Process for preparing an injectable formulation comprising a polyelectrolyte complex GEL of hyaluronic acid and cationic hyaluronic acid, injectable formulation obtained by the process and its use, process for preparing a polyelectrolyte complex GEL of hyaluronic acid and cationic hyaluronic acid, GEL obtained by the process and its use in the manufacture of an injectable formulation

Abstract

The present invention describes a process for preparing an injectable formulation comprising a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid, mannitol, and optionally an anesthetic and / or hyaluronic acid. This process comprises the concomitant reactions of hyaluronic acid cationization and the formation of a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid. The injectable formulation obtained by the process of the present invention exhibits rheological properties comparable to injectable formulations of covalently cross-linked hyaluronic acid and is capable of terminal sterilization, such characteristics being important for its application as an intradermal or intra-articular filler.

Description

PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, INJECTABLE FORMULATION OBTAINED BY THE PROCESS AND ITS USE, PROCESS FOR PREPARING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, GEL OBTAINED BY THE PROCESS AND ITS USE IN THE MANUFACTURE OF AN INJECTABLE FORMULATION.Field of the invention

[0001] The present invention relates to a process for preparing an injectable formulation that concomitantly comprises the reactions of hyaluronic acid cationization and the formation of a polyelectrolyte complex gel of 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 intra-articular filler. The present invention lies in the fields of Chemistry and Pharmaceutical Technology.Background of the invention

[0002] Hyaluronic acid is an anionic polysaccharide composed of N-acetyl-D- glucosamine units linked to D-glucuronic acid, capable of forming intra- and intermolecular hydrogen bonds in solution. It is present in the synovial fluid, vitreous humor, and in the connective collagen tissue of various organisms. Due to its viscoelastic properties, biocompatibility, tolerance, and lack of toxicity, injectable hyaluronic acid is widely used in Medicine in joints and in Aesthetic Medicine as a wrinkle filler or volume promoter. Commercially, there are several products composed of cross-linked hyaluronic acid that provide greater resistance, especially regarding degradation and heat.

[0003] Because of its anionic nature due to the carboxylic acid group, hyaluronic acid has low compatibility with skin and hair, which are negatively charged. A strategy to make anionic polymers or polysaccharides such as hyaluronic acid more adherent to skin or hair is the cationization process.

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

[0005] In the global market, cationic hyaluronic acid is available as a cosmetic-grade ingredient: Hyaloveil-P® (Kewpie, JP); HA Plus™ Hyaluronic Acid (Shandong Focus Freda Biotech, CN); cationHA™ (Bloomage Biotechnology, CN); e Cationic Hyaluronic Acid (Amik, IT). Cationic hyaluronic acid is not available on the market as an injectable-grade ingredient.

[0006] According to the scientific publications of Kamada et al., Wang et al., and Liu et al., the possibility of using cationic hyaluronic acid in the treatment of joint diseases and ophthalmological diseases was evaluated (Kamada, Y. et al. Analysis of the effect of cationic hyaluronic acid on articular cartilage. Abstracts / Osteoarthritis and Cartilage, 29:S10eS432, 2021), (Wang, Y. N. et al. Cationic hyaluronic acid-modified cyclosporin micelles used for ocular drug delivery, Indian Journal of Pharmaceutical Sciences, 86(1): 64-74, 2024), (Liu, Y. et al.; Cationized hyaluronic acid coated spanlastics for cyclosporine A ocular delivery: Prolonged ocular retention, enhanced corneal permeation and improved tear production, International Journal of Pharmaceutics, 565: 133-142, 2019).

[0007] The polymer cationization process is carried out by adding cationic groups to anionic sites using cationizing agents. In a particular case, the cationizing agents are quaternary ammonium salts, such as 2,3-epoxypropyltrialkylammonium halide (also known as glycidyltrialkylammonium halide or GTA) or 3-halo-2- hydroxypropyltrialkylammonium halide. Indeed, under basic conditions, cationizing agents such as 2,3-epoxypropyltrimethylammonium chloride are formed in situ from 3- chloro-2-hydroxypropyltrimethylammonium chloride (see Scheme 1).

[0008] In the hyaluronic acid polysaccharide, the cationization reaction can occur at its four functional groups: carboxylic acid, hydroxyl (including primary and secondaryalcohols), amide (acetamide), and the reduced terminal of the polymer. Document EP2166O22 (Q.P. Corp, JP) reports that in an organic medium the favored hyaluronic acid cationization reaction is the esterification reaction, i.e., nucleophilic substitution occurs at the carboxyl group.

[0009] On the other hand, Prado and Matulewicz report that etherification reaction at the hydroxyl group, predominantly at the primary hydroxyl, is favored, since epoxide opening in alkaline medium occurs through an SN2 mechanism in which the preferred attack occurs at the less substituted carbon (Prado, H. J.; Matulewicz, M. C.; Cationization of polysaccharides: A path to greener derivatives with many industrial applications, European Polymer Journal, 52: 53-75, 2014). In alkaline aqueous medium, the cationization reaction begins 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 alkaline medium attacks the epoxide of the cationizing agent, forming the cationic polysaccharide hydroxypropyltrimethylammonium hyaluronate chloride via an SN2 nucleophilic substitution reaction (see Scheme 1).Scheme 1. Formation of the epoxide in alkaline medium and nucleophilic attack of the alkoxide on the epoxide (Prado e Matulewicz, 2014).

