Nanostructured pharmaceutical composition containing α-bisabol for the treatment of diabetic wounds.

Description

1 / 25 “NANOSTRUCTURED PHARMACEUTICAL COMPOSITION CONTAINING α-BISABOOL FOR THE TREATMENT OF DIABETIC WOUNDS” Field of invention

[001] The present invention relates to lyotropic liquid crystals developed using PEG-40 Hydrogenated Castor Oil and Polysorbate-80 as surfactants forming these systems. The mesophase obtained served as a vehicle for α-bisabolol, a sesquiterpene alcohol with the chemical formula C15H26O, recognized for its therapeutically active properties. More precisely, the present invention is directed towards promoting wound healing in individuals with diabetes, resulting from the healing, antimicrobial, antioxidant and anti-inflammatory activities of the active ingredient α-bisabolol. Fundamentals of the invention

[001] Diabetes Mellitus (DM) is a chronic metabolic disease characterized by persistent hyperglycemia due to a deficiency in insulin production or action. Type 1 DM results from the destruction of pancreatic β cells present in the islets of Langerhans, responsible for the synthesis of endogenous insulin. Whereas, type 2 DM involves a combination of insulin resistance in peripheral tissues and β cell dysfunction, which results in inadequate insulin production. (RODACKI et al., 2022).

[002] Although it is a global health challenge, the greatest burden of diabetes is concentrated in developing countries, where approximately 80% of cases occur. The current prevalence is around 537 million adults aged 20 to 79 years affected by diabetes globally, including 15 million Brazilians. Furthermore, epidemiological projections predict that by 2045 there will be a significant increase to more than 23 million. (SUN et al., 2022). Petition 870240110015, dated 12 / 25 / 2024, page 13 / 49 2 / 25

[003] Based on this, patients living with diabetes often face significant challenges in wound healing due to the complex interaction of pathophysiological factors.

[004] In patients who do not have diabetes, skin lesions represent a discontinuity in the dermal tissue, causing an anatomical disturbance that compromises its physiological functions. After damage occurs, the body initiates a process known as healing to restore the integrity of the skin and mitigate possible secondary complications. This process can be categorized into four distinct phases: hemostasis, inflammatory phase, proliferative phase, and remodeling phase (LEAL; CARVALHO, 2014). During hemostasis, a fibrin clot forms, which performs several essential functions, including the release of pro-inflammatory cytokines and growth factors, as well as inducing vasoconstriction. In the inflammatory phase, there is chemokine-mediated cellular chemotaxis, resulting in the migration of neutrophils and macrophages to the site of injury, where these cells phagocytize pathogens and cellular debris.Simultaneously, enzymes such as elastase and myeloperoxidase are secreted to facilitate the removal of damaged tissue. The proliferative phase is characterized by the deposition of extracellular matrix rich in type III collagen, which is gradually replaced by type I collagen, promoting the formation of granulation tissue. Angiogenesis, mediated by factors such as vascular endothelial growth factor (VEGF), ensures adequate blood supply to the injured area, facilitating healing. Finally, in the remodeling phase, fibroblasts synthesize type I collagen in an organized manner, promoting wound contraction and scar tissue remodeling. This process is crucial for restoring the structural integrity of the skin and re-establishing its normal physiological functions. (COLARES et al., 2019; GOIS et al., 2021).

[005] However, in the case of patients living with diabetes, the healing process is compromised due to several factors, such as coagulation dysfunction due to hyperglycemia, degeneration of the extracellular matrix caused by oxidative stress, and decreased levels of nitric oxide and essential growth factors. (GOIS et al., 2021). Petition 870240110015, dated 12 / 25 / 2024, page 14 / 49 3 / 25

[006] These conditions contribute to a chronic systemic inflammatory response and a reduction in neutrophil activity, increasing susceptibility to infections and hindering the regeneration of damaged tissues (LEAL; CARVALHO, 2014). Chronic hyperglycemia associated with diabetes mellitus compromises angiogenesis and collagen synthesis, which are fundamental for adequate wound healing. Additionally, diabetic neuropathy causes sensory and autonomic neuropathy, resulting in decreased sensitivity and a reduced local inflammatory response (GOIS et al., 2021).

[007] These tissue injuries occur mainly in areas such as the lower limbs, and diabetic neuropathy frequently results in inadvertent injuries among patients. The absence of adequate wound treatment can trigger chronic conditions that, if left uncontrolled, can evolve into more serious complications, including the need for partial and / or total limb amputation. (GOIS et al., 2021). Thus, chronic hyperglycemia in diabetes mellitus negatively interferes with the natural healing process, resulting in delays and complications in injury recovery. This can favor the development of chronic conditions such as diabetic foot, exacerbated by hypoxia due to poor blood perfusion, which amplifies oxidative stress and the production of oxygen free radicals, for example. (LEAL; CARVALHO, 2014).

[008] Faced with this pressing issue, there is a growing demand for the improvement of pharmaceutical formulations and associated technologies, aiming to enhance the pharmacological effects of medications. This is fundamental to ensuring effective treatments, promoting greater clinical adherence, and guaranteeing an adequate quality of life for patients. In this context, we can highlight the application of phytochemicals in therapeutics, which has always been present in humanity. And, currently, there is continuous growth in the development of drugs that employ active principles derived from plant resources, as highlighted by Martelli et al. (2018).

[009] Based on this, we have α-bisabolol, a monocyclic sesquiterpene alcohol molecule with the molecular formula C15H26O, which constitutes approximately 90% of the oil extracted from the bark of the Eremanthus erythropappus tree, known as Candeia. This phytochemical exhibits a variety of Petition 870240110015, dated 12 / 25 / 2024, page 15 / 49 4 / 25 biological activities, including anti-inflammatory, antimicrobial, antiallergic, antiseptic, antioxidant, gastroprotective, neuroprotective and wound healing properties, which makes it attractive for wound treatment. (EL-LAKANY et al., 2019; SANTOS et al., 2019a; XU et al., 2020).

