Lipid-based ophthalmic formulation for topical use that increases aflibercept penetration and bioavailability in the retina

Abstract

The invention relates to an ophthalmic liposomal formulation for topical use that improves paracellular penetration of therapeutic agents such as aflibercept in the eyeball. The formulation includes an optimised mixture of liposomes, non-ionic solubilisers and excipients to facilitate the transport of the drug through the cornea and the controlled release thereof in the posterior segment of the eye. The formulation uses liposomes as a transport system, maximising the ocular bioavailability of aflibercept by means of a synergic combination of emulsifiers and stabilisers, allowing efficient penetration through the corneal barrier and sustained release of the drug in the eye tissue.

Description

[0001] OPHTHALMIC, TOPICAL, LIPID-BASED FORMULATION THAT INCREASES THE PENETRATION AND BIOAVAILABILITY OF AFLIBERCEPT IN THE RETINA.

[0002] FIELD OF INVENTION.

[0003] The invention relates to the field of ophthalmology, focusing on ocular pharmacology. Specifically, it is a lipid-based formulation for topical ophthalmic use that enhances the paracellular penetration of Aflibercept into the eyeball, where Aflibercept blocks vascular endothelial growth factor (VEGF).

[0004] BACKGROUND OF THE INVENTION.

[0005] Posterior segment eye diseases (PSODs) are pathologies with a high functional, economic, and social impact on the global population. Collectively, age-related macular degeneration (AMD), diabetic retinopathy (DR) and its complications, particularly diabetic macular edema (DME), and retinal vascular occlusions (RVOs) are the leading causes of central vision impairment in adults. These pathologies severely affect patients' quality of life and represent a considerable economic, social, and moral burden for their families and healthcare systems worldwide.

[0006] Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people over 65 in developed countries. Its prevalence varies with age, affecting approximately 1% of people aged 65 to 74, 5% of those aged 75 to 84, and 13% of those over 85. Factors such as population aging and the rise in chronic diseases like diabetes and hypertension have contributed to a higher prevalence of AMD, intensifying the burden of this disease.

[0007] Diabetic retinopathy (DR) is a common complication of diabetes mellitus and the leading cause of vision loss in working-age individuals. Globally, it affects more than 30% of diabetics, and between 5% and 10% of them will develop diabetic macular edema (DME). In Mexico, the prevalence of DR is 31.5%, with up to 10% of patients developing DME. According to the National Health and Nutrition Survey (ENSANUT), 54.5% of diabetics report decreased vision, 11.2% show retinal damage, and 9.95% have lost their vision completely due to DR and DME. These conditions represent a considerable burden on healthcare systems, requiring continuous and specialized management to prevent blindness.

[0008] Retinal vascular occlusions (RVOs) include occlusions of the central retinal vein, branch retinal vein, and central retinal artery. They are a significant cause of vision loss in the general population. Central retinal vein occlusion affects approximately 2.5 people per 1,000 inhabitants. Without timely treatment, these conditions can lead to severe and irreversible vision loss.

[0009] Retinal diseases, such as AMD, DR, DME, and VRO, generate a considerable economic, social, and moral burden. In the United States, the annual cost exceeds $139 billion, primarily due to anti-VEGF treatments and surgeries. In Latin America, the per capita cost is significant and higher in developing countries. In Mexico, it is projected that by 2030, the cumulative cost will exceed 100 billion Mexican pesos.

[0010] Socially, vision loss reduces patients' independence and productivity, increasing their dependence on family members and caregivers, and is associated with a higher risk of depression and anxiety. This situation underscores the need for more accessible and effective treatments to reduce both the financial burden and the decline in quality of life.

[0011] Aflibercept as primary treatment for OGTT: Aflibercept has become established as the first-line treatment and a gold standard in the management of OGTT. Its ability to inhibit multiple isoforms of vascular endothelial growth factor (VEGF) has proven highly effective in reducing edema and preserving vision, making it an essential therapeutic tool in ophthalmic practice.

[0012] Aflibercept is a recombinant fusion protein with a molecular weight of approximately 115 kDa. This design allows it to function as a “decoy receptor” that binds to key factors in the pathogenesis of retinal diseases, specifically VEGF and placental growth factor (PGF). Its structure combines selected domains from the VEGFR-1 and VEGFR-2 receptors, fused to the Fe region of human immunoglobulin G1 (IgG1), a configuration that amplifies its affinity and inhibitory capacity.

[0013] Aflibercept binds not only to VEGF-A, but also to VEGF-B and PIGF, differentiating it from other anti-VEGF agents that target only one VEGF isoform. However, its hydrophilic nature poses a challenge to penetrating the ophthalmic barriers of the eye, which has driven research into new delivery routes to maximize its bioavailability in ocular tissue.

[0014] Aflibercept exerts its effect by blocking the angiogenesis and vascular permeability processes induced by VEGF in the retina. It binds with high affinity to the isoforms VEGF-A, VEGF-B, and PIGF, neutralizing these growth factors and preventing their interaction with the endothelial receptors VEGFR-1 and VEGFR-2. This inhibition is fundamental in the treatment of pathologies such as AMD, DR, DME, and VRE, in which the presence of neovascularization and increased capillary permeability impair retinal function.

[0015] Aflibercept was approved by the U.S. Food and Drug Administration (FDA) after multiple studies confirmed its effectiveness in various retinal pathologies. In the treatment of neovascular AMD, the VI EW 1 and VI EW 2 clinical trials demonstrated that a 2 mg dose every two months was equivalent in efficacy to monthly administration of other anti-VEGF agents, stabilizing and even improving visual acuity in a large majority of patients. Regarding DME, the DRCR.net Protocol T study showed that Aflibercept provided a significant improvement in visual acuity compared to other anti-VEGF agents, especially in patients with initially compromised vision. For RVO, Aflibercept has demonstrated significant benefits in reducing macular edema and improving vision in cases of central retinal vein occlusion (CRVO) and branch retinal vein occlusion.

[0016] Intravitreal administration of aflibercept is effective, but it carries risks due to the frequency of injections required to maintain therapeutic levels. These risks include infections (endophthalmitis), retinal detachment, vitreous hemorrhage, traumatic cataracts, severe pain, and intraocular inflammatory reactions. Furthermore, intravitreal injections are expensive, require specialized medical facilities and highly trained personnel, and cause discomfort for patients. The procedure must be performed under aseptic conditions supervised by specialists, which limits access and increases healthcare costs. Psychologically, the need for repeated injections causes anxiety in patients, affecting their adherence to treatment and compromising long-term visual outcomes.

[0017] Topical Administration in Ophthalmology: Advantages and Challenges. Topical drug administration is the ideal route of choice for many ophthalmic therapies due to its simplicity, safety, and ease of application. In the context of retinal and macular diseases, topical administration offers a less invasive approach compared to intravitreal injections, which require procedures in controlled environments and present risks of significant complications, such as endophthalmitis. The advantages of ophthalmic topical administration are:

[0018] 1. Patient Safety and Comfort: Topical administration eliminates the need for invasive procedures, thus reducing the risk of infections or complications associated with injections. For patients, the ability to apply the treatment at home improves adherence and reduces anxiety related to eye treatments.

[0019] 2. Cost Reduction: Topical treatments can be administered without the need for specialized equipment or medical personnel, thus reducing treatment costs. This feature is particularly important in resource-limited countries or in public health systems with high demand.

[0020] 3. Wider Access and Improved Adherence: The ease of use of eye drops facilitates access to treatment for a greater number of patients. This is crucial in chronic diseases, as a convenient dosage can improve adherence and, consequently, the long-term effectiveness of the treatment.

