing agent being 12 times that of 25-hydroxycholesterol;

[0054] a co-solvent, which is liquid polyethylene glycol or glycerol, the content of the co-solvent being 100-1750 times that of 25-hydroxycholesterol, and the solvent being water.

[0055] Further, the pharmaceutically acceptable carrier or adjuvant of the above-mentioned ophthalmic preparation further comprises any one or more of an osmotic pressure regulator, a pH regulator, and a preservative.

[0056] The osmotic pressure regulator is any one or more of glucose, sodium chloride, potassium chloride, mannitol, sorbitol, sodium citrate, potassium citrate, and glycerol.

[0057] The pH regulator 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, sodium dihydrogen phosphate, disodium hydrogen phosphate, boric acid, borax, tartaric acid or a salt thereof.

[0058] The preservative is any one or more of sorbic acid, chlorobutanol, sodium chlorite, sodium perborate, quaternary ammonium salts (including benzalkonium chloride, benzalkonium bromide, polyquaternary salt-1, cetyltrimethylammonium bromide), hydroxybenzoate (including methylparaben, ethylparaben, propylparaben), phenylmercuric nitrate; preferably, the quaternary ammonium salts include benzalkonium chloride, benzalkonium bromide, polyquaternary salt-1, and / or cetyltrimethylammonium bromide, and the hydroxybenzoate includes methylparaben, ethylparaben, and / or propylparaben.

[0059] Further, the above-mentioned ophthalmic preparation comprises a nano-some structure formed by self-assembly of the components of the carrier or adjuvant of the ophthalmic preparation; the nano-some comprises the active substance for treating eye diseases.

[0060] Still further, the above-mentioned nano-some is spherical, and has a particle size of 5-900 nm, preferably 5-50 nm and / or 200-700 nm.

[0061] The present application also provides a method for preparing the above-mentioned preparation, comprising the following steps:

[0062] (1) adding a surfactant and a viscosity-increasing agent to a solvent, and mixing to obtain a mixed solution;

[0063] (2) adding an active substance for treating eye diseases to the mixed solution obtained in step (1), and adding or not adding a co-solvent, and dispersing and mixing to obtain a primary suspension;

[0064] (3) stirring and dispersing and / or homogenously dispersing the initial suspension obtained in step (2) to obtain.

[0065] Further, the dispersing in step (2) is at least one selected from mechanical stirring and dispersing, magnetic stirring and dispersing, vortex shaking and dispersing, shearing and dispersing, homogenously dispersing, grinding and dispersing, and ultrasonic dispersing.

[0066] The present application also provides the use of the above-mentioned preparation in the preparation of a medicine for preventing and treating a lens disease in a human or an animal.

[0067] Further, the above-mentioned medicine is a medicine for preventing and treating cataract, preferably a medicine for reducing the aggregation of lens protein and reducing the turbidity of lens.

[0068] Further, the above-mentioned medicine is a medicine preparation for ocular administration, preferably a medicine for ocular topical administration.

[0069] Oxidized cholesterols including 25-hydroxycholesterol (25-HC) and lanosterol (LAN) can interact with crystallin in the lens, thereby preventing the aggregation of crystallin, improving the shape of the lens, and restoring transparency, and can reverse and restore the transparency of the lens in the case of cataract, thereby achieving the purpose of treating and preventing cataract. Oxidized cholesterols administered systemically by oral administration, intramuscular injection, intravenous injection, and the like participate in the biochemical processes of the whole body and cannot reach an effective therapeutic concentration in the lens; LAN and 25-HC injected into the vitreous body cannot penetrate the lens capsule to enter the lens; and lens injection can cause traumatic or after-cataract.

[0070] The eye drop administration route is a safe and convenient administration method, but in order to treat fundus diseases, the drug needs to penetrate the eye barrier and be safely and effectively delivered to the lesion site of the fundus.

[0071] According to the optimal design principle of the treatment effect / risk ratio, the concentration of the preparation is minimized to minimize the risk on the premise of achieving the treatment effect. Only about 10% of the eye drops remain on the ocular surface due to mechanical actions such as blinking, most of which are discharged from the lacrimal canaliculus with tears, a small part enters the nasal cavity through the nasolacrimal duct, and even is absorbed into the blood circulation through the nasal cavity. The lower the concentration of the preparation, the smaller the risk borne by the tissues and organs, and the smaller the systemic toxic side effects. The concentration of lanosterol in the preparation of the present application (not higher than 4.68 mM) is lower than that claimed in other patents, which can minimize the toxic side effects. At the same time, the concentration of 0.1 μg / μL of the present preparation is dropped into 20 μL of the palpebral conjunctival sac, and about 0.2 μg actually remains on the ocular surface, which is 0.67% of the total amount of the preparation, according to the lens volume of 0.03 cm 3The concentration of LAN in the lens after the eye drops can reach 2.3-2.5 times the original content, and the transmittance can reach 44-52%. Taking a rabbit as an example, 50 uL of the preparation of the application containing 0.11 ug / uL is dropped, and 10% of the preparation stays on the ocular surface, and enters the rabbit lens (diameter = 7.9 mm, volume = 0.258 cm 3 ), the amount of lanosterol entering the lens can reach 3.6 times the original concentration, and the transmittance is almost 100% (A. B. Weir and M. Collins (eds.), Assessing Ocular Toxicology in Laboratory Animals, 1Molecular and Integrative Toxicology, DOI 10.1007 / 978-1-62703-164-6_1, Springer Science+Business Media, LLC 2013; Ling Peixue, editor-in-chief of Ophthalmic Drugs and Formulation, China Light Industry Press, 2010, P6). It can be seen that the utilization rate of the application is very high although the concentration of the used oxidized cholesterols is low, which not only can reduce the potential risk of systemic toxic side effects, but also can fully ensure the drug dosage and efficacy.

