PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, INJECTABLE FORMULATION OBTAINED BY THE PROCESS AND USE THEREOF, PROCESS FOR PREPARING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, GEL OBTAINED BY THE PROCESS AND USE THEREOF IN THE MANUFACTURE OF AN INJECTABLE FORMULATION

Description

1 / 31 PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND ACID Cationic Hyaluronic Acid, Injectable Formulation Obtained by the Process and Use Thereof, Process for Preparing a Polyelectrolyte Complex Gel Between Hyaluronic Acid and Cationic Hyaluronic Acid, Gel Obtained by the Process and Use Thereof in the Manufacture of an Injectable Formulation. Field of invention

[0001] The present invention relates to a process for preparing an injectable formulation comprising simultaneously the cationization reactions of hyaluronic acid and the formation of a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid. The injectable formulation obtained by the process of the present invention is suitable for application as an intradermal or intraarticular filler. The present invention is in the fields of Chemistry and Pharmaceutical Technology. Fundamentals of the invention

[0002] Hyaluronic acid is an anionic polysaccharide, consisting of N-acetyl-D-glucosamine units linked to D-glucuronic acid, capable of forming intra- and intermolecular hydrogen bonds when in solution. It is present in synovial fluid, vitreous humor, and collagenous connective tissue of various organisms. Thanks to its viscoelastic properties, biocompatibility, tolerance, and lack of toxicity, injectable hyaluronic acid is widely used in medicine for joints and in aesthetic medicine as a wrinkle filler or volume enhancer. Commercially, there are several products with a cross-linked hyaluronic acid composition that ensures greater resistance, especially with regard to degradation and heat.

[0003] Due to its anionic nature resulting from the carboxylic acid group, hyaluronic acid has poor compatibility with skin and hair, which are negatively charged. One strategy is to make it into polymers or polysaccharides. Petition 870240109140, dated 12 / 20 / 2024, page 11 / 53 2 / 31 Anionic compounds like hyaluronic acid adhere better to the skin or hair by using the cationization process.

[0004] Cationic hyaluronic acid initially emerged in the cosmetic industry in the "hair care" sector in order to increase the adhesion of this polysaccharide to the hair. Currently, cationic hyaluronic acid has also been used in the "skin care" sector due to its deep hydration and film-forming properties.

[0005] In the global market, cationic hyaluronic acid is available for commercialization as a cosmetic-grade ingredient: Hyaloveil-P® (Kewpie company (JP)); HA Plus™ Hyaluronic Acid (Shandong Focus Freda Biotech company (CN)); cationHA™ (Bloomage Biotechnology company (CN)); and Cationic Hyaluronic Acid (Amik company (IT)). There is no injectable-grade cationic hyaluronic acid ingredient available on the market.

[0006] Based on the work of Kamada and colleagues, Wang and colleagues and Liu and colleagues have verified the possibility of using cationic hyaluronic acid in the treatment of joint diseases and ophthalmological diseases (Kamada, Y. et al.; Analysis of the effect of cationic hyaluronic acid on articular cartilage. Abstracts / Osteoarthritis and Cartilage, 29:S10eS432, 2021), (Wang, YN et al.; Cyclosporine micelles modified by hydrochromic acid used for ocular drug delivery, Indian Journal of Pharmaceutical Sciences, 86(1): 64-74, 2024), (Liu, Y. et al.; Spalastica coated with channeled hyaluronic acid for ocular delivery of cyclosporine A: Prolonged ocular retention, enhanced corneal permeation and improved tear production, International Journal of Pharmaceutics, 565: 133-142, 2019).

[0007] The cationization process of polymers is carried out by adding cation groups to anionic sites, using cationizing agents. In a particular case, the cationizing agents are quaternary ammonium salts, such as 2,3-epoxypropyl trialkyl ammonium halide (also known as glycidyl trialkyl ammonium halide or GTA) or 3-halogen-2-hydroxypropyl trialkyl ammonium halide. In fact, in Petition 870240109140, dated 12 / 20 / 2024, page 12 / 53 3 / 31 In basic medium, cationizing agents such as 2,3-epoxypropyltrimethylammonium chloride are formed in situ from 3-chloro-2-hydroxypropyltrimethylammonium chloride (see Scheme 1).

[0008] In the polysaccharide hyaluronic acid, the cationization reaction can occur in its four functional groups: carboxylic acid, hydroxyl (including primary alcohols and secondary alcohols), amide (acetamide), and the reduced terminal of the polymer. Document EP2166022 (QP Corp (JP)) comments that in organic media the favored cationization reaction of hyaluronic acid is the esterification reaction, i.e., nucleophilic substitution occurs in the carboxylic group.

[0009] On the other hand, Prado and Matulewicz comment that the etherification reaction in the hydroxyl group, predominantly in the primary hydroxyl group, is favored, since the opening of the epoxide in basic medium occurs through an SN2 mechanism, in which the preferential attack occurs on the least substituted carbon (Prado, HJ; Matulewicz, MC; Cationization of polysaccharides: A path to greener derivatives with many industrial applications, European Polymer Journal, 52: 53-75, 2014). In aqueous basic medium, the cationization reaction begins first with the conversion of the quaternary ammonium salt 3-chloro-2-hydroxypropyltrimethylammonium chloride into the epoxide 2,3-epoxypropyltrimethylammonium chloride.Next, the alkoxide formed from the primary hydroxyl group of hyaluronic acid in a basic medium attacks the epoxide of the cationizing agent, forming the cationic polysaccharide hydroxypropyl trimethylammonium hyaluronate chloride, in an SN2 nucleophilic substitution reaction (see Scheme 1). Scheme 1. Epoxide formation in basic medium and nucleophilic attack of the alkoxide on the epoxide (Prado and Matulewicz, 2014). Petition 870240109140, dated 12 / 20 / 2024, page 13 / 53 4 / 31 The cationization of a quaternary ammonium salt as a cationizing agent involves the optimization of several variables. According to Prado and Matulewicz (Prado and Matulewicz, 2014), the presence of a base is fundamental for the cationization reaction. The authors comment that one molar equivalent of sodium hydroxide is necessary to generate the epoxide from 3-chloro-2-hydroxypropyltrimethylammonium chloride. An additional amount of sodium hydroxide is required for the formation of an alkoxide at the primary hydroxyl groups of hyaluronic acid (see Scheme 1), responsible for the nucleophilic attack on the epoxide group, forming cationic hyaluronic acid. Thus, an increase in the concentration of sodium hydroxide increases the degree of cationization. However, an excess of sodium hydroxide can favor the degradation of the polysaccharide by alkaline hydrolysis, as well as the degradation of the epoxide into a diol, decreasing the degree of cationization.In fact, the degradation of epoxide into diol depends on the temperature, the pH of the medium, and the reaction time. At 50 °C, 50% of the epoxide remains in solution after 24 hours.

[0011] Antioxidants can be used to prevent the degradation of hyaluronic acid by reactive oxygen species and hyaluronidase and / or as a thermal protector, especially during thermal sterilization of intradermal and / or intra-articular compositions (W02009071697, WO2019125166, WO2012167079 and W02009024670). Petition 870240109140, dated 12 / 20 / 2024, page 14 / 53 5 / 31

[0012] An increase in the concentration of the cationizing agent provides an increase in the degree of cationization, due to the increased availability of reactant molecules in the vicinity of the polysaccharide. However, this contribution is limited to the amount of sodium hydroxide.

[0013] The effect of temperature on the degree of cationization is controversial. An increase in reaction temperature may initially increase the degree of cationization due to the beneficial effects of temperature on the compatibility of reactants and their kinetic energies. On the other hand, a further increase in temperature may favor the degradation of the polysaccharide and epoxide, reducing the degree of cationization. An increase in reaction time also shows a similar trend, initially increasing the degree of cationization, while longer periods of time may not produce improvements and may even reduce the degree of cationization due to degradation.

