TW202423434A - Eye drop microsuspensions of mtor inhibitors - Google Patents

Abstract

The present invention relates to an aqueous eye drop lipid microsuspension of mTOR inhibitors, e.g. everolimus, and their topical use for treating ocular disorders. More specifically, the ophthalmic composition comprises at least one mTOR inhibitor as the active pharmaceutical ingredient, preferably everolimus, a cyclodextrin, preferably alpha-cyclodextrin, and an oil, preferably castor oil. The invention further relates to the use of the aqueous eye drop lipid microsuspension in treating non-infectious ocular conjunctivitis, in preventing rejection of corneal graft or for treating dry eye disorders and meibomian gland dysfunctions.

Description

mTOR inhibitor eye drops microsuspension

The present disclosure relates to the field of ophthalmic formulations. More specifically, the present disclosure relates to aqueous eyedrop lipid microsuspensions of mTOR inhibitors (e.g., everolimus) and their topical use for treating ocular diseases.

Macrolides with mTOR inhibitory properties have broad therapeutic effects, including the ability to inhibit inflammation, proliferation, angiogenesis, fibrillation, and high permeability. In ophthalmology, these mTOR inhibitor macrolides have been reported to be useful, for example, for activating allergic conjunctivitis, preventing rejection after corneal transplantation, and improving tear secretion in patients with dry eye disease (DED) (Mihir Shah et al., Invest. Oymbol. Vis. Sci., 58(1):372-385, 2017; WO2020018498 A1).

mTOR macrolides are known to cause a variety of systemic side effects; therefore, targeted delivery of macrolides in the form of aqueous eye drops will enhance the therapeutic efficacy of macrolides in ophthalmology (Jorge L. Jacot, David Sherris, Potential Therapeutic Roles for Inhibition of the PI3K/Akt/mTOR Pathway in the Pathophysiology of Diabetic Retinopathy, Journal of Ophthalmology, (2011), 589813).

However, these compounds are very lipophilic, with LogP values ranging from 4.2 to 5.8, and have poor water solubility at room temperature, ranging from 0.004 to 0.25 mg/L.

In addition, these compounds are large macrolides with several asymmetric carbon atoms and double bonds and are therefore very unstable in aqueous solutions (EP2402350B1; Manisha Prajapati et al., International Journal of Pharmaceutics 586 (2020) 119579).

Temsirolimus (4,2-[3-hydroxy-2-(hydroxymethyl)-2-methylpropionate]-rapamycin) and deforolimus (4,2-(dimethylphosphinyl)-rapamycin) are sirolimus derivatives with increased solubility in water, about 7 mg/mL at room temperature, but these derivatives do not improve chemical stability. A topical ophthalmic formulation of tacrolimus has been successfully commercialized with adequate chemical stability, but its stability is improved by non-solubilization of the drug (Talymus ^® -1 mg/mL). The product is a conventional aqueous macrosuspension in which tacrolimus remains as solid particles, physically dispersed in the vehicle. Enhanced chemical stability is achieved by keeping the drug in a solid state. Such formulations do not provide good ocular tolerance, resulting in reduced patient compliance with treatment. In addition, due to the nature of solid drug particles and low drug water solubility, this formulation does not provide rapid ocular release of the drug.

Cyclodextrins and surfactant compounds have been used to improve the chemical stability of macrolides in aqueous solutions, but the stability is still insufficient to allow macrolides to be formulated as aqueous eye drops (Manisha Prajapati et al., International Journal of Pharmaceutics 586 (2020) 119579).

Sirolimus has shown stability in microemulsion eye drops containing about 25% water, but the local bioavailability of the drug in the microemulsion is very low (Guido Buech, Eckart Bertelmann, Uwe Pleyer, Ingo Siebenbrodt, Hans Hubert Borchert, Formulation of sirolimus eye drops and corneal permeation studies. Journal of Ocular Pharmacology and Therapeutics, 23 (2007) 292-303). This study also evaluated the solubility and stability of sirolimus in aqueous media containing cyclodextrin, liposomes, water-soluble mixtures, and poloxamer gels, but the results were minimal, especially in terms of the chemical stability of the dissolved sirolimus.

Therefore, there is still a need to develop stable aqueous eye drops for mTOR inhibitors, especially everolimus.

The present disclosure provides stable aqueous eye drop microsuspensions in which a lipophilic drug (e.g., everolimus) is protected within an oil droplet coated with a selected cyclodextrin to form a lipid microsuspension. The cyclodextrin used is selected based on its relative affinity for the macrolide drug and the oil, wherein the preferred cyclodextrin has a high affinity for the oil (i.e., forms a stable surface-active complex with the oil) and has an exceptionally low, or preferably, negligible affinity for the drug. The formed lipid microsuspension can be further stabilized with a polymer and/or a surfactant to form a physically stable formulation.

When diluted in an aqueous medium, such as tears, the microparticles dissolve, releasing the drug molecules. The water concentration in aqueous eyedrop lipid microsuspensions is generally greater than 70% and usually greater than 85%. The oil concentration in aqueous eyedrop lipid microsuspensions is generally less than 20% and usually less than 10%. The rapid dissolution of the microparticles upon dilution in the medium, the low oil content, and the high water content thus ensure high ocular tolerance when topically applied to the eye.

The present disclosure relates to an ophthalmic composition comprising: - at least one mTOR inhibitor as an effective pharmaceutical ingredient, preferably everolimus, - cyclodextrin, preferably α-cyclodextrin, and - oil, preferably castor oil.

The following embodiments can be implemented separately or in combination with other embodiments as needed:

Embodiment 1: An ophthalmic composition comprising: - at least one mTOR inhibitor as an effective pharmaceutical ingredient, preferably everolimus, - cyclodextrin, preferably α-cyclodextrin, and - oil, preferably castor oil.

Embodiment 2: The ophthalmic composition as described in Embodiment 1, wherein the mTOR inhibitor is selected from the group consisting of compounds having a macrolide structure, for example, selected from the group consisting of everolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus and a combination thereof, in particular everolimus.

Embodiment 3: The ophthalmic composition as described in Embodiment 1 or 2, wherein the concentration of the mTOR inhibitor is 0.01 to 0.1% by weight, particularly 0.02 to 0.08%, more particularly 0.03 to 0.07%, even more particularly 0.04 to 0.06%, for example about 0.05%, based on the weight of the composition.

Embodiment 4: The ophthalmic composition as described in any one of Embodiments 1 to 3, wherein the cyclodextrin is α-cyclodextrin.

Embodiment 5: The ophthalmic composition as described in any one of Embodiments 1 to 4, wherein the concentration of cyclodextrin is 0.5 to 15% by weight, particularly 1 to 10%, more particularly 2 to 8%, even more particularly 3 to 5% by weight based on the weight of the composition.

Embodiment 6: The ophthalmic composition as described in any one of Embodiments 1 to 5, wherein the oil is selected from the group consisting of castor oil, soybean oil, corn oil, olive oil, coconut oil, peanut oil, safflower oil, linseed oil, cottonseed oil, sesame oil, tea oil, coriander oil, rosemary oil, almond oil, safflower oil, linseed oil, rapeseed oil, monoolein, monocaprylin and dicaprylin, monocaprin and dicaprin, and combinations thereof, preferably castor oil.

Embodiment 7: The ophthalmic composition as described in any one of Embodiments 1 to 6, wherein the concentration of the oil is 0.5 to 7% by weight, particularly 1 to 6%, more particularly 1.5 to 5%, even more particularly 2 to 4% oil based on the weight of the composition.

Embodiment 8: The ophthalmic composition as described in any one of Embodiments 1 to 7, wherein the composition is a microsuspension, and wherein the oil and cyclodextrin form microparticles in an aqueous vehicle.

Embodiment 9: The ophthalmic composition as described in Embodiment 8, wherein the microparticles have a diameter D50 of less than 40 μm, particularly less than 25 μm, and more particularly less than 10 μm.

Embodiment 10: The ophthalmic composition as described in any one of Embodiments 1 to 9, further comprising an osmotic agent, which is preferably selected from the group consisting of: salts, such as potassium chloride or sodium chloride; sugars, such as sorbitol, mannitol, dextran or mannitol; or other types of polyols, such as glycerol; and combinations thereof.

Example 11: The ophthalmic composition as described in Example 10, wherein the osmotic agent is glycerol.

Embodiment 12: The ophthalmic composition as described in Embodiment 10 or 11, wherein the concentration of the osmotic agent is 0.1 to 8%, and/or wherein the concentration is adjusted to obtain a final isosmotic property of the composition between 250 and 350 mOsmol/kg, such as about 300 mOsmol/kg.

Embodiment 13: The ophthalmic composition as described in any one of Embodiments 1 to 12, further comprising one or more polymers or one or more stabilizers.

Example 14: The ophthalmic composition as described in Example 13, wherein the polymer is selected from the group consisting of polyoxyethylene fatty acid esters, polyoxyethylene alkylphenyl ethers, polyoxyethylene alkyl ethers, cellulose derivatives, carboxyethylene polymers, polyethylene polymers, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of polyoxypropylene glycol and polyoxyethylene, tyloxapol, and combinations thereof, in particular copolymers of polyoxypropylene glycol and polyoxyethylene.

Embodiment 15: The ophthalmic composition as described in Embodiment 13 or 14, wherein the polymer is poloxamer 407.

Embodiment 16: The ophthalmic composition as described in any one of Embodiments 13 to 15, wherein the concentration of the polymer is 0.1 to 3% by weight, particularly 0.4 to 1.2%, more particularly 0.4 to 1.0% by weight based on the weight of the composition, for example, if it is poloxamer 407, it is about 0.8%.

Embodiment 17: The ophthalmic composition as described in Embodiment 16, wherein the viscosity of the composition is lower than 30 cP, particularly lower than 20 cP, and more particularly between 5 and 20 cP.

Embodiment 18: The ophthalmic composition as described in any one of Embodiments 13 to 17, comprising a stabilizer selected from the group consisting of: polyethylene glycol monostearate, polyethylene glycol distearate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polyoxyethylene lauryl ether, polyoxyethylene octyldodecyl ether, polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, polyoxyethylene oleyl ether, sorbitol esters, polyoxyethylene cetyl ether, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitol ester ... Sugar alcohol fatty acid esters, polyethylene glycol, polyoxyethylene stearate, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cellulose, polyvinyl alcohol (PVA), poloxamer; hydroxylamine, alkyl aryl polyether sulfonate; PEG-modified phospholipids, PEG-modified cholesterol, PEG-modified cholesterol derivatives, PEG-modified vitamin A, PEG-modified vitamin E, random copolymers of vinyl pyrrolidone and vinyl acetate, and combinations thereof.

Example 19: The ophthalmic composition as described in Example 18, which comprises a stabilizer selected from the group consisting of polyoxyethylene castor oil derivatives, preferably polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, and polyoxyl 15 hydroxystearate, and more preferably polyoxyl 15 hydroxystearate.

Example 20: The ophthalmic composition as described in Example 19, comprising polyoxyl 15-hydroxy stearate as a stabilizer, and the concentration of the polyoxyl 15-hydroxy stearate is 0.5 to 2% by weight, such as about 1% based on the weight of the composition.

Embodiment 21: The ophthalmic composition as described in any one of Embodiments 1 to 20, further comprising a complexing agent and/or an antioxidant.

Example 22: The ophthalmic composition as described in Example 21, wherein the complexing agent is EDTA (ethylenediaminetetraacetic acid).

Example 23: The ophthalmic composition as described in Example 22, wherein the composition comprises EDTA and at least one antioxidant, for example, selected from the group consisting of BHA (butylated hydroxymethoxyphenyl), BHT (butylated hydroxytoluene), vitamin E TPGS.

Embodiment 24: The ophthalmic composition as described in Embodiment 21, 22 or 23, wherein the concentration of EDTA is 0.01 to 0.2% by weight, particularly 0.02 to 0.1%, and more particularly 0.03 to 0.07% by weight based on the weight of the composition.

Embodiment 25: An ophthalmic composition as described in any one of Embodiments 1 to 24, which is a microsuspension comprising: - Everolimus; - α-cyclodextrin; - Castor oil; and - Optionally, at least glycerol, - Optionally, at least one polymer, at least one stabilizer, and/or at least one antioxidant or complexing agent.

Embodiment 26: An ophthalmic composition as described in any one of Embodiments 1 to 25, which is a microsuspension comprising: - Everolimus, - α-cyclodextrin; - Castor oil; - Glycerol, and - Optionally, at least one polymer and/or one stabilizer, and/or at least one complexing agent, - Optionally, at least one antioxidant.

Example 27: An ophthalmic composition as described in any one of Examples 1 to 26, which is a microsuspension comprising: - 0.01 to 0.1%, for example 0.05%, of everolimus; - 3 to 5%, for example 4.0%, of α-cyclodextrin; - 2 to 4%, for example 3.0%, of castor oil; - 1 to 3%, for example 2.0%, of glycerol; - 0.5% to 2% of a stabilizer, for example polyoxyl 15 hydroxy stearate, typically 1.0% of polyoxyl 15 hydroxy stearate; - 0.4 to 1.2% of a polymer, for example poloxamer 407, typically 0.8% of poloxamer 407; - 0 to 0.07% of a complexing agent, such as disodium EDTA, typically 0.05% disodium EDTA; - 0 to 0.5% of an antioxidant; wherein the % is by weight based on the weight of the composition.