[0021] However, despite these advantages, topical administration faces significant obstacles due to the complex barriers that protect the eyeball and limit the absorption of pharmacological agents into the retina and vitreous humor.

[0022] The eye presents multiple barriers that hinder the penetration of therapeutic molecules from the ocular surface to the inner layers, such as the retina and vitreous humor. These barriers can be classified as anatomical, physiological, and mechanical.

[0023] 1. Anatomical Barriers: o Cornea: The cornea is a multilayered barrier that protects the eye from external agents. Its highly lipophilic outer epithelium hinders the passage of hydrophilic molecules, while the stromal layer limits the passage of lipophilic molecules. This combination makes drug delivery across the cornea particularly challenging for molecules like Aflibercept, which is hydrophilic and large. o Conjunctiva and Sclera: Although the conjunctiva is more permeable than the cornea, it does not allow effective drug penetration into the deeper layers of the eye. The sclera, on the other hand, acts as a rigid barrier that restricts the penetration of large molecules, reducing the likelihood of them reaching the retina and vitreous humor.

[0024] 2. Physiological Barriers: o Tear Flow and Nasolacrimal Drainage: The constant flow of tears and nasolacrimal drainage rapidly eliminate topically applied drugs. Only a small fraction of the drug remains on the ocular surface before being eliminated, reducing the bioavailability of topical therapy. o Ocular Metabolism: Enzymes present on the ocular surface, such as sterolases and peptidases, can degrade certain drugs before they can cross the ocular barriers, thus decreasing the effectiveness of topical application.

[0025] 3. Mechanical Barriers: o Blinking and Closure Reflex: Constant blinking and the closure reflex protect the eye from foreign bodies, but they also remove eye drops, limiting the contact time of the drug with the ocular surface. o Evaporation: The ocular surface exposed to air undergoes continuous evaporation, which can reduce the effective concentration of the drug in the tear film.

[0026] Conventional topical formulations, such as standard eye drops, have limited penetration into the eyeball, especially when it comes to reaching the inner layers like the retina and vitreous humor. Due to the multiple anatomical, physiological, and mechanical barriers of the eye, less than 5% of the applied dose is absorbed, and of this small fraction, only a minimal amount reaches the retina or vitreous humor at therapeutic concentrations. This low efficacy means that conventional formulations are generally inadequate for treating deep retinal and posterior segment diseases, limiting their use to superficial or anterior segment therapies.

[0027] Strategies to Overcome Ocular Barriers: To improve drug penetration into the eye and optimize topical administration, several innovative strategies have been developed that facilitate the transport of molecules into the eyeball. These innovations represent significant advances in ophthalmology and are fundamental in the formulation of treatments for retinal diseases.

[0028] 1. Nanotechnology in Ophthalmic Formulations: o Liposomes and Lipid Nanoparticles: Liposomes, small vesicles that encapsulate the drug in a lipid bilayer, represent one of the most promising strategies in ophthalmology. Their structure allows the encapsulated molecules to cross the corneal epithelial barrier via the paracellular pathway. Furthermore, liposomes protect the drug from degradation on the ocular surface and allow for controlled release, increasing the duration of the therapeutic effect. o Polymeric Nanoparticles: Nanoparticles based on biocompatible polymers allow for sustained drug release, which is ideal for prolonged treatments. These particles can be designed to adhere to the ocular surface, optimizing drug contact time and absorption.

[0029] 2. Use of barrier permeabilizers: Permeabilizers are compounds that temporarily modify the tight junctions of epithelial cells, facilitating the passage of drugs. In the case of Aflibercept, the formulation with permeabilizers can be particularly useful for increasing its bioavailability in the cornea and aqueous humor.

[0030] 3. Mucoadhesive Release Systems: Mucoadhesive systems adhere to the ocular surface, allowing for gradual drug release and improving absorption. These systems use polymers such as hyaluronic acid, which, in addition to being biocompatible, helps prolong the drug's retention time on the ocular surface.

[0031] 4. Formulation Modification with Penetrating Agents: o The inclusion of excipients such as Kolliphor® 15, which acts as a surfactant and solubilizer, can improve drug penetration through the cornea and conjunctiva by reducing surface tension and facilitating the passage of molecules.

[0032] Application of Strategies in the Liposomal Formulation of Aflibercept. Given the challenge of administering Aflibercept topically to reach the retina and vitreous humor, a liposomal formulation presents itself as an advanced and effective solution. However, currently there is no Aflibercept formulation for topical ophthalmic application, as its absorption into the eyeball is almost negligible and it has no clinical effect in patients with retinal diseases.

[0033] Encapsulating aflibercept in liposomes protects the molecule from degradation on the ocular surface and promotes its absorption via the paracellular pathway. The inclusion of solubilizing and wetting agents, such as Kolliphor® 15 and hyaluronic acid, optimizes both drug retention on the ocular surface and its ability to penetrate external ocular barriers. Compared to intravitreal administration, this formulation represents a less invasive and more convenient alternative for the patient. It not only reduces the risks associated with injections but also improves treatment adherence thanks to its ease of application at home. Furthermore, the liposomal topical formulation could significantly reduce treatment costs and improve access to effective therapies for retinal diseases.

[0034] Innovation and Opportunities in Topical Aflibercept Administration: A topical formulation of aflibercept encapsulated in liposomes offers a safe, accessible, and efficient treatment, eliminating the risks and costs of intravitreal procedures. This technology improves drug penetration and bioavailability in the retina, protecting aflibercept from degradation and allowing for continuous and convenient administration. This advancement promises to revolutionize the treatment of retinal diseases, reducing costs and adverse effects, and optimizing patients' quality of life.

[0035] With respect to prior art, US Patent 11712419, priority date August 21, 2012, describes liposomal formulations designed to enhance the delivery of anti-angiogenic compounds, such as monoclonal antibodies or their fragments, that inhibit vascular endothelial growth factor (VEGF). These formulations utilize liposomes to encapsulate and protect the compounds, improving their stability and controlling their release in the body. Our invention differs in that it focuses on a lipid-based formulation for topical ophthalmic use containing Aflibercept as a bioactive agent, which increases the penetration of Aflibercept into the eyeball to provide adequate and effective concentrations in the vitreous and retina, overcoming ocular barriers without the need for intravitreal injections.

[0036] US patent application 2018 / 0221483, with a priority date of August 16, 2013, describes a system for the sustained, non-invasive delivery of ophthalmic substances into the eye, with the aim of preventing and treating eye diseases and conditions. It utilizes liposomes and inverted micelles as suitable vehicles for the controlled release of the substances. Delivery methods include coating contact lenses or injection into the eye, a process that differs from the method of delivery to the eye, as well as its application to the medical problem addressed by the invention.

[0037] US Patent 10369124 B2, with a priority date of April 30, 2014, describes dendrimer compositions and their use in the treatment of eye diseases. These compositions are designed to enhance drug penetration into the eye, overcoming natural barriers such as the blood-retinal barrier. Dendrimers are branched structures that can transport and release drugs in a controlled manner, improving treatment efficacy and reducing side effects. This invention differs from the invention described herein in terms of process and application.

[0038] US Patent 9962333 B2, priority date August 16, 2013, describes a system for the sustained, non-invasive delivery of ophthalmic substances into the eye for the purpose of preventing and treating eye diseases and conditions. It utilizes liposomes and inverted micelles as suitable vehicles for the controlled release of the substances. Delivery methods include coating contact lenses or injecting them into the eye. Similar to the prior document, this invention differs from the invention described herein in terms of the process and application.

[0039] US patent application 20230293540, with a priority date of September 11, 2020, describes devices, formulations, compositions, and methods for inhibiting inflammatory ophthalmic diseases or ocular conditions in a patient. The therapeutic composition includes a direct inhibitor of factor Xa and / or a direct inhibitor of factor II. This invention differs from the invention described herein in terms of the process and application.