[0010] The process for preparing cationic hyaluronic acid using a quaternary ammonium salt as cationizing agent involves optimizing several variables. According to Prado and Matulewicz (Prado and Matulewicz, 2014), the presence of a base is fundamental for the cationization reaction. The authors state 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 to form the alkoxide on the primary hydroxyls of hyaluronic acid (see Scheme 1), which is responsible for the nucleophilic attack on the epoxide group, forming cationic hyaluronic acid. Thus, an increase in sodium hydroxide concentration increases the degree of cationization. However, an excess of sodium hydroxide can favor polysaccharide degradation by alkaline hydrolysis, as well as degradation of the epoxide into the diol, thereby decreasing the degree of cationization. Indeed, epoxide degradation into diol depends on temperature, pH of the medium, and reaction time. At 50 °C, 50% of the epoxide remains in solution within 24 h.

[0011] Antioxidants can be used to prevent hyaluronic acid degradation by reactive oxygen species and hyaluronidase and / or as thermal protector, especially during thermal sterilization of intradermal and / or intra-articular compositions (W02009071697, WO2019125166, WO2012167079 and W02009024670).

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

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

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

[0015] For example, document EP2166022 (Q.P. Corp, JP) discloses a cationic hyaluronic acid or salt thereof with a degree of cationization of 0.15-0.6, and its preparation method comprising a cationizing agent in alkaline medium at 30-70 °C, wherein the cationizing agent is a 2,3-epoxypropyltrialkylammonium halide or 3-halo-2- hydroxypropyltrialkylammonium halide. Examples 1 and 2 describe preparation of cationic hyaluronic acid from hyaluronic acid with molar mass of 2 MDa in a medium of 65-80% ethanol in water, with reaction times of 0.1-9 h. In organic phase, cationic hyaluronic acid precipitates. For its purification, a sodium chloride solution was used to solubilize the precipitate, followed by ethanol addition to re-precipitate the cationic hyaluronic acid and successive washings of the precipitate with 80% ethanol. Cationic hyaluronic acid was obtained after vacuum drying for 5 h at 60°C as a white powder.

[0016] In turn, document WO2023285663 (Givaudan, CH) discloses hydroxypropyltrimethylammonium hyaluronate or salt thereof with a degree of cationization greater than 1.4, its preparation method, a cosmetic composition and use for hydration and / or UV protection and / or hair repair. In its description, the inventors report that, surprisingly, it was discovered that a relatively low average molar mass, in the range of 10 k-200 kDa, provides a particularly effective cosmetic active, for example, exhibiting better adhesion to hair and deeper penetration into the skin. Furthermore, a relatively low average molar mass facilitates synthesis of hydroxypropyltrialkylammonium hyaluronate and salts thereof. It was found that lower molar mass of hyaluronic acid reacts faster and requires fewer equivalents of reagents to achieve the desired degree of cationization. Without being bound to theory, it isbelieved that the reactive sites of hyaluronic acid with higher average molar mass are less accessible to reactant molecules, both due to lower flexibility of longer polymer chains and increased steric hindrance. Example 1 discloses 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 alkaline medium with sodium hydroxide. Purification was performed by ultrafiltration and the cationic hyaluronic acid was obtained by lyophilization, with degree of cationization of 2.18. Example 2 discloses 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 f in alkaline medium with sodium hydroxide. Purification was performed by dialysis and the cationic hyaluronic acid was obtained by lyophilization, with degree of cationization of 1.47.

[0017] Document EP1961772 (Shiseido, JP) discloses a cationic hyaluronic acid wherein at least part of the hydrogens of hydroxyl groups are substituted by a group with a quaternary ammonium cation, wherein the degree of cationization is greater than 0.1. It further discloses a polyion 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 cationizing agent 2,3- epoxypropyltrimethylammonium chloride in aqueous medium with sodium hydroxide for 3 days at room temperature. Subsequently, the cationic hyaluronic acid was precipitated using methanol, washed with acetone and dried, obtaining cationic hyaluronic acid as a white powder with degree of cationization of 1.4. After purification, a cationic hyaluronic acid solution is combined with a hyaluronic acid solution in equivalent amounts, resulting in the precipitation of a polyion complex due to electrostatic interaction between the polysaccharides.

[0018] Polyelectrolyte complexes, also known as polyion complexes or polysalts, are precipitates formed spontaneously when two polyelectrolytes of opposite chargescome into contact in aqueous solution.