Example 28: A method for preparing an ophthalmic microsuspension, comprising the following steps: a) preparing an aqueous composition A by dissolving α-cyclodextrin in purified water; b) preparing an oily phase composition B comprising an mTOR inhibitor (e.g., everolimus); preferably, the oil is castor oil; c) sterilizing composition B as needed; d) adding the oily phase composition B to the aqueous composition A to obtain a mixture C; and e) homogenizing the mixture C to obtain a microsuspension.

Embodiment 29: The method as described in Embodiment 28, wherein at least one osmotic agent is added to composition A at step a).

Embodiment 30: The method as described in any one of Embodiments 28 or 29, wherein at least one complexing agent (eg, EDTA) is added to composition A at step a).

Embodiment 31: The method as described in any one of embodiments 28 to 30, wherein composition A is heated at a temperature T1 comprised between 100°C and 130°C; in particular between 105°C and 125°C.

Embodiment 32: A method as described in any one of Embodiments 28 to 31, wherein a concentrated aqueous solution of a stabilizer (e.g., polyoxy-15-hydroxystearate) is added to Composition A.

Embodiment 33: The method as described in any one of embodiments 28 to 32, wherein composition A is heated at temperature T1 for a time t, t is comprised between 10 and 40 minutes, in particular between 15 and 35 minutes, more particularly between 20 and 30 minutes.

Embodiment 34: The method as described in any one of embodiments 28 to 33, wherein composition A is cooled to a temperature T2, T2 is comprised between 10°C and 40°C, more particularly between 15°C and 35°C, even more particularly between 20°C and 30°C.

Embodiment 35: The method as described in any one of Embodiments 28 to 34, wherein the oil phase composition B at step b) is prepared by dissolving the mTOR inhibitor (e.g., everolimus) in oil.

Embodiment 36: The method as described in any one of Embodiments 28 to 35, wherein the oil phase composition B at step b) is prepared by dissolving everolimus in oil at a temperature T3 comprised between 20° C. and 50° C., particularly between 25° C. and 45° C., and even more particularly between 30° C. and 40° C. In one embodiment, the oil phase composition B at step b) is prepared by dissolving everolimus in oil at a temperature T3 between 25° C. and 35° C.

Embodiment 37: The method as described in any one of Embodiments 28 to 36, wherein the oil phase composition B is sterilized by filtration.

Embodiment 38: The method as described in any one of embodiments 28 to 37, wherein an aqueous solution of a concentrated polymer (eg, poloxamer 407) is added to the microsuspension after the homogenization step e).

Embodiment 39: The method as described in any one of Embodiments 28 to 38, wherein hydrochloric acid solution and/or sodium hydroxide solution is added to the microsuspension obtained in step e) to adjust the pH value of the microsuspension.

Embodiment 40: The method as described in embodiment 39, wherein the pH value of the microsuspension is comprised between 4 and 8, particularly between 4.5 and 7.5, more particularly between 5 and 7, and preferably between 5.2 and 5.4.

Example 41: A method for preparing an ophthalmic composition, comprising the following steps: a) preparing composition A by dissolving α-cyclodextrin in purified water; b) adding at least one osmotic agent (e.g., glycerol) and at least one complexing agent (e.g., EDTA) to composition A; c) subjecting composition A of step b) to autoclave treatment, for example, at a temperature of 121° C. for a period of 20 minutes; d) adding an aqueous solution of a concentrated stabilizer (e.g., polyoxy-15-hydroxystearate) to the composition of step c); e) dissolving an mTOR inhibitor (e.g., everolimus) in oil, for example, at a temperature between 25° C. and 35° C., preferably in castor oil, to obtain composition B; f) adding composition B to the composition of step e) through a filter with a predetermined porosity to obtain a mixture C; g) homogenizing the mixture C to obtain a microsuspension; h) adding an aqueous solution of a concentrated polymer (e.g., poloxamer 407) to the microsuspension; i) adjusting the pH of the microsuspension to a desired pH, e.g., between 5.0 and 6.0, typically a pH of 5.3±0.1, for example, by adding hydrochloric acid solution and/or sodium hydroxide solution; j) adjusting the final volume or weight of the formulation by adding water, if necessary.

Embodiment 42: The method as described in any one of embodiments 28 to 41, wherein the filter used in the filtering step has a porosity comprised between 1 and 0.01 μm, particularly between 0.7 and 0.05 μm, more particularly between 0.4 and 0.1 μm, even more particularly between 0.3 and 0.15 μm.

Embodiment 43: The method as described in any one of Embodiments 28 to 42, wherein the filter used in the filtering step is a PES filter having a porosity of 0.2 μm.

Embodiment 44: An ophthalmic composition, which can be obtained by the method described in any one of Embodiments 28 to 43.

Embodiment 45: An ophthalmic composition as described in any one of Embodiments 1 to 27 and 44, which is used for treating eye diseases, in particular for treating allergic or atopic conjunctivitis (i.e., vernal conjunctivitis), for preventing rejection after corneal transplantation, for treating dry eye disease (DED), leptomeningeal gland dysfunction, pterygium, corneal endothelial disease or corneal malnutrition, or for postoperative treatment during trabeculectomy.

Embodiment 46: The ophthalmic composition as described in Embodiment 45, wherein the corneal endothelial disease is selected from corneal dystrophy, in particular Fuchs endothelial corneal dystrophy, posterior polymorphic dystrophy, congenital hereditary endothelial dystrophy, corneal endotheliitis, cytomegalovirus corneal endotheliitis, spontaneous corneal endothelial cell disease, post-ophthalmic surgery disease (in particular post-ophthalmic laser surgery disease), trauma and aging.

Embodiment 47: The ophthalmic composition as described in any one of Embodiments 1 to 27 and 44, which is used for preventing rejection of corneal transplantation, wherein the composition is topically applied to the eye in an amount of 1 drop, one to six times a day.

Embodiment 48: The ophthalmic composition as described in any one of Embodiments 1 to 27 and 44, which is used for preventing corneal transplant rejection, wherein the composition is topically applied to the eye in an amount of 1 drop, one to three times a day, starting from 4 weeks after surgery, for 2 to at least 12 months or longer.

Embodiment 49: An ophthalmic composition as described in any one of Embodiments 1 to 27 and 44, which is used to treat dry eyes or blepharoplasty (which may include blepharitis), wherein the composition is topically applied to the eye or eyelid in an amount of 1 drop, one to three times a day, for 2 to at least 12 months or longer.

Embodiment 50: The ophthalmic composition as described in any one of Embodiments 1 to 27 and 44, which is used for treating anterior segment ocular diseases, wherein the composition is topically applied to the eye or eyelid in an amount of 1 drop, one to three times a day, for 2 to at least 12 months or longer.

Embodiment 51: An ophthalmic composition for use as described in any one of Embodiments 45 to 50, wherein the composition comprises 0.01 to 0.1% by weight, particularly 0.02 to 0.08%, more particularly 0.03 to 0.07%, even more particularly 0.04 to 0.06% by weight of an mTOR inhibitor, for example, everolimus, based on the weight of the composition.

Embodiment 52: The ophthalmic composition as described in any one of Embodiments 1 to 27 and 44, which is an aqueous eye drop lipid microsuspension for topical administration of an mTOR inhibitor to the eye.

Embodiment 53: The ophthalmic composition as described in any one of Embodiments 1 to 27 and 44, which is an aqueous eye drop lipid microsuspension for topically administering an mTOR inhibitor to the eye or eyelid of a subject in need thereof.

Embodiment 54: A method for reducing ocular inflammation and/or cellular immunity in a subject in need thereof, the method comprising the step of topically applying an effective amount of the composition as described in any one of Embodiments 1 to 27, 44, 52, and 53 to the eye or eyelid of the subject.

Embodiment 55: A method for treating ocular diseases, particularly for treating allergic conjunctivitis, for preventing rejection after corneal transplantation, for treating dry eye disease (DED) or leucorrhea dysfunction in a subject in need thereof, the method comprising the step of topically administering a therapeutically effective amount of a composition as described in any one of claims 1 to 27 and 44, 51, and 52 to the eye or eyelid of the subject.

Embodiment 56: A composition comprising: - at least one mTOR inhibitor as an effective pharmaceutical ingredient, preferably everolimus, - cyclodextrin, preferably α-cyclodextrin, and - oil, preferably castor oil.

Example 57: The composition as described in Example 56 is used for topical administration to the skin of a subject in need thereof, especially to the eyelid.

Embodiment 58: The composition as described in Embodiment 56 or 57 is used for treating psoriasis, atopic dermatitis and/or other skin diseases sensitive to anti-inflammatory drugs and/or immunosuppressant drugs, especially autoimmune skin diseases or lesions.

Definition

As used herein, the term "about" means approximately, within a certain range, roughly, or around. When the term "about" is used with a numerical range, it modifies the range by expanding the boundaries above and below the stated numerical values. Generally speaking, the term "about" or "approximately" is used herein to modify the numerical values above and below the stated value by a deviation of 10% or at least the deviation associated with the measurement device from which the numerical value is obtained.

The term "dissolved" or "substantially dissolved" as used herein means the dissolution of a solid in a solution. A solid is considered to be "dissolved" or "substantially dissolved" in a solution when the resulting solution is clear or substantially clear.

The term "clear" herein means a translucent or slightly transparent solution. Thus, a "clear" solution has a turbidity of 100 Nephelometric Turbidity Units (NTU), preferably 50 NTU, measured according to ISO standards.

The term "substantially clear" herein means a translucent or slightly transparent solution. Thus, a "substantially clear" solution has a turbidity of 100 Nephelometric Turbidity Units (NTU) measured according to ISO standards.

As used herein, the term "% Compound X by weight based on the weight of the composition" is also abbreviated as "% w/w" and corresponds to the weight of Compound X introduced into the composition, expressed as a percentage of the total weight of the composition.

The term "microsuspension" is intended to mean a composition comprising solid complex particles suspended in a liquid phase. An aqueous lipid microsuspension refers to a composition comprising oil-containing solid complex particles suspended in an aqueous phase. Lipid particles have a higher density than water and therefore will settle out upon storage.

As used herein, the term "aqueous eye drop lipid microsuspension" refers to a microsuspension in which an active pharmaceutical ingredient (e.g., everolimus) is contained in lipid droplets coated with cyclodextrin to form solid complex particles suspended in an aqueous vehicle, which is suitable for use as an eye drop, i.e., for topical administration to the eye or skin, e.g., eyelid, of a subject.

As used herein, the term "microparticle" refers to a particle having a diameter D50 of 1 μm or more to about 200 μm. The term "nanoparticle" refers to a particle having a diameter D50 of less than 1 μm.

As used herein, an "eye disorder" is a disease, illness, or other condition that affects or involves the eye, one of the parts or regions of the eye, or surrounding tissues such as the tear glands. In a broad sense, the eye includes the eyeball and the tissues and fluid that make up the eyeball, the muscles around the eye (such as the oblique muscles and rectus muscles), the portion of the optic nerve in or near the eyeball, and surrounding tissues such as the tear glands and eyelids.

As used herein, an "anterior segment eye disorder" is a disease, condition, or disorder that affects or involves the anterior (i.e., front) eye area or portion of the eye, such as the periocular muscles, eyelids, tear glands, or orbital tissue or fluid located in the posterior wall of the lens capsule or in front of the ciliary muscle.

Thus, anterior segment eye disorders primarily affect or involve one or more of the following: the conjunctiva, cornea, anterior chamber, iris, lens or lens capsule, and the blood vessels and nerves that vascularize or innervate the anterior region or area of the eye. Anterior segment eye disorders are also considered herein to extend to the lacrimal apparatus. Specifically, the lacrimal glands that secrete tears, and the ducts that drain the tears to the surface of the eye.

Additionally, anterior segment eye disorders affect or involve the posterior chamber, which is behind the retina but in front of the back wall of the lens capsule.

"Posterior segment eye disorders" are diseases, conditions, or disorders that primarily affect or involve the posterior region or area of the eye, such as the choroid or sclera (located behind the plane that passes through the posterior wall of the lens capsule), the vitreous body, the vitreous cavity, the retina, the optic nerve (i.e., the optic disc), and the blood vessels and nerves that vascularize or innervate the posterior region or area of the eye.

Thus, posterior segment eye disorders may include the following diseases, conditions or disorders: for example, macular degeneration (such as non-exudative age-related macular degeneration and exudative age-related macular degeneration); choroidal neovascularization; acute maculo-optic retinopathy; macular edema (such as cystoid macular edema and diabetic macular edema); Behcet's disease, retinal disorders, diabetic retinopathy (including proliferative diabetic retinopathy); retinal artery occlusion disease; Central retinal vein occlusion; uveitic retinopathy; retinal detachment; ocular trauma affecting the posterior part or location of the eye; posterior eye disorders caused by or affected by ocular laser therapy; posterior eye disorders caused by or affected by photodynamic therapy; photocoagulation; radiation retinopathy; posterior vitreous disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa, and glaucoma.

