Pharmaceutical preparations for topical use containing nanosystems based on essential oil of Lippia sidoides Cham. (rosemary-pepper) with anti-inflammatory action.

Abstract

This invention relates to topical pharmaceutical preparations containing nanosystems based on Lippia sidoides Cham. (rosemary-pepper) essential oil or thymol, indicated for the treatment of inflammatory diseases. The present invention relates to topical pharmaceutical preparations containing nanosystems such as polymeric nanoparticles or nanoemulsions, based on Lippia sidoides Cham. essential oil, or the isolated compound thymol, carried in pharmaceutical preparations in liquid or semi-solid forms, indicated for the treatment of inflammatory diseases. This invention also contemplates the standardization of the essential oil used as an active ingredient in these preparations, as well as the method of preparing the nanosystems consisting of polymeric nanoparticles or nanoemulsions, as well as their compositions and their potential in the treatment of inflammatory diseases.

Description

Pharmaceutical preparations for topical use containing nanosystems based on essential oil of Lippia sidoides Cham. (rosemary-pepper) with anti-inflammatory action. FIELD OF THE INVENTION

[001] The present invention relates to topical pharmaceutical preparations containing nanosystems such as polymeric nanoparticles or nanoemulsions, based on Lippia sidoides Cham. essential oil, or the isolated compound thymol, carried in pharmaceutical preparations in liquid or semi-solid forms, indicated for the treatment of inflammatory diseases. This invention also contemplates the standardization of the essential oil used as an active ingredient in these preparations, as well as the method of preparing the nanosystems, their compositions, and proof of their potential in the treatment of inflammatory diseases. STATE OF THE ART

[002] Inflammation is a defense response of the body to inflammatory agents, involving various cell types, as well as the important role of the vascular system and numerous chemical mediators, such as cytokines, prostaglandins and protease enzymes (MITCHELL & COTRAN, Eds., Saunders, Philadelphia, Pa, USA, 2003).

[003] The inflammatory response, when inappropriately triggered or exacerbated, plays an important role in the pathophysiology of numerous inflammatory diseases. In this context, anti-inflammatory drugs play an important role in the pharmacotherapy of numerous diseases such as rheumatoid arthritis, asthma, and dermatitis. Non-steroidal and steroidal anti-inflammatory drugs, including corticosteroids, are particularly noteworthy. Petition 870210081630, dated 03 / 09 / 2021, page 10 / 47 2 / 25

[004] Nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the most prescribed and used class of drugs in the world, being the first-line treatment in numerous pathologies such as rheumatoid arthritis, osteoarthritis and other inflammatory diseases. These drugs also have analgesic and antipyretic activity in their vast majority. Their anti-inflammatory activity occurs mainly due to the blocking of the cyclooxygenase enzyme, promoting the inhibition of prostaglandin synthesis (BIA VA et al., J. Med. Chem. v. 50, p. 5403-4 11, 2007, HARIRFOROOSH; ASGHAR; JAMA LI, J Pharm Pharm Sci, v. 16, 8 21- 47, 2013).

[005] Many patients, particularly those with arthritis, are chronic users of NSAIDs. Epidemiological studies have shown that the estimated annual expenditure of patients using NSAIDs is approximately seven billion dollars (COAGHAN, Rheumatol Inter., v. 32, p. 1491-502, 2012).

[006] The adverse effects of this class of drugs are also quite well known, for example, in the recent past, rofecoxib was withdrawn from the market after a randomized placebo-controlled trial found an increased incidence of myocardial infarction and sudden cardiac death among users of this medication (BRESALIER et al., N Engl J Med. v. 352, p. 1092-102, 2005). A subsequent meta-analysis confirmed this conclusion and also found an increased incidence in other users of non-specific NSAIDs. Since both arterial and venous thromboses share several pathophysiological mechanisms, NSAIDs can increase the risk of venous thromboembolism in users by a factor of 1.89 compared to individuals who use this class of medication (UNGPRA ERT et al., Rheumatology, doi: I 0.1 093 / rheumatology / keu 408, 2014). Petition 870210081630, dated 03 / 09 / 2021, page 11 / 47 3 / 25

[007] Another therapeutic resource is the use of glucocorticoids, which are considered potent anti-inflammatory and immunosuppressive drugs, acting on almost all cell types, forming complex interactions with gene expression and the function of various mediators, and more importantly, altering cell differentiation programs (WHITEHOUSE, Inflammopharmacology. v. 19 (!), p. 01-19 201 1).

