COMPOSITION OF NANOSTRUCTURED LIPID CARRIER CONTAINING 1-NITRO-2-PHENYLETANE, PROCESS OF OBTAINING AND USE FOR THE TREATMENT OF DERMATOphytoses

Description

1 / 35 Descriptive Report of Invention Patent Composition of a Nanostructured Lipid Carrier Containing 1-Nitro-2-phenylethane, Process for Obtaining and Use in the Treatment of Dermatophytosis Field of Invention

[0001] This patent application relates to a nanotechnology-based pharmaceutical composition intended for the topical treatment of dermatophytoses. More specifically, the pharmaceutical composition comprises a nanostructured lipid carrier (NLC) containing 1-nitro-2-phenylethane as its main active ingredient, with or without methyleugenol, for the treatment of skin or skin appendage infections caused by dermatophyte fungi. Background of the Invention

[0002] Dermatophytoses are infections of the skin or skin appendages caused by dermatophyte fungi, which consume the keratin present in these areas to maintain their metabolic functions. Dermatophytoses, also called tinea, can occur in various areas of the skin, which gives rise to their clinical classifications, such as on the body (called tinea corporis), on the feet (tinea pedis), on the hands (tinea manus), in the beard area (tinea barbae), on the glabrous skin of the face (tinea faciei), and on the scalp (tinea capitis). In general, skin dermatophytoses are caused by fungi of the genera Trichophyton, Microsporum, and Epidermophyton and generate inflammatory reactions, spots, rashes, scaling, and itching in the affected area, negatively impacting the quality of life of the individual affected by the disease. Dermatophytoses can also affect the nails, being designated as tinea unguium or onychomycosis (DEGREEF, H. Clinical forms of dermatophytosis (ringworm infection).Mycopathology, vol. 166, no. 5, p. 257-265, 2008). Petition 870220112614, dated 02 / 12 / 2022, page 15 / 61 2 / 35

[0003] Onychomycosis is a fungal infection affecting the fingernails or toenails, primarily caused by dermatophyte fungi, but also by non-dermatophyte fungi or yeasts. This disease is considered the most common nail disease, frequently occurring in older individuals. It has a worldwide prevalence of 5.5%, which tends to increase due to the aging population. Risk factors for onychomycosis, in addition to advanced age, include: persistent trauma to the nail plate, use of occlusive shoes, humid environments, tinea pedis (athlete's foot), psoriasis, hyperhidrosis, diabetes, immunosuppressive conditions, smoking, and genetic predisposition (GUPTA, AK et al. Onychomycosis: a review. Journal of the European Academy of Dermatology and Venereology, v. 34, n. 9, p. 1972-1990, 2020).

[0004] Approximately 90% of foot onychomycosis and 75% of hand onychomycosis are caused by dermatophyte fungi, most notably Trichophyton rubrum and Trichophyton mentagrophytes, which are responsible for the vast majority of dermatophytic onychomycosis cases. Non-dermatophyte fungi are responsible for only about 10 to 20% of onychomycosis cases, among which are agents of the Aspergillus and Fusarium species. Onychomycosis caused by yeasts is less common; however, when it occurs, it is usually due to Candida albicans infections (LEUNG, AKC et al. Onychomycosis: an updated review. Recent patents on inflammation & allergy drug discovery, v. 14, n. 1, p. 32-45, 2020).

[0005] The nail apparatus does not possess effective cellular immunity, and onychomycosis is acquired through direct contact of the fungal agent with the nails, considering the risk factors previously mentioned. The fungi that cause this disease produce keratolytic, proteolytic, and lipolytic enzymes, which aid in the degradation of keratin and other constituents of the nail plate, favoring the establishment of the fungal agent in the nails. In this sense, clinically, onychomycosis causes: discoloration Petition 870220112614, dated 02 / 12 / 2022, page 16 / 61 3 / 35 of the nail plate, which acquires a yellowish, whitish, or brownish appearance; detachment of the nail plate from the nail bed (onycholysis); thickening of the nail plate (onycholux); subungual hyperkeratosis; nail fragility; pain in the affected area; and dermatophytomas, which are fungal abscesses that appear as whitish longitudinal streaks or characteristic colors (LIPNER, R.; SCHER, RK Onychomycosis: Clinical overview and diagnosis. Journal of the American Academy of Dermatology, v. 80, n. 4, p. 835-851, 2019).

[0006] In view of its clinical manifestations, onychomycosis negatively impacts the quality of life of the individual affected by this disease, and may cause embarrassment due to the altered appearance of the nail, discomfort due to pain in the area, difficulties in performing daily activities, such as manual tasks and putting on shoes, and difficulties in walking (LIPNER, R.; SCHER, RK Onychomycosis: Clinical overview and diagnosis. Journal of the American Academy of Dermatology, v. 80, n. 4, p. 835-851, 2019).

[0007] The treatment of onychomycosis aims to eliminate the fungal agent and restore the normal appearance of the nails, and can be performed using oral (systemic) or topical medications, laser therapy, photodynamic therapy, or even surgical avulsion of the nail plate in more severe cases. Topical and / or oral drug therapy is the most commonly used treatment method. However, onychomycosis is difficult to treat with conventional medications due to the fungus's establishment in the deeper layers of the nail plate, the long treatment time required, frequent recurrences, the possibility of fungal resistance, and low patient adherence (LIPNER, SR; SCHER, RK Onychomycosis: Treatment and prevention of recurrence. Journal of the American Academy of Dermatology, v. 80, n. 4, p. 853-867, 2019).

[0008] The most commonly used oral medications approved by the National Health Surveillance Agency (ANVISA) include terbinafine, itraconazole, and fluconazole. These medications Petition 870220112614, dated 02 / 12 / 2022, page 17 / 61 4 / 35 exhibit a broad spectrum of action against dermatophytes; however, they require a long treatment period, which can vary from 6 to 12 weeks on average.

[0009] Furthermore, despite their high efficacy compared to other types of treatment, oral antifungal medications cause adverse effects such as headache, gastrointestinal discomfort, abdominal pain, skin rash, elevated transaminase enzymes, and taste disturbances. Additionally, they can cause hepatotoxicity and renal dysfunction, depending on the duration of use. Another disadvantage of systemic therapy is the possibility of drug interactions, which can lead to alterations in the pharmacokinetic profile and, consequently, reduced efficacy or toxicity of the antifungal medication itself or the concomitantly administered medication (LIPNER, SR; SCHER, RK Onychomycosis: Treatment and prevention of recurrence. Journal of the American Academy of Dermatology, v. 80, n. 4, p. 853-867, 2019).

[0010] Topical medications commonly recommended for the treatment of onychomycosis, approved by the National Health Surveillance Agency (ANVISA), include ciclopirox 8% and amorolfine 5%, which should be applied to the nails for an extended period, ranging from 6 to 12 months.

[0011] Topical treatment has the advantage of not causing systemic adverse effects or drug interactions. Additionally, it generally does not present toxicity or contraindications. However, conventional topical medications available are less effective than oral medications and require a longer treatment time, which is attributed to insufficient drug permeation into the nail plate. Nails act as a barrier to the permeation of exogenous compounds due to their highly keratinized composition and the stable interaction networks between keratin molecules, which occur through disulfide and hydrogen bonds (BAROT, BS et al. Drug delivery to the nail: Petition 870220112614, dated 02 / 12 / 2022, page 18 / 61 5 / 35 therapeutic options and challenges for onychomycosis. Critical Reviews™ in Therapeutic Drug Carrier Systems, vol. 31, no. 6, 2014.).

[0012] After treatment of onychomycosis using available medications, relapses (same infection after incomplete cure) or reinfections (same infection after complete cure) occur in 20 to 25% of cases, which may be associated, among other factors, with fungal resistance to conventional drugs (GROVER, C.; KHURANA, A. An update on treatment of onychomycosis. Mycoses, v. 55, n. 6, p. 541-551, 2012).

[0013] The state of the art describes various pharmaceutical compositions, nanostructured or not, that employ essential oils to topically treat fungal infections, such as document BR112019005016 which describes a composition in the form of a hydrophobic gel with antifungal activity for topical application, including the association of melaleuca, oregano and lime essential oils in a vitamin E ester.

[0014] Document BR102019009921 describes a composition of nanostructured lipid carriers with intrinsic anesthetic, bactericidal, antifungal and biopesticidal action, containing the essential oils of garlic, cinnamon, black pepper and peppermint, alone or in combination.

