NANOCRISTAIS DE FIROCOXIBE, MÉTODO DE PREPARO, COMPOSIÇÃO FARMACÊUTICA E USOS

Description

1 / 51 Firocoxib nanocrystals, method of preparation, pharmaceutical composition and uses. FIELD OF THE INVENTION

[001] The present invention belongs to the field of nanotechnology, since it refers to firocoxib nanocrystals, which have potential application in the veterinary treatment of inflammatory and / or pain conditions, for example, associated with osteoarthritis and other inflammatory diseases. The invention also provides a method for obtaining firocoxib nanocrystals using wet milling and the use of said nanocrystals in the preparation of a medicament for the treatment of veterinary inflammatory disorders. BACKGROUND OF THE INVENTION

[002] Osteoarthritis, also known as arthrosis, is the most frequently diagnosed joint disease in veterinary medicine. Among species, it is estimated that 200,000 dogs are affected by osteoarthritis annually in the United Kingdom. In the United States, the disease affects 20% of the adult canine population. Osteoarthritis can be observed in 50% of horses over 15 years of age. The disease is also one of the main causes of chronic pain in cats (ANDERSON et al., 2018; MONTEIRO, 2020; PETTITT; GERMAN, 2016; VAN WEEREN; BACK, 2016).

[003] Osteoarthritis can be associated with obesity and aging, or result from other joint diseases. This condition leads to structural losses in the joint and failure in the repair process. The animal may present difficulties in joint movement and local stiffness. Thus, activities such as walking, running and jumping become arduous tasks. Additionally, chronic pain generates irritability and aggressiveness in the animal (ANDERSON et al., 2020; SHARMA, 2021).

[004] Osteoarthritis also causes overload for caregivers due to the challenges of caregiving, negatively impacting their quality of life (SPITZNAGEL et al., 2022a, 2022b). This Petition 870250006914, dated 01 / 28 / 2025, page 12 / 87 2 / 51 Physical, emotional, and financial burden, as well as animal suffering, increase the euthanasia rate for dogs affected by the disease (KUNTZ et al., 2023; SPITZNAGEL et al., 2022b). After 6 months of diagnosis, it is estimated that 5% of dogs with osteoarthritis are euthanized (BANFIELD PET HOSPITAL, 2021).

[005] With regard to pharmacological treatment, non-steroidal anti-inflammatory drugs (NSAIDs) are the first-line approach (PYE et al., 2022). The efficacy of NSAIDs is related to the inhibition of a significant amount of prostaglandins associated with pain. However, prolonged use of these drugs can cause potential adverse effects. In dogs, gastric mucosal damage, vomiting, impaired renal function, and hepatotoxicity have been observed (PYE et al., 2022). NSAIDs used to treat osteoarthritis in dogs include meloxicam, carprofen, rofenacoxib, cimicoxib, mavacoxib, firocoxib, among others.

[006] Firocoxib (FCX) (3-(cyclopropylmethoxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)furan-2-one) is an NSAID from the class of selective cyclooxygenase-2 (COX-2) inhibitors for strictly veterinary use. This drug is non-ionizable and has a water solubility of 19.6 mg / L at 25 °C (MAGGI et al., 2019).

[007] The absorption of drugs by the body depends on their physicochemical properties, such as lipophilicity, molecular size, polarity and solubility (ANGELES et al., 2019).

[008] Considering the need for the drug to be solubilized in the body fluids to be absorbed, water solubility is a determining factor for its adequate pharmacological and biopharmaceutical profile (CID et al., 2019). Therefore, the low water solubility of FCX poses a challenge in the development of new oral pharmaceutical forms. This fact limits its oral bioavailability in dogs to 36.9% (EMA, 2024). Petition 870250006914, dated 01 / 28 / 2025, page 13 / 87 3 / 51

[009] Like FCX, it is estimated that 40% of approved drugs contain drugs with low water solubility, and that approximately 90% of new chemical entities under development also share this characteristic (VAN DER MERWE et al., 2020).

[0010] The low solubility of drugs in aqueous media can also compromise therapeutic safety. This is due to the need for higher doses to achieve adequate plasma concentration, or a higher frequency of administration (MACEDO et al., 2021). Therefore, it is necessary to seek innovative strategies to improve the oral bioavailability of FCX and the treatment of veterinary osteoarthritis.

[0011] Different strategies are employed to increase the bioavailability of drugs with low water solubility. These strategies allow for an increase in their dissolution rate and saturation solubility. This generates an increase in the concentration of drug dissolved in gastrointestinal fluids and in the amount available to cross membranes. Therefore, it is possible to reach the systemic circulation more easily and increase the bioavailability of these drugs (CID et al., 2019).

[0012] Drug nanocrystals consist of preparations obtained using a non-matrix nanotechnology approach, composed of a drug with a particle size < 1000 nm, surfactants, and an aqueous or non-aqueous vehicle (MELO et al., 2020; PETERS et al., 2021). In the last decade, this pharmaceutical preparation has reached the market in a significant way. The drug nanocrystal market in Europe, Asia, and North America is projected to reach US$ 14.1 billion in 2027 (RESEARCH AND MARKETS, 2020).

[0013] Nanocrystals provide improved physicochemical properties of drugs due to their nanoscale size (FOOD AND DRUG ADMINISTRATION (FDA), Petition 870250006914, dated 01 / 28 / 2025, page 14 / 87 4 / 51 2022) .

[0014] By reducing the particle size of the drug, the surface area to volume ratio increases. This increase allows for a greater number of particles to be available to interact with the body's aqueous fluids. In this way, the rate of dissolution is promoted (NOYES; WHITNEY, 1897).

[0015] With reference to saturation solubility, this is constant as a function of the compound, the dissolution medium, and the temperature. When the drug particle size reaches a size smaller than 1 pm, saturation solubility also depends on this property (LI et al., 2021). Therefore, increasing the dissolution rate and saturation solubility increases absorption, thus improving bioavailability.

[0016] The increased surface area-to-volume ratio of nanocrystals also allows for increased interactions between the drug and the intestinal mucosa, which can prolong its retention time, increase the concentration gradient, and promote intestinal absorption (TIAN et al., 2021). Additionally, nanocrystals are able to minimize the variation in absorption between the fasting and fed states, due to the rapid dissolution of the drug regardless of the presence or absence of food in the gastrointestinal tract (SCHULTZ et al., 2020).

[0017] Nanocrystals result in an unstable system, with a tendency towards sedimentation and aggregation and, consequently, an increase in the particle size of the drug. Furthermore, nanocrystals with different particle sizes result in mass transfer from smaller to larger particles, a phenomenon called Ostwald ripening (LI et al., 2021). In order to overcome this challenge, surfactants are employed to promote the physical stability of drug nanocrystals.

[0018] Cationic and anionic surfactants promote stability through an electrostatic barrier, while stability Petition 870250006914, dated 01 / 28 / 2025, page 15 / 87 Stability 5 / 51 promoted by non-ionic surfactants is achieved by the formation of a steric barrier, preventing the particles from approaching each other (LI et al., 2021). Stability through a steric barrier has the advantage of not being sensitive to changes in the pH of the gastrointestinal tract and being effective in both aqueous and non-aqueous media (MALAMATARI et al., 2018).

[0019] Nanonization strategies are frequently employed in an attempt to improve the solubility profile of COX-2 inhibitor drugs. For example, Ding et al. (2019) describe the preparation of celecoxib nanocrystals using the wet milling technique using polyvinylpyrrolidone and sodium dodecyl sulfate (SDS) as surfactants to stabilize the drug crystals.

[0020] Along the same lines, Arslan and colleagues (2023) also report the preparation of celecoxib nanoformulations, however, using the dry milling technique to promote the nano-ization of the drug. The surfactants used to stabilize the nanocrystals again included SDS.

[0021] However, nanocrystals containing SDS are not applicable to clinical practice since the aforementioned surfactant is not biocompatible. Furthermore, the chemical difference between celecoxib and firocoxib hinders the prompt translation of knowledge gained from nano-izing the former to the latter.

[0022] Each chemical entity exhibits distinct behavior and interactions with the components of the preparation, influenced by its inherent physicochemical properties, the composition of the system, and the conditions of the process used. Such interactions and behavior cannot be predicted without proper experimental evaluation.

[0023] In this sense, previous attempts to improve the solubility of FCX used complex techniques for encapsulating the drug or did not achieve the advantageous properties of structuring at the nanoscale. Petition 870250006914, dated 01 / 28 / 2025, p. 16 / 87 6 / 51

[0024] For example, patent document US20210052489A1 discloses a polymeric nanoparticle whose coating allows penetration into a mucosal layer while the inner layer corresponds to a load of a COX2 inhibitor anti-inflammatory, which may be firocoxib. However, the nanoparticle is not targeted for oral administration of the drug, so it could not be applied to the treatment of veterinary osteoarthritis.

[0025] Finally, Maggi et al. (2019) show a strategy for preparing FCX microfibers using the electrospin technique. The microfibers have an improved dissolution profile compared to the unprocessed drug, and the surfactants used to stabilize the fibers include biocompatible options. However, it does not have the physicochemical and pharmacokinetic advantages of nanometric drugs, and the described technique cannot be applied to the preparation of FCX nanocrystals.

[0026] Given the limitations of the technique to provide a definitive solution to the limited dissolution of FCX and the consequent impacts on the pharmacokinetic profile of the drug, the nanocrystals of the present invention were designed.

[0027] The FCX nanocrystals described here are prepared with minimal percentages of surfactant, which is nevertheless biocompatible, through a preparation method readily adaptable to industrial scale. As a result, the saturation solubility of FCX nanocrystals in water is five times greater than that of the micrometric (unprocessed) drug and at least 6 months of physicochemical stability, i.e., without molecular aggregation at a level detrimental to the nanometric structure and its advantageous properties. SUMMARY OF THE INVENTION

[0028] The invention provides firocoxib nanocrystals, a method for producing them using a wet milling protocol with beads, methods for treating inflammatory and / or pain disorders in animals, and the use of said nanocrystals for Petition 870250006914, dated 01 / 28 / 2025, p. 17 / 87 7 / 51 prepare a remedy.

