Hyaluronic acid hydrogel-based dermal filler compositions and processes for the manufacture thereof

Abstract

A process is provided for manufacturing injectable hyaluronic acid (HA) dermal fillers consisting of cross-linking high molecular weight (MW) HA with the cross-linking agent 1,4-butanediol diglycidyl ether (BDDE) with a relatively small amount of BDDE by reaction in a planetary centrifuge under vacuum or with a relatively large amount of BDDE with mechanical agitation for the reaction and an ultra-turrax device for particle sizing. The processes generate HA hydrogels with only one degree of cross-linking for use in manufacturing HA dermal fillers with varying concentrations of HA and combinations of particle sizes. HA dermal fillers are based on particle balance technology (PBT), in which hydrogels with different physico-chemical and rheological properties are obtained. They can be used in medical surgery and orthopaedic applications, as well as for dermal filling, body and facial contouring, volumisation and skin hydration.

Description

Dermal filler compositions based on hyaluronic acid hydrogels and processes for their manufacture Field of invention

[0001] The present invention relates to dermal filler compositions and, more specifically, to injectable dermal fillers, including hyaluronic acid (HA) hydrogels and their preparation process. Background of the invention

[0002] Hyaluronic acid (HA) is a naturally occurring compound found in various parts of the human body, including the skin, joints, and eyes. Hyaluronic acid belongs to the glycosaminoglycan family and acts as a fundamental component of the extracellular matrix, providing crucial structural support for cellular dynamics and tissue functionality.

[0003] HA has unique physical properties that make it a versatile and essential compound for the skin. HA has a strong capacity to retain water molecules, playing a key role in tissue hydration, thus contributing to skin elasticity, firmness, and overall appearance. Its viscoelastic nature plays a crucial role in tissue lubrication and protection of cellular structures, promoting tissue health and vitality.

[0004] HA signaling stimulates the production of collagen, a protein essential for tissue strength and integrity. This collagen-inducing effect is crucial for skin rejuvenation and revitalization. HA also signals its own endogenous self-renewal production, thus restoring the balance between degradation and synthesis.

[0005] These properties have led to HA being widely used in aesthetic procedures to reduce the appearance of wrinkles and add volume to various areas of the face. Furthermore, HA plays a central role as a common ingredient in a wide variety of skincare products, including moisturizers, serums, and topical beauty formulations.

[0006] The multifaceted potential of HA in hydration, lubrication and cell signaling justifies its relevance in several applications, including, prominently, the object of the present invention.

[0007] The process of preparing HA fillers involves several steps (Rzany B., 2018; Narins RS, 2016; Burgess CM 2018; Micheels P. 2019).

[0008] HA can be obtained from several sources, including fermentation using native or genetically modified bacteria or extraction from gallstone crystals. The most common method used by manufacturers is bacterial fermentation. Typically, HA is a sodium salt (NaAH), which is thermodynamically more stable than its acidic form.

[0009] Once obtained, the HA undergoes a series of purification processes to remove impurities such as proteins, nucleic acids, and endotoxins. Purification methods may include filtration, adsorption, and precipitation. The goal is to obtain a highly pure form of AHA (>99.5% purity).

[0010] HA fillers typically undergo a reaction called crosslinking, which involves the formation of chemical bonds between the HA chains mediated by a crosslinking agent. The crosslinking agents used can vary, but commonly used agents include 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS).

[0011] BDDE is the most widely used dermal filler. It forms covalent bonds with the hydroxyl groups in HA disaccharides, creating a three-dimensional network of interconnected HA chains.

[0012] Cross-linking is a crucial step in preparation because it forms the hydrogels that provide the physicochemical and rheological properties of dermal fillers. It also offers resistance to enzymatic degradation and free radical oxidation, promoting long-lasting effects at the injection site.

[0013] The crosslinking reaction between HA and the crosslinking agent typically occurs under controlled conditions. Factors such as temperature, pH, reaction time, and stirring speed are carefully optimized to ensure the desired level of crosslinking while maintaining the integrity and biocompatibility of the HA filler.

[0014] The degree of crosslinking determines the desired properties of the final HA filler. It is controlled by the molar ratio (also called the molar ratio) of the crosslinking agent to the HA monomer and the reaction conditions. The process yield determines the degree of HA modification, also called the degree of crosslinking (or crosslinking degree), which is the percentage of the polymer chain modified by the crosslinking agent. This modification gives hydrogels distinct physicochemical and rheological properties. Higher degrees of crosslinking generally result in a more robust, stiffer, and longer-lasting filler, while lower levels of crosslinking can produce a softer, more easily moldable product.

[0015] The amount of BDDE in the reaction must be sufficient to ensure an effective crosslinking reaction. The BDDE-to-HA molar ratio (1 BDDE chain to 1 HA disaccharide) typically ranges from 0.1:1 to 2:1, depending on the desired properties of the HA filler, from softer to stiffer, respectively.

[0016] The most common technique for the crosslinking reaction between HA and BDDE is mechanical mixing using an impeller, which can be followed by an oven step. This process can take several hours. However, the crosslinking of HA with BDDE can be enhanced by increased mixing efficiency using a planetary centrifuge, reducing processing time.

[0017] For example, Galderma for Restylane® and Allergan for Juvederm® use their proprietary technologies. Both products preferably use HA in its salt form, e.g., sodium hyaluronate (NaAH). The salt form is then cross-linked in an alkaline medium with a BDDE, which interconnects the HA chains through a chemical reaction with their hydroxyl group (US11058640B1, US20160376382A1, US8394782B2, Kenne L. 2012; Faivre J. 2021; Xue Y. 2020).