[0072] Another feature of the preparation of the application is the formulation advantage. Treatment of cataract is long-term medication, and it is particularly important to avoid active adjuvants in the formulation. For example, a low-concentration formulation with anti-infection activity may cause bacterial resistance on the ocular surface and nasolacrimal duct, and even induce infection. The concentration of the application is low, and the preparation does not need to use preservatives, so there is no potential complication in long-term use.

[0073] It is reported in the literature that oxidized cholesterols have poor water solubility. To prepare an oxidized cholesterols injection or eye drops, solvents such as ethanol and DMSO are often used. Alcohol solvents have a stimulating effect on the eye, and their use amount must be controlled. Using DMSO as a cosolvent can increase the solubility of active substances, but it may allow more active substances to enter the systemic circulation. For example, 0.5% LAN solution prepared with 15% DMSO, 20% ethanol or poly sorbitan 80 solution injected into the vitreous body of rats not only causes vitreous body turbidity, but also has the risk of promoting lens opacity.

[0074] The highly ordered and compact arrangement of crystallin and high transparency, injection will cause its structure damaged, leading to traumatic cataract. It is difficult to deliver LAN to the lens by injection route (Enfu Yin, CN108472303A, 2016; Zheng Qianyuan, Zeyi Dan Gopal, 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).

[0075] The inventors have prepared a solution of oxidized lanosterol or 25-HC in water as the main medium by using non-ionic surfactants as solubilizers, and / or tackifiers, and / or co-solvents, and by using physical mixing, high-speed shearing dispersion, and high-pressure homogenization, and by heating and ultrasonic treatment if necessary. The prepared solution has good stability. The main particle size of the solution is 5-50 nm and / or 200-700 nm, and spherical particles can be observed under an electron microscope. The prepared solution is soluble in tears, and then the oxidized lanosterol passes through the cornea and the lens capsule membrane into the lens. In the in vivo animal eye drop administration and lens absorption test, the concentration of the oxidized lanosterol in the lens is significantly increased after the eye drops are administered to the experimental animals, and the content of the oxidized lanosterol in the aqueous humor and vitreous body is little (in rat test) or not detected (in rabbit test).

[0076] The inventors have observed that the prepared eye drops can delay the occurrence and development of cataract in experimental animals in the rat cataract model test of subcutaneous injection of sodium selenite solution. The prepared LAN eye drops are used for an aged dog (15 years old) with cataract, and the turbidity of the lens of the dog is significantly reduced after 20 days of eye drop administration.

[0077] The eye preparation of the present application can efficiently and accurately deliver the active substance into the lens of experimental animals after eye drop administration, and can prevent the aggregation of crystallin as a chaperone, and can remove cataract, thereby playing a role in treating and preventing cataract.

[0078] In animal experiments, it is unexpectedly found that the eye drop of oxidized cholesterols prepared by the present application has the advantages of safety and effectiveness, and in particular, it is unexpectedly found that after the experimental animals are dropped, the oxidized cholesterols are enriched in the lens; (the New Zealand rabbit eye drop absorption test LAN is enriched in the lens), and the concentration in the aqueous humor and vitreous body is very low, indicating that the eye drop of the present application has high target tissue selectivity.

[0079] The eye drop of the present application can selectively and efficiently deliver the active substance of oxidized cholesterols to the lens through eye drop, supplementing the cataract caused by the lack of oxidized cholesterols; and can also avoid the unpredictable pathophysiological reactions caused by the physiological activity of cholesterols in the body.

[0080] Oxidized cholesterols in the lens can prevent the aggregation of lens proteins and remove cataracts; however, oxidized cholesterols can cause cardiovascular complications in the body and are harmful to the body. So far, there is no technology in the pharmaceutical field to deliver drugs, including oxidized cholesterols, to the lens by non-invasive drug delivery. The novel low-concentration eye drop of the present application realizes the targeted delivery of drugs to the lens, and the most obvious clinical advantage is that the active substance content in the eye drop for cataract is low, and the bioavailability is very high after eye drop administration, effectively increasing the concentration of oxidized cholesterols in the lens and treating cataracts. Because of the low concentration and high ocular local bioavailability, the drugs are greatly reduced to enter the body through the conjunctiva and nasal cavity and produce toxic side effects.

[0081] The active substance for treating eye diseases referred to in the present application is: an active substance for treating eye diseases of humans or animals, which exists or does not exist in the body.

[0082] The nanosome referred to in the present application is: a nanoscale spherical aggregate formed by self-assembly of components of a drug carrier or excipient in a solvent.

[0083] The solvent referred to in the present application is: a liquid capable of dissolving the components of a drug carrier or excipient.

[0084] The surfactant referred to in the present application is: a substance that can significantly reduce the surface tension of a liquid; the non-ionic surfactant referred to in the present application is a surfactant that does not dissociate in water.

[0085] The eye drop administration described in the present application is: a method of administering a liquid into the eye, which belongs to the mucosal administration route.

[0086] The liquid polyethylene glycol (liquid PEG) described in the present application is: a polyethylene glycol that is liquid at normal temperature and pressure, preferably a polyethylene glycol with a weight average molecular weight of not more than 1000.

[0087] Obviously, according to the above content of the present application, other various forms of modifications, replacements or changes can be made according to the common technical knowledge and usual means in the art without departing from the above basic technical idea of the present application.

[0088] The above content of the present application will be further explained in detail through the specific embodiments in the form of examples. However, it should not be understood that the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 Transmission electron microscope image of the sample prepared for Example 3.

[0090] Figure 2 Transmission electron microscope image of the sample prepared for Example 3 after staining with a staining agent.

[0091] Figure 3 Photos showing the lens turbidity of a cataract dog before and after using the ophthalmic preparation of the present application.

[0092] Figure 4 Particle size distribution graph of the sample prepared for Example 5 (0 days).

