Compositions and Methods for Treating Retinopathy

Abstract

Pharmaceutical compositions containing insulin, docosahexaenoic acid (DHA) and coenzyme Q10, as well as methods for making and using the compositions, are provided.

Description

Technical Field

[0001] The present invention relates to a composition for treating retinopathy. Embodiments of the present invention relate to a nanoemulsion containing insulin and / or IGF for treating retinopathy of prematurity (ROP).

[0002] [CROSS - REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 62 / 853,179, filed on May 28, 2019, the entire content of which is hereby incorporated by reference into this application.

Background Art

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

[0004] In humans, the retina develops in the oxygen - poor environment of the uterus. Vascular progenitor cells arise during the gestational period of 12 - 21 weeks and create a scaffold for future vascular development. Retinal angiogenesis begins at approximately 16 weeks of gestation, with new blood vessels budding from 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 abnormal proliferation of retinal blood vessels in an incompletely vascularized retina. ROP occurs mostly in extremely low gestational age neonates (ELGAN) weighing less than 1250 g or born 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 been proven useful in severe late-stage ROP. However, laser photocoagulation destroys major parts of the retina and is a difficult and complex procedure to perform in young infants, while intravitreal injection of VEGF antibodies may cause systemic suppression of blood vessel growth that affects other organs.

[0007] Therefore, there is a need and it is highly advantageous to have a retinopathy treatment method that does not include the above limitations.

Summary of the Invention

[0008] 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 method for treating retinopathy in premature infants, the method comprising administering to the eyes of a 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, a method for preventing or reducing the severity of retinopathy in premature infants, the method comprising administering to the eyes of a 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, a method for reducing retinal hemorrhage in premature infants, the method comprising administering to the eyes of a 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, a method for reducing retinal hemorrhage in a subject experiencing retinopathy, the method comprising administering to the eyes 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 angiogenesis in a subject experiencing retinopathy, the method comprising administering to the eye of the subject a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA), and coenzyme Q10, thereby reducing retinal angiogenesis in the eye of the subject. According to another aspect of the present invention, there is provided a method for increasing retinal vascular coverage in a premature infant, the method comprising: administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA), and coenzyme Q10, thereby increasing retinal vascular coverage in the premature infant (a step of reducing the avascular retinal area). According to another aspect of the present invention, there is provided a method for reducing retinal inflammation in a premature infant, the method comprising 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 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 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 administering to the eye of the premature infant a pharmaceutical composition comprising insulin, docosahexaenoic acid (DHA), and coenzyme Q10, thereby reducing 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 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 following steps: (a) generating a water-in-oil nanoemulsion containing docosahexaenoic acid (DHA) and coenzyme Q10 in an oil phase; (b) binding insulin or IGF-1 to the nano-droplets of the 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0010] Some embodiments of the present invention will be described herein by way of example only with reference to the accompanying drawings. The details shown hereinafter with particular reference to the drawings are for purposes of illustration only and also for purposes of a detailed description of the preferred embodiments of the present invention, and are presented for the purpose of providing what is considered to be the most useful and readily understood description of the principles and conceptual aspects of the present invention. Similarly, no attempt has been made to show the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention. Also, by looking at the description together with the drawings, it will be apparent to those skilled in the art how the embodiments of the present invention can be practiced.

Brief Description of the Drawings

[0011]

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Figure 11A - B

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Mode for Carrying Out the Invention

[0012] The present invention is the composition itself that can be used for treating retinopathy. Specifically, the present invention can be used for treating ROP through the topical administration of a nano - emulsion containing insulin or IGF.

[0013] The principle and implementation of the present invention will be better understood with reference to the drawings and the following description.

[0014] Before explaining in detail at least one embodiment of the present invention, it should be understood that the use of the present invention is not limited in detail as shown in the following description. The present invention allows for other embodiments and can be implemented or executed by various means. It should also be understood that the expressions and terms used in this specification are for the purpose of explanation and should not be regarded as limiting.

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

[0016] The inventor hypothesized that effective treatment of ROP should provide the missing intrauterine factors (insulin and insulin growth factor 1) that can promote physiological vascular development while preventing postnatal toxic effects (e.g., oxygen excess) and minimizing systemic exposure to these factors.