Anterior segment eye disorders include the following diseases, minor conditions or conditions: for example, aphakia; pseudophakia; astigmatism; eyelid spasm; cataracts; conjunctivitis; non-infectious conjunctivitis, including allergic conjunctivitis; atopic conjunctivitis, which may include vernal conjunctivitis; keratitis; corneal ulcers; dry eyes; eyelid diseases, which may include rimitis; tear apparatus diseases; tear duct obstruction; myopia; hyperopia; pupil disorders; refractive disorders, strabismus and glaucoma gland dysfunction. As used herein, anterior segment eye disorders more specifically include: corneal graft rejection prevention, dry eyes, allergic conjunctivitis, glaucoma gland dysfunction, severe keratoconjunctivitis in general and vernal conjunctivitis more specifically. Occasionally, blepharitis is associated with malfunction of the eyelid glands. In this case, treatment for the malfunction of the eyelid glands or blepharitis may also involve topical medications applied to the eyelid.

The present disclosure relates to and is directed to ophthalmic compositions for topical delivery of drugs to the eye and methods for treating eye diseases, such as anterior segment eye diseases or posterior segment eye diseases or eye diseases that can be characterized by both anterior segment eye diseases and posterior segment eye diseases, preferably anterior segment eye diseases, such as corneal transplant rejection. In specific embodiments, the ophthalmic compositions can be topically applied to the eyelid. The ophthalmic compositions of the present disclosure

The present disclosure relates to an ophthalmic composition comprising: - at least one mTOR inhibitor as an effective pharmaceutical ingredient, preferably everolimus, - cyclodextrin, preferably α-cyclodextrin, and, - oil, preferably castor oil.

More specifically, the inventors have successfully designed an aqueous ophthalmic microsuspension consisting of water-insoluble lipid microparticles suspended in an aqueous vehicle. The lipid microparticles are essentially composed of oil coated with a surfactant lipid/cyclodextrin complex.

In certain embodiments, the particles of the microsuspension have a diameter D50 of about 1 μm to about 40 μm, such as about 1 μm to about 25 μm, more particularly about 1 μm to about 10 μm. Such microsuspensions are suitable for use as eye drop formulations in humans and animals, such as mammalian subjects.

Therefore, in a preferred embodiment of the present disclosure, the composition is an aqueous eye drop lipid microsuspension comprising an mTOR inhibitor (preferably everolimus), which comprises lipid microparticles of oil (preferably castor oil) suspended in an aqueous vehicle, wherein the lipid microparticles are coated with α-cyclodextrin.

In a preferred embodiment, the compositions of the present disclosure (usually aqueous eye drops or lipid microsuspensions) are sterile.

In a more specific embodiment, the composition is an aqueous eye drop lipid microsuspension having a shelf life of at least more than 2 years in a refrigerator (i.e., stored at 4 to 8°C) and at least more than 4 weeks at room temperature (i.e., 25°C). Active pharmaceutical ingredient

The aqueous eye drop lipid microsuspension is particularly suitable for preparing an eye drop microsuspension of an mTOR inhibitor, more specifically, an eye drop microsuspension of an mTOR inhibitor having a macrolide structure.

mTOR inhibitors are a class of drugs that inhibit the mechanistic target of rapamycin (mTOR), a serine/threonine-specific protein kinase that belongs to the phosphatidylinositol-3-kinase (PI3K)-related kinase (PIKK) family. mTOR regulates cell metabolism, growth, and proliferation by forming and communicating with two protein complexes, mTORC1 and mTORC2. The most mature mTOR inhibitors are the so-called rapalogs (rapamycin and its analogs), which show immunosuppressive activity and are used to prevent rejection of organ and bone marrow transplants in vivo.

In certain embodiments, these mTOR inhibitors comprise a macrolide ring, typically a 14-, 15-, or 16-membered macrolide ring.

The mTOR inhibitors used in the aqueous eye drop lipid microsuspension of the present disclosure include, but are not limited to, everolimus, pimecrolimus, redaformolimus (also known as defostiolimus), sirolimus (also known as rapamycin), tacrolimus, temsirolimus, umiolimus, and zotarolimus, and combinations thereof.

In a preferred embodiment, everolimus is used as an active pharmaceutical ingredient, either as the sole active pharmaceutical ingredient in a microsuspension or in combination with other drugs.

Everolimus is currently used as both an anticancer drug and an immunosuppressant. Specifically, everolimus is approved as an active ingredient for the treatment of kidney cancer, pancreatic cancer, and breast cancer, and for the prevention of transplant rejection, especially of ear, kidney, and liver transplants.

The chemical formula of everolimus is as follows:

In a specific embodiment, the mTOR inhibitor (preferably everolimus) is contained in the aqueous eye drop lipid microsuspension in an amount between 0.01% and 0.1%, for example 0.05%.

In a specific embodiment, the mTOR inhibitor (preferably everolimus) is stable in the aqueous eye drop lipid microsuspension of the present disclosure for at least 720 days, i.e., its amount is reduced by less than 10% after 720 days at 5° C., as measured by reversed phase liquid chromatography (HPLC) as described in Example 1.

The inventors also show that the eye drop lipid microsuspension of the present disclosure can be used with APIs other than mTOR inhibitors. When the microsuspension is used with a wide variety of drugs, the properties of the microsuspension are actually comparable, as shown in Example 20. Therefore, the composition can be used as a vehicle for delivering drugs such as corticosteroids to the eye.

In one embodiment, the present disclosure relates to an ophthalmic composition comprising: - at least one active pharmaceutical ingredient, - cyclodextrin, preferably α-cyclodextrin, and, - oil, preferably castor oil.

The composition is particularly suitable as a medium for lipophilic drugs, especially drugs with a LogP value of 1.9 or higher (especially a LogP value of 3 or higher), for example, drugs with a LogP value in the range of 4 to 8. Preferably, the active pharmaceutical ingredient is an anti-inflammatory compound. In a specific embodiment, the active pharmaceutical ingredient is selected from mTOR inhibitors, cyclosporine and corticosteroids. In a specific embodiment, the corticosteroid is selected from loteprednol and dexamethasone. In a specific embodiment, the active pharmaceutical ingredient is selected from everolimus, tacrolimus, sirolimus, cyclosporine A, loteprednol and dexamethasone. Cyclodextrin

The aqueous eye drop lipid microsuspension of the present disclosure comprises cyclodextrin. In a specific embodiment, the cyclodextrin forms a lipid/cyclodextrin complex, thereby providing a coating of oil particles of the aqueous eye drop lipid microsuspension of the present disclosure.

Cyclodextrin is a cyclic oligosaccharide containing 6 (α-cyclodextrin), 7 (β-cyclodextrin), and 8 (γ-cyclodextrin) glucopyranose monomers linked by α-1,4-glycosidic bonds. α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin are natural products formed by microbial degradation of starch.

All three natural cyclodextrins (α-, β-, and γ-cyclodextrin) as well as many cyclodextrin derivatives can form inclusion complexes with glycerides, although their affinity may vary depending on the type of triglyceride, diglyceride, and monoglyceride and on the presence of other modifiers in the aqueous medium.

The preferred cyclodextrin used in the present composition, more specifically, when the mTOR inhibitor is everolimus, is α-cyclodextrin or an α-cyclodextrin derivative having the same or lower affinity for everolimus than α-cyclodextrin. Indeed, preferably, cyclodextrin has a low affinity for the active ingredient (e.g., everolimus), but has a good affinity for oil, so that everolimus is retained in lipid particles. In some embodiments, α-cyclodextrin or a derivative thereof is selected from carboxyalkyl-α-cyclodextrin, hydroxyalkyl-α-cyclodextrin, sulfoalkyl ether-α-cyclodextrin, alkyl-α-cyclodextrin, and combinations thereof.

The amount of cyclodextrin (preferably α-cyclodextrin) in the aqueous lipid microsuspension of the present disclosure may be about 1 to about 10%, particularly about 2 to about 8%, based on the weight of the composition, calculated as the weight of cyclodextrin.

In certain embodiments where everolimus is an mTOR inhibitor, the amount of cyclodextrin (usually α-cyclodextrin) in the aqueous eye drop lipid microsuspension is about 3% to about 5%, and the amount of everolimus is about 0.01% to about 0.1%. In other embodiments, the amount of cyclodextrin (usually α-cyclodextrin) in the aqueous eye drop lipid microsuspension is about 4.0%, particularly in combination with an amount of everolimus between 0.01% and 0.1%, for example, in combination with about 0.05% everolimus.

Commercially available pure cyclodextrins are hygroscopic and contain a large amount of water. For example, commercially available α-cyclodextrin contains 10.2% (w/w) water. Therefore, as used herein, the given weight values and % (w/w) are based on the dry weight of the cyclodextrin. Oil

The microparticles of the microsuspension are formed by a cyclodextrin complex and an oil. The oil can be selected from one or more medium-chain or long-chain triglycerides, diglycerides and monoglycerides commonly available in the pharmaceutical field.

For example, the glyceride can be selected from the group consisting of castor oil, soybean oil, corn oil, olive oil, coconut oil, peanut oil, safflower oil, linseed oil, cottonseed oil, sesame oil, tea oil, coriander oil, rosemary oil, almond oil, safflower oil, linseed oil, rapeseed oil, monoolein, mono- and di-caprylin, mono- and di-caprin, and other lipophilic solvents such as silicone oil, mineral oil, and semifluorinated alkanes such as 1-(perfluorobutyl)pentane and 1-(perfluorohexyl)octane. Preferred oils are those that can form multiple hydrogen bonds (i.e., H bonds) with macrolides, such as oils from plant sources.

In embodiments of the present disclosure, the oil is selected from the group consisting of castor oil, soybean oil, corn oil, olive oil, coconut oil, peanut oil, safflower oil, linseed oil, cottonseed oil, sesame oil, tea oil, coriander oil, rosemary oil, almond oil, safflower oil, linseed oil, rapeseed oil, monoolein, mono- and di-caprylin, mono- and di-caprin, and combinations thereof, preferably castor oil. Possible oils are highly refined oils with low acid and peroxide values as defined in the European Pharmacopoeia 11.0 (Sections 2.5.1 and 2.5.5). An example of such a highly refined oil is Super Refined ^™ castor oil from Croda (www.crodapharma.com).

In certain embodiments, the peroxide value of the oil is 0 to 5 meqO ₂ /kg, specifically 0 to 2.5 meqO ₂ /kg, and more specifically 0 to 1 meqO ₂ /kg.

In certain embodiments, the acid value of the oil is 0 to 1 mg KOH/g, specifically about 0.8 mg KOH/g.

In certain embodiments where everolimus is an mTOR inhibitor, the oil is castor oil.

In certain embodiments of everolimus as an mTOR inhibitor, the amount of oil (preferably castor oil) in the aqueous eye drop lipid microsuspension is 2% to 4% w/w, and the amount of everolimus is 0.01% to 0.1% w/w. In other embodiments, the amount of oil (usually castor oil) in the aqueous eye drop lipid microsuspension may be about 3.0% w/w, especially in combination with an amount of everolimus of about 0.01% to about 0.1%, such as about 0.05% w/w.

In certain embodiments, the mole/molar ratio of the amount of cyclodextrin or a derivative thereof to the amount of the mTOR inhibitor is at least about 2:1 to about 500:1.

In certain embodiments, the molar ratio of the amount of α-cyclodextrin or its derivative to the amount of everolimus as the active pharmaceutical ingredient is at least about 2:1 to about 500:1, such as between 40:1 and 120:1, more preferably between 70:1 and 90:1, such as about 80:1. Osmotic agent

In some embodiments, the ophthalmic composition has one or more osmotic agents that can be used to adjust the osmotic properties of the composition (particularly the aqueous eye drop lipid microsuspension disclosed herein, more preferably with everolimus as the active pharmaceutical ingredient).

Suitable osmotic agents include, but are not limited to, salts such as potassium chloride or sodium chloride; sugars such as sorbitol, mannitol, or dextran, or other types of polyols such as glycerol.

The inventors were surprised to note that glycerol has an unexpected but beneficial additional role in the formulation by acting as a co-solvent/co-surfactant that improves the physical state of the microsuspension. As a result, glycerol acts as a stabilizer for the microsuspension by reducing the sedimentation rate of the microparticles. Therefore, in a preferred embodiment, the ophthalmic composition of the present disclosure includes glycerol.

In certain embodiments using everolimus as an mTOR inhibitor, the amount of glycerol in the aqueous eye drop lipid microsuspension is 0.1% to 8% w/w, particularly 0.5 to 6% w/w, more particularly about 1% to about 3% w/w, and the amount of everolimus is between 0.01% and 0.1% w/w. In other embodiments, the amount of glycerol in the aqueous eye drop lipid microsuspension may be between about 1% and 2.5% w/w, particularly in combination with an amount of everolimus of about 0.01% to about 0.1%, such as about 0.05% w/w, so that the osmotic pressure concentration of the formulation is about 250 to about 350 mOsmol/kg, preferably about 300 mOsm/kg. More details are also provided in Example 13. Polymers and Stabilizers

The ophthalmic compositions of the present disclosure may further comprise one or more polymers and/or stabilizers.

In detail, the polymer or stabilizer may be a water-soluble polymer. In a specific embodiment, the polymer may be a viscosity-increasing polymer. The term "viscosity-increasing polymer" is intended to mean a polymer that increases the viscosity of a liquid. The increase in viscosity may result in an increase in the physical stability of the composition. Thus, when the composition includes a polymer, the composition is less susceptible to solid complex sedimentation. Therefore, a polymer may also be considered a stabilizer.