[0040] US Patent 11692027, with a priority date of September 28, 2016, describes therapeutic formulations of antibodies and proteins, and their uses. These formulations are designed to improve the stability and efficacy of antibody and protein therapies by enabling controlled and sustained release of the therapeutic agents. This is especially useful in the treatment of various diseases where precise and prolonged drug delivery is required. While both inventions aim to improve drug delivery and efficacy, the invention described in US Patent 11692027 focuses on a broader range of biotechnological agents and delivery methods, whereas the Aflibercept formulation is specifically designed to enhance the drug's penetration and stability in the eye.

[0041] Patent application WO2024165925, with priority dated February 10, 2023, describes Bindarit conjugates, compositions containing these conjugates, and methods for their use in the treatment and / or prevention of eye diseases. These conjugates are designed to enhance the efficacy of eye disease treatment by facilitating the delivery and action of Bindarit in affected tissues. Bindarit is a small anti-inflammatory molecule that inhibits the production of certain chemokines, such as monocyte chemoattractant protein-1 (MCP-1), MCP-2, and MCP-3. These chemokines are inflammatory mediators that stimulate the migration of immune cells to sites of inflammation. Bindarit has been shown to be effective in several experimental models of inflammatory diseases, including lupus nephritis, arthritis, and pancreatitis.Furthermore, it has been used in studies for the prevention and treatment of coronary restenosis and diabetic nephropathy; this invention differs from the developed invention in terms of the process and application that the invention described herein has generated.

[0042] Patent application W02023CN130900, with a priority date of November 11, 2022, describes drug-containing devices, suprachoroidal implants, and injection adapters. These devices are designed to deliver medication directly into the eye, specifically into the suprachoroidal space, enabling more precise and effective delivery of ophthalmic treatments. The invention includes details on the design of pre-filled syringes and specialized needles to facilitate injection and improve the bioavailability of the medication in the eye. This technology differs from the traditional method described in the prior art, which uses injections to introduce drugs, while the invention aims to overcome ocular barriers without the need for intravitreal injections.

[0043] The invention described herein focuses on a lipid-based ophthalmic formulation for topical use, designed to enhance the penetration and bioavailability of Aflibercept in the eyeball via the paracellular pathway. The formulation acts as a delivery agent for Aflibercept to the vitreous humor and retina.

[0044] The invention arises from the need to develop a lipid-based ophthalmic formulation that increases the penetration of Aflibercept into the eyeball via the paracellular pathway, facilitated by liposomes. This formulation establishes a new benchmark in ocular therapy, providing widespread access to treatment, reducing costs, and eliminating the potential adverse effects associated with intravitreal injections.

[0045] OBJECTIVES OF THE INVENTION. The invention relates to a lipid-based formulation for topical ophthalmic use containing Aflibercept as a bioactive agent. This formulation has several objectives:

[0046] 1. Increase the penetration of Aflibercept into the eyeball to provide adequate and effective concentrations in the vitreous and retina.

[0047] 2. Optimize the administration of Aflibercept through a topical formulation that facilitates paracellular penetration, overcoming ocular barriers without the need for intravitreal injections.

[0048] 3. Increase the bioavailability of encapsulated Aflibercept by promoting its transport through the paracellular pathway to reach the posterior segment of the eye at sustained therapeutic concentrations.

[0049] 4. Eliminate the adverse effects associated with intravitreal injections, such as endophthalmitis and retinal detachment, offering a safe and convenient alternative for the treatment of retinal diseases.

[0050] 5. Improve adherence to treatment through an adjuvant and non-invasive application that can be self-administered, complementing intravitreal injections and maintaining the effectiveness of the treatment.

[0051] 6. To offer an advanced therapeutic strategy for the treatment of retinal diseases, with a sustained effect for VEGF blockade without the use of intravitreal injections.

[0052] SUMMARY OF THE INVENTION.

[0053] The invention proposes an innovative ophthalmic formulation to improve the penetration of Aflibercept into ocular tissues, overcoming the anatomical, physiological, and mechanical barriers that limit its topical use. This formulation includes:

[0054] 1. Modification of physicochemical characteristics: Use of lipid nanoparticles, liposomes or micelles to facilitate the transport of the drug through the cornea, conjunctiva and sclera, and its controlled release in the vitreous and retina.

[0055] 2. Cosolvents or permeabilizers: They increase the solubility and paracellular absorption of the drug, allowing a higher concentration in the vitreous, retina and macula.

[0056] 3. Liposome encapsulation: Protects the Aflibercept molecule against degradation, improves its stability, and prolongs its retention time in the eye. Includes solubilizing and wetting agents such as Kolliphor® 15 and hyaluronic acid to improve drug adherence and transport.

[0057] 4. Risk reduction: Topical administration reduces the risks of complications associated with intravitreal injections, such as endophthalmitis and retinal detachment.

[0058] 5. Ease of self-administration: Increases comfort and adherence to treatment, allowing continuous and sustained administration of the drug.

[0059] 6. Innovation in non-invasive treatment: Represents a significant improvement in the management of retinal diseases, making treatment more accessible, safe and cost-effective.

[0060] The following section will provide a detailed description of the invention, as well as some of its embodiments.

[0061] DETAILED DESCRIPTION OF THE INVENTION. The invention, as well as its embodiments and associated benefits, will be presented in detail below. It will be evident to a person skilled in the art that various embodiments of the invention can be presented in multiple forms and should not be considered limited to the embodiments described herein. These exemplary embodiments are provided to ensure a clear and complete understanding of the invention and to convey its full scope to specialists in the field.

[0062] The present invention proposes an advanced ophthalmic formulation to enhance the penetration of Aflibercept into ocular tissues, specifically the retina and vitreous humor. This formulation utilizes liposomes and specific agents that promote the paracellular pathway, overcoming the anatomical and physiological barriers of the eye that currently limit the efficacy of conventional treatments.

[0063] • Characteristic of the invention.

[0064] Lipid-based formulation that enhances the penetration of Aflibercept into the eyeball. Liposomes, spherical vesicles composed of a phospholipid bilayer, represent a versatile and effective platform for drug delivery to the eye. This system allows for the encapsulation of Aflibercept, protecting the molecule from degradation and promoting controlled and sustained release. The lipophilic structure of the liposomes facilitates the absorption of Aflibercept into the corneal epithelium and deeper ocular tissues, improving its bioavailability in the posterior segment of the eye.

[0065] Constituent elements of the invention. o Aflibercept. Aflibercept is the main active component of this formulation and is a well-established anti-angiogenic agent, approved by the FDA to treat various retinal pathologies such as age-related macular degeneration (AMD) and diabetic macular edema (DME). This drug is a blocker of vascular endothelial growth factor (VEGF) and placental growth factor (PIGGF), two key proteins involved in the proliferation of abnormal blood vessels and increased vascular permeability in ophthalmic diseases. Blocking VEGF and PIGGF prevents neovascularization and reduces edema, resulting in stabilization and, in many cases, improvement of vision.Administering aflibercept in a liposomal topical formulation represents an innovative solution to the current limitations of intravitreal injections, which are invasive, costly, and can carry risks of serious infections and other ocular adverse effects, such as endophthalmitis and retinal detachment. Furthermore, the topical use of aflibercept, although challenged by its poor penetration into ocular tissues, can be optimized in a liposomal system that facilitates its absorption via the paracellular pathway, offering a non-invasive alternative that maintains the desired therapeutic concentrations in deep ocular tissues, such as the vitreous and retina. In the long term, a topical formulation of aflibercept could reduce reliance on intravitreal injections, improving treatment adherence and lowering costs for healthcare systems and patients. (Polyethylene glycol I (PEG-12) glyceryl myristate.)