[0017] While reducing the invention to practice, the inventor formulated a composition that can promote the development of the physiological ocular vascular system 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 (angiogenesis) induced by the oxygen-induced model in rats.

[0018] The expression "promote physiological vascular development" means increasing the flow or passage of oxygen from the optic nerve to the peripheral part of the eye.

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

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

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

[0022] The term "therapeutically effective amount" or "pharmaceutically effective amount" means the dosage of an active ingredient or a composition containing an active ingredient that provides the indicated therapeutic effect.

[0023] The dosage of each active ingredient in the pharmaceutical composition of the present application may depend on many factors, including the subject to be treated, the stage of retinopathy (e.g., ROP), and the route of administration (topical or intraocular).

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

[0025] The composition may be formulated as an oil-in-water nanoemulsion containing docosahexaenoic acid (DHA) and coenzyme Q10 and having nanodroplets that are bound to insulin and / or IGF (e.g., via an amide bond). Figure 1 is a schematic diagram of the present composition showing nanodroplets that are bound to insulin or IGF 12 and contain DHA 12 and coenzyme Q10 14.

[0026] The following example section describes one approach for formulating the present composition.

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

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

[0029] The topical preparation of this pharmaceutical composition can contain carriers such as medium-chain triglycerides (MCT), long-chain triglyceride oils such as castor oil, synthetic oils and semi-synthetic oils such as mineral oil, and unsaturated fatty acids such as oleic acid.

[0030] The intravitreal preparation of this pharmaceutical composition can be formulated as a microemulsion and / or can contain carriers such as liposomes, nanospheres, micelles and nanocapsules.

[0031] The intravitreal preparation can be formulated for sustained or delayed release of the active ingredient using complexing agents, surfactants, and excipients that form inclusion complexes with the active ingredient such as cyclodextrin.

[0032] The pharmaceutical composition can contain the following. (i) Carbohydrates (as stabilizers, lubricants, cryoprotectants): Examples of such carbohydrates include, but are not limited to, monosaccharides (such as glucose and maltose), disaccharides (such as trehalose), oligosaccharides (such as dextrin), cyclodextrins (such as hydroxypropyl-β-cyclodextrin (HPbCD)), polysaccharides (such as dextran), etc. (ii) Emulsifiers: Examples of such emulsifiers include, but are not limited to, nonionic surfactants of natural origin (such as lecithin and egg yolk phospholipid), nonionic surfactants of synthetic origin (such as tyloxapol), and ionic surfactants (such as cetyltrimonium chloride). (iii) Thickening agents: Examples of thickening agents include, but are not limited to, hydrophilic polymers (such as polyvinyl alcohol) or cellulose derivatives (such as hydroxypropylmethylcellulose (HPMC)). (iv) Bioadhesives such as polyamino acids (such as gelatin and human albumin), and polysaccharides such as cellulose derivatives (such as hydroxypropylmethylcellulose (HPMC) and hydroxypropylcellulose (HPC), and hyaluronic acid). (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 can be 0.001 U to 20 U / ml. On the other hand, the concentration of IGF can 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 can be 1 to 4 mg / ml.

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

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

Table 1

[0037] This formulation can be modified to be free of MCT and contains two forms of DHA, as the free acid and as the 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 in which the free acid of DHA to which insulin is bound is included. The second emulsion contains droplets in which Q10 is incorporated into the DHA ethyl ester core. Table 2 below lists the components of this embodiment of the injectable form of this formulation.

Table 2

[0038] The intraocular formulation of the present composition is described in Table 3 below.

Table 3

[0039] To enhance the effectiveness of the composition, a method for producing a nanoemulsion having nanodroplets encapsulating (DHA) and coenzyme Q10 and conjugated with insulin or IGF was developed.

[0040] Accordingly, 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 produced by generating an oil-in-water nanoemulsion containing docosahexaenoic acid (DHA) and coenzyme Q10 in the oil phase and conjugating insulin or IGF-1 to the 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), dialysis. Stabilizers such as cyclodextrin, dextrin, mono- or disaccharides can be added, but are not limited thereto.

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

[0043] The following examples section provides a more detailed description of this formulation approach.

