Methods for preparing polyol-free vegetable oil nanoemulsions using d-phase emulsification, polyol-free vegetable oil nanoemulsions, pharmaceutical compositions, and uses thereof

Abstract

The present invention relates to a method for the preparation of a nanoemulsion comprising a polyol-free vegetable oil using the D-phase emulsification (DPE) method. Furthermore, the present invention relates to a nanoemulsion comprising a polyol-free vegetable oil, a pharmaceutical composition and the use thereof as a medicament in the treatment of atopy in a mammal.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical and nanotechnology, and describes a method for obtaining nanoemulsions preferably comprising polyol-free plant oils, wherein D-phase emulsification (DPE) technology is used. Furthermore, this invention discloses polyol-free plant oil nanoemulsions, pharmaceutical compositions, and the use of said pharmaceutical compositions in the treatment of atopic diseases in mammals. Background Technology

[0002] Plant products (including non-volatile and volatile oils) can contain bioactive substances with the potential to treat various diseases (FAGIONATO MASIERO et al., 2021). Borage oil, derived from the seeds of Borago officinalis, a plant native to the Mediterranean region, contains high levels of essential fatty acids (EFAs), more specifically omega-6, which are important for the structure and function of the skin. They play a crucial role in the fluidity and flexibility of cell membranes and influence the function of membrane-associated proteins such as receptors and enzymes (BACCOUCH et al., 2023). Therefore, this oil can be used as an adjunct in the treatment of skin conditions such as atopic dermatitis.

[0003] Atopic dermatitis is associated with abnormal EFA metabolism, particularly with abnormal production of gamma-linolenic acid (GLA). This characteristic impairs the incorporation of EFA into the phospholipid membrane. EFAs are considered key components in membrane systems. Furthermore, they are metabolized into arachidic acids, particularly prostaglandins and leukotrienes, which are highly active and modulators of inflammatory, immune, and proliferative responses, including those of epidermal cells (DENNIS; NORRIS, 2015). Therefore, the use of ω-6 EFAs can reverse symptoms of atopic dermatitis, such as inflammation, dryness, and itching. The bioavailability of EFAs can be improved by using nanostructured systems containing borage oil.

[0004] Nanoemulsions are thermodynamically unstable oil-in-water (O / W) or water-in-oil (W / O) emulsions. et al., 2008; SOLANS; (2012), with an average particle size of approximately 100 to 1000 nm. Nanoemulsions maintain resistance to sedimentation or emulsion stratification due to their reduced particle size, and ionic and nonionic surfactants are often used in O / W nanoemulsions to stabilize them through steric and electrostatic effects, preventing flocculation, aggregation and agglomeration (ANTON; BENOIT; SAULNIER, 2008).

[0005] The "Ostwald ripening" phenomenon is considered to be the main source of instability in O / W nanoemulsions, caused by the diffusion of smaller particles to larger particles due to Laplace pressure differences (SOLANS). 2012; TAYLOR, 1998). Because nanoemulsions are thermodynamically unstable systems, their formation does not occur spontaneously and requires the application of mechanical or chemical energy. Therefore, unlike microemulsions, obtaining nanoemulsions is strongly constrained by the processes available for reducing particles to nanoscale (SOLANS et al., 2005).

[0006] In the context of obtaining nanoemulsions, the process can be divided into two main mechanisms: mechanical high-energy processes and physicochemical low-energy processes. Although the former has been studied most frequently in the past few decades—employing techniques such as high-pressure homogenization, microfluidization, ultrasonication, and high-amplitude ultrasound—low-energy processes are gaining increasing interest due to their favorable cost-effectiveness, as they do not require specific equipment to prepare nanoemulsions.

[0007] Low-energy processes are considered efficient because they reduce the need for energy costs, especially in industrial-scale production. Among physicochemical methods, the most well-known techniques include phase transition temperature (PIT), phase transition composition (PIC), spontaneous emulsification (SE) (ANTON; BENOIT; SAULNIER, 2008), and more recently, D-phase emulsification (DPE). High-pressure homogenization (as a high-energy method) and D-phase emulsification (as a low-energy method) were chosen to obtain the borage oil nanoemulsion of this invention because they offer specific advantages compared to other processes.

[0008] Conventional (low-energy) physicochemical processes (such as PIT, PIC, and spontaneous emulsification) require precise control of the hydrophilic-lipophilic balance (HLB), temperature, and / or the method of adding different phases to obtain nanoemulsions. These processes require the presence and transformation of microemulsion (ME) or liquid crystal (LC) phases. This means that in addition to requiring high concentrations of surfactant to form LC or ME, time is also needed to regulate the HLB. This characteristic limits these conventional methods for obtaining low-energy nanoemulsions due to the need for high concentrations of surfactant in the formulation and can be highly toxic (ANTON; BENOIT; SAULNIER, 2008). Surfactants are inherently toxic and should be used at the lowest possible concentration. Furthermore, the rigid structure of CL often leads to phase separation at high oil concentrations (e.g., 20-30%) (SAGITANI, 1988).

[0009] D-phase emulsification (DPE), proposed by Sagitani et al. in the 1980s, offers the advantage of obtaining fine and homogeneous emulsions without the need for expensive equipment and with reduced surfactant / stabilizer concentrations.

[0010] Furthermore, compared to conventional phase transition methods, the DPE process offers advantages including the formation of fine emulsions with low surfactant concentrations and the elimination of the need for strict HLB regulation compared to PIC, PIT, and SE. Another advantage of DPE is that, unlike the PIT process, it can form nanoemulsions using a single hydrophilic surfactant (considered safer than lipophilic surfactants) and achieve highly stable emulsions at high temperatures (SAGITANI; NABETA; NAGAI, 1991). Moreover, unlike spontaneous emulsification methods, the DPE process does not require the presence of organic solvents. This characteristic facilitates its industrial-scale application.

[0011] Unlike conventional low-energy methods, in the DPE process, the use of polyols as surfactants is considered an important component of the system, altering the association properties of nonionic surfactants with the medium (MURAKAMI et al., 2005; SAGITANI, 1986). Diols such as 1,3-butanediol increase the HLB of surfactants, directing them to the aqueous phase (forming mixed micelles with nonionic surfactants). Glycerol, D-sorbitol, and polyethylene glycol reduce the HLB of surfactants by directing them to the lipophilic phase, thus decreasing the affinity between nonionic surfactants and water (SAGITANI; IKEDA; OGO, 1984).

[0012] By applying the DPE process, ENDOO;SAGITANI, (1991) successfully obtained an O / W nanoemulsion containing 20.0% (m / m) olive oil and a particle size of 500 nm using a 2.0% (m / m) concentration of hydrophilic surfactant (ethoxylated oleyl alcohol) and a temperature of 60 °C. It is important to emphasize that this achievement was made without the need for any specialized equipment (such as high-pressure or shearing devices). However, it is noteworthy that despite this success, the application of the D-phase emulsification (DPE) process has not been fully utilized in recent decades, and to date, no evidence of progress has been found in the development of nanoemulsions containing more than 20.0% (w / w) borage oil with an average hydrodynamic diameter (AHD) below 500 nm.

[0013] The use of statistical tools such as Design of Experiments (DoE) is crucial for the development and optimization of nanoemulsions. DoE allows for a more comprehensive assessment of the impact of process and formulation variables on quality properties, enabling more efficient optimization with fewer experiments. This quality-derived design approach is essential for ensuring the consistency and efficacy of nanoemulsions, contributing to their clinical application and commercialization.

[0014] Design of Experiments (DoE) is an important statistical tool in the research and development of nanostructured systems using a quality-derived design approach. DoE allows for in-depth understanding of the impact of formulation components and process parameters on key quality properties of the formulation. This tool allows for simultaneous modification of different factors. Therefore, it enables efficient optimization using a reduced number of experiments. et al. (2008).

