Everolimus topical ocular administration formulation

Abstract

An everolimus topical ocular administration formulation, characterized by comprising a therapeutically effective amount of everolimus and following pharmaceutical excipients: (1) a surfactant; (2) water; and (3) other pharmaceutically acceptable pharmaceutical excipients, wherein the surfactant comprises a component A and a component B, the component A being a cyclodextrin excipient and the component B being Tween. Diseases or disorders that may be treated or prevented by the formulation include axial elongation, axial elongation-associated myopia, retinal thinning, retinal degeneration, retinal holes, posterior scleral staphyloma, tessellated fundus, chorioretinal atrophy, macular atrophy, macular retinoschisis, lacquer cracks, Fuchs spots and choroidal neovascularization, and visual impairment associated with these disorders.

Description

An everolimus eye topical administration preparation TECHNICAL FIELD

[0001] The present application relates to the field of ophthalmic pharmaceutical preparations, in particular, an everolimus eye topical administration preparation for treating and preventing myopia and other diseases caused by excessive axial length, and a device and method for treating the diseases. BACKGROUND

[0002] Myopia seriously threatens human visual health. With the increasing prevalence year by year, visual impairment caused by myopia has become an important public health problem. At present, the global myopia population has reached 399 million, and by 2050, half of the global population is expected to have myopia, of which 20% of the population is high myopia (about 938 million people). Most of the myopia in children and adolescents is due to the growth of the axial length, which causes light to focus in front of the retina. At present, myopia has become a major public health and social problem in China, and myopia is becoming younger and more severe. Prevention and control of myopia is a major problem related to human visual health and the national economy and people's livelihood. China is a large country of myopia, and prevention of myopia is a major scientific and social problem related to the national economy and people's livelihood, and it is urgent to develop a safe, effective and convenient new treatment drug regimen to inhibit the occurrence and development of myopia.

[0003] The emmetropization process is regulated by a feedback mechanism composed of receptors and effectors. Animal experiments show that optic nerve transection does not affect the axial elongation of model animals under myopia induction, which suggests that axial elongation is mainly regulated by the mechanism of the eye itself. 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. The mTORC1 signaling pathway in the retinal choroidal tissue of guinea pigs induced by defocus is significantly activated and can be inhibited by intravitreal injection of mTORC1 inhibitor everolimus. Intravitreal injection of everolimus can inhibit the axial elongation, mTORC1 activation, choroidal thinning and expression of hypoxia-inducible factor-1α (HIF-1α) in the scleral tissue induced by defocus. Immunofluorescence shows that retinal pigment epithelial cells are the main location of mTORC1 activation after defocus-induced myopia. Combined with defocus induction and intravitreal injection of MHY1485, it significantly promotes axial elongation, choroidal thinning and peripapillary choroidal atrophy. This indicates that intervention of the retinal mTORC1 signaling pathway will become a key target for potential intervention of 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 after failure of treatment with sunitinib or sorafenib. The approved indications also include prevention of rejection in kidney transplantation, treatment of breast cancer, neuroendocrine cancer, tuberous sclerosis, etc. Compared with rapamycin, there are more clinical data on the systemic application of everolimus, and longer patient follow-up has been completed. Although there is no randomized controlled trial to compare the safety and effectiveness of the two mTORC1 inhibitors, previous studies suggest that everolimus has the same effectiveness and potential better tolerability. The main adverse reactions of oral 10mg / day are non-infectious pneumonia, infection, oral ulcer, renal failure, and some patients show allergic symptoms. The molecular weight of everolimus is 958.22 Da, which is difficult to dissolve in water and easy to dissolve in organic solvents such as ethanol and DMSO. Its solubility in water at room temperature is about 1.63mg / L, and the logP is close to 6, which is extremely lipophilic. Based on in vitro cell experiments, its IC50 for mTORC1 is 1.6-2.4nM (1.5-2.3ug / L).

[0005] Although the existing technology has proved the feasibility of intravitreal injection of everolimus for the treatment of myopia (see Invest Ophthalmol Vis Sci, 2023, 64(10): 24.), intravitreal injection is difficult to operate and has poor patient compliance. It is currently generally used for the treatment of serious 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 drug delivery route and dosage form that is easy for patients to accept.