[0044] As described above, the composition can be used to treat retinopathy, particularly retinopathy of prematurity.

[0045] Accordingly, according to another aspect of the present invention, a method for treating retinopathy in a subject in need thereof, such as a premature infant, is provided. The method is effected by administering the pharmaceutical composition of the present invention to the eye of a subject in need thereof. Such administration can be topical or intravitreal.

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

[0047] The topical formulation (eye drops) of the present composition can be administered to premature infants once or several times a day for a period of 180 days at a dose of 10 microliters to 100 microliters at any time between birth and 6 months of age. The intravitreal formulation of the present composition can be administered to premature infants once every few weeks for a period of 180 days at a dose of 5 to 30 microliters per injection clinically required at any time between birth and 6 months of age.

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

[0049] Further objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following non-limiting examples.

Example

[0050] Next, reference is made to the following examples, which, together with the above description, illustrate the present invention in a non-limiting manner.

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

[0052] Table 4 below lists the components used in the manufacturing process of the composition formulation.

Table 4

[0053] The formulation of the nano-droplets 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, mixed at 900 rpm for 30 minutes at room temperature, added dropwise to 20 ml of a 0.1% w / v aqueous PVA solution, and continuously stirred at 900 rpm for an additional 15 minutes. Thereafter, using a laboratory rotary evaporator, the organic solvents were completely removed under reduced pressure (50 mBar) at room temperature. The resulting emulsion was pre-adjusted to pH 7.4 with 0.5 M NaOH and then subjected to an 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. Then, the pH was 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 set to stir at room temperature for 12 hours. Then, to separate the nano-droplets from smaller-sized particles (e.g., EDC, free active substance molecules), water was used as an eluent and the reaction mixture was loaded onto a gravity flow PD-10 gel filtration column (Sephadex G-25). The excess eluent (water) was removed from the nano-droplet fraction under reduced pressure (50 mBar) and at 37 °C using a laboratory rotary evaporator. Next, the emulsion was mixed with 2-hydroxypropyl-β-cyclodextrin to a final concentration of 2% w / v, filtered through a 0.45 μm PES (polyethersulfone) membrane, dispensed into vials, and then lyophilized. The active ingredients in 1 ml of the reconstituted solution were 0.67 U of rh-insulin, 2 mg of DHA, and 1 mg of CoQ10.

[0055] Example 2 Test 1 The ophthalmic preparation of the composition described in Example 1 (ELGN01 composed of insulin, DHA, and CoQ10) was tested in a rat model of oxygen-induced retinopathy.

[0056] <Procedure> Eighteen offspring of a single pregnant female rat were divided into two groups: Group A - ELGN01 (9 animals), Group B - untreated (9 animals, oxygen chamber without treatment). Three offspring of a single pregnant female rat kept in normal oxygen conditions were used as an additional control.

[0057] Treatment was initiated on days 5 - 14 or 18 (depending on the sacrifice day), first administered under the eyelids using a syringe (local, without damaging the ocular surface), and then instilled into the eyes after eye opening.

[0058] The oxygen administration regimen was as follows: from birth to day 14, high oxygen (50%) for 24 - hour cycles, followed by 24 - hour low oxygen (12%).

[0059] One group of tests ended on day 14 after birth (P14), the second group on day 18 after birth (P18). Evaluation by fundus examination by an ophthalmologist was performed on day 17, and then the samples were evaluated histologically and immunohistologically.

[0060] <Results> Results of in - vivo fundus examination In the treatment group, a total of 12 retinal hemorrhage sites were observed, while in the untreated group, there were 22 sites (significance of treatment with ELGN01, p = 0.04). [Table 5]

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

[0062] Figures 3A - C are images of the retina in the normoxic state, hypoxic state (treated and untreated). In the normoxic state, animals show intact blood vessels in the retina, with no bleeding or ablation. Untreated animals in the hypoxic state exhibit retinal hemorrhage (arrows). Treated animals show a reduction in damage.