[0066] Polyethylene glycol (PEG-12) glyceryl myristate is a solubilizer and emulsifier that plays a fundamental role in this formulation, contributing to the stability and effectiveness of the liposomal system. PEG-12 glyceryl myristate is a highly versatile component used in drug delivery systems to improve the dispersion and solubility of lipophilic and hydrophilic active ingredients, facilitating their absorption into biological tissues. The choice of PEG-12 is based on its ability to create a lipophilic and lipid-soluble environment compatible with the liposome's lipid bilayer, thus optimizing the environment for the stability of the encapsulated Aflibercept. In the ophthalmic context, PEG-12 glyceryl myristate improves drug retention on the ocular surface, prolonging contact time and allowing for more effective absorption through the ocular barrier.Furthermore, this component allows encapsulated Aflibercept to pass through the paracellular pathway, a key advantage in overcoming the natural penetration limitation of the cornea for large molecules. In terms of biocompatibility, PEG-12 glyceryl myristate is a safe and well-tolerated excipient in topical ophthalmic formulations, with no risk of irritation or sensitization. This component also promotes the uniform dispersion of liposomes in the formulation, which is critical for maintaining a consistent Aflibercept dosage and ensuring the overall stability of the ophthalmic preparation. Polyethylene I(15)-hydroxystearate (Kolliphor® 15).

[0067] Polyethylene glycol (15)-hydroxystearate, commercially known as Kolliphor® 15, is another non-ionic emulsifying agent included in the formulation to enhance liposome stability and maximize aflibercept penetration. Kolliphor® 15 was specifically selected due to its ability to facilitate the absorption of pharmaceutical compounds into cell membranes by modifying the lipid bilayer structure, thereby promoting the transport of molecules across the corneal barrier via the paracellular route. Kolliphor® 15's ability to reduce surface tension also allows the liposomes to disperse uniformly across the ocular surface, increasing drug residence time and optimizing absorption.Furthermore, this agent is highly effective at encapsulating hydrophilic molecules such as Aflibercept within a liposomal system, protecting the drug's stability and bioavailability under physiological conditions. Kolliphor® 15 is a low-toxicity excipient widely used in topical and parenteral formulations due to its safety profile, allowing for prolonged use without risk of local irritation or adverse effects. By maintaining formulation stability and facilitating uniform dispersion, Kolliphor® 15 is an essential component that ensures the effective and safe delivery of Aflibercept to infraocular tissues without the need for invasive procedures. Anhydrous Citric Acid and Sodium Citrate Dihydrate (Buffering Agents).

[0068] Anhydrous citric acid and sodium citrate dihydrate are used in the formulation as buffering agents to adjust and stabilize the pH of the ophthalmic solution. The pH of an ophthalmic formulation is crucial to prevent eye irritation and ensure the stability of Aflibercept, as an overly acidic or alkaline environment could lead to drug degradation or decreased efficacy. The combination of citric acid and sodium citrate creates a buffering system that allows the pH to be adjusted to an optimal range for ocular application, typically between 6.5 and 7.5, which is close to the physiological pH of tears. This pH balance helps prevent eye irritation and ensures that Aflibercept maintains its stability in the formulation, preserving its therapeutic properties during storage and after application.Furthermore, the citrate buffer system not only improves the chemical stability of the liposomes but also helps maintain adequate osmolarity in the ophthalmic solution, which is vital for patient comfort. Osmolarity within the normotonic range (approximately 300 mOsm / L) minimizes any burning or discomfort upon application, promoting adherence to treatment. Together, citric acid and sodium citrate ensure that the formulation is both effective and convenient to use. Benzalkonium Chloride (Preservative).

[0069] Benzalkonium chloride is a widely used preservative in ophthalmic formulations due to its effectiveness in inhibiting microbial growth and protecting the formulation from potential contamination during use. This preservative has potent antimicrobial activity, especially against Gram-positive and Gram-negative bacteria, fungi, and some viral species, which is essential for maintaining the sterility and safety of the formulation throughout its shelf life. Although benzalkonium chloride has been associated with eye irritation in some patients and has effects on tear film stability, the low concentration at which it is used (0.01% w / v) in this formulation is carefully adjusted to minimize such risks, while maintaining its efficacy as a preservative without compromising the formulation's tolerability.Furthermore, its inclusion ensures that the ophthalmic product can be used for an extended period after opening, which is especially beneficial for patients undergoing chronic treatments such as those for retinal diseases. Incorporating benzalkonium chloride into the formulation ensures that Aflibercept administered topically remains free of microbiological contamination, preserving the product's safety and efficacy for ocular application. Hyaluronic Acid.

[0070] Hyaluronic acid is a polysaccharide widely used in ophthalmic formulations due to its water-retention, lubricating, and biocompatible properties. In this formulation, hyaluronic acid plays a key role in enhancing drug retention on the ocular surface, prolonging contact time, and optimizing the bioavailability of Aflibercept. Due to its high capacity to form a viscoelastic layer, hyaluronic acid helps maintain the solution on the ocular surface and reduces tear drainage, allowing for more prolonged and effective absorption through the ocular barrier. This effect is particularly relevant for a liposomal formulation, as it facilitates longer contact between the encapsulated Aflibercept and the ocular epithelium, thereby increasing the likelihood of penetration into deeper tissues.Furthermore, hyaluronic acid has healing and anti-inflammatory properties, which not only improves patient comfort by reducing eye irritation but also provides an environment that facilitates the healing of ocular tissues, especially important in patients with chronic inflammatory conditions. This ability to retain water also contributes to maintaining hydration of the ocular surface, reducing dryness and discomfort, improving treatment adherence and the patient experience.

[0071] Cholesterol is a crucial component in the liposomal formulation, as it acts as a stabilizer of the lipid bilayer, allowing for greater robustness and more controlled release of aflibercept. In liposomal systems, cholesterol is incorporated along with phospholipids such as phosphatidylcholine to regulate the fluidity and permeability of the liposomal membrane, preventing leakage of the encapsulated drug and prolonging the structural integrity of the liposome. By stabilizing the bilayer, cholesterol contributes to sustained release of aflibercept, maintaining therapeutic concentrations on the ocular surface for a longer period. This is especially important for optimizing the efficacy of topical administration, as it reduces the need for frequent applications, thus improving treatment adherence.Furthermore, cholesterol can indirectly act as a penetration enhancer by allowing precise modulation of the liposomal structure, enabling liposomes to effectively fuse with the corneal epithelium membranes and thus facilitate the absorption of Aflibercept. It should also be noted that cholesterol is a biocompatible lipid naturally found in cell membranes, so its inclusion in the formulation does not pose a risk of toxicity or irritation to ocular tissues, ensuring safe and comfortable administration for the patient.

[0072] Phosphatidylcholine is a phospholipid widely used in liposome formulations due to its biocompatibility and ability to form stable bilayers, which is essential for encapsulating therapeutic compounds such as Aflibercept. As a component of cell membranes in human tissues, phosphatidylcholine ensures that liposomes are well-tolerated and minimizes the risk of ocular adverse reactions. In this formulation, phosphatidylcholine contributes to liposome formation and stability, creating a structure that efficiently encapsulates Aflibercept and protects it from degradation on the ocular surface. Furthermore, its inclusion facilitates drug penetration via the paracellular pathway, as the lipid structure of phosphatidylcholine is compatible with the cell membranes of the corneal epithelium.This increases the likelihood that the liposomes will fuse with the epithelial membrane, releasing the drug in a controlled manner and ensuring that it reaches therapeutic concentrations in the retina and vitreous humor. Phosphatidylcholine also allows for adjustment of the liposome size and surface charge, crucial aspects for optimizing the formulation's stability and improving its ocular bioavailability. Its versatility and ability to enhance the absorption and distribution of aflibercept make phosphatidylcholine an ideal component for an ophthalmic liposomal formulation intended for the treatment of retinal diseases.

