An ophthalmic topical administration preparation of everolimus

Abstract

The present application provides a topical ophthalmic formulation of everolimus, which is characterized by comprising a therapeutically effective amount of everolimus and the following pharmaceutical excipients: (1) a viscosity-increasing agent; (2) a surfactant; (3) water; (4) other pharmaceutically acceptable pharmaceutical excipients; the surfactant comprises component A and component B, component A is a cyclodextrin-based excipient, and component B is Tween. The diseases or disorders that the formulation can be used to treat or prevent include excessive axial length of the eye, as well as myopia, retinal thinning, degeneration, holes, posterior scleral staphyloma, tigroid fundus, chorioretinal atrophy, macular atrophy, macular splitting, lacquer cracks, Fuchs spots, choroidal neovascularization, and visual impairment associated with these lesions, which are associated with excessive axial length of the eye.

Description

Technical Field

[0001] This application relates to the field of ophthalmic pharmaceutical preparations. Specifically, it relates to a topical ophthalmic administration preparation of everolimus for treating and preventing diseases such as myopia caused by excessive axial length of the eye, and also relates to a device and method for treating the above diseases. Background Art

[0002] Myopia seriously threatens human visual health. With the increasing prevalence year by year, the visual impairment caused by myopia has become a public health problem that cannot be ignored. Currently, the global myopic population has reached 399 million. By 2050, it is estimated that half of the global population will be myopic, and 20% of them will be highly myopic (about 938 million people). The vast majority of myopia in children and adolescents is due to the increase in the axial length of the eye, resulting in light focusing in front of the retina. Currently, myopia has become a major public health and social problem in China, with the trend of younger age and more severe myopia becoming increasingly serious. Preventing and controlling myopia is a major issue related to human visual health and national economy and people's livelihood. China is a big country with a high prevalence of myopia. Preventing myopia is a major scientific and social issue related to national economy and people's livelihood, and there is an urgent need to develop a new, safe, effective, and convenient treatment drug plan to inhibit the occurrence and development of myopia.

[0003] The process of emmetropization of the eyeball is regulated by a feedback mechanism composed of receptors and effectors. Animal experiments have shown that optic nerve transection does not affect the axial elongation of model animals under myopia induction, which indicates that axial elongation is mainly regulated by the eye's own mechanism. Previous studies have found that the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is involved in the occurrence and development of defocus myopia. See Invest Ophthalmol Vis Sci, 2023, 64(10):24. After defocus induction, the mTORC1 signaling pathway in the retina-choroid tissue of guinea pigs is significantly activated, and can be inhibited by intravitreal injection of the mTORC1 inhibitor everolimus. Intravitreal injection of everolimus can inhibit defocus-induced axial elongation, mTORC1 activation, choroidal thinning, and the expression of hypoxia-inducible factor-1α (HIF-1α) in scleral tissue. Immunofluorescence shows that retinal pigment epithelial cells are the main sites of mTORC1 activation after defocus-induced myopia. Combining defocus induction and intravitreal injection of MHY1485 significantly promotes axial elongation, choroidal thinning, and peripapillary choroidal atrophy. This indicates that intervening in the retinal mTORC1 signaling pathway will become a key target for potentially intervening in the occurrence and development of myopia.

[0004] Everolimus is a second-generation inhibitor of mTORC1 developed by Novartis. Everolimus tablets are indicated for the treatment of patients with advanced renal cell carcinoma who have failed treatment with sunitinib or sorafenib. Approved indications also include prevention of rejection in kidney transplantation, treatment of breast cancer, neuroendocrine carcinoma, tuberous sclerosis, etc. Compared with rapamycin, there is more abundant clinical data on systemic application of everolimus, and longer patient follow-up has been completed. Although there are no randomized controlled trials comparing the safety and efficacy of the two mTORC1 inhibitors, previous studies suggest that everolimus has the same efficacy and potentially better tolerance. The main adverse reactions of oral administration at 10 mg / day are non-infectious pneumonia, infection, oral ulcers, and renal failure, and some patients show allergic symptoms. Everolimus has a molecular weight of 958.22 Da, is poorly soluble in water, and is readily soluble in organic solvents such as ethanol and DMSO. Its solubility in water at 20°C room temperature is approximately 1.63 mg / L, and its logP is close to 6, being extremely lipophilic. Based on in vitro cell experiments, its IC50 for mTORC1 is 1.6 - 2.4 nM (1.5 - 2.3 μg / L).

[0005] Although the prior art has demonstrated the feasibility of intravitreal injection of everolimus for the treatment of myopia (see Invest Ophthalmol Vis Sci, 2023, 64(10):24.), intravitreal injection is highly technically demanding and patients have poor compliance. Currently, it is generally used for the treatment of severe diseases such as age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion, and is not suitable for the treatment of myopia. Therefore, it is necessary to develop a dosing route and dosage form that are easily acceptable to patients.