[0063] Neovascularization area The effects on neovascularization are shown in Figures 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 of the disease (progressive phase). At all time points, NV in the treatment group was significantly less than that in the untreated group. At P14, the ELGN01 - administered group showed 0% neovascularization, while the non - administered group showed 0.09% (T - test comparison p = 0.07). At P18, the ELGN01 - administered group showed on average 40% less neovascularization compared to the non - administered group (treated ELGN01 1.35%, untreated 1.89%, T - test comparison p = 0.07, treatment effect 28%).

[0064] Retinal layer The whole paraffin - embedded eye was sectioned and stained with hematoxylin and eosin. Four sections from different locations were collected on one slide. H&E staining was imaged using an optical microscope with a 10x objective lens (4x at some positions).

[0065] Representative staining from the untreated OIR group shows the disintegration of the retinal layer and thickening of the ganglion cell layer as a result of OIR damage. A total of 8 samples were available for each group, i.e., 4 sections per eye and 2 eyes per treatment group (from different animals).

[0066] To evaluate the integrity of the retinal layer, the de - identified H&E - stained images were uploaded to Wimasis Software Image. Each retina was analyzed in a masked state, and 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) was identified and its size was measured. Figure 11A shows the average thickness of the retinal layer.

Table 6

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

[0068] <Procedure> Eighteen offspring of two pregnant female rats each were divided into three treatment groups: Group A ELGN01 (12 rats), Group B ELGN02 (12 rats), and Group C untreated (12 rats). Group D in a normal oxygen state was analyzed as a control.

[0069] Treatment was started on the 5th day and continued until the 14th or 18th day (depending on the sacrifice day). It was first administered under the eyelids with a syringe (local without damaging the eye surface), and then eye drops were administered after eye opening.

[0070] The oxygen regimen involved the following eight intermittent hypoxia events over the first four days: three events of reducing to 12% over 30 minutes, and the remaining time was 50% hyperoxia. From postnatal days 5 to 14, it was a 24-hour cycle of hyperoxia (50%), and then 24-hour hypoxia (12%).

[0071] One group of tests ended on the 14th day, and on the 17th day, evaluation was performed by fundus examination by an ophthalmologist, and then the samples were evaluated histologically and immunohistologically.

[0072] <Results> Isolectin staining Samples were mounted flat and the retina was stained with isolectin GS-IB4. The avascular area (AVA) was manually quantified by an independent expert using images of the isolectin-stained retina.

[0073] Figures 5A - D show the staining of the insulin and IGF treatment groups, the untreated group, and the normoxia group. In the insulin - administered group and the IGF - 1 - administered group (Figures 5A - B), a minimal avascular area with complete central vasculature is observed. In the untreated group (Figure 5C), a large avascular area is observed (arrow). In the normoxia group (Figure 5D), complete vascular coverage is observed.

[0074] Figures 6A - B are graphs representing AVA. At P14, in both treatment groups, AVA decreased by 50% compared to the control group (treatment ELGN01: 2.77%, treatment ELGN02: 3.15%, avascular area: 6.13%). The avascular area of the normoxia group showed 1.4%. When comparing the treatment groups with the untreated animals using a T - Test, the ELGN01 treatment group vs. the untreated group, similar to the ELGN02 treatment group vs. the untreated group (p = 0.03), had a statistically significant difference (p = 0.01).

[0075] For each retina, the vascular density (%, calculated by dividing the number of pixels of blood vessels by the total number of pixels in the target area), total vascular area, number of bifurcation points (where two or more segments converge), number of segments (number of individual vascular segments), and average segment length were analyzed after masking.

[0076] At P14, both treatment groups were superior to the untreated animals in many characteristics. Treatment ELGN01 had a significantly higher vascular density (%) compared to the untreated group (p = 0.051), and similarly, the treatment ELGN02 group (p = 0.032) showed better growth and development of the retinal blood vessels and a smaller avascular area. Also, the treatment groups; ELGN01 (p = 0.073) and the treatment ELGN02 group (p = 0.014) showed a larger vascular area compared to the untreated group. Furthermore, treatment A - ELGN01 had a significantly higher average segment length (p = 0.037) compared to the untreated animals, indicating better vascular continuity.

Table 7

[0077] Figures 7A - D are images of isolectin - B4 staining of P14 per treatment group. ROI (green), vessels in the covered area (blue), vessel skeleton (red), and branch points (white) are shown.