[0010] In Brazil, this species is frequently sighted in the mountainous regions of the Serra da Mantiqueira and in southern Minas Gerais. It is typically found at high altitudes, thriving in shallow, rocky soils known for their low fertility. (FONSECA et al., 2021; ROCHA et al., 2020).

[0011] It is obtained predominantly through direct distillation of candeia essential oil, exhibiting itself as a colorless liquid with a delicate floral aroma. (SANTOS et al., 2019b). Due to its high lipophilicity, it has the ability to penetrate the plasma membrane without adversely affecting cells (CORPASLÓPEZ et al., 2014). This compound is optically active due to the presence of two chiral centers and presents four distinct stereoisomers: (-)-α-bisabolol, (-)-epi-abisabolol, (+)-α-bisabolol and (+)-epi-α-bisabolol, with (-)-α-bisabolol being the predominant and biologically active form. (ALBERTTI et al., 2018a; SANTOS et al., 2019a).

[0012] However, due to its high volatility and susceptibility to degradation from direct exposure to atmospheric oxygen, light, heat and humidity, coupled with potential adverse effects such as skin irritation when applied in pure form to damaged skin, there is a crucial need to deliver this active ingredient in a delivery system that not only transports but also protects and stabilizes its bioactive properties. (BILIA et al., 2014; TUREK; STINTZING, 2013).

[0013] Thus, in the present invention we have lyotropic liquid crystals (LCs), which represent a significant innovation in pharmaceutical nanotechnology, being a promising vehicle for drug delivery. Lyotropic substances modify their structure in response to the addition of lyotropic agents, which can be solvents, surfactants, polymers, and electrolytes, resulting in different molecular arrangements and direct alterations in the physicochemical properties of the system. These systems are composed of an intermediate phase between crystalline solid and isotropic liquid. Petition 870240110015, dated 12 / 25 / 2024, page 16 / 49 5 / 25

[0014] The intermediate structure of these crystals arises due to the intermolecular interactions present in these systems. The overall organization of the system is influenced by the strong hydrogen bonds between the polar portion of the amphiphilic molecule and the solvent (water), while the fluid properties of the system are attributed to the van der Waals interactions that form in the nonpolar portion of the surfactant. (COHEN-AVRAHAMI et al., 2014). In this way, these are systems that have alternating hydrophobic and hydrophilic domains, exhibiting an extensive internal interface that differs in physicochemical properties from the external surface, which offers the advantage of different dielectric constants, enabling the efficient segregation of polar and nonpolar drugs, as described in document BR1020180089684A2.This makes them an excellent alternative for delivering α-bisabolol, as they are able to transport lipophilic substances safely, effectively, sustainably, and while preserving their pharmacological properties, in addition to reducing their volatility and optimizing their stability.

[0015] Document BR1120240014738A2 deals with compositions in the form of lyotropic liquid crystalline nanoparticles. Colloidal nanosystems that encapsulate bioactive macromolecules with peptide bonds for oral administration, which have been shown to be stable in size, size distribution, zeta potential, and to maintain physicochemical stability in both simulated gastric fluid (pH = 1.6) and simulated intestinal fluid (pH = 6.5) over time. Even when administered orally in the cited invention, the liquid crystals (LCs) are able to transport substantial amounts of drugs regardless of their polarity and remain stable, which is advantageous due to the flexibility in choosing the route of administration, including the cutaneous route, used in the present invention.

[0016] Document BR1020180089684A2 deals with liquid crystals for nasal application of isoniazid for the treatment of tuberculosis (TB), where the objective is to obtain phase precursor systems, stabilized by surfactants containing this active ingredient. The inventors highlight that liquid crystals (LCs) have a structure organized in molecular arrangements with hydrophilic and hydrophobic portions, standing out as an ideal pharmaceutical form matrix for the controlled release of drugs. It has a high viscosity, which has the advantage of prolonging drug release. In turn, in the cutaneous route, its Petition 870240110015, dated 12 / 25 / 2024, page 17 / 49 The 6 / 25 viscosity also ensures a protective barrier for open wounds, increasing adhesion to the skin, reducing the risk of infection, and consequently adding benefits for cutaneous administration.

[0017] Furthermore, document GR1010363 (B), which presents lyotropic lipid liquid crystal nanoparticles for use in active drug delivery systems, emphasizes the hierarchically self-organized internal structure of this system, which is ideal for housing and transporting active pharmaceutical molecules. However, currently there is no invention that uses this system to deliver α-bisabolol, making the present invention a groundbreaking innovation in the field of pharmaceutical technology.

[0018] α-bisabolol, due to its incredible pharmacological activities, demonstrates multiple modes of action, for example, as described in document CN117462436 (A), the innovation is situated within the field of cosmetics and presents a nanocomposition with anti-allergic and anti-inflammatory properties, formed by bisabolol, paeonol, glycyrrhetinic acid, an emulsifier, a co-emulsifier, liquid lipid and water. Thus, bisabolol demonstrates effects that include reduction and resistance to inflammation, relief of pain and swelling, promotion of cell regeneration and skin metabolism, as well as improving the skin's ability to resist irritation and repair inflammatory damage. The mass ratio between bisabolol, paeonol and glycyrrhetinic acid used was (4.2-4.8): (2.3-2.7): (0.9-1.1), and significant antiallergic and anti-inflammatory effects were demonstrated, highlighting its potential in wound healing. Furthermore, in several other publications, such as CN117065008 (A), CN116637056 (A), CN117298021 (A), CN115487112 (A), the efficacy of α-bisabolol as a single agent or synergistically in combination with other substances has been demonstrated, exhibiting anti-inflammatory, soothing, skin-repairing and healing properties, evidencing its active pharmacological activity.