[0093] Figure 5 Particle size distribution graph of the sample prepared for Example 5 (15 days at room temperature). DETAILED DESCRIPTION

[0094] The reagents or instruments used in the present application can be purchased through commercially available products, and if the specific conditions are not specified, they are used according to the conventional conditions or the conditions recommended by the manufacturer. Lanosterol (CAS: 79-63-0), content: 99.9% (HPLC, Chengdu Pu Feide Biotechnology Co., Ltd., Chengdu); 25-hydroxycholesterol (Hydroxycholesterol, CAS: 2140-46-7, content ≥98%, Shanghai Maikelin Biotechnology Co., Ltd., Shanghai).

[0095] Some instruments and equipment are as follows:

[0096] ES225SM-DR(E) electronic analytical balance, Precisa Company (Switzerland);

[0097] DF-101S heat collecting constant temperature heating magnetic stirring, Gongyi Yingyu High-tech Instrument Factory (Henan, China);

[0098] WH-2 micro vortex mixer, Shanghai Huxi Analytical Instrument Factory Co., Ltd. (Shanghai, China);

[0099] Dispersion machine: T25 easy clean digital, IKA (Germany);

[0100] Ultrasonic cleaner, Model KQ-500, Kunshan Ultrasonic Instrument Co., Ltd. (Kunshan, China);

[0101] AH-NANO Plus high pressure homogenizer, Antao Nano Technology (Suzhou) Co., Ltd. (China);

[0102] Mettler Toledo FE20 pH meter, Mettler-Toledo (Switzerland);

[0103] NS-90 nanoparticle size analyzer, Zhuhai UMA Instruments Co., Ltd. (Zhuhai, China);

[0104] Agilent 1100 HPLC high performance liquid chromatograph, Agilent Technologies (USA);

[0105] API 4000 triple quadrupole mass spectrometer (Applied Biosystems, USA);

[0106] STY-1A osmotic pressure detector, Tianjin Tianda Tianfa Science and Technology Co., Ltd. (Tianjin, China);

[0107] Zetasizer Nano ZS, nanoparticle size and Zeta potential analyzer, Malvern Instruments (UK).

[0108] The property detection method of the preparation of the present application is as follows

[0109] Particle size detection method:

[0110] 1 mL of the sample prepared in the examples or comparative examples was transferred to the sample cell, the detection temperature was set to 40℃, and the sample cell was placed in the NS-90 nanoparticle size analyzer, and the detection was started. Each sample was detected 3 times, and the average value of the 3 detection results was taken as the particle size (expressed by light intensity distribution and percentage) and polydispersity index (PdI, Polydispersity Index) of the sample.

[0111] Osmotic pressure detection method:

[0112] The freezing point depression of the solution is measured to determine its osmotic pressure molarity. Operation: clean the STY-1A osmotic pressure detector probe: take three 100 μL of distilled water into three sample tubes, after the instrument is preheated, screw the sample tube containing 100 μL of distilled water onto the instrument probe, select cleaning 3 times, click "cleaning", repeat three times. Detection: after filling in the sample information in the instrument information table, click "test"; use a pipette to take 100 μL of the sample into the sample tube, screw the instrument gently, click "start" to detect. Repeat the detection three times, and the average of the three detection results is the detection result. In actual animal experiments, if the osmotic pressure does not reach isotonicity, the osmotic pressure regulator described above is used to make it reach or approach isotonicity.

[0113] pH detection method:

[0114] The FE20 type acidity meter is calibrated with pH buffer solutions (pH is 4.00, 6.86 and 9.18 respectively), the electrode is washed with pure water and the excess water is absorbed with non-fiber paper, and then it is immersed in the liquid sample to be detected to start measurement according to the reading key. The data obtained after the reading is stable is the pH value of the sample.

[0115] If the pH of the solution obtained in the detection example is <5 or >9, it needs to be adjusted to pH 6-8 with acid or base. Common pH regulators are NaOH and HCl, phosphoric acid and phosphate (such as sodium dihydrogen phosphate, disodium hydrogen phosphate), citric acid and citrate (such as sodium citrate), boric acid and borax; in actual animal experiments, if the pH of the obtained liquid does not reach the requirement of the ophthalmic preparation, the pH regulator described above is used for adjustment.

[0116] Example 1, preparation of the ophthalmic preparation of the application

[0117] The materials and ratios used are shown in Table 1, and the preparation process is as follows: firstly, sorbitan 80, PVP K12, HPMC and liquid PEG (PEG400) are weighed into 100 mL polypropylene centrifuge tubes respectively, and an appropriate amount of water for injection is added and stirred for 30 minutes, then 6.0 mg of lanosterol is added, water for injection is added to 60 ml, and stirring and mixing are continued for 10 min to obtain a mixed solution. The mixed solution is dispersed with a dispersion machine at a speed of 12000-15000 rpm for 3 minutes, and after the machine is stopped, the foam is allowed to disappear. The dispersion liquid is transferred to a high-pressure homogenizer, the temperature is controlled at 5±5℃, and the pressure is homogenized at about 400 Bar for 2 minutes, then the pressure is increased to 1200-1400 Bar for homogenization for 20 minutes, and then the pressure is reduced to 500 Bar for 2 minutes, and then the dispersion liquid is discharged. After the foam naturally disappears, a clear homogenate is obtained. The pH value and osmotic pressure are detected, sodium citrate (0.10 g) and sodium chloride (0.4 g) are added, and 0.1N HCl or 0.1N NaOH is used to adjust the pH to 7.0, and the osmotic pressure is 302 mOsmol / kg. The solution is filtered under reduced pressure through a filter membrane to obtain the product as a solution.

[0118] HPLC product concentration detection:

[0119] Detection instrument: Agilent 1100 high performance liquid chromatograph;

[0120] Chromatographic conditions: Agilent ZORBAX Eclipse Plus C18, 4.6x100mm 3.5μm column; flow rate 1.0mL / min, detection wavelength 205nm, mobile phase: MeOH (60%) - acetonitrile (40%) isocratic elution. The sample was diluted 5 times with the mobile phase and then 10μL was injected into the liquid chromatograph. HPLC content detection result: 0.098mg / mL.

[0121] Particle size detection result (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;

[0122] Zeta potential detection average value: -5.03±4.33mV (25℃).