[0006] Ocular topical drug delivery (eye drops or ointments) is restricted by many factors. The anterior segment of the eye consists of the cornea, conjunctiva, iris, ciliary body, and lens, and is filled with aqueous humor. The cornea has no blood vessels and is composed of an epithelial layer, anterior elastic lamina, stromal layer, and endothelial layer. The epithelial layer is hydrophobic; the anterior elastic lamina is a non-cellular colloidal membrane (10 microns); the stromal layer is hydrophilic; and the endothelial layer is a single layer of loosely connected cells with hydrophobicity. This special hydrophobic-hydrophilic-hydrophobic structure makes it difficult for particularly hydrophilic or lipophilic compounds to penetrate the cornea. Generally, compounds with a logP value in the range of 1-3 can penetrate the cornea. The surface of the cornea is also covered by a tear film, which is composed of an oily superficial film that reduces evaporation, an intermediate aqueous layer containing enzymes and bactericidal substances, and a mucous layer containing various proteins that provide lubrication and protection to the cornea. Topical drug delivery formulations will be diluted and excreted by tears. The excretion pathway of tears is through the lacrimal punctum to the lacrimal canaliculus to the lacrimal sac, and then through the nasolacrimal duct to the inferior meatus. The conjunctiva belongs to the adnexa of the eye and is a thin and transparent mucosal tissue. The sac-like space formed by the conjunctiva is the conjunctival sac. The fornix conjunctiva has accessory lacrimal glands. Histologically, the conjunctiva can be divided into an epithelial layer and a lamina propria, and the lamina propria is rich in lymphocytes. The conjunctiva is also rich in small blood vessels. The lens is an elastic and transparent biconvex structure located behind the iris and in front of the vitreous body, and is connected to the ciliary body by the suspensory ligament of the lens to maintain its position. The lens becomes larger and thicker with age, and its elasticity decreases. The posterior segment of the eye accounts for 2 / 3 of the eye and includes the vitreous membrane and all structures behind it, including: the sclera, choroid, retina, vitreous body, and optic nerve. The vitreous cavity is filled with vitreous humor. The vitreous body is a colorless and transparent colloid without blood vessels and nerves and cannot regenerate. Its nutrition comes from the choroid and aqueous humor. The choroid starts from the ora serrata at the front end of the retina and extends around the optic nerve, containing abundant blood vessels to supply nutrition and oxygen to the retina. The retina is a transparent membrane that adheres closely to the inner surface of the choroid. It is mainly composed of retinal pigment epithelial cells, photoreceptor cells, bipolar cells, ganglion cells, horizontal cells, amacrine cells, interplexiform cells, and Muller cells, etc. The central retinal artery supplies nutrition to the inner five layers of the retina and the nerve fibers on the surface of the optic disc. The retina is the most metabolically active tissue in the human body. Currently, ocular topical drug delivery is mainly used to treat diseases on the surface of the eye such as the cornea, glaucoma, and adnexa of the eye, and it is difficult to act on the fundus retina. Drugs used to treat retinal diseases such as macular degeneration or retinal vein occlusion are all administered by intravitreal injection or peribulbar injection.

[0007] Patent No. CN110290835A records an ophthalmic topical drug delivery formulation containing Tween and cyclodextrin. However, the drug with local anesthetic and analgesic effects provided by this patent does not need to be delivered to the retina to take effect. Judging from the data recorded in Example 12 of the specification, the drug is distributed in the cornea and conjunctiva, and a small amount enters the aqueous humor, but it cannot reach the retina. This patent does not give any teaching that the combination of Tween and cyclodextrin can deliver the drug to the retina.

[0008] There are also records in the prior art of using cyclodextrin to deliver dexamethasone (molecular weight 392.461). However, the molecule of everolimus (molecular weight 958.224) is much larger than that of dexamethasone and has a macrocyclic lactone structure. It is found in molecular simulation that its size is larger than the cavity structure of cyclodextrin. Therefore, it is difficult to be encapsulated and delivered. It is also found in experiments that simply using cyclodextrin cannot achieve the delivery of everolimus at all.

[0009] Since the theoretical action target of everolimus is the RPE cells on the retina, if local administration is adopted, it needs to diffuse through the cornea, aqueous humor, lens and vitreous body to reach the posterior segment, and at the same time, it is not desired that its blood drug concentration is too high. Therefore, the technical difficulties to be overcome in developing a local dosage form include but are not limited to: increasing the solubility of everolimus, prolonging the residence time on the eye surface, promoting the penetration of the drug through the cornea, preventing it from entering the blood circulation too much, and prolonging the time to reach the effective concentration at the RPE cells. Summary of the Invention

[0010] To solve the above problems, the present application provides an eye local administration preparation of everolimus, which preparation comprises a therapeutically effective amount of everolimus and the following pharmaceutical excipients:

[0011] (1) Surfactant;

[0012] (2) Water;

[0013] (3) Other pharmaceutically acceptable pharmaceutical excipients;

[0014] The surfactant comprises component A and component B,

[0015] The component A is a cyclodextrin-based excipient, and the component B is Tween.

[0016] The therapeutically effective amount described in the present application means that the concentration of everolimus in the eye local administration preparation is greater than or equal to 0.0001%.

[0017] The eye local administration preparation is selected from eye drops, gels and eye ointments, and eye drops are preferred.

[0018] Preferably, the w / v concentration of everolimus in the preparation is 0.0001% - 0.5%, more preferably 0.0001% - 0.1%, still more preferably 0.0001% - 0.01%, and most preferably 0.001%.

[0019] The cyclodextrin excipients described in this application refer to pharmaceutical excipients containing cyclodextrin structures recorded in various pharmacopoeias, and are selected from one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyalkylidene-β-cyclodextrin, and sulfobutyl-β-cyclodextrin. 2-Hydroxypropyl-β-cyclodextrin (generally abbreviated as HP-β-CD, CAS registration number 128446-35-5) is particularly preferred, with a concentration of 0.5%-10%, and more preferably 5%.

