SUITABLE PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF RETINOPATHY.

Abstract

A pharmaceutical composition including insulin, docosahexaenoic acid (DHA) and coenzyme Q10 and manufacturing procedures and use of the composition are provided.

Description

COMPOSITIONS AND TREATMENT PROCEDURES FOR RETINOPATHY RELATED APPLICATION This application claims the benefit of priority of United States Provisional Patent Application No. 62 / 853.179 filed on May 28, 2019, the contents of which are incorporated in full by reference in this document. BACKGROUND OF THE INVENTION The present invention relates to a composition for the treatment of retinopathy. The embodiments of the present invention relate to a nanoemulsion containing insulin and / or IGF for the treatment of retinopathy of prematurity (ROP). Birth is considered premature when it occurs before the 37th week of pregnancy. The final weeks in the womb are crucial for healthy weight gain and the full development of several vital organs. In humans, the retina develops in utero, where tissue oxygen is low. Vascular precursor cells are deposited from 12 to 21 weeks of gestational age, creating a scaffold for future vessel development. Retinal angiogenesis begins at approximately 16 weeks of gestational age, with new vessels budding from existing ones. The metabolic demands of the developing retina exceed the oxygen supplied by the ceroid circulation, resulting in “physiological hypoxia,” which stimulates angiogenesis. Retinopathy of prematurity (ROP) is a vascular disorder of the ROP is a development characterized by abnormal growth of blood vessels in the retina in an incompletely vascularized retina. ROP occurs primarily in extremely low gestational age (ELGAN) newborns who are 1250 g or less than 28 weeks of gestation at birth and is the most common cause of visual impairment and blindness in children. 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 most of the retina and is a difficult and complicated procedure to perform in young infants, while intravitreal injection of VEGF antibodies can cause systemic suppression of vascular growth, affecting other organs. Therefore, there is a need and it would be very advantageous to have a treatment approach for retinopathy free from the above limitations. BRIEF DESCRIPTION OF THE INVENTION According to one aspect of the present invention, a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA), and coenzyme Q10 is provided. According to another aspect of the present invention, a treatment procedure for retinopathy in premature infants is provided, comprising the administration of a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to an eye of a premature infant to thereby treat retinopathy in premature infants. ML / IZ / ZυZZ / υΊ UOUO According to another aspect of the present invention, a method for preventing or reducing the severity of retinopathy in premature infants is provided, comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to an eye of a premature infant to thereby prevent or reduce the severity of retinopathy in premature infants. According to another aspect of the present invention, a method for reducing retinal hemorrhages in premature infants is provided, comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to an eye of a premature infant to thereby reduce retinal hemorrhages in premature infants. According to another aspect of the present invention, a method for reducing retinal hemorrhages in subjects experiencing retinopathy is provided, comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to one eye of a subject, thereby reducing retinal hemorrhages in the subject's eye. According to another aspect of the present invention, a method for reducing retinal neovascularization in subjects experiencing retinopathy is provided, comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to one eye of a subject, thereby reducing retinal neovascularization in the subject's eye. According to another aspect of the present invention, ivia / t / zuzz / ui youo provides a procedure for increasing retinal vascular coverage in premature infants comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to an eye of a premature infant to thereby increase retinal vascular coverage in premature infants (reduction of avascular retinal areas). According to another aspect of the present invention, a method for reducing retinal inflammation in premature infants is provided, comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to an eye of a premature infant, thereby reducing retinal inflammation in premature infants. According to another aspect of the present invention, a method for reducing retinal oxidative stress in premature infants is provided, comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to an eye of a premature infant to thereby reduce retinal oxidative stress in premature infants. According to another aspect of the present invention, a method for improving the development of the retinal layer in premature infants is provided, comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to an eye of a premature infant to thereby improve the development of the retinal layer in premature infants. According to another aspect of the present invention, ivia / t / zuzz / ui aouo provides a method for reducing vision impairment (incidence or severity) in premature infants comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to one eye of a premature infant to thereby reduce vision impairment in premature infants. According to another aspect of the present invention, a method for increasing the visual field in premature infants is provided, comprising administering a pharmaceutical composition including insulin, docosahexaenoic acid (DHA), and coenzyme Q10 to one eye of a premature infant to thereby increase the visual field in premature infants. According to another aspect of the present invention, a method for formulating a pharmaceutical composition for the topical treatment of retinopathy is provided, comprising: (a) generating an oil-in-water nanoemulsion including docosahexaenoic acid (DHA) and coenzyme Q10 in the oil phase; and (b) conjugating Insulin or IGF-1 with nanodroplets of the nanoemulsion by using an amine coupling reaction. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Although similar or equivalent procedures and materials to those described herein may be used in the practice or testing of the present invention, suitable procedures and materials are described below. In case of conflict, the patent specification, including the definitions, shall prevail. Furthermore, the materials, procedures, and examples are for illustrative purposes only and are not intended to be limiting. ivia / t / zuzz / ui aouo BRIEF DESCRIPTION OF THE FIGURES The invention is described herein, by way of example only, with reference to the accompanying figures. With specific reference now to the detailed drawings, it is emphasized that the details shown are by way of example and for the purpose of an illustrative discussion of preferred embodiments of the present invention only, and are presented to provide what is believed to be the most useful and readily understandable description of the principles and conceptual aspects of the invention. In this respect, no attempt is made to show the structural details of the invention in more detail than is necessary for a fundamental understanding of the invention; the description taken with the figures makes it clear to those skilled in the art how the various embodiments of the invention can be incorporated in practice. In the figures: Figure 1 schematically illustrates the present composition. Figures 2A and 2B are graphs of in vivo fundoscopy results representing total retinal hemorrhages (Figure 2A) and total severe hemorrhages (Figure 2B). Figures 3A to 3C are images of in vivo fundoscopy results from normoxic animals (Figure 3A), untreated hypoxic animals (Figure 3B), and treated animals (Figure 3C). Figures 4A and 4B are graphs showing the effect of treatment on neovascularization on days 14 and 18. Figures 5A to 5D illustrate the isolectin-B4 staining of P14 ivia / t / zuzz / ui eouo for the insulin-treated group (Figure 5A), the IGF-1-treated group (Figure 5B), the untreated group (Figure 5C), and the normoxic (healthy) animals (Figure 5D). Figures 6A and 6B are graphs showing the avascular area of ​​the insulin-treated, IGF-1-treated, untreated, and normoxic groups. Figures 7A to 7D are images showing isolectin-B4 staining of P14, for the insulin (Figure 7), IGF-1 (Figure 7B), untreated (Figure 7C), and normoxic (Figure 7D) groups. The ROI (green), the covered vessel area (blue), the vessel skeleton (red), and the branching points (white) are marked. Figure 8 is a chromatogram of the coupling reaction at a given time with all the reaction components, e.g., reagents (rh-insulin and DHA), an intermediate (DHA-EDC intermediate) and the resulting product (insulin-DHA conjugate). Figure 9 is a chromatogram of insulin-DHA conjugate extracted from the lyophilized emulsion (finished product formulation). Figure 10 is a chromatogram showing the peaks of the constituents of the lyophilized emulsion (finished product formulation). Figures 11A to 11C are graphs that illustrate the results of the automated analysis of retinal layer thickness H&E using Wimretina software (Figure 11A), the results of the analysis of PGE2 biomarker levels carried out in the study, by comparing the different study groups (Figure 11B), and the results of the analysis of 8-iso-PGF2a biomarker levels carried out in the study, by comparing the different study groups (Figure 11C). ML / t / ZUZZ / UI uouo Figures 12 and 13 are chromatograms showing the peaks of the formulation components containing free insulin, DHA, and coenzyme Q10. Figures 14A to 14E and Figures 15A to 15E are cryogenic transmission electron micrographs (TEM) of the composition of the present invention. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition of material that can be used to treat retinopathy. Specifically, the present invention can be used to treat retinopathy of prematurity (ROP) by means of the local administration of a nanoemulsion containing insulin or immunoglobulin (IGF). The principles and operation of the present invention can be better understood with reference to the accompanying drawings and descriptions. Before explaining at least one embodiment of the invention in detail, it should be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is susceptible to other embodiments or can be practiced or carried out in various ways. Furthermore, it should be understood that the phraseology and terminology used herein are for descriptive purposes and should not be considered limiting. Although treatment options exist for ROP, such options are limited by the complexity of administration, side effects, and potential damage to eye tissue. The present inventor postulated that effective treatment of ROP should prevent postpartum toxic influences (e.g., excess oxygen) and IVIA / t / ZUZZ / UI SOUO provides missing intrauterine factors (insulin and insulin-like growth factor 1) that can promote the development of physiological vasculature while minimizing systemic exposure to these factors. While reducing the present invention to practical application, the inventor formulated compositions 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 that follows, the present compositions were effective in stimulating the growth of healthy vessels and preventing and reducing retinal hemorrhages and pathological blood vessel growth (neovascularization) induced by the oxygen-induced model in a rat. The expression “promote physiological vascular development” refers to increasing the flow or passage of oxygen from the optic nerve to the periphery of the eye. The term “retinopathy” refers to any damage to the retina that can lead to vision impairment. This can include, for example, a condition that slows or stops the growth of physiological vasculature (vaso-obliterative or constrictive stage, e.g., stage I of ROP) and abnormal (aberrant) blood vessels that form in response to tissue hypoxia and ischemia. Retinopathy can result from external factors such as radiation or head trauma, or be a manifestation of a systemic disease such as diabetes or hypertension. Retinopathy can also be caused by vascular inflammation and medications (e.g., diabetes medications such as... IVIA / t / ZUZZ / UI uoo exenatide, liraglutide and pramlintide). Therefore, according to one aspect of the present invention, a material composition is provided that includes a therapeutically effective amount of insulin and / or IGF-1, docosahexaenoic acid (DHA) and coenzyme Q10 as active ingredients. According to what is further described in this report, insulin and / or IGF-1 promote physiological vascular development, while DHA reduces the inflammatory response and coenzyme Q10 reduces oxidative stress signaling. The expression “effective quantity for therapeutic use” or “effective quantity for pharmaceutical use” indicates the dose of an active ingredient or a composition comprising the active ingredient that will provide the therapeutic effect for which the active ingredient is indicated. The dosage of each active ingredient in the present pharmaceutical composition may depend on many factors, including the subject being treated, the stage of retinopathy (e.g., ROP), and the route of administration (topical or intraocular). In the case of ROP, progression can be determined by means of somatic effects (e.g., density and coverage of vessels), extent and / or progression of vascularization, or quality of retinal layer development. The material composition can be formulated as a water-in-oil nanoemulsion having