[0014] Therefore, to control the degree of cationization, it is important to optimize the molar ratio between the cationizing agent and the monosaccharide unit, the molar ratio between sodium hydroxide and the cationizing agent, the volume of the medium, the temperature, and the reaction time.

[0015] For example, document EP2166022 (QP Corp (JP)) discloses a cationic hyaluronic acid or its salt, with a degree of cationization of 0.15-0.6, and its preparation method comprising a cationizing agent in a basic medium at 30-70 °C, wherein the cationizing agent is a 2,3-epoxypropyltrialkylammonium halide or a 3-halogen-2-hydroxypropyltrialkylammonium halide. Examples 1 and 2 describe the preparation of cationic hyaluronic acid from hyaluronic acid with a molar mass of 2 MDa, in a medium of 65-80% ethanol in water, with reaction times of 0.1-9 h. In the organic phase, the cationic hyaluronic acid precipitates. For its purification, a sodium chloride solution was used to solubilize the precipitate, followed by the addition of ethanol to precipitate the cationic hyaluronic acid and successive washings of the precipitate with 80% ethanol. The cationic hyaluronic acid was obtained after vacuum drying for 5 hours. Petition 870240109140, dated 12 / 20 / 2024, page 15 / 53 6 / 31 60°C as a white powder.

[0016] In turn, document WO2023285663 (Givaudan (CH)) discloses hydroxypropyl trialkyl ammonium hyaluronate or its salt with a degree of cationization greater than 1.4, its method of preparation, a cosmetic composition and use for hydration and / or UV protection and / or hair repair. In their descriptive report, the inventors report that surprisingly, it was found that a relatively low average molar mass, between 10k-200 kDa, provides a particularly effective cosmetic active, for example, exhibiting better adhesion to hair and penetrating deeper into the skin. Furthermore, a relatively low average molar mass facilitates the synthesis of hydroxypropyl trialkyl ammonium hyaluronate and / or its salt. It was found that hyaluronic acid with a lower average molar mass reacts more quickly and requires fewer equivalents of reagents to obtain the desired degree of cationization.Without being bound by theory, it is believed that the reactive sites of hyaluronic acid with higher average molar mass are less accessible to reactant molecules, both due to the lower flexibility of the longer polymer chains and the increased steric hindrance. Example 1 demonstrates the preparation of hydroxypropyltrimonium hyaluronate using 2,3-epoxypropyltrimonium chloride (also known as 2,3-epoxypropyltrimethylammonium chloride) and hyaluronic acid with a molar mass of 41.5 kDa, at room temperature, for 19 h, in a basic medium with sodium hydroxide. Purification was performed by ultrafiltration, and the cationic hyaluronic acid was obtained via lyophilization, with a degree of cationization of 2.18. Example 2 demonstrates the preparation of hydroxypropyltrimonium hyaluronate using 2-chloro-3-hydroxypropyltrimonium chloride and hyaluronic acid with a molar mass of 37.8 kDa, at room temperature for 21 h in a basic medium with sodium hydroxide.The purification was performed by dialysis and the cationic hyaluronic acid was obtained via lyophilization, with a degree of cationization of 1.47.

[0017] Document EP1961772 (Shiseido (JP)) discloses a cationic hyaluronic acid, in which at least some of the hydrogens of the hydroxyl group are Petition 870240109140, dated 12 / 20 / 2024, page 16 / 53 7 / 31 replaced by a group with a quaternary ammonium cation, where the degree of cationization is greater than 0.1. It also reveals a poly-ionic complex formed between cationic hyaluronic acid and an anionic biocompatible material, which is hyaluronic acid. Example 1 describes the cationization reaction between hyaluronic acid and the cationizing agent 2,3-epoxypropyltrimethylammonium chloride, in aqueous medium with sodium hydroxide, for 3 days at room temperature. Then, the cationic hyaluronic acid was precipitated with methanol, washed with acetone and dried, obtaining cationic hyaluronic acid as a white powder with a degree of cationization of 1.4. After purification, a solution of cationic hyaluronic acid is combined with a solution of hyaluronic acid, in equivalent quantities, resulting in the precipitation of a poly-ionic complex due to the electrostatic interaction between the polysaccharides.

[0018] Polyelectrolyte complexes, also known as polyionic complexes or polysalts, are precipitates that form spontaneously when two polyelectrolytes of opposite charges come into contact in aqueous solution.

[0019] According to Buriuli and Verma, polyelectrolyte complexes exhibit unique physical and chemical properties due to considerably stronger electrostatic interactions compared to most other secondary bonding interactions (Buriuli, M.; Verma, D.; Polyelectrolyte Complexes (PECs) for Biomedical Applications. In: Tripathi, A., Melo, J. (eds) Advances in Biomaterials for Biomedical Applications. Advanced Structured Materials, vol 66. Springer, Singapore, 2017). The main driving force behind the formation of polyelectrolyte complexes is the increase in entropy due to the release of low molar mass counterions bound to polysaccharides of opposite charges. Kulkarni and colleagues mention that the interactions established in the formation of polyelectrolyte complexes include Coulomb interactions, hydrogen bonding, hydrophobic interactions, van der Waals interactions, and dipole-dipole interactions between the polyelectrolytes (Kulkarni, AD et al.).; Polyelectrolyte complexes: mechanisms, critical experimental aspects, and applications, Artificial cells, nanomedicine, and biotechnology, 44(7): 1615-1625, 2016). Petition 870240109140, dated 12 / 20 / 2024, page 17 / 53 8 / 31

[0020] Depending on the compatibility between the reacting polyelectrolytes, the electrostatic interaction between the anionic and cationic groups is stronger than most secondary interactions. Therefore, the formation of polyelectrolyte complexes avoids the use of chemical crosslinking agents, which are notably toxic, thus reducing the toxicity and other harmful effects that can be caused by these agents.

[0021] According to Gucht and colleagues, when aqueous solutions of polycations and polyanions are mixed, a water-insoluble polyelectrolyte complex is formed by complex coacervation (Gucht, J. et al.; Polyelectrolyte complexes: bulk phases and colloidal systems, J. Colloid Interface Sci., 361: 407-422, 2011). Macro-ions in aqueous solution are surrounded by an electrical double layer: a zone with an increased concentration of counterions and a reduced concentration of coions. The double layer has lower energy (the average distance between positive and negative charges is smaller than between positive or negative charges), but it also has lower entropy (small ions have less translational freedom). When two macro-ions with opposite charges form a complex, the double layers are destroyed to some extent and the counterions are released in the form of an ordinary saline solution. This implies changes in both the energy (enthalpy) and entropy of the system.Both contributions vary with the salt concentration (ionic strength).

[0022] Le and Cerf comment that the parameters influencing the characteristics of the polyelectrolyte complex include material parameters (average molar mass and charge density of polyelectrolytes), formulation parameters (total polyelectrolyte concentration, charge ratio, pH, ionic strength, and adjuvant ions), and technical parameters (addition mode, addition order, stirring settings, and temperature) (Le, HV; Cerf, DL; Colloidal Polyelectrolyte Complexes from Hyaluronic Acid: Preparation and Biomedical Applications, Small, 18, 2204283, 2022). All these parameters act interdependently, either synergistically or contradictorily, to govern the properties of the polyelectrolyte complex. Petition 870240109140, dated 12 / 20 / 2024, page 18 / 53 9 / 31

[0023] In general, an increase in the molar mass of polyelectrolytes can promote electrostatic complexation and thus improve the stability of polyelectrolyte complexes. This is attributed to the fact that when a polyelectrolyte with a higher molar mass is transferred from a dilute medium to a concentrated medium (in this case, in the formation of a polyelectrolyte complex), there is less entropy loss. On the other hand, a polyelectrolyte with a low molar mass may be unable to form stable polyelectrolyte complexes. If the molar mass of the polyelectrolyte is too high (i.e., above 1 MDa), precipitation or macrogelation occurs at the macroscopic level of the polyelectrolyte complex.