[0015] Document BR102018072899 describes a pharmaceutical composition in the form of a nanoemulsion, employing Piper aduncum essential oil as the active compound, intended for topical administration in the treatment of fungal or bacterial infections and wounds resulting from cutaneous leishmaniasis.

[0016] Document BR102018016526 describes a micro and / or nanostructured phytotherapeutic composition, in liquid or dry form, containing Lippia sidoides essential oil as the active compound for the treatment of otitis externa in animals and combating the fungal agents Malassezia spp., Candida spp., Trichophyton spp., Neoscytalidium spp. Petition 870220112614, dated 02 / 12 / 2022, page 19 / 61 6 / 35

[0017] Additionally, nanostructured pharmaceutical compositions containing antimicrobial drugs that propose to treat fungal infections are also described.

[0018] Document IN201811021213 describes a nanostructured lipid carrier for controlled release of the drug itraconazole, to be administered via the ocular route in the treatment of fungal keratitis.

[0019] Document WO202028916 describes a nanostructured lipid carrier conjugated with polyethylene glycol (PEG) containing the drug amphotericin B, aiming to treat fungal infections.

[0020] The prior art documents describe pharmaceutical formulations of nanostructured lipid carriers (NLCs) containing essential oils and commercial antifungal drugs. Nanostructured lipid carriers (NLCs) are a type of lipid delivery system for active compounds in which the dispersed phase has a size on the nanometer scale, generally between 10 and 500 nm. They are essentially formed by a mixture of solid (SL) and liquid (LL) lipids at room temperature in a ratio ranging from 70:30 to 99.9:0.1 (SL:LL), surfactants and water (SHARMA, A.; BALDI, A. Nanostructured lipid carriers: A review. Journal of Developing Drugs, v. 7, n. 2, p. 1000191, 2018).

[0021] NLCs overcome the limitations of solid lipid nanoparticles (SNLs), in which the lipid matrix is ​​composed only of a solid lipid at room temperature, resulting in low carrying capacity of the active compound and its expulsion during storage due to crystallization of the solid lipid. Furthermore, NLCs differ from nanoemulsions, in which the lipid matrix is ​​composed only of liquid lipid(s) at room temperature, not comprising a solid lipid, resulting in a nanosystem with distinct properties and applications (PATEL, K. et al. Nanostructured lipid carrier (NLC) a modern approach for topical delivery: a review. World J. Pharm. Pharm. Sci, v. 2, p. 921-938, 2013). Petition 870220112614, dated 02 / 12 / 2022, page 20 / 61 7 / 35

[0022] Lipid-containing liquids (LCLs) constitute a system capable of providing solubilization of hydrophobic active compounds in an aqueous dispersion, controlling release, and increasing the permeability and bioavailability (systemic or topical) of active compounds. These properties are the main reasons for their application in the pharmaceutical field. LCLs can be used both for the delivery of hydrophobic active compounds, which, in this case, are located within the lipid core, and for the delivery of hydrophilic active compounds, which are found next to the surfactant layer, at the oil-water interface.Additionally, the CLN system presents several recognized advantages such as: biocompatibility, the possibility of not using organic solvents for its production, depending on the method employed, ease of scaling and sterilization, the high percentage of encapsulation of the compound of interest within the nanostructure, and the high physicochemical stability of the system (SHARMA, A.; BALDI, A. Nanostructured lipid carriers: A review. Journal of Developing Drugs, v. 7, n. 2, p. 1000191, 2018).

[0023] Furthermore, CLNs provide physicochemical protection for labile compounds. In this sense, they constitute an alternative for the delivery of compounds of natural origin in pharmaceutical formulations, such as essential oils and their components, as they prevent their degradation by hydrolysis, oxidation and / or photolysis, reactions that can be triggered by the action of environmental factors such as humidity, oxygen and light. Additionally, the encapsulation of essential oils or their constituents in a CLN system prevents volatilization, an intrinsic property of these compounds, which limits their use in conventional pharmaceutical formulations (KATOPODI, A.; DETSI, A. Solid Lipid Nanoparticles and Nanostructured Lipid Carriers of natural products as promising systems for their bioactivity enhancement: The case of essential oils and flavonoids. Colloids and Surfaces A: Physicochemical and Engineering Aspects, v. 630, p. 127529, 2021). Petition 870220112614, dated 02 / 12 / 2022, page 21 / 61 8 / 35

[0024] Several techniques are established for the production of NLC, among which we can mention: high-pressure homogenization; high-shear homogenization; high-speed homogenization; ultrasonication; microemulsion; solvent evaporation-emulsification; solvent evaporation injection; multiple emulsion; and phase inversion. Generally, techniques that do not employ organic solvents are performed hot, while techniques that do employ them are operated cold. Furthermore, there may be a combination of different techniques and / or adaptation of existing ones (PATEL, K. et al. Nanostructured lipid carrier (NLC) a modern approach for topical delivery: a review. World J. Pharm. Pharm. Sci, v. 2, p. 921-938, 2013).

[0025] Thus, the present invention relates to a nanostructured lipid carrier containing 1-nitro-2-phenylethane, associated or not with methyleugenol, components that can be found in the essential oil of Aniba canelilla, for the topical treatment of fungal infections such as onychomycosis or dermatophytosis in general.

[0026] Aniba canelilla (Kunth) Mez is a medium-sized aromatic tree belonging to the Lauraceae family. Popularly, it is known as Casca Preciosa, Falsa Canela, Casca do Maranhão or Folha preciosa. This species is native to South American countries (Bolivia, Brazil, Colombia, Ecuador, Peru and Venezuela), being found mainly in the Amazon region (SOUZAJUNIOR, FJC et al. Aniba canelilla (Kunth) Mez (Lauraceae): A review of ethnobotany, phytochemical, antioxidant, anti-inflammatory, cardiovascular, and neurological properties. Frontiers in Pharmacology, v. 11, p. 699, 2020).

[0027] The essential oil of Aniba canelilla (OEAc) has two main constituents, 1-nitro-2-phenylethane and methyleugenol, and is extracted from the trunk, branches, leaves and bark of this tree by steam distillation or hydrodistillation, using a Clevenger-type apparatus (SOUZA-JUNIOR, FJC et al. Aniba canelilla (Kunth) Mez (Lauraceae): A review of ethnobotany, phytochemical, antioxidant, anti Petition 870220112614, dated 02 / 12 / 2022, page 22 / 61 9 / 35 inflammatory, cardiovascular, and neurological properties. Frontiers in Pharmacology, vol. 11, p. 699, 2020).

[0028] 1-Nitro-2-phenylethane is a nitro derivative rarely found in plants, synthesized by the biotransformation of phenylalanine by CYP enzymes. It has the molecular formula C8H9NO2 and a molecular mass of 151.16 g / mol. Methyleugenol, chemically called 1-allyl-3,4-dimethoxybenzene, is formed by the methylation reaction of eugenol, a phenolic compound that may also be present in OEAc. It has the molecular formula C11H14O2 and a molecular mass of 178.23 g / mol (SOUZA-JUNIOR, FJC et al. Aniba canelilla (Kunth) Mez (Lauraceae): A review of ethnobotany, phytochemical, antioxidant, anti-inflammatory, cardiovascular, and neurological properties. Frontiers in Pharmacology, v. 11, p. 699, 2020).

[0029] The technical literature describes the therapeutic properties for the OEAc, among which stand out the antioxidant, hypotensive, bradycardic, anticholinesterase, leishmanicidal, trypanocidal, antibacterial and antifungal activities against different Candida species. However, the state of the art does not describe the use of OEAc and 1-nitro-2-phenylethane in antifungal activity against dermatophytes (DA TRINDADE, RCS et al. Chemical diversity and therapeutic effects of essential oils of Aniba species from the Amazon: a review. Plants, v. 10, n. 9, p. 1854, 2021).