[0019] In light of this, we also have topical treatments currently used for diabetic wounds, which focus on hygiene and the application of dressings and substances adapted to the degree of ulceration. In less severe situations, simple dressings such as sterile gauze with saline solution, hydrocolloid dressings, or essential fatty acids are used. Both aim to maintain local moisture to promote an environment conducive to healing. However, these dressings do not contain Petition 870240110015, dated 12 / 25 / 2024, page 18 / 49 7 / 25 bioactive agents that accelerate the healing process, and in more severe cases, dressings containing calcium and sodium alginate or silver sulfadiazine can be used, and when there are signs of infection, an antimicrobial agent can be added. (SANTOS, 2016). This highlights the advantages of the present invention, since liquid crystalline systems provide the necessary hydration for healing, in addition to the active ingredient acting in accelerating healing while also having antimicrobial effects, without needing to add more therapeutic agents, reducing the difficulty of patient adherence. Furthermore, these systems present rheological properties suitable for cutaneous application, are accessible, low-cost, highly stable and easy to scale, as well as providing a composition conducive to the incorporation of oily substances, highlighting this technology as promising. (LAPTEVA; KALIA, 2017; SHAN et al., 2019). Brief description of the drawings

[0020] Figure 1: Pseudoternary phase diagram, composed of ultrapure water as the aqueous phase, in combination with PEG-40 and Polysorbate-80 (1:1, w / w) as the surfactant phase and TACC as the oily phase, obtained from the point-to-point technique.

[0021] Figure 2: Pseudoternary phase diagram divided by viscosity regions. RBV: Low viscosity region; RMV: Medium viscosity region; RAV: High viscosity region; RLI: Isotropic liquid region; RSNT: Non-transparent system region.

[0022] Figure 3: Photographs of the structures observed under polarized light microscopy of the selected formulations. These are: F1-I: (Inert Formulation 1), F1: (Formulation 1 containing 1% α-bisabolol); F2-I: (Inert Formulation 2), F2: (Formulation 2 containing 1% α-bisabolol); F3-I: (Inert Formulation 1), F3: Formulation 3 containing 1% α-bisabolol); 40X increase.

[0023] Figure 4: SAXS scattering profile of the selected formulations. The arrows represent the scattering peaks (q).

[0024] Figure 5: Photographs of the lesions taken on days 3, 7 and 14 of the experiment. FT - Test formulation, FI - Inert formulation and CN - Negative control. Petition 870240110015, dated 12 / 25 / 2024, page 19 / 49 8 / 25

[0025] Figure 6: Histology images at 100x magnification. Tissue stained with eosin and hematoxylin. FT-Test formulation, Fl- Inert formulation and CN- Negative control. Description of the invention

[0027] In general, the invention aimed to develop liquid crystalline systems containing α-bisabolol for the treatment of diabetic wounds. The liquid crystals were obtained using a ternary phase diagram and were characterized using polarized light microscopy, rheological profile analysis, and small-angle X-ray scattering. The selected formulations were analyzed using in vitro release and skin permeation assays with Franz cells. Finally, the in vivo activity of the selected formulation was performed to evaluate healing at 3, 7, and 14 days, which was determined using an experimental model of streptozotocin-induced Diabetes Mellitus in Wistar rats.

[0028] All components were acquired from domestic suppliers, including α-bisabolol, which was obtained commercially and characterized by gas chromatography coupled to mass spectrometry (GC / MS). Using a GC / MS instrument (Shimadzu QP2010 plus, Tokyo, Japan) equipped with an autosampler (split / splitless).

[0029] Example of implementation 1: Obtaining the liquid crystal type pharmaceutical composition.

[0030] Liquid crystals were synthesized using the pseudoternary phase diagram construction methodology. Using ultrapure water as the aqueous phase, caprylic / capric triglycerides (TACC) as the oil phase, and the combinations shown in Table 1 as the surfactant phase, Table 1. Combinations of different surfactants used in obtaining liquid crystals. Surfactant combinations Peg-40 Hydrogenated Castor Oil: Oil PEG-7 Esters (1:1) Peg-40 Hydrogenated Castor Oil: Polysorbate-80 ____________________________(iro_______________________ Petition 870240110015, dated 12 / 25 / 2024, page 20 / 49 9 / 25 Polysorbate-80: Oil PEG-7 Esters (1:1)

[0031] For the formation of the surfactant phase, a combination of surfactants in a 1:1 (w / w) ratio was used, which was mixed and kept under magnetic stirring for 24 hours to ensure complete homogenization of the components. Subsequently, solutions containing surfactant phase and oil phase were prepared in varying proportions: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9. After 30 minutes of magnetic stirring, the surfactant and oil solutions, still under stirring, were titrated with increasing volumes of water, following a 5-minute interval between each titration to allow the system to stabilize. After each addition of water, the system was macroscopically evaluated for viscosity and transparency (Figure 1), being classified as Transparent Liquid Systems (SLT), Opaque Liquid Systems (SLO), Transparent Viscous Systems (SVT), Opaque Viscous Systems (SVO), or Phase Separation (SF).

[0032] The diagrams were prepared in duplicate. Subsequently, the Transparent Viscous Systems (VSS) region was identified and delimited in the phase diagram (Figure 2), and the surfactant and oil combination that presented the largest area within this region was selected. Consequently, all formulations were prepared separately and subjected to characterization, and based on the results obtained, the formulations for incorporation of α-bisabolol were chosen.

[0033] Results: Through analysis of the diagram, it was observed that using ultrapure water as the aqueous phase, a combination of PEG-40 Hydrogenated Castor Oil and Polysorbate-80 (1:1, w / w) as the surfactant phase, and TACC as the phase, it was possible to obtain a larger region of SVT. SVT was obtained in regions with water content below 50%. At concentrations above this limit, fluid and transparent liquid systems were identified (Figure 1). Thus, in the portion of the diagram corresponding to the range of 10% to 50% water, the transparent systems had significant variations in viscosity, attributes frequently characteristic of liquid crystals (LCs). As shown in Figure 2 Petition 870240110015, dated 12 / 25 / 2024, page 21 / 49 Based on Table 10 / 25 and Table 2, three formulations with distinct viscosity profiles were chosen: Low viscosity region (LVR), Medium viscosity region (MVR), and High viscosity region (HVR). Table 2. Compositions of the selected formulations. System Composition (%) Surfactant Phase Oil Phase Aqueous Phase F1 60 30 10 F2 50 30 20 F3 40 30 30

[0034] Example of implementation 2: Characterization studies of pharmaceutical forms.