[0029] In a central aspect, the invention provides firocoxib nanocrystals comprising firocoxib and at least one pharmaceutically acceptable surfactant.

[0030] In a particular embodiment, the pharmaceutically acceptable surfactant comprising the firocoxib nanocrystals may be selected from poly(ethylene oxide) 80% + poly(propylene oxide) 1800 (Kolliphor® P188, hereinafter P188), poly(ethylene oxide) 70% + poly(propylene oxide) 4000 (Kolliphor® P407, hereinafter P407), vinylpyrrolidone vinyl acetate copolymer (Kollidon® VA64, hereinafter VA64), hydroxypropyl methyl cellulose (HPMC) and polyvinyl caprolactam acetate-polyethylene glycol copolymer (Soluplus®, hereinafter SOL).

[0031] A preferred embodiment provides that the surfactant comprised in the nanocrystals is a copolymer of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (SOL).

[0032] In a particular embodiment, the ratio between firocoxib and polyvinyl caprolactam acetate-polyethylene glycol copolymer surfactant is in the range of 0.33:1 to 3:1.

[0033] Firocoxib nanocrystals have an average particle size, denoted by the hydrodynamic mean diameter (HMD), equal to or less than about 1000 nm. In embodiments of the invention, the HMD of the nanocrystal is less than 400 nm.

[0034] Furthermore, the invention provides that the FCX content in the nanocrystals is at least 90%, at least 95%, or at least 99%.

[0035] In another aspect, the invention provides a wet milling method with beads for preparing firocoxib nanocrystals, comprising: a) prepare a firocoxib suspension (w / w) in a Petition 870250006914, dated 01 / 28 / 2025, p. 18 / 87 8 / 51 aqueous solution of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer; b) add the firocoxib suspension to a grinding chamber containing zirconium oxide spheres; c) complete the volume with purified water qsp; d) subject the mixture to agitation, operating at 800 rpm to 3000 rpm.

[0036] In a particular embodiment of the method for producing firocoxib nanocrystals, the concentrations of firocoxib and polyvinyl caprolactam-polyvinyl polyethylene glycol acetate copolymer (SOL) in the aqueous suspension can be from 1% to 10% (w / w).

[0037] In another aspect, the invention provides a pharmaceutical composition comprising the aforementioned firocoxib nanocrystal and one or more pharmaceutically acceptable excipients.

[0038] In a preferred embodiment of the invention, the pharmaceutical composition is formulated for oral administration to an animal.

[0039] In particular embodiments, the invention provides a method for treating inflammatory and / or pain disorders in animals, wherein such method comprises administering a therapeutically effective amount of firocoxib nanocrystals or the pharmaceutical composition comprising them, preferably orally, to an animal in need of such treatment.

[0040] In a final aspect, the invention provides the use of firocoxib nanocrystals or a composition comprising them in the preparation of a medicament for treating inflammatory and / or pain disorders in animals, particularly canine osteoarthritis. BRIEF DESCRIPTION OF THE FIGURES

[0041] To assist in identifying the main features of the present invention, the following are presented Petition 870250006914, dated 01 / 28 / 2025, page 19 / 87 9 / 51 following figures: Figure 1 presents average hydrodynamic diameter (MHD) values ​​of firocoxib nanocrystals (FCX-NC) as a function of milling time, obtained by small-scale wet milling (n=3). P188: poly(ethylene oxide) 80% + poly(propylene oxide) 1800; P407: poly(ethylene oxide) 70% + poly(propylene oxide) 4000; VA64: vinylpyrrolidone-vinyl acetate copolymer; HPMC: hydroxypropyl methyl cellulose; SOL: polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer.

[0042] Figure 2 illustrates the saturation solubility of micronized firocoxib (FCX micro) and the nanocrystals obtained (FCX-NC) using different surfactants (n=3). P188: poly(ethylene oxide) 80% + poly(propylene oxide) 1800; P407: poly(ethylene oxide) 70% + poly(propylene oxide) 4000; VA64: vinylpyrrolidone-vinyl acetate copolymer; HPMC: hydroxypropyl methyl cellulose; SOL: polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer.

[0043] Figure 3 shows the average hydrodynamic diameter (MHD), polydispersity index (PdI), and zeta potential (ZP) of firocoxib nanocrystals (FCX-NC) for 1 month at 30 °C ± 2 °C and RH 75% ± 5%. (a) P188: poly(ethylene oxide) 80% + poly(propylene oxide) 1800; (b) P407: poly(ethylene oxide) 70% + poly(propylene oxide) 4000; (c) VA64: vinylpyrrolidone-vinyl acetate copolymer; (d) HPMC: hydroxypropyl methylcellulose; (e) SOL: polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer.

[0044] Figure 4 shows time series for evaluating the DHM of FCX-NC for the variables: FCX concentration (% w / w), SOL concentration (% w / w) and agitation time (days).

[0045] Figure 5 represents main effects for evaluating the DHM of FCX-NC for the variables: FCX concentration (% w / w), SOL concentration (% w / w) and agitation time (days). Petition 870250006914, dated 01 / 28 / 2025, page 20 / 87 10 / 51

[0046] Figure 6 illustrates interactions (quadratic effect) for evaluating the DHM of FCX-NC for the following variables: FCX and SOL concentration (% w / w) and agitation time (days).

[0047] Figure 7 shows the response surface plot for evaluating the DHM of FCX-NC containing the variables: FCX and SOL concentration (% w / w) and agitation time (days).

[0048] Figure 8 represents the contour plot for evaluating the DHM of FCX-NC for the variables: FCX and SOL concentration (% w / w) and agitation time (days).

[0049] Figure 9 presents an optimization graph of the average hydrodynamic diameter (MHD) of FCX-NC containing the following variables: FCX concentration 5.0% (w / w), SOL 5.0% (w / w) and agitation time 4.61 days. Prediction for 185 nm for optimized formula 1 (F1).

[0050] Figure 10 presents an optimization graph of the average hydrodynamic diameter (MHD) of FCX-NC containing the following variables: FCX concentration 4.09% (w / w), SOL 6.0% (w / w) and agitation time 3.68 days. Prediction for 195 nm for optimized formula 2 (F2).

[0051] Figure 11 illustrates the stability of firocoxib nanocrystals prepared with surfactant SOL, denoted by hydrodynamic mean diameter (HMD), polydispersity index (PdI) and zeta potential (ZP) of FCX-NC nanocrystals optimized by Box-Behnken (F1) for 6 months at 30 °C ± 2 °C and RH 75% ± 5% (n= 3).

[0052] Figure 12 illustrates the physicochemical stability of FCX-NC nanocrystals from the perspective of the pH of the preparations. pH values ​​at 25 °C during the evaluation of the physicochemical stability of firocoxib nanocrystals (FCX-NC) optimized by Box-Behnken (F1) for 6 months at 30 °C ± 2 °C and RH 75% ± 5% (n= 3).

[0053] Figure 13 shows the mean hydrodynamic diameter (MHD) and polydispersity index (PdI) of FCX-NC during preparation. Petition 870250006914, dated 01 / 28 / 2025, page 21 / 87 11 / 51 of the pilot-scale batch (n= 3).

[0054] Figure 14 shows the physical stability of FCX-NC prepared on a pilot scale, evaluated in terms of average hydrodynamic diameter (DHM), polydispersity index (PdI) and zeta potential (PZ) at room temperature (n= 3).

[0055] Figure 15 shows XRD peaks by powder method of the following samples: firocoxib raw material (FCX), polyvinyl caprolactam-polyvinyl polyethylene glycol acetate copolymer (SOL), physical mixture (MF) of FCX and SOL and firocoxib nanocrystals (FCX-NC).

[0056] Figure 16 shows DSC curves obtained at 10 °C / min and a dynamic nitrogen atmosphere with a flow rate of 50 mL / min of the following samples: firocoxib raw material (FCX), polyvinyl caprolactam-polyvinyl polyethylene glycol acetate copolymer (SOL), physical mixture (MF) of FCX and SOL, and firocoxib nanocrystals (FCX-NC).

[0057] Figure 17 shows TG curves obtained at 10 °C / min in a dynamic nitrogen atmosphere with a flow rate of 100 mL / min of the following samples: firocoxib raw material (FCX), polyvinyl caprolactam-polyvinyl polyethylene glycol acetate copolymer (SOL), physical mixture (MF) of FCX and SOL, and firocoxib nanocrystals (FCX-NC).

[0058] Figure 18 shows electron micrographs obtained by transmission electron microscopy (TEM) of firocoxib raw material (FCX). (a) 2,000x magnification; (b) 4,000x magnification.

[0059] Figure 19 shows electron micrographs obtained by transmission electron microscopy (TEM) of firocoxib nanocrystal particles (FCX-NC). (a) 6,000x magnification; (b) 10,000x magnification.

[0060] Figure 20 shows the in vitro dissolution profile of micronized firocoxib (FCX), physical mixture (MF) and firocoxib nanocrystals (FCX-NC) using apparatus 2, Petition 870250006914, dated 01 / 28 / 2025, page 22 / 87 12 / 51 evaluated in sodium phosphate buffer pH 6.8 (900 mL) containing 1% sodium dodecyl sulfate, at 50 rpm for 60 min (n= 3).

[0061] Figure 21 shows survival and morbidity curves of Galleria mellonella L. larvae treated with negative control (phosphate-buffered saline, PBS - 5 mg / kg), SOL (5 mg / kg), FCX (5 mg / kg) and FCX-NC (5, 10 and 50 mg / kg). (a) Toxicity curve; (b) health index after 5 days of evaluation (n= 20).

[0062] Figure 22 illustrates the mean plasma concentration-time kinetics of FCX-NC and Previcox® after a single oral dose (5 mg / kg) in healthy dogs (mean ± SD, n= 6). DETAILED DESCRIPTION OF THE INVENTION

[0063] Unless otherwise specified, the terms used throughout this descriptive report have their common meanings in the art, within the context of the disclosure, and in the specific context in which each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure. Publications cited herein and the material to which they are cited are specifically incorporated by reference in their entirety.