[008] Until recently, it was believed that the anti-inflammatory suppressive effects of glucocorticoids were dependent on the ability of the glucocorticoid receptor (GR) to inhibit the activity of crucial transcriptional regulators of pro-inflammatory genes, including NF-κB and AP-I, through a mechanism called transrepression. This contrasts with the actions Metabolic mechanisms of glucocorticoids, which require gene activation by GR. This viewpoint has recently been revised with the discovery that the key anti-inflammatory actions of glucocorticoids are promoted through gene activation. Transrepression involves direct protein-protein interaction of GR with other transcription factors, thereby interfering with the pro-inflammatory mechanism of action of the latter. Interaction requires the DNA-binding domain of GR (but not DNA binding itself) and does not require conventional GR homodimerization (CLARK, Mol Cell Endocrinol., v. 8, p. 265-277, 2007; COUTINHO; CHAPMA, Mol Cell Endocrinol, v. 335, p. 02-13, 2011).

[009] Long-term use of glucocorticoids is associated with severe adverse effects, including osteoporosis, metabolic diseases, and an increased risk of cardiovascular disease (DEVRIES et al., J Inter Med., v. 261, p. 170-77, 2007; COUT INHO; CHAPMA, Mol Cell Endocrinol, v. 335 p. Petition 870210081630, dated 03 / 09 / 2021, p. 12 / 47 4 / 25 02-13, 2011

[010] Given the above, it is certainly necessary to invest in the development of drugs that offer therapeutic advantages over current pharmacotherapy for inflammatory diseases. In this context, plant-based products have proven to be a rich source for the discovery of new drugs, including anti-inflammatory drugs (CARVALHO, JCT, Tee Med, p.480, 2004).

[011] Lippia sidoides Cham. (Verbenaceae), popularly known as pepper rosemary, northeastern rosemary, horse strep, wild rosemary (LORENZI & MATOS, Instituto Plantarum, 520 p., 2002) is a shrub found in Northeastern Brazil, mainly in the states of Ceará, Paraíba and Rio Grande do Norte (NUNES et al., Rev. odontol. UNES P;35(4):275-283, Oct.-Dec. 2006). The leaves and flowers are popularly used as a local antiseptic in the form of a steeped tea or tincture, to treat skin and throat infections (ALMEIDA et al., 492p., 2013; LORENZI & MATOS, Instituto Plantarum, 520 p., 2002).

[012] An essential oil is extracted from the leaves of Lippia sidoides, chemically composed of thymol, carvacrol, terpinene, p-cymene-caryophyllene, and other terpenes in smaller quantities (OLIVEIRA Effects of the essential oil of Lippia sidoides Cham. and the fixed oil of Caryocar coriaceum Wittm. on topical inflammation and wound healing, dissertation, 2009). Thymol, the major component of the oil, is one of the most potent natural antiseptics known, exhibiting pronounced antibacterial and antifungal activity (FONTENELLE et al. Journal of Antimicrobial Chemotherapy, v.59, p.934-40, 2007).

[013] 0 essential oil showed topical gastroprotective and anti-inflammatory effect Petition 870210081630, dated 03 / 09 / 2021, p. 13 / 47 5 / 25 when used in different concentrations, which seems to be related to its antioxidant potential (MONTEIRO et al. Journal of Ethnopharmacology, v. 111, p.378-382, 2007). It also has immunomodulatory properties, suppressing humoral and cellular immune responses (LEITE, Anti-inflammatory and immunomodulatory activity of the essential oil and extracts of Lippia sidoides Cham. Dissertation 2003).

[014] Regarding toxicological effects, it was found that acute administration of Lippia sidoides essential oil up to 3 g / kg, orally, in mice did not present evident toxicity, as well as oral administration of this oil (11 7.95 mg / kg / day) in rats for 30 days did not induce significant histological, hematological and serum biochemical alterations (FONTENELLE et al., Journal of Antimicrobial Chemotherapy, v.59, p.934- 40, 2007).

[015] The essential oil of the rosemary-pepper species (Lippia sidoides) or thymol are the subject of some national patents:

[016] The essential oil of the species *Lippia sidoides* is the subject of several national patents, mainly related to the development of products indicated as antimicrobials. Document PI I 005541-0 A2 covers the use of the essential oil alone or in combination with another species (*Lippia salvifolia*) and molecules, thymol and carvacrol, indicated for the treatment of bovine mastitis, while documents PI I 003300-9 A2 and PI 0803306-4 A2 deal with a process for preparing a liquid soap and gel, respectively based on *Lippia sidoides* and *Lippia sidoides*, using nanosystems. In the first document, the product is indicated for purposes Petition 870210081630, dated 03 / 09 / 2021, page 14 / 47 6 / 25 gynecological (antifungal and antibacterial) while the second is intended for the treatment of oral thrush, vaginal candidiasis and fungal skin infections. Another application is described in patent Pl 0602027-5, which describes formulations based on Lippia sidoides essential oil, including a lotion and a suppository, containing 10% Lippia sidoides essential oil indicated for protection against the Aedes aegypti mosquito.

