WO2023113586A1 - Lipid-based formulation for topical ophthalmic use that contains blueberry extract - Google Patents

Abstract

The invention relates to a lipid-based formulation for topical ophthalmic use that acts as a liposomal system for the delivery of bioactive agents contained in blueberry extract, in scleral tissue of the rear portion of the eyeball in human beings. The invention is useful for maintaining health and reducing the risk of the development of chronic degenerative processes in the intraocular tissues, including those of the rear portion of the eyeball, owing to its anti-inflammatory, antiangiogenic, anti-fibrotic and antioxidant activity. The lipid-based formulation has the potential to improve stability, improve the efficiency of penetration of the natural barriers of the eyeball and increase polyphenol bioavailability in the intraocular tissues.

Description

LIPID-BASED FORMULATION FOR TOPICAL OPHTHALMIC USE CONTAINING BLUEBERRY EXTRACT

5

FIELD OF THE INVENTION

The present invention is related to the field of biotechnology and medicine. The invention consists of a lipid-based formulation for topical ophthalmic use that functions as a liposomal delivery system for the bioactive agents contained in the blueberry extract in white tissues of the posterior segment of the eyeball in humans. The invention is useful for maintaining health and reducing the risk of developing chronic-degenerative processes in the infraocular tissues, including those of the posterior segment of the eyeball due to its anti-inflammatory, anti-angiogenic, anti-fibrotic and antioxidant activity.

BACKGROUND OF THE INVENTION

20 The posterior segment of the eye is the part of the eyeball that includes the retina and its blood vessels, as well as the head of the optic nerve. It is in this tissue, and in particular in its central area called the macula, where the vision generation process takes place. Said structure is a complex tissue formed by multiple layers of neurons, which can be functionally divided into two parts: one external and one internal. The external part comprises the Retinal Pigment Epithelium (RPE) and its basal lamina called Bruch's membrane, where both maintain the integrity of the retina. outer blood-retinal barrier. On the other hand, the internal part comprises the photosensitive layer of cones and rods and their nerve connections that capture light and convert it into electrical nerve impulses, which are transmitted through the optic nerve, having in these tissues the vascular structure that forms the internal blood-retinal barrier. These barriers are part of the anatomical and physiological barriers of the eyeball, which limit the penetration and bioavailability of bioactives in the retina when they are administered topically or systemically. The loss of structural integrity and/or function of the tissues of the posterior segment of the ocular globe, secondary to proinflammatory and oxidative processes, leads to pathological states that have a negative impact on visual function.

An extraordinary option for the maintenance of ocular health and the prevention and/or control of abnormal or pathological states, is the use of bioactives with antioxidant, neuroprotective, anti-angiogenic and anti-inflammatory properties. Some of these elements are present in nature, particularly in extracts from some plants, which have become one of the main sources explored by experts in the search for new molecules, whose potential use can provide new alternatives for prevention and/or treatment in conditions that affect the posterior segment of the eyeball and particularly the retina. An example of them are polyphenols, which are micronutrients widely present in food. Multiple scientific publications have shown their great potential as antioxidants, anti-inflammatory and neuroprotective drugs in various pathological conditions in cardiology, oncology, neurology and ophthalmology. However, due to its poor stability at room temperature and its low bioavailability, its application in areas such as ophthalmology is limited, since, although its potentials have been described applications, today an efficient method of delivery of these to tissues of the posterior segment of the ocular globe has not been described and its systemic administration (oral route) requires large doses, most of which are restricted by the different barriers of the eyeball.

For example, various publications have shown that the use of bioactives (polyphenols) contained in blueberry extract, such as anthocyanins (AC), resveratrol (RV) and pteroestil bene (PS), have the potential to maintain health and reduce the risk of developing chronic-degenerative processes in the retina through its anti-inflammatory, anti-angiogenic and antioxidant activity. Both anthocyanins, resveratrol, and pteroestil bene have been found to have a potent anti-inflammatory effect by significantly reducing the expression of anti-inflammatory mediators, tumor necrosis factor alpha (TNF-a), interleukins (IL-10, I L-6, MMP-2), metalloproteinases (MMP-9). Likewise, they significantly reduce the levels of oxidative damage of biomarkers, malondialdehyde (MDA), 4-hydroxynonenalo (4-HNE), aconitase-2 and 8-hydroxydeoxyguanosine (8-OHdG). In addition, anthocyanins and pteroestil beno restore homeostasis between oxygenases and oxidative enzymes by decreasing the expression of cyclooxygenase 2 (COX-2) and restoring the antioxidant activity of enzymes such as superoxide dismutase 1 (SOD1). and peroxiredoxin 4 (PRDX4). Due to the anti-inflammatory, antioxidant and neuroprotective properties of the different blueberry extract compounds described above, different scientific publications have been published in this regard, suggesting their potential use in different conditions of the posterior segment of the eyeball such as diabetic retinopathy, degeneration age-related macular tissue and in different conditions of hypoxia and inflammation. However, they all do emphasis on the limitation of its administration to reach therapeutic doses in the vitreous and retina, since most of them are limited to in vitro studies or predicament studies (in animals) or in systemic administrations (oral route) of the extract or any of it's components.