[0020] The Tween is preferably Tween 20 (TWEEN-20), Tween 21 (TWEEN-21), Tween 40 (TWEEN-40), Tween 60 (TWEEN-60), Tween 61 (TWEEN-61), Tween 80 (TWEEN-80), Tween 81 (TWEEN-81), Tween 85 (TWEEN-85), and more preferably Tween 80. The w / v concentration of Tween 80 is 0.5-10%, and more preferably 2%.

[0021] As a variation of the technical solution of this application, the surfactant further includes component C, and component C is a cationic surfactant with a molecular weight less than 1000. Preferably, component C is benzalkonium chloride and / or benzalkonium bromide, and the w / v concentration of benzalkonium chloride and / or benzalkonium bromide is 0.005%. Specifically, when benzalkonium chloride or benzalkonium bromide is used alone, its w / v concentration is 0.005%. When benzalkonium chloride and benzalkonium bromide are used in combination, the sum of their w / v concentrations is 0.005%.

[0022] As a preference, EDTA is included in the other pharmaceutically acceptable excipients, with a w / v concentration of 0.01%.

[0023] Both component C and EDTA can help break the tear film, thereby accelerating drug delivery. Although drug delivery can also be carried out without using component C and EDTA, it may affect drug compliance. For example, the duration that patients need to wait for drug absorption after each administration is different.

[0024] As an optimization of the technical solution of this application, a thickening agent can also be used. The thickening agent described in this application refers to an excipient that can increase the viscosity of the preparation, slow down the dilution and washing away by tears, and thus extend the adhesion time of the medicament on the eyeball surface. Using a thickening agent can reduce drug loss and improve the absorption rate. Exemplary thickening agents include cellulose-based, hyaluronic acid-based, chitosan-based, and polyethylene glycol-based excipients. The above-mentioned excipients of a certain class include chemically modified compounds recorded in the pharmacopoeia. For example, methylcellulose should be regarded as a kind of "cellulose-based excipient".

[0025] Although drug delivery can be achieved without using a thickening agent, experiments have shown that adding an appropriate amount of thickening agent helps to prolong the residence time of the drug on the ocular surface, thereby facilitating the improvement of drug delivery efficiency. In this application, cellulose 4000 is exemplarily selected, with a concentration not exceeding 0.5%, and the preferred concentration is 0.25%. Since the thickening agent is only used to prolong the residence time of the drug on the ocular surface and does not involve the process of delivering to the fundus, those skilled in the art can independently select the type and concentration of thickening agents with similar viscosities, and such selections should all be regarded as equivalent alternatives to the technical solutions of this application.

[0026] To prevent irritation to the patient's eye, the topical ophthalmic preparation may further comprise an osmotic pressure regulator for adjusting the osmotic pressure to approximately 300 mOsm / L, i.e., an isotonic solution. The osmotic pressure regulator can be, for example, sodium chloride, boric acid, glucose, borax, potassium chloride, glycerol, etc. The eye drop may further comprise a pH regulator for adjusting the pH to approximately 7.4 (e.g., between 7.2 and 7.6). The pH regulator can be, for example, phosphate buffer, borate buffer, Gifford buffer, sodium acetate-boric acid buffer.

[0027] To extend the shelf life, the topical ophthalmic preparation may further comprise a preservative. The dosage of the preservative can refer to the conventional dosage in other topical ophthalmic preparations, and those skilled in the art can also make adjustments independently. This application will not elaborate on this. When using component C benzalkonium chloride and / or benzethonium chloride of this application, since benzalkonium chloride and / or benzethonium chloride itself are preservatives, there is no need to additionally add other preservatives.

[0028] This application also provides a treatment device, characterized in that the treatment device is capable of generating medicament droplets, and the medicament is the topical ophthalmic preparation of this application. The device for generating medicament droplets is common knowledge in the art. For example, it can be a bottle-shaped structure with small holes, and by squeezing the bottle body, the liquid in the bottle is dropped out through the small holes. Those skilled in the art can select various forms of treatment device structures as long as they can perform the function of generating medicament droplets.

[0029] This application also provides the use of everolimus in the preparation of a topical ophthalmic preparation for treating or preventing the following diseases or conditions, which are selected from excessive axial length of the eye, and myopia, retinal thinning, degeneration, holes, posterior scleral staphyloma, tigroid fundus, chorioretinal atrophy, macular atrophy, macular splitting, lacquer crack, Fuchs spot, choroidal neovascularization, and visual impairment associated with these lesions related to excessive axial length of the eye.

[0030] The present application also provides the use of the topical ophthalmic formulation of everolimus in the treatment or prevention of the following diseases or conditions, which are selected from excessive axial length of the eye, and myopia, retinal thinning, degeneration, holes, posterior staphyloma, tigroid fundus, chorioretinal atrophy, macular atrophy, macular splitting, lacquer cracks, Fuchs spots, choroidal neovascularization, and visual impairment associated with these lesions related to excessive axial length of the eye.

[0031] To more clearly describe the technical solutions of the present application, the definitions of some terms are as follows.

[0032] "Everolimus" as described in the present application refers to the compound with the CAS registration number 159351-69-6. During the preparation process, the use of salts of everolimus should also be regarded as the use of everolimus.