[0030] In view of the foregoing, dermatophytoses, especially onychomycosis, lack innovative therapeutic alternatives that aim to overcome the disadvantages of currently available drug therapies. In this sense, the present invention proposes the use of 1-nitro-2-phenylethane, associated or not with methyleugenol, in a nanostructured lipid carrier system. It should be noted that both 1-nitro-2-phenylethane and methyleugenol are commonly found as major constituents of the essential oil of Aniba canelilla; however, they can also be obtained from chemical synthesis. Petition 870220112614, dated 02 / 12 / 2022, p. 23 / 61 10 / 35

[0031] The nanostructured lipid carrier that is the subject of the invention exhibits optimized nail and skin permeation properties, considering its reduced particle size, and avoids the occurrence of systemic adverse effects and drug interactions because it is intended for topical application. Furthermore, the aforementioned nanostructured lipid carrier prevents the volatilization of the active compound and protects it against possible degradation reactions, which would not be possible in a traditional pharmaceutical composition. Moreover, the alternative of lyophilizing the composition that is the subject of the invention, resulting in a lyophilized nanostructured lipid carrier, increases its physicochemical and microbiological stability when compared to a water-based product, which facilitates its commercialization as an intermediate product that can be redispersed in various liquid or semi-solid pharmaceutical vehicles suitable for application to the nails or skin. Summary of the Invention

[0032] The invention describes a nanotechnology pharmaceutical composition comprising a nanostructured lipid carrier containing 1-nitro-2-phenylethane, isolated or associated with methyleugenol, as the main active ingredient.

[0033] The invention describes a nanotechnology pharmaceutical composition potentially useful for the topical treatment of dermatophytoses, more specifically onychomycosis.

[0034] The invention describes a nanotechnological pharmaceutical composition that avoids systemic adverse effects and drug interactions due to topical application, exhibiting optimized nail and skin permeation properties, bypassing the barrier effect of the nail plate and skin and allowing the active ingredient to reach its site of action.

[0035] The invention describes a nanotechnological pharmaceutical composition that uses a nanostructured lipid carrier that prevents the volatilization of the active compound (1-nitro-2-phenylethane isolated or associated with Petition 870220112614, dated 02 / 12 / 2022, page 24 / 61 11 / 35 methyleugenol) and provides protection against possible degradation reactions, such as hydrolysis, oxidation and photolysis, triggered by environmental factors (humidity, oxygen and light).

[0036] The invention describes a nanotechnology pharmaceutical composition that optionally uses a lyophilized nanostructured lipid carrier that provides greater physicochemical and microbiological stability when compared to a water-based product, possessing the potential for a long shelf life.

[0037] The invention describes a nanotechnology pharmaceutical composition that optionally uses a lyophilized nanostructured lipid carrier that provides a versatile intermediate product that can be redispersed into different final liquid or semi-solid pharmaceutical forms. Brief Description of the Figures

[0038] Figure 1A presents the Pareto Chart and Figure 1B presents the Main Effects Plot for the dependent variable particle size (Yi).

[0039] Figure 2A presents the Pareto Chart and Figure 2B presents the Main Effects Plot for the dependent variable polydispersity index (Y2).

[0040] Figure 3A presents the Pareto Chart and Figure 3B presents the Main Effects Plot for the potential zeta dependent variable (Y3).

[0041] Figure 4A presents the Pareto Chart and Figure 4B presents the Main Effects Plot for the dependent variable 1-nitro-2-phenylethane content (Ye).

[0042] Figure 5A presents the Pareto Chart and Figure 5B presents the Main Effects Plot for the dependent variable methyleugenol content (Y7). Petition 870220112614, dated 02 / 12 / 2022, page 25 / 61 12 / 35

[0043] Figure 6A presents the Pareto Chart and Figure 6B presents the Main Effects Plot for the dependent variable 1-nitro-2-phenylethane (Ya) carrying.

[0044] Figure 7A presents the Pareto Chart and Figure 7B presents the Main Effects Plot for the dependent variable methyleugenol carryover (Y9).

[0045] Figure 8A presents the recurrence analysis of the ex vivo assay in pig hooves infected with dermatophyte fungus for Aniba canelilla essential oil diluted to 10%, Figure 8B presents the positive control and Figure 8C presents the negative control.

[0046] Figure 9A presents the relapse analysis of the ex vivo assay in pig hooves infected with dermatophyte fungus for the nanostructured lipid carrier, Figure 9B presents the positive control and Figure 9C presents the negative control. Detailed Description of the Invention

[0047] The nanostructured lipid carrier composition, the subject of this patent, contains between 1.0 and 25.0% w / w of Aniba canelilla essential oil consisting of 1-nitro-2-phenylethane.

[0048] Optionally, the essential oil of Aniba canelilla, in addition to being composed of 1-nitro-2-phenylethane, is also composed of methyleugenol.

[0049] Optionally, the nanostructured lipid carrier composition, the subject of this patent, contains between 1.0 and 25.0% w / w of 1-nitro-2-phenylethane isolated from the essential oil of Aniba canelilla.

[0050] Optionally, the nanostructured lipid carrier composition, the subject of this patent, contains between 1.0 and 25.0% w / w of 1-nitro-2-phenylethane and between 1.0 and 25.0% w / w of methyleugenol, isolated from the essential oil of Aniba canelilla. Petition 870220112614, dated 02 / 12 / 2022, page 26 / 61 13 / 35

[0051] Optionally, 1-nitro-2-phenylethane and methyleugenol are chemically synthesized.

[0052] The nanostructured lipid carrier comprises between 1.0 and 25.0% of a solid lipid; between 1.0 and 8.0% of a non-ionic lipophilic surfactant with a hydrophilic-lipophilic balance (HLB) between 3 and 6; between 1.0 and 8.0% of a non-ionic hydrophilic surfactant with a hydrophilic-lipophilic balance (HLB) between 8 and 18; and water in q.s.

[0053] Optionally, the nanostructured lipid carrier contains between 1.0 and 25.0% of another liquid lipid, in addition to its active compound, which already fulfills this function.

[0054] Solid lipid comprises any lipid raw material with a melting point above 30°C, preferably selected from stearic acid, cetyl palmitate, tristearin, beeswax, tucumã butter, ucuúba butter, carnauba butter, cocoa butter, murumuru butter, shea butter, babassu butter, coconut butter, alone or in combination.

[0055] The lipophilic surfactant is preferably selected from the groups of sorbitan esters and glycol and glycerol esters, such as sorbitan oleate, sorbitan stearate, glyceryl monostearate, glyceryl monooleate and propylene glycol monostearate.

[0056] The hydrophilic surfactant is preferably selected from the polysorbate groups, such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80.

[0057] Optionally, the nanostructured lipid carrier includes another liquid lipid, in addition to the active one, with a melting point below 20°C, such as, but not limited to, oleic acid, isopropyl myristate, caprylic / capric triglycerides, alpha-tocopherol, olive oil, soybean oil, and castor oil.

[0058] Preferably, the nanostructured lipid carrier has a size between 50 and 350 nm, a polydispersity index less than 0.5, and a zeta potential greater than 30 mV in absolute value. Petition 870220112614, dated 02 / 12 / 2022, page 27 / 61 14 / 35

[0059] The process for obtaining the nanostructured lipid carrier comprises a first step of heating and homogenizing the oil phase, which includes Aniba canelilla essential oil (or 1-nitro-2-phenylethane associated or not with methyleugenol), the solid lipid, the lipophilic surfactant and, optionally, the liquid lipid. The oil phase is heated between 40 and 60°C, under stirring, for 3 to 8 minutes, ensuring complete fusion of the solid lipid.

[0060] The components of the aqueous phase, which includes the hydrophilic surfactant and water, are heated under stirring, between 40 and 60°C for 3 to 8 minutes.

[0061] The oil and water phases are mixed by stirring for 1 to 5 minutes, maintaining the temperature between 40 and 60°C.

[0062] Heating of the oil and water phases can be carried out in a thermostatically controlled water bath, for example, and homogenization under magnetic or mechanical stirring. Other suitable heating and / or homogenization mechanisms may also be employed.

[0063] The mixture of the oil and water phases is sheared between 10,000 and The mixture is then mixed at 15,000 rpm for 1 to 5 minutes, using an UltraTurrax® or similar disperser. In the next step, the mixture is subjected to a high-pressure homogenizer for 5 to 8 cycles, operating between 650 and 850 bar, while maintaining a temperature between 40 and 60°C to prevent the solid lipid from solidifying.

[0064] In the cooling stage, the composition is refrigerated to a temperature of 2 to 8°C for 2 to 6 hours in order to promote the solidification of the solid lipid, thus obtaining the nanostructured lipid carrier.

[0065] Optionally, between 1.0 and 25.0% of a cryoprotectant is added to the nanostructured lipid carrier obtained as described, by stirring at room temperature for 30 to 90 minutes.

[0066] Preferably, the cryoprotectant is selected from a carbohydrate, such as: mannitol, sucrose, glucose and trehalose. Petition 870220112614, dated 02 / 12 / 2022, page 28 / 61 15 / 35

[0067] Preferably, the nanostructured lipid carrier containing the cryoprotectant is frozen and kept at a temperature between -15 and -25°C for 12 to 36 hours.