[0035] 2.1 Polarized light microscopy: The isotropy or anisotropy of the studied systems was evaluated using an Olympus® BX51 polarized light microscope with an Evolution LC Color digital camera and Pixel Link software. For the preparation of the microscopic analysis, a small amount of the sample was placed on an appropriate slide and covered with a coverslip. Observations were made at room temperature, using 20x and 40x objectives to observe various fields. The compositions evaluated were: F1-I, F2-I and F3-I inert; F1, F2 and F3 containing 1% abisabolol.

[0036] Results: As demonstrated in Figure 3, the results of the analysis allowed the classification of the samples as anisotropic or isotropic, in addition to showing that the addition of 1% α-bisabolol to the formulations did not cause alterations in the structure of the mesophases. Thus, formulations F1-I and F1 were identified as microemulsions or precursor systems; F2-I and F2 presented areas of hexagonal mesophase anisotropy, identified by their striated texture, in addition to the area of ​​isotropy characteristic of cubic mesophase in formulations F3-I and F3. Petition 870240110015, dated 12 / 25 / 2024, page 22 / 49 11 / 25

[0037] 2.2 Rheology: The systems were analyzed with a rheometer (MCR 302, Anton Paar, Austria) in oscillatory mode using cone-plate geometry (diameter 20 mm, angle 1°, gap of 52 pm). The linear viscoelastic region was determined with a strain variation of 0.1 to 100% at 1 Hz. Frequencies from 0.1 to 100 Hz were swept with fixed strain to evaluate the G' and G'' moduli. Thus, the samples were applied to the center of the plate, waiting 2 minutes before analysis for stabilization. The experiments were conducted at room temperature and the data were collected in triplicate.

[0038] Results: Systems F2 and F3 showed G' greater than G'' (tanδ < 1), while F1 showed the opposite. The G' values ​​followed the order F3 > F2 > F1. F2 and F3 showed similar viscoelasticity profiles, with different magnitudes (F3 > F2), while F1 showed considerably lower viscosity. In F2 and F3, the elastic component showed little variation with increasing frequency, typical of highly organized systems such as liquid crystals. In contrast, F1 showed different behavior, possibly due to the mesophase transition. The incorporation of α-bisabolol did not alter the rheological profiles of the formulations, corroborating the results of polarized light microscopy that identified characteristic structures for F1, F2, and F3.

[0039] 2.3 Small-angle X-ray scattering: SAXS data from the selected formulation samples were obtained using the Nano-InXider (Xenocs) instrument, which includes a monochromator with Cu Kα radiation. A Dectris Pilatus3 detector, positioned 938 mm from the sample, was used to acquire scattering intensity (I(q)) data as a function of the scattering vector q (q = 4π / λ) sin (θ / 2), where θ represents the scattering angle and λ is the wavelength of the X-rays. The analyses were conducted at 25°C and noise signals were subtracted from the total scattering intensity to improve the quality of the data obtained.

[0040] Results: Table 3. Scattering vector (q) values. SP = precursor system, HEX = hexagonal. Position of the peaks (A-Ί) Petition 870240110015, dated 12 / 25 / 2024, page 23 / 49 12 / 25 Formulation q1 q2 q3 Periodicity Structure F1 0.046 - - - SP F2 0.04226 0.07144 0.08266 ^3:2 HEX P6m F3 0.05498 0.09911 0.1335 \3: \6 CUB lm3m

[0041] Through the peaks present, it is possible to relate them to the ordered structure of the mesophase liquid crystal phases. Thus, as shown in Figure 4, sample F1 exhibits a broad peak characteristic of a microemulsion or isotropic system, while F2 and F3 show a first peak followed by two smaller ones, typical of liquid crystals. Therefore, F1 is classified as a precursor or microemulsion, confirmed by other physicochemical analyses described previously, while F2 and F3 are liquid crystals. Furthermore, the analysis of the distance between the Bragg peaks allows the identification of the type of mesophase present in each liquid crystal developed. Sample F2 exhibits a ratio between the three peaks of 3:2 in the SAXS profile; this ratio indicates that the distance between the observed diffraction peaks follows a ratio of 3 to 2.Specifically, in liquid crystal analysis, a 3:2 ratio is indicative of an organizational structure corresponding to the hexagonal mesophase, as corroborated by the results of Polarized Light Microscopy and Rheological Analysis. Whereas, in formulation F3, the correlation between the three observed peaks was 3:6, suggesting the possible formation of a cubic mesophase. This occurs because the cubic mesophase structure tends to exhibit this specific relationship between diffraction peaks due to the three-dimensional organization of molecules or structures in the phase.

[0042] Embodiment example 3: Cytotoxicity assay of bisabolol and the selected formulation.