[0006] The eye is a complex organ with a number of factors that limit the effectiveness of topical ocular drug delivery. The anterior segment of the eye is composed of the cornea, conjunctiva, iris, ciliary body, and lens, and is filled with aqueous humor. The cornea is avascular and is composed of an epithelial layer, an anterior limiting membrane, a stroma layer, and an endothelial layer. The epithelial layer is hydrophobic; the anterior limiting membrane is a non-cellular, gelatinous membrane (10 microns); the stroma layer is hydrophilic; and the endothelial layer is a single layer of loosely connected cells that are hydrophobic. This unique hydrophobic-hydrophilic-hydrophobic structure makes it difficult for either particularly hydrophilic or lipophilic compounds to penetrate the cornea. It is generally accepted that only compounds with a logP in the range of 1-3 can penetrate the cornea. The surface of the cornea is also covered with a tear film, which is composed of an oily surface film that reduces evaporation, an intermediate aqueous layer that contains enzymes and bactericidal substances, and a mucous layer that contains a variety of proteins and provides lubrication and protection to the cornea. Topical formulations are diluted by the tear fluid and are excreted. The tear fluid is excreted through the lacrimal puncta to the lacrimal canaliculi to the lacrimal sac, and then through the nasolacrimal duct to the inferior nasal meatus. The conjunctiva is a thin, transparent mucous membrane that is part of the ocular adnexa. The space formed by the conjunctiva is the conjunctival sac. The palpebral conjunctiva contains the accessory lacrimal glands. The conjunctiva is histologically divided into an epithelial layer and a substantia propria, which is rich in lymphocytes. The conjunctiva is also rich in small blood vessels. The lens is a transparent, elastic, biconvex structure that is located behind the iris and in front of the vitreous body. It is connected to the ciliary body by the zonular suspensory ligaments, which maintain the position of the lens. The lens increases in size and thickness and loses elasticity with age. The posterior segment of the eye occupies 2 / 3 of the eye and includes the vitreous membrane and all of the structures behind it, including the sclera, choroid, retina, vitreous body, and optic nerve. The vitreous body is a colorless, transparent gel that is avascular and non-regenerative and receives its nutrients from the choroid and aqueous humor. The choroid is a vascular layer that supplies nutrients and oxygen to the retina and extends from the ora serrata of the retina to the optic nerve. The retina is a transparent membrane that is attached to the inner surface of the choroid. It is composed of retinal pigment epithelial cells, rods, bipolar cells, ganglion cells, horizontal cells, amacrine cells, interplexiform cells, and Muller cells. The central retinal artery supplies nutrients to the five layers of the retina and the surface of the optic disc. The retina is the most metabolically active tissue in the body. Currently, topical ocular drug delivery is mainly used to treat diseases of the cornea, glaucoma, and ocular adnexa, and it is difficult to act on the retina. Drugs for treating macular degeneration or retinal vein occlusion are injected into the vitreous body or the peribulbar space.

[0007] CN110290835A discloses an ophthalmic topical drug delivery formulation containing Tween and cyclodextrin. However, the topical anesthetic analgesic drug provided in this patent does not need to be delivered to the retina to take effect. As shown in the data in Example 12 of the specification, the drug is distributed in the cornea and conjunctiva and a small amount enters the aqueous humor, but cannot reach the retina. This patent does not teach that the combination of Tween and cyclodextrin can deliver drugs to the retina.

[0008] There are also records in the prior art of using cyclodextrin to deliver dexamethasone (molecular weight 392.461), but everolimus (molecular weight 958.224) is much larger than dexamethasone, and has a macrocyclic lactone structure. When the molecule is simulated, it is found that its size is larger than the cavity structure of cyclodextrin, so it is difficult to be wrapped and delivered. It is also found in experiments that the use of cyclodextrin alone cannot achieve the delivery of everolimus.

[0009] Since the theoretical target of everolimus is the RPE cells on the retina, if local administration is used, it needs to diffuse through the cornea, aqueous humor, lens and vitreous to the posterior segment, and at the same time, it is not desirable for its blood concentration to be too high. Therefore, to develop a local administration dosage form, the technical difficulties to be overcome include but are not limited to: increasing the solubility of everolimus, prolonging the residence time on the surface of the eyeball, promoting the penetration of the drug through the cornea, preventing its excessive entry into the blood circulation, and prolonging the time for it to reach an effective concentration at the RPE cells. SUMMARY

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

[0011] (1) a 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 excipient, and the component B is Tween.

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

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

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

[0019] The cyclodextrin-based excipient described in the present application refers to the pharmaceutical excipient containing cyclodextrin structure recorded in the Pharmacopoeia of various countries, which is selected from one or more of a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyalkylene-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and particularly preferably 2-hydroxypropyl-β-cyclodextrin (commonly abbreviated as HP-β-CD, CAS registration number 128446-35-5), with a concentration of 0.5% to 10%, more preferably 5%.

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

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

[0022] As a preferred, the pharmaceutically acceptable other pharmaceutical excipient includes EDTA, with a w / v concentration of 0.01%.

[0023] Both the component C and EDTA can help break the tear film, thereby accelerating drug delivery. Although delivery can be performed without using the component C and EDTA, it may affect drug compliance, for example, patients need to wait for different lengths of time after each medication for drug absorption.

[0024] As an optimization of the technical solution of the present application, a tackifier can also be used. The tackifier described in the present application refers to an excipient that can increase the viscosity of the preparation, slow down the speed of being diluted and washed away by tears, thereby prolonging the adhesion time of the drug on the eyeball surface. The use of a tackifier can reduce drug loss and improve absorption rate. Exemplary tackifiers include cellulose-based, hyaluronic acid-based, chitosan-based, and polyethylene glycol-based excipients. The above certain class of excipients includes the chemically modified compounds recorded in the Pharmacopoeia, for example, methyl cellulose should be regarded as one of the "cellulose-based excipients".