nanodroplets that include docosahexaenoic acid (DHA) and coenzyme Q10 and are conjugated with insulin and / or IGF (by means of, for example, an amide bond). Figure 1 is a schematic illustration of the present material composition showing ivia / t / zuzz / ui aouo nanodroplets conjugated with insulin or IGF 12 and containing DHA 12 and coenzyme Q10 14. The Examples section that follows describes one approach to formulating the present composition of matter. The material composition can be stored in a lyophilized state and reconstituted with water or saline solution, for example, for use or stored as a ready-to-use pharmaceutical composition. The material composition may be part of a pharmaceutical composition that includes a vehicle formulated for topical or intraocular administration. Topical formulations of the present pharmaceutical composition may include a vehicle 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. The intraocular formulations of the present pharmaceutical composition can be formulated as a microemulsion and / or include a vehicle such as liposomes, nanospheres, micelles, and nanocapsules. The intraocular formulation can be formulated for the slow or delayed release of the active ingredients by using excipients that form inclusion complexes with active ingredients such as chelating agents, surfactants, and cyclodextrins. The pharmaceutical composition may also include: (i) Carbohydrates (as stabilizers, lubricants or cryoprotectants) including, but not limited to, monosaccharides (e.g. glucose, maltose), disaccharides (e.g. trehalose), oligosaccharides (dextrins (by ΜΛ / ΙΖ / ΖυΖ^ / υΊ uouo e.g., maltodextrin), cyclodextrins (e.g., hydroxypropyl-beta-cyclodextrin (HPbCD), polysaccharides (e.g., dextran). (i) Emulsifiers including, but not limited to, non-ionic surfactants of natural origin (e.g., lecithin, egg yolk phospholipids) and synthetic origin (e.g., Tyloxapol) and ionic surfactants (e.g., cetalkonium chloride). (iii) Thickening agents including, but not limited to, hydrophilic polymers (e.g., polyvinyl alcohol) or cellulose derivatives (e.g., hydroxypropyl methylcellulose (HPMC)). (iv) A bioadhesive such as polyamino acids (e.g., gelatin, human albumin) and polysaccharides such as cellulose derivatives (e.g., hydroxypropyl methylcellulose (HPMC) and hydroxypropylcellulose (HPC), hyaluronic acids (v) A gelling agent, such as alginate and polyacrylates, may be added to the pharmaceutical composition to increase the residence time of the active ingredients in the cornea. According to embodiments of the present invention, the concentration of insulin in the pharmaceutical composition can be from 0.001 U to 20 U per mi while the concentration of IGF can be from 0.001 U to 20 U per mi. According to embodiments of the present invention, the concentration of DHA in the pharmaceutical composition can be from 1 to 4 mg / ml. According to embodiments of the present invention, the concentration of coenzyme Q10 in the pharmaceutical composition can be from 1 to 3 mg / ml. ivia / t / zuzz / ui aouo Table 1 below describes a topical formulation of the present composition. Table 1 - Topical Formulation IVIA / t / ZUZZ / UI eouo Ingredient Amount per vial ELGN01 ELGN02 Recombinant Human Insulin (rhInsulin) 0.067 IU - Insulin-like Growth Factor 1 (IGF-1) - 2 IU Cys-4,7,10,13,16,19- Docosahexaenoic Acid (DHA) 2 mg 2 mg Coenzyme Q10 (CoQ10) 1 mg 1 mg Medium-Chain Triglycerides (MCT) 1 mg 1 mg Tyloxapol 0.5 mg 0.5 mg Lipoid E 80 0.5 mg 0.5 mg Polyvinyl Alcohol (PVA) 1 mg 1 mg Hydroxypropyl-beta-cyclodextrin (HPbCD) 2 mg 2 mg The present formulation can be modified to be MCT-free and include DHA in two forms: as free acid and as an ethyl ester. These two forms of DHA replace the MCT in the core of the drop. Two separate emulsions are produced and combined in the final production step. One emulsion contains the drops containing the free DHA acid to which the insulin is conjugated. The second emulsion contains drops in which Q10 is incorporated into the core of the DHA ethyl ester. Table 2 below lists the ingredients for this embodiment of the present injectable formulation. Table 2 ivia / t / zuzz / ui eouo Ingredient Function Amount per 1 ml Recombinant Human Insulin (rh-Insulin) Active ingredient 2 IU (0.07 mg) Cis-4,7,10,13,16,19 Docosahexaenoic Acid* (DHA) Excipient 1.87 mg Coenzyme Q10 (CoQ10) Excipient 1.0 mg Tyloxapol Surfactant 0.5 mg Lipoid E 80 Surfactant 0.5 mg Polyvinyl Alcohol (PVA) Surfactant 1.0 mg Hydroxypropyl-betacyclodextrin (HPbCD) Stabilizing agent 40 mg Sodium Hydroxide*** pH adjustment - Hydrochloric Acid** pH adjustment - Water for Injection Solvent Up to 1 ml * Contains approximately 0.4 mg / ml of free DHA acid and approximately 1.6 mg / ml of DHA ethyl ester (corresponds to 1.47 mg / ml of free DHA acid) ** Sodium hydroxide or hydrochloric acid is used to adjust the pH value and is not included in the sum. An intraocular formulation of the present composition is described in Table 3 below. Table 3 - Infraocular formulation Ingredient Amount per vial ELGN03 Recombinant Human Insulin (rhInsulin) 0.005-0.1 IU Cis-4,7,10,13,16,19- Docosahexaenoic Acid (DHA) 0.2-0.5 mg Coenzyme Q10 (CoQ10) 0.1 mg Medium Chain Triglycerides (MCT) 0.11 mg Tyloxapol 0.05 mg Lipoid E 80 0.05 mg Polyvinyl Alcohol (PVA) 0.1 mg Hydroxypropyl-beta-cyclodextrin (HPbCD) 0.2 mg IVIA / t / ZUZZ / UI uouo In order to improve the efficacy of the present composition, an approach to manufacture a nanoemulsion having nanodroplets that encapsulate (DHA) and Coenzyme Q10 and conjugated with Insulin or IGF. Therefore, according to another aspect of the present invention, a method for formulating a pharmaceutical composition for the topical treatment of retinopathy is provided. The pharmaceutical composition is manufactured by generating an oil-in-water nanoemulsion that includes docosahexaenoic acid (DHA) and coenzyme Q10 in the oil phase and conjugating insulin or IGF-1 with nanodroplets of the nanoemulsion using an amine coupling reaction. After nanoemulsion generation, the nanodroplets can be purified or concentrated using, but not limited to, column chromatography, tangential flow filtration (TFF), or dialysis. A stabilizing agent such as, but not limited to, cyclodextrin, dextrin, or a mono- or disaccharide can be added. The formulation can then be freeze-dried for storage and subsequent reconstitution with saline solution or water before use. The Examples section that follows provides a more detailed description of the present formulation approach. As mentioned above, the present composition can be used to treat retinopathy and, in particular, retinopathy of prematurity (ROP). Therefore, according to another aspect of the present invention, a treatment procedure for retinopathy is provided for a subject in need, such as a premature infant. The procedure is carried out by administering the