[0015] The use of statistical tools in the preparation of nanoemulsions via DPE was designed to optimize the process, which contains a high concentration of borage oil (≥25.0% wt / wt (m / m)) and a low concentration of hydrophilic surfactant (≤4.0% m / m). Furthermore, the tool allowed for the disclosure of the effects of independent variables (oil concentration, surfactant concentration, glycerol concentration, and initial water concentration) on the response (particle size). The target quality characteristics of the system were: AHD (mean hydrodynamic diameter) below 500 nm, PI (polydispersity index) below 0.3, and ζ-potential below -20 mV.

[0016] US Patent 8337931 discloses compositions and methods for preparing foods and beverages containing additives. Additives include nutritional foods, pharmaceuticals, and supplements, such as essential fatty acids, including omega-3 fatty acids, omega-6 fatty acids, conjugated fatty acids, and other fatty acids; phytochemicals, including phytosterols; other oils; and coenzymes, including coenzyme Q10 and other oil-based additives.

[0017] Although US Patent 8337931 discloses a vegetable oil—borose oil—and has a similar particle size range to this invention, there are relevant differences between the two inventions, such as: the US patent method is carried out through high shear and has additional steps involving heating and cooling. Furthermore, the US patent teaches a stirring speed range between 850 and 1200 rpm, and reducing the speed to 25 or 50 rpm to reduce air incorporation into the nanoemulsion.

[0018] Furthermore, US Patent 8337931 teaches a different sequence for preparing nanoemulsions than this invention, in which a surfactant is added first, followed by a preservative, then a solvent, then a co-surfactant, a nonpolar compound, and finally an emulsion stabilizer. The present invention uses only borage oil, a surfactant, and water in the formulation, and the stirring speed is limited to 300 rpm.

[0019] Patent document CN105963335 discloses a nano-sized borage oil microemulsion and a method for preparing the nano-sized borage oil microemulsion. The nano-sized borage oil microemulsion is prepared from the following components: 2.5 to 5 parts borage oil, 3 to 6 parts glyceryl citrate, 3 to 7 parts sorbitan monooleate, 2 to 5 parts sodium cocoyl hydroxyethyl sulfonate, 0.5 to 3 parts isopropyl myristate, 3 to 6 parts polyglycerol-10 monostearate, 2 to 5 parts N-oleoyl sarcosine, 2 to 4 parts polyglycerol fatty acid ester, 0.5 to 2 parts sucrose isobutyrate acetate, 6 to 10 parts glycerol, 2 to 5 parts polyethylene glycol 400, and 100 to 140 parts water. The disclosed preparation method includes the following steps: heating, mechanical stirring, homogenization, and high-shear processing. The nano-sized borage oil microemulsion prepared and obtained through patent document CN105963335 has the advantages of small average particle size and strong solubility. Furthermore, according to the Chinese patent document, this preparation method is characterized by its simple steps and ease of industrial production.

[0020] However, unlike the present invention, patent document CN105963335 discloses a method for preparing microemulsions using high-speed shearing between 5000 and 7000 rpm, which is considered a high-energy method. Furthermore, the method described in this Chinese patent document includes steps not present in the present invention: a heating step and a microemulsion formation step with a stirring speed between 50 and 150 rpm.

[0021] In contrast, this invention discloses a minimal formulation containing only three components: borage oil, a surfactant, and water, which has shown remarkable results in physicochemical tests. This contrasts sharply with Chinese patent document CN105963335, which describes a microemulsion using borage oil, similar to that in this invention, but including additional components such as glyceryl citrate, sorbitol monooleate, sodium cocoyl hydroxyethyl sulfonate, isopropyl myristate, decaglyceryl monostearate, N-oleoyl sarcosine, glycerol, propylene glycol, and water.

[0022] US Patent 5690947 discloses a composition containing hydroxy acids and / or retinoids, and also borage seed oil as an anti-irritant. This US patent describes the discovery that borage seed oil is highly effective in reducing irritation caused by hydroxy acids and / or retinoids (compared to gamma-linolenic acid or other anti-irritants containing gamma-linolenic acid or other known anti-irritants). Although this US patent uses borage oil to prepare an emulsion, there are significant differences between this patent document and the present invention.

[0023] First, the present invention is not an emulsion, but a nanoemulsion for oral use; therefore, its particle size is much smaller than that of patent US5690947, which is intended for topical application. Furthermore, in the present invention described herein, borage oil is used at a high concentration of 50% m / m, while in patent US5690947 it is used at a low concentration between 0.05 and 10% m / m.

[0024] As observed, existing technologies will benefit from providing solutions for the preparation of nanoemulsions containing a high concentration of borage oil greater than 20 to 50% m / m, preferably 50%, without requiring strict HLB regulation and costly equipment, keeping the surfactant at the lowest possible concentration (maximum 4.0% m / m), while simultaneously achieving the necessary physical stability. Summary of the Invention

[0025] This invention relates to a method for preparing stable vegetable oil nanoemulsions having a high oil concentration of greater than 20% to 50%, preferably 50.0% (w / w), without the use of polyols, and an average hydrodynamic diameter (AHD) between 200 and 400 nm, obtained by means of a DPE process using a single hydrophilic surfactant at a concentration of 3% to 5% (w / w), preferably 4.0% (w / w). These characteristics represent a surfactant to oil ratio of 1:12 to 1:16, thus exhibiting a low surfactant concentration. The plant oils mentioned include, for example, avocado oil (Persea americana), sweet almond oil (Prunus dulcis), neem oil (Carapa guianensis), argan oil (Argania spinosa), hazelnut oil (Corylus avellana), babassu oil (Orbignya phalerata), baobab oil (Adansonia digitata), brickberry oil (Mauritia flexuosa), camellia oil (Camellia oleifera), canola oil (Brassica napus), Brazil nut oil (Bertholletia excelsa), and Maranhã nut oil (…). Nut oil (Sterculia striata), Safflower oil (Carthamus tinctorius), Chia oil (Salvia hispanica), Coconut oil (Cocosnucifera), Bitter pepper oil (Copaifera spp.), Sea cabbage oil (Crambeabyssinica), Almond oil (Prunus armeniaca), Cactus oil (Opuntia ficus-indica), Rice bran oil (Oryza sativa), Raspberry oil (Rubus idaeus), Sunflower oil (Helianthus annuus), Sesame oil (Sesamum indicum), Guarana oil (Paullinia cupana), Shea butter (Vitellaria paradoxa), Lavender oil (Lavandula) Flaxseed oil (Linum usitatissimum), macadamia oil (Macadamia integrifolia), castor oil (Ricinus communis), passion fruit oil (Passiflora edulis), marula oil (Sclerocarya birrea), tea tree oil (Melaleuca alternifolia), corn oil (Zea mays), moringa oil (Moringa oleifera), mustard oil (Brassica juncea), neem oil (Neem... zPecan oil (Carya illinoinensis), olive oil (Olea europaea), pequi oil (Caryocar brasiliense), patauá oil (Oenocarpus bataua), pracaxi oil (Pentaclethra macroloba), rosehip oil (Rosa rubiginosa), pumpkin seed oil (Cucurbita pepo), cottonseed oil (Gossypium hirsutum), grapeseed oil (Vitis vinifera), tamanu oil (Calophylluminophyllum), tucuma oil (Astrocaryum vulgare), cashew oil (Anacardium) occidentale), large flower cocoa butter ( Oils include: (Theobroma grandiflorum) (cocoa oil), (Theobromacacao) (cocoa), (Amburana cearensis) (emburana oil), (Arachis hypogaea) (peanut oil), (Ribes nigrum) (blackcurrant oil), (Schizochytrium spp.) (algae oil), (Euphausia superba) (krill oil), (borose oil), (Foeniculum vulgare) (fennel oil), (Rosmarinus officinalis) (rosemary oil), (Silybummarianum) (milk thistle oil), (Prickly pear cactus oil), (Bixaorellana) (annatto oil), and (Matricaria) (chamomile oil). Peppermint (recutita), peppermint oil (Menthapiperita), ginger oil (Zingiber officinale).

