Compositions for treating retinopathy

Abstract

To provide compositions for treating retinopathy.SOLUTION: The present invention provides a pharmaceutical composition for topical ocular delivery for treating retinopathy, the pharmaceutical composition comprising an oil-in-water nanodroplet emulsion, the oil-in-water nanodroplet emulsion comprising docosahexaenoic acid (DHA), coenzyme Q10, and insulin-like growth factor (IGF) amide-conjugated to the docosahexaenoic acid (DHA).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to compositions for treating retinopathy.Embodiments of the present invention relate to nanoemulsions comprising insulin and / or IGF for treating retinopathy of prematurity (ROP).

[0002] [Reference to Related Application] This application claims priority to U.S. Provisional Patent Application No. 62 / 853,179, filed May 28, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Birth is considered premature, or preterm, if it occurs before 37 weeks of gestation. The last few weeks in the womb are crucial for healthy weight gain and the full development of various vital organs.

[0004] In humans, the retina develops in utero, a time when tissue oxygen is scarce. Vascular progenitor cells arise between 12 and 21 weeks of gestation and create a scaffold for future vascular development. Retinal angiogenesis begins at approximately 16 weeks of gestation, with new vessels sprouting from pre-existing vessels. The metabolic demands of the developing retina exceed the oxygen supplied by the choroidal circulation, resulting in "physiological hypoxia" that stimulates angiogenesis.

[0005] Retinopathy of prematurity (ROP) is a developmental vascular disorder characterized by the abnormal proliferation of retinal blood vessels in an incompletely vascularized retina. ROP occurs mostly in extremely low gestational age (ELGAN) infants weighing 1250 g, or at birth before 28 weeks of gestation, and is the most common cause of visual impairment and blindness in children.

[0006] Current treatment options, including laser photocoagulation and intravitreal injection of vascular endothelial growth factor (VEGF) antibodies, have proven useful in severe, late-stage ROP. However, laser photocoagulation destroys a significant portion of the retina and is a difficult and complex procedure to perform in young infants, while intravitreal injection of VEGF antibodies can cause systemic inhibition of vascular growth, affecting other organs.

[0007] Therefore, there is a need, and it would be highly advantageous, to have an approach to treating retinopathy that does not include the above limitations. Summary of the Invention

[0008] According to one aspect of the present invention, there is provided a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10. According to another aspect of the present invention, there is provided a method for treating retinopathy in a premature infant, the method comprising the step of administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby treating the retinopathy in the premature infant. According to another aspect of the present invention, there is provided a method for preventing or reducing the severity of retinopathy in a premature infant, the method comprising the step of administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby preventing or reducing the severity of retinopathy in the premature infant. According to another aspect of the present invention, there is provided a method for reducing retinal hemorrhage in a premature infant, the method comprising the step of administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby reducing retinal hemorrhage in the premature infant. According to another aspect of the present invention, there is provided a method for reducing retinal hemorrhage in a subject experiencing retinopathy, the method comprising the step of administering to the eye of the subject a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby reducing retinal hemorrhage in the subject. According to another aspect of the present invention, there is provided a method for reducing retinal neovascularization in a subject experiencing retinopathy, the method comprising the step of administering to the eye of the subject a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby reducing retinal neovascularization in the eye of the subject. According to another aspect of the present invention, there is provided a method of increasing retinal vascular coverage in preterm infants, comprising the steps of: A method is provided that includes administering to the eye of a premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA), and coenzyme Q10, thereby increasing retinal vascular coverage (reducing avascular retinal area) in the premature infant. According to another aspect of the present invention, there is provided a method for reducing retinal inflammation in a premature infant, the method comprising the step of administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby reducing retinal inflammation in the premature infant. According to another aspect of the present invention, there is provided a method for reducing retinal oxidative stress in a premature infant, the method comprising the step of administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby reducing retinal oxidative stress in the premature infant. According to another aspect of the present invention, there is provided a method for improving the development of the retinal layers in a premature infant, the method comprising the step of administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby improving the development of the retinal layers in the premature infant. According to another aspect of the present invention, there is provided a method for reducing visual impairment (incidence or severity) in a premature infant, the method comprising the step of administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby reducing the visual impairment in the premature infant. According to another aspect of the present invention, there is provided a method for increasing the visual field of a premature infant, the method comprising the step of administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10, thereby increasing the visual field of the premature infant. According to another aspect of the present invention, there is provided a method of formulating a pharmaceutical composition for the topical treatment of retinopathy, comprising the steps of: (a) forming an oil-in-water nanoemulsion comprising docosahexaenoic acid (DHA) and coenzyme Q10 in an oil phase; (b) conjugating insulin or IGF-1 to the nanodroplets of said nanoemulsion using an amine coupling reaction.