[0033] "Pharmaceutical excipients" or "excipients" as described in the present application refer to the excipients that are allowed to be added to drugs in the pharmacopoeias or official documents with similar effects issued by the drug regulatory departments of countries or regions such as China, the United States, Europe, and Japan. When there are conflicts among the pharmacopoeias of different countries, the regulations of the jurisdiction where the corresponding patent of the present application is located shall prevail. It is easy for those skilled in the art to understand that based on the above limitations, although "cyclodextrin excipients", "cellulose excipients", etc. in the present application are not limited to substituents, they also have clear meanings. For example, within the territory of China, their meanings are limited to the types recorded in the pharmacopoeia.

[0034] The terms "surfactant" and its subordinate concepts such as "cationic surfactant" and "nonionic surfactant" as described in the present application have accurate definitions in pharmaceutics. For example, reference can be made to the definitions in Section 3 of Chapter 3 of the textbook "Pharmaceutics" (ISBN978-7-122-29868-3) published by Chemical Industry Press. No detailed explanation will be given in the present application. In case of disputes over the definition, this book shall prevail for interpretation.

[0035] Tween as described in the present application refers to polysorbate, or polyoxyethylene sorbitan fatty acid ester, which is a nonionic surfactant. Tween is widely used as an emulsifier and solubilizer for oily substances. Polysorbate is generally considered to be a non-toxic and non-irritating material. Since Tween is an ester formed by sorbitol and different higher fatty acids, Tween is actually a series of products of the same type. For example, Tween-60 is a stearate ester; Tween-80 is an oleate ester; Tween-20 is a laurate ester, which is a mixture of polyoxyethylene sorbitan monolaurate and a part of polyoxyethylene disorbitan monolaurate.

[0036] The concentration as described in the present application, unless otherwise specified, is the w / v concentration. For example, 1% means that 10 mg of solute is contained in 1 ml of the preparation.

[0037] EDTA is ethylenediaminetetraacetic acid, and the phosphate buffer is sodium dihydrogen phosphate or potassium dihydrogen phosphate, which is obtained by adding a small amount of sodium hydroxide or potassium hydroxide to adjust the pH. As common knowledge in the art, an exemplary preparation method for phosphate buffer (pH 7.4) is as follows: Take 1.36 g of potassium dihydrogen phosphate, add 79 ml of 0.1 mol / L sodium hydroxide solution, and dilute with water to 200 ml to obtain it.

[0038] For various compounds described in this application, such as EDTA, they may form salts at different pH values. Since the actually acting components remain unchanged, they should still be considered as falling within the protection scope of this application.

[0039] The construction and detection methods of the animal model in this application basically refer to the published journal literature "mTORC1 Signaling and Negative Lens-Induced Axial Elongation", Ruiheng Zhang et al., Invest Ophthalmol Vis Sci. 2023; 64(10):24. For the convenience of expression, when "IOVS paper" is mentioned in this application, it specifically refers to this literature.

[0040] Compared with the prior art, the progress of this application lies in:

[0041] 1. By using Tween and cyclodextrin excipients, everolimus is delivered to the retinal pigment epithelium (RPE) cells in the fundus through topical ocular administration. Without using cyclodextrin excipients, or replacing Tween with other non-ionic surfactants, everolimus cannot be delivered. This specific combination has technical effects unexpected by those skilled in the art.

[0042] 2. By adopting the preferred technical solution of this application, even when the concentration of everolimus is as low as 0.0001%, it can still reach the effective treatment concentration, thus eliminating the need for intravitreal injection and significantly reducing the usage risk and treatment burden compared with intraocular injection.

[0043] 3. High safety. Even at the highest tested concentration of 0.1%, the blood drug concentration is only equivalent to that of oral tablets. At the preferred concentration of 0.001% and lower formulation concentrations, the safety is greatly improved.

[0044] 4. Based on the above characteristics, the technical solution of this application is particularly suitable for topical ocular administration. When using the preferred solution, even due to individual differences (such as blinking or more excretion through the lacrimal duct) or patient misuse (for example, squeezing out multiple drops at once, or using more than the recommended dose in the instruction manual multiple times in a day) resulting in significant differences in drug metabolism, it is not likely to cause ineffectiveness or serious side effects. Description of the Drawings

[0045] Other features, objectives, and advantages of the present application will become more apparent by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0046] Figure 1 It is a diagram of the axial length of the guinea pig eye after applying a 0.5% concentration of everolimus preparation according to 2.2 of Example 2 of the present application;

[0047] Figure 2 It is a diagram of the axial length of the guinea pig eye according to 2.2 of Example 2 of the present application;

[0048] Figure 3 It is a diagram of the change in the axial length of the guinea pig eye according to 2.2 of Example 2 of the present application;

[0049] Figure 4 It is a diagram of the change in the depth of the guinea pig cornea + anterior chamber according to 2.2 of Example 2 of the present application;

[0050] Figure 5 It is a diagram of the change in the thickness of the guinea pig lens according to 2.2 of Example 2 of the present application;

[0051] Figure 6 It is a diagram of the change in the depth of the guinea pig vitreous body according to 2.2 of Example 2 of the present application;

[0052] Figure 7 It is a diagram of the change in the refractive power of the guinea pig eye according to 2.2 of Example 2 of the present application;

[0053] Figure 8 It is a fundus image of the simple induction group after 3 weeks of defocus-induced myopia according to 2.3 of Example 2 of the present application;

[0054] Figure 9 It is a fundus image after 3 weeks of combined application of defocus-induced myopia and low-concentration everolimus eye drops according to 2.3 of Example 2 of the present application;

