Ophthalmic compositions

Abstract

The present invention relates to a pharmaceutical compositions comprising human insulin that are preservative free, sterile, and stable for prolonged shelf-life for the treatment of refractory neurotrophic corneal ulcers.

Description

DESCRIPTIONOPHTHALMIC COMPOSITIONSFIELD OF INVENTION

[0001] Provided are ophthalmic compositions comprising insulin for the treatment and / or mitigation of ophthalmic diseases and disorders associated with corneal lesions or ulcers.BACKGROUND

[0002] When an injury occurs, the cornea normally regenerates within a few days. However, some cases may require more time to heal. In cases where a corneal injury does not heal after two weeks, it is considered that there is a persistent corneal epithelial defect. Standard treatment includes eye lubricants or ointments, which often fail, requiring more complex therapies, including surgery. Corneal epithelial defects are damaged areas of the corneal epithelium as a result of an injury. The existence of insulin and insulin-like growth factor receptors in corneal keratocytes and epithelial cells may explain the therapeutic usefulness of insulin in increasing corneal epithelial healing rates.

[0003] The standard medical treatment for the treatment of many of the aforementioned ophthalmic conditions is topical cyclosporine emulsion in eye drops, namely for the treatment of severe keratitis in adult patients, which has frequent adverse reactions such as ocular pain (19.0%), ocular irritation (17.5%), ocular hyperemia (5.5%), increased lacrimation (4.9%) and erythema of the eyelid (1.7%), which are generally transient and occur during instillation. These adverse reactions are consistent with those reported during post-marketing experience. A frequently reported local adverse reaction associated with the use of, for example, IKERVIS® during clinical trials. Patients undergoing immunosuppressive therapy, including cyclosporine, have a higher risk of developing infections. Both generalized and localized infections can occur. This medicine may temporarily induce blurred vision or other visual disturbances. Topical corticosteroids in eye drops such as dexamethasone phosphate can also be used. Like all drugs, this drug can cause side effects, the most common (>1 / 100, <1 / 10) being discomfort, burning, stinging sensation and less common (>1 / 1.000, <1 / 100), dryness around the eyes; eye irritation; tearing; glaucoma; increased intraocular pressure (pressureinside the eye); cataract formation, particularly in diabetics; increased risk of hyperglycemia (high blood glucose levels) in diabetic patients; risk of opportunistic infections (infections that appear when the body's defense reactions are diminished) and risk of keratic calcification.

[0004] Recent studies suggest that insulin and insulin receptors are present in the eye, and are important for the regulation of metabolism, circadian rhythm, autophagy, differentiation and migration of corneal epithelial cells, as well as the restoration of corneal enervation; however, an uncomfortable burning sensation is associated with the use of insulin.

[0005] The patent application US 2021 / 0236604 Al discloses reagents, pharmaceutical compositions and methods for the treatment and prevention of disorders, dermatoses and diseases related to defects in the trigeminal innervation of the cornea, stem cell deficiency, non-healing epithelial wounds and particularly diseases such as neurotrophic keratopathy. This patent application does not disclose the compositions of the present invention.

[0006] EPl 192947 discloses ophthalmic pharmaceutical compositions for treating and / or preventing clinical ophthalmological signs and symptoms in Sjogren's syndrome. This patent does not disclose the compositions of the present invention, nor does it give any indication of the possibility of using the composition of the said patent in conditions or diseases other than Sjogren's syndrome.

[0007] EP3110425 discloses an ophthalmic composition for topical application, intended in particular for the treatment of dry eye. This composition comprises a lubricating polymer of the hyaluronic acid type, with which an oligosaccharide is associated. This patent does not disclose the compositions of the present invention or the possibility of their use in ocular conditions other than dry eye.

[0008] In the study, Serrano-Gimenez R, Contreras-Macias E, Garcia-Bernal A, Fobelo-Lozano MJ. Insulin eye drops for treating corneal ulcer in a non-diabetic patient: regarding a case. Farm Hosp. 2020 Oct 6;44(6):297-299 which discloses the use of topical insulin for the treatment of corneal ulcer in a non-diabetic patient. However, it does not disclose compositions such as those of the present invention. This document alsosuggests further studies to find an ophthalmic composition comprising insulin that has greater stability. It is an object of the present invention to provide such a composition.

[0009] In the clinical study Soares RJDSM, Arede C, Sousa Neves F, da Silva Fernandes J, Cunha Ferreira C, Sequeira J. Topical Insulin-Utility and Results in Refractory Neurotrophic Keratopathy in Stages 2 and 3. Cornea. 2022 Aug l;41(8):990- 994 which discloses the usefulness of insulin in the therapy of neurotrophic keratopathy. However, this study does not disclose the compositions of the present invention, nor does it mention the efficacy of using insulin in other types of eye disease apart from neurotrophic keratopathy.

[0010] In the study Wang AL, Weinlander E, Metcalf BM, Barney NP, Gamm DM, Nehls SM, Struck MC. Use of Topical Insulin to Treat Refractory Neurotrophic Corneal Ulcers. Cornea. 2017 Nov;36(ll): 1426-1428 which discloses the use of topical insulin in the treatment of neurotrophic keratopathy. This study does not disclose the compositions of the present invention, nor does it mention the possibility of using insulin in other types of ophthalmic diseases apart from neurotrophic keratopathy. It also mentions that formulations with optimal insulin concentrations should be developed, which is one of the aims of the invention.

[0011] There is therefore a need to develop ophthalmic formulations comprising human insulin or analogs thereof, such as insulin aspart, that have good stability allowing the user to administer regular doses, such as daily doses, without causing discomfort during or after treatment, that are stable such that the same dosage unit can be used to administer the daily doses for days or weeks at a time, and that can be applied to treat the various ophthalmic conditions described herein. The compositions of the present invention are easy to dilute and have good stability, remaining effective for 30 days when stored at a temperature between 2 ° and 8 °C.

[0012] There is a need for shelf stable, sterile formulation of insulin for selfadminister for one month or longer for the treatment of refractory neurotrophic corneal ulcers.SUMMARY

[0013] The present invention provides topical ocular pharmaceutical compositions of human insulin and analogs thereof suitable for the treatment of various serious ophthalmological conditions for which there is inadequate medical therapy to date. The formulation provided are cost-effective, easy to administer, and are stable in terms of the composition itself, including being shelf stable for ease of daily self-administration for the patient over a course of treatment, such as over a 30 day period. The present invention as well as improving the adverse effects reported in studies already carried out with similar compositions.