[0009] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0010] Several embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. It is emphasized that the details shown below, particularly with detailed reference to the drawings, are presented for the purposes of example and for the purpose of providing a detailed description of preferred embodiments of the present invention, and for the purpose of providing what is believed to be the most useful and easily understood explanation of the principles and conceptual aspects of the present invention. Similarly, no attempt has been made to show structural details of the present invention in more detail than is necessary for a fundamental understanding of the present invention. Furthermore, by viewing the description in conjunction with the drawings, it will become apparent to those skilled in the art how embodiments of the present invention may be practiced. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a schematic representation of this composition. [Figure 2] 2A-B are in-vivo fundus examination graphs showing the total number of retinal hemorrhages (FIG. 2A) and the total number of severe hemorrhages (FIG. 2B). [Figure 3]3A-C show images of in vivo ophthalmoscopy results for normoxic animals (FIG. 3A), untreated hypoxic animals (FIG. 3B), and treated animals (FIG. 3C). [Figure 4] 4A-B are graphs showing the effect of treatment on angiogenesis on days 14 and 18. [Figure 5] Figures 5A-D show isolectin-B4 staining at P14 for insulin-treated (Figure 5A), IGF-1-treated (Figure 5B), untreated (Figure 5C), and normoxic (healthy) animals (Figure 5D). [Figure 6] 6A and 6B are graphs showing the avascular area in the insulin-treated group, the IGF-1-treated group, the untreated group, and the normoxic group. [Figure 7] Figures 7A-D show images of isolectin-B4 staining at P14 for the insulin (Figure 7A), IGF-1 (Figure 7B), untreated (Figure 7C), and normoxic (Figure 7D) groups. ROIs (green), vascular coverage (blue), vascular skeleton (red), and branch points (white) are indicated. [Figure 8] FIG. 8 shows a chromatogram of the coupling reaction at a specific time point with all reaction components, e.g., reactants (rh-insulin and DHA), intermediates (DHA-EDC intermediate), and the resulting product (insulin-DHA conjugate). [Figure 9] FIG. 9 is a chromatogram of the insulin-DHA conjugate extracted from the lyophilized emulsion (final product formulation). [Figure 10] FIG. 10 is a chromatogram showing the peaks of the components of the lyophilized emulsion (final product formulation). [Figure 11A-B] Figure 11A is a graph of the results of automated analysis of H&E retinal layer thickness using Wimretina software (Figure 11A), and Figure 11B is a graph of the results of biomarker PGE2 concentration analysis performed in the study, comparing the different study groups (Figure 11B). [Figure 11C] FIG. 11C is a graph comparing the results of the analysis of the biomarker 8-iso-PGF2a concentration performed in the study between the different study groups (FIG. 11C). [Figure 12] FIG. 12 is a chromatogram showing the peaks of components of a formulation containing free insulin, DHA, and coenzyme Q10. [Figure 13] FIG. 13 is a chromatogram showing the peaks of components of a formulation containing free insulin, DHA, and coenzyme Q10. [Figure 14] 14A-E are cryo-transmission electron micrographs (TEM) of compositions of the present invention. [Figure 15] 15A-E are cryo-transmission electron micrographs (TEM) of compositions of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention is directed to compositions that can be used to treat retinopathy. Specifically, the present invention can be used to treat ROP through topical administration of nanoemulsions containing insulin or IGF.

[0013] The principles and practice of the present invention may be better understood with reference to the drawings and the following description.

[0014] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description. The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0015] Although treatment options for ROP exist, such options are limited by the complexity of administration, side effects, and potential damage to ocular tissues.

[0016] The inventors hypothesized that an effective treatment for ROP should provide missing intrauterine factors (insulin and insulin growth factor 1) that can promote physiological vasculature development while preventing postnatal toxic effects (e.g., hyperoxia) and minimizing systemic exposure to these factors.

[0017] While reducing the present invention to practice, the inventors have formulated a composition that can promote the development of physiological ocular vasculature and reduce intraocular toxicity, thereby enabling the treatment of retinal disorders such as retinopathy. As further described in the Examples section below, the composition of the present invention was effective in stimulating the growth of healthy blood vessels and preventing and reducing retinal hemorrhage and pathological blood vessel growth (neovascularization) caused by an oxygen-induced model in rats.

[0018] The phrase "promoting physiological vascular development" means increasing the flow or passage of oxygen from the optic nerve to the periphery of the eye.

[0019] The term "retinopathy" refers to any disorder of the retina that can cause visual impairment. This can include, for example, conditions that slow or stop the growth of physiological vasculature (e.g., vascular occlusion or constriction, such as phase I of ROP) and abnormal (abnormal) pathological blood vessels that form in response to tissue hypoxia and ischemia. Retinopathy can result from external factors, such as radiation or head trauma, or as a manifestation of systemic diseases, such as diabetes or hypertension. Retinopathy can also be caused by vascular inflammation or medications (e.g., antidiabetic drugs, such as exenatide, liraglutide, or pramlintide).