[0055] Figure 10 It is a fundus image after 3 weeks of combined application of defocus-induced myopia and medium-concentration everolimus eye drops according to 2.3 of Example 2 of the present application;

[0056] Figure 11 It is a fundus image after 3 weeks of combined application of defocus-induced myopia and high-concentration everolimus eye drops according to 2.3 of Example 2 of the present application;

[0057] Figure 12 It is a cross-sectional structure diagram of the fundus retina, choroid, and sclera centered on the optic disc according to 2.4 of Example 2 of the present application;

[0058] Figure 13 It is a diagram of the change in the thickness of the guinea pig retina according to 2.4 of Example 2 of the present application;

[0059] Figure 14 is the graph of the change in the choroid thickness of a guinea pig according to 2.4 of Embodiment 2 of the present application;

[0060] Figure 15 is the graph of the change in the sclera thickness of a guinea pig according to 2.4 of Embodiment 2 of the present application. Detailed implementation manners

[0061] To better understand the present application, the technical solutions of the present application will be described in more detail with reference to the accompanying drawings. It should be understood that these detailed descriptions are only descriptions of the exemplary embodiments of the present application and are not intended to limit the scope of the present application in any way. Throughout the specification, the same reference numerals refer to the same elements. The expression "and / or" includes any combination or all combinations of one or more of the listed items associated therewith.

[0062] In the accompanying drawings, for the sake of convenience of illustration, the dimensions, scales, and shapes of the legends have been slightly adjusted. The accompanying drawings are only examples and are not drawn strictly to scale. As used herein, terms such as "substantially", "about", and similar terms are used as terms indicating approximation and not as terms indicating degree, and are intended to account for the inherent deviations in measured or calculated values that would be recognized by a person of ordinary skill in the art.

[0063] It should also be understood that expressions such as "comprises", "comprising", "has", "including", and / or "including having" are open-ended rather than closed-ended expressions in this specification, which mean the presence of the stated features, elements, and / or components, but do not exclude the presence of one or more other features, elements, components, and / or combinations thereof. In addition, when an expression such as "at least one of..." appears after a list of listed features, it modifies the entire list of features rather than just a single feature in the list. In addition, when describing the embodiments of the present application, the use of "may" means "one or more embodiments of the present application". And the term "exemplary" is intended to refer to an example or illustration.

[0064] Unless otherwise defined, all terms used herein (including engineering terms and scientific and technical terms) have the same meaning as commonly understood by a person of ordinary skill in the art to which the present application pertains. It should also be understood that, unless clearly stated otherwise in the present application, words defined in a commonly used dictionary should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and should not be interpreted in an idealized or overly formal sense.

[0065] It should be noted that, without conflict, the features in the embodiments and examples of this application can be combined with each other. Additionally, unless clearly defined or inconsistent with the context, the specific steps included in the methods described in this application do not have to be limited to the recorded order, but can be executed in any order or executed in parallel. The following will detail this application with reference to the accompanying drawings and in combination with the embodiments.

[0066] Experimental method description:

[0067] 1. Construction of animal model

[0068] In this application, guinea pigs are used as experimental animals, and the animal model is constructed using the method described in the IOVS paper in the literature.

[0069] 2. Preparation and administration of preparations

[0070] Unless otherwise specified, all ophthalmic topical administration preparations in the examples of this application are diluted with physiological saline containing phosphate buffer. After dilution, the osmotic pressure is approximately 300 mOsm / L, and the pH is adjusted to 7.4.

[0071] When detecting tissue concentration and blood drug concentration, for each sample, 3 experiments are carried out in parallel. For each experiment, the drug is administered at a dose of 1 drop once a day for 10 days, and the volume of each drop is approximately 30 μL. When detecting in 2.2 - 2.4 of Example 2, for each sample, 8 experiments are carried out in parallel, and the drug is administered continuously for three weeks. The drug is administered at a dose of 1 drop once a day, and the volume of each drop is approximately 30 μL.

[0072] 3. Detection of the content of everolimus in animal tissues

[0073] The content of everolimus in animal tissues is detected by Tianjin Puri Huasheng Medical Laboratory Co., Ltd. using a high performance liquid chromatography - triple quadrupole tandem mass spectrometer HPLC - MS / MS API3200MD.

[0074] Instrument model: HPLC - MS / MS (shimadzu LC20AD - API 3200MD TRAP);

[0075] Methanol, acetonitrile, etc. are all purchased from sigma.

[0076] Sample pretreatment: For the solid samples submitted for inspection, after weighing an appropriate sample and grinding it, 100 ul of methanol - water (8:2) is added, vortex - shaken for 5 min, centrifuged at 13200 rpm for 6 minutes in the cold, 50 ul of the supernatant is taken and 150 ul of methanol is added to precipitate proteins, vortex - shaken for 1 min, centrifuged at 13200 rpm for 6 minutes in the cold, and 80 ul is taken for testing on the machine.