[0024] When the charge densities of the polyelectrolytes in polyelectrolyte complexes increase and / or become comparable to each other, their electrostatic attraction should be stronger and subsequently should increase phase separation, favoring precipitation. A change in pH can lead to an increase in the ionization rate and therefore to a higher charge density, as well as an increase in the stiffness of the polymer chains, favoring the precipitation of the polyelectrolyte complex.

[0025] An increase in the concentration of polyelectrolytes favors the formation of polyelectrolyte complexes, due to the formation of larger particles and a higher particle yield, or macroaggregation, thanks to the involvement of more polymer chains. Macroaggregation is also favored if the molar mass of the polyelectrolytes is relatively high or if the concentration of polyelectrolytes is extremely high.

[0026] An increase in ionic strength reduces the absolute value of the zeta potential of the particles in polyelectrolyte complexes, favoring a higher yield due to particle aggregation or even macroscopic precipitation, given the lower interparticle electrostatic repulsion (weaker repulsive force). However, an extremely high ionic strength can prevent the formation of polyelectrolyte complexes or cause their... Petition 870240109140, dated 12 / 20 / 2024, page 19 / 53 10 / 31 disintegration, because the electrostatic attraction between polyelectrolytes with opposite charges is reduced (Le and Cerf, 2022).

[0027] According to Kulkarni and colleagues (Kulkarni et al., 2016), polyelectrolyte complexes may have applications in different technological fields because they are biodegradable, biocompatible, and non-toxic. These authors also mention the limitation of large-scale commercial production of polyelectrolyte complexes and detected few patent documents related to the subject. In fact, no processes and formulations comprising polyelectrolyte complexes between hyaluronic acid and cationic hyaluronic acid of pharmaceutical grade for injectable use were identified in the state of the art. On the other hand, there are several commercial injectable products related to cross-linked hyaluronic acid for intradermal or intra-articular purposes, with extensive patent and non-patent literature.As previously mentioned, thanks to their rheological properties provided by covalent bonds, cross-linked hyaluronic acid compositions are more resistant than non-cross-linked hyaluronic acid compositions, especially with regard to thermal degradation during steam sterilization. A major challenge in the case of injectable formulations comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid may be stability against thermal degradation during steam sterilization, since their viscoelastic properties result from electrostatic interactions.Thus, there is a need in the state of the art for processes for preparing formulations comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, applicable on an industrial scale, where the processes allow obtaining pharmaceutical-grade formulations for injectable use in order to enable their intradermal or intra-articular application. Therefore, the present invention aims to provide a process for preparing formulations comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid that surpasses the needs of the state of the art. Surprisingly, the process of... Petition 870240109140, dated 12 / 20 / 2024, page 20 / 53 11 / 31 The present invention comprises the concomitant reactions of cationization of hyaluronic acid or its salt and formation of a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt; the formulation obtained by the process can be terminally sterilized and exhibits properties suitable for the intended application.

[0028] The aspects and embodiments of the present invention will become clear from the detailed description that follows. Brief description of the figures

[0029] Figure 1 shows: Frequency sweep as a function of the G', G (Pa) modules of injectable polyelectrolyte complex gel formulations between hyaluronic acid and cationic hyaluronic acid prepared: in the absence of heat protectant and without sterilization (Test A, orange); in the absence of heat protectant and after sterilization (Test B, blue); with 0.7% mannitol and without sterilization (Test C, green); and with 0.7% mannitol and after sterilization (Test D, gray).

[0030] Figure 2 shows: Frequency sweep as a function of the G', G (Pa) modules of injectable polyelectrolyte complex gel formulations between hyaluronic acid and cationic hyaluronic acid prepared: with 1.2% mannitol and without sterilization (Test E, green); and with 1.2% mannitol and after sterilization (Test F, gray).

[0031] Figure 3 shows: Frequency sweep as a function of the G', G (Pa) modules of the injectable formulation of a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, prepared with 1.2% sorbitol and after sterilization (Assay G).

[0032] Figure 4 shows: Frequency sweep as a function of the G', G (Pa) modules of injectable formulations prepared with the following proportions between polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, and hyaluronic acid with a molar mass of 1.4 MDa, with 1.2% mannitol, after sterilization: 1:0 Petition 870240109140, dated 12 / 20 / 2024, page 21 / 53 12 / 31 (Test H, blue); 1:1 (Test I, gray); 1:2 (Test J, green); and 2:1 (Test K, orange).

[0033] Figure 5 shows: Evaluation of degradation kinetics against reactive oxygen species performed on Lot 1 (light blue), Lot 2 (green), Lot 3 (blue) and commercial comparator Juvéderm® Voluma (black). Summary of the invention

[0034] The present invention describes a process for preparing an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, mannitol, and optionally an anesthetic and / or hyaluronic acid with a molar mass of 1-6 MDa. The process comprises the concomitant reactions of hyaluronic acid cationization and the formation of a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid. The injectable formulation obtained by the process of the present invention exhibits rheological properties comparable to covalently cross-linked hyaluronic acid injectable formulations and is capable of terminal sterilization, characteristics that are important for its application as an intradermal or intra-articular filler.

[0035] The present invention discloses as its first object a process for preparing an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, comprising the steps of: (a) homogenizing hyaluronic acid or its salt, an alkali metal halide salt, an antioxidant, in an aqueous basic medium, for 1-3 h, at 20-30 °C; (b) adding a cationizing agent and homogenizing for 1-3 h, at 20-30 °C; (c) keeping the reaction mixture at rest for 1-24 h, at 40-60 °C, in which the concomitant reactions of cationization of hyaluronic acid or its salt, and formation of a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt occur; (d) purify the gel via dialysis with buffered saline solution (PBS) for 48-72 h at 2030 °C, with 4-10 washouts, dry the gel under vacuum and adjust the content of Petition 870240109140, dated 12 / 20 / 2024, page 22 / 53 13 / 31 total hyaluronic acid to 15-30 mg / mL with buffered saline solution (PBS); (e) extrude the gel obtained in step (d) add mannitol and optionally an anesthetic and / or hyaluronic acid or its salt, adjust the pH to 6.5-7.8, homogenize the formulation for 2-24 h at 20-30 °C, fill into a pre-filled syringe and sterilize under steam at 100-124 °C for 1-5 minutes of sterilization.

[0036] The present invention discloses as a second object an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt, mannitol, optionally lidocaine hydrochloride anesthetic, optionally hyaluronic acid or its salt with an average molar mass of 1-6 MDa and a pharmaceutically acceptable carrier, which is buffered saline solution (PBS), obtained according to the process disclosed in the present invention.

[0037] The present invention discloses as a third object the use of an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, obtained according to the process disclosed in the present invention, for application as an intradermal or intra-articular filler.

[0038] The present invention discloses as a fourth object a process for preparing a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, comprising the steps of: (a) homogenizing hyaluronic acid or its salt, an alkali metal halide salt, an antioxidant, in an aqueous basic medium, for 1-3 h, at 20-30 °C; (b) adding a cationizing agent and homogenizing for 1-3 h, at 20-30 °C; (c) keeping the reaction mixture at rest for 124 h, at 40-60 °C, in which the concomitant reactions of cationization of hyaluronic acid or its salt, and formation of a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt occur; (d) purify the gel via dialysis with buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 washout changes, dry the gel under vacuum and adjust the total hyaluronic acid content to 15-30 mg / mL with buffered saline (PBS). Petition 870240109140, dated 12 / 20 / 2024, page 23 / 53 14 / 31

[0039] The present invention discloses as a fifth object a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt, obtained according to the process disclosed in the present invention.