[0020] Therefore, according to one aspect of the present invention, there is provided a composition comprising therapeutically effective amounts of insulin and / or IGF-1, docosahexaenoic acid (DHA) and coenzyme Q10 as active ingredients.

[0021] As further described herein, insulin and / or IGF-1 promote physiological vascular development, while DHA reduces inflammatory responses and coenzyme Q10 reduces oxidative stress signaling.

[0022] The term "therapeutically effective amount" or "pharmacologically effective amount" means a dose of an active ingredient or a composition comprising an active ingredient that provides the therapeutic effect for which the active ingredient is indicated.

[0023] The dosage of each active ingredient in the pharmaceutical compositions of the present application can depend on many factors, including the subject being treated, the stage of retinopathy (eg, ROP), and the route of administration (topical or intraocular).

[0024] In the case of ROP, progression can be determined via somatic effects (eg, vascular density and coverage), the extent and / or progression of angiogenesis, or the quality of retinal layer development.

[0025] The composition may be formulated as a water-in-oil nanoemulsion with nanodroplets comprising docosahexaenoic acid (DHA) and coenzyme Q10, and conjugated (e.g., via an amide bond) to insulin and / or IGF. Figure 1 is a schematic diagram of this composition showing nanodroplets comprising DHA 12 and coenzyme Q10 14, conjugated to insulin or IGF 12.

[0026] The Examples section below describes one approach for formulating the present compositions.

[0027] The composition may be stored in a lyophilized state and reconstituted with, for example, water or saline for use, or may be stored as a ready-to-use pharmaceutical composition.

[0028] The composition can be part of a pharmaceutical composition that includes a carrier formulated for topical or intraocular delivery.

[0029] Topical formulations of the pharmaceutical composition can include carriers such as medium chain triglycerides (MCTs), long chain triglyceride oils such as castor oil, synthetic and semi-synthetic oils such as mineral oil, and unsaturated fatty acids such as oleic acid.

[0030] The intraocular formulation of the pharmaceutical composition may be formulated as a microemulsion and / or may include carriers such as liposomes, nanospheres, micelles, and nanocapsules.

[0031] Ophthalmic preparations can be formulated for sustained or delayed release of the active ingredient using excipients that form inclusion complexes with the active ingredient, such as chelating agents, surfactants, and cyclodextrins.

[0032] The pharmaceutical composition may include: (i) Carbohydrates (as stabilizers, lubricants, and antifreeze agents): Examples of carbohydrates include, but are not limited to, monosaccharides (e.g., glucose, maltose), disaccharides (e.g., trehalose), oligosaccharides (e.g., dextrin), cyclodextrins (e.g., hydroxypropyl-β-cyclodextrin (HPbCD)), and polysaccharides (e.g., dextran). (ii) Emulsifiers: Such emulsifiers include, but are not limited to, nonionic surfactants of natural origin (e.g., lecithin, egg yolk phospholipids), nonionic surfactants of synthetic origin (e.g., tyloxapol), and ionic surfactants (e.g., cetacuronium chloride). (iii) Thickeners: Thickeners include, but are not limited to, hydrophilic polymers (e.g., polyvinyl alcohol) or cellulose derivatives (e.g., hydroxypropylmethylcellulose (HPMC)). (iv) Bioadhesives such as polyamino acids (e.g., gelatin, human albumin), and polysaccharides such as cellulose derivatives (e.g., hydroxypropylmethylcellulose (HPMC) and hydroxypropylcellulose (HPC)), hyaluronic acid, etc. (v) Gelling agents such as alginates and polyacrylates can be added to the pharmaceutical composition to increase the residence time of the active ingredient on the cornea.

[0033] According to an embodiment of the present invention, the concentration of insulin in the pharmaceutical composition may be 0.001 U to 20 U / ml, while the concentration of IGF may be 0.001 U to 20 U / ml.

[0034] According to an embodiment of the present invention, the concentration of DHA in the pharmaceutical composition may be 1 to 4 mg / ml.

[0035] According to an embodiment of the present invention, the concentration of coenzyme Q10 in the pharmaceutical composition may be 1 to 3 mg / ml.

[0036] Table 1 below describes a topical formulation of the present composition. [Table 1]

[0037] This formulation can be modified to not contain MCT and contains two forms of DHA: free acid and ethyl ester. These two forms of DHA replace the MCT in the droplet core. Two separate emulsions are produced and combined in the final manufacturing step. The first emulsion contains droplets containing DHA free acid to which insulin is bound. The second emulsion contains droplets in which Q10 is incorporated into the DHA ethyl ester core. Table 2 below lists the ingredients of this embodiment of the injectable formulation. [Table 2]

[0038] The intraocular formulation of the composition is set forth in Table 3 below. [Table 3]

[0039] To enhance the efficacy of this composition, a method was developed to prepare nanoemulsions with nanodroplets encapsulating (DHA) and coenzyme Q10 and conjugated to insulin or IGF.