[0014] The ophthalmic conditions for which the use of the compositions of the present invention is suitable are selected from corneal epithelial defects and / or corneal epithelial cell deficiency, non-healing epithelial lesions caused by diseases such as neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, application after corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases.

[0015] The compositions of the present invention are intended to overcome and minimize the discomfort of undesirable side effects. The ophthalmic compositions disclosed herein comprise, in addition to insulin (including human insulin or analogs thereof such as insulin aspart), sodium hyaluronate and trehalose, as well as other excipients as described herein.

[0016] The present invention relates to a pharmaceutical compositions comprising human insulin (including human insulin aspart) that are preservative free, sterile, and stable for prolonged shelf-life for the treatment of refractory neurotrophic corneal ulcers and other ophthalmic diseases and disorders.

[0017] Thus, disclosed are pharmaceutical compositions comprising a therapeutically effective amount of human insulin (including insulin aspart), including 1 unit / ml or 1 lU / ml, 2 unit / ml or 2 lU / ml, 3 unit / ml or 3 lU / ml, 4 unit / ml or 4 lU / ml, or 5 unit / ml or 5 lU / ml, hyaluronic acid and trehalose formulated at pH 6.5 to 7.5 and, in embodiments,osmolarity of 275 to 325 mOsm, for topical ocular administration, for example, by eye drops.

[0018] In embodiments, provided are pharmaceutical compositions comprising 4 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metacresol, pH 7.0 in purified water.

[0019] Also disclosed are pharmaceutical compositions comprising 4 lUmL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol, 0.001 %^IN of sodium borate, and 0.7% w / v sodium chloride (or amounts of sodium borate to achieve a pH ranging from 6.5 to 7.5, including pH 7.0) and amounts of sodium chloride to achieve an osmolarity of 275 to 325 mOsm) in purified water.

[0020] Also disclosed are pharmaceutical compositions comprising 1 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metacresol in purified water, including at a pH ranging from 6.5 to 7.5, preferably to pH 7.0.

[0021] Also disclosed are pharmaceutical compositions comprising 1 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metacresol, 0.0013 %^IN of sodium borate, and 0.7% w / v sodium chloride in purified water, including at a pH ranging from 6.5 to 7.5, preferably to pH 7.0.

[0022] Also disclosed are pharmaceutical compositions for use in the treatment of an ocular disease or disorder in a subject, said pharmaceutical composition comprising a disclosed pharmaceutical composition, wherein said pharmaceutical compositions are administered topically to the eye of said subject. In embodiments, the composition is administered daily to the eye of the subject for 30 days or more using the same dosage form unit.

[0023] Also disclosed are multidose vials referred here as unit dosage forms comprising of between 5 mL to 15 mL and / or single doses forms comprising of between 0.4mL to 1 mL of a disclosed pharmaceutical composition.

[0024] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

[0026] FIG. 1 shows representative data for the flow behavior with low-gradient flow for Formulation 9.

[0027] FIG. 2 shows representative data for the flow behavior with low-gradient flow after shearing for Formulation 9.

[0028] FIG. 3 shows representative data comparing the flow behavior with low- gradient flow for Formulation 10.

[0029] FIG. 4 shows representative data for the flow behavior with low-gradient flow after shearing comparing Formulation 9 and Formulation 10.

[0030] FIG. 5 shows representative data for the flow behavior with low-gradient flow for Formulation 9 at 35 °C with artificial tears.

[0031] FIG. 6 shows representative data for simulated eye blinks at 35°C with artificial tears for Formulation 9.

[0032] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attainedby means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0033] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0034] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein may be different from the actual publication dates, which can require independent confirmation.A. DEFINITIONS

[0035] The ophthalmic pharmaceutical compositions of the present invention can be prepared using commonly used pharmaceutically acceptable vehicles in order to mix them with an effective amount of human insulin or analog thereof, sodium hyaluronate, trehalose and other components to suit the desired formulation. The vehicles used for ophthalmic solutions and eye drops include any of those commonly used for this, usually purified water. These ophthalmic pharmaceutical compositions can still be prepared according to conventional methods and should preferably be sterilized before use by conventional methods using membrane filters, autoclaves, etc.

[0036] As used herein, the term “viscosity” refers to the dynamic viscosity (q) of the pharmaceutical composition. The dynamic viscosity may, for example, be measured by a high-performance rheometer, such as an Anton Paar MCR92 Rheometer. Other methods of measuring viscosity may also be applicable.

[0037] As used herein, the term “human insulin” refers to the human hormone whose structure and properties are well-known. Human insulin has two polypeptide chains(chains A and B) that are connected by disulphide bridges between cysteine residues, namely the A-chain and the B-chain. The A-chain is a 21 amino acid peptide and the B- chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges: one between the cysteines in position 6 and 11 of the A-chain; the second between the cysteine in position 7 of the A-chain and the cysteine in position 7 of the B- chain; and the third between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain.

[0038] As used herein, the terms “analogue of insulin” and “insulin analogue” refers to a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, for example that of human insulin, by deleting and / or exchanging at least one amino acid residue occurring in the naturally occurring insulin and / or adding at least one amino acid residue. The added and / or exchanged amino acid residue can either be codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues. Examples of analogues of insulin include, but are not limited to, the following: (i). ‘Insulin aspart’ is created through recombinant DNA technology so that the amino acid B28 in human insulin (i.e. the amino acid no. 28 in the B chain of human insulin), which is proline, is replaced by aspartic acid and it is marketed as NOVOLOG® or ACTRAPID®; (ii). ‘Insulin lispro’ is created through recombinant DNA technology so that the penultimate lysine and proline residues on the C-terminal end of the B-chain of human insulin are reversed (human insulin: ProB28LysB29; insulin lispro: LysB28ProB29); (iii). ‘Insulin glulisine’ differs from human insulin in that the amino acid asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid; (iv). “Insulin glargine” differs from human insulin in that the asparagine at position A21 is replaced by glycine and the B chain is extended at the carboxy terminal by two arginines.

[0039] Human insulin is produced by recombinant DNA technology, but there are non-human insulins and analogues. Insulin aspart is rapid acting human insulin analog homologous with regular human insulin with the exception of a single substitution of the amino acid proline by aspartic acid in position B28, and is reproduced by recombinant DNA technology utilizing Saccharomyces cerevisiae (baker’s yeast). Insulin aspart has the empirical formula C256H381N65O79S6 and a molecular weight of 5825.8 Ka.