In particular, the polymer or stabilizer may be a surfactant polymer. The term "surfactant polymer" is intended to mean a polymer that exhibits surfactant properties. For example, a surfactant polymer may include a hydrophobic chain grafted to a hydrophilic backbone polymer; a hydrophilic chain grafted to a hydrophobic backbone; or alternating hydrophilic and hydrophobic segments. The first two types are called graft copolymers, and the third type is called a block copolymer.

In one embodiment, the ophthalmic composition of the present disclosure comprises a polymer or stabilizer selected from the group consisting of: polyoxyethylene fatty acid esters; polyoxyethylene alkylphenyl ethers; polyoxyethylene alkyl ethers; cellulose derivatives such as alkyl cellulose, hydroxyalkyl cellulose and hydroxyalkyl alkyl cellulose; carboxyvinyl polymers such as carbomers, for example, ^Carbopol® 971 and ^Carbopol® 974; polyethylene polymers; polyvinyl alcohol; polyvinyl pyrrolidone; copolymers of polyoxypropylene glycol and polyoxyethylene; tyloxapol; and combinations thereof.

Examples of suitable polymers or stabilizers include, but are not limited to, polyethylene glycol monostearate, polyethylene glycol distearate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polyoxyethylene lauryl ether, polyoxyethylene octyldodecyl ether, polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, polyoxyethylene oleyl ether, sorbitan esters, polyoxyethylene cetyl ether (e.g., cetomacrogol 1000), polyoxyethylene castor oil derivatives (e.g., polyoxyl 35 castor oil: ^Kolliphor® EL, polyoxyl 40 hydrogenated castor oil: ^Kolliphor® RH40, polyoxyl 15 hydroxy stearate: ^Kolliphor® HS15-BASF), polyoxyethylene sorbitan fatty acid esters (e.g., ^Tween® 20 and ^Tween® 80 (ICI Specialty Chemicals); polyethylene glycols (e.g., Carbowax ^™ 3550 and 934 (Union Carbide)), polyoxyethylene stearate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cellulose, polyvinyl alcohol (PVA), poloxamers (e.g., ^Pluronics® F68 and FI08, which are block copolymers of ethylene oxide and propylene oxide); polydimethyl methacrylate (e.g., ^Tetronic® 908, also known as polydimethyl methacrylate 908, which is a tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Wyandotte Corporation, Parsippany, NJ)); ^Tetronic® 1508 (T-1508)( BASF Wyandotte Corporation), Tritons X-200, which is an alkyl aryl polyether sulfonate (Rohm and Haas); PEG-modified phospholipids, PEG-modified cholesterol, PEG-modified cholesterol derivatives, PEG-modified vitamin A, PEG-modified vitamin E, random copolymers of vinyl pyrrolidone and vinyl acetate, combinations thereof, and the like.

More specifically, the copolymer of polyoxypropylene and polyoxyethylene may be a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration.

Particularly useful polymeric stabilizers are poloxamers. Poloxamers may include any type of poloxamer known in the art. Poloxamers include poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 236, poloxamer 237, poloxamer 238, poloxamer 239, poloxamer 240, poloxamer 241, poloxamer 242, poloxamer 243, poloxamer 244, poloxamer 245, poloxamer 246, poloxamer 247, poloxamer 248, poloxamer 249, poloxamer 250, poloxamer 251, poloxamer 252, poloxamer 253, poloxamer 254, poloxamer 255, poloxamer 256, poloxamer 257, poloxamer 258, poloxamer 259, poloxamer 260, poloxamer 261, poloxamer 262, poloxamer 263, poloxamer 264, poloxamer 265 Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407, Poloxamer 105 benzoate, and Poloxamer 182 dibenzoate. Poloxamers are also referred to by their trade name, Pluronic, such as Pluronic 10R5, Pluronic 17R2, Pluronic 17R4, Pluronic 25R2, Pluronic 25R4, Pluonic 31R1, Pluronic F 108 Cast Solid Surfacta, Pluronic F 108 NF, Pluronic F 108 Pastille, Pluronic F 108NF Prill Poloxamer 338, Pluronic F 127, Pluronic F 127 NF, Pluronic F 127 NF 500 BHT Prill, Pluronic F 127 NF Prill Poloxamer 407, Pluronic F 38, Pluronic F 38 Pastille, Pluronic F 68, Pluronic F 68 Pastille, Pluronic F 68 LF Pastille, Pluronic F 68 NF, Pluronic F 68 NF Prill Poloxamer 188, Pluronic F 77, Pluronic F 77 Micropastille, Pluronic F 87, Pluronic F 87 NF, Pluronic F 87 NF Prill Poloxamer 237, Pluronic F 88, Pluronic F 88 Pastille, Pluronic F 98, Pluronic L 1 0, Pluronic L 101, Pluronic L 121, Pluronic L 31, Pluronic L 35, Pluronic L 43, Pluronic L 44 NF Poloxamer 124, Pluronic L 61, Pluronic L 62, Pluronic L 62 LF, Pluronic L 62D, Pluronic L 64, Pluronic L 81, Pluronic L 92, Pluronic L44 NF INH surfactant Poloxamer 124 View, Pluronic N 3, Pluronic P 103, Pluronic P 104, Pluronic P 105, Pluronic P 123 Surfactant, Pluronic P 65, Pluronic P 84, Pluronic P 85, combinations thereof, and the like.

A preferred polymer for use in the compositions of the present disclosure is Poloxamer 407.

The amount of polymer in the composition of the present disclosure, for example, poloxamer 407, can be 0.1% to 5.0% (w/w). In a preferred embodiment, the amount of polymer is about 0.4% to about 1.2%, more particularly about 0.8%, based on the weight of the composition.

In a specific embodiment, the ophthalmic composition comprises poloxamer 407 as a preferred polymer and/or polyoxy 15-hydroxy stearate as a stabilizer, and preferably, the composition comprises a mixture of poloxamer 407 and polyoxy 15-hydroxy stearate, for example in a ratio of 0.8:1, respectively.

Antioxidants and complexing agents

One of the known degradation pathways of mTOR inhibitor macrolides is oxidation, therefore, the ophthalmic compositions of the present disclosure may include one or more antioxidants or complexing agents.

Suitable examples of complexing agents include, but are not limited to, EDTA (ethylenediaminetetraacetic acid). The amount of complexing agent in the composition of the present disclosure may be 0 to about 0.1%, particularly 0.001 to 0.07%, more particularly 0.03 to 0.07%, even more particularly about 0.05%, based on the weight of the composition.

Suitable examples of antioxidants include, but are not limited to, BHA (butylated hydroxymethoxyphenyl) and BHT (butylated hydroxytoluene), and sulfur derivatives such as sodium thiosulfate, sodium bisulfite, sodium metabisulfite, thiourea, α-tocopherol and vitamin E TPGS.

The amount of antioxidant in the composition of the present disclosure may be 0 to about 1%, specifically 0.05 to 0.5%, by weight of antioxidant, based on the weight of the composition.

In a specific embodiment, the composition includes at least one complexing agent and does not contain an antioxidant. In a specific embodiment, the composition includes at least one complexing agent, such as EDTA, and at least one antioxidant, such as selected from the group consisting of BHA (butylated hydroxymethoxyphenyl) and BHT (butylated hydroxytoluene), and sulfur derivatives such as sodium thiosulfate, sodium bisulfite, sodium metabisulfite, thiourea, alpha-tocopherol and vitamin E TPGS.

In a specific embodiment, the composition does not include any complexing agent, but includes at least one antioxidant, such as selected from the group consisting of BHA (butylated hydroxymethoxyphenyl) and BHT (butylated hydroxytoluene), and sulfur derivatives such as sodium thiosulfate, sodium bisulfite, sodium metabisulfite, thiourea, α-tocopherol and vitamin E TPGS. pH of the composition

In a specific embodiment, especially when the active pharmaceutical ingredient is everolimus, the pH of the composition is maintained between 5.0 and 6.0, preferably between 5.2 and 5.4, for example about 5.3 .

In a specific embodiment, the ophthalmic composition is an aqueous eye drop lipid microsuspension, which contains at least: - 0.01 to 0.1%, for example, about 0.05% of everolimus; - α-cyclodextrin; and - castor oil; wherein the oil and α-cyclodextrin form microparticles in an aqueous medium, and the microparticles have a diameter of about 1 μm to 40 μm, preferably about 1 μm to 10 μm.

In a preferred embodiment, the ophthalmic composition is an aqueous eye drop lipid microsuspension, which contains at least: - 0.01 to 0.1%, such as about 0.05%, of everolimus; - 3 to 5%, such as about 4.0%, of α-cyclodextrin; and - 2 to 4%, such as about 3.0%, of castor oil; wherein the % is % by weight based on the weight of the composition.

In a further preferred embodiment, the ophthalmic composition is an aqueous eye drop lipid microsuspension, which contains at least: - 0.01 to 0.1%, such as about 0.05%, of everolimus; - 3 to 5%, such as about 4.0%, of α-cyclodextrin; and - 2 to 4%, such as about 3.0%, of castor oil; and - 1 to 3%, such as about 2.0%, of glycerol; wherein the % is % by weight based on the weight of the composition.

For example, the ophthalmic composition is an aqueous eye drop lipid microsuspension, which contains at least: - 0.01 to 0.1%, such as about 0.05% of everolimus; - 3.5 to 5.5%, such as about 4.0% of α-cyclodextrin; and - 2 to 4%, such as about 3.0% of castor oil; - 1 to 3%, such as about 2.0% of glycerol; - 0.5% to 2% of a stabilizer, such as polyoxyl 15-hydroxy stearate, typically about 1.0% of polyoxyl 15-hydroxy stearate; - 0.4 to 1.2% of a polymer, such as poloxamer 407, typically about 0.8% of poloxamer 407; - 0 to 0.07% of a complexing agent, such as disodium EDTA, typically about 0.05% of disodium EDTA; - an antioxidant as required; wherein the % is by weight based on the weight of the composition. Method for preparing the aqueous eye drop lipid microsuspension of the present disclosure

The composition of the present disclosure can be obtained by the following method:

In a specific embodiment, a method for preparing an ophthalmic microsuspension comprises the following steps: a) preparing an aqueous composition A by dissolving cyclodextrin in purified water; b) preparing an oil phase composition B containing an mTOR inhibitor; preferably the oil is castor oil, c) sterilizing the composition B by filtration as needed; d) adding the oil phase composition B to the aqueous composition A to obtain a mixture C; e) preferably homogenizing the mixture C with a high energy homogenizer to obtain a microsuspension; and f) adding a concentrated aqueous solution of a polymer to the microsuspension as needed.

Step a) of preparing the aqueous composition A comprises dissolving cyclodextrin (preferably α-cyclodextrin) to obtain an aqueous α-cyclodextrin solution. In a specific embodiment, a complexing agent such as EDTA and/or an osmotic agent such as glycerol are added to the composition A.

The thus obtained composition A containing dissolved cyclodextrin (preferably α-cyclodextrin) and optionally an appropriate amount of EDTA and/or glycerol can then be autoclaved for sterilization purposes (e.g. at 121° C. for 20 minutes).

A concentrated aqueous solution of a stabilizer (e.g., polyoxy-15-hydroxystearate) may also be added to the autoclaved composition A. The aqueous solution of the stabilizer may be sterilized by autoclaving together with phase A, but preferably, it may be sterilized separately by filtration.

Step b) of preparing the oil phase composition B comprises dissolving the mTOR inhibitor (e.g., everolimus), usually by heating at a temperature of up to 25° C. to 40° C., preferably between 25° C. and 35° C. for about 10 minutes. The oil solution with the active pharmaceutical ingredient can then be sterilized by filtration before being introduced into the aqueous composition A.

Then, in step d), the aqueous composition A prepared as above and the oil phase B containing the mTOR inhibitor prepared as above are gently mixed to obtain a mixture C.

The mixture C is then homogenized, for example, using a high-energy homogenizer to obtain a microsuspension. As used herein, the term "high-energy homogenizer" refers to a component that performs homogenization under speed and time conditions suitable for obtaining a microsuspension having microparticles (which preferably have a D50 diameter of less than 40 μm, preferably less than 10 μm). For example, the high-energy homogenizer can be any high-pressure homogenizer that uses different settings with respect to time and pressure but drives to equivalent results with respect to the composition quality.

In a specific embodiment, a concentrated solution of a polymer such as poloxamer 407 may then be added to the microsuspension.

In a specific embodiment, the pH value can be adjusted to the desired pH value, preferably 5.3±0.1, using, for example, concentrated hydrochloric acid solution and/or sodium hydroxide solution. Finally, if necessary, the weight of the composition can be adjusted with water.

Therefore, in a more specific embodiment, the method for preparing the aqueous eye drop lipid microsuspension disclosed herein comprises the following steps: a) preparing composition A by dissolving cyclodextrin (preferably α-cyclodextrin) in purified water; b) adding at least one osmotic agent (e.g., glycerol) and at least one antioxidant or complexing agent to composition A; c) mixing the composition of step b) and autoclaving, for example, at a temperature of 121°C for a time t (20 minutes); d) adding a sterile aqueous solution of a concentrated polymer (e.g., polyoxy-15-hydroxystearate) to the sterile composition of step c); e) Dissolve the mTOR inhibitor (preferably everolimus) in oil (e.g. castor oil) at a temperature between 25°C and 45°C, in particular between 25°C and 35°C for about 10 minutes to obtain composition B; f) Add composition B sterilized by filtration to the composition of step d) to obtain mixture C; g) Homogenize mixture C to obtain a microsuspension; h) Adjust the pH of the microsuspension to the desired pH, preferably 5.3±0.1, for example by adding hydrochloric acid solution and/or sodium hydroxide solution; and i) If necessary, adjust the final volume or weight of the composition by adding an appropriate amount of water.