[0019] According to Buriuli and Verma, polyelectrolyte complexes exhibit unique physical and chemical properties due to the considerably stronger electrostatic interactions compared to most other secondary binding 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 of polyelectrolyte complex formation is the increase in entropy due to the release of the low molar mass counterions bound to polysaccharides of opposite charges. Kulkarni et al. mention that interactions established in the formation of polyelectrolyte complex include Coulomb's interactions, hydrogen bonding, hydrophobic interactions, van der Waals interactions and dipole-dipole interactions between the polyelectrolytes (Kulkarni, A. D. et al. Polyelectrolyte complexes: mechanisms, critical experimental aspects, and applications, Artificial cells, nanomedicine, and biotechnology, 44(7): 1615-1625, 2016).

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

[0021] According to Gucht et al., 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 electric double layer: a zone with increased concentration of counterions and reduced concentration of co-ions. The double layer has lower energy (the average distance between positive and negative charges is smaller than between positives or between negatives), but also 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 energy (enthalpy) and entropy of the system. Both contributions vary with salt concentration (ionic strength).

[0022] Le and Cerf comment that parameters affecting polyelectrolyte complex characteristics 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, H. V.; Cerf, D. L.; 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.

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

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

[0025] Increasing polyelectrolyte concentration favors polyelectrolyte complex formation, due to formation of larger particles and higher yield of particles or macroaggregation thanks to the involvement of more polymer chains. Macroaggregation is also favored if the molar mass of polyelectrolyte is relatively high or if the concentration of polyelectrolyte is extremely high.

[0026] Increasing ionic strength reduces the absolute value of zeta potential of polyelectrolytes complex particles, favoring higher yield due to particle aggregation or even macroscopic precipitation, given the weakened interparticle electrostatic repulsion (weaker repulsive force). However, extremely high ionic strength can prevent polyelectrolyte complex formation or cause its disintegration because electrostatic attraction between polyelectrolytes with opposite charge is reduced (Le and Cerf, 2022).

[0027] According to Kulkarni et al. (Kulkarni et al., 2016), polyelectrolyte complexes can have applications in various technological fields as they are biodegradable, biocompatible and non-toxic. These authors also mention the limitation of large-scale commercial production of polyelectrolyte complexes and identified few patent documents related to the subject. Indeed, no processes and formulations comprising polyelectrolyte complexes of hyaluronic acid and cationic hyaluronic acid in pharmaceutical grade for injectable use have been identified in the prior 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 commented, due to their rheological properties provided by covalent bonds, cross-linked hyaluronic acid compositions are more resistant compared to non-cross-linked hyaluronic acid compositions, especially regarding thermal degradation during steam sterilization. A major challenge for injectable formulations comprising a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid may be the stability against thermal degradation during steam sterilization because their viscoelastic properties arise from electrostatic interactions. Thus, there is a need in the prior art for processes for preparing formulations comprisinga polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid, applicable to industrial scale, wherein 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 of hyaluronic acid and cationic hyaluronic acid that overcomes the need of the prior art. Surprisingly, the process of the present invention comprises the concomitant reactions of cationization of hyaluronic acid or salt thereof and formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof; 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 below.Brief description of the drawings

[0029] Figure 1 shows: Frequency sweep as a function of modules G' and G" (Pa) of injectable formulations of polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid prepared: without thermal protector and without sterilization (Test A, orange); without thermal protector 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 modules G' and G" (Pa) of injectable formulations of polyelectrolyte complex gel of 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 modules G' and G" (Pa) of injectable formulation of polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid prepared with 1.2% sorbitol and after sterilization (Test G).

[0032] Figure 4 shows: Frequency sweep as a function of modules G' and G" (Pa) of injectable formulations prepared with the following ratios of polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid and hyaluronic acid with molar mass of 1.4 MDa, with 1.2% mannitol, after sterilization: 1:0 (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 Batch 1 (light blue), Batch 2 (green), Batch 3 (blue) and commercial comparator Juvederm® Voluma (black).Summary of the invention

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

[0035] In a first aspect, the present invention discloses a process for preparing an injectable formulation comprising a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid comprising the steps of: (a) homogenizing hyaluronic acid or salt thereof, an alkali metal halide salt, an antioxidant, in alkaline aqueous 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) maintaining the reaction mixture at rest for 1-24 h at 40-60 °C, wherein the concomitant reactions of cationization of hyaluronic acid or salt thereof and formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronicacid or salt thereof occur; (d) purifying the gel via dialysis with phosphate-buffered saline (PBS) for 48-72 h at 20-30 °C with 4-10 exchanges of washing solution, drying the gel under vacuum and adjusting the total hyaluronic acid content to 15-30 mg / mL with phosphate-buffered saline (PBS); (e) extruding the gel obtained in step (d), adding mannitol and optionally an anesthetic and / or hyaluronic acid or salt thereof, adjusting the pH to 6.5-7.8, homogenizing the formulation for 2-24 h at 20-30 °C, filling into a prefilled syringe and sterilizing under steam at 100-124 °C for 1-5 minutes of sterilization.