[0068] Preferably, the nanostructured lipid carrier containing cryoprotectant is dried by lyophilization at a temperature between -40 and -60°C for 24 to 48 hours.

[0069] Optionally, the lyophilized nanostructured lipid carrier composition is redispersed in a liquid or semi-solid pharmaceutical vehicle suitable for administration to the skin or nails, with a pH preferably between 4.0 and 6.0.

[0070] Preferably, the lyophilized nanostructured lipid carrier redispersed in a liquid or semi-solid pharmaceutical vehicle retains its original properties, exhibiting: size on the nanometric scale, between 50 and 350 nm, polydispersity index less than 0.5 and zeta potential greater than 30 mV in modulus.

[0071] Preferably, the amount of liquid or semi-solid pharmaceutical carrier in the composition is equivalent to the amount of water (by mass) removed during the lyophilization process.

[0072] The aforementioned liquid pharmaceutical vehicle comprises an aqueous glaze, an aqueous solution or an aqueous dispersion.

[0073] The aforementioned semi-solid pharmaceutical vehicle comprises a gel, a cream, a lotion or an ointment.

[0074] The lyophilized nanostructured lipid carrier composition is redispersed in the liquid or semi-solid pharmaceutical vehicle by stirring, at room temperature, for 30 to 120 minutes. For this purpose, it is considered that the stirring may be magnetic, mechanical or of any other appropriate nature. Furthermore, the required stirring time will depend on the rheological characteristics and other physicochemical properties of the pharmaceutical vehicle, and must be sufficient to ensure a homogeneous dispersion. Petition 870220112614, dated 02 / 12 / 2022, page 29 / 61 16 / 35

[0075] Specific embodiments of the invention will be demonstrated below. However, the examples presented here are intended only to illustrate some of the numerous ways of carrying out the invention, without limiting its scope. Modifications to the embodiments disclosed here are suggested to a person skilled in the art and are included within the scope of the invention. Example 1: Extraction and characterization of Aniba canelilla essential oil

[0076] For the extraction of Aniba canelilla essential oil (OEAc), a specimen was used whose proof was deposited in the herbarium of the National Institute of Amazonian Research (INPA), under number 18138.

[0077] The bark of Aniba canelilla was dried at room temperature and ground. Subsequently, 100 g of the material was subjected to hydrodistillation in a modified Clevenger apparatus for 4 hours. After extraction, the Clevenger apparatus was washed with dichloromethane to remove the essential oil adhering to the walls of the apparatus. The mixture was separated and dried with anhydrous sodium sulfate. After drying, the obtained OEAc was stored in an amber bottle in a freezer at -20°C for later analysis. The yield of the extraction procedure was 0.5%.

[0078] The extracted OEAc was diluted with hexane and analyzed in a Gas Chromatograph (5975C) coupled to a flame ionization detector and a mass spectrometry detector (GC-FID / MS, Agilent Technologies, CA, USA) and equipped with an autosampler, using a previously developed and validated method.

[0079] The components of the essential oil were determined by their retention times and linear retention index data. The linear retention index (LRI) was calculated for all volatile constituents using a homologous series of n-alkanes. The identification of the major components of the essential oil, 1-nitro-2-phenylethane and methyleugenol, was made by comparing their mass spectra and LRI with those contained in the NIST Mass database. Petition 870220112614, dated 02 / 12 / 2022, page 30 / 61 17 / 35 Spectral Search Program 2.0 and in the literature. In addition, the retention times obtained by GC-FID / MS of 1-nitro-2-phenylethane and methyleugenol contained in OEAc were compared with the retention time of an isolated 1-nitro-2-phenylethane standard (purity > 99%) and with the retention time of a commercial methyleugenol standard (purity > 98%, Sigma Aldrich, Brazil).

[0080] Thus, the composition obtained for the extracted OEAc was as follows: 86.637% 1-nitrophenylethane, 12.7% methyleugenol and 0.663% benzaldehyde, totaling 100% of identified compounds. Example 2: Production of lyophilized nanostructured lipid carriers containing Aniba canelilla essential oil and their redispersions in aqueous glaze.

[0081] For the production of lyophilized nanostructured lipid carriers containing Aniba canelilla essential oil and their subsequent redispersions in an aqueous glaze (pharmaceutical vehicle), a statistical design of the 23 Full Factorial Design type (with the inclusion of 2 central points) was carried out using Minitab® 17 software (State College, PA, USA). The aforementioned statistical design resulted in ten compositions to be prepared by crossing the pre-established independent variables at different levels.

[0082] The following factors and levels were used as independent variables: the concentration of solid lipid (Xi), varied at levels of 5% (-1), 7.5% (0) and 10% (+1); the concentration of cryoprotectant (X2), also varied at levels of 5% (-1), 7.5% (0) and 10% (+1); and the redispersion time of the lyophilized nanostructured lipid carrier in the pharmaceutical vehicle (X3), varied at levels of 30 min. (-1), 60 min. (0) and 90 min. (+1). Where “-1” represents a low level, “0” represents a medium level and “+1” represents a high level for each factor studied. Table 1 presents the statistical combination of the independent variables at their different levels, which resulted in ten different compositions, as mentioned. Petition 870220112614, dated 02 / 12 / 2022, pp. 31 / 61 18 / 35

[0083] Table 1 - Combination of independent variables at their different levels in the Full Factorial Design 23. Composition Xi: Solid lipid concentration (%) X2: Cryoprotectant concentration (%) X3: Redispersion time (min.) 1 10 5 90 2 7.5 7.5 60 3 10 10 90 4 7.5 7.5 60 5 5 5 90 6 5 10 90 7 5 10 30 8 10 10 30 9 10 5 30 10 5 5 30

[0084] For the preparation of the compositions, Aníba canelilla essential oil containing mainly 1-nitro-2-phenylethane (86.637%) and methyleugenol (12.7%) was used, extracted and characterized as described previously. Furthermore, the concentrations of Aníba canelilla essential oil, lipophilic surfactant, and hydrophilic surfactant were kept constant. Similarly, the process parameters adopted for the production of the lyophilized nanostructured lipid carrier were also kept constant. In this sense, the inputs used, as well as their functions and concentrations, are presented in Table 2 below.

[0085] Table 2 - Inputs and their concentrations for the preparation of ten compositions of lyophilized nanostructured lipid carrier in the 23 Full Factorial Design. Raw Material Function Concentration (m / m) Aniba canelilla essential oil consisting of 1-nitro- Active compound and liquid lipid 10% Petition 870220112614, dated 02 / 12 / 2022, pp. 32 / 61 19 / 35 2-phenylethane and methyleugenol Tucumã butter Solid lipid Varied in levels 5, 7.5 and 10% Sorbitan oleate High HLB lipophilic surfactant 2.5% Polysorbate 20 High HLB hydrophilic surfactant 2.5% Water* Dispersant Quantity sufficient for 100% Mannitol** Cryoprotectant Varied in levels 5, 7.5 and 10%

[0086] *Removed during the lyophilization process; **Added after the formation of the nanostructured lipid carrier, prior to the lyophilization step; Tucumã butter (solid lipid) corresponds to Xi of the Complete Factorial Design 23; Mannitol (cryoprotectant) corresponds to X2 of the Complete Factorial Design 23.

[0087] The preparation process for the ten compositions will be described below. The raw materials were weighed according to the concentrations discussed in Table 2.

[0088] Heating and homogenization of the oil phase: the oil phase, consisting of Aníba canelilla essential oil, tucumã butter and sorbitan oleate, was heated in a thermostatic bath at 45°C under magnetic stirring for 5 minutes, ensuring complete fusion of the solid lipid.

[0089] Heating and homogenization of the aqueous phase: in parallel and similarly, the aqueous phase, consisting of polysorbate 20 and water, was heated in a thermostatic bath at 45°C under magnetic stirring for 5 minutes.

[0090] Pouring one phase over the other: The aqueous phase was poured over the oily phase, and magnetic stirring was maintained for another 3 minutes at a temperature of 45°C in a thermostatically controlled bath.

[0091] High shear agitation: the compositions were subjected to the Ultra-turrax® disperser (T8, IKA, Germany) at 13,500 rpm for 1 minute. Petition 870220112614, dated 02 / 12 / 2022, pp. 33 / 61 20 / 35

[0092] High-pressure homogenization: the compositions were then placed in a High-Pressure Homogenizer (Emulsiflex-C®, Avestin, Canada), which operated at 750 bar for 6 cycles. During this process, the system temperature was maintained at 45°C in order to prevent the solidification of the solid lipid.

