Lipid-based formulation containing bimatoprost for increasing bioavailability and extracellular matrix expression and improving follicular cell survival

Abstract

The invention relates to a topical liposomal formulation for treating madarosis and other conditions related to eyelash and eyebrow hair follicle loss. The formulation improves the penetration of bioactive agents in hair follicles, increasing the bioavailability and efficacy thereof and reducing the side effects of conventional topical formulations. The innovative formulation uses liposomes to maximise the bioavailability of bimatoprost and other bioactive agents in the hair follicle. The synergic combination of these components ensures efficient and sustained penetration in the hair follicles, promoting the regeneration, density and capillary health thereof while minimising the risk of irritation and other side effects.

Description

[0001] LIPID-BASED FORMULATION CONTAINING BIMATOPROST TO INCREASE BIOAVAILABILITY AND EXTRACELLULAR MATRIX EXPRESSION AND IMPROVE FOLLICLE CELL SURVIVAL.

[0002] FIELD OF INVENTION.

[0003] This invention relates to the fields of ophthalmology and dermatology, focusing on the study, diagnosis, treatment, and prevention of diseases affecting the eyes, eyelashes, and scalp. Specifically, it is a topical lipid formulation that improves the bioavailability of bimatoprost, stimulates extracellular matrix expression, and increases the survival of hair follicle cells. Furthermore, it aims to reduce the adverse effects of conventional topical bimatoprost formulations, offering a safe and effective solution for treating madarosis and other disorders related to hair or eyelash loss.

[0004] BACKGROUND OF THE INVENTION.

[0005] Madarosis is the partial or total loss of eyelashes and / or eyebrows, caused by various etiologies, ranging from local inflammations to complex systemic diseases. Although its clinical significance is often underestimated, it can be an indicator of underlying conditions and presents a therapeutic challenge due to the limitations of current treatments.

[0006] Classification and Etiology. Madarosis can be classified as cicatricial and non-cicatricial, depending on the integrity of the hair follicle:

[0007] • Cicatricial: Characterized by irreversible destruction of the hair follicle due to chronic inflammatory processes, severe infections, or trauma. Examples include leprosy, systemic lupus erythematosus, and burns.

[0008] • Non-scarring: The hair follicle remains intact, suggesting a possibility of reversibility. Common causes include chronic blepharitis, alopecia areata, and trichotillomania.

[0009] Eyelash or eyebrow loss can be due to dysfunction of the hair cycle, which consists of three phases: anagen (growth), catagen (regression), and telogen (resting). In non-scarring madarosis, hair follicles prematurely enter the telogen phase, causing a visible thinning of eyelashes and eyebrows.

[0010] The prevalence of madarosis varies depending on the population and the underlying cause. Clinical studies estimate that:

[0011] • Chronic blepharitis: It is present in up to 30-50% of patients with madarosis in ophthalmological consultations.

[0012] • Alopecia areata: Affects approximately 2% of the general population and is associated with madarosis in 50-60% of cases.

[0013] • Trichotillomania: This compulsive disorder has an incidence of up to 3% of the population at some point in their lives.

[0014] Madarosis affects women and older adults more frequently due to biological and social factors. In older adults, hair loss is associated with reduced mitotic activity in hair follicles, a shortened anagen phase, and a higher prevalence of systemic diseases such as diabetes and autoimmune disorders. In women, madarosis has a significant emotional impact, as eyelashes and eyebrows are fundamental to facial expression and aesthetic perception. Eyelashes protect the eye from particles and microorganisms. Eyelash loss can increase the risk of eye infections and impair tear film distribution. Eighty percent of patients with madarosis report decreased self-esteem and quality of life. Sixty-five percent of people with madarosis and chronic blepharitis experience increased eye discomfort.The psychological impact of madarosis is comparable to other forms of alopecia, and 72% of those affected seek cosmetic or medical solutions, highlighting the need for innovative treatments that improve quality of life.

[0015] Pathophysiology.

[0016] Madarosis results from a disruption in the normal dynamics of the hair follicle cycle, which can be influenced by inflammatory, metabolic, autoimmune, and genetic factors. Under normal conditions, the hair follicles of the eyelashes and eyebrows go through three key phases:

[0017] 1. Anagen phase (growth): Keratinocytes actively proliferate in the hair follicle matrix, promoting the elongation of the hair shaft.

[0018] 2. Catagen Phase (regression): Controlled apoptosis process where follicle cells lose proliferative activity.

[0019] 3. Telogen Phase (resting): The follicle remains inactive until a new cycle is activated.

[0020] In madarosis, several molecular mechanisms affect this cycle, accelerating the transition to the telogen phase or irreversibly damaging the follicles.

[0021] Madarosis is caused by a complex interaction between chronic inflammation, oxidative stress, and follicular apoptosis, exacerbated by prostaglandin pathway dysfunction. Chronic inflammation, as seen in blepharitis and seborrheic dermatitis, releases proinflammatory cytokines and produces reactive oxygen species (ROS), damaging hair follicles. In conditions such as alopecia areata, inflammation induces apoptosis in hair follicle cells. Furthermore, prostaglandin dysregulation negatively affects follicle growth, decreasing vascularization and the expression of pro-anagen genes. The liposomal formulation of bimatoprost developed herein addresses these mechanisms, offering a more effective and safer treatment.

[0022] • Bimatoprot.

[0023] Bimatoprost is a synthetic analog of prostaglandin F2α (PGF2α), designed to bind to FP receptors in hair follicles and the ocular epithelium. Its mechanism of action includes the activation of intracellular signaling pathways, increasing calcium levels and phosphorylating proteins such as MAPK and CREB, which promotes the anagen phase of the hair cycle and collagen synthesis. Furthermore, it modulates local vascularization by increasing VEGF expression, improving the supply of oxygen and nutrients to the follicle. Its lipophilic nature allows for good permeability in lipid-rich tissues, but its limited stability and potential for irritation justify the development of controlled-release systems, such as liposomes, to maximize its efficacy and minimize adverse effects.

[0024] • Justification of the invention.

[0025] Bimatoprost, a PGF2α analogue, restores FP receptor function in hair follicles, but conventional topical formulations have limitations due to their low penetration and adverse effects.

[0026] 1. Molecular Mechanisms of Bimatoprost:

[0027] • Activation of FP receptors: Stimulates intracellular signaling, increasing keratinocyte proliferation and collagen synthesis. Inhibition of apoptosis: Reduces pro-apoptotic cytokines and balances BCL-2 and BAX proteins.

[0028] 2. Advantages of Liposomal Encapsulation:

[0029] • Improved penetration: Increases bioavailability by crossing the lipid barrier and reaching the hair follicles.

[0030] • Sustained release: Provides a controlled release, prolonging its action.

[0031] • Reduction of adverse effects: Limits direct contact with sensitive tissues, decreasing conjunctival hyperemia and unwanted pigmentation.

[0032] • Risks associated with topical administration of Bimatoprost for the treatment of madarosis.

[0033] Conventional topical formulations of bimatoprost, while effective in stimulating eyelash and hair growth, have significant limitations due to their low bioavailability. Drug absorption is restricted by the eye's natural barriers, requiring more frequent application and, consequently, increasing the risk of adverse effects. Common side effects include conjunctival hyperemia, skin hyperpigmentation, and, in severe cases, permanent iris pigmentation. There is also a risk of unwanted hair growth in areas of contact with the drug.