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

[0037] In a third aspect, the present invention discloses the use of an injectable formulation comprising a polyelectrolyte complex gel of 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] In a fourth aspect, the present invention discloses a process for preparing a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid comprising the steps of: (a) homogenizing hyaluronic acid or salt thereof, an alkali metal halide salt, an antioxidant, in alkaline aqueous 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) maintaining the reaction mixture at rest for 1-24 h at 40-60 °C, wherein the concomitant reactions of cationization of hyaluronic acid or salt thereof and formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof occur; (d) purifying the gel via dialysis with phosphate-buffered saline (PBS) for 48-72 h at 20-30 °C with 4-10 exchanges of washing solution, drying the gel under vacuum and adjusting the total hyaluronic acid content to 15-30 mg / mL with phosphate-buffered saline (PBS).

[0039] In a fifth aspect, the present invention discloses a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof, obtained according to the process disclosed in the present invention.

[0040] In a sixth aspect, the present invention discloses the use of a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid, obtained according to the process disclosed in the present invention, for the manufacture of an injectable formulation intended 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, concomitantly prepares cationic hyaluronic acid and a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid, in alkaline aqueous medium, without the presence of organic solvents. Furthermore, the present invention relates to an injectable formulation comprising the polyelectrolyte complex gel, mannitol and optionally an anesthetic and / or hyaluronic acid with molar mass of 1-6 MDa, wherein exhibits rheological properties comparable to injectable formulations of covalently cross-linked hyaluronic acid.

[0042] According to a first aspect, the present invention relates to a process for preparing an injectable formulation comprising a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid comprising the steps of:(a) homogenizing hyaluronic acid or salt thereof with an average molar mass of 1-6 MDa in alkaline aqueous 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) maintaining the reaction mixture of step (b) at rest for 1-24 h at 40-60 °C, wherein the concomitant reactions occur:i. cationization of hyaluronic acid or salt thereof; and ii. formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof occurs;(d) purifying the polyelectrolyte complex gel obtained in step (c) by dialysis with phosphate-buffered saline (PBS) for 48-72 h at 20-30 °C with 4-10 exchanges of washing solution, drying the gel under vacuum and adjusting the total hyaluronic acid content to 15-30 mg / mL with phosphate-buffered saline (PBS);(e) extruding the gel obtained in step (d), adding mannitol and optionally an anesthetic and / or hyaluronic acid or salt thereof with average molar mass of 1-6 MDa, adjusting the pH to 6.5-7.8, homogenizing the formulation for 2-24 h at 20-30 °C, filling into a prefilled syringe and sterilizing 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, sodium hyaluronate, calcium hyaluronate, magnesium hyaluronate, potassium hyaluronate, zinc hyaluronate, commercially available, having an average molar mass of 1-6 MDa. Preferably, sodium hyaluronate is employed as starting material at 4-14% (w / w) 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 use of high molar mass of hyaluronic acid (1-6 MDa) in the process of the present invention promotes greater entanglement of the polysaccharide chains, however, it results in a significant increase in the viscosity of reaction mixture and, consequently hinders homogenization and cationization due to steric hindrance. Therefore, an anchor-type impeller was used in steps (a) and (b) to assist homogenization and avoid shearing of the hyaluronic acid chains.

[0045] Two homogenization steps were performed: the first homogenization step, step (a), is required to ensure the formation of an alkoxide group on the primary hydroxyls of hyaluronic acid; the second homogenization step, step (b), favors a betterdistribution of the cationizing agent in the vicinity of the hydroxyls.

[0046] The cationization reaction and concomitant formation of polyelectrolyte complex according to the process of the present invention involve hyaluronic acid or salt thereof with average molar mass of 1-6 MDa and a cationizing agent, in alkaline aqueous 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 alkaline with pH between 9-13, a base is added wherein is selected from inorganic bases such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide and mixtures thereof, or organic bases such as ethylenediamine, triethylamine and trimethylamine, at 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, but not limited to, ascorbic acid, citric acid, sodium metabisulfite, tocopherol, butylhydroxytoluene, butylhydroxyanisole, propyl gallate, histidine, methionine, cysteine and polyol 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 of 2 to 6 units and mixtures thereof, in a concentration range of 1- 5 % (w / w). Preferably, the antioxidant is mannitol.

[0049] The ionic strength in the reaction medium of the present invention is ensured by the presence of an alkali metal halide salt selected from halides fluoride, chloride, bromide or iodide combined with alkali metals sodium, potassium or lithium, and mixtures thereof, but not limited to these ones, in an amount range of 1-5 % (w / w). Preferably, the alkali metal halide salt is sodium chloride.