[0077] Liquid phase conditions:

[0078] Chromatographic column: Agela Venusil MP C18 (100*4.6mm, 3um), column temperature: 50°C, flow rate: 1.0 ml / min,

[0079] Mobile phase: A organic phase: methanol (2 mmol / L ammonium acetate + one-thousandth formic acid)

[0080] B aqueous phase: water (2 mmol / L ammonium acetate + one-thousandth formic acid)

[0081] Sample injection volume: 10 ul

[0082] Gradient: isocratic elution

[0083] 1 2 3 4 5 6 7 8 Retention 0.01 0.70 0.71 1.9 1.91 3.0 3.01 4.0 ％A 35 35 95 95 100 100 35 35 ％B 65 65 5 5 0 0 65 65

[0084] Mass spectrometry conditions:

[0085] Ion source: +ESI electrospray ionization source, IS: +5000V (spray voltage), GS1: 50 psi (nebulizing gas), GS2: 60 psi (auxiliary gas), scanning mode: MRM multiple reaction monitoring, CAD: 3Medium (collision gas), TEM: 550°C (nebulization temperature), CUR: 20 psi (curtain gas), CXP: +2.0 (collision cell exit pressure), EP: +10 (injection voltage).

[0086] Since it is impossible to accurately isolate and detect RPE cells in the experiment, the drug content in the retinal choroid tissue located at the fundus of the eye is detected instead.

[0087] The detection limit of drug concentration is 0.0001 ng / mg, and values lower than this cannot be detected and are recorded as 0. In this application, the experimental results are retained to four significant figures.

[0088] 4. Detection method and description of the drawings

[0089] To detect the axial length of the eye, tessellated lesions, retina, choroid, scleral thickness, etc., the detection methods in the IOVS papers are used.

[0090] Unless otherwise specified, in the drawings of the specification, the five data in the same group are, from left to right, the blank control group, the simple induction group, the low concentration group, the medium concentration group, and the high concentration group. The number of * represents the P value. 1 * indicates P < 0.05 with statistical significance, 2 * indicates P < 0.01 with significant statistical significance, and 3 * indicates P < 0.001 with statistical significance.

[0091] Example 1

[0092] Prepare eye drops as shown in Table 1.

[0093]

[0094]

[0095] After weighing the above components, dilute with physiological saline containing phosphate buffer salts, adjust the osmotic pressure to about 300 mOsm / L, and adjust the pH to 7.4.

[0096] Example 2 Determination of the Everolimus Dose

[0097] 2.1 Choroidal Drug Concentration

[0098] As shown in Table 2, prepare the following everolimus ophthalmic topical administration preparations with reference to Example 1, but change the concentration of everolimus. After 10 days of administration, sacrifice the guinea pigs and detect the choroidal drug concentration and blood drug concentration.

[0099] Table 2 Determination of the Everolimus Dose

[0100]

[0101] As can be seen from Table 2, the concentration of everolimus in the choroid can already reach the therapeutic level at a concentration of 0.0001%. As the concentration of everolimus in the eye drops increases, the concentration of everolimus in the retina-choroid tissue further increases; in contrast, the increase in blood drug concentration is more rapid. The reason may be that the eye drops are absorbed by the periorbital blood vessels or absorbed after entering the lacrimal duct, thus increasing the blood drug concentration, while the structure inside the eyeball is complex and the penetration speed is slow.

[0102] In order to shorten the axial length of the eye, we hope to deliver more everolimus to the retina-choroid. Since this drug has systemic side effects, a lower blood drug concentration is beneficial to its medication safety. A concentration of 0.0001% - 0.001% is more ideal.

[0103] To further determine the everolimus dose, with reference to Example 1, preparations with ultra-high concentrations of 0.5%, 0.1% (high dose), 0.01% (medium dose), and 0.001% (low dose) were prepared. Preparations without everolimus and non-myopia-induced guinea pigs were used as the blank control group, and preparations without everolimus and the myopia-induced guinea pig model were used as the simple induction group (LIM + Vehicle) group. Parallel tests were conducted with the high, medium, and low dose groups (using the myopia-induced guinea pig model), and observations were made for 3 weeks. The results are shown in 2.2 - 2.4 of this example.

[0104] 2.2 Changes in Axial Length of the Eye

[0105] See Figure 1As shown, compared with the blank control group, the axial length of the guinea pig eyes was significantly prolonged after myopia induction. After applying 0.5% everolimus, the axial length elongation caused by defocus myopia was significantly alleviated, preliminarily demonstrating that topical administration of everolimus can achieve pharmacological effects.

[0106] To further determine the effective concentration range downward, three groups of high, medium, and low concentrations were used for exploration.

[0107] See Figure 2 , compared with the blank control group and the defocus induction group, there was no significant difference in the axial length of the left and right eyes. After applying low (0.001%), medium (0.01%), and high concentration (0.1%) everolimus eye drops to the right eye during defocus induction, and applying the carrier solution without everolimus to the left eye, the axial length elongation of the right eye of the guinea pig was significantly inhibited, manifested as the axial length of the right eye being significantly shorter than that of the left eye.

[0108] See Figure 3 , compared with the blank control group, the axial length of the guinea pig eyes was significantly prolonged after myopia induction. After applying low (0.001%), medium (0.01%), and high concentration (0.1%) everolimus eye drops to the right eye during defocus induction, the axial length elongation was significantly alleviated.

[0109] As can be seen from the figure, one week later, the axial length of the three concentration groups showed an obvious shortening trend compared with the defocus myopia group. As the administration time became longer, the shortening trend became more obvious. There was no obvious difference among the three dose groups.

[0110] See Figure 4 , compared with the blank control group, there was no obvious change in the corneal + anterior chamber depth of the guinea pig eyes after myopia induction. After applying low (0.001%), medium (0.01%), and high concentration (0.1%) everolimus eye drops to the right eye during defocus induction, there was no obvious change in the corneal + anterior chamber depth.