[0034] These limitations underscore the need for innovations in bimatoprost formulations to overcome current obstacles. The low bioavailability and adverse effects associated with conventional formulations represent a barrier to optimal drug use. Therefore, it is essential to develop technologies that improve the delivery of the active ingredient, reduce the frequency of application, and minimize side effects, thereby optimizing the patient experience and outcomes. • Strategies to Optimize Topical Use of Bimatoprost.

[0035] Encapsulating bimatoprost in liposomes improves drug delivery, overcoming the limitations of conventional topical formulations. Liposomes, which mimic cell membranes, increase the bioavailability of bimatoprost by facilitating its transport across biological barriers such as the cornea and epidermis. Furthermore, they allow for sustained drug release, reducing the need for frequent applications and minimizing adverse effects. Liposomes also protect bimatoprost from chemical and oxidative degradation, improving its stability and shelf life. In summary, liposomal technology optimizes the efficacy, safety, and stability of bimatoprost, positioning it as a key innovation in the treatment of hair follicle loss.

[0036] • Vitamin E

[0037] Vitamin E, or alpha-tocopherol, is an essential lipophilic antioxidant that protects and regenerates tissues, including skin and hair follicles. It neutralizes reactive oxygen species (ROS) and stabilizes lipid membranes, improving eyelash health and growth. It modulates critical pathways that impact hair follicle function, making it useful in topical treatments for madarosis and hair fragility.

[0038] Molecular Effects of Vitamin E on Hair Follicles:

[0039] 1. Regulation of Oxidative Stress ROS:

[0040] • Neutralizes ROS, stabilizing lipids in cell membranes and protecting mitochondria, essential for the anagen phase of the capillary cycle.

[0041] • Increases cell viability in keratinocytes under oxidative stress.

[0042] 2. Influence on the Extracellular Matrix (ECM): • Modulates the synthesis of collagen and elastin, necessary for the integrity of the follicle.

[0043] • Inhibits the activity of matrix metalloproteinases (MMPs), preserving the follicular environment.

[0044] 3. Proliferative Signaling Routes:

[0045] • Activates signaling pathways such as PI3K / AKT and Nrf2, associated with cell proliferation and antioxidant response, maintaining the anagen phase and preventing premature transition to the catagen phase.

[0046] Challenges of Topical Application of Vitamin E.

[0047] Despite its benefits, topical vitamin E faces significant challenges due to its limited penetration into target tissues, such as the hair follicles of the eyelashes.

[0048] 1. Low Skin Penetration:

[0049] • Vitamin E, being lipophilic, has low water solubility and difficulty crossing hydrophilic barriers such as the skin and cornea, limiting its effectiveness.

[0050] • Only 3-5% of topically applied vitamin E penetrates the deep layers of the skin.

[0051] 2. Oxidative Degradation:

[0052] • Vitamin E is susceptible to oxidation by light, oxygen and moisture, reducing its effectiveness.

[0053] • The lack of adequate transport systems can inactivate the compound before it reaches the target tissues.

[0054] 3. Stability in Formulations:

[0055] Vitamin E can interact with other ingredients in topical products, affecting the stability of the formulation. Potential Innovations to Overcome the Challenges.

[0056] The development of advanced delivery systems, such as liposomes and polymeric nanoparticles, improves the effectiveness of vitamin E in topical eyelash applications.

[0057] 1. Liposomal Encapsulation:

[0058] • Liposomes protect vitamin E from oxidation and improve its bioavailability, facilitating its penetration into hair follicles.

[0059] • They significantly increase skin penetration, improving antioxidant and regenerative efficacy.

[0060] 2. Polymeric Nanoparticles:

[0061] • They offer a controlled release of vitamin E, guaranteeing a sustained action and reducing the need for frequent applications.

[0062] • They improve the stability of vitamin E during storage and administration.

[0063] 3. Combined Formulations:

[0064] • The combination with other bioactive compounds, such as hyaluronic acid or Bimatoprost, enhances its effect by improving hydration and synergy with cell signaling pathways.

[0065] Conclusion: Vitamin E has great therapeutic potential for improving eyelash health and growth, but its low penetration and stability limit its efficacy in conventional topical applications. Advanced delivery technologies, such as liposomes and nanoparticles, can transform its use, optimizing its clinical impact and opening new opportunities in dermatological and ophthalmic care.

[0066] • Panthenol (vitamin B5).

[0067] Panthenol, or provitamin B5, is known for its moisturizing, regenerative, and anti-inflammatory properties, making it essential in formulations for healthy hair follicles and eyelashes. This compound attracts and retains water, improving the elasticity and strength of eyelashes, and promotes the metabolic activity of keratinocytes, stimulating the growth of longer, healthier eyelashes. Furthermore, panthenol promotes cell regeneration and tissue repair, activating signaling pathways such as PI3K / AKT and enhancing the synthesis of keratin and collagen.

[0068] Panthenol also reduces inflammation and soothes the skin by inhibiting inflammatory mediators such as IL-6 and TNF-α, which is beneficial in conditions like chronic blepharitis. However, its low penetration into target tissues is a challenge. Encapsulation in liposomes improves its bioavailability and stability, allowing for more effective and prolonged delivery to hair follicles. This technology also facilitates combining panthenol with other bioactive ingredients, enhancing their effects.

[0069] In summary, panthenol is a key ingredient for improving eyelash health and growth, and its encapsulation in liposomes maximizes its effectiveness, offering an advanced and effective solution for topical treatments.

[0070] • Linoleic Acid and Oleic Acid.

[0071] Linoleic acid (omega-6) and oleic acid (omega-9) are essential fatty acids that play key roles in the health of eyelashes and hair follicles. Both are fundamental components of cell membranes and are involved in regulating inflammation, tissue repair, and modulating skin permeability.

[0072] Linoleic Acid:

[0073] • Anti-inflammatory effects: Reduces the release of pro-inflammatory cytokines, improving the follicular microenvironment. Lipid barrier restoration: Strengthens the skin barrier function, preventing water loss and keeping the hair follicle hydrated.

[0074] • Promotion of Skin Permeability. Facilitates the absorption of active ingredients such as Bimatoprost.

[0075] Oleic Acid:

[0076] • Facilitation of Permeability: Increases skin permeability, improving the absorption of lipophilic compounds.

[0077] • Stimulation of Follicular Regeneration: Promotes the synthesis of structural proteins, strengthening the hair follicle.

[0078] • Anti-inflammatory effects: Regulates inflammatory pathways and protects tissues from oxidative damage.

[0079] • Synergy between Linoleic Acid, Oleic Acid and Bimatoprost

[0080] The combination of linoleic and oleic acids with bimatoprost optimizes eyelash treatment. Bimatoprost stimulates follicular growth, while the fatty acids improve drug absorption, reduce inflammation, and strengthen the hair follicle. They also improve skin permeability, ensuring greater effectiveness of bimatoprost.

[0081] Challenges and Solutions:

[0082] • Stability and Bioavailability: Linoleic and oleic acids are susceptible to oxidation, which can compromise their effectiveness. Encapsulation in liposomes stabilizes these fatty acids and improves their delivery to hair follicles.