[0025] Although the drug delivery can be accomplished without using a viscosity-increasing agent, experiments show that adding an appropriate amount of viscosity-increasing agent helps prolong the time of the drug on the surface of the eyeball, thus improving the efficiency of drug delivery. The application exemplarily selects cellulose 4000, with a concentration not exceeding 0.5%, and a preferred concentration of 0.25%. Since the viscosity-increasing agent is only used to prolong the time of the drug on the surface of the eyeball and does not involve the process of delivery to the fundus, the person skilled in the art can select the type and concentration of the viscosity-increasing agent with similar viscosity by himself / herself, and such selection shall be regarded as an equivalent alternative of the technical solution of the application.

[0026] In order to prevent irritation to the patient's eye, the eye topical administration preparation can further comprise an osmotic pressure regulator for adjusting the osmotic pressure to about 300 mOsm / L, i.e. an isotonic solution, which can use, for example, sodium chloride, boric acid, glucose, borax, potassium chloride, glycerol, etc. The eye drops can also comprise a pH regulator for adjusting the pH to about 7.4 (e.g. between 7.2-7.6), which can use, for example, phosphate buffer, borate buffer, Gifford buffer, sodium acetate-boric acid buffer.

[0027] In order to prolong the shelf life, the eye topical administration preparation can further comprise a preservative, which can refer to the conventional amount in other eye topical administration preparations, and the person skilled in the art can also adjust it by himself / herself, which will not be described herein. When the component C benzalkonium chloride and / or benzalkonium bromide described in the application is used, since benzalkonium chloride and / or benzalkonium bromide itself is a preservative, it can not be necessary to additionally add other preservatives.

[0028] The application also provides a treatment device, characterized in that the treatment device can generate droplets of a medicament, and the medicament is the eye topical administration preparation described in the application. The device for generating droplets of a medicament is common in the art, for example, a bottle-shaped structure with a small hole, by squeezing the bottle body to make the liquid in the bottle drip through the small hole. The person skilled in the art can select various forms of treatment device structures as long as they can generate droplets of a medicament.

[0029] The application also provides the use of everolimus in the preparation of an eye topical administration preparation for treating or preventing the following diseases or conditions: axial length overgrowth, myopia associated with axial length overgrowth, retinal thinning, degeneration, hole, posterior scleral staphyloma, leopard-shaped fundus, choroid retinal atrophy, macular atrophy, macular splitting, lacquer crack, Fuchs spot, choroidal neovascularization and visual impairment associated with these lesions.

[0030] The application also provides use of the above-mentioned eye local administration formulation of everolimus in treating or preventing the following diseases or conditions: axial length overgrowth, myopia associated with axial length overgrowth, retinal thinning, degeneration, retinal breaks, posterior scleral staphyloma, leopard-like fundus, chorioretinal atrophy, macular atrophy, macular foveoschisis, lacquer cracks, Fuchs spot, choroidal neovascularization, and visual impairment associated with these lesions.

[0031] In order to more clearly express the technical solutions of the application, some terms are defined as follows.

[0032] "Everolimus" as used in the present application refers to the compound with CAS No. 159351-69-6. The use of a salt of everolimus in the preparation process should also be regarded as the use of everolimus.

[0033] "Pharmaceutically acceptable excipient" or "excipient" as used in the present application refers to an excipient allowed to be added in a drug by a pharmacopoeia or an official document with similar power issued by a drug regulatory department of a country or region such as China, the United States, Europe, Japan, etc. When the pharmacopoeias of different countries conflict, the provisions of the jurisdiction of the same family patent of the present application shall prevail. It is easy for those skilled in the art to understand that based on the above definition, "cyclodextrin excipient", "cellulose excipient" and the like in the present application also have clear meanings although the substituents are not limited, for example, in China, its meaning is limited to the types recorded in the pharmacopoeia.

[0034] "Surfactant" and its sub-concepts "cationic surfactant", "non-ionic surfactant" and the like as used in the present application have their accurate definitions in pharmacy, for example, the definition in the textbook "Pharmacy" (ISBN 978-7-122-29868-3) published by the Chemical Industry Press, which will not be explained in detail in the present application. If there is any dispute over the definition, the book shall be used for interpretation.

[0035] Tween as used in the present application refers to polysorbate, also known as polysorbate, polyoxyethylene sorbitan fatty acid ester, which is a non-ionic surfactant. Tween is widely used as an emulsifier and a solubilizer for oil substances. Polysorbate is generally considered to be a non-toxic, 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; Tween-80 is an oleate; Tween-20 is a laurate, which is a mixture of polyoxyethylene sorbitan monolaurate and a part of polyoxyethylene sorbitan monolaurate.