[0040] Conventional insulin concentration units (International Units / mL or lU / mL or just U / mL) are bioefficacy based. lU / mL and U / mL are interchangeable for a form of insulin or analog, for example lU / ml of ACTRAPID® is interchangeable with units / mL of NOVOLOG®.

[0041] As used herein, the term “hyaluronic acid” or “hyaluronate” is a non-sulphated glycosaminoglycan (GAG) and is composed of repeating polymeric disaccharides of D- glucuronic acid and N-acetyl-D-glucosamine linked by a glucuronidic P (1— 3) bond. Hyaluronic acid (HA) can have various formulations and can be provided at various concentrations and molecular weights. The terms “hyaluronic acid,” “hyaluronan,” “hyaluronate,” and “HA” are used interchangeably herein to refer to hyaluronic acids or salts of hyaluronic acid, such as the sodium, potassium, magnesium, and calcium salts, among others.

[0042] Hyaluronic acid can be used in the compositions and methods of the present invention at various molecular weights. Since hyaluronic acid is a polymeric molecule, the hyaluronic acid component can exhibit a range of molecular weights, and almost any average of modal molecular weight formulation of hyaluronic acid can be used in the compositions and methods of the present invention, including Low Molecular Weight (“LWM”) Hyaluronan (about 500 to 700 kilodaltons (kDa), Medium Molecular Weight (“MMW”) Hyaluronan (700-1000 kDa), and High Molecular Weight (“BMW”) Hyaluronan (1.0-6.0 million daltons (MDa)). In certain exemplary embodiments, the hyaluronic acid has a molecular weight of at least about 700 kDa, and in certain embodiments, the hyaluronic acid is a High Molecular Weight (“HWM”) hyaluronic acid having a molecular weight of at least about 1 MDa. The molecular weight can be, for example, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400,4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800,5900, 6000 kDa or more, or any range derivable therein. Preferably the hyaluronic acid molecular weight is 1.45MDa.

[0043] Sodium hyaluronate is considered by ophthalmologists to be the standard substance for moisturizing the ocular surface. It is a polysaccharide of natural origin thatis found in the human eye and helps to retain water, moisturizing and lubricating the surface of the eye. In relation to hyaluronic acid, however, there is the problem that it has a limited acting time and the patient needs to repeat applications on a regular basis.

[0044] As used herein, the term “trehalose” is a naturally occurring, non-reducing disaccharide, comprises two glucopyranosyl units connected via an a, a-1, 1-glycosidic bond. Useful forms of trehalose can include trehalose dihydrate (TD) which is crystalline, amorphous trehalose (AT) which is a vitreous form, and the anhydrous forms of trehalose, anhydrous amorphous trehalose (AAT) and anhydrous crystalline trehalose (ACT). Powdered anhydrous trehalose may contain AAT and / or ACT. The term “trehalose,” as used herein, refers to any physical form of trehalose including anhydrous, partially hydrated, fully hydrated and mixtures and solutions thereof.

[0045] Trehalose protects, moisturizes and has antioxidant properties. Trehalose is a naturally occurring bioprotective disaccharide that is present in numerous nonmammalian species, enabling survival in osmotically unfavorable environments by playing an important role in anhydrobiosis, desiccation and protection against oxidative and other stresses.

[0046] It acts as an osmoprotector that is associated with a reduced concentration of intracellular inorganic salts, helping to control the osmotic gradient between the extracellular and intracellular environments. It is also a bioprotector which helps to maintain the integrity of cell membranes, protects proteins, promotes homeostasis, and activates autophagy, reducing cell death associated with apoptosis and inflammation. It has several medical applications, such as cryopreservation and organ transplantation or improving the stability of antibodies.

[0047] The pharmaceutical compositions may further comprise one or more stabilizing agents, such as zinc, sodium chloride, calcium chloride, and arginine. Zinc is a commonly used stabilizing agent. Zinc is preferably added as a salt. Representative examples of zinc salts include zinc acetate, zinc bromide, zinc chloride, zinc fluoride, zinc iodide and zinc sulfate. The skilled artisan will recognize that there are many other zinc salts which also might be used in the process of the present invention. Preferably, zinc chloride is used because this salt does not add new chemical ions to commercially accepted processes.

[0048] The pharmaceutical compositions are sterile when first produced. When the composition is provided in a multi-use vial or cartridge, an anti-microbial preservative compound or mixture of compounds that is compatible with the other components of the formulation is typically added at sufficient strength. Preferred preservatives are aryl acids and phenolic compounds, or mixtures of such compounds. Effective concentrations can be ascertained readily using the methods referenced above. Preservatives most commonly used in insulin products are phenol, metacresol, and benzyl alcohol. Preferably, the preservative is metacresol.

[0049] It is desirable to approximately match the tonicity (i.e., osmolality) of body fluids at the site as closely as possible when administering the pharmaceutical compositions because solutions that are not approximately isotonic with body fluids can produce a painful stinging sensation when administered. Thus, it is desirable that the compositions be approximately isotonic with body fluids at the treatment sites. Typical tonicity agents are glycerol (glycerin) and sodium chloride. The amount of tonicity agent to add is readily determined using standard techniques. Remington: The Science and Practice of Pharmacy, David B. Troy and Paul Beringer, eds., Lippincott Williams & Wilkins, 2006, pp. 257-259; Remington: Essentials of Pharmaceutics, Linda Ed Felton, Pharmaceutical Press, 2013, pp. 277-300.B. PHARMACEUTICAL COMPOSITIONS

[0050] In one aspect, the pharmaceutical composition comprises a therapeutically effective amount of human insulin, hyaluronic acid and trehalose formulated at pH 6.5 to 7.5 for topical ocular administration. The pH of the formulation may be adjusted using a buffer and / or salt, including, for example sodium borate. The pH of the formulation may be pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 and may be, in embodiments, preferably at pH 7.0.

[0051] In various aspects, the human insulin is recombinant human insulin or insulin aspart.

[0052] In various aspects, the pharmaceutical composition further comprises zinc chloride and / or metacresol.

[0053] In various aspects, the concentration of zinc chloride is from 0.0005% w / v to 0.005% w / v. In a further aspect, the zinc chloride concentration is 0.001% w / v. Theconcentration of zinc chloride may be 0.0005% w / v, 0.001% w / v, 0.0015% w / v, 0.002% w / v, 0.0025% w / v, 0.003% w / v, 0.0035% w / v, 0.004% w / v, 0.0045% w / v, 0.005% w / v.