Step e) may be performed at any time from before step a) until step d) is completed.

The homogenization step can be performed using a suitable homogenization technique to obtain a microsuspension with particles of appropriate size, preferably particles having a D50 diameter ranging from 1 to 40 μm, more preferably 1 to 10 μm. High energy homogenization parameters should be fine-tuned according to the mixing technology used, the formulation composition, the manufacturing batch capacity and the design of the formulation equipment. Example 17 describes the high shear homogenization parameters used to make a typical 100 g composition including 3.6% α-cyclodextrin (dry basis) and 1% castor oil in a 125 mL container using an Ultra-Turrax high shear homogenizer (i.e., 8000 RPM / 10 minutes). In Example 19, a 260 g batch capacity formulation was made in a 280 mL container using the same Ultra-Turrax equipment with a setting of 12000 RPM / 10 minutes using a formulation comprising 3% castor oil and 4.0% α-cyclodextrin. Example 23 describes an optimized high shear homogenization process for a composition comprising 3.0% castor oil and 4.0% α-cyclodextrin using a high shear L5M Silverson homogenizer operating at 9000 RPM for 9 minutes using a "square hole screen" probe (Silverson Reference No.: 7250-HQ0005).

Further details or examples of manufacture are provided in the examples. Uses of aqueous lipid microsuspensions

mTOR inhibitors with a macrolide ring structure have both immunosuppressive and anti-inflammatory activities. These two pharmacological activities are widely involved in ophthalmic diseases in both the anterior and posterior parts of the eye.

Anterior or anterior segment diseases are associated with physiological disorders at the level of the ocular surface, including the cornea and sclera. Posterior or posterior segment diseases are associated with physiological disorders inside the eye, usually more specifically at the level of the retina.

Therefore, examples of ocular diseases that can be treated by the mTOR inhibitor macrolide are all inflammatory diseases of the ocular surface, such as dry eye disease (DED), leptomeningeal gland dysfunction and any ocular keratitis.

This class of drugs is particularly interesting for the treatment of both anterior and posterior segment fibrosis or neurodegenerative diseases.

Therefore, the compositions of the present disclosure, particularly the preferred aqueous eye drops lipid microsuspension disclosed herein, can be used to treat a disease selected from the group consisting of allergic or atopic conjunctivitis (i.e., vernal conjunctivitis), post-corneal transplant rejection, dry eye disease (DED), and chondroitinib dysfunction.

The compositions of the present disclosure, especially the preferred aqueous eye drops lipid microsuspension disclosed herein, can also be used to treat or prevent corneal endothelial disorders, such as those selected from the group consisting of: corneal endothelial disorders (e.g., selected from corneal dystrophy, especially Fuchs endothelial corneal dystrophy, posterior polymorphic dystrophy, congenital hereditary endothelial dystrophy), corneal endotheliitis, cytomegalovirus corneal endotheliitis, spontaneous corneal endothelial cell disorders, post-ophthalmic surgery disorders, especially post-ophthalmic laser surgery disorders, trauma and aging.

As used herein, the term "treating" or "treatment" of a disease, disorder, or syndrome includes (i) preventing the disease, disorder, or syndrome from occurring in a subject, i.e., causing one or more clinical symptoms of the disease, disorder, or syndrome to not develop in an animal that may be exposed to or susceptible to the disease, disorder, or syndrome but does not yet experience or display one or more symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, or at least one or more symptoms of the disease, disorder, or syndrome, or (iii) alleviating the disease, disorder, or syndrome or one or more of its symptoms, i.e., causing regression of the disease, disorder, or syndrome.

Anti-inflammatory properties are also of particular interest. The anti-fibrotic properties of some specific mTOR inhibitor macrolides (such as everolimus) may further play a role in ophthalmologically related cancer diseases. This anti-fibrotic activity also appears to be of particular interest in preventing scarring during filtering surgery for glaucoma. The compositions of the present disclosure may also be used in trabeculectomy, typically as a postoperative treatment. The compositions may be used in trabeculectomy to prevent clogging of the orifice during the postoperative wound healing process. The anti-fibrotic properties of mTOR inhibitors such as everolimus may also be used in the treatment of pterygium. In specific embodiments, the present disclosure therefore relates to the use of the compositions in the treatment of pterygium. The addition of both the antifibrotic and immunosuppressive properties of everolimus makes this particular compound a particularly good candidate for reducing the risk of corneal transplant rejection after ocular surgery. Therefore, the compositions of the present disclosure, particularly the preferred aqueous eyedrop lipid microsuspensions disclosed herein, can be used to prevent corneal transplant rejection in subjects in need thereof, particularly high-risk corneal transplants. In this particular application, everolimus is expected to be superior to tacrolimus, which has been used in some topical ophthalmic formulations for many years.

As used herein, the term "subject" refers to a mammal, preferably a human subject.

The compositions of the present disclosure may also be used to treat uveitis in subjects in need thereof, as topical ophthalmic formulations are preferred for administration of everolimus, particularly by improving patient treatment compliance as a more patient-friendly and less risky medical practice.

The present disclosure also encompasses the use of the ophthalmic composition of the present disclosure as an eye drop solution.

In various embodiments, the compositions of the present disclosure, particularly any of the preferred aqueous eye drops lipid microsuspensions disclosed in the above sections, are administered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to any amount that results in improved treatment, cure, prevention or amelioration of a disease or condition, or a reduced rate of progression of a disease or condition, compared to a corresponding subject who has not received the amount, or includes an amount that effectively enhances normal physiological function.

In some embodiments, the compositions herein are used in a method for treating or reducing ocular inflammation in a subject in need thereof, the method comprising topically administering an effective amount of a composition disclosed herein to the eye or lid of the subject.

For example, in a preferred embodiment, the composition of the present disclosure is generally a preferred everolimus-containing aqueous eye drop lipid microsuspension disclosed in the previous section, which is used to prevent corneal transplant rejection. In such preferred embodiments, the microsuspension can be topically applied to the eye in an amount of 1 drop starting 4 weeks after surgery, one to three times a day, for 2 to at least 12 months or longer. In other embodiments, the microsuspension is topically applied to the eye in an amount of 1 drop, one to six times a day.

In other embodiments, the composition of the present disclosure is generally a preferred everolimus-containing aqueous eye drop lipid microsuspension disclosed in the previous section for the treatment of dry eye or blepharoplasty (possibly including blepharitis). In such embodiments, the microsuspension is topically applied to the eye or eyelid in an amount of 1 drop, one to three times a day, for 2 to at least 12 months or longer.

In some embodiments, the compositions herein are used in a method for treating or reducing ocular cellular immunity in a subject in need thereof, the method comprising topically administering an effective amount of a composition disclosed herein to the eye or lid of the subject.

The compositions of the present disclosure are also useful for treating skin diseases, including inflammatory or immune-related skin disorders. For example, the compositions may be administered to treat psoriasis, atopic dermatitis, and/or other skin diseases that are sensitive to anti-inflammatory drugs and/or immunosuppressants. In such applications, the compositions are topically applied to the skin of a subject. Examples Example 1: Methods for characterizing microsuspensions Determination of particle diameter in microsuspensions

The diameter of particles such as microparticles of mTOR inhibitors in oil and cyclodextrin/lipid complexes can be correlated to the D50 diameter of the particles. Diameter D50 is also called the median diameter or the median value in the particle size distribution. Diameter D50 corresponds to the value of the particle diameter at 50% in the cumulative distribution. For example, if D50 is 5 μm, 50% of the particles in the sample are larger than 5 μm; and 50% of the particles are smaller than 5 μm. Diameter D50 is often used to indicate the size of a group of particles.

The diameter and/or size of the particles can be measured according to any method known to those of ordinary skill. For example, the diameter D50 is measured by laser diffraction particle size analysis. In general, there are a limited number of techniques for measuring/evaluating the diameter and/or size of cyclodextrin/drug particles or complexes. In particular, it is known to those of ordinary skill that physical properties (e.g., particle size, diameter, average diameter, average particle size, etc.) are typically evaluated/measured using such limited, typically known techniques.

For example, such known techniques are described in Int. J. Pharm. 493 (2015) pp. 86-95 cited in the above paragraph [00076], which is incorporated herein by reference in its entirety. In addition, such limited, known measurement/assessment techniques are known in the art, as demonstrated by other technical references, such as, for example, the European Pharmacopoeia (2.9.31 Particle size analysis by laser diffraction, January 2010), and Saurabh Bhatia's Nanoparticles types, classification, characterization, fabrication methods and drug delivery applications, Chapter 2, Natural Polymer Drug Delivery Systems, pp. 33-94, Springer, 2016, which is also incorporated herein by reference in its entirety.

In a specific embodiment, the particle size is measured by laser diffraction particle size analysis according to European Pharmacopoeia 2.9.31, with the following parameters: - System: Malvern Mastersizer 3000 with hydraulic MV disperser, - Fraunhofer approximation - Dispersant: water - Refractive index of dispersant: 1.33 - Measuring time: 1 second - Measuring time of background: 10 seconds - Stirrer speed: 3500 rpm - Shading range: 1-20% - Mode: Standard - Sample preparation: Homogenize the eye drop by shaking - Sample size: Add 0.5 ml of eye drop to the dispersion - Cleaning: Rinse twice with dispersant (water) and start measuring, confirm that the beam intensity in the first channel is less than 120 units, and load the background. Stability determination of active pharmaceutical ingredients in microsuspensions

The quantitative determination of everolimus was performed on a reverse phase liquid chromatography system (HPLC system) consisting of a pump operated at 1.1 mL/min, an autosampler, a column compartment operated at 50°C, a UV-Vis detector operated at 210 nm and 275 nm, and a Thermo BDS Hypersil ^® C18 (250x3.0 mm, 5 μm column). The injection volume was 10 μL. The operation time was 23 minutes. The gradient mobile phase system consisted of A: 2 mM potassium dihydrogen phosphate and B: acetonitrile: Table 1 : Time(min) Mobile phase A (%) Mobile phase B (%) 0 50 50 7 35 65 10 0 100 15 50 50 twenty three 50 50

Preparation of standard solution: Accurately weigh approximately 25 mg of the active pharmaceutical ingredient and transfer to a 50 mL volumetric flask, dissolve and fill to volume with methanol. Take 1 mL of the reserve solution, transfer to a 10 mL volumetric flask, and fill to volume with methanol (final concentration is 0.05 mg/mL).

Preparation of test solution: Transfer 1 mL of the microsuspension into a 10 mL volumetric flask and fill up to volume with methanol.

Typically, to evaluate the stability of everolimus in a medium, an in-medium drug substance assay is performed by HPLC at different time points, while different samples from the same raw product are stored for a period of time under different storage conditions (i.e., different temperatures). Example 2: Solubility of Everolimus in Different Media

Excess Everolimus was added to each medium and the samples were stirred continuously overnight at room temperature. The next day, the samples were filtered through a 0.2 μm PES membrane filter and the concentration of dissolved Everolimus was determined by HPLC as described in Example 1. The results are shown in Table 2. The visual appearance of the samples was then recorded after two days of storage at 70°C. Table 2. Medium composition and solubility of Everolimus at room temperature. Solubilizer Brand Name Medium Verolimus Solubility (% w/w) Polysorbate 80 Tween ^® 80 10% (w/w) aqueous solution 1.3 ^a Polyoxyethylene 40 Hydrogenated Castor Oil Kolliphor ^® RH40 10% (w/w) aqueous solution ^1.6a Polyoxyethylene 35 Castor Oil Kolliphor ^® EL 10% (w/w) aqueous solution 1.7 ^b Polyoxyl 15-hydroxy stearate Kolliphor ^® HS15 10% (w/w) aqueous solution 1.5 ^c Polyoxyethylene 40 stearate Myrj ^TM S40 10% (w/w) aqueous solution 1.3 ^a Polyvinyl alcohol Polyvinyl alcohol 40/88 1.4% (w/w) aqueous solution Below detection limit Phosphatidylcholine Lipoid ^® S100 5% (w/w) aqueous solution ^1.0d PEG 400 Kollisolv ^® PEG400 100% 19.5 ^days Poloxamer 407 Kolliphor ^® P407 10% (w/w) aqueous solution 1.3 ^{c a} Precipitation after two days of storage at 70°C. ^b Turned milky white after two days of storage at 70°C. ^c No change in solution during two days of storage at 70°C. ^d The solution turned yellow after two days of storage at 70°C. Example 3 : Simple surfactant solubilization of everolimus is not sufficient to achieve acceptable stability

The stability of everolimus was determined in 1% (w/w) aqueous solutions of selected surfactants. The surfactants were dissolved in water containing 0.1 M citric acid/phosphate buffer at pH 5.0. These buffered surfactant solutions were filled in 5 mL transparent sealed glass bottles and heated to 70°C. Then 0.1% (w/w) everolimus was added at zero time. The concentration of everolimus in the solution was measured at zero time and then after 24 hours and 72 hours. Simple surfactant solubilization of everolimus did not achieve acceptable stability of the drug. Table 3. Concentration of everolimus as a percentage of initial concentration. The initial everolimus concentration was 0.1% (w/w), the medium was a buffered aqueous solution with a pH of 5.0, and the storage temperature was 70°C. Solubilizer Brand Name Everolimus concentration (% of initial concentration) 0:00 ​ 24 hours 72 hours 1% (w/w) Polyoxyethylene 35 Castor Oil Kolliphor ^® EL 100 90.7 72.7 1% (w/w) Polyoxyl 15-hydroxy stearate Kolliphor ^® HS15 100 95.9 85.4 1% (w/w) phospholipid acylcholine Lipoid ^® S100 100 65.1 4.1 1% (w/w) Poloxamer 407 Kolliphor ^® P407 100 93.5 74.5 Example 4 : Natural γ- cyclodextrin has the highest affinity for everolimus, and α- cyclodextrin has the lowest affinity

Phase solubility of everolimus was determined in pure aqueous cyclodextrin solutions at 25°C. The concentration range of cyclodextrin was 0 to 10% (w/w), but the concentration range of β-cyclodextrin was 0 to 1.50% (w/w). Everolimus was added in excess to each cyclodextrin solution, and the samples were stirred continuously on a magnetic stirrer for 24 hours. The next day, the samples were filtered through a 0.45 μm PES syringe filter, and the concentration of dissolved everolimus was determined by HPLC as described in Example 1.