[0026] Furthermore, this invention discloses a plant oil nanoemulsion, which serves as a platform for encapsulating drugs with low water solubility, the drugs being selected from the group consisting of: immunosuppressants such as azathioprine, cyclosporine, tacrolimus, sirolimus (rapamycin), everolimus, tamsulolimus, zotamolimus, leflunomide, mycophenolate mofetil, mycophenolate sodium, guaniolimus, pimecrolimus, fingolimod, baricitinib, ruxolitinib, tofacitinib, abatacept, and beraccept. Circocillin and lacquimod; nonsteroidal anti-inflammatory drugs (NSAIDs) such as aceclofenac, mefenamic acid, niflufenic acid, tiprofen, guanethidine, benzoxam, bromofenac, carbofen, celecoxib, ketoprofen, clonisin, dextro ibuprofen, dextro ketoprofen, diclofenac, diflunisal, etodoxacin, etofenamic acid ester, phenylbutazone, flurbiprofen, ibuprofen, indomethacin, ketorazole, lornoxicam, phenoxicam, meclofenamic acid, meloxicam, nabumetole Ketoconazole, naproxen, nimesulide, oxapezil, parecoxib, piroxicam, propylmetamethasone, rofecoxib, sulindac, tenoxicam, vardicoxib, tometidine, azaprozin, and feprazosin; steroidal anti-inflammatory drugs, such as dexamethasone, betamethasone, budesonide, clobetasol, difluralasone, fluocinolone acetonide, fluocinolone acetonide, fluticasone, fluticasone, halcinonide, hydrocortisone, methylprednisolone, mometasone, peramisone, prednisolone, prednisolone, triamcinolone. Dragon, desoxymethasone, cyclohexanolone, beclomethasone, and aclomethasone; antihistamines such as astemizole, bilastine, ebastine, loratadine, desloratadine, mizolastine, terfenadine, chlorpheniramine, dextromethorphan, brompheniramine, diphenhydramine, promethazine, doxylamine, cyproheptadine, meclomethasone, hydroxyzine, triprolidine, tripiramine, dimethinidine, oxamide, and nitrogen Stine, bucrorazine, chlorcyclorhizine, ketotifen, alimazine, antazoline, trimethoprim, fenamiphos, and dextrobromophenamine; and antibiotics such as azithromycin, clarithromycin, erythromycin base, telithromycin, clindamycin, lincomycin, rifampin, rifabutin, rifapentine, isoniazid, ethionamide, para-aminosalicylic acid, dapsone, chloramphenicol, tiamulin, vornimulin, bacitracin, bacitracin, polymyxin B, colistin, and ash. Flavomycin, fusidic acid sodium, streptomycin, amikacin, capreomycin, spectinomycin, cefadroxil, cefaclor, cefixime, cefdinir, cefpodoxime, cefbufen, minocycline, doxycycline, tetracycline, tigecycline, ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, besifloxacin, gatifloxacin, gemifloxacin, sitafloxacin, prulifloxacin, finafloxacin, naflufloxacin, and enoxacin.

[0027] The present invention also discloses pharmaceutical compositions comprising the plant oil nanoemulsion as a platform preferably used for 1% (w / w) cyclosporine.

[0028] The present invention also relates to the use of the pharmaceutical composition in the preparation of a medicament for treating atopic dermatitis, hypercholesterolemia, premenstrual syndrome, insulin resistance, supporting wound healing, diabetic neuropathy, metabolic syndrome, preferably atopic dermatitis, in mammals (i.e., humans or animals), wherein the mammals are selected from the group consisting of: dogs, cats, horses, cattle, sheep, goats, pigs, rabbits, hamsters, guinea pigs, rats, preferably dogs and cats.

[0029] Another embodiment relates to a method of treating atopic dermatitis in mammals (i.e., humans or animals, preferably dogs and cats) using the pharmaceutical composition.

[0030] Finally, the present invention also relates to the use of the pharmaceutical composition for the treatment of atopic dermatitis.

[0031] The use of statistical tools in this invention allows for the revelation of surprising and unpredictable phenomena that are fundamentally important for the full acquisition of nanoemulsions.

[0032] The low-energy method used in this invention, as presented herein, allows for the development of nanoemulsions with low surfactant content, high oil concentration, and without the use of polyols and organic solvents—a surprising feature in nanoemulsion development. Furthermore, the low-energy method allows for scalability and industrial application. Attached Figure Description

[0033] The subject matter of the invention will become fully clear in its technical aspects through a detailed description based on the accompanying drawings listed below.

[0034] Figure 1A The structure and visual appearance of the DPE process at 25°C are shown, displaying an isotropic solution containing surfactant and initial water.

[0035] Figure 1B The structure and visual appearance of the DPE process at 25°C are shown, revealing the translucent / transparent viscous structure obtained after the addition of oil.

[0036] Figure 1C The diagram shows the structure and visual appearance of the DPE process at 25°C, showing a white and flowing O / W emulsion after the final addition of water.

[0037] Figure 2A The image shows the visual appearance of the DPE process at 25°C, with an enlarged view of the viscous structure.

[0038] Figure 2B The image shows the visual appearance of the DPE process at 25°C on a scaled-up O / W emulsion.

[0039] Figure 3A The response surface and contour plot of the AHD of borage oil nanoemulsion are shown, considering the following variables: borage oil concentration, polysorbate 80, and initial water content. The response surface and contour plot represent the potential relationships between these variables by displaying the three-dimensional relationships between the three variables in two dimensions. Therefore, Figure 3A The changes in particle size (AHD) are shown when using different concentrations of borage oil and polysorbate 80.

[0040] Figure 3B The response surface and contour plot of the AHD of borage oil nanoemulsion are shown, considering the following variables: borage oil, polysorbate 80, and initial water concentrations. The response surface and contour plot represent the potential relationships between these variables by displaying the three-dimensional relationships between the three variables in two dimensions. Therefore, Figure 3B The changes in particle size (AHD) are shown when using different concentrations of borage oil and initial water.

[0041] Figure 3C The response surface and contour plot of the AHD of borage oil nanoemulsion are shown, considering the following variables: borage oil, polysorbate 80, and initial water concentrations. The response surface and contour plot represent the potential relationships between these variables by displaying the three-dimensional relationships between the three variables in two dimensions. Therefore, Figure 3C The changes in particle size (AHD) are shown when using different concentrations of polysorbate 80 and initial water.

[0042] Figure 4 The diagram shows the main effects of AHD as a function of the composition and preparation variables used in the DPE process.

[0043] Figure 5A The study demonstrates the toxicity assay of a polyol-free borage oil nanoemulsion prepared by the DPE method in the invertebrate model *Galleria mellonella*, showing the health index (A) of *Galleria mellonella* larvae injected with the following substances: PBS: phosphate-buffered saline solution, pH 7.4; 50% borage oil nanoemulsion.