[0040] The present invention discloses as its sixth object the use of a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, obtained according to the process disclosed in the present invention, in the manufacture of an injectable formulation for application as an intradermal or intra-articular filler. Detailed description of the invention

[0041] The present invention relates to a process that, unexpectedly and surprisingly, simultaneously prepares cationic hyaluronic acid and a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, in a basic aqueous medium, without the presence of organic solvents. Furthermore, the present invention relates to an injectable formulation comprising said polyelectrolyte complex gel, mannitol, and optionally an anesthetic and / or hyaluronic acid with a molar mass of 1-6 MDa, which exhibits rheological properties comparable to injectable formulations of covalently cross-linked hyaluronic acid.

[0042] According to a first object, the present invention relates to a process for preparing an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid comprising the steps of: (a) homogenize hyaluronic acid or its salt with an average molar mass of 16 MDa, in an aqueous basic medium comprising a base, an alkali metal halide salt and an antioxidant, for 1-3 h, at 20-30 °C; (b) add a cationizing agent to the reaction mixture from step (a) and homogenize for 1-3 h at 20-30 °C; (c) Keep the reaction mixture from step (b) at rest for 1–24 h at 4060 °C, where the concomitant reactions of: Petition 870240109140, dated 12 / 20 / 2024, page 24 / 53 15 / 31 i. cationization of hyaluronic acid or its salt; and ii. formation of a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt; (d) purify the polyelectrolyte complex gel obtained in step (c) via dialysis with buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 washouts, dry the gel under vacuum and adjust the total hyaluronic acid content to 15-30 mg / mL with buffered saline (PBS); (e) extrude the gel obtained in step (d), add mannitol and optionally an anesthetic and / or hyaluronic acid or its salt with an average molar mass of 1-6 MDa, adjust the pH to 6.5-7.8, homogenize the formulation for 2-24 h at 20-30 °C, fill into a pre-filled syringe and sterilize under steam at 100-124 °C for 1-5 minutes.

[0043] The hyaluronic acid or its pharmaceutically acceptable salt used as starting material in the process of the present invention is selected from, but not limited to, commercially available sodium hyaluronate, calcium hyaluronate, magnesium hyaluronate, potassium hyaluronate, zinc hyaluronate, having an average molar mass of 1-6 MDa. Preferably, sodium hyaluronate is used as starting material at 4-14% by weight relative to the weight of the reaction mixture of step (c).

[0044] The homogenization steps are fundamental for obtaining and stabilizing the polyelectrolyte complex. The high molar mass of hyaluronic acid (1-6 MDa) used in the process of the present invention provides greater entanglement of the polysaccharide chains; however, this results in a significant increase in the viscosity of the reaction mixture and, consequently, hinders homogenization and cationization due to steric hindrance. Therefore, an anchor-type impeller was used in steps (a) and (b), which assists homogenization and prevents shearing of the hyaluronic acid chains.

[0045] Two homogenization stages were carried out: the first stage of Petition 870240109140, dated 12 / 20 / 2024, page 25 / 53 16 / 31 homogenization, step (a), is necessary to ensure the formation of an alkoxide group in the primary hydroxyl groups of hyaluronic acid; the second homogenization step, step (b), favors a better distribution of the cationizing agent in the vicinity of the hydroxyl groups.

[0046] The cationization reaction and the concomitant formation of the polyelectrolyte complex according to the process of the present invention involve hyaluronic acid or its salt with an average molar mass of 1-6 MDa and a cationizing agent, in an aqueous basic medium comprising a base, an antioxidant and an alkali metal halide salt, at rest, at 40-60 °C.

[0047] To maintain the aqueous reaction medium basic with a pH between 9-13, a base is added which is selected from an inorganic base such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide and mixtures thereof, or an organic base such as ethylenediamine, triethylamine and trimethylamine, in a concentration ranging from 0.10-1.25 M. Preferably, the base is 0.25 M sodium hydroxide.

[0048] In order to avoid alkaline hydrolysis of hyaluronic acid, an antioxidant was used in the reaction medium, selected from ascorbic acid, citric acid, sodium metabisulfite, tocopherol, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, histidine, methionine, cysteine ​​and polyols such as mannitol, sorbitol, xylitol, lactitol, maltitol, erythritol, inositol, glucose, fructose, xylose, trehalose, maltose, sucrose, lactose, glycerol, ethylene glycol, propylene glycol, butylene glycol, diethylene glycol; polyglycerol from 2 to 6 units and mixtures thereof, but not limited to these, in a concentration ranging from 1-5% by weight. Preferably, the antioxidant is mannitol.

[0049] The ionic strength is ensured in the reaction medium of the present invention by the presence of an alkali metal halide salt selected from the halides fluoride, chloride, bromide or iodide combined with the alkali metals sodium, potassium or lithium, and mixtures thereof, but not limited to these, in an amount ranging from 1-5% in Petition 870240109140, dated 12 / 20 / 2024, page 26 / 53 17 / 31 weight. Preferably, the alkali metal halide salt is sodium chloride.

[0050] The cationizing agent functions to preferentially add a positive charge to the primary hydroxyl groups of hyaluronic acid when in aqueous solution. Examples of cationizing agents include, but are not limited to: 3-halogen-2-hydroxypropyl trialkyl ammonium halide, 2-halogen-3-hydroxypropyl trialkyl ammonium halide, 3-chloro-2-hydroxypropyltrimethyl ammonium chloride, 2,3-epoxypropyltrimethyl ammonium chloride, 2-chloro-3-hydroxypropyltrimethyl ammonium chloride, 3-chloro-2-hydroxypropyldimethyldodecyl ammonium chloride, 3-chloro-2-hydroxypropylcocoalkyldimethyl ammonium chloride, 3-chloro-2-hydroxypropyldimethylstearyl ammonium chloride. According to the process of the present invention, the amount of cationizing agent is determined from the molar ratio between hyaluronic acid and the cationizing agent of 1:1 to 1:20, considering the molar mass of the hyaluronic acid disaccharide unit to be 401.3 g / mol.Preferably, the cationizing agent is 3-chloro-2-hydroxypropyltrimethylammonium chloride.

[0051] Unexpectedly and surprisingly, it was observed that under the conditions of the present invention process, as its formation occurs, cationic hyaluronic acid immediately and spontaneously forms a polyelectrolytic complex in contact with hyaluronic acid that has an anionic character in a basic medium. It is important to mention that hyaluronic acid has a very low charge density (only one negative charge per disaccharide), but this was not limiting for the formation of the polyelectrolytic complex with cationic hyaluronic acid, evidencing its behavior as a polycation.

[0052] As mentioned earlier, given the high molar mass of hyaluronic acid, the reaction medium is viscous. The formation of the polyelectrolyte complex between hyaluronic acid and cationic hyaluronic acid, which is a water-insoluble gel, makes the medium even more viscous. Faced with this challenge, the concomitant reactions of cationization and polyelectrolyte complex gel formation were carried out at rest to avoid the breakdown of the formed gel, considering the steps of Petition 870240109140, dated 12 / 20 / 2024, page 27 / 53 18 / 31 prior homogenization to ensure process efficiency.

[0053] The polyelectrolyte complex gel obtained according to the process of the present invention, having a pH between 9-13, is purified using the phosphate-buffered saline (PBS) dialysis technique for 48-72 h at 20-30 °C, with 4-10 washout changes. To avoid abrupt pH changes, pH adjustment was performed slowly during dialysis using phosphate-buffered saline (pH 7.2) without apparent destabilization of the product, and when necessary, the pH was adjusted in the formulation step to the range 6.5-7.8.

[0054] Next, the washing solution is drained. The gel is dried under vacuum so that the water not covalently bound to the gel is removed.