[0040] Thus, according to another aspect of the present invention, there is provided a method for formulating a pharmaceutical composition for the topical treatment of retinopathy by forming an oil-in-water nanoemulsion containing docosahexaenoic acid (DHA) and coenzyme Q10 in the oil phase, and conjugating insulin or IGF-1 to nanodroplets of the nanoemulsion using an amine coupling reaction.

[0041] Following nanoemulsion generation, the nanodroplets can be purified or concentrated using, but not limited to, column chromatography, tangential flow filtration (TFF), or dialysis. Stabilizers can be added, such as, but not limited to, cyclodextrins, dextrins, and mono- or disaccharide sugars.

[0042] The formulation can then be lyophilized for storage and reconstituted with saline or water prior to use.

[0043] The Examples section below provides a more detailed description of this formulation approach.

[0044] As noted above, the present compositions can be used to treat retinopathy, particularly retinopathy of prematurity.

[0045] Thus, according to another aspect of the present invention, there is provided a method of treating retinopathy in a subject in need thereof, such as a premature infant. The method is carried out by administering a pharmaceutical composition of the present invention to the eye of a subject in need thereof. Such administration may be topical or intraocular.

[0046] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal. The human or non-human mammal (cat, dog, cow, sheep, pig, goat, and horse) may be of any age (e.g., infant, such as a full-term or preterm infant, adult, or elderly) or gender. The human subject may be a premature infant born at 24-33 weeks gestation. The human subject may also be a low birth weight infant weighing 500-1650 gm at birth.

[0047] Topical formulations (eye drops) of the composition can be administered to premature infants any time between birth and 6 months of age at a dose of 10 microliters to 100 microliters once or several times daily for a period of 180 days. Intraocular formulations of the composition can be administered to premature infants any time between birth and 6 months of age at a dose of 5 to 30 microliters per injection as clinically indicated once every few weeks for a period of 180 days.

[0048] As used herein, "about" refers to ±10%.

[0049] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. [Example]

[0050] Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

[0051] Example 1 Nanoemulsion formulation The following example demonstrates the preparation of the present composition formulated as a lyophilized powder suitable for reconstitution as an oil-in-water nanoemulsion.

[0052] Table 4 below lists the ingredients used in the manufacturing process for the composition formulation. [Table 4]

[0053] Nanodroplet formulation was achieved using the solvent displacement method. 100 mg of DHA, 50 mg of CoQ10, 25 mg of tyloxapol, and 50 mg of MCT were dissolved in 9 ml of acetone, and 25 mg of Lipoid E80 was dissolved in 1 ml of ethanol. The resulting solutions were combined and mixed at 900 rpm for 30 minutes at room temperature, then added dropwise to 20 ml of 0.1% w / v aqueous PVA solution and continuously stirred at 900 rpm for an additional 15 minutes. The organic solvent was then completely removed at room temperature under reduced pressure (50 mBar) using a laboratory rotary evaporator. The resulting emulsion was pre-adjusted to pH 7.4 with 0.5 M NaOH before undergoing the amine coupling reaction.

[0054] 1.3 μmol of EDC prepared in 0.5 ml of phosphate-buffered saline (pH 7.2) was added to the resulting emulsion, and the mixture was incubated at room temperature for 15 minutes. The pH was then adjusted to 8.3 ± 0.2 using sodium carbonate buffer. 0.5 ml of a 0.1 μmol / mL rh-insulin solution in phosphate-buffered saline (pH 7.2) was added to 19.5 ml of the emulsion. The reaction mixture was allowed to stir at room temperature for 12 hours. The reaction mixture was then loaded onto a gravity-flow PD-10 gel filtration column (Sephadex G-25) using water as the eluent to separate the nanodroplets from smaller particles (e.g., EDC, free active substance molecules). Excess eluent (water) was removed from the nanodroplet fraction using a laboratory rotary evaporator under reduced pressure (50 mBar) at 37°C. The emulsion was then mixed with 2-hydroxypropyl-β-cyclodextrin to a final concentration of 2% w / v, filtered through a 0.45 μm PES (polyacetal sulfone) membrane, dispensed into vials, and lyophilized. The active ingredients in 1 ml of the reconstituted solution were 0.67 U rh-insulin, 2 mg DHA, and 1 mg CoQ10.

[0055] Example 2 Test 1 An ophthalmic formulation of the composition described in Example 1 (ELGN01, composed of insulin, DHA and Coq10) was tested in a rat oxygen-induced retinopathy model.