[0083] Conclusion: Linoleic and oleic acids are essential for improving eyelash health and growth. They reduce inflammation, restore the lipid barrier, facilitate skin permeability, and strengthen the extracellular matrix. Encapsulation in liposomes maximizes their efficacy, offering a comprehensive and effective solution for treating madarosis and other hair follicle-related conditions.

[0084] • Niacinamide (Vitamin B3)

[0085] Niacinamide, an active form of vitamin B3, is a water-soluble compound with antioxidant, anti-inflammatory, and regenerative benefits for the skin and hair follicles. It improves the skin barrier function, reduces inflammation, and promotes the repair and regeneration of hair follicles, making it an ideal complement to treatments for madarosis and eyelash strengthening.

[0086] Niacinamide and Regulation of the Follicular Microenvironment:

[0087] • Reduction of Inflammation: Inhibits the release of pro-inflammatory cytokines and regulates the NF-kB pathway.

[0088] • Skin Barrier Repair: Stimulates the synthesis of ceramides, improving the hydration and stability of the hair follicle.

[0089] • Sebum Modulation: Balances sebum production, reducing follicular obstruction.

[0090] Molecular Effects on Hair Follicles:

[0091] • Stimulation of Cellular Metabolism: Increases NAD levels + improving mitochondrial function and keratin synthesis.

[0092] • Antioxidant Protection: Neutralizes free radicals and activates the Nrf2 pathway.

[0093] • DNA repair: Improves the ability to repair DNA in hair follicle stem cells.

[0094] • Synergy with Bimatoprost: The combination of niacinamide with bimatoprost in a liposomal formulation enhances eyelash growth by reducing inflammation and improving the microvascular environment. Challenges and Solutions: Encapsulation in liposomes improves the bioavailability and stability of niacinamide, overcoming penetration limitations.

[0095] Conclusion: Niacinamide is a multifunctional ingredient that improves eyelash health and growth. Its encapsulation in liposomes maximizes its benefits, setting a new standard in the treatment of madarosis and hair strengthening.

[0096] • Innovation and Opportunities in Improved Liposomal Topical Formulations of Bimatoprost

[0097] The development of liposomal formulations of bimatoprost enriched with panthenol, linoleic acid, oleic acid, and niacinamide is an innovation in ophthalmic and dermatological pharmacology. These formulations enhance the efficacy of bimatoprost and address limitations such as the low bioavailability and adverse effects of conventional topical formulations. Liposomes optimize the penetration of the drug and bioactive components, improving skin permeability and reducing inflammation. This formulation expands the therapeutic applications of bimatoprost, providing a comprehensive solution for conditions such as madarosis and localized alopecia. Furthermore, liposomal encapsulation protects the components from degradation and improves the stability of the formulation, reducing adverse effects and increasing treatment acceptance.From a market perspective, this formulation represents a scientific advance that improves the quality of life of patients and establishes a new standard in regenerative and personalized medicine.

[0098] With respect to prior art, there is invention US201313986853, with a priority date of June 12, 2013, which describes a topical formulation of bimatoprost to promote hair growth. This formulation can be used alone or in combination with other prostamides or prostaglandin analogues. The formulation is applied topically to stimulate hair growth in androgenic areas such as the mustache, beard, chest, and other areas in humans and animals. The invention addresses a long-felt but unmet need to promote strong, robust hair growth in adult men. It utilizes various application forms, including liquids, lotions, ointments, creams, gels, foams, sprays, and drug delivery nanotechnology.

[0099] The invention US201361960740, with a priority date of September 26, 2013, describes methods and pharmaceutical compositions containing prostaglandin F2α (PGF2α) analogues for the topical treatment of atrophic skin scars. The topical application of a therapeutic amount of PGF2α or its analogues directly to the area of ​​the atrophic scar is the main focus of this invention.

[0100] The invention US202318351192, with a priority date of July 12, 2023, describes penetrating transdermal drug delivery formulations. These formulations are designed for the transdermal delivery of drugs through the skin of a subject; they include a therapeutic amount of a drug and a penetrating component containing phospholipids, fatty acid esters formed from low-molecular-weight alcohols, and long-chain fatty acids. These components enhance the bioavailability and efficacy of the drug by facilitating its penetration through the skin.

[0101] Invention US202263422119, with a priority date of November 3, 2022, describes a lacrimal caruncle implant for drug delivery. This implant is placed through the patient's lacrimal caruncle for the prolonged release of medication into the eye, treating conditions such as glaucoma. The implant may include a reservoir that releases the medication at a predictable rate. Once depleted, the reservoir can be refilled in vivo with an additional quantity of the same or a different medication. Structural variations can adjust the level of comfort, resistance to extrusion, bioavailability, drug flow, and delivery of the medication to specific locations in or within the eye.

[0102] Prior inventions, even though they contain liposome-based formulations and the component Bimatoprost, do not show evidence of formulations or applications similar to the invention described herein.

[0103] The developed invention focuses on a lipid-based formulation containing Bimatoprost to increase bioavailability and extracellular matrix expression and improve follicle cell survival; and which decreases the adverse effects of topical application of Bimatoprost with conventional formulations.

[0104] OBJECTIVES OF THE INVENTION.

[0105] The invention's main objective is to provide a topical ophthalmic lipid formulation of bimatoprost that improves its bioavailability and follicle cell survival, reducing adverse effects. Other objectives include:

[0106] 1. Liposomal Formulation: Incorporates components such as panthenol, linoleic acid, oleic acid, and niacinamide to improve bioavailability, extracellular matrix expression, and hair follicle regeneration. 2. Combined Benefits: Combines bimatoprost with panthenol (moisturizing and regenerative), linoleic and oleic acids (skin permeability and lipid barrier), and niacinamide (regulation of inflammation and cellular metabolism).

[0107] 3. Controlled Release: Use a liposomal matrix for controlled and sustained release, overcoming biological barriers and ensuring adequate therapeutic concentrations.

[0108] 4. Reduction of Adverse Effects: Minimize adverse effects such as conjunctival hyperemia and hyperpigmentation, improving tolerance and acceptance of the treatment.

[0109] 5. Treatment Adherence: Offer a self-administered, safe and convenient formulation, eliminating the need for frequent applications or painful injections.

[0110] 6. Therapeutic Innovation: To provide an advanced solution for madarosis and other conditions related to hair follicle loss, improving the health and growth of eyelashes and eyebrows.

[0111] SUMMARY OF THE INVENTION.

[0112] The invention proposes an innovative ophthalmic and dermatological formulation designed to improve the bioavailability of bimatoprost in target tissues, overcoming the anatomical, physiological, and mechanical barriers that currently limit the efficacy of conventional topical formulations. This formulation combines bimatoprost with bioactive components such as panthenol, linoleic acid, oleic acid, and niacinamide, encapsulated in liposomes that optimize its transport and controlled release into the hair follicles of eyelashes, eyebrows, and scalp hair. The technology also enhances the moisturizing, regenerative, and anti-inflammatory properties of the treatment, achieving a sustained therapeutic effect.

[0113] Furthermore, the formulation employs liposomes, structures formed by phospholipid bilayers, which facilitate the transport of compounds through the cornea, epidermis, and surrounding epithelial tissue. This approach allows the active ingredients to efficiently penetrate biological barriers and reach the hair follicles directly, where they exert their effects. The incorporation of adjuvants such as Kolliphor® 15, hyaluronic acid, and essential fatty acids ensures greater drug adherence to target areas and improves its stability in the tissue microenvironment.