[0055] After adjusting the total hyaluronic acid content to 15-30 mg / mL with buffered saline solution (PBS) and extruding the resulting gel, an injectable formulation is prepared comprising said polyelectrolyte complex gel, mannitol and optionally an anesthetic and / or hyaluronic acid with a molar mass of 16 MDa.

[0056] In injectable formulations, mannitol acts as a heat protectant, particularly during steam sterilization, and is present at 0.5-4.0% by weight.

[0057] Additionally, the injectable formulation optionally comprises an anesthetic selected from benzocaine, bupivacaine, lidocaine, ropivacaine, tetracaine or their salts, but not limited to these, in an amount ranging from 0.1 to 0.4% by weight. Preferably, the optional anesthetic is lidocaine hydrochloride.

[0058] Additionally, the injectable formulation optionally comprises hyaluronic acid with a molar mass of 1-6 MDa, in order to enhance the mechanical properties of the injectable formulation, as well as increase the fluidity and spreadability of the final product, in an amount ranging from 0.1-0.4% by weight.

[0059] In view of the foregoing, unexpectedly and surprisingly, the present invention reveals a process for preparing an injectable formulation capable of preparing Petition 870240109140, dated 12 / 20 / 2024, page 28 / 53 19 / 31 simultaneously a cationic hyaluronic acid and a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, steps (a) to (d), and an injectable formulation comprising said polyelectrolyte complex gel, mannitol and optionally an anesthetic and / or hyaluronic acid with a molar mass of 1-6 MDa, step (e).

[0060] According to one embodiment of the present invention, the process for preparing an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid comprises the following steps: (a) homogenize hyaluronic acid or its salt with an average molar mass of 16 MDa, sodium chloride and mannitol, in an aqueous basic medium comprising sodium hydroxide, for 1-3 h, at 20-30 °C; (b) add the cationizing agent 3-chloro-2-hydroxypropyltrimethylammonium chloride to the reaction mixture from step (a) and homogenize for 1-3 h at 20-30 °C; (c) Keep the reaction mixture from step (b) at rest for 1–24 h at 4060 °C, where the concomitant reactions of: i. cationization of hyaluronic acid or its salt; and ii. formation of a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt; (d) purify the polyelectrolyte complex gel obtained in step (c) via dialysis with buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 washouts, dry the gel under vacuum and adjust the total hyaluronic acid content to 15-30 mg / mL with buffered saline (PBS); (e) Extrude the gel obtained in step (d), add mannitol and optionally lidocaine chloride anesthetic and / or hyaluronic acid or its salt with an average molar mass of 1-6 MDa, adjust the pH to 6.5-7.8, homogenize the formulation for 2-24h, a Petition 870240109140, dated 12 / 20 / 2024, page 29 / 53 20 / 31 20-30 °C, fill into a pre-filled syringe and sterilize under steam at 100-124 °C for 1-5 minutes.

[0061] Scheme 2 shows the reactions according to the process of the present invention considering the quaternary ammonium salt 3-chloro-2-hydroxypropyltrimethylammonium chloride as the cationizing agent. Scheme 2. Reactions that may occur during the cationization of hyaluronic acid, according to the present invention: (a) formation of 2,3-epoxypropyltrimethylammonium chloride in alkaline medium (NaOH) from the cationizing agent 3-chloro-2-hydroxypropyltrimethylammonium chloride; (b) alkaline hydrolysis of 2,3-epoxypropyltrimethylammonium chloride; (c) cationization reaction between hyaluronic acid (HA-OH) and 2,3-epoxypropyltrimethylammonium chloride in alkaline medium to form cationic hyaluronic acid; (d) alkaline hydrolysis of cationic hyaluronic acid; (e) formation of a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid.

[0062] 3-Chloro-2-hydroxypropyltrimethylammonium chloride in alkaline medium forms Petition 870240109140, dated 12 / 20 / 2024, page 30 / 53 21 / 31 2,3-epoxypropyltrimethylammonium chloride, which is an epoxide. Hyaluronic acid in an alkaline medium forms the alkoxide at the primary hydroxyl group. Thus, the epoxide can bind with the alkoxide of hyaluronic acid through an etherification reaction, forming cationic hyaluronic acid.

[0063] A sodium hydroxide concentration of 0.25M ensures a pH greater than 9, necessary for the formation of O- without significant degradation by alkaline hydrolysis with a drastic reduction in the molar mass of hyaluronic acid. The residence time of hyaluronic acid and quaternary ammonium salt in alkaline medium and the temperature of the homogenizations of steps (a) and (b) of the process were also selected to avoid alkaline hydrolysis of hyaluronic acid with chain breakage and the epoxide formed from 3-chloro-2-hydroxypropyltrimethylammonium chloride with the formation of the diol, which result in the reduction of cationization and consequently the formation of the polyelectrolyte complex.Homogenization was performed for an extended period of 1 hour to ensure homogenization efficiency without favoring the aforementioned hydrolysis; the homogenization steps were carried out at room temperature (~25°C) to reduce the hydrolysis of hyaluronic acid and the epoxide formed in situ, and to prevent the premature onset of cationization reactions / formation of the polyelectrolyte complex, which were conducted statically to avoid breaking the formed gel. Sodium chloride provides the desired ionic strength and thus acts as a potentiator of the cationization reaction / formation of the polyelectrolyte complex, while the antioxidant mannitol acts as a stabilizer / thermoprotector of the hyaluronic acid chains.

[0064] In a second object, the present invention relates to an injectable formulation, obtained according to the process defined in the first object and its embodiments, comprising a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt, mannitol, optionally an anesthetic and / or hyaluronic acid or its salt with an average molar mass of 1-6 MDa and a pharmaceutically acceptable carrier which is buffered saline solution (PBS). Petition 870240109140, dated 12 / 20 / 2024, page 31 / 53 22 / 31

[0065] According to one embodiment, the injectable formulation, obtained according to the process disclosed in the present invention, comprises: (a) 15-30 mg / mL of a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt with a degree of cationization of 0.2-0.7; (b) 0.5-4.0 % by weight of mannitol; (c) optionally 0.1-0.4% by weight of lidocaine hydrochloride anesthetic; (d) optionally 0.1-0.4% by weight of hyaluronic acid or its salt with an average molar mass of 1-6 MDa; (e) a pharmaceutically acceptable carrier, which is buffered saline solution (PBS), with the pH of the formulation in the range between 6.5 and 7.8.

[0066] According to one embodiment of the present invention, said polyelectrolyte complex is between sodium hyaluronate and hydroxypropyl trimethylammonium hyaluronate chloride.

[0067] According to one embodiment, the injectable formulation obtained according to the process disclosed in the present invention comprises: (a) 20-25 mg / mL of a polyelectrolyte complex gel between sodium hyaluronate and hydroxypropyl trimethylammonium hyaluronate chloride with a degree of cationization of 0.2-0.7; (b) 1.2% by weight of mannitol; (c) 0.3% by weight of lidocaine hydrochloride; (d) a pharmaceutically acceptable carrier, which is buffered saline solution (PBS), with the pH of the formulation in the range between 6.5 and 7.8.

[0068] The presence of mannitol in the injectable formulation of the present invention is essential to prevent thermal degradation caused by the sterilization process. The Petition 870240109140, dated 12 / 20 / 2024, page 32 / 53 23 / 31 tests performed on the present invention show that sorbitol, which is also a known thermal protectant of the same class as mannitol, failed to protect the formulation from thermal degradation caused by the sterilization process. Therefore, unexpectedly, mannitol proved capable of thermally protecting the injectable formulation.

[0069] The injectable formulation obtained by the process of the present invention has rheological properties comparable to injectable formulations of covalently cross-linked hyaluronic acid and is capable of terminal sterilization, these characteristics being important for its application as an intradermal or intra-articular filler.