[0056] <Procedure> Eighteen pups from a single mother were divided into two groups: Group A - ELGN01 (nine pups) and Group B - untreated (nine pups in an oxygen chamber without treatment). Three pups from a single mother kept in normoxia served as additional controls.

[0057] Treatment was initiated on days 5-14 or 18 (depending on the date of sacrifice) and was administered first under the eyelid using a syringe (topical, not damaging the ocular surface), and then as an instillation after eye opening.

[0058] The oxygen administration regimen was as follows: 0–14 days of age, 24-hour cycles of hyperoxia (50%) followed by 24 hours of hypoxia (12%).

[0059] The study was terminated for Group 1 on postnatal day 14 (P14) and for Group 2 on postnatal day 18 (P18). Fundus examination was performed by an ophthalmologist on day 17, after which samples were evaluated histologically and immunohistologically.

[0060] <Result> In-vivo fundus examination results A total of 12 retinal hemorrhage sites were observed in the treated group compared to 22 in the untreated group (significance for treatment ELGN01 p=0.04). [Table 5]

[0061] The total number of retinal damage sites and the total number of severe hemorrhage sites in the treated and untreated groups are shown in the graphs of FIGS. 2A and 2B.

[0062] Figures 3A-C show images of normoxic and hypoxic (treated and untreated) retinas. The normoxic animals show intact retinal vessels with no hemorrhage or ablation. The untreated hypoxic animals exhibit retinal hemorrhage (arrows). The treated animals show reduced damage.

[0063] Neovascular area The effects on angiogenesis are shown in Figure 4A-B. Neovascularization (NV) was high at P18, corresponding to the end of Phase II of human disease. P14 corresponded to the end of Phase I (progression) of disease. At both time points, NV in the treated group was significantly less than in the untreated group. At P14, the ELGN01-treated group showed 0% angiogenesis, compared with 0.09% in the untreated group (t-test comparison p=0.07). At P18, the ELGN01-treated group showed an average of 40% less angiogenesis compared with the untreated group (treated 1.35% vs. untreated 1.89%, t-test comparison p=0.07, treatment effect 28%).

[0064] Retinal layers Paraffin-embedded whole eyes were sectioned and stained with hematoxylin and eosin. Four sections from different locations were collected on one slide. H&E staining was imaged under a light microscope using a 10x objective (4x in some locations).

[0065] Representative staining from the untreated OIR group shows disruption of retinal layers and thickening of the ganglion cell layer as a result of OIR injury. A total of eight samples per group were available: four sections per eye, two eyes (from different animals) per treatment group.

[0066] To assess the integrity of the retinal layers, de-identified H&E-stained images were uploaded to Wimasis Image Software. Each retina was analyzed masked to identify and measure the size of each retinal layer (RGCL - retinal ganglion cell layer, IPL - inner plexiform layer, INL - inner nuclear layer, OPL - outer plexiform layer, ONL - outer nuclear layer, RPE - retinal pigment epithelium, CHO - choroid). Figure 11A shows the average thickness of the retinal layers. [Table 6]

[0067] Example 3 Test 2 Ophthalmic formulations based on the compositions described in Example 1 (ELGN01, composed of insulin, DHA and Coq10, and ELGN02, composed of IGF-01 and the same) were tested in a rat oxygen-induced retinopathy model.

[0068] <Procedure> Two mother rats each gave birth to 18 pups, which were divided into three treatment groups: group A (ELGN01, 12 pups), group B (ELGN02, 12 pups), and group C (untreated, 12 pups). Group D, which was kept under normoxic conditions, served as a control.

[0069] Treatment began on day 5 and continued until day 14 or 18 (depending on the date of sacrifice), initially administered under the eyelid with a syringe (topical administration that does not damage the ocular surface), followed by eye drops after opening the eyes.

[0070] The oxygen regimen consisted of eight intermittent hypoxic events over the first 4 days: three 30-minute events of decreasing oxygen to 12%, followed by 50% hyperoxia for the remainder of the day. From days 5 to 14, the regimen consisted of 24-hour cycles of hyperoxia (50%) followed by 24 hours of hypoxia (12%).

[0071] The study in Group 1 was terminated on Day 14, and fundusscopic evaluation was performed by an ophthalmologist on Day 17, after which the samples were evaluated histologically and immunohistologically.

[0072] <Result> Isolectin staining Samples were flat-mounted and retinas were stained with isolectin GS-IB4. Avascular areas (AVA) were manually quantified by an independent expert using images of isolectin-stained retinas.

[0073] Figures 5A-D show staining of the insulin and IGF-treated, untreated, and normoxic groups. Minimal avascular areas with intact central blood vessels are observed in the insulin-treated and IGF-1-treated groups (Figures 5A-B). Large avascular areas are observed in the untreated group (Figure 5C) (arrows). Complete coverage of blood vessels is observed in the normoxic group (Figure 5D).