[0050] The function of the cationizing agent is to add positive charge preferably to the primary hydroxyls of hyaluronic acid in aqueous solution. Examples of cationizingagents include, but are not limited to: 3-halo-2-hydroxypropyltrialkylammonium halide,2-halo-3-hydroxypropyltrialkylammonium halide, 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- hydroxypropyldimethylstearyl ammonium chloride. According to the process of the present invention, the amount of cationizing agent is determined based on the molar ratio of hyaluronic acid and cationizing agent ranging from 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 process of the present invention, as it is formed, the cationic hyaluronic acid immediately and spontaneously forms a polyelectrolyte complex when in contact with hyaluronic acid which has an anionic character in alkaline 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 formation of the polyelectrolyte complex with cationic hyaluronic acid, evidencing its behavior as a polycation.

[0052] As previously mentioned, the reaction medium is viscous due to the high molar mass of hyaluronic acid. Formation of the polyelectrolyte complex of hyaluronic acid and cationic hyaluronic acid, which is water-insoluble gel, makes the medium even more viscous. Facing 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 prior homogenization steps to ensure the efficiency of the process.

[0053] The polyelectrolyte complex gel obtained according to the process of the present invention, having pH in the range 9-13, is purified using dialysis with phosphate- buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 exchanges of washing solution.To avoid abrupt pH changes, pH adjustment was performed slowly during dialysis using phosphate-buffered saline (pH 7.2) without apparent product destabilization, 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 water not covalently bound to the gel is removed.

[0055] After adjusting the total hyaluronic acid content to 15-30 mg / mL with phosphate-buffered saline (PBS) and extruding the obtained gel, an injectable formulation is prepared comprising the polyelectrolyte complex gel, mannitol and optionally an anesthetic and / or hyaluronic acid with molar mass of 1-6 MDa.

[0056] In the injectable formulation, mannitol has the function of a thermal protector, particularly during steam sterilization, present at 0.5-4.0% (w / w).

[0057] Additionally, the injectable formulation optionally comprises an anesthetic selected from, but not limited to, benzocaine, bupivacaine, lidocaine, ropivacaine, tetracaine or their salts, in an amount range of 0.1-0.4 % (w / w). Preferably, the optional anesthetic is lidocaine hydrochloride.

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

[0059] In view of the foregoing, unexpectedly and surprisingly, the present invention discloses a process for preparing an injectable formulation capable of concomitantly preparing a cationic hyaluronic acid and a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid - steps (a) to (d) - and an injectable formulation comprising the polyelectrolyte complex gel, mannitol and optionally an anesthetic and / or hyaluronic acid with molar mass of 1-6 MDa - step (e).

[0060] According to an embodiment of the present invention, the process for preparing an injectable formulation comprising a polyelectrolyte complex gel ofhyaluronic acid and cationic hyaluronic acid comprises the steps of:(a) homogenizing hyaluronic acid or salt thereof with an average molar mass of 1-6 MDa, sodium chloride and mannitol, in alkaline aqueous 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;(c) maintaining the reaction mixture of step (b) at rest for 1-24 h at 40-60 °C, wherein the concomitant reactions occur: i. cationization of hyaluronic acid or salt thereof; and ii. formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof;(d) purifying the polyelectrolyte complex gel obtained in step (c) via dialysis with phosphate-buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 exchanges of washing solution, drying the gel under vacuum and adjusting the total hyaluronic acid content to 15-30 mg / mL with phosphate-buffered saline (PBS);(e) extruding the gel obtained in step (d), adding mannitol and optionally the anesthetic lidocaine hydrochloride and / or hyaluronic acid or its salt with an average molar mass of 1-6 MDa, adjusting the pH to 6.5-7.8, homogenizing the formulation for 2-24 h at 20-30 °C, filling into a prefilled syringe and sterilizing 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 the polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid.

[0062] 3-chloro-2-hydroxypropyltrimethylammonium chloride in alkaline medium forms 2,3-epoxypropyltrimethylammonium chloride, which is an epoxide. Hyaluronic acid in alkaline medium forms the alkoxide at the primary hydroxyl group. Thus, the epoxide can bind with the alkoxide of hyaluronic acid by an etherification reaction, forming cationic hyaluronic acid.

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

[0064] In a second aspect, the present invention discloses an injectable formulation obtained according to the process defined in the first aspect and its embodiments, comprising a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof, mannitol, optionally an anesthetic and / or hyaluronic acid or salt thereof with an average molar mass of 1-6 MDa and a pharmaceutically acceptable carrier, which is phosphate-buffered saline (PBS).

[0065] According to an 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 of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof with degree of cationization of 0.2- 0.7;(b) 0.5-4.0 % (w / w) of mannitol;(c) optionally 0.1-0.4 % (w / w) of anesthetic lidocaine hydrochloride;(d) optionally 0.1-0.4 % (w / w) of hyaluronic acid or salt thereof with average molar mass of 1-6 MDa;(e) a pharmaceutically acceptable carrier, which is a phosphate-buffered saline (PBS), wherein the pH of the formulation is in the range of 6.5-7.8.

[0066] According to an embodiment of the present invention, the injectable formulation comprises a polyelectrolyte complex of sodium hyaluronate and hydroxypropyltrimethylammonium hyaluronate chloride.