[0054] In various aspects, the concentration of metacresol is from 0.01% w / v to 0.05% w / v. In an exemplary embodiment, the metacresol concentration is 0.012% w / v. The concentration of metacresol may be 0.01% w / v, 0.011% w / v, 0.012% w / v, 0.013% w / v, 0.014% w / v, 0.015% w / v, 0.016% w / v, 0.017% w / v, 0.018% w / v, 0.019% w / v, 0.020% w / v.

[0055] In various aspects, the pharmaceutical composition further comprises an amount of sodium borate to make the composition ranging from pH 6.5 to 7.5. In a further aspect, the pharmaceutical composition further comprises an amount of sodium borate to make the composition pH 7.0.

[0056] In various aspects, the pharmaceutical composition further comprises an amount of sodium chloride to make the pharmaceutical composition between 275 and 325 mOsm. In a further aspect, the pharmaceutical composition further comprises an amount of sodium chloride to make the composition between 275 to 325 mOsm. The osmolarity of the formulation may be 275 mOsm, 280 mOsm, 285 mOsm, 290 mOsm, 295 mOsm, 300 mOsm, 305 mOsm, 310 mOsm, 315 mOsm,, 320 mOsm or 325mOsm.

[0057] In various aspects, the hyaluronic acid is high molecular weight hyaluronic acid and is present in a concentration between 0.1% and 0.4% w / v. In a further aspect, the sodium hyaluronate has a concentration of 0.2% w / v. Suitable concentrations of sodium hyaluronate include 0.1% w / v, 0.2% w / v, 0.3% w / v, and 0.4% w / v.

[0058] In various aspects, the trehalose is trehalose dihydrate and is present in a concentration between 2% and 5% w / v. In a further aspect, the trehalose dihydrate has a concentration of 2% w / v. Suitable concentrations of trehalose dihydrate include 2% w / v, 3% w / v, 4% w / v, and 5% w / v.

[0059] In various aspects, the human insulin is present at 1 lU / mL to 5 lU / mL or between 0.0037% to 0.0185% w / v. In a further aspect, the human insulin is present at 1 lU / mL. Suitable concentrations of human insulin is present at 1 lU / mL, 2 lU / mL, 3 lU / mL, 4 lU / mL, and 5 lU / mL.

[0060] In various aspects, the human insulin is insulin aspart and is present at 1 lU / mL to 4 lU / mL or between 0.0037% to 0.015% w / v. In a further aspect, the insulin aspart ispresent at 4 lU / mL. In a further aspect, the insulin aspart is present at 1 lU / mL. Suitable concentrations of insulin aspart is present at 1 lU / mL, 2 lU / mL, 3 lU / ml, 4 lU / mL.

[0061] In various aspects, the pharmaceutical composition comprises 4 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metacresol, 0.0013 %^IN of sodium borate, and 0.7% w / v sodium chloride in purified water at pH 7.0 (including between pH 6.5 to 7.5) and in embodiments osmolarity between 275 to 325 mOsm.

[0062] In various aspects, the pharmaceutical composition comprises 1 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metacresol, 0.001 %^IN of sodium borate, and 0.7% w / v sodium chloride at pH 7.0 (including between pH 6.5 to 7.5) and in embodiments osmolarity between 275 to 325 mOsm.

[0063] In various aspects, the pharmaceutical composition has a pH between pH 6.5 to 7.5 and osmolarity between 275 to 325 mOsm after storage at 2 °C to 5 °C or at room temperature for 3 weeks or 4 weeks, that is the pH and osmolarity of the formulation does not increase or decrease by more than 1%, 5% or 10% after storage at 2 °C to 5 °C or at room temperature for 3 weeks or 4 weeks .

[0064] In various aspects, the pharmaceutical composition retains 90% or 95% insulin activity after storage at 2 °C to 5 °C or at room temperature for 3 weeks or 4 weeks.

[0065] In various aspects, the pharmaceutical composition has a non-Newtonian flow behavior.

[0066] An ophthalmic composition of the present invention comprises between 10 IU and 50 IU of human insulin (including 1 lU / mL to 5 lU / mL) and may be recombinant human insulin or an analog thereof such as insulin aspart, between 0.15% and 0.30% by weight of high molecular weight sodium hyaluronate and trehalose between 2% and 5% by weight, in a total of 10 mL of final ophthalmic solution.

[0067] An ophthalmic composition of the invention comprises for 10 mL of ophthalmic solution, human insulin between 10 IU and 50 IU, and may be recombinant human insulin or an analog thereof such as insulin aspart, sodium hyaluronate at a concentration between 0.1% and 0.4% by weight.

[0068] An ophthalmic composition of the invention comprises, for 10 mL of ophthalmic solution, insulin between 10 IU and 50 IU, and may be recombinant human insulin or an analog thereof such as insulin aspart, and trehalose in a concentration between 2% and 5% by weight.

[0069] In one aspect the ophthalmic compositions of the invention comprise excipients selected from sodium chloride, potassium chloride, sodium dihydrogen phosphate, sodium borate, D-glucose, magnesium sulphate, sodium citrate, sterilized refined water, or combinations thereof.

[0070] In one embodiment, the ophthalmic compositions of the invention comprise sodium borate between 0.015g and 0.050g per lOmL, between 0.01% and 0.1% by weight of sodium chloride and sterilized refined water. The amount of sodium borate may be adjusted to bring the pH of the formulation to pH 7.0 (or ranging from pH 6.5 to 7.5). The amount of sodium chloride may be adjusted to bring the osmolarity of the formulation to between 275 to 325 mOsm.

[0071] The ophthalmic compositions of the present invention can be prepared as nonoral formulations, for example, ophthalmic solutions, eye ointments, prolonged local release implants, parenteral solutions and eye drops, where the preferred form of administration is eye drops.

[0072] In one aspect, the ophthalmic solutions of the present invention are prepared in multidose vials referred here as unit dosage forms of between 5 mL and 15 mL and / or in single doses forms of between 0.4 mL and 1 mL.C. METHODS OF TREATING AN OCULAR DISEASE OR DISORDER IN ASUBJECT

[0073] In one aspect, the pharmaceutical composition is for use in the treatment of an ocular disease or disorder in a subject, said pharmaceutical composition comprising a disclosed pharmaceutical composition wherein said pharmaceutical composition is administered topically to the eye of said subject.

[0074] In various aspects, ocular disease or disorder is a corneal ulcer, neurotrophic keratitis, herpetic and non-herpetic keratitis, and ulcers due to trauma or infection, dry eye, severe dry eye and may also include use to address lesions after corneal surgery,infections, trauma and general inflammatory processes or those resulting from systemic diseases.