Anthocyanins, resveratrol, and pterostilbene are not only found in blueberry extract, as there are other natural sources. In addition to blueberry extract (Vaccinium Myrtillys and Vaccinium Corymbosum), anthocyanins can be found in high amounts in blueberry extract (Vaccinium Macrocarpon and Vaccinium Oxycoccos) and raspberry (Rubus Ideaeus) extract. Resveratrol is present not only in blueberry extract (Vaccinium Myrtillys and Vaccinium Corymbosum), but also in other extracts such as grape (Vitris Viniforme) and raspberry (Rubus Ideaeus) extract. Likewise, Pterostilbene can be found in large amounts in blueberry extract (Vaccinium Myrtillys and Vaccinium Corymbosum), almond (Prunus Dulcís) and raspberry (Rubus Ideaeus) extract.

Other compounds have also been described for their potential use in the preservation of ocular health, as well as in various ocular pathologies. Such is the case of vitamins A, C, D and E, carotenoids such as zeaxanthin and lutein and some minerals such as zinc and magnesium.

Vitamin C, also known as ascorbic acid by its chemical formulation, is a compound commonly found in fresh fruits, as well as vegetables. This acid compound is subjected to oxidation becoming dehydroascorbic acid, which is why it participates as a cofactor of multiple hydroxylation reactions, thus involved in the biosynthesis of collagen, which is the main structural protein of connective tissue. That is the main reason, not the only one, why it is associated with the prevention of capillary fragility, its requirements are 50 mg per day. Vitamin E is a fat-soluble vitamin that has antioxidant effects and works uniquely by breaking the lipid peroxidation chain reaction. This action is crucial to stabilize cell membranes and has been shown to prevent changes to the ocular surface and retina. Vitamin E can regenerate other antioxidants such as ascorbic acid or glutathione. A high concentration of vitamin E similar to that found in the AREDS (Age-Related Eye Disease Studies) formula may decrease the risk of progression of age-related macular degeneration. Vitamin E exists naturally as eight different fat-soluble compounds of tocopherols and tocotrienols. Sunflower seeds and walnuts, as well as almonds, spinach and those dark leafy vegetables are rich sources of vitamin E. Vitamin D has shown important effects as an antioxidant in retinal vasculopathies such as diabetic retinopathy.

In the case of micronutrients such as magnesium and zinc, their use has been extended for pathologies such as age-related macular degeneration. Magnesium is an essential cation as a cofactor in phosphate-using enzymatic metabolic pathways. Hypomagnesemia, related to increased losses or decreased income, is closely related to the metabolic syndrome observed during diabetes or its complications; in fact, it is considered to be the most common electrolyte disturbance in diabetic subjects. Since magnesium is predominantly an intracellular ion, the plasma concentration may not reflect the status of magnesium content. of the organism. Magnesium deficiencies can cause alterations in the activity of the enzyme tyrosine kinase and in the number of insulin receptors and an increase in the concentration of intracellular calcium, all of which is related to resistance to the action of insulin.

Other studies have associated low levels of magnesium with elevated levels of tumor necrosis factor and C-reactive protein, which could explain low-grade inflammatory processes. Zinc has the potential to have positive effects on retinal cells, so its absence is related to dark adaptation defects.

No less important is to mention the effect of carotenoids such as lutein and zeaxanthin, which are yellow pigments found in vegetables and other plants. Both have the potential to absorb excess light to prevent damage to the neurosensory retina, especially short-wavelength light rays such as violet and blue. Its use is widely used and recognized in ophthalmology as preventive and/or conservative of retinal health in patients with age-related macular degeneration. Regardless of their origin, these compounds of vegetable and mineral origin could be administered for use in ophthalmology, seeking adequate concentrations for the preservation of the health of the various structures of the eye and/or in some pathologies.

Topical instillation is the non-invasive route preferred by ophthalmology specialists worldwide for the administration of drugs and molecules for the treatment of diseases of the anterior segment of the eyeball. This route is non-invasive, with few adverse effects, lower costs and better patient adherence, since it does not require complex facilities or highly specialized human capital for its administration.