[0064] It will be appreciated that the same thing can be said in different ways. Consequently, alternative language and synonyms may be used for any or more of the terms discussed here. No special significance should be placed on whether a term is elaborated or discussed here. Synonyms for certain terms are provided, but the exemplification of some synonyms does not preclude the potential use of others not listed here.

[0065] In one embodiment of the invention, firocoxib nanocrystals, also designated as FCX-NC, are provided to Petition 870250006914, dated 01 / 28 / 2025, page 23 / 87 13 / 51 throughout the present description. As used in this invention, the term nanocrystal denotes firocoxib particles with a particle size of less than 1000 nanometers (nm) as measured by conventional particle size measurement techniques well known to those skilled in the art, such as, for example, laser diffraction or dynamic light scattering.

[0066] As used herein, the term pharmaceutically acceptable surfactant denotes a pharmaceutically acceptable substance, or a combination thereof, that reduces the surface tension of a liquid and decreases the interfacial tension between two liquids. Surfactants are generally organic compounds that are amphipathic, meaning that they contain hydrophobic groups (their tails) and hydrophilic groups (their heads). Therefore, they are typically moderately soluble in organic solvents and water.

[0067] A surfactant can be classified by the presence or absence of formally charged groups in its head. A non-ionic surfactant has no charged groups in its head. The head of an ionic surfactant carries a net charge; if the charge is negative, the surfactant is anionic; if the charge is positive, it is cationic; if it contains a head with two groups of opposite charges, it is zwitterionic.

[0068] In the context of this invention, surfactants and stabilizers are synonyms used interchangeably throughout the description. Surfactants applicable in the context of the invention include 80% poly(ethylene oxide) + 1800 poly(propylene oxide) copolymer, 70% poly(ethylene oxide) + 4000 poly(propylene oxide) copolymer, vinylpyrrolidone-vinyl acetate copolymer, hydroxypropyl methylcellulose (HPMC), and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. A surfactant particularly relevant to the invention is polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. Petition 870250006914, dated 01 / 28 / 2025, page 24 / 87 14 / 51

[0069] The firocoxib nanocrystals of this invention can be prepared by a method comprising the steps of dispersing firocoxib in a pharmaceutically acceptable surfactant (i.e., stabilizer), in the presence or absence of a buffer at pH 7.0, and applying mechanical milling means to reduce the particle size of firocoxib to an average particle size of less than about 1000 nm.

[0070] In some embodiments of the invention, the firocoxib nanocrystals have an average particle size, denoted by the hydrodynamic mean diameter (HMD), measured, for example, by dynamic light scattering, equal to or less than about 1000 nm. In embodiments of the invention, the HMD is from about 100 to about 200 nm, from about 200 to about 300 nm, from about 300 to about 400 nm, from about 400 to about 500 nm, from about 500 to about 600 nm, from about 600 to about 700 nm, from about 700 to about 800 nm, from about 800 to about 900 nm, from about 900 to about 999 nm. In a preferred embodiment of the invention, the DHM of the nanocrystal is less than 400 nm.

[0071] To achieve stability of firocoxib nanocrystals, specific components must be combined in appropriate concentrations and processed in a particular way. Thus, each formula is unique and can therefore only be achieved through empirical and exhaustive testing.

[0072] Among these factors, the appropriate ratio of surfactant(s) and firocoxib is of fundamental importance for the stability of the nanocrystals. The main instability phenomena, resulting from inappropriate combinations of drug excipients, refer to coalescence, flocculation, Ostwald ripping and sedimentation (ALI et al., 2017).

[0073] The term stability, as used throughout the text, refers to the maintenance of physical characteristics of firocoxib nanocrystals, such as size, Petition 870250006914, dated 01 / 28 / 2025, page 25 / 87 15 / 51 Morphology and uniformity of particle size distribution of nanocrystals. Methods for evaluating each of these parameters are widely known to those skilled in the art and are particularly summarized in the work of Phan and Haes (2019).

[0074] Mechanical means applied to reduce the particle size of the drug can conveniently take the form of a dispersion mill. Suitable dispersion mills include a ball mill, an attrition mill, a vibratory mill, and medium mills, such as a sand mill and a sphere mill. A medium mill, with spheres, is preferred due to the relatively shorter grinding time required to provide the intended result, i.e., the desired reduction in particle size.

[0075] The grinding medium for the particle size reduction step can be selected from a rigid medium, preferably spherical or in the form of particles with an average size of less than about 3 mm and, more preferably, less than about 1 mm. Such media can desirably provide the nanocrystals of the invention with shorter processing times and cause less wear on the grinding equipment. Zirconium oxide spheres with a size of 0.1 mm to 0.2 mm constitute the preferred grinding medium.

[0076] The grinding speed may vary depending on the mechanical means and processing conditions selected. For ball grinding as described in the present invention, grinding speeds of at least 800 rpm to at least 3000 rpm may be required. In preferred embodiments, the grinding speed is 1200 rpm to 2600 rpm.

[0077] In one embodiment, the invention provides pharmaceutical compositions comprising the firocoxib nanocrystals described herein, a carrier and one or more pharmaceutically acceptable excipients. Such compositions may be prepared and formulated Petition 870250006914, dated 01 / 28 / 2025, p. 26 / 87 16 / 51 employing conventional methods and excipients, as disclosed, for example, in the British (21), European (22) and United States Pharmacopoeias (23), Remington's Pharmaceutical Sciences (24), Martindale: The Extra Pharmacopoeia (25) and in Prista's Pharmaceutical technology (26).

[0078] Pharmaceutical compositions may be formulated for any route of administration including, for example, topical, oral, nasal, rectal or parenteral administration. The term parenteral, as used in this document, includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal and intraperitoneal injections, as well as any similar injection or infusion technique.

[0079] Preferred pharmaceutical compositions may be formulated for oral administration.

[0080] In a further embodiment, the invention provides the use of firocoxib nanocrystals to prepare a medicament for treating inflammatory conditions and / or musculoskeletal pain in animals. In the context of this invention, the term "preparation of a medicament" includes the use of the components of the invention directly as a medicament, as well as their use at any stage of the preparation of such a medicament.

[0081] In a preferred embodiment, the drug is intended for the veterinary treatment of osteoarthritis.

[0082] As used herein, the term treat includes extinguishing, substantially inhibiting, delaying or reversing the progression of a disease or disorder, substantially improving the clinical symptoms of a disease or disorder, or substantially preventing the appearance of clinical symptoms of a disease or disorder.

[0083] In another embodiment of the invention, a method is provided for treating inflammatory diseases and / or musculoskeletal pain in animals comprising administering Petition 870250006914, dated 01 / 28 / 2025, page 27 / 87 17 / 51 a therapeutically effective amount of firocoxib nanocrystals or the pharmaceutical composition comprising them, as described above, to a mammal in need of treatment.

[0084] In preferred modalities, the inflammatory disease is osteoarthritis and the mammal is a dog.

[0085] As used herein, the term therapeutically effective denotes an effective amount of an active ingredient (e.g., firocoxib nanocrystals) to achieve a desired clinical effect. A therapeutically effective amount varies with the nature of the condition being treated, the length of time the activity is desired, the age and condition of the subject, and is ultimately determined by the healthcare professional.

[0086] The following examples are intended to be purely illustrative of the invention. They are presented in order to provide the person skilled in the art with a complete description of how the nanocrystals of this invention are prepared, evaluated and employed. A person skilled in the art, in the light of this disclosure, will recognize that many changes can be made to the specific embodiments disclosed and still obtain a similar or equivalent result without departing from the spirit and scope of the invention. EXAMPLES

[0087] The embodiments of the invention are organized into three stages, illustrating from conception to experimental demonstration of the technical advantages of the firocoxib nanocrystals described herein. The illustrations are preceded by a description of the material and the tests employed. Material

[0088] The material used to obtain the nanocrystals refers to the drug Firocoxib 99.5% (FCX) (Sichuan Qingmu Pharmaceutical Co., China) and the following Petition 870250006914, dated 01 / 28 / 2025, page 28 / 87 18 / 51 Excipients: Kolliphor® P188 (P188) (poly(ethylene oxide) 80% + poly(propylene oxide) 1800), Kolliphor® P407 (P407) (poly(ethylene oxide) 70% + poly(propylene oxide) 4000), Kollidon® VA64 (VA64) (vinylpyrrolidone-vinyl acetate copolymer) and Soluplus® (SOL) (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer) supplied by BASF (BASF, Germany). Hydroxypropyl methylcellulose (HPMC) was supplied by IFF (Methocel™ E3, International Flavors & Fragrances Inc., USA). Screening of stabilizers for the preparation of FCX-NC by wet milling on a laboratory scale.

[0089] Initially, an exploratory assay was performed to determine the milling time for obtaining FCXNC. The following stabilizers were evaluated: Kolliphor® P188 (P188), Kolliphor® P407 (P407), Kollidon® VA64 (VA64), hydroxypropyl methyl cellulose (HPMC), and Soluplus® (SOL). The conditions employed for obtaining FCX-NC by wet milling on a laboratory scale are presented in Table 1. The milling medium consisted of a 7 mL glass container filled with yttrium-stabilized zirconium beads 0 = 0.1 mm (Zetabeads Plus, Netzsch, Germany) and cross-shaped magnetic stir bars (Romero et al., 2016). The drug and aqueous stabilizer solution were transferred to the milling medium and stirred on a magnetic stirrer plate (IKA Werke, Germany) at room temperature. The resulting formulation was collected and separated from the grinding medium using a pipette. The grinding conditions used are presented in Table 1. Table 1 - Preparation process conditions for determining the grinding time during the production of firocoxib nanocrystals (FCX-NC) by small-scale wet milling. Process parameters Conditions employed Drug concentration (% w / w) 3.0 Petition 870250006914, dated 01 / 28 / 2025, page 29 / 87 19 / 51 Surfactant concentration (%) 3.0 m / m Zirconium spheres 0.1 mm (%) 42.0 Grinding speed (rpm) 800 Mean hydrodynamic diameter (MHD), polydispersity index (Pdl) and apparent zeta potential (PZ)

[0090] The particle size (hydrodynamic mean diameter, DHM) and PdI of FCX-NC were determined by dynamic light scattering using the Zetasizer Nano ZS90 instrument (Malvern Instruments, UK). 20 pL aliquots of nanocrystal samples were added to cuvettes, and a 1:100 dilution was performed using purified water. The DHM was obtained by considering the diameter of a sphere that diffuses at the same speed as the particle to be measured. The PZ was determined using the Zetasizer Nano ZS90 instrument (Malvern Instruments, UK). The medium consisted of purified water with conductivity adjusted to 50 pS / cm by the addition of 0.9% (w / v) NaCl solution. The PZ calculation in the instrument was performed from electrophoretic mobility measurements. Saturation solubility

[0091] The saturation solubility of FCX-NC using different stabilizers, as well as micronized FCX, was evaluated by the shake-flask method. Excess nanocrystals and micronized drug were transferred to an Erlenmeyer flask containing 10 mL of purified water (pH 6.73). The assay was performed on an orbital shaker (Tecnal TE 4080, Tecnal, Brazil) at 100 rpm, 39 °C for 24 h. Aliquots were withdrawn and centrifuged at 15,000 x g for 10 min (Microcentrifuge 5425R, Eppendorf, Germany).