[017] Among the Brazilian patents related to thymol, some stand out concerning the use of free thymol or thymol associated with other components for therapeutic purposes, such as the use of thymol as an isolated antiseptic agent (PI 0707569-3 A2) or in combination with plant essential oils (PI 0210327-3 A2) or synthetic antibiotic or antifungal actives (PI 0610125-9 A2; PI 0501494-8 A2). Another document (PI 9813729-8 A2) covers the use of thymol in the treatment of gingivitis in combination with eucalyptol.There is also a patent for thymol as an insect repellent (PI 1000539-0 A2), in addition to other specific indications, such as the use of thymol and carvacrol for application in alcoholic fermentation aimed at controlling bacterial and fungal growth in the culture medium (PI 0905003-5 A2); a composition for animal feed indicated for improving weight gain and / or modulating the intestinal flora of animals (PI 0804911-4 A2); animal feed indicated for the therapy of coccidiosis and clostridial disease (PI 0704238-8 A2) and agricultural use for pest control (PI 0417150-0 B1). The present invention differs from these documents and others similarly related in several aspects, such as the fact that it uses only thymol or essential oil of Lippia sidoides on a nanometric scale as the active ingredient. These products (formulations) incorporate technological and biological advantages that are not currently available. Petition 870210081630, dated 03 / 09 / 2021, page 15 / 47 7 / 25 up to that point, and which provide greater product stability with reduced risk of active ingredient degradation, the absence of risk of interactions between thymol or essential oil with other active ingredients in the formula, in addition to reduced toxicity and proven anti-inflammatory activity, which justifies the potential of these products in the treatment of inflammatory diseases.

[018] Document MX2013000640 describes compositions containing one or more derivatives of essential oil compounds for use in personal care, including mouth, throat and skin care. The present invention differs from this in that the present invention relates to nanosystems consisting of Lippia sidoides essential oil standardized to 65% thymol marker.

[019] Document RU2228168 deals with the use of thymol and other components for the prevention and treatment of inflammatory diseases of the oral cavity mucosa. The present invention differs from this due to the fact that the thymol is carried in standardized nanosystems in the form of nanoemulsions and nanocapsules, which represents a major technological and inventive difference.

[020] Studies conducted by Oliveira (Oliveira, EF; Paula, Haroldo; Paula, Regina CM Alginate / cashew gum nanoparticles for essential oil encapsulation. Colloids and Surfaces B:Biointerfaces 113 (2014) 146-151), Abreu (Abreu, Flávia; Oliveira, EF; Paula, Haroldo; Paula, Regina CM Chitosan / cashew gum nanogels for essential oil encapsulation. Carbohydrate Polymers 89 (2012) 1277-1282), and Paula (Paula, Haroldo; Sombra, Fernanda; Abreu, Flávia; Paula, Regina. Lippia sidoides Essential Oil Encapsulation by Angico Gum / Chitosan Nanoparticles. J. Braz. Chem. Soc., Vol. 21, No. 12, 2359-2366) describe the encapsulation of Lippia sidoides essential oil. sidoides in the form of alginate, cashew gum, or chitosan nanoparticles through a spraying technique. Petition 870210081630, dated 03 / 09 / 2021, page 16 / 47 8 / 25 solution followed by high-temperature evaporation using spray dryer equipment, i.e., at 160 to 170°C, to obtain nanoparticles. This technology differs from the present invention because in the present invention the method of obtaining nanoparticles is by emulsification at a median temperature of 45 to 65°C followed by evaporation of the organic solvent, i.e., the process of preparing and obtaining the nanoparticles is totally different, and the advantage of the present invention is that it uses a lower temperature for preparing the nanosystems, which prevents losses related to thymol evaporation.

[021] Ensuring the safety, quality and efficacy of a pharmaceutical preparation depends fundamentally on the proper development of the formulation, which includes the choice of active ingredients, excipients and delivery systems.

[022] The development of pharmaceutical formulations based on nanocarriers has demonstrated numerous advantages over conventional formulations, such as increased stability and bioavailability, and reduced active ingredient dosage, making this line of research a promising and innovative area of ​​the pharmaceutical sector (ATON et al., J. Control. Release, v. 1 28, p. 185 - 199, 2008).