[0093] Cooling: the compositions were refrigerated at 4°C for 4 hours, in order to promote the solidification of the solid lipid.

[0094] Addition of cryoprotectant: mannitol was added by magnetic stirring for 60 minutes at room temperature, ensuring its complete solubilization.

[0095] Freezing: the compositions were frozen between -20 ± 5°C for 24 hours.

[0096] Lyophilization: Subsequently, they were subjected to the lyophilization process in the Lyophilizer equipment (MicroModulyo, Savant Thermo Electron Corporation, Waltham, USA), which operated at -50°C for 24 hours. At the end of this step, the lyophilized nanostructured lipid carrier compositions were obtained.

[0097] As a pharmaceutical vehicle for redispersing the lyophilized nanostructured lipid carrier, an aqueous enamel containing the Eudragit® polymer and other necessary additives, free of organic solvents, was used. The redispersion time of the nanostructured lipid carrier in the pharmaceutical vehicle (X3) was varied at 30, 60, and 90 minutes, comprising one of the independent variables of the 23 Full Factorial Design, as discussed in Table 1. Redispersion was carried out by magnetic stirring at room temperature, and the amount of pharmaceutical vehicle used was the same amount (by mass) of water removed by the lyophilization process.

[0098] The ten compositions obtained of lyophilized nanostructured lipid carrier dispersed in aqueous enamel were evaluated for: particle size (Y1) in nm; polydispersity index (Y2); zeta potential (Y3) in mV; the encapsulation efficiency, expressed for 1-nitro-2-phenylethane (Y4) and Petition 870220112614, dated 02 / 12 / 2022, pp. 34 / 61 21 / 35 for methyleugenol (Y5) in %; the content, expressed for 1-nitro-2-phenylethane (Yβ) and for methyleugenol (Y7) in mg / g; and the carrying capacity of the active compound, expressed for 1-nitro-2-phenylethane (Ye) and for methyleugenol (Y9) in %. All these parameters (Yi to Y9) constituted the dependent variables of the 2³ Full Factorial Design and were statistically analyzed using Minitab® 17 software (State College, PA, USA). In this way, it was possible to evaluate the impact of the independent variables (X1 to X3) on the dependent variables (Y1 to Y9). The methodology adopted for determining the aforementioned dependent variables, as well as the results obtained for each composition, are presented in the following section. Example 3: Characterization of lyophilized nanostructured lipid carriers containing Aniba canelilla essential oil redispersed in aqueous enamel.

[0099] Particle size (nm) and polydispersity index were determined in triplicate by the dynamic light scattering method at 25°C using a Nanosizer® ZS90 instrument (Malvern Instruments, UK). Samples were diluted 1000 times in ultrapure water and filtered at 0.22 µm. Results were expressed as the average of the three determinations. Zeta potential (mV) was determined in triplicate by the principle of mobility electrophoresis at 25°C using the same instrument, with samples prepared by dilution 1000 times in 0.1 mM NaCl solution and filtered at 0.22 µm. Results were also expressed as the average of the three determinations.

[0100] To determine the content (mg / g) of 1-nitro-2-phenylethane and methyleugenol, a Gas Chromatograph (Shimadzu GC 2010, Kyoto, Japan) with a split / splitless manual injector, equipped with a flame ionization detector (FID), was used. The analytes were extracted from the sample by solid-phase microextraction (headspace). The operating conditions adopted followed the specifications of a previously developed and validated method according to international guidelines. Petition 870220112614, dated 02 / 12 / 2022, pp. 35 / 61 22 / 35

[0101] The injection was performed in sp / it / ess mode and the chromatographic system was equipped with a DB-5 fused silica capillary column (30 m χ 0.25 mm χ 0.25 pm). The carrier gas was nitrogen at a flow rate of 1 mL / min and a linear velocity of 40 cm / s. The oven temperature was programmed as follows: from 60°C to 155°C at 15°C / min, from 155°C to 165°C at 3°C / min, and from 165°C to 260°C at 40°C / min, completing the chromatographic run in 14 minutes. The injector and detector temperatures were set at 250°C and 300°C, respectively. The signal was processed using LabSo / utions GCSo / ution software.

[0102] For solid-phase microextraction of the analytes, a hand support and a 100 µm thick polydimethylsiloxane-coated fiber (Sigma-Aldrich, São Paulo, Brazil) were used. In order to promote volatilization and adsorption of the analytes to the fiber, 5% (mass / volume) NaCl was added to the samples, which were then heated to 53°C in a thermostatic bath and simultaneously stirred on a magnetic stirrer for 21 minutes, using a sealed via / with a septum. After 21 minutes, the fiber was removed from the via / and inserted into the injection portion of the chromatograph. The analyte desorption time was 4 minutes.

[0103] Samples were prepared by diluting 250 mg of the composition in methanol, followed by dilution in ultrapure water, taking 1 mL of the final solution obtained for analysis (solid-phase microextraction followed by gas chromatography).

[0104] Encapsulation efficiency (%) and active compound carrying capacity (%) were determined by an indirect method, measuring the amount of 1-nitro-2-phenylethane and methyleugenol in the aqueous phase of the composition. For this purpose, centrifugation-ultrafiltration was performed using a centrifuge filter (Amicon® Ultra 15, Millipore) with a molecular weight cutoff of 100 kDa. A volume of 0.5 mL of the composition was placed in the inner compartment of a centrifuge tube and centrifuged at 4000 rpm for 1.5 hours at 4°C. The analytes free in the aqueous phase were collected in the outer compartment of the Petition 870220112614, dated 02 / 12 / 2022, pp. 36 / 61 23 / 35 centrifuge tube filter unit, diluted and quantified by Gas Chromatography, adopting the same procedure described above for content determination.

[0105] Encapsulation efficiency (EE), which corresponds to the amount of compound retained in the lipid matrix (nanoencapsulated) in relation to the total amount of compound contained in the composition, was calculated using Equation 1.

[0106] Equation 1: EE (%) of 1-nitro-2-phenylethane or methyleugenol = ((Qtotal compound - Qfree compound) Q total compound ) X 100

[0107] Where:

[0108] Qcomposto total corresponds to the total quantity of the compound dosed in the composition;

[0109] Qfree compound corresponds to the amount of compound dosed in the aqueous phase.

[0110] The carrying capacity of the active compound, which corresponds to the percentage of the amount of compound carried in the lipid matrix in relation to the amount of compound initially added to prepare the nanosystem, was calculated according to Equation 2.

[0111] Equation 2: Carrying (%) of 1-nitro-2-phenylethane or methyl eu enol “ ( (Q total compound - Q free compound) \ * XQQ g \Q compound initially added )X

[0112] Where:

[0113] Q total compound corresponds to the total quantity of the compound dosed in the composition;

[0114] Qfree compound corresponds to the amount of compound dosed in the aqueous phase;

[0115] Qcompound added initially corresponds to the amount of compound weighed for the preparation of the nanosystem.

[0116] The results obtained were submitted to statistical evaluation by Analysis of Variance (ANOVA) and Tukey's test (significance level: p < 0.05) were performed using Minitab® 17 software (State College, PA, USA). The model Petition 870220112614, dated 02 / 12 / 2022, pp. 37 / 61 24 / 35 The mathematical model that best fit the results was selected based on the p-value. The significance for each response was expressed by the coefficient of determination (R²) of the polynomial regression equation capable of describing the relationship between the independent variables, considering the individual terms and their interactions. Additionally, Pareto and Main Effects charts for each dependent variable studied were plotted to support the results of the polynomial equation, which are presented in the Figures.

[0117] The results of the dependent variables (Yi, Y2, Y3, Y4, Ys, Yθ, Yϊ, Ys and Yθ) obtained for the ten compositions produced in the 23 Full Factorial Design are expressed in Table 3 below, in which the respective independent variables (Xi, X2 and X3) that generated these results can also be observed.