[0111] See Figure 5 , compared with the blank control group, there was no obvious change in the lens thickness of the guinea pig eyes after myopia induction. After applying low (0.001%), medium (0.01%), and high concentration (0.1%) everolimus eye drops to the right eye during defocus induction, there was no obvious change in the lens thickness.

[0112] See Figure 6 , compared with the blank control group, the vitreous depth of the guinea pig eyes was significantly increased after myopia induction. After applying low (0.001%), medium (0.01%), and high concentration (0.1%) everolimus eye drops to the right eye during defocus induction, the increase in vitreous depth was significantly alleviated.

[0113] Figures 4 - 6Note that after the induction of defocus myopia, the vitreous depth mainly increases, without affecting the lens thickness or anterior chamber depth. After the application of eye drops at three concentrations, the vitreous depth showed an obvious tendency to shorten compared with that in the defocus myopia group. There was no obvious difference among the three concentration groups.

[0114] See Figure 7 , through streak retinoscopy, guinea pigs showed an obvious tendency of myopia (decrease in diopter) after the induction of defocus myopia. After the application of eye drops at each concentration, the degree of myopia in guinea pigs showed an obvious tendency to slow down compared with that in the defocus myopia group..

[0115] 2.3 Fundus morphology (leopard pattern lesions)

[0116] See Figure 8 , after 3 weeks of defocus induction, strip leopard pattern lesions widely distributed in the posterior pole could be seen in the fundus images of the simple induction group.

[0117] See Figure 9 , after 3 weeks of combined application of low-concentration everolimus eye drops in defocus induction, the fundus images showed that the strip leopard pattern lesions widely distributed in the posterior pole were less than Figure 8 .

[0118] See Figure 10 , after 3 weeks of combined application of medium-concentration everolimus eye drops in defocus induction, the fundus images showed that the strip leopard pattern lesions widely distributed in the posterior pole were less than Figure 9 .

[0119] See Figure 11 , after 3 weeks of combined application of high-concentration everolimus eye drops in defocus induction, the fundus images showed that the strip leopard pattern lesions widely distributed in the posterior pole were less than Figure 10 .

[0120] 2.4 Retina, choroid and sclera thickness study

[0121] Three weeks after administration, see Figure 12 , through OCT examination (the method is shown in the IOVS article), cross-sectional structure diagrams of the fundus retina, choroid and sclera centered on the optic disc can be obtained. In the figures, the membranes of each layer in defocus myopia (induction group) were thinned.

[0122] To more intuitively show the thickness changes of each layer of the membrane, the thickness values of each layer of the membrane were further measured and shown in Figures 13 - 15 .

[0123] See Figure 13, measurements were taken at the superior, inferior, nasal, and temporal retinas at a position 3 disc diameters centered on the optic disc. Compared with the blank control group, the retinal thickness in each direction was significantly thinned after simple myopia induction, and this change was closely related to myopia progression. After applying low (0.001%), medium (0.01%), and high-concentration (0.1%) everolimus eye drops to the right eye during defocus induction, the thinning of the retina at the same position was significantly alleviated.

[0124] See Figure 14 , measurements were taken at the superior, inferior, nasal, and temporal choroids at a position 3 disc diameters centered on the optic disc. Compared with the blank control group, the choroidal thickness in each direction was significantly thinned after simple myopia induction, and this change was closely related to myopia progression. After applying low (0.001%), medium (0.01%), and high-concentration (0.1%) everolimus eye drops to the right eye during defocus induction, the thinning of the choroid at the same position was significantly alleviated.

[0125] See Figure 15 , measurements were taken at the superior, inferior, nasal, and temporal scleras at a position 3 disc diameters centered on the optic disc. Compared with the blank control group, the scleral thickness in each direction was significantly thinned after simple myopia induction, and this change was closely related to myopia progression. After applying low (0.001%), medium (0.01%), and high-concentration (0.1%) everolimus eye drops to the right eye during defocus induction, the thinning of the sclera at the same position was significantly alleviated.

[0126] Example 3 Determination of the amount of thickening agent

[0127] As shown in Table 3, the following everolimus ophthalmic topical administration preparations were prepared with reference to Example 1, but the amount of the thickening agent was changed. The guinea pigs were sacrificed 10 days after administration, and the choroidal drug concentration was detected.

[0128] Table 3 Determination of the thickening agent concentration

[0129]

[0130] Within a certain range, increasing the amount of the thickening agent can prolong the residence time of the drug on the eye surface, which is beneficial to improving the absorption rate. However, too much thickening agent will reduce the fluidity of the preparation to form a gel or paste, making it difficult to control the administration dose. Moreover, everolimus encapsulated by cyclodextrin and surfactant has a relatively large collision volume and is difficult to migrate freely in the reticular structure of the colloid, resulting in a decrease in the absorption rate. In this application, through experimental tests, the preferred amount of methylcellulose 4000 is: 0.25%.

[0131] Example 4 Optimization of the type and concentration of cyclodextrin excipients

[0132] As shown in Table 4, the following topical ophthalmic formulations of everolimus were prepared with reference to Example 1, but the types and concentrations of cyclodextrin excipients were changed. After 10 days of administration, the guinea pigs were sacrificed, and the drug concentrations in the choroid and blood were measured.