pharmaceutical composition of the present invention to the eye of the subject in need. Such administration may be topical or intraocular. As used in this document, the term “subject in need” refers to any human or non-human mammal. A human or non-human mammal (cats, dogs, cows, sheep, pigs, goats, and horses) may be of any age (e.g., infants such as full-term or premature babies, adults, or the elderly) or sex. A human subject may be a premature infant born at a gestational age of 24 to 33 weeks. A human subject may also be a low birth weight infant weighing 500 to 1650 g at birth. A topical formulation (eye drops) of the present composition may be administered to a premature infant at any time between birth and 6 months of age once or several times daily for a period of 180 days at a dose of 10 microliters to 100 microliters. An intraocular formulation of the present composition may be administered to a premature infant at any time between birth and 6 months of age once every few weeks for a period of 180 days, as clinically required, at a dose of 5 to 30 microliters per injection. According to the usage in this report, the term “approximately” refers to ± 10%. Additional novel features, advantages, and objects of the present invention will become evident to those skilled in the art upon examination of the following examples, which are not intended to be limiting. EXAMPLES Reference is now made to the following examples, which together with the descriptions above illustrate the invention in a non-limiting manner. EXAMPLE 1 Nanoemulsion Formulation The following example demonstrates the manufacture of the present composition of matter formulated as a freeze-dried powder suitable for reconstitution as an oil-in-water nanoemulsion. Table 4 below lists the ingredients used in the manufacturing process of the material composition formulation. Table 4 - components of the ivia / t / zuzz / ui youo formulation ML / IZ / ZυZZ / υΊ UOUO Ingredient Function Recombinant Human Insulin (rhInsulin) 0 Insulin-like Growth Factor 1 (IGF-1) 1 Active ingredient 5 Cis-4,7,10,13,16,19- Docosahexaenoic Acid (DHA) Active ingredient Synthetic Coenzyme Q10 (Q10) Active ingredient Medium Chain Triglycerides (MCT) Oil Phase Tyloxapol Non-Lipoid Ionic Surfactant E 80 Amphiphilic Surfactant 10 Polyvinyl Alcohol (PVA) Non-Lipoid Ionic Surfactant NaOH 0.5 N 1 Reagent, pH adjustment Water (DDW) 2 Aqueous Phase Acetone 2 Solvent Ethanol 2 Solvent 15 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide Hydrochloride (EDC) 2' 3 Coupling Reagent Phosphate-buffered saline (PBS), pH 7.2 Buffer Sodium carbonate buffer Reagent, pH adjustment 2-Hydroxypropyl-3-cyclodextrin (HPbCD) Stabilizer, cryoprotectant 1. Example 1 describes a certain manufacturing process for a formulation containing insulin. 2. Removed during the process 3. Crosslinking reagent used in the amine coupling reaction and subsequently removed The nanodroplet formulation was achieved using a solvent displacement procedure. 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 were dissolved in 1 mL of ethanol. The resulting solutions were combined, mixed at 900 rpm for 30 minutes at room temperature, and added dropwise to 20 mL of 0.1% w / v aqueous PVA solution, stirred continuously at 900 rpm for an additional 15 minutes. Subsequently, the organic solvents were completely removed at room temperature under reduced pressure (50 mBar) using a laboratory rotary evaporator, and the resulting emulsion was subjected to an amine coupling reaction after a preliminary pH adjustment to 7.4 with 0.5 M NaOH. 1.3 pmol of EDC prepared in 0.5 mL of phosphate-buffered saline (pH 7.2) was added to the resulting emulsion. The mixture was incubated at room temperature for 15 minutes, and then the pH was adjusted to 8.3 ± 0.2 using sodium carbonate buffer. 0.5 mL of rh-insulin solution (0.1 pmol / mL in phosphate-buffered saline, pH 7.2) was added to 19.5 mL of the emulsion. The reaction mixture was stirred for 12 hours at room temperature. The reaction mixture was then loaded onto a PD-10 gravity-flow gel filtration column (Sephadex G25) 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 at reduced pressure (50 mBar) and 37 °C using a laboratory rotary evaporator.The emulsion was then mixed with 2-hydroxypropyl-3-cyclodextron to a final concentration of 2% w / v, filtered through a 0.45 µm PES (polyethersulfone) membrane, dispensed into vials, and lyophilized. The contents of. ΜΛ / ιζ / ζυζ^ / υΊ eouo active components in 1 ml of the reconstituted solution was 0.67 U of rhinsulin, 2 mg of DHA and 1 mg of CoQ10. EXAMPLE 2 Study 1 An ophthalmic formulation (ELGN01 consisting of insulin DHA and Coq10) of the material composition described in Example 1 was tested in the oxygen-induced retinopathy model in rats. Procedure A single rat mother, with a litter of 18 pups, was divided into two groups: Group A - ELGN01 (9), Group B - untreated (9, in the oxygen chamber without treatment). As an additional control, a single mother with 3 pups was placed under normoxic conditions. Treatment was initiated on day 5 to 14 or 18 (according to the day of sacrifice), first via administration under the eyelid with a syringe (topical, without damaging the ocular surface) and then with eye drops after opening the eye. The oxygen regimen was as follows: from day 0 to 14 of life, 24-hour cycles of hyperoxia (50%), then hypoxia (12%) for 24 hours. The study of group 1 was completed on day 14 (P14) of life and of group 2 on day 18 (P18) of life and an ophthalmologist carried out a fundoscopy evaluation on day 17, then the samples were evaluated histologically and immunologically. Results Results of In Vivo Fundoscopy In the treated group, a total of 12 hemorrhages were observed. ML / E / ZυZZZ / υΊ youo retín ¡anas, compared to 22 in the untreated animal group (ELGN01 treatment significance p = 0.04). Table 5 - in vivo fundoscopy results - retinal hemorrhages by group ML / E / ZuZZ / u eouo Treatment ELGN01 Untreated Normoxic Number of all retinal hemorrhages 12* 22 0 Number of severe retinal hemorrhages (Medium-Large) 1 11 0 Average number of retinal hemorrhages per eye 1.4 2.75 0 Data are mean ± SD; (f) p < 0.1, (*) p < 0.05, (**) p < 0.01 by means of T test compared to the control group without treatment. Total retinal damage and total severe hemorrhages in the treated and untreated groups are shown in the graphs in Figures 2A to B. Figures 3A to C are retinal images of normoxic animals and hypoxic animals (treated and untreated). Normoxic animals show intact retinal vessels, without hemorrhages or ablation. Untreated hypoxic animals show retinal hemorrhages (arrows). Treated animals show reduced damage. Neovascular area The effect on neovascularization is shown in Figures 4A to 4B. Neovascularization (NV) was greatest at P18, corresponding to the end of phase II of the human disease. P14 corresponded