[0075] In various aspects, the administration ameliorates symptoms of the disease or disorder, wherein said symptoms include pain or itching of the eye, foreign body sensation.

[0076] In various aspects, the pharmaceutical composition is administered daily for 30 days from the same unit dosage comprising the pharmaceutical composition each day or from single doses.

[0077] In one aspect, the pharmaceutical composition for use in the treatment of an ocular disease or disorder in a subject comprises 4 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol in purified water at pH 7.0. In embodiments, the treatment is topical administration to the eye of the composition. In embodiments, the ocular disease or disorder is neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, lesions or ulcers caused by corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases. In embodiments, the pharmaceutical composition is administered daily in the eye affected by the disease or disorder and in embodiments is administered for 5 days, 10 days, 20 days, 25 days or 30 days.

[0078] In one aspect, the pharmaceutical composition for use in the treatment of an ocular disease or disorder in a subject comprises 4 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol, 0.001 %^IN of sodium borate, and 0.7% w / v sodium chloride in purified water at pH 7.0. In embodiments, the treatment is topical administration to the eye of the composition. In embodiments, the ocular disease or disorder is neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, lesions or ulcers caused by corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases. In embodiments, the pharmaceutical composition is administered daily in the eye affectedby the disease or disorder and in embodiments is administered for 5 days, 10 days, 20 days, 25 days or 30 days.

[0079] In one aspect, the pharmaceutical composition for use in the treatment of an ocular disease or disorder in a subject comprises 1 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol in purified water at pH 7.0. In embodiments, the treatment is topical administration to the eye of the composition. In embodiments, the ocular disease or disorder is neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, lesions or ulcers caused by corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases. In embodiments, the pharmaceutical composition is administered daily in the eye affected by the disease or disorder and in embodiments is administered for 5 days, 10 days, 20 days, 25 days or 30 days.

[0080] In one aspect, the pharmaceutical composition for use in the treatment of an ocular disease or disorder in a subject comprises 1 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol, 0.0013 %^IN of sodium borate, and 0.7% w / v sodium chloride in purified water at pH 7.0. In embodiments, the treatment is topical administration to the eye of the composition. In embodiments, the ocular disease or disorder is neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, lesions or ulcers caused by corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases. In embodiments, the pharmaceutical composition is administered daily in the eye affected by the disease or disorder and in embodiments is administered for 5 days, 10 days, 20 days, 25 days or 30 days. In one aspect, provided are methods of treating, preventing or ameliorating the symptoms of an ocular disease or disorder in a subject comprising administering a pharmaceutical composition comprising 4 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol in purified water at pH 7.0. In embodiments, thetreatment is topical administration to the eye of the composition. In embodiments, the ocular disease or disorder is neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, lesions or ulcers caused by corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases. In embodiments, the pharmaceutical composition is administered daily in the eye affected by the disease or disorder and in embodiments is administered for 5 days, 10 days, 20 days, 25 days or 30 days.

[0081] In one aspect, provided are methods of treating, preventing or ameliortating the symptoms of an ocular disease or disorder in a subject comprising administering a pharmaceutical composition comprising 4 lUmL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol, 0.001 %^IN of sodium borate, and 0.7% w / v sodium chloride in purified water at pH 7.0. In embodiments, the treatment is topical administration to the eye of the composition. In embodiments, the ocular disease or disorder is neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, lesions or ulcers caused by corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases. In embodiments, the pharmaceutical composition is administered daily in the eye affected by the disease or disorder and in embodiments is administered for 5 days, 10 days, 20 days, 25 days or 30 days.

[0082] In one aspect, provided are methods of treating, preventing or ameliortating the symptoms of an ocular disease or disorder in a subject comprising administering a pharmaceutical composition comprising 1 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol in purified water at pH 7.0. In embodiments, the treatment is topical administration to the eye of the composition. In embodiments, the ocular disease or disorder is neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, lesions or ulcers caused by corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases. Inembodiments, the pharmaceutical composition is administered daily in the eye affected by the disease or disorder and in embodiments is administered for 5 days, 10 days, 20 days, 25 days or 30 days.

[0083] In one aspect, provided are methods of treating, preventing or ameliortating the symptoms of an ocular disease or disorder in a subject comprising administering a pharmaceutical composition comprising 1 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid (high molecular weight), 0.001% w / v zinc chloride, 0.012% w / v metracresol, 0.0013 %^IN of sodium borate, and 0.7% w / v sodium chloride in purified water at pH 7.0. In embodiments, the treatment is topical administration to the eye of the composition. In embodiments, the ocular disease or disorder is neurotrophic keratopathy, herpetic and non-herpetic keratitis, recurrent and / or persistent corneal ulcers, erosion or abrasion of corneal epithelial cells caused by ocular surface diseases, dry eye, lesions or ulcers caused by corneal surgery, infections, trauma and general inflammatory processes or those resulting from systemic diseases. In embodiments, the pharmaceutical composition is administered daily in the eye affected by the disease or disorder and in embodiments is administered for 5 days, 10 days, 20 days, 25 days or 30 days.D. EXAMPLES

[0084] The following examples are intended to better illustrate certain aspects of the ophthalmic compositions of the invention. The following examples are given by way of illustration and should not be regarded as limiting.

[0085] The quantity of insulin aspart for the formulations was calculated according to the monography of insulin aspart where 1 IU is equivalent to 0.0350 mg of insulin aspart.

[0086] The quantity to weigh to obtain 1 lU / mL is 0.037 mg / mL and the quantity to weigh to obtain 4 lU / mL is 0.148 mg / mL.