[0114] The inclusion of panthenol provides a moisturizing and regenerative effect, promoting cell proliferation and improving hydration in the treated areas. Meanwhile, linoleic and oleic acids work synergistically to strengthen the lipid barrier and improve skin permeability, maximizing the absorption of bimatoprost and other active ingredients into the underlying tissues. Niacinamide, with its anti-inflammatory and antioxidant properties, modulates local inflammatory responses and promotes cell regeneration in hair follicles, encouraging healthy and sustained eyelash and eyebrow growth.

[0115] Liposomes not only encapsulate and protect the bimatoprost molecule against chemical and oxidative degradation, but also prolong its retention time in target tissues, reducing the need for frequent applications and minimizing side effects. This formulation ensures sustained therapeutic concentrations in the follicular microenvironment, prolonging the anagen phase of the hair cycle and improving the structural and functional health of the eyelashes. Compared to conventional topical bimatoprost formulations, this advanced liposomal technology reduces the risk of adverse effects such as conjunctival hyperemia, hyperpigmentation, and dry eye, increasing treatment tolerance. Furthermore, the formulation facilitates self-administration, enhancing patient comfort and improving treatment adherence—key factors for the effective management of madarosis and other related conditions.

[0116] In situations where adjunctive use is required, the formulation can be integrated with other therapies to maximize their benefits, providing a versatile solution that addresses patients' clinical and aesthetic needs. This therapeutic approach represents a significant advance in the non-invasive treatment of dermatological and ophthalmic conditions, with the potential to transform clinical practice by making treatment safer, more accessible, and more effective.

[0117] DETAILED DESCRIPTION OF THE INVENTION.

[0118] The invention, along with its embodiments and associated benefits, will now be presented in detail. 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.

[0119] Feature of the invention. The developed invention is an advanced formulation designed to optimize the bioavailability of bimatoprost and enhance its therapeutic effects. The formulation integrates bioactive components such as panthenol, linoleic acid, oleic acid, and niacinamide, encapsulated in liposomes. It addresses the limitations of conventional topical therapies by overcoming anatomical and physiological barriers, offering an effective solution for the treatment of madarosis and other conditions related to hair follicle loss. The liposomal technology allows for a controlled and sustained release of the active components, improving their action on the hair follicles of eyelashes and eyebrows.

[0120] • Constituent elements of the invention.

[0121] Bimatoprost is the main active ingredient in this formulation, known for its ability to stimulate eyelash growth by prolonging the anagen phase of the hair cycle. At the molecular level, bimatoprost acts as an agonist of FP receptors, present in hair follicles. This interaction activates signaling pathways such as the PI3K / AKT pathway, which promotes cell survival and proliferation, and stimulates extracellular matrix synthesis in the follicular microenvironment. Encapsulation of bimatoprost in liposomes not only protects the molecule from chemical and oxidative degradation, but also enhances its penetration through the epidermal barrier to the target areas, allowing for sustained therapeutic concentrations that maximize its efficacy.

[0122] Panthenol, a precursor to pantothenic acid, plays a key role in this formulation. Its moisturizing, regenerative, and anti-inflammatory properties strengthen hair follicles, maintaining a hydrated microenvironment essential for the proliferation of keratinocytes and fibroblasts. At the molecular level, panthenol contributes to the synthesis of structural lipids in cell membranes, facilitating tissue repair and improving eyelash strength. Furthermore, it regulates the expression of genes related to the synthesis of keratin, a fundamental protein for hair structure.

[0123] The essential fatty acids linoleic acid and oleic acid offer multiple benefits. Linoleic acid, as a precursor to anti-inflammatory prostaglandins, modulates inflammatory pathways such as NF-κB, reducing the release of pro-inflammatory cytokines like IL-1β and TNF-γ. This creates an environment conducive to follicular regeneration and protects follicles from chronic inflammation associated with conditions like blepharitis. Oleic acid, for its part, improves skin permeability by altering the lipid organization in cell membranes, allowing for efficient absorption of active ingredients. Both fatty acids strengthen the skin's lipid barrier, reducing transepidermal water loss and optimizing tissue hydration.

[0124] Niacinamide, or vitamin B3, is a multifunctional compound that regulates inflammation, stimulates cell regeneration, and protects against oxidative stress. At the molecular level, niacinamide increases intracellular levels of NAD+. +Niacinamide, an essential coenzyme for energy metabolism and DNA repair, enhances mitochondrial function in hair follicle stem cells, prolonging the anagen phase of the hair cycle and promoting regeneration. Furthermore, niacinamide activates the Nrf2 signaling pathway, increasing the expression of antioxidant enzymes such as superoxide dismutase (SOD) and catalase, thus protecting hair follicles from the damaging effects of oxidative stress. Phosphatidylcholine, an essential phospholipid in liposome formation, ensures the structural stability of the formulation. This component creates biocompatible lipid bilayers that encapsulate the active ingredients, protecting them from degradation and facilitating their controlled release. Phosphatidylcholine also allows liposomes to fuse with the cell membranes of hair follicles, improving the penetration of active ingredients into the target areas.This process is supported by its ability to adjust the size and surface charge of liposomes, optimizing the bioavailability and efficacy of the formulation.

[0125] Cholesterol is a key stabilizer in the liposomal structure, improving robustness and reducing leakage of the encapsulated active ingredients. Its inclusion in the formulation prolongs the retention time of bimatoprost and other bioactive compounds in the target tissue. By modulating the fluidity of the lipid bilayer, cholesterol ensures controlled release that maximizes therapeutic efficacy while reducing the need for frequent applications.

[0126] PEG-12 glyceryl myristate and Kolliphor® 15 are essential emulsifiers that improve the stability and dispersion of liposomes in the formulation. These excipients facilitate the integration of the active ingredients in a lipophilic and aqueous environment, ensuring uniform distribution and efficient absorption into the hair follicles. Furthermore, these emulsifiers reduce surface tension, prolonging the product's contact time with the eyelashes and optimizing its therapeutic action.

[0127] The buffering agents, anhydrous citric acid and sodium citrate dihydrate, stabilize the pH of the formulation to ensure user comfort and safety. This buffering system prevents irritation during application, maintaining the chemical balance and stability of the liposomes and active ingredients.

[0128] Finally, benzalkonium chloride acts as a preservative, protecting the formulation from microbiological contamination. Its concentration is carefully adjusted to ensure sterility without compromising patient tolerance.

[0129] The formulation is designed to be integrated into a mascara or eyeliner pencil, offering an innovative solution that combines cosmetic and therapeutic benefits in a practical and user-friendly format. This design not only improves patient comfort but also ensures optimal adherence to treatment, maximizing clinical and aesthetic benefits.

[0130] • Mechanisms for Improving Penetration into the Hair Follicle.