[0067] According to an 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 of sodium hyaluronate and hydroxypropyltrimethylammonium hyaluronate chloride with a degree of cationization of 0.2-0.7;(b) 1.2 % (w / w) of mannitol;(c) 0.3 % (w / w) of lidocaine hydrochloride;(d) a pharmaceutically acceptable carrier, which is a phosphate-buffered saline (PBS), wherein the pH of the formulation is 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. Tests performed in the present invention show that sorbitol, which is also a known thermal protector of the same class as mannitol, failed to protect the formulation from thermal degradation induced by the sterilization process. Thus, unexpectedly, mannitol proved to be capable of thermally protecting the injectable formulation.

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

[0070] In a third aspect, the present invention discloses the use of an injectable formulation comprising a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid, defined in the second aspect 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 of hyaluronic acid and cationic hyaluronic acid, defined in the first aspect of the present invention and its embodiments, can be carried out in its entirety or, alternatively, the polyelectrolyte complex gel of 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 discloses as a fourth aspect a process for preparing a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid, comprising the steps of:(a) homogenizing hyaluronic acid or salt thereof, an alkali metal halide salt, an antioxidant, in alkaline aqueous 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) maintaining the reaction mixture at rest for 1-24 h at 40-60 °C, wherein the concomitant reactions occur: i. cationization of hyaluronic acid or salt thereof; and ii. formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof;(d) purifying the gel via dialysis with phosphate-buffered saline (PBS) for 48- 72 h at 20-30 °C, with 4-10 exchanges of washing solution, drying the gel under vacuum and adjusting the total hyaluronic acid content to 15-30 mg / mL with phosphate- buffered saline (PBS).

[0073] In a fifth aspect, the present invention discloses a polyelectrolyte complexgel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof, obtained according to the process disclosed in the present invention.

[0074] According to an embodiment, the present invention discloses a polyelectrolyte complex gel of sodium hyaluronate and hydroxypropyltrimethylammonium hyaluronate chloride, obtained according to the process disclosed in the present invention.

[0075] In a sixth aspect, the present invention discloses the use of a polyelectrolyte complex gel of 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 an embodiment, the present invention discloses the use of a polyelectrolyte complex gel of sodium hyaluronate and hydroxypropyltrimethylammonium 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 additionally comprises mannitol and optionally an anesthetic and / or hyaluronic acid or salt thereof with an average molar mass of 1-6 MDa.Examples

[0077] The following non-limiting examples serve to illustrate aspects of the present invention.EXAMPLE 1 - Preparation of an injectable formulation comprising a polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid; evaluation of the effect of thermal protector 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 into 10 mL of 0.25 M sodium hydroxide, according to the conditions described in Table 1, under homogenization for 1 h at 25 °C using an anchor-type impeller at 300 rpm. After this period, 60 % of 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) was added to the reactor according to conditions described in Table 1, under homogenization for 1 h at 25 °C using an anchor-type impeller at 300 rpm. Next, the reaction mixture was maintained at rest for 4.5 h at 52 °C. During this period, in alkaline aqueous medium, the cationization of hyaluronic acid occurs, forming hydroxypropyltrimethylammonium hyaluronate chloride and the concomitant formation of a polyelectrolyte complex gel of sodium hyaluronate and hydroxypropyltrimethylammonium hyaluronate chloride which exhibits rheological behavior typical of a weak gel with G' > G" over the complete frequency axis.Table 1. Description of reagents used in the cationization reaction.

[0079] In a previous study, purification of the polyelectrolyte complex gel was carried out by successive washings with phosphate-buffered saline (PBS). However, a mass loss was observed, evidenced by an increase in the viscosity of the washing solutions after purification. To solve this problem, dialysis technique was used to purify the polyelectrolyte complex gel in a dialysis bag with molar mass cut-off of 12-16 MDa and porosity of 25 A, with 6 exchanges of phosphate-buffered saline (PBS) with pH 7.2 ± 0.1, for 48 h at 25 °C, in order to ensure the absence of cationizing agent. Then, the gel drying was carried out under vacuum. 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 pm mesh.

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

[0081] As previously mentioned, the sterilization process is critical for the rheological characteristics of the injectable formulation of the present invention. In order to evaluate the effects of the presence or absence of a thermal protector or different thermal protectors 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 thermal protector on the injectable formulation of polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid with respect to degradation, based on rheological data (see Figures 1 to 3).

[0082] As can be seen in Figure 1, the injectable formulation of Test B, prepared without adding a thermal protector at the formulation step and with sterilization, exhibited rheological behavior of a polymer solution (G" modulus (viscous) > G' modulus (elastic)), characterized by the presence of a cross-over at 3.59 Hz, indicating degradation of the polyelectrolyte complex.