[0036] The concentration as used in the present application is w / v concentration unless otherwise specified, for example, 1% means 10 mg of solute 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 after adding a small amount of sodium hydroxide or potassium hydroxide to adjust the pH. As common knowledge in the art, an exemplary preparation method is phosphate buffer (pH 7.4): 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.

[0038] The various compounds described in the present application, such as EDTA, can form salts at different pH values, and since the actual active ingredient does not change, they should still be considered to fall within the scope of protection of the present application.

[0039] The animal model construction and detection method described in the present application basically refers to the published journal article mTORC1 Signaling and Negative Lens-Induced Axial Elongation, Ruiheng Zhang et al., Invest Ophthalmol Vis Sci. 2023; 64(10): 24; for convenience of expression, the present application refers to the "IOVS paper" when referring to this article.

[0040] Compared with the prior art, the progress of the present application is:

[0041] 1. Using Tween and cyclodextrin adjuvants, the use of eye local administration to deliver everolimus to RPE cells in the fundus, and the use of cyclodextrin adjuvants or the replacement of Tween with other non-ionic surfactants cannot deliver everolimus, and this specific combination has unexpected technical effects for those skilled in the art.

[0042] 2. Using the preferred technical solution of the present application, everolimus can still reach an effective treatment concentration as low as 0.0001%, thus eliminating the need for intravitreal injection, and significantly reducing the risk and treatment burden compared to intravitreal injection;

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

[0044] 4. Based on the above characteristics, the technical solution of the present application is particularly suitable for eye local administration, and when the preferred solution is used, even if there are large differences in drug metabolism due to individual differences (such as blinking or more excretion through the tear duct) or patient misuse (such as squeezing out more drops at a time, or using more than the recommended dose multiple times within a day), it is not easy to cause invalidation or serious side effects. BRIEF DESCRIPTION OF DRAWINGS

[0045] Other features, objects, and advantages of the application will become more apparent from the following detailed description when read in conjunction with the accompanying drawings:

[0046] Figure 1 is a graph of axial length of guinea pigs after application of an everolimus formulation at a concentration of 0.5% according to Example 2.2 of the present application;

[0047] Figure 2 is a graph of axial length of guinea pigs according to Example 2.2 of the present application;

[0048] Figure 3 is a graph of change in axial length of guinea pigs according to Example 2.2 of the present application;

[0049] Figure 4 is a graph of change in corneal + anterior chamber depth of guinea pigs according to Example 2.2 of the present application;

[0050] Figure 5 is a graph of change in lens thickness of guinea pigs according to Example 2.2 of the present application;

[0051] Figure 6 is a graph of change in vitreous depth of guinea pigs according to Example 2.2 of the present application;

[0052] Figure 7 is a graph of change in refractive power of guinea pigs according to Example 2.2 of the present application;

[0053] Figure 8 is an image of the fundus of the simple induction group after 3 weeks of myopic induction according to Example 2.3 of the present application;

[0054] Figure 9 is an image of the fundus after 3 weeks of myopic induction combined with the use of a low concentration of everolimus eye drops according to Example 2.3 of the present application;

[0055] Figure 10 is an image of the fundus after 3 weeks of myopic induction combined with the use of a medium concentration of everolimus eye drops according to Example 2.3 of the present application;

[0056] Figure 11 is an image of the fundus after 3 weeks of myopic induction combined with the use of a high concentration of everolimus eye drops according to Example 2.3 of the present application;

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

[0058] Figure 13 is a graph of the change in retinal thickness of guinea pigs according to Example 2.4 of the present application;

[0059] Figure 14 is a graph of the change in choroidal thickness of guinea pigs according to Example 2.4 of the present application;

[0060] Figure 15 is a graph of the change in scleral thickness of guinea pigs according to Example 2.4 of the present application;

[0061] Figure 16 is a graph showing the pharmacokinetic analysis of adult New Zealand rabbits after a single administration of 0.005% everolimus eye topical administration preparation and 0.01% everolimus eye topical administration preparation according to Embodiment 6 of the present application. DETAILED DESCRIPTION

[0062] For a better understanding of the present application, reference will be made to the detailed description of the technical solutions of the present application in conjunction with the accompanying drawings. It should be understood that the detailed description is only a description of exemplary embodiments of the present application, and is not intended to limit the scope of the present application in any way. Throughout the description, 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 associated listed items.

[0063] In the drawings, the size, proportions and shapes of the figures have been slightly adjusted for ease of illustration. The drawings are merely examples and are not strictly drawn to scale. As used in this document, the terms "approximately", "about", and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent deviations in a measured or calculated value that would be recognized by those of ordinary skill in the art.

[0064] It should also be understood that expressions such as "include", "including", "have", "has", "contain" and / or "containing" and the like, are open-ended expressions that are used to specify the presence of stated features, elements, and / or components, but do not preclude the presence or addition of one or more other features, elements, components, and / or combinations thereof. In addition, when expressions such as "at least one of" appear after a list of items, they modify the entire list of items and do not modify the individual items in the list. Furthermore, when describing embodiments of the present application, the use of "may" means "one or more embodiments of the present application". Also, the word "exemplary" is intended to mean an example or an illustration.