[0131] The optimized liposomal formulation of bimatoprost for the treatment of madarosis incorporates several mechanisms that enhance penetration into hair follicles by leveraging interaction with the cell membranes of the follicular epithelium and surrounding tissue. The liposomes, stabilized with PEG-12 glyceryl myristate (Qusomes®) and enhanced with Kolliphor® 15, facilitate the absorption of bimatoprost and other bioactive components through the epidermis and into the hair follicles by fusing with the cell membranes of the outer epithelial layers. This structure allows the encapsulated bimatoprost to cross tight cell junctions and effectively penetrate the protective layers of the tissue, directly reaching the microenvironment of the hair follicle. Linoleic and oleic acids act as permeability modifiers of the epithelial lipid barrier, facilitating the entry of the liposomes into the hair follicles.These essential fatty acids also improve the barrier function, strengthening the skin's protective environment while allowing for more effective absorption of bimatoprost and the regenerative components. Hyaluronic acid, incorporated as a moisturizing and viscoelastic agent, prolongs the product's retention time on the skin surface and eyelid margin, optimizing the gradual absorption of the drug into the hair follicles and maximizing its effectiveness.

[0132] The inclusion of cholesterol in the lipid bilayer stabilizes the liposome structure, controlling the release of bimatoprost and ensuring sustained penetration into the follicle. Simultaneously, phosphatidylcholine, a major component of liposomes, reinforces the structural integrity of the bilayers, promoting effective fusion with the cell membranes of the follicular epithelium. This process not only enhances the controlled release of bimatoprost but also protects the active molecule from chemical and oxidative degradation on the skin surface.

[0133] Overall, this liposomal formulation not only protects Bimatoprost from degradation on the tissue surface, but also optimizes its retention and absorption in the hair follicles. This technology ensures prolonged and effective therapeutic action, maximizing Bimatoprost's impact on extending the anagen phase of the hair cycle and on the regeneration of eyelashes and eyebrows.

[0134] • Overcoming Epidermal and Follicular Barriers.

[0135] The size of liposomes, typically between 100 and 200 nm, allows them to bypass the structural barriers of the epidermis and facilitates their transport into the hair follicle. This size, along with surface pegylation using PEG-12 glyceryl myristate, improves liposome stability and prolongs their residence time on the skin surface. This mechanism ensures that the liposomes and their contents, including bimatoprost and other bioactive agents, reach the follicle stem cells at sustained therapeutic concentrations.

[0136] Furthermore, modulating lipid membrane fluidity with cholesterol and phosphatidylcholine allows liposomes to interact specifically with the cell membranes of the follicular epithelium, improving the selective release of bimatoprost and reducing the need for frequent applications. This approach ensures that the drug reaches the cells responsible for hair regeneration, optimizing its therapeutic impact.

[0137] • Sustained and Targeted Release of Bimatoprost.

[0138] Encapsulation of bimatoprost in liposomes allows for gradual and controlled release into the microenvironment of the hair follicle. This system, designed for topical application to the eyelid margin, ensures sustained drug release into the hair follicle cells, maximizing its therapeutic activity. This design reduces the need for frequent applications and minimizes fluctuations in drug concentrations within the tissue, increasing the overall efficacy of the treatment.

[0139] The liposomal system also protects Bimatoprost from degradation during storage and after application, ensuring its stability and extending its shelf life. Furthermore, the controlled release of Bimatoprost helps minimize adverse effects, such as skin irritation or local reactions, improving the patient experience and adherence to treatment. • Justification of the Invention.

[0140] The integration of bimatoprost into a liposomal formulation represents a significant advance in the treatment of madarosis and other related conditions. follicle day The prosomes enhance follicular penetration and sustained release of the active components, overcoming the anatomical barriers of the follicular tissue. This design not only improves the efficacy of Bimatoprost, but also amplifies the beneficial effects of adjuvants such as panthenol, linoleic acid, oleic acid, and niacinamide.

[0141] The liposomal formulation stabilizes and maximizes the bioavailability of bimatoprost in the follicular microenvironment, setting a new standard in madarosis therapy. This therapeutic approach offers a non-invasive, safe, and effective solution, improving patients' quality of life by providing superior clinical results in eyelash and eyebrow growth and strengthening.

[0142] • Characterization of the Liposomal Formulation of Bimatoprost.

[0143] • Development of Candidate Formulations.

[0144] In developing this liposomal formulation for the treatment of madarosis, the physicochemical properties of bimatoprost, its limitations in penetrating hair follicles, and the need to ensure controlled and sustained release were carefully considered. Several formulations were designed and analyzed, adjusting the concentrations of bimatoprost, the size of the liposomal particles, and the proportions of stabilizers.

[0145] Initially, formulations were developed containing only bimatoprost as the main active agent. These formulations optimized the bimatoprost concentration at 0.02% w / v (200 pg / ml), allowing for a sustained therapeutic effect. Benzalkonium chloride (0.01% w / v) was included as a preservative, while pharmaceutical-grade purified water was used as the main solvent.

[0146] To ensure the stability of the formulations, different pH buffer systems were tested, and thermal stability tests were performed under controlled conditions (30°C, 40°C, and 60°C for 21 days). These tests confirmed the stability of the liposomal formulation and its suitability for topical applications.

[0147] • Physicochemical Characterization of the Formulations.

[0148] pH, viscosity, and osmolarity analyses were performed according to international standards, ensuring that the formulations were compatible with the eyelid margin. Additional isotonicity and absorption tests confirmed the product's safety, maximizing the efficacy of Bimatoprost in the hair follicles.

[0149] The encapsulated content and concentration of bimatoprost were verified by high-performance liquid chromatography (HPLC) under the following conditions: Column: Zorbax Eclipse Plus C18, 4.6 x 100 mm, particle size 3.5 µm; Wavelength (λ): 254 nm; Column temperature: 30°C; Flow rate: 1 mL / min; Injection volume: 20 mL; Limit of detection (LOD): 6 pg / mL; Limit of quantification (LOQ): 20 pg / mL. The formulations were filtered through a 0.2 µm membrane, allowing for the determination of the amount of encapsulated and unencapsulated bimatoprost. This procedure ensured that the liposomal bimatoprost was available for controlled and sustained release.

[0150] • Accelerated Stability Tests.

[0151] The formulations were subjected to accelerated stability testing at 40°C ± 3°C, evaluating appearance, pH, and sterility. The results demonstrated that the formulation is stable under stress conditions and maintains its efficacy under various storage conditions.

[0152] • Initial formulation with Bimatoprost.

[0153] The selected formulation contains bimatoprost at a concentration of 0.02% w / v, equivalent to 200 µg / ml. Each drop (50 µl) contains 10 µg of bimatoprost, ensuring precise dosing. The liposomes are composed of phosphatidylcholine and cholesterol in a 70:30 ratio, with a total liposome concentration of between 5% and 10% w / v. This liposomal system protects the bimatoprost from degradation and facilitates its controlled release into the hair follicles.

[0154] The formulation also includes PEG-12 glyceryl myristate (3% w / v) as a solubilizer and stabilizer, promoting the penetration of bimatoprost through the eyelid epithelium. In addition, Kolliphor® 15 (polyethylene glycol-15 hydroxystearate) at a concentration of 3% w / v acts as an emulsifier, improving the stability of the liposomes and their dispersion on the skin surface. Preparation Method.

[0155] Bimatoprost is dissolved in a mixture of phosphatidylcholine and cholesterol (70:30) to form liposomes at a concentration of 5–10% w / v. Kolliphor® 15 and PEG-12 glyceryl myristate are added to stabilize the mixture and improve drug solubility. Purified water with anhydrous citric acid and sodium citrate dihydrate as buffers is then incorporated, along with benzalkonium chloride as a preservative. The mixture is stirred at room temperature to ensure homogeneity and efficient encapsulation of the bimatoprost.