[0018] Galderma has NASHA (Non-Animal Stabilized Hyaluronic Acid) technologies, which use viscous high molecular weight (MW) HA, physically cross-linked only by the intertwining of its chains or with very low chemical cross-linking to obtain firmer textures and a more pronounced lifting (Micheels P., 2016; Edsman K., 2012; Kablik J., 2016).

[0019] In addition, Galderma also uses OBT (Optimal Balance Technology), which combines the balance between physically cross-linked HAs and chemically cross-linked viscoelastic HA with BDDE to obtain softer textures, which allow for smoother integration with the tissue, providing a more natural appearance (Micheels P., 2016).

[0020] Allergan uses Hylacross and Vycross technologies in its product line. Hylacross technology uses a high ratio of high MW HA to low MW HA, with different degrees of crosslinking and BDDE-to-HA molar ratio (0.1:1 to 0.5:1), resulting in smooth, pliable gels with homologous consistency (Brandt, 2008; Duranti, 1998; US6921819B2).

[0021] Vycross technology uses a higher proportion of low-PM HA compared to high-PM HA, resulting in efficient crosslinking. The product has low swelling capacity, improving smoothness and longer durability (Segurado, 2021; Brandt 2008; Fallacara, 2017).

[0022] The Galderma brand uses various degrees of crosslinking in low and high PM HAs, obtaining different particle sizes using sieving (low shear) (Fundarò, 2022).

[0023] The Allergan brand uses a single degree of crosslinking in low and high molecular weight HAs, with the low molecular weight HA being in greater proportion, generating firmer particles by crosslinking the softer, low molecular weight HA with the crosslinking of the high molecular weight HA (US7741476B2).

[0024] After the cross-linking reaction, the resulting HA hydrogel undergoes a critical series of fundamental processes that determine its composition and properties. First, the hydrogel is broken down (micronized) by low-shear mixing, washed with phosphate-buffered saline (PBS) to remove excess BDDE, and swollen (absorbed water) until it reaches the hydration equilibrium that defines its final mass. This final mass defines the HA concentration in the hydrogel and plays a key role in shaping the hydrogel's properties.

[0025] Subsequently, the swollen HA hydrogel can be subjected to processing using instrumental techniques to obtain cross-linked particles with different microscopic sizes, creating products in the same line that differ in texture, cohesion, viscoelasticity and flexibility and, thus, meeting specific applications and / or functions.

[0026] The most frequently employed techniques are sieving, jet milling and high shear dispersion using ultra-turrax equipment.

[0027] Sieving involves passing the washed and swollen HA hydrogel through meshes of specific sizes, producing particles of varying diameters and isolating those within a desired size range (in other words, the desired diameter range). This process is essential to obtain a consistent particle size distribution (which should preferably be understood as the number of particles characterized by different average sizes or average diameters), a factor that profoundly influences the properties and performance of the resulting dermal filler.

[0028] Jet milling, on the other hand, uses high-velocity jets of gas or compressed air to break larger particles into smaller, more uniform particles. This milling technique can achieve specific particle size distributions in HA hydrogel preparations.

[0029] High-shear dispersion with ultra-turrax equipment operates based on the rotor-stator system. Here, high axial velocity attracts the hydrogel toward the head of the dispersing element, propelling it forcefully with the help of the stator teeth. This dynamic process results in a hydrogel dispersion, with the shear force exerted at high speeds adjustable according to the duration and intensity of the application. This feature allows for control of particle size, i.e., the particle size distribution to be achieved in the process.

[0030] In this context, a high degree of hydrogel crosslinking can produce a product line with firmer textures, varying the particle diameters of the formulations. Conversely, a low degree of crosslinking can produce a product line with softer textures, altering the particle diameters of the formulations. Differences in their cohesion, viscosity, viscoelasticity, durability, and flexibility properties influence the texture of the product lines (Choi, 2020).

[0031] Galderma also combines various degrees of crosslinking with various particle sizes (calibration) obtained through sieving, which distinguishes products for deeper and more superficial fillers. Thus, thicker or thinner hydrogels are obtained by varying the particle size calibration, and firmer or softer hydrogels by varying the degree of crosslinking.

[0032] To further improve dermal filler extrusion and protect HA hydrogel particles from degradation, non-crosslinked HA can be introduced into the HA hydrogel formulation.

[0033] Galderma, Allergan, and other brands use high and low MW fillers and combine chemically crosslinked and non-crosslinked fillers. As is clear to the skilled artisan, these fillers containing HA can be supplied in pre-filled syringes. The total mass of hyaluronic acid in the syringe is adjusted with non-crosslinked HA. For commercial products with high and low MW and crosslinking, with low MW crosslinking being greater than high MW, the change in rheological properties is monitored with a concentration of crosslinked and non-crosslinked HA. Typically, 1% w / w of non-crosslinked HA is added.

[0034] Other commercial products use varying degrees of crosslinking for a given PM and adjust rheological properties with HA concentration; others use methods that involve several sequential crosslinking steps. These strategies alter the viscoelasticity of the rheological property, directing the products toward various functions, such as volumizing, surface wrinkling, and so on.

[0035] These brands of hydrogels contain an anesthetic. Typically, the anesthetic is introduced at the end of the process. For example, lidocaine in an acidic medium (lidocaine HCl) is added and vigorously homogenized into the hydrogels, increasing their cohesion and providing patient comfort by relieving pain during application.

[0036] The final step in the manufacturing process involves sterilizing the HA hydrogel to ensure its safety. Sterilization methods typically include gamma irradiation or autoclaving, which effectively eliminate potential contaminants.

[0037] The sterile filler is then packaged into pre-filled syringes or vials, ready for clinical use. This comprehensive process ensures that the HA hydrogel meets the highest quality and safety standards, making it suitable for both medical and aesthetic applications.