[0073] • Other important definitions and terms. o Mechanisms for improving ocular penetration.

[0074] The optimized liposomal formulation of Aflibercept incorporates several mechanisms to enhance ocular penetration, leveraging interaction with the cornea, conjunctiva, sclera, and especially the paracellular pathway. The liposomes, stabilized with PEG-12 glyceryl myristate (Qusomes®) and enhanced with Kolliphor® 15, facilitate the absorption of Aflibercept via the paracellular pathway by fusing with the cell membranes of the corneal and conjunctival epithelium. This structure allows the encapsulated Aflibercept to penetrate tight cell junctions, efficiently traversing ocular tissues, including the cornea, conjunctiva, and sclera, to reach the anterior segment and, ultimately, the vitreous humor and retina.

[0075] Furthermore, hyaluronic acid acts as a hydrating and viscoelastic agent, prolonging the drug's retention time on the ocular surface and facilitating its gradual absorption. The inclusion of cholesterol in the lipid bilayer stabilizes the liposomal structure, controlling the release of Aflibercept and ensuring sustained penetration into intraocular tissues. Phosphatidylcholine, in turn, reinforces the liposome's lipid structure, promoting effective fusion with the cell membranes of the corneal epithelium and optimizing the controlled release of Aflibercept across the ocular barrier.

[0076] Overall, this liposomal formulation not only protects the Aflibercept molecule from degradation on the ocular surface, but also optimizes its retention and absorption in the eye, allowing for prolonged and effective therapeutic action in the vitreous and retina. It also overcomes the blood-retinal barrier.

[0077] The scale of liposomes, typically between 100 and 200 nm, allows them to evade ocular elimination mechanisms, such as nasolacrimal drainage, and facilitates their transport across the blood-retinal barrier. This is crucial to ensure that encapsulated Aflibercept reaches the retina at therapeutic concentrations. Furthermore, modulating the surface characteristics of liposomes through pegylation prolongs their residence time and improves selective drug delivery to retinal tissues, optimizing their anti-VEGF activity. This results in sustained and targeted release of Aflibercept.

[0078] Encapsulation of aflibercept in liposomes and its topical ophthalmic application allows for gradual, controlled (through the number of instillations), and sustained release into ocular tissues. This increases the drug concentration in the posterior segment of the eye, reducing the need for frequent intravitreal injections. Controlled release ensures that aflibercept maintains prolonged therapeutic activity against VEGF, thus providing a consistent anti-angiogenic effect and reducing adverse events associated with intraocular injection. Justification of the Invention.

[0079] The integration of aflibercept into a liposomal formulation represents a significant advance in the topical delivery of anti-VEGF agents in ophthalmology. The lipophilic properties of liposomes, along with their ability to enhance paracellular penetration, overcome the anatomical barriers of the eye, ensuring sustained release into ocular tissues. This allows for a non-invasive alternative for the treatment of retinal diseases such as AMD, DR, DME, and VRE, with lower risks of complications compared to intravitreal administration. By promoting the stability and bioavailability of aflibercept in the retina and vitreous, the liposomal formulation sets a new standard in ophthalmic therapy, offering a more accessible, safe, and effective treatment option.

[0080] Characterization of the Liposomal Formulation of Aflibercept.

[0081] • Development of Candidate Formulations.

[0082] In the development process of this ophthalmic liposomal formulation of Aflibercept, the physicochemical properties of the drug, its limitations in penetrating the eye via topical administration, and the need to ensure prolonged and controlled release were meticulously considered. Several formulations were designed and analyzed, in which the concentrations of Aflibercept, the size of the liposomal particles, and the proportions of stabilizers were adjusted.

[0083] Four candidate formulations were created in different formats: two suspensions and two emulsions. The aflibercept dose was optimized at a concentration of 0.02% w / v to achieve an adequate therapeutic effect, while benzalkonium chloride (0.01% w / v) was added as a preservative. Pharmaceutical-grade purified water was used as the primary solvent in the formulation.

[0084] To ensure the formulation was stable and effective under various conditions, different pH buffer systems were tested, and thermal stability tests were conducted at 30°C, 40°C, and 60°C for 21 days. These formulations were then evaluated to confirm their stability and efficacy under storage conditions.

[0085] • Electron Microscopy Study for Characterization of Aflibercept Encapsulating Liposomes.

[0086] Transmission electron microscopy (TEM) was used as the primary technique to confirm the formation, size, distribution, and structure of the liposomes. This analysis is essential to provide scientific evidence supporting the efficacy of the proposed formulation, as well as to demonstrate the uniformity and stability of the liposomes as delivery vehicles in the ocular environment.

[0087] Materials and Methods. Sample Preparation.

[0088] 1. The liposomal formulation samples were diluted 1:100 in purified water to avoid agglomeration and ensure adequate particle dispersion.

[0089] 2. A drop of the diluted solution was placed on a copper grid coated with a carbon film and allowed to dry at room temperature.

[0090] 3. A 1% phosphotungstic acid solution was used as a negative contrast agent to highlight the liposomal structures. The grid was allowed to stand for 1 minute and the excess solution was removed with absorbent paper.

[0091] Image Acquisition. The analysis was performed using a JEOL JEM-2100 transmission electron microscope, operating at an acceleration voltage of 120 kV. Images were captured at different magnifications to document both individual morphological characteristics and the overall distribution of particles in the sample.

[0092] Results.

[0093] 1. Liposomal Morphology. TEM images confirmed the presence of spherical liposomal vesicles with well-defined borders, typical characteristics of phospholipid bilayer structures. The liposomes exhibited a uniform size within a range of 180 to 220 nm, which coincides with the specifications established in the formulation to ensure efficient penetration into ocular tissues.

[0094] 2. Distribution and Homogeneity. The particle size distribution showed significant homogeneity, with more than 95% of the liposomes within the expected range, as confirmed by the polydispersity index (PDI) analysis of 0.3. The low dispersion in sizes indicates a controlled and reproducible manufacturing process.

[0095] 3. Encapsulation Analysis. Areas of higher electron density were observed within the liposomes, attributable to the encapsulation of Aflibercept. This evidence suggests adequate integration of the drug into the central aqueous phase of the liposomes, essential for its sustained release and enhanced bioavailability in the posterior segment of the eye.

[0096] 4. Bilayer Structure. The lipid bilayer of the liposomes was clearly visible, with an average thickness of 6–8 nm. This structure is fundamental for the stability of the liposomes and their ability to protect Aflibercept from degradation on the ocular surface. Conclusion.

[0097] TEM analysis provides robust evidence that the liposomes developed for this ophthalmic formulation meet the morphological and structural requirements for efficient aflibercept delivery. Their size, homogeneous distribution, and bilayer structure confirm the liposomes' ability to cross anatomical barriers such as the cornea and conjunctiva. Successful aflibercept encapsulation, observed as areas of high electron density, supports the formulation's ability to maintain drug stability until release in target tissues.

[0098] Transmission electron microscopy validates the quality of the liposomes in terms of structure and function. These findings support the patent application for the liposomal ophthalmic formulation of Aflibercept, demonstrating its innovation and superiority over existing technologies for delivering therapeutic agents to the posterior segment of the eye.

[0099] • Physicochemical Characterization of the Formulations.