[0123] The product was placed at 40℃ in the dark for 30 days, and there was no obvious change in appearance. The particle size detection result was 40.68nm (88.8%), PdI was 0.249, and the HPLC content detection result was 0.093mg / mL.

[0124] Animal eye drop absorption test result: three rats (6 eyes) were taken, both eyes were given 20μL / eye, 1.5 hours later, the animals were euthanized and the vitreous and lens were quickly taken, and the content of LAN was detected. The detection result was: lanosterol content, in the lens: 5.16±1.90(μg / mL); in the vitreous: 0.045±0.091(μg / mL).

[0125] Example 2, preparation of the ophthalmic preparation of the application

[0126] The materials and ratios used are shown in Table 1, and the preparation process is the same as in Example 1, to obtain a solution;

[0127] Particle size detection result: particle size 441.8nm (100.0%), PdI: 0.190;

[0128] HPLC content detection:

[0129] Detection instrument: Agilent 1100 high performance liquid chromatograph;

[0130] Chromatographic conditions: Agilent ZORBAX Eclipse Plus C18, 4.6x100mm 3.5μm column; flow rate 0.8 mL / min, detection wavelength: 205 nm, mobile phase: MeOH (85%) - 0.1% H3PO4 (15%) isocratic elution. The sample was diluted with 5 times mobile phase and 10 μL was injected into the HPLC. The HPLC content detection result: 0.095 mg / mL.

[0131] The product was placed at 40°C in the dark for 15 days, and no obvious change in appearance was observed. The particle size detection result was 413.2 nm (94.5%), the PdI was 0.214, and the HPLC content detection result was 0.090 mg / mL.

[0132] Example 3, Preparation of the ophthalmic preparation of the application

[0133] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0134] The particle size detection result was a particle size of 416.3 nm (100.0%), the PdI was 0.214, and the HPLC content was 0.078 mg / mL.

[0135] The product was placed at 2-8°C in the dark for 30 days, and no obvious change in appearance was observed. The particle size detection result was 478.5 nm (98.2%), the PdI was 0.245, and the HPLC content detection result was 0.077 mg / mL.

[0136] Animal lens eye drop absorption experiment result: 2 New Zealand rabbits (4 eyes) were given eye drops, 50 μL / eye, and after 1.5 hours, the animals were euthanized and the aqueous humor, vitreous body and lens were quickly taken and the content of LAN therein was detected; the detection result: 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); the lanosterol content in the lens of the blank control animal (1 New Zealand rabbit, 2 eyes, no drug) was 0.76±0.01 (μg / mL).

[0137] Example 4, Preparation of the ophthalmic preparation of the application

[0138] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0139] The particle size detection result was a particle size of 416.3 nm (100.0%), the PdI was 0.214, and the HPLC content was 0.078 mg / mL.

[0140] The product was placed at room temperature in the dark for 15 days, and no obvious change in appearance was observed. The particle size detection results were: 613.2 nm (100.0%), Pdl: 0.350, and the HPLC content detection results were: 0.064 mg / mL.

[0141] Example 5, Preparation of the ophthalmic preparation of the application

[0142] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0143] The particle size detection results were: particle size, 401.3 nm (98.8%); Pdl: 0.221; and HPLC content: 0.063 mg / mL.

[0144] The product was placed at room temperature in the dark for 15 days, and no obvious change in appearance was observed. The particle size detection results were: 613.2 nm (100.0%), Pdl: 0.350, and the HPLC content detection results were: 0.064 mg / mL.

[0145] The animal eye drop lens absorption test results were as follows: three rats (6 eyes) were given eye drops, 20 μL / eye, and 1.5 hours later, the animals were euthanized and the lenses were quickly removed, and the content of lanosterol in the lenses was detected; the detection results were: the content of lanosterol in the lenses was 2.89 ± 0.60 (μg / mL).

[0146] Example 6, Preparation of the ophthalmic preparation of the application

[0147] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0148] The particle size detection results were: particle size, 401.3 nm (98.8%); Pdl: 0.221; and HPLC content: 0.063 mg / mL.

[0149] The product was placed at room temperature in the dark for 15 days, and no obvious change in appearance was observed. The particle size detection results were: 613.2 nm (100.0%), Pdl: 0.350, and the HPLC content detection results were: 0.064 mg / mL.

[0150] The animal lens absorption test results were as follows: three rats (6 eyes) were given eye drops, 20 μL / eye, and 1.5 hours later, the animals were euthanized and the lenses were quickly removed, and the content of lanosterol in the lenses was detected; the detection results were: the content of lanosterol in the lenses was 4.83 ± 2.15 (μg / mL).

[0151] Example 7, Preparation of the ophthalmic preparation of the application

[0152] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0153] Particle size detection result: particle size 472.5 nm (99.4%); Pdl: 0.205; HPLC content: 0.072 mg / mL.

[0154] The product was placed in the dark at 2-8°C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 513.2 nm (97.1%), Pdl was 0.235, and the HPLC content detection result was 0.070 mg / mL.

[0155] Example 8, Preparation of an ophthalmic preparation of the application

[0156] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0157] Particle size detection result: particle size 508.5 nm (97.7%); Pdl: 0.245; HPLC content: 0.080 mg / mL.

[0158] The product was placed in the dark at 40°C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 423.5 nm (90.1%), Pdl was 0.223, and the HPLC content detection result was 0.078 mg / mL.

[0159] Animal lens absorption test result: three rats (6 eyes) were given eye drops, 20 μL / eye, and 1.5 hours later, the animals were euthanized and the lenses were quickly removed, and the content of lanosterol therein was detected. The detection result was that the content of lanosterol in the lenses was 5.68 ± 1.60 (μg / mL).

[0160] Example 9, Preparation of an ophthalmic preparation of the application

[0161] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0162] Particle size detection result: particle size 508.5 nm (97.7%); Pdl: 0.245; HPLC content: 0.080 mg / mL.