[0038] The current preparation of HA for dermal fillers is a time-consuming process. Starting from a HA previously manufactured by fermentation or extraction, highly purified and with its average molecular weight and distribution characterized, the preparation of dermal fillers involves several steps, such as cross-linking, washing, particle sizing, final mass adjustment with non-cross-linked HA, sterilization, and packaging into syringes.

[0039] Despite these advances in the field, there is still a need for alternative processes and optimizations in the preparation steps of dermal fillers. Summary of the invention

[0040] The present invention relates to advances in the manufacturing of HA hydrogels, focusing on their compositions and manufacturing processes (i.e., production / preparation processes) for injectable dermal fillers using innovative "Particle Balance Technology" (PBT).

[0041] PBT uses HA (preferably in the form of a salt, in particular its sodium salt, i.e. NaAH; it should be understood that preferably the MW values ​​refer to the MW of the said NaAH) with only one MW of HA (e.g. with an average MW in the range of 10 5 Give it a 10 6 For example, the PM with an average of 10 6 Da). PBT uses AH with an average PM of the order of 10 6 Da, which is composed of HA fractions with PMs in the range of 10 5 to 10 7 Yes, preferably in the range of 10 5 to 10 6There is a degree of crosslinking (in a single crosslinking step) with different particle size ratios (which can be understood, for example, as the ratio between the number of particles of a specific defined size and the number of particles of another specific defined size). It is particularly preferable for the HA to be in its salt form, particularly its sodium salt, i.e., NaAH, for the crosslinking reaction. It should be understood here that reference to MW or molecular weight (which can also be referred to as average MW) preferably refers to the average molecular weight. As is clear to those skilled in the art, the average molecular weight can be determined, for example, by mass spectrometry (MS) or size exclusion chromatography (SEC) relative to a standard. Measurements using SEC are particularly preferred, which determines the average MW of the HA and the percentage of its fractions.The balance of particle sizes (i.e., the presence of particles of different average sizes - or different average diameters - and the specific ratio of their numbers) controls the essential features of hydrogels, such as texture, degree of hydration, cohesion and viscoelasticity, allowing them to offer a wide range of rheological and physicochemical properties to dermal fillers.

[0042] According to the invention, the properties of the hydrogels are determined by the manufacturing process comprising specific steps of the crosslinking reaction method, which mainly depends on the AH - BDDE ratio and the efficiency of the reaction, obtaining the particles and producing the PBT hydrogels.

[0043] In a first manufacturing process, the present invention uses a planetary centrifugal mixing equipment under vacuum to intensify the crosslinking reaction, allowing a wide range of rheological and physicochemical properties with a smaller amount of the crosslinker BDDE, such as 0.075:1 (molar ratio between crosslinker and HA), and shorter reaction times due to the high mixing efficiency, compared to commercial dermal fillers.

[0044] The low amount of BDDE avoids the adverse effects caused by the crosslinker, in addition to reducing or eliminating the residual non-crosslinked HA, as well as avoiding exhaustive washing after crosslinking, which results in better quality products.

[0045] Particles are obtained by sieving using different mesh sizes to achieve a balance of different proportions of large and small particles of the desired sizes. Flexible particles have an irregular shape with low polydispersity compared to spherical shapes, characterized by an average diameter, as shown in Table 1.2, and a smooth surface.

[0046] The final product also includes the addition of uncrosslinked HA to improve gel cohesion and extrusion. Typically, approximately 1% w / w of uncrosslinked HA is added.

[0047] These innovations in manufacturing processes make the manufacture of dermal fillers more efficient and less time-consuming.

[0048] The products of the present invention are cohesive and allow the use of a wide range of needles (27-32G), with extrusion force suitable for dermal filler application, as shown in Table 3.

[0049] In a second manufacturing process, the crosslinking reaction is performed using the usual mechanical mixing method, such as a turbine impeller in a temperature-controlled tank. The molar ratios of BDDE-HA are in the range of commercial dermal fillers, from 0.1:1 to 0.5:1. The particles are obtained by high-shear dispersion using ultra-turrax equipment at different speeds and application times. The particles are of the "crunch" type, with less sphericity compared to the sieving method and a rough surface due to the high shear forces applied. The final product also includes the addition of non-crosslinked HA to achieve the total HA mass in the syringe with the desired physicochemical and rheological properties.

[0050] Products from both manufacturing processes may incorporate an anesthetic medication, such as lidocaine, to minimize pain during application, in addition to the effects of its gradual release from the hydrogels.

[0051] The present invention is further described by the following embodiments.

[0052] In a first embodiment, the present invention relates to a process for manufacturing a crosslinked hyaluronic acid (HA) hydrogel comprising a mixture of two particle sizes of 150 µm and 400 µm to 800 µm and with a HA concentration of 12 to 24 mg / ml, the process comprising the following steps: crosslinking the HA characterized by the average MW of 10 6Give with a molar ratio of the crosslinking agent 1,4-butanediol diglycidyl ether (BDDE) to HA of 0.075:1; conduct the reaction of HA and BDDE in a planetary centrifuge under vacuum for less than 1 hour; wash the crosslinked and swelled HA to reach a HA concentration of 12 to 24 mg of HA per g of gel; homogenize the crosslinked and swelled HA into particles using sieves of different mesh sizes and obtain gel particles of two specific diameters between 150 and 800 µm, as measured by optical microscopy; isolate a batch of HA particles with an average diameter of 150 µm and a batch of HA particles with an average diameter ranging from 400 to 800 µm; combine particles characterized by a diameter of 150 µm with particles of a diameter of 400 to 800 µm to fabricate the hydrogels; mix particles of different sizes (i.e., different average diameters) in a planetary centrifuge.