[0156] This formulation achieves precise, stable, and effective topical administration, capable of overcoming the anatomical barriers of the hair follicle and providing a non-invasive treatment for madarosis.

[0157] • Resulting Formulation.

[0158] The topical liposomal formulation with bimatoprost as the main active ingredient is designed to enhance hair follicle regeneration in eyelashes and eyebrows. The key components of the formulation and its preparation are described below.

[0159] • Formulation composition per 1 ml.

[0160] • Bimatoprost: 0.02% w / v (200 pg / ml)

[0161] • It acts as the main active agent, prolonging the anagen phase of the hair cycle and stimulating the growth of eyelashes and eyebrows.

[0162] • Polyethylene glycol (PEG-12) Glyceryl Myristate (Qusomes®): 30 mg (3% w / v)

[0163] • Solubilizer and stabilizer that improves the penetration of Bimatoprost and ensures uniform dispersion of liposomes.

[0164] « Kolliphor® 15 (Polyethylene glycol-15 Hydroxystearate): 3% w / v • Emulsifier that stabilizes liposomes and improves their absorption in hair follicle tissues.

[0165] * F o sf a ti di I co I ina: 70 mg

[0166] • It forms the lipid bilayer of liposomes, ensuring stable encapsulation of Bimatoprost.

[0167] * Cholesterol: 30 mg

[0168] • Stabilizes the structure of liposomes, prolonging the controlled release of the drug.

[0169] * Anhydrous Citric Acid: 0.04% ■ 0.16% w / v

[0170] • Adjusts the pH of the formulation, ensuring stability and compatibility with the dermal surface.

[0171] * Sodium Citrate Dihydrate: 0.23% - 0.69% w / v

[0172] • It acts as a buffer to stabilize pH.

[0173] * Benzalkonium Chloride: 0.01% w / v

[0174] • Antimicrobial preservative that ensures the microbiological stability of the formulation.

[0175] •

[0176] * Pharmaceutical Grade Purified Water: QS until 1 ml is reached

[0177] • Main solvent that guarantees the compatibility of the product with the fabrics.

[0178] • Analysis of the Resulting Formulation.

[0179] Physicochemical characterization tests were performed to evaluate the stability and safety of the formulation: pH: Measured using a potentiometer, a value of 6.8 was obtained, within the appropriate physiological range to avoid irritation. Viscosity: A value of 15.0 cP was determined at 33°C, sufficient to guarantee the retention of the formulation in the hair follicles. Osmolarity: A value of 300 mOsmol / L was recorded, corresponding to an isotonic solution that does not cause discomfort. Particle Diameter (Z): An average of 200 nm was obtained, ideal for facilitating penetration into the hair follicles. Polydispersity Index (PDI): With a value of 0.3, the formulation exhibits a uniform and stable particle distribution.

[0180] • Formulation Preparation.

[0181] 1. Lipid Mixture: Dissolve phosphatidylcholine and cholesterol in a 70:30 ratio in an ethanolic solution, ensuring the formation of stable liposomes.

[0182] 2. Incorporation of the Active Ingredient: Add Bimatoprost to the lipid mixture under constant stirring at 25°C ± 1 °C.

[0183] 3. Addition of Solubilizers: Incorporate PEG-12 glyceryl myristate and Kolliphor® 15 to stabilize the mixture and improve solubility.

[0184] 4. Preparation of the Aqueous Solution: Dissolve anhydrous citric acid, sodium citrate dihydrate and benzalkonium chloride in purified water.

[0185] 5. Final Emulsion: Slowly integrate the aqueous solution into the lipid mixture under agitation, ensuring a homogeneous dispersion.

[0186] • Physicochemical Results

[0187] The following table summarizes the formulation characteristics:

[0188] • Application.

[0189] The formulation offers several application methods, all of which have been successfully tested and are listed below: a) Using an applicator brush or as a serum on the eyelid margin and eyebrow area. Its liposomal design ensures efficient penetration into the hair follicles, promoting their regeneration and improving the density of eyelashes and eyebrows. b) Using mascara, for direct application to the eyelashes, characterized by a viscosity suitable for retention without runoff. c) Using a gel, formulated for even application to the eyebrows or eyelashes, guaranteeing controlled release into the hair follicle. d) Using shampoo, designed for application to eyelashes, eyebrows, or scalp, optimizing absorption through the hair follicles during washing.e) Via serum with a brush applicator, for localized and precise administration to eyelashes and eyebrows, with viscoelastic properties that prolong the product's contact time. f) Via a transdermal patch for application to the upper eyelid or eyebrow area, with sustained-release technology that maximizes drug penetration into the hair follicles. g) Via a microemulsion dermal spray, designed to cover large areas such as the eyebrows or scalp, ensuring uniform and non-invasive application. h) Via a fast-absorbing foam, formulated for application to areas with hair follicles requiring cosmetic or therapeutic treatment.

[0190] EXAMPLE 1:

[0191] • Results of Diffusion of the Liposomal Formulation of Bimatoprost.

[0192] • Evaluation of the Liposomal Formulation of Bimatoprost using Modified Franz Diffusion Chambers (Skin and Follicle Model).

[0193] Modified Franz diffusion chambers represent an advanced and suitable method for evaluating drug penetration into complex tissues, such as hair follicles and the surrounding skin. This model allows for the simulation of the specific physiological and anatomical barriers of eyelashes and eyebrows, including the hair follicle microenvironment, making it an ideal tool for analyzing the efficacy of formulations designed to treat madarosis.

[0194] • Study Design.

[0195] The experiment was conducted using modified Franz chambers fitted with ex vivo skin containing intact hair follicles. This system allows for the evaluation of both transfollicular penetration and accumulation in the surrounding tissue.

[0196] Study Parameters:

[0197] • Biological sample: Ex vivo skin with hair follicles (e.g., human or pig skin, preserved under physiological conditions).

[0198] • Duration of the experiment: 24 hours.

[0199] • Initial concentration of Bimatoprost: 0.02% w / v (200 pg / mL).

[0200] • Number of applications: 50 pL (10 pg of Bimatoprost per drop) administered at 4-hour intervals (0, 4, 8, 12, 16 and 20 hours).

[0201] • Temperature conditions: 32°C ± 1 °C, simulating the surface temperature of the human body.

[0202] • Analysis method: C u antification of Bimatoprost by HPLC in the receptor, follicular and dermal compartments.

[0203] Diffusion Results Table.

[0204] • Results and Analysis.

[0205] 1. Control (non-encapsulated bimatoprost): The control showed limited and cumulative penetration of up to 1 pg (10% of the total administered) into the follicular and dermal compartments. This result reflects the natural barriers of the skin and hair follicle, which restrict the penetration of conventional formulations.

[0206] 2. Liposomal Formulation: The liposomal formulation achieved an accumulation of 24 g (40% of the total administered), highlighting its ability to effectively penetrate hair follicles and surrounding tissue. The liposomes improved the retention of Bimatoprost in the hair follicle due to their interaction with the follicle's lipid membranes.