to the end of phase I of the disease (progression stage). At both time points, the treatment group showed significantly less NV than the untreated animals. At P14, the ELGN01 treatment group showed 0% neovascularization compared to 0.09% in the untreated group (T-test comparison, p = 0.07). At P18, the ELGN01 treatment group showed 40% less neovascularization on average compared to the untreated group (ELGN01 treatment 1.35%, untreated 1.89%, T-test comparison, p = 0.07, a treatment effect of 28%). Layers of the retina Whole eyes embedded in paraffin were sectioned and stained with hematoxylin and eosin. Four sections from different locations were collected on one slide. H&E stains were imaged using a light microscope with a 10x objective (4x in some areas). Representative stains from the untreated OIR group show disorganization of the retinal layers and thickening of the ganglion cell layer as a result of OIR damage. A total of 8 samples per group were available: 4 sections per eye, 2 eyes per treatment group (from different animals). To assess the integrity of the retinal layers, anonymized H&E-stained images were loaded into the Wimasis software image. Each retina was analyzed in a masked manner by identifying and measuring 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), and RPE (retinal pigment epithelium). IVIA / t / ZUZZ / UI uouo retinal pigment epithelium, CHO - choroid. Figure 11A shows the mean thickness of the retinal layers. Table 6 - Average thickness [px] of retinal layers ML / E / ZuZz / u SOUO GCL Layer [1] IPL Layer [2] INL Layer [3] OPL Layer [4] ONL Layer [5] IS Layer [6] ELGN01 Treatment P18 118.8 0 180.2 3 203.7 3 32.02 298.7 1 103.9 5 Untreated P18 121.9 1 128.1 0 157.9 0 21.33 270.2 0 90.71 Normoxic P18 137.0 4 192.4 2 203.6 1 28.32 298.3 8 97.35 EXAMPLE 3 Study 2 Ophthalmic formulations based on the material composition described in Example 1 (ELGN01 consisting of Insulin DHA and Coq10, and ELGN02 consisting of IGF-01 and the same) were tested in the Oxygen Induced Retinopathy model in rats. Procedure Two rat mothers, each with 18 pups, were divided into three treatment groups: Group A ELGN01 (12), Group B ELGN02 (12), and Group C untreated (12). Group D Normoxic was analyzed as a control. The treatment was started on day 5, the treatment was administered to the animals until day 14 or day 18 (according to the day of slaughter), first under the eyelid with a syringe (topical, without damaging the ocular surface), then with eye drops after opening the eyes. The oxygen regimen was as follows: In the first 4 days, 8 intermittent hypoxia events were carried out, with 3 reductions to 12% during the 30-minute event and 50% hyperoxia for the remainder of the time. From days 5 to 14 of life, 24-hour cycles of hyperoxia (50%) followed by hypoxia (12%) were administered. The Group 1 study was completed on day 14 of life and an ophthalmologist performed a fundoscopy evaluation on day 17, then the samples were evaluated histologically and immunologically. Results Isolectin staining The samples were mounted in planar fashion and the retinas were stained with Isolectin GS-IB4. Avascular areas (AVA) were manually quantified by independent experts using images of isolectin-stained retinas. Figures 5A to D illustrate the staining of the insulin- and IGF-1-treated groups, the untreated group, and the normoxic group. In the insulin- and IGF-1-treated groups (Figures 5A to B), minimal avascular areas with complete central blood vessels are observed. In the untreated group (Figure 5C), large avascular areas are observed (arrows). In the normoxic group (Figure 5D), complete coverage of the blood vessels is observed. Figures 6A to 6B are graphs representing AVA. At P14, both treatment groups showed a 50% reduction in AVA compared to the control group (2.77% for treatment ELGN01, 3.15% for treatment ELGN02, and 6.13% for untreated avascular area). The normoxic group had 1.4% avascular area. When the t-test was used to compare the treatment groups with the untreated animals, the results showed a 50% reduction in AVA. ML / E / ZuZZ / uΊ 8000 treat, Treatment-ELGNOIvs. the untreated had a statistically significant difference (p = 0.01), as well as Treatment-ELGNO2vs. untreated (p = 0.03). Each retina was analyzed in a masked manner to determine vascular density (%, calculated by dividing the number of vessel pixels by the total number of pixels in the region of interest), total vascular area, number of branching points (where two or more segments converge), number of segments (number of individual vessel segments), and mean segment length. At P14, both treatment groups outperformed untreated animals in numerous characteristics. Treatment ELGN01 had a significantly higher vessel density (%) compared to untreated animals (p = 0.051), as did treatment ELGN02 (p = 0.032), indicating improved growth and development of blood vessels in the retina, as well as fewer avascular areas. The treatment groups also showed a larger vascular area compared to the untreated group: treatment ELGN01 (p = 0.073) and treatment ELGN02 (p = 0.014). Furthermore, Treatment A – ELGN01 had a significantly longer mean segment length (p = 0.037) compared to untreated animals, indicating better blood vessel continuity. Table 7 - quantification of retinal vasculature in P14 ivia / t / zuzz / ui uouo ML / E / ZuZz / u UOUO Treatment -ELGN01 Treatment -ELGN02 Untreated Normoxic Vessel Density (%) 57.04 ± 2.15 t 57.3 ±1.5* 54.1 + 2.82 65.47 ±0.31 Total Vascular Area (in units of 1,000,000 px) 3.826 ± 0.23 t 3.978 ±0.2 * 3.571 + 0.26 4.28 ± 0.05 Total Branching Points 6065 ±1039 6958 ± 407 6700 ±555 9710 ±97 Data are mean ± SD; (t) p < 0.1, (*) p < 0.05, (**) p < 0.01 by means of T-test compared to the untreated control group. N = 5 per group Figures 7A to D are isolectin-B4 staining images of P14, by treatment group. The ROI (green), vessel-covered area (blue), vessel skeleton (red), and branching points (white) are marked. According to Table 8 below, in the P18 treatment group, ELGN01 had a significantly higher vessel density (%) compared to the untreated group (p = 0.007). The ELGN02 treatment group showed no significant trend. The percentage of vessel density reflects the amount of retina that is vascularized compared to the non-vascularized area. A higher vessel density without neovascularization indicates better growth and development of blood vessels in the retina, as well as less avascular areas. The treatment groups also showed a trend toward a larger vascular area compared to the untreated group. The ELGN01 treatment group showed a greater number of branching points compared to the untreated group (p = 0.02). In addition, the ELGN01 treatment group showed a greater number of blood vessels compared to the untreated group (p = 0.04). Table 8 - Quantification of retinal vasculature in P18 ML / E / ZuZz / u