[0065] Unless otherwise defined, all terms used in this document, including engineering and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. It should also be understood that, unless explicitly stated otherwise, words defined in commonly used dictionaries 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.

[0066] It should be noted that the features of the embodiments and examples in the present application can be combined with each other as long as there is no conflict. In addition, unless explicitly limited or contradictory in context, the specific steps contained in the methods described in the present application do not have to be limited to the order described, but can be performed in any order or in parallel. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0067] Experimental method:

[0068] 1. Construction of animal model

[0069] The guinea pigs were used as experimental animals in the present application, and the animal model was constructed using the method described in the literature IOVS paper.

[0070] 2. Preparation of preparation and administration

[0071] Unless otherwise specified, all eye topical administration preparations in the examples of the present application were diluted with physiological saline containing phosphate buffered saline, and the osmotic pressure after dilution was about 300 mOsm / L, and the pH was adjusted to 7.4.

[0072] When detecting tissue concentration and blood concentration, for each sample, 3 experiments were carried out in parallel, each experiment was administered for 10 days, once a day, 1 drop of dose each time, and the volume of each drop was about 30 μL. In the detection of 2.2-2.4 in Example 2, 8 experiments were carried out in parallel for each sample, and the continuous administration lasted for three weeks. Once a day, 1 drop of dose each time, and the volume of each drop was about 30 μL.

[0073] 3. Detection of everolimus content in animal tissues

[0074] The content of everolimus in animal tissues was detected by high performance liquid chromatography-triple quadrupole tandem mass spectrometer HPLC-MS / MS API3200MD of Tianjin Puhua Wealth Medical Testing Co., Ltd.

[0075] Instrument model: HPLC-MS / MS (shimadzu LC20AD-API 3200MD TRAP);

[0076] Methanol, acetonitrile and the like were purchased from sigma.

[0077] Sample pretreatment: For the solid sample to be tested, weigh the appropriate sample after grinding, add 100ul of methanol water (8:2), vortex for 5min, centrifuge at 13200rpm for 6min, take 50ul of supernatant, add 150ul of methanol to precipitate protein, vortex for 1min, centrifuge at 13200rpm for 6min, and take 80ul for testing.

[0078] Liquid phase conditions:

[0079] Chromatographic column: Agela Venusil MP C18 (100*4.6mm, 3um), column temperature: 50℃, flow rate: 1.0ml / min,

[0080] Mobile phase: A organic phase: methanol (2mmol / L ammonium acetate + one thousandth formic acid)

[0081] B aqueous phase: water (2mmol / L ammonium acetate + one thousandth formic acid)

[0082] Injection volume: 10ul

[0083] Gradient: isocratic elution

[0084] Mass spectrometry conditions:

[0085] Ion source: +ESI electrospray ion source, IS: +5000V (spray voltage), GS1: 50psi (atomizing gas), GS2: 60psi (auxiliary gas), scanning mode: MRM multiple reaction monitoring, CAD: 3Medium (collision gas), TEM: 550℃ (atomizing temperature), CUR: 20psi (gas curtain gas), CXP: +2.0 (collision chamber ejection pressure), EP: +10 (injection voltage).

[0086] Because it is not possible to accurately separate RPE cells in the experiment and detect them, the drug content in the retinal choroidal tissue located in the fundus is detected instead.

[0087] The detection limit of drug concentration is 0.0001 ng / mg, and below this value cannot be detected, recorded as 0, and the experimental results in this application are retained to four significant digits.

[0088] 4. Detection method and figure description

[0089] The axial length, leopard-like lesions, retina, choroid, sclera thickness, etc. are detected using the detection method in the IOVS paper.

[0090] Without special instructions, in the drawings of the specification, the five data in the same group from left to right are blank control group, simple induction group, low concentration group, medium concentration group and high concentration group, the number of * represents P value, 1 * represents P <0.05 with statistical difference, 2 * represents P <0.01 with significant statistical difference, and 3 * represents P <0.001 with statistical difference.

[0091] Example 1

[0092] The eye drops as shown in Table 1 are prepared.

[0093] Table 1 Eye drop composition table

[0094] After weighing the above ingredients, dilute with physiological saline containing phosphate buffered saline, adjust the osmotic pressure to about 300 mOsm / L, and adjust the pH to 7.4.

[0095] Example 2 Everolimus dose determination

[0096] 2.1 Choroidal drug concentration

[0097] As shown in Table 2, the following everolimus eye topical administration formulations were prepared according to the preparation method of Example 1, but the concentration of everolimus was changed. The guinea pigs were sacrificed after 10 days of administration, and the choroid drug concentration and blood drug concentration were detected.