[0044] Figure 5B The toxicity assay of a polyol-free borage oil nanoemulsion prepared by the DPE method in the invertebrate model *Heliotropium indicum* is shown, and the survival rate of *Heliotropium indicum* larvae injected with the following substance (B): PBS: sodium phosphate buffer, pH 7.4; 50% borage oil nanoemulsion. Detailed Implementation

[0045] This invention relates to a method for preparing vegetable oil nanoemulsions using DPE based on the ENDOO method (SAGITANI, (1991)), wherein three components are used: water, a nonionic surfactant, and vegetable oil. The nonionic surfactant includes, for example, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sorbitol monolaurate, sorbitol monopalmitate, sorbitol monooleate, sorbitol trioleate, lecithin, glyceryl stearate, glyceryl laurate, glyceryl oleate, glyceryl cetearate, and caprylyl / caprylglucoside. Decyl glucoside, lauryl polyglucoside, glyceryl isostearate, cetyl ethoxylate, octyl phenyl alcohol-9, nonyl phenyl alcohol ether-9, poloxamer 188, poloxamer 407, poloxamine 904, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 3350, PEG-8 polydimethylsiloxane, PEG-12 polydimethylsiloxane, lauryl methacrylate, cholesterol ethoxylate, methyl reduced glucosamine, polyethylene glycol distearate, methyl glucoside ester, methyl glucoside phosphate, polyglycerol-4 ester, polyglycerol-10 lauryl ester, glyceryl castor oil, preferably polysorbate 80;The plant oils mentioned include avocado oil, sweet almond oil, neem oil, argan oil, hazelnut oil, babassu oil, baobab oil, brighamb oil, camellia oil, rapeseed oil, Brazil nut oil, and sterculia nut oil. striata), safflower oil, chia oil, coconut oil, tartare oil, sea cabbage oil, almond oil, prickly pear oil, rice bran oil, raspberry oil, sunflower oil, sesame oil, guarana oil, shea butter, lavender oil, flaxseed oil, macadamia nut oil, castor oil, passion fruit oil, marula oil, tea tree oil, corn oil, moringa oil, mustard oil, neem oil, pecan oil, olive oil, Brazilian nectarine oil, patauá Oils including oyster palm oil, bakas fruit oil, rosehip oil, pumpkin seed oil, cottonseed oil, grapeseed oil, tamanu oil, tucuma oil, cashew oil, cocoa butter, cocoa oil, emburana oil, peanut oil, blackcurrant oil, seaweed oil, krill oil, borage oil, fennel oil, rosemary oil, milk thistle oil, cactus oil, annatto oil, chamomile oil, peppermint oil, and ginger oil are prepared at temperatures ranging from 10°C to 50°C, preferably 25°C.

[0046] In a second embodiment, the present invention discloses a plant oil nanoemulsion, which is a platform for encapsulating drugs with low water solubility, said drugs being selected from the group consisting of: immunosuppressants such as azathioprine, cyclosporine, tacrolimus, sirolimus (rapamycin), everolimus, tamsulolimus, zotamolimus, leflunomide, mycophenolate mofetil, mycophenolate sodium, guanitrolimus, pimecrolimus, fingolimod, baricitinib, ruxolitinib, tofacitinib, etc. Abatacept, beracide, vorciclosporine, and laquimod; nonsteroidal anti-inflammatory drugs (NSAIDs) such as aceclofenac, mefenamic acid, niflufenicol, tiprofen, guanethidine, benzoxetine, bromofenac, carbofen, celecoxib, ketoprofen, clonisin, dextro ibuprofen, dextro ketoprofen, diclofenac, diflunisal, etodoxacin, etofenadine, phenylbutazone, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lornoxicam, phenoxicam, metronidazole Fenamic acid, meloxicam, nabumetone, naproxen, nimesulide, oxapezil, parecoxib, piroxicam, propylmetamethasone, rofecoxib, sulindac, tenoxicam, vardicoxib, tometidine, azaprozin, and feprazolam; steroidal anti-inflammatory drugs, such as dexamethasone, betamethasone, budesonide, clobetasol, difluralasone, fluocinolone acetonide, fluocinolone acetonide, fluticasone, halcinonide, hydrocortisone, methylprednisolone, mometasone, and paracetamol. Prednisolone, triamcinolone, hydroxymethasone, cyclohexanone, beclomethasone, and aclomethasone; antihistamines such as astemizole, bilastine, ebastine, loratadine, desloratadine, mizolastine, terfenadine, chlorpheniramine, dextromethorphan, brompheniramine, diphenhydramine, promethazine, doxylamine, cyproheptadine, meclomethasone, hydroxyzine, triprolidine, tropineamine, dimethinidine, oxamidine, and nitrogen Stine, bucrorazine, chlorcyclorhizine, ketotifen, alimazine, antazoline, trimethoprim, fenamiphos, and dextrobromophenamine; and antibiotics such as azithromycin, clarithromycin, erythromycin base, telithromycin, clindamycin, lincomycin, rifampin, rifabutin, rifapentine, isoniazid, ethionamide, para-aminosalicylic acid, dapsone, chloramphenicol, tiamulin, vornimulin, bacitracin, bacitracin, polymyxin B, colistin, and ash. Flavomycin, fusidic acid sodium, streptomycin, amikacin, capreomycin, spectinomycin, cefadroxil, cefaclor, cefixime, cefdinir, cefpodoxime, cefbufen, minocycline, doxycycline, tetracycline, tigecycline, ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, besifloxacin, gatifloxacin, gemifloxacin, sitafloxacin, prulifloxacin, finafloxacin, naflufloxacin, and enoxacin.

[0047] In a third embodiment, the present invention relates to a pharmaceutical composition comprising a plant oil nanoemulsion and an immunosuppressant.

[0048] In a fourth embodiment, the present invention discloses the use of the pharmaceutical composition in the preparation of a drug for treating atopic dermatitis, hypercholesterolemia, premenstrual syndrome, insulin resistance, supporting wound healing, diabetic neuropathy, and metabolic syndrome in mammals, preferably atopic dermatitis, wherein the mammals are selected from the group consisting of dogs, cats, horses, cattle, sheep, goats, pigs, rabbits, hamsters, guinea pigs, and rats, preferably dogs and cats.

[0049] - Development of Nanoemulsions Using DPE Process — Nanoemulsion Preparation

[0050] The process begins with the preparation of an aqueous phase comprising 0.5 to 1.5% initial water, 1 to 5%, preferably 4.0% (w / w) nonionic surfactant, preferably polysorbate 80, homogenized at a temperature of 10 to 50°C, preferably 25°C, under stirring at 50 to 500 rpm, preferably 300 rpm. Using a magnetic or mechanical stirrer at a controlled speed of 50 to 500 rpm, preferably 300 rpm, vegetable oil, preferably borage oil, is added dropwise to the aqueous phase at 5% to 50%, preferably 50% of its total volume. After the oil has been completely added, the system is maintained under stirring for 10 to 30 minutes, preferably 20 minutes. The process then proceeds with the addition of 46.5% to 43.5% of the remaining water. Following this final step, the oil-in-water (O / W) nanoemulsion is maintained under constant stirring at 50 to 500 rpm, preferably 300 rpm, for 10 to 30 minutes, preferably 20 minutes.

[0051] - To prepare nanoemulsions on a large scale

[0052] A process was developed on a laboratory scale for its expansion, aiming to achieve a yield of 1 kg. Used in… Nanoemulsion Preparation The same components and parameters were used as mentioned in the previous article. The changes involved the type of stirrer (this time a mechanical stirrer (IKA, RW20 digital display)) and the amount prepared (1 kg). Borage oil was added slowly, but not in a dropwise manner as in the previous model; due to the higher preparation volume, the required volume of oil was added using a peristaltic pump (flow rate of 5 to 100 mL / min, preferably 10 mL / min). Since it is a physicochemical method, the water flow rate did not affect the quality characteristics of the nanoemulsion.

[0053] Box-Behnken Experimental Design

[0054] The effects of nanoemulsion components on AHD were analyzed using the Box-Behnken response surface methodology (three factors at three levels: +1; 0; -1). A total of 15 preparations were performed, including three center points (formulations 5, 10, and 11) (Table 1 / 2). The independent variables were borage oil (25.0; 37.5; 50.0% m / m), polysorbate 80 (2.0; 3.0; 4.0% m / m), and initial water (0.5; 1.0; 1.5% m / m). The dependent variable, or response, was the AHD of the nanoemulsion. Matrix was generated using the statistical software Minitab 17 (State College PA, USA).

[0055] Table 1 - Box-Behnken experiments used to evaluate the average hydrodynamic diameter and polydispersity index obtained by DPE process without polyols.