youo Treatment ELGN01 Treatment - ELGN02 Untreated Normoxic Vessel Density (%) 62.24 ± 1.15 ** 60.38 ± 1.67 59.46 ± 1.6 65.83 ± 0.87 Total Vascular Area (in units of 1,000,000 px) 4.44 ± 0.29 4.41 ± 0.16 4.26 + 0.32 4.91 ± 0.6 Total Branching Points 7898 ± 491 * 7250 ± 438 7023 ± 668 8649 ± 242 Number of Segments 8415 ± 458* 7741 ± 445 7629 ± 732 9044 ±280 Data are mean ± SD; (t) p < 0.1, (*) p < 0.05, (**) p < 0.01 by means of T-test compared to the untreated control group. N = 5 per group Biomarker activity Eye samples were collected at P14 and P18, homogenized, and centrifuged to compare the levels of different biomarkers in the tissue of the different study groups. Samples included four different rats per group, and three samples were analyzed. Contents were normalized to the total protein concentration in each sample. The biomarkers analyzed were 8-isoprostane and 8-isoPGF2α, a commonly studied drug abundantly generated in vivo during oxidative stress and lipid peroxidation, and a reliable and proven biomarker for oxidative stress (Beharry 2017). Additionally, PGE2, a biomarker of inflammatory processes, was also measured. PGE2 has dual, opposing effects on endothelial cells (Figure 11B), mediating both vasoconstriction and vasodilation (via different receptors).PGE2 is the main metabolite of the COX-2 isoform that is activated by cytokines and growth factors, and is heavily involved in angiogenesis (Beharry 2017). The results showed reduced levels of 8-isoPGF2α at P14 and P18 compared to the untreated group, indicating a preventive effect against oxidative stress damage, as demonstrated by the animal model (Figure 11C). This effect was observed in both the first and second stages of the disease (P14 and P18). PGE2 levels were higher at P14 in all groups compared to normoxic individuals. The treatment groups showed a decrease at P18, while the untreated group showed a significant increase, related to the inflammatory stage of the pathology (Figure 11B). EXAMPLE 4 Study 3 An ophthalmic formulation (ELGN01) that included insulin, DHA, and Coq10 (described in Example 1) was administered to newborn rats to determine the concentration of insulin in the eye after administration. Procedure Two rat mothers with litters of 18 pups were divided into two groups: Group A - ELGN01 Normoxic Group (18), Group B - ELGN01 Group ML / t / ZUZZ / UI uouo Hypoxia (18). A single mother with 2 offspring placed in normoxic conditions was used as a control. Treatment began on day 5 and continued for 4 days by administering the composition (a 10 pl dose containing 0.0067 units of insulin) under the eyelid using a syringe (topical). Rats were sacrificed at 30, 60, and 120 minutes post-administration (N = 3 per T). Whole eyes were homogenized and evaluated by ELISA (Quantikine® ELISA). Results The results shown in Table 9 below indicate that 8 to 16% of the administered insulin was absorbed into the eye tissues within the first two hours. Table 9 - Averages per time point (± SDV) ML / E / ZuZz / u aOUQ Time Insulin Concentration (pmol / l) 30 5.52 ± 2.05 60 4.66 ± 0.92 120 4.80 ± 1.15 EXAMPLE 5 Nanoemulsion formulations The following example demonstrates an alternative approach to manufacturing the material composition of the present invention. The present invention discloses the conjugation of insulin in a vessel with oily nanodroplets directly during the formulation process. The conjugation is carried out by coupling insulin to DHA carboxyl groups in an aqueous medium using the crosslinking reagent N-(3-dimethylaminopropyl)-N'ethylcarbodiimide (EDC). The process for making an insulin-DHA conjugate uses the following general steps: - Formation of nanodroplets by means of the solvent displacement procedure - Activation of the DHA carboxyl group with EDC by means of the formation of an active O-acylisourea DHA-EDC ester. - Conjugation of insulin with the DHA carboxyl group by means of the formation of an amide bond with the primary amine groups of insulin accompanied by the release of an EDC byproduct as Ninsubstituted soluble urea. - Purification of the reaction mixture from the EDC by-product by means of ultrafiltration through 30,000 to 100,000 MWCO membranes Materials and procedures Preparation of the organic phase: 347 mg of free DHA, 75 mg of Tyloxapol, and 75 mg of Lipoid E80 were dissolved in 25 mL of ethanol. The mixture was added dropwise through a 21G needle to 100 mL of doubly deionized water and mixed continuously at 350 RPM at room temperature. The resulting emulsion was mixed for an additional 10 minutes, and then the organic solvent was completely removed under reduced pressure using a laboratory rotary evaporator (40 ± 2 °C, 50 mBar). ivia / t / zuzz / ui aouo The resulting emulsion was subjected to an amine coupling reaction after a preliminary adjustment of the pH to 4.5 with 0.1 N HCl. 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 intermediate DHA-EDC ester was complete. 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 carried out for 1 hour, maintaining the pH between 6.3 and 6.4 during coupling. The reaction was monitored by HPLC (Dionex Ultimate 3000). The procedure conditions and the chromatogram of the reaction mixture at the 30-minute mark are shown in Figure 8. Once the reaction was complete, the mixture was diluted 1:2 with doubly deionized water and transferred through a 100,000 MWCO Hydrosart ultrafiltration cassette (Sartoñus) using a peristaltic pump. The insulin conjugate content in 150 ml of the resulting retained was 0.037 mmol, yielding 82% calculated for insulin content. The osmolarity of the emulsion was 301 mosm / kg. Figure 8 presents a chromatogram of the coupling reaction mixture; the components and conditions are listed in Table 10 below. Table 10 IVIA / t / ZUZZ / UI youo Procedure Parameter Adjustment Value Column Thermo Hypersil GoldC18 Column (3 x 50 mm, 3 pm) Column Temperature 45 °C Injection Volume 5 pl Flow Rate 1.0 ml / min UV Detector 208 nm Mobile Phase A 1000 ml of water: 1 ml of TFA Mobile Phase B 750 ml of Acetonitrile: 150 ml of Methanol: 100 ml of IPA: 0.5 ml of TFA Degradation 0 to 7.5 min 30% of B to 100% of B 7.5 to 11.0 min 100% of B Another composition of material was manufactured as a freeze-dried powder suitable for reconstitution into an oil-in-water nanoemulsion. Table 11 below lists the components used in the manufacturing process. Table 11 Ingredient Function Recombinant Human Insulin (rh-insulin) Active ingredient Cis-4,7,10,13,16,19- Docosahexaenoic acid (DHA), as free acid and as ethyl ester Active ingredient Coenzyme Q10 Active ingredient Tyloxapol Non-ionic surfactant Lipoid E 80 Amphiphilic surfactant Polyvinyl alcohol (PVA) Non-ionic surfactant NaOH 0.1 N Reagent, pH adjustment HCl 0.1 N Reagent, pH adjustment Water (DDW)1 Aqueous phase Ethanol1 Solvent Acetone1 Solvent 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)12 Reagent 2-Hydroxypropyl-8-cyclodextrin (HPbCD) Stabilizer, cryoprotectant 1Removed during the process. 