[0078] As summarized 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 retina with angiogenesis compared to areas without angiogenesis. A high vascular density without angiogenesis indicates good growth and development of retinal blood vessels and fewer avascular areas. Also, the treated groups showed a tendency for a larger vascular area compared to the untreated group. The treated ELGN01 group showed a higher number of branch points compared to the untreated group (p = 0.02). Furthermore, the treated ELGN01 group had a greater amount of blood vessels 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 the tissues of different study groups. Samples included 4 different rats per group and were tested in triplicate for each. The content was normalized to the total protein concentration of each sample. The biomarkers analyzed were 8 - isoprostane or 8 - isoPGF2α, which are well - studied, reliable, and proven biomarkers of oxidative stress that are abundantly produced in vivo during oxidative stress and lipid peroxidation (Beharry 2017). Furthermore, PGE2, a biomarker of the inflammatory process, was also measured (Figure 11B). This PGE2 has a dual opposite effect on endothelial cells. It mediates both vasoconstriction and vasodilation (via different receptors). PGE2 is the major metabolite of the COX - 2 isoform activated by cytokines and growth factors and is deeply involved in angiogenesis (Beharry 2017).

[0080] The results showed that at P14 and P18, compared with the untreated group, there was a decrease in 8-isoPGF2α levels, indicating a preventive effect against oxidative stress damage created by the animal model (Figure 11C). This effect was seen in both the first and second stages of the disease (P14 and P18). At P14, the level of PGE2 was higher in all groups compared to normoxia, and at P18, the level of PGE2 showed a decrease in the treated group and a significant increase related to the inflammatory stage of pathology in the untreated group (Figure 11B).

[0081] Example 4 Test 3 An ophthalmic preparation (ELGN01 containing insulin, DHA, and CoQ10 as described in Example 1) was administered to neonatal rats, and the insulin concentration in the eyes after administration was measured.

[0082] <Procedure> Eighteen offspring of two pregnant female rats each were divided into two groups: Group A - ELGN01 normoxia group (18), Group B - ELGN01 hypoxia group (18). Two offspring of a single pregnant female rat placed in normoxia were used as controls.

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

[0084] <Results> As shown in Table 9 below, 8 - 16% of the administered insulin was absorbed into the eye tissue within the first 2 hours. [Table 9]

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

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

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

[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 was added dropwise to 100 ml of double-deionized water with a 21G needle and continuously mixed at room temperature and 350 rpm. The resulting emulsion was further mixed for 10 minutes and then 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 an 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 completed within 1 hour, and the pH was maintained at 6.3 - 6.4 during the coupling. The reaction was monitored by HPLC (Dionex Ultimate 3000), and the method conditions and the chromatogram of the reaction mixture at the 30 - minute time point are shown in Figure 8.

[0091] At the end of the reaction, 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 150 ml of the resulting retentate was 0.037 mmol, and the yield calculated based on the insulin content was 82%. The molar osmotic concentration of the emulsion was 301 mosm / kg.

[0093] The 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 prepared as a lyophilized powder suitable for reconstitution into an oil - in - water nanoemulsion. The components used in the manufacturing process are listed in Table 11 below.

Table 11

[0095] The formulation process involves the preparation of two separate emulsions: the first emulsion incorporates coenzyme Q10 into DHA nano - droplets, and the second emulsion contains insulin bound to DHA nano - droplets. The emulsions were prepared separately by the displacement method and combined before 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% PVA aqueous solution through a 21G needle and continuously mixed at room temperature and 350 rpm. The obtained emulsion was further mixed for 10 minutes, and then 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 room temperature and 350 rpm. The obtained emulsion was further mixed for 10 minutes, and then the organic solvent was completely removed under reduced pressure using a laboratory rotary evaporator (40 ± 2 °C, 50 mBar).

[0098] The obtained emulsion was pre-adjusted to pH 4.5 with 0.1 N HCl and then an amine coupling reaction was carried out.

[0099] 0.11 mmol of EDC dissolved in 1 ml of water was added to the obtained emulsion, and the mixture was incubated at room temperature for 1.25 hours until the formation of the DHA-EDC intermediate ester was completed. 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 completed within 1 hour, and pH 6.2 - 6.4 was maintained during the coupling. The reaction was monitored by HPLC (Dionex Ultimate 3000), and the method conditions and a typical chromatogram of the reaction mixture are provided in Figure 8.