[0043] Isolated α-bisabolol was tested, as well as formulations F1 and F1-Inert. Formulation F1 was chosen due to its higher surfactant concentration, indicating a greater potential for cytotoxicity. They were subjected to MTT assays using L929 fibroblast cell lines in culture plates maintained for 24h at 37°C / 5% CO2. Petition 870240110015, dated 12 / 25 / 2024, page 24 / 49 13 / 25 Immediately after, the fibroblasts were treated with concentrations of 50, 100, and 200 μg / mL for 24 hours. After the treatment period, the cells were washed twice with PBS pH 7.4 and 200 μL of a 5 mg / mL MTT solution diluted 10x in PBS, and subsequently placed in contact with the cells, which were incubated at 37 °C for 3 hours. After removing the MTT, 200 μL of DMSO was added to solubilize the formazan. Optical density readings were taken after 10 minutes using an automated plate reader, ELISA, at a wavelength of 570 nm. Cell viability analysis was performed according to the following equation: Abs (treated cells) - Abs (DMSO) Cell viability (%) =------------------------------------ x 100 Abs (negative cells) - Abs (DMSO)

[0044] Results: Cell viability was close to 100% for all formulations tested, regardless of concentration, while isolated α-bisabolol, at a concentration of 200 pg / mL, showed lower cell viability. Therefore, the formulations tested did not prove to be cytotoxic at the concentrations tested, as evidenced by the high cell viability observed.

[0045] Example of implementation 4: Permeation and release study using Franz cells.

[0046] In vitro release and permeation assays were performed using modified Franz diffusion cells with a diffusion area of ​​1.77 cm2. To investigate the release and permeation profile of α-bisabolol from the selected systems, a cellulose membrane (45 µm pore) was used as a physical barrier in the release tests, and pig ear skin was used in the permeation assays, placed directly in contact with the receptor medium. The formulations were administered into the donor compartment and, at predetermined intervals of 0, 0.5, 1, 2, 4, 6, 9, 12, and 24 hours, 1.0 mL aliquots of the receptor medium were collected, followed by replacement with the same volume of receptor medium. The collected samples were quantified by High-Performance Liquid Chromatography (HPLC). The receiving medium used consisted of phosphate buffer pH 7.4 containing 30% ethanol to ensure sink conditions.After the skin permeation experiment, the skin was removed from the Franz cell, washed to remove any excess formulation, cut, homogenized in acetonitrile, and... Petition 870240110015, dated 12 / 25 / 2024, page 25 / 49 14 / 25 was subjected to an ultrasound bath for 30 minutes. The resulting dispersion was centrifuged, filtered, and quantified by HPLC. (LIRA et al., 2008).

[0047] Results: The control with 10% Polawax™ GP-200 showed approximately 80% release of the active ingredient at the end of the experiment, more than the formulations tested. Among formulations F1, F2, and F3, the results showed that F1 released 9.20% (± 0.82), F2 released 34.99% (± 0.15), and F3 released 22.80% (± 1.50) of the active ingredient at the end of the experiment. There was no statistically significant difference between the formulations in the first 9 hours, but after the twelfth hour, there was a difference between the release profiles of the different mesophases. However, in general, it can be concluded that the selected formulations were able to provide sustained release of the active ingredient when compared to the control formulation. Regarding permeation and skin retention, it was not possible to detect the active ingredient in the samples even after 24 hours of the assay.

[0048] Example of implementation 5: In vivo study in Wistar rats using a DM1 model.

[0049] The study employed 15 Wistar rats, after approval from the Ethics Committee on the Use of Animals of the Federal University of Sergipe. The animals were kept in accordance with the guidelines of the National Council for the Control of Animal Experimentation (CONCEA), housed in the Bioterium of the Integrated Laboratory of Experimental Biology - Libex1 of UFS. They were housed in specific boxes containing wood shavings as substrate, with suitable metal grids, under controlled environmental conditions of temperature (22°C ± 3°C) and light and dark cycle (12 hours of light and 12 hours of darkness). In addition, the animals had free access to food and water.

[0050] 5.1 Induction of diabetes:

[0051] The study used the method described by JUNIOR et al (2024) to induce experimental diabetes in animals. Induction was performed by intraperitoneal administration of Streptozocin dissolved in 0.05 M citrate buffer solution (pH 4.5), at a single dose of 40 mg / kg body weight, after a 24-hour fasting period. After administration of the substance, the animals were fed, and the Petition 870240110015, dated 12 / 25 / 2024, page 26 / 49 Blood glucose levels were monitored 72 hours after induction (15 / 25). Blood was collected for analysis by caudal puncture using lancets and an Accu-Check Go glucometer to measure blood glucose. Animals with fasting blood glucose levels equal to or greater than 200 mg / dL were selected to participate in the study.

[0052] 5.2 Characterization of wounds and treatment with liquid crystal:

[0053] Diabetic animals were divided into three groups: negative control (NC), inert formulation (IF), and test formulation (TF), identified externally on the cages. The NC group received saline solution, while the IF group received a formulation without α-bisabolol, and the TF group received a formulation with α-bisabolol. Before applying the formulations, complete hair removal was performed in the area of ​​interest (trichotomy). To create the wounds, the rats were anesthetized with Ketamine (80 mg / kg), Midazolam (3 mg / kg), and Tramadol Hydrochloride (2 mg / kg) intraperitoneally, with maintenance of anesthesia using isoflurane. Antisepsis was performed on the dorsal region, and the cutaneous wounds were made with an 8.0 mm punch, exposing the dorsal muscle fascia. After hemostasis control, the injured areas were treated with 600 mg of the respective treatments every 48 hours for 14 days. (GARROS et al„ 2006; DOS SANTOS, 2016).

[0054] Implementation example 6: Healing assessment parameters.

[0055] 6.1 Evolution of wound healing:

[0056] After creating wounds in the animals, images of the lesions were captured on days 0, 7, and 14 of the experiment using a digital camera positioned 30 cm away from the lesions, before the daily application of the treatment. The area of ​​the wounds was measured on days 0, 3, 7, 10, and 14 using a digital caliper. To calculate the percentage of wound closure over time, the elliptical area formula was applied: Larger diameter Smaller diameter Area = [(------------------) x (------------------) x π] 2

[0057] The percentage of lesion contraction was determined using the equation: % = [1 - current lesion area / initial lesion area x 100]. The data were then statistically analyzed using GraphPad Prism software and ANOVA. Petition 870240110015, dated 12 / 25 / 2024, page 27 / 49 16 / 25 followed by Tukey's test for comparison between groups. Differences were considered statistically significant when p < 0.05 (FALCÃO et al., 2016).