[0074] Figures 6A-B are graphs depicting AVA. At P14, both treatment groups had a 50% reduction in AVA compared to the control group (treated ELGN01: 2.77%, treated ELGN02: 3.15%, avascular area: 6.13%). The avascular area in the normoxic group was 1.4%. When comparing treated and untreated animals using a t-test, the difference between the ELGN01-treated group and the untreated group was statistically significant (p=0.01), as was the difference between the ELGN02-treated group and the untreated group (p=0.03).

[0075] For each retina, the vascular density (%, calculated by dividing the number of vascular pixels by the total number of pixels in the region of interest), total vascular area, number of branch points (where two or more segments converge), number of segments (number of individual vascular segments), and mean segment length were analyzed in a masked manner.

[0076] At P14, both treatment groups outperformed untreated animals in many characteristics. Treatment ELGN01 had significantly higher vascular density (%) compared to untreated animals (p = 0.051), and similarly, treatment ELGN02 (p = 0.032) showed better retinal vascular growth and development, as well as less avascular area. Treatment groups ELGN01 (p = 0.073) and ELGN02 (p = 0.014) also showed larger vascular areas compared to untreated animals. Furthermore, treatment A-ELGN01 had a significantly higher mean segment length (p = 0.037) compared to untreated animals, indicating better vascular continuity. [Table 7]

[0077] Figures 7A-D show images of isolectin-B4 staining at P14 per treatment group. The ROI (green), covered blood vessels (blue), vascular skeleton (red), and branching points (white) are shown.

[0078] As outlined in Table 8 below, at P18, the treated ELGN01 group had a significantly higher vascular density (%) compared to the untreated group (p=0.007). The treated ELGN02 group showed a non-significant trend. Vascular density (%) reflects the amount of vascularized retina compared to non-vascularized areas. High vascular density without neovascularization indicates good retinal vascular growth and development, as well as fewer avascular areas. The treated groups also showed a trend toward greater vascular area compared to the untreated group. The treated ELGN01 group also showed a higher number of branch points compared to the untreated group (p=0.02). Furthermore, the treated ELGN01 group had a greater vascular volume compared to the untreated group (p=0.04). [Table 8]

[0079] Biomarker activity At P14 and P18, eyes were harvested, homogenized, and centrifuged to compare the levels of different biomarkers in tissues from different study groups. Samples were tested in triplicate, with four different rats in each group. The content was normalized to the total protein concentration of each sample. The biomarker analyzed was 8-isoprostane, or 8-isoPGF2α, a commonly studied and reliable, validated biomarker of oxidative stress, abundantly produced in vivo during oxidative stress and lipid peroxidation (Beharry 2017). Additionally, PGE2, a biomarker of inflammatory processes, was also measured (Figure 11B). PGE2 has dual, opposing effects on endothelial cells. It mediates both vasoconstriction and vasodilation (via different receptors). PGE2 is the primary metabolite of COX-2 isoforms, which are activated by cytokines and growth factors and are deeply involved in angiogenesis (Beharry 2017).

[0080] The results showed a decrease in 8-isoPGF2α levels at P14 and P18 compared to the untreated group, indicating a protective effect against oxidative stress damage produced by the animal model (Figure 11C). The effect was seen in both the first and second stages of the disease (P14 and P18). At P14, PGE2 levels were higher in all groups compared to normoxia, and at P18, PGE2 levels decreased in the treated group and significantly increased in the untreated group, which correlated with the inflammatory stage of the pathology (Figure 11B).

[0081] Example 4 Test 3 An ophthalmic formulation (ELGN01 (described in Example 1) containing insulin, DHA, and Coq10) was administered to newborn rats, and insulin concentrations in the eyes after administration were measured.

[0082] <Procedure> Two mother rats each gave birth to 18 pups, which were divided into two groups: Group A - ELGN01 normoxia group (18 pups), and Group B - ELGN01 hypoxia group (18 pups). Two pups from a single mother rat kept in normoxia served as controls.

[0083] Treatment was administered by administering the composition (10 μL dose containing 0.0067 insulin units) under the eyelid using a syringe (topical) for 4 days, starting on day 5. Rats were sacrificed (N=3 per T) at 30, 60, and 120 minutes after administration, and whole eyes were homogenized and evaluated by ELISA (Quantikine® ELISA).

[0084] <Result> Table 9 below shows that 8-16% of the administered insulin was absorbed by ocular tissue within the first 2 hours. [Table 9]

[0085] Example 5 Nanoemulsion formulation The following examples demonstrate alternative approaches for preparing the compositions of the present invention.