[0133] Table 4 Optimization of the types and concentrations of cyclodextrin excipients

[0134]

[0135] The data in No. 3 of Table 4 directly adopted the values of Example 1. As can be seen from Table 4, at lower concentrations, the dosage of cyclodextrin had little effect on the delivery efficiency, and the best effect was achieved at about 5%. When the concentration was too high, the effect was reduced instead. γ-Cyclodextrin also had a delivery effect, but the delivery effect was not as good as that of HP-β-CD at the same concentration, and the whole preparation was milky white and opaque, which might cause transient blurred vision. The data in No. 6 proved that the delivery could not be completed without using cyclodextrin excipients.

[0136] Example 5 Adjustment of the types and dosages of surfactants

[0137] As shown in Table 5, the following topical ophthalmic formulations of everolimus were prepared with reference to Example 1, but the types and dosages of non-ionic surfactants were changed. As a control, Span 60 and Transcutol P, which are commonly used in ophthalmic administration, were selected. After 10 days of administration, the guinea pigs were sacrificed, and the drug concentration in the choroid was measured.

[0138] Table 5

[0139]

[0140] Note: Transcutol P is P, with the chemical name of diethylene glycol monoethyl ether, is a potent solubilizer and is commonly used as a penetration enhancer for topical preparations. In order to avoid irritating the surface of the eyeball, its concentration in approved eye drops generally does not exceed 0.03%. See Effects of Transcutol P on the corneal permeability of drugs and evaluation of its ocular irritation of rabbit eyes, Liu, Zhidong et al, Journal of Pharmacy and Pharmacology, 2006, 58(1): 45-50. The non-ionic surfactant was not used in the column where No. 7 is located.

[0141] This example demonstrated that non-ionic surfactants of the Tween type, especially Tween 80, in combination with cyclodextrins could effectively deliver everolimus, while the use of other non-ionic surfactants had no delivery effect at all.

[0142] Further experiments also show that although EDTA, benzalkonium bromide, and benzalkonium chloride can break the tear film and promote drug delivery, they are not necessary. When their content is 0, as long as there are cyclodextrin excipients and Tween present, the delivery of everolimus can also be achieved. Since the functions of components C such as EDTA, benzalkonium bromide, and benzalkonium chloride are all to break the tear film and improve drug delivery efficiency, any one of them can be selected, or they can be used simultaneously. The principle of using both EDTA and component C in Example 1 of this application is that using only EDTA has no antibacterial and preservative effect, and other preservatives need to be added to ensure sterility. However, adding too much benzalkonium bromide or benzalkonium chloride will cause irritation and affect the comfort of use.

[0143] The above description is only for the implementation mode of this application and the explanation of the technical principles applied. Those skilled in the art should understand that the scope of protection involved in this application is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the technical concept. For example, the technical solutions formed by mutually replacing the above features with (but not limited to) the technical features with similar functions disclosed in this application.

Claims

1. An ophthalmic topical administration preparation of everolimus capable of delivering a therapeutic level concentration of everolimus to retinal pigment epithelial cells, characterized in that, Comprising a therapeutically effective amount of everolimus and the following pharmaceutical excipients: (1) Surfactant; (2) Water; (3) Other pharmaceutically acceptable pharmaceutical excipients; The surfactant comprises component A and component B, Component A is hydroxypropyl-β-cyclodextrin or γ-cyclodextrin excipient, component B is Tween, and the Tween is Tween 40, Tween 60 or Tween 80; The eye topical administration preparation is an eye drop.

2. The ophthalmic topical administration preparation according to claim 1, wherein The w / v concentration of everolimus in the preparation is 0.0001% - 0.5%.

3. The ophthalmic topical administration preparation according to claim 1, wherein Component A is hydroxypropyl-β-cyclodextrin, and the w / v concentration of hydroxypropyl-β-cyclodextrin is 0.5% - 10%.

4. The ophthalmic topical administration preparation according to claim 1, wherein The Tween is Tween 80, and the w / v concentration of Tween 80 is 0.5 - 10%.

5. The ophthalmic topical administration preparation according to claim 1, characterized in that, The surfactant further comprises component C, and component C is a cationic surfactant with a molecular weight less than 1000.

6. The ophthalmic topical administration preparation according to claim 5, wherein Component C is benzalkonium chloride and / or benzalkonium bromide, and the w / v concentration of benzalkonium chloride and / or benzalkonium bromide is 0.005%.

7. The ophthalmic topical administration preparation according to claim 1, wherein, The pharmaceutical excipients further comprise a thickening agent, and the thickening agent is selected from one or more of cellulose-based, hyaluronic acid-based, chitosan-based, polyethylene glycol-based excipients.

8. The ophthalmic topical administration preparation according to claim 7, wherein, The thickening agent is methylcellulose 4000, and the concentration is 0.25%.

9. The ophthalmic topical administration preparation according to claim 1, wherein, The other pharmaceutically acceptable pharmaceutical excipients include EDTA, and the w / v concentration is 0.01%.

10. A treatment device, characterized in that, The treatment device is capable of generating medicament droplets, and the medicament is the eye topical administration preparation according to any one of claims 1 - 9.

11. Use of everolimus in the preparation of an eye topical administration preparation for preventing the following diseases or conditions, wherein the diseases or conditions are selected from excessive eye axis length, and myopia, retinal thinning, retinal degeneration, retinal tear, posterior scleral staphyloma, tigroid fundus, chorioretinal atrophy, macular atrophy, macular splitting, lacquer crack, Fuchs spot, choroidal neovascularization and visual impairment associated with these lesions related to excessive eye axis length, and the preparation is the eye topical administration preparation according to any one of claims 1 - 9.

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