However, the bioavailability of the drugs administered by this route in the case of diseases that affect the posterior segment of the ocular globe is almost nil. In the case of drugs for the treatment of diseases that affect the retina, different routes of administration, such as intravitreal (infraocular) injections, periocular injections, and systemic administration have been reported; however, the presence of static barriers (cornea, sclera, Bruch's membrane and retinal pigment epithelium) prevent the passage of drugs and molecules into the vitreous cavity and into the retina. Likewise, the dynamic barriers of the eye (blinking, tear film, lacrimal duct drainage system, conjunctival, episcleral and choroid dispersion, uveoscleral drainage, hydrostatic and osmotic pressure, as well as the different flow pumps and transporter proteins of the retinal pigment epithelium) facilitate the elimination of the poor concentrations that manage to reach the interior of the eyeball. Finally, metabolic barriers (cytochrome P450 and lysosomal enzymes) end up metabolizing the remainder of these concentrations.

Due to all of the above, the use of intravitreal injection is the delivery method most used by specialists in ophthalmology and retina to be able to deliver adequate and clinically effective concentrations for the treatment of diseases that affect the posterior segment of the eyeball. However, its use is related to serious ocular adverse events such as endophthalmitis, retinal detachment, cataracts, and vitreous hemorrhage, among others. Likewise, this treatment modality is expensive, painful, with little access to the general population (requiring hospital facilities and highly trained human capital for its administration), and generates little attachment and great discomfort both to the user (patients) and to the medical team. In the case of systemic administration (oral route), which is the most commonly used for the use of herbal extracts (including blueberry extract), high doses are required, which are often insufficient or even toxic with adverse events. serious reported.

On the other hand, blindness is currently one of the most relevant disabilities in the world, since the human being is primarily a visual entity, so its development, in addition to being an individual catastrophe, represents a family, social and economic burden. relevant. Conditions such as diabetic retinopathy have a high cost in their treatment. In the United States, the annual expense per eye for the treatment of diabetic macular edema ranges from USD 14,000 to USD 25,000 and in Mexico from USD 66,00 to USD 114,000. Age-Related Macular Degeneration currently represents a US$16 billion market. Additionally, the negative experience, and the discomfort generated by the repeated application of drug injections, added to the essential use of facilities and specialized human capital, result in poor adherence and access to treatment, as well as saturation in health services, being It is essential to seek treatment alternatives and above all prevention.

In summary, the eyeball presents significantly greater challenges than other organs for the delivery of medicines, molecules and herbal extracts, given the large number of anatomical and physiological barriers that it presents, so the greatest challenge for the release of drugs, molecules and extracts herbalists is to find a delivery method that is highly effective, with a high safety profile, increases patient adherence, and has mass access to it.

Previously, the research group author of this invention developed a lipid-based formulation, designed for the delivery of drugs, spheroids, proteins, and peptides to infraocular tissues, which is referenced in the following publication: [Altamirano-Vallejo JC. , Navarro-Partida., Santos A., et al. Characterization and Pharmacokinetics of Triamcinolone Acetonide-Loaded Liposomes Topical Formulations for Vitreoretinal Drug Delivery: Characterization and Pharmacokinetics of Triamcinolone Acetonide-Loaded Liposomes Topical Formulations for Vitreoretinal Drug Delivery. J of Ocul Pharmacol and Ther. 2018]. However, this development did not include a nanosystem for the delivery of micronutrients present in food useful for the preservation of ocular health, such as the polyphenols present in herbal extracts, particularly in blueberry extract.

For this reason, having a topical delivery system for the polyphenols with bioactive properties contained in the blueberry extract would be the ideal system.

Justification for the development of the invention.

It is estimated that around 1.3 billion people worldwide suffer from visual impairment problems. Currently, the main causes of non-reversible blindness are those that affect target tissues in the posterior segment of the eyeball. Conditions such as Macular Degeneration Related to Age (AMD) and Diabetic Retinopathy (DR) comprise more than 80% of the cases of non-reversible blindness in the group of patients of productive age and in the group of elderly patients in the western world. Particularly, Diabetic Retinopathy is a problem in Latin American countries and in Mexico given that it disables economically active individuals, resulting in an impact on the economy of health systems and countries. This makes the field of ophthalmic therapeutics particularly interesting for scientific-technological researchers and entrepreneurs.

Unfortunately, delivering therapies to the eye is extremely challenging due to the anatomical and physiological characteristics that the eyeball possesses. Anatomical barriers include structures such as the corneal epithelium, conjunctiva, sclera, and the two blood-retinal barriers; and physiological barriers include processes such as the tear film, its drainage systems, and blinking, as well as the aqueous humor production, flow, and absorption system, and the composition of the vitreous gel. Said barriers limit the systemic penetration that one or several bioactives present in a herbal extract could have.