[0163] The product was placed in the dark at 2-8°C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 513.2 nm (97.1%), Pdl was 0.235, and the HPLC content detection result was 0.070 mg / mL.

[0164] Animal lens absorption test result: three rats (6 eyes) were given eye drops, 20 μL / eye, and 1.5 hours later, the animals were euthanized and the lenses were quickly removed, and the content of lanosterol therein was detected. The detection result was that the content of lanosterol in the lenses was 5.68 ± 1.60 (μg / mL).

[0165] Example 10, Preparation of the ophthalmic formulation of the present application

[0166] The materials and ratios used are shown in Table 1, the preparation process and content determination are the same as in Example 1, to obtain a solution;

[0167] The particle size detection result is 12.8 nm (99.0%); Pdl: 0.175; HPLC content: 0.073 mg / mL.

[0168] The product was placed in the dark at 40°C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 13.2 nm (91.5%); Pdl: 0.222; HPLC content detection result: 0.072 mg / mL.

[0169] Example 11, Preparation of the ophthalmic formulation of the present application

[0170] The materials and ratios used are shown in Table 1, the preparation process is the same as in Example 1, and the content determination is the same as in Example 2, to obtain a solution;

[0171] The particle size detection result is 455.6 nm (99.7%); Pdl: 0.249; HPLC content: 0.051 mg / mL.

[0172] The product was placed in the dark at 40°C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 216.8 nm (95.6%); Pdl: 0.351; HPLC content detection result: 0.049 mg / mL.

[0173] Example 12, Preparation of the ophthalmic formulation of the present application

[0174] The materials and ratios used are shown in Table 1, the preparation process and content determination are the same as in Example 1, to obtain a solution;

[0175] The particle size detection result is 420.0 nm (100.0%); Pdl: 0.205; HPLC content: 0.037 mg / mL.

[0176] The product was placed in the dark at 40°C for 30 days, and no obvious change in appearance was observed. The particle size detection result was 452.0 nm (94.0%); Pdl: 0.256; HPLC content detection result: 0.033 mg / mL.

[0177] Example 13, Preparation of the ophthalmic formulation of the present application

[0178] The materials and ratios used are shown in Table 1, the preparation process is the same as in Example 1, and the content determination is the same as in Example 2, to obtain a solution;

[0179] Particle size detection result: particle size 474.3 nm (98.7%); Pdl: 0.229; HPLC content: 0.089 mg / mL.

[0180] The product was placed in the dark at 40 °C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 425.2 nm (88.6%), the Pdl was 0.245, and the HPLC content detection result was 0.087 mg / mL.

[0181] Example 14, Preparation of an ophthalmic formulation of the application

[0182] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0183] Particle size detection result: particle size 474.3 nm (98.7%); Pdl: 0.229; HPLC content: 0.089 mg / mL.

[0184] The product was placed in the dark at 40 °C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 425.2 nm (88.6%), the Pdl was 0.245, and the HPLC content detection result was 0.087 mg / mL.

[0185] Example 15, Preparation of an ophthalmic formulation of the application

[0186] The materials and ratios used are shown in Table 1. The preparation process was the same as in Example 1, and the content detection was the same as in Example 2, and a solution was obtained.

[0187] Particle size detection result: particle size 474.3 nm (98.7%); Pdl: 0.229; HPLC content: 0.089 mg / mL.

[0188] The product was placed in the dark at 40 °C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 425.2 nm (88.6%), the Pdl was 0.245, and the HPLC content detection result was 0.087 mg / mL.

[0189] Example 16, Preparation of an ophthalmic formulation of the application

[0190] The materials and ratios used are shown in Table 1. The preparation process and content detection were the same as in Example 1, and a solution was obtained.

[0191] Particle size detection result: particle size 474.3 nm (98.7%); Pdl: 0.229; HPLC content: 0.089 mg / mL.

[0192] The product was placed in the dark at 40 °C for 15 days, and no obvious change in appearance was observed. The particle size detection result was 425.2 nm (88.6%), the Pdl was 0.245, and the HPLC content detection result was 0.087 mg / mL.

[0193] Example 17, Preparation of the ophthalmic formulation of the application

[0194] The materials and ratios used are shown in Table 1, the preparation process and content determination are the same as in Example 1, to obtain a solution;

[0195] The particle size detection result is 527.5 nm (99.4%); PdI: 0.240; HPLC content: 0.058 mg / mL.

[0196] The product was placed in the dark at 40°C for 15 days, and no obvious change in appearance was observed. The particle size detection result is 161.3 nm (100.0%); PdI: 0.505; HPLC content detection result: 0.052 mg / mL.

[0197] Comparative Example 1

[0198] The materials and ratios used are shown in Table 1, the preparation process and content determination are the same as in Example 1, to obtain a solution;

[0199] The particle size detection result is 16.86 nm (69.0%); PdI: 1.000; HPLC content: 0.051 mg / mL.

[0200] Precipitation was generated after standing overnight at room temperature, and this formulation has poor stability.

[0201] Comparative Example 2

[0202] The materials and ratios used are shown in Table 1, the preparation process and content determination are the same as in Example 1, to obtain a solution containing flocculent suspensions;

[0203] The particle size detection result is 369.8 nm (82.8%); PdI: 0.554; HPLC content of the supernatant of the example: 0.025 mg / mL.

[0204] The product was placed in the dark at 40°C for 15 days, and flocculent suspensions precipitated. The particle size detection result is 462.1 nm (80.2%); PdI: 0.979; HPLC content detection result of the supernatant of the example: 0.027 mg / mL.

[0205] Comparative Example 3

[0206] The materials and ratios used are shown in Table 1, the preparation process and content determination are the same as in Example 1, to obtain a solution containing flocculent suspensions;

[0207] The particle size detection result is 313.7 nm (74.9%); PdI: 0.587; HPLC content of the supernatant of the example: 0.029 mg / mL.

[0208] The product was placed in the dark at 40℃ for 15 days, and the flocculent suspension precipitated. The particle size detection results were: 161.4 nm (100.0%), PdI: 0.231, and the supernatant HPLC content detection results were: 0.023 mg / mL, with low active substance content.