[0058] Results: Based on the results of the percentage of lesion contraction, which are: FT Group: 15.3% ± 9.3% (day 3), 56.5% ± 8.7% (day 7), 90.6% ± 2.2% (day 10), 96% ± 1.7% (day 14); FI Group: 11.9% ± 7.2% (day 3), 34.5% ± 9.6% (day 7), 89.3% ± 0.7% (day 10), 96% ± 2.9% (day 14); CN Group: 7.9% ± 3.4% (day 3); 44.7% ± 2.7% (day 7), 85.5% ± 5.2% (day 10), 90.5% ± 2.2% (day 14). Combined with the images of the lesions (Figure 5), a statistically significant difference was observed on days 3 and 7 between the evaluated groups. This effect can be attributed to the anti-inflammatory properties of α-bisabolol, which act especially during the initial inflammatory phase of the healing process. These findings suggest that the tested substance may reduce inflammation and, consequently, accelerate the healing process in the first seven days of the experiment.

[0059] 6.2 Histological evaluation:

[0060] At the end of the 14-day experiment, the animals were euthanized for histological evaluation of the wounds. Tissue from the affected area was collected and fixed in 10% buffered formalin. After paraffin embedding, the skin was sectioned into 5 µm slices, perpendicular to the lesion surface. The samples were deparaffinized in ethanol and xylene, stained with hematoxylin and eosin, and analyzed by microscopy (OLYMPUS BX 45, OLYMPUS). Images of the sections were documented. (CHATTERJEE et al., 2022; DARA et al., 2021).

[0061] Results: Morphologically, histological analysis of the tissues revealed comparable re-epithelialization between the groups after 14 days of treatment (Figure 6), which is consistent with the percentage reduction in lesions. Additionally, there was a decrease in inflammatory infiltrate in the tissues of animals in the FT group compared to the CN and FI groups, possibly attributed to the anti-inflammatory action of α-bisabolol. (KAZEMI et al., 2020).

[0062] 6.3 Biochemical and hematological evaluation of the animals:

[0063] On the last day of the experiment (day 14), blood was collected from the animals for biochemical and hematological analysis. Part of the samples was collected in Petition 870240110015, dated 12 / 25 / 2024, page 28 / 49 17 / 25 tubes containing sodium EDTA were used for the evaluation of hematological parameters, including total red blood cell count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), total platelets, total leukocytes, total lymphocytes, total eosinophils, and total monocytes. The remaining samples were collected in dry tubes for the determination of biochemical parameters such as glycated hemoglobin (HbA1c), blood glucose, aspartate aminotransferase (AST or TGO), alanine aminotransferase (ALT or TGP), uric acid, C-reactive protein (CRP), and cholesterol (FARIA, 2022).

[0064] Results:

[0065] Thus, through the evaluation of hematological parameters of the red blood cell series, it was observed that most values, such as red blood cells, hemoglobin, hematocrit, MCH, MCHC, and monocytes, were close to the reference standards. Regarding the results of the evaluation of the analyzed biochemical parameters, it was observed that blood glucose levels were elevated in all groups (Table 4), which is consistent with the condition of diabetes in rats. Furthermore, the presence of liver damage is suggested in animals from all groups, although AST levels were within normal values, ALT values ​​were elevated, as were cholesterol levels. These findings probably result from the damage associated with diabetes, where the persistence of high glucose levels can induce this type of liver damage. Additionally, uric acid and CRP values ​​are above the reference values ​​in all three groups, with the CN group showing the highest levels.These markers are associated with inflammation, suggesting that inflammation in the CN group is more intense compared to the other two groups, which is consistent with histological findings that demonstrated a greater inflammatory infiltrate in the CN group. (NERY et al., 2015). Table 4. Biochemical parameters. The reference values ​​adopted were taken from the study by Lima et al. Group FT Group FI Group CN Reference Values Blood glucose 138 ± 30.17 374.8 ± 155.2 536 ± 60 460 ± 99 (mg / dL) Petition 870240110015, dated 12 / 25 / 2024, page 29 / 49 18 / 25 TGO (U / L) 131.33 ± 43 127.8 ± 41 94.9 ± 4.9 115.6 ± 19.1 TGP (U / L) 57.5 ± 11.95 124.8 ± 41 91.9 ± 4.9 112.6 ± 19 Uric acid 1.81 ± 0.67 3.6 ± 0.3 3.1 ± 0.2 19.8 ± 0.4 (mg / dL) Cholesterol 60.68 ± 6.51 104.2 ± 16 96.1 ± 2.9 99.8 ± 14 (mg / dL) CRP (mg / dL) 7.2 ± 0.4 31.2 ± 5 37 ± 3 48.4 ± 12.5

[0066] Conclusion:

[0067] In summary, the results of this study highlight the viability and efficacy of liquid crystals as vehicles for α-bisabolol in topical applications for the treatment of cutaneous wounds. The formulated systems (F2 and F3) not only allowed for the controlled and sustained release of the active ingredient, but also demonstrated a positive influence on healing, accelerating the contraction of lesions and reducing inflammatory infiltrate in the tissues. These findings not only reinforce the therapeutic potential of lyotropic liquid crystals, but also emphasize the importance of protecting this technology through patents, since formulations that show promising results in in vivo studies are crucial for future clinical and commercial applications. Petition 870240110015, dated 12 / 25 / 2024, pp. 30 / 49 19 / 25 References: ALBERTTI, L. A. G. et al. Identification of the bisabolol synthase in the endangered candeia tree (eremanthus erythropappus (dc) mcleisch). Frontiers in Plant Science, v. 9, n. September,p. 1-7, 2018a. ALBERTTI, L. A. G. et al. Identification of the bisabolol synthase in the endangered candeia tree (eremanthus erythropappus (dc) mcleisch). Frontiers in Plant Science, v. 9, 19 set. 2018b.ALVES, A. Q. et al. The Fatty Acid Composition of Vegetable Oils and Their Potential Use inWound Care. Advances in skin & wound care, v. 32, p. 1-8, 2019. AMARAL, V. DO et al. Alfa-bisabolol: uma revisão de propriedades bioativas antiflogísticas. Research, Society and Development, v. 10, n. 12, p. e447101220618, 26 set. 2021. BANERJEE, K. et al. Journal of Drug Delivery Science and Technology Azadirachta indica A. Juss ( Neem ) oil topical formulation with liquid crystals ensconcing depot water for anti- inflammatory , wound healing and anti-methicillin resistant Staphylococcus aureus activities. Journal of Drug Delivery Science and Technology, v. 64, n. November 2020, p. 102563, 2021. BILIA, A. R. et al. Essential oils loaded in nanosystems: A developing strategy for a successful therapeutic approach. Evidence-based Complementary and Alternative MedicineHindawi Limited, , 2014. BRASIL. RDC N. 166, de 25 de Julho de 2017. Agência Nacional de Vigilância Sanitária, v.2017, p. 1-21,2017. CARVALHO, A. L. M. et al. Evaluation of Microemulsion and Lamellar Liquid Crystalline Systems for Transdermal Zidovudine Delivery. Journal of Pharmaceutical Sciences, v. 105,n. 7, p. 2188-2193, 2016. CARVALHO, F. C. et al. Development and In Vitro Evaluation of Surfactant Systems for Controlled Release of Zidovudine. Journal of pharmaceutical sciences, v. 99, n. 7, p. 2367- 2374, 2010. CHAIYANA, W.; RADES, T.; OKONOGI, S. Characterization and in vitro permeation study of microemulsions and liquid crystalline systems containing the anticholinesterase alkaloidal extract from Tabernaemontana divaricata. International Journal of Pharmaceutics , v. 452, n. 1-2, p. 201-210, ago. 2013. Petição 870240110015, de 25 / 12 / 2024, pág. 31 / 49 20 / 25 CHATTERJEE, N. S. et al. Nano-encapsulation of curcumin in fish collagen grafted succinyl chitosan hydrogel accelerates wound healing process in experimental rats. Food Hydrocolloidsfor Health, v. 2, p. 100061, dez. 2022. CHAVDA, V. P. et al. Lyotropic liquid crystals for parenteral drug delivery. Journal of Controlled ReleaseElsevier B.V., , 1 set. 2022. COHEN-AVRAHAMI, M. et al. On the correlation between the structure of lyotropic carriersand the delivery profiles of two common NSAIDs. Colloids and Surfaces B: Biointerfaces, v. 122, p. 231-240, out. 2014. COLARES, C. M. P. et al. Cicatrização E Tratamento De Feridas: a Interface Do ConhecimentoÀ Prática Do Enfermeiro. Enfermagem em Foco, v. 10, n. 3, 2019. CORPAS-LÓPEZ, V. et al. ( - )- α -Bisabolol, a Promising Oral Compound for the Treatmentof Visceral Leishmaniasis. 2014. COUILLAUD, B. M. et al. State of the Art of Pharmaceutical Solid Forms: from Crystal Property Issues to Nanocrystals Formulation. ChemMedChemJohn Wiley and Sons Ltd, ,8 jan. 2019. DARA, P. K. et al. Biocompatibility and histopathological evaluation of chitosan nanoparticles grafted fish gelatin bio-nanocomposite membranes in rats. Iranian Polymer Journal (EnglishEdition), v. 30, n. 9, p. 953-964, 1 set. 2021. DE ARAUJO, GRS et al. In situ microemulsion-gel obtained from bioadhesive hydroxypropyl methylcellulose films for transdermal administration of zidovudine. Colloids and Surfaces B: Biointerfaces, vol. 188, 1st Apr. 2020. DE OLIVEIRA, AC et al. Quality of life of people with chronic wounds. ACTA Paulista de Enfermagem, v. 32, n. 2, p. 194-201, 2019. DE SÁ, A. Á. M. et al. Evaluation of the incorporation of essential oils in microemulsions as a promising formulation in the inhibition of tyrosinase. Cultures and Industrial Products, v. 154,n. May, p. 112654, 2020. D'EMERY, MB EVALUATION OF ESSENTIAL OIL FROM Curcuma longa LEAVES IN THE HEALING OF SKIN WOUNDS IN RATS. Recife: FEDERAL RURAL UNIVERSITY OF PERNAMBUCO, 2020. DO PRADO, AH et al. Bioadhesive liquid crystal systems for octyl methoxycinnamate skin delivery. Journal of Molecular Liquids, vol. 345, 1 Jan. 2022. EL-LAKANY, SA et al. α-Bisabolol-loaded cross-linked zein nanofibrous 3D Petition 870240110015, dated 12 / 25 / 2024, pp. 32 / 49 21 / 25 scaffolds for accelerating wound healing and tissue regeneration in rats. International Journal of Nanomedicine, vol. 14, p. 8251-8270, 2019. ESPOSITO, E. et al. Monoolein liquid crystalline phases for topical delivery of crocetin. Colloids and Surfaces B: Biointerfaces, v. 171, p. 67-74, 2018. FAGIONATO, JM et al. Vegetable oils in pharmaceutical and cosmetic lipidbased nanocarriers preparations. Industrial Crops and ProductsElsevier BV, , 15 Oct. 2021. FARIA, TF. STUDY OF THE HEALING ACTIVITY OF GREEN PROPOLIS EXTRACT IN THE TREATMENT OF INDUCED SKIN LESIONS IN ADULT WISTAR RATS. Brasilia: UNIVERSITY OF BRASÍLIA, 2022. FERREIRA, S. G. et al. An environmentally safe larvicide against Aedes aegypti based on in situ gelling nanostructured surfactant systems containing an essential oil. Journal of Colloid and Interface Science, v. 456, p. 190-196, 2015. FONSECA, A. L. C. et al. Rescue and vegetative propagation of Eremanthus erythropappus ( DC .) MacLeish in natural stand. Brazilian Journal of biology, v. 6984, p. 1-9, 2021. FONSECA-SANTOS, B.; GREMIÃO, M. P. D.; CHORILLI, M. A simple reversed phase high-performance liquid chromatography (HPLC) method for determination of in situ gelling curcumin-loaded liquid crystals in in vitro performance tests. Arabian Journal of Chemistry,v. 10, n. 7, p. 1029-1037, 2017. FORMARIZ, TP et al. Microemulsions and liquid crystalline phases as drug delivery systems. Brazilian Journal of Pharmaceutical Sciences, v. 41, n. 03, p. 301-313, 2005. GAINAR, A. et al. Incompatibility-Driven Self-Organization in Polycatenar Liquid Crystals Bearing Both Hydrocarbon and Fluorocarbon Chains. Journal of Physical Chemistry B, v.121, n. 37, p. 8817-8828, 21 September. 2017. GARROS, IDC et al. Passiflora edulis extract in the healing of open skin wounds in rats: a morphological and histological study. Acta Brazilian Surgical Journal, v. 21, n. 3, 2006. GOIS, T. DA S. et al. Physiopathology of healing in patients with diabetes mellitus. Brazilian Journal of Health Review, v. 4, no. 4, p. 14438-14452, 2021. Petition 870240110015, dated 12 / 25 / 2024, pp. 33 / 49 22 / 25 GOTO, P. L.; SIQUEIRA-MOURA, M. P.; TEDESCO, A. C. Application of aluminum chloride phthalocyanine-loaded solid lipid nanoparticles for photodynamic inactivation of melanoma cells. International Journal of Pharmaceutics, v. 518, n. 1-2, p. 228-241,25 fev.2017. HUANG, Y.; GUI, S. Factors affecting the structure of lyotropic liquid crystals and the correlation between structure and drug diffusion. RSC Advances, v. 8, n. 13, p. 6978-6987, 2018a. HUANG, Y.; GUI, S. Factors affecting the structure of lyotropic liquid crystals and the correlation between structure and drug diffusion. RSC AdvancesRoyal Society of Chemistry, , 2018b. JANG, H. J.; SHIN, C. Y.; KIM, K. B. Safety evaluation of polyethylene glycol (PEG) compounds for cosmetic use. Toxicological Research, v. 31, n. 2, p. 105-136, 2015. KAZEMI, M. et al. Deep skin wound healing potential of lavender essential oil and licorice extract in a nanoemulsion form: Biochemical, histopathological and gene expression evidences. Journal of Tissue Viability, vol. 29, no. 2, p. 116-124, 1 May 2020. KUROKAWA, SSS. Obtaining and studying supra-amphiphilic meglumine carboxylic acid derivatives in aqueous phase for the development of biomaterials. Araraquara: Universidade Estadual Paulista, September 14, 2018. 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Claims