To administer bioactives in the anterior segment of the eye, the ideal route of administration is topical, since it is easy for the patient to use, with few adverse effects and with adequate control of the frequency used. However, the bioavailability of bioactives from herbal extracts, after topical ophthalmic administration of conventional formulations, is inefficient for target tissues of the posterior segment of the eyeball, since penetration is usually less than 5%, which constitutes a very limited limitation. important and with obtaining poor bioavailability of these. For the above, those conditions that affect the health of the target tissues in the posterior segment of the eyeball, such as the retina, are not candidates for this route of administration.

Since in nature there are herbal extracts that contain bioactives with an anti-inflammatory, anti-angiogenic and antioxidant effect on some target tissues, their use becomes an option in conditions that affect the health of the retina. Extracts such as blueberry that have an additive effect of bioactives AC, RV, and PS can be administered topically, with adequate penetration efficiency and bioavailability by using a lipid-based formulation.

At present there are no commercially available products of natural origin such as herbal extracts that serve as auxiliaries in the prevention and/or modulation of the progression of these conditions responsible for the main causes of non-reversible blindness.

previous art.

Based on the analysis of prior art documents, some inventions involving blueberry were found, such as the case of invention CN104147081 A, dated August 20, 2014, which describes a soft capsule of blueberry lutein to improve the view and a method of making it. They mention that cranberry lutein is a nutrient that is suitable for improving eyesight, but excess cranberry lutein can cause eye problems. The cranberry lutein softgel consists of a capsule shell and a content, in which the content is stores in capsule shell; the content is composed of the following components in parts by weight: 43 to 47 parts of cranberry extract, 20 to 23 parts of zinc gluconate, 14 to 16 parts of vitamin C, 1 1 to 14 parts of powdered lutein, 3 to 4 parts vitamin E, 0.2 to 1 part vitamin A, 143 to 146 parts soybean oil, and 6 to 9 parts beeswax; and the capsule shell is composed of the following components in parts by weight: 190 to 210 parts of gelatin, 70 to 90 parts of glycerin, 190 to 210 parts of purified water, and 9 to 10 parts of caramel color. The capsule is used to improve eyesight. This invention is different in content and application from the developed formulation.

The invention CN104606565A, dated February 15, 2015, describes a traditional Chinese medicine and in particular refers to capsules for preventing and treating ophthalmic diseases. The capsule contents comprise 40-45 parts of a traditional Chinese medicine extract, 5-7 parts of semen boitae, 8-12 parts of orris, 4-5 parts of allium victorialis, 20 parts of peony seed oil and 5-10 parts of blueberries. They mention that the anthocyanin contained in blueberries is an important element to maintain eye health and prevent visual impairment; and peony seed oil has the effect of improving autoimmunity, lowering blood fat and blood pressure, preventing diabetes, preventing and treating cancers, cleaning up dangerous substances in the blood, preventing and treating heart disease, refreshing and improving intelligence, improve attention span and memory and beautification. The medications are compatible and have the effect of treating both the symptoms and the root causes of eyestrain. TCM capsules have rapid pharmacological effects and good curative effects and have no side effects or toxic effects, and the effective rate is 100 percent. Based on the foregoing, this invention is different from the developed formulation both in content and application.

The invention WO2015177353A1, dated May 22, 2014, describes a composition that contains, as active principle, ectoin, hydroxyectoin and/or salts, esters or amides of these compounds, for the treatment and/or prophylaxis of eye diseases associated with an alteration of the tear film. In particular, this can be keratoconjunctivitis sicca. Unexpectedly, it has been shown that these compounds can prevent the breakdown of the outer lipid layer of the tear film and thus prevent undesired rapid evaporation of tear fluid. Based on the foregoing, this invention is different from the developed formulation both in content and application.

The invention CN 103860625A, dated February 20, 2013, describes a cranberry extract ophthalmic preparation and a preparation method and uses thereof and belongs to the technical field of ocular ophthalmic preparations. The technical problem to be solved is to provide a blueberry extract ophthalmic preparation. The cranberry extract ophthalmic preparation comprises a cranberry extract active ingredient. The eye ophthalmic preparation can effectively treat, prevent or alleviate cataracts, glaucoma, vitreous opacity, macular degeneration, retinopathy, optic atrophy, keratitis, wind-induced epiphora, presbyopia, myopia, eyestrain, blurred vision, conjunctival burn, conjunctivitis, corneal ulcer , eyelid eczema, floaters or trachoma and other eye diseases. A new preparation is provided for the treatment of cataracts, glaucoma, vitreous opacity, macular degeneration, retinopathy, optic atrophy, keratitis, wind-induced epiphora, presbyopia, myopia, visual fatigue, vision blur, conjunctival burn, conjunctivitis, corneal ulcer, eyelid eczema, floaters or trachoma and other eye diseases, and meet the strong demand to protect eyes. Based on the foregoing, this invention is different from the formulation developed both in content and application, this based on the capacity of the formulation object of this invention.