[0118] Table 3 > - Results obtained for the dependent variables evaluated in the Complete Factorial Design 23. Composition Independent variables Dependent variables Xi X2 X3 Yi Y2 Y3 Y4 Y5 Y6 Y? Ys Y9 1 10 5 90 147.144 0.336 -69.60 100 100 41.87 6.60 49.59 53.36 2 7.5 7.5 60 126.45 0.223 -63.45 100 100 26.89 5.08 31.65 43.86 3 10 10 90 137.478 0.256 -69.08 100 100 36.53 6.11 43.27 49.43 4 7.5 7.5 60 129.93 0.212 -65.21 100 100 27.14 5.19 32.24 45.12 5 5 5 90 119.756 0.125 -69.02 100 100 16.11 4.42 20.79 38.99 6 5 10 90 119.378 0.124 -68.02 100 100 15.60 3.98 20.13 35.04 7 5 10 30 116.222 0.126 -66.77 100 100 19.69 4.83 25.42 42.52 8 10 10 30 134.278 0.292 -60.88 100 100 33.31 5.54 39.45 44.82 9 10 5 30 135.689 0.305 -64.76 100 100 32.90 5.46 38.96 44.14 10 5 5 30 117.444 0.160 -65.31 100 100 22.70 5.03 29.30 44.30

[0119] Xi: concentration of solid lipid (%); X2: concentration of cryoprotectant (%); X3: redispersion time of lyophilized nanostructured lipid carrier (min); Y1: particle size (nm); Y2: polydispersity index; Y3: zeta potential (mV); Y4: encapsulation efficiency of 1-nitro-2-phenylethane (%); Ys: encapsulation efficiency of methyleugenol (%); Yθ: 1-nitro-2-phenylethane content Petition 870220112614, dated 02 / 12 / 2022, pp. 38 / 61 25 / 35 phenylethane (mg / g); Y7: Methyleugenol content (mg / g); Ys: Carriage of 1-nitro2-phenylethane (%); Y9: Methyleugenol carriage (%).

[0120] As can be observed in Table 3, the particle size (Yi) for the produced compositions presented values ​​ranging from 116 to 147 nm, not being influenced by any of the independent variables studied (Xi, X2 and X3), which is confirmed by the plotted Pareto Chart (Figure 1A), since the proposed model did not present a significant p-value (p > 0.05). Despite this, the Main Effects Plot for particle size (Figure 1B) shows a tendency for this parameter to increase when a higher concentration of solid lipid (X1) is added to the nanosystem, which can be attributed precisely to a greater amount of solid lipid incorporated into the dispersed phase. Due to the reduced particle size that the compositions presented (from 116 to 147 nm), it can be inferred that they have a high permeation potential, an important property when aiming for cutaneous and nail administration routes.

[0121] The polydispersity index (PDI) (Y2) ranged from 0.124 to 0.336 for the prepared compositions and, based on factorial regression and Pareto chart (Figure 2A), this parameter is influenced by the concentration of solid lipid (X1) (p-value <0.021). A higher PDI was found for the compositions containing a higher percentage of solid lipid, as can be seen in Table 3 and the Main Effects Plot (Figure 2B), which probably occurred due to the increase in particle size of these nanosystems. PDI values ​​between 0.1 and 0.25 indicate a narrow distribution in nanoparticle size, favoring long-term stability for nanometric dispersions. On the other hand, PDI values ​​above 0.5 suggest a wide distribution in nanoparticle size, indicating nanosystem instability.Therefore, a monodispersity is suggested for all prepared compositions, considering an IPD below 0.35, indicating homogeneity in the size of the nanoparticles, which is desirable in terms of stability. Petition 870220112614, dated 02 / 12 / 2022, pp. 39 / 61 26 / 35

[0122] The zeta potential (Y3) for the produced compositions ranged from -60.8 mV to -69.9 mV, suggesting physical stability. Additionally, as can be seen in the Pareto Chart (Figure 3A), this dependent variable was not influenced by any formulation or process parameter studied (X1, X2, and X3), since no statistical significance was detected in the proposed factorial model (p-value > 0.05). Despite a suggestive relationship between the increase in redispersion time and the increase in zeta potential in absolute value in the Main Effects Plot (Figure 3B), no statistical significance was detected (p > 0.05). The zeta potential interferes with the physical stability of dispersed systems; the higher its absolute value, the greater the electrostatic repulsion and the distance between the nanoparticles, and therefore, the greater the stability of the nanosystem.

[0123] The encapsulation efficiency (Y4 and Y5) for both major components of Aniba canelilla essential oil, 1-nitro-2-phenylethane and methyleugenol, was 100% for all compositions produced, as shown in Table 3. This result means that the total amount of 1-nitro-2-phenylethane and methyleugenol contained in the composition is incorporated into the lipid matrix, with no presence of these compounds in the aqueous phase, which is desirable because they must be trapped in the lipid nanoparticle to be protected from volatilization and external environmental factors. The high encapsulation efficiency obtained can be attributed to the lipophilicity of 1-nitro-2-phenylethane and methyleugenol. Pareto and Main Effects charts were not generated for the dependent variables encapsulation efficiency of 1-nitro-2-phenylethane (Y4) and methyleugenol (Y5), since the results were the same for all prepared compositions (100%).

[0124] The content of 1-nitro-2-phenylethane (Y6), as can be seen in The Pareto chart (Figure 4A) was affected by the following independent variables, which showed statistical significance (p-value <0.05): solid lipid concentration (X1), cryoprotectant concentration (X2), interaction Petition 870220112614, dated 02 / 12 / 2022, pp. 40 / 61 27 / 35 between the solid lipid concentration (Xi) and the redispersion time (X3) and the interaction between the solid lipid concentration (X1), the cryoprotectant concentration (X2) and the redispersion time (X3). The ME content (Y7), in turn, was influenced by the independent variables: solid lipid concentration (X1) and the interaction between the solid lipid concentration (X1) and the redispersion time (X3) (p-value <0.05), as can be seen in the Pareto Chart (Figure 5A).

[0125] The Main Effects Plots for the content of 1-nitro-2-phenylethane (Figure 4B) and methyleugenol (Figure 5B) indicate that the higher the concentration of solid lipid, the higher the content of these components, which can be attributed to the greater solidification capacity of the nanoparticles and, consequently, the immobilization of the incorporated compounds.

[0126] The carrying of 1-nitro-2-phenylethane (Ye) was statistically significantly affected by the solid lipid concentration (X1) and by the interaction between the solid lipid concentration (Xi) and the redispersion time (X3) (p-value <0.05), as shown in the Pareto Chart (Figure 6A). Complementarily, the Main Effects Plot for 1-nitro-2-phenylethane (Figure 6B) shows an increase in the carrying of this component as the solid lipid concentration increases.

[0127] Methyleugenol (Y9) carrying, on the other hand, was not affected by any of the independent variables studied (solid lipid concentration, cryoprotectant concentration, and redispersion time, X1, X2, and X3, respectively), considering the p-value >0.05 obtained, as can be seen in the Pareto Chart (Figure 7A). However, in the Main Effects Plot (Figure 7B), it is possible to observe a trend in the increasing carrying of methyleugenol as the solid lipid concentration (X1) increases, as was also observed for 1-nitro-2-phenylethane.

[0128] Finally, the independent variables of the Factorial Design Complete 23, namely: the concentration of solid lipid (X1), the concentration of cryoprotectant (X2) and the redispersion time (X3), were optimized Petition 870220112614, dated 02 / 12 / 2022, pages 41 / 61 28 / 35 statistically using a desirability function with Minitab® 17 software (State College, PA, USA), aiming to determine which values ​​of Xi, X2, and X3 generated the best results for the dependent variables. To this end, the following were considered: a maximized response for the content and carrying capacity of 1-nitro-2-phenylethane and methyleugenol (ME) (Ye, Y7, Y8, and Y9); a minimized response for particle size (Y1) and polydispersity index (Y2); and a maximized response for the zeta potential (Y3) in absolute value. Encapsulation efficiency (Y4 and Y5) was not considered for optimization, as it was 100% for all prepared compositions, both for 1-nitro-2-phenylethane and methyleugenol.A weight of 2 and 3 was assigned to the content of 1-nitro-2-phenylethane and methyleugenol and to the carrier of 1-nitro-2-phenylethane and methyleugenol, respectively, considering that these parameters directly impact the effectiveness of the composition.

[0129] In this way, composition 3 (Table 3) was the one that generated the best results for the dependent variables studied, which was produced containing 10% solid lipid (Xi) and 10% cryoprotectant (X2) and redispersed in a pharmaceutical vehicle for 90 minutes (X3). The dependent variables obtained for composition 3, prepared according to the conditions mentioned, were: particle size (Y1) of 137.48 nm; polydispersity index (Y2) of 0.25e; zeta potential (Y3) of -e9.09; encapsulation efficiency of 1-nitro-2-phenylethane (Y4) of 100%; encapsulation efficiency for methyleugenol (Y5) of 100%; 1-nitro-2-phenylethane content (Ye) of 3e.53 mg / g; methyleugenol (Y7) content of e.11 mg / g; 1nitro-2-phenylethane (Y8) loading of 43.27%; and methyleugenol (Y9) loading of 49.43%.These conditions yielded a desirability value of 0.5250, which is considered appropriate, given that the desirability function (D) comprises a scale from 0 to 1, where 1 is the ideal value.