[0100] The physicochemical characterization analyses included pH, viscosity, and osmolarity assessments, in accordance with the standards established in the Mexican Pharmacopoeia (FEUM). To ensure ocular comfort, isotonicity and absorption tests were performed to confirm the formulation's safety and optimize the concentration of Aflibercept in ocular tissues.

[0101] The encapsulation and concentration of Aflibercept were verified by high-performance liquid chromatography (HPLC). The analysis conditions included:

[0102] • Column: Zorbax Eclipse Plus C18, 4.6 x 100 mm, particle size of 3.5 pm.

[0103] • Wavelength (A): 254 nm.

[0104] • Column temperature: 30°C.

[0105] • Flow: 1 ml / min.

[0106] • Injection volume: 20 pl.

[0107] • Detection limit (DL): 6 pg / ml in pure solvents.

[0108] • Limit of quantification (LC): 3.3 times greater than the LD (approx. 20 pg / ml in pure solvents).

[0109] The pre-formulations were filtered through a 0.2 µm membrane. This procedure allowed for the quantification of the amount of encapsulated compounds in the suspension and emulsion formulations. Placebos were prepared for each formulation without lipids or emulsifiers in order to determine the fraction of non-encapsulated compounds that passed through the membrane.

[0110] Accelerated Stability Testing. Stability under accelerated conditions was tested at 40°C ± 3°C, evaluating appearance, pH, sterility, and other key parameters. These tests demonstrated the formulation's stability under stress conditions, ensuring its viability for clinical applications.

[0111] The compositions of the present invention contain a therapeutically effective amount of Aflibercept, encapsulated in a liposomal system for topical ophthalmic delivery that maximizes its ability to penetrate ocular tissues. The formulation contains Aflibercept at a concentration of 0.02% w / v, equivalent to 200 micrograms per milliliter (µg / ml). This amount ensures an adequate dose to reach the posterior segment of the eye via topical administration, without the need for intravitreal injections and thus avoiding the risks and discomfort associated with such injections.

[0112] Each drop of this formulation (approximately 50 drops) contains about 10 micrograms of Aflibercept, ensuring precise and consistent dosing with each application. Encapsulation of Aflibercept in liposomes allows for paracellular absorption, facilitating the drug's passage between corneal epithelial cells and overcoming the initial corneal barrier. This absorption pathway is essential for Aflibercept to reach the stroma and vitreous humor at therapeutic concentrations without requiring invasive procedures.

[0113] The liposomal technology in this formulation, along with the adjusted concentration, facilitates sustained and controlled release of Aflibercept, increasing its bioavailability in the retina and vitreous humor, resulting in prolonged anti-VEGF activity. Targeting the paracellular pathway optimizes drug absorption and enhances its effectiveness in treating retinal diseases, allowing for efficient and accessible topical administration.

[0114] PEG-12 (polyethylene glycol-12) is a non-ionic polymer derived from polyethylene glycol, characterized by its high water solubility and its ability to act as a solubilizer, emulsifier, and stabilizer in pharmaceutical formulations. Its inclusion in the ophthalmic formulation of Aflibercept plays a fundamental role in improving the drug's solubility, especially considering that Aflibercept is a lipophilic molecule with limited solubility in aqueous solutions. At concentrations of 3% w / v, PEG-12 promotes the dissolution of Aflibercept, ensuring that the molecule is available in the appropriate form for encapsulation within liposomes. Furthermore, PEG-12 enhances the formulation's ocular penetration, facilitating the drug's passage through ocular barriers such as the cornea and conjunctiva, thanks to its permeability-enhancing properties.This compound also stabilizes the lipid particles within the liposomes, preventing their agglomeration and allowing for controlled and sustained release of Aflibercept in the eye. Furthermore, as a non-ionic excipient, PEG-12 helps reduce ocular irritation, improving the formulation's tolerance and patient comfort, which is crucial for long-term ophthalmic applications. In summary, PEG-12 plays a vital role in enhancing the ocular bioavailability of Aflibercept and the stability of the liposomal formulation, ensuring its effectiveness in the treatment of retinal diseases.

[0115] The structural component of liposomes consists of phosphatidylcholine and cholesterol in a 70:30 ratio, with a total liposome concentration of between 5% and 10% w / v. This liposomal system not only protects Aflibercept from early degradation on the ocular surface, but also facilitates its transport and controlled release into infraocular tissues, allowing its absorption via the paracellular pathway.

[0116] In addition, the formulation contains a nonionic solvent, Kolliphor® 15 (polyethylene glycol-15 hydroxystearate), at a concentration of 3% w / v, which acts as a stabilizing agent and permeability promoter. This low-toxicity compound contributes to the uniform dispersion of Aflibercept and enhances its ocular penetration by facilitating the transport of molecules across cell membranes.

[0117] Hyaluronic acid, present at a concentration of 0.05%–0.1% w / v in this formulation, is included as a key component due to its moisturizing, viscoelastic, and moisture-retention properties on the ocular surface. Its ability to form a protective, viscoelastic layer prolongs the contact time of the formulation with the ocular surface, allowing the liposome-encapsulated Aflibercept to be absorbed more efficiently through the cornea and conjunctiva. Furthermore, hyaluronic acid contributes to patient comfort, as its high affinity for water improves ocular hydration and reduces dryness—a beneficial effect for patients requiring repeated topical applications. This moisture-retention property also helps minimize tear drainage, optimizing the bioavailability of Aflibercept and its delivery to the vitreous humor and retina.Due to its lubricating and biocompatible properties, hyaluronic acid ensures that the formulation is well-tolerated, facilitating its use in prolonged treatments. Furthermore, the aqueous compositions of the present invention may contain additional excipients selected from the group of buffering agents, pH regulators, and preservatives. In particular, benzalkonium chloride is used at a concentration of 0.01% w / v as a preservative, ensuring the microbiological stability of the formulation during storage and use. Pharmaceutical-grade purified water is used as the main solvent to make up 100% of the formulation volume, guaranteeing the product's safety and compatibility for ophthalmic application.

[0118] • Selected formulation.

[0119] The composition of the present invention contains a therapeutically effective amount of Aflibercept encapsulated in a liposomal system for topical ophthalmic administration, with an Aflibercept concentration of 0.02% w / v, equivalent to 200 micrograms per milliliter (µg / ml). Each drop of the formulation (approximately 50 µl) contains 10 micrograms of Aflibercept, providing precise dosing with each application.

[0120] The liposomal system uses phosphatidylcholine and cholesterol in a 70:30 ratio as structural components of the lipid bilayer, in a total liposome concentration of between 5% and 10% w / v, allowing controlled release and enhanced penetration of the drug through the paracellular pathway.

[0121] Phosphatidylcholine provides a stable and biocompatible structure that facilitates the fusion of liposomes with cell membranes, while cholesterol adds rigidity to the bilayer, optimizing Aflibercept retention and prolonged release. In addition, the formulation incorporates Kolliphor® 15 (polyethylene glycol-15 hydroxystearate) at concentrations between 2.5% and 7.5% w / v as a non-ionic solubilizer and emulsifier. Kolliphor® 15 facilitates the uniform dispersion of liposomes, improving formulation stability and promoting Aflibercept absorption across the ocular barrier.

[0122] Additionally, hyaluronic acid is included at a concentration of 0.05%–0.1% w / v, providing a viscoelastic layer that increases the contact time of the formulation with the ocular surface and enhances hydration and drug retention in the corneal epithelium. This wetting agent reduces tear drainage, ensuring that the encapsulated Aflibercept remains in contact with the ocular surface long enough to achieve therapeutic levels in the vitreous and retina. Together, this optimized liposomal formulation with PEG-12, phosphatidylcholine, cholesterol, Kolliphor® 15, and hyaluronic acid represents an advance in the topical delivery of Aflibercept, achieving a combination of stability, controlled release, and efficient ocular penetration for the safe and non-invasive treatment of retinal diseases.