[0209] Comparative Example 4

[0210] The materials and ratios used are shown in Table 1, and the preparation process and content detection were the same as in Example 1, to obtain a solution with flocculent suspension. After the product was placed in the dark at room temperature for 15 days, the flocculent suspension aggregated to precipitate.

[0211] Comparative Example 5

[0212] The materials and ratios used are shown in Table 1, and the preparation process and content detection were the same as in Example 1, to obtain a solution;

[0213] The particle size detection results were: particle size 292.2 nm (72.6%); PdI: 1.000, and HPLC content: 0.029 mg / mL.

[0214] The product was placed in the dark at 40℃ for 15 days, and there was no obvious change in appearance. The particle size detection results were: 188.3 nm (100.0%), PdI: 0.217, and the supernatant HPLC content detection results were: 0.026 mg / mL, with reduced active substance content.

[0215] Animal lens absorption test results: 2 rats (4 eyes) were given eye drops, 20 μL / eye, and after 1.5 hours, the animals were euthanized and the lenses were quickly removed, and the content of LAN therein was detected; the detection results were: the content of lanosterol in the lenses was 2.00 ± 0.41 (μg / mL), which was almost at the same level as the blank control eye, indicating that the sample of the present comparative example could not effectively enter the animal lens.

[0216] Table 1: Dosage information for examples and comparative examples

[0217]

[0218]

[0219] Note: 1, HPMC: hydroxypropyl methylcellulose; 2, PVP: polyvinylpyrrolidone; 3, HPC: hydroxypropyl cellulose; 4, PEG: polyethylene glycol with an average molecular weight of ≤5000 Da; 5, CMC-Na: carboxymethyl cellulose sodium salt; 6, PEG-60 hydrogenated castor oil: polyoxyethylene hydrogenated castor oil.

[0220] As can be seen from the results of the examples and comparative examples, the eye drop system of the present application contains at least 1 surfactant, 2 tackifiers, and an appropriate amount of a cosolvent, the prepared product is stable, and the animal lens absorption rate is high;

[0221] When the eye drop system of the present application contains one surfactant, no or only one thickening agent; or no surfactant, the prepared products are all relatively poor in stability (see Comparative Examples 2, 3 and 4).

[0222] If an ionic polymer is added in the formulation, the prepared product will produce a precipitate after a short period of placement, and is not good in stability (see Comparative Example 1).

[0223] If the amount of surfactant exceeds the range of the present application, the prepared product will have a great influence on the absorption of the lens of animals, and will result in that the active substance cannot effectively enter the lens (Comparative Example 5).

[0224] The beneficial effects of the present application are proved by the following experimental examples.

[0225] Experimental Example 1, SD rat eye absorption test (LAN)

[0226] Six healthy adult SD rats, SPF level, 180-220 g, all male, after respiratory anesthesia, 20 μL (concentration 0.1 mg / mL) of the tested preparation (Example 1) was respectively dropped into the eyes of each rat, 1.5 hours after the eye dropping, the animals were euthanized, and the lens and vitreous body were immediately collected and stored at -80℃ for testing. The content of LAN in the lens and vitreous body was detected by LC / MS / MS method.

[0227] The animal samples were treated as follows:

[0228] After homogenization of the vitreous body sample of the animal, 10 μL was taken, 40 μL of 70% methanol was added, ultrasonic was performed for 2 min, vortex was performed for 1 min, 175 μL of methanol was added, vortex mixing was performed for 2 min, centrifugation was performed at 4℃ at a speed of 12000 rpm for 10 min, and the supernatant was used for LC-MS / MS analysis.

[0229] After homogenization of the lens sample of the animal (physiological saline was added to the lens at a ratio of 1:4, and homogenization was performed), 50 μL was taken, 175 μL of methanol was added, vortex mixing was performed for 2 min, centrifugation was performed at 4℃ at a speed of 12000 rpm for 10 min, and the supernatant was used for LC-MS / MS analysis.

[0230] The LC-MS / MS test conditions are as follows, LC-20AD high performance liquid chromatography system (SHIMADZU) - API4000 triple quadrupole mass spectrometer (Applied Biosystems Corporation), Fortis Pace C185UM 2.1X30mm chromatographic column is configured, the column temperature is 40℃, the mobile phase is methanol: water (95:5), the flow rate is 0.4mL / min, the injection volume is 10μL; the mass spectrometer selects the atmospheric pressure chemical ionization source (Atomsperic Pressure Chemical Ionization, APCI). The mass spectrometry conditions are shown in the following table:

[0231]

[0232]

[0233] The detection results are shown in Table 2:

[0234] Table 2 Lan content in rabbit lens and vitreous after eye drops (Mean ± SD)

[0235]

[0236] The background value of rats is detected, 2 SD rats (4 eyes) are euthanized and the lens is quickly taken, the sample is treated in the same way and the content of LAN is detected. The detection results are as follows: lanosterol content: 2.23±0.86 (μg / mL). It can be seen that the ophthalmic preparation of the application can effectively transport lanosterol through the lens barrier by eye drop administration, and enrich in the lens.

[0237] The above results show that the ophthalmic preparation of the application can effectively transport lanosterol through the lens barrier by eye drop administration, and enrich in the lens, and almost not remain in the vitreous.

[0238] Experimental example 2, New Zealand rabbit eye absorption test (LAN)

[0239] Healthy adult New Zealand rabbits, SPF level, 2-2.5kg, all male, a total of 7, 6 rabbits are selected for eye drop administration of the test substance (example 3), all are single administration of 50μL, the remaining 1 rabbit is not administered in both eyes, as a background control; 1.5 hours after administration, the animals are euthanized, the aqueous humor, lens and vitreous are collected, and the drug content in the aqueous humor, lens and vitreous is detected, the sample treatment and LC / MS / MS detection method are the same as the rat eye absorption test, and the detection results are shown in Table 3:

[0240] Table 3 Lan content in rabbit lens, aqueous humor and vitreous after eye drops (Mean ± RSD)

[0241]

[0242]

[0243] Note: BLOQ: below the limit of detection (LOQ = 0.001 μg / mL), not detected.