1 / 2 CLAIMS 1. Pharmaceutical composition of the lyotropic liquid crystal type, characterized by comprising a surfactant phase formed by a mixture of non-ionic surfactants, an oily phase, an aqueous phase and a wound-healing active ingredient.

2. A lyotropic liquid crystal type pharmaceutical composition, according to claim 1, characterized by comprising a surfactant phase formed by a combination of PEG-40 Hydrogenated Castor Oil and Polysorbate-80 in a 1:1 (w / w) ratio, an oily phase composed of caprylic / capric triglycerides (CACC), and an aqueous phase composed of ultrapure water and α-bisabolol.

3. A lyotropic liquid crystal pharmaceutical composition, according to claims I and II, characterized by comprising a surfactant phase formed by a combination of PEG-40 Hydrogenated Castor Oil and Polysorbate-80 in a 1:1 (w / w) ratio at a concentration ranging from 20% to 70% relative to the total weight of the system, an oily phase composed of caprylic / capric triglycerides (CACC) at a proportion of 5% to 50% of the total weight of the system, an aqueous phase composed of ultrapure water at a proportion of 10% to 50% of the total weight of the system, and α-bisabolol at a concentration ranging from 0.5% to 5% relative to the total weight of the system.

4. A lyotropic liquid crystal pharmaceutical composition, according to claims I to III, characterized by containing in the final formulation a surfactant phase formed by a combination of PEG-40 Hydrogenated Castor Oil and Polysorbate-80 in a 1:1 (w / w) ratio at a concentration of 50%, an oily phase composed of caprylic / capric triglycerides (CACC) at a concentration of 30%, an aqueous phase composed of ultrapure water at a concentration of 19% and α-bisabolol at a concentration of 1%.

5. Pharmaceutical composition of the lyotropic liquid crystal type according to claim IV, characterized by containing anisotropic hexagonal structures, with sustained release of the active ingredient. Petition 870260005525, dated 01 / 21 / 2026, page 4 / 5 2 / 2 6. Method for preparing a lyotropic liquid crystal pharmaceutical composition, characterized by comprising the formulation of a mixture obtained by incorporating the active ingredient into a liquid crystal system comprising at least one fatty acid ester and a liquid crystal forming agent, followed by the addition of an aqueous solvent to form a controlled-release base and subsequent homogenization until a homogeneous formulation with uniform distribution of the active ingredient is obtained.

7. Use of a lyotropic liquid crystal type pharmaceutical composition, characterized by comprising the composition described in claim IV, in which α-bisabolol in combination with a liquid crystalline system results in a pharmaceutical formulation intended for the topical treatment of diabetic wounds.

8. Use of the pharmaceutical composition according to claim IV, characterized by being applied directly to the wound with a frequency that may vary from daily use to every 48 hours, accelerating healing and reducing inflammatory infiltrate. Petition 870260005525, dated 01 / 21 / 2026, page 5 / 5

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