[023] Nanosystems can be prepared by mechanical, physicochemical and chemical methods (COUVREUR et al, J. Solid State Chem., v.34, n. 2-4, p. 231-235, 2006). Fessi and colleagues (Int J Pharm., v. 55, n. l, p. 1-4, 1989) proposed a method for preparing polymeric nanoparticles, which consists of the interfacial deposition of preformed polymers. In this method, the active compound to be encapsulated is dissolved in an organic solvent along with the polymer, the oily component and the surfactant. Petition 870210081630, dated 03 / 09 / 2021, page 17 / 47 9 / 25 lipophilic. This oily phase is poured, under moderate agitation, onto the aqueous phase, which is composed of water and a hydrophilic surfactant. The mixture spontaneously produces polymeric nanoparticles with average diameters between 200 and 500 nanometers. The advantage of this method is the spontaneous, simple, efficient, and reproducible production of nanoparticles with a high capacity for encapsulating drugs (FESSI et al., I Int J Pharm., v. 55, n. I, p.1-4,989; GUTERRES et al., Drug Target Insights., v. 2, n. 2, p. 147-157, 2007). DESCRIPTION OF THE INVENTION

[024] The present invention relates to topical pharmaceutical preparations that can be prepared in liquid or semi-solid form, and which contain as active ingredients nanosystems consisting of polymeric nanoparticles or nanoemulsions containing the essential oil of Lippia sidoides Cham. (rosemary-pepper), or the isolated compound thymol, these preparations being indicated for the treatment of inflammatory diseases.

[025] Therefore, the present invention relates to the standardization of Lippia sidoides essential oil, the encapsulation of the essential oil, or of the isolated active thymol, in nanosystems, and the incorporation of these into pharmaceutical preparations in different physical forms (liquid and semi-solid), for the purpose of treating various inflammatory diseases.

[026] The novelty of this invention lies in associating an essential oil (Lippia sidoides) rich in thymol, or the active ingredient thymol itself, in nanosystems and incorporating these nanosystems into topical pharmaceutical preparations indicated for the treatment of inflammatory diseases. This represents a major innovation, as the conventional treatment of inflammatory diseases, whether they Petition 870210081630, dated 03 / 09 / 2021, page 18 / 47 10 / 25 topical or not, has been performed especially with NSAIDs and / or corticosteroids.

[027] Among the disadvantages of NSAID treatment are that non-selective COX inhibitors promote changes in the gastrointestinal tract, mainly leading to bleeding; selective COX 2 inhibitors promote cardiovascular changes in individuals predisposed to these diseases.

[028] Treatment with corticosteroids, on the other hand, promotes deleterious effects on the body such as: hyperglycemia, altered blood pressure, difficulty in healing, among others, and its prolonged use can cause Cushing's Syndrome.

[029] The present innovation represents a new therapeutic option, since it leads to a reduction in the inflammatory process with improved pharmacokinetics, mainly in relation to toxicity studies, where the incorporation of Lippia sidoides essential oil or the isolated active ingredient thymol in nanosystems showed a reduction in toxicity without altering the anti-inflammatory and antioxidant activity when compared to the essential oil or thymol not encapsulated in nanosystems. All these characteristics, related to the controlled release of the active ingredients conferred by the nanosystems, represent an important innovation in the pharmacotherapy of inflammatory diseases, including topical ones.

[030] Both the essential oil of Lippia sidoides Cham. and the isolated active ingredient thymol constitute the pharmacologically active compounds of the nanosystems proposed in the present invention. However, these compounds will not be combined in the same formulation; that is, the present invention relates to nanosystems produced from essential oil of Petition 870210081630, dated 03 / 09 / 2021, page 19 / 47 11 / 25 Lippia sidoides, or nanosystems produced from the isolated active ingredient thymol.

[031] As Lippia sidoides essential oil is a derivative of a natural product, and as such is constituted of a complex mixture that may undergo qualitative and quantitative variations, the standardization of the essential oil in relation to thymol content is also an object of the present invention. Lippia sidoides essential oil has thymol as one of its major components, responsible at least in part for the pharmacological properties attributed to the essential oil, such as anti-inflammatory and antiseptic properties. This justifies the choice of this terpene as an active marker in the quality control of Lippia sidoides essential oil. The identification and determination of thymol in the essential oil was carried out using the analytical method - High Performance Liquid Chromatography (HPLC) validated.

[032] The thymol concentration in the essential oil should be within the range of 585 to 715 milligrams of thymol per gram of Lippia sidoides essential oil. This value corresponds to 65±10% thymol in the essential oil, and was established based on studies quantifying the essential oil at different collection sites, as well as preliminary pharmacological studies which demonstrated that essential oil containing around 60-70% thymol has antiseptic activity. Therefore, the Lippia sidoides essential oil used in the preparation of the nanosystems of the present invention was standardized to 65% thymol.

[033] For the incorporation of standardized Lippia sidoides essential oil, the subject of this invention, into pharmaceutical preparations, a nanotechnology-based delivery system was developed, which consists of encapsulating the essential oil in polymeric nanoparticles or in Petition 870210081630, dated 03 / 09 / 2021, page 20 / 47 12 / 25 nanoemulsion, in order to modulate and optimize the expected pharmacological effect for these preparations.