[0053] In a second embodiment, the present invention provides a process for manufacturing a cross-linked hyaluronic acid (HA) hydrogel, which further comprises step h) of cross-linking the HA (average MW of 10 6 Da) with the crosslinking agent 1,4-butanediol diglycidyl ether (BDDE) in the range of 0.1:1 to 0.5:1 molar ratios BDDE-AH containing only one degree of crosslinking and preparation of hydrogels.

[0054] In a third embodiment, the present invention relates to a crosslinked HA hydrogel with a particle size balance that can be obtained according to any of the processes described above.

[0055] In a fourth embodiment, the present invention relates to an aqueous composition comprising the crosslinked HA hydrogel of the present invention and, optionally, a buffering agent and / or a tonicity agent.

[0056] In a fifth embodiment, the present invention relates to a pre-filled syringe, which is pre-filled with the cross-linked HA hydrogel of the present invention or with the aqueous composition of the present invention.

[0057] In a sixth embodiment, the present invention relates to the crosslinked HA hydrogel of the present invention for use in cosmetic surgery (e.g., in dermal filling, body contouring, or facial contouring), in medical surgery (e.g., dermal filling, body contouring, prevention of tissue adhesion, channel formation, incontinence treatment, or orthopedic applications), or for hydration and / or vitalization of the skin.

[0058] In a seventh embodiment, the present invention relates to a cosmetic (non-therapeutic) use of the cross-linked HA hydrogel of the present invention as a dermal filler for the treatment (or reduction) of wrinkles.

[0059] In an eighth embodiment, the present invention relates to a dermal filler containing the crosslinked HA hydrogel of the present invention. Description of figures

[0060] [Fig. 1A] shows images of different particle diameters obtained after homogenization of HA gel in different sieves and [Fig. 1B] shows PBT hydrogels resulting from the combination of small and large particles with different sizes in a ratio of 1:4. Scale = 500 µm.

[0061] [Fig. 2] shows the schematic of the general PBT process.

[0062] [Fig. 3] shows the spectra of NMR 1 H of the degraded PBT hydrogel, showing its components. Detailed description of the invention

[0063] Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing contained herein should be construed as an admission that the present invention does not have a claim to prior invention. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0064] In the event of a conflict, this specification, including the definitions, shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the subject matter pertains. The following definitions are provided to facilitate understanding of the present invention.

[0065] The manufacturing processes according to the invention, based on Particle Balance Technology (PBT), allow obtaining HA particles with different diameters, from 150 to 800 µm, and then combining HA gel particles of two different sizes to obtain a hydrogel with the required physicochemical and rheological properties.

[0066] In the first manufacturing process (or, in other words, in the first stage or in the first step of the process of the invention) to obtain said hydrogels, a low BDDE-HA molar ratio is initially used, preferably in the range of 0.05:1.0 to 0.1:1.0, more preferably in the range of 0.07:1.0 to 0.08:1.0, such as 0.075:1.0, and the crosslinking reaction is carried out, for example, in a planetary centrifuge under vacuum (i.e., under pressure below 1 atm, more preferably 0.1 atm), which intensifies the reaction and optimizes the time, allowing a fast process (e.g., 45 min). The hydrogel is washed to remove residual BDDE (so that the residual content is up to 2 ppm) for 96 h and swollen until reaching the desired HA concentration.The hydrogel is then homogenized through sieves of different mesh sizes (for a non-limiting example, as exemplified herein, the swollen hydrogel is homogenized into large particles using 60, 40, or 20 mesh sieves to obtain particles with 400, 600, or 800 µm in diameter, respectively), and the particles are mixed in a planetary centrifuge for 10 minutes. Non-crosslinked HA (1% w / w) is added to the mixture and incorporated into the particles using a planetary centrifuge for 10 minutes.

[0067] Particles can also be obtained, as an alternative to the planetary centrifuge described above, using an ultra-turrax device, also known as a high-speed homogenizer or rotor-stator homogenizer. This device is commonly used in laboratories to mix, emulsify, disperse, and homogenize various substances, including hyaluronic acid.

[0068] These products can also optionally incorporate (i.e., include) an anesthetic, such as lidocaine, to minimize pain during application. Incorporating lidocaine into hydrogels composed of different particle sizes can generate different controlled-release profiles (immediate release and modified release, for example, with a specific duration), providing greater patient comfort during application.

[0069] The manufacturing processes according to the invention allow for a variety of particle combinations and HA concentrations in hydrogels. The balance of particle sizes (i.e., defined by the particle size distribution) controls essential hydrogel characteristics, such as texture, cohesion, viscoelasticity, and flexibility, making them unique in terms of specific functions for dermal filler application sites.

[0070] Diameter and polydispersity measurements are made using optical microscopy. Both diameter and polydispersity are average values ​​because the particles are flexible, not rigid. Therefore, as should be understood in this document, a reference to particle size (or mean particle size) can be understood as a reference to its mean, as shown in Table 1.2. As is evident to the expert, this parameter can be measured, for example, using optical microscopy or in a dynamic light scattering (DLS) experiment. Preferably, this parameter (or mean particle size / mean particle diameter) is measured using optical microscopy.