[0087] Initial formulations were utilized to evaluate the behavior and solubility of insulin aspart in the aqueous medium and in aqueous medium with sodium borate. Insulin dosage used for these formulations was 4 lU / mL. These formulations determined the sodium borate to add to the formulation in order to obtain a pH target of 7 for an ophthalmic utilization.Example 1

[0088] The composition of Formulation 1 ((SX01118-E001)) is shown in Table 1. Insulin aspart (0.0739 g) was added to WFI (499.6 g) and the mixture was stirred at 700 rpm for 10 minutes providing a slightly cloudy, visually homogeneous solution. The solution was prepared to evaulate the behavior and solubility of insulin aspart in the aqueous medium and monitoring the appearance, pH and osmolarity over 7 days. The solubilization of insulin aspart in water is quick and the pH in water is close to the target for an ophthalmic utilization (6.69 for a target of 7.0). The insulin aspart in water solution does not provide osmolarity to the formulation (0 mOsm).TABLE 1.Example 2

[0089] The composition of Formulation 2 ((SX01118-E002)) is shown in Table 2. Sodium borate (7.5 g) was added to WFI (333.0 g) and the mixture was stirred at 650 rpm for 10 minutes providing a clear, visually homogeneous solution. Insulin aspart (0.0740 g) was added to solution and addition WFI (169.9 g) was added and the mixture was stirred at 700 rpm for 10 minutes providing a clear, visually homogeneous solution. ThepH for this formulation was 9.25 and is above the target for an ophthalmic utilization.The osmolarity for the formulation was 181 mOsm.TABLE 2.Example 3

[0090] The composition of Formulation 3 (SX01118-EOO3) is shown in Table 3. Sodium borate (25 g) was added to WFI (333.0 g) and the mixture was stirred at 650 rpm for 38 minutes.TABLE 3.Example 4

[0091] The composition of Formulation 4 (SX01118-E004) is shown in Table 4. Sodium borate (15 g) was added to WFI (333.0 g) and the mixture was stirred at 650 rpm for 10 minutes providing a clear, visually homogeneous solution. Insulin aspart (0.0740 g) was added to solution and addition WFI (169.9 g) was added and the mixture was stirred at 700 rpm for 10 minutes providing a clear, visually homogeneous solution. The osmolarity for the formulation was 306 mosm.TABLE 4.Example 5

[0092] The composition of Formulation 5 (SX01118-E005) is shown in Table 5. Sodium borate (0.25 g) was added to WFI (333.0 g) and the mixture was stirred at 650 rpm for 10 minutes providing a clear, visually homogeneous solution. Insulin aspart (0.0738 g) wasadded to solution and addition WFI (166.74 g) was added and the mixture was stirred at 700 rpm for 10 minutes providing a clear, visually homogeneous solution. The osmolarity for the formulation was 8 mOsm.TABLE 5.Example 6

[0093] The composition of Formulation 6 (SX01118-E006) is shown in Table 6. Sodium borate (0.005 g) was added to WFI (333.0 g) and the mixture was stirred at 650 rpm for 10 minutes providing a clear, visually homogeneous solution. Insulin aspart (0.0738 g) was added to solution and additional WFI (167.1 g) was added and the mixture was stirred at 700 rpm for 10 minutes providing a clear, visually homogeneous solution. The pH of this formulation was 6.98 and is close to the target for an ophthalmic utilization. The osmolarity for the formulation was 1 mOsm.TABLE 6.Example 7

[0094] The composition of Formulation 7 (SX01118-E007) is shown in Table 7. After the adjustment of the pH with the addition of sodium borate, the formulation of Example 7 (SX01118-E007) was prepared to evaluate the quantity of sodium chloride to be added to be at the target of an ophthalmic eye drop for the osmolarity (between 275 and 325 mosm). Insulin aspart (0.0738 g) was added to solution and additional WFI (333.0 g) was added and the mixture was stirred at 700 rpm for 10 minutes providing a clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 7.13 and osmolarity was 196 mOsm.TABLE 7.Example 8

[0095] The composition of Formulation 8 (SXO1118-EOO8) with no insulin aspart is shown in Table 8. Trehalose dihydrate (2.0 g) was added to WFI (67.0 g) and the mixture was stirred at 450 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.200 g) was added to solution and the mixture was stirred at 600 rpm to 1100 rpm for 360 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 6.68 and osmolarity was 359 mOsm.TABLE 8.Example 9

[0096] The composition of Formulation 9 (SX01118-E009) is shown in Table 9. Sodium hyaluronate (0.2 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm to 1100 rpm for 360 minutes providing a viscous, clear,visually homogeneous solution. Insulin aspart (0.0148 g) was added and the mixture was stirred at 1100 rpm for 10 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 6.67 and osmolarity was 334 mOsm.TABLE 9.Example 10

[0097] The composition of Formulation 10 (SX01118-E010) is shown in Table 10. Trehalose dihydrate (2.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.20 g) was added to the mixture and stirred at 600 rpm to 1100 rpm for 360 minutes providing a viscous, clear, visually homogeneous solution. Insulin aspart (0.0148 g) was added and the mixture was stirred at 1100 rpm for 10 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 6.85 and osmolarity was 292 mOsm.TABLE 10.Example 11

[0098] The composition of Formulation 11 (SX01118-E011) is shown in Table 11. Trehalose dihydrate (2.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.20 g) was added to the mixture and stirred at 600 rpm to 1100 rpm for 360 minutes providing a viscous, clear, visually homogeneous solution. Insulin aspart (3.7 mg) was added and the mixture was stirred at 1100 rpm for 10 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 6.94 and osmolarity was 277 mOsm.TABLE 11.Example 12

[0099] The composition of Formulation 12 (SX01118-E012) is shown in Table 12. Trehalose dihydrate (2.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Zinc chloride, as a 0.01% aqueous solution, (0.168 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Metacresol (12 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.20 g) was added to the mixture and stirred at 600 rpm to 1100 rpm for 361 minutes providing a viscous, clear, visually homogeneous solution. Insulin aspart (0.0148 g) was added and the mixture was stirred at 1100 rpm for 10 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 7.14 and osmolarity was 291 mOsm.TABLE 12.Example 13

[0100] The composition of Formulation 13 (SX01118-EO13) without insulin aspart is shown in Table 13. Trehalose dihydrate (2.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Zinc chloride, as a 0.01% aqueous solution, (0.168 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Metacresol (12 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.20 g) was added to the mixture and stirred at 600 rpm to 1100 rpm for 361 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 6.77 and osmolarity was 268 mOsm.TABLE 13.Example 14

[0101] The composition of Formulation 14 (SX01118-E014) is shown in Table 14. Trehalose dihydrate (2.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Zinc chloride, as a 0.01% aqueous solution, (0.168 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Metacresol (12 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.20 g) was added to the mixture and stirred at 600 rpm to 1100 rpm for 361 minutes providing a viscous, clear, visually homogeneous solution. Insulin aspart (3.7 mg) was added and the mixture was stirred at 1100 rpm for 10 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 6.59 and osmolarity was 314 mOsm.TABLE 14.Example 15

[0102] The composition of Formulation 15 (SX01118-E015) is shown in Table 15. Trehalose dihydrate (2.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Metacresol (12 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.20 g) was added to the mixture and stirred at 600 rpm to 1100 rpm for 361 minutes providing a viscous, clear, visually homogeneous solution. Insulin aspart (3.7 mg) was added and the mixture was stirred at 1100 rpm for 10 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 6.88 and osmolarity was 289 mOsm.TABLE 15.Example 16