[0098] Table 2: Comparison of everolimus dosage

[0099] As can be seen from Table 2, the concentration of everolimus in the choroid at a concentration of 0.0001% can already reach the therapeutic level, and with the increase of the concentration of everolimus in the eye drops, the concentration of everolimus in the retina and choroid tissue is further increased; in contrast, the blood drug concentration increases more rapidly, which may be due to the absorption of the eye drops by the blood vessels around the eye, or the absorption after entering the tear duct, thereby increasing the blood drug concentration, and the intraocular structure is complex, and the penetration speed is slow.

[0100] In order to shorten the axial length, we hope to deliver more everolimus to the retina and choroid, and since the drug has systemic side effects, lower blood drug concentration is beneficial to its safety. The concentration of 0.0001%-0.01% is more ideal.

[0101] In order to further determine the dosage of everolimus, according to Example 1, 0.5% ultra-high concentration, 0.1% (high-dose), 0.01% (medium-dose), 0.001% (low-dose) formulations were prepared, and the formulations without adding everolimus and the guinea pigs without myopia induction were used as blank control group, and the formulations without adding everolimus and the guinea pigs model with myopia induction were used as simple induction group (LIM+Vehicle) group, and the three high, medium and low dose groups (using guinea pigs model with myopia induction) were used for parallel test, and the observation was carried out for 3 weeks, and the results are shown in Examples 2.2-2.4.

[0102] 2.2 Change of axial length

[0103] As shown in Figure 1, compared with the blank control group, the axial length of the guinea pigs was significantly prolonged after myopia induction, and the axial length prolongation caused by defocus myopia was significantly alleviated after the application of 0.5% everolimus, which preliminarily proved that the eye administration of everolimus can achieve pharmacological effect.

[0104] In order to further determine the effective concentration range, high, medium and low groups were used for exploration.

[0105] As shown in FIG. 2, compared with the blank control group and the defocus-induced group, there was no significant difference in the eye axis of the right and left eyes. After the defocus-induced group was treated with low (0.001%), medium (0.01%), and high (0.1%) concentrations of eye drops of everolimus, the right eye axis of the guinea pigs was significantly inhibited, and the right eye axis was significantly shorter than the left eye axis.

[0106] As shown in FIG. 3, compared with the blank control group, the eye axis of the guinea pigs was significantly elongated after myopia induction. After the defocus-induced group was treated with low (0.001%), medium (0.01%), and high (0.1%) concentrations of eye drops of everolimus, the elongation of the eye axis was significantly alleviated.

[0107] As shown in the figure, after one week, the eye axis length of the three concentration groups had a significant shortening trend compared with the defocus myopia group, and the shortening trend became more obvious as the administration time became longer. No significant difference was found among the three dose groups.

[0108] As shown in FIG. 4, compared with the blank control group, the corneal + anterior chamber depth of the guinea pigs did not change significantly after myopia induction. After the defocus-induced group was treated with low (0.001%), medium (0.01%), and high (0.1%) concentrations of eye drops of everolimus, the corneal + anterior chamber depth did not change significantly.

[0109] As shown in FIG. 5, compared with the blank control group, the lens thickness of the guinea pigs did not change significantly after myopia induction. After the defocus-induced group was treated with low (0.001%), medium (0.01%), and high (0.1%) concentrations of eye drops of everolimus, the lens thickness did not change significantly.

[0110] As shown in FIG. 6, compared with the blank control group, the vitreous depth of the guinea pigs was significantly deepened after myopia induction. After the defocus-induced group was treated with low (0.001%), medium (0.01%), and high (0.1%) concentrations of eye drops of everolimus, the deepening of the vitreous depth was significantly alleviated.

[0111] FIGS. 4-6 show that defocus myopia induction mainly causes an increase in vitreous depth, without affecting lens thickness or anterior chamber depth. The vitreous depth of the guinea pigs treated with the three concentrations of eye drops had a significant shortening trend compared with the defocus myopia group. No significant difference was found among the three concentration groups.

[0112] As shown in FIG. 7, through retinoscopy, the guinea pigs showed a significant myopic trend (decrease in diopter) after defocus myopia induction. The myopia degree of the guinea pigs treated with the eye drops of different concentrations had a significant delay trend compared with the defocus myopia group.

[0113] 2.3 Fundus morphology (tiger stripe lesions)

[0114] Referring to Figure 8, after 3 weeks of defocus-induced, the fundus image of the simple induction group can see the posterior pole of the wide distribution of strip leopard-like lesions.

[0115] Referring to Figure 9, after 3 weeks of defocus-induced combined with low concentration of everolimus eye drops, the fundus image can see that the posterior pole of the wide distribution of strip leopard-like lesions is less than that of Figure 8.

[0116] Referring to Figure 10, after 3 weeks of defocus-induced combined with medium concentration of everolimus eye drops, the fundus image can see that the posterior pole of the wide distribution of strip leopard-like lesions is less than that of Figure 9.

[0117] Referring to Figure 11, after 3 weeks of defocus-induced combined with high concentration of everolimus eye drops, the fundus image can see that the posterior pole of the wide distribution of strip leopard-like lesions is less than that of Figure 10.