[0056]

[0057]

[0058] BO: Borage oil; T80: Polysorbate 80; H2O i Initial water

[0059] - Mean hydrodynamic diameter (AHD) and polydispersity index (PI)

[0060] The AHD and PI of the droplets were determined by dynamic light scattering in freshly prepared ultrapure water. Samples were diluted 1:100 and observed at a 90° angle under scattered light. Particle size distribution and polydispersity index were determined using a Zetasizer Nano ZS90 instrument (Malvern Instruments, Malvern, UK). Mean diameter (based on the Stokes-Einstein equation) and polydispersity index (PI) were determined by three separate measurements. The polydispersity index (Equation 1) was defined as the standard deviation of the particle size distribution (σ) divided by the mean particle size (2a).

[0061]

[0062] -ζ potential (ZP)

[0063] The zeta potential was determined using an electrophoretic mobility method with a Zetasizer ZS90 instrument (Malvern Instruments, Malvern, UK), whereby the electrophoretic mobility was converted into the zeta potential (ζ) (mV) using the Henry equation (Equation 2). Prior to measurement, the sample was... Dilute in ultrapure water.

[0064]

[0065] Where η and ε are the viscosity and dielectric constant of the solvent at 25 °C, respectively. UE is the electrophoretic mobility, and ζ is the zeta potential. The applied electric field strength is 20 V / cm. The conditions used to determine the zeta potential were determined using a conductivity versus zeta potential curve. The conductivity range evaluated using a 0.9% NaCl solution (m / V) was 50 μS / cm. The pH was adjusted to 5.5 ± 0.2 by adding 0.01 N HCl or 0.01 N NaOH.

[0066] -Nanoemulsion optimization

[0067] Response optimization was performed using the expectation function method with Minitab 17 statistical software (State College, PA, USA). This tool allows for the determination of the maximum concentration of borage oil and the minimum concentration of surfactant in the formulation that allows for formulation formation. Furthermore, the average hydrodynamic diameter (AHD) of less than 500 nm, preferably 350 nm, was sought. An expectation function (d) was established in the range of 0 to 1, where 1 corresponds to 100% expectation.

[0068] - Validation of the mathematical model generated through statistical analysis

[0069] Based on the optimization procedure, a new formulation was obtained. Observed and predicted AHD values ​​of the nanoemulsion were determined to evaluate the adequacy of the mathematical model.

[0070] -Physical stability

[0071] The stability of the nanoemulsion was evaluated by analyzing the mean hydrodynamic diameter (AHD), polydispersity index (PI), and zeta potential (ZP), as well as by visual observation of the formulation during a storage period of 12 months at 25 ± 0.5 °C. The formulation was stored in hermetically sealed borosilicate glass vials in a climate chamber (Climacell MMM Group).

[0072] - Toxicity in alternative models of the giant wax moth

[0073] Toxicity tests were conducted using larvae of the large wax moth, ranging in length from 2.0 to 2.5 cm and in weight from 150 to 200 mg. Samples evaluated included borage oil (BO) and borage oil nanoemulsions prepared via the DPE method (BO = 25% w / w; BO = 50.0% w / w). A 10 μL volume of the formulation was injected into the last proleg of each larva (n = 16 larvae per group). Injection of sodium phosphate buffer at pH 7.4 served as a control for mechanical damage, and survival and morbidity were monitored daily for five days.

[0074] The following examples provide the results of the following experiments: preparation of polyol-free nanoemulsions by the DPE method (Example 1); large-scale preparation of polyol-free nanoemulsions by the DPE method (Example 2); Box-Benhken experimental design experiment of polyol-free formulations prepared by the DPE method (Example 3); regression equation experiment of variables of polyol-free formulations prepared by the DPE method (Example 4); optimization experiment of polyol-free nanoemulsions and validation of mathematical models generated by statistical analysis for polyol-free formulations prepared by the DPE method (Example 5); physical stability experiment of polyol-free formulations prepared by the DPE method (Example 6); toxicity experiment of polyol-free formulations prepared by the DPE method in an alternative model of giant wax moth (Example 7); experiment on the physical stability optimization of polyol-free formulations (Example 8); and encapsulation experiment of polyol-free borage oil formulation and cyclosporine in nanoemulsions obtained by the DPE method (Example 9).

[0075] Example 1 - Preparation of polyol-free nanoemulsions for formulations prepared by the DPE method

[0076] Isotropic solutions containing surfactants and an aqueous phase ( Figure 1A During stirring, the oil phase exhibits rapid dispersion upon addition. As oil concentration increases, the initially clear and isotropic solution gradually becomes turbid, leading to an increase in viscosity. The system gradually evolves towards a semi-transparent state, characterized by a high viscosity system ( Figure 1B Even in the presence of a high concentration of oil phase, it is easily incorporated into the system without phase separation. Then, by continuously adding the remaining water, the fine oil particles of the inner phase generated from phase B are dispersed in the continuous aqueous phase, resulting in the final oil-in-water (O / W) nanoemulsion. Figure 1C The formation of ).

[0077] The rapid dispersion of the oil phase in the isotropic solution is explained by the low interfacial tension between the added oil phase and the isotropic solution. The observed phenomena indicate the presence of an oil-in-surfactant (O / S) phase, consistent with the procedure outlined by ENDOO; SAGITANI, (1991). The subsequently added water readily disperses in the O / S phase, resulting in the formation of a final emulsion with a milky white appearance. This phenomenon suggests that the external continuous phase is not composed of an oil phase (and therefore, is neither characteristic of water-in-oil emulsions (W / O) nor of surfactant-in-oil emulsions (S / O)). In short, during the continuous addition of residual water, small oil droplets previously formed in the O / S phase disperse in the aqueous phase, leading to the formation of an oil-in-water (O / W) nanoemulsion. These characteristics are consistent with the findings of SAGITANI, (1986), reinforcing the correct application of the DPE process in this experiment, although polyols were excluded from the formulation.

[0078] Example 2 - Scale-up preparation of polyol-free nanoemulsions for formulations prepared using the DPE method

[0079] Nanoemulsions were prepared on a scale-up basis according to the method described in Example 1 to verify the feasibility of transferring the method to industrial applications. With the addition of oil, the color of the previously transparent isotropic mixture was observed to gradually change to milky white and light green. Figure 2A After the borage oil is fully added, the viscous substance is kept under stirring for 20 minutes, and then the final water is added slowly. Then, by continuously adding the remaining water, the fine oil particles of the inner phase generated from the B phase are dispersed in the continuous aqueous phase, resulting in the final oil-in-water (O / W) nanoemulsion. Figure 2B The nanoemulsion formed after preparation. It had an AHD of 339.2±3.8 nm, a PI of 0.118±0.090, and a ZP of -35.7±0.6 mV.

[0080] Example 3 - Box-Behnken design experiment on polyol-free formulations prepared using the DPE method.

[0081] The experiment was conducted using statistical methods with three center points (preparations with identical composition: 5, 10, and 11) to increase the probability of detecting the significance of the input variable, estimate variability (pure error), and statistically determine whether the relationship between the independent variable and the response is linear (MONTGOMERY, 2019). Furthermore, a total of 15 experiments were randomly conducted, eliminating the need for additional replicates.

[0082] Response surface methodology (RSM) was applied to evaluate the effects and interactions of the following independent variables: borage oil concentration, surfactant concentration, and initial water concentration, with AHD as the output response (dependent variable). The experiments resulted in formulations exceeding specifications (no emulsion formation), which did not allow for subsequent statistical analysis of the data. However, these results guided decisions for further process improvements. Furthermore, they allowed us to identify factors contributing to the unpredictability of the formulation in this exploratory phase of the experimental design in the DPE process. This step in the experimental design determined the optimal range of the independent variables and improved the understanding of the process, as described below, allowing the output response to remain within the specified quality assessment (CQA) range.

[0083] Table 2 - Evaluation of the average hydrodynamic diameter and polydispersity index obtained by the DPE process without polyols - First Box-Behnken experiment.

[0084]

[0085]

[0086] BO: Borage oil; T80: Polysorbate 80; H2O i Initial water; AHD: Mean hydrodynamic diameter; PI: Polydispersity index; ZP: Zeta potential.