[0092] The solubility of FCX was determined according to the previously validated method by UV-Vis spectrophotometry (Evolution 201, Thermo Fisher Scientific, USA) at 290 nm. A stock solution of FCX (400 pg / mL) was prepared by dissolving the drug in acetonitrile and purified water. The linearity range Petition 870250006914, dated 01 / 28 / 2025, page 30 / 87 20 / 51 encompassed the range of 4-28 pg / mL, resulting in the equation: y = 0.0226x + 0.1812 (R² = 1). The limits of detection and quantification were 1.10 pg / mL and 3.67 pg / mL, respectively. The intra-day and inter-day coefficients of variation for accuracy were 4.13% and 3.20%, respectively. The mean recovery was 97.69 ± 4.73%. Optimization of the FCX-NC preparation process using Box-Behnken experimental design (BBD)

[0093] The influence of independent variables on the DHM of FCX-NC, on a laboratory scale, was evaluated using Box-Behnken (BBD) experimental design. The selected variables were: FCX concentration (% w / w), SOL concentration (% w / w) and agitation time (days). Table 2 presents the minimum and maximum levels of FCX and SOL concentration (4 and 6% w / w) and agitation time (3 and 5 days) used for the assay matrix, which was performed using Minitab® 20 software (Minitab Inc., USA). Six central points were included, resulting in 18 formulations. From this experimental design, the polynomial equation was obtained (Eq. 1): Yi =a0+ al xl+ a2x2+ a3x3+ al2xlx2+ al3xlx3+ a23x2x3+ allxl2 + a22x22 + a33x32 (1)where Yi is the dependent variable (DHM); a0 is the intercept; a1 to a33 are the regression coefficients; and x1 (FCX % m / m), x2 (SOL % m / m) and x3 (agitation time in days) are the independent variables. Table 2 - Matrix of tests for optimizing the preparation process of FCX-NC obtained by wet milling on a laboratory scale, using Box-Behnken (BBD). F Order FCX (% m / m) SUN (% m / m) Time (days) 10 1 5 6 3 2 2 6 4 4 9 3 5 4 3 13 4 5 5 4 Petition 870250006914, dated 01 / 28 / 2025, page 31 / 87 21 / 51 6 5 6 5 3 18 6 5 5 4 5 7 4 5 3 15 8 5 5 4 4 9 6 6 4 17 10 5 5 4 14 11 5 5 4 11 12 5 4 5 16 13 5 5 4 8 14 6 5 5 7 15 4 5 5 1 16 4 4 4 3 17 4 6 4 12 18 5 6 5 Exploratory study of transposing FCX-NC preparation to the pilot scale

[0094] The transposition of the FCX-NC preparation method to the pilot scale was carried out using a high-energy recirculation mill (LabStar®, Netzsch, Germany). The milling medium consisted of a 470 mL capacity milling chamber, agitator shaft (Neos®, Netzsch, Germany) and 85% 0.2 mm zirconium beads (Zetabeads Plus, Netzsch, Germany).

[0095] The concentration of FCX and SOL was 3% w / w each, and purified water was added to obtain a 3000 g batch. This dispersion was subjected to milling at a speed of 2600 rpm, a flow rate of 48 kg / h and a pump speed of 85 rpm. Aliquots of nanocrystals were removed every 30 minutes over 3 hours, and the DHM and PdI at each time point were determined. The physical stability of the preparation was evaluated for 6 months at room temperature, obtaining the DHM and PdI (Zetasizer Nano ZS90, Malvern Instruments, United Kingdom). Relative density

[0096] To determine the relative density of FCX-NC, Petition 870250006914, dated 01 / 28 / 2025, page 32 / 87 22 / 51 the pycnometer method was used (BRASIL, 2019). The masses of the empty pycnometer, filled with purified water and with FCXNC were obtained, and the relative density was calculated by the ratio of the mass of the pycnometer filled with the formulation and with water. Firocoxib content in FCX-NC

[0097] For content determination, 396 pL of FCX-NC were transferred to a 50 mL volumetric flask. 25 mL of acetonitrile were added and the solution was subjected to an ultrasonic bath for 5 minutes. The volume of the flask was completed with purified water. The concentration of FCX in the solution was determined by UV-Vis spectrophotometry (Evolution Series 201, Thermo Fischer Scientific Inc., USA) at a wavelength of 290 nm, using the developed and validated method mentioned in Section 5.2.1.2., and the values ​​were expressed as mean ± standard deviation (n= 3). X-ray diffraction (XRD)

[0098] XRD was performed using the powder method as described in the Brazilian Pharmacopoeia 6th ed. (BRASIL, 2019) employing the Bruker D8 Advance diffractometer (Bruker Co., USA). Samples FCX, SOL, their physical mixtures (PM) and FCX-NC were subjected to 40 kV Cu Ka radiation. Samples were evaluated between 2 and 50° (2θ) at a step of 0.02 and a speed of 0.5° / min. Differential scanning calorimetry (DSC) and thermogravimetry (TG)

[0099] In the thermal analysis by DSC and TG, the STA 7200 equipment (Hitachi, Japan) was used. For DSC, the samples: firocoxib (FCX) (3.50 mg), SOL (3.87 mg), physical mixture (MF) (4.32 mg, 1:1) and firocoxib nanocrystals (FCX-NC) (1.16 mg) were carefully weighed and transferred to a hermetically sealed aluminum crucible. These samples were subjected to a heating rate of 10 °C / min at a temperature of 30-255 °C, using a dynamic nitrogen atmosphere of 50 mL / min. Petition 870250006914, dated 01 / 28 / 2025, page 33 / 87 23 / 51

[00100] For TG, samples FCX (1.75 mg), SOL (4.48 mg), MF (2.85 mg, 1:1) and FCX-NC (0.49 mg) were evaluated at temperatures of 30-500 °C at 10 °C / min, in a dynamic nitrogen atmosphere of 100 mL / min. Mass loss was recorded as a function of temperature. Transmission electron microscopy (TEM)

[00101] The morphology of FCX and FCX-NC was evaluated by TEM (JEM 2100, JEOL, Japan). A drop of the samples, previously diluted in purified water, was added to copper grids with a 200-mesh opening, covered with a parlodium membrane. After 30 min for adsorption, the excess was removed with filter paper and the samples were subjected to natural drying for 24 h. The samples were negatively stained using 1% (w / v) uranyl acetate for 2 min. FCX and FCX-NC were observed under a transmission electron microscope at 200 kV acceleration. Dissolution

[00102] Dissolution was evaluated using apparatus II (paddles), considering a rotation of 50 rpm and a temperature of 39 ± 0.5 °C (VK-7010 dissolution apparatus, Agilent Technologies, USA). The medium consisted of 900 mL of sodium phosphate buffer pH 6.8, containing 1% sodium dodecyl sulfate. Aqueous suspensions of micronized FCX, physical mixture (PM), and FCX-NC were prepared, and an amount equivalent to 57 mg of the drug was gently added to the dissolution vessel. 5 mL aliquots were withdrawn, without replacement, at predetermined times: 5, 10, 15, 30, 45, and 60 min, and centrifuged at 15,000 x g for 10 min (Rotofix 32A, Hettich, Germany). The amount of dissolved FCX was determined using a spectrophotometric method at 290 nm (Evolution 201, Thermo Fisher Scientific, USA), and the values ​​were expressed as mean ± standard deviation (n= 3). Toxicity assessment in Galleria mellonella L.

[00103] Toxicity tests were performed on an invertebrate model using Galleria mellonella L. larvae. Petition 870250006914, dated 01 / 28 / 2025, page 34 / 87 24 / 51 Larvae measuring 2 cm in length and weighing 110-130 mg were divided into six groups: negative control (phosphate-buffered saline, PBS, at 5 mg / kg), micronized FCX (5 mg / kg), SOL (5 mg / kg), and FCX-NC (5 mg / kg, 10 mg / kg, and 50 mg / kg), and each sample was injected into the last left proleg (n=20).

[00104] The larvae were incubated at 35 °C, and survival and health index were observed daily for 5 days and morbidity was determined by the score on the health index.

[00105] Larval survival (%) was determined using the Log-rang test (Mantel-Cox), while the health index was assessed using two-way ANOVA, both tests considering a p-value <0.05 (α= 0.05) (GraphPad Prism 10, GraphPad Software Inc., USA). Pharmacokinetics in Beagle dogs

[00106] A crossover pharmacokinetic study was conducted in healthy beagle dogs, in accordance with the National Council for the Control of Animal Experimentation (CONCEA), and the study protocol was approved by the Ethics Committee on the Use of Animals (CEUA) of Synergy Animal Research (Vila Velha, Brazil) (certificate 015 / 2023, as per Annex). The dogs involved in the study consisted of 3 males and 3 females, with body weights ranging from 9.4 to 17.7 kg.

[00107] The animals were assigned to receive, orally, a single dose of 5 mg / kg of the commercial product (Previcox® 57 mg, Boehringer Ingelheim, Germany) or FCX-NC, with at least a 4-day washout period (10 half-lives of FCX, considering t1 / 2 7.6 ± 1.5h (EMA, 2024)) (n= 6).