[034] The delivery of assets in nanosystems has several advantages, including: - Protecting the asset against degradation - possibility of sustained or controlled release of the asset - possibility of sterilization by filtration when in liquid form - even distribution of the product on the skin, in the case of products for topical application. - reducing the dose and maintaining or optimizing the pharmacological effect - reduction of the inherent toxicity of the active ingredient - increased asset stability during preparation

[035] However, developing a pharmaceutical product based on nanotechnology is not a simple task, as it involves several aspects, such as: the choice of formulation constituents, the interaction between them, the appropriate proportion of each, and the preparation method. It is the combination of all these factors that will guarantee the formation of the desired system and the maintenance of its stability, efficacy, safety, and quality.

[036] Both Lippia sidoides essential oil and the active compound thymol, although they have proven anti-inflammatory and antiseptic action, are potentially irritating when applied topically.

[037] The present invention offers the novelty of incorporating these active ingredients (thymol or essential oil of Lippia sidoides) in the form of polymeric nanoparticles or nanoemulsion, so that these compounds, when carried in these nanostructures, exhibit high safety by significantly reducing the possibility of causing toxicity reactions, as has been demonstrated. Petition 870210081630, dated 03 / 09 / 2021, page 21 / 47 13 / 25 experimentally. Therefore, this is one of the great technological advantages attributed to this invention.

[038] The nanoparticulate system may consist of solid polymeric nanoparticles that encapsulate the essential oil within the polymeric structure or of nanoemulsion, which encapsulates the essential oil inside nanoglobules formed by surfactants.

[039] The nanoparticles that constitute the present invention are made of biodegradable polymer such as PCL, or PLGA, or Eudragit responsible for the formation of the polymeric envelope, and surfactants such as phosphatidylcholine, polysorbate, or poloxamer may also be present in the formulation, which have surfactant action and favor the stabilization of lipophilic actives. Additionally, the nanosystem formulations may contain a fatty component such as caprylic / capric triglycerides, which acts in the dispersion of the active and contributes to the structuring of the nanosystem.

[040] Nanoemulsions, which also constitute the present invention, may be composed of the surfactants phosphatidylcholine, polysorbate, sorbitan monooleate, or poloxamer, associated or not, and may also contain a fatty component such as caprylic / capric triglycerides, which acts in the dispersion of the active ingredient and contributes to the structuring of the nanosystem.

[041] The method for preparing these nanosystems is based on the interfacial deposition technique of the pre-formed polymer, initially proposed by Fessi, 1989. This method consists of dissolving the polymer, the oil, the lipophilic drug, and the lipophilic surfactant in a water-miscible organic solvent such as acetone or ethanol (this phase constitutes the organic phase), and separately dispersing the hydrophilic surfactant in water (this phase constitutes the aqueous phase). The organic phase is then added to the aqueous phase under moderate stirring, and the excess of Petition 870210081630, dated 03 / 09 / 2021, page 22 / 47 14 / 25 of the solvent is evaporated.

[042] The modification that characterizes process improvement consists of heating the organic and aqueous phases to a temperature of 45 to 65°C, and pouring the organic phase over the aqueous phase in a controlled flow (3-5 milliliters per minute), under agitation at 8,000 revolutions per minute, maintaining agitation and heating for 5 to 10 minutes after the incorporation of the phases. Heating favors a greater interaction between the components, that is, it increases the solubility of the molecules of the organic and aqueous phases, and consequently favors the interaction of the different components dispersed in the medium.When the organic phase is added to the aqueous phase, spontaneous emulsification of the lipophilic components present in the organic phase occurs due to the diffusion of the water-miscible organic solvent generating interfacial turbulence. This causes a decrease in the oil / water interfacial tension, favoring a curvature angle close to zero. This leads to the formation of nanometric globules, through molecular interactions between the lipophilic components and the surfactants present in the medium; or of polymeric nanoparticles, when a lipophilic polymer is present in the medium. Due to molecular affinity, this polymer interacts with the lipophilic components present, engulfing them, and solidifies on the particle surface due to the polymer's insolubility in water. Therefore, this method can produce both nanoemulsions and polymeric nanoparticles, depending on the formulation constituents.The high agitation imposed on the system during the emulsification process favors a narrower size distribution, that is, it decreases the polydispersity index, leaving the system with greater size uniformity, which favors stability over time, as demonstrated in experiments comparing the characteristics of systems produced with moderate agitation (approximately 400 revolutions per minute) and with ultra-high agitation (8,000 to 10,000 revolutions per minute). The organic solvent residue is removed from the system by evaporation at reduced pressure in an evaporator. Petition 870210081630, dated 03 / 09 / 2021, page 23 / 47 15 / 25 rotary, temperature from 25 to 30°C.

[043] The characterization of the nanosystems of the present invention was carried out by determining the average particle diameter, determining the zeta potential and the polydispersity index, using the hydrodynamic light scattering technique in a Zetasizer apparatus. The morphology of the system was also characterized using an atomic force microscope.