[0103] The composition of Formulation 16 (SX01118-E016) is shown in Table 16. Trehalose dihydrate (3.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visuallyhomogeneous solution. Zinc chloride, as a 0.01% aqueous solution, (0.252 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Metacresol (18 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.30 g) was added to the mixture and stirred at 600 rpm to 1100 rpm for 361 minutes providing a viscous, clear, visually homogeneous solution. Insulin aspart (22.14 mg) was added and the mixture was stirred at 1100 rpm for 10 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 7.19 and osmolarity was 282 mOsm.TABLE 16.Example 17

[0104] The composition of Formulation 17 (SX01118-E017) is shown in Table 17. Trehalose dihydrate (3.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Zinc chloride, as a 0.01% aqueous solution, (0.063 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneoussolution. Metacresol (4.5 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.20 g) was added to the mixture and stirred at 600 rpm to 1100 rpm for 367 minutes providing a viscous, clear, visually homogeneous solution. Insulin aspart (0.0148 g) was added and the mixture was stirred at 1100 rpm for 10 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 7.10 and osmolarity was 285 mOsm.TABLE 17.Example 18

[0105] The composition of Formulation 18 (SXO1118-EO18) is shown in Table 18. Trehalose dihydrate (3.0 g) was added to solution and addition WFI (90.0 g) was added and the mixture was stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Zinc chloride, as a 0.01% aqueous solution, (0.252 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Metacresol (18 mg) was added and stirred at 600 rpm for 10 minutes providing a clear, visually homogeneous solution. Sodium hyaluronate (0.20 g) was added to themixture and stirred at 600 rpm to 1100 rpm for 360 minutes providing a viscous, clear, visually homogeneous solution. Final adjustments were made to pH, osmolarity and volume by addition of sufficient quantities of sodium borate, sodium chloride and WFI. The pH was 7.06 and osmolarity was 291 mOsm.TABLE 18.Example 19

[0106] Formulations 1, 2, 4 and 6 were placed under different conditions and evaluated after 3 days and 1 week to assess their stability in terms of appearance, pH and osmolarity as shown in Table 19.TABLE 19.NSC - no significant change

[0107] No visual change and variation in osmolarity were observed on the 4 Formulations after 7 days. The pH increases when only insulin is present water (Formulation 1). The addition of sodium borate in high quantity avoids the pH variation for 7 days (Formulation 2 and Formulation 4). In Formulation 6, the lowest quantity addedof sodium borate doesn't allow to minimize pH variation as observed in previous trials and the pH increases in the same way when there is only insulin with water.Example 20

[0108] A second study was carried out with Formulations 9, 10, 11, 12, 14 and 15 in order to understand the role of each excipient of the formulation. Samples of the formulations were placed in a refrigerator at 2-5 °C, at room temperature (ambient temperature), and at 30 °C and appearance, pH and osmolarity were measured after 3 weeks and 4 weeks in terms of appearance, pH and osmolarity as shown in Table 20.TABLE 20NVC - No Visual Change

[0109] No change in appearance was observed on these Examples, except for Formulation 15 containing only metacresol without zinc chloride, where black filaments were present in suspension after 4 weeks at 30°C / 65% RH. No significative osmolarity variation was observed. A slight decrease in osmolarity was observed on Formulation 14 (insulin aspart at 1 lU / mE, with metacresol and zinc chloride) compared with initial, but this remained stable for all temperature conditions for 4 weeks. The pH of each batch tends to increase compared with initial, except for Formulation 12 (4 lU / mE, with metacresol and zinc chloride), whose pH remains stable in the fridge, and Formulation 14 (1 lU / mE, with metacresol and zinc chloride), whose pH remains stable at all three temperatures.

[0110] Assays were performed after 2 months on Formulation 12, the results are showing storage for two months at 2-5°C and ambient temperature results of : 97.2% and 96.1% respectively. Storage at 30°C / 65%RH for two months is 82.2%. Compliant results were obtained for the storage in the fridge and at room temperature after 2 months.Example 21

[0111] A study of the product's rheological behavior was carried out on Formulation 9 containing sodium hyaluronate but without trehalose dihydrate. Formulation 10 wasalso evaluated in order to compare the impact of other excipients into the formulation rheological behavior.

[0112] The protocol applied was as follows:Samples are left at room temperature for 1 hour before analysis (samples were stored in the fridge), and rehomogenized just before analysis. If artificial tears are required, they are added directly to the sample at the time of measurement (ratio 7 / 30: artificial tear s / s ample). These analyses are carried out using an Anton Paar MCR92 Rheometer.The formulation of the artificial tears used is shown in Table 21:TABLE 21.

[0113] The first characterization consisted in assessing the product's flow behavior and viscosity at 25°C without artificial tears.

[0114] For this purpose, two analyses were carried out with the following parameters:- Flow behavior with low -gradient flow:- Geometry: CP50-1, diameter: 50 mm, air gap: 0.101 mm,- Temperature: 25°C + / - 0.1 °C,- Measurement: shear gradient: o Interval 1: shear gradient: 0.1 s’1to 100 s’1, o Interval 2: constant shear at 100 s’1,o Interval 3: shear gradient: 100 s’1to 0.1 s’1.- Flow behavior with low -gradient flow after shearing:- Geometry: CP50-1, diameter: 50 mm, air gap: 0.101 mm,- Temperature: 25°C + / - 0.1 °C,- Measurement: shear gradient: o Interval 1: constant shear at 5000 s’1, o Interval 2: shear gradient: 100 s’1to 0.1 s’1.

[0115] The product's behavior at low shear at 25 °C is non-Newtonian and shearthinning with pseudo plastic and thixotropic behavior is observed (FIG.l - 4). Indeed, the product fluidizes with increasing shear rate, flows by gravity without the need for a flow threshold, and returns to its initial state after shearing. FIG. 1 shows the flow behavior with low-gradient flow for Formulation 9. FIG. 2 shows the flow behavior with low- gradient flow after shearing for Formulation 9. FIG. 3 shows the flow behavior with low- gradient flow for Formulation 10. A similar behavior is observed between the Example 9 (gray) and Example 10 (black) as shown in FIG. 4, confirming the point that only sodium hyaluronate has a role on the rheological behavior of the product.