[00108] Blood samples (4 mL) were collected from the cephalic vein at: 0 (pre-treatment), 0.5, 1, 1.5, 2, 4, 6, 8, 12 and 24 hours after administration. These samples were centrifuged at 1200 x g for 15 min and stored at -20 °C for analysis by liquid chromatography coupled to mass spectrometry (LCPetition 870250006914, dated 01 / 28 / 2025, page 35 / 87). 25 / 51 MS / MS).

[00109] For non-compartmental pharmacokinetic analysis, the PkSolver extension for Microsoft Excel was used to calculate the following pharmacokinetic parameters: maximum plasma concentration (Cmax), time to reach Cmax (Tmax), area under the curve from 0 to 24h (AUC 0-24h), and from 0 to infinity (AUC 0"), and half-life (t.1 / 2).

[00110] The difference between FCX-NC and the commercial product in pharmacokinetics was considered statistically significant when p-value <0.05 (α= 0.05) using the paired t-Student test, and the data are presented as mean ± standard deviation (n= 6) (Minitab®20, Minitab Inc., USA). EXAMPLE I — DESIGN OF FIROCOXIB NANOCRYSTALS Determination of milling time for obtaining FCX-NC

[00111] After 24 hours of processing, FCX-NC using surfactants P188 and P407 showed DHM above 1000 nm. After 48 hours, the DHM was 656.2 ± 74.07 nm and 762.9 ± 123.3 nm for P188 and P407, respectively. With reference to the polydispersity index (PdI), the values ​​were close to 1.0. The polydispersity index (PdI) is an indicative value of the quality of the DHM distribution of the preparation, where values ​​close to 0 refer to a sample with a uniform distribution of its particle size, while values ​​close to 1 indicate multiple DHM populations, which may compromise the physical stability of the preparation (DANAEI et al., 2018). After 72 hours, the milling process resulted in an increase in the DHM of FCX-NC for these surfactants, with final particle sizes of 751.0 ± 133.7 nm and 844.8 ± 81.59 nm for P188 and P407, respectively (Figure 1).

[00112] With reference to the VA64 surfactant, in the first 24 hours of milling the nanocrystals revealed a DHM of 328.2 ± 19.76 nm. At 72 hours, the DHM was 237.1 ± 3.001 nm, which subsequently increased to 310.6 ± 20.40 nm at the end of 96 hours, with Petition 870250006914, dated 01 / 28 / 2025, p. 36 / 87 26 / 51 Pdl value of 0.137 ± 0.049 (Figure 1). For HPMC, the stabilizer reached the smallest particle size after 96h, with DHM equal to 310.6 ± 20.40 nm and Pdl 0.137 ± 0.049. Similarly, the smallest particle size of FCX-NC with the surfactant SOL was also obtained at the end of 96h, being 207.7 ± 1.804 nm, PdI 0.151 ± 0.009 (Figure 1).

[00113] Considering the stabilizers tested, the appropriate milling time to obtain FCX-NC with DHM < 400 nm by wet milling on a laboratory scale was between 72h and 96h, depending on the stabilizer adopted. Based on the results presented, the saturation solubility and physical stability of these preparations were evaluated in order to select the most suitable surfactant to compose FCX-NC. Saturation solubility

[00114] Figure 2 and Table 3 show the solubility of FCX-NC obtained with different surfactants. All FCX-NC preparations showed increased water saturation solubility compared to the micronized drug (FCX micro), regardless of the stabilizer tested (Figure 2). This increase was surprising for the nanocrystals obtained using the surfactant SOL, which was 5 times greater compared to FCX micro (Table 3). The water solubility of FCX-NC containing the stabilizers HPMC and VA64 was similar, representing approximately 2 times the value obtained for FCX micro (Table 3). Both preparations containing the surfactants P188 and P407 increased saturation solubility by 1.42 times, representing the smallest increase in solubility among the stabilizers tested (Figure 2). Table 3 - Saturation solubility in water using the equilibrium method, at 39 °C for 24h of micronized firocoxib (FCX micro) and nanocrystals (FCX-NC) obtained with different stabilizers (n= 3). P188: Kolliphor® P188; P407: Kolliphor® P407; VA64: Kollidon® VA64; HPMC: hydroxypropyl methylcellulose; SOL: Soluplus®. Petition 870250006914, dated 01 / 28 / 2025, page 37 / 87 27 / 51 Solubility in water Preparations (mg / mL) FCX-NC (SOL) 0.070 ± 0.001 FCX-NC (HPMC) 0.031 ± 0.006 FCX-NC (VA64) 0.028 ± 0.002 FCX-NC (P407) 0.020 ± 0.002 FCX-NC (P188) 0.020 ± 0.003 FCX micro 0.014 ± 0.002 Physical stability

[00115] The physical stability of FCX-NC using different surfactants was evaluated at 30 °C ± 2 °C and RH 75% ± 5% for 1 month. The DHM after 1 week of preparation was 965.5 ± 209.2 nm for the surfactant P188, and 994.1 ± 164.8 nm for the surfactant P407. At the end of 1 month of physical stability, the DHM of the nanocrystals with the surfactant P188 increased to 2638.0 ± 832.9 nm (Figure 3a), while FCX-NC with P407 showed a DHM of 1348.0 ± 200.2 nm (Figure 3b). The large particle size and PdI of these preparations, associated with the smaller increase in aqueous solubility, led to the exclusion of surfactants P188 and P407 from the FCX-NC preparation.

[00116] With reference to FCX-NC containing VA64, after 1 week, the DHM was 376.6 ± 35.2 nm and PdI 0.552 ± 0.417. This result represents an increase of approximately 140 nm when compared to the initial particle size of the preparation. After 1 month of physical stability, the DHM increased to 983.0 ± 274.7 nm (Figure 3c). The presence of visible particles around the vial (data not shown) and the prominent increase in the DHM of the preparation at the end of 1 month of stability resulted in the exclusion of the surfactant VA64 in the development of FCX-NC.

[00117] For FCX-NC containing HPMC and SOL, in 1 week, the particle size of the preparation containing HPMC increased by 44 nm during the period evaluated during physical stability, obtaining a DHM equal to 349.8 ± 3.6 nm at the end of 1 month (Figure 3d). Petition 870250006914, dated 01 / 28 / 2025, page 38 / 87 28 / 51

[00118] Regarding FCX-NC with the surfactant SOL, the variation during this period was approximately 2.0 nm, with a DHM of 213.1 ± 2.3 nm, after 1 month of physical stability (Figure 3e). Since the preparation containing surfactant SOL surprisingly showed the lowest particle size variability during physical stability (2.0 nm) and the greatest increase in water solubility (5x) compared to nanocrystals containing HPMC, SOL was selected as the surfactant. EXAMPLE II — OPTIMIZATION AND CHARACTERIZATION OF FIROCOXIB NANOCRYSTALS Preparation of firocoxib nanocrystals (FCX-NC)

[00119] The methodology for obtaining the nanocrystals was wet milling on a laboratory scale as described by Romero and colleagues (ROMERO; KECK; MÜLLER, 2016). The drug firocoxib was used at a micrometric scale (initial particle size: D50 = 17.54 pm) and an aqueous solution of the surfactant Soluplus®. These components were then transferred to glass vials containing zirconium spheres, at an appropriate concentration, and subjected to subsequent magnetic stirring. Optimization of the FCX-NC preparation process using Box-Behnken experimental design (BBD)

[00120] The Box-Behnken (BBD) methodology allows the identification of interactions, possible quadratic effects, and critical process factors in the preparation of FCX-NC, providing a rational scientific basis for understanding the reduction of drug particle size as a function of independent variables.

[00121] Eighteen experiments were conducted, including six central points, to evaluate the influence of FCX and SOL concentration (% w / w) and agitation time (days) on the drug's DHM. The minimum and maximum levels of the independent variables were: FCX concentration 4 and 6% (w / w) and SOL concentration 4 and 6% (w / w), and agitation time was 3 and 5 days, while the dependent variable Petition 870250006914, dated 01 / 28 / 2025, page 39 / 87 29 / 51 (answer) was the DHM of the preparation.

[00122] Table 4 presents the DHM results for the 18 experiments, with minimum and maximum values ​​of 154.9 and 198.0 nm, respectively. The significance of the independent variables was assessed using analysis of variance (ANOVA), where the model and its terms were considered statistically significant when p-values ​​<0.05 (α= 0.05). A model suitable for the experimental data can be determined by significant regression and non-significant lack of fit (FUKUDA et al., 2018). Table 4 - Mean hydrodynamic diameter (MHD) of firocoxib nanocrystals (FCX-NC) in the Box-Behnken experimental design (BBD) assay matrix. F Test order FCX (% mm) SOL (% mm) Time (days) DHM (nm) 10 1 5 6 3 184.7 2 2 6 4 4 183.7 9 3 5 4 3 191.0 13 4 5 5 4 185.6 6 5 6 5 3 175.0 18 6 5 5 4,186.3 5 7 4 5 3,198.0 15 8 5 5 4,188.0 5 5 4 189.9 8 14 6 5 5 170.9 7 15 4 5 5 185.8 1 16 4 4 4 184.9 3 17 4 6 4 193.9 12 18 5 6 5 178.0

[00123] Table 5 presents the ANOVA results for Petition 870250006914, dated 01 / 28 / 2025, page 40 / 87 30 / 51 the DHM response. The concentration of FCX and SOL (% w / w), and the agitation time (days) were significant, as were the quadratic terms of FCX*FCX (% w / w) and SOL*SOL (% w / w) and the interaction between the concentration of FCX and SOL (% w / w) (p-value <0.005, α= 0.05). These results cannot be inferred from existing knowledge. Furthermore, the lack of fit was non-significant (p= 0.169, α= 0.05). Table 5 - ANOVA to test the significance of the regression to evaluate the average hydrodynamic diameter (MHD) of firocoxib nanocrystals (FCX-NC), for the following variables: FCX and SOL concentration (% w / w) and agitation time (days) (α=0.05). Source GL SQ (Aj. ) MQ (Aj. ) Test F P-value Model 6 1541.23 256.872 58.40 0.001 Linear 3 1004.74 334.914 76.15 0.001 FCX (% mm) 1 762.45 762.451 173.35 0.001 SUN (% mm) 1 103.68 103.680 23.57 0.001 Time (days) 1 138.61 138.611 31.52 0.001 Square 2 179.28 89.639 20.38 0. 001 FCX (% mm)*FCX mm) (% 1 102.92 102.918 23.40 0.001 SOL (% mm)*SOL mm) (% 1 58.98 58.985 13.41 0.004 Interaction with Factors 2 1 357.21 357.210 81.22 0.001 FCX (% mm)*SOL mm) (% 1 357.21 357.210 81.22 0.001 Error 11 48.38 4.398 Lack of fit 6 36.21 6.034 2.48 0.169 Pure error 5 12.18 2.435 * * Total 17 1589.61 SD= 2.09720 R 2 = 96.96% R2—aj = 95.30% R2- prev= 88.55% Petition 870250006914, dated 01 / 28 / 2025, page 41 / 87 31 / 51 FCX: firocoxib. SOL: Soluplus®. Time: agitation time. GL: degrees of freedom. SS: adjusted sum of squares. MS: adjusted mean square.