[044] The results demonstrated that the proposed formulations and technique are capable of forming nanosystems composed of polymeric nanoparticles or nanoemulsions with average sizes of 130 to 200 nanometers, polydispersity indices < 0.2 and zeta potential between -14.0 and -42.0 millivolts. Atomic force microscopy analysis demonstrated that the produced systems have spherical particles without agglomeration. Stability studies revealed that the nanosystems remain intact in terms of macroscopic appearance, showing no signs of creaming or precipitation, maintaining the characteristics of size, zeta potential, pH and polydispersity index within the pre-established limits.

[045] After preparation and characterization, the nanosystems formed can be delivered in liquid or semi-solid pharmaceutical forms, thus constituting the pharmaceutical preparations that are the subject of the present invention.

[046] Liquid pharmaceutical forms may contain the following components in their formulation: butylene glycol, glycerin, sorbitol, carboxymethylcellulose, hydroxypropylmethylcellulose, chitosan, ethoxylated lanolin, ethoxylated glycerin, water, silicone, imidazolidinyl urea, methylparaben, hyaluronic acid, lactic acid, aminomethylpropanol, and nanosystems consisting of nanoemulsion containing Lippia sidoides essential oil, or nanoparticles containing Lippia sidoides essential oil, or nanoemulsion containing thymol or nanoparticles containing thymol. Petition 870210081630, dated 03 / 09 / 2021, page 24 / 47 16 / 25

[047] Semi-solid pharmaceutical forms may contain the following components in their formulation: xanthan gum, hydroxyethylcellulose, hydroxypropylmethylcellulose, chitosan, carboxyvinyl polymer, butylene glycol, glycerin, sorbitol, ethoxylated lanolin, ethoxylated glycerin, water, silicone, imidazolidinyl urea, methylparaben, hyaluronic acid, lactic acid, aminomethylpropanol, and nanosystems consisting of nanoemulsion containing Lippia sidoides essential oil, or nanoparticles containing Lippia sidoides essential oil, or nanoemulsion containing thymol or nanoparticles containing thymol.

[048] Therefore, this invention also contemplates the development of various liquid and semi-solid formulations, with different classes of polymers and different nanosystems (nanoparticles containing Lippia sidoides essential oil, nanoparticles containing thymol, nanoemulsion containing Lippia sidoides essential oil and nanoemulsion containing thymol) as a new therapeutic option for the treatment of inflammatory diseases.

[049] In order to achieve this purpose, in vitro studies were carried out with cell lines, primary cell culture and in vivo studies to determine the safety and anti-inflammatory potential, defining the effective concentrations or doses of nanosystems containing Lippia sidoides essential oil or thymol. In the safety assessment of the products, different types of polymers were investigated, defining their safe concentrations for the development of nanosystems.

[050] The nanosystems that are the subject of the present invention have a narrow size distribution, as can be seen in figures 1 and 2, where the size distribution in nanometers by intensity of nanoparticles produced from Lippia sidoides essential oil (figure 1) and nanoemulsion produced from the active ingredient thymol (figure 2) is represented. Petition 870210081630, dated 03 / 09 / 2021, page 25 / 47 17 / 25

[051] The stability of these preparations was evaluated for 60 days at room temperature 30±2°C, and all preparations evaluated showed no significant changes in size, zeta potential or polydispersity parameters. Figure 2 shows the stability graph over time for the preparation of a nanosystem consisting of Lippia sidoides essential oil nanoparticles.

[052] The components that make up the nanosystem formulation and their concentration range are shown in the table below. Phase Components Concentration Range* Organic Lippia sidoides essential oil 50 - 300pL Thymol 30-200μL Sorbitan esters (sorbitan monooleate) 30 - 150mg Phospholipids (phosphatidylcholine (98%), phosphatidylserine, cholesterol) 40 - 90mg Capric / caprylic triglycerides 50 - 300pL Polycaprolactone (PCL) 30 - 100mg Polylactic-coglycolic acid (PLGA) 20 - 100mg Acetone 10-40 mL Petition 870210081630, dated 03 / 09 / 2021, page 26 / 47 18 / 25 Ethanol 10-40 mL Aqueous Copolymer Polypropylene Glycol Polyethylene Glycol 40 - 200mg Polysorbate 80 30 - 200mg Water - 100mL * for preparation of a nanosystem containing a final volume of 10 mL

[053] Nanosystems (nanoemulsions or nanoparticles of Lippia sidoides or thymol) are added in the concentration range between 10 and 50%, preferably 50%, to pharmaceutical preparations in liquid (lotion) or semi-solid (gel) form. Preparation examples Example 1

[054] Example of preparation of a thymol-containing nanoemulsion Phase Component Concentration* Organic Thymol 100 pL Phospholipids (phosphatidylcholine (98%) phosphatidylserine, cholesterol 40 mg Petition 870210081630, dated 03 / 09 / 2021, page 27 / 47 19 / 25 Caprylic / Capric Triglycerides 100 pL Acetone 15 mL Aqueous Polypropylene Glycol Copolymer 130 mg Water 30 mL *The concentrations described in this example refer to a preparation with a final volume of 10 mL.