[0087] A new experimental design was generated using Minitab software, and the results are presented in Table 3, where the AHD ranges from 257.8 to 505.1 nm (PI values ​​are between 0.036 and 0.183), showing a narrow and unimodal distribution.

[0088] Table 3 - Average hydrodynamic diameter and polydispersity index obtained by DPE process without polyols.

[0089]

[0090] BO: Borage oil; T80: Polysorbate 80; H2Oi: Initial water; AHD: Mean hydrodynamic diameter; PI: Polydispersity index; ZP: Zeta potential.

[0091] Table 4 - Analysis of variance for different fitted response models and quadratic regression of the average hydrodynamic diameter of nanoemulsions.

[0092]

[0093]

[0094] Analysis of variance (Table 4) showed that the independent variables and their interactions were statistically significant (p < 0.05; ∝ = 0.05). The quadratic effect of initial water on the AHD of the nanoemulsion was also significant (p < 0.05; ∝ = 0.05). The lack of adjustment was not significant (p-value = 0.188), indicating that the proposed quadratic polynomial model is sufficient. The effects describing AHD are shown in the response surface and contour plot (Figure 3).

[0095] As can be observed from the graph in Figure 3, AHD decreases with increasing initial water concentration. A secondary effect of decreasing AHD was also observed between polysorbate 80 and initial water.

[0096] Example 4 - Regression equation for variables in a polyol-free formulation prepared by the DPE method

[0097] The quadratic regression model demonstrated a determination coefficient (R²) of 97.48% for AHD, indicating that the response value can be attributed to a definite independent variable. R² reflects the correlation between the experimental and predicted values. 2 adj The result is 94.95%. This is close to the R² value, indicating a good statistical model. The coefficient of determination (R²) for the prediction... 2 pred The regression rate was 84.17%, indicating the model's adequacy in predicting responses to new observations. The regression equations for the uncoded factors are shown below:

[0098] Equation 1:

[0099] AHD(nm)=-735-14.4BO+867T80-853 H2Oi+1922H2O i *H2O i +12.12

[0100] BO*T80-18.93BO*H2O i -941 T80*H2O i

[0101] This equation describes the average particle size, taking into account linearity, quadratic terms, and interactions. The linear coefficients indicate the direct effect of each component on particle size. For example, the negative coefficient for BO (-14.4) indicates that increasing BO concentration is associated with decreasing particle size, while the positive coefficient for T80 (867) indicates that increasing the concentration of this component increases particle size. Furthermore, the quadratic term 1922H2O... i *H2O i The existence of this reveals a nonlinear relationship between H2Oi concentration and particle size. Therefore, increasing the initial water concentration increases particle size. In this case, the value of the coefficient (1922) has a striking effect on particle size. Interaction terms, such as +12.12BO*T80 and -18.93BO*H2O, also influence particle size.i This indicates how the combination of different components affects particle size, increasing or decreasing it. Such phenomena are unpredictable. For example, the positive coefficient of BO*T80, +12.12, indicates that a combined increase in the concentrations of BO and T80 leads to an increase in particle size. And BO and H2O... i Increasing the concentration will reduce the particle size. These phenomena are disclosed specifically for this formulation and method and cannot be predicted or inferred from the prior art.

[0102] Example 5 - Optimization of polyol-free nanoemulsions and their use in preparing polyol-free nanoemulsions via the DPE method Validation of the mathematical model generated through statistical analysis of the formulation

[0103] Based on regression equations, optimized conditions for nanoemulsion preparation yielded nanoemulsions with high borage oil concentrations using low-concentration surfactants. By combining a borage oil concentration of 31.0% (m / m), a surfactant concentration of 3.8% (m / m), and an initial water concentration of 1.5% (m / m), the optimal conditions within the desired function were revealed (1.0)( Figure 4 This results in a theoretical AHD of 350.0 nm. The experimental value obtained under these conditions is 300.5 ± 2.2 nm. As shown in Table 5, similar predicted and observed AHD values ​​obtained from the optimized formulation validate the proposed model.

[0104] Table 5 - Theoretical and experimental values ​​of AHD for particles of the optimized formulation.

[0105]

[0106] In this invention, the use of statistical methods revealed an optimized combination of independent variables that, surprisingly, allowed for the acquisition of nanoemulsions with an AHD close to that of nanoemulsions previously studied by the inventors of this invention (glycerol-containing formulations, MASIERO, Jéssica Fagionato et al. Unveiling the optimal path: High-pressure homogenization and D-phase emulsification methods for borage oil nanoemulsion design. Industrial Crops and Products, Vol. 208, p. 117853, 2024.), maintaining a satisfactory borage oil content (31.0% m / m) with a low surfactant concentration (3.8% m / m). Similar to such studies, the effect of initial water and its interaction with surfactants was statistically confirmed in reducing AHD and subsequently forming nanoemulsions without the need for HLB adjustment.

[0107] Example 6 - Physical stability of polyol-free formulations prepared by the DPE method

[0108] The physical stability of nanoemulsions is a critical aspect of drug development, and is particularly challenging due to the Ostwald ripening phenomenon, which can occur over extended periods (KOROLEVA; NAGOVITSINA; YURTOV, 2018). This phenomenon refers to the gradual decrease in small droplets and the gradual increase in larger droplets during storage, eventually leading to the complete dissolution of the small droplets in the external phase (ROCCHIO et al., 2017). In this invention, polysorbate 80 is chosen as the preferred surfactant to counteract this instability effect and promote stability.

[0109] For the DPE method, the AHD immediately after preparation was 328.9 ± 7.1 nm, and the final AHD after 12 months was 339.9 ± 5.2 nm, representing a 3% change. These results indicate that the formulation remained stable during the 12-month evaluation period, with no signs of phase separation. The initial zeta potential immediately after preparation was -31.2 ± 3.5 mV, and it was -29.1 ± 3.1 mV after 12 months.

[0110] In summary, the stability evaluation of the polyol-free borage oil nanoemulsion using the DPE method demonstrates its potential suitability for industrial applications.

[0111] Example 7 - Toxicity of polyol-free formulations prepared by the DPE method in a giant wax moth substitution model

[0112] Toxicity tests were conducted using larvae of the large wax moth, measuring 2.0 to 2.5 cm in length and weighing 150 to 200 mg. Samples evaluated included pure borage oil, crude borage oil emulsion, and a polyol-free borage oil nanoemulsion (BO = 50% w / w) prepared via the DPE method. A 10 μL volume of the formulation was injected into the last pseudopodia of each larva (n = 16 larvae per group). Sodium phosphate buffer (pH 7.4) was injected as a mechanical damage control, and survival and health indices were monitored daily for five days.

[0113] The toxicity of borage oil nanoemulsions (50% w / w BO) prepared by DPE without polyols was evaluated. The group tested with nanoemulsions prepared by the DPE method did not show significant toxicity. Figure 5A and Figure 5B The health index of the large wax moth larvae was close to the maximum measurement, and the survival rate of the experimental group was 100%. The health index was assessed according to a scoring system.

[0114] The assessment was based on four categories: activity, cocoon formation, melanosis, and survival, with a specific score assigned to each criterion. In the activity category, larvae that did not show any movement received 0 points. Larvae that showed minimal movement only when stimulated received 1 point. Larvae that moved only when stimulated, but in a more obvious manner, received a score of 2 points. Larvae that moved spontaneously without stimulation received the highest score of 3 points.

[0115] The evaluation also assessed cocoon formation. Larvae showing no signs of cocoon formation received 0 points. Larvae in the process of cocoon formation but incomplete received 0.5 points. Finally, larvae forming a complete cocoon received 1 point.