[00124] Overall performance can be assessed by the coefficient of determination, or R2, which demonstrates how much the response is explained by the factors. Together, the adjusted R2 (R2-aj) allows comparison of the explanatory power of the regression, including different numbers of terms. The predictive R2 (R2-prev) indicates how well the regression predicts the response for new observations. Therefore, for an adequate statistical model, R2 and R2-aj should not differ drastically (FUKUDA et al., 2018; MONTGOMERY, 2016).

[00125] According to Table 4, the R2 and R2aj values ​​were similar, and the prediction of the adjusted model (R2-prev) was equal to 88.55%. This demonstrates that the regression is significant and adequate, and the mathematical model can be used for the optimization of the composition and preparation process of FCX-NC.

[00126] The uncoded Equation 2 presents the model and coefficients that describe the influence of FCX and SOL concentration (% w / w) and agitation time (days) on the DHM of FCXNC. Surprisingly, the FCX concentration (% w / w) contributes significantly to the increase in particle size (coefficient + 85.9). In contrast, the square of the FCX concentration (% w / w) results in a reduction in particle size (coefficient -4.84).

[00127] Similarly, the concentration of SOL (% w / w) also results in an increase in the DHM of the preparation (coefficient +80.3), while the square of the concentration of this surfactant reduces the particle size of the nanocrystals (coefficient 3.66). The stirring time (days) also contributes to the reduction of the DHM (coefficient -4.162). Additionally, synergy was observed between the concentration of FCX (% w / w) and the concentration Petition 870250006914, dated 01 / 28 / 2025, page 42 / 87 32 / 51 of SOL (% w / w). This synergy resulted in a reduction in particle size (coefficient -9.45). These results are unexpected and not deducible from the knowledge existing to date. DHM = 177.7 + 85.9 FCX + 80.3 SOL - 4.162 T - 4.84 FCX * FCX - 3.66 SOL * SOL - 9.45 FCX* SOL (2) where: FCX = firocoxib (% m / m); SOL = Soluplus® (% m / m); T = stirring time (days)

[00128] With reference to the central points, they presented values ​​equal to 185.6 nm, 186.3 nm, 188.0 nm, 188.5 nm, 188.2 nm, and 189.9 nm (Figure 4). The replicates of the central points provide independent estimates of the experimental error. The difference between the values ​​found was 4.3 nm, revealing the reduced variability of the test. Unexpectedly, the central points are displaced in the upper part of the graph, indicating a quadratic effect of the independent variables.

[00129] Figure 5 shows the main effects of the independent variables on the DHM of FCX-NC. All three variables influenced the DHM values. In the interaction plot (Figure 6), the non-parallel lines show the interaction between FCX ​​and SOL concentration (% w / w) and agitation time (days).

[00130] The surface (Figure 7) and contour (Figure 8) plots reveal the optimal region for response maximization (DHM) as a function of the independent variables. For an FCX ​​concentration of 5% (w / w), it is possible to obtain nanocrystals with a particle size between 180 and 190 nm independent of the SOL concentration under the study conditions (Figure 8). Optimization of the mathematical model for the mean hydrodynamic diameter (MHD)

[00131] The influence of FCX concentration (% w / w) and SOL concentration (% w / w) and agitation time (days) was investigated to promote optimal DHM (target: lower DHM). For the formulation Petition 870250006914, dated 01 / 28 / 2025, page 43 / 87 For optimized formulation 1 (F1), with a predicted wavelength of 185.0 nm, the values ​​were: FCX 5.0% (w / w), SOL 5.0% (w / w) and a stirring time of 4.61 days (Figure 9). For optimized formulation 2 (F2), with a predicted wavelength of 195.0 nm, the values ​​were: FCX 4.09% (w / w), SOL 6.0% (w / w) and a stirring time of 3.68 days (Figure 10).

[00132] To verify the adequacy of the model, FCX-NC were prepared under the conditions described in Figures 9 and 10. Under these conditions, the experimental DHMs were 184.4 ± 2.1 nm (F1) and 194.6 ± 6.9 nm (F2), respectively for optimized formulas 1 and 2 (Table 6). These values ​​are within the predicted range calculated with α = 0.05, demonstrating that the mathematical model is valid for optimizing the DHM of FCX-NC. Table 6 - Average hydrodynamic diameter (DHM), polydispersity index (Pdl) and apparent zeta potential (PZ) of FCX-NC after optimization of the mathematical model containing the variables: FCX concentration (% w / w), SOL concentration (% w / w) and agitation time (days). F theoretical DHM (nm) practical DHM (nm) Pdl PZ (mV) F1 185.0 184.4 ± 2.1 0.190 ± 0.015 -5.05 ± 0.55 F2 195.0 194.6 ± 6.9 0.188 ± 0.021 -4.57 ± 0.28

[00133] Based on the optimization of the FCX-NC preparation process, a prospective study was carried out to obtain these nanocrystals using different agitation speeds (rpm) and concentrations of FCX and SOL (% w / w) distinct from the experimental design presented in Table 4. In this study, the agitation time (4 days), the number of spheres (42% w / w) and the batch size (7 g) were kept fixed. The DHM (nm) and Pdl values ​​for these combinations are presented in Table 7, while Table 8 shows the physical stability of the nanocrystals obtained at 30 °C ± 2 °C and RH 75% ± 5% for 1 month.

[00134] The concentrations of FCX and SOL varied between 1 and 3% w / w in different proportions, while the speed of Petition 870250006914, dated 01 / 28 / 2025, page 44 / 87 34 / 51 agitation was adjusted between 800 and 1200 rpm. Under these conditions, FCX-NC showed DHM between 191.0 and 360.7 nm, and PdI values ​​below 0.3 (Table 7). Regarding physical stability, the obtained nanocrystals maintained DHM < 350 nm and PdI < 0.3 after 1 month of storage. The greatest variation in particle size was observed for preparation F6, with a decrease of 53.5 nm (Table 8). These results indicate that the composition of FCX and SOL is capable of maintaining the physical stability of the nanocrystals at these concentrations used (concentration of 1 to 3% w / m and speed of 800 to 1200 rpm). Table 7 - DHM (nm) and PdI of firocoxib nanocrystals (FCXNC) from different combinations between FCX ​​and SOL concentration (1 to 3% w / w) and stirring speed (800 to 1200 rpm) (n= 3). Formulation FCX ​​(% mm) SOL (% mm) Speed ​​(rpm) DHM (nm) PdI 269.8 0.196 ± 1 3 1 1000 ± 8.6 0.023 230.1 0.174 ± 2 2 2 1000 ± 9.1 0.003 299.2 0.232 ± 3 2 1 800 ± 8.3 0.016 257.0 0.190 ± 4 2 1 1200 ± 11.0 0.010 198.4 0.154 ± 5 ​​3 2 1200 ± 8.2 0.011 302.9 0.165 ± 6 3 2 800 ± 9.0 0.019 203.7 0.172 ± 7 1 3 1000 ± 2.2 0.006 360.7 0.282 ± 8 1 2 800 ± 5.7 0.037 9 2 3 1200 201.5 0.145 ± Petition 870250006914, dated 01 / 28 / 2025, page 45 / 87 35 / 51 ± 3.2 0.020 191, 0 0, 158 ± 1 2 1200 ± 2.2 0.034 Table 8 - Physical stability of firocoxib nanocrystals (FCX-NC) obtained from different combinations of FCX and SOL concentration (1 to 3% w / w) and stirring speed (800 to 1200 rpm), evaluated for 1 month at 30 °C ± 2 °C and RH 75% ± 5% (n= 3). F _ DHM 1 week DHM 2 weeks 1 month DHM (nm) Pdl (nm) Pdl (nm) Pdl 1 294, 9, 4 0 ± 0.204 ± 0.028 246, 11 6, 4 ± 0.163 ± 0.010 292, 1 6, 6 ± 0.210 ± 0.014 2 217, 4, 9 8 ± 0.152 ± 0.002 221, 2, 7 3 ± 0.135 ± 0.027 234, 3 5, 7 ± 0.139 ± 0.014 3 261, 5, 3 6 ± 0.181 ± 0.020 315, 2, 4 6 ± 0.190 ± 0.032 265.8 6.0 ± 0.188 ± 0.015 4 239, 7, 5 5 ± 0.162 ± 0.031 271, 6, 0 7 ± 0.203 ± 0.008 244.6 4.3 ± 0.170 ± 0.031 5 231, 6, 0 4 ± 0.187 ± 0.009 201, 3, 9 6 ± 0.116 ± 0.034 222.4 6.0 ± 0.129 ± 0.036 6 269, 3, 8 3 ± 0.152 ± 0.029 289, 3, 5 2 ± 0.158 ± 0.011 249.4 3.0 ± 0.209 ± 0.016 7 210, 11 7 ,0 ± 0.154 ± 0.026 194, 5, 2 3 ± 0.145 ± 0.026 227.9 9.7 ± 0.117 ± 0.048 8 318, 7, 6 0 ± 0.192 ± 0.023 307, 7, 6 7 ± 0.189 ± 0.015 317.1 10.2 ± 0.116 ± 0.035 9 201.5 ± 0.112 ± 195.5 ± 0.075 ± 200.6 ± 0.087 Petition 870250006914, dated 01 / 28 / 2025, page 46 / 87 36 / 51 8.9 0.011 4.4 0.037 4.6 ± 0.003 0.142 229.5 ± 0.139 ± 195.2 ± 0.118 ± 229.6 ± 10 ± 7.8 0.035 3.9 0.001 5.9 0.029

[00135] In another prospective study, the production of these nanocrystals was evaluated using concentrations of 10% w / w of FCX and SOL (1:1). The process conditions, such as batch size (7 g), number of spheres (42%), stirring time (4 days) and stirring speed (1200 rpm) were maintained. The size of FCX-NC at this concentration was 227.5 ± 2.2 nm, PdI equal to 0.124 ± 0.019, and PZ equal to -7.42 ± 0.67 mV.