2Crosslinking reagent used in the amine coupling reaction and subsequently removed. The formulation process includes the preparation of two separate emulsions: a first emulsion incorporates Coenzyme Q10 into DHA nanodroplets, and a second emulsion incorporates insulin conjugated with DHA nanodroplets. The emulsions were prepared separately using the displacement method and combined before the purification stage. 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 through a 21G needle to 250 mL of a 0.1% aqueous PVA solution, continuously mixed at 350 RPM at room temperature. The resulting emulsion was mixed for an additional 10 minutes, and then the organic solvents were completely removed under reduced pressure using a laboratory rotary evaporator (45 ± 2 °C, 50 mBar). Emulsion 2 125 mg of free DHA, 25 mg of Tyloxapol, and 25 mg of Lipoid E80 were dissolved in 12 mL of ethanol. The mixture was added dropwise through a 21G needle to 50 mL of double-deionized water, continuously mixed at 350 RPM at room temperature. The resulting emulsion was mixed for an additional 10 minutes, and then the organic solvents were completely removed under reduced pressure using a laboratory rotary evaporator (40 ± 2 °C, 50 mBar). The resulting emulsion was subjected to an amine coupling reaction after a preliminary adjustment of the pH to 4.5 with 0.1 N HCl. 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 hours until the formation of the intermediate DHA-EDC ester was complete. The pH of the reaction mixture was adjusted to 6.2, and 0.015 mmol of insulin dissolved in 18 mL of water (pH 4.2) was added. The reaction was carried out for 1 hour, maintaining a pH between 6.2 and 6.4 during coupling. The reaction was monitored by HPLC (Dionex Ultimate 3000). The procedure conditions and a typical chromatogram of the reaction mixture are shown in Figure 8. Once the reaction was complete, the mixture was combined with Emulsion No. 1 and then diluted 1:2 with a 0.1% aqueous PVA solution (osmolality <5 mosm / kg) and transferred through a 30,000 MWCO Hydrosart ultrafiltration cassette (Sartorius) using a pump ML / E / ZυZZZ / υΊ UOUO peristaltic, the final retained volume was 250 ml (the theoretical conjugated insulin content was 0.06 pmol / ml). 4 g of HPBCD dissolved in 8 ml of water were 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, introduced into glass vials (4 ml, 0.5 ml per vial), and lyophilized. The osmolarity of the finished bulk product was 376 mosm / kg. The theoretical content of insulin conjugate per vial was 0.024 pmol / vial, the observed content was 0.017 pmol / vial; the yield of conjugated insulin was 72%. The average size Z of the bulk and dry liquid product were 119.9 nm (polydispersity index 0.137) and 243 nm (polydispersity index 0.342), respectively. The conjugated insulin content, as well as DHA and Coenzyme Q10, of the lyophilized powder was controlled by RP-HPLC. The chromatograms of the finished lyophilized product are shown in Figure 10. Table 12 below provides the chromatographic conditions used to test the lyophilized formulation. IVIA / t / ZUZZ / UI eouo Table 12 Procedure Parameter Setting Value Column Thermo column, Hypersil Gold-C18 (3 x 50 mm, 3 pm) Column temperature 45 °C Injection volume 5 µL Flow rate 1.0 ml / min UV detector 208 nm for conjugated insulin monitoring 220 nm for DHA and coenzyme Q10 monitoring Mobile Phase A 1000 ml of water: 1 ml of TFA Mobile Phase B 750 ml of Acetonitrile: 150 ml of Methanol: 100 ml of IPA: 0.5 ml of TFA Degradation 0 to 3.0 min 30% of B to 42% of B 3.0 to 8.5 min 42% of B to 100% of B 8.5 to 12.5 min 100% of B ivia / t / zuzz / ui youo Figures 14A to E and 15A to E show typical Cryo transmitting electron micrographs (TEM) of the drug product produced according to what is described in Example 5. EXAMPLE 6 Gl Formulation An oral emulsion was developed for the local treatment of intestinal malabsorption in premature infants. The formulation contains three active ingredients: rh-insulin, DHA, and coenzyme Q10. In the reconstituted formulation, the insulin exists as a free protein, while the DHA and coenzyme Q10 are incorporated into the oil droplets. The formulation process included the following general steps: - Formation of DHA and Coenzyme Q10 emulsion using the solvent displacement procedure - Addition of rh-insulin and cryoprotectant - Filtration and freeze-drying Materials and procedures 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 through a 21G needle to 350 mL of a 0.1% aqueous PVA solution, continuously mixed at 350 rpm at room temperature. The resulting emulsion was mixed for an additional 10 minutes, and then the organic solvents were completely removed under reduced pressure using a laboratory rotary evaporator (45 ± 2 °C, 50 mBar). One milliliter of insulin solution in water (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. The resulting solution was added to a continuously mixed emulsion and mixed for 20 minutes. The emulsion was filtered through a 0.22 µm PES membrane, introduced into 4 mL glass vials (0.5 mL fill volume / vial), and lyophilized. The osmolarity of the finished bulk product was 358 mOsm / kg. Each vial contained 0.65 U of rh-insulin, 0.9 mg of DHA, and 0.5 mg of coenzyme Q10. Table 13 below lists the components of the formulations. Chromatograms of the lyophilized product are provided in Figures 12 and 13. IVIA / t / ZUZZ / UI eouo Table 13 ML / E / ZuZz / u UOUO Ingredient Amount per 1 ml of reconstituted product Recombinant Human Insulin (rhInsulin) 1.3 IU (0.045 mg) Cis-4,7,10,13,16,19- Docosahexaenoic Acid (DHA) 1.9 mg Coenzyme Q10 (CoQ10) 1.1 mg Tyloxapol 0.4 mg Lipoid E 80 0.4 mg Polyvinyl Alcohol (PVA) 0.8 mg Hydroxypropyl-beta-cyclodextrin (HPbCD) 6.7 mg Maltodextrin 80.0 mg Sodium Hydroxide* - Hydrochloric Acid* - Water for Injection Up to 1 ml * Sodium hydroxide or hydrochloric acid are used to adjust the pH value and are not included in the sum. It is apparent that certain features of the invention, which, for clarity, are described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, several features of the invention, which, for brevity, are described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. Although the invention has been described along with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to encompass all alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, and patent applications mentioned in this specification are incorporated herein in their entirety by reference, to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated for inclusion as a reference herein. Furthermore, the 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. Furthermore, any priority document for this application is incorporated in its entirety as a reference in this report.