[0116] Melanosis is an important indicator of immune response and potential infection, scored as follows: Completely black larvae receive 0 points, as this may indicate a high level of infection or necrosis. Larvae that are black but have brown spots receive 1 point. Larvae that are beige with three or more dark spots receive 2 points. Larvae that are beige with fewer than three spots receive 3 points. The highest score, 4 points, is awarded in the best case scenario where there are no signs of melanosis. Finally, survival is assessed in a binary manner. Dead larvae receive 0 points, while live larvae receive 2 points.

[0117] Based on this system, larvae considered healthy, i.e., those without infection or significant damage, have a total score between 9 and 10 (LOH et al., 2013).

[0118] Example 8 - Polyol-free formulation optimized for physical stability

[0119] To evaluate physical stability in order to achieve the longest possible shelf life and to facilitate scale-up, formulations with higher oil concentrations were prepared in addition to the optimized formulations (Table 6).

[0120] Table 6 - Formulations developed at laboratory and scale-up scales in stability assessments.

[0121]

[0122] BO: Borage oil; T80: Polysorbate 80; H2Oi: Initial water; H2Of: Final water; Ot.F.: Optimized formulation; Max.F.: Formulation with maximum borage oil concentration.

[0123] These formulations were kept in a climate chamber at 30°C ± 2°C and 75% humidity for 12 months and evaluated periodically (12 months - laboratory scale, 6 months - scale-up scale), and they presented AHD, PI and ZP according to Table 7.

[0124] Table 7 - AHD, PI, and ZP of stable formulations evaluated after a 12-month period.

[0125]

[0126]

[0127] Ot.F.: Optimized formulation; Max.F.: Formulation with maximum borage oil concentration; AHD: Mean hydrodynamic diameter; PI: Polydispersity index; ZP: Zeta potential.

[0128] Example 9 - Encapsulating a borage oil formulation containing cyclosporine but without polyols in a container obtained by the DPE method. In nanoemulsions (Example 9)

[0129] In addition to its inherent properties mentioned above, borage oil nanoemulsions also constitute a platform for encapsulating drugs with low water solubility. Using borage oil as the lipid phase, nanoemulsions containing cyclosporine were prepared via the DPE method. Initially, two formulations were prepared using the DPE method: one containing 10% (m / m) cyclosporine (100 mg / mL), and the other containing 1% (m / m) cyclosporine (10 times lower, 10 mg / mL). This concentration was chosen because 100 mg / mL is the concentration used in veterinary medicine for treating chronic atopic dermatitis in dogs and for symptomatic treatment of chronic allergic dermatitis in cats. The rationale for reducing the concentration is based on the inherent properties of nanoparticles, which have a larger area / surface area to volume ratio, requiring a lower drug concentration to achieve the desired therapeutic effect.

[0130] In the prepared formulation, cyclosporine was dissolved in borage oil for approximately 1 hour at room temperature with magnetic stirring (300 rpm). The formulation was then prepared according to the steps described in the section on "Preparation of Nanoemulsions".

[0131] In formulations containing 10% (w / w) cyclosporine, turbidity was observed in the oil after the drug dissolved. A polyol-free formulation was prepared and showed a good appearance after preparation, with an AHD of 285.3 nm, a PI of 0.44, and a ZP of -26.6 mV. However, sedimentation was observed at the bottom of the vial after 24 hours, which may be related to cyclosporine, which may not have been completely dissolved in borage oil.

[0132] Subsequently, another polyol-free formulation was prepared, containing ten times less cyclosporine, i.e., 1% (w / w). During the drug dissolution process in borage oil, the oil remained completely translucent, indicating complete dissolution of cyclosporine. The polyol-free formulation was prepared and showed an AHD of 223.5 nm, a PI of 0.16, and a ZP of -27.0 mV.

[0133] It should be understood that this description does not limit this application to the details described herein, and the invention is capable of other embodiments within the scope of the claims and can be practiced or performed in various ways. Although specific terminology has been used, such terminology should be interpreted in a general and descriptive sense and is not intended to limit the scope of the invention.

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Claims

1. A method for preparing vegetable oil nanoemulsions, characterized in that, It does not contain polyols and uses D-phase emulsification (DPE). The method includes the following steps: a) Homogenize an aqueous phase containing 0.5 to 1.5% initial water and 1 to 5% nonionic surfactant under stirring at 50 to 500 rpm, preferably 300 rpm, and at a temperature of 10 to 50°C, preferably 25°C; b) Add 5 to 50% by volume of vegetable oil to the aqueous phase at a controlled speed while stirring at 50 to 500 rpm, preferably 300 rpm; c) Add the remaining volume of vegetable oil and stir at 50 to 500 rpm, preferably 300 rpm, for 10 to 30 minutes, preferably 20 minutes. d) Add the remaining water to the total volume of the formulation; and e) The vegetable oil nanoemulsion is stirred at a constant speed of 50 to 500 rpm, preferably 300 rpm, for 10 to 30 minutes, preferably 20 minutes.

2. The method according to claim 1, characterized in that, The nonionic surfactant is selected from the group consisting of: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sorbitol monolaurate, sorbitol monopalmitate, sorbitol monooleate, sorbitol trioleate, lecithin, glyceryl stearate, glyceryl laurate, glyceryl oleate, glyceryl cetearate, capryloyl / decanoyl glucoside, decyl glucoside, lauryl polyglucoside, glyceryl isostearate, cetyl alcohol ethoxylate, octylphenyl polyol- 9. Nonoxynol-9, Poloxamer 188, Poloxamer 407, Poloxamer 904, Polyethylene Glycol 400, Polyethylene Glycol 600, Polyethylene Glycol 1000, Polyethylene Glycol 3350, PEG-8 Polydimethylsiloxane, PEG-12 Polydimethylsiloxane, Laureth Methacrylate, Cholesterol Ethoxylate, Methyl Reduced Glucosamine, Polyethylene Glycol Distearate, Methyl Glucoside Ester, Methyl Glucoside Phosphate, Polyglyceryl-4 Ester, Polyglyceryl-10 Laurate, Glyceryl Castor Oil.

3. The method according to claim 2, characterized in that, The nonionic surfactant is preferably polysorbate 80.

4. The method according to claim 1, characterized in that, The plant oils may be selected from the following group: avocado oil (Persea americana), sweet almond oil (Prunus dulcis), neem oil (Carapaguianensis), argan oil (Argania spinosa), hazelnut oil (Corylus avellana), babassu oil (Orbignya phalerata), baobab oil (Adansonia digitata), brickberry oil (Mauritia flexuosa), camellia oil (Camellia oleifera), rapeseed oil (Brassica napus), Brazil nut oil (Bertholletia excelsa), Maranhã nut oil (Sterculia striata), safflower oil (Carthamus tinctorius), chia oil (Salvia hispanica), coconut oil (Coconut). Oils containing various species including *Crambe abyssinica*, *Prunus armeniaca*, *Opuntia ficus-indica*, rice bran (*Oryza sativa*), raspberry (*Rubus idaeus*), sunflower (*Helianthus annuus*), sesame (*Sesamumindicum*), guarana (*Paullinia cupana*), shea butter (*Vitellaria paradoxa*), lavender (*Lavandula angustifolia*), flaxseed (*Linum usitatissimum*), macadamia nut (*Macadamia integrifolia*), and castor oil (*Ricinus spp.*). Oils containing various plant species include: * *Citrus communis* (commons), passion fruit oil (Passiflora edulis), marula oil (Sclerocarya birrea), tea tree oil (Melaleuca alternifolia), corn oil (Zea mays), moringa oil (Moringa oleifera), and mustard oil (Brassica oleracea).juncea), neem oil (Azadirachta indica), pecan oil (Carya illinoinensis), olive oil (Olea europaea), Brazilian nectarine oil (Caryocar brasiliense), patauá oil (Oenocarpus bataua), bacas fruit oil (Pentaclethra macroloba), rosehip oil (Rosa rubiginosa), pumpkin seed oil (Cucurbita pepo), cottonseed oil (Gossypium hirsutum), grapeseed oil (Vitis vinifera), tamanu oil (Calophylluminophyllum), tucuma oil (Astrocaryum vulgare), cashew oil (Anacardium) occidentale), large-flowered cocoa oil (Theobroma grandiflorum), cocoa oil (Theobroma cacao), emburana oil (Amburana cearensis), peanut oil (Arachis hypogaea), blackcurrant oil (Ribes nigrum), seaweed oil (Schizochytrium spp.), krill oil (Euphausia superba), borage oil (Borago officinalis), fennel oil (Foeniculum vulgare), rosemary oil (Rosmarinus officinalis), milk thistle oil (Silybum marianum), cactus oil (Pyracantha fortuneana), annatto oil (Bixa) orellana), chamomile oil (Matricaria recutita), peppermint oil (Mentha piperita), ginger oil (Zingiber officinale).