The results are shown in Figure 1. As shown in the figure, native γ-cyclodextrin has the highest affinity for everolimus and α-cyclodextrin has the lowest affinity. Although native γ-cyclodextrin has the highest affinity for everolimus, about 80 mM of γ-cyclodextrin is required to dissolve 1 mM everolimus. Example 5 : Everolimus has the greatest stability between pH 4 and 5

4 mg of everolimus was dissolved in 800 μL of acetonitrile, and 1.2 mL of McIlvaine buffer in the pH range of pH = 4 to pH = 8 was added to make the initial everolimus concentration 2 mg/mL, and the solution was kept constant at 70°C. The everolimus content in the sample was quantified as the percentage of the peak area of the initial value.

The results in Figure 2 show that the degradation of everolimus in pure aqueous solution follows first-order kinetics, with maximum stability at pH values below about 4 to 6. Example 6 : Simple cyclodextrin complexation of everolimus does not result in acceptable stability of the drug

The chemical stability of everolimus in a pure aqueous solution of everolimus saturated with 15% (w/w) γ-cyclodextrin was studied. The degradation rate of everolimus was determined at 70°C and a pH of about 4.2. Complexation with γ-cyclodextrin resulted in an approximately three-fold increase in the chemical stability of everolimus compared to the degradation rate in 40% acetonitrile aqueous solution (Example 5). However, the shelf life (i.e., time to 10% degradation or _t90 ) was only 1 day. Accordingly, the _t90 was estimated to be only 2 to 6 months at 25°C and 8 to 24 months at 5°C. Everolimus degrades via an acid-catalyzed pathway as well as by oxidation. Simple cyclodextrin complexation of everolimus does not provide acceptable stability for the drug. Example 7 : Dissolution of Everolimus in Castor Oil and Medium Chain Triglycerides

The ability of castor oil and medium chain triglycerides (European Pharmacopoeia (Kollisolv ^® MCT 70)) to dissolve 5% (w/w) everolimus was determined. 75 mg of everolimus was suspended in 1.5 mL of both castor oil and medium chain triglycerides. Everolimus dissolved relatively quickly in medium chain triglycerides, while castor oil took some time to dissolve, probably due to its higher viscosity. The samples were stirred at room temperature for 24 hours. After 24 hours, the castor oil formed a clear solution of everolimus, while the previously clear medium chain triglyceride solution turned into a white viscous gel. The castor oil solution was observed for several days, during which time it remained stable. Further studies showed that 7.5% (w/w) everolimus formed a stable solution in castor oil. Example 8 : Everolimus is more stable in microsuspension with α- cyclodextrin and oil

The chemical stability of 0.05% everolimus was evaluated in three castor oil/cyclodextrin suspensions and two reference solutions, one containing 15% (w/w) γ-cyclodextrin and the other containing 1% tyloxapol (a nonionic surfactant polymer with an HLB value of 12.9). The suspensions and reference solutions were stored in sealed glass containers at 60°C, and the everolimus concentration at each time point was determined by HPLC as described in Example 1. The composition and everolimus concentration on day 0, day 1, and day 3 are shown in Table 4. β-cyclodextrin has low water solubility, therefore, only 1% (w/w) β-cyclodextrin suspension was evaluated. The 2% oil/8% α-cyclodextrin suspension showed the highest stability of everolimus. As the concentrations of oil and α-cyclodextrin decreased, the stability decreased, which may be due to the fact that the reduced portion of everolimus was located within the oil droplets (or oil particles) . Solutions without oil were much less effective in protecting everolimus from degradation. However, the physical stability of cyclodextrin-based suspensions is limited. Table 4. Compositions of lipid suspensions and aqueous reference solutions and the concentration of everolimus remaining when stored at 60°C. Cyclodextrin or solubilizer castor oil Remaining everolimus (mg/mL) Day 0 Day 1 3rd day 8% (w/w) α-cyclodextrin 2% (w/w) 0.501 0.503 0.505 4% (w/w) α-cyclodextrin 1 % (w/w) 0.492 0.495 0.454 1% (w/w) β-cyclodextrin 0.80% (w/w) 0.504 0.500 0.478 15% (w/w) γ-cyclodextrin - 0.444 0.417 0.345 1% (w/w) Tyloxapol - 0.465 0.460 0.442 Example 9 : Insufficient stability of everolimus in the emulsion

The chemical stability of 0.05% everolimus in a known emulsion was evaluated and compared to a solution including a surfactant. The solution contained 0.1% everolimus dissolved in water containing 1% Kolliphor ^® HS15 and a 0.1M citric acid/phosphate buffer at pH 5.0. The emulsion contained everolimus (0.05%), castor oil (0.8%), glycerol (2.5%), EDTA (0.05%), and Kolliphor ^® HS15 (1%). The emulsion was prepared at room temperature by dissolving everolimus in the oil followed by high speed mixing with an aqueous solution of Kolliphor ^® HS15. An Ultra Turax ^® T-25 Classic homogenizer (Ika Werke) with a S25KD-18G mixing probe was used for this experiment. The mixing speed started at 11,000 RPM and increased to 24,000 RPM for 2 minutes. Then, the solution containing the remaining other ingredients was added to the previous emulsion and homogenized again with a high-speed homogenizer as before. The two samples were then filled into sealed glass bottles and stored at 70°C.

After one week of storage, the everolimus concentration was determined as described in Example 1. The concentration of everolimus in the solution decreased by about 35%, while the concentration of everolimus in the emulsion decreased by about 27%. Even though the stability of everolimus in this emulsion appears to be better than that in aqueous solution, the stability of everolimus in the emulsion is still unacceptable. Example 10 : Everolimus is not stable in aqueous solution after autoclaving

The chemical stability of aqueous solutions of everolimus after sterilization by autoclave was evaluated. A 15% (w/w) γ-cyclodextrin stock solution was prepared without buffer. An excess of everolimus (approximately 10 mg/mL) was added to the stock solution. The resulting suspension was subjected to mild sonication for approximately 5 minutes, vortexed, and mixed on an electromagnetic stirrer at 25°C overnight. The next day, the suspension was filtered using a 0.45 μm syringe filter (PES material from Agilent). A portion of the filtered solution was saved for analysis, and the other portion was filled in a sealed glass bottle and exposed to an autoclave treatment cycle (121°C for 20 minutes). Two samples were then tested by HPLC according to Example 1. The sample without autoclaving contained 1.4 mg/mL everolimus at a pH of 4.9. After autoclaving, 90.8% of everolimus was lost. Example 11 : Chemical stability of everolimus in microsuspension increases with increasing oil concentration

Two eye drop formulations were prepared and the chemical stability of everolimus was determined at 5°C, 25°C, and 40°C. The compositions of the eye drops are shown in Table 5. The results of the chemical stability studies are shown in Table 6. Increasing the castor oil concentration from 1% (composition A) to 3% (composition B) resulted in a significant increase in the chemical stability of everolimus or a decrease in degradation of about 2.5 to 3 times . Samples of both composition A and B were prepared according to Example 17. Table 5. Compositions and stability study results of aqueous eye drops containing everolimus in lipid microsuspension (% w/w). Composition A Composition B Everolimus 0.05% 0.05% castor oil 1.0% 3.0% α-Cyclodextrin 3.6% 3.6% glycerin 2.0% 2.0% Kolliphor ^® HS15 1.0% 1.0% Poloxamer 407 0.8% 0.8% Ethylenediaminetetraacetic acid (EDTA) disodium 0.05% 0.05% Purified water qs 100 qs 100 Hydrochloric acid or sodium hydroxide qs pH 5.3 ± 0.1 qs pH 5.3 ± 0.1 pH 5.4 5.4 Viscosity 4.5 cP 7.7 cP Osmotic pressure concentration 279 mOsmol/kg 287 mOsmol/kg Particle size Dx(50) 6.5 μm 8.5 μm Table 6. Degradation of Everolimus in Compositions A and B. Percentage of Everolimus remaining after storage at 5°C, 25 ° C, and 40°C when stored in Type I glass primary containers. Composition A 5±2℃ 25℃/60%RH 40℃/25%HR Day 0 100% 100% 100% 45 days 99.9% 99.2% 95.4% 90 days 99.8% 98.2% 90.9% 180 days 99.4% 96.8% 83.8% Composition B Day 0 100% 100% 100% 45 days 99.9% 99.6% 98.4% 90 days 99.9% 99.4% 96.3% 180 days 99.6% 98.5% 93.6% Example 12 : Eye drops vehicles A and B are well tolerated in the human body

Two eyedrop vehicles of identical composition were the same as described in Table 5 but did not contain everolimus. Two healthy male volunteers instilled one drop of vehicle A in the left eye and 24 hours later, one drop of vehicle B in the left eye. Slight blurring of vision was observed approximately 10 seconds after the eyedrop was applied. No burning sensation, itching or other side effects were observed. The eyedrop was well tolerated. Example 13 : Effect of glycerol on microsuspension

In a specific experiment, different concentrations of glycerol were added to the formulation in order to study its possible effects. The samples were prepared according to Example 17.

The permeability of the samples increases in proportion to their glycerol content. In addition, the physical state of the formulations appears to be significantly different in the absence of glycerol: Formulation #1, which does not contain any glycerol, has a different physical appearance compared to Formulations #2 and #3. Moreover, when exposed to the centrifugation test, the total content of precipitated lipid microparticles (the white product portion at the bottom of the centrifuge tube) is approximately 20% lower in quantity compared to the two other samples, indicating that the formulation precipitates faster. This can be inferred as a product with lower physical stability, because this sample without glycerol precipitates faster than the other samples during long-term static storage under normal storage conditions. Table 7. Composition and physicochemical properties of lipid microsuspensions containing different amounts of glycerol . Element Preparation 1 Preparation 2 Preparation 3 glycerin 0 1% 2.5% castor oil 1% 1% 1% Kolliphor ^® HS15 1% 1% 1% Poloxamer 407 0.8% 0.8% 0.8% α-Cyclodextrin 3.6% 3.6% 3.6% Everolimus 0.05 % 0.05 % 0.05 % EDTA (Na ₂ ) 0.05% 0.05% 0.05% HCl/NaOH (0.25N) QS pH 5.3 QS pH 5.3 QS pH 5.3 Purified water QS 100 QS 100 QS 100 Trial Sedimentation rate(*) 35% 45% 45% pH 5.4 5.4 5.4 Permeability 25 mOsmol/kg 143 mOsmol/kg 357 mOsmol/kg particle Dx10 (μm) 1.3 1.3 1.2 Dx50 (μm) 5.7 4.9 4.4 Dx90(μm) 18 18 16 (*) Sedimentation (solid material) from product sample after centrifugation at 13500 RPM for 20 minutes. Example 14 : Effect of high shear mixing speed at constant mixing time

The mixing speed implemented during the high shear homogenization step of a formulation manufacturing process using an Ultra- ^Turrax® tool was evaluated as described in Example 17. Different samples were prepared and a high shear homogenization step was performed for 10 minutes at a specific mixing speed (ranging from 4000 RPM to 20000 RPM) for each of them.

The results show that the physical appearance of the formulation is improved, generally resulting in an increase in the sedimentation rate of the formulation and a decrease in particle size. The higher the mixing speed, the smaller the particle size and the improved (more homogeneous) particle size distribution curve.