[0116] In order to simulate the behavior of the formulation after application into the eye, the flow behavior method with low-gradient flow method with artificial tears at 35 °C was applied to Example 9:- Geometry: CP50-1, diameter: 50 mm, air gap: 0.101 mm,- Temperature: 35 °C + / - 0.1 °C,- Measurement: shear gradient: o Interval 1: shear gradient: 0.1 s’1to 100 s’1, o Interval 2: constant shear at 100 s’1, o Interval 3: shear gradient: 100 s’1to 0.1 s’1.

[0117] Similar behavior was observed at 35 °C, with a slight drop in viscosity, potentially due to dilution of the sample during introduction of the artificial tears as shown in FIG. 5. The product does not seem to gel on contact with tears.

[0118] A second characterization consisted in assessing Formulation 9 resistance to simulated eye blinks.

[0119] Analyses were performed after the addition of artificial tears by rotational measurements. The analysis parameters for rotational measurements were as follows:- Geometry: CP50-1, diameter: 50 mm, air gap: 0.101 mm,- Temperature: 35 °C + / - 0.1 °C,- Measurement: shear gradient: o Interval 1: Constant shear at 5000 s’1, o Interval 2: Constant shear at 1 s’1for 5 points, o Interval 2 and 3 repeated 4 times, o Interval 4: Constant shear at 5000 s’1, o Interval 5: Constant shear at 1 s’1for 60 points, o Interval 4 and 5 repeated 4 times.

[0120] Distinct blinks are observed, and the product withstands repeated blinking without losing viscosity as shown in FIG. 6.Example 22

[0121] The appearance, pH, osmolarity, assay and microbiological analysis of Formulations 16, 17 and 18 are shown in Table 22.TABLE 22.STABILLITY TESTSStability tests were performed to Formulation 016 (SX01118-E016) and 012 (SX01118- E012) both with 4 lU / mL of the present invention. The stability data are shown in the Table 23 below at indicated storage temperatures for 2 months (T2M) or 3 months (T3M).TABLE 23.As can be seen the pharmaceutical compositions of the present invention maintains stable after 3 months storage at 5 °C or 25 °C.

Claims

AMENDED CLAIMS received by the International Bureau on October 9, 2024 (09.10.2024)CLAIMS1. A pharmaceutical composition comprising a therapeutically effective amount of human insulin, hyaluronic acid and trehalose formulated at pH 6.5 to 7.5 for topical ocular administration, wherein:- The human insulin is is recombinant human insulin or insulin aspart present in an amount of 1 to 5 lU / ml,- The hyaluronic acid has a molecular weight of 1.0 to 6.0 million Daltons (MDa) and is present in a concentration of 0.1% to 0.4% w / v,- The trehalose is trehalose or dehydrated trehalose and is present in a concentration of 2% to 5% w / vThe composition further comprising at least one stabilizing agent selected from zinc, sodium chloride, calcium chloride, sodium borate or arginine or combinations thereof,- a tonicity agent selected from glycerol, glycerin or sodium chloride, and- has an osmolarity of 275 to 325 mOsm having a non-Newt onian flow behavior.

2. The pharmaceutical composition according to claim 1 having a pH value of 7.0.

3. A pharmaceutical composition according to claim 1 wherein the hyaluronic acid molecular weight is 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200,3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200,4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200,5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000 kDa, preferably the hyaluronic acid molecular weight is 1.45MDa.45AMENDED SHEET (ARTICLE 19)4. A pharmaceutical composition according to claim 1 wherein the hyaluronic acid is present in a concentration of 0.1% w / v, 0.2% w / v, 0.3% w / v, and 0.4% w / v, preferably in a concentration of 0.2%.

5. A pharmaceutical composition according to claim 1 wherein trehalose is trehalose dihydrate and is present in a concentration of 2% w / v, 3% w / v, 4% w / v, and 5% w / v, preferably in a concentration of 2%.

6. A pharmaceutical composition according to claim 1 wherein the human insulin is insulin aspart and is present in a concentration of 1 lU / mL, 2 lU / mL, 3 lU / mL, 4 lU / mL, and 5 lU / mL, preferably in a concentration of 0.0037% to 0.015% w / v .

7. A pharmaceutical composition according to claim 1 wherein the sodium borate is present in said composition in an amount of 0.01% and 0.1% by weight of sodium chloride.

8. A pharmaceutical composition according to any of the claims 1 to 7 comprising 4 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid, 0.001% w / v zinc chloride, 0.012% w / v metacresol, 0.001 %w / v of sodium borate, and 0.7% w / v sodium chloride.

9. A pharmaceutical composition according to any of the claims 1 to 7 comprising 1 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid, 0.001% w / v zinc chloride, 0.012% w / v metacresol at a pH of 6.5 to 7.5, preferably to pH 7.0.

10. A pharmaceutical composition according to any of the claims 1 to 7 comprising 1 lU / mL insulin aspart, 2.0% w / v trehalose46AMENDED SHEET (ARTICLE 19)dihydrate, 0.2% hyaluronic acid, 0.001% w / v zinc chloride, 0.012% w / v metacresol, 0.0013 %w / v of sodium borate, and 0.7% w / v sodium chloride at a pH of 6.5 to 7.5, preferably at a pH of 7.0.

11. A pharmaceutical composition according to any of the claims 1 to 7 comprising 4 lU / mL insulin aspart, 2.0% w / v trehalose dihydrate, 0.2% hyaluronic acid, 0.001% w / v zinc chloride, 0.012% w / v metacresol.

12. A pharmaceutical composition according to any of the claims 1 to 10 in the form of a solution or powder.

13. A pharmaceutical composition as described in any of the claims 1 to 11 for use in the treatment of an ocular disease or disorder in a subject, wherein said pharmaceutical composition is administered topically to the eye of said sub ect .

14. A pharmaceutical composition as described in any of the claims 1 to 11 for use for use in the treatment of an ocular disease or disorder in a subject wherein, said ocular disease or disorder is a corneal ulcer, neurotrophic keratitis, herpetic and non-herpetic keratitis or ulcers.

15. The pharmaceutical composition as described in any of the claims 1 to 11 wherein the administration ameliorates symptoms of the disease or disorder, wherein said symptoms include pain or itching of the eye, foreign body sensation.

16. A unit dosage form comprising between 5 to 15 ml and / or a single dosage form between 0.4 to 1 ml of the pharmaceutical composition described in any of claims 1 to 11.47AMENDED SHEET (ARTICLE 19)7. The pharmaceutical composition as described in any of the claims 1 to 11 for being administered daily for 30 days from the same unit dosage comprising the pharmaceutical composition each day.48AMENDED SHEET (ARTICLE 19)

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