[0083] The effect of sterilization in the presence of 0.7 % mannitol reduced 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 1.2 % mannitol, wherein 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, Test G (Figure 3), prepared with the addition of 1.2% sorbitol in the formulation step and with sterilization, exhibited a polymer solution rheological behavior (G">G') and the presence of cross-over, i.e., sorbitol failed to prevent thermal degradation of the injectable formulation, although there are several reports in the state of the art mentioning the use of sorbitol as a thermal protector 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 of hyaluronic acid and cationic hyaluronic acid, and hyaluronic acid of 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 sterilized under steam, in order to evaluate the contribution of added hyaluronic acid to the rheological behavior of the final product.Table 3. G' and G" values of injectable formulations comprising different mass proportions of polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid, and hyaluronic acid of molar mass of 1.4 MDa, sterilized under steam.

[0086] Figure 4 shows the frequency sweep of Tests H-K according to Table 3. It can be seen that G' increased from 176 Pa in Test H with ratio 1:0 (blue) to 1291 Pa in Test I with ratio 1:1 (gray), confirming the positive character of the polyelectrolyte complex gel of the present invention.

[0087] In Test J with ratio 1:2, 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 positive charge in the polyelectrolyte complex gel to interact with 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 relative to Test I, indicating that in this mass ratio all negative charges of unmodified hyaluronic acid were occupied, i.e., they formed electrostatic interactions with the positive charges of the polyelectrolyte complex gel, leaving positive charges remaining in the product of Test K.EXAMPLE 3 - Evaluation of process reproducibility.

[0089] Three batches were prepared according to Test F procedure of Example 1 and analyzed in order to evaluate process reproducibility. Table 4 presents resultsobtained for physicochemical characterization of Batches 1 to 3.Table 4. Physicochemical characterization of injectable formulations of polyelectrolyte complex gel of hyaluronic acid and cationic hyaluronic acid, prepared according to Test F of Example 1.*Using 231 / 2G needle (Terumo) and 1 mL syringe (Schott).

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

[0091] Total hyaluronic acid contents are close to the theoretical value (24.1 mg / mL) which was adjusted considering the water content of the hyaluronic acid raw material with 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® Puresense Ultimate (Teoxane) was used, which has an extrusion force of 25 N.

[0093] The G' (elastic modulus) and G" (viscous modulus) values at 1 Hz obtained in frequency sweeps show that Batches 1-3 maintained weak gel behavior with G' > G" and both modules parallel to the frequency axis, even after sterilization, demonstrating that there was no thermal degradation of the injectable formulations of polyelectrolyte complex gel of 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 surface charge of particles in solution. In the case of the present invention, since the injectable formulation is insoluble in water, zeta potential results are only indicative (not conclusive).

[0095] Zeta potential results for Batches 1-3 presented in Table 4 (mean value -13±1 mV) showed reduction of negative charge density relative to unmodified hyaluronic acid (-40 mV), indicating the occurrence of hyaluronic acid cationization reaction and formation of a polyelectrolyte complex of hyaluronic acid and cationic hyaluronic acid through the carboxyl 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 Juvederm® Voluma (hyaluronic acid covalently cross-linked with BDDE, at 20 mg / mL, free of mannitol) 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 Flegeau et al. (Flegeau et al.; Multidose Hyaluronidase Administration as an Optimal Procedure to Degrade Resilient HyaluronicAcid Soft Tissue Fillers, Molecules, 28: 1003, 2023). To that end, degradation kinetics of the products in the presence of 30% hydrogen peroxide was performed, at a proportion of 10:1, evaluating G' as a function of time for 20 minutes.

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

[0098] It should be understood that the embodiments described above are merely illustrative and that any modifications may be made thereto by a person skilled in the art. Consequently, the present invention should not be considered limited to the embodiments described in this report.

Claims

CLAIMS1. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID characterized by comprising the steps of:(a) homogenizing hyaluronic acid or salt thereof, with an average molar mass of 1-6 MDa, in an alkaline aqueous 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) maintaining the reaction mixture of step (b) at rest for 1-24 h at 40-60 °C, wherein the concomitant reactions occur: i. cationization of hyaluronic acid or salt thereof; and ii. formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof;(d) purifying the polyelectrolyte complex gel obtained in step (c) by dialysis with phosphate-buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 exchanges of washing solution, drying the gel under vacuum, and adjusting the total hyaluronic acid content to 15-30 mg / mL with phosphate-buffered saline (PBS);(e) extruding the gel obtained in step (d), adding mannitol and optionally an anesthetic and / or hyaluronic acid or salt thereof with an average molar mass of 1- 6 MDa, adjusting the pH to 6.5-7.8, homogenizing the formulation for 2-24 h at 20-30 °C, filling into a prefilled syringe, and sterilizing under steam at 100-124 °C for 1-5 minutes.

2. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized by the hyaluronic acid or salt thereof is selected from sodium hyaluronate, calcium hyaluronate, magnesium hyaluronate, potassium hyaluronate, and zinc hyaluronate, preferably sodium hyaluronate, with anaverage molar mass of 1-6 MDa, at 4-14% (w / w) relative to the mass of the reaction mixture of step (c).

3. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized by the base is selected from inorganic bases such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide and mixtures thereof, or organic bases such as ethylenediamine, triethylamine, and trimethylamine, preferably sodium hydroxide, in order to maintain the alkaline aqueous reaction medium with a pH between 9 andl3.

4. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized by the antioxidant is selected from ascorbic acid, citric acid, sodium metabisulfite, tocopherol, butylhydroxytoluene, butyl 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, polyglycerols containing 2 to 6 units, and mixtures thereof, preferably mannitol, at 1-5% (w / w).

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

6. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING APOLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized by the cationizing agent is 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 based on a molar ratio between hyaluronic acid and cationizing agent ranging from 1:1 to 1:20.

7. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING APOLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized by mannitol in step (e) has the function of a thermal protector of the injectable formulation, at 0.5-4.0 % (w / w).

8. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, according to claim 1, characterized by the anesthetic is selected from benzocaine, bupivacaine, lidocaine, ropivacaine, tetracaine or their salts, preferably lidocaine hydrochloride, at 0.1-0.4% (w / w).

9. PROCESS FOR PREPARING AN INJECTABLE FORMULATION COMPRISING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID characterized by comprising the steps of:(a) homogenizing hyaluronic acid or salt thereof with an average molar mass with 1- 6 MDa, sodium chloride, and mannitol, in an alkaline aqueous 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;(c) maintaining the reaction mixture of step (b) at rest for 1-24 h at 40-60 °C, wherein the concomitant reactions occur: i. cationization of hyaluronic acid or salt thereof; andii. formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof;(d) purifying the polyelectrolyte complex gel obtained in step (c) via dialysis with phosphate-buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 exchanges of washing solution, drying the gel under vacuum, and adjusting the total hyaluronic acid content to 15-30 mg / mL using phosphate-buffered saline (PBS);(e) extruding the gel obtained in step (d), adding mannitol and optionally the anesthetic lidocaine hydrochloride and / or hyaluronic acid or salt thereof with an average molar mass of 1-6 MDa, adjusting the pH to 6.5-7.8, homogenizing the formulation for 2-24 h at 20-30 °C, filling into a prefilled syringe, and sterilizing under steam at 100-124 °C for 1-5 minutes.

10. INJECTABLE FORMULATION, obtained according to the process defined in claim1, characterized by comprising:(a) 15-30 mg / mL of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof, with a degree of cationization of 0.2- 0.7;(b) 0.5-4.0 % (w / w) of mannitol;(c) optionally 0.1-0.4 % (w / w) of anesthetic lidocaine hydrochloride;(d) optionally 0.1-0.4 % (w / w) of hyaluronic acid or salt thereof with an average molar mass of 1-6 MDa; and(e) a pharmaceutically acceptable carrier, which is phosphate-buffered saline (PBS), wherein the pH of the formulation is in the range between 6.5 and 7.8.

11. INJECTABLE FORMULATION, according to claim 10, characterized by comprising a polyelectrolyte complex of sodium hyaluronate and hydroxypropyltrimethylammonium hyaluronate chloride.

12. INJECTABLE FORMULATION, obtained according to the process defined in claim9, characterized by comprising:(a) 20-25 mg / mL of a polyelectrolyte complex gel of sodium hyaluronate and hydroxypropyltrimethylammonium hyaluronate chloride with a degree of cationization of 0.2-0.7;(b) 1.2 % (w / w) of mannitol;(c) 0.3 % (w / w) of anesthetic lidocaine hydrochloride; and(d) a pharmaceutically acceptable carrier, which is phosphate-buffered saline (PBS), wherein the pH of the formulation is in the range between 6.5 and 7.8.

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

14. PROCESS FOR PREPARING A POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID characterized by comprising the steps of:(a) homogenizing hyaluronic acid or salt thereof with an average molar mass of 1-6 MDa, in a alkaline aqueous 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) maintaining the reaction mixture of step (b) at rest for 1-24 h at 40-60 °C, in which the concomitant reactions occur: i. cationization of hyaluronic acid or salt thereof; and ii. formation of a polyelectrolyte complex gel of hyaluronic acid or salt thereof and cationic hyaluronic acid or salt thereof;(d) purifying the polyelectrolyte complex gel obtained in step (c) via dialysis with phosphate-buffered saline (PBS) for 48-72 h at 20-30 °C, with 4-10 exchanges of washing solution, drying the gel under vacuum, and adjusting the total hyaluronic acid content to 15-30 mg / mL using phosphate-buffered saline (PBS).

15. POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, obtained according to the process defined in claim 14, characterized by comprising a polyelectrolyte complex of hyaluronic acid or salt thereof, preferably sodium hyaluronate, and cationic hyaluronic acid or salt thereof, preferably hydroxypropyltrimethylammonium hyaluronate chloride.

16. USE OF POLYELECTROLYTE COMPLEX GEL OF HYALURONIC ACID AND CATIONIC HYALURONIC ACID, obtained according to the process defined in claim 15, characterized by being for the manufacture of an injectable formulation intended for application as intradermal or intra-articular filler.

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