[0244] The above results further confirm that the ophthalmic preparation of the present application can effectively deliver lanosterol through the lens barrier, enrich in the lens, and not enrich in the vitreous body and aqueous humor by the way of eye drop administration.

[0245] Experimental Example 3, SD Rat Eye Absorption Test (25-HC)

[0246] Six healthy adult SD rats, SPF level, 180-220 g, all male, after respiratory anesthesia, each rat was given 20 μL of the test preparation (Example 15) (concentration 0.1 mg / mL) in both eyes, and immediately after euthanasia at each time point, the lens and vitreous body were collected, and the drug content in the lens and vitreous body was detected. Since the animal lens itself almost does not contain 25-HC, no control eye was set.

[0247] Specifically, after respiratory anesthesia of rats, each group of animals was given 20 μL of the test preparation (Example 15) (concentration 0.1 mg / mL) in both eyes, 1.5 h after administration, 3 animals were euthanized with carbon dioxide, and the lens of both eyes was collected rapidly and stored at -80°C for testing. The sample was treated with an equal volume of 0.10 mM silver acetate methanol solution, and other treatment and test conditions were the same as in Experimental Example 2.

[0248]

[0249] The test results are shown in Table 4:

[0250] Table 4 Content of 25-Hydroxycholesterol (25-HC) in the Lens of Rats after Eye Drops

[0251]

[0252] No 25-hydroxycholesterol was detected in the aqueous humor and vitreous body samples (below the limit of detection, LCQ = 0.001 μg / mL). The above results confirm that the ophthalmic preparation of the present application can effectively deliver 25-hydroxycholesterol through the lens barrier, enrich in the lens.

[0253] Experimental Example 4, Animal Absorption Utilization Rate of Part of the Preparations of the Present Invention

[0254] After eye drops, only about 10% of the drug enters the internal eye, the rest of the majority through the conjunctiva and nasal cavity into the body system (Ling Peixue, editor in chief of "ophthalmic drugs and preparations", China Light Industry Press, 2010, P6), therefore, the utilization rate of the preparation of the present application is calculated according to the following standard:

[0255] For the examples of rats or rabbits as experimental animals, the total absorption rate is A% = [(C-C0) V 晶状体 / V 给药 ]*100%, and the effective absorption rate is A% / 10%.

[0256] Wherein, C is the concentration of active substance in the lens after eye drops, and C0 is the concentration of active substance in the lens without eye drops (μg / mL). Rat lens: diameter = 3.87mm, V 晶状体 =0.03cm 3 ;

[0257] Rabbit lens: diameter = 7.9mm, V 晶状体 =0.258cm 3 ;

[0258] The LAN concentration of examples 1, 3 and 8 is 0.1mg / mL, i.e. 0.1μg / μL; the eye drop administration amount: rat: V 给药 =20uL*0.1μg / μL=2μg; rabbit: V 给药 =50μL*0.1μg / μL=5μg.

[0259] Therefore, the total absorption rate and effective absorption rate of the preparation of examples 1, 3 and 8 are calculated as shown in table 5:

[0260] Table 5 animal absorption rate of part of the preparation of the present application

[0261]

[0262] It can be seen that the preparation of the present application can achieve very high lens absorption rate on the basis of low active substance concentration, and the effective absorption rate of rabbit lens is even as high as 100%. It is shown that the utilization rate of the preparation of the present application is very high, and the active substance for treating cataract is delivered to the lens, which effectively increases the concentration of oxidized cholesterols in the lens, treats cataract, and avoids systemic absorption and side effects.

[0263] Experimental example 5, cataract dog administration observation test

[0264] A Poodle (age 15 years, male, weight 4.0 kg) was kept in an indoor home environment, and was fed and given water on a regular and fixed schedule, and was treated as a pet throughout the experiment. The dog was given one drop of about 30 μL of the preparation of Example 3 per eye once a day. After 20 days of eye drop administration, the dog's autonomous activity range was significantly increased; photographic comparison showed partial regression of the cataract after 20 days Figure 3

[0265] Experimental Example 6, Rat Pharmacodynamic Test

[0266] 1. Test Method A

[0267] Nine-day-old newborn SD rats were selected, and the opening of the eyes was observed daily. When slight opening of the eyes occurred (about 13 days old), the rats were subcutaneously injected with a sodium selenite solution (Na2SeO3, 20 μmol / kg) at a volume of 2 ml / kg, and the same dose of sodium selenite solution was injected again one day later. After the first injection of the sodium selenite solution, the animals were randomly divided into two groups, a control group and a drug administration group, wherein the eyes of each rat in the control group were used as a control, and were administered with a physiological saline (NS) eye drop, and the eyes of each rat in the drug administration group were administered with a test drug (Example 3) eye drop. The administration schedule is shown in Table 6:

[0268] Table 6 Administration Schedule

[0269]

[0270] 2. Cataract Observation and Grade Scoring

[0271] The test animals were anesthetized by inhalation of isoflurane, and the lens turbidity in the eyes of the rats was observed after topical anesthesia with lidocaine, the cataract formation time was recorded, and was scored according to the following criteria:

[0272] Grade 6: Mature cataract, involving the entire lens.

[0273] Grade 5: Nucleus opacity not involving the lens cortex.

[0274] Grade 4: Partial nucleus opacity.

[0275] Grade 3: Diffuse nucleus opacity with some cortical scattering.

[0276] Grade 2: Slight nucleus opacity, swelling fibers or posterior infrarenal opacity produce scattering after 2 to 3 days of injection of selenite.

[0277] Grade 1: Initial signs of nucleus opacity.