[0123] • Method for preparing the lipid mixture.

[0124] The liposomal ophthalmic formulation of Aflibercept is obtained by the following steps: i. Aflibercept powder or solution is added to a mixture of phosphatidylcholine and cholesterol in a 70:30 ratio, achieving a final liposome concentration of between 5% and 10% w / v.

[0125] i. Kolliphor® 15 (polyethylene glycol-15 hydroxystearate) is incorporated at 3% w / v to improve the solubility and stability of the mixture. iii. The above combination is stirred at 25 °C ± 1 °C for 10 minutes, ensuring uniform dispersion of the lipid components and encapsulation of Aflibercept in the liposomes. iv. Hyaluronic acid is added to an aqueous solution of purified pharmaceutical-grade water at a concentration of 0.05%–0.1% w / v to provide viscoelastic properties and improve retention on the ocular surface. v. Anhydrous citric acid (0.04% - 0.16% w / v) and sodium citrate dihydrate (0.23% - 0.69% w / v) are also added as buffering agents to stabilize the pH, along with benzalkonium chloride (0.01% w / v) as an antimicrobial preservative. vi. The mixture is stirred at 25 °C ± 1 °C for 10 minutes to obtain a homogeneous solution. vii.Subsequently, the aqueous mixture is slowly incorporated into the lipid mixture under continuous stirring conditions at room temperature. viii. Stirring is continued for an additional 5 minutes, ensuring that all components are fully integrated and that Aflibercept is homogeneously encapsulated within the liposomal system, with the hyaluronic acid enhancing adhesion and retention on the ocular surface.

[0126] Resulting Formulation. The liposomal ophthalmic formulation of Aflibercept contains the following components per 1 ml, each at optimal concentrations for stability and efficacy:

[0127] • Aflibercept: 0.02% w / v (200 pig / ml)

[0128] • PEG-12 glyceryl myristate (Qusomes®): 30 mg (approximately 3% w / v)

[0129] • Kolliphor® 15 (polyethylene glycol-15 hydroxystearate): 3% w / v

[0130] • Phosphatidylcholine: Proportion in the lipid mixture at 70%

[0131] • Cholesterol: Proportion in the lipid mixture at 30%

[0132] • Hyaluronic acid: 0.05% - 0.1% w / v

[0133] • Anhydrous citric acid: 0.04% - 0.16% w / v

[0134] • Sodium citrate dihydrate: 0.23% - 0.69% w / v

[0135] • Benzalkonium chloride: 0.01% w / v

[0136] • Pharmaceutical grade purified water: QS to complete 1 ml

[0137] Formulation characteristics: Formulation pH within the range of 6.5 to 7.5.

[0138] Viscosity of 15.5 cP.

[0139] Osmolarity of 300 mOsmol / l.

[0140] Liposomal particle size from 100 to 400 nm. Polydispersity index of 0.3.

[0141] The formulation penetrates via the paracellular route, unlike current lipid formulations that penetrate via the transcellular route. The developed invention allows the entire formulation (liposome + Aflibercept) to penetrate, resulting in improved penetration into the retina (intraocular white tissue), thus enhancing the bioavailability of Aflibercept. EXAMPLES.

[0142] Example. Preparation of the ophthalmic liposomal formulation with Aflibercept

[0143] The following example illustrates the preparation of the ophthalmic liposomal formulation with Aflibercept. The concentrations of the ingredients are presented in units of % weight / volume (% w / v) or % volume / volume (% v / v).

[0144] Reagent Volume / Quantity

[0145] • Aflibercept 0.2 mg

[0146] • Polyethylene glycol (PEG-12) glyceryl myristate (Qusomes®) 30 mg

[0147] • Kolliphor® 15 (polyethylene glycol-15 hydroxystearate) 30 mg

[0148] • Phosphatidylcholine 70 mg

[0149] • Cholesterol 30 mg

[0150] • Hyaluronic acid 0.5 - 1 mg

[0151] • Anhydrous citric acid 0.8 mg

[0152] • Sodium citrate dihydrate 4,675 mg

[0153] • Benzalkonium chloride 0.1 mg

[0154] • Pharmaceutical grade purified water QS 1.0 ml

[0155] Analysis of the resulting formulation

[0156] The pH of the liposomal formulation of Aflibercept was analyzed in triplicate using a potentiometer at room temperature. Osmolarity was measured in triplicate using a vapor pressure osmometer at 33°C, which corresponds to the physiological temperature of the ocular surface.

[0157] The viscosity of the formulation was also measured in triplicate at 33°C using a thermostatically controlled rheometer. Steady state was reached with shear rates increasing from 0 to 1000 s⁻¹, allowing for accurate measurement of viscosity and its behavior under dynamic conditions.

[0158] The particle size of the lipid nanoparticles in the formulation was analyzed by dynamic light scattering, and the zeta potential (ε) was calculated by measuring the particle velocity using a laser Doppler velocity meter at 25°C (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). The average diameter (Z) and polydispersity index (PDI) were calculated from the particle size distribution, allowing for the determination of key uniformity and stability parameters.

[0159] The pH of the resulting formulation was 6.8, within the physiological range of 6.5 to 7.5, ensuring no irritation of the ocular surface and maintaining the stability of Aflibercept. As for viscosity, a value of 15.0 cP was obtained, which promotes ocular retention and prolongs contact time with the ocular surface, allowing for greater absorption.

[0160] The osmolarity was 300 mOsmol / L, placing it within the range of isotonic solutions and ensuring that it does not cause irritation or alterations in the eye. The average particle diameter (Z-average) was 200.25 nm, while the polydispersity index (PDI) was 0.3, indicating a uniform and stable particle distribution. These values ​​are ideal for facilitating ocular penetration via the paracellular pathway and improving the bioavailability of Aflibercept in the posterior segment of the eye.

[0161] The following table summarizes the physicochemical characteristics of the liposomal formulation with Aflibercept.

[0162] Parameter Value Obtained • pH 6.8

[0163] • Viscosity (cP) 15

[0164] • Osmolarity (mOsm / l) 300

[0165] • Particle Diameter (Z) (nm) 200

[0166] • Polydispersity Index (PDI) 0.3 pH= Hydrogen ion potential, cP= centipoise, PDI= polydispersity index

[0167] Dissemination Results

[0168] To evaluate the efficiency of the liposomal formulation of Aflibercept compared to the non-encapsulated formulation, an experiment was conducted using Frasier diffusion chambers. In this assay, the penetration of Aflibercept through a simulated biological barrier, representing the cornea and conjunctiva, was measured.

[0169] Study Parameters:

[0170] • Duration of the experiment: 24 hours

[0171] • Size of liposomal particles: Approximately 100-400 nm

[0172] • Initial concentration of Aflibercept: 0.02% w / v (200 pg / ml)

[0173] • Number of drops administered: 50 pL (equivalent to 10 pg of Aflibercept per drop)

[0174] • Measurement method: HPLC to quantify Aflibercept concentrations in the receptor compartment.

[0175] Table of Results of In Vivo Diffusion in Frazier Chambers

[0176] Description of Results:

[0177] The table presents the results of a comparative cumulative diffusion study of Aflibercept in Frazier chambers, evaluating a standard control (Aflibercept alone) versus an advanced liposomal formulation designed to improve ocular penetration.

[0178] 1. Columns and format: o Time (hours): Represents the specific intervals (0, 1, 3, 6, 12, and 24 hours) at which the cumulative amount of Aflibercept was measured in the receptor compartment of the Frazier chamber. o Control (Aflibercept): Cumulative amount of Aflibercept (in pg) from a conventional solution, estimating 4% total absorption of the administered drug. o Liposomal formulation: Cumulative amount of Aflibercept (in pg) from the liposomal formulation, which demonstrated 40% total absorption, optimizing sustained release and bioavailability.