[0130] In this sense, the results obtained and presented here demonstrate the viability of the present invention, with regard to its composition, its Petition 870220112614, dated 02 / 12 / 2022, pp. 42 / 61 29 / 35 preparation process and its physicochemical characteristics. The nanostructured lipid carrier containing Aniba canelilla essential oil (mainly composed of 1-nitro-2-phenylethane and methyleugenol), after being lyophilized and redispersed in aqueous enamel, presented desirable quality characteristics. Example 4: In vitro minimum inhibitory concentration assay with dermatophyte fungi

[0131] In order to ascertain the antifungal activity against dermatophyte fungi of Aniba canelilla essential oil (OEAc) and 1-nitro-2-phenylethane, its main constituent, an in vitro minimum inhibitory concentration assay was performed using the broth microdilution method, adopting the method recommended by CLSI Protocol M38-A2 for filamentous fungi (CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi, Approved Standard - Second Edition. CLSI Document M38A2. Wayne, PA: Clinical and Laboratory Standards Institute, 2008).

[0132] For this purpose, the OEAc containing 1-nitro-2-phenylethane (86.637%) and methyleugenol (12.7%), extracted and characterized in accordance with the description in Example 1: Extraction and characterization of Aniba canelilla essential oil, and a high purity (100%) standard of isolated 1-nitro-2-phenylethane was tested.

[0133] For this assay, four clinically isolated strains of Trichophyton mentagrophytes (named TME 40, TME 60, TME 46 and TME 1), three clinically isolated strains of Trichophyton rubrum (named TRU 45, TRU 51 and TRU 47) and two clinically isolated strains of Microsporum canis (named MCA 29 and MCA 38) were used, totaling nine clinically isolated strains from three different species that cause fungal nail infections.

[0134] Serial dilutions were performed in RPMI 1640 culture medium in order to achieve different concentrations for OEAc and 1-nitro-2-phenylethane. The experiments were performed in triplicate. The minimum inhibitory concentration was defined as the lowest concentration of the compound in Petition 870220112614, dated 02 / 12 / 2022, pp. 43 / 61 30 / 35 that the tested microorganisms did not show visible growth after 96 hours. The drug ciclopirox olamine was used as a reference antifungal for dermatophyte fungi. A negative control for sterility (drug-free medium) and a positive control for fungal cell viability were performed in parallel.

[0135] Table 4 below shows the minimum inhibitory concentration (pg / mL) results obtained for OEAc and 1-nitro-2-phenylethane, considering the nine clinical strains of dermatophyte fungi that were tested.

[0136] Table 4 - Minimum inhibitory concentration (pg / mL) of Aniba canelilla essential oil and 1-nitro-2-phenylethane against dermatophyte fungi. Dermatophyte (n=9) OEAc 1N2FE CPX TME 60 256 221.7 2 TME 40 128 110.8 2 TME 1 256 221.7 1 TME 46 64 27.7 1 TRU 45 32 55.4 2 TRU 51 32 27.7 2 TRU 47 128 110.8 1 MCA 29 >256 >221.7 1 MCA 38 64 27.7 1

[0137] OEAc: Aniba canelilla essential oil, 1N2FE: 1-nitro-2- phenylethane; CPX:ciclopirox olamine; TME: Trichophyton mentagrophytes', TRU: Trichophyton rubrunr, MCA: Microsporum canis.

[0138] According to the data presented, OEAc was able to inhibit the growth of the dermatophyte fungi isolates tested, with a minimum inhibitory concentration ranging from 32 to 128 pg / mL for Trichophyton rubrum, from 64 to 256 pg / mL for Trichophyton mentagrophytes and from 64 pg / mL for Microsporum canis (MCA 38).

[0139] Similarly, 1-nitro-2-phenylethane was also able to inhibit the growth of the tested isolated dermatophyte fungi, with a Petition 870220112614, dated 02 / 12 / 2022, pp. 44 / 61 31 / 35 minimum inhibitory concentration ranging from 27.7 to 110.8 pg / mL for Trichophyton rubrum, from 27.7 to 221.7 pg / mL for Trichophyton mentagrophytes and from 27.7 pg / mL for Microsporum canis (MCA 38).

[0140] These results confirm the antifungal activity of OEAc (containing mainly 1-nitro-2-phenylethane and methyleugenol) and 1-nitro-2-phenylethane (isolated) against fungi that cause dermatophytosis and onychomycosis. Example 5: ex vivo assay in pig hooves infected with dermatophyte fungus

[0141] In order to prove the antifungal activity against onychomycosis of the nanostructured lipid carrier compositions that are the subject of the present invention and of Aniba canelilla essential oil (OEAc), containing mainly 1-nitro-2-phenylethane and methyleugenol, an ex vivo assay was performed on pig nails infected with a dermatophyte fungus, based on the methodology described by Quatrin et al. (QUATRIN, PM et al. Ex vivo nail infection as an effective preclinical method for screening of new topical antifungals. Journal de Mycologie Médicale, v. 30, n. 2, p. 100938, 2020).

[0142] For the execution of this assay, discs of 9.5 mm in diameter and mm in thickness were formed from pig hooves. The resulting discs were then placed in Potato Dextrose Agar culture medium and infected with a clinically isolated strain of Trichophyton rubrum (named TRU 47), the main causative agent of onychomycosis, by inoculating 100 pL of a fungal suspension at 10⁵ CFU / mL, followed by incubation in an oven at 32°C for 5 days. In this way, an ex vivo model of onychomycosis was obtained, aiming to simulate what occurs with human nails when affected by this disease.

[0143] After the aforementioned incubation period, the infected (pig hoof) discs were transferred to a Sabouraud Dextrose Agar culture medium, and 20 pL of test sample (detailed later) was pipetted daily for 7 days onto each infected disc. Three discs per test sample were considered, and they were stored in the same culture medium. The media Petition 870220112614, dated 02 / 12 / 2022, pp. 45 / 61 32 / 35 of the cultures were kept in an incubator at 32°C throughout the treatment period, that is, throughout the entire period they received the test sample. For the execution of the assay, a positive control was considered, in which the discs were infected and did not receive any treatment (no test sample was pipetted), and a negative control, in which the discs were neither infected nor received treatment.

[0144] After 7 days, each disc was individually transferred to a tube containing 2 mL of saline solution, which was vortexed for 1 minute and then ultrasonically heated for 20 minutes. Next, 20 pL of the saline solution was pipetted onto Potato Dextrose Agar. The 20 pL of saline solutions used to wash discs from the same test sample were pipetted into the same culture medium. The culture media were then incubated at 32°C for 5 days. After the incubation period, the fungal cell count (CFU) was performed and the percentage of fungal cell removal from the test samples was calculated in relation to the positive control.

[0145] Finally, after being subjected to the saline washing process, as described, the discs were placed back in Potato Dextrose Agar culture medium and incubated at 32°C for 3 days to analyze recurrence, that is, whether there would be fungal growth after the treatment period. In this case, fungal growth was assessed visually.

[0146] For the described assay, the following test samples were used: (1) the OEAc containing mainly 1-nitro-2-phenylethane (86.637%) and methyleugenol (12.7%), extracted and characterized as presented in Example 1: Extraction and characterization of Aniba canelilla essential oil, diluted to 10% with mineral oil (inert input), so as to be at the same concentration as the nanostructured lipid carrier compositions; (2) the nanostructured lipid carrier; (3) the lyophilized nanostructured lipid carrier redispersed in water; and (4) the lyophilized nanostructured lipid carrier redispersed in aqueous enamel (containing Eudragit®). Petition 870220112614, dated 02 / 12 / 2022, pp. 46 / 61 33 / 35

[0147] All three nanostructured lipid carrier compositions mentioned (test samples 2 to 4) contained in their composition 10% of OEAc (which had 86.637% 1-nitro-2-phenylethane and 12.7% methyleugenol) extracted and characterized as presented in Example 1: Extraction and characterization of Aniba canelilla essential oil. Furthermore, the nanostructured lipid carrier compositions were prepared as described in Example 2: Production of lyophilized nanostructured lipid carriers containing Aniba canelilla essential oil and their redispersions in an aqueous glaze containing, in addition to the aforementioned essential oil, 10% tucumã butter, 2.5% sorbitan oleate, and 2.5% polysorbate 20. Additionally, the lyophilized compositions were supplemented with 10% mannitol prior to drying and subsequently redispersed in the pharmaceutical vehicle for 90 minutes.