[055] Preparation method: - Heat the components of the organic phase in a glass or stainless steel container to 45-65°C until completely dissolved. Separately, heat the components of the aqueous phase to 45-65°C in a glass or stainless steel container until completely dissolved. - While still at a temperature of 45 to 65°C, pour the organic phase over the aqueous phase at a controlled flow rate (3-5 milliliters per minute), while stirring at 8,000 revolutions per minute (rpm), maintaining stirring and heating for 5 to 10 minutes after the phases have been incorporated. - Proceed with the evaporation of the solvent residue in a rotary evaporator in order to obtain a final volume of 10 mL. Example 2 [ 056] Example of preparation of nanoparticles containing Lippia essential oil. Petition 870210081630, dated 03 / 09 / 2021, page 28 / 47 20 / 25 sidoides Phase Component Concentration* Organic Lippia sidoides essential oil 85 pL Phospholipids (phosphatidylcholine (98%), phosphatidylserine, cholesterol) 30 mg Polylactic-co-glycolic acid (PLGA) 40 mg Acetone 20 mL Aqueous Polypropylene glycol copolymer Polyethylene glycol 180 mg Water 60 mL *The concentrations described in this example refer to a preparation with a final volume of 10 mL.

[057] Preparation method: - Heat the components of the organic phase in a glass or stainless steel container to 45-65°C until completely dissolved. Separately, heat the ingredients to 45-65°C in a glass or stainless steel container. Petition 870210081630, dated 03 / 09 / 2021, page 29 / 47 21 / 25 components of the aqueous phase, until complete solubilization. - While still at a temperature of 45 to 65°C, pour the organic phase over the aqueous phase at a controlled flow rate (3-5 milliliters per minute), while stirring at 8,000 revolutions per minute (rpm), maintaining stirring and heating for 5 to 10 minutes after the phases have been incorporated. - Proceed with the evaporation of the solvent residue in a rotary evaporator in order to obtain a final volume of 10 mL. Example 3 [ 058] Example of preparation of a pharmaceutical preparation in semi-solid pharmaceutical form containing thymol nanoemulsion Phase Component Concentration*(%) A Xanthan gum 1.0 g Hydroxypropylmethylcellulose 1.0 g Glycerin 5.0 mL Ethoxylated lanolin 0.3 g B Lactic acid 0.2 g Hyaluronic acid 1.8 g Water 45 mL C Imidazolidinyl urea 0.1 g Water 5.53 mL D Thymol nanoemulsion 40 mL Petition 870210081630, dated 03 / 09 / 2021, page 30 / 47 22 / 25 Aminomethylpropanol 0.07 mL *The concentrations described in this example refer to a preparation with a final mass of 100g.

[059] Preparation method: - Homogenize the components of phase A, in order to obtain a paste. - Gradually add the previously dissolved phase B to phase A, mixing until a homogeneous gel is obtained. Add the previously dissolved phase C, phases A+B, and homogenize. Add phase D to phases A, B, and C and homogenize. Add Phase E to A+B+C+D and homogenize to adjust the pH. It should be around 6.5. Example 4

[060] Example of preparation of a pharmaceutical preparation in liquid pharmaceutical form containing Lippia sidoides essential oil nanoparticles Phase Component Concentration*(%) A Hydroxypropylmethylcellulose 0.12 g Chitosan 0.35 g Glycerin 3.0 mL Butylene glycol 2.0 mL Ethoxylated lanolin 0.45 g B Lactic acid 0.2 Petition 870210081630, dated 03 / 09 / 2021, page 31 / 47 23 / 25 Hyaluronic acid 2.5g Water 55mL c Imidazolidinyl urea 0.1g Water 6.21mL D Lippia sidoides essential oil nanoparticles 30mL E Aminomethylpropanol 0.07mL *The concentrations described in this example refer to a preparation with a final mass of 100g.