[0071] In one embodiment of the process according to the invention, the preparation of a hydrogel comprising a mixture of two particles (i.e. two different types of particles) of HA with diameters of 150 to 800 µm and HA concentration of 12 to 24 mg / g comprises the following steps: crosslinking of the HA characterized by MW (to be understood as average molecular weight) in the range of 10 5 to 10 6 From (preferably 10) 6Da) with the crosslinking agent BDDE, obtained using a molar ratio of BDDE / HA between 0.05:1 and 0.10:1, preferably 0.075:1, preferably in which the reaction of HA and BDDE is conducted in a planetary centrifuge under vacuum for less than 1 hour; wash the crosslinked HA and swell to reach a HA concentration of 12 to 24 mg / g (i.e., mg per g of gel); homogenize the crosslinked and swollen HA gel in sieves of different mesh sizes to obtain particles between 150 and 800 µm in diameter, as measured by optical microscopy; isolate HA particles with different diameters to obtain a batch of HA particles of larger diameter, i.e., large particles, and a batch of HA particles of smaller diameter, i.e., small particles, in which the average diameter of the large particles is larger than the average diameter of the small particles; small particles;(it should be understood that a batch of HA particles with an average diameter of 150 µm and a batch of HA particles with an average diameter ranging from 400 to 800 are obtained) combine HA particles of different sizes (i.e., different average diameters) in the range of 150 to 800 µm to HA hydrogels; and mix the particles of different sizes from both batches obtained in and, preferably in a ratio of 1:4, understood as the numerical ratio of small to large particles in a planetary centrifuge.;

[0072] Optionally, the process may further include the step of mixing non-crosslinked HA (1% w / w) in a planetary centrifuge in the composition of g).

[0073] According to the invention, the process allows the rapid and reproducible obtaining of a wide variety of hydrogels and the fine control of the steps to obtain a HA mixture with the expected physicochemical and rheological requirements.

[0074] The preferred source of HA is bacterial fermentation. HA is classified as high PM, with an average PM of 10 6 From (range of 10) 5 to 10 7 From the).

[0075] In an alternative embodiment, the process of the present invention further comprises steps h) of reticularizing AH (average MW 10 6 Da) with the crosslinking agent 1,4-butanediol diglycidyl ether (BDDE) in the range of BDDE-AH molar ratios from 0.1:1 to 0.5:1 and preparation of hydrogels using only 1 degree of crosslinking.

[0076] The HA and BDDE reaction should preferably be conducted in a mechanically stirred reactor at a controlled temperature and with a processing time of more than 1 hour. This process is followed by particle production using a high-shear homogenizer, such as an Ultra-Turrax, with pre-calibrated rotation speed and application time.

[0077] Optionally, the process may also include the step of mixing non-crosslinked HA (1% w / w) in a planetary centrifuge in the composition obtained.

[0078] The present invention, in one embodiment, relates to a crosslinked HA hydrogel that can be obtained by any of the methods of the present invention, provided herein.

[0079] According to the present invention, hydrogels can be sterilized inside syringes in an autoclave, for example, at 121°C for 5 minutes. It was found that a product treated in this way preserved its rheological properties and was not susceptible to the growth of bacteria and fungi.

[0080] Cellular characterizations should be different, such as particle-mediated signaling for endogenous HA and collagen production. Signaling efficiency depends on particle size and surface properties.

[0081] In the subject of the invention, PBT products have the following advantages in terms of process and product.

[0082] Process-wise, the invention provides highly efficient crosslinking between HA and BDDE using a smaller amount of crosslinking agent (in particular, a BDDE / HA molar ratio of 0.075:1) compared to Galderma and Allergan. The reaction is intensified in a planetary centrifuge under vacuum, as provided herein, which optimizes the reaction time from several hours to less than an hour.

[0083] The present invention also provides, in one embodiment, a pre-filled syringe, which is pre-filled with a cross-linked HA product (or a cross-linked HA hydrogel) or an aqueous composition thereof.

[0084] The present invention also provides, in one embodiment, the use of a cross-linked HA hydrogel product in cosmetic surgery, e.g., dermal filler, body contouring, and facial contouring; in medical surgery, e.g., dermal filler, body contouring, tissue adhesion prevention, channel formation, incontinence treatment, and orthopedic applications; and for skin hydration and / or revitalization. Thus, in one embodiment, the present invention relates to the cross-linked HA hydrogel of the present invention for use in cosmetic surgery, e.g., dermal filler, body contouring, and facial contouring. The present invention relates to the cross-linked HA hydrogel of the present invention for use in medical surgery, e.g., dermal filler, body contouring, tissue adhesion prevention, channel formation, incontinence treatment, and orthopedic applications.The present invention also relates to the cross-linked HA hydrogel of the present invention for use in hydrating and / or revitalizing the skin. Thus, the present invention also relates to the use of the cross-linked HA hydrogel of the present invention for hydrating and / or revitalizing the skin.

[0085] The low amount of BDDE and only one degree of crosslinking allow the manufacture of products in a wide range of HA concentrations (i.e., only one percentage of BDDE-modified HA produces different products), such as the range of 12 to 24 mg / g, with varied viscoelastic properties and applications.

[0086] In PBT technology, the final products, which will be placed in the syringe, are formulated with a mixture of two types of particles, characterized by two different average diameters, in a ratio of small particles to large particles of 1:4 (preferably understood as the numerical ratio), wherein the small particles are characterized by an average diameter of about 150 µm (wherein the term "about" is generally understood to mean ± 10% of the given numerical value, more preferably ± 5% of the given numerical value, even more preferably ± 2% of the given numerical value, even more preferably ± 1% of the given numerical value, again most preferably the given numerical value) in diameter and the large particles are characterized by an average diameter ranging from 400 to 800 µm.The distribution and composition of the particles, in particular the balance of size in the formulation, control the texture, cohesion, viscoelasticity, durability and flexibility of the products, differentiated to meet the specific functions of the dermal filler application sites.

[0087] With a wider and more controlled range of particle sizes, PBT technology can offer a greater variety of textures, cohesion, viscosity, viscoelasticity, durability and flexibility to your products.

[0088] In addition to textures, cohesion, viscosity, durability, and PBT viscoelasticity, formulations can also be characterized by their flexibility. This property determines the behavior of hydrogels after application—their ability to withstand greater or lesser stresses from facial movements without disrupting their structure.