Figure 3 shows the particle size evolution of a formulation containing 1% oil (see Composition A in Example 11) at different high shear mixing speeds. While on a laboratory scale, the ideal high shear mixing speed should be higher than 8000 RPM (for 1% oil) or 12000 RPM (for 3% oil), these speeds can be further increased and optimized in larger industrial-scale batch production and depend on the geometry of the high shear homogenizer used (e.g., IKA, Silverson, or other homogenizer brands), or the mixing technology: high shear vs. high pressure homogenizer (i.e., IKA, Hommac, GEA, or other homogenizer brands). Example 15 : Effect of high shear mixing time at constant mixing speed

The mixing time implemented during the high shear homogenization step of the formulation manufacturing process using the Ultra- ^Turrax® tool was evaluated as described in Example 17. Different samples were prepared and the high shear homogenization step was performed for each of them at a constant mixing speed of 8000 rpm for a specific mixing period (ranging from 4 minutes to 20 minutes). The results showed that the physical appearance of the formulation was improved, generally resulting in an increase in the sedimentation rate of the formulation and a decrease in particle size. The longer the mixing period, the smaller the particle size, and the particle size distribution curve was improved (more homogeneous). The curve in Figure 4 shows the particle size evolution at different high shear mixing periods at 8000 RPM for a formulation containing 1% oil (see Composition A of Example 11). If the ideal high shear mixing time is higher than 10 minutes (for both 1% and 3% oil) at laboratory scale, this time can be further increased and optimized in larger industrial scale batch production and depends on the geometry of the high shear homogenizer (e.g., IKA, Silverson, or other homogenizer brands) used, or the mixing technology: high shear vs. high pressure homogenizer (i.e., IKA, Hommac, GEA, or other homogenizer brands). Example 16 : Maximum stability of everolimus at pH between 5.2 and 5.4

Everolimus, like other macrolide mTOR inhibitors, is sensitive to hydrolysis. When dissolved in aqueous solution, everolimus can degrade rapidly. This degradation is affected by the pH of the aqueous environment. The pH for optimal stability was determined by dissolving everolimus (0.025% w/w) in a 14% (w/w) 2-hydroxypropyl-β-cyclodextrin aqueous solution containing 0.1% (w/w) tyloxapol. The pH of the final solution was adjusted with concentrated hydrochloric acid solution and/or concentrated sodium hydroxide solution. The final everolimus solution was stored in a sealed glass container at 40°C and analyzed at different time points as described in Example 1. The results shown in Figure 5 below demonstrate that in aqueous solution, everolimus has maximum stability between pH 5.2 and 5.4.

The solution was also heated in an autoclave at 121°C for 20 minutes in a sealed glass container, and the pH and concentration of everolimus were measured before and after heating (Figure 6). The maximum stability was observed between pH 5.3 and 5.4. Example 17 : Mixing order of components of lipid microsuspension

The following is a general procedure for preparing non-sterile lipid microsuspensions at laboratory scale with a total batch capacity of 100 g:

First, transfer an appropriate amount of castor oil containing the dissolved drug substance (i.e., everolimus) to a glass container. Second, add an aqueous solution of α-cyclodextrin to the container while stirring with an electromagnetic stirrer. Third, add an aqueous solution containing EDTA and glycerol. Fourth, add an aqueous solution of Kolliphor HS15. Mixing is continued for about 10 minutes before removing the stirring bar, and the resulting suspension is mixed for about 10 minutes using a high shear homogenizer probe (Ultra-Turrax ^® T25 homogenizer from IKA Werke, Germany, with probe IKA-S-25N-18G). Then add an aqueous poloxamer 407 solution and mix carefully for about 30 minutes with an electromagnetic stirrer. Adjust the pH to 5.3±0.1 with concentrated hydrochloric acid solution and/or sodium hydroxide solution. Finally, if necessary, add purified water to adjust the final weight of the sample to 100%.

In this example, Poloxamer 407 is preferably not introduced before high shear homogenization because it will produce a lot of foam, which prevents adequate homogenization of the formulation. This will affect the rheological properties and particle size distribution of the formulation. On the other hand, Kolliphor HS15 is preferably introduced before the high shear homogenization step. If it is performed after homogenization, the lipid microsuspension may not have the targeted physical properties. Oil should be introduced before high shear homogenization. Example 18 : Sterilization of microsuspensions at industrial production scale

Topical eye drops must be sterile pharmaceutical products. This means that their manufacturing process must include a sterilization step to provide a sterile product in the primary container so that its integrity is maintained until the liquid formulation is administered to the patient.

Typically, sterile liquids are sterilized by heat, using terminal sterilization of the solution by autoclaving (e.g., 121° C. for 20 minutes). Previous examples have shown that everolimus, like other macrolide mTOR inhibitors, is highly sensitive to heat and degrades during autoclaving. Therefore, one of the challenges of manufacturing lipid microsuspensions containing macrolide mTOR inhibitors is the sterilization of the final ophthalmic solution. One of the advantages of the formulation is that the sterile pharmaceutical formulation can be produced without exposing everolimus, or other mTOR macrolides, to thermal sterilization. To this end, an aseptic manufacturing process including both autoclaving and aseptic filtration steps is used, as described below.

On an industrial scale, the formulation is manufactured using double-jacketed stainless steel tanks. The tank is equipped with a known mixing system, either by stirring blades, or by electromagnetic stirrers. The tank is also equipped with an embedded high shear homogenization tool (either high shear, or high pressure). A portion of purified water is introduced into the industrial stainless steel tank, in which the alpha-cyclodextrin is dissolved. Then, both EDTA and glycerol are added. The tank is then closed and the solution is autoclaved (20 minutes at 121°C). The autoclave step allows to sterilize all parts of the manufacturing equipment that are in direct contact with the product. A concentrated aqueous solution of Kolliphor HS15 is prepared in parallel and, after autoclaving, introduced into the tank through a PES filter of 0.2 μm porosity when the temperature drops to 25°C. PES Supor ^® filters (Pall Corporation, USA) or equivalents may be used. In the next step, the active pharmaceutical ingredient (i.e., everolimus) is dissolved in oil in a separate stainless steel container, which may be slightly heated to a temperature in the range of 25°C-35°C for about 10 minutes to facilitate dissolution of the drug. The oil solution, still at a temperature of at least 30°C, is then added to the tank through another PES filter of 0.2 μm porosity. The 0.2 μm filtration step allows the liquid to be sterilized before it is introduced into the tank containing the sterile bulk solution. Next, a high shear homogenizer installed in the tank and sterilized during the previous autoclave treatment is started and a microsuspension is formed. Next, a concentrated aqueous solution of poloxamer 407 is introduced into the tank through another 0.2 μm PES filter. The pH of the microsuspension is adjusted to 5.3±0.1 with concentrated hydrochloric acid solution and/or sodium hydroxide solution and introduced into the tank through a 0.2 μm PES filter. Finally, the weight of the aqueous microsuspension is adjusted by introducing the remaining amount of purified water through a 0.2 μm PES filter. The final bulk formulation is left in a sterile tank for at least one hour with moderate mixing at a temperature not exceeding 25°C. The tanks containing the sterile formulation are finally connected aseptically to a blow-fill-seal machine capable of filling single-dose eye drop containers (i.e., Rommelag Kunststoff-Maschinen Vertriebsgesellschaft mbH, Germany, or Weiler Engineering, USA, or equivalent). Example 19 : Aqueous Everolimus Lipid Microsuspension Containing 3% Castor Oil

The composition of the aqueous everolimus lipid microsuspension containing 3% castor oil is shown in Table 8. The lipid microsuspension was prepared as described in Example 17, but the high shear homogenization step was performed at 12000 rpm for 10 minutes (Ultra-Turrax ^® ) . Table 8. Composition of the aqueous 0.05% everolimus lipid microsuspension containing 3% castor oil. Element Concentration Everolimus 0.05% castor oil 3.0% α-Cyclodextrin 4.0% glycerin 2.0% Kolliphor ^® HS15 1.0% Poloxamer 407 0.8% Ethylenediaminetetraacetic acid (EDTA) disodium 0.05% Purified water qs100 Hydrochloric acid or sodium hydroxide qs pH 5.3 ± 0.1 pH 5.3 Viscosity 11 cP Osmotic pressure concentration 284 mOsmol/kg Particle size Dx(50) 5.0 μm Example 20 : Eye drop media is suitable for a wide variety of drugs

Eye drops containing a given drug (everolimus, tacrolimus, sirolimus, cyclosporine A, loteprednol, or dexamethasone) were prepared in 250 mL glass bottles (all w/w %) as follows: 8 g of castor oil containing the drug (0.05 to 0.1%) was added to 133.4 g of an aqueous stock solution containing 8.0% α-cyclodextrin, 4.0% glycerol, and 0.1% EDTA, followed by 58.7 g of an aqueous stock solution containing 4.55% Kolliphor ^® HS15 and 0.3595% vitamin E-TPGS. The obtained mixture was homogenized at 12000 rpm for 10 minutes using an Ultra-Turrax ^® high shear homogenizer with a S25N-18G probe at 12000 rpm for 10 minutes. Then 66.7 g of an aqueous 3.217% poloxamer 407 solution was added under stirring. The resulting lipid microsuspension was stirred at room temperature for 30 minutes by an electromagnetic stirrer. Finally, the pH was adjusted by adding hydrochloric acid and/or sodium hydroxide. The final composition of the aqueous lipid microsuspension vehicle was as follows: castor oil 3.00%, EDTA*Na ₂ 2H ₂ O 0.05%, glycerol 2.00%, α-cyclodextrin 4.00%, Kolliphor ^® HS15 1.00%, poloxamer 407 0.80%, vitamin E-TPGS 0.079%, and HCl/NaOH qs for adjusting pH in purified water.

All evaluated drugs, with low water solubility and LogP values ranging from 1.9 to 7.5, were formulated as aqueous eyedrop lipid microsuspensions. In the vehicle, they all formed lipid microsuspensions with suitable appearance, suitable particle size profile, acceptable rheological behavior, and no significant effect on osmotic pressure concentration (Table 9). Therefore, aqueous eyedrop lipid microsuspensions can be used to deliver a wide variety of lipophilic drugs. Table 9. Physicochemical properties of lipid microsuspensions containing different drugs . Parameters Everolimus Tacrolimus Sirolimus Cyclosporine A Lotiprednol Dexamethasone Drug-free Drug LogP 5.9 2.7 6.0 7.5 3.9 1.9 - Drug concentration (% w/w) 0.05 0.05 0.05 0.1 0.05 0.05 - pH ​ 5.2 5.0 5.9 5.9 6.5 6.4 5.3 Osmotic pressure concentration (mOsmol/kg) 291 304 309 292 282 297 293 Viscosity at 15 rpm (cP) 19.4 15.7 16.5 13.6 16.1 17.7 8.7 Particle size (μm) Dx(10) 1.6 1.5 1.5 2.4 1.5 1.4 1.3 Dx(50) 6.6 5.3 5.7 5.5 6.2 4.6 4.3 Dx(90) 26 twenty two 25 36 twenty four 16 17 Example 21 : Effects of polyols on the physical and chemical properties of eye drops

The eye drops were prepared as described in Example 20, but glycerol was omitted or replaced with another type of polyol. The composition of the aqueous lipid microsuspension vehicle was as follows: castor oil 3.00%, EDTA*Na ₂ 2H ₂ O 0.05%, α-cyclodextrin 4.00%, ^Kolliphor® HS15 1.00%, poloxamer 407 0.80%, vitamin E-TPGS 0.079%, and HCl/NaOH qs for pH adjustment in purified water. The formulations contained no polyol, 2.00% glycerol, 2.00% sorbitol, 2.00% propylene glycol (PG), or 2.00% polyethylene glycol 400 (PEG 400) (all in % w/w). Glycerol showed the best curve in terms of, for example, osmotic pressure concentration and particle size distribution. Table 10. Physicochemical properties of lipid microsuspensions containing different polyols . Sample Polyol-free glycerin Sorbitol PG PEG 400 Parameters pH ​ 5.3 5.3 5.4 5.3 5.2 Osmotic pressure concentration (mOsmol/kg) 27 293 169 334 80 Viscosity at 15 rpm (cP) 12 9 12 18 7 Particle size (μm) Dx(10) 1.3 1.3 1.5 1.4 1.4 Dx(50) 5.6 4.2 8.5 4.4 12 Dx(90) 17 17 31 17 71 Particle size distribution Double peak type Single peak Double peak type Single peak Double peak type Δ Settlement (%)* 21.2 17.1 19.4 19.4 25.0 (*) Relative sedimentation is expressed as the percentage of Δ when the sample is centrifuged at 4500 rpm and 13500 rpm. The lower value is the best. Example 22 : In vivo test in rabbits

The purpose of this study was to compare the concentration of everolimus in ocular tissues and to evaluate the potential irritation of a single application of 0.05% everolimus eye drops in the conjunctival sac of the left eye of female New Zealand white rabbits. An aqueous 0.05% everolimus eye drop lipid microsuspension was prepared as described in Example 20. The composition of the eye drop was as follows (all w/w %): 0.05% everolimus in a vehicle consisting of 3.00% castor oil in purified water, 0.05% EDTA*Na ₂ 2H ₂ O, 2.00% glycerol, 4.00% α-cyclodextrin, 1.00% Kolliphor ^® HS15, 0.80% poloxamer 407, and 0.079% vitamin E-TPGS. Adjust pH to 5.4 with HCl/NaOH. Osmotic pressure concentration of eye drops is 291 mOsmol/Kg.