[061] Preparation method: - Homogenize the components of phase A, in order to obtain a paste. - Gradually add the previously dissolved phase B to phase A, mixing until a homogeneous solution is obtained. Add the previously dissolved phase C, phases A+B, and homogenize. Add phase D to phases A+B+C and homogenize. Add Phase E to A+B+C+D and homogenize to adjust the pH. It should be around 6.5. Pharmacological Studies 1. In vitro studies with human neutrophils 1.1. Cytotoxicity assessment Petition 870210081630, dated 03 / 09 / 2021, pp. 32 / 47 24 / 25 1.1.1. Lactate dehydrogenase (LDH) enzyme activity

[062] Figures 3, 4 and 5 show the effect of Lippia sidoides essential oil (OELS), thymol (TIM) and Lippia sidoides essential oil nanosystem (NSLS), respectively, on neutrophil toxicity measured by the LDH enzyme. It was observed that OELS (Figure 3) after 30 minutes of incubation promoted an increase in LDH activity at concentrations of 50 and 100 μg / mL, (44.67 ± 2.11 and 58.77 ± 5.46 %) when compared to the Control group (DMSO 1%; 24.90 ± 1.60 %). This relative cytotoxicity of OELS was no longer observed when OELS was evaluated incorporated into a nanosystem - NSLS (Figure 5), at concentrations of 10, 50 and 100 μg / mL (LDH activity percentage: 9.32 ± 0.86; 13.01 ± 0.41 and 11.73 ± 2.48%, respectively) in relation to the control group (nanosystem (NS) without OELS: 12.01 ± 1.61%).The addition of TIM (Figure 4) (1, 10, 50 and 100 μg / mL) to the neutrophil suspension did not interfere with cell viability, showing a percentage of LDH activity (6.60 ± 1.23; 6.24 ± 0.83; 5.85 ± 0.89; 7.15 ± 1.20%, respectively) even lower than the control group.

[063] Figures 6 and 7 show the effects of OELS and NSLS, respectively, on neutrophil toxicity measured by LDH enzyme activity through the construction of a concentration-time-response curve. It was observed that treatment of cells with OELS (50 and 100 μg / mL) promoted a significant increase in LDH enzyme activity from 15 minutes of incubation (OELS: 44.67 ± 2.11 and 58.77 ± 5.46%, respectively) when compared to the control group (DMSO 1%: 7.54 ± 0.41%) (Figure 6). On the other hand, incubation of NSLS (10, 50 and 100 μg / mL) in neutrophil suspension for up to 60 minutes did not promote an increase in LDH activity (60 min: 12.20 ± 0.88; 11.85 ± 1.18 and 14.31 ± 0.99%, respectively) when compared to the control group (NS without OELS: 16.76 ± 1.52%) (Figure 7). Petition 870210081630, dated 03 / 09 / 2021, pp. 33 / 47 25 / 25 1.1.2. MTT Test

[064] Figure 8 shows that the addition of OELS to human neutrophils significantly reduced cell viability as assessed by the MTT assay at concentrations of 100 and 200 μg / mL (56.64 ± 1.56; 23.44 ± 2.40%, respectively) when compared to the control group (DMSO 1%: 97.86 ± 2.34%). TIM (1 and 10 μg / mL) also did not significantly reduce cell viability (110.01 ± 10.55; 101.40 ± 7.46%, respectively) compared to the control group (DMSO 1%: 101.80 ± 3.08%) (Figure 9). When incubated with NSLS (10, 50 and 100 μg / mL), neutrophil viability was not significantly reduced (88.50 ± 2.57; 93.31 ± 5.11; 96.69 ± 2.13 %; respectively) when compared to the control group (nanosystem (NS) without OELS: 102.1 ± 4.93 %) (Figure 10). 1.2. Anti-inflammatory activity: Degranulation of human neutrophils activated by PMA, through measurement of the myeloperoxidase enzyme.

[065] Figures 11, 12, and 13 show the effects of OELS, TIM, and NSLS, respectively, on the release of the MPO enzyme resulting from PMA-induced neutrophil degranulation. It was observed that after 30 minutes of incubation, OELS (2, 5, 10, and 20 μg / mL) promoted inhibition of MPO release by up to 65% (Figure 11). TIM (1, 10, 50, and 100 μg / mL) promoted a maximum inhibition of 78.5% (Figure 12). NSLS (10, 50, and 100 μg / mL) also reduced the degranulation of stimulated neutrophils by up to 52% (Figure 13). Indomethacin (36 μg / mL), the reference drug, inhibited human neutrophil degranulation by 76% (76.3 ± 1.06%).

Claims

1. Manufacturing process for nanosystems containing Lippia sidoides essential oil or thymol as the active ingredient, characterized by: a) Heat the organic phase consisting of thymol or Lippia sidoides essential oil, together with the organic solvent, the fatty component, the lipophilic surfactant and the biodegradable polymer, to 45 to 65°C; b) Separately, heat the components of the aqueous phase, which are water and the water-soluble surfactant, to 45-65°C; c) While still at a temperature of 45 to 65°C, pour the organic phase over the aqueous phase at a controlled flow rate (3-5 milliliters per minute), while stirring at 8,000 revolutions per minute, maintaining stirring and heating for 5 to 10 minutes after the phases have been incorporated; d) Proceed with the evaporation of the solvent residue in a rotary evaporator.

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