[0089] Flexibility is defined as the greatest stress the hydrogel can withstand before its structure ruptures. In an intact hydrogel, viscoelasticity is greater than viscosity due to the high elastic portion over the fluid portion, provided by crosslinking. This situation is reversed at the rupture stress, with a decrease in viscoelasticity and an increase in viscosity.

[0090] Another objective of the invention is to use the hydrogel formulation as a dermal filler for the treatment of wrinkles. Thus, in another embodiment, the present invention relates to a cosmetic (non-therapeutic) use of the cross-linked HA hydrogel of the present invention, in particular as a dermal filler for the treatment of wrinkles. Furthermore, in one embodiment, the present invention relates to a dermal filler comprising the cross-linked HA hydrogel of the present invention. In accordance with the above, the present invention also relates to the use of the cross-linked HA hydrogel of the present invention in the manufacture of a dermal filler of the present invention.

[0091] As should be understood here, wrinkle treatment can refer to either the reduction or the removal of wrinkles, preferably the reduction of wrinkles (i.e., the reduction of their number, size and / or extent).

[0092] The present invention also provides an aqueous composition comprising a crosslinked HA product of the present invention and, optionally, a buffering agent and / or a tonicity agent.

[0093] The present invention also provides pre-filled syringes, which are pre-filled with a cross-linked HA product or an aqueous composition thereof.

[0094] The present invention also provides a crosslinked HA hydrogel product of the present invention for use in cosmetic surgery, e.g., dermal filler, body contouring, and facial contouring, in medical surgery, e.g., dermal filler, body contouring, prevention of tissue adhesion, channel formation, incontinence treatment, and orthopedic applications, and for hydration and / or revitalization of skin.

[0095] The term "washing," as used here, is conventional and apparent to the skilled artisan. In particular, the skilled artisan is in a position to perform washing as defined here until excess BDDE is removed from the material.

[0096] The invention is illustrated in the following examples. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention, which is defined by the appended claims. Examples

[0097] Example 1 – Synthesis of HA dermal filler with low concentration of BDDE

[0098] 0.25 mol / L NaOH is previously prepared in distilled water, and 9 mL of the alkaline solution is transferred to a container of the SMIDA TMV-200T planetary centrifuge. Then, 1 g of NaAH powder (MW 10 6Da) is weighed and transferred to the vessel containing NaOH for dissolution in a planetary centrifuge for 20 minutes at 2000 rpm and under 10 kPa vacuum. After complete dissolution, a highly viscous, homogeneous and transparent NaAH solution (~12%, w / w) is obtained.

[0099] Crosslinking is done by pipetting 34 µL of BDDE into the alkaline NaAH solution (BDDE-AH molar ratio of 0.075:1) and mixing in a planetary centrifuge for 45 minutes at 2000 rpm and under 10 kPa vacuum. The resulting mixture is a highly crosslinked gel with a compact and yellowish appearance, with a temperature of 45 ± 5 ºC and a weight of 3.1 ± 0.7 g. The crosslinked hydrogel is then immersed in 20 mL of phosphate buffer (PBS) at 4 ºC to stop the reaction and stabilize the pH.

[0100] The partially swollen hydrogel and PBS are transferred to a dialysis membrane with a 14 kDa cutoff and an available volume of approximately 94 cm 3The membrane is then placed in a tank containing 500 ml of PBS, and the hydrogel is dialyzed against PBS for 48 hours to remove unreacted BDDE. The washing step is performed in a continuous-flow system that includes inlet and outlet pumps connected to the tank at 2 ml / min.

[0101] After dialysis, the hydrogel is highly cohesive and has a neutral pH (7.2 to 7.4). The hydrogel is then weighed and cut into small pieces. The HA concentration is adjusted with water for injection (WFI), allowing it to swell overnight. The final hydrogel mass is adjusted according to the desired product, which can range from 12 to 24 mg / g (Table 1.1), conferring different properties to the products.

[0102] The swollen hydrogel is homogenized into large particles using 60-, 40-, or 20-mesh sieves to obtain particles with diameters of 400, 600, or 800 µm, respectively (Table 1.2). Then, approximately 24% of the particulate hydrogel is homogenized into small particles using a 140-mesh sieve to obtain particles with diameters of 150 µm ().

[0103] PBT hydrogel is manufactured by mixing small and large particles in a 1:4 ratio (w / w), with the hydrogel consisting of 20% small particles and 80% large particles (). The mixing is done in a planetary centrifuge for 10 minutes at 1000 rpm.

[0104] Uncrosslinked HA is added to the mixture to ensure greater cohesion stability and facilitate product extrusion. A previously prepared solution of NaAH powder in API is then transferred to the PBT hydrogel to obtain a final free (uncrosslinked) HA concentration of 1% (w / w). The mixture is blended in a planetary centrifuge for 10 minutes at 1000 rpm.

[0105] After this step, an anesthetic, such as lidocaine, can be added to the mixture to the desired concentration, e.g., 3 mg / mL (0.3%, w / w), followed by pH correction to 7.2 to 7.4. Mixing can be done in a planetary centrifuge for 10 minutes at 1000 rpm.

[0106] The resulting PBT hydrogel is filled into sterile 1 mL syringes and autoclaved for 5 minutes at 121 °C.