[0070] In a third object, the present invention relates to the use of an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, defined in the second object and its embodiments, for application as an intradermal or intra-articular filler.

[0071] The process for preparing an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, defined in the first object of the present invention and in its embodiments, can be carried out in its entirety or, alternatively, the polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid obtained in step (d) of the process can be stored for later preparation of the injectable formulation.

[0072] In this sense, the present invention presents as its fourth object a process for preparing a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, comprising the steps of: (a) homogenize hyaluronic acid or its salt, an alkali metal halide salt, an antioxidant, in aqueous basic medium, for 1-3 h, at 20-30 °C; (b) add a cationizing agent and homogenize for 1-3 h at 20-30 °C; (c) keep the reaction mixture at rest for 1-24 h, at 40-60 °C, where Petition 870240109140, dated 12 / 20 / 2024, page 33 / 53 24 / 31 concomitant reactions occur of: i. cationization of hyaluronic acid or its salt; and ii. formation of a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt; (d) purify the gel via dialysis with buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 washout changes, dry the gel under vacuum and adjust the total hyaluronic acid content to 15-30 mg / mL with buffered saline (PBS).

[0073] In a fifth object, the present invention relates to a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt, obtained according to a process disclosed in the present invention.

[0074] According to one embodiment, the present invention relates to a polyelectrolyte complex gel between sodium hyaluronate and hydroxypropyl trimethylammonium hyaluronate chloride, obtained according to the process disclosed in the present invention.

[0075] In a sixth object, the present invention relates to the use of a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, obtained according to the process disclosed in the present invention, in the manufacture of an injectable formulation for application as an intradermal or intra-articular filler.

[0076] According to one embodiment, the present invention relates to the use of a polyelectrolyte complex gel between sodium hyaluronate and hydroxypropyl trimethylammonium hyaluronate chloride, obtained according to the process disclosed in the present invention, in the manufacture of an injectable formulation for application as an intradermal or intra-articular filler, wherein the formulation further comprises mannitol and optionally an anesthetic and / or hyaluronic acid or its salt with an average molar mass of 1-6 MDa. Examples

[0077] The following examples serve to illustrate aspects of the present invention. Petition 870240109140, dated 12 / 20 / 2024, page 34 / 53 25 / 31 without, however, having any limiting character. EXAMPLE 1 - Preparation of an injectable formulation comprising a polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid; evaluation of the effect of the thermoprotective agent on the formulation.

[0078] In a reactor, 1.2 g of sodium hyaluronate (NaHA) with an average molar mass of 4.3 MDa, sodium chloride, and mannitol were added to 10 mL of 0.25 M sodium hydroxide, according to the conditions described in Table 1, with homogenization for 1 h at 25 °C, using an anchor-type impeller at 300 rpm. After this period, 60% 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) was added to the reactor, according to the conditions described in Table 1, with homogenization for 1 h at 25 °C, using an anchor-type impeller at 300 rpm. The reaction mixture was then allowed to stand for 4.5 h at 52 °C.During this period, in a basic aqueous medium, cationization of hyaluronic acid occurs, forming hydroxypropyl trimethylammonium hyaluronate chloride, and concomitant formation of a polyelectrolyte complex gel between sodium hyaluronate and hydroxypropyl trimethylammonium hyaluronate chloride, which exhibits typical weak gel rheological behavior with G' > G across the entire frequency axis. Table 1. Description of the reagents used in the cationization reaction. Cationization reaction Assays NaCl (g) Mannitol (g) 60% CHPTAC (mL) molar ratio NaHA:CHPTAC AD 0.200 0.200 5.0 (3.462g) 1:6.15 EF 0.500 0.500 10.0 (6.924g) 1:12.31

[0079] In a previous study, the purification of polyelectrolyte complex gel was performed by successive washes with saline buffer (PBS). However, Petition 870240109140, dated 12 / 20 / 2024, page 35 / 53 In sample 26 / 31, a mass loss was observed, evidenced by an increase in the viscosity of the washing solutions after purification. To solve this problem, the dialysis technique was used to purify the polyelectrolyte complex gel in a dialysis bag with a cut-off molar mass of 12-16 MDa and a porosity of 25 Å, with 6 exchanges of buffered saline solution (PBS) with pH 7.2 ± 0.1, for 48 h at 25 °C, in order to guarantee the absence of a cationizing agent. Subsequently, the gel was dried under vacuum. The total hyaluronic acid content of the polyelectrolyte complex gel was adjusted to 15-30 mg / mL and the purified gel was extruded through a 750 µm mesh.

[0080] In the formulation step, 60.0 g of purified polyelectrolyte complex gel comprising hyaluronic acid and cationic hyaluronic acid, and mannitol or sorbitol (see Table 2) were added to a reactor, where the pH of the medium was adjusted to 6.5-7.8 with 0.1 M sodium hydroxide or 0.1 M hydrochloric acid. The formulation was then homogenized for 4 h at 25 °C and 300 rpm. After this step, the formulation was packaged in pre-filled syringes and steam-sterilized at 121 °C for 2 minutes (total time 15-25 minutes). The final total hyaluronic acid content is 20-25 mg / mL.

[0081] As mentioned earlier, the sterilization process is critical to the rheological characteristics of the injectable formulation of the present invention. In order to evaluate the effects of the presence or absence of a heat protectant or different heat protectants in the injectable formulation before and after the sterilization process, several tests were carried out, as shown in Table 2. Table 2. Evaluation of the effect of the thermoprotective agent in the injectable formulation of a polyelectrolyte complex gel comprising hyaluronic acid and cationic hyaluronic acid against degradation, based on rheological data (see Figures 1 to 3). Injectable formulation Assay Mannitol (g) Mannitol % (w / w) Sorbitol (g) Sorbitol % (w / w) Sterilization Rheological data Petition 870240109140, dated 12 / 20 / 2024, page 36 / 53 27 / 31 A Exempt Exempt No In orange, Figure 1 B Exempt Exempt Yes In blue, Figure 1 C 0.400 0.7 Exempt No In green, Figure 1 D 0.400 0.7 Exempt Yes In gray, Figure 1 E 0.700 1.2 Exempt No In green, Figure 2 F 0.700 1.2 Exempt Yes In gray, Figure 2 G Exempt 0.700 1.2 Yes In orange, Figure 3

[0082] As can be seen in Figure 1, the injectable formulation of Assay B, prepared without the addition of a heat protectant in the formulation step and with sterilization, showed a rheological behavior of a polymeric solution (G modulus (viscous) > G' modulus (elastic)), characterized by the presence of cross-over at 3.59 Hz, indicating the degradation of the polyelectrolyte complex.

[0083] The effect of sterilization in the presence of 0.7% mannitol reduced the G' values ​​from 305 Pa (Test C, Figure 1) to 97 Pa (Test D, Figure 1), but the weak gel behavior (G' > G'') was maintained, indicating that 0.7% mannitol thermally protected the injectable formulation from sterilization conditions. The same protective effect occurred with the presence of 1.2% mannitol, where the G' values ​​decreased from 653 Pa (Test E, Figure 2) to 361 Pa (Test F, Figure 2), demonstrating the importance of mannitol as a thermal protector in the injectable formulation of the present invention against thermal degradation caused by the sterilization process.

[0084] On the other hand, Assay G (Figure 3), prepared with the addition of 1.2% sorbitol in the formulation step and with sterilization, showed a polymeric solution rheological behavior (G''>G') and the presence of cross-over, that is, the sorbitol failed to prevent the thermal degradation of the injectable formulation, although there are several reports in the state of the art that mention the use of sorbitol as Petition 870240109140, dated 12 / 20 / 2024, page 37 / 53 28 / 31 thermoprotective agent in injectable formulations comprising hyaluronic acid. EXAMPLE 2 - Preparation of injectable formulations with different proportions of polyelectrolyte complex gel and added hyaluronic acid.