5. The method according to claim 4, characterized in that, The preferred vegetable oil is borage oil.

6. The method according to claim 1, characterized in that, The D-phase emulsification (FDE) process uses low energy.

7. A vegetable oil nanoemulsion, characterized in that, The concentration of the vegetable oil is higher than 5% to 50% w / w, preferably 50% w / w, and the concentration of the nonionic surfactant is at most 2% to 5% w / w, preferably 4.0% w / w, and the nonionic surfactant is selected from the group consisting of: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sorbitol monolaurate, sorbitol monopalmitate, sorbitol monooleate, sorbitol trioleate, lecithin, glyceryl stearate, glyceryl laurate, glyceryl oleate, glyceryl cetearate, capryloyl / decyl glucoside, decyl glucoside, lauryl oleate, etc. Polyglucoside, glyceryl isostearate, ethoxylated cetyl alcohol, octylphenyl polyol-9, nonylphenyl alcohol ether-9, poloxamer 188, poloxamer 407, poloxamer 904, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 3350, PEG-8 polydimethylsiloxane, PEG-12 polydimethylsiloxane, lauryl methacrylate, cholesterol ethoxylate, methyl reduced glucosamine, polyethylene glycol distearate, methyl glucoside ester, methyl glucoside phosphate, polyglycerol-4 ester, polyglycerol-10 lauryl ester, glyceryl castor oil, preferably polysorbate 80.

8. The vegetable oil nanoemulsion according to claim 7, characterized in that, The vegetable oil may be selected from the following group: avocado oil, sweet almond oil, neem oil, argan oil, hazelnut oil, babassu oil, baobab oil, brighamb oil, camellia oil, rapeseed oil, Brazil nut oil, and sterculia nut oil. Striata, safflower oil, chia oil, coconut oil, tartare oil, sea cabbage oil, almond oil, prickly pear oil, rice bran oil, raspberry oil, sunflower oil, sesame oil, guarana oil, shea butter, lavender oil, flaxseed oil, macadamia nut oil, castor oil, passion fruit oil, marula oil, tea tree oil, corn oil, moringa oil, mustard oil, neem oil, pecan oil, olive oil, Brazilian nectarine oil. Nectarine, Patauá oil, Bacas fruit oil, Rosehip oil, Pumpkin seed oil, Cottonseed oil, Grape seed oil, Tamanu oil, Tucuma oil, Cashew oil, Giant Coconut oil, Coconut oil, Emburana oil, Peanut oil, Blackcurrant oil, Algae oil, Krill oil, Borage oil, Fennel oil, Rosemary oil, Milk thistle oil, Cactus oil, Napier oil, Chamomile oil, Peppermint oil, Ginger oil.

9. The vegetable oil nanoemulsion according to claim 8, characterized in that, The vegetable oil mentioned is borage oil.

10. The vegetable oil nanoemulsion according to any one of claims 7 to 9, characterized in that, It has an average hydrodynamic diameter of less than 500 nm, preferably 350 nm, a polydispersity index between 0.05 and 0.300, and a zeta potential between -48.9 and -29.1 mV.

11. The vegetable oil nanoemulsion according to any one of claims 7 to 10, characterized in that, It is a platform for encapsulating drugs with low water solubility, the drugs being selected from the group consisting of immunosuppressants, anti-inflammatory drugs, antihistamines, and antibiotics.

12. The vegetable oil nanoemulsion according to claim 11, characterized in that, The immunosuppressants are selected from the following group: azathioprine, cyclosporine, tacrolimus, sirolimus (rapamycin), everolimus, tamsulolimus, zotamolimus, leflunomide, mycophenolate mofetil, mycophenolate sodium, guaniolimus, pimecrolimus, fingolimod, baricitinib, ruxolitinib, tofacitinib, abatacept, beraccept, vorciclosporine, and laquimod; the steroidal anti-inflammatory drugs are selected from the following group: dexamethasone, Betamethasone, budesonide, clobetasol, difluralasone, fluocinolone acetonide, flucloxason, fluticasone, halcinonide, hydrocortisone, methylprednisolone, mometasone, peramisone, prednisolone, prednisolone, triamcinolone, deshydroxymethasone, cyclohexanone, beclomethasone, aclomethasone; nonsteroidal anti-inflammatory drugs (NSAIDs) selected from the following group: aceclofenac, mefenamic acid, niflufenicol, tiprofen, guanethidine, benzoxam. Bromfenac, Carbofenac, Celecoxib, Ketoprofen, Clonisin, Dextrobufen, Dextrokeprofen, Diclofenac, Diflunisal, Etodoxacin, Etofenamate, Phenytasol, Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen, Lornoxicam, Lumixicam, Meclofenamic acid, Meloxicam, Nabumetone, Naproxen, Nimesulide, Oxaprazin, Parecoxib, Piroxicam, Promethazine, Rofecoxib, Sulindac Tenoxacin, vardicoxib, tometidine, azapromide, feprasone; antihistamines selected from the following group: astemizole, bilastine, ebastine, loratadine, desloratadine, mizolastine, terfenadine, chlorpheniramine, dextrochlorpheniramine, brompheniramine, diphenhydramine, promethazine, doxylamine, cyproheptadine, meclopramide, hydroxyzine, triprolidine, tripiramine, dimethinidine, oxamide, nitrogen Stine, bucrorazine, chlorcyclorhizine, ketotifen, alimazine, antazoline, trimethoprim, fenamirine, dextrobrompheniramine; and antibiotics selected from the following groups: azithromycin, clarithromycin, erythromycin base, telithromycin, clindamycin, lincomycin, rifampin, rifabutin, rifapentine, isoniazid, ethionamide, para-aminosalicylic acid, dapsone, chloramphenicol, tiamulin, vornimulin, bacitracin, bacitracin, polymyxin B, colistin Griseofulvin, fusidic acid sodium, streptomycin, amikacin, capreomycin, spectinomycin, cefadroxil, cefaclor, cefixime, cefdinir, cefpodoxime, cefbufen, minocycline, doxycycline, tetracycline, tigecycline, ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, besifloxacin, gatifloxacin, gemifloxacin, sitafloxacin, prulifloxacin, finafloxacin, naflufloxacin, enoxacin.

13. The vegetable oil nanoemulsion according to claim 11, characterized in that, The immunosuppressant is cyclosporine.

14. A pharmaceutical composition, characterized in that, It contains 1% (w / w) cyclosporine dissolved in a borage oil nanoemulsion that is free of polyols as defined in any one of claims 7 to 13.

15. The composition according to claim 14, characterized in that, It includes an AHD of 223.5nm, a PI of 0.16, and a ZP of -27.0mV.

16. Use of the pharmaceutical composition as defined in claim 14 or 15 for the preparation of a medicament for the treatment of atopic dermatitis, hypercholesterolemia, premenstrual syndrome, insulin resistance, support for wound healing, diabetic neuropathy, metabolic syndrome, preferably atopic dermatitis in mammals.

17. The use according to claim 16, characterized in that, The mammals are selected from humans and animals, and the animals are selected from the group consisting of: dogs, cats, horses, cattle, sheep, goats, pigs, rabbits, hamsters, guinea pigs, and rats, with dogs and cats being preferred.

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