[0107] Table 1.1. Final hydrogel mass according to the HA concentration of the desired product.HA concentration (mg / g)Final gel mass (g)12,083,315,066,717,557,120,050,024,041,7Table 1.2. Mean particle size ± standard deviation and polydispersity after sieving swollen hydrogels using different mesh sizes20 mesh40 mesh60 mesh140 meshParticle size (µm)800.6 ± 61.3603.4 ± 49.8455.7 ± 32.3156.9 ± 20.6Polydispersity0.0060.0070.0050.017Example 2 – Degree of modification (MOD) of PBT dermal fillers

[0108] HA dermal fillers with particle balance technology (PBT) rely on a single degree of crosslinking, using a low amount of BDDE (BDDE-to-HA molar ratio of 0.075:1) to obtain a wide variety of products. The following sterile PBT formulations were tested for the degree of crosslinking, analyzed by NMR spectroscopy. 1 H.

[0109] The PBT hydrogel was weighed (1 g) and transferred to a 15 ml tube. The enzyme hyaluronidase was pipetted into the gel to a final concentration of 30 U per 0.1 g of hydrogel. The tube was left at 37 °C until the gel was completely degraded, and the enzyme was denatured at 100 °C for 30 minutes. The hydrogel was filtered through a 0.2 µm filter, frozen at -80 °C, and lyophilized for 24 h. The degraded hydrogel was resuspended in D2O, and NMR spectra were obtained. 1 H were recorded on a Bruker 500 MHz instrument at 30 °C.

[0110] MoD was calculated by integrating signals at δ 2 ppm of N-acetyl disaccharide units and at δ 1.6 ppm of BDPE residues. Then, the ratio between the integrals was calculated according to the equation: MoD (%) = (I δ1.6 ppm / 4) / (I δ2 ppm / 3) * 100.

[0111] Sample NMR spectrum 1H of the degraded PBT gel, with an intense peak at 2 ppm and a low-intensity peak at 1.6 ppm. The peaks were integrated and the MoD was calculated. The MoD was found to be 6.15 ± 0.7%.

[0112] Example 3 – Viscoelastic properties of PBT dermal fillers

[0113] The properties of HA products manufactured according to the method described here are shown in Table 2. Viscoelastic properties were measured with an Anton Paar MCR92 rheometer and 25 mm parallel plate geometry at 25 °C. Oscillatory measurements were performed in the linear region at a stress of 1.188 Pa in a frequency range of 0.1 to 10 Hz.

[0114] PBT hydrogels comprise a wide range of products that can vary from 12 to 24 mg of HA per g of gel and different combinations of particle sizes, all manufactured using only one degree of crosslinking.

[0115] Table 2 presents the range of the storage modulus (G'), related to the elastic behavior of the hydrogel, and the loss modulus (G"), related to the viscous behavior of the hydrogel. Because the samples have a gel-like behavior, they will present a G' > G". The extent of the difference between the G' and G" values ​​is represented by tan δ (G" / G'). Values ​​below 1 mean that the sample is more elastic than fluid. However, the lower the tan δ, the more stable the sample. That is, as tan δ increases, the ability of the dermal filler to return to its initial shape during facial movements decreases and its spread increases.

[0116] As the HA concentration in PBT gels increases, G' increases, while tan δ decreases due to the greater elastic properties of hydrogels with a high HA content in the formulation. Hydrogels with low HA concentrations exhibit more fluid behavior, as observed by the increase in their tan δ. For the same HA concentration, the viscoelastic properties of the hydrogels were modulated by the particle size ratio, with elasticity increasing with decreasing diameter of larger particles.

[0117] The technology presented here allows the manufacture of HA dermal fillers with a wide range of properties for different applications.

[0118] Table 2. Viscoelasticity of PBT dermal fillers crosslinked using a low amount of BDDE (BDDE-to-HA molar ratio of 0.075:1) for different HA concentrations and particle size combination.HA concentration (mg / g)Particle size ratio 1:4 (µm)G' at 1 Hz (Pa)G" at 1 Hz (Pa)tan δ at 1 Hz12150:400423,477,90,18150:600397,974,90,19150:800272,6246,50,1715150:400493.4 84,20,17150:600424,360,60,14150:800444,856,30,1317,5150:400603,290,10,15150:600 451,270,70,16150:800541,170,10,1320150:400683,294,90,14150:600634,588,40,14150:800544,171,10,1324150:400727,4110,60,15150:600892,5122,80,14150:800849,588,40,10 Example 4 – Extrusion force of PBT hydrogels

[0119] The injectability of PBT gels was tested using their extrusion force through needles of different internal diameters. The tests were performed on home-made equipment, and the force was expressed in newtons (N), directly affected by the degree of crosslinking. All PBT hydrogels were manufactured with only one degree of crosslinking, and yet the injection force varied with other gel characteristics and properties, such as HA concentration and particle balance.

[0120] The PBT gels were filled into 1 ml syringes, autoclaved, and connected to sterile 27- to 32-gauge needles. The syringes were then connected to the home-made equipment and compressed at 12.5 mm / min until the hydrogel was extruded. The force values ​​are shown in Table 3 and represent the highest peak measured during extrusion.

[0121] PBT hydrogels with a HA concentration of 12 mg / g have a more viscous portion and were therefore extruded through a 32G needle, while PBT hydrogels with 15 and 17.5 mg / g HA were extruded through a 30G needle. PBT gels with the highest HA concentration, 20 and 24 mg / g, have less viscous portions and are stiffer; therefore, they were extruded through a 27G needle.

[0122] Particle size directly influenced hydrogel extrusion, and the force required to extrude gels composed of balanced 150 / 800 µm particles was much greater than that required for formulations composed of smaller particles, especially through 32G and 30G needles. Therefore, a 27G needle would be recommended for this condition. PBT gels with higher HA concentrations, 20 and 24 mg / g, were easily extruded through 27G needles and exhibited extrusion forces between 10 and 30 N, depending on the particle size combination.