Claims

1. A pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA) and coenzyme Q10.

2. The pharmaceutical composition according to claim 1, further comprising an insulin-like growth factor (IGF).

3. The pharmaceutical composition according to claim 1, further comprising a vehicle formulated for topical administration.

4. The pharmaceutical composition according to claim 1, further comprising a vehicle formulated for ocular administration.

5. The pharmaceutical composition according to claim 4, wherein said vehicle includes a surfactant.

6. The pharmaceutical composition according to claim 1, formulated as an oil-in-water nanodroplet emulsion with said insulin conjugated with said nanoparticles.

7. The pharmaceutical composition according to claim 6, wherein said insulin is amide-conjugated with said nanodroplets.

8. The pharmaceutical composition according to claim 6, wherein said nanodroplets include said docosahexaenoic acid (DHA) and said coenzyme Q10.

9. The pharmaceutical composition according to claim 8, wherein said insulin is conjugated with docosahexaenoic acid (DHA) amide.

10. The pharmaceutical composition according to claim 1, wherein a concentration of said insulin is from 0.001 U to 20 U per mi.

11. The pharmaceutical composition according to claim ML / E / ZuZZ / uJ 80U0 1, wherein the concentration of said DHA is 1 to 3 mg / ml.

12. The pharmaceutical composition according to claim 1, wherein a concentration of said coenzyme Q10 is from 1 to 3 mg / ml.

13. The pharmaceutical composition according to claim 5 2, wherein a concentration of said IGF is from 0.001 U to 20 U per mi.