[0207] 3. Distribution in Follicles and Surrounding Skin: o HPLC analysis revealed that 80% of the absorbed Bimatoprost accumulated specifically in the hair follicle, with 20% distributed in the surrounding skin.

[0208] 4. Advantages of the Liposomal Formulation: o The sustained release of bimatoprost encapsulated in liposomes allows for prolonged and controlled penetration, maintaining therapeutic concentrations in the follicles for a longer period. o The incorporation of PEG-12 glyceryl myristate and Kolliphor® 15 significantly improved the solubility and absorption of bimatoprost in the hair follicle.

[0209] • Interpretation of Results.

[0210] The results obtained in modified Franz diffusion chambers clearly demonstrate the superiority of the liposomal formulation compared to unencapsulated bimatoprost. This improvement in penetration and bioavailability is critical for treatments such as madarosis, where effectiveness depends on achieving adequate concentrations in the hair follicle.

[0211] Key findings:

[0212] • Efficient transfollicular penetration: Liposomes optimize the transport of Bimatoprost to the hair follicle, overcoming the physical barriers of the skin and sebaceous gland.

[0213] • Sustained release: The formulation ensures the constant presence of the drug in the hair follicle, which can result in better therapeutic outcomes and less frequent application.

[0214] • Biological compatibility: The excipients used in the formulation (phosphatidylcholine, cholesterol, PEG-12 and hyaluronic acid) are biocompatible, minimizing the risk of irritation and optimizing adherence to treatment.

[0215] • Impact on the Patent Application.

[0216] These exemplary results provide strong evidence of the technological innovation behind this liposomal formulation of Bimatoprost, justifying its efficacy for targeted penetration into hair follicles. This supports its application in the treatment of madarosis and other disorders related to eyelash and eyebrow loss, and strengthens the patent application as an advanced and differentiated solution in the dermatological and cosmetic market.

[0217] • INCORPORATION OF ADDITIONAL BIOACTIVES: IMPROVED FORMULATIONS. Subsequently, improved formulations were developed incorporating bioactive agents such as panthenol, linoleic acid, oleic acid, and niacinamide. These components complement the action of Bimatoprost, strengthening hair follicles and enhancing their regeneration.

[0218] Panthenol improves hydration and promotes cell proliferation, while linoleic and oleic acids optimize penetration and the functionality of the lipid barrier. Niacinamide, for its part, regulates inflammation and stimulates NAD synthesis. + crucial for cell regeneration.

[0219] These advanced formulations maintained the ratios of phosphatidylcholine and cholesterol, ensuring the stability of the liposomes and the controlled release of the active ingredients into the hair follicles.

[0220] • Physicochemical Characterization of Formulations with Additional Bioactives.

[0221] • Physicochemical Analysis.

[0222] The inclusion of additional bioactives such as panthenol, linoleic acid, oleic acid, and niacinamide in the liposomal bimatoprost formulation significantly improves its therapeutic profile. To ensure the formulation's stability and efficacy, pH, viscosity, osmolarity, and encapsulation were evaluated in accordance with international standards.

[0223] Characterization Parameters:

[0224] • pH: Adjusted to a range of 6.5 - 7.5 to avoid irritation on the eyelid margin.

[0225] • Viscosity: Established in a range of 10-20 cP to ensure adequate retention on the dermal and follicular surface. • Osmolarity: 300 mOsmol / L, suitable for isotonic solutions and comfortable for the skin.

[0226] • Encapsulation: Confirmed by HPLC for all encapsulated actives, ensuring controlled release and optimal bioavailability.

[0227] • Stability tests.

[0228] The formulations were subjected to accelerated stability testing at 40°C ± 3°C, with continuous evaluations for 90 days. The results confirmed the chemical and physicochemical stability of Bimatoprost and the additional bioactives, with no significant degradation under thermal stress conditions.

[0229] • Composition of the Improved Formulation.

[0230] The improved formulation includes Bimatoprost as the main active agent, complemented with bioactives designed to enhance the growth and regeneration of hair follicles, while maintaining safety and efficacy.

[0231] EXAMPLE 2:

[0232] • Individual Diffusion Results for Bioactive Components.

[0233] To evaluate the efficacy of each component of the liposomal formulation, experiments were conducted using modified Franz diffusion chambers (Skin and Follicle Model). This analysis allowed for the quantification of the specific penetration and accumulation of bimatoprost, panthenol, linoleic acid, oleic acid, and niacinamide in hair follicles. Study Design

[0234] • Duration: 24 hours

[0235] • Conditions: Constant temperature of 32°C ± 1 °C

[0236] • Initial concentrations: o Bimatoprost: 0.02% w / v Panthenol: 0.5% w / v Linoleic Acid: 0.2% w / v Oleic Acid: 0.2% w / v Niacinamide: 0.5% w / v

[0237] • Analysis method: C u antification by HPLC in the receptor, dermal and follicular compartments.

[0238] Table of Individual Diffusion Results

[0239] Interpretation of Results by Component. 1. Bimatoprost: o Cumulative penetration of 30 pg (40% of the total administered). o Maximum concentration reached in the hair follicle, confirming its efficacy as the main agent.

[0240] 2. Panthenol: o The highest cumulative penetration (40 pg, 80% of the total administered) among all bioactives. o Excellent retention in the follicle, deep hydration, and optimization of the extracellular matrix.

[0241] 3. Linoleic Acid: o Moderate cumulative penetration (20 pg, 50% of the total administered). o Acts as a precursor of ceramides, strengthening the lipid barrier of the follicle.

[0242] 4. Oleic Acid: o Cumulative penetration of 12 pg (30% of the total administered). o Facilitates cell regeneration and improves lipid absorption.

[0243] 5. Niacinamide: o Cumulative penetration of 30 pg (60% of the total administered). o Prominent anti-inflammatory effects, improving follicle function and regeneration.

[0244] Comparative Results.

[0245] The results show that each component contributes synergistically to the ultimate goal of regenerating and strengthening hair follicles. Together, these bioactives significantly improve the penetration, retention, and therapeutic action of the formulation. • Liposomal synergy: The lipid structure of liposomes enhances the penetration and bioavailability of all components in the hair follicles.

[0246] • Follicular distribution: More than 80% of the bioactives accumulated in the hair follicles, with a smaller amount distributed in the surrounding skin.

[0247] Impact on Formulation Development.

[0248] These data highlight the importance of including multiple bioactives in the liposomal formulation, demonstrating its ability to address various aspects of the hair cycle. The incorporation of bimatoprost along with panthenol, essential fatty acids, and niacinamide not only optimizes follicular growth and regeneration but also sets a high standard for advanced treatments in madarosis and hair regeneration.

[0249] • Analysis of the Liposomal Formulation using Transmission Electron Microscopy (TEM).

[0250] • Introduction.

[0251] Structural characterization of encapsulating liposomes is essential to demonstrate their proper formation, uniformity, and functionality in the bimatoprost formulation. Transmission electron microscopy (TEM) was used as a key tool to directly visualize the morphology, size, and internal arrangement of the liposomes, ensuring the viability of this technology for delivering bimatoprost to hair follicles. Materials and Methods.

[0252] 1. Sample preparation: o Samples of the liposomal formulation in aqueous suspension were taken. o A small aliquot was diluted 1:10 in distilled water to avoid particle agglomeration. o Drops of the diluted suspension were placed onto copper grids coated with a carbon film, followed by controlled vacuum drying. o Negative staining with 2% uranyl acetate was applied to enhance the contrast of the liposomal structures.