[00136] The preliminary physical stability of the resulting preparation was evaluated for 1 month at 30 °C ± 2 °C and RH 75% ± 5% (Table 9). The maximum DHM variation during the evaluated period was 7.3 nm. These results, combined with process optimization, demonstrate the feasibility of obtaining FCX-NC using the following conditions: FCX and SOL concentration between 1 and 10% w / w; drug-to-surfactant ratio of 0.33:1 to 3:1 (or vice versa); agitation speed between 800 and 1200 rpm; agitation time of 3 to 5 days. Table 9 - Physical stability of firocoxib nanocrystals (FCX-NC) at 30 °C ± 2 °C and RH 75% ± 5%, using an FCX ​​and SOL concentration of 10% w / w (n= 3). Stability DHM (nm) Pdl PZ (mV) Day 0 227.5 ± 2.2 0.124 ± 0.019 -7.42 ± 0.67 1 week 222.3 ± 1.7 0.119 ± 0.010 -7.62 ± 0.91 2 weeks 234.8 ± 6.0 0.080 ± 0.020 -6.63 ± 0.56 1 month 234.8 ± 4.0 0.103 ± 0.034 -4.43 ± 0.55 Physicochemical stability of FCX-NC Petition 870250006914, dated 01 / 28 / 2025, page 47 / 87 37 / 51

[00137] The physicochemical stability of FCX-NC containing SOL surfactant and obtained after process optimization by BoxBehnken (F1) was evaluated for 6 months in a climatic chamber at 30 °C ± 2 °C and RH 75% ± 5%. After 6 months, the DHM was 181.7 ± 1.595 nm, PdI 0.199 ± 0.011 and PZ -9.37 ± 0.757 mV (Figure 11). PdI values ​​< 0.3 indicate a narrow particle size distribution. Regarding the pH of the preparation, it varied between 6.36 and 6.90 during the evaluated period (Figure 12). Combined with the reduced variation of the DHM over the 6-month period, the results reveal an important indication of the physicochemical stability of the obtained nanocrystals. Exploratory study of the transposition of the FCX-NC preparation method to the pilot scale.

[00138] The production of FCX-NC on a pilot scale was carried out using a high-energy mill. The scale-up of the preparation was from 7 g to 3000 g. In this method, the drug and surfactant suspension is kept in a dispersion chamber under constant agitation. The material is propelled by a pneumatic pump to the grinding chamber, being agitated there by the rotation of the internal shaft. The material is then propelled back to the dispersion chamber, operating continuously until the end of the process.

[00139] The following composition was used: FCX 3% (w / w) and SOL 3% (w / w) and purified water qsp 3,000 g, and the process conditions are presented in Table 10. Figure 13 shows the monitoring of the particle size of FCX-NC during the process. In the first 30 minutes, a reduction in DHM to < 470 nm was observed. After 2 h, no significant reduction in the particle size of the drug was observed, obtaining 3,000 g of FCX-NC with a final batch DHM of 357.7 ± 7.5 nm, PdI 0.071 ± 0.008. Therefore, 2 h of processing time was established as the appropriate time to obtain FCX-NC in this mill. Table 10 - Comparison between the process conditions of Petition 870250006914, dated 01 / 28 / 2025, page 48 / 87 38 / 51 Preparation of FCX-NC by wet milling on a laboratory and pilot scale. Laboratory Process Conditions (7 g) (3000 g) Pilot Scale Grinding Speed ​​(rpm) 800 2600 Pump Speed ​​(rpm) 85 Zirconium Spheres (mm) 0.1 0.2 Sphere Quantity (% m / m) 42 85 Grinding Time (h) 96 2 DHM (nm) 207.7 357.7

[00140] The nanocrystals obtained on a pilot scale had their physical stability evaluated for 6 months at room temperature (Figure 14). The DHM of the preparation remained < 400 nm during the evaluated period. This fact highlights the adequate steric stability conferred to FCX-NC by the surfactant SOL. This stability is attributed to the molecular interactions between the hydrophilic and hydrophobic chains of this copolymer and the drug, and its high molecular mass, conferring adequate steric stability to the system.

[00141] The success in obtaining FCX-NC on a pilot scale with reduced DHM can be attributed to the high grinding speed and the proportion of zirconia spheres (Table 10). These process parameters allow for increased collision of the drug with the components of the grinding medium, favoring the reduction of particle size.

[00142] Obtaining FCX-NC on a pilot scale with a distinct DHM (357.7 nm) from the formulation obtained on a laboratory scale (207.7 nm) is justified by the differences in energy transfer to the milling medium and size reduction kinetics. Petition 870250006914, dated 01 / 28 / 2025, page 49 / 87 39 / 51 of particle size. In this sense, the reduction of the particle size of nanocrystals on a laboratory scale is achieved by continuous collision between the drug particles, the grinding medium, and the wall of the grinding vessel until the end of the process. In mills employing recirculation, the collision is performed in fractions with each pass of the material through the grinding chamber, which has a capacity of 470 mL.

[00143] Delineating the interactions and process conditions in this mill using a statistical approach presents itself as an alternative to optimize the DHM of FCX-NC on a pilot scale, aiming to achieve a particle size reduction < 300 nm. Relative density of FCX-NC

[00144] To determine the relative density of FCXNC obtained on a laboratory scale, the pycnometer method was used. Table 11 shows the masses of the empty pycnometer, the pycnometer filled with purified water, and the pycnometer filled with FCX-NC. By means of the ratio between the mass of the pycnometer filled with FCX-NC and with purified water, it was possible to calculate the relative density of the preparation, which was 1.01 ± 0.05 g / cm3. Table 11 - Masses of the empty pycnometer, pycnometer filled with purified water, and pycnometer filled with FCX-NC for determining the relative density of the preparations (n=3). Pycnometer (10.123 cm3) Mass (g) Empty 17.13 ± 0.07 With purified water 27.51 ± 0.01 With FCX-NC 27.81 ± 0.02 Firocoxib content in FCX-NC

[00145] The FCX content in the nanocrystals was determined by UV-Vis spectrophotometry at a wavelength of 290 nm. Table 12 presents the FCX-NC (F1) content obtained by wet milling on a laboratory scale, this value being close to Petition 870250006914, dated 01 / 28 / 2025, page 50 / 87 40 / 51 100% of the stated content (50.5 mg / mL). Table 12 - Firocoxib content in FCX-NC obtained by wet milling on a laboratory scale (n= 3). Concentration Content Sample (mg / mL) (%) FCX-NC 50.03 ± 6.93 99.07 (F1) X-ray diffraction (XRD)

[00146] Figure 15 shows the diffraction patterns of firocoxib raw material (FCX), Soluplus® surfactant (SOL), their physical mixtures (MF) and firocoxib nanocrystals (FCX-NC). The drug shows intense and narrow peaks at 9.877°, 19.746° and 21.524° (2θ), with intensities 1261, 1697 and 1117, respectively, relative to the polymorphic form B (CALAIS; CHASSAGNEUX; BONARD, 2004).

[00147] Polymorphic form B has low solubility in water and is the form frequently obtained during FCX synthesis (EMA, 2024). The surfactant SOL is amorphous, evidenced by the absence of peaks in the diffractogram. The amorphous nature of this copolymer is well established in the literature (NANDI et al. 2021; JIA et al. 2022; DIOGO; RAMOS 2022; MACEDO et al. 2022).

[00148] In the physical mixture (PM) of FCX and SOL, the FCX peaks were preserved. With reference to FCX-NC, the characteristic drug peaks at 9.877°, 19.746°, and 21.524° (2θ) were maintained, but with reduced intensities at 337, 827, and 922, respectively, indicating partial amorphization of the drug in FCX-NC. This partial amorphization may be related to the milling process, which can induce cleavage of the intrinsic crystalline network of the drug in weakened areas (YANG et al., 2014). Additionally, the surfactant SOL has a solubilizing nature for compounds with low water solubility, which suggests the dissolution of FCX in this copolymer, contributing to Petition 870250006914, dated 01 / 28 / 2025, page 51 / 87 41 / 51 the reduction in drug crystallinity in FCX-NC (LIU et al., 2020). Differential scanning calorimetry (DSC) and thermogravimetry (TG)

[00149] Figure 16 presents the DSC curves of firocoxib raw material (FCX), Soluplus® copolymer (SOL), their physical mixtures (MF), and firocoxib nanocrystals (FCX-NC), while Table 13 reveals the thermal properties of these samples. In the evaluated temperature range, FCX has a single thermal event related to drug melting, with a peak at 118.6 °C and enthalpy ΔH = 92.90 J / g. This data corroborates the polymorphic form B of this drug, which presents a characteristic endothermic peak at approximately 120 °C related to its melting (CALAIS; CHASSAGNEUX; BONARD, 2004; EMA, 2024). In the TG curve (Figure 17), the thermal decomposition process occurs in a single step at 289 °C (Δm = 98.5%). Table 13 - Thermal properties of firocoxib raw material (FCX), Soluplus® surfactant (SOL), their binary physical mixtures (MF) and firocoxib nanocrystals (FCX-NC). DSC TG / DTG Typical ΔH Event Διη Event Sample ΔT (°C) (°C) (J / g) thermal (%) thermal Endothermic FCX 118.6 92.90 (melting point) 187-294 98.5 Decomposition 255-340 20 Decomposition Transition 1 SOL 63.2 6.25 glassy 340-458 73 Decomposition 2 Endothermic 194-301 76 Decomposition (melting point) FCX and SOL MF 114.1 41.67 301-443 21 Decomposition FCX) SOL FCX-NC 101.9 24.43 Endothermic 236-327 52 Decomposition Petition 870250006914, dated 01 / 28 / 2025, page 52 / 87 42 / 51 (FCX point and SOL decomposition fusion) 327-469 32 FCX) SUN DSC: differential scanning calorimetry; TG / DTG: Thermogravimetry; Tpico: peak temperature; AH: enthalpy change; AT: temperature change; Am: mass change.