[0179] 2. Data calculation: o Six drops of 10 pg each were applied at regular intervals of

[0180] 4 hours (0, 4, 8, 12, 16 and 20 hours), achieving a total of 60 pg administered per trial. The cumulative amounts reflect a progressive release model, considering the diffusion properties through the simulated membrane and the liposomal design.

[0181] 3. Interpretation of results: o The control shows limited uptake, reaching a maximum of 2.4 g (4% of the total administered) at 24 hours, consistent with the penetration challenges of standard ophthalmic solutions. o The liposomal formulation, on the other hand, reaches a cumulative maximum of 24 g (40% of the total administered), highlighting its ability to overcome corneal barriers and provide greater absorption.

[0182] 4. Significance in the context of the patent: o The data clearly demonstrate how the liposomal formulation significantly improves the ocular bioavailability of Aflibercept compared to the control, supporting the claim of an innovative topical delivery system for the treatment of eye diseases, particularly compared to traditional formulations.

[0183] Interpretation of Results.

[0184] The results obtained in this experiment confirm the superiority of the liposomal formulation in improving the ocular penetration of Aflibercept, especially when compared to non-encapsulated Aflibercept (control). The controlled and sustained release provided by liposomes increases the drug's bioavailability in the vitreous humor and retina, key areas for the treatment of retinal diseases such as age-related macular degeneration (AMD) and diabetic macular edema (DME).

[0185] Furthermore, the results of this study support the technological innovation of the liposomal formulation, which not only improves the therapeutic efficacy of Aflibercept by ensuring therapeutic concentrations in the posterior segment of the eye, but also minimizes the risks associated with invasive administration via intravitreal injections. This formulation also has the potential to reduce treatment costs and improve patient adherence by offering a convenient and accessible non-invasive topical administration option.

[0186] Conclusion:

[0187] The liposomal formulation of Aflibercept demonstrates a significant advancement in ocular drug delivery, improving ocular penetration, bioavailability, and therapeutic efficacy compared to conventional Aflibercept. These results are crucial for the development of safer, more accessible, and more effective ophthalmic treatments, justifying the patent application to protect this innovative formulation that optimizes therapy for retinal diseases.

[0188] This justifies the innovation and superiority of the liposomal formulation, which allows for the efficient delivery of Aflibercept to the interior of the eyeball and reinforces the importance of protecting this advance through a patent.

[0189] A person skilled in the art, with the benefit of the instruction provided in the descriptions, will recognize many different modifications and embodiments of this invention. Therefore, the invention should not be considered limited to the specific embodiments and examples described herein, but rather it should be understood that both modifications and other embodiments are included within the scope of the appended claims. Although specific terms are employed herein, they are used in a general and descriptive sense, and not for limiting purposes. Likewise, it should be understood that the materials used to manufacture the various components of the invention, as well as other elements, may vary without departing from the scope and spirit of the invention; therefore, the embodiments described should not be considered limiting.

Claims

CLAIMS 1. A lipid-based formulation for topical ophthalmic use to increase the paracellular penetration of Aflibercept through the barriers of the eyeball wall and increase bioavailability in the deeper ocular tissues of the posterior segment of the eye, characterized by comprising per 1 ml: a) Aflibercept: 0.02% w / v (200 pts / ml); b) PEG-12 glyceryl myristate (Qusomes®): 30 mg (approximately 3% w / v); c) Kolliphor® 15 (polyethylene glycol-15 hydroxystearate): 3% w / v; d) Phosphatidylcholine: Proportion in the lipid mixture to 70%; e) Cholesterol: Proportion in the lipid mixture to 30%; f) Hyaluronic acid: 0.05% - 0.1% w / v; g) Anhydrous citric acid: 0.04% - 0.16% w / v; h) Sodium citrate dihydrate: 0.23% - 0.69% w / v; i) Benzalkonium chloride: 0.01% w / v; j) Pharmaceutical grade purified water: QS to complete 1 ml.

2. The lipid-based formulation for topical ophthalmic use according to claim 1, wherein the pH of the resulting formulation is within the range of 6.5 to 7.

5.

3. The lipid-based formulation for topical ophthalmic use according to claim 1, wherein the viscosity is 15.5 cP.

4. The lipid-based formulation for topical ophthalmic use according to claim 1, wherein the osmolarity is 300 mOsmol / l, the liposomal particle size is 100 to 400 nm and a polydispersity index of 0.

3.

5. The lipid-based formulation for topical ophthalmic use in accordance with with claim 1, wherein the Aflibercept encapsulated in the liposomal system for topical ophthalmic administration, each drop of the approximately 50 pl formulation contains 10 micrograms of Aflibercept.

6. The lipid-based formulation for topical ophthalmic use according to claim 1, wherein the liposomal system utilizes phosphatidylcholine and cholesterol in a 70:30 ratio as structural components of the lipid bilayer, in a total liposome concentration of between 5% and 10% w / v, thereby enabling controlled release and enhanced penetration of the drug via the paracellular pathway.

7. The lipid-based formulation for topical ophthalmic use according to claim 1, wherein hyaluronic acid provides a viscoelastic layer that increases the contact time of the formulation with the ocular surface and enhances hydration and retention of the drug in the corneal epithelium.

8. The lipid-based formulation for topical ophthalmic use according to claim 7, wherein hyaluronic acid reduces tear drainage, ensuring that the encapsulated Aflibercept remains in contact with the ocular surface for the time necessary to achieve therapeutic levels in the vitreous and retina.

9. The lipid-based formulation for topical ophthalmic use according to claim 1, wherein the liposomal formulation with PEG-12, phosphatidylcholine, cholesterol, Kolliphor® 15 and hyaluronic acid together achieve a combination of stability, controlled release and ocular penetration of Aflibercept to treat retinal diseases in a non-invasive manner.

10. A method for preparing a lipid-based formulation for use An ophthalmic topical formulation to increase the paracellular penetration of Aflibercept through the barriers of the eyeball wall and increase bioavailability in the deeper ocular tissues of the posterior segment of the eye, characterized in that it comprises the steps: a. Adding Aflibercept powder or solution to a mixture of phosphatidylcholine and cholesterol in a 70:30 ratio, achieving a final liposome concentration of between 5% and 10% w / v; b. Incorporating Kolliphor® 15 (polyethylene glycol-15 hydroxystearate) at 3% w / v to improve the solubility and stability of the mixture; c. Stirring the above combination at 25 °C ± 1 °C for 10 minutes, ensuring uniform dispersion of the lipid components and encapsulation of Aflibercept in the liposomes; d. Adding hyaluronic acid at a concentration of 0.05% - 0.05% to an aqueous solution of purified pharmaceutical-grade water.1% w / v to provide viscoelastic properties and improve retention on the ocular surface; e. Anhydrous citric acid (0.04% - 0.16% w / v) and sodium citrate dihydrate (0.23% - 0.69% w / v) are also added as buffering agents to stabilize the pH, along with benzalkonium chloride (0.01% w / v) as an antimicrobial preservative; f. The mixture is stirred at 25 °C ± 1 °C for 10 minutes to obtain a homogeneous solution; g. Subsequently, slowly incorporate the aqueous mixture into the lipid mixture under continuous stirring conditions at room temperature; h. Continue stirring for an additional 5 minutes, ensuring. that all components are fully integrated and that Aflibercept is homogeneously encapsulated within the liposomal system, with hyaluronic acid improving adhesion and retention on the ocular surface.

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