The invention LT2017521 A, dated July 28, 2017, describes eye drops, which contain extracts of carrot and blueberry, with application for the conservation of visual acuity, and the production method thereof. The ophthalmic pharmaceutical formulation contains extracts of carrot and blueberry used with any basic mono or multicomponent solution of neutral acidity, where the extracts of carrot and blueberry in the solution do not exceed 10% by weight of the solution, the proportion of extracts of carrot and cranberry in the solution is from 3: 7 to 7: 3, and the pH of the resulting preparation is in the range from 6.9 to 7.6. Based on the foregoing, this invention is different from the formulation developed both in content and application.

The invention MX2017016991 A, dated December 20, 2017, describes a nutraceutical formulation of cranberry extract, fish oil (omega-3 and omega-6), recombinant human lactoferrin, vitamin A and vitamin E, for human oral consumption. The invention belongs to the field of ophthalmology and has been developed as an adjuvant to preserve the health of the precorneal film and the ocular surface. This formulation contains an extract of natural origin (Vaccinium myrtillus L) with antioxidant and anti-inflammatory properties; also uses eicosapentaenoic acid (EPA), omega-6, and docosahexaenoic acid (DHA), omega-3, obtained from fish oil and which, together with lactoferrin, vitamin A and vitamin E, it improves tear quality, since these compounds have antimicrobial and anti-inflammatory properties. The formulation has been designed as an adjuvant to preserve the health of the precorneal film. There is no formulation in the state of the art for the oral administration of a blueberry extract together with lactoferrin, in combination with vitamin A, vitamin E and fish oil (eicosapentaenoic acid and docosahexaenoic acid), nor the use of this formulation. as an adjuvant to preserve the health of the precorneal film and/or the surface of the eye. Based on the foregoing, this invention is different from the formulation developed both in content and application, because it does not have a liposomal application.

Regarding the analysis of the state of the art, it was found that there are no inventions related to the object of the invention developed in this document, even when cranberry is used with ophthalmological applications, until now there are no lipid-based applications for topical ophthalmic use that function as a liposomal delivery system of the bioactive agents contained in the blueberry extract in white tissues of the posterior segment of the eyeball in humans.

The technical details of the developed invention are described below.

OBJECT OF THE INVENTION.

The object of this invention is to make available a lipid-based formulation for topical ophthalmic use that functions as a liposomal delivery system for the bioactive agents contained in the blueberry extract (EA) in tissues. white of the posterior segment of the eyeball in humans. The formulation comprises a combination of polyphenol-rich blueberry extract as active ingredient, polyethylene glycol glyceryl dimyristate (PEG-12) as structural component of liposomes, ethyl alcohol as organic solvent for liposome generation, Kolliphor® HS 15 as penetration enhancer, anhydrous citric acid and dehydrated sodium citrate as buffers and grade 2 purified water as inorganic solvent.

Therefore, the formulations of the present invention are useful to maintain health and reduce the risk of developing chronic-degenerative processes in the infraocular tissues, including those of the posterior segment of the eyeball due to their anti-inflammatory, anti-angiogenic, anti-inflammatory, and anti-inflammatory activity. fibrotic and antioxidant.

BRIEF DESCRIPTION OF THE INVENTION.

In general, the invention comprises a topical ophthalmic formulation of lipid-based blueberry extract where the value lies in allowing more efficient penetration and bioavailability of its b¡oactives in white tissues of the posterior segment of the eyeball in humans.

It is important to mention that the use of natural extracts for beneficial purposes on ocular health becomes relevant when it is considered that all the bioactives contained in it will have a synergistic therapeutic effect, derived from the sum of the different mechanisms of action.

Due to the high predisposition of several of these bioactives present in the blueberry extract, they have a high predisposition for degradation, with a rather low solubility in water, so their bioavailability is consequently quite poor, therefore, this lipid-based formulation represents an advantage for the topical use of these bioactives; so that the invention consists of a lipid-based formulation for topical ophthalmic use that functions as a liposomal delivery system for the bioactive agents contained in the blueberry extract in white tissues of the posterior segment of the eyeball in humans.

The invention is useful for maintaining health and reducing the risk of developing chronic-degenerative processes in the infraocular tissues, including those of the posterior segment of the eyeball due to its anti-inflammatory, anti-angiogenic, anti-fibrotic and antioxidant activity.

Due to the above, and being an efficient option for use as a delivery system for bioactive polyphenols such as anthocyanins, resveratrol and pteroestil not contained in blueberry extract, a lipid-based ophthalmic topical formulation has the potential to be a release method that makes the penetration of the barriers of the eyeball more efficient, thus achieving an increase in the bioavailability of bioactives in the white tissues of the posterior segment of the eyeball, such as the retina, minimizing the risks associated with other forms of administration local and/or systemic.