[00150] In the DSC curve for the SOL copolymer, the glass transition temperature (Tg) of the species is observed to be 63.2 °C (Table 13) (DIOGO; RAMOS, 2022). The Tg of this copolymer is frequently observed at 70 °C, and this value may vary depending on the humidity and aging of the material (SINGH et al., 2016).

[00151] No endothermic melting event was observed for the SOL copolymer, indicating the amorphous nature of the material (Figure 16). In the TG curve, the copolymer shows two decomposition steps, whose values ​​are consistent with the literature (Table 13) (MACEDO et al., 2022).

[00152] In the DSC curve for the MF (FCX and SOL), the drug melting occurs at 114.1 °C, with an enthalpy of AH = 41.67 J / g (Table 13). In this assay, the endothermic glass transition (Tg) event of the SOL copolymer was not observed (Figure 16). In the TG curve, the sample showed two decomposition steps, the first related to the overlap of the decompositions of FCX and SOL, and the second attributed to the decomposition of the SOL copolymer (Figure 17). The thermal analyses performed for the MF reveal that the drug and copolymer are compatible.

[00153] The DSC curve for FCX-NC shows that the characteristic endothermic melting peak of the drug occurs at 101.91 °C, with AH = 24.43 J / g (Table 13). As with MF, the TG curve for the nanocrystals shows an initial mass loss of 52% related to the overlap of the drug and copolymer decomposition steps (Table 13). Subsequently, a second mass loss of 32% is observed, attributed to the second decomposition step of the SOL copolymer (Figure 17). Petition 870250006914, dated 01 / 28 / 2025, page 53 / 87 43 / 51

[00154] The advancement of the endothermic melting peak, along with the drastic reduction in fusion enthalpy, from ΔH = 92.90 J / g (FCX) to ΔH = 24.43 J / g (FCX-NC), are related to the partial amorphization of the nanocrystal, as mentioned in the XRD results. This phenomenon can be attributed to the milling process and the partial solubilization of the drug in the surfactant (LAKSHMAN et al., 2020). Transmission electron microscopy (TEM)

[00155] Figures 18 and 19 show the morphology of FCX and FCX-NC, respectively. The micronized drug has rod-shaped and flake-shaped particles (Figure 18a). Figure 18b shows the characteristic of firocoxib to form aggregates. This property represents a challenge in the development of nanostructured systems containing this drug.

[00156] Regarding nanocrystals, these exhibit irregular morphology, with rounded, rectangular, and flake-shaped particles (Figure 19). This irregularity is attributed to the milling process, in which the reduction of the drug particle size due to collision, shear forces, and friction in various directions can affect the shape of the particles obtained (BHUYAN et al., 2020). Furthermore, this irregular morphology may be related to the partial amorphization of the drug within the nanocrystals, as mentioned previously. In vitro dissolution

[00157] The in vitro dissolution of FCX, MF, and FCX-NC is shown in Figure 20. The dissolution of FCX and MF in sodium phosphate buffer (pH 6.8) under sink conditions was 59.4% and 41% in the first 5 min of testing, respectively. During this same period, the nanocrystals released 89.3% of their content, maintaining this high level until the end of the experiment. The release of FCX-NC represents a 1.46x increase compared to the conventional drug, and 1.37x greater than MF. Petition 870250006914, dated 01 / 28 / 2025, page 54 / 87 44 / 51

[00158] According to the results presented, FCX-NC has the potential to increase oral absorption and efficacy of firocoxib. The rapid release of the drug from the nanocrystals is attributed to the Noyes-Whitney equation, in which reducing the particle size of the drug to the nanometer scale promotes an increase in its surface area to volume ratio (NOYES; WHITNEY, 1897). Considering that FCX is a non-steroidal anti-inflammatory drug used in the treatment of pain and inflammation, increasing the dissolution rate of FCX-NC in aqueous media is fundamental to promoting a rapid onset of action and relief of these symptoms. EXAMPLE III — SAFETY AND PHARMACOKINETIC EVALUATIONS OF FIROCOXIB NANOCRYSTALS Evaluation of the toxicity of FCX-NC in Galleria mellonella L.

[00159] Toxicity assays were performed on an invertebrate model using Galleria mellonella L. larvae. According to Figure 21a, no toxicity was observed for FCX-NC at the different doses administered to Galleria mellonella L. larvae when compared to the PBS control group (p= 0.942). Furthermore, FCX and SOL evaluated at a dose of 5 mg / kg also showed no toxicity. With reference to the larval health index, there were no significant variations over the 5 days of testing (Figure 21b). Thus, FCX-NC revealed little or no preliminary toxicity in this invertebrate model considering the dosage (5 mg / kg), double that dose (10 mg / kg), and five times the dose (50 mg / kg). These results indicate potential therapeutic safety for patients. Pharmacokinetic analysis in beagle dogs

[00160] Table 14 presents the pharmacokinetic parameters obtained. Figure 22 shows the mean plasma concentration of FCX-NC and Previcox® at the evaluated times. Considering the dose of 5 mg / kg, the highest mean plasma concentration for the Previcox® product was 0.580 ± 0.265 pg / mL, reached after 1 hour of administration, followed by a decrease to Petition 870250006914, dated 01 / 28 / 2025, page 55 / 87 45 / 51 0.098 ± 0.081 pg / mL after 24h (Figure 22).

[00161] Regarding FCX-NC, its highest mean plasma concentration was observed after 30 min of administration (1.105 ± 0.219 pg / mL), with a decay similar to Previcox® after 24h (0.113 ± 0.089 pg / mL) (Figure 22). In this sense, it is noted that it took half the time for the nanocrystals to reach almost double the plasma concentration of the commercial product.

[00162] Compared to Previcox®, FCX-NC showed a 1.97x higher Cmax, a larger total AUC (1.4x), and lower Tmax and t1 / 2 (both 1.3x lower), although only the Cmax values ​​were considered statistically different (p-value 0.002, α= 0.05). These surprising results indicate a significantly faster action of the firocoxib nanocrystal compared to the conventional product. Table 14 - Pharmacokinetic parameters of FCX-NC and commercial firocoxib (Previcox 57 mg), after administration in a single oral dose (5 mg / kg) (mean ± SD, n= 6). Previcox® FCX-NC Parameters Cmax (pg / mL) 1.214 ± 0.616 ± 0.177* 0.312 Tmax (h) 0.92 ± 0.66 1.17 ± 0.52 T1 / 2 (h) 8.91 ± 3.73 11.46 ± 6.11 AUC 0-t (pg / mL*h) 8.252 ± 5.831 ± 2.482 3.609 AUC 0-inf (pg / mL*h) 10.112 ± 7.531 ± 4.356 6.082 *p < 0.05 vs Previcox®.

[00163] The high mean plasma concentration for FCX-NC is consistent with the in vitro dissolution results, which showed rapid drug release (around 90%) in the first 5 min of the assay. This demonstrates that firocoxib nanocrystals have a better oral absorption profile compared to the unmodified drug. It is worth mentioning that, the Petition 870250006914, dated 01 / 28 / 2025, pp. 56 / 87 46 / 51 Despite the surprising increase in Cmax, no adverse effects were observed in any of the animals included in the study. BIBLIOGRAPHIC REFERENCES

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Claims

1 / 2 CLAIMS 1. Firocoxib nanocrystal characterized in that it comprises firocoxib and at least one pharmaceutically acceptable surfactant.

2. Nanocrystal, according to claim 1, characterized in that the pharmaceutically acceptable surfactant is selected from the group consisting of poly(ethylene oxide) 80% + poly(propylene oxide) 1800, poly(ethylene oxide) 70% + poly(propylene oxide) 4000, hydroxypropyl methylcellulose (HPMC), vinylpyrrolidone-vinyl acetate copolymer and polyvinyl caprolactam-polyvinyl polyethylene glycol acetate copolymer.

3. Nanocrystal, according to claim 1 or 2, characterized in that the surfactant is a copolymer of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol.

4. Nanocrystal, according to claim 1, characterized in that the ratio between firocoxib and surfactant is in the range between 0.33:1 and 3:

1.

5. Nanocrystal, according to claim 4, characterized in that the average hydrodynamic diameter is less than 400 nm.

6. Nanocrystal, according to any one of claims 1 to 4, characterized in that the firocoxib content in the nanocrystal is greater than 95%.

7. Wet milling method with beads for preparing firocoxib nanocrystal, as defined in claims 1 to 6, characterized in that it comprises the steps of: a) preparing a firocoxib suspension (w / w) in an aqueous solution of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer; b) adding the firocoxib suspension to a milling chamber containing zirconium oxide beads; c) completing the volume with purified water; d) subjecting the mixture to agitation, operating from 800 RPM to 3000 RPM.

8. Method according to claim 7, characterized in that the concentrations of firocoxib and polyvinyl caprolactam-polyvinyl polyethylene glycol acetate copolymer (SOL) in the aqueous suspension can be from 1% to 10% (w / w).

9. Pharmaceutical composition characterized by comprising firocoxib nanocrystal, as defined in any one of claims 1 to 6, and one or more pharmaceutically acceptable excipients.

10. Use of firocoxib nanocrystal, as defined in any one of claims 1 to 6, or of the composition, as defined in claim 9, characterized in that it is for the preparation of a medicament to treat inflammatory and / or pain disorders in animals.

11. Use, according to claim 10, characterized in that the inflammatory and / or pain disorder in the animal is canine osteoarthritis. Petition 870250006914, dated 01 / 28 / 2025, pp. 64 / 87

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