[0278] 3. Test Observation Results are Shown in Table 7

[0279] Table 7 Test Observation Results

[0280] ​

[0281] The results of the test showed that on the 2nd day of starting eye drops, all of the 18 eyes of the model control group (dropping normal saline) animals developed grade 4 cataract (100%); while in the administration group (dropping the test drug), 10 / 16 eyes (62.5%) developed grade 4 cataract; on the 6th day, 66.7% of the test eyes of the model group developed grade 5 cataract, and the proportion of the test eyes of the test group developing grade 5 cataract was 50%.

[0282] 4. Test method B

[0283] Select 9-day-old newborn SD rats to observe the open-eye condition every day, when slight open-eye appears (about 13 days old), subcutaneously inject sodium selenite solution (Na2SeO3, 20 μmol / kg), the injection volume is 2 ml / kg. Randomly divide the animals into a model group and an administration group, the animals in the model group are given 10 μL of normal saline (NS) eye drops for each eye, 3 times / day; the animals in the administration group are given 10 μL of the test drug (Example 3) eye drops for each eye, 3 times / day, the administration scheme is shown in Table 6. The test observation results are shown in Table 8:

[0284] Table 8 Test observation results

[0285]

[0286] On the 15th day (D15) of eye drop administration to the test rats, the incidence of grade 3 cataract in the eyes of the model group rats was 68.75%, and 1 / 16 eyes developed grade 4 cataract; the incidence of grade 3 cataract in the eyes of the administration group rats was 28.57%, and no eye developed grade 4 cataract.

[0287] The above results prove that the ophthalmic preparation of the present application can effectively enrich the active substance in the lens by eye drop administration, and has the effects of preventing cataract, delaying the development process of cataract and improving cataract.

[0288] In summary, the present application provides an ophthalmic preparation for preventing and treating cataract by eye drop administration, the ophthalmic preparation of the present application has excellent stability, after eye drop administration, the active substance is enriched in the lens of the test animals, and the effects of treating and preventing cataract are achieved. Moreover, the active substance is not detected in the aqueous humor and vitreous body around the lens, the systemic toxic side effects are avoided, the technical problem that people in the field of ophthalmic drug delivery have always desired to solve but have always failed to successfully solve is solved, and the present application has extremely high clinical application value.

Claims

1. An ophthalmic preparation for eye drop administration, characterized in that, It consists of active substances for treating eye diseases and pharmaceutically acceptable excipients; The active substance for treating eye diseases is lanosterol or 25-hydroxycholesterol, and the content of the active substance for treating eye diseases is 0.01~0.2 mg / mL; The pharmaceutically acceptable excipients are surfactants, thickeners, cosolvents, and solvents; The ophthalmic formulation is composed of polysorbate 80, hydroxypropyl methylcellulose, PVP K12, liquid PEG, lanosterol, and water, wherein the weight ratio of polysorbate 80, hydroxypropyl methylcellulose, PVP K12, liquid PEG, and lanosterol is 100:24:24:1000:1; or, The ophthalmic formulation is composed of polysorbate 80, hydroxypropyl methylcellulose, PVP K12, liquid PEG, lanosterol, and water, wherein the weight ratio of polysorbate 80, hydroxypropyl methylcellulose, PVP K12, liquid PEG, and lanosterol is 25:6:6:500:1; or, The ophthalmic formulation is composed of polysorbate 80, hydroxypropyl methylcellulose, PVP K17, PEG-60 hydrogenated castor oil, lanosterol, and water, wherein the weight ratio of polysorbate 80, hydroxypropyl methylcellulose, PVP K17, PEG-60 hydrogenated castor oil, and lanosterol is 25:6:6:600:1; or, The ophthalmic formulation is composed of polysorbate 80, hydroxypropyl methylcellulose, PVP K30, liquid PEG, lanosterol, and water, wherein the weight ratio of polysorbate 80, hydroxypropyl methylcellulose, PVP K30, liquid PEG, and lanosterol is 25:6:6:500:1; or, The ophthalmic formulation is composed of polysorbate 80, hydroxypropyl cellulose, PVP K15, propylene glycol, lanosterol, and water, wherein the weight ratio of polysorbate 80, hydroxypropyl cellulose, PVP K15, propylene glycol, and lanosterol is 200:5:6:2500:1; or, The ophthalmic formulation is composed of polysorbate 80, hydroxypropyl methylcellulose, PVP K90, liquid PEG, 25-hydroxycholesterol and water, wherein the weight ratio of polysorbate 80, hydroxypropyl methylcellulose, PVP K90, liquid PEG and 25-hydroxycholesterol is 25:6:6:300:

1.

2. The formulation according to claim 1, characterized in that, The pharmaceutically acceptable excipients in the formulation also include any one or more of the following: osmotic pressure regulators, pH regulators, and preservatives; 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 adjuster is any one or more of 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 or its salt; The preservative is any one or more of the following: sorbic acid, chlorobutanol, sodium chlorite, sodium perborate, benzalkonium chloride, benzalkonium bromide, polyquaternium-1, hexadecyltrimethylammonium bromide, methylparaben, ethylparaben, propylparaben, and phenylmercuric nitrate.

3. A method for preparing the formulation according to claim 1 or 2, characterized in that, Includes the following steps: (1) Add surfactant and thickener to solvent and mix to obtain a mixture; (2) Add the active substance for treating eye diseases to the mixture obtained in step (1), add a solubilizer, and disperse and mix to obtain a preliminary suspension; (3) Stir and disperse and / or homogenize the initial suspension obtained in step (2) to obtain the final product.

4. The method according to claim 3, characterized in that, The dispersion in step (2) is selected from at least one of mechanical stirring dispersion, magnetic stirring dispersion, vortex shaking dispersion, shearing dispersion, grinding dispersion, and ultrasonic dispersion.

5. Use of the formulation according to claim 1 or 2 in the preparation of a medicament for the prevention and treatment of cataracts in humans or animals.

6. The use according to claim 5, characterized in that, The drug is a pharmaceutical preparation for ocular administration.

7. The use according to claim 6, characterized in that, The drug is a drug for topical ocular administration.

Images