[0148] The results of the ex vivo assay are presented in Table 5 below, expressed as a percentage of removal of Trichophyton rubrum fungal cells (inoculum of 100 pL at 105 CFU / mL) in relation to the positive control group, in which the discs were infected and did not receive any treatment. The negative control showed no fungal growth, as expected, considering that its discs were not infected.

[0149] Table 5 - Percentage of removal of Trichophyton rubrum fungal cells (inoculum of 100 pL at 105 CFU / mL) for the test samples in relation to the positive control. Test sample (group) Removal of fungal cells Aniba canelilla essential oil diluted to 10% 97.44% Nanostructured lipid carrier 100% Lyophilized nanostructured lipid carrier redispersed in water 94.45% Lyophilized nanostructured lipid carrier redispersed in aqueous glaze 86.76% Petition 870220112614, dated 02 / 12 / 2022, pp. 47 / 61 34 / 35

[0150] As shown in Table 5, all test samples considered for the assay proved effective in combating fungal cells in an ex vivo model of onychomycosis, given that they presented high removal percentages. These results demonstrate the antifungal potential of the invention for the topical treatment of onychomycosis.

[0151] OEAc was able to eliminate 97.44% of fungal cells, even when diluted to a concentration of 10%, which proves its antifungal activity, and it can be inferred that it is a promising active ingredient to be used in topical pharmaceutical compositions for the treatment of onychomycosis. This finding is attributed to its major component, 1-nitro-2-phenylethane, which, therefore, also shows potential to be used as an antifungal active ingredient, alone or in combination with methyleugenol.

[0152] The nanostructured lipid carrier composition (non-lyophilized) demonstrated a 100% fungal cell removal rate, meaning it was able to combat all infecting fungal cells on the discs. This result indicates that delivering the essential oil used and / or its predominant components in a lipid nanosystem is a suitable approach. Furthermore, the lyophilized nanostructured lipid carrier composition redispersed in water showed a 94.45% fungal cell removal rate, ensuring that the lyophilization and subsequent redispersion process does not compromise the antifungal activity of the nanoparticles.

[0153] The lyophilized nanostructured lipid carrier redispersed in aqueous enamel showed a lower percentage of fungal cell removal, but still indicative of efficacy (86.76%), compared to the nanostructured lipid carrier (100%) and lyophilized nanostructured lipid carrier redispersed in water (94.45%) groups. This finding is probably due to the polymeric composition of the enamel base, which may lead to a slower release of the nanoparticles from the pharmaceutical form.

[0154] Regarding the recurrence analysis, there was no fungal growth in the pig hoof discs treated with OEAc diluted to 10%, as can be seen Petition 870220112614, dated 02 / 12 / 2022, pp. 48 / 61 35 / 35 visualized in Figure 8A, which can be compared with Figure 8B, which shows fungal growth on the positive control discs, and with Figure 8C, which demonstrates the absence of fungal growth on the negative control discs. In this sense, the antifungal potential of this essential oil and its major component can again be seen.

[0155] Furthermore, in the recurrence analysis, there was also no fungal growth in the discs treated with the nanostructured lipid carrier composition, as shown in Figure 9A, which can be compared with Figure 9B, which shows fungal growth in the positive control discs, and with Figure 9C, which shows the absence of fungal growth in the negative control discs. This result corroborates the 100% removal of fungal cells for this group, as previously presented.

[0156] For the discs treated with lyophilized nanostructured lipid carrier redispersed in water and lyophilized nanostructured lipid carrier redispersed in an aqueous enamel base, fungal growth was observed in the recurrence analysis, which likely occurred due to the lower percentages of fungal cell removal that they presented, even if significant, compared to the other two groups.

[0157] However, fungal growth in the recurrence analysis may be attributed to the fact that a longer treatment period with these compositions and / or a higher daily dose is necessary, considering that in this assay only 20 pL of test sample was pipetted once a day for 7 days onto the infected discs.

[0158] In this sense, the results presented demonstrate the antifungal potential of the invention and its active ingredients against dermatophyte fungi. Petition 870220112614, dated 02 / 12 / 2022, pp. 49 / 61

Claims

1 / 3 Claims 1. NANOSTRUCTURED LIPID CARRIER COMPOSITION characterized by including: a. a nanostructured lipid carrier having a size between 50 and 350 nm, a polydispersity index less than 0.5 and a zeta potential greater than 30 mV in modulus, which includes: i. between 1.0 and 25.0% of a solid lipid; ii. between 1.0 and 8.0% of a non-ionic lipophilic surfactant with a hydrophilic-lipophilic balance (HLB) between 3 and 6; iii. between 1.0 and 8.0% of a non-ionic hydrophilic surfactant with a hydrophilic-lipophilic balance (HLB) between 8 and 18; iv. between 1.0 and 25.0% of a liquid lipid selected from a lipid feedstock with a melting point below 20°C; v. Water as needed; b. Between 1.0 and 25.0% w / w of Aniba canelilla (Kunth) Mez essential oil consisting of 1-nitro-2-phenylethane.

2. NANOSTRUCTURED LIPID CARRIER COMPOSITION characterized by including: a. a nanostructured lipid carrier having a size between 50 and 350 nm, a polydispersity index less than 0.5 and a zeta potential greater than 30 mV in modulus, which includes: i. between 1.0 and 25.0% of a solid lipid; ii. between 1.0 and 8.0% of a non-ionic lipophilic surfactant with a hydrophilic-lipophilic balance (HLB) between 3 and 6; iii. between 1.0 and 8.0% of a non-ionic hydrophilic surfactant with a hydrophilic-lipophilic balance (HLB) between 8 and 18; iv. between 1.0 and 25.0% of a liquid lipid selected from a lipid raw material with a melting point below 20°C; v. water q.s.; b. between 1.0 and 25.0% w / w of 1-nitro-2-phenylethane. Petition 870260079260, dated 07 / 08 / 2026, page 7 / 12 2 / 3 3. NANOSTRUCTURED LIPID CARRIER COMPOSITION, according to claim 2, characterized by including between 1.0 and 25.0% w / w of methyleugenol.

4. NANOSTRUCTURED LIPID CARRIER COMPOSITION, according to claims 2 and 3, characterized in that 1-nitro-2-phenylethane and methyleugenol are obtained from the essential oil extracted from the plant Aniba canelilla (Kunth) Mez.

5. NANOSTRUCTURED LIPID CARRIER COMPOSITION, according to claims 1 and 2, characterized in that the solid lipid comprises any lipid raw material with a melting point above 30°C.

6. NANOSTRUCTURED LIPID CARRIER COMPOSITION, according to claims 1 and 2, characterized by including between 1.0 and 25.0% of a cryoprotectant selected from a carbohydrate and by being lyophilized.

7. PROCESS FOR OBTAINING A NANOSTRUCTURED LIPID CARRIER added to the compositions claimed in 1 and 2, characterized by comprising the following steps: a. heating and homogenization of the oil phase between 40 and 60°C, under agitation, for 3 to 8 minutes; b. heating and homogenization of the aqueous phase between 40 and 60°C for 3 to 8 minutes; c. mixing of the oil and aqueous phases by stirring for 1 to 5 minutes, maintaining the temperature between 40 and 60°C; d. shearing of the mixture of oil and aqueous phases between 10,000 and 15,000 rpm, for 1 to 5 minutes; e. high-pressure homogenization of the mixture of oil and aqueous phases for 5 to 8 cycles, between 650 and 850 bar, maintaining the temperature between 40 and 60°C; f. Cooling to a temperature of 2 to 8°C, between 2 and 6 hours. Petition 870260079260, dated 07 / 08 / 2026, p. 8 / 12 3 / 3 8. PROCESS FOR OBTAINING A NANOSTRUCTURED LIPID CARRIER, according to claim 7, characterized by including the following steps necessary to obtain a lyophilized nanostructured lipid carrier: a. addition of a cryoprotectant by stirring at room temperature for 30 to 90 minutes; b. freezing at a temperature between -15 and -25°C for 12 to 36 hours; c. drying between -40 and -60°C for 24 to 48 hours.

9. USE of the compositions claimed in 1, 2 and 3, characterized by being for preparing a product for the topical treatment of dermatophytoses. Petition 870260079260, dated 07 / 08 / 2026, page 9 / 12

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