[0118] 2.4 Retinal, choroidal and scleral thickness study

[0119] After three weeks of administration, referring to Figure 12, the cross-sectional structure of the fundus retina, choroid and sclera centered on the optic disc can be obtained by OCT examination (method see IOVS article), and it can be seen from the figure that the layers of the defocus myopia (induction group) are thinned.

[0120] In order to more intuitively show the thickness change of each layer of film, the thickness value of each layer of film was further determined and shown in Figures 13-15.

[0121] Referring to Figure 13, the retina at the upper, lower, nasal and temporal retina positions of the three disc diameters centered on the optic disc was measured, and compared with the blank control group, the retina thickness in each direction was significantly thinned after simple myopia induction, and the change was closely related to the progression of myopia. After defocus-induced combined with low (0.001%), medium (0.01%) and high concentration (0.1%) everolimus eye drops in the right eye, the retina thinning at the same position was significantly alleviated.

[0122] Referring to Figure 14, the choroid at the upper, lower, nasal and temporal retina positions of the three disc diameters centered on the optic disc was measured, and compared with the blank control group, the choroid thickness in each direction was significantly thinned after simple myopia induction, and the change was closely related to the progression of myopia. After defocus-induced combined with low (0.001%), medium (0.01%) and high concentration (0.1%) everolimus eye drops in the right eye, the choroid thinning at the same position was significantly alleviated.

[0123] Referring to FIG. 15, the scleral thickness at the positions of the optic disc, 3 optic disc diameters above, below, nasal, and temporal, was measured. Compared with the blank control group, the scleral thickness in each direction was significantly thinner after simple myopia induction, and this change was closely related to myopia progression. After the right eye was applied with low (0.001%), medium (0.01%), and high (0.1%) concentration of eye drops containing everolimus during the defocus induction, the scleral thinning at the same positions was significantly alleviated.

[0124] Example 3 Determination of the amount of tackifier

[0125] As shown in Table 3, the following everolimus eye topical administration preparation was prepared according to the preparation method of Example 1, but the amount of tackifier was changed. The guinea pigs were sacrificed after 10 days of administration, and the choroidal drug concentration was detected.

[0126] Table 3 Comparison table of tackifier concentration

[0127] Within a certain range, increasing the amount of tackifier can prolong the residence time of the drug on the surface of the eyeball, thereby facilitating the improvement of the absorption rate. However, too much tackifier will make the preparation flowability low to form a gel or paste, thereby making it difficult to control the administration dose. In addition, the everolimus wrapped by cyclodextrin and surfactant has a large collision volume and is difficult to migrate freely in the network structure of the gel, and the absorption rate will decrease instead. In the present application, it is preferred that the amount of methyl cellulose 4000 is 0.25%.

[0128] Example 4 Optimization of the type and concentration of cyclodextrin adjuvant

[0129] As shown in Table 4, the following everolimus eye topical administration preparation was prepared according to the preparation method of Example 1, but the type and concentration of cyclodextrin adjuvant were changed. The guinea pigs were sacrificed after 10 days of administration, and the choroidal drug concentration and blood drug concentration were detected.

[0130] Table 4 Comparison table of the type and concentration of cyclodextrin adjuvant

[0131] The data in Table 4, No. 3, directly uses the data of Example 1. As can be seen from Table 4, at a lower concentration, the amount of cyclodextrin has little effect on the delivery efficiency, and the effect is best at about 5%. When the concentration is too high, the effect is actually reduced. γ-cyclodextrin also has a delivery effect, but the delivery effect is not as good as HP-β-CD at the same concentration, and the entire preparation is milky white and opaque, which may cause temporary blurred vision. The data in No. 6 proves that the delivery cannot be completed without using cyclodextrin adjuvant.

[0132] Example 5 Adjustment of the type and amount of surfactant

[0133] As shown in Table 5, the following everolimus eye topical administration preparation was prepared according to the reference example 1, but the type and amount of non-ionic surfactant were changed. As a control, Span 60 and Transp commonly used in ophthalmic administration were selected. The guinea pigs were sacrificed after 10 days of administration, and the choroid drug concentration was detected.

[0134] Table 5 Comparison table of surfactant type and amount

[0135] Note: Transp is P, the chemical name of which is diethylene glycol monoethyl ether, is a strong solubilizer and is often used as a penetration enhancer for external preparations. In order to avoid irritating the eyeball surface, its concentration in the approved eye drops is generally not more than 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 column in which the number 7 is located does not use non-ionic surfactants.

[0136] This example proves that the Tween non-ionic surfactant, especially Tween 80, can effectively deliver everolimus when used in combination with cyclodextrin, while other non-ionic surfactants have no delivery effect at all.