DETAILED DESCRIPTION OF THE INVENTION

The characteristic details of the lipid-based formulation for ophthalmic topical use, which contains blueberry extract as the main component to make more efficient the penetration and bioavailability of its bioactives in infraocular white tissues in humans, they are clearly shown in the following description.

• Components.

The compositions of the present invention contain an effective amount of blueberry extract. The concentration of the blueberry extract in the lipid-based formulations varies from 0.3 to 2 mg, that is to say, from 0.03 to 2% (p/v). Blueberry extract has a large amount of polyphenol bioactive agents such as anthocyanins, resveratrol and bene pteroestil that have the potential to preserve health and reduce the risk of developing chronic-degenerative processes in intraocular tissues, including those of the posterior segment of the eyeball due to its anti-inflammatory, anti-angiogenic, anti-fibrotic and antioxidant activity.

Polyethylene glycol (PEG-12) glyceryl myristate is used as a structural component of liposomes at a concentration of 5-15% (w/v) and ethyl alcohol is used as an organic solvent for liposome generation at a concentration of 0.7 to 2.1% (v/v).

In addition, the formulation contains polyethylene glycol (15) -hydroxystearate or Kolliphor® HS15 from 2.5 to 7.5% (w/v), as a potent non-ionic solubilizer and emulsifying agent, with low toxicity proposed to act as a permeability enhancer. Kolliphor® HS15 also promotes the transport of molecules across cell membranes by increasing the rate of endocytosis and stimulates the translocation of drugs through the pathway to cellular (affecting actin organization in the cell cytoskeleton with subsequent opening of tight junctions between cells).

Likewise, the aqueous compositions of the present invention optionally comprise further excipients selected from the group consisting of buffering agents, pH adjusting agents and preservatives. Anhydrous citric acid (0.04 to 0.16%) and Sodium Citrate Dihydrate (0.23 to 0.69%) are used as buffers.

The lipid-based compositions of the present invention can be prepared by conventional methods of preparing pharmaceutical suspension compositions.

• Preparation method.

According to the preferred method, blueberry extract (0.3 to 2 mg/mL) is first added to a lipid mixture containing PEG-12 glyceryl dimistrate (5 to 15%), polyethylene glycol (15)-hydroxystearate (Kolliphor® HS15) (2.5 to 7.5%) and ethyl alcohol (0.7 to 2.1%) at 25 °C ±1 °C, combining the components by shaking for 10 minutes ±1 minute.

On the other hand, an aqueous base mixture composed of grade 2 purified water (Q.S), anhydrous citric acid (0.04 to 0.16%) and sodium citrate dihydrate (0.23 to 0.69%) at 25 °C ±1 °C is prepared. , combining the components by shaking for 10 minutes ± 1 minute.

The aqueous mixture is added to the lipid mixture at 25 °C ±1 °C and shaken. for 5 minutes ±1 minute to thus obtain the final formulation.

• Resulting formulation. The resulting lipid-based formulation object of this invention is made up as follows:

The formulation makes the penetration and bioavailability of bioactives present in the blueberry extract more efficient in the white tissues of the eyeball due to the use of advanced delivery methods and systems, such as lipid-based systems.

Some of these lipid systems include lipid nanoparticles, micelles, and liposomes. Lipid nanoparticles are aqueous colloidal dispersions composed of solid lipids as matrix material with the ability to encapsulate both hydrophilic and lipophilic agents, which ultimately result in thermodynamically stable compounds, with protection for the encapsulated agent, controlled release and biocompatible with the barriers of the globe ocular, easy to produce and with the possibility of being sterilized in an autoclave.

However, some of its limitations are lower encapsulation and release capacity for both hydrophilic and lipophilic agents. Polymeric micelles (10-1 OOnm) comprise amphiphilic block copolymers above the critical micelle concentration with a hydrophobic core and a hydrophilic shell. The micelles prevent or minimize the degradation of the molecules, increase their penetration into the infraocular tissues and also increase their solubility.

Additionally, the resulting formulation can include the following micronutrients:

• Vitamin E which is a fat-soluble vitamin that has antioxidant effects and works uniquely by breaking the lipid peroxidation chain reaction. This action is crucial to stabilize cell membranes and has been shown to prevent changes to the ocular surface and retina. This vitamin can regenerate other antioxidants such as ascorbic acid or glutathione.

• Vitamin D since it has shown important effects as an antioxidant in retinal vasculopathies such as diabetic retinopathy.

• Magnesium and Zinc, since its use has spread for pathologies such as age-related macular degeneration.