[0123] Table 3. Extrusion force of PBT gels with different HA concentrations and particle size equilibrium. HA concentration (mg / g) Particle size ratio 1:4 (µm) Needle (G) Extrusion force (N) 12 150:400 32 36.5 150:600 35.6 150:800 78 15 150:400 30 25.6 150:600 18.5 150:800 57 150:400 30 22.3 150:600 3 3,1150:80048,620150:4002718,9150:60013,0150:80023,924150:4002712,5150:60016,3150:80029,3Example 5 – Synthesis of HA hydrogels with higher BDDE concentrations and particle sizes from an ultra-turrax equipment

[0124] This example shows PBT technology applied to HA hydrogels with BDDE-HA molar ratios of 0.1:1 to 0.5:1. The hydrogels were obtained with high MW HA, average of 10 6 Da and a degree of crosslinking (same BDDE-HA ratio); adjustment of total HA concentration with crosslinked HA and free HA (i.e., non-crosslinked HA).

[0125] Table 4.1 shows the size combinations (600 µm and 150 µm) in various ratios, resulting in a wide range of viscoelasticity represented by G', the storage modulus, characterizing the viscoelasticity of the hydrogels. Other size combinations and ratios can be obtained, expanding the range of properties. Therefore, according to the invention, the technology offers opportunities for the fabrication of dermal fillers with different properties for a wide range of applications.

[0126] Table 4.1BDDE-AH Molar RatioHA Concentration (mg / mL)Free HA (%)Particle Size Ratio (600 to 150 µm)Gauge NeedleG' 0.1 to 10 Hz (Pa)0.1:112170:1324 to 850.1:112171:130190 to 3200.1:112174:127500 to 6800.1:115331:130140 to 2750.1:115334:127300 to 4800.5:117.5141:130580 to 11700.5:117.5144:127785 to 13200.5:120331:127550 to 11400.5:120334:127820 to 12000.5:12437.51:027840 to 1370

[0127] Table 4.2 shows the Ultra Turrax parameters, such as speed and time, calibrated for the fabrication of hydrogels with particles of specific diameter for the BDDE-AH molar ratio of 0.1:1 and 0.5:1.

[0128] Table 4.2Speed ​​(rpm)Time (min)Particle size (µm)30003600 ± 12040005500 ± 150400010400 ± 110800020250 ± 701000020150 ± 45 References

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Claims

Manufacturing process of crosslinked hyaluronic acid (HA) hydrogels comprising a mixture of two particle sizes of 150 µm and 400 µm to 800 µm and with HA concentration of 12 to 24 mg / g, the process comprising the following steps: crosslinking the HA with an average MW of 10 6Give with a molar ratio of the crosslinking agent 1,4-butanediol diglycidyl ether (BDDE) to HA of 0.075:1; conduct the reaction of HA and BDDE in a planetary centrifuge under vacuum for less than 1 hour; wash the crosslinked and swelled HA to reach a HA concentration of 12 to 24 mg of HA per g of gel; homogenize the crosslinked and swollen HA into particles using sieves of different mesh sizes and obtain gel particles of two specific diameters between 150 and 800 µm, as measured by optical microscopy; isolate a batch of HA particles with an average diameter of 150 µm and a batch of HA particles with an average diameter ranging from 400 to 800 µm; combine the particles characteristic of a diameter of 150 µm with particles of a diameter of 400 to 800 µm to manufacture the hydrogels; mix the particles of different sizes in a planetary centrifuge. Manufacturing process of cross-linked hyaluronic acid (HA) hydrogels, comprising the steps of: cross-linking the HA with a MW of 10 6 With the crosslinking agent 1,4-butanediol diglycidyl ether (BDDE) in the range of BDDE-HA molar ratios from 0.1:1 to 0.5:1 containing only 1 degree of crosslinking; conduct the reaction of HA and BDDE in a reactor with mechanical stirring at a controlled temperature and a processing time of more than 1 hour; and obtain the particles using a high-shear homogenizer, for example, an ultra-turrax equipment, with pre-calibrated rotation speed and application time. Process according to claim 1, in which particles of different average diameters are mixed and combined in step f) with a ratio of 1:4 of small particles, which have an average diameter of 150 µm, to large particles, which have an average diameter ranging from 400 to 800 µm, and then subjected to intensified mixing in step g) for homogenization of the particles. The process according to claim 1 or 3, wherein the particles combined in step f) are supplemented with about 1% w / w of free, non-crosslinked HA. Process according to claim 1, 3 or 4, wherein the HA used is in the form of sodium salt of HA (NaAH) with an average MW of 10 6 From the. Process according to any one of claims 1 to 5, wherein the crosslinking and mixing of particles is carried out in a planetary centrifuge. The method according to any one of claims 1 to 6, wherein an anesthetic, such as lidocaine, is incorporated into the hydrogel. Process according to any one of claims 1 to 7, wherein the hydrogel obtained is sterilized, for example sterilized in an autoclave. Crosslinked HA hydrogel obtained by the process as defined in any one of claims 1 to 8. An aqueous composition comprising a crosslinked HA hydrogel as defined in claim 9 and, optionally, a buffering agent and / or tonicity agent. A pre-filled syringe, wherein the syringe is pre-filled with the cross-linked HA product as defined in claim 9 or with the aqueous composition thereof as defined in claim 10. Crosslinked HA hydrogel according to claim 9 for use in cosmetic surgery, e.g., in dermal filling, body contouring, and facial contouring; in medical surgery, e.g., dermal filling, body contouring, prevention of tissue adhesion, channel formation, incontinence treatment, and orthopedic applications; or for hydration and / or revitalization of the skin. Cosmetic use of the crosslinked HA hydrogel as defined in claim 9 or the composition as defined in claim 10 as a dermal filler for the treatment of wrinkles. Dermal filler containing a crosslinked HA hydrogel as defined in claim 9.

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