[0085] Injectable formulations according to the present invention comprising different mass ratios of polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, and hyaluronic acid with a molar mass of 1.4 MDa, as shown in Table 3, were prepared according to Test F of Example 1, with 1.2% mannitol and steam sterilized, in order to evaluate the contribution of the added hyaluronic acid to the rheological behavior of the final product. Table 3. G' and G values ​​of injectable formulations comprising different mass ratios of polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, and of hyaluronic acid with a molar mass of 1.4 MDa, steam-sterilized. Assay Mass ratio of polyelectrolyte complex: hyaluronic acid G' (Pa) at 1 Hz G' (Pa) at 1 Hz H 1:0 (Figure 4, blue) 176.4 80.6 I 1:1 (Figure 4, gray) 1.291 651.2 J 1:2 (Figure 4, green) 6.561 2.760 K 2:1 (Figure 4, orange) 586.7 293.8

[0086] Figure 4 shows the frequency sweep of the HK tests, as per Table 3. It can be observed that there was an increase in G' from Test H with a 1:0 ratio (blue) from 176 Pa to 1291 Pa in Test I with a 1:1 ratio (gray), confirming the positive character of the polyelectrolyte complex gel of the present invention.

[0087] In the 1:2 ratio J assay, when there was an increase in the amount of unmodified hyaluronic acid relative to the polyelectrolyte complex, a much higher G' value (6561 Pa) was observed, indicating that there was still a positive charge remaining from the gel. Petition 870240109140, dated 12 / 20 / 2024, page 38 / 53 29 / 31 polyelectrolyte complex to interact with the negative charges of unmodified hyaluronic acid.

[0088] On the other hand, when the amount of polyelectrolyte complex gel was increased, Test K, there was a reduction of G' to 587 Pa in relation to Test I, indicating that at this mass ratio all the negative charges of the unmodified hyaluronic acid were occupied, that is, they formed electrostatic interactions with the positive charges of the polyelectrolyte complex gel, with positive charges still remaining in the product of Test K. EXAMPLE 3 - Evaluation of process reproducibility.

[0089] Three batches were prepared, according to the procedure of Test F of Example 1, and analyzed in order to evaluate the reproducibility of the process. Table 4 presents the results obtained for the physicochemical characterization of Batches 1 to 3. Table 4. Physicochemical characterization of injectable polyelectrolyte complex gel formulations between hyaluronic acid and cationic hyaluronic acid, prepared according to Test F of Example 1. Parameters Lot 1 Lot 2 Lot 3 Average pH 7.48 7.40 7.49 7.46+0.05 Appearance Transparent, homogeneous gel without particles. Total hyaluronic acid content (mg / mL) 22.7 22.6 23.2 22.9±0.4 Degree of cationization calculated from total nitrogen content 0.46 0.46 0.54 0.49±0.05 Extrusion force (N)* 19.5 19.9 19.4 19.6±0.6 G' (Pa) at 1Hz 400.1 460.2 464.5 441±36 G (Pa) at 1Hz 235.9 209.8 209.2 218±15 Zeta potential (mV) -13 -14 -12 -13±1 Petition 870240109140, dated 12 / 20 / 2024, pp. 39 / 53 30 / 31 *Using a 231 / z G needle (Terumo) and a 1mL syringe (Schott).

[0090] In general, all parameters evaluated showed acceptable repeatability, demonstrating the reproducibility of the process of the present invention. Batches 1-3 presented a transparent, homogeneous gel appearance without particles, as desired for the intended applications. The pH values ​​are within the specification for dermal filler products (pH between 6.5-7.8).

[0091] The total hyaluronic acid content is close to the theoretical value (24.1 mg / mL), which was corrected considering the water content of the hyaluronic acid raw material with a molar mass of 4.3 MDa (12.8%). The values ​​obtained are within the specification range of 21.7-26.5 mg / mL (90-110%).

[0092] As a reference for extrusion force, the product Teosyal® was used. Puresense Ultimate (Teoxane) which has an extrusion force of 25 N.

[0093] The values ​​of G' (elastic modulus) and G (viscous modulus) at 1 Hz obtained in the frequency sweeps demonstrate that Batches 1-3 maintained the weak gel behavior with G'>G and with both moduli parallel to the frequency axis, even after sterilization, demonstrating that there was no thermal degradation of the injectable formulations of polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid obtained according to the process disclosed in the present invention.

[0094] The zeta potential technique is indicated for evaluating the surface charge of particles in solution. In the case of the present invention, since the injectable formulation is insoluble in water, the zeta potential results are only indicative (not conclusive).

[0095] The zeta potential results of Batches 1-3 presented in Table 3 (mean value of -13±1 mV) showed a reduction in negative charge density compared to unmodified hyaluronic acid (-40 mV), indicating the occurrence of the cationization reaction of hyaluronic acid and the formation of a polyelectrolytic complex between the Petition 870240109140, dated 12 / 20 / 2024, pp. 40 / 53 31 / 31 hyaluronic acid and cationic hyaluronic acid, through the carboxylic group (pKa 2.9) which is ionized at pH 6.5-7.8 (Gatej et al.; Role of the pH on Hyaluronan Behavior in Aqueous Solution, Biomacromolecules, 6(1): 61-67, 2005). EXAMPLE 4 - Evaluation of degradation kinetics against reactive oxygen species.

[0096] A commercial comparator Juvéderm® Voluma (covalently crosslinked hyaluronic acid with BDDE, 20 mg / mL, mannitol-free) and Batches 1-3 prepared in Example 3 were evaluated for in vitro performance under oxidative stress with free radical (hydrogen peroxide), according to Conrozier et al. (Conrozier et al.; Mannitol Preserves the Viscoelastic Properties of Hyaluronic Acid in an In Vitro Model of Oxidative Stress, Rheumatol Ther, 1(1): 45-54, 2014) and Flégeau et al. (Flégeau et al.; Multidose Hyaluronidase Administration as an Optimal Procedure to Degrade Resilient Hyaluronic Acid Soft Tissue Fillers, Molecules, 28: 1003, 2023). For this purpose, the degradation kinetics of the products were carried out in the presence of 30% hydrogen peroxide, in a 10:1 ratio, evaluating G' as a function of time for 20 minutes.

[0097] Figure 5 shows the degradation kinetics curves against reactive oxygen species performed with Lot 1 (dark green), Lot 2 (light green), Lot 3 (orange) and the commercial comparator Juvéderm® Voluma (black). It was observed that, even being a polyelectrolyte complex, Lots 1-3 did not show pronounced degradation of G' (elastic modulus) and G” (viscous modulus) in the presence of free radicals, indicating that mannitol protects the β-1,3 and β-1,4 glycosidic bonds of hyaluronic acid in the polyelectrolyte complex.

[0098] It should be understood that the embodiments described above are merely illustrative and that any modification thereof may occur for a person skilled in the art. Consequently, the present invention should not be considered limited to the embodiments described in this report. Petition 870240109140, dated 12 / 20 / 2024, pp. 41 / 53

Claims

1 / 6 CLAIMS 1. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID characterized in that it comprises the steps of: (a) homogenizing hyaluronic acid or its salt with an average molar mass of 1-6 MDa, in an aqueous basic medium comprising a base, an alkali metal halide salt and an antioxidant, for 1-3 h, at 20-30 °C; (b) adding a cationizing agent to the reaction mixture of step (a) and homogenizing for 1-3 h, at 20-30 °C; (c) keep the reaction mixture from step (b) at rest for 1-24 h, at 40-60 °C, where the concomitant reactions of: i. cationization of hyaluronic acid or its salt; and ii.(d) Purify the polyelectrolyte complex gel obtained in step (c) via dialysis with buffered saline (PBS) for 48-72 h at 20-30 °C with 4-10 washouts, dry the gel under vacuum and adjust the total hyaluronic acid content to 15-30 mg / mL with buffered saline (PBS); (e) extrude the gel obtained in step (d), add mannitol and optionally an anesthetic and / or hyaluronic acid or its salt with an average molar mass of 1-6 MDa, adjust the pH to 6.5-7.8, homogenize the formulation for 2-24 h at 20-30 °C, fill into a pre-filled syringe and sterilize under steam at 100-124 °C for 1-5 minutes.

2. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized in that said hyaluronic acid or its salt is selected from sodium hyaluronate, calcium hyaluronate, magnesium hyaluronate, potassium hyaluronate, zinc hyaluronate, preferably sodium hyaluronate, with an average molar mass between 1-6 MDa, at 4-14 % by weight relative to the weight of the reaction mixture of step (c).

3. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized in that said base is selected from an inorganic base such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide and mixtures thereof, or an organic base such as ethylenediamine, triethylamine and trimethylamine, preferably sodium hydroxide, to maintain the aqueous reaction medium basic with a pH between 9-13.

4. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized in that said antioxidant is selected from ascorbic acid, citric acid, sodium metabisulfite, tocopherol, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, histidine, methionine, cysteine ​​and polyols such as mannitol, sorbitol, xylitol, lactitol, maltitol, erythritol, inositol, glucose, fructose, xylose, trehalose, maltose, sucrose, lactose, glycerol, ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, polyglycerol 2 to 6 units, and mixtures thereof, preferably mannitol, at 1-5% by weight.

5. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized in that said alkali metal halide salt is selected from the halides fluoride, chloride, bromide or iodide combined with the alkali metals sodium, potassium or lithium, and mixtures thereof, preferably sodium chloride, at 1-5% by weight.

6. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized by the fact that Petition 870240109140, dated 12 / 20 / 2024, page. 43 / 53 3 / 6 the said cationizing agent being selected from 3-chloro-2-hydroxypropyltrimethylammonium chloride, 2,3-epoxypropyltrimethylammonium chloride, 2-chloro-3-hydroxypropyltrimethylammonium chloride, 3-chloro-2-hydroxypropyldimethyldodecylammonium chloride, 3-chloro-2-hydroxypropylcocoalkyldimethylammonium chloride, 3-chloro-2-hydroxypropyldimethylstearylammonium chloride, preferably 3-chloro-2-hydroxypropyltrimethylammonium chloride, wherein the amount of cationizing agent is determined from the molar ratio between hyaluronic acid and the cationizing agent from 1:1 to 1:

20.

7. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized in that said mannitol in step (e) has the function of a thermoprotector of the injectable formulation, at 0.54.0% by weight.

8. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTE COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized in that said anesthetic is selected from benzocaine, bupivacaine, lidocaine, ropivacaine, tetracaine or their salts, preferably lidocaine hydrochloride, at 0.1-0.4% by weight.

9. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID characterized by comprising the steps of: (a) homogenizing hyaluronic acid or its salt with an average molar mass of 1-6 MDa, sodium chloride and mannitol, in an aqueous basic medium comprising sodium hydroxide, for 1-3 h, at 20-30 °C; (b) adding the cationizing agent 3-chloro-2-hydroxypropyltrimethylammonium chloride to the reaction mixture of step (a) and homogenizing for 1-3 h, at 20-30 °C; Petition 870240109140, dated 12 / 20 / 2024, p. 44 / 53 4 / 6 (c) keep the reaction mixture from step (b) at rest for 1-24 h, at 40-60 °C, where the concomitant reactions of: i. cationization of hyaluronic acid or its salt; and ii.(d) Purify the polyelectrolyte complex gel obtained in step (c) via dialysis with buffered saline (PBS) for 48-72 h at 20-30 °C with 4-10 washouts, dry the gel under vacuum and adjust the total hyaluronic acid content to 15-30 mg / mL with buffered saline (PBS); (e) extrude the gel obtained in step (d), add mannitol and optionally lidocaine chloride anesthetic and / or hyaluronic acid or its salt with an average molar mass of 1-6 MDa, adjust the pH to 6.5-7.8, homogenize the formulation for 2-24h at 20-30 °C, fill into a pre-filled syringe and sterilize under steam at 100-124 °C for 1-5 minutes.

10. INJECTABLE FORMULATION, obtained according to the process defined in claim 1, characterized by comprising: (a) 15-30 mg / mL of a polyelectrolyte complex gel between hyaluronic acid or its salt and cationic hyaluronic acid or its salt with a degree of cationization of 0.2-0.7; (b) 0.5-4.0% by weight of mannitol; (c) optionally 0.1-0.4% by weight of lidocaine hydrochloride anesthetic; (d) optionally 0.1-0.4% by weight of hyaluronic acid or its salt with an average molar mass of 1-6 MDa; and (e) a pharmaceutically acceptable carrier, which is buffered saline solution (PBS), the pH of the formulation being in the range between 6.5 and 7.

8.

11. INJECTABLE FORMULATION, according to claim 10, characterized in that said polyelectrolyte complex is between sodium hyaluronate and hydroxypropyl trimethylammonium hyaluronate chloride. Petition 870240109140, dated 12 / 20 / 2024, page 45 / 53 5 / 6 12. INJECTABLE FORMULATION obtained according to the process defined in claim 9, characterized by comprising: (a) 20-25 mg / mL of a polyelectrolyte complex gel between sodium hyaluronate and hydroxypropyl trimethylammonium hyaluronate chloride with a degree of cationization of 0.2-0.7; (b) 1.2% by weight of mannitol; (c) 0.3% by weight of lidocaine hydrochloride anesthetic; and (d) a pharmaceutically acceptable carrier, which is buffered saline solution (PBS), the pH of the formulation being in the range between 6.5 and 7.

8.

13. USE OF AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, as defined in any one of claims 10 to 12, characterized by being for application as an intradermal or intra-articular filler.

14. PROCESS FOR PREPARING A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID characterized by comprising the steps of: (a) homogenizing hyaluronic acid or its salt with an average molar mass of 1-6 MDa, in an aqueous basic medium comprising a base, an alkali metal halide salt and an antioxidant, for 1-3 h, at 20-30 °C; (b) adding a cationizing agent to the reaction mixture of step (a) and homogenizing for 1-3 h, at 20-30 °C; (c) keeping the reaction mixture of step (b) at rest for 1-24 h, at 40-60 °C, in which the concomitant reactions of: i. cationization of hyaluronic acid or its salt; and ii. (d) Purify the polyelectrolyte complex gel obtained in step (c) via dialysis with buffered saline solution (PBS) for 48-72 h at 20-30 °C with 4-10 exchanges of Petition 870240109140, dated 12 / 20 / 2024, page 1.46 / 53 6 / 6 washing solution, dry the gel under vacuum and adjust the total hyaluronic acid content to 15-30 mg / mL with buffered saline solution (PBS).

15. A polyelectrolyte complex gel between hyaluronic acid and cationic hyaluronic acid, obtained according to the process defined in claim 14, characterized by comprising a polyelectrolyte complex between hyaluronic acid or its salt, preferably sodium hyaluronate, and cationic hyaluronic acid or its salt, preferably hydroxypropyl trimethylammonium hyaluronate chloride.

16. USE OF A POLYELECTROLYTIC COMPLEX GEL BETWEEN HYALURONIC ACID AND CATIONIC HYALURONIC ACID, obtained according to the process defined in claim 15, characterized by being used in the manufacture of an injectable formulation for application as an intradermal or intra-articular filler. Petition 870240109140, dated 12 / 20 / 2024, pp. 47 / 53

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