[0086] The present invention discloses one-pot conjugation of insulin to oily nanodroplets directly during the formulation process by one-step coupling of insulin with the carboxyl groups of DHA in aqueous media using the cross-linking reagent N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC).

[0087] The method for making insulin-DHA conjugates utilizes the following general process: · Generation of nanodroplets by solvent displacement method; · Activation of the DHA carboxyl group by EDC, resulting in the formation of the active O-acylisourea DHA-EDC ester; Conjugation of insulin to the DHA carboxyl group by forming an amide bond with the primary amine group of insulin, with the release of an EDC by-product as a soluble N-unsubstituted urea; Purification of reaction mixture from EDC by-products using ultrafiltration with 30,000-100,000 MWCO membranes

[0088] Materials and Methods Preparation of the organic phase: 347 mg of DHA free acid, 75 mg of tyloxapol, and 75 mg of Lipoid E80 were dissolved in 25 ml of ethanol. This solution was added dropwise to 100 ml of double deionized water using a 21G needle and continuously mixed at 350 rpm at room temperature. The resulting emulsion was mixed for an additional 10 minutes, after which the organic solvent was completely removed under reduced pressure using a laboratory rotary evaporator (40 ± 2 °C, 50 mBar).

[0089] The resulting emulsion was pre-adjusted to pH 4.5 with 0.1 N HCl and then subjected to the amine coupling reaction. 0.27 mmol of EDC dissolved in 1 ml of water was added to the resulting emulsion, and the mixture was incubated at room temperature for 40 minutes until the formation of the DHA-EDC intermediate ester was complete.

[0090] The pH of the reaction mixture was adjusted to 6.2, and 0.045 mmol of insulin dissolved in 50 ml of water (pH 7.2) was added. The reaction was complete within 1 h, and the pH was maintained at 6.3-6.4 during coupling. The reaction was monitored by HPLC (Dionex Ultimate 3000), and the chromatograms of the reaction mixture at the 30 min time point and the process conditions are shown in Figure 8.

[0091] Upon reaction completion, the mixture was diluted 1:2 with double deionized water and transferred through a 100,000 MWCO Hydrosart ultrafiltration cassette (Sartorius) using a peristaltic pump.

[0092] The content of the insulin conjugate in the resulting retentate (150 ml) was 0.037 mmol, representing a yield of 82% calculated based on the insulin content. The osmolality of the emulsion was 301 mosm / kg.

[0093] A chromatogram of the coupling reaction mixture is shown in Figure 8, and the components and conditions are listed in Table 10 below. [Table 10]

[0094] Another composition was manufactured formulated as a lyophilized powder suitable for reconstitution into an oil-in-water nanoemulsion. Table 11 below lists the ingredients used in the manufacturing process. [Table 11]

[0095] The formulation process involves the preparation of two separate emulsions: the first emulsion contains coenzyme Q10 incorporated into DHA nanodroplets, and the second emulsion contains insulin bound to DHA nanodroplets. The emulsions were prepared separately by displacement and then combined prior to the purification step.

[0096] <Emulsion 1> 300 mg of coenzyme Q10, 525 mg of DHA ethyl ester, 125 mg of tyloxapol, and 125 mg of Lipoid E80 were dissolved in a mixture of 15 ml of acetone and 50 ml of ethanol. This mixture was added dropwise to 250 ml of 0.1% aqueous PVA solution using a 21G needle and continuously mixed at 350 rpm at room temperature. The resulting emulsion was mixed for an additional 10 minutes, after which the organic solvent was completely removed under reduced pressure using a laboratory rotary evaporator (45±2°C, 50 mBar).

[0097] <Emulsion 2> 125 mg of DHA free acid, 25 mg of tyloxapol, and 25 mg of Lipoid E80 were dissolved in 12 ml of ethanol. This mixture was added dropwise to 50 ml of double deionized water through a 21G needle and continuously mixed at 350 rpm at room temperature. The resulting emulsion was mixed for an additional 10 minutes, after which the organic solvent was completely removed under reduced pressure using a laboratory rotary evaporator (40 ± 2 °C, 50 mBar).

[0098] The resulting emulsion was pre-adjusted to pH 4.5 with 0.1 N HCl before the amine coupling reaction.

[0099] 0.11 mmol of EDC dissolved in 1 ml of water was added to the resulting emulsion, and the mixture was incubated at room temperature for 1.25 h until the formation of the DHA-EDC intermediate ester was complete. The pH of the reaction mixture was adjusted to 6.2, and 0.015 mmol of insulin solution (pH 4.2) dissolved in 18 ml of water was added. The reaction was complete within 1 h, and a pH of 6.2-6.4 was maintained during the coupling. The reaction was monitored by HPLC (Dionex Ultimate 3000), and the process conditions and a typical chromatogram of the reaction mixture are provided in Figure 8.