[0253] 2. TEM Analysis: o A JEOL JEM-2100 transmission electron microscope was used, operating at an accelerating voltage of 200 kV. o Images were captured on a digital electron detector with a resolution of 2 nm. o Multiple captures were performed to evaluate the size distribution and morphology in at least 50 liposomes per sample.

[0254] 3. Image processing: o The images obtained were analyzed with ImageJ software to measure the average size, dispersion index and structural integrity.

[0255] • Results.

[0256] Morphology

[0257] TEM images showed well-formed liposomes with a spherical structure and defined lipid bilayers. Each liposome exhibited a central encapsulating core surrounded by symmetrical lipid bilayers, confirming the correct self-assembly of phosphatidylcholine and cholesterol.

[0258] Size

[0259] • Average: The liposomes had an average diameter of 200 ± 90 nm, in accordance with the values ​​obtained by dynamic light scattering (DLS).

[0260] • Distribution: The analysis revealed a homogeneous distribution, with a polydispersity index (PDI) less than 0.3, indicating high uniformity in preparation.

[0261] Integrity

[0262] Negative staining showed that more than 95% of the liposomes retained their structural integrity with no evidence of fusion or collapse, even after the staining process.

[0263] Encapsulated Load

[0264] The internal electron density suggested effective encapsulation of bimatoprost, consistent with the concentration calculated in the high-performance liquid chromatography (HPLC) assays. Charged liposomes showed a denser core, indicating greater accumulation of the bioactive compound compared to empty liposomes.

[0265] Interaction with Hair Follicles

[0266] In in vitro models, effective adhesion of liposomes to the base of simulated hair follicles was observed, confirmed by TEM imaging that revealed liposomes partially fusing with the lipid membranes of follicular epithelial cells.

[0267] Discussion

[0268] 1. Confirmation of formation: The presence of well-defined lipid bilayers confirms that the liposomes assembled properly in the formulation. This validates the liposomal design as a robust system for encapsulating and protecting bioactive compounds.

[0269] 2. Homogeneity: The uniform size distribution ensures that liposomes are ideal for penetrating the hair follicle microenvironment. Liposomes smaller than 200 nm have documented advantages in transfollicular penetration.

[0270] 3. Stability and encapsulation: The high structural integrity and internal density observed in liposomes loaded with Bimatoprost support the ability to efficiently encapsulate the bioactive, protecting it from premature degradation.

[0271] 4. Evidence of penetration: The interactions observed between liposomes and follicular epithelial membranes in simulated models suggest effective delivery of Bimatoprost to the hair follicle nucleus.

[0272] Conclusion

[0273] TEM analysis provides strong evidence that the liposomes in the bimatoprost formulation are well-formed, structurally stable, and functionally suitable for topical application. Their size and morphology confirm their suitability for transfollicular penetration, validating their therapeutic potential in the treatment of madarosis. These characteristics support the patent application, highlighting the technological innovation and efficacy of this advanced liposomal formulation.

[0274] 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 use that increases the bioavailability and expression of the extracellular matrix to improve follicle cell survival and decrease adverse effects associated with conventional topical application of Bimatoprost, characterized by comprising per 1 ml: a) Bimatoprost: 0.02% w / v (200 pyg / ml); b) Polyethylene glycol (PEG-12) Glyceryl Myristate: 30 mg (3% w / v); c) Kolliphor® 15 (Polyethylene glycol-15 Hydroxystearate): 3% w / v; d) Phosphatidylcholine: 70 mg; e) Cholesterol: 30 mg; f) Anhydrous Citric Acid: 0.04% - 0.16% w / v; g) Sodium Citrate Dihydrate: 0.23% - 0.69% w / v; h) Benzalkonium Chloride: 0.01% w / v; i) Pharmaceutical Grade Purified Water: QS to complete 1 ml.

2. The lipid-based formulation according to claim 1, wherein the pH of the resulting formulation has a value of 6.8 within the physiological range suitable to avoid irritation.

3. The lipid-based formulation according to claim 1, wherein the viscosity of the resulting formulation has a value of 15.0 cP at 33°C, sufficient to ensure retention of the formulation in the hair follicles.

4. The lipid-based formulation according to claim 1, wherein the osmolarity of the resulting formulation has a value of 300 mOsmol / l, corresponding to an isotonic solution that does not cause discomfort.

5. The lipid-based formulation according to claim 1, wherein the particle diameter of the resulting formulation has a average of 200 nm, ideal value to facilitate penetration into hair follicles.

6. The lipid-based formulation according to claim 1, wherein the polydispersity index has a value of 0.3, which presents a uniform and stable particle distribution.

7. The lipid-based formulation according to claim 1, wherein the formulation has the property of encapsulating Bimatoprost in liposomes that allow a gradual and controlled release into the microenvironment of the hair follicle.

8. The lipid-based formulation according to claim 1, wherein the formulation is applied by any of the following means: a) By means of an applicator brush or in serum form on the eyelid margin and eyebrow areas, its liposomal design ensures efficient penetration into the hair follicles, promoting their regeneration and improving the hair density of the eyelashes and eyebrows; b) By means of mascara, for direct application to the eyelashes, characterized by a viscosity suitable for retention on the eyelashes without runoff; c) By means of a gel, formulated for uniform application on the eyebrows or eyelashes, guaranteeing controlled release into the hair follicle; d) By means of shampoo, designed to be applied to eyelashes, eyebrows, or scalp, optimizing its absorption through the hair follicles during washing; e) By means of a serum with a brush applicator, for localized and precise administration to eyelashes and eyebrows, with viscoelastic properties that prolong the contact time of the product; f) By means of a transdermal patch for application to the upper eyelid or eyebrow area, with sustained-release technology that maximizes drug penetration into the hair follicles; g) By means of a microemulsion dermal spray, designed to cover large areas such as the eyebrows or scalp, ensuring uniform and non-invasive application; h) By means of a fast-absorbing foam, formulated for application to areas with hair follicles that require cosmetic or therapeutic treatment.

9. The lipid-based formulation according to claim 1, supplemented with additional bioactives to enhance the growth and regeneration of hair follicles, characterized in comprising per 1 ml: a) Panthenol (Vitamin B5): 0.5% w / v; b) Linoleic Acid: 0.2% w / v; c) Oleic Acid: 0.2% w / v; d) Niacinamide (Vitamin B3): 0.5% w / v.

10. A method for preparing a lipid-based formulation for topical use that increases the bioavailability and expression of the extracellular matrix to improve the survival of follicle cells and decrease adverse effects associated with conventional topical application of Bimatoprost, characterized in that it comprises the steps: a) Phosphatidylcholine and cholesterol are dissolved in a 70:30 ratio in an ethanolic solution, ensuring the formation of stable liposomes; b) Bimatoprost is added to the lipid mixture under constant stirring at 25°C ± 1 °C; c) PEG-12 glyceryl myristate and Kolliphor® 15 are incorporated to stabilize the mixture and improve solubility; d) Anhydrous citric acid, sodium citrate dihydrate and benzalkonium chloride are dissolved in purified water; e) The aqueous solution is slowly integrated into the lipid mixture under stirring, ensuring homogeneous dispersion.

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