• Carotenoids such as Lutein and Zeaxanthin, which are yellow pigments found in vegetables and other plants. Both have the potential to absorb excess light to prevent damage to the neurosensory retina, especially short-wavelength light rays such as violet and blue. Its use is widely used and recognized in ophthalmology. as preventives and/or preservers of retinal health in patients with age-related macular degeneration.

Finally, liposomes are biocompatible vesicles composed of a phospholipid bilayer with a structural similarity to the cell membrane, which form small spheroids capable of transporting both hydrophilic and lipophilic molecules. They are biocompatible, thermostable and can present a high percentage of encapsulation efficiency of the different compounds.

A lipid-based formulation has the potential to improve stability, make penetration of the natural barriers of the eyeball more efficient, and increase the bioavailability of polyphenols in infraocular tissues.

• Example.

The following example is intended to illustrate, but not limit, the present invention. Ingredient concentrations are reported in units of % weight/volume (% w/v) or % volume/volume (% v/v).

The final concentration of blueberry extract in the resulting dispersion in the example is 0.3 mg/ml (0.03%). The composition of the formulation of liposomes loaded with blueberry extract (EA-LF), hereinafter EA-LF is described below.

• Analysis of the resulting formulation.

The pH of EA-LF was analyzed by potentiometer in triplicate at room temperature. Osmolarity was measured by a vapor pressure osmometer in triplicate at 33°C (the ocular surface temperature).

The viscosity was also measured in triplicate at 33°C. Viscosity was measured using a thermostatically controlled rheometer when steady state was reached with shear rates increasing from 0 to 1000 s-1.

The particle size of the liposomes contained in EA-LF was analyzed by dynamic light scattering and the zeta potential was calculated by measuring the velocity of the particles using a Doppler laser velocimeter at 25°C (Zetasizer Nano ZS, Malvern Instruments). , Malvern, UK).

The diameter Z (mean particle diameter) and the polydispersity index (PDI) were calculated from the particle size distribution. The pH of EA-LF was 6.5. The physiological pH range of the tear is between 6.5 and 7.6.

On the other hand, the viscosity value of TA-LF was 70 cP (elevated), which could favor their permanence on the ocular surface. The osmolarity was

354 mOsmol/l being in the isotonic margins.

The average particle diameter (z-average) was 167.8 nm, while its PDI was 1 16 and its zeta potential was 0.554, therefore, there is a risk of crystallization of the TA encapsulated in the micelles. The physicochemical characteristics of EA-LF are summarized below.

Physicochemical characteristics of EA-LF.

Formulation Physicochemical parameters pH Viscosity Osmolarity Z PDI

(cP) (mOsm/L) (nm) (nm)

EA-LF 6.5 70 354 167.8 1 16 pH= Potential of hydrogen ions, cP= centipoise, PDI= polydispersity index

To assess the stability of EA-LF, a heat stress test was performed. EA-LF samples were kept at 30°C, 40°C and 60°C for three weeks. The pH and the encapsulation efficiency were evaluated on days 0 (baseline) 7, 14 and 21 of thermal incubation.

To assess the encapsulation efficiency, we measured the PS concentration in the filtered solution obtained after EA-LF was extruded through a 0.22 µm size polycarbonate membrane. The concentration of PS in EA-LF samples was subtracted from the concentration of control samples (liposomal solution without EA or cLF). The determination of the PS concentration was performed by high performance liquid chromatography (HPLC) using the Varian 920 LC equipment (Aligent Technologies, Santa Clara, CA, USA) with a Zorbax Eclipse Plus C18 column, 4.6 x 100 mm and 3.5 pm column (Agilent, Santa Clara, CA, USA) at a temperature of 30°C.

Samples (20 μl) were eluted from the column in a mobile phase composed of water:methanol (30:70) at a flow rate of 1 ml/min. Detection was performed at 320 nm. The retention time and the detection limit were 6.8 min and 0.004 mg/ml, respectively. The TA standard curve was linear from 0.004 to 0.100 mg/mL (correlation > 0.99). After 21 days of heat stress, EA-LF maintained its stable pH at 30 and 40°C and even improved encapsulation at higher temperatures (60°C). The results related to the stability tests are shown below.