One drop (30 μl) was applied to the left eye and drug levels were measured 2 hours after application. Six female rabbits were used and the results are mean ± SD. The right eye was used as a reference eye. Everolimus concentrations were measured in the cornea, aqueous humor, vitreous, sclera, and plasma in both eyes. Everolimus levels in plasma and ocular tissue supernatants were quantified using the RGA-2 (Research Grade Assay-2) LC-MS/MS method using a surrogate matrix matched calibration curve and QC samples. Surrogate matrix suitability experiments were performed to demonstrate the appropriate use of this method. RGA-2 Standard: Overall back calculation of the bracket curve, at least 75% of the calibration samples must be within 25% of their actual concentration (LLOQ (lower limit of quantification) is 30%), and at least 66% of the QC samples must be within 25% of their actual concentration. For everolimus analysis, an aliquot of each prepared sample was injected onto the HPLC column by an automatic sample injector (Shimadzu, Japan, SIL30-AC). Chromatographic separation was performed on a Kinetix ^® C18 column (2.1x50 mm, 2.6 μm; Phenomenex) maintained at 50°C. The components were separated by gradient separation using ultrapure water and methanol, both containing 0.1% trifluoroacetic acid, at a flow rate of 0.4 mL/min. MS analysis was performed using an API 4500 MS/MS system (Sciex, USA). The instrument was operated in multiple-reaction-monitoring (MRM) mode. Data acquisition was performed in positive ionization mode and optimized for the test items. Data were acquired and processed using the Analyst ^TM data system (Sciex, USA, version 1.7.2) . Table 11. Concentration of 0.05% ( w /w) everolimus in ocular tissue two hours after topical application of aqueous everolimus lipid microsuspension. organization Everolimus concentration (pmol/g), mean ± SD LLOQ ^* cornea 91.6 ± 24.6 0.8 pmol/g Conjunctiva 159 ± 103 0.8 pmol/g Sclera 7.34 ± 1.49 0.8 pmol/g Aqueous humor ＜ 0.45 0.4 pmol/g Vitreous Below LLOQ 0.4 pmol/g Plasma Below LLOQ 0.2 nM ^(*) Lower limit of quantitation (LLOQ)

Everolimus levels in the reference eye (i.e., right eye) were below the LLOQ. Treatment with 0.05% everolimus ophthalmic solution was well tolerated, and studies have shown that 0.05% everolimus ophthalmic solution lipid microsuspension is negligible in irritation. Quantifiable concentrations of everolimus were observed in the aqueous humor, cornea, sclera, and conjunctiva, but not in the vitreous and plasma. The highest everolimus concentrations were found in the conjunctiva, ranging from 47.6 to 308 pmol/g. The cornea contained the second highest concentration, ranging from 67.2 to 127 pmol/g, while other ocular tissues contained significantly lower everolimus concentrations. Therefore, 0.05% everolimus eye drops lipid microsuspension targets the anterior segment of the eye tissue, making it an ideal drug for the treatment of anterior segment eye diseases. Example 23 : Process using a high shear Silverson homogenizer

The formulation manufacturing process described in Example 17 was carried out in an optimized manner using a high shear L5M Silverson homogenizer using a "square hole screen" probe (Silverson Ref. No. 7250-HQ0005) at 9000 RPM for 9 minutes. Table 12. Formulation compositions and physicochemical properties. Element Quantitative formula (% w/w) Everolimus 0.05 α-Cyclodextrin 4.00( ^* ) castor oil 3.0 glycerin 2.0 Kolliphor HS 15 1.0 Poloxamer 407 0.8 EDTA, Na ₂ , 2 H ₂ O 0.05 Purified water QS100 Sedimentation rate 52.0% particle Dx50 (μm) 1.1 Dx10 (μm) 2.5 Dx90 (μm) 7.9 ^(*) Amount corrected for water content The process using the L5M Silverson homogenizer enables the production of lipid microsuspensions with improved homogeneity and smaller particles. Example 24 : Stabilizing effect of EDTA

Two everolimus formulations were prepared according to the method described in Example 17 using an Ultra-Turrax ^® T25 homogenizer at 12000 RPM for about 10 minutes. One contained no EDTA (Formulation A) and the other contained 0.05% (w/w) EDTA (Formulation B). The formulations were stored in glass containers at 40°C for 3 months and the loss of everolimus, pH change and total impurities were monitored (Table 13). In addition, the concentration of butylated hydroxymethoxybenzene (BHT) in Formulation A decreased significantly during storage, while the concentration of BHT in Formulation B remained constant. The addition of EDTA to the formulation stabilizes the pH and reduces the degradation of everolimus during storage. Table 13. Formulation composition (% w /w) and physicochemical properties. Formulation A Formulation B Everolimus (with 2% BHT) 0.05% 0.05% castor oil 3.0% 3.0% α-Cyclodextrin (dry matter) ^(*) 4.0% 4.0% glycerin 2.0% 2.0% Kolliphor ^® HS15 1.0% 1.0% Poloxamer 407 0.8% 0.8% Ethylenediaminetetraacetic acid (EDTA) disodium 0% 0.05% Purified water qs 100 qs 100 Hydrochloric acid or sodium hydroxide qs pH 5.3 ± 0.1 qs pH 5.3 ± 0.1 After 3 months storage at 40℃: pH 4.93 5.27 Everolimus concentration 2.54% 2.84% Total impurities (increase from zero time %) 872% 831% ^(*) : Amount corrected for water content. Example 25 : Stability in plastic containers

The eye drops were prepared according to the method described in Example 17 using an Ultra-Turrax ^® T25 homogenizer at 12000 RPM for about 10 minutes. The composition of the aqueous lipid microsuspension eye drops was as follows: everolimus 0.05%, castor oil 3.00%, α-cyclodextrin 4.00%, glycerol 2.0%, Kolliphor ^® HS15 1.00%, poloxamer 407 0.80%, ethylenediaminetetraacetic acid (EDTA) disodium 0.05%, and HCl/NaOH qs for adjusting the pH value to 5.3 in purified water. The formulation was stored in a primary plastic container, i.e., an ophthalmic plastic bottle with a dropper and cap made of LDPE (pharmaceutical grade low-density polyethylene), 5 mL bottle, completely filled with the formulation. Two sterilization methods were applied, namely , by gamma irradiation at 25 kGy (container A) and by ethylene oxide treatment (container B). Table 14. Physicochemical properties of eye drops during storage. Storage conditions: 5℃ (± 3℃) 25℃ and 60% relative humidity 40℃ and ＜25% relative humidity container: A B A B A B Storage time pH initial 5.4 5.4 5.4 5.4 5.4 5.4 3 months 5.4 5.4 5.3 5.2 5.1 4.9 6 months 5.3 5.2 5.2 5.0 4.8 4.6 Osmolarity (mOsmol/kg) initial 284 284 284 284 284 284 3 months 287 285 283 287 291 291 6 months 287 287 287 290 297 302 Particle size (Dx50 (mm)) initial 6.6 6.6 6.6 6.6 6.6 6.6 3 months 5.5 5.5 6.6 6.2 2.7 2.8 6 months 5.8 5.4 6.2 6.2 2.6 2.6 PSD curve(*) initial conform to conform to conform to conform to conform to conform to 3 months conform to conform to conform to conform to conform to conform to 6 months conform to conform to conform to conform to conform to conform to Everolimus assay (HPLC) (% of labeled amount 0.05% w/w) initial 100.0 100.0 100.0 100.0 100.0 100.0 3 months 97.3 97.3 95.6 96.6 94.1 93.9 6 months 97.0 96.0 94.5 94.3 88.2 88.1 Total impurities test (HPLC) (%) initial 0.2 0.2 0.2 0.2 0.2 0.2 3 months 0.3 0.3 0.8 0.7 3.0 2.6 6 months 0.7 0.5 2.0 1.9 6.5 6.3 (*): Particle size distribution curve: "Conforms" means: "similar to the initial time, with a Gaussian curve".

without

Other features, details and advantages will be seen in the following detailed description and in the accompanying drawings, which show:

Fig . 1. Phase solubility diagram of everolimus in aqueous cyclodextrin solutions: α - cyclodextrin (●), β-cyclodextrin (■), γ-cyclodextrin (□), and 2-hydroxypropyl-γ-cyclodextrin (○). The pH of the unbuffered solution was 4.3±0.1 (SD; γ-cyclodextrin).

Figure 2. Semi - logarithmic plots of the percentage of everolimus remaining in solution versus time at pH 4 (♦), pH 5 (○), pH 6 (□), pH 7 (■), and pH 8 (●) .

Figure 3. Effect of mixing speed on particle size when a high shear homogenizer probe (Ultra-Turrax ^® T25 homogenizer with probe IKA-S-25N-18G from IKA Werke, Germany) is operated for a constant time of 10 minutes .

Fig . 4. Effect of mixing time on particle size using a high shear homogenizer probe (Ultra-Turrax ^® T25 homogenizer with probe IKA-S-25N-18G, IKA Werke , Germany) operating at 8000 rpm.

Fig . 5. Degradation of everolimus in aqueous solutions at different pH values and 40°C.

Fig . 6. Concentration of everolimus before (●) and after (○) heating in an autoclave at 121°C for 20 minutes.

Figure 7. Particle size distribution curve of the sample produced by L5M Silverson homogenizer and measured by Mastersizer analyzer (see Example 1).

Domestic storage information (please note in the order of storage institution, date, and number) None Foreign storage information (please note in the order of storage country, institution, date, and number) None

Claims (15)

An ophthalmic composition comprising, - at least one mTOR inhibitor as an effective pharmaceutical ingredient, preferably everolimus, - a cyclodextrin, preferably α-cyclodextrin, and - an oil, preferably castor oil. The ophthalmic composition as described in claim 1, wherein the cyclodextrin is α-cyclodextrin. The ophthalmic composition as claimed in claim 1 or 2, wherein the composition is a microsuspension, and wherein the oil and the cyclodextrin form a plurality of microparticles in an aqueous medium. The ophthalmic composition as claimed in claim 3, wherein the microparticles have a diameter D50 of less than 40 μm, particularly less than 25 μm, and more particularly less than 10 μm. The ophthalmic composition as described in claim 1, further comprising glycerin as an osmotic agent. The ophthalmic composition as described in claim 1, further comprising poloxamer 407 as a polymer. The ophthalmic composition as described in claim 1 comprises a stabilizer selected from the group consisting of a variety of polyoxyethylene castor oil derivatives, preferably polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil, and polyoxyethylene 15 hydroxy stearate, and more preferably polyoxyethylene 15 hydroxy stearate. The ophthalmic composition as described in claim 1 is a microsuspension, which contains: - 0.01 to 0.1%, such as 0.05% of everolimus; - 3 to 5%, such as 4.0% of α-cyclodextrin; - 2 to 4%, such as 3.0% of castor oil; - 1 to 3%, such as 2.0% of glycerol; - 0.5% to 2% of a stabilizer, such as polyoxyl 15-hydroxy stearate, usually 1.0% of polyoxyl 15-hydroxy stearate; - 0.4 to 1.2% of a polymer, such as poloxamer 407, usually 0.8% of poloxamer 407; - 0 to 0.07% of a complexing agent, such as disodium EDTA, typically 0.05% disodium EDTA; - 0 to 0.5% of an antioxidant, wherein the % is a weight % based on the weight of the composition. A method for preparing an ophthalmic microsuspension, the method comprising the following steps: a) preparing an aqueous composition A by dissolving α-cyclodextrin in purified water; b) preparing an oil phase composition B containing an mTOR inhibitor (e.g., everolimus); preferably, the oil is castor oil, c) sterilizing the composition B as needed; d) adding the oil phase composition B to the aqueous composition A to obtain a mixture C; and e) homogenizing the mixture C to obtain a microsuspension. A method for preparing an ophthalmic composition as described in claim 9, the method comprising the following steps: a) preparing a composition A by dissolving α-cyclodextrin in purified water; b) adding at least one osmotic agent (e.g., glycerol) and at least one complexing agent (e.g., EDTA) to the composition A; c) subjecting the composition A of step b) to an autoclave, for example, at a temperature of 121°C for a period of 20 minutes; d) adding an aqueous solution of a concentrated stabilizer (e.g., polyoxy-15-hydroxy stearate) to the composition of step c); e) Dissolving the mTOR inhibitor (e.g., everolimus) in an oil (preferably castor oil), for example, at a temperature between 25°C and 35°C, to obtain a composition B; f) adding the composition B to the composition of step e) through a filter to obtain a mixture C; g) homogenizing the mixture C to obtain a microsuspension; h) adding an aqueous solution of a concentrated polymer (e.g., poloxamer 407) to the microsuspension; i) adjusting the pH of the microsuspension to a desired pH, for example, between 5.0 and 6.0, typically a pH of 5.3±0.1, for example, by adding a hydrochloric acid solution and/or a sodium hydroxide solution; and j) If necessary, adjust the final volume or weight of the formulation by adding water. An ophthalmic composition obtainable by the method of claim 9 or 10. The ophthalmic composition as described in claim 1 is used for treating an ocular disease, in particular for treating allergic or atopic conjunctivitis (i.e., vernal conjunctivitis), for preventing rejection after corneal transplantation, for treating dry eye disease (DED), leptomeningeal gland dysfunction, pterygium, corneal endothelial disease or corneal malnutrition, or as a postoperative treatment in trabeculectomy. The ophthalmic composition as described in claim 1, which is used for preventing corneal transplant rejection, wherein the composition is topically applied to the eye in an amount of 1 drop, one to three times a day, starting from 4 weeks after surgery, for 2 to at least 12 months or longer. An ophthalmic composition as claimed in claim 1, which is used to treat dry eyes or blepharoplasty (which may include blepharitis), wherein the composition is topically applied to the eye or eyelid in an amount of 1 drop, one to three times a day, for 2 to at least 12 months or longer. An ophthalmic composition for use as described in claim 1, wherein the composition comprises 0.01 to 0.1%, particularly 0.02 to 0.08%, more particularly 0.03 to 0.07%, even more particularly 0.04 to 0.06% by weight of an mTOR inhibitor, such as everolimus, based on the weight of the composition.

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