[0100] After the reaction was completed, the mixture was combined with Emulsion #1, then diluted 1:2 with a 0.1% aqueous PVA solution (osmolarity < 5 mosm / kg), and transferred through a 30,000 MWCO hydro-sart filter cassette (Sartorius) using a peripheral pump. The final volume of the residue was 250 ml (the theoretical content of conjugated insulin was 0.06 μmol / ml).

[0101] 4 g of HPBCD dissolved in 8 ml of water was added to 80 ml of the emulsion, and the volume was adjusted to 100 ml. The emulsion was filtered through a 0.22 μm PES membrane, 4 ml was filled into glass vials (0.5 ml per vial), and cooled. The osmolarity 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 size of the liquid bulk and dry final product was 119.9 nm (polydispersity index 0.137) and 243 nm (polydispersity index 0.342), respectively.

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

Table 12

[0105] Typical cryo-transmission electron micrographs (TEM) of the manufactured formulation are shown in Figures 14A - E and 15A - E as described in Example 5.

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

[0107] The compounding process included the following general steps: - Generation of DHA and coenzyme Q10 emulsion using the solvent replacement method, - Addition of rh-insulin and cryoprotectant, - Filtration and lyophilization.

[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 dropped into 350 ml of 0.1% PVA aqueous solution with a 21G needle and continuously mixed at 350 rpm at room temperature. The obtained emulsion was further mixed for 10 minutes, and then the organic solvent was completely removed under reduced pressure using a laboratory rotary evaporator (45 ± 2 °C, 50 mBar).

[0109] 1 ml of insulin solution (2.7 mg / ml, pH 8.5) was mixed with 14 ml of cryoprotectant solution containing 28.6 mg / ml of HPBCD and 343 mg / ml of maltodextrin. The obtained 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, 4 ml was filled into glass vials (filling volume 0.5 ml / vial), and lyophilized. The molar osmotic concentration 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 components. The chromatograms of the lyophilized product are shown in Figures 12 and 13.

Table 13

[0112] It should be understood that certain features of the invention that have been described in connection with separate embodiments for clarity may also be provided in combination in one embodiment. Conversely, the multiple features of the invention that have been described in connection with one embodiment for brevity may also be provided separately or in any suitable partial combination.

[0113] Although the invention has been described in connection with its specific embodiments, 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 included within the spirit and broad scope of the appended claims. All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be incorporated by reference. In addition, any reference or identification of a reference in this application should not be construed as an admission that such reference is available as prior art to the present invention.

[0114] Furthermore, the documents related to the basic application of this application are also hereby incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical composition for topical delivery to the eye for treating retinopathy, wherein the pharmaceutical composition comprises an oil-in-water nano-droplet emulsion, the oil-in-water nano-droplet emulsion comprises docosahexaenoic acid (DHA), coenzyme Q10, and insulin amide-bonded to the docosahexaenoic acid (DHA), pharmaceutical composition.

2. further comprising a carrier formulated for delivery to the eye, the pharmaceutical composition according to claim 1.

3. wherein the carrier comprises a surfactant, the pharmaceutical composition according to claim 2.

4. wherein the concentration of the insulin is 0.001 U to 20 U per ml, the pharmaceutical composition according to claim 1.

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

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

7. A method for preparing a pharmaceutical composition for topical treatment of retinopathy, comprising the following steps: (a) preparing an oil-in-water nano-emulsion containing docosahexaenoic acid (DHA) and coenzyme Q10 in an oil phase; (b) binding insulin or IGF-1 to the nano-droplets of the nano-emulsion using an amine coupling reaction.

8. further comprising the step of purifying the nano-droplets to which the insulin or IGF-1 is bound, the method according to claim 7.

9. subsequent to step (c), further comprising the step of adding a stabilizer in step (d), the method according to claim 8.

10. wherein the stabilizer is cyclodextrin, the method according to claim 9.

11. subsequent to step (d), further comprising the step of lyophilization, the method according to claim 9.

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