Results of the stability study

EA-LF EA-LF EA-LF

Parameter Time 30°C 40°C 60°C

(days)

0 6.55 6.5 6.55 pH 7 6.55 6.55 6.45

14 6.45 6.35 6.45

21 6.5 6.55 6.5**

Encapsulation 0 0.1 0.1 0.1 of EA (%) 7 0.21 * 0.47** 0.45**

14 0 0 0.02 21 0 0 0.05

The asterisks indicate values statistically different from the values of day 0. *= P< 0.05, **= P< 0.01 Additionally, diffusion chambers and New Zealand white rabbit corneas were used to perform in vitro diffusion experiments (BW200S Chemotaxis Chambers, NeuroProbe, Gaithersburg, MD, USA). The central section of the cornea (6 mm in diameter) was located between the upper and lower compartments of the diffusion chambers with the purpose of acting as a diffusion barrier for EA-LF. The upper compartment was filled with 180 µl of balanced salt solution (BSS) while the lower compartment was filled with 200 µl of EA-LF. To prevent evaporation, the diffusion chambers were placed indoors in a humid chamber at 37 °C. To carry out the analysis of the diffusion of EA-LF, the concentration of pteroestil beno (PS), contained in the EA, was determined by means of high performance liquid chromatography (HPLC) in the upper compartment at 2, 4, 6 and 8 o'clock. hours after starting the diffusion assay, Regarding the in vitro diffusion analysis, it was observed that EA-LF reached the highest PS concentrations after 8 hours of follow-up. The results of the in vitro diffusion study are presented below.

difusión Pteroestil beno (PS) concentration in the upper compartment of the chamber

diffusion

Time (hours) PS (pg/mL)

2 0.28

3 0.37

4 0.55

5 0.75

6 0.82

7 0.98

8 1.32 Values represent the average of three measurements.

The above description of the disclosed definitions is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these definitions and/or implementations will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but should be accorded the broadest scope consistent with the following claims and the principles and novel features disclosed herein.

Claims

28 CLAIMS Having sufficiently described my invention, I consider as a novelty and therefore claim as my exclusive property, what is contained in the following clauses: 1. A lipid-based formulation for topical ophthalmic use, containing blueberry extract as the main component to make the penetration and bioavailability of its bioactives in infraocular white tissues in humans more efficient, characterized by comprising: a. Blueberry Extract from 0.3 to 2 mg, that is, from 0.03 to 2% (w/v), where this component has a large amount of polyphenol bioactive agents such as anthocyanins, resveratrol and beno pteroestil that have the potential to preserve health. and reduce the risk of developing chronic-degenerative processes in the infraocular tissues, b. Polyethylene glycol (15) -hydroxystearate from 2.5 to 7.5% (w/v) where this component is used as a potent non-ionic solubilizer and emulsifying agent, with low toxicity proposed to act as a permeability enhancer, c. PEG-12 glyceryl dimistrate from 5-15% (w/v) where this component is used as a structural component of liposomes, d. Ethyl alcohol from 0.7 to 2.1% (v/v) where this component is used as an organic solvent for the generation of liposomes, e. Anhydrous citric acid from 0.04 to 0.16% where this component is used as a buffer, f. Sodium citrate dihydrate from 0.23 to 0.69% where this component is used as a buffer, g. Purified water grade 2 with QS 1.0 ml. A method of preparing the formulation according to claim 1 comprising the following steps: a. According to the preferred method, cranberry extract (0.3 to 2 mg/mL) is first added to a lipid mixture containing PEG-12 glyceryl dimistrate (5 to 15%), polyethylene licol (15)-hydroxystearate (2.5 to 7.5%) and ethyl alcohol (0.7 to 2.1%) at 25 °C ±1 °C, combining the components by shaking for 10 minutes ±1 minute, b. On the other hand, an aqueous base mixture composed of grade 2 purified water (QS), anhydrous citric acid (0.04 to 0.16%) and sodium citrate dihydrate (0.23 to 0.69%) at 25 °C ±1 °C is prepared. , combining the components by shaking for 10 minutes ± 1 minute, c. The aqueous mixture is added to the lipid mixture at 25 °C ±1 °C, and stirred for 5 minutes ±1 minute to thus obtain the final formulation. The formulation according to claim 1, which is characterized by: a. Possessing the potential to improve stability, make the penetration of the natural barriers of the eyeball more efficient, and increase the bioavailability of polyphenols in infraocular tissues. The formulation according to claim 1, which can include micronutrients such as the following: a. Vitamins: Vitamin E which is a fat-soluble vitamin that has antioxidant effects and works uniquely by breaking the chain reaction of lipid peroxidation this action is crucial to stabilize cell membranes and has been shown to prevent changes in the ocular surface and in the retina. b. Vitamin D since it has shown important effects as an antioxidant in retinal vasculopathies such as diabetic retinopathy, c. Minerals: Magnesium and Zinc since its use has been extended for pathologies such as age-related macular degeneration, d. Carotenoids: Lutein and Zeaxanthin which are yellow pigments found in vegetables and other plants. Both have the potential to absorb excess light to prevent damage to the neurosensory retina, especially short-wavelength light rays such as violet and blue. Its use is widely used and recognized in ophthalmology as preventive and/or conservative of retinal health in patients with age-related macular degeneration.