[0137] Table 6 Comparison table of everolimus delivery efficiency

[0138] As shown in Table 6, further experiments also show that although EDTA and benzalkonium bromide, benzalkonium chloride can break the tear film and promote delivery, they are not essential. When the content of EDTA and benzalkonium bromide, benzalkonium chloride is 0, as long as there are cyclodextrin adjuvants and Tween, the delivery of everolimus can also be achieved. Since the functions of EDTA and benzalkonium bromide, benzalkonium chloride and other components C are to break the tear film and improve the drug delivery efficiency, any one of them can be selected, or they can be used at the same time. The principle of using EDTA and component C at the same time in the example 1 is that only EDTA has no antibacterial and preservative effect, and in order to ensure sterility, other preservatives also need to be added. However, too much benzalkonium bromide and benzalkonium chloride will cause irritation and affect the comfort of use.

[0139] Example 6 Everolimus eye topical administration preparation has unexpected technical effects

[0140] The everolimus eye local administration preparation provided in the application can not only deliver the therapeutic level concentration of everolimus to the choroid, but also ensure that the therapeutic level concentration of everolimus is maintained on the choroid for a long time.

[0141] Specifically, in the absorption phase, in the peak concentration layer of the tissue, as shown in the following figure, the peak concentration of 0.005% everolimus eye local administration preparation in the choroid tissue is 9.92±1.55 ng / g; the peak concentration of 0.01% everolimus eye local administration preparation in the choroid tissue is 12.87±2.43 ng / g. The peak concentration of everolimus on the choroid of the 0.005% everolimus eye local administration preparation provided in the application has reached the therapeutic level concentration after the first drop, which has achieved an unexpected technical effect compared with the prior art.

[0142] According to the adult New Zealand rabbit single-dose 0.005% and 0.01% pharmacokinetic analysis data obtained by the applicant through the experiment of everolimus concentration change in the animal body after single-dose administration of the everolimus eye local administration preparation, in the drug elimination phase, the drug elimination time in the choroid is longer. The drug half-life of the 0.005% everolimus eye local administration preparation in the choroid elimination phase is 20.45 hours, and the 24-hour drug concentration area under the curve (AUC) is 141.3±39.38; the drug half-life of the 0.01% everolimus eye local administration preparation in the choroid elimination phase is 24.74 hours, and the 24-hour drug concentration area under the curve (AUC) is 207.3±48.90. The above experimental data show that the everolimus eye local administration preparation provided in the application can maintain the therapeutic level concentration on the choroid for a longer time.

[0143] The above description is only an embodiment of the application and an explanation of the technical principles used. Those skilled in the art should understand that the protection scope of the application is not limited to the technical solutions formed by the specific combinations of the above technical features, and should also cover other technical solutions formed by any combinations of the above technical features or their equivalent features without departing from the technical concept. For example, the above features are replaced with the technical features disclosed in the application (but not limited to) having similar functions to form technical solutions.

Claims

1. An eye topical administration preparation of everolimus, characterized by, The preparation comprises a therapeutically effective amount of everolimus and the following pharmaceutical excipients: (1) a surfactant; (2) water; (3) pharmaceutically acceptable other pharmaceutical excipients; The surfactant comprises component A and component B, The component A is a cyclodextrin excipient, and the component B is Tween.

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

3. The ophthalmic preparation according to claim 1, wherein The cyclodextrin excipient is selected from one or more of β-cyclodextrin, γ-cyclodextrin, sulfobutyl-β-cyclodextrin or hydroxyalkylene-β-cyclodextrin.

4. The ophthalmic preparation according to claim 3, wherein The cyclodextrin excipient is selected from hydroxypropyl β-cyclodextrin, and the w / v concentration of the hydroxypropyl β-cyclodextrin is 0.5%-10%.

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

6. The ophthalmic preparation according to claim 1, wherein The surfactant further comprises component C, which is a cationic surfactant with a molecular weight less than 1000.

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

8. The ophthalmic preparation according to claim 1, wherein The pharmaceutical excipients further comprise a viscosity-increasing agent selected from one or more of cellulose, hyaluronic acid, chitosan, and polyethylene glycol excipients.

9. The ophthalmic preparation according to claim 8, wherein The viscosity-increasing agent is methyl cellulose 4000, and the concentration is 0.25%.

10. The ophthalmic preparation according to claim 1, wherein The pharmaceutically acceptable other pharmaceutical excipients comprise EDTA, and the w / v concentration of EDTA is 0.01%.

11. A treatment device, characterized by The therapeutic device is capable of generating droplets of a medicament, and the medicament is the eye topical administration preparation according to any one of claims 1-10.

12. Use of everolimus in the preparation of an eye topical administration preparation for treating or preventing a disease or condition selected from the group consisting of axial length overgrowth, myopia associated with axial length overgrowth, retinal thinning, retinal degeneration, retinal breaks, posterior scleral staphyloma, leopard-spot fundus, chorioretinal atrophy, macular atrophy, macular foveoschisis, lacquer crack, Fuchs spot, choroidal neovascularization, and visual impairment associated with these pathologies, the preparation comprising everolimus, a cyclodextrin excipient, and Tween.

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