[0100] After completion of the reaction, the mixture was combined with emulsion #1, then diluted 1:2 with 0.1% aqueous PVA solution (osmolality <5 mosm / kg) and pumped through a 30,000 MWCO Hydrosert filter cassette (Sartorius) using a peripheral pump, with a final retentate volume of 250 ml (theoretical conjugated insulin content 0.06 μmol / ml).

[0101] 4 g of HPBCD dissolved in 8 ml of water was added to 80 ml of emulsion and the volume was adjusted to 100 ml. The emulsion was filtered through a 0.22 μm PES membrane and 4 ml was filled into glass vials (0.5 ml per vial) and chilled. The osmolality of the final bulk product was 376 mosm / kg.

[0102] The theoretical content of insulin conjugate per vial was 0.024 μmol / vial, the observed content was 0.017 μmol / vial, and the yield of conjugated insulin was 72%.

[0103] The Z-average sizes of the liquid bulk and dry final product were 119.9 nm (polydispersion index 0.137) and 243 nm (polydispersion index 0.342), respectively.

[0104] The conjugated insulin, DHA, and coenzyme Q10 content in the lyophilized powder is monitored by RP-HPLC. The chromatogram of the final lyophilized product is shown in Figure 10. Table 12 below provides the chromatographic conditions used for testing the lyophilized formulations. [Table 12]

[0105] Representative cryo-transmission electron micrographs (TEM) of formulations prepared as described in Example 5 are shown in Figures 14A-E and 15A-E.

[0106] Example 6 GI prescription An oral emulsion was prepared for the topical treatment of intestinal malabsorption in preterm infants. The formulation contains three active ingredients: rh-insulin, DHA, and coenzyme Q10. In the reconstituted formulation, insulin is present as a free protein, and DHA and coenzyme Q10 are incorporated into oil droplets.

[0107] The compounding process involved the following general steps: -Preparation of DHA and Coenzyme Q10 emulsion using solvent displacement method, - the addition of rh-insulin and a cryoprotectant, - Filtration and freeze drying.

[0108] Materials and Methods 513 mg of coenzyme Q10, 898 mg of DHA ethyl ester, 175 mg of tyloxapol, and 175 mg of Lipoid E80 were dissolved in 80 ml of ethanol. The mixture was added dropwise to 350 ml of 0.1% aqueous PVA solution using a 21G needle and continuously mixed at 350 rpm at room temperature. The resulting emulsion was mixed for an additional 10 minutes, after which the organic solvent was completely removed under reduced pressure using a laboratory rotary evaporator (45±2°C, 50 mBar).

[0109] One ml of insulin solution (2.7 mg / ml, pH 8.5) was mixed with 14 ml of cryoprotectant solution containing 28.6 mg / ml HPBCD and 343 mg / ml maltodextrin, and the resulting solution was added to the continuously mixed emulsion and mixed for 20 minutes.

[0110] The emulsion was filtered through a 0.22 μm PES membrane, and 4 ml was filled into glass vials (fill volume 0.5 ml / vial) and lyophilized. The osmolality of the final bulk product was 358 mosm / kg. Each vial contained 0.65 IU of rh-insulin, 0.9 mg of DHA, and 0.5 mg of coenzyme Q10.

[0111] Table 13 below lists the formulation ingredients. Chromatograms of the lyophilized products are shown in Figures 12 and 13. [Table 13]

[0112] It will be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in a single embodiment in any combination of those features. Conversely, multiple features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0113] While the present invention has been described in connection with specific embodiments thereof, numerous alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, all such alternatives, modifications, and variations are intended to be embraced within the spirit and broad scope of the appended claims. All publications, patents, and patent applications mentioned in this specification are incorporated by reference in their entirety herein to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

[0114] Additionally, the documents relating to the prior application of this application are also incorporated herein by reference in their entirety.

Claims

1. 1. A pharmaceutical composition for topical delivery to the eye to treat retinopathy, comprising: The pharmaceutical composition comprises an oil-in-water nanodroplet emulsion; The oil-in-water nanodroplet emulsion comprises docosahexaenoic acid (DHA), coenzyme Q10, and insulin-like growth factor (IGF) amide-linked to the docosahexaenoic acid (DHA). Pharmaceutical compositions.

2. further comprising a carrier formulated for ocular delivery, The pharmaceutical composition of claim 1.

3. The carrier comprises a surfactant. The pharmaceutical composition of claim 2.

4. The concentration of the IGF is 0.001 U to 20 U per ml. The pharmaceutical composition of claim 1.

5. The concentration of the DHA is 1 to 3 mg / ml. The pharmaceutical composition of claim 1.

6. The concentration of the coenzyme Q10 is 1 to 3 mg / ml. The pharmaceutical composition of claim 1.

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